BER Research Highlights

Search Date: August 16, 2017

523 Records match the search term(s):


June 19, 2017

Isotope Delivery in Lignin: Not an Easy Path

Scientists attempt to overcome challenge of limited deuterium uptake by lignin for studies of biomass breakdown pathways.

The Science
Isotopic labeling of biological molecules has long been used to investigate complex chemical and structural interactions. In a previous study, deuterium (D) was successfully incorporated into a 50% solution of deuterated water (D2O) in three grass species and Lemna duckweed. However, while isotopic labeling with deuterium using 50% D2O in higher plants is promising for understanding plant cell wall structure, it has exhibited low deuterium uptake in lignin while higher D2O concentrations inhibit growth. In this study, the objective was to determine if deuterium isotopic labeling can be targeted to lignin through the absorption of deuterated phenylalanine by roots of growing whole plants.
The Impact
Lignin plays key roles in biomass recalcitrance, pyrolysis, biochar, manufacture of carbon fiber and other products, therefore imaging of lignin in biomass is an important research tool. By enabling deuteration of natural lignocellulosic plant biomass, unique insights can be found using nuclear magnetic resonance (NMR) and small-angle neutron scattering (SANS) and a host of other bioimaging techniques.

Summary
Researchers at Oak Ridge National Laboratory (ORNL) examined the effects of phenylalanine and deuterated phenylalanine in four species of monocotyledonous plants: two annual grasses, one perennial grass, and duckweed. Switchgrass, a dedicated bioenergy perennial crop, was observed to grow at a similar rate to the control plants when in a 2mM deuterated phenylalanine concentration well. Similarly, winter rye grain, a forage and winter cover crop, was able to tolerate deuterated phenylalanine at the same concentration. Annual ryegrass, a forage and amenity grass also used for phytoremediation and toxicity studies, had significantly reduced growth rates with phenylalanine—less inhibition was observed with deuterated phenylalanine. Duckweed, a small aquatic plant commonly used for toxicity tests, exhibited toxic effects with both phenylalanine and deuterated phenylalanine. Overall, deuterium was not incorporated at a high enough level (30-40%) for lignocellulosic neutron scattering studies. However, the observed 0.5-3% levels of deuterium incorporation may be high enough for discovery of metabolic pathways through mass spectroscopy or other imaging techniques. This research aligns with DOE’s bioenergy and environmental missions.

PM Contact
Roland Hirsch Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
Roland.Hirsch@science.energy.gov

PI Contact
Barbara Evans
Chemical Sciences Division
Oak Ridge National Laboratory
evansb@ornl.gov

Funding
This research was supported by the U. S. Department of Energy, Office of Science, through the Genomic Science Program, Office of Biological and Environmental Research, under Contract FWP ERKP752. The research at Oak Ridge National Laboratory’s Center for Structural Molecular Biology (CSMB) was supported by the U. S. Department of Energy, Office of Science, through the Office of Biological and Environmental Research under Contract FWP ERKP291, using facilities supported by the Office of Basic Energy Sciences, U. S. Department of Energy. R. Shah was partly supported by the graduate fellowship program of the Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville. C. Howard, F. Lavenhouse, and D. Ramirez, with K. Ramey as teacher-mentor, were supported by the Siemens Foundation through the Siemens Teachers As Research Scientists (STARS) summer 2014 program administered by Oak Ridge Institute of Science and Education, Oak Ridge Associated Universities. V. Cangemi, B. Kinney, C. Partee, and T. Ware were participants in the Appalachian Regional Commission/Oak Ridge National Laboratory Summer Math Science Technology Institute 2015 summer program. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U. S. Department of Energy under Contract DE-AC05-00OR22725.

Publications
B.R. Evans, G. Bali, A. Ragauskas, R. Shah, H. O’Neill, C. Howard, F. Lavenhouse, D. Ramirez, K. Weston, K. Ramey, V. Cangemi, B. Kinney, C. Partee, T. Ware, and B. Davidson, “Allelopathic effects of exogenous phenylalanine: A comparison of four monocot species” Planta (2017). [DOI:10.1007/s00425-017-2720-x] (Reference link)

Related Links
Science Focus Area: Oak Ridge National Laboratory (ORNL) Biofuels Program  

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Switchgrass was grown hydroponically in perdeuteration chambers on deuterated phenylalanine to label lignin and on 50% D2O to label the entire biomass. [Image courtesy of Barbara Evans, Oak Ridge National Laboratory (ORNL)]



May 30, 2017

Unlocking the Potential of Fungal Enzymes to Break Down Plant Cell Walls

Biomass-degrading enzyme complexes could improve biofuel production.

The Science
A major bottleneck in biofuel production is the difficulty of breaking down lignocellulose—the primary building block of plant cell walls. A recent study suggests that unlike bacterial enzyme complexes for breaking down lignocellulose, fungal enzyme complexes have a more diverse functionality.

The Impact
The findings highlight the powerful degradation activity of fungal lignocellulose-degrading enzymes, which could be harnessed to develop novel strategies for efficient biofuel production.

Summary
Gut microbes play a major role in helping ruminants such as cows, goats and sheep break down lignocellulose-rich plant matter in their diet. Anaerobic bacteria and fungi inhabiting the ruminant gut have evolved a suite of lignocellulose-degrading enzymes, whose activity supports microbial metabolism while supplying nutrients to ruminants. These enzymes often assemble together in large, multi-protein complexes called cellulosomes, which enhance the ability of gut microbes to degrade lignocellulose by confining all the enzymes in one place. Although bacterial cellulosomes now serve as a standard model for biomass conversion and synthetic biology applications, fungal cellulosomes have not been well characterized due to the lack of genomic and proteomic data, despite their potential value for biofuel and bio-based chemical production. To address this knowledge gap, a collaborative effort by researchers from the University of California, Santa Barbara, the Environmental Molecular Sciences Laboratory ( EMSL); the Department of Energy Joint Genome Institute (DOE JGI); Pacific Northwest National Laboratory; Centre National de la Recherche Scientifique; French National Institute for Agricultural Research; Radboud University; King Abdulaziz University; and the University of California, Berkeley combined next-generation sequencing with functional proteomics to describe the comprehensive set of proteins that play a role in fungal cellulosome assembly. This research was performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and used resources at DOE JGI and EMSL, which are DOE Office of Science User Facilities. This analysis revealed a new family of genes that likely serves as scaffolding proteins critical for cellulosome assemblies across diverse species of anaerobic gut fungi. Unlike bacterial cellulosomes, which have high species specificity, fungal cellulosomes are likely a composite of enzymes from several species of gut fungi. Although many bacterial and fungal plant biomass-degrading enzymes have shared similarities, the fungal cellulosomes were found to contain additional lignocellulose-degrading enzymes not found in bacterial cellulosomes. These features may not only confer a selective advantage of fungi over bacteria in the ruminant gut, but also impart fungal cellulosomes with great potential for biomass conversion. Taken together, the findings highlight key differences in bacterial and fungal cellulosomes and suggest enzyme connections (known as tethering) play such an important role in plant cell wall degradation.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contact
Michelle A. O’Malley
University of California, Santa Barbara
momalley@engineering.ucsb.edu

EMSL Contact
Sam Purvine
EMSL
samuel.purvine@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy’s Office of Science (Office of Biological and Environmental Research), including support of the Environmental Molecular Sciences Laboratory (EMSL) and the DOE Joint Genome Institute (DOE JGI), DOE Office of Science User Facilities; U.S. Department of Agriculture; National Science Foundation; U.S. Army; University of California, Santa Barbra and Berkeley; and California NanoSystems Institute.

Publication
C.H. Haitjema, S.P. Gilmore, J.K. Henske, K.V. Solomon, R. de Groot, A. Kuo, S.J. Mondo, A.A. Salamov, K. LaButti, Z. Zhao, J. Chiniquy, K. Barry, H.M. Brewer, S.O. Purvine, A.T. Wright, M. Hainaut, B. Boxma, T. van Alen, J.H.P. Hackstein, B. Henrissat, S.E. Baker, I.V. Grigoriev, and M.A. O’Malley, “A Parts List for Fungal Cellulosomes Revealed by Comparative Genomics.” Nature Microbiology (2017). DOI 10.1038/nmicrobiol.2017.87 (Reference link)

Related Links
Unlocking the Potential of Fungal Enzymes to Break Down Plant Cell Walls EMSL science highlight
Fungal Enzymes Team Up to More Efficiently Break Down Cellulose DOE JGI news release
Biofuel breakdown science highlight

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Lignocellulose-degrading enzymes in fungal cellulosomes have great potential for biomass conversion. [Image courtesy of EMSL]



May 23, 2017

First Look at a Living Cell Membrane

Neutrons provide the solution to nanoscale examination of living cell membrane and confirm the existence of lipid rafts.

The Science
The cell membrane, a thin bilayer of lipid molecules with embedded proteins, provides the essential function of protecting the cell from its outside environment and controlling the movement of substances in and out of the cell. However, much about this thin bilayer of lipid molecules has remained a mystery despite being extensively studied. This has been due to the difficulty of viewing a living cell membrane; previous methods employed to investigate membrane structure, such as X-rays and electron beams, were not well suited for studying living cells due to their high-energy nature (>5,000 eV) that damage membranes. Using cold neutrons with a low kinetic energy (<0.025 eV), for the first time, researchers performed direct nanoscale examination of a living cell membrane.

The Impact
Using isotopes to create internal contrast within living cells the membrane structure and thickness of the bacterium, B. subtilis, was determined. In addition, the researchers were able to confirm the existence of the long hypothesized presence of lipid rafts, tightly packed free-floating membrane lipids and proteins thought to be important to cell signaling and facilitating movement of essential biomolecules in and out of the cell, along with a variety of other functions. The methods developed may prove valuable in areas of interest to DOE such as biomass feedstock and biofuel production, in which bacteria have an important role.

Summary
Examining a living cellular membrane has remained an unsolved challenge up to this point due to the dynamic, chemically diverse, and fragile nature of living cells. Too small to be seen by a traditional optical microscope, neutrons emerged as the solution to studying a living lipid bilayer at nanoscale without damaging the cell. Neutrons can be used as a probe for characterizing biological materials because a neutron beam scattered by a biological sample creates a pattern that is dependent on the material’s isotopic composition and reflects the material’s structural arrangement. Deuterium is an isotope of a highly abundant atom in biological matter, hydrogen. It contains a neutron and a proton, in contrast to hydrogen, which contains a single proton but no neutron. This seemingly small difference makes substituting deuterium for hydrogen an ideal approach to studying membranes and other nanoscale biological systems.  Cells perceive little difference between hydrogen and its isotope, deuterium, while the isotopes appear very differently using the neutron scattering technique. A team of researchers at Oak Ridge National Laboratory (ORNL) was able to introduce enough deuterium into the membrane of the bacterium B. subtilis to differentiate it from other cell components. Further, the team was able to tune the specific proportions of deuterium and hydrogen by introducing into the cell two fatty acid (the molecules that comprise the membrane lipids) types, with unique isotope ratios. The cell incorporated the specific mix of isotope-labeled fatty acids into its membrane and a non-uniform distribution of lipids was observed, confirming the lipid raft hypothesis. These experiments answer some of biology’s longest-standing questions, aligning with the DOE Office of Science mission of providing fundamental science research to address some of the most pressing challenges of our time.

PM Contact
Amy Swain, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
Amy.Swain@science.doe.gov

PI Contact
John Katsaras
katsarasj@ornl.gov

Funding
This research was sponsored by the Laboratory Directed Research and Development Program (grant number 6988) of Oak Ridge National Laboratory (ORNL), managed by UT-Battelle, LLC, for the U. S. Department of Energy (DOE) under Contract No. DE-AC05-00OR22725. Support for J.K. provided by the DOE Office of Basic Energy Sciences, Scientific User Facilities Division and for R.F.S by the DOE Office of Biological and Environmental Research (grant number ERKP-851). This research used resources of the Oak Ridge Leadership Computing Facility at ORNL, supported by the DOE Office of Advanced Scientific Computing Research, Facilities Division. Small-angle neutron scattering was performed at ORNL using the Bio-SANS instrument at the High Flux Isotope Reactor, supported by the DOE Office of Biological and Environmental Research, Biological Systems Science Division, through the ORNL Center for Structural Molecular Biology, and the EQ-SANS instrument at the Spallation Neutron Source, supported by the DOE Office of Basic Energy Sciences, Scientific User Facilities Division (grant number ERKP-SNX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Publications
J.D. Nickels, S. Chatterjee, C.B. Stanley, S. Qian, X. Cheng, D.A.A. Myles, R.F. Standaert, J.G. Elkins, and J. Katsaras, “The in vivo structure of biological membranes and evidence for lipid domains,” PLoS Biology 5, 15. DOI:10.1371/journal.pbio.2002214. (Reference link)

Related Links
ORNL Press Release: Neutrons provide the first nanoscale look at a living cell membrane

Topic Areas:



Neutron scattering is a valuable technique for studying cell membranes, but signals from the cell’s other components such as proteins, RNA, DNA and carbohydrates can get in the way (left). An ORNL team made these other components practically invisible to neutrons by combining specific levels of heavy hydrogen (deuterium) with normal hydrogen within the cell (right). [Image courtesy of ORNL/Xiaolin Cheng and Mike Matheson]



May 23, 2017

Modifications to the Bacterial Cell Envelope Increase Lipid Production

A new strategy significantly increases the production and secretion of microbial lipids in bacteria that can be grown at industrial scale.

The Science
High-yield microbial production of lipids presents a significant challenge, often falling short of what can be theoretically obtained. This study characterized high-lipid (HL) mutant variants of Rhodobacter sphaeroides and showed that alterations to the bacterial cell envelope can result in increased accumulation of lipids relative to the parent strain.

The Impact
Knowledge of the mechanisms that limit microbial lipid production can reveal new strategies to increase lipid yield and the economic viability of alternatives to fuels or chemicals currently derived from petroleum.

Summary
Microbial lipids are potential replacements for petroleum-based fuels and chemicals; however, their production often falls short of theoretical yield, and improvement strategies are needed. Researchers from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, and Environmental Molecular Sciences Laboratory (EMSL; a DOE Office of Science user facility) advanced their research on microbial lipid production by examining a new class of Rhodobacter sphaeroides mutants that exhibited enhanced lipid accumulation relative to the parent strain. The researchers used EMSL’s FEI Tecnai T-12 cryo-transmission electron microscope and structured illumination super resolution fluorescence microscope, in which chemical sensitivity profiles indicated HL mutants were sensitive to drugs that target the cell envelope. Changes in cell shape were also observed, suggesting that previously undescribed alterations in the bacterial cell envelope could be used to increase bacterial lipid production. Importantly, a subset of the HL mutants were able to secrete lipids, two of which accumulated approximately 60 percent of their total lipids extracellularly, potentially enabling easy product recovery from a bioreactor. When one of the highest lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to oleaginous microbes, defined as those accumulating 20 percent or more of their dry cell weight as lipid. Knowledge of the biological mechanisms that limit lipid production can inform new genetic engineering and growth strategies and enable this important class of molecules to be adopted as fuels or chemicals on a larger scale.

BER PM Contacts
Paul Bayer, SC-23.1, 301-903-5324
Kent Peters, SC-23.2, 301-903-5549

PI Contact
Timothy J. Donohue
University of Wisconsin-Madison
tdonohue@bact.wisc.edu

EMSL Contacts
Alice Dohnalkova
EMSL
Alice.Dohnalkova@pnnl.gov

Dehong Hu
EMSL
Dehong.Hu@pnnl.gov

Galya Orr
EMSL
Galya.Orr@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, including support of the Environmental Molecular Sciences Laboratory and Great Lakes Bioenergy Research Center.

Publication
Lemmer, K. C., W. Zhang, S. J. Langer, A. C. Dohnalkova, D. Hu, R. A. Lemke, J. S. Piotrowski, G. Orr, D. R. Noguera, and T. J. Donohue. 2017. “Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production,” mBio 8(3), e00513-17. DOI: 10.1128/mbio.00513-17. (Reference link)

Related Links
Modifications to the Bacterial Cell Envelope Increase Lipid Production, EMSL science highlight
Modifying Cell Wall Can Increase Bacterial Lipids, Great Lakes Bioenergy Research Center highlight
Lipid Biofuels EMSL science highlight
Enhancing Microbial Lipid Production, Great Lakes Bioenergy Research Center highlight

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 19, 2017

High Yield Biomass Conversion Strategy Ready For Commercialization

Researchers demonstrate 80% of biomass is converted into high-value products.

The Science
Researchers show that the three main components of plant biomass can be converted to high value products in economically favorable yields. Using the concept of an integrated biorefinery multiple products streams are produced, comparable to the process of a petroleum refinery, making lignocellulosic (nonedible) biomass a promising alternative source of carbon. The products produced--dissolving pulp, furfural, carbon foam, and battery anodes--have established markets, minimizing market risk for the first commercial plant.

The Impact
This technology can be expanded upon to produce fermentable sugars, advanced biofuels, or specialty chemicals, and could enable the concept of an integrated renewable biorefinery that is cost-competitive with petroleum. The techno-economic analysis estimates an overall revenue of $500 per dry megaton; this results in an internal rate of return over 30%, thereby making the technology attractive for investment.

Summary
The production of renewable chemicals and biofuels must be cost-competitive with petroleum-derived equivalents to be accepted by markets. At the Great Lakes Bioenergy Research Center (GLBRC), one of three DOE Bioenergy Research Centers (BRCs), scientists propose a biomass conversion strategy that maximizes the conversion of lignocellulosic biomass. Using this method, up to 80% of the biomass can be converted into high value products that can be commercialized, providing the opportunity for successful translation to a viable commercial process. Their fractionation method preserves the value of all three primary biomass components: cellulose, which is converted into dissolving pulp for fibers and chemical production; hemicellulose, which is converted into furfural, a building block chemical; and lignin, which is converted into carbon products (carbon foam, fibers, or battery anodes). Since these products are all existing targets for pulp mills, they can be directly introduced into current markets, minimizing market risk for the first commercial plant. The overall revenue of the process is about $500 per dry megaton of biomass, which combined with low total cost, results in an internal rate of return of over 30%. Once de-risked, the technology can be extended to produce fermentable sugars, advanced biofuels, or other specialty chemicals. This research aligns closely with DOE’s environmental and energy independence missions.

Contact
N. Kent Peters
Program Manager, Office of Biological and Environmental Research
kent.peters@science.doe.gov, 301-903-5549

(PI Contacts)
James A. Dumesic
University of Wisconsin - Madison
dumesic@engr.wisc.edu

Christos T. Maravelias
University of Wisconsin - Madison
christos@engr.wisc.edu

Troy Runge
University of Wisconsin - Madison
trunge@wisc.edu

Funding
This work was funded in part by NSF SBIR 1602713 and 1632394 and by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). Additional funding provided by Glucan Biorenewables LLC.

Publications
Alonso, D.M. et al, “Increasing the revenue from lignocellulosic biomass: Maximizing feedstock utilization.” Science Advances (2017), DOI: 10.1126/sciadv.1603301 (Reference link)

Related Links
University of Wisconsin-Madison Press Release: Triple play boosting value of renewable fuel could tip market in favor of biomass
Great Lakes Bioenergy Research Center (GLBRC)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



UW-Madison researchers and collaborators have developed a new “green” technology for converting non-edible biomass into three high-value chemicals that are the basis for products traditionally made from petroleum. [Image courtesy of Phil Biebl/UW-Madison College of Engineering]



May 08, 2017

Fungi: Gene Activator Role Discovered

High levels of DNA base modification reported in early-derived fungi.

The Science
DNA, the molecule carrying instructions for development, growth, function, and reproduction, is made up of four bases: cytosine (C), guanine (G), adenine (A), and thymine (T)–defining the genetic code. When the organism’s genetic code is modified by changing a single base this can cause changes in protein structure and function, impacting an organism’s traits. However, there are other subtler changes which can affect the activity of a DNA segment without changing the sequence. One of the most common examples involves the methylation (addition of a methyl group) of cytosine (C) on the 5th position of its carbon ring (5mC). This research explores one of the other less known modifications, adding a methyl group to base 6 of adenine (6mA) in early-diverging fungi.

The Impact
In comparison to other lineages, early-diverging fungi have not been well studied or understood. However, many of these fungi are powerful plant biomass degraders with potential bioenergy applications. In this study, the discovery of adenine methylation associated with effects on gene expression in early-diverging fungi may explain the historic difficulty in altering the DNA of these early-diverging fungi, and aid in the development of future tools for their genetic modification.

Summary
The Fungi kingdom is estimated to be ~1 billion years old; the first six phyla comprise the ‘early-diverging’ fungi and the last two phyla make-up the Dikarya, which evolved ~500 million years ago.   In this study, for the first time, 6mA base modification was identified as a widespread marker for transcriptionally active genes in early diverging fungi. The researchers examined long-read sequences from 16 diverse fungal genomes for the presence of adenine methylation. In the early-diverging fungi up to 2.8% of adenines were methylated, much higher than is seen in comparison to the eukaryotes and the more derived fungi (both less than 0.4%). Interestingly, despite fungi and animals’ closer phylogenetic relation, early-deriving fungi and algae-two distantly related kingdoms-are more similar in 6mA profiles than their more recently derived- (but more closely related)- fungi and animals. In early-derived fungi and algae, 6mA’s presence signals gene expression, while the role appears reversed in animals. This significant finding suggests 6mA’s association with gene expression is ancestral to the eukaryotic domain of life. This research also represents a previously uncharacterized difference between the role of 6mA in early-derived fungi and Dikarya of gene suppression and expression. More broadly this research highlights the variation in how 6mA is used to modify gene expression across eukaryotes, further defining the collective understanding of transcriptional regulation in this domain of life.

Contact
BER PM
Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
US Department of Energy
daniel.drell@science.doe.gov

PI
Igor V Grigoriev, Ph.D.
Fungal Genomics Program Lead, DOE Joint Genome Institute
ivgrigoriev@lbl.gov

Funding
Work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. This work was partially supported by funding from the National Science Foundation (DEB-1441715 to JES, DEB-1441604 to J.W.S. and DEB-1354625 to T.Y.J. and I.V.G.). This work was further supported by the Office of Science (BER), US Department of Energy (DE-SC0010352) and the Institute for Collaborative Biotechnologies through grant W911NF-09- 0001. R.J.S. is supported by funding from the Office of the Vice President of Research at UGA as well as the Pew Charitable Trusts.

Publications
S.J. Mondo, R.O. Dannebaum, R.C. Kuo, K.B. Louie, A.J. Bewick, K. LaButti, S. Haridas, A. Kuo, A. Salamov, S.R. Ahrendt, R. Lau, B.P. Powen, A. Lipzen, W. Sullivan, B.B. Andreopoulos, A. Clum, E. Lindquist, C. Daum, J. Magnuson, T.Y. James, M.A. O’Malley, J.E. Stajich, J.W. Spatafora, A. Visel, I.V. Grigoriev, “Widespread adenine N6-methylation of active genes in fungi” Nature Genetics (2017). [DOI: 10.1038/ng.3859] (Reference link)

Related Links
JGI Press Release: Finding a New Major Gene Expression Regulator in Fungi

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The genome of the Linderina pennispora, a fungus belonging to the earliest branches of the fungal kingdom, was sequenced and annotated as part of the Nature Genetics study. [Image courtesy of (ZyGoLife Research Consortium, Flickr, CC BY-SA 2.0)]



April 18, 2017

A Gene that Influences Grain Yields in Grasses

Genetic screen identifies mutations that impact green foxtail panicle formation

The Science
Through deep sequencing of the model grass green foxtail (Setaria viridis), researchers pinpointed a gene critical for the development of flowers that give rise to the grain. Using this information, a homologous gene in maize was identified as playing a similar role highlighting the utility of S. virdis as a model crop.

The Impact
Maize, an important food and bioenergy crop, has been limited in the progress of gene discovery due to its large and complex genome. Recently, S. virdis has been proposed as a model crop for maize. In a new study, researchers screening for mutants of the model grass green foxtail identified several mutations that disrupt the regular pattern of panicle development, the panicle is the spear-shaped flowering cluster at the tip of each branch necessary for reproduction.

Summary
Setaria species, among them green foxtail (S. viridis) and foxtail millet (S. italica), are related to several candidate bioenergy grasses including switchgrass and Miscanthus and serve as grass model systems to study grasses that photosynthetically fix carbon from CO2 through a water-conserving (C4) pathway. The genomes of both green foxtail and foxtail millet have been sequenced and annotated through the DOE JGI’s Community Science Program. A team led by Tom Brutnell at the Donald Danforth Plant Science Center and including researchers at the U.S. Department of Energy Joint Genome Institute (DOE JGI), a DOE Office of Science User Facility, reported in Nature Plants, that they had identified genes that may play a role in flower development on the panicle of green foxtail.

The team identified four recessive mutants, tagged spp1 through spp4, that lead to panicles with reduced and uneven flower clusters. Focusing on the spp1 mutation, they performed deep sequencing to specifically locate the genes that cause the mutation, narrowing their search down to a 1-million base sequence. They ultimately identified the SvAUX1 gene in green foxtail as one critical for flower cluster development in green foxtail. Panicle development is critical for determining grain yield that is crucial to food crops as well as candidate crops for producing renewable and sustainable fuels.  A homologous gene in maize was identified as playing a similar role, illustrating the value of model systems in finding genes involved in important properties in potential bioenergy-relevant plants.

BER Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
US Department of Energy
daniel.drell@science.doe.gov

Pablo Rabinowicz, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
US Department of Energy
Pablo.Rabinowicz@science.doe.gov

PI Contacts
Jeremy Schmutz
Plant Program Head
DOE Joint Genome Institute
jschmutz@hudsonalpha.com

Thomas Brutnell
Donald Danforth Plant Science Center
tbrutnell@danforthcenter.org

Funding
This work was conducted by the U.S. Department of Energy’s (DOE) Joint Genome Institute, a DOE Office of Science user facility (contract number DE-AC02-05CH11231). This work was also supported by a Department of Energy grant to T. Brutnell (DE-SC0008769), and a National Science Foundation grant to E.A. Kellogg (IOS-1413824).

Publications
P. Huang, H. Jiang, C. Zhu, K. Barry, J. Jenkins, L. Sandor, J. Schmultz, M.S. Box, E.A. Kellogg, and T.P. Brutnell “Sparse panicle1 is required for inflorescence development in Setaria viridis and maize” Nat. Plants. (2017) DOIi: 10.1038/nplants.2017.54 (Reference link)

Related Links
Green foxtail Community Science Program proposal
Foxtail millet Community Science Program proposal
Foxtail millet genome publication
Community Science Program
Setaria italica on Phytozome

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Traits including its relatively small genome and short life cycle make green foxtail (Setaria viridis) a model grass system for finding genes involved in important properties in potential bioenergy-relevant plants. (Jose Sebastian, Wikimedia Commons, CC BY-SA 3.0)



April 17, 2017

Biosynthetic Pathway to Nylons

Biological production of cyclic precursors of nylon and related products avoids harsh chemicals and high heat.

The Science
Nylons are a class of widely used synthetic compounds known for their extreme toughness, strength, and elasticity. There are millions of tons produced annually for commercial applications as fibers (apparel, rubber reinforcement, flooring), shapes (electrical equipment, parts for cars, etc.) and films (mostly food packaging). Five- and six-member ring lactams are important commodity chemicals used as precursors in the manufacture of nylons. (Precursors are compounds that participate in a chemical reaction producing another compound). However, biological production of these highly-valued chemicals has been limited due to a lack of appropriate enzymes that form these lactam rings. An enzyme in the bacteria Streptomyces aizunensis has been enlisted to fulfill a crucial step in biological synthesis of these precursors.  

The Impact
Current commercial nylon production methods start with crude oil that requires energy intensive processes and harsh acidic reaction conditions. This is the first study to demonstrate a suitable enzyme for synthesis of lactams under microbial fermentation conditions for the manufacture of nylon. A Streptomyces enzyme enables five-membered, six-membered and even seven-membered ring formation at mild temperatures, resulting in the production of important industrial lactams via fermentation from biological molecules avoiding petroleum, harsh chemicals and heat.

Summary
Five- and six-member ring lactams are important commodity chemicals because they are used as precursors in the manufacture of nylons with millions of tons produced annually requiring harsh conditions. Biological production of these highly-valued precursors will reduce the need of petroleum and avoid toxic harsh conditions. However, biological production has been limited due to a lack of enzymes that carry out crucial steps at room temperature and pressure. DOE Joint BioEnergy Institute (JBEI) researchers demonstrated production of these precursors using an acyl-CoA ligase from Streptomyces aizunensis. This enzyme has a broad substrate spectrum and can cyclize linear amino acids into their corresponding cyclic lactam when expressed in Escherichia coli. Further metabolic engineering of the pathway lead to production of the lactams directly from the common amino acid lysine. This research advances DOE’s environmental and energy missions.

PM Contact
N. Kent Peters
Program Manager, Office of Biological and Environmental Research
kent.peters@science.doe.gov, 301-903-5549

PI Contact
Jay Keasling
CEO, Joint BioEnergy Institute, Emeryville, California 94608, United States
jdkeaslilng@lbl.gov

Funding
This work was funded by the Joint BioEnergy Institute (http:// www.jbei.org/), which is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy and The Synthetic Biology Engineering Research Center (SynBERC) through National Science Foundation grant NSF EEC 0540879

Publications
J. Zhang, J.F. Barajas, M. Burdu, G. Wang, E.E. Baidoo, and JD. Keasling (2017) “Application of an Acyl-CoA Ligase from Streptomyces aizunensis for Lactam Biosynthesis” ACS Synthetic Biology [DOI: 10.1021/acssynbio.6b00372] (Reference link)

Related Links
JBEI Website

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Biological production of lactams has been limited due to a lack of enzymes that cyclize ?-amino fatty acid precursors to corresponding lactams under ambient conditions.  In this study, we demonstrated production of these chemicals using ORF26, an acyl-CoA ligase involved in the biosynthesis of ECO-02301 in Streptomyces aizunensis. [Image courtesy of JBEI]



April 07, 2017

Tracking Genome Expansion in Giant Viruses

Piecemeal acquisition of genes from hosts may explain the rise of giant viruses.

The Science
The number of microbes in, on, and around the planet is said to outnumber the stars in the sky. The number of viruses found worldwide is at least an order of magnitude greater. As their name suggests, giant viruses are larger than many bacterial and eukaryotic cells. They were first discovered in 2003, and the true breadth of their diversity remains unknown. Researchers recently uncovered a new group of giant viruses after sifting through metagenomic datasets. Dubbed Klosneuviruses, these giant viruses contain a more complete set of translation machinery genes than any other virus known to date.  

The Impact
Contrary to popular belief, most viruses do not affect humans. They do, however, impact microbes, which regulate biogeochemical cycles. Protists such as algae, for example, sequester large fractions of carbon in the atmosphere and are key components of the global carbon cycle. Viruses can significantly impact the productivity of the protist population, reducing their capabilities in regulating global cycles. As protists are thought to be the host of these Klosneuviruses, a better understanding of how viruses impact microbial survival and community interactions is relevant to Department of Energy (DOE) missions in bioenergy and environment.

Summary
While sifting through metagenomic sequence datasets for a DOE Joint Genome Institute (JGI) Community Science Program project, DOE JGI researchers identified genome sequences typically found in giant viruses. A group of giant viruses called Mimiviruses was first discovered in 2003, and a handful of such groups have been reported since. DOE JGI researchers assembled a 1.57-million base (Megabase) genome of a putative virus they called Klosneuvirus, and further searching through the metagenomic datasets uncovered three more related giant virus genomes. Three of the four Klosneuviruses were found with representatives of the protist phylum Cercozoa. This is unusual because until now, all giant viruses had been recovered with Acanthamoeba (amoebas found in soils and fresh waters), which was not seen with the Klosneuviruses. The team also found that the Klosneuviruses encoded components for a far more expansive translation system than had been seen with other giant viruses. Aside from increasing the known gene pool of giant viruses by nearly 2,500 additional gene families, comparing the genes to previously discovered giant viruses revealed that the Klosneuviruses are a subfamily of Mimiviruses. Starting then from their last shared ancestor with the Mimiviruses, the researchers suggest that over time, the Klosneuviruses picked up genes from various different hosts. Overall, the team’s findings lend credence to the theory that giant viruses evolved from much smaller viruses, rather than aligning with theories that they may instead be descended from a cellular ancestor. The consequences of Klosneuvirus infection of protist hosts remains to be explored.

Contacts (BER PM)
Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
daniel.drell@science.doe.gov 

(PI Contact)

Tanja Woyke
Microbial Genomics Program Lead
DOE Joint Genome Institute
twoyke@lbl.gov

Funding
This work was conducted by the U.S. Department of Energy’s (DOE) Joint Genome Institute, a DOE Office of Science user facility (contract number DE-AC02-05CH11231). Additional support was provided by the U.S. Department of Health and Human Services, European Research Council, Austrian Science Fund, and John Templeton Foundation.

Publication
Schulz, F., N. Yutin, N. N. Ivanova, D. R. Ortega, T. K. Lee, J. Vierheilig, H. Daims, M. Horn, M. Wagner, G. J. Jensen, N. C. Kyrpides, E. V. Koonin, and T. Woyke. 2017. “Giant Viruses with an Expanded Complement of Translation System Components,” Science 356(6333), 82-85. DOI: 10.1126/science.aal4657. (Reference link)

Related Links
JGI Earth’s viral diversity
JGI IMG/VR database
JGI Surveying viral populations
JGI Community Science Program

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Giant virus acquiring genes from different eukaryotic host cells. [Image courtesy Ella Maru Studio]



March 23, 2017

Small Proteins Secreted by Poplar Roots Form Communication Route with Associated Fungal Communities

The Science  
Small proteins that are secreted from the plant roots were found to act as communication signals between the plant and associated beneficial fungus. These molecular cues have the potential to alter development of both beneficial and pathogenic fungi.

The Impact
This work elucidates the complex communications that occur between mutualistic plant-microbe interactions and the influence of such associations on both partners, opening new avenues for development of high-yielding, sustainable bioenergy field crops.

Summary
Microbial communities surrounding plant roots can form symbiotic associations with the plant, an interaction that requires complex communications between both organisms. Mutualistic associations offer several benefits to the plant such as enhanced growth and tolerance to drought. Mutualistic fungi have evolved elaborate protein-based signals (effectors) that communicate their metabolic requirements to their plant hosts; in turn, root exudates contain small secreted proteins (SSPs) that influence mutualism with the microbes and could function as effector proteins during symbiotic interactions. While many new SSPs have been discovered through annotation of plant genome sequences, their roles as secreted effector proteins during mutualistic symbiosis was uncertain. Researchers at the Oak Ridge National Laboratory, supported by the DOE BER Plant-Microbe Interfaces Scientific Focus Area, used computational prediction and experimentation to identify SSPs in the bioenergy tree Populus trichocarpa and elucidate their effect during mutualistic symbiosis with the ectomycorrhizal fungus, Laccaria bicolor. Of the 2,819 Populus protein-encoding genes that were identified as differentially expressed across all stages of mycorrhizal root tip development during symbiosis between P. trichocarpa and L. bicolor, 417 were predicted to be SSPs (=250 aa in length). Experimentation verified that a subset of these SSPs were able to enter and accumulate in L. bicolor, then alter the development of multiple ectomycorrhizal and pathogenic fungi. This study demonstrates that SSPs in Populus can function as effector proteins during symbiotic interactions, highlighting a novel avenue by which plants communicate with and possibly influence their mutualistic microbial partners.

Contacts (BER PM)
Cathy Ronning 
SC 23.2
catherine.ronning@science.doe.gov

(PI Contact)
Xiaohan Yang
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN
yangx@ornl.gov

Funding
Genomic Science Program, US Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant-Microbe Interfaces Scientific Focus Area (http://pmi. ornl.gov)

Publications
J.M. Plett, H. Yin, R. Mewalal, R. Hu, T. Li, P. Ranjan, S. Jawdy, H.C. DePaoli, G. Butler, T.M. Burch-Smith, H-B Guo, C.J. Chen, A Kohler, I.C. Anderson, J.L. Labbé, F. Martin, G.A. Tuskan, and X. Yang, “Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis.” Scientific Reports 7:382 (2017). [DOI:10.1038/s41598-017-00400-8]. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The Populus-Laccaria association established in vitro. Photo credits: Anne Jambois, INRA.



March 17, 2017

Grasses: The Secrets Behind Their Stomatal Success

Finding a grass gene impacting stomatal morphology underscores the importance of developing a mutant gene index.

The Science
The evolution of adjustable pores, or stomata, enables plants to modify their stomatal pore size to control the amount of CO2 that enters and water that escapes. Plants have evolved two kidney shaped guard cells that swell to create the stomate. In grasses, however, they have further evolved with the addition of two subsidiary cells flanking the guard cells, which may be linked to improved stomatal physiology. In a recent study, researchers identified a transcription factor needed for subsidiary cell formation using a genetic screen.

The Impact
Subsidiary cells, unique to grasses, have been linked to improved physiological performance. These cells enable a greater range of pore size and quicker stomatal responsiveness. The ability to better control water loss and increase carbon assimilation in plants could affect its ability to handle stressors such as drought and play a role in the health and yields of candidate bioenergy feedstocks. Understanding the water management mechanism could aid the identification and selection of individuals better suited for growing in otherwise marginal soils.

Summary
Brachypodium distachyon is a small, rapidly growing grass that serves as a model for candidate bioenergy grasses such as Miscanthus and switchgrass. For this reason, in 2010, the B. distachyon genome was sequenced and annotated as part of the Community Science Program of the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI), a DOE Office of Science user facility. To further accelerate research in the development of biofuel feedstocks, a project to sequence thousands of B. distachyon mutants was selected for the 2015 CSP portfolio. This library of sequenced mutants will aid researchers in studying and rapidly identifying and ordering plants with mutations in any gene in their genomes.

Using a forward genetic screen, a Stanford University team identified a B. distachyon subsidiary cell identify defective (sid) mutant; as a result, the mutant is unable to produce subsidiary cells. In comparing the whole genome sequence of B. distachyon with the sid mutant, a 5-base pair deletion that encodes for the transcription factor BdMUTE was discovered. Further, BdMUTE was identified as a mobile transcription factor responsible for coordinating the development of subsidiary and guard cell complexes. The unique subsidiary cells in grasses may enable enhanced performance when stressors such as increased temperature or drought are placed on the plant. Though his contribution to the work predates his time at DOE JGI, JGI’s Plant Functional Genomics lead and study co-author John Vogel provided the team with the mutant population and showed them how to manipulate the plant for their studies.

Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Science Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
daniel.drell@science.doe.gov

John Vogel
Plant Functional Genomics Lead
DOE Joint Genome Institute
jpvogel@lbl.gov

Funding
U.S. Department of Energy Office of Science
Swiss National Science Foundation
The Gordon and Betty Moore Foundation
National Science Foundation
Howard Hughes Medical Institute

Publication
Raissig, M. T., J. L. Matos, M. X. A. Gil, A. Kornfeld, A. Bettadpur, E. Abrash, H. Allison, G. Badgley, J. P. Vogel, J. A. Berry, and D. C. Bergmann. 2017. “Mobile MUTE Specifies Subsidiary Cells to Build Physiologically Improved Grass Stomata,” Science 35(6330), 1215â€"18. DOI: 10.1126/science.aal3254. (Reference link)

Related Links
Stanford Press Release: Scientists reveal how grass developed a better way to breathe
JGI Brachypodium Resources
JGI Plant Flagship Genomes
JGI News Release: First Wild Grass Species and Model System for Energy Crops Sequenced
Brachypodium distachyon on Phytozome portal
JGI: Indexed Collection of Brachy Mutants

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Researcher John Vogel with Brachypodium plants at the Department of Energy’s Joint Genome Institute. [Image courtesy Lawrence Berkeley National Laboratory]



March 15, 2017

Phosphate Stress and Immunity Systems in Plants are Orchestrated by the Root Microbial Community

Better understanding of these plant-microbe interactions could lead to improved bioenergy feedstocks.

The Science 
The microbial community associated with plant roots coordinates the simultaneous response of plants to both nutrient stress and disease. In a recent study, researchers established that a genetic network controlling the phosphate stress response influences how the root microbiome community is structured, even under nonstress phosphate conditions.

The Impact
Understanding how plants interact with beneficial soil microbial communities may lead to novel approaches for breeding high-yielding bioenergy feedstocks on marginal lands with few inputs. This study, for the first time, provides evidence that genes controlling phosphate starvation response (PSR) and plant defense regulation are coordinated.

Summary
To become a sustainable and viable source of biofuels, biomass feedstock crops must be capable of high productivity on marginal lands not fit for food crop production. Nutrients such as phosphorus are critical to plant productivity but are scarce in low-fertility soils, so breeding biomass plants that efficiently utilize nutrients even in nutrient-depleted soils is critical to their use as a sustainable and cost-effective bioenergy resource. Plants form intimate associations with the soil microbial communities that surround their root systems. These communities are diverse and can contain both pathogenic microbes that compete with the plant for nutrients as well as beneficial microbes that increase plant health, vigor, and productivity. Soil nutrient content can influence the composition of the microbial community, but the mechanisms are unknown. Researchers at the University of North Carolina at Chapel Hill, with partial funding from the U.S. Department of Energy-U.S.Department of Agriculture Plant Feedstocks Genomics for Bioenergy program, used mutants of the model plant Arabidopsis thaliana with altered PSR to show that genes controlling PSR contribute to normal root microbiome assembly. They discovered that the regulatory gene PHR1 can fine-tune this response. They further showed that PSR regulation and pathogen defense are coordinated, providing insight into the coordinated interchange of plant response to nutritional stress, the plant immune system, and the root microbiome, as well as a foundational basis for using the soil microbiome to enhance phosphate use efficiency in plants.

Contacts (BER PM)
Cathy Ronning
SC-23.2
catherine.ronning@science.doe.gov

(PI Contact)
Jeffery L. Dangl
University of North Carolina at Chapel Hill
dangl@email.unc.edu

Funding
Partial support for this work was provided by the U.S. Department of Energy-U.S. Department of Agriculture Plant Feedstock Genomics for Bioenergy (award DE-SC001043) and National Science Foundation INSPIRE grant IOS-1343020.

Publication
Castrillo, G., P. J. P. L. Teixeira, S. H. Paredes, T. F. Law, L. de Lorenzo, M. E. Feltcher, O. M. Finkel, N. W. Breakfield, P. Mieczkowski, C. D. Jones, J. Paz-Ares, and J. L. Dangl. 2017. “Root Microbiota Drive Direct Integration of Phosphate Stress and Immunity,” Nature 543, 513-18. DOI: 10.1038/nature21417. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Synthetic Bacterial Community Induces Typical Phosphate Starvation Phenotypes in Arabidopsis. Phenotypes of plants lacking phosphate in the presence of a 35-member synthetic community (+ Synthetic Community) or in matching axenic conditions (No Bacteria). Typical responses to phosphate starvation, including shorter primary roots and stunted shoots, are exacerbated in the presence of the bacterial community. [Image courtesy Castrillo et al. 2017. “Root Microbiota Drive Direct Integration of Phosphate Stress and Immunity,” Nature 543, 513-518. DOI: 10.1038/nature21417.]



March 14, 2017

Unplugging the Cellulose Bottleneck

Molecular-level understanding of cellulose structure reveals why it resists degradation and could lead to cost-effective biofuels.

The Science
A major bottleneck hindering cost-effective production of biofuels and many valuable chemicals is the difficulty of breaking down cellulose—an important structural component of plant cell walls. A recent study addressed this problem by characterizing molecular features that make cellulose resistant to degradation.

The Impact
The findings reveal for the first time structural differences between surface layers and the crystalline core of the two types of cellulose found in plant cell walls. These insights could help researchers develop efficient, cost-effective strategies for breaking down cellulose for renewable energy production and other industrial applications.

Summary
A molecular-level understanding of the resistance of cellulose to degradation is a key step toward overcoming the fundamental barrier to making biofuels cost-competitive. However, significant questions remain with respect to cellulose’s structure, particularly its surface layers and crystalline core. To address this knowledge gap, researchers from Washington State University; the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility ; and Pacific Northwest National Laboratory developed a novel high-resolution technique called Total Internal Reflection Sum Frequency Generation Vibrational Spectroscopy (TIR-SFG-VS) and combined it with conventional non-TIR SFG-VS to characterize molecular structures of cellulose’s surface layers and crystalline bulk, respectively. The researchers used Sum Frequency Generation for Surface Vibrational Spectroscopy at EMSL. The findings revealed for the first time the structural differences between the surface layers and the crystalline core of cellulose. By revealing cellulose’s conformation and non-uniformity, the results challenge the traditional understanding of cellulose materials and showcase the strong value of powerful spectroscopic tools in advancing knowledge about the structure of cellulose.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contact
Bin Yang
Washington State University
bin.yang@wsu.edu

Funding
This work was supported by the U.S. Department of Energy’s Office of Science (Office of Biological and Environmental Research), including support of the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science User Facility; a DARPA Young Faculty Award; and the U.S. National Science Foundation.

Publication
L. Zhang, L. Fu, H.-F. Wang, and B. Yang, “Discovery of Cellulose Surface Layer Conformation by Nonlinear Vibrational Spectroscopy.” Scientific Reports 7:44319 (2017). DOI: 10.1038/srep44319 (Reference link)

Related Links
Unplugging the Cellulose Bottleneck EMSL science highlight
New way to characterize cellulose, advance bioproducts WSU News Post

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



A research collaboration between WSU Tri-Cities, EMSL and PNNL substantially increased understanding of cellulosic biomass recalcitrance, which not only challenges traditional understanding but provides further insight into the molecular structure of cellulose that will advance bioproducts. [Image courtesy of EMSL]



February 23, 2017

Microbes Drive Methane Release from Wetlands

Study reveals how shallow wetlands act as hotspots for greenhouse gas generation.

The Science
Inland waters and wetlands are increasingly recognized as critical sites of methane emissions to the atmosphere, but little is known about the biological and geochemical processes driving the release of this powerful greenhouse gas from these ecosystems. A new study of microbial and geochemical processes in shallow wetlands known as “potholes” reveals that these wetlands are biogeochemical hot spots for some of the highest methane fluxes to the atmosphere ever reported.

The Impact
The study's findings reveal high concentrations of carbon and sulfur compounds in the Prairie Pothole Region wetlands of North America and that these wetlands support microorganisms that generate high levels of methane. Moreover, the results show that this region is a hot spot of geochemical and microbial activity and plays an important role in regional elemental cycling—the flow of chemical elements and compounds between living organisms and the physical environment.

Summary
Small ponds and lakes recently have been found to play an oversized role in degrading carbon and catalyzing fluxes of greenhouse gases such as methane and carbon dioxide to the atmosphere. The Prairie Pothole Region is a huge wetland ecosystem containing thousands of shallow wetlands that span five states in the United States and two provinces in Canada. This region's wetland sediments contain some of the highest concentrations of dissolved organic carbon and sulfur compounds ever recorded in terrestrial aquatic environments. The observations suggest that these wetlands likely support high levels of microbial activity, which, in turn, could account for substantial greenhouse gas emissions from this ecosystem. To explore this possibility, researchers from The Ohio State University; Environmental Molecular Sciences Laboratory (EMSL), a Department of Energy Office of Science user facility; and the U.S. Geological Survey conducted one of the first studies of coupled geochemical and microbial processes driving methane emissions from Prairie Pothole Region wetlands. They collected sediment and pore water samples from these wetlands; used chemical analysis techniques to measure the concentrations of carbon, sulfur and methane; and conducted gene sequencing to identify members of the microbial community. They also performed in-depth chemical analysis of the dissolved carbon pools using 600-MHz nuclear magnetic resonance (NMR) spectrometers and the 12 Tesla Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer at EMSL. The findings suggest that conversion of abundant carbon pools into methane in the Prairie Pothole Region results in some of the highest fluxes of this greenhouse gas to the atmosphere ever reported. Moreover, high levels of carbon and sulfur compounds support some of the highest sulfate reduction rates ever measured in terrestrial aquatic environments. Taken together, the findings reveal a significant and previously underappreciated role for this ecosystem in supporting extremely high levels of microbial activity that directly impact terrestrial elemental cycling. As such, the results offer novel insights into how Prairie Pothole Region wetlands and other small inland waters act as hot spots for greenhouse gas generation.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contact
Michael J. Wilkins
Ohio State University
wilkins.231@osu.edu

EMSL Contacts
Malak Tfaily
malak.tfaily@pnnl.gov
David Hoyt
david.hoyt@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy’s Office of Science (Office of Biological and Environmental Research), including support of the Environmental Molecular Sciences Laboratory (EMSL) and the DOE Joint Genome Institute, both DOE Office of Science User Facilities; U.S. Geological Survey Climate and Land Use Change R&D Program; and National Science Foundation.

Publication
P. Dalcin Martins, D.W. Hoyt, S. Bansal, C.T. Mills, M. Tfaily, B.A. Tangen, R.G. Finocchiaro, M.D. Johnston, B.C. McAdams, M.J. Solensky, G.J. Smith, Y-P Chin, and M.J. Wilkins, “Abundant carbon substrates drive extremely high sulfate reduction rates and methane fluxes in Prairie Pothole Wetlands.” Global Change Biology (2017). [DOI: 10.1111/gcb.13633] (Reference link)

Related Links
EMSL Science Highlight: Microbes Drive Methane Release from Wetlands

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


February 10, 2017

Poplar Gene Enhances Lateral Root Formation and Biomass Growth Under Drought Stress

The Science 
A newly characterized poplar gene expressed primarily in roots influences the plant’s root development and drought resistance.

The Impact
This discovery will facilitate the development of bioenergy poplar trees with enhanced drought resistance, a key trait for the sustainable growth of bioenergy feedstocks on marginal lands.

Summary
Developing crops with improved drought resistance and water use efficiency is important for sustainable agriculture. These traits are particularly critical for plants to be grown as dedicated biomass feedstocks on marginal lands with little or no inputs such as irrigation. Since water is taken up by the roots, root architecture is directly related to the plant’s ability to tolerate drought conditions, and researchers have found several genomic regions (quantitative trait loci, or QTL) for root traits associated with drought resistance. However, the multigenic nature of many of these traits make using these QTL in a breeding program difficult, and few specific genes have been identified. Recently, scientists at Michigan Technological University and Oak Ridge National Laboratory used a powerful forward genetics approach known as activation tagging in the bioenergy crop poplar to identify a specific transcription factor gene (PtabZIP1-like), predominately expressed in poplar roots, that moderates the development of lateral roots and drought resistance through multiple metabolic pathways. The discovery of this gene provides a path to further knowledge of the functional mechanism of drought resistance, which could, in turn, offer potential new approaches to breeding more sustainable bioenergy feedstocks.

Contacts (BER PM)
Cathy Ronning
Biological Systems Science Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
catherine.ronning@science.doe.gov

(PI Contact)
Victor Busov
Michigan Technological University, Houghton MI
vbusov@mtu.edu

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, Genomic Science program, Plant Feedstock Genomics (DE-SC0008462); and U.S. Department of Agriculture (USDA), National Institute of Food Agriculture, Institute  of Bioenergy, Climate and Environment (grant number 2009-65504-05767). This work was also sponsored in part by DOE’s Genomic Science program (Science Focus Area ‘Plant-Microbe Interfaces’ at Oak Ridge National Laboratory) under contract DE-AC05-00OR22725  and USDA National Institute of Food Agriculture (MICW-2011-04378).

Publication
Dash, M., Y. S. Yordanov, T. Georgieva, T. J. Tschaplinski, E. Yordanova, and V. Busov. 2017. “Poplar PtabZIP1-Like Enhances Lateral Root Formation and Biomass Growth Under Drought Stress,” The Plant Journal 89(4), 692-705. DOI: 10.1111/tpj.134. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 30, 2017

Vitamin B12 Plays Broad Role in Cellular Metabolism

Scarce compound is key for microbial growth and may help shape microbial communities.

The Science
Vitamin B12 regulates the production of deoxyribonucleic acid (DNA) and many proteins required for normal cellular function. A recent study revealed that this compound plays an even greater role in cellular metabolism and growth than previously thought, and may even coordinate the behavior of complex microbial communities.

The Impact
The findings suggest that vitamin B12 helps shape microbial communities, which affect wide-ranging processes including energy and food production, the environment, and human health.

Summary
Vitamin B12 is used by all domains of life to control the production of DNA and a variety of proteins that support cellular function, but this vitamin is only produced by certain bacterial and archaeal species. A recent study showed that this compound has an unexpectedly broad influence on metabolic processes important for synthesis of DNA, ribonucleic acid (RNA), and proteins. To explore vitamin B12’s role in a variety of cellular processes, researchers from Pacific Northwest National Laboratory, Sanford-Burnham-Prebys Medical Discovery Institute, and Polytech Nice-Sophia set out to identify which proteins bind to vitamin B12. To do so, they first developed a chemical probe that mimics vitamin B12 and then directly applied the probe to live Halomonas bacterial cells. The researchers next analyzed the probe-labeled proteins using an Orbitrap mass spectrometer at the Environmental Molecular Sciences Laboratory, a Department of Energy Office of Science user facility. They found that the vitamin B12-mimicking probe interacted with 41 different proteins, including enzymes involved in the synthesis and metabolism of another B vitamin called folate, an amino acid called methionine, and a compound called ubiquinone. These metabolic processes, in turn, increase the production of DNA, RNA, and proteins. The findings reveal vitamin B12 plays a more pivotal role in cellular growth and metabolism than previously thought. As a result, this scarce compound may facilitate the coordination of cell behavior in complex microbial communities, shaping their structure, stability, and overall function.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contact
Lee Ann McCue
Environmental Molecular Sciences Laboratory
leeann.mccue@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, including support of the Environmental Molecular Sciences Laboratory, a DOE Office of Science user facility; Genome Science Program Foundational Scientific Focus Area at Pacific Northwest National Laboratory; Russian Foundation for Basic Research; and Russian Academy of Sciences.

Publication
M. Romine, D. Rodionov, Y. Maezato, L. Anderson, P. Nandhikonda, I. Rodionova, A. Carre, X. Li, C. Xu, T. Clauss, Y.-M. Kim, T. Metz, and A. T. Wright, “Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism.” Proceedings of the National Academy of Sciences (USA) 114(7), E1205-E1214 (2017). DOI: 10.1073/pnas.1612360114. (Reference link)

Related Links
EMSL Article
PNNL News Release

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER



Researchers explore functions controlled by vitamin B12 and the importance for microbial communities. [Image courtesy Department of Energy’s Environmental Molecular Sciences Laboratory]



January 25, 2017

Using Microbial Community Gene Expression to Highlight Key Biogeochemical Processes

A study of gene expression in an aquifer reveals unexpectedly diverse microbial metabolism in biogeochemical hot spots.

The Science
Researchers conducted a study of naturally reduced zones (NRZs)—biogeochemical hot spots—in the Rifle, Colo., aquifer, a legacy Department of Energy uranium mill site. They performed a state-of-the-art analysis of gene expression in the aquifer’s microbial communities, elucidating metabolic pathways and organisms underlying observed biogeochemical phases as well as revealing unexpected metabolic activities.

The Impact
NRZs, organic-rich deposits heterogeneously distributed in alluvial aquifers, modulate aquifer redox status and influence the speciation and mobility of metals. Overall, NRZs have an outsized effect on groundwater geochemistry. This study’s results highlight the complex nature of organic matter transformation in NRZs and the microbial metabolic pathways that interact to mediate redox status and elemental cycling.

Summary
Organic matter deposits in alluvial aquifers have been shown to result in the formation of NRZs, which can modulate aquifer redox status and influence the speciation and mobility of metals, significantly affecting groundwater geochemistry. In this study, researchers sought to better understand how natural organic matter fuels microbial communities within anoxic biogeochemical hot spots (or NRZs) in a shallow alluvial aquifer at the Rifle site. The researchers conducted an anaerobic microcosm experiment in which NRZ sediments served as the sole source of electron donors and microorganisms. Biogeochemical data indicated that native organic matter decomposition occurred in different phases, beginning with the mineralization of dissolved organic matter (DOM) to carbon dioxide (CO2) during the first week of incubation. This was followed by a pulse of acetogenesis that dominated carbon flux after two weeks. DOM depletion over time was strongly correlated with increases in the expression of many genes associated with heterotrophy (e.g., amino acid, fatty acid, and carbohydrate metabolism) belonging to a Hydrogenophaga strain that accounted for a relatively large percentage (roughly 8%) of the metatranscriptome. This Hydrogenophaga strain also expressed genes indicative of chemolithoautotrophy, including CO2 fixation, dihydrogen (H2) oxidation, sulfur compound oxidation, and denitrification. The acetogenesis pulse appeared to have been collectively catalyzed by a number of different organisms and metabolisms, most prominently pyruvate:ferredoxin oxidoreductase.  Unexpected genes were identified among the most highly expressed (more than 98th percentile) transcripts, including acetone carboxylase and cell-wall-associated hydrolases with unknown substrates.  Many of the most highly expressed hydrolases belonged to a Ca. Bathyarchaeota strain and may have been associated with recycling of bacterial biomass. Overall, these results highlight the complex nature of organic matter transformation in NRZs and the microbial metabolic pathways that interact to mediate redox status and elemental cycling.

Contacts (BER PM)
David Lesmes
SC-23
david.lesmes@science.doe.gov

(PI Contact)
Harry R. Beller
Senior Scientist, Lawrence Berkeley National Laboratory
HRBeller@lbl.gov

Funding
This work was supported as part of the Subsurface Biogeochemical Research Scientific Focus Area funded by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under award number DE-AC02-05CH11231. This work used the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley, supported by the National Institutes of Health S10 instrumentation grants S10RR029668 and S10RR027303.  

Publication
Jewell, T. N. M., U. Karaoz, M. Bill, R. Chakraborty, E. L. Brodie, K. H. Williams, and H. R. Beller. 2017. “Metatranscriptomic Analysis Reveals Unexpectedly Diverse Microbial Metabolism in a Biogeochemical Hot Spot in an Alluvial Aquifer,” Frontiers in Microbiology, DOI: 10.3389/fmicb.2017.00040. (Reference link)

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


January 16, 2017

Scientists Program Yeast to Turn Plant Sugars into Biodiesel

Redox metabolism was engineered in Yarrowia lipolytica to increase the availability of reducing molecules needed for lipid production.

The Science  
With the depletion of fossil fuels, biodiesel precursors produced by oleaginous (oil-producing) yeast from renewable carbohydrates are a promising alternative to fossil- and food-crops-derived fuels. However, production yields are still too low to be commercially competitive. In a new study, researchers achieved a 25% improvement in lipid production, relative to existing oil-producing yeast strains, by rewiring the metabolism in a naturally high lipid producing yeast.

The Impact
Diesel is used to power large vehicles (e.g., trucks) and is a sought after fuel source due to its high fuel efficiency and energy density. Until now, advances in microbial production of biodiesel were not significant enough to make it close to a commercially viable option. The titer, yield, and productivity of oil achieved in an engineered strain of Y. lipolytica using sugar as substrate are now close to those required to make microbial carbohydrate conversion into fuels commercially viable.

Summary
Researchers at the Massachusetts Institute of Technology used a mathematical model to identify the oil production bottlenecks in the industrial yeast Y. lipolytica. With information provided by the model, they designed several metabolic engineering strategies to increase conversion of surplus NADH (a product of glucose degradation) to NADPH, which is needed for lipid biosynthesis. Of the strategies tested, a combination of two were the most effective in lipid yield improvement. By introducing heterologous yeast and bacterial glyceraldhyde-3-phosphate dehydrogenase (GDP) genes that utilize NADP+ instead of NAD+ w into Y. lipolytica and overexpressing a bacterial malic enzyme (MCE2) in the GDP-expressing strain, an improvement of 25% over previously engineered yeasts was observed. In addition, as the engineered Y. lipolytica required less oxygen, it could be grown at higher density in the bioreactor, further increasing biomass and lipid yields. The redox engineering approach reported in this work could be optimized for converting plant biomass into biofuel precursors and other Department of Energy-relevant bioproducts.

(BER Contact)
Pablo Rabinowicz
Biological Systems Science Division
Office Biological and Environmental Research
U.S. Department of Energy
pablo.rabinowicz@science.doe.gov

(PI Contact)
Gregory Stephanopoulos
Department of Chemical Engineering
Massachusetts Institute of Technology
Cambridge, Massachusetts
gregstep@mit.edu

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under award DE-SC0008744.  

Publication
K. Qiao, T. M. Wasylenko, K. Zhou, P. Xu, and G. Stephanopoulos, “Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism.” Nature Biotechnology 35, 173 (2017). [DOI: 10.1038/nbt.3763] (Reference link)

Related Links
MIT Press Release: A step towards renewable diesel

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Yarrowia cells, at the initiation of lipogenesis, metabolically engineered to overproduce oil. [Image courtesy Stephanopoulos Lab, Massachussetts Institute of Technology]



January 09, 2017

Microbial Communities Thrive by Transferring Electrons

A newly discovered microbial metabolic process linking different microbes in a community could enhance bioenergy production.

The Science
Photosynthetic bacteria are major primary producers on Earth, using sunlight to convert inorganic compounds in the environment into more complex organic compounds that fuel all living systems on the planet. A team of researchers recently discovered a new microbial metabolic process called syntrophic anaerobic photosynthesis, which could represent an important, widespread form of carbon metabolism in oxygen-depleted zones of poorly mixed freshwater lakes.

The Impact
The discovery of syntrophic anaerobic photosynthesis reveals new possibilities for bioengineering microbial communities for use in waste treatment and bioenergy production.

Summary
Almost all life on Earth relies directly or indirectly on primary production—the conversion of inorganic compounds in the environment into organic compounds that store chemical energy and fuel the activity of organisms. Nearly half of global primary productivity occurs through photosynthetic carbon dioxide (CO2) fixation by sulfur bacteria and cyanobacteria. In oxygen-depleted environments, photosynthetic bacteria use inorganic compounds such as water, hydrogen gas, and hydrogen sulfide to provide electrons needed to convert CO2 into organic compounds. These organic compounds also make their way into the food web, where they support the growth of heterotrophs—organisms that cannot manufacture their own food. A recent study revealed a new metabolic process, called syntrophic anaerobic photosynthesis, in which photosynthetic and heterotrophic bacteria cooperate to support one another’s growth in oxygen-depleted environments. Researchers from Washington State University, Pacific Northwest National Laboratory (PNNL), China University of Geoscience, and Southern Illinois University made this discovery using the Quanta scanning electron microscope and the FEI Tecnai T-12 cryo-transmission electron microscope at the Environmental Molecular Sciences Laboratory (EMSL), a Department of Energy Office of Science user facility. Their analysis revealed that a heterotrophic bacterial species, Geobacter sulfurreducens, directly transfers electrons to a photosynthetic bacterial species, Prosthecochloris aestuarii, which uses electrons to fix CO2 into cell material. At the same time, donating electrons allows G. sulfurreducens to support its own metabolic needs by converting acetate into CO2 and water. This potentially widespread, symbiotic form of metabolism, which links anaerobic photosynthesis directly to anaerobic respiration, could be harnessed to develop new strategies for waste treatment and bioenergy production.

BER PM Contacts
Roland Hirsch (FSFA), SC-23.2, 301-903-9009
Paul Bayer (EMSL), SC-23.1, 301-903-5324

PI Contacts
Haluk Beyenal
Washington State University
beyenal@wsu.edu

Alice Dohnalkova
EMSL
Alice.Dohnalkova@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Biological and Environmental Research, Genomic Science program and is a contribution of the PNNL Foundational Scientific Focus Area (FSFA). A portion of this work was conducted at EMSL, a DOE Office of Science user facility.

Publication
P. T. Ha, S. R. Lindemann, L. Shi, A. C. Dohnalkova, J. K. Fredrickson, M. T. Madigan, and H. Beyenal, “Syntrophic anaerobic photosynthesis via direct interspecies electron transfer.” Nature Communications 8 (2017). DOI:10.1038/ncomms13924. (Reference link)

Related Links
EMSL Article
WSU Article

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER



Electron microscopy image of two distinct microbes that can, when in close association, produce electric current. Researchers studied metabolic processes in microbes for potential applications to waste treatment and bioenergy production. [Image courtesy Ha et al. 2017. DOI: 10.1038/ncomms13924. (CC-BY 4.0)]



January 03, 2017

Modified Switchgrass: Success in Biofuel-relevant Characteristics

Introduced traits remain stable for improved biofuel production in a field setting.

The Science  
The development of near-term fossil fuel alternatives is needed to reduce carbon emissions and to ensure U.S. energy security. Switchgrass, a biofuel feedstock (biological material that can be converted into a fuel), is a perennial grass that is able to grow on marginal lands and has served as an adaptable lignocellulosic bioresource. Due to its complex composition, the plant cell wall is resistant to deconstruction to its component sugars, known as recalcitrance. Overcoming this recalcitrance is necessary to enable the economic feasibility of biofuel production. In this study, researchers evaluated the physiological and chemical effects of genetically modified switchgrass lignin in a three year field study.

The Impact
Using a genetically modified line of switchgrass a team of scientists was able to demonstrate that biofuel-relevant characteristics remained stable while recalcitrance was reduced after three generations in the field. This is a vital step towards understanding how to overcome the recalcitrance problem and thus has the potential to reduce economic barriers to cost-effective biofuel production.
 
Summary
The plant cell wall is primarily made up of three biopolymers: lignin, hemicellulose, and cellulose. Lignin’s complex architecture provides structural support and pathogen defense, but it is due to these functions lignin is considered a major contributor to recalcitrance. Researchers at the Department of Energy’s (DOE) BioEnergy Science Research Center (BESC) silenced the caffeic acid O-methyltransferase (COMT) gene in the lignin biosynthesis pathway and demonstrated over three growing seasons that the genetically modified plants retained both reduced cell wall recalcitrance and lignin content in comparison to the non-transgenic controls. A 35-84% higher sugar release was reported in the lignin modified plants after a 72-h enzymatic hydrolysis without pretreatment and a 25-32% increase in enzymatic sugar release (after hydrothermal pretreatment). For years 2 and 3 in the field, lignin modified plants had 12% and 14% reduced lignin content, respectively. This study demonstrated the important traits associated with the COMT-silenced field-grown switchgrass are an increase in cell wall accessibility for sugar release and a reduction in lignin content. These traits were able to remain durable in the field for 3 years in field trials. This research helps to provide a mechanistic understanding of lignin modified switchgrass relevant to DOE’s energy and environmental missions.

Contact
Kent Peters, Ph.D.
Program Manager Biological Systems Sciences
Division Office of Biological and Environmental Research
Office of Science 
U.S. Department of Energy
kent.peters@science.energy.doe.gov

(PI Contact)
Arthur Ragauskas
aragausk@utk.edu

Funding
This work was supported by the BioEnergy Science Center, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science under Contract No. DE-AC05-00OR22725.

Publications
M. Li, Y. Pu, C. Yoo, E. Gjersing, S.R. Decker, C. Doeppke, T. Shollenberger, T.J. Tschaplinski, N.L. Engle, R.W. Sykes, M.F. Davis, H.L. Baxter, M. Mazarei, C. Fu, R.A. Dixon, Z. Wang, C.N. Stewart, and A.J. Ragauskas, “Study of traits and recalcitrance reduction of field-grown COMT down-regulated switchgrass.” Biotechnology for Biofuels 10, 12 (2017) [DOI: 10.1186/s13068-016-0695-7] (Reference link)

Related Links
BESC: Biomass formation and modification

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Field site for studying switchgrass species with enhanced biofuel-relevant characteristics [Image courtesy of BESC]



December 27, 2016

Microbial Community Interactions Drive Methane Consumption in Lakes

Understanding interactions among organisms in complex microbial communities sheds new light on globally significant environmental processes.

The Science  
Large amounts of methane, a potent greenhouse gas, are produced as a byproduct during decomposition of plant matter in the sediments of lakes and wetlands. Bacteria known as methanotrophs consume much of this methane before it can enter atmosphere. In a recent study, researchers examined community interactions among methanotrophs and other types of microbes that control this important process.

The Impact
The biological mechanisms underlying many important environmental processes can be understood only by examining cooperative processes performed by diverse communities of microbes. This study uses an elegantly constructed model experiment and genomic analysis to examine the genetic basis of these interactions and determine how they influence microbial consumption of methane in lake sediments.

Summary
Several decades of research have demonstrated the importance of bacterial methanotrophs in carbon cycling processes of lakes, wetlands, and a variety of other environments. However, methanotrophs exist as members of diverse communities of regularly co-occurring non-methanotrophic microbes, and the roles of these organisms in methane cycling are not well understood. In a recent study, researchers at the University of Washington assembled an experimental model community of methanotrophs and associated non-methanotrophic microbes previously isolated from lake sediments. Using a community-scale metaomics analysis of shifts in gene expression, the team tracked how the associated organisms influenced each other during methane-driven growth. The presence of non-methanotrophs was shown to trigger an enzymatic and metabolic shift in the methanotrophs, resulting in conversion of a portion of the available methane into methanol, which was released to fuel the growth of these microbes. Not yet clear is if the methanotrophs derive some form of reciprocal benefit from this “cross-feeding,” or if this represents a type of parasitism. In either case, these findings considerably alter current understanding of methanotrophy as it occurs in complex environmental communities and suggest that much remains to be learned about the basic biological mechanisms driving an important element of the global carbon cycle.

Contacts (BER PM)
Dr. Joseph Graber
DOE Office of Biological and Environmental Research, Biological Systems Science Division
joseph.graber@science.doe.gov

(PI Contact)
Dr. Mary Lidstrom
University of Washington
lidstrom@u.washington.edu

Funding
This study was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science program under award DE-SC-0010556.

Publication
S. M. B. Krause, T. Johnson, Y. S. Karunaratne, Y. Fu, D. A. C. Beck, L. Chistoserdova, and M. E. Lidstrom, “Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions.” Proceedings of the National Academy of Sciences (USA) 114(2), 358-63 (2017). DOI: 10.1073/pnas.1619871114. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Diverse microbial communities consume methane produced as a byproduct during decomposition of plant matter in lake sediments. Image courtesy of iStock



December 22, 2016

Metagenomics Leads to New CRISPR-Cas Systems

Researchers discover the first CRISPR-Cas9 system in archaea.

The Science
Using large amounts of metagenomic data generated by the Department of Energy’s Joint Genome Institute (DOE JGI), researchers analyzed 155 million protein coding genes from uncultivated microbial communities. This work led to the discovery of the first CRISPR- (clustered regularly interspaced short palindromic repeats) Cas9 protein in the archaeal domain, as well as two previously unknown simple bacterial CRISPR-Cas systems.

The Impact
Microbes play key roles in the planet’s cycles, and characterizing them helps researchers work toward solutions for energy and environmental challenges. Examining environmental microbial communities has enabled access to an unprecedented diversity of genomes and CRISPR-Cas systems that have many applications, including biological research. The combined computational-experimental approach that was successful in this study can be used to investigate nearly all environments where life exists.

Summary
Microbes heavily influence the planet’s cycles, but only a fraction have been identified. Characterizing the abundant but largely unknown extent of microbial diversity can help researchers develop solutions to energy and environmental challenges. In microbes, CRISPR-Cas systems provide a form of adaptive immunity, and these gene-editing tools are the foundation of versatile technologies revolutionizing research. Thus far, CRISPR-Cas technology has been based only on systems from isolated bacteria. In a study led by longtime DOE JGI collaborator Jill Banfield of the University of California, Berkeley, researchers discovered, for the first time, a CRISPR-Cas9 system in archaea, as well as simple CRISPR-Cas systems in uncultivable bacteria. To identify these new CRISPR-Cas systems, the team harnessed more than a decade’s worth of metagenomic data from samples sequenced and analyzed by DOE JGI, a DOE Office of Science user facility. The CasX and CasY proteins were found in bacteria from groundwater and sediment samples. The archaeal Cas9 was identified in samples taken from the Iron Mountain Superfund site as part of Banfield’s pioneering metagenomics work with DOE JGI. Both CasX and CasY are among some of the most compact systems ever identified. This application of metagenomics validates studies of CRISPR-Cas proteins using living organisms.

Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Science Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
daniel.drell@science.doe.gov

David Lesmes, Ph.D.
Program Manager
Climate and Environmental Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
david.lesmes@science.doe.gov

Jill Banfield
University of California, Berkeley
jbanfield@berkeley.edu

Funding
DOE Office of Science, National Science Foundation, EMBO, German Science Foundation, Paul Allen Institute, and Howard Hughes Medical Institute 

Publication
Burstein, D., et al., “New CRISPR-Cas systems from uncultivated microbes.” Nature (2016). [DOI: 10.1038/nature21059] (Reference link)

Related Links

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER



An artistic representation of the Tree of Life, with the many groups of bacteria and archaea at the upper left and eukaryotes, which include humans, at the lower right. Department of Energy Joint Genome Institute scientists are using gene-editing tools to explore microbial “dark matter.” [Artistic representation of Fig. 1 from Hug et al., “A new view of the tree of life.” Nature Microbiology 1 (2016). DOI: 10.1038/nmicrobiol.2016.48. Courtesy of Jill Banfield, University of California, Berkeley]



December 12, 2016

A New High-Throughput Genome Editing Technique to Generate Mutant Bacterial Strains

Using computer-aided design to develop a CRISPR/Cas9-based approach to cause thousands of mutations and map their effects to the mutated genes.

The Science
The generation of large collections of mutant bacterial strains is limited due to low mutagenic efficiencies and the difficulty of tracking diverse types of mutations or their combinations. Researchers at the University of Colorado in Boulder and their collaborators have taken advantage of the high editing efficiency of the CRISPR (clustered regularly interspaced short palindromic repeats) -Cas9 system, combined with synthetic bar-codes, to develop a method that can mutate thousands of genes and easily track the mutated genes to determine their effect on the bacterial physiology.    

The Impact
This new editing technique, for the first time, makes it possible to induce individual mutations throughout a bacterial genome in parallel, and associate each mutation with the resulting phenotype at single-nucleotide resolution in a single experiment. This method gives researchers the ability to design and modify microorganisms in a genome-wide manner allowing them to engineer new metabolic pathways for the production of biofuels and other relevant industrial products.

Summary
A CRISPR-enabled trackable genome engineering (CREATE) cassette was developed to include a targeting guide RNA (gRNA), a DNA sequence homologous to a given target locus in the genome, and a unique bar code to tack each mutation. A computationally designed library of over 50,000 CREATE cassettes targeting multiple genome locations was synthesized and used to induce specific mutations in a bacterial population. The resulting mutant strains were tracked by genomic sequencing showing an average editing efficiency of 70%. The CREATE library was tested on a bacterial culture under thermal stress and several hundred mutants that had previously been identified as adaptations to heat were also identified with CREATE, in addition to 140 new mutations in genes involved in the bacterial response to high temperature. Furthermore, several strains that showed high stress tolerance were the result of combinations of two or more single-nucleotide mutations that would not have been detected in normal mutagenesis experiments. The potential of CREATE to identify improved mutant strains can be used to develop new and enhanced biosynthetic abilities for the biological production of fuels and relevant chemicals.

Contacts (BER PM)
Pablo Rabinowicz
Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
Ryan Gill
Department of Chemical and Biological Engineering
University of Colorado
Boulder, Colorado
rtggtr@me.com

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DE-SC0008812. The authors also acknowledge support from the CAPES foundation.

Publications
Andrew Garst, Marcelo Bassalo, Gur Pines, Sean Lynch, Andrea Halweg-Edwards, Rongming Liu, Liya Liang, Zhiwen Wang, Ramsey Zeitoun, William Alexander, and Ryan Gill, “Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.” Nature Biotechnology 35, 48 (2017). [DOI: 10.1038/nbt.3718] (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 06, 2016

New Software Tools Streamline DNA Sequence Design-and-Build Process

These enhanced tools will accelerate gene discovery and characterization.

The Science                       
Synthetic DNA enables scientists to expand the breadth and depth of their genomic research. In a recent study, researchers developed a suite of build optimization software tools (BOOST) to streamline the design-build transition in synthetic biology engineering workflows. BOOST can automatically detect “difficult” sequences of nucleotides and redesign them for DNA synthesis, addressing DNA sequences with certain problematic characteristics (e.g., extreme % guanine-cytosine content, sequence patterns, and repeats), which decrease the success rate of DNA synthesis.

The Impact
By improving the design and manufacture of synthetic DNA through enhanced tools, scientists can accelerate gene discovery and gene characterization toward practical applications for energy and the environment.

Summary
The ability to design and manufacture synthetic DNA has opened tremendous possibilities in genomic research. In addition to providing access to samples that are difficult to find in nature (as well as crafting genomic sequences not known to occur in the natural world), manufacturing DNA enables scientists to test any sequence in a wide variety of contexts and environments. Biological computer-aided design and manufacture (bioCAD/CAM) software tools help researchers design sequences that can be critical to discovering new solutions for energy and the environment. So far, however, the software has not been able to automatically fix problematic sequences, slowing down the transition from the design to the manufacturing process and delaying the synthesis of designed DNA.

To solve this problem, researchers at the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI), a DOE Office of Science user facility, developed the BOOST suite to automate the synthetic DNA design process—and do away with the trial-and-error process scientists currently utilize to determine a sequence that can be synthesized.

The new suite of tools is available as a web application, an executable JAVA Archive (JAR), and as a representational state transfer application program interface (RESTful API). Ultimately, BOOST will accelerate the use of synthetic DNAs to explore gene functions relevant to DOE’s energy and environmental missions.

Contact (BER PM)
Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
US Department of Energy
daniel.drell@science.doe.gov

(PI Contact)
Samuel Deutsch
DOE Joint Genome Institute SDeutsch@lbl.gov

Funding
U.S. Department of Energy Office of Science

Publication
E. Oberortner, J.F. Cheng, N.J. Hillson, and S. Deutsch, “Streamlining the design-to-build transition with build-optimization software tools.” ACS Synthetic Biology (2016). DOI:10.1021/acssynbio.6b00200. (Reference link)

Related Links
BOOST
JGI: DNA Synthesis Science Program

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The goal of build optimization software tools is to streamline, in a scalable fashion, the process of designing readily synthesizable DNA fragments. [Image courtesy of the Department of Energy’s Joint Genome Institute]



November 29, 2016

A synthetic microbial ecosystem helps understand the behavior of bacterial communities


A bacterial co-culture was engineered to force two bacterial species to depend on each other to grow, shedding light on mutualism dynamics.

The Science
Researchers designed a stable co-culture in which Escherichia coli consumed sugar and produced organic acids to feed a Rhodopseudomonas palustris mutant strain that fixed and provided nitrogen for both microbes. A mathematical model was developed for this system, and the model accurately predicted how the co-culture would reach a new equilibrium when one of the microbes was genetically modified.

The Impact
Artificial co-cultures of two or more microbial species are useful tools for understanding how microbial communities behave in their natural habitat. However, the instability of co-culture systems has limited their utility for both fundamental and biotechnological studies. This research developed a microbial cross-feeding system that maintains its species composition over multiple generations, constituting a novel and important tool for understanding mutualistic relationships in natural environments and how to manipulate microbial communities for useful purposes.     

Summary
A mutant strain of R. palustris that can fix nitrogen gas and excrete ammonium was cultured together with E. coli in the presence of glucose as the only carbon source. R. palustris cannot consume glucose, but it feeds on the organic acids excreted by E. coli after it metabolizes glucose. In turn, E. coli obtains its nitrogen from the ammonium excreted by the R. palustris mutant. This cross-feeding dependency forced the co-culture to stabilize at a ratio of one E. coli cell to nine R. palustris cells, regardless of the proportion of each strain in the initial inoculum. The researchers at Indiana University also developed a mathematical model that enabled them to successfully predict the co-culture composition if the amounts of nutrients excreted by the microbes were altered. To test the model's accuracy, the investigators made a new R. palustris mutant that excreted three times more ammonium than the original strain. When this new mutant was co-cultured with E. coli, the system reached equilibrium at a ratio of one-to-one, as the model predicted. These results demonstrate the utility of stable co-cultures to understand cross-feeding relationships in ecosystems relevant for the global carbon cycle, or to engineer microbial systems for practical applications.

Contact (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
pablo.rabinowicz@science.doe.gov

(PI Contact)
James McKinlay
Joint BioEnergy Institute
Department of Biology
Indiana University
Bloomington, IN
jmckinla@indiana.edu

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research Early Career Research Program award number DE-SC0008131 to James McKinlay. Authors also acknowledge partial support from the U.S. Army Research Office.

Publication
LaSarre, B., A. McCully, J. Lennon, and J. McKinlay. 2017. “Microbial Mutualism Dynamics Governed by Dose-Dependent Toxicity of Cross-Fed Nutrients,” The ISME Journal 11, 337–48. DOI: 10.1038/ismej.2016.141.

Reference link: http://www.nature.com/ismej/journal/v11/n2/abs/ismej2016141a.html.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Microbial interactions, including mutualistic nutrient exchange, underpin the flow of energy and materials in all ecosystems. [Image courtesy J. B. McKinlay]



November 21, 2016

A Big Step Forward in Designing Drought-Tolerant Bioenergy Crops

Day and night patterns of gene activity in agave reveal key genes involved in a type of photosynthesis that maximizes water-use efficiency.

The Science
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis found in plants adapted to hot and arid conditions. CAM photosynthesis differs from the more common C3 and C4 photosynthesis types in that it inverts the day and night pattern of stomata opening to capture carbon dioxide (CO2) at night and avoid water evaporation through stomata opening during the day. Researchers at the University of Nevada and Oak Ridge National Laboratory conducted metabolomics, proteomics, and transcriptomics analyses of the desert plant agave across a diel cycle to identify genes involved in the CAM photosynthesis process and its higher water-use efficiency.

The Impact
As the photosynthetic machinery of most bioenergy crops is adapted to temperate and humid environments, carbon fixation and, therefore, biomass accumulation are less efficient and require more water than CAM plants adapted to hot and dry conditions. For that reason, introducing CAM photosynthesis into bioenergy crops would enable them to grow in marginal environments, but the molecular and genetic basis of CAM photosynthesis are not well enough understood to do this. This research identified candidate genes responsible for several aspects of the CAM process that can be used to design bioenergy crops with increased water-use efficiency and tolerance to extreme environmental conditions. 

Summary
A comparison of diel metabolic profiles of the CAM photosynthesis plant agave and the C3 photosynthesis plant Arabidopsis showed that metabolites involved in the redox reactions required for photosynthesis are found at different times of the day in each plant. Consistent with those results, transcription and protein profiling confirmed that the expression patterns of genes necessary for redox balance were shifted between agave and Arabidopsis through the day and night cycle. Furthermore, cell signaling genes in the guard cells that form the stomata, as well as CO2-sensing genes responsible for the closing of stomata and ion channels that participate in stomata opening, also showed the same opposite expression patterns between the two photosynthetic modes. This research provides strong evidence that bioengineering CAM in a C3 plant will require temporal reprogramming and identifies potential key targets for engineering this mode of photosynthesis in C3 plants, such as poplar and other selected bioenergy crops.     

Contacts (BER PM)
Pablo Rabinowicz
DOE Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contacts)
Xiaohan Yang
Biosciences Division
Oak Ridge National Laboratory, Oak Ridge, TN
yangx@ornl.gov

John Cushman
Department of Biochemistry and Molecular Biology
University of Nevada, Reno, NV
jcushman@unr.edu

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DE-SC0008834.

Publication
Abraham, P. E., H. Yin, A. M. Borland, D. Weighill, S. D. Lim, H. C. De Paoli, N. Engle, P.C. Jones, R. Agh, D. J. Weston, S. D. Wullschleger, T. Tschaplinski, D. Jacobson, J. C. Cushman, R. L. Hettich, G. A. Tuskan, and X. Yang. 2016. “Transcript, Protein, and Metabolite Temporal Dynamics in the CAM Plant Agave,” Nature Plants 2(16178), DOI: 10.1038/nplants.2016.178. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 16, 2016

Bacteria Living Within Plant Roots Affect Where and How Plants Allocate Carbon for Growth

Bacteria within plant root tissues influence the size and shape of plant leaves and roots, as well as how plants allocate carbon toward leaves, stems, or roots.

The Science
Plant traits, such as root and leaf area, influence how plants interact with their environment, and bacteria living within plant tissues can determine morphology (plant form and structure) and physiology (how they function). To understand how different microbes shaped plant morphology and physiology, researchers inoculated cottonwood seedlings with three different strains of root-dwelling bacteria. They found that the bacteria did not change photosynthesis rates or total biomass, but bacteria regulated where carbon was allocated and how plants used it. Additionally, the researchers found closely related bacteria can have vastly different effects on plant growth.

The Impact
Since plants interact with their environment through their traits, bacteria may be an important middleman in determining how plants will respond to changing environmental conditions.

Summary
Bacteria living within plant tissues (endophytes) can change how plants express traits such as root and leaf growth rates and the ratio of root to leaves. Small changes in these traits could build up to alter how plants survive, adapt, and compete within their environment. In a recent study, researchers either inoculated cottonwood seedlings with one of three endophytic bacterial stains or left the plant un-inoculated as a control. They then looked at several responses including root and leaf growth rate, plant biomass, photosynthetic rate, and the ratio of roots to leaves. They found that inoculation was linked to an increase in root and leaf growth rate, but that this increase in growth rate did not lead to an increase in plant biomass or photosynthetic efficiency. These findings indicate bacterial endophytes can change where and how carbon is used in a plant, but may not increase the overall amount of carbon fixed by photosynthesis and stored in the plant’s biomass.

Contacts (BER PM)
Daniel Stover, SC-23.1, Daniel.Stover@science.doe.gov, 301-903-0289

(PI Contact)
Aimee T. Classen      
University of Vermont
Aimee.Classen@uvm.edu

Funding
Funding was provided by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER), Genomic Science program as part of the Plant-Microbe Interfaces Scientific Focus Area project at Oak Ridge National Laboratory. Additional funding was provided by BER’s Terrestrial Ecosystem Science program under award number DE-SC0010562.

Publication
Henning, J., et al. 2016. “Root Bacterial Endophytes Alter Plant Phenotype, but not Physiology,” PeerJ  4, e2606. DOI: 10.7717/peerj.2606. (Reference link)

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER


November 11, 2016

Nitrogen Uptake Between Fungi and Orchids

Fungal and plant gene expression provides clues to nitrogen pathways.

The Science                       
Orchids are an example of an experimentally tractable plant that is highly dependent on its relationship with its mycorrhizal fungal partners for nutrient supply. In a recent study, researchers, for the first time, identified some genetic determinants potentially involved in nitrogen uptake and transfer in orchid mycorrhizas.

The Impact
This study provides a model system, amenable to experimental manipulation, for plant-fungi nutrient exchanges on a symbiotic level. It also offers insights into how host plants benefit from the mutualistic relationships formed with soil fungi that can expand their habitat range. Understanding these vital relationships may shed light on microbial symbioses applicable to growing bioenergy feedstock plants.

Summary
Orchids, like the majority of terrestrial plants, form symbiotic relationships between their plant roots and soil fungi, known as mycorrhizal associations. However, unlike other terrestrial plants, orchids rely on their mycorrhizal fungal partners for nutrient supply during the feed germination and development stages. Following these stages, most orchid species develop leaves and are capable of self-nourishment, whereas some species continue to rely on their fungal partners for an organic carbon supply. In this study, a team led by University of Turin researchers investigated the orchid mycorrhizal fungus Tulasnella calospora as both a free-living mycelium and in symbiosis with the photosynthetic orchid long-lipped serapias, or Serapias vomeracea. For the first time, researchers looked at the fungal genes that may have been involved in both the uptake and transfer of nitrogen to the host plant. RNA sequencing for the project was performed at the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI), a DOE Office of Science user facility.

The team also used JGI’s fungal genome database MycoCosm to identify fungal genes coding for proteins that were involved in nitrogen uptake and transfer. They found that the T. calospora genome has two genes coding for ammonium transporters and several genes coding for amino acid transporters, proteins that play roles in the nitrogen nutrient pathway. Overall, the orchid mycorrhizal fungi’s use of nitrogen may broaden the habitat ranges of orchids, allowing them to grow in a variety of soil types. Of more general interest to the DOE, this study provides important insights for this process and furthers understanding of plant-microbial symbioses that are vital for plant health and may inform understanding of microbial symbioses relevant to bioenergy feedstock plants.

Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
daniel.drell@science.doe.gov

Silvia Perotto
University of Turin
silvia.perotto@unito.it

Funding
U.S. Department of Energy Office of Science
Ministry of Education, Universities, and Research (Italy)
University of Turin
‘Compagnia di San Paolo’ (Torino, Italy)
Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE)

Publication
Fochi, V., W. Chitarra, A. Kohler, S. Voyron, V. Singan, E. Lindquist, K. Berry, M. Girlanda, I. V. Grigoriev, F. Martin, R. Balestrini, and S. Perotto. 2017. “Fungal and Plant Gene Expression in the Tulasnella calosporaSerapias vomeracea Symbiosis Provides Clues About Nitrogen Pathways in Orchid Mycorrhizas,” New Phytologist 213(1), 365-79. DOI: 10.1111/nph.14279. (Reference link)

Related Links
JGI MycoCosm Fungal Genomic Resource
JGI MycoCosm Fungal Genomic Resource: Tulasnella calospora
JGI Community Science Plans FY 2013: The Mycorrhizal Genomics Initiative
JGI Community Science Plan FY 2016: Microbial Mutualism with Orchids
JGI MycoCosm Fungal Genomic Resource: Sebacina vermifera
JGI MycoCosm Fungal Genomic Resource: Sebacina vermifera
JGI MycoCosm Fungal Genomic Resource: Ceratobasidium

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Researchers investigated Tulasnella calospora as both a free-living mycelium and in symbiosis with Serapias vomeracea (pictured). [Image courtesy Ziegler175 Wikimedia Commons, CC BY-SA 3.0]



November 06, 2016

Consequences of Drought Stress on Biofuel Production

Switchgrass cultivated during a year of severe drought inhibited microbial fermentation.

The Science
Investment in plant-derived sustainable biofuel sources could contribute to a near-term solution toward U.S. energy security and independence. However, weather conditions have the potential to greatly affect yearly biomass production. When plants are grown under water-stressed conditions, reduction in photosynthesis and slower growth are exhibited, leading to decreased biomass production. In this study researchers examined the effect of weather on biofuel production by comparing switchgrass and corn stover harvested after a year of major drought and after 2 years of normal precipitation (2010 and 2013).

The Impact
The study is the first linking variation in environmental conditions during bioenergy crop growth to potential detrimental effects on fermentation during biofuel production. This underscores the need for the development of biofuel production systems able to tolerate changes in precipitation and water availability as well as robust fermentation processes.

Summary
In response to the 2012 severe Midwestern drought, soluble sugar accumulated in switchgrass at significantly higher levels in comparison to non-drought period years. These sugars were chemically changed during the pretreatment stage, the step which opens up the physical structure of the plant cell wall. The soluble sugars chemically changed by reacting with the ammonia-based pretreatment chemicals to form highly toxic compounds known as imidazoles and pyrazines. The formation of toxic compounds during the pretreatment stage inhibited conversion, the final step where intermediates such as sugars are fermented into biofuel by microorganisms, such as the microbe S. cerevisiae. However, it may be possible to overcome this issue by 1) removing the soluble sugars prior to pretreatment or 2) using microbial strains resistant to the toxic effects of imidazoles and pyrazines. This study demonstrates that while there are benefits to growing bioenergy crops on marginal lands to avoid competition with food crops, the plants grown there may experience higher levels of stress resulting in deleterious impacts on microbes during biofuel production. To develop sustainable biofuel production systems, the deleterious effects of stress, such as fluctuations in precipitation and water availability, must be mitigated. This research helps to provide an understanding of the effects of drought stress on switchgrass and is relevant to DOE’s energy and environmental missions.

Contact
Kent Peters, Ph.D.
Program Manager Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
kent.peters@science.energy.doe.gov

(PI Contact)
Rebecca Garlock Ong
Assistant Professor, Chemical Engineering - Michigan Technological University
rgong1@mtu.edu

Funding
This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). Additional funding for L.G.O. is under DOE OBP Office of Energy Efficiency and Renewable Energy (DE-AC05-76RL01830).

Publications
R.G. Ong, A. Higbee, S. Bottoms, Q. Dickinson, D. Xie, S.A.Smith, J. Serate, E. Pohlmann, A.D. Jones, J.J. Coon, T.K. Sato, G.R. Sanford, D. Eilert, L.G. Oates, J.S. Piotrowski, D.M. Bates, D. Cavalier, and Y. Zhang, “Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate.” Biotechnology for Biofuels 9, 237 (2016) [DOI: 10.1186/s13068-016-0657-01] (Reference link)

Related Links
Great Lakes Bioenergy Research Center

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



From field to fuel: Illustration of switchgrass conversion process. [Image courtesy of Matthew Wishiewski]



October 26, 2016

New System for Introducing Genetic Pathways into Plants, Making Them More Productive

A yeast DNA recombination system facilitates the transference and expression of entire heterologous metabolic pathways into plant genomes.

The Science
Researchers have adapted a DNA recombination system from yeasts to facilitate the construction of large stretches of DNA and their introduction into plant genomes. This technology, called jStack, advances the engineering of new, complex functionality into plants by enabling the expression of heterologous multigene pathways.

The Impact
Engineering more productive and resilient crops requires the introduction of new biological functions into plants. Often, multiple genes from different organisms need to be transferred to a given crop to provide new desirable properties, but assembling and introducing multiple genes into plant crops is difficult. The jStack technology will make it easier to combine genes from different sources and incorporate them into the genome of engineered crops to improve their performance. 

Summary
Researchers at Lawrence Berkeley National Laboratory modified plasmid vectors commonly used for plant transformation so that they can be replicated and selected in yeasts, in addition to the Escherichia coli and plant hosts, to create a multigene plant transformation system called jStack. The system also includes yeast DNA sequences required for homologous recombination so that multiple DNA elements can be assembled into a single DNA molecule in yeast intermediary hosts in vivo. The resulting recombinant vectors can then be selected and purified for introduction in the desired plant host. In an attempt to standardize plant genetic engineering, the jStack system was designed to be compatible with commonly used cloning systems. Furthermore, a publicly available library of over 100 compatible promoters, genes, and terminator sequences was created to encourage collaboration and innovation within the plant synthetic biology community. The utility of the jStack technology was validated by introducing the entire pathway of the pigment violacein from the soil bacterium Chromobacterium violaceum into a model plant, as well as the metabolic pathway required to produce bisabolene, a precursor to bisabolane and a potential biodiesel component.    

Contacts (BER PM)
Pablo Rabinowicz
DOE Office Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
Dominique Loqué
Joint BioEnergy Institute
Biological Systems and Engineering Division
Lawrence Berkeley National Laboratory
Berkeley, CA
dloque@lbl.gov

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science Early Career Research Program award to D. Loqué, and by contract DE-AC02-05CH11231.

Publication
Shih, P., K. Vuu, N. Mansoori, L. Ayad, K. Louie, B. Bowen, T. Northen, and D. Loqué. 2016. “A Robust Gene-Stacking Method Utilizing Yeast Assembly for Plant Synthetic Biology,” Nature Communications 7(13215), DOI: 10.1038/ncomms13215. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 24, 2016

Metabolic Handoffs Among Microbial Community Members Drive Biogeochemical Cycles

Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system.

The Science
2,540 genomes that represent the majority of known bacterial phyla and 47 new phylum-level lineages were reconstructed from sediment and groundwater collected from a semi-arid floodplain near Rifle, CO. Analyses showed that inter-organism interactions are required to turn the carbon, sulfur and nitrogen biogeochemical cycles and revealed that complex patterns of community assembly are likely key to ecosystem functioning and resilience.

The Impact
The research almost doubled the number of major bacterial groups and provided detailed information about the ecosystem roles of organisms from these groups. The research dramatically increased understanding of subsurface biology and motivates new approaches to ecosystem modeling. The genomes represent a treasure-trove that will be mined for biotechnology.

Summary
The subterranean world hosts up to one fifth of all biomass, including microbial communities that drive transformations central to Earth’s biogeochemical cycles. However, little is known about how complex microbial communities in such environments are structured, and how inter-organism interactions shape ecosystem function. Terabase-scale cultivation-independent metagenomics was applied to aquifer sediments and groundwater and 2,540 high-quality near-complete and complete strain-resolved genomes that represent the majority of known bacterial phyla were constructed.  Some of these genomes derive from 47 newly discovered phylum-level lineages. Metabolic analyses spanning this vast phylogenetic diversity and representing up to 36% of organisms detected in the system were used to document the distribution of pathways in coexisting organisms. Consistent with prior findings indicating metabolic handoffs in simple consortia, it was shown that few organisms within the community conduct multiple sequential redox transformations. As environmental conditions change, different assemblages of organisms are selected for, altering linkages among the major biogeochemical cycles.

BER PM Contact
David Lesmes, SC-23.1, 301-903-2977

Contact
Susan Hubbard
Lawrence Berkeley National Laboratory
sshubbard@lbl.gov

Funding: This work was supported by Lawrence Berkeley National Laboratory’s Sustainable Systems Scientific Focus Area funded by the US Department of Energy, Office of Science, Office of Biological and Environmental Research.  Terabase-scale sequencing critical for this work was provided by the Joint Genome Institute via Community Science Program allocations.

Publication
K. Anantharaman, C. T. Brown, L. A. Hug, I.Sharon, C. J. Castelle, A. J. Probst, B. C. Thomas, A. Singh, M. J. Wilkins, U. Karaoz, E. L. Brodie, K. H. Williams, S. S. Hubbard, and J. F. Banfield. “Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system”. Nature Communications 7, ncomms13219 (2016). [DOI: 10.1038/ncomms13219]. (Reference link)

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER



Tree showing all of bacterial diversity that is now represented by genomes, with the major lineages indicated by wedges. Research on the microbiology of the Rifle aquifer has provided new genomic information within previously identified groups (black wedges). In addition, many major bacterial groups were first identified and via study of the Rifle site (red and purple wedges). Red wedges indicate many major lineages that were first identified in the current study. Colored dots indicate the genomically predicted roles of members of these newly defined bacterial lineages in geochemical cycling. Remarkably, few major bacterial lineages have not been genomically sampled at this site (olive green wedges). [Image courtesy of Anantharaman et al. 2016. DOI: 10.1038/ncomms13219. Reprinted under CC by 4.0.]



October 13, 2016

Database of DNA Viruses and Retroviruses Debuts on Integrated Microbial Genomes Platform

The publicly accessible database promotes comparative analyses and ground-breaking discoveries through biological translation of sequence data.

The Science
A new database dedicated to global viral diversity has been developed by the Department of Energy Joint Genome Institute (DOE JGI). This database is the largest publicly available database for viruses, with 3,908 isolate reference DNA viruses and 264,413 computationally identified viral contigs from more than 6,000 ecologically diverse metagenomic samples. In a series of four articles recently published in Nucleic Acids Research, DOE JGI researchers also report on the latest updates to several publicly accessible databases and computational tools that benefit the global community of microbial researchers.

The Impact
Microbes play key roles in maintaining the planet’s biogeochemical cycles. Viruses, thought to outnumber microbes by 10-fold, exert major influences on microbial survival and community interactions. Advances in sequencing technologies have generated vast amounts of data about these viruses, requiring tools to manage and interpret the information. Recent updates focus on database analytical tools for microbial genomics and viruses relevant to DOE missions in bioenergy and environment.

Summary
Providing high-quality, publicly accessible sequence data goes hand-in-hand with developing and maintaining the databases and tools that the research community can harness to help answer scientific questions. In a recent series of articles published in Nucleic Acids Research, researchers at DOE JGI, a national scientific user facility, describe a database called Integrated Microbial Genomes with Virus Samples (IMG/VR). IMG/VR is a comprehensive computational platform integrating all the sequences in the database with associated metadata and analytical tools. IMG/VR follows on the heels of a recent DOE JGI viral diversity study report in Nature. Additional articles in the same issue describe updates to several publicly accessible, interactive databases since the last set of reports published in 2014. For example, as of July 2016, there were 47,516 archaeal, bacterial, and eukaryotic genomes in the IMG with Microbiome Samples (IMG/M) system, with researchers noting that number “represents an over 300% increase since September 2013.” IMG/M contains annotated DNA and RNA sequence data of archaeal, bacterial, eukaryotic, and viral genomes from cultured organisms; single cell genomes (SCG) and genomes from metagenomes from uncultured archaea, bacteria, and viruses; and metagenomes from environmental, host-associated, and engineered microbiome samples. Another paper concerns the Genomes OnLine Database (GOLD), a manually curated data management system that catalogs sequencing projects with associated metadata from around the world. In the current version of GOLD (v.6), all projects are organized based on a four-level classification system in the form of a study, organism (for isolates) or biosample (for environmental samples), sequencing project, and analysis project. A fourth paper focuses on the IMG Atlas of Biosynthetic gene Clusters (IMG-ABC). Launched in 2015, IMG-ABC enables researchers to search for biosynthetic gene clusters and secondary metabolites. Their latest update now incorporates ClusterScout, a tool for targeted identification of custom biosynthetic gene clusters across several thousand isolate microbial genomes, as well as a new search capability.

Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Science Division
Office of Biological and Environmental Research
Office of Science, U.S. Department of Energy
daniel.drell@science.doe.gov

Nikos Kyrpides
Prokaryote Super Program Head
DOE Joint Genome Institute
nckyrpides@lbl.gov

Funding
U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research
U.S. National Institutes of Health Data Analysis and Coordination Center

Publications

I.-M. A. Chen, et al., “IMG/M: Integrated genome and metagenome comparative data analysis system.” Nucleic Acids Research (2016). [DOI:10.1093/nar/gkw929] (Reference link)

S. Mukherjee, et al., “Genomes OnLine Database (GOLD) v.6: Data updates and feature enhancements.” Nucleic Acids Research (2016). [DOI: 10.1093/nar/gkw992] (Reference link)

D. Paez-Espino, et al., “IMG/VR: A database of cultured and uncultured DNA Viruses and retroviruses.” Nucleic Acids Research (2016). [DOI: 10.1093/nar/gkw1030] (Reference link)

M. Hadjithomas, et al., “IMG-ABC: New features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes.” Nucleic Acids Research (2016). [DOI: 10.1093/nar/gkw1103] (Reference link)

Related Links

IMG

GOLD

IMG/VR

IMG-ABC

JGI News Release: Unveiled: Earth's Viral Diversity

JGI Science Highlight: First Public Resource for Secondary Metabolites Searches

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Geographic distribution of biosamples and organisms encompassed by the Genomes OnLine Database. Organism location of isolation is marked with pink dots, biosample location with blue dots. [Image courtesy of Mukherjee et al., Nucleic Acids Research (2016). DOI: 10.1093/nar/gkw992]



October 12, 2016

Unraveling the Molecular Complexity of Cellular Machines and Environmental Processes

State-of-the-art mass spectrometer delivers unprecedented capability to users.

The Science
Two recent studies demonstrate the enormous potential for scientists to explore extremely complex molecular mixtures and systems frequently encountered in environmental, biological, atmospheric, and energy research.

The Impact
The Environmental Molecular Sciences Laboratory (EMSL), a Department of Energy Office of Science user facility, has an unprecedented ability to routinely analyze large intact proteins, precisely measure the fine structure of isotopes, and extract more information from complex natural organic matter mixtures. One of the world’s most powerful mass spectrometry instruments, a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T FTICR MS), is now available to the scientific community. Illustrating the power of this new instrument for biogeochemical research, EMSL scientists were able to make over 8,000 molecular formula assignments from dissolved organic matter mixtures using the 21T FTICR MS. In another study, EMSL users rapidly identified and discovered new types of metal-binding molecules known as siderophores, which are produced by bacterial cells.

Summary
As the highest-performance mass spectrometry technique, the FTICR MS has become increasingly valuable in recent years for various research applications. The FTICR MS determines the mass-to-charge ratio of ions by measuring the frequency at which ions rotate in a magnetic field, providing ultra-high resolution and mass measurement accuracy. The 21T FTICR MS, which is one of only two in the world with this high magnetic field strength, went online at EMSL in 2015. In a recent study, a team of EMSL scientists evaluated performance gains produced by this high magnetic field strength. They found this next-generation instrument empowers routine analysis of large intact proteins, precisely measures the fine structure of isotopes, and elicits more information than ever before from complex natural organic matter mixtures. The initial performance characterization of the 21T FTICR MS demonstrates enormous potential for future applications to extremely complex molecular mixtures and systems frequently encountered in environmental, biological, atmospheric, and energy research. Moreover, this unprecedented level of mass resolution and accuracy will help promote widespread use of top-down proteomics—an approach that enables accurate characterization of different protein variants with different biological activity. As a result, this transformative instrument will enable users from around the world to tackle previously intractable questions related to atmospheric, terrestrial, and subsurface processes; microbial communities; biofuel development; and environmental remediation.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contact
Ljiljana Paša-Tolic
Environmental Molecular Sciences Laboratory
ljiljana.pasatolic@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, including support of the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility, and the "High Resolution and Mass Accuracy Capability" development project at EMSL.

Publications
J. B. Shaw, T.-Y. Lin, F. E Leach III, A. V. Tolmachev, N. Tolic, E. W. Robinson, D. W. Koppenaal, and L. Paša-Tolic, “21 Tesla Fourier transform ion cyclotron resonance mass spectrometer greatly expands mass spectrometry toolbox.” Journal of the American Society for Mass Spectrometry 27(12), 1929-36 (2016). DOI: 10.1007/s13361-016-1507-9. (Reference link)

L. R. Walker, M. M. Tfaily, J. B. Shaw, N. J. Hess, L. Pasa-Tolic, and D. W. Koppenaal, “Unambiguous identification and discovery of bacterial siderophores by direct injection 21 Tesla Fourier transform ion cyclotron resonance mass spectrometry.” Metallomics (2017). DOI: 10.1039/c6mt00201c. (Reference link)

Related Links
Unraveling Molecular Complexity of Natural Systems
Top-down Proteomics: Onward and Upward

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER



The 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer will propel the future direction of environmental, biological, atmospheric, and energy research. [Image courtesy Pacific Northwest National Laboratory]



September 27, 2016

Oleaginous Yeasts Move One Step Closer to Becoming Industrial Biodiesel Producers

Engineering metabolic pathways and enzyme subcellular localization enables efficient production of fatty acids and other green chemicals.

The Science
Using a combination of different genetic engineering strategies, scientists were able to make oleaginous yeasts convert low-value carbon compounds into different fatty acids and alcohols that can be used for diesel-like fuel production and other industrial applications. The high levels of product achieved with this approach bring the development of a yeast biorefinery platform for high-value fuel and oleochemical production closer to reality.

The Impact
Oleaginous microorganisms, such as Yarrowia lipolytica, have great potential as industrial producers of biofuels and bioproducts due to their high lipid biosynthetic capacity. However, lipid metabolic engineering in eukaryotes is not advanced enough to take advantage of these organisms. This research demonstrates that a deeper understanding of different aspects of lipid metabolism, from genetic regulation to metabolic compartmentalization to enzyme structure, enables the design and engineering of new strains to substantially increase lipid production.  

Summary
Researchers at the Massachusetts Institute of Technology applied a multipronged strategy to engineer Y. lipolytica to produce several lipid molecules with applications as biofuels and other oleochemicals such as fatty acid ethyl esters, fatty alkanes, fatty acids, fatty alcohols, and triacylglycerides. This strategy included engineering Y. lipolytica lipid metabolism by expressing enzymes from other microorganisms within specific subcellular compartments within the yeast cells where specific lipids or their precursors are metabolized. Another approach was to engineer a chimeric enzyme to regulate the chain length of specific fatty acids. Finally, to increase the availability of acetyl-CoA building blocks for fatty acid synthesis, alternative acetyl-CoA pathways were designed to avoid the normal repression of acetyl-CoA synthesis by low nitrogen concentration in the medium. Production of different lipid molecules in these engineered strains was increased between 2 and 20 fold, paving the way toward developing industrial strains for commercial production of biodiesel and bioproducts from renewable sources.     

Contacts (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
Gregory Stephanopoulos
Department of Chemical Engineering
Massachusetts Institute of Technology
Cambridge, MA
gregstep@mit.edu

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science program (award DE-SC0008744).  

Publication
P. Xu, K. Qiao, W. S. Ahn, and G. Stephanopoulos, “Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals.” Proceedings of the National Academy of Sciences (USA) 113(39), 10848-853 (2016). [DOI: 10.1073/pnas.1607295113] (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Oleaginous yeasts such as Yarrowia lipolytica (pictured) can accumulate more than 80 percent of their dry weight as lipids, making them promising organisms for biodiesel production. [Image courtesy Massachusetts Institute of Technology/Peng Xu]



September 27, 2016

The Brown Rot Two-Step

Understanding the biomechanisms brown rot fungi use to degrade wood could lead to new tools for more efficient biofuel production. 

The Science
Wood’s complex structure of cellulose, long chains of linked sugar molecules, embedded in a scaffolding of polyaromatic lignin makes it highly resistant to biological or chemical decomposition. Brown rot fungi, however, possess a unique ability to attack the cellulose fraction of wood while avoiding the surrounding lignin. This study provides evidence that brown rot fungi accomplish this using a two-step process: (1) by secreting a set of chemicals and enzymes that open up the lignin framework, and then (2) releasing a second set of enzymes that break down the cellulose chains into sugars that are absorbed by the fungi.

The Impact
Understanding the newly discovered two-step mechanism of this degradation process could lead to the development of new biotechnology approaches for efficient and cost-effective conversion of wood cellulose into biofuels or bioproducts while leaving the lignin intact as a potential useful byproduct.

Summary
Wood-decomposing fungi are essential players in breaking down plant biomass in forest ecosystems and could provide important clues on how to more efficiently convert lignocellulose—the primary building block of wood cell walls—to biofuels and other products. Among these organisms, brown rot fungi are unique in their ability to selectively degrade the cellulose in wood while leaving the lignin portion mainly intact.To accomplish this task, these fungi generate highly reactive oxygen species that alter the chemical structure of wood and work in tandem with enzymes that break down cellulose chains. However, reactive oxygen species could just as easily damage the fungal enzymes as the wood structure, so researchers have long hypothesized that the fungi spatially segregate the oxidant generation process from the secreted enzymes using sets of chemical barriers. However, in this study, scientists found evidence that brown rot fungi separate the oxidants and enzymes in time rather than in space. This two-step wood decomposition mechanism was discovered by designing a simple, yet elegant experiment: brown rot fungi were grown in one direction along thin wood specimens separating the stages of wood decay linearly across the substrate. The wood was then cut into sections and analyzed for patterns of gene expression using whole-transcriptome shotgun sequencing (RNA-seq), assayed for relevant enzyme activity, and imaged using confocal and fluorescence microscopy. The researchers found that at early time points in the brown rot colonization, there was evidence of lignocellulose oxidation by reactive oxygen species and an increase in expression of genes important for plant cell wall-swelling. Both of these activities would weaken the structural integrity of wood and make it easier for enzymes to access cellulose chains. Only at later time points of colonization did brown rot fungi begin to produce glycoside hydrolase enzymes that break down cellulose chains into their component sugars. This unique fungal “pretreatment” strategy predates chemical pretreatment approaches used in industrial biomass processing by millions of years and could provide important new clues for improved conversion of woody plant materials into renewable cellulosic biofuels.

Contacts
(BER PM)
Dawn M. Adin
Program Manager, Office of Biological and Environmental Research
dawn.adin@science.doe.gov
Paul Bayer
Program Manager, Office of Biological and Environmental Research
paul.bayer@science.doe.gov

(PI Contact)
Jonathan S. Schilling
University of Minnesota
schillin@umn.edu  

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research under award numbers DE-SC0004012 (DOE Early Career Research) and DE-SC0012742. This research also used resources at the Environmental Molecular Sciences Laboratory, which is a DOE Office of Science user facility.

Publication
Zhang, J., G. N. Presley, K. E. Hammel, J.-S. Ryu, J. R. Menke, M. Figueroa, D. Hu, G. Orr, and J. S. Schilling. 2016. “Localizing Gene Regulation Reveals a Staggered Wood Decay Mechanism for the Brown Rot Fungus Postia placenta,” Proceedings of the National Academy of Sciences (USA) 113(39), 10968-973. DOI: 10.1073/pnas.1608454113. (Reference link)

Related Links
EMSL Highlight

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Image courtesy of Jonathan S. Schilling (University of Minnesota)
Pictured is a brown rot wood-degrading fungus Laetiporus sulphureus commonly known as the “chicken of the woods” fruiting on a hardwood log.



September 05, 2016

Microbial Metabolism Impacts Sustainability of Fracking Efforts

Through hydraulic fluids, surface microbes are colonizing the deep subsurface where they are adapting and thriving.

The Science
Hydraulic fracturing (“fracking”) is the industry standard for extracting hydrocarbons from shale formations, which provide one-third of natural gas energy resources worldwide. Poorly understood, however, are the biogeochemical changes that this process induces in the deep subsurface. In a recent study, researchers, for the first time, were able to reconstruct microbial genomes from shale formations that are being drilled for natural gas. Coupled with microbial metabolic information, the data shed considerable light on the impacts to microbial communities in the deep subsurface, as well as on the sustainability of energy extraction through this approach.

The Impact
Microbial metabolism and growth in hydrocarbon reservoirs are known to have both positive and negative impacts on energy recovery, but little is known about the structure, function, and activity of microorganisms in hydraulically fractured shale. This study provides evidence for microbial degradation of chemical additives and the potential for microbially induced corrosion and formation of biogenic methane. These findings could be used to develop strategies to reduce the risk of fracking-related environmental contamination and to improve long-term sustainability of energy extraction.

Summary
Hydraulic fracturing uses high-pressure injection of fresh water and chemical additives deep into the earth to generate extensive fractures in the shale matrix, thereby releasing hydrocarbons trapped in tiny pore spaces. A recent study—led by researchers from The Ohio State University, Department of Energy’s (DOE) Environmental Molecular Sciences Laboratory (EMSL), DOE Joint Genome Institute (JGI), and University of Maine—found that along with these fluids, microbes from the surface are also being injected and colonizing the deep subsurface, 2.5 km underground. To find out how this process may be impacting resident microbial community structure, function, and activity, the research team conducted metagenomic and metabolite analyses on input and produced fluids from gas wells for up to a year after hydraulic fracturing at two Appalachian basin shales: the Marcellus and Utica/Point Pleasant formations. The researchers used several nuclear magnetic resonance instruments at EMSL and high-throughput DNA sequencing technologies at JGI, both of which are DOE Office of Science user facilities. By reconstructing the first genomes of microbes in fractured shale, researchers discovered remarkable adaptations by microorganisms to survive the extreme chemical conditions produced by fracking. For example, microbes in fractured shales commonly consume injected chemical additives and produce an amino acid derivative called glycine betaine, which protects against high salinity by balancing the osmotic difference between the cell's surroundings and the internal cytoplasm. Glycine betaine is then taken up and used as a source of energy by other microbes, which, in turn, release metabolites that support methane-producing bacteria known to enhance energy recovery. On the other hand, salt-loving bacterial strains that synthesize glycine betaine also produce hydrogen sulfide, which contributes to equipment corrosion, risks environmental contamination, and decreases profits. Additional analysis revealed the majority of archaeal and bacterial genomes reconstructed from fluid samples showed evidence of acquired immunity against viruses, which actively infect other microbes vulnerable to fracking-related environmental stressors. Taken together, these findings illustrate the role of microbial communities resident in oil-bearing shales and begin to reveal a wide range of factors supporting long-term microbial persistence and adaptation to extreme environmental conditions in hydraulically fractured shales.

BER PM Contacts
Paul Bayer, SC-23.1, 301-903-5324
Dan Drell, SC-23.2, 301-903-4742

PI Contacts
Rebecca A. Daly
The Ohio State University
daly.130@osu.edu

David Hoyt
DOE Environmental Molecular Sciences Laboratory
david.hoyt@pnnl.gov

Susannah Tringe
DOE Joint Genome Institute
sgtringe@lbl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (BER), and used resources at DOE’s Joint Genome Institute and Environmental Molecular Sciences Laboratory, which are DOE Office of Science user facilities. Both facilities are sponsored by BER and operated under contract numbers DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). Additional funding was provided by the National Science Foundation’s Dimensions of Biodiversity (award number 1342701).

Publication
Daly, R. A., M. A. Borton, M. J. Wilkins, D. W. Hoyt, D. J. Kountz, R. A. Wolfe, S. A. Welch, D. N. Marcus, R. V. Trexler, J. D. MacRae, J. A. Krzycki, D. R. Cole, P. J. Mouser, and K. C. Wrighton. 2016. “Microbial Metabolisms in a 2.5-KM-Deep Ecosystem Created by Hydraulic Fracturing in Shales,” Nature Microbiology, DOI: 10.1038/nmicrobiol.2016.146. (Reference link)

Related Links
EMSL Highlight
JGI Highlight

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER



Oil and gas well site in the Appalachian Basin similar to the well sites where researchers conducted metagenomic and metabolite analyses on hydraulic fluids. [Image courtesy of the Marcellus Shale Energy and Environment Laboratory]



September 01, 2016

Genomics Helps Advance Understanding of How an Important Bioenergy Feedstock Tolerates Environmental Stresses

First comprehensive study of an important protein family in the perennial woody plant Populus lays the foundation for functional characterization.

The Science
A genome-wide characterization of a family of plant-specific receptor proteins in the bioenergy feedstock Populus revealed tissue-specific expression and suggests a possible function in tolerance to environmental stresses.

The Impact
This comprehensive study of lectin receptor-like kinases (LecRLKs) in a woody plant provides the foundation for functional characterization of an important protein family.

Summary
Cell-surface receptor proteins play an important role in signal perception and processing, which, in turn, influence growth and development. The membrane-bound LecRLKs comprise a large family of such proteins. LecRLKs are specific to plants and are believed to be involved in responses to external stimuli such as pathogens and environmental stresses. Scientists with Oak Ridge National Laboratory’s Plant-Microbe Interface project report the first genome-wide analysis and classification of LecRLKs in the perennial woody model plant Populus, a bioenergy feedstock tree important for carbon sequestration, ecological systems studies, and biomass production. The researchers found that the LecRLK family was greatly expanded in Populus, with notably high levels of expression in the roots as compared with other plant tissues. They hypothesize that since the root system provides the interface for soil microbes, LecRLKs expressed in the roots may function to perceive microbial signals, which, in turn, influence plant health and tolerance of biotic and abiotic stresses. This first comprehensive study of LecRLKs in a woody plant lays the basis for functional characterization of an important protein family.

Contacts (BER PM)
Cathy Ronning 
SC-23.2
catherine.ronning@science.doe.gov

(PI Contact)
Jin-Gui Chen
Biosciences Division, Oak Ridge National Laboratory
chenj@ornl.gov

Funding
This work was supported by the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science program, Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy [(DOE); DE-AC05-00OR22725], as well as DOE’s Joint Genome Institute, an Office of Science user facility (DE-AC02-05CH11231).

Publication
Yang, Y., J. Labbé, W. Muchero, X. Yang, S. S. Jawdy, M. Kennedy, J. Johnson, A. Sreedasyam, J. Schmutz, G. A. Tuskan, and J.-G. Chen. 2016. “Genome-Wide Analysis of Lectin Receptor-Like Kinases in Populus,” BMC Genomics 17, 699. DOI 10.1186/s12864-016-3026-2. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 23, 2016

Bacterial Protein Shows Promise for Efficiently Converting Plant Biomass to Biofuels

Enzyme has the highest known activity for hydrolyzing recalcitrant crystalline cellulose found in plant cell walls.

The Science
Glycoside hydrolases are microbial enzymes that play a key role in nutrient acquisition through the breakdown of cellulose—a major component of plant cell walls. A recent study showed that a protein from the bacterial glycoside hydrolase family 12 plays an unexpectedly important role in converting the hard-to-degrade crystalline form of cellulose and that it does so through a random mechanism unlike other hydrolases.

The Impact
The discovery of a glycoside hydrolase protein that is highly effective at breaking down rigid plant cell wall components could be harnessed to develop more efficient strategies for converting plant biomass to fuels and chemicals.

Summary
Microbes such as fungi and bacteria produce enzymes called glycoside hydrolases to acquire nutrients through the degradation of cellulose—carbohydrates that make up plant cell walls. Some of these enzymes are capable of breaking down the rigid, crystalline form of cellulose and, therefore, could be especially effective at efficiently converting tough plant biomass to fuels and chemicals. However, they have largely been studied in pure cultures of microorganisms, even though microorganisms break down cellulose as communities in the environment. To address this knowledge gap, a multi-institutional team of researchers led by scientists at the Department of Energy’s (DOE) Joint BioEnergy Institute (JBEI) combined comparative proteomics with biochemical measurements. They then assessed differences in glycoside hydrolases produced by diverse microbes in communities cultivated from green waste compost and grown on crystalline cellulose. The team used several mass spectrometry instruments at the Environmental Molecular Sciences Laboratory (EMSL) and high-throughput DNA sequencing technologies at the Joint Genome Institute, both of which are DOE Office of Science user facilities. Their analysis revealed that a glycoside hydrolase family 12 protein, produced by the bacterium Thermobispora bispora, plays a previously underappreciated important role in breaking down crystalline cellulose. The new findings suggest this protein could be especially effective at converting plant biomass to fuels and chemicals. More broadly, the study illustrates the power of comparative community proteomics to reveal novel insights into microbial proteins that could be harnessed for fuel production from renewable energy sources. This research represents collaboration among JBEI, Lawrence Berkeley National Laboratory, Sandia National Laboratories, Pacific Northwest National Laboratory, EMSL, and University of Applied Sciences Mannheim.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contacts
Steven Singer
Lawrence Berkeley National Laboratory/Joint BioEnergy Institute
SWSinger@lbl.gov

Errol (Robby) Robinson
Pacific Northwest National Laboratory
errol.robinson@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (BER). Furthermore, this work was performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and used resources at DOE’s Environmental Molecular Sciences Laboratory and Joint Genome Institute, which are DOE Office of Science user facilities sponsored by BER.

Publication
J. Hiras, Y. W. Wu, K. Deng, C. D. Nicora, J. T. Aldrich, D. Frey, S. Kolinko, E. W. Robinson, J. M. Jacobs, P. D. Adams, T. R. Northen, B. A. Simmons, and S. W. Singer, “Comparative community proteomics demonstrates the unexpected importance of actinobacterial glycoside hydrolase family 12 protein for crystalline cellulose hydrolysis.” mBio 7(4), e01106-16 (2016). [DOI: 10.1128/mBio.01106-16]. (Reference link)

Related Links
Steve Singer bio
EMSL News

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER



A glycoside hydrolase protein is highly effective at breaking down rigid plant cell wall components and could be used to develop more efficient strategies for converting plant biomass to fuels and chemicals. [Image courtesy Department of Energy Environmental Molecular Sciences Laboratory]



August 19, 2016

Advancing Toward Construction of a Bacterial Recoded Genome

Initial testing of a synthetic bacterial genome that uses 57 of the 64 natural codons showed minimal fitness impairment.

The Science
Taking advantage of the genetic code’s redundancy, a collaborative project led by researchers at Harvard University synthesized a bacterial genome that uses 57 of the 64 natural codons; the remaining seven codons were reassigned to nonstandard amino acids that can be used to develop novel protein functions. So far, DNA segments that span over 60% of the synthetic genome and contain over half the essential genes have been introduced into living cells to test for deleterious effects, and only very few cases showed significant growth defects.

The Impact
Reassigning several of the 64 natural codons in a bacterial genome enables the development of microbial strains with multiple combinations of proteins that can perform novel functions, while preventing the engineered strain from surviving if it escapes laboratory conditions. This large-scale, genome-wide recoding required developing design tools that must be fine-tuned after testing and gaining knowledge of rules that must be observed to synthesize functional genetic elements and networks. This research shows that recoding essential genes is possible and has uncovered fundamental design principles.    

Summary
To construct a completely recoded Escherichia coli genome, the researchers first used computational tools to design a genomic sequence lacking all instances of seven redundant codons and synthesized the genome in 87 fragments spanning 50 kb each. Testing of 55 of these fragments, which contain 63% of the genome and 52% of essential genes, showed that most of them caused limited or no change in growth and transcription levels. The recoded version of one gene resulted in severe fitness impairment, but the researchers were able to redesign the gene, allowing the strain to survive. At the same time, the researchers were able to optimize the design tools to further reduce potential growth defects in recoded microbes. This research demonstrates the feasibility of high-level recoding of microbial organisms to confer new functionality such as the development of new bioproducts. It also shows that genome-wide engineering approaches provide new knowledge on the fundamental principles that drive biological systems.  

Contacts (BER PM)
Pablo Rabinowicz
pablo.rabinowicz@science.doe.gov
(PI Contact)
George M. Church
Department of Genetics, Harvard Medical School
Wyss Institute for Biologically Inspired Engineering
Harvard University
Boston, MA
gchurch@genetics.med.harvard

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science (award DEFG02-02ER63445). Authors also acknowledge support from the U.S. Department of Defense and National Science Foundation.

Publication
Ostrov, N., M. Landon, M. Guell, G. Kuznetsov, J. Teramoto, N. Cervantes, M. Zhou, K. Singh, M. Napolitano, M. Moosburner, E. Shrock, B. Pruitt, N. Conway, D. Goodman, C. Gardner, G. Tyree, A. Gonzales, B. Wanner, J. Norville, M. Lajoie, and G. Church. 2016. “Design, Synthesis, and Testing Toward a 57-Codon Genome,” Science 353(6301), 819-22. DOI: 10.1126/science.aaf3639. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



By recoding bacterial genomes such as Escherichia coli (pictured), it is possible to create organisms that can potentially synthesize products not commonly found in nature. Image courtesy of iStock



August 02, 2016

Grasses Fight Drought by Squelching Root Growth

Shoot-borne roots that normally start developing in grasses weeks after germination are suppressed under drought conditions.  

The Science
Using fluorescent imaging technologies and direct observation of green foxtail roots excavated from soil, researchers discovered that root growth normally initiated from the crown (belowground shoot-root joint) is inhibited when water is scarce.  

The Impact
Drought tolerance is an important trait needed in bioenergy crops to enable their cultivation in marginal lands. As roots are the main conduit for water acquisition, understanding their biology is critical to discovering ways to improve bioenergy crops. The root system of potential bioenergy crops in the Poaceae family, such as switchgrass and sorghum, and the model grass Setaria viridis (green foxtail) is composed mostly by crown roots that emerge days or weeks after germination. However, little is known about their development under drought conditions. The discovery of a widespread mechanism of crown root suppression in grass species opens new avenues for improving bioenergy crop performance in dry environments.

Summary
A detailed study of root growth using traditional and new fluorescent imaging technologies in the model bioenergy crop Setaria showed that the crown (shoot-root node found belowground) senses the level of water conditions immediately surrounding the plant. At low soil humidity, root growth is arrested shortly after initiation, while root growth is rapidly resumed when water availability increases. Researchers from the Carnegie Institution for Science and international collaborators observed that drought-induced inhibition of root growth is also present in several other grasses, including the bioenergy crops sorghum and switchgrass and corn wild relatives, but not in highly domesticated corn lines. Furthermore, a corn mutant that lacks crown roots retains more water in the stem. These results suggest that grasses are adapted to inhibit root growth to preserve water and to induce crown root growth in response to precipitation to maximize water absorption in wet conditions. Genetic and transcriptomics analyses showed that oxidative-stress response genes may be involved in the process. The identification of the genes responsible for this phenomenon will be critical targets for engineering drought tolerance in bioenergy grasses.

Contacts (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
José Dinneny
Department of Plant Biology
Carnegie Institution for Science, Stanford, CA 94305
jdinneny@carnegiescience.edu

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under award DE-SC0008769. Additional support was provided by the National Science Foundation.

Publications
Sebastian, J., M. Yee, W. Viana, R. Rellán-Álvarez, M. Feldman, H. Priest, C. Trontin, T. Lee, H. Jiang, I. Baxter, T. Mockler, F. Hochholdinger, T. Brutnell, and J. Dinneny. 2016. “Grasses Suppress Shoot-Borne Roots to Conserve Water During Drought,” Proceedings of the National Academy of Sciences (USA) 113(31), 8861-66. DOI: 10.1073/pnas.1604021113. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 02, 2016

LuxR Homolog in a Cottonwood Tree Endophyte Activates Gene Expression in Response to Plant Signal or Specific Peptides

This new gene discovery opens the door for investigating other signals involved in plant-bacteria interactions.

The Science                       
Scientists discovered a gene in the bacterium Pseudomonas sp. GM79, a beneficial microbe commonly found within the roots of Populus trees (cottonwood), that is activated by signals exuded from the plant. 

The Impact
The study’s findings provide a model for investigating a possible new family of signals involved in plant-bacteria interactions that are present in dozens of bacterial species associated with economically important plants. 

Summary
Many beneficial soil bacteria are associated with plant roots, both outside the root (rhizosphere) and within (endophytic microbes). In Populus, a candidate bioenergy feedstock, the endophyte- and rhizosphere-associated communities are distinct, with a- and ?-Proteobacteria dominating the endophyte communities and Acidobacteria and a-Proteobacteria predominant within the rhizosphere. Proteobacteria isolated from Populus roots have been shown to possess acyl-homoserine lactone (AHL)-type quorum sensing (QS) activity, a cell-to-cell signaling system among bacteria that is dependent on cell density. The AHL QS system includes both signal synthases (encoded by luxI-type genes) and signal receptors (encoded by luxR-type genes), but some of the LuxR proteins have been found to respond instead to plant-derived chemical elicitors. Scientists at Oak Ridge National Laboratory, as part of the Plant-Microbe Interfaces Scientific Focus Area within the Department of Energy’s Office of Biological and Environmental Research, discovered a gene in a Proteobacteria Pseudomonas spGM79 isolated from Populus roots that is a plant signal-activated “orphan” member of the LuxR family of regulatory genes. The gene, pipR, is often flanked by predicted peptidase and peptide transporter genes and is closely related to a gene present in plant pathogens that similarly responds directly to plant-derived signals. Studies support the hypothesis that active transport of a peptide-like signal is required for the signal to interact with PipR, which then activates peptidase gene expression. The identification of a peptide ligand for PipR provides a foundation to identify plant-derived signals for orphan LuxR family proteins.

Contacts (BER PM)
Cathy Ronning
SC-23.2
catherine.ronning@science.doe.gov
(PI Contact)
Caroline Harwood
University of Washington, Seattle, WA
csh5@uw.edu

Funding
This work was funded by the Genomic Science program, Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy, as part of the Plant-Microbe Interfaces Scientific Focus Area (http://pmiweb.ornl.gov/).

Publications
Schaefer, A. L., Y. Oda, B. G. Coutinho, D. Pelletier, J. Weiburg, V. Venturi, E. P. Greenberg, and C. S. Harwood. 2016. “A LuxR Homolog in a Cottonwood Tree Endophyte that Activates Gene Expression in Response to a Plant Signal or Specific Peptides,” mBio 7(4), e01101-16. DOI: 10.1128/mBio.01101-16. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 19, 2016

Diverse Fungi Secrete Similar Suite of Decomposition Enzymes

Genomic and proteomic analyses reveal diversity in carbon turnover and other degradation processes.

The Science
Soil fungi secrete a wide range of enzymes that play an important role in biogeochemistry as well as in biofuel production and bioremediation of metal-contaminated soils and water. A recent study sheds new light on a suite of enzymes secreted by diverse fungal species commonly found in soil ecosystems worldwide.

The Impact
The findings reveal different fungal species secrete a rich set of enzymes that share similar functions, despite species-specific differences in the amino acid sequences of these enzymes. This information enhances understanding of the role fungi play in biogeochemical processes occurring in soil and could be used to engineer fungal enzymes for biofuel production and bioremediation efforts.

Summary
Fungi secrete a diverse repertoire of enzymes that break down tenacious plant material. These powerful enzymes degrade plant cell wall components such as cellulose and lignin, resulting in the release of carbon dioxide from soils with dead plant material into the atmosphere. As such, fungal enzymes are not only critical drivers of climate dynamics, but they also hold promise for cost-effective development of alternative transportation fuels. Moreover, the manganese [Mn(II)]-oxidizing capacity of certain fungal species can be harnessed to remove toxic metals from contaminated soils and water. Yet few studies have characterized enzymes secreted by diverse Mn(II)-oxidizing fungi that are commonly found in the environment. To address this knowledge gap, a team of researchers recently used liquid chromatography-tandem mass spectrometry (LC-MS/MS), genomic, and bioinformatic analyses to characterize and compare enzymes secreted by four Mn(II)-oxidizing Ascomycetes species. These four species were recently isolated from coal mine drainage treatment systems and a freshwater lake contaminated with high concentrations of metals and are associated with varied environments and common in soil ecosystems worldwide. The researchers performed LC-MS/MS-based comparative proteomics using the Linear Ion Trap Quadrupole Orbitrap Velos mass spectrometer at the Department of Energy’s (DOE) Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility. This analysis revealed that fungi secrete a rich yet functionally similar suite of enzymes, despite species-specific differences in the amino acid sequences of these enzymes. These findings enhance understanding of the role Ascomycetes species play in biogeochemistry and climate dynamics and reveal lignocellulose-degrading enzymes that potentially could be engineered for renewable energy production or bioremediation of metal-contaminated waters. This study represents a collaboration among scientists from Harvard University, EMSL, Pacific Northwest National Laboratory, Smithsonian Institution, DOE Joint Genome Institute (JGI), Centre National de la Recherche Scientifique and Aix-Marseille Université, King Abdulaziz University, University of Minnesota, and Woods Hole Oceanographic Institution.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contact
Carolyn Zeiner
Harvard University
zeiner@bu.edu

Funding
This work was supported by DOE’s Office of Science, Office of Biological and Environmental Research, including support of EMSL and JGI, Office of Science user facilities, and Harvard University.

Publication
Zeiner, C. A., S. O. Purvine, E. M. Zink, L. PaÅ¡a-Tolić, D. L. Chaput, S. Haridas, S. Wu, K. LaButti, I. V. Grigoriev, B. Henrissat, C. M. Santelli, and C. M. Hansel. 2016. “Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi,” PLOS ONE 11(7), e0157844. [DOI:10.1371/journal.pone.0157844]. (Reference link)

Related Links
EMSL science highlight
JGI science highlight

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER



Researchers compared fungal secretions to enhance understanding of the role Ascomycetes species play in soil biogeochemistry and climate dynamics. Image courtesy of the Department of Energy’s Environmental Molecular Sciences Laboratory



July 02, 2016

Unraveling the Complex Metabolism of a Potential Biofuels-Producing Green Alga

A genome-scale metabolic model of a green microalga is providing strategies for improving its growth.

The Science  
The green microalga, Chlorella vulgaris, has the potential to act as a cell factory in the production of biofuels and bioproducts. To better understand the complex and diverse metabolic capabilities of this green microalga, researchers transformed the organism’s genomic data into a mathematical model. This model enabled the researchers to understand and systematically analyze how the alga is able to grow in a variety of conditions including with just sunlight and carbon dioxide. The model then provided guidance on modifying the conditions to enhance growth performance.

The Impact
An in-depth understanding of how microorganisms use nutrients and grow is essential to improving the production of desired products, including biofuels and bioproducts. The developed model simulated different growth parameters simultaneously (e.g., nutritional resources, genetic modifications, and light source and availability) so that optimal conditions can be predicted. Optimizing growth conditions maximizes the probability of obtaining the desired experimental result, while also saving valuable time and resources.

Summary
The global movement toward more green-energy opportunities is resulting in the development of new approaches for producing renewable fuels in economical ways. The green microalga, C. vulgaris, is recognized as a promising candidate for biofuel production due to its ability to store high amounts of lipids and its natural metabolic versatility. However, many fundamental questions remain on how this alga and other microorganisms can more efficiently use nutritional sources not just for the organism’s growth, but also for sustainable and efficient production of biofuel and bioproducts. Researchers from the University of California, San Diego; Johns Hopkins University; University of Delaware; and National Renewable Energy Laboratory wanted to develop a way to more efficiently modify C. vulgaris to improve growth productivity. To do this, the scientists developed a compartmentalized genome-scale metabolic model that enabled quantitative insight into the organism’s metabolism. The model accurately predicted phenotypes under a variety of growth conditions including photoautotrophic, heterotrophic, and mixotrophic conditions. Model validation was performed using experimental data, laying the foundation for model-driven strain design and growth medium alteration to improve biomass yield. Model prediction of growth rates under various medium compositions and subsequent experimental tests showed an increased growth rate with the addition of the amino acids tryptophan and methionine. The reconstruction represents the most comprehensive model of eukaryotic photosynthetic organisms to date, based on genome size and number of genes in the reconstruction. With this metabolic model, researchers should be able to improve experimental design strategies for strain, production process, and final product yield optimization.

Contact (BER PM)
Dawn Adin, Ph.D.
Program Manager, Office of Biological and Environmental Research
dawn.adin@science.doe.gov  

Contacts (PIs)
Michael J. Betenbaugh
Department of Chemical and Biomolecular Engineering
Johns Hopkins University
beten@jhu.edu    

Karsten Zengler
Department of Bioengineering
University of California, San Diego
kzengler@ucsd.edu

Funding
This work was supported by the National Science Foundation (grant number 1332344); U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (grant number DE-SC0012658); and Mexican National Research Council (fellowship number 237897 to C.Z.).

Publication
C. Zuñiga, C.-T. Li, T.Huelsman, J. Levering, D. C. Zielinski, B. O. McConnell, C. P. Long, E. P Knoshaug, T. G. Guarnieri, M. R. Antoniewicz, M. J. Betenbaugh, and K. Zengler, “Genome-scale metabolic model for the green alga Chlorella vulgaris UTEX 395 accurately predicts phenotypes under autotrophic, heterotrophic, and mixotrophic growth conditions.” Plant Physiology 172, 589-602 (2016). [DOI: 10.1104/pp.16.00593] (Reference link)

Related Links
Zengler Laboratory Website
Betenbaugh Laboratory Website

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The polytrophic metabolism of photosynthetic microorganisms such as Chlorella vulgaris (pictured in test tubes) enables them to take advantage of different energy sources for growth. [Image courtesy of the National Renewable Energy Laboratory]



April 26, 2016

Poplar-Associated Bacterial Isolates Induce Additive Favorable Responses in a Constructed Plant-Microbiome System

These findings suggest microbiome phenotype can be predicted from phenotypes of individual community members.

The Science
A recent study showed that two species of plant growth-promoting bacteria enhanced beneficial plant traits such as root growth and photosynthetic potential in poplar trees, both by themselves and, in combination, in an additive manner.

The Impact
The effects observed in this constructed microbial community study suggest that microbiome function may be predicted in these systems from the additive functions of selected individual microbial species.

Summary
The diverse microbial communities that inhabit the zones within and surrounding the roots of plants, the “root microbiome,” have a significant influence on the host plant’s health and vitality. The root microbiome of Populus, a genus of trees that are a potential bioenergy feedstock, contains a high abundance of microbes known as β- and γ-Proteobacteria. Both of these classes include multiple bacterial species known to promote plant growth. To understand the contribution of individual microbiome members in a community, researchers at Oak Ridge National Laboratory (ORNL), funded by the Department of Energy’s (DOE) Plant-Microbe Interfaces Science Focus Area and U.S. Department of Agriculture-DOE Plant Feedstocks Genomics for Bioenergy program, studied a simplified community consisting of Pseudomonas (γ-Proteobacteria) and Burkholderia (β-Proteobacteria) bacterial strains inoculated on sterile Populus cuttings under controlled laboratory conditions. Alone and in combination, the two species increased root growth and photosynthetic potential and activated unique pathways relative to uninoculated controls.   Complementary data such as photosynthetic efficiency, gene expression, and metabolite expression data, in individual and in mixed inoculated treatments, indicate that the molecular effects of these bacterial strains are unique and additive. This work is the first constructed community study to show the additive host effects of bacteria, and the results suggest that microbiome function may be predicted from the synergistic effects of individual members of the microbial community.

Contacts
(BER PM)
Cathy Ronning, SC-23.2, catherine.ronning@science.doe.gov, 301-903-9549

(PI Contact)
Collin Timm
Biosciences Division, ORNL
timmcm@ornl.gov

Funding
This work was funded by DOE’s Office of Science, Office of Biological and Environmental Research, Biological Systems Science Division, Genomic Science and Plant Feedstock Genomics for Bioenergy programs (DE-SC0010423); and Plant-Microbe Interfaces Science Focus Area at Oak Ridge National Laboratory.

Publications
Timm, C. M., D. A. Pelletier, S. S. Jawdy, L. E. Gunter, J. A. Henning, N. Engle, J. Aufrecht, E. Gee, I. Nookaew, Z. Yang, T. Lu, T. J. Tschaplinksi, M. J. Doktycz, G. A. Tuskan, and D. J. Weston. 2016. “Two Poplar-Associated Bacterial Isolates Induce Additive Favorable Responses in a Constructed Plant-Microbiome System,” Frontiers in Plant Sciences 7:497. DOI: 10.3389/fpls.2016.00497. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 11, 2016

A New View of the Tree of Life

Access to a wealth of environments and the ability to reconstruct genomes for previously unknown and uncultured lineages has greatly expanded understanding of the diversity of life on earth.

The Science
A comprehensive three domain tree of life was constructed from all lineages for which sequenced genomes are available. The tree highlights the diversity contained in candidate phyla: lineages with no cultivated representatives for which genome sequences are derived from environmental surveys.

The Impact
The tree of life is one of the most important organizing principles in biology. The new depiction will be useful not only to biologists who study microbial ecology, but also to biochemists searching for novel genes and researchers studying evolution and earth history. This updated view highlights the weight of diversity found within the bacteria and within lineages with no cultured representatives.

Summary
This tree presents a new view of the diversity of life from a genome perspective. Exploration of new environments and deeper sequencing of well-studied systems continue to uncover new organisms and lineages on the tree. To construct a comprehensive tree of life, researchers gathered 3,085 genomes representing all genera for which genomes are available and including over 1,000 newly reconstructed genomes targeting candidate phyla representatives. Sample sites for new genomes included extreme environments like Chile’s Atacama Desert salt flats and Yellowstone National Park hot springs, but also more common environments such as groundwater, estuarine sediment, meadow soil, and dolphin oral microbiomes. The tree inferred from this genomic perspective shows the predominance of bacterial diversity compared to the divergence seen in the Archaea and Eukarya.  Collapsing the tree based on sequence divergence rather than taxonomy highlighted the amount of diversity found within candidate phyla, emphasizing the importance of environmental surveys for discovery of organisms not tractable in laboratory experiments.

Contacts (BER PM)
Todd Anderson
Todd.Anderson@science.doe.gov

David Lesmes
David.Lesmes@science.doi.gov

(PI Contact)
Jillian Banfield
University of California Berkeley
jbanfield@berkeley.edu

Funding
This research was largely supported by Lawrence Berkeley National Laboratory’s (LBNL) Genomes to Watershed Scientific Focus Area funded by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (BER) under contract DE-AC02-05CH11231. Additional support was provided by LBNL EFRC award DE-AC02-05CH11231; National Aeronautics and Space Administration NESSF grant 12 PLANET12R-0025 and National Science Foundation DEB grant 1406956; DOE BER grant DOE-SC10010566; Office of Naval Research grants N00014-07-1-0287, N00014-10-1-0233, and N00014-11-1-0918; and the Thomas C. and Joan M. Merigan Endowment at Stanford University. In addition, funding was provided by the Ministry of Economy, Trade, and Industry of Japan, and metagenome sequence was generated by DOE’s Joint Genome Institute via the Community Science Program.

Publication
Hug, L. A., B. J. Baker, K. Anantharaman, C. T. Brown, A. J. Probst, C. J. Castelle, C. N. Butterfield, A. W. Hernsdorf, Y. Amano, K. Ise, Y. Suzuki, N. Dudek, D. A. Relman, K. M. Finstad, R. Amundson, B. C. Thomas, and J. F. Banfield. 2016. “A New View of the Tree of Life,” Nature Microbiology 1(16048), DOI: 10.1038/nmicrobiol.2016.48. (Reference link)

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


April 04, 2016

A One-Pot Recipe for Making Jet Fuel

Researchers use engineered bacteria to simplify biofuels production, potentially lowering cost.

The Science
Researchers isolated an Escherichia coli mutant that tolerates a liquid salt used to break apart plant biomass into sugary polymers. Because the salt solvent, known as ionic liquid (IL), interferes with the later stages of biofuels production, it has to be removed before proceeding, a process that requires time and money. The researchers genetically engineered the E. coli strain to excrete an IL-tolerant cellulase and used the resulting sugars to synthesize d-limonene, a jet fuel precursor.

The Impact
IL-tolerant bacteria enable a “one-pot” method for producing advanced biofuels from a slurry of pretreated plant material, helping to streamline the production process, which is critical to making biofuels a viable competitor with fossil fuels.

Summary
Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multistep process. Each of the steps—deconstruction of the cellulose, hemicellulose, and lignin that are bound together in the plant cell wall; addition of enzymes to release sugars; and conversion into the desired biofuel—is done in separate pots. Significant effort has gone into developing efficient solutions to these discrete steps, but few studies report the consolidation of the multistep workflow into a single pot reactor system. Researchers at the Department of Energy’s (DOE) Joint BioEnergy Institute (JBEI) demonstrate a one-pot biofuel production process that uses an IL (1-ethyl-3-methylimidazolium acetate) for pretreating switchgrass biomass. This IL is highly effective in deconstructing lignocellulose, but leaves behind a residue that is toxic to standard cellulase and the microbial production host. JBEI scientists established that an amino acid mutation in the gene rcdA leads to an E. coli strain that is highly tolerant to ILs. To develop a strain for a one-pot process, they engineered this IL-tolerant strain to express a d-limonene production pathway. The JBEI researchers also screened previously reported IL-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the IL-tolerant E. coli strain to secrete this IL- tolerant cellulase. The final strain was found to digest pretreated biomass and use the liberated sugars to produce the jet fuel candidate precursor d-limonene in a one-pot process.

Contacts (BER PM)
N. Kent Peters
Program Manager, Office of Biological and Environmental Research
kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
Aindrila Mukhopadhyay
Joint BioEnergy Institute, Emeryville, CA, USA
amukhopadhyay@lbl.gov

Funding
This work was part of the Joint BioEnergy Institute supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research through contract DE-AC02-05CH11231.

Publication
Frederix, M., et al. 2016. “Development of an E. coli strain for One-Pot Biofuel Production from Ionic Liquid Pretreated Cellulose and Switchgrass,” Green Chemistry, DOI: 10.1039/c6gc00642f. (Reference link)

Related Links
News release

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Researchers in a microbiology lab at the Joint BioEnergy Institute are working to streamline the biofuels production process. Image courtesy Lawrence Berkeley National Laboratory



March 30, 2016

Engineering Intracellular Organelles to Increase Production of Useful Chemicals by Confining Their Metabolic Pathways

Scientists move closer to engineering metabolic pathways within yeast intracellular compartments tailored for desired purposes.

The Science
Engineering microbes to produce valuable bioproducts or biofuels is often complicated, because the product is deleterious to the cell or the chemical conditions in the cytosol are not favorable for the required chemical reactions. To overcome this challenge, researchers at the University of California (UC), Berkeley, have developed a system to facilitate the introduction of enzymes into the yeast peroxisome, isolating the selected metabolic pathway from the cytosolic environment.

The Impact
The development of a versatile intracellular organelle to confine engineered metabolic pathways will facilitate metabolic engineering. This research advances the repurposing of the yeast peroxisome to isolate synthetic metabolic pathways that would be inefficient if expressed dissolved in the cytosol. This system will make it possible to engineer eukaryotic cells to produce high yields of useful chemicals that cannot be achieved in traditional biological systems.

Summary
High-yield production of bioproducts and fuels in microbial systems requires metabolic flux to be directed toward an engineered pathway. However, this redirection of metabolic flux is difficult to achieve because cells tend to divert metabolic flux toward native cellular processes. Engineered metabolic pathways have been confined to organelles such as the mitochondrion or the vacuole to isolate them from the host's metabolism, but the cell needs those organelles for its normal functions and, therefore, they cannot be completely repurposed. On the other hand, yeast can live without peroxisomes, making this an ideal organelle to isolate newly designed metabolic pathways and their products. A research team at UC Berkeley has discovered a protein signal that allows the efficient targeting of engineered proteins into the peroxisome. The researchers also devised a high-throughput method to measure the efficiency of the process and demonstrated the feasibility of the approach by introducing a simple metabolic pathway that produces a colored compound into the yeast peroxisome. The strategy can now be used to sequester useful metabolic pathways into the peroxisome to produce high yields of valuable chemicals and fuels.

Contacts (BER PM)
Pablo Rabinowicz
Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
John Dueber
Bioengineering Department
University of California, Berkeley
jdueber@berkeley.edu

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science under Early Career Research Program award DE-SC0008084. Authors also acknowledge support from the National Science Foundation and U.S. Department of Defense.

Publications
DeLoache, W. C., Z. N. Russ, and J. E. Dueber. 2016. “Towards Repurposing the Yeast Peroxisome for Compartmentalizing Heterologous Metabolic Pathways,” Nature Communications 7, 11152. DOI: 10.1038/ncomms11152. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 25, 2016

Improving Lipid Yields for Biofuel Production

New insights into lipid metabolism in yeast could benefit biofuel production.

The Science
Using a comprehensive system-wide approach, researchers identified regulators of metabolic pathways that drive lipid accumulation in a genetically tractable yeast species.

The Impact
A better understanding of the metabolic pathways that regulate lipid accumulation in yeast could be harnessed to improve lipid yields and enhance the efficiency of biofuel production.

Summary
The yeast Yarrowia lipolytica is capable of accumulating a large amount of lipids when nitrogen is limited. This ability, along with its amenability to genetic methods, has made Y. lipolytica an attractive model for generating high-value lipids for biofuel production. However, relatively little is known about the factors that regulate enzymatic pathways responsible for lipid accumulation in this species. To address this knowledge gap, a team of researchers from Pacific Northwest National Laboratory (PNNL) integrated metabolome, proteome, and phosphoproteome data to characterize lipid accumulation in response to limited nitrogen in Y. lipolytica. The researchers used a microscopy system that integrates nonlinear two-photon excitation, laser-scanning confocal microscopy, and fluorescence lifetime imaging at the Environmental Molecular Sciences Laboratory (EMSL), a U.S. Department of Energy (DOE) scientific user facility. In this first global study of protein phosphorylation in Y. lipolytica, the researchers focused their analysis on changes in the expression and phosphorylation state of regulatory proteins, including kinases, phosphatases, and transcription factors. They found that lipid accumulation in response to nitrogen limitation results from two distinct processes: (1) higher production of malonyl-CoA from excess citrate increases the pool of building blocks for lipid production, and (2) decreased capacity for β-oxidation reduces lipid consumption. These findings provide new genetic targets that could be manipulated to improve lipid yields in future metabolic engineering efforts.

Contacts
(BER PM Contact)
Paul Bayer, SC-23.1, 301-903-5324

(PI Contact)
Scott E. Baker
EMSL
Scott.Baker@pnnl.gov
509-372-4759

Funding
This work was supported by DOE’s Office of Science, Office of Biological and Environmental Research (BER), including support of EMSL, an Office of Science user facility; BER Genomic Science program; William Wiley Distinguished Postdoctoral Fellowship; and BER-funded Pan-omics program at PNNL.

Publication
Pomraning, K. R., Y.-M. Kim, C. D. Nicora, R. K. Chu, E. L. Bredeweg, S. O. Purvine, D. Hu, T. O. Metz, and S. E. Baker. 2016. “Multi-Omics Analysis Reveals Regulators of the Response to Nitrogen Limitation in Yarrowia lipolytica,” BMC Genomics 17(138). DOI: 10.1186/s12864-016-2471-2. (Reference link).

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


February 18, 2016

Biofuel Tech Straight from the Farm

Herbivore digestion of lignocellulosic biomass involves a large variety of enzymes.

The Science
Herbivores eat many types of lignocellulosic plants, and fungi digest this material in the animals’ guts. A new study has characterized several fungi involved in this digestion process, identifying a large number of enzymes that work synergistically to degrade the raw biomass.

The Impact
Industry is exploring strategies to more effectively turn biomass like wood and grasses into fuel or chemicals. Because the matrix of complex molecules found in plant cell walls—lignin, cellulose, and hemicellulose—is difficult to break down using biological methods, costly pretreatments with heat or chemicals are necessary. The discovery of new, highly effective biomass-degrading enzymes in anaerobic fungi could accelerate the development of a process to convert lignocellulose feedstocks into fermentable sugars without pretreatment, potentially leading to more efficient conversion of raw biomass to biofuels and biobased products.

Summary
Scientists have long known that anaerobic fungi living in the guts of herbivores play a significant role in helping those animals digest plants. However, culturing these fungi in the lab is difficult because they cannot survive in the presence of oxygen and must be grown in sealed containers. A research team led by Michelle O’Malley at the University of California, Santa Barbara, isolated three species of these fungi in feces from goats, horses, and sheep. The enzymes expressed by these fungi work together to break down crude, untreated plant biomass. The research showed that the fungi adapt their enzymes to the different kinds of plant materials eaten by these animals, so that wood, grass, or agricultural waste all can be efficiently digested. Each of the fungi studied was found to contribute in a characteristic way, tailoring their combined action to the particular type of biomass being digested. These findings could help in identifying distinctive enzymes from other anaerobic gut fungi, with potential applications for biomass processing and sustainable biofuel production.

Contacts
PM Contact
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

PI Contact
Michelle A. O’Malley
Department of Chemical Engineering
University of California, Santa Barbara
momalley@engineering.ucsb.edu

Funding
This work was supported by the Office of Biological and Environmental Research (BER) within the U.S. Department of Energy’s (DOE) Office of Science under Early Career Research Program award DE-SC0010352. A portion of this research was performed under the JGI-EMSL Collaborative Science Initiative and used resources at DOE’s Joint Genome Institute (JGI) and Environmental Molecular Sciences Laboratory (EMSL), which are DOE Office of Science user facilities and sponsored by BER. Authors also acknowledge support from the U.S. Department of Agriculture (Award 2011-67017-20459) and Institute for Collaborative Biotechnologies through grant W911NF-09-0001.

Publications
Solomon, K., et al. "Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes." Science (2016). [DOI: 10.1126/science.aad1431].   (Reference link

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Microbes found in the digestive tract of large herbivores offer attractive enzyme platforms for lignocellulosic processing. [Image courtesy of iStock]



February 08, 2016

New Real-Time Approach for Monitoring Chemical Production by Genetically Engineered Microbes

Fluorescent sensors were developed to measure the productivity of bacteria engineered to synthesize precursors for plastics and other materials.  

The Science      
Metabolic engineering of microbes has great potential for sustainable and environmentally friendly production of industrial chemicals. Researchers at Harvard University have designed molecular tools (sensors) that enable them to follow the production of precursors for plastics and other chemicals in engineered microorganisms. These sensors produce increasing fluorescence as the amount of the desired product augments (i.e., "sensing" the presence of the product), making it possible to rapidly select the genetic modifications that result in the highest chemical yields.

The Impact
Constructing new microorganisms that make high amounts of desired compounds requires designing, modifying, and testing many different strains to ultimately select the best producer. Those tests use laborious and costly analytical techniques. The fluorescent sensors developed by this research enable rapid detection of individual strains that produce the largest amounts of desired chemicals by just measuring fluorescence. Coupled with cell sorting technologies, these sensors will enable the testing of millions of engineered strains in a single day.      
 
Summary
This research has resulted in the development of a genetic sensor that provides a fluorescent readout proportional to the intracellular concentration of 3-hydroxypropionate, a valuable plastic precursor also called 3HP. This sensor required the introduction of several enzymes into the model bacterium Escherichia coli to convert 3HP into acrylate (another plastic precursor). Next, the gene for a fluorescent reporter whose expression is activated by acrylate also was introduced into the same E. coli strain so that when acrylate is produced, fluorescence can be detected and used as proxy for the amount of 3HP synthesized. With this system, the researchers could easily identify a strain and culture conditions that produced over 20 times more 3HP than previously achieved. At the same time, this research demonstrated the first heterologous pathway for microbial production of acrylate. The investigators proved the flexibility of the approach by designing a similar sensor to monitor muconate (used to make nylon) and glucarate (needed for manufacturing detergents and other chemicals). The fluorescent biosensors developed by this research combined with fluorescence-based cell sorting will accelerate the development of sustainable production of relevant chemicals such as biofuels and biopolymers in engineered microbial systems.

Contacts (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
George M. Church
Wyss Institute for Biologically Inspired Engineering
Harvard University
Boston, MA
gchurch@genetics.med.harvard

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DEFG02-02ER63445. Authors also acknowledge support from the National Science Foundation.  

Publications
Rogers, J. K., and G. M. Church. 2016. “Genetically Encoded Sensors Enable Real-Time Observation of Metabolite Production,” Proceedings of the National Academy of Sciences (USA) 113(9), 2388-93. DOI: 10.1073/pnas.1600375113. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 05, 2016

New Understanding of One of Nature’s Best Biocatalysts for Biofuels Production

Discovery of a new enzyme system sheds further light on a microbe’s ability to efficiently break down inedible plant matter for conversion to biofuels and biobased chemicals.

The Science
Researchers found that Clostridium thermocellum—an anaerobic, thermophilic microorganism—uses a previously unknown mechanism to degrade cellulose (scaffolded cellulase enzymes not attached to the bacterial cell wall), in addition to other known degradation mechanisms (cellulosomes and free enzymes).

The Impact
This discovery helps explain C. thermocellum’s superior ability to digest biomass and demonstrates the highly diverse strategies evolved in nature for biomass conversion. Researchers are using the study’s findings to develop optimal systems for breaking down lignocellulosic biomass to produce biofuels and biobased chemicals.

Summary
Lignocellulosic biomass is the largest source of organic matter on Earth, making it a promising renewable feedstock for producing biofuels and chemicals. Currently, however, the main bottleneck in biofuel production is the low efficiency of cellulose conversion, which leads to high production costs. To date, C. thermocellum is the most efficient microorganism known for solubilizing lignocellulosic biomass. Its high cellulose digestion capability has been attributed to the organism’s efficient cellulases consisting of both a free enzyme system and a tethered cellulosomal system, wherein multiple carbohydrate active enzymes are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. U.S. Department of Energy (DOE) BioEnergy Science Center (BESC) researchers recently discovered that C. thermocellum also expresses a type of cellulosomal system that is not bound to the cell wall, a “cell-free” cellulosomal system. Researchers believe the cell-free cellulosome complex functions as a “long-range” cellulosome because it can diffuse away from the cell and degrade polysaccharide substrates distant from the bacterial cells. This discovery reveals that C. thermocellum utilizes not only all the previously known cellulase degradation mechanisms (cellulosomes and free enzymes), but also a new category of scaffolded enzymes not attached to the cell. This unexpected finding explains C. thermocellum’s superior performance on biomass, demonstrating that nature’s strategies for biomass conversion are not yet fully understood and could provide further opportunities for microbial enzyme discovery and engineering efforts.

Contacts (BER PM)
N. Kent Peters
Program Manager, Office of Biological and Environmental Research
kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
Yannick Bomble
Research Scientist, Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, and BESC, Oak Ridge National Laboratory, Oak Ridge, Tennessee
yannick.bomble@nrel.gov

Funding
This work was supported by BESC, a DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research within DOE's Office of Science. A portion of this work also was supported by the United States-Israel Binational Science Foundation, Jerusalem, Israel; Israel Science Foundation, Israeli Center of Research Excellence; European Union NMP.2013.1.1-2: CellulosomePlus Project 8 number 604530; and the ERA-IB Consortium (EIB.12.022) FiberFuel. 

Publications
Xu, Q., et al. 2016. “Dramatic Performance of Clostridium thermocellum Explained by Its Wide Range of Cellulase Modalities,” Science Advances 2(2), e1501254. DOI: 10.1126/sciadv.1501254. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The microbe Clostridium thermocellum (stained green) is seen growing on poplar tissue. [Image courtesy of Oak Ridge National Laboratory]



January 12, 2016

Using Bacteria to Achieve High Solubilization of Biomass with Minimal Pretreatment

Thermophilic bacteria prove to be efficient biocatalysts for biomass solubilization.

The Science
A comprehensive comparison of lignocellulosic solubilization by various thermophilic bacteria to standard enzyme treatment found microbial solubilization of cellulosic biomass to be more effective, and enhanced by mechanical disruption.

The Impact
Using thermophilic bacteria instead of expensive yeast enzymes to decompose biomass into its sugars for fermentation into biofuels will greatly reduce costs and potentially simplify the process.

Summary
Feedstock recalcitrance is the greatest barrier to cost-effective production of cellulosic biofuels. To overcome this recalcitrance, existing commercial cellulosic ethanol facilities employ thermochemical pretreatment with subsequent addition of fungal cellulase. However, processing cellulosic biomass without thermochemical pretreatment may be possible using thermophilic, cellulolytic bacteria. Researchers at the Department of Energy’s (DOE) BioEnergy Science Center (BESC) examined the ability of various thermophilic bacteria to solubilize autoclaved, but otherwise unpretreated cellulosic biomass. Carbohydrate solubilization of mid-season harvested switchgrass after 5 days ranged from 24 percent to 65 percent, with Clostridium thermocellum showing the best results among the four thermophiles tested. This finding was as much as fivefold better than with the standard method using a fungal cellulase cocktail and yeast fermentation. Other findings showed that there was equal fractional solubilization of glucan and xylan, and, importantly, that there was no biological solubilization of the noncarbohydrate fraction of biomass. A fivefold improvement over standard treatment was observed when using the most effective biocatalyst. Using thermophilic bacteria in biomass-solubilizing systems may enable a reduction in the amount of nonbiological processing required and, in particular, substitution of cotreatment for pretreatment.

Contacts (BER PM)
N. Kent Peters, SC-23.2, kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
Lee Lynd
Professor, Thayer School of Engineering, Dartmouth College
lee.lynd@dartmouth.edu

Funding
This research was sponsored by BESC, a DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research within DOE's Office of Science. TYN was supported by the National Science Foundation. The generation of the CCRC series of plant cell wall glycan-directed monoclonal antibodies used was supported by NSF's Plant Genome Program.

Publication
Paye, J. M. D., et al. 2016. “Biological Lignocellulose Solubilization: Comparative Evaluation of Biocatalysts and Enhancement via Cotreatment,” Biotechnology for Biofuels 9(8), DOI 10.1186/s13068-015-0412-y. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 07, 2016

A Novel Lipid Pathway Makes Massive Quantity of Surface Wax on Bayberry Fruit

Pathway gives metabolic engineers new tools for producing high-value lipids.

The Science
Bayberry fruits produce the highest amount of surface wax known in nature. Recent biochemical and gene expression data have revealed a novel biosynthesis pathway for the waxy layer that is more closely related to cutin biosynthesis than conventional triacylglyceride biosynthesis.

The Impact
The discovery of how the Bayberry fruit produces massive amounts of unique surface wax aids in understanding the plant’s mechanism for producing and secreting nonmembrane glycerolipids and suggests ways to engineer pathways for high-value waxy lipid production in biomass crops.

Summary
Scientists from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) studied how Bayberry fruits accumulate massive quantities of a unique surface wax with a structure similar to triacylglycerol seed oils. Research on plants that produce such large amounts of surface lipids is providing insights into the molecular features and biochemical pathways for plant lipid secretion and thus may help in developing strategies to engineer lipid production in non-seed tissues. The GLBRC scientists examined changes in fruit anatomy and details of the chemical structures secreted by Bayberry fruits, and quantified the accumulation of wax through fruit development. Biochemical pathway analysis by [14C]-labeling and transcript analysis by RNA-seq revealed features of Bayberry wax accumulation that are distinctly different from conventional triacylglycerol production. Together, these results indicate that the extracellular glycerolipids in Bayberry wax are synthesized by a novel pathway that differs from previously defined triacylglycerol biosynthesis pathways. An increased understanding of this process may prove useful in engineering plants for secretion of high-energy and high-value lipids, particularly those that have toxic or negative consequences when accumulated inside cells.

Contacts
(BER PM)

N. Kent Peters, SC-23.2, kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
John B. Ohlrogge
Michigan State University
ohlrogge@msu.edu

Funding
This work was funded by GLBRC (DOE, Office of Science, Office of Biological and Environmental Research DE-FC02-07ER64494) and a National Science and Engineering Research Council of Canada postgraduate fellowship (PGS-D3).

Publications
Simpson, J. P., and J. B. Ohlrogge. 2016. “A Novel Pathway for Triacylglycerol Biosynthesis Is Responsible for the Accumulation of Massive Quantities of Glycerolipids in the Surface Wax of Bayberry (Myrica pensylvanica) Fruit, The Plant Cell 28(1), 248–64. DOI: 10.1105/tpc.15.00900. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 04, 2016

Increased Production of Bioplastics in Engineered Bacteria

Metabolic engineering doubles the production of ethylene in Escherichia coli.

The Science
Researchers analyzed growth media and nutrient supplements to identify candidate genes that affect yields in an engineered E. coli strain that produces ethylene, a hydrocarbon used in the production of a wide range of chemicals and plastics. Guided by the results of those analyses, the researchers further engineered the bacterial strain, altering several metabolic and regulatory genes to more than double the original ethylene production levels.

The Impact
Ethylene is currently derived from fossil fuels through an energy-intensive process called steam cracking. The production of plastics and many other products and chemicals creates huge demand for ethylene, so biological production of ethylene has great potential to reduce the industry's carbon footprint. Ethylene biosynthesis has been engineered in microbial systems, but with low yields. The identification and engineering of selected E. coli metabolic and regulatory genes in this research has resulted in a substantial increase in ethylene yield, advancing the sustainable bioproduction of this critical hydrocarbon.

Summary
Ethylene is one of the most industrially important chemicals derived from petroleum. Therefore, scientists have been trying to develop biological systems to produce ethylene in a sustainable way. Expression of a heterologous bacterial ethylene-forming enzyme (EFE) in E. coli has resulted in the production of ethylene, but the yields were too low for industrial purposes. Researchers at the National Renewable Energy Laboratory and University of Colorado Boulder conducted a study of the effects of different nutrients and substrates present in the growth medium for the EFE-expressing E. coli strain to be able to predict which genes significantly affect ethylene yields. Guided by those findings, they re-engineered E. coli to minimize competing pathways within central metabolism and to overproduce key enzymes predicted to increase ethylene productivity. The re-engineered strain produced more than twice as much ethylene relative to the original EFE-expressing E. coli strain. Those yields can be further improved by identifying and engineering additional enzymes and regulatory factors that prevent higher metabolic flow toward ethylene biosynthesis. This work advances the development of a sustainable ethylene production industry that is not dependent on fossil fuels.  

Contacts (BER PM)
Pablo Rabinowicz, SC-23.2, pablo.rabinowicz@science.doe.gov, 301-903-0379

(PI Contact)
Pin–Ching Maness
National Renewable Energy Laboratory, Golden, Colorado
pinching.maness@nrel.gov

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science under award DE-SC008812.

Publications
Lynch, S., C. Eckert, J. Yu, R. Gill, and P. C. Maness. 2016. “Overcoming Substrate Limitations for Improved Production of Ethylene in E. coli,” Biotechnology for Biofuels 9:3. DOI: 10.1186/s13068-015-0413-x. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 14, 2015

Optimizing Microbial Bioproduction of Fuels

Identifying factors that contribute to cell-to-cell variability in lipid production.

The Science
Microbial strains engineered to produce a large amount of lipids hold tremendous promise for the production of biofuels and chemicals. A recent study shed light on underlying causes of microbial cell-to-cell variability in lipid production.

The Impact
The findings revealed that conditions within cells and in the surrounding environment interact to contribute to variability in lipid production. The new insights could lead to strategies that optimize the use of engineered microbial strains for the production of important biofuels and chemicals.

Summary
The microbial production of biofuels and chemicals often does not reach the theoretical maximum yield, even for engineered strains, thereby limiting the reliability of large-scale bioprocessing. To understand the limitations, scientists have started to investigate the reasons for phenotypic diversity of cells within a culture. A team of scientists from the University of Idaho, Environmental Molecular Sciences Laboratory (EMSL), and Massachusetts Institute of Technology used advanced microfluidics combined with Epifluorescent and Raman microscopy at EMSL to study differences in the ability of individual cells of low-yield and high-yield strains of the fungus Yarrowia lipolytica to produce lipids. The researchers found lipid production fluctuated sporadically with time in both strains. The researchers labeled this newly discovered phenomenon “bioprocessing noise.” Furthermore, the high-yield fungal strain showed reduced bioprocessing noise in lipid production than the low-yield fungal strain. This finding indicates differences in the activity of key metabolic genes that contribute to bioprocessing noise and thus cellular diversity in lipid production. Moreover, this variability was amplified by environmental factors such as chemical gradients of nutrients or waste products surrounding cells. Taken together, these findings show extracellular and intracellular fluctuations interact to place an upper limit on the reliability of lipid production and total yield of lipids. This research could pave the way for new strategies to improve the reliability and efficiency of using engineered microbial strains for the production of lipids that could then be converted to valuable biofuels or chemicals.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contacts
Andreas Vasdekis
University of Idaho
andreasv@uidaho.edu

Gregory Stephanopoulos
Massachusetts Institute of Technology
gregstep@mit.edu

Funding
This work was supported by the U.S. Department of Energy’s Office of Science, Office of Biological and Environmental Research, including support of EMSL, a DOE Office of Science user facility; National Institute of General Medical Sciences of the National Institutes of Health; and a Linus Pauling Fellowship from Pacific Northwest National Laboratory.

Publications
Vasdekis, A. E., A. M. Silverman, and G. Stephanopoulos. 2015. “Origins of Cell-to-Cell Bioprocessing Diversity and Implications of the Extracellular Environment Revealed at the Single-Cell Level,” Nature Scientific Reports 5(17689), DOI: 10.1038/srep17689. (Reference link)

Related Links
EMSL article

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


December 04, 2015

Characterizing the Structural Basis of Stereospecificity in Enzymatic Cleavage of Lignin Bonds

Understanding how bacteria digest plant lignin informs engineering efforts to extract value from lignin.  

The Science
To determine the structural basis for stereospecificity of bacterial enzymes involved in lignin bond cleavage, researchers solved the crystal structures of the enzymes involved. The detailed structural and biochemical characterization of the lignin degradation pathway members reveals important new aspects of the enzyme mechanisms and determinants of substrate specificity.

The Impact
Lignin is a combinatorial polymer comprised of monoaromatic units and is a potential source of valuable aromatic chemicals. However, its recalcitrance to chemical or biological digestion presents a major obstacle to the production of second-generation biofuels and other valuable bioproducts. These collaborative studies elucidating mechanisms of lignin degradation may enable the development of efficient pathways for converting lignin into components of advanced biofuels and other bioproducts.      

Summary
Lignin’srecalcitrance to chemical or biological digestion presents a major obstacle to the production of second-generation biofuels and valuable coproducts from lignin’s monoaromatic units. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin was reported in the bacterium Sphingobium sp. SYK-6. In a collaborative effort, researchers from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) and Joint BioEnergy Institute (JBEI) determined the X-ray crystal structures and biochemical characterizations of several glutathione-dependent β-etherases that participate in the cleavage of lignin. Results from these studies reveal important new aspects of the enzyme mechanisms and the determinants of substrate specificity. As β-aryl ether bonds account for 50 percent to 70 percent of all inter-unit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on lignin valorization.

Contacts
(BER PM)

N. Kent Peters, SC-2.32, kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
George N. Phillips, Jr.
Rice University
georgep@rice.edu

Funding
This work was funded by GLBRC and JBEI (DOE Office of Science, Office of Biological and Environmental Research DE-FC02-07ER64494 and DE-AC02-05CH11231, respectively), additional grants from DOE (Office of Science, Office of Basic Energy Sciences, Contract No. DE-AC02-05CH11231 and DE-AC02-06CH11357), grants from the National Institutes of Health (AGM-12006, GM109456, GM098248, P41GM103399, and S10RR027000), the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817), National Cancer Institute (ACB-12002), and University of Wisconsin-Madison.  

Publications
Helmich, K., et al. 2015.  “Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-aryl Ether Bonds in Lignin,” The Journal of Biological Chemistry, DOI: 10.1074/jbc.M115.694307. (Reference link)
Pereira, J. H., et al. 2016. “Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-aryl Ether Cleavage Pathway from Sphingobium sp SYK-6,” The Journal of Biological Chemistry, DOI: 10.1074/jbc.M115.700427. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 27, 2015

Mass Spectrometry Deduces Selectivity of Glycoside Hydrolases for Degrading Biomass Polysaccharides

Improving the annotation of glycoside hydrolases and their phylogenetic trees.

The Science
Multiple classes of polysaccharide-degrading enzymes are used to hydrolyze plant biomass into fermentable sugars for conversion to biofuels. However, there are large numbers of suspected polysaccharide-degrading enzymes whose activities have not been determined biochemically. Researchers have now determined the reaction specificity and other parameters for several of these uncharacterized enzymes using a special mass spectroscopy system along with artificial substrates.

The Impact
Improving the annotation of glycoside hydrolase (GH) phylogenetic trees will improve understanding of the function, synergy, and stability of these enzymes and thereby the creation of biomass-degrading enzymatic cocktails.  

Summary
Researchers at the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) have used chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides to study the reactivity of several enzymes representative of GH function. Patterns of reactivity identified with these NIMS probes provide a diagnostic approach to assess reaction selectivity as well as comparative apparent rate information. Their results show diagnostic patterns for reactions of a β-glucosidase, relaxed but varied specificity of several endoglucanases, and high specificity of a cellobiohydrolase with the model substrate. The researchers also modeled time-dependent reactions of these enzymes by numerical integration, providing a quantitative basis to make functional distinctions among reactive properties, thus providing a new approach to enhance the annotation of GH phylogenetic trees with functional measurements. This research was carried out in collaboration with researchers at DOE’s Joint BioEnergy Institute (JBEI).

Contacts (BER PM)
N. Kent Peters, SC-23.2, kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
Brian Fox
University of Wisconsin-Madison
bgfox@biochem.wisc.edu

Funding
GLBRC and JBEI are supported by DOE’s Office of Science, Office of Biological and Environmental Research through contracts DE-FC02-07ER64494 and DE-AC02-05CH11231, respectively.

Publications
Deng, K., T. E. Takasuka, C. M. Bianchetti, L. F. Bergeman, P. D. Adams, T. R. Northen, and B. G. Fox. 2015. “Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases,” Frontiers in Bioengineering and Biotechnology 3(165), DOI: 10.3389/fbioe.2015.00165. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 13, 2015

Cyanobacterial Alkanes: Today’s Bacterial Antifreeze, Tomorrow’s Fuel

A promising biofuel molecule helps the photosynthetic bacteria that naturally produce it tolerate cold temperatures.

The Science
Cyanobacteria, photosynthetic microorganisms, contain a unique and universal pathway that converts fatty acids to alkanes, a promising biofuel candidate. Recent spectroscopic and modeling studies illuminated how the produced alkanes allow cyanobacteria to adjust photosynthetic activity to tolerate cold temperatures.

The Impact
Understanding the natural function of alkanes in cyanobacteria may lead to production of these molecules as biofuels. These bacteria-produced alkanes are excellent fuel candidates because they have high-energy content and are highly compatible with existing infrastructure for petroleum-based fuel distribution and use.

Summary
Cyanobacteria are photosynthetic bacteria that, like plants, consume carbon dioxide and produce oxygen through photosynthesis. All cyanobacterial membranes contain diesel-range C15-C19 hydrocarbons in high concentration and the production pathways for these metabolites are exclusive to cyanobacteria. In this study, the model cyanobacterium, Synechocystis sp. PCC 6803, was modified to produce no alkanes, and the resulting strain grew poorly at low temperatures. To understand the growth defect, the researchers assessed the redox kinetics of how cyanobacteria convert solar energy into chemical energy in the form of adenosine triphosphate (ATP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). ATP and NADPH are produced using a linear and a cyclic pathway with the pigment-protein complex, photosystem I (PSI), as the hub to both. The modified strain made greater use of the cyclic pathway, which raises the ATP:NADPH ratio, especially at low temperature. This use helps to balance reductant requirements and maintain the redox poise of the electron transport chain. While previous theories held that the cyclic pathway was used in a fixed ratio to the linear pathway, the researchers demonstrated that the cyclic pathway responds dynamically to the environment and that alkanes play a role in this response. Flux balance computational analysis showed that an intermediate use of the cyclic pathway (circa one-fourth that of the linear pathway) maximized growth as well. From this analysis, the team concluded that the lack of membrane alkanes required greater use of the cyclic pathway, presumably to maintain redox poise. In turn, such an increase compromises growth by activating energy-inefficient pathways. This study highlights the unique and universal role of medium-chain hydrocarbons in cyanobacteria: they regulate redox balance and reductant partitioning in these photosynthetic cells under stress.

Contacts
(BER PM)
Dawn Adin, SC-23.2, dawn.adin@science.doe.gov, 301-903-0570

(PI Contact)
Himadri B. Pakrasi  
Washington University in St. Louis
pakrasi@wustl.edu

Funding
This work was funded by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Biological Systems Science Division, Genomic Science Program.

Publications
Berla, B., R. Saha, C. Maranas, and H. Pakrasi. 2015. “Cyanobacterial Alkanes Modulate Photosynthetic Electron Flow to Assist Growth under Cold Stress,” Scientific Reports 5, 14894. DOI: 10.1038/srep14894. (Reference link)

Related Links
Pakrasi Laboratory Website
Maranas Laboratory Website

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Cyanobacteria produce alkanes that modulate the balance between different energy production pathways. This feature allows these photosynthetic bacteria to thrive across a range of environmental temperatures.

Image modified from Berla, B., et al. 2015. “Cyanobacterial Alkanes Modulate Photosynthetic Electron Flow to Assist Growth under Cold Stress,” Scientific Reports 5, 14894. DOI: 10.1038/srep14894.



September 22, 2015

Identifying Specific Preferences in Organic Compound Consumption by Desert Soil Microbes

Every natural soil ecosystem hosts a great diversity of microbes that consume complex organic matter and transform it to simpler small carbon compounds (metabolites) or gaseous endproducts such as carbon dioxide. This decompositional microbial activity transforms organic compounds in the soil, playing a critical role in the global carbon cycle. To determine the functional characteristics of a microbial community’s different members, it is necessary to understand the complex mixture of metabolites present in their environment and to determine which compounds are preferentially consumed by each microorganism. Researchers at Lawrence Berkeley National Laboratory and collaborating institutions have used new exometabolomics techniques to quantitatively analyze the compounds consumed by seven bacterial species isolated from soil crusts in the desert environment of the Colorado Plateau. In these arid environments, most of the organic matter is produced by photosynthetic bacteria and released in the form of metabolites that other microbes can consume and further transform. The investigators discovered that each of the seven species consumes only 13% to 26% of the nearly 500 metabolites produced by these bacteria, and only 0.4% of the metabolites are used by all of them. These different feeding habits may represent a form of ecological niche specialization and may play important roles in maintaining non-overlapping diversity within microbial consortia. This study constitutes a significant advance in our understanding of how microbes in terrestrial ecosystems transform soil organic matter and may affect atmospheric carbon dioxide levels.

Reference: Baran, R., E. Brodie, J. Mayberry-Lewis, E. Hummel, U. N. Da Rocha, R. Chakraborty, B. Bowen, U. Karaoz, H. Cadillo-Quiroz, F. Garcia-Pichel, and T. Northen. 2015. “Exometabolite Niche Partitioning Among Sympatric Soil Bacteria,” Nature Communications 6(8289), DOI:10.1038/ncomms9289. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 21, 2015

Neutron Crystallography Visualizes How Nature’s Most Efficient Enzyme Works

Enzymes play a critical role in all aspects of life by speeding up specific chemical reactions in living cells. The glycoside hydrolases (GHs) are a group of enzymes that catalyze the breakdown of large quantities of organic matter in nature, specifically cellulose and hemicellulose, and that are being applied industrially to the conversion of biomass to useful products. GHs speed up the cleavage of an otherwise very stable chemical bond through a complex process that is not well understood. New research led by scientists at Oak Ridge National Laboratory (ORNL) on the key steps in the action of xylanase, a GH that cuts xylan chains in hemicellulose (a major component of biomass) into smaller units, has shown how this enzyme coordinates the movement of hydrogen ions to speed up the breakdown process. The scientists combined information from several neutron and X-ray crystallography experiments to visualize the exact atomic structure of the xylanase during the initial steps of the reaction. They found that a side chain of the enzyme amino acid residue that is key to its activity moves between two orientations to first accept a hydrogen ion and then deliver it to the place where the xylan is to be cut. In the former orientation, the side chain is more basic and thus is able to grab a hydrogen ion from water, whereas in the latter it becomes more acidic and ready to initiate the catalytic process. This publication is the first from the new Macromolecular Neutron Diffractometer (MaNDi) at ORNL’s Spallation Neutron Source. Scientists at Los Alamos National Laboratory, Argonne National Laboratory, the University of Toledo, and universities and user facilities in the People’s Republic of China, Sweden, and Germany collaborated in the research.

Reference: Wan, Q., et al. 2015. “Direct Determination of Protonation States and Visualization of Hydrogen Bonding in a Glycoside Hydrolase with Neutron Crystallography,” Proceedings of the National Academy of Sciences (USA) 112(40), 12,384–389. DOI: 10.1073/pnas.1504986112. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 16, 2015

Novel Biological Wiring System Detected in a Methane-Consuming Microbial Symbiosis

Every year, large amounts of methane (CH4) are produced in coastal wetlands and deep ocean sediments through the decay of organic material or seepage from geological reservoirs. Fortunately, microbes consume the majority of this potent greenhouse gas before it reaches Earth’s atmosphere. Although these subsurface environments are typically depleted of oxygen, methane can still be oxidized by symbiotic partnerships between methane-consuming archaea and sulfate-reducing bacteria that collaboratively transfer electrons from methane to sulfate (rather than O2) to generate useful energy. Observed near sites of environmental CH4 production, consortia of cells performing anaerobic oxidation of methane (AOM) form mixed balls composed of tens to hundreds of cells, but the exact mechanism by which they consume CH4 and share energy is not fully understood. In a new study, scientists at the California Institute of Technology used high-resolution microscopy paired with mass spectrometry (NanoSIMS) to examine the relationship between spatial distribution of microbes and metabolic processes in AOM consortia. To their surprise, the researchers found that metabolically active partner microbes did not need to be closely associated with each other, even though each organism performs only half of the critical methane-consuming reaction. Using data from these studies, the team constructed a computational model of consortial metabolism that predicted an extracellular conduit allowing direct transfer of electrons between the organisms. By re-examining the genomes of both microbes, the team identified a previously overlooked set of genes in the archaeal partner encoding an electron transfer system similar to those observed in known electroconductive bacteria. Histological staining was then used to detect this system in active AOM consortia, revealing components arrayed across the extracellular space between the microbes. These results indicate the presence of a biological wiring system within AOM consortia that allows the two partners to more efficiently consume methane, share resulting energy, and form larger consortial structures than would otherwise be possible. These findings reveal another new aspect of the diverse metabolic capacities present in the microbial world and considerably advance our understanding of a key microscale mechanism driving a carbon cycle process of global significance.

Reference: McGlynn, S. E., G. L. Chadwick, C. P. Kempes, and V. J. Orphan. 2015. “Single Cell Activity Reveals Direct Electron Transfer in Methanotrophic Consortia,” Nature, DOI: 10.1038/nature15512. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 12, 2015

Elimination of Non-Productive Fermentation Products Boosts Cellulosic Ethanol Production in Consolidated Bioprocessing

Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. In addition to ethanol, however, C. thermocellum produces a number of unwanted fermentation products such as organic acids and gaseous hydrogen, which divert energy and carbon from the desired fermentation product, ethanol. Researchers at the Department of Energy’s BioEnergy Science Center sought to eliminate these non-target fermentation products in order to increase ethanol yields. In doing so, they created C. thermocellum strain AG553 by deleting genes involved in the production of acetate, formate, lactate, and hydrogen gas. Strain AG553 showed a two- to three-fold increase in ethanol yield relative to the wild type on all substrates tested. When grown in a defined medium with 5 g/L of soluble disaccharide cellobiose as the carbon source, the mutant strain produced greater than two-fold more ethanol than the wild type strain. It exceeded 70% of theoretical ethanol yield with no appreciable amounts of other fermentation products detected and H2 production reduced five-fold. Wild type C. thermocellum will naturally acidify a non-buffered medium during fer­mentation by production of organic acids and limit ethanol production by limiting growth. The elimination of organic acid production suggested that strain AG553 might be capable of growth under higher substrate loadings in the absence of pH control. The maximum titer of wild type C. thermocellum was only 14.1 mM ethanol on 10 g/L Avicel. For strain AG553, final ethanol titer peaked at 73.4 mM in on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to carbon dioxide accumulation. With the elimination of the non-target fermentation metabolic pathways, AG553 is the best ethanol-yielding CBP strain to date. It will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass into advanced biofuels other than ethanol.

Reference: Papanek, B., R. Biswas, T. Rydzak, and A. M. Guss. 2015. “Elimination of Metabolic Pathways to All Traditional Fermentation Products Increases Ethanol Yields in Clostridium thermocellum,”Metabolic Engineering, DOI: 10.10/16/j.ymben.2015.09.002. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 27, 2015

Eucalyptus Trees with Reduced Lignin Content Display Reduced Recalcitrance

Lignocellulosic materials offer an attractive replacement for food-based crops used to produce ethanol, but understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of lignocellulosic biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. Changes to lignin gene expression in switchgrass and Populus have shown increased sugar release and reduced recalcitrance. Researchers at the Department of Energy’s BioEnergy Science Center have sought to transfer these results to eucalyptus, a fast-growing, warm climate, woody biofeedstock also suitable for cellulosic biofuel production. The researchers genetically engineered reduced gene expression of two key lignin biosynthesis enzymes, cinnamate 4-hydroxylase (C4H) and p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H), in eucalyptus. The engineered plants were evaluated for cell wall composition and reduced recalcitrance. Eucalyptus trees with down-regulated C4H or C3'H expression displayed lowered overall lignin content than the control samples. The C3'H and C4H down-regulated lines also had different lignin compositions when compared to the control eucalyptus trees. Both the C4H and C3'H down-regulated lines had reduced recalcitrance as indicated by increased sugar release, which was determined using enzymatic conversion assays utilizing both no pretreatment and a hot water pretreatment. Lowering lignin content rather than altering lignin content was found to have the largest impact on reducing recalcitrance of the transgenic eucalyptus variants. The development of lower recalcitrance trees opens up the possibility of using alternative pretreatment strategies in biomass conversion processes that can reduce processing costs.

Reference: Sykes, R. W., E. L. Gjersing, K. Foutz, W. H. Rottmann, S. A. Kuhn, C. E. Foster, A. Ziebell, G. B. Turner, S. R. Decker, M. A. W. Hinchee, and M. F. Davis. 2015. “Down Regulation of P-Coumaroyl Quinate/Shikimate 3'-Hydroxylase (C3'H) and Cinnamate 4-Hydroxylase (C4H) Genes in the Lignin Biosynthetic Pathway of Eucalyptus urophylla x E. grandis Leads to Improved Sugar Release,” Biotechnology for Biofuels 8,128. DOI: 10.1186/s13068-015-0316-x. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 27, 2015

Structural Characterization of Isolated Poplar and Switchgrass Lignins During Dilute Acid Treatment

A key step in converting cellulosic biomass into sustainable fuels and chemicals is thermochemical pretreatment to reduce plant cell wall recalcitrance. An improved understanding of the chemistry of lignin as it undergoes this processing is critical to the development of renewable biofuel production. Researchers at the Department of Energy’s BioEnergy Science Center (BESC) have studied the behavior of lignin during dilute acid pretreatment (DAP). They isolated lignin from poplar and switchgrass using a cellulolytic enzyme system and then treated it under DAP conditions. Results highlighted that lignin is subjected to depolymerization reactions within the first 2 minutes of DAP, and these changes are accompanied by increased generation of aliphatic and phenolic hydroxyl groups of lignin. These developments are followed by a competing set of depolymerization and repolymerization reactions that lead to a decrease in the content of guaiacyl lignin units and an increase in condensed lignin units as the reaction residence time is extended beyond 5 minutes. A detailed comparison of changes in functional groups and molecular weights of cellulolytic enzyme lignins demonstrated that several structural parameters related to lignin’s recalcitrant properties are altered during DAP conditions. This deeper understanding of the chemical structure of lignin as it undergoes processing is an important step toward the goal of efficient conversion of lignocellulose into renewable biofuel products.

Reference: Sun, Q., Y. Pu, X. Meng, T. Wells, and A. J. Ragauskas. 2015. “Structural Transformation of Isolated Poplar and Switchgrass Lignins During Dilute Acid Treatment,” ACS Sustainable Chemistry and Engineering 3(9), 2203-10. DOI: 10.1021/acssuschemeng.5b00426. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 13, 2015

Enhancing a microbe’s cellulolytic ability for biomass deconstruction

The in vitro activity of the Caldicellulosiruptor bescii secretome to digest lignocellulosic biomass was significantly increased with the addition of the E1 endoglucanase from Acidothermus cellulolyticus.

The Science
The most effective commercial enzyme cocktails currently used to deconstruct biomass in vitro are derived from fungal cellulase components. These fungal cellulases consist of cellobiohydrolases, endoglucanases, and β-D-glucosidases that act synergistically to release sugars from biomass for microbial conversion to products. However, these fungal cellulase components contribute significantly to overall deconstruction costs. As a potentially cost-effective alternative, C. bescii, a cellulolytic thermophile, is a prime candidate for effective consolidated bioprocessing as it contains more than 50 glycoside hydrolases including CelA, a multidomain enzyme. C. bescii’s ability to solubilize lignocellulose could be enhanced with engineering to include an endonuclease with additional activities such as E1 fromA. cellulolyticus, another cellulolytic thermophile.

The Impact
This work provides an understanding of the action and limitations of the CelA enzyme and demonstrates that CelA can act synergistically with the E1 protein to digest cellulose. These results contribute to the knowledgebase that enables enzyme engineering to generate novel enzyme mixtures for biomass deconstruction. The new information could lead to a more economical means of converting biomass to simple sugars for bioproducts production.

Summary
The most effective commercial enzyme cocktails of carbohydrate-active enzymes (CAZymes) used in vitro to pretreat biomass are derived from fungal cellulases. These cellobiohydrolases, endoglucanases, and β-d-glucosidases act synergistically to release sugars for microbial conversion. The genome of the thermophilic bacterium C. bescii encodes a potent set of CAZymes, found primarily as multidomain enzymes. This set of CAZymes exhibits high cellulolytic and hemicellulolytic activity on and allows utilization of a broad range of substrates, including plant biomass, without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in a single protein. E1 is an endo-1,4-β-glucanase from A. cellulolyticus linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates and has been shown in vitro to work synergistically with CelA. To test if the addition of E1 to the C. bescii secretome would improve its cellulolytic activity, U.S. Department of Energy (DOE) BioEnergy Science Center (BESC) scientists cloned and expressed the E1 gene in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 percent at 65 °C, and 12.6 percent at 75 °C. Activity against Avicel increased on average 17.5 percent at 65 °C and 16.4 percent at 75 °C. Thus, expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome in agreement with in vitro evidence that E1 acts synergistically with CelA to digest cellulose. This result offers the possibility of engineering additional enzymes for improved biomass deconstruction into C. bescii effectively.

Contacts (BER PM)
N. Kent Peters, SC-23.2, kent.peters@science.doe.gov, 301-903-5549

PI Contact
Janet Westpheling
Department of Genetics, University of Georgia, Athens, GA, and BESC, Oak Ridge National Laboratory, Oak   Ridge, TN
janwest@uga.edu

Funding
This research was supported as a subcontract by BESC, a DOE Bioenergy Research Center funded by the Office of Biological and Environmental Research within DOE’s Office of Science (DE-AC05-000R22725).

Publication
Chung, D., J. Young, M. Cha, R. Brunecky, Y. J. Bomble, M. E. Himmel, and J.Westpheling. 2015. “Expression of the Acidothermus cellulolyticus E1 Endoglucanase in Caldicellulosiruptor bescii Enhances Its Ability to Deconstruct Crystalline Cellulose,” Biotechnology for Biofuels 8:113. DOI: 10.1186/s13068-015-0296-x. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 13, 2015

Expression of Heterologous Endoglucanases in Caldicellulosiruptor bescii Enhances Secretome Activity

Currently, the most effective commercial enzyme cocktails of carbohydrate-active enzymes (CAZymes) used in vitro to pretreat biomass are derived from fungal cellulases. These cellobiohydrolases, endoglucanases, and β-d-glucosidases act synergistically to release sugars for microbial conversion. The genome of the thermophilic bacterium, Caldicellulosiruptor bescii, encodes a potent set of CAZymes, found primarily as multidomain enzymes. This set of CAZymes exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in a single protein. E1 is an endo-1,4-β-glucanase from Acidothermus cellulolyticus linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates and has been shown in vitro to work synergistically with CelA. To test if the addition of E1 to the C. bescii secretome would improve its cellulolytic activity, the E1 gene was cloned and expressed in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 % at 65 °C and 12.6 % at 75 °C. Activity against Avicel increased on average 17.5 % at 65 °C and 16.4 % at 75 °C. Thus, expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome in agreement with in vitro evidence that E1 acts synergistically with CelA to digest cellulose. This result offers the possibility of effectively engineering additional enzymes for improved biomass deconstruction into C. bescii.

Reference: Chung, D., J. Young, M. Cha, R. Brunecky, Y. J. Bomble, M. E. Himmel, and J. Westpheling. 2015. “Expression of the Acidothermus cellulolyticus E1 Endoglucanase in Caldicellulosiruptor bescii Enhances Its Ability to Deconstruct Crystalline Cellulose,” Biotechnology for Biofuels 8, 113. DOI: 10.1186/s13068-015-0296-x. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 10, 2015

Hybrid Spectroscopy Helps Elucidate Fine Cell Wall Structure

A key obstacle to large-scale production of biofuels is the resistance of biomass to deconstruction into simple biomolecules that can be converted to the desired fuels. This so-called recalcitrance is being studied intensively at the cellular level. Non-destructive, simultaneous chemical and physical characterization of materials at the nanoscale is a highly sought-after capability for understanding the underlying mechanisms of this cell wall recalcitrance to deconstruction. However, a combination of physical limitations of existing nanoscale technologies has made achieving this goal challenging. To overcome these obstacles, researchers at the Department of Energy’s BioEnergy Science Center (BESC) have developed a hybrid approach for nanoscale material characterization based on nanomechanical force microscopy in conjunction with infrared photoacoustic spectroscopy. The researchers targeted the outstanding problem of spatially and spectrally resolving plant cell walls. Nanoscale characterization of plant cell walls and the effect of complex phenotype treatments on biomass are challenging but necessary in the search for sustainable and renewable bioenergy. The BESC scientists were able to reveal both the morphological and compositional substructures of the cell walls. They found that the measured biomolecular traits are in agreement with the lower-resolution chemical maps obtained with infrared and confocal Raman microspectroscopies of the same samples. These results should prove relevant in fields such as energy production and storage, as well as medical research, where morphological, chemical, and subsurface studies of nanocomposites, nanoparticle uptake by cells, and nanoscale quality control are in demand.

Reference: Tetard, L., A. Passian, R. H. Farahi, T. Thundat, and B. H. Davison. 2015 “Opto-Nanomechanical Spectroscopic Material Characterization,” Nature Nanotechnology, DOI: 10.1038/NNANO.2015.168. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 22, 2015

Engineered Furfural Tolerance in Caldicellulosiruptor bescii, a Consolidated Bioprocessing Thermophile

Harsh pretreatments are often used to make lignocellulose sugars more accessible for conversion to biofuels. These pretreatments can cause problems for subsequent stages of biofuel production. For example, dilute-acid pretreatment of lignocellulosic biomass creates potent inhibitors of microbial growth and fermentation such as furfural and 5-hydroxymethyl-furfural (5-HMF). The enzymatic reduction of these furan aldehydes to their corresponding less toxic alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenic Escherichia coli. However, this approach has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP), such as Caldicellulosiruptor bescii. To test if C. bescii could be engineered to be more tolerant of these inhibitors, researchers from the Department of Energy’s BioEnergy Science Center (BESC) constructed a strain of C. bescii using a butanol dehydrogenase encoding gene from Thermoanaerobacter pseudethanolicus 39E (BdhA), which had previously been shown to have furfural and 5-HMF reducing capabilities. Heterologous expression of the NADPH-dependent BdhA enzyme conferred increased resistance of the engineered strain to both furfural and 5-HMF relative to the wild-type and parental strains. Further, when challenged with 15 mM concentrations of either furan aldehyde, the ability to eliminate furfural or 5-HMF from the culture medium was significantly improved in the engineered strain. This study represents the first example of engineering furan aldehyde resistance into a CBP-relevant thermophile and further validates C. bescii as being a genetically tractable microbe of importance for lignocellulosic biofuel production.

Reference: Chung, D., T. J. Verbeke, K. L. Cross, J. Westpheling, and J. G. Elkins. 2015. “Expression of a Heat-Stable NADPH-Dependent Alcohol Dehydrogenase in Caldicellulosiruptor bescii Results in Furan Aldehyde Detoxification,” Biotechnology for Biofuels 8,102. DOI: 10.1186/s13068-015-0287-y. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 10, 2015

Consolidated Bioprocessing of Cellulose to an Advanced Biofuel Using a Cellulolytic Thermophile

Consolidated bioprocessing (CBP) has the potential to reduce biofuel and biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously, without the addition of premanufactured cellulases and other hydrolytic enzymes. In particular, Clostridium thermocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Toward this end, researchers at the Department of Energy’s BioEnergy Science Center (BESC) researchers engineered C. thermocellum to produce isobutanol, an advanced biofuel. Metabolic engineering for isobutanol production in C. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. Engineering of the isobutanol pathway into C. thermocellum was facilitated by first cloning plasmids into Escherichia coli before transforming these constructs into C. thermocellum for testing and optimization. Among these engineered strains, the best isobutanol producer was selected. Interestingly, both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (KIVD) were expressed and found to be responsible for isobutanol production. A single crossover integration of the plasmid into the chromosome resulted in a stable strain not requiring antibiotic selection. This strain produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50°C within 75 hours, corresponding to 41% of theoretical yield. While there is significant room for further optimization, this initial engineering of a cellulolytic thermophile to produce an advanced biofuel demonstrates the potential of this strategy to help create a sustainable and commercially viable biofuel.

Reference: Lin, P. P., L. Mi, A. H. Morioka, K. M. Yoshino, S. Konishi , S. C. Xu , B. A. Papanek, L. A. Riley, A. M. Guss, and J. C. Liao. 2015. “Consolidated Bioprocessing of Cellulose to Isobutanol Using Clostridium thermocellum,” Metabolic Engineering 31, 44-52. DOI:10.1016/j.ymben.2015.07.001. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 04, 2015

Engineering Restricted Lignin and Enhanced Sugar Deposition in Secondary Cell Walls Enhances Monomeric Sugar Release

Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. However, the lignin component, a complex and interlinked phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis to convert them to sugars. Therefore, before enzymatic hydrolysis, biomass must first be pretreated to make it more susceptible to saccharification and release high yields of fermentable sugars. To reduce the impact of lignin on limiting saccharification, researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI) engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the plant’s vessels, and polysaccharide deposition was enhanced in fiber cells. Growth of these engineered plants showed little to no apparent negative impact on growth phenotype. Analyses of these engineered Arabidopsis plants were conducted to determine if the engineered plants would yield more sugars than wild type. Both wild type and engineered plant biomasses were treated with an ionic liquid at either 70°C for 5 hours or 140°C for 3 hours. After pretreatment at 140°C and subsequent saccharification, the relative peak sugar recovery from biomass of engineered plants and wild type was not statistically different. However, reducing the pretreatment temperature to 70°C resulted in a higher peak sugar recovery for the engineered lines, but a significant reduction in the peak sugar recovery obtained from the wild type. These results demonstrate that employing cell wall engineering to decrease the recalcitrance of lignocellulosic biomass has the potential to drastically reduce the energy required for effective pretreatment.

Reference: Scullin, C., A. G. Cruz, Y.-D. Chuang, B. A. Simmons, D. Loque, and S. Singh. 2015. “Restricting Lignin and Enhancing Sugar Deposition in Secondary Cell Walls Enhances Monomeric Sugar Release After Low Temperature Ionic Liquid Pretreatment,” Biotechnology for Biofuels 8, 95. DOI: 10.1186/s13068-015-0275-2. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 29, 2015

Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by Rhodopseudomonas palustris

A major barrier to efficient conversion of lignocellulosic materials to biofuels is the sensitivity of microbes to inhibitory compounds formed during biomass pretreatment. Aromatics derived from lignocellulose are a major class of inhibitors that typically are not metabolized by microbes commonly used as biocatalysts. However, the purple nonsulfur bacterium Rhodopseudomonas palustris is known to utilize aromatic compounds such as benzoate or p-hydroxybenzoate under anaerobic conditions. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center (GLBRC) have now shown that R. palustris is able to remove a majority of the aromatic compounds present in corn stover hydrolysates while leaving the sugars intact. The conditioned hydrolysate supported improved growth of a second microbe that was not able to grow in untreated hydrolysate. GLBRC researchers also found that most of the aromatic compounds were metabolized via the known R. palustris benzoyl-coenzyme A (CoA) pathway. Furthermore, the use of benzoyl-CoA pathway mutants prevents complete degradation of the aromatics and allows for production of selected products that may be recovered as coproducts from fermentations. This work presents the first demonstration of a microbe’s ability to metabolize and remove mixed aromatics in biomass hydrolysate, compounds that are detrimental to most microbes and generally unsuitable as carbon sources. This knowledge may inform the design of new microbes for bioconversion that can generate valuable coproducts from fermentation of sugars in lignocellulosic biomass.

Reference: Austin, S., W. S. Kontur, A. Ulbrich, J. Z. Oshlag, W. Zhang, A. Higbee, Y. Zhang, J. J. Coon, D. B. Hodge, T. J. Donohue, and D. R. Noguera. 2015. “Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by Rhodopseudomonas palustris,” Environmental Science and Technology 49(14), 8914–22. DOI: 10.1021/acs.est.5b02062. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 17, 2015

Long-Term Study Alleviates Water-Use Concern for Biofuel Crops

Potential water requirements are a significant concern for large-scale production of biofuel crops. Studying water use for plant communities across years of varying water availability can indicate how terrestrial water balances will respond to climate change and variability as well as to land cover change. Perennial biofuel crops, likely grown mainly on marginal lands of limited water availability, provide an example of a potentially extensive future land-cover conversion. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center measured growing-season evapotranspiration based on daily changes in soil profile water contents in five perennial systems—switchgrass, Miscanthus, native grasses, restored prairie, and hybrid poplar—and in annual maize (corn) in a temperate humid climate (Michigan, USA). Three study years (2010, 2011, and 2013) had normal growing-season rainfall, whereas 2012 was a drought year with about half to a third normal rainfall. Overall growing-season mean evapotranspiration for the four years did not vary significantly among corn and the perennial systems. Differences in biomass production largely determined variation in water-use efficiency. Miscanthus had the highest water-use efficiency in both normal and drought years, followed by maize; the native grasses and prairie were lower and poplar was intermediate. Measured water use by perennial systems was similar to maize across normal and drought years and contrasts with earlier modeling studies suggesting that rain-fed perennial biomass crops in this climate have little impact on landscape water balances, whether replacing rain-fed maize on arable lands or successional vegetation on marginal lands. Results also suggest that crop evapotranspiration rates, and thus groundwater recharge, streamflow, and lake levels, may be less sensitive to climate change than has been assumed.

Reference: Hamilton, S. K., M. Z. Hussain, A. K. Bhardwaj, B. Basso, and G. P. Robertson. 2015. “Comparative Water Use by Maize, Perennial Crops, Restored Prairie, and Poplar Trees in the U.S. Midwest,” Environmental Research Letters 10, 064015. DOI:10.1088/1748-9326/10/6/064015. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 15, 2015

New Microfluidics DNA Assembly Platform

Microbes are being engineered for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful for this process, further improvements are needed to lower the barriers scientists face if they plan to enter this growing field. Researchers at the Department of Energy’s Joint BioEnergy Institute have developed an innovative microfluidic platform for assembling DNA fragments, a critical step in the entire process. The new system uses volumes 10 times lower than current microfluidic platforms and has integrated region-specific temperature control and on-chip transformation. Integration of these steps in a single device minimizes the loss of reagents and products compared to conventional methods, which require, for example, multiple pipetting steps. For assembling DNA fragments, researchers implemented three commonly used DNA assembly protocols on the new microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). Assembly of two combinatorial libraries of 16 plasmids each demonstrated the utility of these microfluidic methods. Each DNA plasmid was transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. This platform likely will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids, helping to expedite the overall synthetic biology process for biofuels development.

Reference: Shih, S. C. C., G. Goyal, P. W. Kim, N. Koutsoubelis, J. D. Keasling, P. D. Adams, N. J. Hillson, and A. K. Singh. 2015. “A Versatile Microfluidic Device for Automating Synthetic Biology,” ACS Synthetic Biology, DOI: 10.1021/acssynbio.5b00062. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 12, 2015

Phenolic Amides are Potent Inhibitors of de novo Nucleotide Biosynthesis

Lignocellulose-derived hydrolysates contain several different inhibitors (collectively called lignotoxins or LTs) that arise during pretreatment of biomass. Determining the mechanisms by which yeast or bacteria are adversely affected by LTs is a key step toward improving the efficiency of fermentation and bioconversion. Prior work has established that LTs present in ammonia pretreated corn stover hydrolysates inhibit growth and sugar utilization in Escherichia coli. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center (GLBRC) have now keyed in on two phenolic amine LTs, feruloyl amide (FA) and coumaroyl amide (CA). These inhibitors are important because these two alone are sufficient to recapitulate the inhibitory effects of all LTs present. Analysis of the metabolome in untreated versus treated cells indicated that these phenolic amides cause rapid accumulation of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis. Moreover, isotopic tracer studies confirmed that carbon and nitrogen flux into nucleotides is inhibited by the amides, suggesting that these phenolic amines are potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Biochemical studies showed that the amides directly inhibit glutamine amidotransferases, with FA acting as a competitive inhibitor of the E. coli enzyme responsible for the first committed step in de novo purine biosynthesis. Supplementation of cultures with nucleosides was sufficient to reverse the effect of the amides, suggesting the ability to bypass the block in de novo nucleotide biosynthesis via salvage pathways. Collectively, these results provide a direct mechanism for the inhibitory effects of phenolic amides, knowledge that will inform future design of biocatalysts for improved bioconversion.

Reference: Pisithkul, T., T. B. Jacobson, T. J. O'Brien, D. M. Stevenson, and D. Amador-Noguez. 2015. “Phenolic Amides are Potent Inhibitors of De Novo Nucleotide Biosynthesis,” Applied and Environmental Microbiology 81(17), 5761-72. DOI: 10.1128/AEM.01324-15. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 14, 2015

N2O Emissions During Establishment Phase of Various Bioenergy Cropping Systems

As bioenergy cropping systems are developed, their greenhouse gas (GHG) emissions will be a key component of sustainability evaluations. Nitrous oxide (N2O) is a potent GHG and a substantial proportion of the total GHG footprint associated with feedstock production. N2O emitted from soils is primarily the result of microbial activities, which are influenced by various environmental factors including temperature and oxygen and water availability. The impact of each of these factors differs among various cropping systems. To understand how traditional and biomass feedstock cropping systems might vary with regard to N2O emissions, researchers at the Department of Energy’s Great Lakes Bioenergy Research Center compared the establishment phase N2O emissions of annual monocultures of continuous corn and corn-soybean-canola rotations; perennial monocultures of switchgrass, Miscanthus, and hybrid poplar; and perennial polycultures of early successional species, native grasses, and native prairie species. Measurements were done over a 2- to 4-year period following planting over which several perennial crops attained “full capacity” biomass production. They found that during the establishment phase, perennial bioenergy crops emit less N2O than annual crops, especially when not fertilized. Emissions for perennials were about three times less than for annuals on a per hectare basis. N2O peak fluxes were associated with periods of rain following fertilizer application. And finally, the results show that simulation models trained on single systems performed well in most monocultures but worse in polycultures, which means models including N2O emissions should be parameterized specifically for particular plant systems. The results suggest that perennial biomass feedstock cropping systems have the potential for a lower GHG burden even during their establishment phase.

Reference: Oates, L. G., D. S. Duncan, I. Gelfand, N. Millar, G. P. Robertson, and R. D. Jackson. 2015. “Nitrous Oxide Emissions During Establishment of Eight Alternative Cellulosic Bioenergy Cropping Systems in the North Central United States,” Global Change Biology Bioenergy, DOI: 10.1111/gcbb.12268. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 12, 2015

Using Natural Microbial Communities as Biosensors for Environmental Contaminants

Microbial communities are highly attuned to changes in environmental conditions, rapidly sensing and responding to shifts in temperature, pH, nutrient availability, toxin levels, and dozens of other variables. For decades, scientists have studied the abilities of microbes to survive exposure to (and in some cases make use of) environmental contaminants such as heavy metals, radionuclides, and hydrocarbons. However, microbial communities can contain hundreds of different species, and this complexity makes it extremely difficult to quantitatively measure community-level responses to contaminant exposure. In a new study, a team of researchers from Lawrence Berkeley National Laboratory’s ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) science focus area developed a new computational approach for the analysis and computational modeling of microbial community responses to environmental contaminants. Using direct sequencing of DNA from environmental samples, the team examined the microbial community of a subsurface aquifer in Oak Ridge, Tennessee, that had been contaminated with uranium, nitrate, and a variety of other compounds. Drawing on this data, a modeling framework was constructed to enable prediction of the types and amounts of contaminants that had been experienced by the microbial community based on known physiological characteristics of detected bacterial species. The predictions of this model strongly correlated with amounts of uranium, nitrate, and a variety of other geochemical factors measured at the sampling sites. To test the utility of this approach using an independent dataset, the team applied the model to microbial DNA samples collected during the Deepwater Horizon oil spill in 2010. Again, the model accurately predicted which samples had experienced oil contamination based on microbial DNA sequences and suggested that the community fingerprint retained a “memory” of exposure even after oil was no longer detectable. The results of this study provide a powerful new approach for not only the identification of contaminants in environmental samples, but also the microbial processes that are acting on them and potentially impacting their movement and/or longevity in the environment.

Reference: Smith, M. B., A. M. Rocha, C. S. Smillie, S. W. Oleson, C. J. Paradis, L. Wu, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, S. P. Preheim, M. S. Sanders, J. Yang, M. A. Mueller, S. C. Brooks, D. B. Watson, P. Zhang, Z. He, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, J. Zhou, E. J. Alm, and T. C. Hazen. 2015. “Natural Bacterial Communities as Quantitative Biosensors,” mBio 6(3), e00326-15. DOI: 10.1128/mBio.00326-15. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 01, 2015

Mechanisms of Limonene Toxicity and Tolerance Elucidated

Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. Limonene has antimicrobial properties, but also has potential as a biofuel component, making it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity was unknown. Researchers at the Department of Energy’s Joint BioEnergy Institute have characterized a limonene-tolerant strain of Escherichia coli and found a mutation in a gene encoding alkyl hydroperoxidase, which alleviates limonene toxicity. They found that the acute toxicity previously attributed to limonene was largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. The researchers found that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. These results have implications for both the renewable production of limonene and limonene’s applications as an antimicrobial.

Reference: Chubukov, V., F. Mingardon, W. Schackwitz, E. E. Baidoo, J. Alonso-Gutierrez, Q. Hu, T. S. Lee, J. D. Keasling, and A. Mukhopadhyay. 2015. “Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC,” Applied and Environmental Microbiology 81, 4690-6. DOI: 10.1128/AEM.01102-15. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 23, 2015

Using Metatranscriptomics to Understand Carbon Decomposition in Forest Soils

Decomposition of plant materials in soils is accomplished by a complex and highly diverse community of microorganisms. The vast majority of these microbes cannot be grown in laboratories, and the roles of different species in decomposition and responses to changing environmental conditions are not well understood. Ecologists have demonstrated that the addition of nitrogen to forest soils significantly slows the rate of carbon decomposition, but it is not well understood why this change occurs. Recent advances in soil metatranscriptomics, the direct analysis of microbial community gene expression in environmental samples, have provided researchers with a more sophisticated set of tools to track changes in microbial community structure and function. In a new study, a collaborative team of scientists at Los Alamos National Laboratory and the University of Michigan have completed a metatranscriptomic analysis of forest soils at a long-term ecological experiment examining impacts of nitrogen addition. By developing a new technique for metatranscriptomic sampling, the team was able to complete a much deeper analysis of community metabolic potential than has been previously attempted. Using this approach, fungal and bacterial genes involved in degradation of plant lignocellulose were determined to undergo large changes in expression at two separated sites with elevated nitrogen. Overall pattern shifts were consistent with decreased carbon decomposition rates, but specific mechanisms appeared to vary between the different forest sites. As climate change processes shift environmental variables and agricultural practices continue to alter nitrogen inputs in terrestrial soils, understanding their coupled impacts on microbial community activities will be crucial to more confidently modeling and predicting impacts on different ecosystems.

Reference: Hesse, C. N., R. C. Mueller, M. Vuyisich, L. Gallegos-Graves, C. D. Gleasner, D. R. Zak, and C. R. Kuske. 2015. “Forest Floor Community Metatranscriptomics Identify Fungal and Bacterial Response to N Deposition in Two Maple Forests,” Frontiers in Microbiology, DOI: 10.3389/fmicb.2015.00337. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 20, 2015

Hardwood Lignin Engineered into Softwoods

Conifer (softwood) biomass naturally has high lignin content and is more difficult to process than biomass from hardwood species because softwoods lack syringyl units in their lignins. Using genetic engineering strategies, researchers from the Department of Energy’s Great Lakes Bioenergy Research Center transformed into Pinus radiata two enzyme functions necessary to produce syringyl units in order to metabolicly engineer syringyl lignin production into conifers. Analytical methods performed on the transformed P. radiata showed evidence that the new enzymatic activities were being expressed—namely, ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT)—and that sinapyl alcohol was being incorporated into the lignin polymer. These results provide the proof of concept that generating a lignin polymer containing syringyl units is possible in softwood species such as P. radiata. Additionally, these results suggest that retaining the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods more favorable for industrial processing also may be possible.

Reference: Wagner, A., Y. Tobimatsu, L. Phillips, H. Flint, B. Geddes, F. Lu, and J. Ralph. 2015. “Syringyl Lignin Production in Conifers: Proof of Concept in a Pine Tracheary Element System,” Proceedings of the National Academy of Sciences (USA), DOI: 10.1073/pnas.1411926112. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 15, 2015

Determining Sugar Content of Plant Biomass

Assessing biomass recalcitrance in large populations of both natural and transgenic plants is important to identify promising candidates for lignocellulosic biofuel production. To properly test and optimize biofuel production parameters, the starting sugar content must be known to calculate percent sugar yield and conversion efficiencies. The current standard procedure is both labor- and time-intensive, requiring gram quantities of biomass and taking close to 2 weeks for the full analysis. Pyrolysis molecular beam mass spectrometry (py-MBMS) has been used as a high-throughput method for determining lignin content and structure, and researchers at the Department of Energy’s BioEnergy Science Center are demonstrating its applicability for deter­mining glucose, xylose, arabinose, galactose, and mannose content in biomass. Py-MBMS measure­ments of sugars in the biomass from conifers, hardwoods, and herbaceous species give similar values to those measured using standard high-performance liquid chromato­graphy, indicating that py-MBMS provides an accurate quantification of total sugar content for a range of biomass types. With data collection for py-MBMS taking only 1.5 minutes per sample, py-MBMS is a rapid high-throughput method for quantifying sugar content in biomass. This improved rate of analysis will help in evaluating approaches to overcoming biomass recalcitrance.

Reference: Sykes, R. W., E. L. Gjersing, C. L. Doeppke, and M. F. Davis. 2015. “High-Throughput Method for Determining the Sugar Content in Biomass with Pyrolysis Molecular Beam Mass Spectrometry,” BioEnergy Research, DOI: 10.1007/s12155-015-9610-5. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 15, 2015

Heterologous Orthogonal Fatty Acid Biosynthesis System in Escherichia coli for Oleochemical Production

Producing biofuels and bioproducts from biomass requires the construction of efficient biosynthetic pathways. The introduction of heterologous enzymes into the well-established model microbe, Escherichia coli, can have the benefits of expanding the metabolite produced while avoiding feedback inhibition. Researchers at the Department of Energy’s Joint BioEnergy Institute expressed several heterologous type I fatty acid synthases (FAS) in E. coli that functioned in parallel with the native FAS. The most active heterologous FAS expressed in E. coli was Corynebacterium glutamicum FAS1A and resulted in the production of oleochemicals including fatty alcohols and methyl ketones. Chain length distribution of fatty alcohols produced shifted with coexpression of FAS1A with the acyl carrier protein/coenzyme A (CoA)-reductase from Marinobacter aquaeolei (Maqu2220). Coexpression of FAS1A with the Micrococcus luteus acyl-CoA-oxidase (FadM, FadB) resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work is believed to be the first example of in vivo function of a heterologous FAS in E. coli. Functional expression of these large enzyme complexes in E. coli will enable their study without the need to culture the native organisms as well as enable the study of FAS from uncultured organisms. In addition, using FAS1 enzymes for oleochemical production has several potential advantages, and further optimization of this system could lead to strains with more efficient conversion of biomass to desired biofuels and bioproducts.

Reference: Haushalter, R. W., D. Groff, S. Deutsch , L. The, T. A. Chavkin, S. F. Brunner, L. Katz, and J. D. Keasling. 2015. “Development of an Orthogonal Fatty Acid Biosynthesis System in Escherichia coli for Oleochemical Production,” Metabolic Engineering 30, 1-6. DOI: 10.1016/j.ymben.2015.04.003. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 14, 2015

Methane Consumption by Microbes in High Arctic Soils

As global climate change warms Arctic ecosystems, organic carbon locked in frozen soils thaws and becomes susceptible to decomposition by microbes. Major uncertainties remain regarding what fraction of this carbon will be released as carbon dioxide (CO2) versus methane (CH4), especially in different types of environments. Both CO2 and CH4 act as greenhouse gases, but with different intensities and residence times in the atmosphere. Various microbes can either produce methane (methanogens) or consume it (methanotrophs), so understanding the roles played by these organisms in different Arctic habitats is critical in determining potential outcomes of warming scenarios. In a recent study, a collaborative team of researchers used a combination of systems biology tools and biogeochemical process measurements to examine methanogenic and methanotrophic microbes in soils on Axel Heiberg Island in the Canadian high Arctic. In a surprising finding, the low nutrient mineral soils found on the island acted a methane sink, actively removing CH4 from the atmosphere. Metagenomic profiling of core samples taken from these soils identified a specific subclass of high-affinity methanotrophs capable of growth on very low CH4 concentrations. Targeted metatranscriptomic and metaproteomic profiling demonstrated that these organisms are not only present in these samples, but are actively expressing the genes and protein involved in high-affinity CH4 uptake. In a series of microcosm experiments using intact soil cores from the island, the team subjected the samples to warming and moisture additions consistent with current climate change projections for the region. Although rates of CH4 production by methanogens increased in deeper layers of the samples, there was no net release of CH4, suggesting that it was completely consumed by methanotrophs and converted to CO2. These results are very different from observations in more nutrient-rich permafrost ecosystems, where warming typically results in significant CH4 releases. As predictions of climate change impacts continue to improve, these findings highlight the importance of understanding the complex set of interrelationships between microbial community members and habitat-specific environmental conditions.

Reference: Lau, M. C. Y., B. T. Stackhouse, A. C. Layton, A. Chauhan, T. A. Vishnivetskaya, K. Chourey, J. Ronholm, N. C. S. Mykytczuk, P. C. Bennett, G. Lamarche-Gagnon, N. Burton, W. H. Pollard, C. R. Omelon, D. M. Medvigy, R. L. Hettich, S. M. Pfiffner, L. G. Whyte, and T. C. Onstott. 2015. “An Active Atmospheric Methane Sink in High Arctic Mineral Cryosols,” The ISME Journal, DOI: 10.1038/ismej.2015.13. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 30, 2015

Promoter Set for Heterologous Gene Expression in Clostridium thermocellum

For successful fermentation of biofuels and bioproducts from biomass, using microorganisms for which fewer genetic tools have been developed might be the most effective approach. To date, most metabolic engineering work in Clostridium thermocellum has focused on gene deletion, but many metabolic engineering strategies require well controlled heterologous gene expression, which requires a collection of well characterized and understood promoters. Researchers from the Department of Energy’s BioEnergy Science Center sought to identify new promoters for predictable gene expression in C. thermocellum. For this work, 17 different C. thermocellum promoters were tested with two different reporter genes (LacZ and AdhB) to ensure the activity of the target promoter was not gene-specific. Putative promoters were chosen by analyses of published C. thermocellum gene expression datasets. Promoter activity in both C. thermocellum and Escherichia coli were testedbecauseideally a promoter would not be strongly expressed in E. coli to avoid toxicity problems during cloning. Several useful promoters were identified (eno, cbp, cbp_2, 815, 966, 2638, and 2926), which showed high expression and high enzymatic activity of both reporter genes in C. thermocellum. Other promoters were not useful, showing no heterologous gene activity or negatively impacting plasmid stability. These results provide several new good promoters for C. thermocellum. This improved understanding of promoter function will enhance efforts to express heterologous genes important for improved biofuel production in C. thermocellum.

Reference: Olson, D. G., M. Maloney, A. A. Lanahan, S. Hon, L. J. Hauser, and L. R. Lynd. 2015. “Identifying Promoters for Gene Expression in Clostridium thermocellum,” Metabolic Engineering Communications, DOI: 10.1016/j.meteno.2015.03.002. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 30, 2015

The Priming Effect: How Plant Root Exudates Make Soil Carbon More Susceptible to Microbial Degradation

Rates of decompositional processes performed by soil microbes are influenced by a variety of factors including temperature, water availability, and the presence of minerals. As plant materials are broken down by microbes, released organic carbon compounds can bind to soil minerals, becoming much less accessible to further decomposition. These bound pools of organic carbon can be stored in soils for years, decades, or centuries depending on local site conditions. However, microbiologists have long observed a phenomenon known as “the priming effect,” in which the addition of small amounts of unbound organic carbon results in microbial degradation of older pools of mineral-bound soil carbon. Elevated atmospheric CO2 levels recently have been shown to cause plant roots to increase their secretion of small carbon molecules (“exudates”), which has significantly increased the importance of understanding how the priming effect works. In a recent study, a team of scientists co-led by Lawrence Livermore National Laboratory and Oregon State University used a combination of microbial community analysis and high-resolution mass spectrometry (NanoSIMS) to examine the mechanistic basis of the priming effect in soil microcosms. When a variety of different carbon compounds associated with root exudates were added to the soils via an artificial root system, they were shown to directly disrupt associations between older carbon and soil minerals. Liberated carbon was rapidly consumed by soil microbes, and the team was able to follow correlated shifts in microbial community composition and elevated CO2 production. Different types of exudate compounds had varying degrees of ability to strip stored carbon from minerals, a particularly significant observation since elevated atmospheric CO2 shifts both the amounts and types of exudates that plants produce. These results represent a new breakthrough in understanding the molecular-scale mechanisms underlying the priming effect and could significantly advance our ability to predict impacts of climate change on carbon cycling in terrestrial ecosystems.

Reference: Keiluweit, M., J. J. Bougoure, P. S. Nico, J. Pett-Ridge, P. K. Weber, and M. Kleber. 2015. “Mineral Protection of Soil Carbon Counteracted by Root Exudates,” Nature Climate Change, DOI: 10.1038/NCLIMATE2580. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 27, 2015

Making Sense of Genomic Networks

Genomes contain the information underlying an organism’s molecular functions. One way to compare the entire genomes of different organisms is to compare their gene-family content profiles, which is effectively a comparison of their functional potential. Standard networks, when used to model phylogenomic similarities, are not capable of capturing some of the underlying complexity of the relationships between genomes. To address this limitation, scientists at Oak Ridge National Laboratory, funded through the Department of Energy’s Plant-Microbe Interfaces Science Focus Area, developed a new three-way similarity metric and constructed three-way networks modeling the relationships among 211 bacterial genomes. They found that such three-way networks find cross-species genomic similarities that would otherwise have been missed by simpler models such as standard networks. Interactions within and between the multiple species that make up the complex microbial communities associated with plant roots are believed to influence the plant’s overall health and vigor and may contribute to the plant’s ability to survive adverse environmental conditions. This research is the first time the concept of three-way networks has been applied in the field of comparative genomics. These networks will be a useful tool to model and reveal complex interspecific bacterial relationships that are not found using the conventional two-way network models, and could pave the way toward deciphering intricate plant-microbe and microbe-microbe interactions.

Reference: Weighill, D. A., and D. A. Jacobson. 2015. “Three-Way Networks: Application of Hypergraphs for Modelling Increased Complexity in Comparative Genomics,” PLoS Computational Biology 11(3), e1004079. DOI: 10.1371/journal.pcbi.1004079. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 23, 2015

New Technology Tracks Cells Containing Multiple Mutations Within a Cellular Population

Different techniques to generate large collections of cells intentionally mutated in a number of targeted genes are currently available, and specific mutants in those collections can be readily identified. However, to manipulate complex traits involving multiple genes, it is necessary to identify individual cells that contain several mutated genes. Tracking individual cells that harbor specific combinations of two or more mutations separated by long distances within their genome is a time-consuming and effort-intensive process. In a recent study, researchers at the University of Colorado in Boulder reported the development of a new method called "TRACE" that allows the identification of single bacterial or eukaryote cells with mutations in about six targeted genes. The technique uses mathematical modeling to design short DNA fragments (or primers) that specifically bind to the targeted mutation sites. These primers are synthesized in a way that allows amplification of the targeted regions and subsequent joining of the amplification products into a single DNA molecule. By performing the amplification and joining of the DNA products in an emulsion where each cell in the population is confined to a single droplet, the six targeted sites can be analyzed by high-throughput sequencing to identify which cells contain mutations in one or more of the sites. In proof-of-concept experiments, the team used TRACE to identify a combination of mutant genes that confer the bacterium Escherichia coli tolerance to the toxicity of cellulose hydrolysate and the biofuel isobutanol. Because of the much higher throughput of TRACE relative to other genotyping methods, this technology will substantially accelerate the engineering of microbes for the production of biofuels and other chemicals.

Reference: Zeitoun, R. I., A. D. Garst, G. D. Degen, G. Pines, T. J. Mansell, T. Y. Glebes, N. R. Boyle, and R. T. Gill. 2015. “Multiplexed Tracking of Combinatorial Genomic Mutations in Engineered Cell Populations,” Nature Biotechnology, DOI: 10.1038/nbt.3177. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 10, 2015

Comparative Genomics Reveal Functional Diversification of the Methanogen Methanosarcina mazei

Methanogenic archaea play a major role in global carbon cycle processes, participating in the conversion of organic carbon to the greenhouse gas methane in oxygen-limited environments such as waterlogged soils and wetland sediments. Different types of methanogens are capable of converting either hydrogen and carbon dioxide or intermediate fermentation products (e.g., ace­tate and methanol) into methane; both processes are key components of carbon decomposi­tion food webs. In a new study, researchers at the University of Illinois have completed a compara­tive genomics study on 56 different isolates of the metabolically versatile methanogen Methanosarcina mazei cultivated from sediments of the Columbia River in Oregon. While all isolates are members of the same species, they showed a surprising degree of genomic diversity and formed a distinct pattern of subgroups (i.e., clades) based on their site of isolation. The investigators were able to identify a core genome shared by all isolates, but other genetic elements were variable in distribution and showed evidence of transfer between different clades of M. mazei. Several of the variable genes encoding proteins involved the methanogenic metabol­ism, cofactor utilization, and (most intriguingly) uptake of organic substrates. These observations led the researchers to hypothesize that M. mazei has evolved into strains optimized for specific ecological niches in the sedimentary environments, a phenomenon that has been observed in environmental populations of bacteria. This hypothesis was supported by physiological experi­ments showing that isolates from different M. mazei clades varied in their ability to use the organic compound trimethylamine for methanogensis. These results advance our mechanistic understanding of a key step in the global carbon cycle and highlight the importance of analyzing metabolically significant differences that occur in microbes at the subspecies level.

Reference: Youngblut, N. D., J. S. Wirth, J. R. Henriksen, M. Smith, H. Simon, W. W. Metcalf, and R. J. Whitaker. 2015. “Genomic and Phenotypic Differentiation Among Methanosarcina mazei Populations from Columbia River Sediment,” The ISME Journal, DOI: 10.1038/ismej.2015.31. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 09, 2015

New Antifungal Agents from Lignocellulose Hydrolysate

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides to protect bioenergy crops. Scientists at the Department of Energy’s Great Lakes Bioenergy Research Center discovered that poacic acid found in lignocellulosic hydrolysates of grasses functions as a potent antifungal compound. Several lines of evidence pointed toward fungal cell wall synthesis as the point of action of poacic acid. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. In addition, morphological analysis of cells treated with poacic acid revealed morphologies similar to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development on soybean leaves. The discovery of poacic acid as a natural antifungal agent targeting ß-1,3-glucan further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates. This research highlights the potential use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds.

Reference: Piotrowski, J. S., H. Okada, F. Lu, S. C. Li, L. Hinchman, A. Ranjan, D. L. Smith, A. J. Higbee, A. Ulbrich, J. J. Coon, R. Deshpande, Y. V. Bukhman, S. McIlwain, I. M. Ong, C. L. Myers, C. Boone, R. Landick, J. Ralph, M. Kabbage, and Y. Ohya. 2015. “Plant-Derived Antifungal Agent Poacic Acid Targets ß-1,3-Glucan,” Proceedings of the National Academy of Sciences (USA), DOI: 10.1073/pnas.1410400112. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 02, 2015

Regulation of Lipid Accumulation in a Photosynthetic Bacterium

Lipids serve important functions in living systems, either as structural components of membranes or as a form of carbon storage. Understanding the mechanisms of lipid accumulation in microorganisms is important for providing insight into the assembly of biological membranes and additionally has important applications in the production of renewable fuels and chemicals. Researchers at the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) in collaboration with DOE’s Environmental Molecular Sciences Laboratory (EMSL) have investigated the ability of Rhodobacter sphaeroides to increase membrane production at low O2 tensions in order to house its photosynthetic apparatus. They found that this bacterium has a mechanism to increase lipid content in response to decreased O2 tension and identified a specific transcription factor necessary for this response. This finding is significant because it identifies a transcriptional regulatory pathway that can increase microbial lipid content and has applications for increasing biofuel production

Reference: Lemmer, K. C., A. C. Dohnalkova, D. R. Noguera, and T. J. Donohue. 2015. “Oxygen Dependent Regulation of Bacterial Lipid Production,” Journal of Bacteriology 197(9), 1649-58. DOI: 10.1128/JB.02510-14. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 02, 2015

Understanding and Enhancing Microbial Lipid Production for Biofuels

Lipids derived from oil-rich microorganisms such as bacteria, yeast, and microalgae offer a promising source of renewable fuels and chemicals. However, genetic and biochemical mechanisms regulating lipid accumulation in microorganisms are poorly understood. A recent study revealed a novel molecular pathway involved in microbial lipid accumulation. Researchers from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) and the University of Wisconsin-Madison used the cryotransmission electron microscope at the DOE Environmental Molecular Sciences Laboratory to study lipid accumulation in the microbe Rhodobacter sphaeroides. Using fatty acid levels to assess membrane lipid content, the team found that the total fatty acid content per cell increased three-fold under low oxygen and anaerobic conditions compared to high oxygen conditions. They also found that the microbes’ lipid and pigment accumulation processes were separable, and they identified a transcription factor called PrrBA that is required for fatty acid accumulation in response to low oxygen levels. This new approach to maximize lipid production through an alteration in the activity of a single transcriptional regulator could lead to the development of strategies for engineering this microbe to increase yields for large-scale production of lipids for biofuels and chemicals.

Reference: Lemmer, K. C., A. C. Dohnalkova, D. R. Noguera, and T. J. Donohue. 2015. “Oxygen-Dependent Regulation of Bacterial Lipid Production,” Journal of Bacteriology, DOI: 10.1128/JB.02510-14. (Reference link)

Contact: Paul E. Bayer, SC-23.1, (301) 903-5324, Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 26, 2015

Novel Noncatalytic Cellulase-Binding Proteins Identified in Caldicellulosiruptor

Lignocellulose-degrading microorganisms often produce cellulosomes, which are protein complexes containing cellulase enzymes and noncatalytic binding modules. However, the genus Caldicellulosiruptor does not encode for cellulosomes, indicating that this genus uses alternative attachment mechanisms. To look for cellulose-binding proteins in Caldicellulosiruptor kronotskyensis, researchers from the Department of Energy’s BioEnergy Science Center performed a proteomic screen to detect proteins enriched in a cellulose-bound fraction. A comparison of amino acid sequences from the cellulose-binding proteins to the C. kronotskyensis genomic sequence identified the likely encoding gene and a closely related gene. These genes, subsequently named tapirins, are unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. In addition, no genes homologous to these tapirin genes were found outside of the genus Caldicellulosiruptor. Heterologously expressed tapirin gene products demonstrated binding to insoluble substrates such as Avicel, switchgrass, and Populus biomass, with a high affinity and specificity. Crystallization of a cellulose-binding truncation from one tapirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face and are structurally unique and define a new class of polysaccharide adhesins. Thus, the tapirins establish a new paradigm for how cellulolytic bacteria adhere to cellulose and may be used in engineering more efficient cellulase enzymes for more efficient lignocellulose deconstruction.

Reference: Blumer-Schuette, S. E., M. Alahuhta, J. M. Conway, L. L. Lee, J. V. Zurawski, R. J. Giannone, R. L. Hettich, V. V. Lunin, M. E. Himmel, and R. M. Kelly. 2015. “Discrete and Structurally Unique Proteins (Tapirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose,”The Journal of Biological Chemistry, DOI: 10.1074/jbc.M115.641480. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 23, 2015

Elucidating the Evolution of Mutualistic Plant Fungi

The large variety of fungi that exist in forest soils play diverse and important roles when in association with plant roots. One such type, the ectomycorrhizal (ECM) fungi, is a beneficial mutualist. ECM fungi obtain carbon compounds from the host plant, and in doing so provide critical ecological services such as decomposing lignocellulose and promoting plant growth. To unravel the mechanisms of nutrient cycling in forests, a better understanding of ECM fungi is needed. As part of a consortium investigating mycorrhizal fungal genomics, scientists at Oak Ridge National Laboratory, funded through the Department of Energy’s (DOE) Plant-Microbe Interfaces Science Focus Area, and DOE’s Joint Genome Institute performed phylogenomic and comparative genomic analyses of newly sequenced fungal genomes, including 13 ECM fungi, to elucidate the genetic bases of mycorrhizal lifestyle evolution. They found that although the ECM fungi have a reduced complement of genes encoding plant cell-wall degrading enzymes, those enzymes that were retained made up a distinct suite, indicating that they possess diverse capabilities to decompose lignocellulose. They also found that the symbiosis that develops between ECM fungi and the host plant and contributes to plant development and immunity requires lineage-specific fungal genes, including genes that code for mycorrhiza-induced small secreted proteins. The researchers conclude that convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a “symbiosis toolkit”, with reduced numbers of plant cell-wall degrading enzymes and lineage-specific suites of mycorrhiza-induced genes. Studies designed to predict the response of ECM and other mycorrhizal fungi to fluctuations in the environment will benefit from these genomic resources.

Reference: Kohler, A., A. Kuo, L. G. Nagy, E. Morin, K. W. Barry, F. Buscot, B. Canbäck, C. Choi, N. Cichocki, A. Clum, J. Colpaert, A. Copeland, M. D. Costa, J. Doré, D. Floudas, G. Gay, M. Girlanda, B. Henrissat, S. Herrmann, J. Hess, N. Högberg, T. Johansson, H.-R. Khouja, K. LaButti, U. Lahrmann, A. Levasseur, E. A. Lindquist, A. Lipzen, R. Marmeisse, E. Martino, C. Murat, C. Y. Ngan, U. Nehls, J. M. Plett, A. Pringle, R. A. Ohm, S. Perotto, M. Peter, R. Riley, F. Rineau, J. Ruytinx, A. Salamov, F. Shah, H. Sun, M. Tarkka, A. Tritt, C. Veneault-Fourrey, A. Zuccaro, Mycorrhizal Genomics Initiative Consortium, A. Tunlid, I. V. Grigoriev, D. S. Hibbett, and F. Martin. 2015. “Convergent Losses of Decay Mechanisms and Rapid Turnover of Symbiosis Genes in Mycorrhizal Mutualists,” Nature Genetics 47, 410-15, DOI:, DOI: 10.1038/ng.3223. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 15, 2015

Newly Identified Archaea Involved in Anaerobic Carbon Cycling

Archaea, a domain of single-celled microorganisms, represent a significant fraction of Earth’s biodiversity, yet much less is known about Archaea than bacteria. One reason for this lack of knowledge is relatively poor genome sampling, which has limited accuracy for the Archaeal phylogenetic tree. To obtain a better understanding of the diversity and physiological functions of members of the Archaea domain, a team of scientists from the University of California, Berkeley, The Ohio State University, Columbia University, Lawrence Berkeley National Laboratory, the Department of Energy’s (DOE) Joint Genome Institute, Pacific Northwest National Laboratory, and DOE Environmental Molecular Sciences Laboratory used genome-resolved metagenomics analyses to investigate the diversity, genome sizes, metabolic capabilities, and potential environmental niches of Archaea from the Rifle, Colorado, uranium mill tailings site. The team used DOE JGI to sequence DNA from Rifle sediment and groundwater samples, and they not only identified new sequences for more than 150 Archaea but were able to reconstruct the complete genomes of two Archaea that were demonstrated to be representative of two different phyla. Transcriptomic studies conducted using EMSL capabilities on one of these microbes demonstrate that they have small genomes and limited metabolic capabilities; however, these metabolic capabilities are associated with carbon and hydrogen metabolism. These results suggest that these Archaea are either symbionts or parasites that depend on other organisms for some of their metabolic requirements. This research approximately doubled the known genomic diversity of Archaea, reconstructed the first complete genomes for Archaea using cultivation-independent methods, and enabled an extensive revision of the Archaeal tree of life. In addition, these findings can be incorporated into genome-resolved ecosystem models to more accurately reflect the role played by Archaea in the global carbon cycle.

References: Castelle, C. J., K. C. Wrighton, B. C. Thomas, L. A. Hug, C. T. Brown, M. J. Wilkins, K. R. Frischkorn, S. G. Tringe, A. Singh, L. M. Markillie, R. C. Taylor, K. H. Williams, and J. F. Banfield. “Genomic Expansion of Domain Archaea Highlights Roles for Organisms from New Phyla in Anaerobic Carbon Cycling,” Current Biology 25(6), 690-701. DOI: 10.1016/j.cub.2015.01.014. (Reference link)
(See also)

Contact: Paul E. Bayer, SC-23.1, (301) 903-5324, Dan Drell, SC-23.2, (301) 903-4742, David Lesmes, SC 23.1, (301) 903-2977, Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER


February 11, 2015

Use of Co-Solvent Saves on Cost and Enzymes

Production of cost-effective biofuels from lignocellulosic biomass must overcome lignocellulose recalcitrance. Current processes to release sugars for viable biochemical conversion to biofuels requires energy-intensive pretreatment and large amounts of expensive enzymes. Researchers from the Department of Energy’s BioEnergy Science Center (BESC) have discovered that a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) reduces enzyme costs dramatically, resulting in high sugar yields from hemicellulose and cellulose. CELF employs tetrahydrofuran (THF), which is miscible with aqueous dilute acid, and gives up to 95% of the theoretical yield of glucose, xylose, and arabinose from corn stover even when coupled with enzymatic hydrolysis at only 2 mg enzyme/g glucan—an unusually low concentration of enzymes. The unusually high saccharification with such low enzyme loadings can be attributed to very high lignin removal, which was evidenced by compositional analysis, fractal kinetic modeling, and scanning electron microscopy imaging. Subsequently, nearly pure lignin product was precipitated giving a clean lignin stream for valorization. THF was efficiently recovered and recycled by evaporation of the volatile solvent. Simultaneous saccharification of CELF-pretreated solids with low enzyme loadings and fermentation by Saccharomyces cerevisiae produced twice as much ethanol as that from dilute acid-pretreated solids after being optimized for corn stover. Thus, CELF offers efficient lignocellulosic biomass pretreatment and saccharification with reduced costs relative to current processes.

Reference: Nguyen, T. Y., C. M. Cai, R. Kumar, and C. E. Wyman. 2015 “Co-Solvent Pretreatment Reduces Costly Enzyme Requirements for High Sugar and Ethanol Yields from Lignocellulosic Biomass,” ChemSusChem, DOI: 10.1002/cssc.201403045. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 30, 2015

Systems Biology of a Cyanobacterial Chassis for Photosynthetic Biosynthesis

Cyanobacteria, a broadly distributed class of photosynthetic bacteria, are attractive candidates for development as “chassis organisms” for production of biofuels and other products. In comparison to photosynthetic algae, cyanobacteria grow more quickly, are capable of growth in a broad range of conditions, and possess much simpler (and thus more easily engineered) genomes. However, developing systems-level understanding of integrated metabolic networks in cyanobacteria will be necessary before more sophisticated bioengineering approaches can be applied to further optimize performance or more easily introduce new biosynthetic modules. A new study by researchers at Washington University examines systems biology properties of the recently discovered cyanobacterial strain Synechococcus elongatus UTEX 2973, which grows at double the rates of other members of this species under high light intensities. Using a comparative genomics approach, the team was able to identify a surprisingly small set of genetic differences between UTEX 2973 and slower growing S. elongatus strains, amounting to 55 amino acid substitutions and a small missing region encoding six genes seen in the slower growing strains. Leveraging capabilities at the Department of Energy’s Environmental Molecular Sciences Laboratory, these findings were validated using global proteomics analysis, confirming predicted amino acid substitutions and showing that UTEX 2973 is missing five of the six predicted proteins. Although these proteins are currently of unknown function, UTEX 2973 fails to form cytoplasmic glycogen granules observed during growth of the other strains. This observation suggests that UTEX 2973 may not store photosynthetically fixed carbon, but instead immediately uses it as substrate fueling accelerated growth. UTEX 2973 can be genetically manipulated using tools developed for related cyanobacterial strains, and the team currently is developing a mutant library to explore the specific mechanistic basis of the UTEX 2973’s rapid growth phenotype. These findings expand our knowledge of cyanobacterial systems biology and present Synechococcus elongatus UTEX 2973 as a promising potential biotechnological chassis organism for the direct conversion of sunlight and CO2 into biofuels and other compounds.

Reference: Yu, J., M. Liberton, P. F. Cliften, R. D. Head, J. M. Jacobs, R. D. Smith, D. W. Koppenaal, J. J. Brand, and H. B. Pakrasi. 2015. “Synechococcus elongatus UTEX 2973, a Fast Growing Cyanobacterial Chassis for Biosynthesis Using Light and CO2,” Scientific Reports 5: 8132. DOI: 10.1038/srep08132. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 22, 2015

Using a Designer Synthetic Media to Study Inhibitors Effect in Biomass Conversion

The biofuels industry has devoted significant efforts to converting lignocellulosic substrates into sugars that can be fermented into biofuels or other bioproducts. However, one of the major bottlenecks for cost-effective conversion in biorefineries has been the fermentation inhibition of yeast or bacteria by pretreatment degradation products. To engineer microbial strains for improved conversion, it is important to understand the inhibition mechanisms that affect the fermentative organisms in the presence of a lignocellulosic hydrolysate. One way in which these processes can be understood is by developing a synthetic hydrolysate media with a composition similar to the one that will be found after pretreating lignocellulosic biomass. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center characterized the plant-derived decomposition products present in ammonia fiber expansion (AFEX) pretreated corn stover hydrolsate (ACH), and a synthetic hydrolysate (SH) was formulated based on that ACH composition. The SH was used to evaluate the inhibitory effects of various families of decomposition products during fermentation using Saccharomyces cerevisiae strain 424A (LNH-ST). The SH did not entirely match the ACH performance; however, the major groups of inhibitory compounds were identified and used for further evaluation and comparison. Their characterization showed that the compounds present in ACH that were most inhibitory to fermentation were nitrogenous compounds, especially amides, though this result is associated with a concentration effect, given that nitrogenous compounds were the most abundant. Comparing inhibition due to amides in AFEX pretreatment versus inhibition due to carboxylic acids and other compounds formed in alternative pretreatment methods, they discovered that amides are significantly less inhibitory to both glucose and xylose fermentation. This means that ACH would be easily fermentable by yeast without any further detoxification. These studies help to map where to focus research efforts to overcome pretreatment byproduct inhibition of fermentation.

Reference: Tang, X., L. daCosta Sousa, M. Jin, S. P. S. Chundawat, C. K. Chambliss, M. W. Lau, Z. Xiao, B. E. Dale, and V. Balan. 2015. “Designer Synthetic Media for Studying Microbial-Catalyzed Biofuel Production,” Biotechnology for Biofuels 8:1. DOI: 10.1186/s13068-014-0179-6. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 13, 2015

Diversion of Lignin Precursor Reduces Content and Improves Biomass Saccharification Efficiency

Lignin confers recalcitrance to plant biomass used for producing biofuels and bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically have employed gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI) employed a new strategy using gain of function. In this method, expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) was targeted to the plastids of Arabidopsis to convert 3-dehydroshikimate—an intermediate of the shikimate pathway—into protocatechuate. This enzymatic conversion diverted lignin precursor into protocatechuate and related molecules and away from lignin precursors. Compared to wild-type plants, Arabidopsis lines expressing QsuB contained reduced levels of lignin deposition in the cell walls. Because this strategy is a gain of function, its expression can be controlled by selective promoters, thus offering better spatiotemporal control of lignin deposition than the gene knockout or gene silencing strategies. Finally, biomass from these engineered Arabidopsis lines exhibits more than a twofold improvement in saccharification efficiency. This result confirms that QsuB expression in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.

Reference: Eudes, A., N. Sathitsuksanoh, E. E. Baidoo, A. George, Y. Liang, F. Yang, S. Singh, J. D. Keasling, B. A. Simmons, and D. Loqué. 2015. “Expression of a Bacterial 3-Dehydroshikimate Dehydratase Reduces Lignin Content and Improves Biomass Saccharification Efficiency,” Plant Biotechnology Journal, DOI: 10.1111/pbi.12310. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 29, 2014

Statistics to Help Optimize Engineered Heterologous Pathways

For metabolic engineering to reach its full potential, systematic pathway optimization approaches are needed for biofuel production. In previous work, Department of Energy Joint Bioenergy Institute (JBEI) researchers assembled a set of nine heterologous genes in Escherichia coli to produce from glucose the monoterpene limonene, a potential biofuel. While they were able to achieve 435 mg/L of limonene production, they believed further optimization was possible. In a new research article, the JBEI scientists present and demonstrate a computational tool (principal component analysis of proteomics; PCAP) that uses quantitative targeted proteomics data to guide metabolic engineering and achieve higher production of target molecules from heterologous pathways. Counterintuitively, PCAP suggested that an overexpression of the terpene synthase combined with a balanced expression of the remaining enzymes was key to improving limonene production. The PCAP-guided engineering resulted in a more than 40% improvement in the production of limonene and a second valuable terpene. Thus, PCAP could be broadly applied to heterologous pathways for optimized biofuel production.

Reference: Alonso-Gutierrez, J., E.-M. Kim, T. S. Batth, N. Cho, Q. Hu, L. J. G. Chan, C. J. Petzold, N. J. Hillson, P. D. Adams, J. D. Keasling, H. G. Martin, and T. S. Lee. 2015. “Principal Component Analysis of Proteomics (PCAP) as a Tool to Direct Metabolic Engineering,” Metabolic Engineering 28, 123–33. DOI: 10.1016/j.ymben.2014.11.011. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 24, 2014

Elucidating Control of Secondary Cell Wall Synthesis

The plant cell wall plays an important role in cell function and environmental response by providing both mechanical support and a barrier against invading pathogens. Furthermore, the highly-abundant secondary cell walls, which are composed of cellulose, hemicelluloses and lignin, are an important source of dietary fiber, raw material for paper and pulp, and feedstock for biofuel production. Despite the importance of the plant secondary cell wall for renewable resources, knowledge of the precise mechanisms that regulate these critical functions is limited. New research results published in the journal Nature report the identification of a gene network in the model plant Arabidopsis thaliana that controls synthesis of the biopolymers that comprise the secondary cell wall. Instead of using a gene-by-gene approach, the scientists undertook a comprehensive, large-scale analysis, which revealed a highly integrated network involving hundreds of genes and protein-DNA interactions. Furthermore, they found that the extremely large number of combinatorial possibilities provided by this arrangement allows for subtle adaptation to specific abiotic stresses such as salt stress and iron deprivation. These findings provide a framework for future work to dissect and refine specific gene functions, enabling targeted manipulation of the network to produce high-yielding plant feedstocks for bioenergy production. The Nature paper is accompanied by a commentary by two prominent plant scientists. This research was supported by the U.S. Department of Agriculture-Department of Energy Plant Feedstocks Genomics for Bioenergy program.

References: Taylor-Teeples, M., L. Lin, M. de Lucas, G. Turco, T. W. Toal, A. Gaudinier, N. F. Young, G. M. Trabucco, M. T. Veling, R. Lamothe, P. P. Handakumbura, G. Xiong , C. Wang, J. Corwin, A. Tsoukalas, L. Zhang, D. Ware, M. Pauly, D. J. Kliebenstein, K. Dehesh, I. Tagkopoulos, G. Breton, J. L. Pruneda-Paz, S. E. Ahnert, S. A. Kay, S. P. Hazen, and M. Brady.   2014. “An Arabidopsis Gene Regulatory Network for Secondary Cell Wall Synthesis,” Nature 517, 571–75. DOI: 10.1038/nature14099.   (Reference link)

Bishopp, A., and M. J. Bennett. 2015. “Seeing the Wood and the Trees,” Nature 517, 558–59. DOI:10.1038/nature14085. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 19, 2014

Field Production of Novel Plant Oils in Camelina

Some plants synthesize acetyl-triacylglycerols (acetyl-TAGs), some of which are suitable as ‘drop-in’ biodiesel. A diacylglycerol acetyltransferase from Euonymus alatus, EaDAcT, synthesizes such acetyl-TAGs when expressed in Arabidopsis, Camelina, and soybean. Compared to most vegetable oils, acetyl-TAGs have reduced viscosity and improved cold temperature properties that confer advantages in applications as biodegradable lubricants, food emulsifiers, plasticizers, and ‘drop-in’ fuels for some diesel engines. Previously, researchers in the Department of Energy’s Great Lakes Bioenergy Research Center (GLBRC) engineered a Camelina line producing high levels of oleic to express the EaDAcT gene to produce acetyl-TAG oils with fatty acid compositions and physiochemical properties complementary to wild-type acetyl-TAG. In field-grown engineered Camelina, the acetyl-TAGs accumulated to 70 mol% of seed TAG and had minor or no effect on seed weight, oil content, harvest index, and seed yield. The total moles of TAG increased up to 27%, reflecting the ability to synthesize more acetyl-TAG from the same supply of long-chain fatty acid. The crystallization temperature of high-oleic acetyl-TAG was reduced by 30? C compared to control TAG. The viscosity of high-oleic acetyl-TAG was 27% lower than TAG from the high-oleic control, and the caloric content was reduced by 5%. Field production of T4 and T5 transgenic plants yielded over 250 kg seeds for oil extraction and analysis. These results demonstrate that high-oleic Camelina lines can be engineered to produce desirable oils for ‘drop-in’ biodiesel and that establishing crop production of Camelina acetyl-TAG will enable sufficient quantities of acetyl-TAG to be produced for further agronomic and commercial development.

Reference: Liu, J., H. Tjellstrom, K. McGlew, V. Shaw, A. Rice, J. Simpson, D. Kosma, W. Ma, W. Yang, M. Strawsine, E. Cahoon, T. P. Durrett, and J. Ohlrogge. 2015 “Field Production, Purification, and Analysis of High-Oleic Acetyl-Triacylglyceros from Transgenic Camelina sativa,” Industrial Crops and Products 65, 259-68. DOI: 10.1016/j.indcrop.2014.11.019. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 16, 2014

Key Transcription Factor in Plant Senescence Regulates Chlorophyll Degradation and Abscisic Acid Biosynthesis

The timing of plant senescence can have a significant impact on the yield and quality of bioenergy feedstocks. Therefore, more knowledge is welcome on the regulation of and genes involved in plant senescence. Department of Energy BioEnergy Science Center researchers have gained new understanding of senescence in the experimentally tractable plant Arabidopsis thaliana. Chlorophyll degradation is an important part of leaf senescence, but the underlying regulatory mechanisms are largely unknown. The researchers found that the dark, excised leaves of an Arabidopsis thaliana transcription factor mutant (nap) exhibit a stay-green phenotype. This finding is correlated with lower transcript levels of several known chlorophyll degradation genes, and higher chlorophyll retention than the wild type during dark-induced senescence. Several plant hormones play a role in senescence; one of them, abscisic acid (ABA), is known to induce leaf senescence. Transcriptome coexpression analysis revealed that ABA metabolism/signaling genes were disproportionately represented among those positively correlated with expression of the NAP transcription factor. To further investigate ABA’s role in senescence and the stay-green phenotype, ABA was applied exogenously to excised NAP mutant leaves. Transcript levels of several chlorophyll degradation enzymes increased and the stay-green phenotype was suppressed. Collectively, the results show that the NAP transcription factor promotes chlorophyll degradation by enhancing transcription of the ABA biosynthesis gene, AAO3, which leads to increased levels of the senescence-inducing hormone ABA. This new understanding will be helpful in improving yields of bioenergy feedstocks by controlling senescence.

Reference: Yang, J., E. Worley, and M. Udvard. 2014. “A NAP-AA03 Regulatory Module Promotes Chlorophyll Degradation via ABA Biosynthesis in Arabidopsis leaves,” The Plant Cell 26, 4862–74. DOI: 10.1105/tpc.114.133769. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 18, 2014

A Fungal Garden’s Microbial Makeup

Leafcutter ants (Atta cephalotes) are of interest to bioenergy researchers because they farm gardens made up of communities of bacteria and fungi that break down plant biomass. Beetles and termites have similar symbiotic relationships with microbial communities in the gardens they cultivate for food, suggesting that different insect hosts have exploited microbes more than once as a strategy for breaking down biomass. In a recent collaboration, scientists from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center and DOE Joint Genome Institute used genomic techniques to analyze the composition of microbial communities in these fungal gardens. They found that regardless of their geographic location, these gardens have a similar microbial makeup. The high whole-genome similarity across distantly related insect hosts that reside thousands of miles apart shows that these bacteria are an important and underappreciated feature of diverse, fungus-growing insects. Because of the similarities in the agricultural lifestyles of these insects, this is an example of convergence between both the life histories of the host insects and their symbiotic microbiota. These results may point the way to both bacteria and fungi that are predisposed to having genes for enzymes and pathways useful for breaking down biomass to potential bioenergy feedstock sources.

Reference: Aylward, F. O., G. Suen, P. H. W. Biedermann, A. S. Adams, J. J. Scott, S. A. Malfatti, T. Glavina del Rio, S. G. Tringe, M. Poulsen, K. F. Raffa, K. D. Klepzig, and C. R. Curriea. 2014. “Convergent Bacterial Microbiotas in the Fungal Agricultural Systems of Insects,” mBio 5(6), e02077-14. DOI: 10.1128/mBio.02077-14. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 12, 2014

Genomic Selection to Accelerate Switchgrass Breeding

The perennial grass switchgrass (Panicum virgatum L.) shows great promise as a biofuel feedstock due to its ability to produce high biomass yields with relatively few inputs, and on lands not typically used for agricultural crops. The high genetic variability among different switchgrass accessions indicates that varieties with improved biomass quality traits could be developed through traditional breeding programs. However, this potential has been largely unattained due to the lengthy breeding cycle as well as a need for accurate measurement of biomass yield. A new approach known as genomic selection, which uses whole-genome, high-density molecular markers developed with high-throughput genotyping, has been used successfully with livestock and forest trees. Taking advantage of available genomic resources for switchgrass, including a reference genome, researchers have evaluated the accuracy of three genomic selection models in predicting phenotypic values of seven morphological and 13 biomass quality traits in a switchgrass association panel. Most traits were predicted with high accuracy, suggesting that the application of genomic selection to switchgrass breeding would be highly beneficial. Rather than waiting until the plant reaches adulthood, accurate prediction of biomass yield will allow DNA marker-based selection of seedlings, thus greatly accelerating breeding and potentially transforming switchgrass improvement efforts. The research was funded in part by the U.S. Department of Agriculture-Department of Energy Plant Feedstock Genomics for Bioenergy program.

Reference: Lipka, A. E., F. Lu, J. H. Cherney, E. S. Buckler, M. D. Casler, and D. E. Costich.  2014. “Accelerating the Switchgrass (Panicum virgatum L.) Breeding Cycle Using Genomic Selection Approaches,” PLoS ONE 9(11), e112227. DOI: 10.1371/journal.pone.0112227. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 03, 2014

High-Temperature Microbe Metabolically Engineered to Produce Biofuel Alcohols

The U.S. bioethanol industry depends largely on glucose conversion by yeast wherein pyruvate is decarboxylated to acetaldehyde and then reduced to the 2-carbon biofuel, ethanol. Interest is growing, however, in microorganisms that produce longer-chain alcohols with superior characteristics as fuel molecules compared to ethanol. Examples include microbial strains engineered to produce a specific alcohol such as isopropanol, n-butanol, or isopentanol. Much of the research to date focuses on engineered organisms that operate at ambient temperatures (e.g, 37°C), but the ability to produce bioalcohols at temperatures above 70°C has several advantages over ambient-temperature processes, including lower risk of microbial contamination, higher diffusion rates, and lower cooling and distillation costs. Researchers at the Department of Energy’s BioEnergy Science Center describe the metabolic engineering of a hyperthermophilic archaeon, Pyrococcus furiosus, to produce not only ethanol but a range of alcohols at 70-80°C via a synthetic pathway not known in nature and fundamentally different from those previously described. Specifically, the researchers engineered P. furiosus to produce various alcohols from their corresponding organic acids by constructing a novel synthetic route termed the aldehyde ferredoxin oxidoreductase (AOR)/alcohol dehydrogenase (AdhA) pathway. For example, in addition to converting acetate to ethanol, the synthetic pathway was shown to convert longer chain acids such as propionate to propanol, isobutyrate to isobutanol, and phenylacetate to phenylalcohol. This study is the first example of significant alcohol formation in an archaeon, emphasizing the biotechnological potential of novel microorganisms for biofuel production.

Reference: Basen, M., G. J. Schut, D. M. Nguyen, G. L. Lipscomb, R. A. Benn, C. J. Prybol, B. J. Vaccaro, F. L. Poole, R. M. Kelly, and M. W. W. Adams. 2014. “Single Gene Insertion Drives Bioalcohol Production by a Thermophilic Archaeon,” Proceedings of the National Academy of Sciences (USA) 111(49), 17,618-623. DOI: 10.1073/pnas.1413789111. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 02, 2014

Improved Lignin Depolymerization for Higher-Value Products

Lignin is a heterogeneous aromatic biopolymer that accounts for nearly 30% of the organic carbon on Earth and is one of the few renewable sources of aromatic chemicals. As the most recalcitrant of the three components of lignocellulosic biomass (cellulose, hemicellulose, and lignin), lignin has been treated as a waste product in the pulp and bioenergy industries, where it is sometimes burned to provide energy. Creation of higher-value bioproducts from lignin will increase the economic viability of integrated biorefineries. Depolymerization is an important starting point for many lignin valorization strategies, because it can generate valuable aromatic chemicals and provide a source of low-molecular-mass feedstocks suitable for downstream processing. Commercial precedents show that certain types of lignin (lignosulphonates) may be converted into vanillin and other marketable products, but new technologies are needed to enhance the lignin value chain. Lignin’s complex, irregular structure complicates chemical conversion efforts, and known depolymerization methods typically afford ill-defined products in low yields (that is, less than 10-20 wt%). Researchers of the Department of Energy’s Great Lakes Bioenergy Research Center describe a method for the depolymerization of oxidized lignin under mild conditions in aqueous formic acid that results in more than 60 wt% yield of low molecular-mass aromatics. This facile C-O cleavage method was used to depolymerize aspen lignin, providing mechanistic insights into the reaction. Efficient lignin depolymerization and biomass refining have the potential to contribute to the commercial and economic viability of lignocellulosic biofuels.

Reference: Rahimi, A., A. Ulbrich, J. J. Coon, and S. S. Stahl. 2014. “Formic-Acid-Induced Depolymerization of Oxidized Lignin to Aromatics,” Nature 515, 249-52. DOI: 10.1038/nature13867. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 31, 2014

Unraveling Nitrogen Cycling by Soil Microbes

Large amounts of nitrogen enter soil ecosystems as nitrate (NO3-) fertilizers. In addition to being available as a nitrogen source, a variety of soil microbes can generate energy from NO3- either by (1) denitrification, the conversion of NO3 to ammonia (NH4+), or (2) respiratory ammonification, which results in a mixture of nitrous oxide and dinitrogen gas (N2O and N2). NH4+ remains in soil while N2O and N2 are lost to the atmosphere, where N2O acts as a potent greenhouse gas. Understanding the microorganisms that perform these competing pathways is important for both agriculture and global climate change. Researchers at the University of Tennessee and Oak Ridge National Laboratory examined the systems biology properties of an unusual microbe able to perform both of these processes. While the majority of microbes utilizing NO3- as an energy source perform either denitrification or ammonification, the bacterium Shewanella loihica was shown to possess both pathways and thus offers a unique opportunity to examine the specific environmental factors that result in production of NH4+ versus N2O and N2. Using a series of careful physiological studies coupled to measurements of gene expression, the team determined that S. loihica activates the most energetically favorable pathway depending on its growth conditions, with the ratio of available carbon substrates to nitrogen availability (C/N ratio) playing the most influential role in the organism opting for either ammonification or denitrification. Recent studies have shown that organisms like S. loihica that are capable of both denitrification and ammonification are far more common in soil ecosystems than previously suspected. As such, this study’s findings have major implications for predicting climate change impacts on terrestrial biogeochemical cycles and designing more sustainable bioenergy agriculture practices.

Reference: Yoon, S., C. Cruz-Garcia, R. Sanford, K. M. Ritalahti, and F. E. Loffler. 2014. “Denitrification Versus Respiratory Ammonification: Environmental Controls of Two Competing Dissimilatory NO3-/NO2- Reduction Pathways in Shewanella loihica Strain PV-4,” The ISME Journal, DOI: 10.1038/ismej.2014.201. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 23, 2014

Microbial Community Dynamics Dominate Greenhouse Gas Production in Thawing Permafrost

Northern permafrost ecosystems are changing rapidly, with rising temperatures causing the transition of many previously frozen environments to wetlands. As permafrost thaws, the trapped organic carbon is accessible to decomposition by microbes and can be released to the atmosphere as greenhouse gases (GHGs). Understanding of these communities is limited, especially the specific nature of processes that impact rates of carbon decomposition and the balance of the carbon dioxide (CO2) versus methane (CH4) released to the atmosphere. Although both gases are GHGs, CH4 is much more potent in the short term, so understanding the microbial mechanisms driving these large-scale processes would significantly improve predictions of possible climate change impacts.

An interdisciplinary team of researchers led by the University of Arizona has examined microbial community dynamics at a site in northern Sweden that occupies a natural temperature gradient. Northern portions of this site are frozen permafrost while southern areas are thawed fens. Over several years, the team measured CO2 and CH4 production along the gradient, examined isotopic signatures of gases characteristic of distinct microbial processes, and correlated the data with measured shifts in microbial community composition and abundance. Only small amounts of GHGs were released from frozen permafrost, but in progressively more thawed sites, CH4 was the dominant product released. The team was able to link these observations with extensive shifts in microbial community composition, revealing a reproducible succession pattern of different types of CH4-producing microbes (methanogens) across the thaw gradient. Surprisingly, a single methanogen species, Candidatus Methanoflorens stordalenmirensis, was dominant in recently thawed sites and its relative abundance strongly correlated with the magnitude and specific type of CH4 produced at any given site.

The striking dominance of a single microbial species in mediating a large-scale carbon cycle process is highly unusual and provides an opportunity to more effectively track and predict the impacts of climate change across an entire region. The team has begun to incorporate integrated datasets on biogeochemical process measurements and microbial community patterns into ecosystem-scale models of carbon cycle processes. This effort represents a significant advance in understanding and more accurately representing critical biogeochemical processes in permafrost that are performed by microbes, improving predictions of climate change impacts on these delicate ecosystems and their potential atmospheric consequences.

Reference: McCalley, C. K., B. J. Woodcroft, S. B. Hodgkins, R. A. Wehr, E.-H. Kim, R. Mondav, P. M. Crill, J. P. Chanton, V. I. Rich, G. W. Tyson, and S. R. Saleska. 2014. “Methane Dynamics Regulated by Microbial Community Response to Permafrost Thaw,” Nature 514, 478-81. DOI: 10.1038/nature13798. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 21, 2014

Microbial Community Dynamics Impacting Methane Consumption in Freshwater Lakes

Decay of plant material in oxygen-limited sediments of lakes and wetlands results in the production of massive amounts of methane (CH4), a potent greenhouse gas. However, only a fraction of the CH4 produced in these environments enters the atmosphere due to the metabolic activities of microbial methanotrophs. Methanotrophs are a class of bacteria capable of consuming CH4 and using it as both a source of carbon and energy to fuel their growth. Understanding even the basic physiology of methanotrophs remains limited, as evidenced by the recent discovery of a new fermentative mode of methanotrophic metabolism in organisms that were previously thought to strictly require oxygen for growth. In a new study by researchers at the University of Washington, experimental microcosms established with lake sediments were used to examine methanotrophic communities and their response to varying levels of oxygen. By tracking community composition through DNA pyrosequencing, the team determined that the methanotroph community features a nonrandom assemblage of organisms, with specific types adapted to either high or low oxygen levels. When the methanotroph community shifted in response to oxygen availability, an array of nonmethanotrophic microbes also changed. Preliminary evidence suggests that these organisms are metabolically partnered with methanotrophs, exchanging nutrients and facilitating methanotrophic processes. These results represent the first detailed examination of microbial community dynamics in a methanotrophic ecosystem and suggest a high degree of complexity in their response to shifting environmental variables. Gain­ing a more sophisticated understanding of microbial community dynamics influencing methano­trophs in natural settings will help to facilitate more accurate predictions of environmental CH4 production and consumption.

Reference: Oshkin, I. Y., D. A. C Beck, A. E. Lamb, V. Tchesnokova, G. Benuska, T. L. McTaggart, M. G. Kalyuzhnaya, S. N. Dedysh, M. E. Lidstrom, and L. Chistoserdova. 2014. “Methane-Fed Microbial Microcosms Show Differential Community Dynamics and Pinpoint Taxa Involved in Communal Response,” The ISME Journal, DOI: 10.1038/ismej.2014.203. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 17, 2014

Enhancing Microbial Pathways for Biofuel Production

Produced in microbes and plants, terpenes are high-energy compounds that could be used for producing biofuels. For example, U.S. Department of Energy (DOE) researchers at the Joint BioEnergy Institute (JBEI) had reported that bisabolane, a biofuel derived from the sesquiterpene precursor bisabolene, could serve as an alternative to diesel fuel. Enhancing terpene yields in suitable microbes and plants is thus an important step toward commercial-scale production of these biofuels. Terpene synthesis in the majority of bacterial species, as well as in plant plastids, takes place via a pathway in which one-sixth of the carbon in the starting metabolites is lost as carbon dioxide (CO2). JBEI researchers wanted to improve terpene production in Escherichia coli by developing a pathway that would not result in any carbon loss as CO2. To do this, they focused on using a novel route that would form terpenes from 5-carbon (C5) sugars such as xylose, which is a breakdown product of hemicellulose. The researchers created a mutant in the metabolism of C5 sugars and then selected for complementary mutants that could grow on the C5 sugar xylose. E. coli colonies that were able to grow under this selective pressure were sequenced at DOE’s Joint Genome Institute and all were found to have mutations in the ribB gene. The researchers then inserted the pathway for bisabolene production into the strains able to grow on xylose, and they found bisabolene production in these strains. Further manipulation of the pathways by gene fusion and varying the gene order enhanced bisabolene yields several fold. These results demonstrate that biosynthetic pathways that are not found in nature may be constructed by selection and targeted engineering. This pathway is can now be further optimized for terpene yield in preparation for commercial-scale production.

Reference: Kirby, J., M. Nishimoto, R. W. N. Chow, E. E. K. Baidoo, G. Wang, J. Martin, W. Schackwitz, R. Chan, J. L. Fortman, and J. D. Keasling. 2015. “Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate,” Applied and Environmental Microbiology 81,130–8. DOI: 10.1128/AEM.02920-14. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 14, 2014

New Target for Engineering Lignin for Biofuel Production

Plant cell walls contain polysaccharides that can be hydrolyzed into fermentable sugars, but this process is inhibited by lignin. Altering lignin composition or structure can reduce the amount of effort needed to release glucose from cellulose, thus improving the economics of cellulosic biofuels production. Department of Energy Great Lakes Bioenergy Research Center (GLBRC) researchers John Ralph and Hoon Kim and their colleagues at Ghent University and Flanders Institute of Biology have a goal of understanding the control points in the lignin biosynthetic pathway and how to use them to improve biomass properties. They identified a new target for engineering lignin for biofuel production by using transcriptomics and microarray studies to identify genes that co-express with other known lignin biosynthesis genes. In the model plant Arabidopsis, there are three cytochrome P450 reductase genes, and one of these three genes controls an enzyme (ATR2) that is co-expressed with lignin biosynthetic genes. By studying mutant plants in which the atr2 gene was down-regulated via T-DNA insertion, researchers found that the atr2 mutants had increased glucose release from cellulose relative to the wild type following base pretreatment. This increase in saccharification appeared to result from both altered lignin structure and altered lignin content. The results support the contention that ATR2 is involved in the lignin pathway and is thus a target for engineering plant cell walls that are better suited for biofuels applications. The study also suggests additional candidates in the lignin pathway for future study.

Reference: Sundin, L., R. Vanholme, J. Geerinck, G. Goeminne, R. Höfer, H. Kim, J. Ralph, and W. Boerjan. 2014. “Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification,”Plant Physiology 166, 1956–71. DOI: 10.1104/pp.114.245548. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 10, 2014

Investigating Nitrogen Fixation in a Photosynthetic Microbial Community

Photosynthetic microbial mats dominated by cyanobacteria achieve high rates of productivity using little more than sunlight, atmospheric gases (CO2 and N2), and trace nutrients. These complex, stratified ecosystems thus can provide experimentally tractable models to investigate functional properties of microbial communities and serve as valuable analogues for bioenergy production systems. The high rates of photosynthetic productivity observed in microbial mats are made possible by microbial nitrogen fixation, the process of converting N2 gas into biologically useful forms of nitrogen. Identifying which community members perform this process would provide a key to understanding overall community function. A team of investigators led by Lawrence Livermore National Laboratory scientists have reported new findings on nitrogen fixation in photosynthetic microbial mats using a combination of community gene expression analysis (metatranscriptomics), high-resolution microscopy, and nanoscale mass spectrometry (nanoSIMS). Metatranscriptomic analysis provided an overview of metabolically active community members capable of N2 fixation, thus providing an initial roster of target species worthy of further examination. Microscopically enabled nanoSIMS then provided the capability to narrow the search, tracking isotopically labeled nitrogen through the community at the scale of single cells. By coupling these two technologies, the team was able to identify specific members of the cyanobacterial portion of the community as the dominant N2 fixers and examine their spatial relationships within the overall community structure. These findings highlight the importance of pairing omics-driven techniques with complementary approaches that provide validation of functional predictions. By coupling cutting-edge experimental capabilities, researchers are developing a more sophisticated understanding of the biological rules that govern community structure and function, potentially enabling construction of analogous systems devoted to high-efficiency bioenergy production.

Reference: Woebken, D., L. C. Burow, F. Behnam, X. Mayali, A. Schintlmeister, E. D. Fleming, L. Prufert-Bebout, S. W. Singer, A. Lopez Cortes, T. M. Hoehler, J. Pett-Ridge, A. M. Spormann, M. Wagner, P. K. Weber, and B. M. Bebout. 2015. “Revisiting N2 Fixation in Guerrero Negro Intertidal Microbial Mats with a Functional Single-Cell Approach,” The ISME Journal 9, 485–96. DOI: 10.1038/ismej.2014.144. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 29, 2014

Tracking the Evolution of a Methane-Producing Symbiosis in Real Time

Just below the surface of soils and sediments, large portions of Earth’s biosphere exist in the absence of oxygen. The microbial inhabitants of these anoxic environments drive planetary biogeochemical cycles, and their metabolic activities impact the bioavailability of nutrients, metals, and environmental contaminants. To survive in these energy-limited habitats, many microbial species have evolved collaborative symbiotic lifestyles that allow two organisms to perform metabolic processes that neither would be capable of independently (i.e., “mutualistic syntrophy”). In a new study by Lawrence Berkeley National Laboratory scientists, an experimental evolutionary system was constructed that pairs a common sulfate-reducing bacterium, Desulfovibrio vulgaris, with a methane-producing archaea, Methanococcus maripaludis, neither of which is known to grow via mutualistic syntrophy in nature. Experimental conditions were manipulated so that neither organism would have access to an energy source it could use independently. In 21 independent experiments over 1,000 generations, mutualistic syntrophies that closely resembled associations observed in nature evolved between the two organisms 13 times. In these syntrophies, consumption of lactate (a common product of fermentation in anoxic environments) by D. vulgaris provided hydrogen and carbon dioxide to M. maripaludis, which, in turn, produced methane and maintained an energetic environment favorable to continued consumption of lactate by D. vulgaris. The partners quickly improved their performance efficiency for coupled syntrophic growth, but in many cases, D. vulgaris lost its ability to grow in the absence of M. maripaludis even under normal growth conditions. By sequencing the genomes of the evolved strains from the various experimental replicates, it was determined that D. vulgaris quickly accumulated loss of function mutations, particularly in three key sulfate reduction genes needed for independent growth. The team currently is examining the relationship between the loss of capacity for independent growth and improved symbiotic performance. These results provide a fascinating glimpse at the molecular underpinnings of a natural selection process and demonstrate the importance of tradeoffs between growth efficiency and metabolic flexibility during the evolution of a symbiotic partnership. In the broader sense, understanding the molecular factors governing the formation of these associations and their performance under changing environmental conditions could provide valuable new insights into the way that carbon and energy flow through anoxic environments.

Reference: Hillesland, K. L., S. Lim, J. Flowers, S. Turkarslan, N. Pinel, G. Zane, N. Elliott, Y. Qin, L. Wu, N. Baliga, J. Zhou, J. Wall, and D. Stahl. 2014. “Erosion of Functional Independence Early in the Evolution of a Microbial Mutualism,” Proceedings of the National Academy of Sciences (USA) 111(41), 14822-827. DOI: 10.1073/pnas.1407986111. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 22, 2014

Integration of Carbon, Sulfur, and Iron Cycling in Anaerobic Methane Oxidation

Coastal wetlands and ocean sediments are significant sites of methane (CH4) production, either through decomposition of organic material or via natural seepage from deeper geological reservoirs. These environments are home to unique microbial communities capable of converting CH4 to carbon dioxide (CO2) even in the absence of oxygen, which does not penetrate below the top few centimeters of sediment. No individual microbe or microbial species can generate enough energy to survive using this mode of metabolism. However, symbiotic partnerships between methane-consuming archaea and sulfate-reducing bacteria thrive using a collaborative metabolism called anaerobic oxidation of methane (AOM). In this mode of growth, electrons freed during CH4 oxidation by archaea are transferred to sulfate (SO4) by the bacterial partner, generating energy for both organisms. Since this process results in the conversion of up to 90% of available CH4 to CO2 (a much less potent greenhouse gas) in some environments, studying its mechanistic basis and the organisms performing it could have major implications for understanding the global carbon cycle and potential climate change impacts. Researchers at the California Institute of Technology and partner institutions in the United Kingdom and Israel have uncovered new evidence of a significant role for iron minerals in accelerating the rates of AOM processes. Sediments with higher levels of iron oxides had decreased rates of methane release and increases in AOM processes. By using a series of microcosm experiments and carefully tracking conversion of isotopically labeled CH4 and SO4 in the presence of varying concentrations of the iron mineral hematite, the team determined that the presence of iron oxides stimulated bacterial sulfate reduction, facilitating recycling of reduced sulfur compounds back to SO4, and driving increased rates of methane consumption by archaea. These findings reveal new biological linkages in the biogeochemical cycling of carbon, sulfur, and iron and will have important implications in predicting the contribution of AOM processes to the global carbon cycle.

Reference: Sivan, O., G. Antler, A. V. Turchyn, J. J. Marlow, and V. J. Orphan. 2014. “Iron Oxides Stimulate Sulfate-Driven Anaerobic Methane Oxidation in Seeps,” Proceedings of the National Academy of Sciences (USA), DOI:10.1073/pnas.1412269111. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 20, 2014

Identification of Two Key Enzymes in Xylan Synthesis and Acetylation in Plant Cell Walls

Only a few dozen of the thousands of genes involved plant cell wall biosynthesis have been identified and confirmed. Xylan, a part of hemicellulose, is a major component of plant cell walls and the third most abundant polysaccharide on Earth. The key enzymes responsible for elongation of the xylan backbone and addition of acetyl groups had not been identified, but researchers from the BioEnergy Science Center of Oak Ridge National Laboratory recently identified two key enzymes for the synthesis of this polysaccharide and confirmed their function biochemically. Mutations that impair synthesis of the xylan backbone give rise to plants with collapsed xylem cells and poor growth. Phenotypic analysis of these mutants has implicated many possible proteins in xylan biosynthesis. To further investigate the role of the mutant genes in xylan biosynthesis, recombinant tagged proteins encoded by the Arabidopsis thaliana genes, IRX10-L and ESK1/TBL29, were expressed in vitro and purified. Enzymatic activity of these proteins was inferred from the similarity of their primary amino acid sequence to enzymes of known function. Their enzyme activity was analyzed in vitro by mass spectroscopy and nuclear magnetic resonance. This direct biochemical evidence confirmed the A. thaliana protein IRX10-L enzyme as the xylan synthase and ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase. Thus, two key enzymes for two critical process in xylan (and secondary plant cell wall) synthesis now have been identified, purified, and confirmed. These findings will accelerate understanding of and the ability to manipulate plant cell wall structures for advanced renewable feedstocks for conversion into sugars and fuels or into valuable products such as biomaterials.

Reference: Urbanowicz, B. R., M. J. Peña, H. A. Moniz, K. W. Moremen, and W. S. York. 2014. “Two Arabidopsis Proteins Synthesize Acetylated Xylan In Vitro,” The Plant Journal 80(2), 197-206. DOI:10.1111/tpj.12643. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 24, 2014

Identifying Representative Corn Rotation Patterns in the U.S. Western Corn Belt

To accurately assess the impacts of biofuel crop production on regional ecosystem services such as crop yields, carbon and nutrient cycling, soil erosion, water quality, and pest and disease control, it is necessary to have an accurate picture of which crop rotation systems are utilized by growers. Despite the availability of databases such as the Cropland Data Layer (CDL), which provide remotely sensed data on U.S. crop types on a yearly basis, crop rotation patterns remain poorly mapped due to the lack of tools that allow for efficient and consistent analysis of multiyear CDLs. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center created an algorithm that can select representative crop rotation systems by combining and analyzing multiyear CDLs. Among the findings using this algorithm is that only 82 representative crop rotations accounted for over 90% of the spatiotemporal variability of the more than 13,000 rotations in the Western Corn Belt; it also can detect pronounced shifts from grassland to monoculture corn and soybean cultivation. Furthermore, the area estimates of the rotation systems are comparable to those obtained from agricultural census data. Given this algorithm’s novel capability to flexibly and efficiently derive representative crop rotation patterns in a spatially and temporally explicit manner, it is expected to be a useful tool for providing input data to drive agro-ecosystem models and for detecting shifts in cropping patterns in response to environmental and socio-economic changes.

Reference: Sahajpal, R., X. Zhang, R. C. Izaurralde, I. Gelfand, and G. C. Hurtt. 2014.   “Identifying Representative Crop Rotation Patterns and Grassland Loss in the U.S. Western Corn Belt,” Computers and Electronics in Agriculture 108, 173–82. DOI: 10.1016/j.compag.2014.08.005. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 21, 2014

New Technologies Facilitate Investigation of Wood Formation

Woody plants are an important source of renewable biomass for bioenergy feedstocks. Wood formation is a complex, highly regulated process generating key sources of material for bioenergy and bioproducts. Understanding the gene regulatory networks underlying wood formation would facilitate efforts to develop higher biomass yielding, sustainable trees as bioenergy feedstocks. However, the nature of woody material makes it recalcitrant to genetic manipulation, presenting a significant challenge. Researchers funded by the Department of Energy’s Genomic Science program report the development of two new methods optimized for woody material and expediting molecular genetic approaches for investigating wood formation in Populus trichocarpa, a model woody plant and bioenergy feedstock. They detail systematic and extensive modification of the chromatin immunoprecipitation (ChIP) procedure, widely used to identify chromatin-associated DNA-protein interactions in nonwoody plants and animals, making it usable for the first time with wood-forming tissues. Using this new protocol, the researchers identified genome-wide specific transcription factor-DNA interactions associated with the regulation of wood formation. They also describe a new higher-yielding and faster method for the isolation and transfection of high-quality protoplasts from P. trichocarpa wood-forming tissue. Protoplasts are useful for transient transgene expression-based studies, particularly for woody plants that are difficult to genetically transform and for which mutants are unavailable. Both methods should be broadly applicable to other woody species, enabling comparative analyses of the evolution of the genetic regulation and epigenetic modifications of wood formation. These advances will facilitate essential genome-wide studies of wood formation and biomass productivity in woody feedstocks.

References: Li, W., Y.-C. Lin, Q. Li, R. Shi, C.-Y. Lin, H. Chen, L. Chuang, G.-Z. Qu, R. R. Sederoff, and V. L. Chiang. 2014. “A Robust Chromatin Immunoprecipitation Protocol for Studying Transcription Factor-DNA Interactions and Histone Modifications in Wood-Forming Tissue,” Nature Protocols 9(9), 2180-93. DOI:10.1038/nprot.2014.146. (Reference link)

Lin, Y.-C., W. Li, H. Chen, Q. Li, Y.-H. Sun, R. Shi, C.-Y. Lin, J. P. Wang, H.-C. Chen, L. Chuang, G.-Z. Qu, R. R. Sederoff, and V. L. Chiang. 2014. “A Simple Improved-Throughput Xylem Protoplast System for Studying Wood Formation,” Nature Protocols 9(9), 2194-2205. DOI:10.1038/nprot.2014.147. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 18, 2014

Ionic Liquids Provide Effective Biomass Pretreatment

Ionic liquids (ILs) have been shown to be an excellent pretreatment solvent for biomass in preparation for hydrolysis into its component sugars. However, IL availability and high cost remain an issue. Researchers from the Department of Energy’s Joint BioEnergy Institute sought to decrease the cost of ILs by synthesizing new ILs directly from lignin monomers and hemicellulose, which are found in the biomass. Tertiary amine-based ILs were synthesized from aromatic aldehydes derived from lignin and hemicellulose. Molecular modeling was used to compare IL solvent parameters with experimentally obtained compositional analysis data.

Effective pretreatment using these new ILs of switchgrass was investigated by powder X-ray diffraction showing structural changes in cellulose and glycome profiling showing changes in the extractability of hemicellulose epitopes. Deriving ILs from lignocellulosic biomass shows significant potential for the realization of a “closed-loop” process for future lignocellulosic biorefineries and has far-reaching economic impacts for other IL-based conversion technology currently using ILs synthesized from petroleum sources. IL synthesis by reductive animation of aromatic aldehydes, followed by treatment with phosphoric acid, provided three biomass-derived ILs in excellent yields without the need for chromatographic purification. When these renewable biomass-derived ILs were used in pretreatment of switchgrass biomass, comparable high yields of sugar were generated and saccharification was comparable to current imidazolium-based ILs. Cost projections of renewable ILs are $4/kg, much lower than top performing conventional ILs, improving the economic viability of lignocellulosic-derived sugars.

Reference: Socha, A. M., R. Parthasarathia, J. Shia, S. Pattathil, D. Whyte, M. Bergeron, A. George, K. Tran, V. Stavila, S. Venkatachalam, M. G. Hahn, B. A. Simmons, and S. Singh. 2014. “Efficient Biomass Pretreatment Using Ionic Liquids Derived from Lignin and Hemicellulose,” Proceedings of the National Academy of Sciences (USA) 111(35), E3587-95. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 06, 2014

Evolution of Substrate Specificity in Bacterial Lytic Polysaccharide Monooxygenases

Cellulose is one of the most abundant polysaccharides in nature and one of the primary components of plant cell walls. The biofuels industry has devoted significant efforts to establish processes to convert these energy-rich molecules into sugars that can be fermented into biofuels or other bioproducts. However, the hydrolysis of these polysaccharides, a key step in converting them to biofuels, is difficult due to their crystalline structure, the stability of some bonds within their structure, and how closely they are associated with structure-modifying molecules such as hemicellulose and lignin. Efficient hydrolysis requires a cocktail of different enzymes. Enzymes capable of hydrolyzing these polymers have been identified in various organisms, especially bacteria and fungi, but the pathways for deconstruction of certain polysaccharides, such as cellulose and chitin, are only partially understood. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center analyzed the sequences, structures, and evolution of two families of enzymes, fungal AA9 and bacterial AA10, both lytic polysaccharide monooxygenases (LPMOs), to understand the factors that influence substrate specificity in these families and to characterize the selective pressures that may have led to their functional diversification. Their sequence similarity suggests that both families share a distant common ancestor and that certain clades within the AA10 family are specialized for different substrates, while others went through a diversifying selection at surface-exposed regions of the protein. Understanding the diversity of these lignocellulosic-degrading enzymes in nature provides information that can help improve enzymatic cocktails used in the biofuels industry.

Reference: Book, A. J., R. M. Yennamalli, T. E. Takasuka, C. R. Currie, G. N. Phillips, and B. G. Fox. 2014. “Evolution of Substrate Specificity in Bacterial AA10 Lytic Polysaccharide Monooxygenases,” Biotechnology for Biofuels 7,109. DOI: 10.1186/1754-6834-7-109. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 25, 2014

Role of Post-Translational Protein Modification in Community-Scale Processes

Although biological processes are often modulated by the direct regulation of gene expression, post-translational modifications (PTM) of expressed proteins frequently play an equally important regulatory role. PTM occurs when protein function is altered by the addition of a phosphate, acetate, or other small molecule in response to a sensed environmental cue. These alterations create rippling signal cascades, often leading to pervasive changes in cellular metabolism and functional properties. PTM-based regulation has been extensively studied in individual organisms, but the role of this regulatory mechanism at the scale of complex communities remains poorly understood. In a new study, a collaborative team of researchers at the University of California, Berkeley, and Oak Ridge National Laboratory developed a novel technique that allows PTM analysis in proteins collected from an intact microbial community (i.e., the metaproteome) using high-resolution mass spectrometry coupled to high-performance computing. The investigators examined PTM in a model biofilm community found in a highly acidic environment and were able to link this regulatory mechanism to several community-scale phenotypes that could not be explained by observed changes in gene expression. Community-level attributes associated with PTM in this study included alterations in community structure, nutrient acquisition strategies, and resistance to viral invasion. This finding represents a considerable advance in the application of systems biology approaches to community-level analysis. The team now is working to scale up this technique to enable investigations of more complex communities and environments.
Reference: Li, Z., Y. Wang, Q. Yao, N. B. Justice, T.-H. Ahn, D. Xu, R. L. Hettich, J. F. Banfield, and C. Pan. 2014. “Diverse and Divergent Protein Post Translational Modifications in Two Growth Stages of a Natural Microbial Community,” Nature Communications 5, 4405. DOI: 10.1038/ncomms5405. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 20, 2014

Characterization of Poplar Budbreak Gene Enhances Understanding of Spring Regrowth

Trees in temperate climates undergo annual cycles of growth and dormancy corresponding to summer and winter seasons, a critical strategy that allows perennial plants to survive cold and dehydration during the winter months. These important transitions are controlled by photoperiod and temperature, but the exact mechanisms by which key physiological processes are initiated are still poorly understood. Researchers at Michigan Technological University and Oregon State University have identified and functionally characterized a gene in the bioenergy feedstock tree Populus called Early Bud-Break 1 (EBB1). EBB1 serves as a “master regulator” in the timing of spring growth reactivation, or budbreak. In addition, the protein encoded by EBB1 was found to function in many other processes critical to poplar survival, including nutrient cycling and root growth. These results enhance understanding of dormancy release in woody perennial plants and will enable new approaches for breeding trees better adapted to changing environments such as a warmer climate. The research was supported by the U.S. Department of Agriculture-Department of Energy Plant Feedstock Genomics for Bioenergy Program. (Reference link)

Reference: Yordanov, Y. S., C. Ma, S. H. Strauss, and V. G. Busov. 2014. “Early Bud-Break 1 (EBB1) is a Regulator of Release from Seasonal Dormancy in Poplar Trees,” Proceedings of the National Academy of Sciences (USA) 111(27), 10,001-10,006. DOI: 10.1073/pnas.1405621111. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 02, 2014

Ethanol Produced from Switchgrass Biomass Without Pretreatment

One strategy for reducing costs associated with biomass deconstruction and fermentation of sugars to biomass into advanced biofuels is consolidated bioprocessing (CBP). In CBP, non-pretreated biomass is converted to a biofuel in a single process by a cellulolytic microbe that breaks down the biomass and ferments the sugars. U.S. Department of Energy BioEnergy Research Center (BESC) scientists have been working toward CBP by looking at a variety of thermophilic cellulolytic bacteria. A candidate CBP microbe is Caldicellulosiruptor bescii, a natural thermophilic cellulolytic bacterium for which BESC researchers have developed genetic tools for gene insertion and deletion. In this study, BESC researchers demonstrate the successful CBP of switchgrass cellulosic biomass using an engineered strain of C. bescii.

C. bescii had been shown to ferment untreated switchgrass biomass, but it lacked the genes to make ethanol. As C. bescii is a thermophile and CBP is carried out at elevated temperatures, a gene for a heat-stable enzyme for ethanol synthesis was needed. A candidate gene was identified in Clostridium thermocellum and cloned into C. bescii. The engineered C. bescii strain now produced ethanol from cellobiose, Avicel, and switchgrass. To optimize the fermentation of ethanol, two genes were deleted that would otherwise divert fermentation products. In this new C. bescii strain, roughly 30% of biomass was fermented and 1.7 moles of ethanol was produced for each mole of glucose, close to the theoretical 2.0 moles of ethanol per mole of glucose. While there are opportunities to further improve efficiencies, this is an important step in actualizing the CBP’s potential and provides a platform for engineering the production of advanced biofuels and other bioproducts directly from cellulosic biomass without harsh and expensive pretreatment.

Reference: Chung, D., M. Cha, A. M. Guss, and J. Westpheling. 2014. “Direct Conversion of Plant Biomass to Ethanol by Engineered Caldicellulosiruptor bescii,” Proceedings of the National Academy of Sciences (USA) 111, 8931–36. DOI:10.1073/pnas.1402210111. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 01, 2014

Evolution of Potential Energy Grass Genome Structure

The Saccharinae group of grasses contains two members that are potentially important sources of sugar and lignocellulosic biomass for bioenergy, due at least in part to highly efficient C4 photosynthesis. These grasses are the warm temperate-tropical sugarcane (Saccharum officinarum) and Miscanthus spp., which can yield high levels of biomass at temperate latitudes. A close relative is sorghum (Sorghum bicolor), also grown as a bioenergy feedstock in addition to its use as food and feed. In contrast to sorghum, the Saccharinae grasses are known for polyploidy and possess high chromosome numbers, offering an opportunity to investigate the evolutionary processes of genome duplication, genome structure, and the implications for crop improvement strategies. Researchers funded by the joint U.S. Department of Agriculture-Department of Energy Plant Feedstock Genomics for Bioenergy program have applied genome sequencing and global comparative analyses of Miscanthus, Saccharum, and sorghum to gain insight into the different evolutionary fates of Miscanthus and Saccharum after they diverged from sorghum. The researchers report evidence for the existence of a genome duplication shared between Saccharum and Miscanthus as well as an additional Saccharum-specific duplication event. Understanding the genome structure of these two complex grasses in relation to the closely related and fully sequenced sorghum genome will facilitate breeding efforts to improve bioenergy-relevant traits such as biomass yield and adaptation to changing environments.

Reference: Kim, C., X. Wang, T.-H. Lee, K. Jakob, G.-J. Lee, and A. H. Paterson. 2014. “Comparative Analysis of Miscanthus and Saccharum Reveals a Shared Whole-Genome Duplication but Different Evolutionary Fates,” Plant Cell 26, 2420-29. DOI:10.1105/tpc.114.125583. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 01, 2014

Poplar Tree Root Response to Symbiotic Fungus Determines Success of Fungal Colonization

Microbial communities sharing the soil environment with plant roots can have a pro­found influence on the overall health and vitality of the plant. One well-known example of a beneficial relationship is that formed between forest trees and shrubs and a type of mutualistic fungi known as ectomycorrhizal fungi (ECM). In a compatible reaction, ECM facilitate the plant’s access to nutrients and increase its tolerance to biotic and abiotic stress through formation of an “organ” between fungal hyphae and plant roots called the ECM root tip. However, little is known about the metabolic reprogramming that leads to the development of this hybrid tissue. Researchers at Oak Ridge National Laboratory, funded through the Department of Energy’s Plant-Microbe Interfaces Science Focus Area, characterized the metabolic changes taking place during the interaction between the ECM fungus Laccaria bicolor and two different species of the bioenergy feedstock tree Populus. They found that when P. trichocarpa is colonized by the fungus shifts occurred in aromatic acid, organic acid, and fatty acid metabolism. On the contrary, this metabolic reprogramming was repressed in the incompatible P. deltoides interaction, which was instead characterized by the production of more defense-related secondary metabolites. The results highlight distinct differences in mechanisms control­ling compatibility between beneficial and nonbeneficial inter­actions, and increase under­standing of how plant roots respond to the presence of L. bicolor, which determines the out­come of the fungal-host interaction.

Reference: Tschaplinski, T. J., J. M. Plett, N. L. Engle, A. Deveau, K. C. Cushman, M. Z. Martin, M. J. Doktycz, G. A. Tuskan, A. Brun, A. Kohler, and M. Martin. 2014. “Populus trichocarpa and Populus deltoides Exhibit Different Metabolomic Responses to Colonization by the Symbiotic Fungus Laccaria bicolor,” Molecular Plant-Microbe Interactions 27(6), 546-56. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 23, 2014

Microbes Disprove Long-Held Assumption that All Organisms Share a Common Vocabulary

Four letters—A, C, G, and T—make up the DNA bases in all organisms on Earth. The particular order, or sequence, of these same four letters genetically defines an organism and is a main reason that determining the genome sequence is now a foundational starting point for many biological investigations. Within this sequence are shorter, three-letter groups called codons that represent amino acids, the building blocks of proteins that carry out the myriad functions critical to life and biology. There are 64 of these codons and, routinely, 61 of them code for the 20 known amino acids. Three of these triplets function as stop signals and are used to mark the end of protein generation. Given that all organisms have genomes built on the same four letters, scientists had long assumed that they also all shared the same vocabulary and the 64 codons would be interpreted the same way across the board. However, a recent study from the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI) shows that for some organisms the instructions for these three codons mean anything but stop. The researchers focused on uncultivated microbes, whose genomes had been described through single-cell genomics and metagenomics, and on a collection of viral sequences. Nearly six trillion bases of sequence data were analyzed from 1,776 samples collected from the human body and several sites around the world. The study found that these stop codons often were reassigned to code for amino acids. This work builds on a previous study in which DOE JGI researchers successfully employed single-cell genomics to shed insight on a plethora of microbes representing 29 “mostly uncharted” branches on the tree of life.

Reference: Ivanova, N., et al. 2014. “Stop Codon Reassignments in the Wild,” Science 344, 909–13. DOI:10.1126/science.1250691. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 20, 2014

Developing Synthetic Microbial Communities to Improve Predictions of Their Behavior

Microbial communities populate every natural environment, playing critical roles in fundamen­tal biological and environmental processes such as food webs and carbon cycling. Members of microbial communities interact with each other both as competitors and collabora­tors. Understanding the complex interactions within these communities is necessary to predict and eventually manipulate their behavior for biotechnol­ogy applica­tions. Studying natural microbial consortia is extremely challenging, so simple microbial cocultures are often used to gain insights on microbial crossfeeding and communication. However, such studies rarely represent natural systems, and, therefore, more complex synthetic microbial communities are needed to model the development and evolution of microbial populations. Researchers at Harvard and Columbia universities report the development of a system of synthetic microbial communities composed of up to 14 Escherichia coli mutants, each one incapable of synthesizing a different amino acid. Using this system, the investigators could experimentally determine the behavior of the different members of the consortium, identifying mutants that act as keystone species or that promote positive or negative interactions. After several generations, these bacterial populations tend to become enriched in mutants that cannot produce metabolically costly amino acids (those that require more energy to synthesize). The authors hypothesize that such mutants persist in the population by crossfeeding from less abundant microbes that provide needed amino acids. This hypothesis was supported by the observation that the majority of the microorganisms whose genomes have been sequenced do not have the metabolic capacity to produce costly amino acids. These results will enable develop­ment of more accurate predictive models of microbial communities and their iterative improve­ment by experimentation, advancing toward a more comprehensive understanding of microbial communities such as those involved in carbon cycling.   

Reference: Meea, M. T., J. J. Collins, G. M. Church, and H. H. Wang. 2014. “Syntrophic Exchange in Synthetic Microbial Communities,” Proceedings of the National Academy of Sciences (USA) 111(20), E2149-56. DOI:10.1073/pnas.1405641111. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 20, 2014

Fungal Protein Allows Beneficial Colonization in Populus

The soil environment surrounding plant roots is filled with bacteria and fungi, both harmful and beneficial, many of which attempt to colonize root tissues to gain access to and use plant nutrients. In response, plant hormones such as jasmonic acid (JA) mediate the plant’s defense signaling system. By altering this pathway, some microorganisms can gain entry into the plant root cells and promote colonization. Investigating the symbiotic relationship between the bioenergy feedstock tree Populus trichocarpa and the beneficial fungus Laccaria bicolor, researchers at Oak Ridge National Laboratory found that a fungal protein essential for root establishment (called MiSSP7; Mycorrhiza-induced Small Secreted Protein 7) interacts with a plant-produced protein within the host plant nuclei to promote symbiosis. While both pathogenic and mutualistic fungi use fungal “effector” proteins to facilitate colonization, the results suggest how the mechanisms used to overcome the plant’s defenses differ between these two types of organisms, furthering understanding of how L. bicolor alters the plant’s response to JA and allows formation of symbiotic relationships.

Reference: Plett, J. M., Y. Daguerre, S. Wittulsky, A. Vayssières, A. Deveau, S. J. Melton, A. Kohler, J. L. Morrell-Falvey, A. Brun, C. Veneault-Fourrey, and F. Martin. 2014. “Effector MiSSP7 of the Mutualistic Fungus Laccaria bicolor Stabilizes the Populus JAZ6 Protein and Represses Jasmonic Acid (JA) Responsive Genes,” Proceedings of the National Academy of Sciences (USA) 111(22), 8299-304. DOI: 10.1073/pnas.1322671111. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 21, 2014

Engineered Switchgrass Shows Increased Ethanol Production During 2-Year Field Trial

A major assumption in much plant-focused bioenergy research is that key plant cell wall traits can be genetically manipulated to reduce recalcitrance and increase biofuel yields per unit of biomass. A number of greenhouse experiments have shown promise, but few field studies have been completed to assess this assumption. Researchers at the BioEnergy Science Center (BESC) are the first to report a field study evaluating the biofuel potential of genetically engineered switchgrass (Panicum virgatum L.). BESC researchers previously had used RNAi (inhibitory RNA) to down-regulate caffeic acid O-methyltransferase (COMT), a key enzyme in the synthesis of lignin precursors. Switchgrass plants engineered in this way and grown in the greenhouse had less lignin and a shift in the quality of lignin to a more hydrolysable form. These plants showed less recalcitrance and a greater percentage of cell wall sugars being converted to ethanol than control plants. However, greenhouse results do not always replicate in the field, so researchers were anxious to learn if COMT-engineered switchgrass would show reduced recalcitrance and increased ethanol production when grown in the field.      

The 2-year field trial in large part recapitulated the greenhouse results. Namely, the transgenic switchgrass plants had a reduction in the quantity of lignin and a shift in the quality of lignin. A greater percentage of the cell wall sugars were released with pretreatment, and ethanol yield increased by as much as 28% in the transgenic lines relative to controls. These results were with senescent tissues, whereas the greenhouse studies had only looked at green tissues. Importantly for agronomic applications, the transgenic plants were not more susceptible to rust (Puccinia emaculata) or other plant pests. This important 2-year field study affirms genetic engineering of the plant cell wall as a viable strategy to improve plant biomass for the production of high-energy biofuels.  

Reference: Baxter, H. L.; M. Mazarei; N. Labbe; L. M. Kline; Q. Cheng; M. T. Windham; D. G. J. Mann; C. Fu; A.  Ziebell; R. W. Sykes; M. Rodriguez, Jr.; M. F. Davis; J. R. Mielenz; R. A. Dixon; Z. W. Wang; and C. N. Stewart, Jr. 2014. “Two-Year Field Analysis of Reduced Recalcitrance Transgenic Switchgrass,” Plant Biotechnology Journal 1–11. DOI:10.1111/pbi.12195. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 04, 2014

Engineered Poplar Lignin Improves Wood Degradability

Lignin is an irregular phenolic plant cell wall polymer that is integral to plant strength and function. It is important in bioprocessing of plant biomass because it inhibits deconstruction of plant cell wall sugar polymers, such as cellulose and hemicellulose, into sugar monomers, a key step in biofuel production. The irregular structure and types of linkages among the phenolic monolignol precursors contribute to lignin’s recalcitrance to cleavage and hydrolysis. Interestingly, the enzymes that polymerize lignin are known to be promiscuous and can incorporate nonstandard monolignols if alternate precursors are supplied. Exploiting this promiscuity to construct a lignin more amenable to hydrolysis, Great Lakes Bioenergy Research Center (GLBRC) researchers genetically engineered poplar—an attractive biofuels feedstock—to biosynthesize ferulate conjugated monolignols in the developing cell wall of plant tissues that contain significant amounts of lignin. The ferulate monolignols are of particular interest because they form ester bonds in lignin that are more hydrolysable than the typical ether bonds that normally connect lignin monolignols. The modified lignin altered the amount of sugars released from the poplar cell walls, and the researchers found that mild alkaline pretreatment released as much as double the glucose compared to the unmodified poplar lignin. These studies demonstrate the usefulness of modifying plant lignin as a means to simplify and improve processing of plant biomass and increasing sugar yields from plant biomass for biofuel production. These improvements are important advances in overcoming the technical barriers to an economically viable and sustainable biofuels industry.

Reference: Wilkerson, C. G., S. D. Mansfield, F. Lu, S. Withers, J.-Y. Park, S. D. Karlen, E. Gonzales-Vigil, D. Padmakshan, F. Unda, J. Rencoret, and J. Ralph. 2014. “Monolignol Ferulate Transferase Introduces Chemically Labile Linkages into the Lignin Backbone,” Science 344, 90–93. DOI:10.1126/science.1250161. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 26, 2014

Engineering Escherichia coli to Tolerate Ionic Liquids for Biofuel Production

Ionic liquids (IL) are a class of environmentally friendly solvents that are effective at loosening cellulose from lignin in plant biomass. This is an important step in the production of biofuels as it makes cellulose available for breakdown into its component sugars. The sugars are fermented into biofuels by microbes such as Escherichia coli. While most of the IL is recovered from the processed biomass, some remains and can inhibit the growth of E. coli and the enzymes that convert cellulose into biofuel, greatly reducing yields of biofuel product. To address this inhibition, scientists at the U.S. Department of Energy’s Joint BioEnergy Institute (JBEI) looked for genes that might confer tolerance on the E. coli to the ILs. They looked to Enterobacter lignolyticus, a bacterium known to grow in the presence of ILs. First, they moved large parts of the E. lignolyticus genome into E. coli and asked the E. coli to grow in the presence of the IL. Several colonies were found to now tolerate the IL; each colony had two E. lignolyticus genes in common, an efflux pump gene and its regulator. Efflux pumps confer tolerances by transporting toxic compounds out of the cell into the medium. To determine if the tolerance conferring efflux pump could improve biofuel synthesis in the presence of IL, the efflux pump genes were placed together in a strain of E. coli engineered to produce a biofuel precursor, bisabolene. The resulting strain was able to produce more bisabolene in the presence of much greater amounts of IL than the E. coli strain without the efflux pump. An E. coli strain that tolerates ILs and synthesizes bisabolene means that ILs can be used to treat biomass to free cellulose from lignin without negatively impacting subsequent biofuel production. This can reduce biofuel production costs because extra expense is not needed to remove the last amounts of IL from the processed biomass. As cellulosic biofuel production plants come online, such adaption of technological advances like these that will improve their economic viability.

Reference: Rüegg, T. L., E.-M. Kim, B. A. Simmons, J. D. Keasling, S. W. Singer, T. S. Lee, and M. P. Thelen. 2014. “An Auto-Inducible Mechanism for Ionic Liquid Resistance in Microbial Biofuel Production,” Nature Communications 5. DOI:10.1038/ncomms4490. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 20, 2014

Microbes in Antarctic Lake Divvy Up the Waters

Four microbes, recently sequenced at the U.S. Department of Energy’s Joint Genome Institute, dominate in Antarctica’s Deep Lake, making up 70% of the microbial community. They belong to a group called haloarchaea, which require high salt concentrations to grow and are naturally adapted to extreme conditions that would prove lethally cold to other organisms. In a recent study, researchers found that three of the four haloarchaea are adapted to niche environments within the lake. The most abundant of the four, strain tADL (44% of the lake community), has genes for light harvesting and gas vesicles that help it float near the light-rich surface. The second most abundant haloarchaea, strain DL31 (18% of the community), appears to be adept at metabolizing proteins and peptides. H. lacusprofundi (10% of the lake community)appears to be a more versatile generalist that can feed on a variety of nutrients. The least abundant, strain DL1 (0.3% of the lake community), shows a taste for amino acids and is the only one without genes for using glycerol as a nutrient. The next step is to use metaproteomics (study of proteins in an environmental sample) to investigate whether protein abundance in Deep Lake supports the research team’s hypothesis about niche specialization. Understanding how haloarchaea thrive in extreme polar niches could be used to improve the role of microbes in contaminated site cleanup in permanently or seasonally cold regions. Also, the genes that allow them to adapt to select conditions can be re-tooled for use in industrial or environmental remediation settings.

Reference: Williams, T. J., et al. 2014. “Microbial Ecology of an Antarctic Hypersaline Lake: Genomic Assessment of Ecophysiology Among Dominant Haloarchaea,” The ISME Journal 8, 1645-58. DOI:10.1038/ismej.2014.18. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 19, 2014

Floating Water Weed Could Be Used as Biofuels Feedstock

Duckweed is one of the world’s smallest and fastest-growing flowering plants and can be a hard-to-control weed in ponds and small lakes. It shows great promise as a biofuel feedstock, however, and private companies are already exploring its use in fuel production. Researchers at Rutgers University, the Department of Energy’s Joint Genome Institute, and several other facilities recently sequenced the complete genome of Greater Duckweed (Spirodela polyrhiza) and analyzed it in comparison with several other plants, including rice and tomato. S. polyrhiza’s very small genome is missing many genes for plant maturation and production of cellulose and lignin but has more genes than comparable plants for starch production. Determining which genes produce desirable traits will allow researchers to create new varieties of duckweed with enhanced biofuel traits.

Reference: Wang, W., et al. 2014. “The Spirodela polyrhiza Genome Reveals Insights into Its Neotenous Reduction Fast Growth and Aquatic Lifestyle,” Nature Communications 5, 3311. DOI: 10.1038/ncomms4311. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742, John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Comparison of Fronds and Turions. Duckweed is a relatively simple plant with fronds that float on the surface and roots that extend into the water (right). In the flask on the left, dormant phase turions have dropped to the bottom. [Image credit: Rutgers University]



February 18, 2014

Understanding Ecological Forces Governing Assembly and Function of Microbial Communities

A complex, dynamic, and interactive set of ecological forces governs the assembly of a microbial community in any given environment. The composition and structure of the resulting community in turn controls functional biological processes performed at the site, influencing biogeochemical cycling of nutrients, transport of contaminants, and interactions with other organisms. As such, understanding the rules that govern assembly and successional change of microbial communities in different types of environments is critical to predicting changes in ecosystem-scale processes under changing environmental conditions. In a new study by Lawrence Berkeley National Laboratory’s ENIGMA Science Focus Area, researchers examined mechanisms driving microbial community assembly and succession in an experimentally manipulated groundwater ecosystem. The team tested a set of theoretical models to compare the relative importance of stochastic (i.e., random) and deterministic processes in shaping community structure after an environmental change (in this case, the addition of nutrients). Community assembly and succession were found to be driven by a dynamic, time-dependent interaction of stochastic and deterministic processes, with stochastic forces dominating. By identifying the mechanisms controlling microbial community assembly and succession, this study makes an important contribution to the mechanistic understanding essential for a predictive microbial ecology of natural and managed ecosystems.

Reference: Zhou, J., Y. Deng, P. Zhang, K. Xue, Y. Liang, J. D. Van Nostrand, Y. Yang, Z. He, L. Wu, D. A. Stahl, T. C. Hazen, J. M. Tiedje, and A. P. Arkin. 2014. “Stochasticity, Succession, and Environmental Perturbations in a Fluidic Ecosystem,” Proceedings of the National Academy of Sciences (USA), DOI: 10.1073/pnas.1324044111. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 14, 2014

Novel Methanogenic Microbe Discovered in Thawing Permafrost

Northern high-latitude ecosystems are undergoing rapid changes with rising temperatures catalyzing the transition of many permafrost sites to wetlands. As the organic carbon locked in permafrost thaws, it becomes accessible to decomposition by microbial communities. Understanding of these communities is limited, especially regarding functional processes that impact rates of carbon degradation and the balance of carbon dioxide (CO2) versus methane (CH4) released to the atmosphere. In a new U.S. Department of Energy Genomic Science Program study led by researchers at the University of Arizona, a combination of metagenomics, metaproteomics, and geochemical flux measurements were used to characterize microbial community structure and function at a thawing permafrost site in northern Sweden. A new species of archaea, Candidatus Methanoflorens stordalenmirensis, was found to dominate methanogen populations in the thawing active layer of permafrost. Using deep metagenomic sequencing, the team was able to assemble a nearly complete genome from this organism and identify the metabolic pathway for methanogenesis—consumption of hydrogen and CO2 and production of CH4. Measurements of CH4 flux at the thawing permafrost site and quantitative in situ detection of M. stordalenmirensis methanogensis proteins suggest that this organism may perform the majority of methane production at these sites, especially during thawing. The team also searched published metagenomic libraries collected from permafrost sites across the northern hemisphere and detected closely related methanogens at high numbers in the majority of sites. The dominance of a single organism in methane production is a surprising finding. Given evidence for the global distribution of this type methanogen in thawing permafrost sites, these results may have wide-ranging implications for understanding of climate change impacts.

Reference: Mondav, R., B. J. Woodcroft, E.-H. Kim, C. K. McCalley, S. B. Hodgkins, P. M. Crill, J. Chanton, G. B. Hurst, N. C. VerBerkmoes, S. R. Saleska, P. Hugenholtz, V. I. Rich, and G. W. Tyson. 2014. “Discovery of a Novel Methanogen Prevalent in Thawing Permafrost,” Nature Communications 5, DOI: 10.1038/ncomms4212. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 16, 2014

Genome Watch Highlights DOE JGI Explorations of Microbial “Dark Matter.”

Adam Walker of the Wellcome Trust’s Sanger Institute has published an analysis of the Department of Energy’s Joint Genome Institute’s (DOE JGI) explorations of “microbial dark matter” metagenomics and single cell genomics. Four recent publications are highlighted, two directly from DOE JGI and two involving past and present collaborators. All used novel technologies to characterize microbes and microbial communities refractory to standard culture in the lab and involved in mission-relevant activities such as bioenergy and bioremediation. Most microbes cannot be readily grown in culture, so they are difficult to study with molecular and genetic approaches that require large amounts of starting genomic material. With the advent of single cell techniques, it is now possible to derive information about the genome of single isolated cells without a cultivation step. Furthermore, with the massive sequencing throughput available at DOE JGI, the DNA from bulk environmental samples can be characterized and a “fingerprint” of the sampled environment can be studied and compared, for example, both before and after perturbations. Even some whole microbial genomes can be assembled from the sequence fragments. These genomes, as noted by Walker, can provide new opportunities for biochemistries relevant to bioenergy, environmental remediation, and carbon and nutrient processing.

Reference: Walker, A. 2014. “Adding Genomic ‘Foilage’ to the Tree of Life,” Nature Reviews Microbiology 12, 78. DOI:10.1038/nrmicro3203. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 02, 2014

Understanding Mineral Transport in Switchgrass for Enhanced Sustainability

A viable bioenergy industry will depend on the development of sustainably grown feedstocks, which are bioenergy crops that yield high amounts of biomass with minimal inputs of water, fertilizer, and other chemicals. The efficient acquisition and mobilization of mineral nutrients by feedstocks are key to their sustainability. Additionally, the platform used to produce biofuel from plant feedstocks (e.g., pyrolysis and thermochemical) is affected by biomass minerals (e.g., high levels of silicon in ash decreases conversion efficiency). In perennial bioenergy plants such switchgrass, certain minerals are recycled—mobilized from senescing tissues in the autumn to perennial crowns, rhizomes, and roots for winter storage, and remobilized and translocated to growing stem and leaf tissues in the spring. This seasonal storage and recycling of minerals depends on specific transporters for movement into and out of cells, a poorly understood process. With funding from the joint U.S. Department of Agriculture-Department of Energy Plant Feedstocks Genomics for Bioenergy activity, researchers combined bioinformatics and real-time qRT-PCR approaches to classify mineral transporter genes and gene families in switchgrass and to discern differential expression of these genes during the growing season. In this first molecular study of mineral transporter genes in switchgrass, 520 genes in 40 different families were identified and both tissue and temporal specificity of expression was observed. These results provide the foundation for correlating expression of specific genes with mineral translocation. This will facilitate functional characterization of genes critical for efficient nutrient transport and use and will lead to the development of sustainable, high-yielding switchgrass cultivars.

Reference: Palmer, N. A., A. J. Saathoff, B. M. Waters, T. Donze, T. M. Heng-Moss, P. Twigg, C. M. Tobias, and G. Sarath. 2014. “Global Changes in Mineral Transporters in Tetraploid Switchgrasses (Panicum virgatum L.),” Frontiers in Plant Science 4, DOI: 10.3389/fpls.2013.00549. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 24, 2013

Discovery of Key Brachypodium Regulators May Help Improve Bioenergy Feedstocks

The wild grass Brachypodium distachyon is a model system for temperate grasses, including biofuel plants such as switchgrass and Miscanthus. Because of its relatively small, sequenced genome and a large and growing number of genetic and genomic resources, Brachypodium is useful for studying bioenergy-relevant traits such as grass cell wall characteristics and regulation of plant processes. One key type of regulator is microRNAs (miRNAs), short RNA moleculas involved in many processes such as development and stress response. miRNAs regulate expression of specific genes by pairing with target mRNAs. While many miRNAs have been identified in plants, little is known about these critical regulators in temperate grasses. With funding from the joint U.S. Department of Agriculture-Department of Energy Plant Feedstocks Genomics for Bioenergy program, researchers sequenced small RNAs from different tissues and environmental stress-treated Brachypodium plants and identified miRNAs using a computational approach. Both conserved, newly discovered miRNAs and nonconserved miRNAs not found in other plants were detected. Newly identified regulation of a flowering time gene was found, as well as miRNAs differentially expressed in various tissues. The results improve understanding of the role of miRNAs and their target-specific regulation in Brachypodium and related grasses, and may suggest strategies for bioenergy crop improvement.

Reference: Jeong, D.-H., S. A. Schmidt, L. A. Rymarquis, S. Park, M. Ganssmann, M. A. German, M. Accerbi, J. Zhai, N. Fahlgren, S. E. Fox, D. F. Garvin, T. C. Mockler, J. C. Carrington, B. C. Meyers, and P. J. Green. 2013. “Parallel Analysis of RNA Ends Enhances Global Investigation of microRNAs and Target RNAs of Brachypodium distachyon,” Genome Biology 14, R145. DOI: 10.1186/gb-2013-14-12-r145. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 03, 2013

New Metabolic Pathway Discovered in Methane-Consuming Bacteria

Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases. Major uncertainties remain as to how global climate change will impact the release of carbon stored in ecosystems, particularly in terms of the balance between CO2 and methane entering the atmosphere. Recent technological advances in natural gas extraction from the deep subsurface also have vastly increased the supply of methane for energy production and potentially as an alternate carbon source for synthesis of fuels and other value-added chemicals. These developments have focused increased attention on biological processes that involve methane. For example, aerobic methane-consuming bacteria (methanotrophs) perform key ecosystem processes that affect methane release and represent a potential biological platform for methane-based industrial biocatalysis. In a new study, U.S. Department of Energy investigators at the University of Washington used a multifaceted systems biology approach to examine methane utilization by the methanotrophic bacterium Methylomicrobium alcaliphilum. Their results reveal a previously unknown metabolic pathway in which methane uptake is tightly coupled with glycolytic carbon metabolism, resulting in a novel form of fermentation-based methanotrophy. Under oxygen-limited conditions, this pathway produces acetate and other organic compounds as endproducts rather than CO2, which had been thought to be the sole product of methanotrophic metabolism. This discovery significantly alters our understanding of the role of methanotrophs in environmental carbon cycle processes and presents new opportunities for metabolic engineering of these organisms as platforms for biological conversion of methane to advanced biofuels and other products.

Reference: Kalyuzhnaya, M. G., S. Yang, O. N. Rozova, N. E. Smalley, J. Clubb, A. Lamb, G. A. Nagana Gowda, D. Raftery, Y. Fu, F. Bringel, S. Vuilleumier, D. A. C. Beck, Y. A. Trotsenko, V. N. Khmelenina,  and M. E. Lidstrom. 2013. “Highly Efficient Methane Biocatalysis Revealed in a Methanotrophic Bacterium,” Nature Communications 4, 2785. DOI: 10.1038/ncomms3785. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 26, 2013

New Method for Identifying Genetic Regulatory Networks in Poplar

Wood is an important renewable material for bioenergy and other industrial products, but its formation, a complex process regulated at many levels, is poorly understood. Such processes often involve interactions between regulatory genes known as transcription factors (TFs) and their direct DNA targets. These TF-DNA interactions constitute a regulatory hierarchy. To begin to understand these systems in poplar trees, researchers at North Carolina State University funded by the Department of Energy’s Genomic Science Program developed a robust, high-throughput pipeline to study the hierarchy of genetic regulation of wood formation using tissue-specific single cells known as protoplasts. A new method for isolating protoplasts from the wood-forming stem differentiating xylem (SDX) tissues of Populus trichocarpa was developed and used to study the expression of a specific poplar TF affecting wood formation. By integrating this novel system with computational approaches, a hierarchical layer of genes was inferred that was then functionally validated in SDX. This approach will be particularly useful in studying complex processes in plant species that lack mutants and a stable transformation system. It also can be used to improve forest tree productivity with more precise genetic approaches.

Reference: Lin, Y.-C., W. Li, Y.-H. Sun, S. Kumari, H. Wei, Q. Li, S. Tunlaya-Anukit, R. R. Sederoff, and V. L. Chiang VL. 2013. “SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa,” Plant Cell 25, 4324-41. DOI: 10.1105/tpc.113.117697. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 22, 2013

Predictive Modeling of Microbial Partnerships

Many biogeochemical processes involved in the global carbon cycle are not performed by individual organisms, but rather by collaborative partnerships between two or more microbes. Referred to as “syntrophy,” these partnerships often involve consumption of carbon compounds that cannot be used by any individual organism, but yield sufficient energy for growth when paired organisms couple their metabolic capabilities. These associations are critical to carbon decomposition processes and are particularly important in oxygen-limited environments such as wetlands, sediments, and subsurface aquifers. In a new study funded by the Department of Energy’s Genomic Science Program, a team of researchers has developed a novel genome-scale, multi-omics based modeling approach to investigate the systems biology of syntrophic microbial partnerships. The team focused on Geobacter metallireducens and Geobacter sulfurreducens, two microbes that are capable of syntrophically consuming ethanol and formate (two major products of carbon decomposition). By examining the flow of metabolites within and between the partners, and coupling this information to genome-wide analysis of shifts in gene expression, a new model was developed that enabled the team to test the hypothesis that direct transfer of electrons between the two species permits this mode of metabolism. The study’s results shed new light on a poorly understood aspect of carbon cycle processes. They also represent a significant advance in our ability to extend genome scale systems biology modeling approaches to multispecies microbial consortia. This publication was selected as a research highlight in the January 2014 issue of the journal Nature Reviews Microbiology.

Reference: Nagarajan, H., M. Embree, A.-E. Rotaru, P. M. Shrestha, A. M. Feist, B. Ø. Palsson, D. R. Lovley, and K. Zengler. 2013. “Characterization and Modelling of Interspecies Electron Transfer Mechanisms and Microbial Community Dynamics of a Syntrophic Association,” Nature Communications 4, 2809. DOI:10.1038/ncomms3809. (Reference link)

Research Highlight: Molloy, S. 2013. “Disentangling Syntrophy,” Nature Reviews Microbiology 12, 7. DOI:10.1038/nrmicro3194. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 29, 2013

Understanding Thermal Pretreatment of Lignocellulosic Biomass

Plants contain substantial amounts of cellulose, hemicellulose, and lignins. Much research is being devoted to developing ways to convert these materials (commonly called ‘lignocellulose’) into fuels. The first step, breaking down the biomass into these three constituents, is particularly difficult to study due to the complexity of ways in which they are entangled in biomass. A new approach has been developed that combines x-ray and neutron beam studies with advanced computational modeling to visualize the breakdown of biomass in wood chips from aspen trees. The research, led by scientists at Oak Ridge National Laboratory, studied the wood chips as they were exposed to a variety of treatments, including steam explosion pretreatment, dilute acid pretreatment, and ammonia fiber expansion. The experiments visualized the structural changes in the biomass during the processing, showing for example how porosity of the cell walls and extent of hydration of the different biomass components changes as treatments proceed. The key mechanisms responsible for structural changes are the dehydration of cellulose fibers and lignin-hemicellulose phase separation. These fundamental insights will guide the development of more efficient pretreatments. The research was featured on the January 2014 cover of Green Chemistry.

Reference: Langan, P., L. Petridis, H. M. O'Neill, S. Venkatesh Pingali, M. Foston, Y. Nishiyama, R. Schulz, B. Lindner, B. L. Hanson, S. Harton, W. T. Heller, V. Urban, B. R. Evans, S. Gnanakaran, A. J. Ragauskas, J. C. Smith, and B. H. Davison. 2014. Common Processes Drive the Thermochemical Pretreatment of Lignocellulosic Biomass,Green Chemistry 16, 63–68. DOI:10.1039/C3GC41962B. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 27, 2013

Faster, Bigger, Stronger: Genome Database Improvements

The Department of Energy’s Joint Genome Institute (DOE JGI) maintains the Integrated Microbial Genomes (IMG) data warehouse, which contains a rich collection of genomes from all three domains of life. IMG/M provides a similar collection of partially assembled genome reads from microbial communities (metagenomes). Both have recently been upgraded to address the increase in genome sequences and provide more options for users. IMG was introduced in 2005. Since the last published report in 2012, both systems have grown and improved. The improvements for both systems are described in a pair of reports in the Jan. 1, 2014, issue of Nucleic Acids Research.

The late 2013 version of IMG contains more than 16,000 genome datasets with more than 42 million protein-coding genes. Most (nearly 12,000) are bacterial, archaeal, and eukaryotic genomes. The number of genomes is more than three times the number two years ago. IMG also includes thousands of viral genomes, plasmids that did not come from a specific microbial genome sequencing project, and hundreds of genome fragments. Also in late 2013, IMG/M contained 3,328 metagenome datasets from 460 metagenome studies, with more than 19.5 billion protein coding genes.

Both systems have enhanced analysis tools for publicly available datasets. The latest version of IMG includes tools for recording and analyzing single cell genomes, RNA sequencing data, and gene clusters coding for synthesis of complex organic molecules (biosynthetic clusters).
Both systems are continually being improved to keep up with recent advances in genomics. Future advances will include incorporating pangenomic data (genes that make up the core genes common to all individuals in a species as well as variant genes to enable some individuals to adapt to different environments) and analysis tools for IMG and metaproteomics datasets (protein samples collected from environmental sources) in IMG/M.

References: Markowitz, V. M., et al. 2013. “IMG 4 Version of the Integrated Microbial Genomes Comparative Analysis System,” Nucleic Acids Research 42(D1), D560–67. DOI:10.1093/nar/gkt963. (Reference link)

Markowitz, V. M., et al. 2013. “IMG/M 4 Version of the Integrated Metagenome Comparative Analysis System,” Nucleic Acids Research 42 (D1), D568–73. DOI:10.1093/nar/gkt919. (Reference link)

Related Links:
IMG website: https://img.jgi.doe.gov/
IMG/M website: https://img.jgi.doe.gov/cgi-bin/m/main.cgi

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 18, 2013

Recoding a Bacterial Genome Allows Biosynthesis of Proteins with New Functions

Engineered bacteria are used in biotechnology for producing enzymes and other proteins as well as the biological molecules they synthesize. However, the spectrum of possible proteins that can be biotechnologically produced is limited by the 20 amino acids in the genetic code. One way to expand the possibilities of potential engineered protein functions is to add more amino acids to the repertoire that can be incorporated into proteins. In a recent article published in Science, researchers at Yale and Harvard Universities altered the genome of the model bacterium Escherichia coli so that one of the three stop codons (three-letter words that constitute the genetic code) is no longer used. In this recoded E. coli strain, the freed stop codon (UAG) could now be used to incorporate new amino acids by providing the necessary machinery (a modified tRNA that recognizes UAG and a special aminoacyl–tRNA synthetase, the enzyme that loads amino acids onto the tRNA). With these tools, the researchers showed that they can incorporate novel amino acids into a selected protein without affecting the rest of the bacterial proteins, while maintaining a normal cellular physiology. In addition, the recoded cells are less susceptible to viral infection, and the risk of transferring altered DNA to other organisms is minimized because the normal protein synthesis machinery will not work properly with the recoded genes from the recoded strain. This work has tremendous implications for engineering new organisms that can be used for producing novel proteins that perform new functions needed in DOE-relevant processes such as biofuels production.    

Reference: Lajoie, M. J., A. J. Rovner, D. B. Goodman, H.-R. Aerni, A. D. Haimovich, G. Kuznetsov, J. A. Mercer, H. H. Wang, P. A. Carr, J. A. Mosberg, N. Rohland, P. G. Schultz, J. M. Jacobson, J. Rinehart, G. M. Church, and F. J. Isaacs. 2013. “Genomically Recoded Organisms Expand Biological Functions,” Science 342, 357-60. DOI:10.1126/science.1241459. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 16, 2013

Root Microbial Populations May Enhance Tree Productivity

Bacterial and fungal communities inhabiting the soil around a plant’s roots (the rhizosphere) as well as within the roots (the endosphere) can signifi­cantly benefit the plant’s overall health and productivity, especially in long-lived perennials such as trees. However, the molecular mechanisms that regulate these very complex interactions between plants and microbes are difficult to study and poorly understood. To gain insight into these interactions, researchers at Oak Ridge National Laboratory conducted a detailed study of the rhizosphere and endosphere “microbiomes” of the Eastern Cottonwood tree (Populus deltoides), a promising bioenergy feedstock candidate, from two natural settings in North Carolina and Tennessee and over two seasons. While much of the observed variation is still to be explained, the group did find significant differences in microbial communities between the two locations and between the fall and spring seasons. Additionally, they found that microbes within roots were very different from those just outside the roots, indicating that selection for specific, rather than random, microbes to colonize plant roots may occur. The results suggest that these beneficial microbes might be manipulated to enhance plant growth and productivity as well as increase resistance and adaptability to environmental stresses.

Reference: Shakya, M., N. Gottel, H. Castro, Z. K. Yang, L. Gunter, J. Labbé, W. Muchero, G. Bonito, R. Vilgalys, G. Tuskan, M. Podar, and C. W. Schadt. 2013 “A Multifactor Analysis of Fungal and Bacterial Community Structure in the Root Microbiome of Mature Populus deltoides Trees,” PLoS ONE 8(10), e76382. DOI:10.1371/journal.pone.0076382. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 26, 2013

Discovery of a New Way that Bacteria Regulate Their Genes

The amino acid composition of proteins is encoded in DNA in the form of three-letter words (codons). Each amino acid can be coded for by more than one codon and, for a given amino acid, different organisms use one codon more frequently than the alternatives. This codon usage preference, particularly near the start of genes, has a strong influence in gene expression, but the causes and precise effects of such codon preference are unclear. Scientists at Harvard University analyzed thousands of synthetic gene constructs containing either frequent or infrequent codons toward their start. Using next-generation sequencing to determine gene expression and fluorescent cell sorting to assess protein abundance, the investigators concluded that the presence of infrequent codons near the start of genes dramatically increases protein expression. Furthermore, using computational methods to predict RNA structure, the authors demonstrated that the three-letter sequence of infrequent codons reduces the formation of secondary structures in the messenger RNA (mRNA) molecule involved in the protein synthesis process, facilitating the translation of the DNA sequence of genes into proteins. This mRNA structural modification is in large part responsible for the observed increase in expression of genes with infrequent codons. These results have important implications for the design of synthetic genes that can be more efficiently expressed in engineered organisms for the production of new biomolecules such as biofuels.      

Reference: Goodman, D. B., G. M. Church, and S. Kosuri. 2013. “Causes and Effects of N-Terminal Codon Bias in Bacterial Genes,” Science 342(6157), 475 €"79. DOI:10.1126/science.1241934. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 19, 2013

Diverse Microbial Community Found on Sea Squirt Coat

The sea squirt Ciona intestinalis is a well-studied model organism in developmental biology. Ciona is the closest invertebrate relative to the chordate (backboned) lineage to which humans and other primates belong. Little is known about its associated bacterial community in spite of growing evidence that microbes play key roles in organisms from plants to humans. New research supported by the Department of Energy’s Joint Genome Institute (DOE JGI) combined several technologies to characterize the bacteria living inside and on the exterior coating, or tunic, of C. intestinalis adults. The Ciona tunic is a complex cellulose and mucopolysaccharide envelope; the sequencing data demonstrates that the bacterial community structure on Ciona’s tunic differs from that of bacteria in the surrounding seawater. The observed tunic bacterial consortium contains a shared community of less than 10 abundant bacterial phylotypes across three individuals. The relatively simple bacterial community and availability of dominant community members in culture make C. intestinalis a promising system in which to investigate functional interactions between host-associated microbiota and bacterial enzymes that could digest or alter celluloses. Leveraging the original sequencing work of the C. intestinalis by DOE JGI, this work was supported by an interagency program, the International Collaborative Biodiversity Group program administered by the National Institutes of Health’s Fogarty International Center, in which multiple agencies participated.

Reference: Blasiak, L. C., S. H. Zinder, D. H. Buckley, and R. T. Hill. 2014. “Bacterial Diversity Associated with the Tunic of the Model Chordate Ciona intestinalis,” The ISME Journal 8, 309–20. DOI:10.1038/ismej.2013.156. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 09, 2013

Tropical Soil Bacterium Frees Plant Sugars for Biofuels

The lignocellulose component of plants could be a sustainable alternative fuel if it could be easily degraded and transformed for use in biofuels. In a recent study, a team of scientists—from the University of Massachusetts, Amherst; U.S. Department of Energy (DOE) Joint BioEnergy Institute; and DOE Environmental Molecular Sciences Laboratory (EMSL)—used proteomic, transcriptomic, and metabolomic approaches at EMSL to examine the ability of Enterobacter lignolyticus SCF1 to degrade lignocellulose. SCF1 is found in tropical forest soils and is known to rapidly decompose leaf litter. This study demonstrated that this bacterium can degrade the lignin portion of plant cellulosic biomass by both assimilatory and dissimilatory pathways. By breaking down the lignin, SCF1 is able to free the cellulosic sugars found in plant cells, thereby making those sugars available for use in biofuels. These research findings are the first to demonstrate that an anaerobic soil bacterium can use both assimilatory and dissimilatory pathways to reduce lignocellulose, as well as demonstrating the importance of a multi-omics, holistic approach to studying biochemical processes in microbes.

Reference: DeAngelis, K. M., D. Sharma, R. Varney, B. Simmons, N. G. Isern, L. M. Markillie, C. Nicora, A. D. Norbeck, R. C. Taylor, J. T. Aldrich, and E. W. Robinson. 2013. “Evidence Supporting Dissimilatory and Assimilatory Lignin Degradation in Enterobacter lignolyticus SCF1,” Frontiers in Microbiology 10.3389/fmicb.2013.00280. (Reference link)
Related link

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER


September 04, 2013

Candidate Genes Involved in Lignin Degradation Found in Wood-Boring Beetle’s Mid Gut

The Asian longhorned beetle (Anoplophora glabripennis ) is an invasive species first discovered in the United States in 1996. It attacks both healthy and stressed hardwood trees, including the bioenergy candidate feedstocks poplar and willow, and has no natural enemies in this environment. The microbial community in the beetle’s midgut is capable of breaking down the lignin, cellulose, and hemicellulose in the trees to acquire needed nutrients, but little is known about the processes involved. To learn more about how microbial communities in the guts of such wood-boring insects break down these woody tissues, a team including researchers from the Department of Energy’s (DOE) Joint Genome Institute (JGI) sequenced, assembled, and analyzed the Asian longhorned beetle’s midgut metagenome.

In the study published in Plos ONE , the team compared the metagenome assembly from the wood beetles to annotated assemblies in DOE JGI’s IMG/M database. These datasets came from microbial communities associated with herbivores that feed to plant tissues, insects that feed on specific plant tissues, and insects (e.g., termites) that feed on woody tissues. The findings revealed that the beetle’s midgut contained a community dominated by aerobes, which research­ers expected, noting that large-scale lignin-degrading reactions require oxygen and have only been demonstrated in aerobic environments. They identified several genera of fungi and bacteria in the assembly; many of the microbes have been associated with break down of lignocellulose, hemicellulose, and other similar compounds. The metagenome assembly also led to the identifi­ca­tion of candidate genes for a variety of functions, including lignin-degrading enzymes, cellu­lases, xylose utilization, and fermentation as well as for nitrogen and nutrient acquisition.

This study is the first large-scale functional metagenomic analysis of the midgut micro­bial community of a beetle with known lignin-degrading capabilities. Lignin is one of the most recalcitrant components of plant biomass. The candidate genes identified from by the functional profile could lead to novel enzymes that might either be useful for industrial biofuels applications or else be used to control this invasive insect.

Reference: Scully, E. D., et al. 2013. “Metagenomic Profiling Reveals Lignocellulose Degrading System in a Microbial Community Associated with a Wood-Feeding Beetle,” PLoS ONE 8(9), e73827. DOI: 10.1371/journal.pone.0073827. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742, John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The Asian longhorn beetle is an invasive pest that can break down lignin in host deciduous trees. (Wikimedia Commons)



September 02, 2013

POPSEQ for Plant Genome Assembly: New Approach Allows Researchers to Work on Many Species Regardless of Sequence Resources

One of the challenges in assembling plant genome “contigs,” fragments of the entire genome that are identified by the assembly algorithms, is that they are not easily linked together or even placed in their proper order. In an effort to mitigate this problem, researchers with the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI) teamed with other researchers to develop another approach for assembling contigs.

In a study published in The Plant Journal, the team reports on the results of testing the approach they call POPSEQ with the barley genome. The plant was selected for DOE JGI’s 2011 Community Sequencing Program portfolio in part for its potential as a bioenergy feedstock crop. Grown on four million acres in the United States, the crop could be used to produce cellulosic ethanol from the straw. More than 80 percent of the 5.1 billion-base genome is composed of repeats, adding to its complexity.

Using POPSEQ, researchers assembled the barley genome while testing a number of variables. For example, they used datasets obtained from different mapping populations, or, in another case, assembled the genome based solely on short reads. The team reported that the results from these tests were comparable with the assembly previously produced by the International Barley Sequencing Consortium. “By comparison,” they wrote, “POPSEQ is inexpensive, rapid, and conceptually simple, the most time-consuming step being the construction of a mapping population…The method is independent of the need for any prior sequence resources,” and this proof of principle demonstrates that POPSEQ can be effectively applied to many species.

Reference: Mascher, M., et al. 2013. “Anchoring and Ordering NGS Contig Assemblies by Population Sequencing (POPSEQ),” The Plant Journal, DOI: 10.1111/tpj.12319. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742, John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Image: Cultivated barley is the fourth most abundant crop in the world and a model for plant genetics research.
(Image courtesy of freefotouk, Flickr CC BY 2.0)



August 15, 2013

New Gene Discovery Clarifies Lignin Biosynthetic Pathway

Lignin is integral to plant cell wall strength and function. It is also important in bioprocessing of plant biomass, because it inhibits deconstruction of plant cell wall sugar polymers such as cellulose and hemicellulose into sugar monomers—a key step in the production of biofuels. Lignin’s irregular polymeric structure has made it difficult to establish a clear biosynthetic pathway for its formation, making it a challenging target for genetic engineering of plants for enhanced bioprocessing of plant biomass. Recently, scientists at the U.S. Department of Energy’s Great Lakes Bioenergy Research Center (GLBRC) identified a new enzyme in the biosynthetic pathway of lignin monomers. The enzyme caffeoyl shikimate esterase (CSE) was found to catalyze a previously unidentified step in the biosynthesis of lignin monomers. Analysis of plant lines with a mutation in CSE demonstrated altered accumulation of lignin precursors consistent with its hypothesized activity and position in the lignin biosynthetic pathway. This enzymatic step is important, leading to a lignin that is less inhibitory to deconstruction than wild type lignin. In fact, one CSE mutant showed significantly more saccharification (78%) than wild type (18%), though plant growth was stunted. The discovery of this previously unknown enzymatic step highlights the success of genomics, global gene expression studies, data sharing, and bioinformatics, because the gene was found by searching publicly available gene expression databases for genes of unknown function that are co-expressed with other known lignin biosynthesis genes. This more complete knowledge of the lignin biosynthesis pathway will enable more intelligent engineering of lignin biosynthesis that may lead to more efficient bioprocessing without negatively impacting plant growth and viability. The GLBRC research was carried out in collaboration with an international team of scientists from Belgium and the United Kingdom.

Reference: Vanholme, R., I. Cesarino, K. Rataj, Y. Xiao, L. Sundin, G. Goeminne, H. Kim, J. Cross, K. Morreel, P. Araujo, L. Welsh, J. Haustraete, C. McClellan, B. Vanholme, J. Ralph, G. G. Simpson, C. Halpin, and W. Boerjan. 2013. “Caffeoyl Shikimate Esterase (CSE) is an Enzyme in the Lignin Biosynthetic Pathway,” Science 341, 1103–06. DOI: 10.1126/science.1241602. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 07, 2013

Novel Bioengineering Technique for Genome-Scale Tuning of Gene Expression

Introduction of new genes encoding desired functional attributes has long been a central tool for metabolic engineering and synthetic biodesign of microorganisms. However, difficulties in accurately predicting the expression levels of these genes in their new hosts significantly slow the design cycle and hinder progress. This is particularly problematic in synthetic biology, where large genetic constructs containing multiple genes are often introduced. Now researchers present a novel technique to more accurately predict gene expression levels in engineered biosystems by combining recent advances in DNA synthesis with novel, multiplexed methods for measuring DNA, RNA, and protein levels simultaneously using next-generation sequencing. This new technique allowed the team to simultaneously measure transcription and translation rates of thousands of synthetic regulatory elements introduced into the model microbe Escherichia coli . The resulting dataset was then used to model gene and protein expression levels under various sets of regulatory elements and “compose” a designed regulatory strategy that enables accurate prediction of expression levels of introduced genetic elements. This new technique has the potential to allow much more sophisticated forward design of genetic engineering strategies to improve production of biofuels and other bioproducts.

Reference: Kosuri, S. D. B. Goodman, G. Cambray, V. K. Mutalik, Y. Gao, A. P. Arkin, D. Endy, and G. M. Church. 2013. “Composability of Regulatory Sequences Controlling Transcription and Translation in Escherichia coli ,” Proceedings of the National Academy of Sciences USA 110 , 14024–29. DOI: 10.1073/pnas.1301301110. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 05, 2013

Microbes from Phyla Chloroflexi Provide Clues to Carbon Cycling, Respiration in Sediments

Through metagenomics, researchers sequenced 86 organisms from the phylum Chloroflexi, representing 15 distinct lineages, to discover the secrets of microbial life within terrestrial aquifer sediment deposits.

These Chloroflexi microbes were found to have metabolic processes involved in plant biomass degradation, which could be useful for biofuels production, as well as a better understanding of the subsurface nitrogen and carbon cycles. Microorganisms in aquifer sediments are responsible for subterranean carbon turnover and the degradation of organic contaminants. Consequently, these microorganisms can heavily impact the quality of underground drinking water. In earlier studies, it was determined that Chloroflexi represent a significant amount of the microbial population in sediments. However, these microbes are poorly understood, as only six of about 30 Chloroflexi classes have been sequenced. For this reason, a team of researchers including scientists from the Department of Energy’s Joint Genome Institute (DOE JGI) conducted a study on the microbial composition of these aquifer sediments to gain a broader knowledge of the metabolic characteristics of Chloroflexi microbes.

The researchers were able to reconstruct three near-complete Chloroflexi genomes from the metagenomic data collected at the Integrated Field-Scale Subsurface Research Challenge Site in Colorado as part of a DOE JGI Community Sequencing Program project led by Jill Banfield of the University of California, Berkeley. Metabolic analyses revealed that Chloroflexi can break down plant mass, influence subsurface carbon and nitrogen cycles, and adapt to changing oxygen levels. These traits, the researchers noted, were likely to apply to Chloroflexi in other sediment environments, making these microbes good candidates for mining useful enzymes and pathways for DOE missions of bioenergy and carbon processing as well as for biodegradation.

Reference: Hug, L. A., et al. 2013. “Community Genomic Analyses Constrain the Distribution of Metabolic Traits Across the Chloroflexi Phylum and Indicate Roles in Sediment Carbon Cycling,” Microbiome 1(22), DOI:10.1186/2049-2618-1-22. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 05, 2013

Using Mass Spectrometry to Localize Lipid Metabolites in Camelina Seeds

Camelina sativa is a nonfood oilseed crop that, because of relatively low production costs and potential for use in a number of industrial applications, shows promise as a bioenergy feedstock. Additionally, the relative ease with which the plant can be genetically modified offers potential for altering the seed oil composition through engineering of the lipid and fatty acid metabolic pathways. To do this, however, it is important to understand how these pathways are regulated in different seed tissues. With funding from the Department of Energy’s Office of Science Genomic Science Program, researchers from the University of North Texas used mass spectrometry imaging techniques to show that the distribution of various lipid-related metabolites and precursors are specific to certain distinct tissues within the seed embryo. This high-resolution metabolite mapping in Camelina seeds can be used to reveal new insights into tissue-based variation and illustrates the importance of considering spatial heterogeneity when designing metabolic engineering strategies for manipulating seed lipid composition. This work will facilitate more refined and accurate targeting when engineering plants for optimal seed oil composition.
 
Reference: Horn, P. J., J. E. Silva, D. Anderson, J. Fuchs, L. Borisjuk, T. J. Nazarenus, V. Shulaev, E. B. Cahoon, and K. D. Chapman. 2013. “Imaging Heterogeneity of Membrane and Storage Lipids in Transgenic Camelina Sativa Seeds with Altered Fatty Acid Profiles,” The Plant Journal 76(1), 138 €"50. DOI:10.1111/tpj.12278. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 01, 2013

Hope for Reestablishing Microbial Populations in the Gulf of Mexico

Researchers used metatranscriptomic analyses to compare the microbial populations in the Gulf of Mexico before and after the Deepwater Horizon oil spill to learn more about the impact of   petroleum being spilled into the waters. Though the oil spill reduced the diversity of the microbial communities in the Gulf of Mexico, some microbial populations remain unchanged suggesting that they may be important in reestablishing the original microbial community.
One of the first studies published in the aftermath of the Deepwater Horizon oil spill involved the Department of Energy’s Joint Genome Institute (DOE JGI) researchers and confirmed that microbial communities did play a role in dispersing the hydrocarbons from the waters. A second study released in 2012 tracked the populations of several microbial species in the Gulf of Mexico as they dominated in the waters at various time points to remove different fractions of the oil.

DOE JGI associated researchers recently carried out a new study of the microbial populations in the Gulf of Mexico, this time focusing on the expressed genetic information of an ecosystem, its metatranscriptomes. They examined species in the bathypelagic zone at depths of 1,000 to 4,000 meters underwater where no sunlight penetrates. The analysis of roughly 66 million transcripts sequenced for the study attributes 40% of the reads to just six genomes from Gammaproteobacteria known to be capable of breaking down methane and petroleum. The findings confirm that the diversity of microbes and their functional roles in the waters have decreased since the oil spill. However, the team also found that some microbial populations did not appear to be affected by the events that took place three years ago, as their numbers remain similar both before and after 2010.

“Despite the enormous bloom of hydrocarbon-degrading Gammaproteobacteria that increased bacterial cell counts by two orders of magnitude, members of the natural microbial community persisted at their pre-bloom activity levels and may be important in reestablishing the original microbial community,” the researchers concluded.

Reference: Rivers A.R., et al. 2013. “Transcriptional Response of Bathypelagic Marine Bacterioplankton to the Deepwater Horizon Oil Spill,” The ISME Journal 7, 2315-29. DOI:10.1038/ismej.2013.129. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Comparing the expressed genetic information from microbial communities before and after the 2010 Deepwater Horizon spill indicated that while the overall population diversity has changed, some microbes were unaffected. (Image courtesy of Green Fire Productions, Flickr CC BY 2.0)



July 28, 2013

New Understanding of Microbial Community Processes Improves Carbon Cycle Models

Current Earth system models (ESMs) draw on soil carbon cycle models that use relatively simple representations of the biogeochemical processes performed by microbial communities. Now, investigators at the University of California, Irvine, have developed a new module for the Community Land Model (CLM) that attempts to more accurately represent the distribution of soil microbial communities and their functional processes related to carbon degradation. Projections of climate change impacts on soil carbon stocks using this module showed improved agreement with results observed during experimental studies. Developing improved models of microbial processes will generate more accurate projections of soil carbon feedbacks on climate change and reduce a source of uncertainty in current ESMs.

Reference: W. R.Wieder, G. B. Bonan, and S. D. Allison. 2013. “Global Soil Carbon Projections Are Improved by Modeling Microbial Processes,” Nature Climate Change. DOI: 10.1038/NCLIMATE1951. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 27, 2013

Higher Yields of Advanced Biofuels from Genetically Engineered Yeast

The development of renewable substitutes for fuels and chemicals supplied by petroleum is an important aspect of achieving energy security. Currently, the United States annually produces more than 10 billion gallons of the biofuel ethanol from microbial fermentation of corn sugars using yeast. As this industry has matured, it has become clear that ethanol is not an ideal gasoline replacement due to its low-energy density, handling challenges, and limited compatibility with the current transportation fleet. Focus therefore has shifted to the production of advanced biofuels, designed to be “drop-in” fuels, having the same properties as gasoline, diesel, or jet fuel. Researchers at the Joint BioEnergy Institute (JBEI) recently achieved the highest ever reported yields of drop-in fuel precursors in yeast. Diesel fuels are composed mainly of long-chain hydrocarbon esters, similar to the fatty acids produced by yeast and other microorganisms for construction of their cell membranes. Overproduction of fatty acids in yeast is no easy task as elaborate regulatory and feedback systems exist to prevent excessive accumulation of these building blocks. To overcome this hurdle, the JBEI researchers replaced the highly-regulated native promoters for fatty acid production machinery with new high-intensity promoters. These promoters are effectively always “on,” directing the cell to make more fatty acid assembly machinery. The researchers also engineered cellular machinery to reroute fatty acids from cell membrane manufacture to free fatty acids that can be transformed through industrial processes to drop-in biofuels. These engineering changes led to an over 500-fold increase in production of free fatty acids when compared to the native strain. Strains also were engineered to produce drop-in biofuels directly, rerouting fatty acids into fatty alcohols and fatty acid ethyl esters that can be used in diesel engines. With these increased yields of fatty alcohols and fatty acid ethyl esters, this work represents a major advance toward production of next generation drop-in biofuels.

Reference: Runguphan, W., and J. D. Keasling. 2013. “Metabolic Engineering of Sacchromyces cerevisiae for Production of Fatty Acid-Derived Biofuels and Chemicals,” Metabolic Engineering 21, 103 €"13. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 23, 2013

Physcomitrella Moss Genome Expected to Help In Understanding Potential Climate Change

An international team of scientists has re-annotated the genome of Physcomitrella patens, a moss sequenced by the Department of Energy’s Joint Genome Institute (DOE JGI) that contains about 10,000 more genes than humans. It is widely believed that the information contained in the P. patens genome can help researchers improve crop yields, disease and insect resistance, drought tolerance, and more efficient biofuel production. Researchers were able to provide a functional analysis of many of its previously unknown genes, adding to its value as a model plant and for interpreting other sequenced plant genomes.

P. patens has long been the experimental moss of choice for researchers around the world and was first sequenced by DOE JGI in 2007. P. patens can be more efficiently studied than other plants, mainly due to its accelerated lifecycle, hence short generation time. An international team of researchers from Germany, Belgium, and Japan has worked with the genes of what DOE JGI refers to as a “flagship genome,” a term meaning that sustained and significant computational and experimental resources are directed to this organism. By using the sequencing information from DOE JGI, the team was able to suggest potential functions for 58% of all the genes identified, a large increase over the 41% in the earlier publication.

“One of our intriguing findings is that 13% of the Physcomitrella genes have no clear relatives in any other sequenced organism so far. Analyzing these orphan genes more deeply will reveal the hidden treasures of the moss genome,” said University of Freiburg Chair of Plant Biotechnology Ralf Reski, a senior coordinator on the study. The study’s findings were made available at www.cosmoss.org, as well as further information regarding moss genomes through DOE JGI’s Phytozome.

Reference: Zimmer, A. D., et al. 2013. “Reannotation and Extended Community Resources for the Genome of the Non-Seed Plant Physcomitrella patens Provide Insights into the Evolution of Plant Gene Structures and Functions,”BMC Genomics 14, DOI:10.1186/1471-2164-14-498. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 14, 2013

Illuminating Biology’s Dark Matter

In cosmology, dark matter is said to account for the majority of mass in the universe. Its presence, however, is inferred by indirect effects rather than detected through telescopes. The biological equivalent is “microbial dark matter,” a largely unexplored realm of microbial life on Earth that can profoundly influence key environmental processes such as plant growth, nutrient cycles, the global carbon cycle, and climate processes. An international collaboration, led by the U.S. Department of Energy’s Joint Genome Institute (DOE JGI) where the sequencing of genomes isolated from single cells was carried out, targeted uncultivated microbial cells from nine diverse habitats, derived from 28 major, but previously uncharted branches of the tree of life. The results fall into three main areas: 1) metabolic features previously only seen in bacteria are also found in Archaea, such as an enzyme used by bacteria to “thin out” their protective cell wall so that the cell can expand during cell division ; 2) the ability to correctly assign d ata from 340 million DNA fragments from other habitats to the proper lineage, linking these fragments to organisms and particular ecosystems, as well as providing insights into possible functional roles; and 3) the ability to more accurately resolve microbial taxonomical relationships within and between microbial phyla, which is critical to predict ecological niches and capabilities. The new results will enable scientists to better predict metabolic properties and other useful traits of different microbial groups. The Nature publication builds upon a DOE JGI pilot project, the Genomic Encyclopedia of Bacteria and Archaea (GEBA: http://www.jgi.doe.gov/programs/GEBA/).

Reference: Rinke, C., P. Schwientek, A. Sczyrba, N. N. Ivanova, I. J. Anderson, J.-F. Cheng, A. Darling, S. Malfatti, B. K. Swan, E. A. Gies, J. A. Dodsworth, B. P. Hedlund, G. Tsiamis, S. M. Sievert, W.-T. Liu, J. A. Eisen, S. Hallam, N. C. Kyrpides, R. Stepanauskas, E. M. Rubin, P. Hugenholtz, and T. Woyke. 2013. “Insights into the Phylogeny and Coding Potential of Microbial Dark Matter,” Nature 499 , 431–37. DOI: 10.1038/nature12352. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 12, 2013

Unraveling Plant-Microbe Communication

The soil environment contains a complex of microbial communities living in close association with plants, both outside the root (rhizosphere) and within (endosphere). These interactions between plants and microbes can significantly influence plant growth and development and, in the case of beneficial microorganisms, increase plant health and yield. These complex interactions involve cell-to-cell communication, but very little is known about how these signals are triggered and regulated. To better understand the dynamics of these systems, scientists at Oak Ridge National Laboratory have undertaken an extensive survey of the “microbiome” of the woody perennial Populus , a tree that has intimate associations with many types of beneficial fungi and bacteria and is a potential biofuel feedstock for cellulosic ethanol production. Focusing on a specific type of sensing molecule known as acyl-homoserine lactone (AHL), the researchers screened 129 bacterial isolates from P. deltoides (Eastern cottonwood) and found that 40% were AHL positive. Furthermore, they found a subgroup of AHL-controlled regulators that respond to unknown plant-derived signals rather than bacterial AHLs. The results indicate that the microbiota that comprises the Populus root zone has substantial capacity for cell-to-cell communication, furthering our understanding of the role these microbial signaling molecules play in the plant’s biology.

Reference: Schaefer, A. L., C. R. Lappala, R. P. Morlen, D. A. Pelletier, T.-Y. S. Lu, P. K. Lankford, C. S. Harwood, and E. P. Greenberg. 2013. “LuxR- and LuxI-type Quorum Sensing Circuits Are Prevalent in Members of the Populus deltoides Microbiome,” Applied and Environmental Microbiology 79 , 5745–52. DOI: 10.1128/AEM.01417-13. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 02, 2013

Lipid Droplet-Associated Proteins in Plant Tissues

Lipid droplets (“oil bodies”) are found within the cells of all multicellular organisms, and they provide storage of high-energy carbon reserves. These subcellular organelles are well characterized in seeds, but they also occur in nearly all plant cells, although little is known about the proteins associated with nonseed lipid droplets. To elucidate the mechanisms involved in lipid droplet metabolism in nonseed plant tissues, researchers at the University of North Texas in collaboration with the U.S. Department of Energy’s Great Lakes Bioenergy Research Center used a multi-pronged approach to investigate lipid-associated proteins in the oil-rich tissues of avocado, a fruit widely used as a model system to study lipid synthesis. They identified a new class of lipid droplet-associated proteins (LDAPs) in nonseed tissues very similar to small rubber particle proteins found in rubber-producing plants; these LDAPs may be important to lipid particle binding and stabilization. The results further understanding of the subcellular processes involved with lipid metabolism and will be useful for endeavors to increase concentrations of energy-dense lipids in plants that may serve as bioenergy crops.

Reference: Horn, P. J., C. N. James, S. K. Gidda, A. Kilaru, J. M. Dyer, R. T. Mullen, J. B. Ohlrogge, and K. D. Chapman. 2013. “Identification of a New Class of Lipid Droplet-Associated Proteins in Plants,” Plant Physiology 162 , 1926–36. DOI: 10.1104/pp.113.222455. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 22, 2013

How Amines Penetrate Cellulose Fibers and Make Cellulose Accessible for Bioconversion

Cellulose is a major component of biomass and the primary biomass component being studied for biofuel production. However, cellulose fibers are extremely resistant to solvents, preventing enzymes, which are needed for conversion to products, from entering the fibers. Ammonia and simple organic amine molecules are well-known exceptions to this rule, but the mechanism by which they make cellulose fibers accessible is not understood. New research by an international team led by scientists at Oak Ridge National Laboratory (ORNL) combines neutron fiber diffraction and computational simulation to show how ethylene diamine (EDA, a representative amine solvent) binds to cellulose fibers. Experimental neutron diffraction data for EDA-cellulose complexes were the starting point for quantum chemical construction of optimized atomic-level structures that were then studied using computational molecular dynamics simulations. The results show how EDA disrupts normal hydrogen bonding in cellulose fibers, and the MD simulations explain the dynamic nature of EDA action. These results will help optimize techniques for breakdown of cellulose fibers to convert them on a large scale to biofuels and other renewable products. The research is featured on the cover of the August 2013 issue of the journal Cellulose and was carried out at ORNL; French National Center for Scientific Research (CNRS) and Institut Laue Langevin in Grenoble, France; Los Alamos National Laboratory; Keele University; University of Tokyo; and Kyung Hee University in the Republic of Korea.

Reference: Sawada, D., Y. Nishiyama, L. Petridis, R. Parthasarathi, S. Gnanakaran, V. T. Forsyth, M. Wada, and P. Langan. 2013. “Structure and Dynamics of a Complex of Cellulose with EDA: Insights into the Action of Amines on Cellulose,” Cellulose 20 , 1563–71. DOI: 10.1007/s10570-013-9974-7. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 12, 2013

Algal Pan-Genome Fills Gap in Tree of Life

To World War II soldiers, “The White Cliffs of Dover” was a morale-boosting song that lifted spirits in dark times. To scientists, the white cliffs are towering structures made of the chalky, white calcium carbonate exoskeleton that envelop the single-celled photosynthetic alga known as Emiliania huxleyi or “Ehux.” In some marine ecosystems, Ehux can trap as much as 20 percent of organic carbon derived from CO2 , making it a critical player in the marine carbon cycle . The Department of Energy’s Joint Genome Institute (DOE JGI) has sequenced the Ehux genome and compared it with sequences from other algal isolates. The Ehux genome turned out to be large and complex. Also, Ehux does not exist as a clearly defined species with a uniform genome, but as a more diffuse community—a “pan-genome”—with different individuals possessing a shared core of genes, supplemented by different gene sets to cope with the particular challenges of a local environment. DOE JGI and its collaborators compared 13 Ehux strains, revealing the first ever algal pan-genome. Ehux ’s genomic variability helps explain its ability to thrive in oceans from the equator to the subarctic. The researchers found that the core gene sets include genes that enable Ehux to survive in low levels of phosphorus and to assimilate and break down nitrogen-rich compounds. Additionally, the algal genome offers hints that Ehux may be involved in the global sulfur cycle, as it is able to produce a compound that can influence cloud formation and the climate.

Reference: Read, B. A., J. Kegel, M. J. Klute, A. Kuo, S. C. Lefebvre, F. Maumus, C. Mayer, J. Miller, A. Monier, A. Salamov, J. Young, M. Aguilar, J.-M. Claverie, S. Frickenhaus, K. Gonzalez, E. K. Herman, Y.-C. Lin, J. Napier, H. Ogata, A. F. Sarno, J. Shmutz, D. Schroeder, C. de Vargas, F. Verret, P. von Dassow, and et al. 2013. “Pan Genome of the Phytoplankton Emiliania Underpins Its Global Distribution,” Nature 499 , 209–13. DOI DOI: 10.1038/nature12221. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 07, 2013

Emerging Discipline of Structural Systems Biology Reveals E. coli Heat Tolerance

Microbial sensitivity to heat, or thermosensitivity, depends on the stability of cellular proteins and their ability to remain in an active, folded state. Research to improve microbial survival and function at higher temperatures has mainly focused on strategies for increasing the structural stability of individual proteins. A new approach called structural systems biology directly assesses the genome-scale metabolic potential of a model organism, E. coli, for thermostability. Using this approach, metabolic reactions of E. coli were integrated with three-dimensional structures of each catalytic enzyme. To simulate E. coli growth at various temperatures, protein (structural) activity functions were defined to impose temperature constraints on the metabolic models. This combined metabolic-structural method allows researchers to integrate temperature-dependent information about enzyme function with simulations of microbial metabolic growth. This approach enabled simulation of E. coli growth under various temperature conditions that was in good agreement with experimental growth data. It also provided mechanistic interpretations of mutations that conferred greater thermostability in E. coli. This new approach has important implications for developing industrial microbes as biocatalysts.

Reference: Chang, R. L., K. Andrews, D. Kim, Z. Li, A. Godzik, and B. O. Palsson. 2013. “Structural Systems Biology Evaluation of Metabolic Thermotolerance in Escherichia coli,” Science 340, 1220–23. DOI: 10.1126/science/1234012. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 03, 2013

Engineering Thermophilic Bacteria for Efficient Fermentation of Plant Biomass

Higher temperatures make plant biomass more accessible for processing, so thermophilic bacteria, which are active at higher temperatures than other bacteria, are promising candidates for biofuel production systems. To take full advantage of their potential in consolidated bioprocessing, efficient genetic tools are needed to metabolically engineer the thermophile. Researchers at the U.S. Department of Energy’s BioEnergy Science Center have been developing a series of genetic tools to manipulate Caldicellulosiruptor bescii. C. bescii is one of the most promising thermophiles for deconstructing and fermenting lignocellulose from nonfood plants. New research demonstrates a gene replacement strategy used to delete the lactate dehydrogenase gene from C. bescii. Because the plasmid contains a gene for which there is both positive and negative selection, it is possible to select first for recombination of the deleted ldh gene and then for loss of the plasmid sequences. This method allows clean genetic insertions and deletions, leaving no residual genetic material so that the method can be used repeatedly for adding and subtracting genes for metabolic engineering. The C. bescii strain containing the ldh gene deletion exhibited the expected metabolism changes, namely the engineered strain no longer produced lactate and had increased acetate and H2 production. This gene replacement demonstration paves the way for further genetic manipulation of C. bescii to produce desired biofuel fermentation products directly from plant biomass.

Reference: Cha, M., D. Chung, J. G. Elkins, A. M. Guss, and J. Westpheling. 2013. “Metabolic Engineering of Caldicellulosiruptor bescii Yields Increased Hydrogen Production from Lignocellulosic Biomass,” Biotechnology for Biofuels 6, 85. DOI: 10.1186/1754-6834-6-85. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 18, 2013

Bioinformatics Web Tool Aids Functional Annotation of Plant and Microbial Genomes

Gene sequencing has become very fast and inexpensive, yet the bottleneck of producing reliable functional annotations of gene sequences remains a challenge. Functional annotations commonly use a protocol based on pairwise sequence comparison algorithms such as the Basic Local Alignment Search Tool (BLAST). However, these methods can miss important phylogenetic relationships such as orthology. Phylogenetic methods that explicitly reconstruct evolutionary relationships in multigene families have a higher precision for whole genome functional annotation. A new phylogenetic web server and analysis platform, PhyloFacts, integrates experimental and annotation data from different resources including SwissProt, Gene Ontology, Pfam, BioCyc, Enzyme Commission, and third-party orthology databases. These data are then used to provide functional annotations for user-inputted protein sequences. PhyloFacts also allows users to drill down and view provenance and supporting data for functional annotations. PhyloFacts makes use of Hidden Markov Model (HMM) algorithms to place user-submitted sequences into precalculated phylogenetic relationships, or trees. As a result, its functional subclassifications have greater precision when compared with other orthology web services. Funding for PhyloFacts was provided as part of the Department of Energy’s Systems Biology Knowledgebase (KBase) enabling tools program and will be a component of future KBase services.

Reference: Afrasiabi, C., B. Samad, D. Dineen, C. Meacham, and K. Sjölander. 2013. “The PhyloFacts FAT-CAT Web Server: Ortholog Identification and Function Prediction Using Fast Approximate Tree Classification,” Nucleic Acids Research, 1–7. DOI: 10.1093/nar/gkt399. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 17, 2013

Thermophilic Bacterium Efficiently Deconstructs All Major Plant Biomass Components

Conversion of plant biomass to biofuels holds great promise for developing renewable and secure energy sources. However, the presence of lignin in plant biomass creates problems because of its recalcitrance to solubilization and because it limits access to energy-rich polysaccharides, cellulose, and hemicellulose. New research has iden­tified a thermophilic bacterium, Caldicellulosiruptor bascii, that can solubilize the lignin under the same conditions used for degradation of cellulose and hemicellulose, allowing efficient use of plant biomass for microbial growth and biosynthesis of fermentation products. This finding could enable the development of more economical and environ­mental­ly sustainable biomass conversion processes. This research was carried out by a team of scientists at the University of Georgia as part of the U.S. Department of Energy’s BioEnergy Science Center.

Reference: Kataevaa, I., M. B. Foston, S.-J. Yang, S. Pattathil, A. K. Biswal, F. L. Poole II, M. Basen, A. M. Rhaesa, T. P. Thomas, P. Azadi, V. Olman, T. D. Saffold, K. E. Mohler, D. L. Lewis, C. Doeppke, Y. Zeng, T. J. Tschaplinsk, W. S. York, M. Davis, D. Mohnen, Y. Xu, A. J. Ragauskas, S.-Y. Ding, R. M. Kelly, M. G. Hahn, and M. W. W. Adams. 2013. “Carbohydrate and Lignin Are Simultaneously Solubilized from Unpretreated Switchgrass by Microbial Action at High Temperature,” Energy and Environmental Science 6, 2186–95. DOI: 10.1039/C3EE40932E. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 05, 2013

New Technique for Improved Microbial Genome Assembly

In addition to sequencing the genomes of microbes, plants, fungi, and metagenomes, the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI) develops tools to improve the assembly and analysis of the DNA sequences that it generates. One tool, HGAP (Hierarchical Genome Assembly Process), provides a fully automated workflow for users of the Pacific Biosciences’ single molecule, real-time DNA sequencing machine. The “PacBio” sequencer generates initial DNA sequences up to 10 or more times longer than those provided by other technologies, which is a great assistance in the assembly of sequences into more complete genomes, but at a higher cost and lower accuracy. Competing sequencing technologies involve creating multiple DNA libraries, conducting multiple runs, and combining the data. I n contrast, HGAP requires just a single, long-insert, shotgun DNA library, enabling the resolution of long regions of repeated DNA sequence that often complicate other assembly methods. This new assembly method was tested using three microbes previously sequenced by DOE JGI. The HGAP produced final assemblies with >99.999% accuracy when compared to the reference sequences for these microbes. Next steps in the project will focus on extending HGAP’s utility beyond microbes to the larger genomes of more complex organisms. By improving sequence assemblies in this way, sequencing information can more readily be developed into understanding the role of biological processes and genes in DOE bioenergy and environmental missions.

Reference: Chin, C.-S., D. H. Alexander, P. Marks, A. K. Klammer, J. Drake, C. Heiner, A. Clum, A. Copeland, J. Huddleston, E. E. Eichler, S. W. Turner, and J. Korlach. 2013. “Nonhybrid, Finished Microbial Genome Assemblies from Long-Read SMRT Sequencing Data,” Nature Methods 10, 563–69. DOI: 10.1038/nmeth.2474. (Reference link)

For more information, see: http://bit.ly/JGI-Assembly.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 25, 2013

Carbon-11 Azelaic Acid as a Signaling Molecule for Mechanistic Studies in Plants

When a pathogen attacks a plant, the plant mounts an immune response that alerts the rest of the plant, a response called systemic acquired resistance (SAR). The chemical compound(s) responsible for inducing the immunity is a topic of intense interest for agriculture, including for bioenergy crops. For example, the application of a 9-carbon-atom-chain (C-9) dicarboxylic acid, azaleic acid, induces immunity, but the similar C-8 and C-10 diacids do not. One hypothesis is that the azaleic acid, but not the related acids, moves to distant parts of the plant. New radiochemistry imaging research at Brookhaven National Laboratory has developed a rapid method to label these three acids with Carbon-11 (11C, half-life of 20.4 min) for short-term (minutes to hours) tracking of their movement within the plant, and with Carbon-14 (14C, half-life of 5730 years) for long-term (hours to days) studies. When applied to a leaf, [11C]-azaleic acid shows substantial movement within an hour. When [14C]-azaleic acid is applied to the roots, it distributes throughout the whole plant within a day. These studies demonstrate that azaleic acid has the potential to be a mobile signaling molecule. The radioactive-carbon labeled diacids will have utility as scientific tools to unravel SAR mechanisms and other phenomena that impact production of robust bioenergy crops.

References: Yu, K., J. M. Soares, M. K. Mandal, C. Wang, B. Chanda, A. N. Gifford, J. S. Fowler, D. Navarre, A. Kachroo, and P. Kachroo. 2013. “A Feedback Regulatory Loop Between G3P and Lipid Transfer Proteins DIR1 and AZI1 Mediates Azelaic-Acid-Induced Systemic Immunity,” Cell Reports 3, 1266–78. DOI: 10.1016/j.celrep.2013.03.030. (Reference link)

Best, M., A. N. Gifford, S. W. Kim, B. Babst, M. Piel, F. Roesch, J. S. Fowler. 2012. “Rapid Radiosynthesis of [11C] and [14C]Azaleic, Suberic, and Sebacic Acids for in vivo Mechanistic Studies of Systemic Acquired Resistance in Plants,” Journal of Labelled Compounds and Radiopharmaceuticals 55, 39-43. DOI: 10.1002/jlcr.1951. (Reference link)

Contact: Prem Srivastava, SC-23.2, (301) 903-4071
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 16, 2013

Challenging Traditional Understanding of Microbial Gene Regulation

The traditional view of adaptive gene regulation is that bacteria adapt to sense their environment and then selectively tune the expression of their genes for optimal growth efficiency and survival (i.e., fitness) under those conditions. Numerous observations of seemingly nonoptimal gene expression in various microbes suggest, however, that reality is more complex. Researchers at Lawrence Berkeley National Laboratory’s ENIGMA Science Focus Area are gaining a more sophisticated understanding of bacterial gene regulation by examining over a thousand different combinations of gene expression patterns and growth conditions to determine their relation to overall fitness. Four genetically tractable bacterial species representing a broad diversity of microbial lifestyles have been studied: the aquatic metal-reducing environmental microbe Shewanella oneidensis, common intestinal bacterium Escherichia coli, ethanol-producing bacterium Zymomonas mobilis, and anaerobic sulfate-reducing bacterium Desulfovibrio alaskensis. In all four organisms, evidence of adaptive gene regulation was observed for only a small minority of genes; most gene expression was determined to be neutral or even detrimental to growth efficiency and fitness under experimental conditions. While these observations need testing in more realistic environmental settings and in microbial communities, the team concludes that under laboratory conditions, most gene expression is nonadaptive and reflects some form of indirect control unrelated to functional properties of specific genes. These study results add a new layer of complexity to our knowledge of the forces governing gene expression in microorganisms. They have important implications in understanding fundamental systems biology of microbes and attempts to engineer organisms with modified functional capabilities. This publication was selected as a research highlight in the June 2013 issue of Nature Reviews Microbiology.

Reference: Price, M. N., A. M. Deutschbauer, J. M. Skerker, K. M. Wetmore, T. Ruths, J. S. Mar, J. V Kuehl, W. Shao, and A. P. Arkin. 2013. “Indirect and Suboptimal Control of Gene Expression Is Widespread in Bacteria,” Molecular Systems Biology 9(660), DOI: 10.1038/msb.2013.16. (Reference link)

Research Highlight: Hofer, U. 2013. “Unfit Expression,” Nature Reviews Microbiology 11, 362–63. DOI: 10.1038/nrmicro3035. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 14, 2013

Understanding Genome Evolution with the Help of Plasmid Gene Pools

Understanding how genomes of organisms change over time underlies much of biology and its practical applications. Plasmids are DNA molecules that can replicate independently of chromosomal DNA in a cell. This enables organisms to "collect" and move genes to other organisms through lateral gene transfer (like “genomic email”) and contributes to prokaryotic genome evolution. To understand the depth and breadth of the prokaryote plasmid gene pool, scientists have isolated, sequenced, and compared plasmids from two wastewater sludge communities. The authors studied the “mobilome,” a name for the mobile elements in a community genome, by specifically targeting, separating, and purifying closed circular supercoiled DNAs (CCSD) originating from the plasmids. They found that the plasmids isolated from the sludge wastewater microbial communities turned out to contain primarily uncharacterized coding sequences. Besides lending credence to the idea that plasmids are crucial to genome innovation, evolution, and community structure and functioning, this study generated a large library of new genes involved in wastewater sludge degradation and processing that could enable new approaches to microbial wastewater cleanup. The study was enabled by the DOE Joint Genome Institute.

Reference: Sentchilo, V., A. P. Mayer, L. Guy, R. Miyazaki, S. G. Tringe, K. Barry, S. Malfatti, A. Goessmann, M. Robinson-Rechavi, and J. R. van der Meer. 2013. “Community-Wide Plasmid Gene Mobilization and Selection,” The ISME Journal, DOI: 10.1038/ismej.2013.13. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 06, 2013

New Method Reveals Bacterial Diversity in Subsurface Sediments

A fundamental question in microbial ecology is how do community diversity and composition change in response to perturbations. Most ecological studies have a limited ability to deeply sample community structure or a limited taxonomic resolution to track changing microbial diversity. To address this issue, researchers at the University of California, Berkeley, developed a method to assemble full length 16S rRNA sequences from short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. This approach, termed EMIRGE and optimized for large sequencing data size, allows researchers to differentiate the community composition among samples acquired before and after an environmental perturbation. Briefly, EMIRGE relies on a database of candidate 16S sequences for a template-guided assembly. An iterative method, sequencing reads are first aligned and probabilistically attributed to candidate 16S genes. Subsequently, candidate gene abundances and consensus sequences are adjusted based on the calculated probabilistic read attribution. The results were highly reproducible across very high alpha microbial diversity and abundant organisms from phyla that do not have cultivated representatives. This method allows for sensitive, accurate profiling of the “long tail” of low-abundance organisms that exist in many microbial communities and can resolve population dynamics in response to environmental change.

Reference: Miller, C. S., K. M. Handley, K. C. Wrighton, K. R. Frischkorn, B. C. Thomas, and J. F. Banfield. 2013. “Short-Read Assembly of Full-Length 16S Amplicons Reveals Bacterial Diversity in Subsurface Sediments,” PLoS ONE 8(2), e56018. DOI: 10.1371/journal.pone.0056018. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 06, 2013

Understanding Enzymes that Help Convert Biomass to Biofuels

A key step in the production of biofuels from biomass is hydrolytic breakdown of cellulose, a major component of all plants, into simple, fermentable sugars. Many natural systems carry out this breakdown, and much research is devoted to find systems that are highly efficient and thus candidates for inclusion in a biofuel production system. A new study of a subfamily of glucosidase enzymes (6-P-β-glucosidases), critical to efficient hydrolysis of cellulose, uses x-ray crystallography to determine their structures and how they bind to cellulose molecules. The researchers isolated these enzymes from two bacteria commonly found in the digestive tracts of many mammals, including humans: Lactobacillus plantarum and Streptococcus mutans. They obtained structures of the enzymes alone and bound to key cellulose breakdown molecules, using the Structural Biology Center’s stations at Argonne National Laboratory’s Advanced Photon Source. Different bacteria show different P-β-glucosidase and P-β-galactosidase activities. The structures and functional studies enabled the scientists to define structural features shared by glucosidases and galactosidases and those that are unique to the 6-P-β-glucosidases subfamily. Both enzymes show hydrolytic activity against 6’-P-β-glucosides but exhibit surprisingly different kinetic properties and affinities for substrates. Considering the conservation of the overall structures and active sites of various 6-P-β-glucosidases, the differences at their ligand binding subsites and the entrance to the active site are likely the determinants of their substrate specificities. These new findings will help scientists studying the design of efficient enzyme systems for biofuel production and will also have implications for human health.

Reference: Michalska, K., K. Tan, H. Li, C. Hatzos-Skintges, J. Bearden, G. Babnigg, and A. Joachimiak. 2013. “GH1-Family 6-P-ß-Glucosidases from Human Microbiome Lactic Acid Bacteria,” Acta Crystallographica Section D: Biological Crystallography 69, 451–63. DOI: 10.1107/S0907444912049608. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 01, 2013

Improving Cyanobacterial Synthesis of Alkanes

Cyanobacteria are important photoautotrophic organisms that can capture carbon dioxide and convert it into a suite of organic compounds such as high-density liquid fuels. Using synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy as a high-throughput imaging method, researchers tracked metabolic phenotypes of Synechocystis 6803, which was engineered for enhanced production of alkanes and free fatty acids. Multivariate SR-FTIR data analysis revealed biochemical shifts in the engineered cells. These results demonstrate the applicability of SR-FTIR spectromicroscopy for rapid metabolic screening and phenotyping of live individual cells. The research was conducted using resources at the Advanced Light Source at Lawrence Berkeley National Laboratory.

Reference: Hu, P., et al. 2013. “Metabolic Phenotyping of the Cyanobacterium Synechocystis 6803 Engineered for Production of Alkanes and Free Fatty Acids,” Applied Energy 102, 850–59. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Alkane Biosynthesis. Comparison of visible and infrared images shows localized production of alkanes adhering to a cyanobacterium’s outer cell surface (represented by rainbow-colored speckles; red = maximum). more...

Image Credit: Lawrence Berkeley National Laboratory



January 30, 2013

Plants, Fungi, and Microbes: Symbiosis in Carbon and Nitrogen Cycling

Arbuscular mycorrhizal (AM) fungi form intimate affiliations with the roots of many plant types. This classic example of symbiosis is commonly understood to involve AM fungi helping the plants take up soil nutrients. In exchange, the fungi receive some of the sugars generated by the plants from photosynthesis. Although AM fungi play a large role in carbon and nitrogen cycling in terrestrial environments, details of how they actually function remain poorly understood. In particular, the impact of AM fungi on soil microbe communities has not been examined in detail due to the difficulty of tracking nanoscale processes in complex soil habitats. U.S. Department of Energy researchers at the University of California Berkeley and Lawrence Livermore National Laboratory used a combination of "omics" tools and nanoscale tracking of isotopically labeled compounds to dissect interactions of AM fungi and soil microbial communities in carefully constructed soil microcosms. Plant-affiliated AM fungi were allowed to colonize small chambers containing soil samples and radiolabelled dead plant material ("litter"). The team found that the AM fungi have a significant impact on surrounding microbial community composition, increasing the abundance of microbes involved in plant litter degradation. During degradation of litter in soil, microbes play an important role in liberating nitrogen compounds bound in dead plant matter. The team observed significant uptake of microbially released nitrogen (but not carbon) by the AM fungi. These findings reveal another layer of complexity in this symbiotic system and yield another important puzzle piece towards understanding the complex routes by which carbon and nitrogen flow through ecosystems.

Reference: Nuccio, E. E., A. Hodge, J. Pett-Ridge, D. J. Herman, P. K. Weber, and M. K. Firestone. 2013. "An Arbuscular Mycorrhizal Fungus Significantly Modifies the Soil Bacterial Community and Nitrogen Cycling During Litter Decomposition," Environmental Microbiology, DOI: 10.1111/1462-2920.12081. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 17, 2013

New Analytical Tool Enables Switchgrass Improvement

Switchgrass (Panicum virgatum L.) is a prime bioenergy feedstock candidate due to its high biomass yields, minimal input requirements, broad adaptability, and perenniality. However, its large genome size, complicated genetics, and lack of a reference genome make efforts to improve switchgrass extremely challenging. Some of these difficulties can be overcome with genotyping-by-sequencing (GBS), a relatively low-cost method that targets a fraction of the genome for sequencing. GBS has already been used in many plant species to find molecular markers called single nucleotide polymorphisms (SNPs). To be both accurate and economical, however, this strategy requires a fully sequenced and assembled reference genome. To respond to this challenge, researchers funded in part by the joint U.S. Department of Agriculture-U.S. Department of Energy Plant Feedstocks Genomics for Bioenergy Program used GBS to develop a SNP discovery platform that does not require a reference genome and that can be applied to any complex plant species. This pipeline, called the Universal Network-Enabled Analysis Kit (UNEAK), was validated with maize and then successfully tested on switchgrass. Over one million SNPs were discovered in the switchgrass collection and used to construct high-density linkage maps, providing insight into the genetic diversity, population structure, phylogeny, and evolution of this species. UNEAK is providing an invaluable resource for switchgrass improvement programs.

Reference: Lu, F., A. E. Lipka, J. Glaubitz, R. Elshire, J. H. Cherney, M. D. Casler, E. S. Buckler, and D. E. Costich. 2013. "Switchgrass Genomic Diversity, Ploidy, and Evolution: Novel Insights from a Network-Based SNP Discovery Protocol," PLoS Genetics 9(1), e1003215. DOI: 10.1371/journal.pgen.1003215. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 16, 2013

Marginal Lands: A Valuable Resource for Sustainable Bioenergy Production

Growing plants on marginal lands, or lands unsuitable for conventional agricultural crops, is a promising route towards attaining sufficient cellulosic biomass for the production of biofuels without compromising food crops. However, both the availability of such lands as well as the potential environmental impacts (e.g., greenhouse gas emissions) resulting from widespread biofuel crop production remain uncertain. Researchers at the U.S. Department of Energy's Great Lakes Bioenergy Research Center (GLBRC) report results from the first assessment of the total biomass potential of these lands, including an estimate of greenhouse gas benefits and the productivity potential of unmanaged lands. Using 20 years of data from 10 Midwest states, the researchers compared both productivity and greenhouse gas impacts of several potential biofuel feedstocks, including corn, poplar, alfalfa, and old field vegetation, and then used supercomputers to model the biomass production required to support local biorefineries. The assessment shows that if properly managed, marginal lands could provide sufficient biomass to support a viable cellulosic biofuel production industry while benefiting conservation efforts and the environment.

Reference: Gelfand, I., R. Sahajpal, X. Zhang, R. C. Izaurralde, K. L. Gross, and G. P. Robertson. 2013. "Sustainable Bioenergy Production from Marginal Lands in the U. S. Midwest," Nature, DOI: 10.1038/nature11811. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 14, 2012

Metabolic Imaging: Watching Sugars Move in Plants

Fluorine-18 is a radioactive isotope that emits positrons. Using positron emission tomography (PET), scientists can image the movement and localization, in living organisms, of molecules that contain fluorine-18. Fluorine-18-labeled-fluorosugars, that is, natural sugars into which fluorine-18 atoms have been incorporated, enable study of the mechanisms by which living organisms use and process these biomolecules and offer opportunities to observe sugar distribution and metabolism in real time. Fluorine-18 fluoro-deoxyglucose (FDG) has already been established as an important PET imaging agent in human medicine. It is well known that vascular plants transport the bulk of their carbohydrate load in the form of sucrose. Now, U.S. Department of Energy scientists at the University of Missouri—Columbia have synthesized fluorine-18-fluoro-deoxy-sucrose (FDS) and used it to obtain the first images of corn plant leaves that demonstrate realtime transport of the sugar. Their results will enable investigators to image sucrose metabolism in living plants and, from these images, gain insight into metabolic pathways in plants with potential value for biofuel production.

Reference: Gaddam, V., and M. Harmata. 2013. “Synthesis of 6′-Deoxy-6′-Fluorosucrose,” Carbohydrate Research 369, 38–41. DOI: 10.1016/j.carres.2012.12.001. (Reference link)

Contact: Prem Srivastava, SC-23.2, (301) 903-4071
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 09, 2012

Using Synchrotron Spectroscopy to Understand How a Protein Evolves

A major challenge in research to enable large-scale production of biofuels is developing enzymes that are highly efficient in converting biomass components into usable fuels. Enzymes are proteins that are configured to catalyze such conversions. Many protein structures are known, including those of many valuable enzymes. Much less is known about how small changes in a protein’s composition can change its three-dimensional structure and control its catalytic efficiency, or even convert a protein with no catalytic function into one that is an efficient catalyst. New research shows the structural basis for conversion by directed evolution of a non-catalytic small protein into an enzyme that is an effective catalyst for linking RNA molecules. The scientists used an Extended X-ray Absorption Fine Structure (EXAFS) station at the Stanford Synchrotron Radiation Lightsource (SSRL) to determine the active-site structure of the newly synthesized enzyme. The EXAFS experiments were able to show the exact chemical environment of each zinc atom in the new enzyme, leading to an explanation of why it had developed the catalytic activity. The research was carried out by a team of scientists from the University of Minnesota and SSRL and is published in Nature Chemical Biology.

Reference: Chao, F.-A., et al., 2013. “Structure and Dynamics of a Primordial Catalytic Fold Generated by In Vitro Evolution,” Nature Chemical Biology 9, 81–83. DOI: 10.1038/nchembio.1138. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 07, 2012

Finding a Steady-State Solution in Dynamical Biological Networks

Cellular biochemical networks govern biological function and are strongly influenced by the exchange of molecules between the cell and its environment. Modeling this exchange process and its impact on cellular networks for whole microbial cells will be a key step in developing biology-based applications in bioenergy and other Department of Energy (DOE) mission areas. However, it has been a problem to represent nutrient exchange with the environment for genome-scale kinetic models, in a manner consistent with the existence of a steady state. New research has developed a mathematical model that establishes sufficient conditions for a non-equilibrium steady-state for cellular biochemical networks. The research proves the theorem that reactions conserving mass and kinetic rate laws are sufficient conditions for the existence of a non-equilibrium steady state. The new study demonstrates how to mathematically model the exchange of molecules between any cell and its environment. The results of this DOE Scientific Discovery through Advanced Computing (SciDAC) research by Fleming and Thiele of the University of Iceland are foundational for future efforts to computationally model non-equilibrium steady states as part of  whole cell microbial models.

Reference: Fleming, R. M. T., and I. Thiele. 2012. “Mass Conserved Elementary Kinetics Is Sufficient for the Existence of a Non-Equilibrium Steady State Concentration,” Journal of Theoretical Biology 314, 173–81. DOI: 10.1016/j.jtbi.2012.08.021. (Reference link)

Contact: Christine Chalk, SC-21.1, (301) 903-5152, Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 07, 2012

Increased Nitrogen Deposition Slows Carbon Decomposition in Forest Soils

Global production of agricultural fertilizers has vastly increased the amount of nitrogen compounds entering natural terrestrial ecosystems. Although it is clear that increased nitrogen availability boosts primary productivity (i.e., plant growth) in ecosystems, the impacts of this nitrogen influx on the decomposition of dead plant material by soil microbes remain poorly understood. A collaborative team of U.S. Department of Energy researchers at the Universities of Michigan and Oklahoma examined carbon decomposition by soil fungi and bacteria at an experimental forest site in Michigan. GeoChip 4.0, a DNA microarray containing probes for thousands of functional genes, was used to measure expression of genes involved in degradation of complex carbon compounds in soil samples from sites that have been exposed to elevated nitrogen input for the past 18 years. Compared to nearby control plots, sites with elevated nitrogen showed significant decreases in the diversity and overall expression levels of fungal and actinobacterial genes involved in deconstruction of cellulose, lignin, and other plant compounds. This finding correlates with a long-term observation of decreased carbon decomposition rates in soils at the nitrogen-elevated sites and points to the specific mechanism underlying this shift. These findings shed new light on poorly understood processes occurring in forest soils and improve our ability to better predict how ecosystems will respond to changing environmental variables.

Reference: Eisenlord, S. D., Z. Freedman, D. R. Zak, K. Xue, Z. He, and J. Zhou. 2013. "Microbial Mechanisms Mediating Increased Soil Carbon Storage under Elevated Atmospheric Nitrogen Deposition," Applied Environmental Microbiology 79, 1191–99. DOI: 10.1128/AEM.03156-12. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 23, 2012

Watching Plant Biomass Breakdown to Improve Biofuel Production

Sustainable and cost-effective production of biofuels from plant biomass is hindered by the cost of pretreatment and low sugar yields after enzymatic hydrolysis of plant cell wall polysaccharides. Many studies have looked at enzymatic action on individual biomass components, but in nature, the plant cell wall is a complex, networked structure that interacts concertedly with pretreatment enzymes. To fully understand the mechanisms of enzymatic plant cell wall deconstruction for optimal production of bioenergy from biomass, it is imperative to understand the whole system. Scientists at the U. S. Department of Energy’s (DOE) BioEnergy Science Center (BESC) and DOE National Renewable Energy Laboratory (NREL) have addressed this problem by using a combination of advanced microscopic imaging methods in a correlative, real-time manner to examine both fungal and bacterial enzyme systems. With this new technology, they are able to localize the enzymatic sites of action without compromising the cell wall’s structural integrity. The results suggest that an optimal strategy for enhancing fermentable sugar yield from enzymatic deconstruction is to modify lignins to be more amenable to removal through pretreatment while maintaining polysaccharide integrity, improving accessibility to enzyme action.

Reference: Ding, S.-Y., Y.-S. Liu, Y. Zeng, M. E. Himmel, J. O. Baker, and E. A. Bayer. 2012. “How Does Plant Cell Wall Nanoscale Architecture Correlate with Enzymatic Digestibility?” Science 338(6110), 1055–60. DOI: 10.1126/science.1227491. (Reference link).

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 22, 2012

Biochemistry of a Mysterious Microbial Community

Subsurface microbial communities are highly diverse and comprise an enormous fraction of Earth’s biomass, but lack of knowledge related to their ecological function makes understanding their ongoing biogeochemical processes difficult. Using synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy to probe biofilm samples from a cold subsurface sulfur spring, researchers recently determined how bacteria and archaea work together to influence global sulfur and carbon cycles. By revealing the bright spectral signals of akylic and methyl groups, together with sulfur functional groups, SR-FTIR unambiguously identified the bacteria’s sulfur-oxidizing metabolic activity. Archaeal cells, which were the dominant population in this biofilm, showed no such activity, suggesting a thriving mutual metabolism of archaea and bacteria. The research was conducted using resources at the Advanced Light Source at Lawrence Berkeley National Laboratory.

Reference: Probst, A. J., et al. 2012. “Tackling the Minority: Sulfate-Reducing Bacteria in an Archaea-Dominated Subsurface Biofilm,” The ISME Journal 7, 635–51. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 12, 2012

Engineering Secondary Cell Walls in Plants

The polysaccharide polymers of plant cell walls provide a carbon and energy source for biofuel production, but they are embedded in lignin, which gives plants their required rigidity but is also primarily responsible for the recalcitrance of plant biomass to enzymatic hydrolysis. Previous attempts to engineer reduced lignin content in plants were imprecise and resulted in unacceptable negative impacts on plant growth because of vessel integrity loss. In this work, researchers engineered lignin and polysaccharide biosynthesis in a cell-type specific manner such that lignin was greatly reduced in the normally lignin-rich fiber cells, and the amount of polysaccharide polymers was much greater in vessel cells. The resulting plants were viable and grew normally. When biomass from these engineered plants was subjected to enzymatic digestion, more sugars were released than from wild-type plants, a desirable trait for biofuels production.

Reference: Yang, F., P. Mitra, L. Zhang, L. Prak, Y. Verhertbruggen, J.-S. Kim, L. Sun, K. Zheng, K. Tang, M. Auer, H. V. Scheller, and D. Loque. 2013. “Engineering Secondary Cell Wall Deposition in Plants,” Plant Biotechnology Journal 11, 325–335. DOI: 10.1111/pbi.12016. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 12, 2012

Soil Microbes Eat Nitrous Oxide, a Potent Greenhouse Gas

The use of large amounts of nitrogen fertilizer in modern agriculture has resulted in massive releases of nitrous oxide (N2O) into the atmosphere. Although shorter lived than CO2, N2O is over 300 times more potent as a greenhouse gas, so understanding its role and behavior in global climate change is important. Soil microbes naturally consume ammonia in fertilizers, converting it into N2O or dinitrogen gas (N2), a harmless component of the atmosphere. Previous attempts to estimate the abundance of microbes that perform these processes have significantly overestimated N2O production, suggesting that a large, but undetected group of microbes is converting ammonia to N2. In a new study, researchers have used a comparative genomics approach to identify new gene sequences involved in conversion of ammonia to N2 and demonstrated that this genetic pathway is present in several abundant groups of soil microbes not previously thought to be involved in nitrogen conversion. Preliminary experiments suggest that these organisms are capable of this form of metabolism in the laboratory and that the relevant genes are present in soil samples. These results have revealed an important missing piece in our understanding of the terrestrial nitrogen cycle. Further research on the physiology of these organisms and determination of their environmental abundance should improve model predictions for release of greenhouse gasses from soils of bioenergy landscapes or other agricultural systems.

Reference: Sanford, R. A., D. D. Wagner, Q. Wu, J. C. Chee-Sanford, S. H. Thomas, C. Cruz-García, G. Rodríguez, A. Massol-Deyá, K. K. Krishnani, K. M. Ritalahti, S. Nissen, K. T. Konstantinidis, and F. E. Löffler. 2012. “Unexpected Nondenitrifier Nitrous Oxide Reductase Gene Diversity and Abundance in Soils,” Proceedings of the National Academy of Sciences USA 109(48), 19709–714. DOI: 10.1073/pnas.1211238109. (Reference link).

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 06, 2012

Identifying the Best Biofuel-Producing Microbes

To use a microbe as a factory to make a desired product, a bacterial strain that already produces the compound is treated to generate many mutants, some of which may produce more of the product. From all these new variants, the challenge is to identify those microbes that make the largest amounts of the desired compound. This is particularly difficult when the target compound (e.g., a biofuel) does not confer any selective advantage to the microbe. To solve this problem, researchers at the U.S. Department of Energy’s (DOE) Lawrence Berkeley National Laboratory and DOE Joint BioEnergy Institute designed a “biosensor”—a genetic regulator that “senses” the presence of the desired product (e.g., butanol). The expression of a gene that confers an advantage to the microbe, such as resistance to the antibiotic tetracycline, is then induced by the presence of the biosensor. Butanol biosensor-containing Escherichia coli cells, for example, grow in the presence of the antibiotic only if the medium also contains butanol. Finally, plasmids capable of synthesizing various amounts of butanol were introduced into E. coli containing the butanol biosensor and growing in tetracycline-containing medium. High butanol-producing cells could readily be identified by their faster growth rates. This approach will facilitate the selection of microbial strains that produce large quantities of any small molecule, an important step toward the development of renewable biofuels.

Reference: Dietrich, J. A., D. L. Shis, A. Alikhani, and J. D. Keasling. 2012. “Transcription Factor-Based Screens and Synthetic Selections for Microbial Small-Molecule Biosynthesis,” ACS Synthetic Biology, DOI: 10.1021/sb300091d. (Reference link).

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 01, 2012

Impacts of Elevated CO2 on Photosynthetic Microbes in Arid Ecosystems

In many harsh desert environments, microbial “biocrust” communities dominated by photosynthetic bacterial species (cyanobacteria) cover up to 70% of the land surface and play important roles in nutrient cycling, water retention, and stabilizing soil against erosion. These communities are highly adapted to the specific environmental conditions of arid ecosystems, and it is unclear what impacts climate change processes may have on them. Operating in collaboration with DOE’s long-term Free-Air CO2 Enrichment (FACE) program, researchers at Los Alamos National Laboratory have published new findings on the effects of 10 years of controlled elevated CO2 exposure on cyanobacterial biocrusts using environmental metagenomics. Natural biocrusts exposed to elevated CO2 (550 ppmv) were shown to have significantly reduced abundance of cyanobacteria relative to plots exposed to ambient CO2 concentrations (360 ppmv). These findings were correlated with an observed loss of biocrust coverage in the elevated CO2 plots, although curiously, total soil biomass measurements did not change significantly. Loss of cyanobacterial abundance appears to be at partially related to increased damage from oxidative stress, with genes involved in resistance to this kind of stress appearing more frequently in the elevated CO2 samples. Although more study is needed, these results present preliminary evidence suggesting that increasing atmospheric CO2 concentrations have a deleterious impact on desert biocrusts and may result in decreased performance by these communities.

Reference: Steven, B., L. Gallegos-Graves, C. M. Yeager, J. Belnap, R. D. Evans, and C. R. Kuske. 2012. “Dryland Biological Soil Crust Cyanobacteria Show Unexpected Decreases in Abundance Under Long-Term Elevated CO2,” Environmental Microbiology, DOI: 10.1111/1462-2920.12011. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 19, 2012

Marine Ecosystems More Complex Than Previously Thought

The tiny cyanobacterium Prochlorococcus is among the most abundant and important in the oceans, and distinct variants (“ecotypes”) exist at different water depths. An estimated 100 million cells of this unicellular organism can be found in a single liter of seawater. These cyanobacteria help remove some 10 billion tons of carbon from the atmosphere each year. New research addresses a long-held assumption that the size of a microbial population in the marine community corresponds to its level of activity in terms of carbon uptake and growth rate, thus determining its impact on global biogeochemical cycles. Researchers, including scientists at the U.S. Department of Energy’s Joint Genome Institute, studied the activity levels of several Prochlorococcus ecotypes at several locations in the Pacific and Atlantic oceans. The results suggest that the theory does not fully explain the link between abundance levels and activity. In their article, the authors state: “Our results suggest that low abundance microbes may be disproportionately active in certain environments and that some of the most abundant may have low metabolic activity.” “We observed uncoupling of abundance and specific activity of Prochlorococcus in the Sargasso Sea depth profile, which highlights deficiencies in our understanding of marine microbial ecology and population structure.” They conclude that marine ecosystem functioning is likely to be more complex and dynamic than previously thought. This finding has significant implications for understanding the role of the oceans in the global carbon cycle.

Reference: Hunt, D. E., Y. Lin, M. J. Church, D. M. Karl, S. G. Tringe, L. K. Izzo, and Z. I. Johnson. 2012. “The Relationship Between Abundance and Specific Activity of Two Bacterioplankton in Open Ocean Surface Waters,” Applied Environmental Microbiology, DOI: 10.1128/AEM.02155-12. (Reference link).

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 16, 2012

Proteome Atlas for the Poplar Tree

Populus, a fast-growing perennial tree, holds potential as a bioenergy crop due to its ability to produce large amounts of biomass on non-agricultural land. For woody perennial plants such as poplar, there is a tight coupling between growth and photosynthesis during the plant's lifetime. To understand this process, researchers at the U.S. Department of Energy's BioEnergy Science Center (BESC) have measured more than 11,000 proteins in different tissues of poplar, including mature leaves, young leaves, roots, and stems. They have developed a poplar proteome atlas that shows which proteins are present in the various tissue types at a given point in time. By mapping the proteins back to tissue-specific metabolic pathways, the BESC scientists demonstrated that the same organ can participate in two different growth stages. Their findings confirm prior hypotheses that mature leaves appear to function primarily in the generation of energy via photosynthesis while young leaves partition resources between growth and photosynthesis. This study illustrates that a comprehensive systems approach to proteomics can yield valuable information on the lifecycle of bioenergy-related plants. The paper is the cover article for the latest issue of Molecular and Cellular Proteomics.

Reference: Abraham, P., R. J. Giannone, R. M. Adams, U. Kalluri, G. A. Tuskan, and R. L. Hettich. 2013. "Putting the Pieces Together: High-Performance LC-MS/MS Provides Network-, Pathway-, and Protein-Level Perspectives in Populus, "Molecular and Cellular Proteomics 12, 106–119. DOI: 10.1074/mcp.M112.022996. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 10, 2012

Rapid Mapping of All Atoms in Biochemical Reactions

In the design and bioengineering of metabolic pathways for clean bioenergy and other applications, it is important to understand and eventually manipulate the movement of atoms in these biochemical reactions. For example, assessing how a reactant compound is transformed into a targeted product allows researchers to optimize for efficiency in the pathways. A new computational system (Minimum Weighted Edit-Distance or MWED) allows mapping of all the non-hydrogen atoms in biochemical reactions from the initial reactants to the final products. MWED relies on predicting the propensity of forming or breaking chemical bonds during a biochemical reaction. It then calculates and optimizes all possible solutions to the reaction of interest. Because it also uses a mixed-integer linear programming technique, it is three-fold faster than other, similar techniques. The MWED all atom pathway mapping was benchmarked on 2,446 manually curated biochemical reactions from the KEGG database. The researchers found that only 22 MWED-predicted reactions were in error (error rate of 0.9%) due mainly to difficulties in representing stereochemistry in the reactions. MWED offers research scientists an extremely fast and highly accurate method to model all atoms in biochemical reactions, both for novel bioengineering as well as for tracking isotopically labeled atoms in metabolic experiments.

Reference: Latendresse, M., J. P. Malerich, M. Travers, and P. D. Karp. 2012. “Accurate Atom-Mapping Computation for Biochemical Reactions,” Journal of Chemical Information and Modeling, DOI: 10.1021/ci3002217. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 04, 2012

Locating Hydrogen Atoms in a Protein Using Neutron Crystallography

Hydrogen atoms are notoriously difficult to locate in proteins, yet they are key atoms in many of the chemical reactions of life and comprise one-half of a protein's atoms. X-ray crystallography has been used to determine the atomic structure of many proteins and macromolecular complexes, but only a small fraction of the hydrogen atoms in these molecules can be located using this technique. In contrast, neutrons are scattered by hydrogen atoms, enabling determination of the position of these atoms in a protein molecule, though usually only to a medium resolution of about 2Å. Now, scientists at the Los Alamos Neutron Science Center have used the Protein Crystallography Station to determine the structure of a protein with the positions of its hydrogen atoms defined to an ultrahigh resolution of 1.1Å, the highest resolution ever for a neutron structure of a protein. They were able not only to locate nearly 95% of the hydrogen atoms in the protein at this resolution, but could determine the location of the hydrogen bonds that help determine the three-dimensional structure of the folded protein, and in some cases see how individual hydrogen atoms vibrate about their position in the protein. This new capability will improve understanding of the activity of many proteins, as well as guide computational modeling of systems such as protein-substrate and protein-drug complexes. The research was a collaboration of scientists at the University of Toledo, Los Alamos National Laboratory, and Oak Ridge National Laboratory.

Reference: Chen, J. C.-H., B.L. Hanson, S.Z. Fisher, P. Langan, and A.Y. Kovalevsky. 2012. "Direct Observation of Hydrogen Atom Dynamics and Interactions by Ultrahigh Resolution Neutron Protein Crystallography," Proceedings of the National Academy of Sciences (USA) 109(38), 15301–306. DOI: 10.1073/pnas.1208341109. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 04, 2012

Structural Patterning in Bacteria May Improve Their Bioenergy Uses

In comparison to multicellular plants and animals, bacteria are relatively simple, typically existing as single cells. However, some bacteria cooperate to form surprisingly sophisticated structures. The photosynthetic microbe Nostoc punctiforme forms long filaments of connected cells. At regular spacing along these filaments, individual cells differentiate to form heterocysts, non-photosynthetic cells that convert nitrogen gas into biologically useful nitrogen compounds. This patterning allows these microbes to separately perform both photosynthesis (which produces O2 as byproduct) and "fix" nitrogen using enzymes that are poisoned by oxygen, cooperatively exchanging the resulting nutrients between the cell types. In a new study, U.S. Department of Energy (DOE) researchers at the University of California, Davis, describe genetic mechanisms responsible for the establishment and maintenance of this distinctive pattern in growing filaments. When the expression of a series of regulatory genes (the "pat system") was experimentally manipulated, filaments formed with abnormal distributions of heterocysts. By analyzing these patterns and tracking the distribution of related proteins in dividing cells, the investigators were able to develop a new model describing the regulatory interactions resulting in the pattern that allows optimal photosynthesis and nitrogen fixation in the filaments. The results of this study provide valuable new insights into the mechanisms used by microbes to tune their functional attributes through the use of structural patterns and could lead to the development of new tools for optimizing processes in biological systems engineered for bioenergy applications.

Reference: Risser, D. D., F. C. Y. Wong, and J. C. Meeks. 2012. "Biased Inheritance of the Protein PatN Frees Vegetative Cells To Initiate Patterned Heterocyst Differentiation," Proceedings of the National Academy of Sciences (USA) 109, 15342-347. DOI: 10.1073/pnas.1207530109. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 31, 2012

New Computational Method To Simulate Behavior of Cellulose Fibers

Cellulose fibers provide the structural framework for plant cell walls and are critical for plant growth, stability, and normal function. These same properties of cellulose fibers are also the main obstacle for efficient conversion of biomass to biofuels. Molecular dynamic simulations can aid in understanding cellulose fiber crystallinity and its resilience to deconstruction; however, since the fibers are very large, realistic molecular simulations require extensive run times on leadership-class supercomputers. Recently Scientific Discovery through Advanced Computing (SciDAC) supported researchers at Oak Ridge National Laboratory, in collaboration with RIKEN National Lab in Japan, developed a coarse-grained simulation method termed REACH (Realistic Extension Algorithm via Covariance Hessian) that will enable more efficient simulation of large cellulose fibers. The REACH method reduces the complexity of the simulation (coarse graining) and directly relates molecular force parameters from the more complex all-atom simulation to the faster REACH simulation. Using this method, the researchers simulated the behavior of a cellulose fiber of 36 chains and 40 to 160 degrees of polymerization with a speed of up to 24 nanoseconds per day of computation. The REACH simulations are in agreement with previous findings that the hydrophobic face of the cellulose fiber is more easily deconstructed than the hydrophilic face. An extension of REACH is now being developed that will account for larger amplitude strand separation motions of the fibers thought to precede subsequent deconstruction.

Reference: Glass, D. C., K. Moritsugu, X. Cheng, and J. C. Smith. 2012. "REACH Coarse-Grained Simulation of a Cellulose Fiber," Biomacromolecules 13(9), 2634-44. DOI: 10.1021/bm300460f. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 31, 2012

Understanding Enzyme Specificity Through Systems-Level Metabolic Modeling

In biology, some enzymes are highly specialized and catalyze specific reactions with a few or only one substrate, while other enzymes are promiscuous and can catalyze reactions using a variety of substrates. This phenomenon also has been observed experimentally for microbes involved in bioenergy-related processes. What is not understood, however, is why, within an organism, some enzymes are highly specialized while others remain generalists. Recently, researchers addressed this question using whole genome metabolic reconstructions and analysis, including dynamical simulations of environmental changes to understand microbial responses. Their findings indicate that enzymes with very specialized function maintain a higher flux, or processing rate, and require more regulation of their activities. This higher flux and higher regulation allows these enzymes to be more responsive and adaptive to environmental surroundings and changes then their less specialized counterparts. This work also illustrates that understanding environmental cellular physiology is greatly enhanced when using a systems biology approach rather than approaches that are focused on single enzyme simulations. These new results offer a means of translating genomic information into functional capabilities, with particular relevance for microbes involved in biofuel production.

Reference: Nam, H., N. E. Lewis, J. A. Lerman, D.-H. Lee, R. L. Chang, D. Kim, and B. O. Palsson. 2012. "Network Context and Selection in the Evolution to Enzyme Specificity," Science 337(6098), 1101-04. DOI: 10.1126/science.1216861. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 24, 2012

Understanding How Microbial Membrane Transporters Work

Membrane transport proteins play a key role in controlling the movement of a wide variety of carbon sources into microbial cells, including complex sugars and plant structural polymers derived from lignin. The transporter profile also influences the composition and structure of microbial communities in soils. However, the functioning of these proteins has not been adequately characterized. Researchers at Argonne National Laboratory have studied a specific type of transporter called the ATP-binding cassette (ABC) proteins. Using a combination of functional characterization (ligand-binding thermal screens), analytical tools for structural analysis (x-ray crystallography), and a computational framework, the functions of ABC transporters have been identified and better defined. The binding strength of various ABC transporters to aromatic products of lignin degradation was determined, and a set of ABC microbial transporters not previously identified with aromatic product transport was found. High-resolution crystal structures were produced for seven of the strongly bound molecular complexes, providing insights into the molecular basis for the observed strong binding. They revealed essential details about the modes of molecular interactions (e.g., hydrogen bonds) and the physical configuration of the active binding site. Knowledge derived from these experiments creates a foundation for developing a sequence-based computational method to predict what molecules will bind similar, but uncharacterized transporters in other microbes.

Reference: Michalska, K., C. Chang, J. C. Mack, S. Zerbs, A. Joachimiak, and F. R. Collart. 2012. “Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins,” Journal of Molecular Biology 423(4), 555–75. DOI: 10.1016/j.jmb.2012.08.017. (Reference link)

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 22, 2012

New Genetic Tools for Engineering a Biomass-Degrading Microbe

Achieving efficient and cost-effective breakdown of cellulosic plant biomass remains a significant barrier to the development of economically competitive biofuels that do not compete with food supplies. The hot spring bacterium Caldicellulosiruptor has been shown to efficiently degrade biomass (e.g., switch grass and corn stover) at temperatures over 160° Fahrenheit, but further characterization and engineering of this organism for biofuel production has proven challenging due to a lack of tools for genetic manipulation. Researchers at the DOE BioEnergy Science Center (BESC) have now developed the first system allowing the stable introduction of foreign DNA elements into this microbe. This breakthrough is based on the identification of a Caldicellulosiruptor "immune system" that normally protects the bacterium from viral infection, destroying outside DNA before it can be integrated into the host genome. The BESC team was able develop a set of targeted nucleic acid modifications that protects DNA from the host immune system and allows the introduction of new genes and regulatory elements into the organism. Now that Caldicellulosiruptor is a step closer to the model status of an easily manipulated microbe like E. coli, the team can more effectively study the organism's unique cellulose-degrading properties and engineer new metabolic pathways that would allow direct conversion of plant biomass into next-generation biofuels.

Reference: Chung, D., J. Farkas, J. R. Huddleston, E. Olivar, and J. Westpheling. 2012. "Methylation by a Unique a-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725," PLoS ONE 7(8), e43844. DOI: 10.1371/journal.pone.0043844. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 22, 2012

Regulation of Wood Formation Characterized in Populus

Poplar is a promising bioenergy feedstock due to its rapid growth and large biomass, and because sugars extracted from the lignocellulosic biomass (wood) of these native trees can be fermented to form renewable biofuels. These sugars are embedded within lignin, a complex, rigid structure that is critical to the overall health of the plant but that also impedes extraction of the sugars. New U.S. Department of Energy research is providing insight into how the lignocellulosic material forms in poplar. The process involves the expression of a cascade of genes whose regulation is poorly understood. The researchers at North Carolina State University report their discovery of a single protein ("controller" protein) that regulates this cascade on multiple levels to ensure normal growth, doing so in a way never before seen in plants. The controller protein was found outside the cell nucleus. In the presence of one of four other related proteins, it is carried into the nucleus where the two proteins bind. The newly formed molecule then suppresses expression of the regulatory gene cascade. This discovery helps define how wood formation occurs at the molecular level, furthering our understanding of a process critical to plant growth. The results will help guide research to optimize bioenergy production from biomass.

Reference: Li, Q., Y.-C. Lin, Y.-H. Sun, J. Song, H. Chen, X.-H. Zhang, R. R. Sederoff, and V. L. Chiang. 2012. "Splice Variant of the SND1 Transcription Factor Is a Dominant Negative of SND1 Members and Their Regulation in Populus trichocarpa," Proceedings of the National Academy of Sciences (USA) 109(36), 14699-704. DOI: 10.1073/pnas.1212977109. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Poplar Trees provided by North Carolina State University



August 14, 2012

New Method for Delivering Biologically Active Molecules into Algae Cells

Algae can produce a wide variety of biofuels, chemical building blocks, nutrients, and proteins using sunlight as an energy source and carbon dioxide or other simple carbon compounds. DOE scientists at Lawrence Berkeley Lab have developed a new method to deliver radioactive or fluorescently labeled small molecules or protein probes into algal cells to monitor cellular messengers such as mRNA, gene expression or to develop biosensors. A molecular probe's ability to pass through the cell membrane is often restricted by its water and lipid solubility. The new method overcomes these restrictions, enabling transport of molecules across the cell wall and membrane barriers. The transporter technology is broadly applicable and can be used for the delivery of labeled probes into algal cells for the development of sensitive biological assays for dynamic imaging of gene expression. The technique is being further developed to transport genetic materials and for probing changes in the carbon metabolism of these cells. These advances will enable scientists to improve algae as a tool for a wide variety of applications.

Reference: Hyman, J. M., E. I. Geihe, B. M. Trantow, B. Parvin, and P. A. Wender. 2012 "A Molecular Method for the Delivery of Small Molecules and Proteins Across the Cell Wall of Algae Using Molecular Transporters," Proceedings of the National Academy of Sciences (USA) 109(33), 13,225-230. DOI: 10.1073/pnas.1202509109. (Reference link)

Contact: Prem Srivastava, SC-23.2, (301) 903-4071
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 07, 2012

Bacterium with Improved Hydrogen Production from Sunlight

One challenge to the commercialization of microbial production of hydrogen using sunlight is that the oxygen produced by photosynthesis decreases hydrogen production. Various biological mechanisms have evolved to separate the two reactions and scientists have been looking for engineering solutions, but the challenge is not yet solved. Scientists at the Pacific National Northwest Laboratory now have shown for the first time that a single-celled cyanobacterium, Cyanothece, is able to produce hydrogen and oxygen simultaneously without interruption for at least 100 hours. The bacteria produce hydrogen at relatively high rates without high cell density or inducing circadian rhythms, as required in studies by other researchers. Furthermore, there is little photo-damage and decay of the photosynthesis apparatus, perhaps enabled by the removal of excess electrons by the hydrogen production. These results and the improved understanding of the underlying cyanobacterial physiology will help advance the biotechnology of microbial hydrogen production.

Reference: Melnicki, M. R., et al. 2012. "Sustained H2 Production Driven by Photosynthetic Water Splitting in a Unicellular Cyanobacterium," mBio 3(4), e00197-12. DOI:10.1128/mBio.00197-12. (Reference link)

Contact: John Houghton, SC-23.2, (301) 903-8288, John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Marine Cyanobacterium Cyanothece
The single-celled marine cyanobacterium Cyanothece 51142, captured by a light microscope. Source: Washington University, St. Louis



August 03, 2012

Resequencing Poplar To Improve Its Use as a Bioenergy Feedstock

The fast-growing black cottonwood (Populus trichocarpa), a fast-growing tree that inhabits stream and river banks across a long north-south range of western North America, has been identified as a promising bioenergy crop. Many genetic and genomic resources for Populus have been developed and are being used to study the molecular basis of desirable traits such as biomass yield, cell wall characteristics, and environmental adaptation. To develop superior Populus cultivars for bioenergy feedstocks, it is necessary to understand the genetic and genomic structure of the Populus population to reliably detect phenotype-genotype associations, which informs suitable breeding approaches. Researchers at the DOE BioEnergy Research Center (BESC), together with the DOE Joint Genome Institute (DOE JGI), sequenced the genomes of 16 different black cottonwood varieties, broadly spanning north to south of the species' native range, and determined the population structure and genetic variation on a geographic scale. They found that significant genetic differentiation existed and was strongly correlated with latitudinal location of the sampled trees, suggesting that this species may have survived the past glaciation in multiple locations along the northwest of North America. The study demonstrates that advanced population genetics approaches should be more feasible in Populus than previously thought, increasing the potential for genetic improvement of Populus as a biofuel feedstock.

Reference: Slavov, G. T., S. P. DiFazio, J. Martin, W. Schackwitz, W. Muchero, E. Rodgers-Melnick, M. F. Lipphardt, C. P. Pennacchio, U. Hellsten, L. A. Pennacchio, T. C. Mockler, M. Freitag, A. Geraldes, Y. A. El-Kassaby, S. D. Mansfield, Q. C. B. Cronk, C. J. Douglas, S. H. Strauss, D. Rokhsar, and G. A. Tuskan. 2012. "Genome Resequencing Reveals Multiscale Geographic Structure and Extensive Linkage Disequilibrium in the Forest Tree Populus trichocarpa," New Phytologist, DOI: 10.1111/j.1469-8137.2012.04258.x. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 26, 2012

Switchgrass Chromosome Structure Revealed

Switchgrass is considered to be a promising biofuel feedstock because of its ability to produce high biomass yields on marginal lands with minimal inputs. Several efforts to improve switchgrass as a dedicated bioenergy crop have been initiated, but breeding efforts are hampered by the outbred, tetraploid nature of this species and by limited knowledge of its chromosome architecture. Researchers at the USDA-Agricultural Research Service have used sophisticated molecular, cytological, and imaging techniques to tease apart and unambiguously identify the nine relatively small and otherwise undistinguishable base chromosomes of a dihaploid switchgrass line, producing the first karyotype (systematized arrangement of the total chromosome complement) of this bioenergy crop. The scientists were able to distinguish the two switchgrass ecotypes as well as the two basic subgenomes using this resource. This new capability will greatly facilitate identification of specific gene pools (e.g., regionally adapted cultivars) for switchgrass improvement toward the goal of making it a productive biomass crop. The research was supported in part by the joint USDA-DOE Plant Feedstocks Genomics for Bioenergy Program.

Reference: Young, H. A., G. Sarath, and C. M. Tobias. 2012. "Karyotype Variation Is Indicative of Subgenomic and Ecotypic Differentiation in Switchgrass," BMC Plant Biology 12, 117. DOI: 10.1186/1471-2229-12-117. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 02, 2012

How a Surface Protein Enables Metabolism of a Methane-Generating Microbe

Methanogenic microbes known as Archaea carry out many chemical transformations essential for anaerobic carbon recycling in virtually all environments. However, little is known about how raw materials for, and products of, these transformations are transported between an Archaeal cell and its environment. Research now has determined the structure of a key surface-layer protein of a methane-generating microbe, Methanosarcina acetivorans, enabling new insights into how this microbe communicates with its surroundings. The new information enables construction of a diagram of the cell envelope's surface layer, showing the pores through which chemical species move back and forth. Three types of pores with distinctly different sizes and shapes were identified. All of them are small and highly negatively charged, which means that they are highly selective about which substances can pass through the layer into the cell. DNA sequencing of several related species of Methanosarcinales suggests that the structures of their surface layer proteins are similar to the one in M. acetivorans. These results provide valuable information for understanding the role of these microbes in producing methane in natural environments, a potentially major factor in global carbon cycling. The research was led by Robert Gunsalus of the UCLA-DOE Institute of Genomics and Proteomics.

Reference: Arbing, M. A., et al. 2012. "Structure of the Surface Layer of the Methanogenic Archaean Methanosarcina acetivorans," Proceedings of the National Academy of Sciences of the USA 109(89), 11,812-817. DOI: 10.1073/pnas.1120595109. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 29, 2012

Fungal End to Coal and the Carboniferous Period: A Possible Solution for Biofuels?

Much of the world's coal was generated 300-360 million years ago during the Carboniferous period. Wood (a major pool of organic carbon that is highly resistant to decay largely due to its lignin content) was deposited, transformed to peat, and eventually transformed to coal. But coal formation may also have declined from an unlikely source: fungi. These fungi had enzymes (ligninases) capable of degrading lignin, a category of enzyme important for the Department of Energy's bioenergy mission, since lignin in plant biomass hinders biomass conversion to biofuels. An international team of scientists from Clark University and DOE's Joint Genome Institute has proposed that a species of fungus, first appearing at about the end of the Carboniferous period, could more efficiently break down dead plant matter, possibly leading to the decline in coal formation. By comparing the genomic sequences of 31 fungi, including 12 sequenced for this study, the researchers showed that genes able to degrade lignin first appeared at the end of this period. Instead of becoming coal, the plant biomass decayed and the carbon was released into the atmosphere as carbon dioxide. This research provides insights into the origin of ligninases that can be used to develop processes for converting plant and tree biomass into bioenergy products.

Reference: Floudas, D., et.al. 2012. "The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes," Science 336, 1715-19. DOI: 10.1126/science.1221748. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Amanita muscaria of the Class Agaricomycetes
Source: Lawrence Berkeley National Laboratory



June 28, 2012

Understanding How microbes Work Together: Methane Production by Partnered Microbes

Methanogenic archaea and sulfate-reducing bacteria (SRBs) both play important roles in the carbon cycle of soils, wetlands, and other environments with limited oxygen availability. SRBs are versatile consumers of a variety of organic compounds, while methanogens primarily convert hydrogen and CO2 into methane. Neither of these organisms is capable of independent growth on lactate, a small organic compound that is an important intermediate in food webs, but can consume it when working together in a partnership called syntrophy. Researchers at the University of Washington and Lawrence Berkeley National Laboratory have published a new study that helps explain how this partnership works. They carried out a high-resolution transcriptomic study of changes in gene expression of the methanogen Methaococcus maripaludis during syntrophic growth on lactate with the SRB Desulfovibrio vulgaris as a partner. The methanogen shows a substantial shift in genes associated with conversion of hydrogen to methane, switching over to a parallel set of enzymes that may be better adapted to low rates of hydrogen production and other conditions associated with syntrophy. These results advance our understanding of microbial production of a potent greenhouse gas and highlight the important role of subtle interactions between organisms that influence environmental processes.

Reference: Walker, C. B., A. M. Redding-Johanson, E. E. Baidoo, L. Rajeev, Z. He, E. L. Hendrickson, M. P. Joachimiak, S. Stolyar, A. P. Arkin, J. A. Leigh, J. Zhou, J. D. Keasling, A. Mukhopadhyay, and D. A. Stahl. 2012. "Functional Responses of Methanogenic Archaea to Syntrophic Growth," The ISME Journal 6, 2045-2055. DOI: 10.1038/ismej.2012.60. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 18, 2012

Genomic Encyclopedia of Bacteria and Archaea Finds More Cellulases

The biotechnology and biofuels industries are particularly interested in cellulases, enzymes that break down cellulose, the most abundant organic compound on Earth and the component that makes up 33 percent of all plant matter. Cellulases from a group of aerobic bacteria called Actinobacteria are of special interest as sources of enzymes useful for biofuel production from lignocellulosic biomass. They have distinct features and cellular organization when contrasted to those in anaerobic bacteria (such as the Clostridia). The DOE Joint Genome Institute (JGI) has sequenced the genomes of 11 diverse strains of these bacteria. Comparative analysis using the JGI's Integrated Microbial Genomes system followed by experimental verification identified eight cellulolytic Actinobacterial species that were not previously known to degrade cellulose. Of seven organisms tested, six showed activity in assays for cellulases. One organism, Catenulispora acidiphilia, previously unknown to break down cellulose, has 15 predicted cellulases and may be used in future biofuel production. This work, conducted under the umbrella of the JGI's Genomic Encyclopedia of Bacteria and Archaea (GEBA) project, broadens the repertoire of useful enzymes beyond those previously recognized.

Reference: Anderson, I., B. Abt, A. Lykidis, H.-P. Klenk, N. Kyrpides, and N. Ivanova. 2012. "Genomics of Aerobic Cellulose Utilization Systems in Actinobacteria," PLoS ONE 7(6), e39331. DOI: 10.1371/journal.pone.0039331. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 08, 2012

Watching Carbon Dioxide Move in Plant Leaves

U.S. Department of Energy (DOE) plant biology research seeks to optimize plant productivity, both for biofuel development and for carbon sequestration in biomass. Taking a lesson from medical technology, plant biologists are now using sophisticated imaging technology to learn more about nutrient utilization in plants by watching the movement of those nutrients in real time. Positron emission tomography (PET) imaging has been used to study carbon transport in live plants using 11CO2, but because plants typically have very thin leaves, littlemedium is availablefor the emitted positronsto undergo an annihilation event within the plant leaf resulting in limited sensitivity for PET imaging.To address this problem DOE’s Thomas Jefferson Laboratory has developed a compact beta-positive, beta-minus particle imager (PhytoBeta imager) for 11CO2 leaf imaging. The detector is equipped with a flexible arm to allow its placement on or under a leaf while maintaining its original orientation. The detector has been used to generate dynamic images of carbon translocation in a leaf of the spicebush (Lindera benzoin) under two transient light conditions. The PhytoBeta detector system and methodology opens new possibilities for short-lived radioisotope use in plant biology research,especially for problems relatedto carbon utilization, transport, and sequestration.

Reference: Weisenberger, A. G., B. Kross, S. Lee, J. McKisson, J. E. McKisson, W. Xi, C. Zorn, C. D. Reid, C. R. Howell, A. S. Crowell, L. Cumberbatch, B. Fallin, A. Stolin, and M. F. Smith. 2012. “PhytoBeta Imager: A Positron Imager for Plant Biology,” Physics in Medicine and Biology 57(13), 4195–210. DOI: 10.1088/0031-9155/57/13/4195. (Reference link)

Contact: Dean Cole, SC 23.2, (301) 903-3268
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 07, 2012

Improving the Reliability of Metagenomic Sequencing Data

Natural microbial communities usually are made up of a large variety of species. Knowing the community's composition is important for addressing DOE energy and environmental missions. Sequencing of the community's combined genome (the ‘metagenome') is now the best way to characterize these communities, but to make sense of the data, it is important to accurately account for all of the experimental and instrumental errors in the process. Up to now, the instrumental errors have been routinely estimated, but not the sample collection and preparation errors. As part of the DOE Systems Biology Knowledgebase project, researchers at Argonne National Laboratory have developed an open-source program called DRISEE (duplicate read inferred sequencing error estimation) to account for both types of errors. DRISEE identifies errors that could be due to sample collection, intermediary DNA processing techniques, or to the instruments themselves. Using DRISEE, the authors reproduce known error rates from a given set of standard data. They then apply this method to show that many factors can contribute to errors in sequencing including read length and sample preparation. Although this method so far only applies to 454 and Illumina sequencing, it will provide valuable assistance to scientists trying to assemble genomes from metagenomic data by helping them determine if the sequence data has a true error and should be disregarded or if it is a natural sequence variation and should be included.

Reference: Keegan, K. P., W. L. Trimble, J. Wilkening, A. Wilke, T. Harrison, M. D'Souze, and F. Meyer. 2012. "A Platform-Independent Method for Detecting Errors in Metagenomic Sequencing Data: DRISSE," PLoS Computational Biology 8(6), e1002541. DOI: 10.1371/journal.pcbi.1002451. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 25, 2012

New Clues to Cold Tolerance and Lipid Production for Biofuels in Polar Alga

Algae are of major interest to researchers who are developing alternative energy sources. For example, lipids making up algal membranes can be transformed into biodiesel. One photosynthetic alga, Coccomyxa subellipsoidea C-169, was recently isolated in Antarctica and now is the first alga from a polar region to have its genome sequenced. Surprisingly, the alga thrives at temperatures close to 20°C, though it is tolerant of the cold temperatures in the Antarctic. C. subellipsoidea was sequenced by the DOE Joint Genome Institute, and its predicted protein families were compared with those from several other sequenced green algae. The researchers found that the polar alga had more enzymes involved in lipid metabolism, such as those that desaturate fatty acids. This greater versatility of lipid metabolism is thought to have contributed to its adaptation to cold. The research will provide insights on novel enzymes that may prove useful to researchers working to harness algae for biodiesel production.

Reference: Blanc, G., et al. 2012. "The Genome of the Polar Eukaryotic Microalga Coccomyxa subellipsoidea Reveals Traits of Cold Adaptation," Genome Biology 13, R39. DOI: 10.1186/gb-2012-13-5-r39. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 24, 2012

Understanding Plant Hormones

Plants respond to developmental cues and environmental stresses by controlling both the level and activity of various hormones. A highly adaptable scaffold enables the evolution of promiscuous activity within the auxin-responsive GH3 enzyme family, leading to diversification of substrate specificity and evolution of metabolic control systems. Newly reported crystal structures provide a glimpse into substrate recognition and control of hormones involved in plant growth, development, and defense, enabling deeper understanding of plant metabolism intricacies. The research was conducted using resources at the Advanced Photon Source at Argonne National Laboratory.

Reference: Westfall, C. S., et al. 2012. “Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins,” Science 336, 1708–11. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Comparison of GH3 Protein Binding Sites. In plants, GH3 proteins act as molecular on/off switches that control bioactive plant hormone formation by catalyzing the addition of specific amino acids to jasmonic acid, auxin, and benzoates. X-ray structures of GH3 proteins reveal a common three-dimensional fold but variability in the hormone binding site. This figure shows the variation in the jasmonic acid binding site of Arabidopsis thaliana GH3.11/JAR1 (gold) and the salicylic acid binding site of A. thaliana GH3.12/PBS3 (green). more...

Image credit: Argonne National Laboratory



May 14, 2012

Ionic Liquids: Degrading Biomass but Not Biofuel-Producing Microbes

A major hurdle to the development of economically competitive biofuels remains the difficulty of separating long sugar chains from plant biomass (cellulose and hemicellulose) from the tough network of lignin that gives strength and resilience. Pretreatment of plant material by ionic liquids (ILs), a class of salts that are molten at room temperature, is highly effective in disrupting biomass structure and liberating cellulose chains for subsequent conversion to biofuel compounds by fermentative microbes. However, residual IL molecules are highly toxic to biofuel-producing microbes and must be fully removed from the cellulose fraction prior to conversion, an expensive and time-consuming process. To understand this IL toxicity and enable development of resistant strains of microbes, researchers at the Joint Bioenergy Institute (JBEI) examined shifts in gene expression of a novel biomass-degrading bacterium when exposed to an IL. Enterobacter lignolyticus was surprisingly resistant to IL exposure, altering its cell membrane composition, activating a series of pumps to remove IL from the cell interior, and balancing osmotic pressure across the cell membrane. Many of the response mechanisms were specific to IL exposure and were not triggered by exposure to standard salts. These findings provide new insights into the mechanisms used by microbes to tolerate exposure to ionic liquids and may lead to the improvement of IL tolerance in biofuel-producing microbes through targeted genetic engineering.

Reference: Khudyakov, J. I., P. D'haeseleer, S. E. Borglin, K. M. DeAngelis, H. Woo, E. A. Lindquist, T. C. Hazen, B. A. Simmons, and M. P. Thelen. 2012. "Global Transcriptome Response to Ionic Liquid by a Tropical Rain Forest Soil Bacterium, Enterobacter lignolyticus," Proceedings of the National Academy of Sciences of the USA 109(32), E2173-E2182. DOI: 10.1073/pnas.1112750109. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 10, 2012

Insights into Transport of Lignin-Degradation Compounds in Biofuel-Producing Microbes

Understanding how lignin degradation compounds are transported into microbial cells for further processing into biofuels and for other biotechnology purposes is essential. Using the bacterium Rhodopseudomonas palustris as a model to study the transport of these compounds, researchers from Argonne and Brookhaven national laboratories have applied high-throughput genomic and biophysical approaches to determine the characteristics of the proteins that bind the lignin-degradation products. These binding proteins are part of a large complex, the ABC transporter, that moves chemical compounds through the cell membrane into the cell. The researchers found that the proteins bind aromatic compounds with high affinity and tested the physical configuration of these binding proteins with and without the aromatic degradation products present. The results suggested that the shape of the proteins does not change, but that local changes do occur in the tertiary structure where degradation compounds bind. This molecular reconfiguration could position the aromatic compounds to be more easily transported through the cell membrane. The combination of theoretical models validated by these studies and experimental approaches should be applicable to other organisms relevant to biofuels research.

Reference: Pietri, R., S. Zerbs, D. Corgliano, M. Allaire, F. Collart, and L. Miller. 2012. "Biophysical and Structural Characterization of a Sequence-Diverse Set of Solute-Binding Proteins for Aromatic Compounds," Journal of Biological Chemistry 287, 23748-56. DOI: 10.1074/jbc.M112.352385. (Reference link)

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 16, 2012

New Method to Compare Organism Functionality

Systems biology approaches to bioenergy and environmental research are enabled by reliable models of processes in living cells. Advances in genome sequencing and computational modeling have led to the development of over 100 genome-scale network reconstructions (constraint-based models). Rapid increases in this number are expected, so methods that use algorithms to compare functional characteristics between organisms will be increasingly important. Scientists at the University of Wisconsin have reported a novel approach that embeds two constraint-based models into an optimization model. This combination identifies those genes and reaction pathways that contribute most to differences in metabolic functionality. The authors identified several differences in metabolism in two cyanobacteria that have potential for biofuel production, Synechococcus and Cyanothece. For example, they demonstrated the necessity for a particular protein (plastocyanin) for photosynthesis in Cyanothece, but not in Synechococcus. The new approach also aids the curation of constraint-base models by identifying pathways that are coded by the organism, but that are missing from the model.

Reference: Hamilton, J. J., and J. L. Reed. 2012. "Identification of Functional Differences in Metabolic Networks Using Comparative Genomics and Constraint-Based Models," PLoS ONE 7(4), e34670. DOI:10.1371/journal.pone.0034670. (Reference link)

Contact: John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 12, 2012

Switchgrass Sequencing Provides Insight into Genome Structure and Organization

Perennial switchgrass (Panicum virgatum L.) is capable of producing high biomass yields with low inputs on marginal lands, making it one of the most promising candidate bioenergy feedstocks. Breeding programs are underway to enhance and improve switchgrass as a viable agricultural crop, but these efforts are hampered by the limited genetic and genomic information currently available. The switchgrass genome is now being sequenced, but its highly complex structure makes assembly difficult. Researchers at the DOE Joint Genome Institute (JGI) and the DOE Joint BioEnergy Institute (JBEI) report on the construction, sequencing, and analysis of two "Bacterial Artificial Chromosome" (BAC) libraries from switchgrass. These libraries contain relatively large DNA segments and represent essentially a random sampling of the genome, allowing the researchers to analyze structure and function at a genome-wide scale. Comparisons with sequences from other bioenergy-relevant grasses reveal that switchgrass is closely related to sorghum, indicating that the fully sequenced sorghum genome would serve as a good reference for assembling switchgrass gene space. The resources generated here will have utility for a number of applications, including identification of switchgrass gene functions relevant to bioenergy production.

Reference: Sharma, M. K., R. Sharma, P. Cao, J. Jenkins, L. E. Bartley, M. Qualls, J. Grimwood, J. Schmutz, D. Rokhsar, and P. C. Ronald. 2012. "A Genome-Wide Survey of Switchgrass Genome Structure and Organization," PLoS ONE 7(4), e33892, DOI: 10.1371/journal.pone.0033892. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 05, 2012

Understanding How Bacteria Use Sunlight

Cyanobacteria are prime candidates for the biological production of biofuels, especially hydrogen. They photosynthesize in sunlight, have relatively fast growth rates, are tolerant to extreme environments, and can accumulate high amounts of intracellular compounds and produce large quantities of H2. New research has combined a new genome-scale, constraint-based model of the cyanobacterium Cyanothece with experiments in a novel photobioreactor. The model and experiments provide new insights into the effect of light quality on metabolism and the bacteria's mechanisms for balancing reductant and electron flows. The model differs from similar models of other cyanobacteria in its detailed treatment of the photosynthesis and respiratory systems. The photobioreactor features dual sources of monochromatic light that can vary photon flux with wavelengths that are tuned to the two bacterial photosynthesis systems. The results will guide development of genome-scale metabolic models for other cyanobacteria and may help with the genetic manipulation of photosynthetic microorganisms to improve biofuel production. These findings were presented by a team of DOE scientists led by Pacific Northwest National Laboratory and the University of Wisconsin.

Reference: Vu, T. T, et al. 2012. "Genome-Scale Modeling of Light-Driven Reductant Partitioning and Carbon Fluxes in Diazotrophic Unicellular Cyanobacterium Cyanothece sp. ATCC 51142," PLoS Computational Biology 8(4), e1002460, DOI:10.1371/journal.pcbi.1002460. (Reference link)

Contact: John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Pacific Northwest National Laboratory Photobioreactor



March 27, 2012

Using Systems Biology to Understand Complex Microbial Communities

The ability to effectively model and predict integrated functional properties across complex groups of microbes is critical to understanding major environmental processes. Advances in this area would also facilitate development of novel bioengineering approaches utilizing the unique functional compartmentalization that enables microbial communities to efficiently perform complex cooperative processes. In a new perspective essay, DOE researchers Karsten Zengler and Bernhard Palsson of the University of California San Diego describe a conceptual approach to extend systems biology tools developed to understand metabolic functions of single organisms to more complex multispecies communities. This is a considerable challenge since detailed physiological information is only available for the small fraction of microbes that can be cultivated. Cultivation independent approaches such as metagenomics provide a snapshot of overall functional potential but little information on dynamic processes or interactions between members. Building on preliminary successes with modeling interactions in simple two member partnerships, the authors suggest that a combination of these "bottom up" and "top down" approaches that incorporates efficient targeting of organisms performing processes of interest, high-resolution imaging of spatial process relationships, and more refined environmental 'omics techniques could yield predictive computational models of microbial community function.

Reference: Zengler, K., and B.O. Palsson. 2012. "A Roadmap for the Development of Community Systems (CoSys) Biology," Nature Reviews Microbiology, DOI:10.1038/nrmicro2763. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 20, 2012

White Rot Fungus Sequence Provides New Understanding of Lignin Degradation

Lignin is a key building block in plant cell walls and one of the two most abundant biopolymers on Earth. It is also highly resistant to breakdown, complicating efforts to use plant biomass for producing biofuels. No animals and few fungi or bacteria are able to degrade lignin. However, the white rot fungus Ceriporiopisis subvermispora not only degrades lignin but leaves the cellulose in biomass intact. An international team of scientists has sequenced and annotated (assigned possible functions to genes) the genome of this fungus to learn more about its mechanisms of lignin degradation. Using experiments and a comparison with the sequence of its more studied relative Phanaerochaete chrysosporium, the scientists identified differences in the degradation genes between the two fungi and developed new hypotheses about the mechanisms that enable these fungi to target lignin but not cellulose. These results may assist in the development of improved pathways for the conversion of biomass to biofuels as well as provide improvements in deconstruction of wood for the pulp and paper industry. The study included researchers at the DOE's Joint Genome Institute (DOE-JGI).

Reference: Fernandez-Fueyo, E., et al. 2012. "Comparative Genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium Provide Insight into Selective Ligninolysis," Proceedings of the National Academy of Sciences of the United States of America 109(14), 5458-63. DOI:10.1073/pnas.1119912109. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 11, 2012

Understanding the Roles Played by Hydrosulphide Membrane Channel and Its Relatives in Living Systems

The hydrosulphide ion (HS–), a critical element in the origin of life on Earth, is important in physiology and cellular signaling. The HS– species is also the terminal product when an anaerobic bacterium derives its oxidative power from sulphate instead of oxygen. A recent study conducted on beamlines at the National Synchrotron Light Source revealed the structure of the hydrosulphide ion channel (HSC), a membrane-pore molecule, elucidating how HS– is able to escape from pathogenic Clostridium difficile cells. In the same protein family, the formate channel (FocA), which has a fold similar to HSC, has been shown to play two other roles related to bioenergy and environmental science. In the first case, hydrogen gas production in Escherichia coli depends on the selective decomposition of formate, whose concentration depends on FocA. In the second, when Euglena experiences long-term chronic exposure to cadmium ions, it overexpresses a FocA protein. This protein has been proposed as a marker for long-lasting cadmium pollution in water.

Reference: Czyzewski, B. K., and D.-N. Wang. 2012. “Identification and Characterization of a Bacterial Hydrosulphide Ion Channel,” Nature 483, 494–97. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 02, 2012

Microbes Stress Out During Conversion of Pretreated Biomass to Biofuels

Chemical pretreatment of plant biomass prior to enzymatic breakdown significantly improves the release of sugar molecules, which are subsequently converted to biofuel compounds by fermentative microbes. However, pretreatment also introduces a variety of stress factors that can interfere with these fermentative organisms, including residual chemicals, toxins released from the biomass, high concentrations of sugars, and production of biofuels themselves. Researchers at the DOE Great Lakes Bioenergy Research Center (GLBRC) describe the integration of gene expression and physiological stress responses in an ethanol-producing strain of Escherichia coli during growth on corn stover that had been pretreated using ammonia fiber expansion (AFEX) and enzymatic digestion. Their results indicate that osmotic pressure resulting from high sugar concentrations and toxicity due to ethanol production were the two most important stressors to E. coli under these conditions, and that the cells activated a cascade of carefully timed stress tolerance pathways in response to these factors. Identification of these pathways provides new targets for metabolic engineering to improve stress tolerances of biofuel-producing microbes, leading to the development of more sophisticated approaches to leverage microbes' natural abilities to sense and respond to environmental stress.

Reference: Swalbach, M. S., et al. 2012. "Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen," Applied and Environmental Microbiology 78, 3442-57, DOI: 10.1128/AEM.07329-11. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 01, 2012

Microbial Communities Help Solve Environmental Contamination

Microbes are very effective at carrying out a wide range of chemical reactions, even ones that involve substances toxic to higher life forms. Many groundwater sites contaminated with compounds such as trichloroethene (TCE), a pervasive groundwater pollutant often used by industry as cleansers or degreasers, are decontaminated by microbes. Dehalococcoides are the only family of bacteria known to break down TCE to ethene, a harmless chemical compound often used to help ripen fruits. A team of researchers has conducted a metagenomic analysis of a stable dechlorinating community derived from sediment collected at the Alameda Naval Air Station (ANAS) in California. The team identified the other members of this microbial community, since microbes such as Dehalococcoides are known to dechlorinate chemicals more effectively in the presence of other microorganisms. This study showed that all of the genes that code for enzymes involved in dechlorination were associated with Dehalococcoides, emphasizing its importance as the dominant dechlorinating microbe in the ANAS microbial community. Understanding the composition and functioning of communities such as this one will contribute to similar remediation efforts on a variety of cleanup challenges that DOE faces, as well as other processes (e.g., plant nutrition, carbon processing) that microbial communities carry out. The research was based on sequencing carried out by the DOE Joint Genome Institute (JGI).

Reference: Brisson, V. L. et al. 2012. "Metagenomic Analysis of a Stable Trichloroethene-Degrading Microbial Community," The ISME Journal, 1–13. DOI:10.1038/ismej.2012.15. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 16, 2012

Using High-Performance Computing to Study the Hydration of Cellobiose

Cellobiose, the two glucose basic repeat unit of cellulose, is formed during enzymatic or acidic hydrolysis of plant biomass, an early step in the production of biofuels. DOE researchers at the University of California, Irvine, have investigated the stability of cellobiose in water using high-level quantum molecular dynamics at DOE's NERSC high-performance computing facility. The results from these simulations suggest that water dynamics play a leading role in stabilizing cellobiose in particular low energy states. The findings also indicate that long-range interactions between the water molecules and the sugar give rise to collective motions that could impact downstream enzymatic functions in the production of biofuels. These results provide new insight into a key step in the conversion of biomass to fuel molecules.

Reference: Pincu, M., and R. B. Gerber. 2012. "Hydration of Cellobiose: Structure and Dynamics of Cellobiose-(H2O)n, n=5 to 25," Chemical Physics Letters 531, 52–58, DOI: 10.1016/jcplett.2012.02.019. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 09, 2012

Understanding How Plants Sense Ultraviolet Light

Sunlight is essential for plant development and growth, yet many details of the mechanisms by which plants respond to sunlight are poorly understood. A recent study published in Science provides new information about the molecular changes initiated by exposure to the UV-B portion of sunlight. The research used small-angle x-ray scattering (SAXS) experiments to characterize how the plant photoreceptor UVR8 changes shape when exposed to UV-B radiation. Two UVR8 molecules are complexed together as a dimer in plant cells and break apart on exposure to UV-B. The separate molecules then interact with a series of proteins in the cell to signal the presence of solar radiation. A specific mutation in UVR8 was found to "retune" the molecule's response from UV-B to UV-C radiation. The results will be useful in understanding how to optimize biomass crop growth. The SAXS studies were carried out at the SIBYLS experimental station at the Advanced Light Source at the Berkeley Lab. The study was led by Elizabeth Getzoff of the Scripps Research Institute.

Reference: Christie, J. M., et al. 2012. "Plant UVR8 Photoreceptor Senses UV-B by Tryptophan-Mediated Disruption of Cross-Dimer Salt Bridges," Science 335, 1492&ndash96. DOI: 10.1126/science.1218091. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 08, 2012

New Approach for Converting Plant Biomass to Ethanol

The conversion of plant biomass to liquid transportation fuel using consolidated bioprocessing (CBP) technology is a promising, cost-efficient strategy to develop energy from renewable sources. CBP takes advantage of the ability of certain microbes to convert sugars contained within the plant cell wall to high-energy chemicals such as ethanol or butanol, but the efficiency can be hampered by the recalcitrance of certain plant materials to deconstruction. While plant cell wall composition and corresponding resistance to breakdown varies considerably within plant species, this genetic diversity can potentially be exploited if plant material is efficiently screened for such properties. Researchers at the U.S. Department of Energy’s (DOE) BioEnergy Science Center (BESC), together with scientists funded by the U.S. Department of Agriculture-DOE Plant Feedstocks Genomics for Bioenergy program, report the development of a robust assay for biomass digestibility and conversion using the anaerobic bacterium Clostridium phytofermentans. This bacterium is capable of directly converting a wide array of fermentable biomass components to ethanol without the addition of costly, exogenous, deconstruction enzymes. The assay, which measures ethanol production under the influence of different variables, was tested on both herbaceous grasses and woody plants. Significant differences in ethanol production within individual plant species were found, indicating detection of subtle genetic differences. This method provides a means of assessing feedstock quality for digestibility and ethanol production that will facilitate genetic analysis of energy crops for amenability to biological conversion.

Reference: Lee, S. J., et al. 2012. “Biological Conversion Assay Using Clostridium phytofermentans to Estimate Plant Feedstock Quality,” Biotech for Biofuels 5:5, DOI: 10.1186/1754-6834-5-5. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 25, 2012

Miscanthus Genetic Map Provides Resource for Crop Improvement

Perennial grasses are a potential source of feedstocks for "second-generation" cellulosic bioethanol because they efficiently accumulate large amounts of biomass and can be grown on marginal lands not suitable for conventional agricultural food crops. Among these grasses, Miscanthus is one of the most promising bioenergy crops in the Midwest because of its extremely high biomass yields, in particular the species Miscanthus x giganteus. However, efforts to breed improved varieties of Miscanthus are hampered by its complicated genome structure and lack of genetic tools. With support from the Joint USDA-DOE Plant Feedstocks Genomics for Bioenergy program, researchers report the first genetic linkage maps of Miscanthus using molecular markers derived from the closely related sugarcane grass. Genetic similarity between Miscanthus, sorghum, and sugarcane allowed comparative studies between the three species, revealing information into the genomic relationships among them and also allowing the first genetic map length estimate of Miscanthus. These resources provide a framework that will significantly enhance Miscanthus improvement efforts by facilitating identification of biomass-relevant genes and marker-assisted selection in this important bioenergy crop.

Reference: Kim, C., D. Zhang, S. A. Auckland, L. K. Rainville, K. Jakob, B. Kronmiller, E. J. Sacks, M. Deuter, and A. H. Paterson. 2012. "SSR-Based Genetic Maps of Miscanthus sinensis and M. sacchariflorus and Their Comparison to Sorghum," Theoretical and Applied Genetics, DOI:10.1007/s00122-012-1790-1. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 17, 2012

New Type of Lignin Discovered in Vanilla Plant

Found within the plant cell wall, lignin is a complex polymeric compound that provides the plant with both mechanical support and protection from pests and pathogens. However, the structural rigidity of this compound also inhibits efficient conversion of the sugars within plant cell walls into biofuels, making lignin a major obstacle to the efficient production of biofuels from cellulosic feedstocks. Three types of lignin are usually found in nature: H-, G-, and S-lignins. They are synthesized by polymerization of their respective monolignol units. However, lignin biosynthesis can be relatively flexible, sometimes allowing different and more unusual monolignols to be incorporated. Researchers at the DOE BioEnergy Science Center (BESC) and DOE Great Lakes Bioenergy Research Center (GLBRC) report the identification and characterization of a new type of polymer, C-lignin, composed almost exclusively of caffeyl units. Detected in the Vanilla orchid, a few related orchids, and some cactus species, this unique new lignin was found only in the seed coats, with more conventional lignins observed in other plant tissues. These results may lead to a greater understanding of the lignin biosynthetic pathway, as well as new approaches for engineering biomass that can be more easily and efficiently digested for conversion into biofuels.

Reference: Chen, F., Y. Tobimatsu, D. Havkin-Frenkeld, R. A. Dixon, and J. Ralph. 2012. "A Polymer of Caffeyl Alcohol in Plant Seeds," Proceedings of the National Academy of Sciences of the United States of America 109(5), 1772-77. DOI: 10.1073/pnas.1120992109. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 15, 2012

Bioenergy Plants Database

Plant feedstocks for next-generation biofuels (e.g., lignocellulosic biomass) will come from many different sources depending on the geographic region and will likely include high biomass-producing species such as switchgrass, pine, poplar, and sorghum. Genome-enabled tools promise to facilitate breeding efforts to maximize biomass quality and yield in these plants; however, most of these species lack a complete genome sequence and many have only limited genetic tools available. To enable genome-based improvement of lignocellulosic biofuel feedstock species, researchers at Michigan State University, with support from the joint USDA-DOE Plant Feedstocks Genomics for Bioenergy program, have developed the Biofuel Feedstock Genomics Resource (BFGR). This web-based portal and database contains data from 54 bioenergy-relevant plant species, together with annotation and tools that allow identification and analysis of genes important for improvement of bioenergy traits, molecular marker analysis, and mapping to specific biochemical and metabolic pathways. Importantly, the database provides comparative analysis tools to allow scientists investigating species that lack a genome sequence to identify critical genes and develop experimentation to determine gene function. The BFGR will provide a valuable resource for plant breeders to use in improving bioenergy feedstocks for biofuel production.

Reference: Childs, K. L., K. Konganti, and C. R. Buell. 2012. "The Biofuel Feedstock Genomics Resource: A Web-Based Portal and Database To Enable Functional Genomics of Plant Biofuel Feedstock Species," Database, DOI:10.1093/database/bar061. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 11, 2012

Helping Researchers Find Bioenergy-Related Data

A systems biology approach to biological research requires ready access to information from many investigators conducting a wide variety of experiments. DOE's BioEnergy Science Center (BESC) is undertaking large experimental campaigns to understand the biosynthesis and biodegradation of biomass and to develop biofuel solutions. BESC is generating large volumes of diverse data, including genome sequences, omics data, and diverse assay results. To assist the community of bioenergy researchers, BESC has developed a public Knowledgebase repository (besckb.ornl.gov) that they describe in the journal Bioinformatics. The BESC Knowledgebase serves as a central repository for experimentally generated data and provides an integrated, interactive, and user-friendly analysis framework. The Knowledgebase portal makes tools available for visualization, integration, and analysis of data produced by BESC or obtained from external resources. The aim of this database is to provide a resource for a systems-level understanding of cellular processes involved in plant formation, degradation, and biofuel production. The BESC Knowledgebase fits within the scope of a larger Knowledgebase activity across the DOE Genomic Science Program (http://genomicscience.energy.gov/compbio/).

Reference: Syed, M. H., T. V. Karpinets, M. Parang, M. R. Leuze, B. H. Park, D. Hyatt, S. D. Brown, S. Moulton, M. D. Galloway, and E. C. Uberbacher. 2012. "BESC Knowledgebase Public Portal," Bioinformatics, DOI: 10.1093/bioinformatics/bts016. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 08, 2012

Combining Crystallography and Visible Spectroscopy to Understand Enzymes

Structure and function are intimately linked but do not necessarily predict the other. For example, X-ray crystallography provides 3-D atomic structural information about biological macromolecules but does not define important details about metal ions. However, the oxidation state of metal ions at an enzyme's active site has a critical effect on enzyme behavior. Thus, an enzyme's catalytic function derives from the electronic structure of those atoms influencing or directly participating in the reaction, information not revealed by the scattering methods used in X-ray crystallography. A new technology has been developed that simultaneously carries out crystallography and UV-visible and Raman spectroscopy to determine the atomic structure of the entire protein, and electronic and vibrational structures of the metal ions or cofactors within. The combined instrumentation has been used to study the process of demethylation of an organic substrate molecule by an enzyme whose active site includes an iron-sulfur cluster. The authors used spectroscopy to follow the change in the oxidation state of the cluster during the crystallography data collection and to formulate a mechanism for the process. The results provide insight into an important class of phenomena that control cellular behavior. The technology was developed by scientists at the Protein Crystallography Research Resource at the National Synchrotron Light Source at Brookhaven National Laboratory. The new study was led by Allen M. Orville of Brookhaven and Pinghua Liu and Karen N. Allen of Boston University and is published in the Journal of the American Chemical Society.

Reference: Daughtry, K. D., et al. 2012. "Quaternary Ammonium Oxidative Demethylation: X-Ray Crystallographic, Resonance Raman, and UV-Visible Spectroscopic Analysis of a Rieske-Type Demethylase," Journal of the American Chemical Society 134(5), 2823-2834. DOI: 10.1021/ja2111898. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 05, 2012

Nanowire pH Sensor for Biological Applications

A cell's internal and external pH plays a critical role in influencing many cellular chemical reactions and functions. Yet measuring pH without the appearance of artifacts in these challenging cellular and extracellular nanoscale environments is very difficult. New silicon nanowire (SiNW) pH sensors that possess long-term stability in these difficult environments have been developed by scientists at Lawrence Berkeley National Laboratory and their collaborators. The sensors were produced using a top-down fabrication process combining electron beam lithography (EBL) with conventional photolithography. A passivation layer (silicon nitride applied using plasma enhanced chemical vapor deposition) is coated on the SiNW's surface to enhance electrical insulation and ion-blocking properties. This study shows that the application of these techniques results in improved stability of the sensor and enhances its performance. The paper explains how to achieve reliable performance in biological systems and discusses the trade-off between stability and pH sensitivity of the sensor response.

Reference: Choi, S., I. Park, Z. Hao, H.-Y. Holman, and A. P. Pisano. 2012. "Quantitative Studies of Long-Term Stable, Top-Down Fabricated Silicon Nanowire pH Sensors," Applied Physics A: Materials Science & Processing, DOI: 10.1007/s00339-011-6754-9. (Reference link)

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 18, 2011

Understanding Impacts of Climate Change on Carbon Cycling by Soil Microbes

Quantifying feedbacks between terrestrial carbon cycling and changing climate conditions remains one of the major sources of uncertainty in predicting climate change impacts. A lack of mechanistic understanding of biogeochemical processes mediated by soil microbes and how they are affected by climate change variables is a significant element of this problem. New 'omics techniques for high-throughput characterization of microbial community structure and function are now providing powerful tools to examine these processes in intact ecosystems. Researchers at the University of Oklahoma have studied the impacts of long-term warming experiments (10+ years) on soil microbes at a grassland field site. The study describes compositional and functional shifts in the microbial communities related to elevated temperature and resulting changes in overlying vegetation and soil moisture. These effects were correlated with an increase in CO2 efflux from soils, which was tied to stimulation of microbial community members and enzyme activities associated with degradation of labile (but not recalcitrant) soil carbon sources. The team also observed an accelerated microbial cycling of nitrogen, phosphorous, and other soil nutrients that appeared to help stimulate plant growth and at least partially ameliorate the net loss of carbon from the system. These findings point to the complex role of microbial communities in climate impacted ecosystem processes. Further study will be needed to tease apart their net effects on carbon feedbacks.

Reference: Zhou, J., K. Xue, J. Xie, Y. Deng, L. Wu, X. Cheng, S. Fei, S. Deng, Z. He, J. D. Van Nostrand, and Y. Luo. 2011. "Microbial Mediation of Carbon-Cycle Feedbacks to Climate Warming," Nature Climate Change 2, 106-10. DOI: 10.1038/nclimate1331. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 12, 2011

Understanding Winter Hardiness in Switchgrass

The nation's dependence on imported fossil fuels could be alleviated, at least in part, by the domestication of dedicated bioenergy crops such as native perennial switchgrass for lignocellulosic ethanol production. Switchgrass is a promising feedstock candidate because it produces high yields of biomass on marginal lands unsuitable for production of food crops. In addition, perenniality (the ability of a plant to survive over winter and resume growth in the spring) is important for sustainability, since the unharvested below-ground tissues help maintain the integrity and nutrient status of the soil. Perennial biomass cultivars will need to tolerate fluctuations in temperature and rainfall, traits influenced by the overall health of below-ground tissues. Research¬ers at the USDA-ARS in Lincoln, Nebraska, with funding from the joint USDA-DOE Plant Feedstocks Genomics for Bioenergy Program, analyzed changes in gene expression patterns in below-ground tissues (crowns and rhizomes) of the switchgrass cultivar 'Summer' to gain insight into the genetic mechanisms regulating these processes. The results revealed that these tissues are metabolically active, including pathways involved in basal cell metabolism and stress response. In addition, several novel gene sequences of unknown function were identified, which may represent genes specific to these tissues and with unique functions. These analyses should yield further insights into perenniality that will improve switchgrass as a sustainable bioenergy feedstock.

Reference: Palmer, N. A., A. J. Saathoff, J. Kim, A. Benson, C. M. Tobias, P. Twigg, K. P. Vogel, S. Madhavan, and G. Sarath. 2011. "Next-Generation Sequencing of Crown and Rhizome Transcriptome from Upland, Tetraploid Switchgrass," BioEnergy Research, DOI: 10.1007/s12155-011-9171-1. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 09, 2011

Microbial Carboxysomes: Key to Understanding Ocean Carbon Cycle

Bacteria play a key role in sequestering carbon dioxide (CO2) in the oceans. In particular, Prochlorococcus cyanobacteria are considered the world's most abundant photosynthetic organisms, able to convert sunlight to energy at ocean depths of up to 200 meters. Despite their small size, they are estimated to contribute up to half of all marine biological carbon sequestration. This microbe's ability to use carbon is attributed in part to the RuBisCO enzymes that fix CO2 and are stored in microcompartments known as carboxysomes. Learning about these tiny cellular structures can help researchers understand how their composition and design support their function, contributing to a better understanding of the ocean carbon cycle. Scientists at the University of Mississippi, the DOE Joint Genome Institute (JGI), and University of California at Berkeley report the first successful purification and characterization of these carboxysomes from a strain of P. marinus. Comparisons against 29 cyanobacterial genomes in a phylogenetic assay suggested, based on the numbers and types of genes that the team identified, that the carboxysome's structure is more complex than had been previously assumed. "Our findings have important implications for the structure, function, and regulation of α-carboxysomes and suggest that the protein composition of these important bacterial organelles warrants a closer look beyond what was assumed to be a solved problem," the team concluded.

Reference: Roberts, E. J., et al. 2012. "Isolation and Characterization of the Prochlorococcus Carboxysome Reveal the Presence of the Novel Shell Protein CsoS1D," Journal of Bacteriology 194(4), 787-95. DOI: 10.1128/JB.06444-11. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 02, 2011

Genome-Scale Modeling of Methane-Producing Microbes

Methane-producing microbes (i.e., methanogens) play a key role in the global carbon cycle and could significantly contribute to climate change due to the potent greenhouse gas properties of methane. These organisms occupy a central place in the biogeochemistry of soils, wetlands, and permafrost. However, it remains difficult to predict how they may respond to changing environmental conditions due to limited understanding of their biology. In a new study by DOE investigators at the University of Illinois, the first fully curated genome-scale metabolic model has been assembled for the methanogen Methanosarcina acetivorans. M. acetivorans is unique among methanogens in its ability to convert organic compounds such as acetate to methane, but it cannot perform the more traditional conversion of hydrogen and CO2. The new model's predictions have been validated using flux balance analysis and gene knockouts. The model provides new information on the integration of central and peripheral metabolic pathways, an important step in developing a systems biology approach to understanding this methanogen's behavior. These findings significantly increase our predictive understanding of this important class of microbes providing a powerful new tool to test hypotheses on their potential roles in climate change.

Reference: Benedict, M. N., M. C. Gonnerman, W. W. Metcalf, and N. D. Price. 2012. "Genome-Scale Metabolic Reconstruction and Hypothesis Testing in the Methanogenic Archaeon Methanosarcina acetivorans C2A," Journal of Bacteriology 194, 855-65, DOI: 10.1128/JB.06040-11. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 30, 2011

Protein Complex Within Plant Cell Wall Associated with Secondary Cell-Wall Synthesis

The plant cell wall polysaccharide pectin is often associated with the tissue softening that occurs during fruit ripening. However, this complex compound is also involved in secondary cell-wall synthesis in grasses and woody plants, helping to give the plant rigidity, but also impeding the deconstruction of plant biomass and hence its conversion into biofuels. Researchers at the DOE BioEnergy Research Center (BESC) have discovered that the pectin-synthesizing enzyme GAUT1 forms an unusual, two-protein complex with a similar protein (GAUT7) that constitutes a critical part of a pectin-synthesizing protein complex. They also showed that this complex plays a role in secondary cell-wall synthesis. Manipulating the formation of this complex may provide a way to modify secondary cell walls, which could either increase available biomass or improve its digestibility for biofuel production.

Reference: Atmodjo, M. A., Y. Sakuragi, X. Zhu, A. J. Burrell, S. S. Mohanty, J. A. Atwood III, R. Orlando, H. V. Scheller, and D. Mohnen. 2011. "Galacturonosyltransferase (GAUT)1 and GAUT7 Are the Core of a Plant Cell Wall Pectin Biosynthetic Homogalacturonan: Galacturonosyltransferase Complex," Proceedings of the National Academy of Sciences of the United States of America 108(50), 20225-230. DOI: 10.1073/pnas.11128116108. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 28, 2011

Designing Low Lignin, High Biomass Yielding Plants

The major barrier to the efficient conversion of biomass from plant feedstocks to biofuels is breaking down the plant cell wall so that the sugars locked within can be released. This barrier is due to the presence of lignin, a complex compound that cross links the walls and provides rigidity to the plant. Plants that are genetically modified to have less lignin can be broken down more easily, but often these plants show severely stunted growth. Plants have a stress hormone (salicylic acid (SA)) that is known to impact plant growth and development and whose levels are inversely proportional to lignin levels. Researchers at the DOE BioEnergy Science Center (BESC) have found that genetically removing SA from Arabidopsis plants that were also modified to produce low levels of lignin restores normal growth to these plants while maintaining low lignin content. These results support the hypothesis that low lignin, high biomass yielding plants can be engineered to produce sustainable biofeedstocks for biofuel production.

Reference: Gallego-Giraldo, L., L. Escamilla-Trevino, L. A. Jackson, and R. A. Dixon. 2011. "Salicylic Acid Mediates the Reduced Growth of Lignin Down-Regulated Plants," Proceedings of the National Academy of Sciences of the United States of America 108(51), 20814-19. DOI: 10.1073/pnas.1117873108. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 28, 2011

Microbial Conversion of Switchgrass to Multiple Drop-In Biofuels

The low efficiency and high cost of enzymes used to break down plant material into sugars remains a major barrier to economically competitive production of cellulosic biofuels. Consolidated biomass processing, in which a single microorganism both produces cellulose-degrading enzymes and converts the resulting sugars to a desired biofuel, presents a promising alternative to improve efficiency and reduce costs, but few organisms naturally possess both capabilities. Researchers at the Joint Bioenergy Institute (JBEI) have now engineeered a modified strain of the workhorse industrial microbe E. coli that expresses a tailored set of cellulases, allowing it to degrade both the cellulose and hemicellulose chains released from switchgrass pretreated with ionic liquid. This was accomplished by cloning cellulase genes from Cellvibrio japonicus, a soil microbe with similar protein secretion systems to E. coli, and modifying the genes to allow proper timing and level of cellulase expression in the host. The team then added metabolic pathways that allowed E. coli to convert resulting sugars to either of two drop-in automotive biofuels (biodiesel and butanol) or a jet fuel precursor terpene compound. This presents a promising new advance in consolidated biomass processing, and, given the relative ease of genetic modification in E. coli, offers tremendous potential for subsequent engineering to increase conversion efficiency or synthesize a broader range of fuels.

Reference: Bokinsky, G., P. P. Peralta-Yahyn, A. George, B. M. Holmes, E. J. Steen, J. Dietrich, T. S. Lee, D. Tullman-Ercek, C. A. Voigt, B. A. Simmons, and J. D. Keasling. 2011. "Synthesis of Three Advanced Biofuels from Ionic Liquid-Pretreated Switchgrass Using Engineered Escherichia coli," Proceedings of the National Academy of Sciences of the United States 108(50), 19949-54. DOI: 10.1073/pnas.1106958108. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 23, 2011

Structure of Essential Malaria Parasite Enzyme Determined

The three-dimensional structures of proteins and other macromolecules often provide a starting point for designing new approaches to solving problems in a wide range of applications from bioenergy to medicine. The high-resolution structure of a specific protein can be used to identify small molecules that would bind to the protein and increase or decrease its activity to achieve a desired change in a biological system. A new study has determined the structures of an enzyme found in the malaria parasite (Plasmodium falciparum). The enzyme is not found in humans but is required by the parasite for the formation of its outer membrane. Several high-resolution structures were obtained for the enzyme in several stages of its functioning as well as with a small molecule that inhibits it. The structural information helped identify the enzyme’s active site and will be used as a starting point to seek drugs to treat infections by the malaria parasite. The results, published in the Journal of Biological Chemistry, were obtained by scientists from Washington University at the highly productive beamline 19ID of the DOE Structural Biology Center at Argonne National Laboratory’s Advanced Photon Source.

Reference: Lee, S. G., Y. Kim, T. D. Alpert, A. Nagata, and J. M. Jez. 2012. "Structure and Reaction Mechanism of Phosphoethanolamine Methyltransferase from the Malaria Parasite Plasmodium falciparum," Journal of Biological Chemistry 287, 1426-1434, DOI: 10.1074/jbcM111.315267. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 22, 2011

How do Microbes Adapt to Diverse Environments?

Earth's microbes live in staggeringly diverse environments, colonizing habitats with extremes of temperature, pH, salt concentration, or presence of toxic compounds. Archaea, a domain of single-celled microbes sharing traits with bacteria and simple eukaryotes, are well known for their ability to thrive in harsh environments. How this impressive adaptive capability is achieved has remained a mystery. Now, a team of investigators at the Institute for Systems Biology has completed a groundbreaking study on the role of gene regulation in environmental niche adaptation by Halobacterium salinarum, an archaeal microbe that grows in high salt environments. Using a combination of comparative genomics and hypothesis-driven molecular biology experiments, the team found that a specific class of regulatory genes had been duplicated during the archaea's evolution and controls a nested set of "niche adaptation programs." These programs control cascades of gene expression essential for adaptation to particular environments. Diversification of these control elements has resulted in a "division of labor" such that overlapping regulatory networks flexibly balance large-scale functional shifts under changing conditions, where rapid adaptation increases fitness. Describing mechanisms that control niche adaptation in microbes allows us to better understand how microbial communities function in natural environments, and provides an intriguing glimpse into fundamental design rules governing biological systems.

Reference: Turkarslan, S., D. J. Reiss, G. Gibbins, W. L. Su, M. Pan, J. C. Bare, C. L. Plaisier, and N. S. Baliga. 2011. “Niche Adaptation by Expansion and Reprogramming of General Transcription Factors,” Molecular Systems Biology 7, Article 554. DOI:10.1038/msb.2011.87. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 18, 2011

Microbes Solve Environmental Contamination Problems

Microbes carry out a wide range of chemical transformations. Understanding the mechanisms of these processes can lead to new biological insights and practical applications. For example, removal of polycyclic aromatic hydrocarbons (PAHs) from contaminated soils is facilitated by microbial degradation. The PAH phenanthrene can be broken down by Arthrobacter phenanthrenivorans, a bacterium isolated from a creosote-polluted site in Greece and that uses phenanthrene as a carbon and energy source. A team of researchers, including a collaborator from the DOE Joint Genome Institute, has purified and analyzed two phenanthrene-breakdown enzymes from this microbe. Based on the similarity of the two genes' sequences and their common expression in the presence of the PAH, the authors suggest that one of the genes is a duplication of the other even though they are located in very different parts of the genome. Similar results are found in other related bacteria. These types of comparative studies may aid in the design of strategies using microbes for DOE missions or other applications, such as wastewater treatment, biodegradation, and biocatalysis.

References: Vandera, E., K. Kavakiotis, A. Kallimanis, N. Kyrpides, C. Drainas, and A. Koukkoua. 2012. "Heterologous Expression and Characterization of Two 1-Hydroxy-2-Naphthoic Acid Dioxygenases from Arthrobacter phenanthrenivorans," Applied and Environmental Microbiology 78(3), 621-27. DOI: 10.1128/AEM.07137-11. (Reference link)

Kallimanis, A., et al. 2011. "Complete Genome Sequence of Arthrobacter phenanthrenivorans Type Strain (Sphe3)," Standards in Genomic Sciences 4, 123-30. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 06, 2011

Permafrost Microbes Could Make Impacts of Arctic Warming Worse

In Earth’s Arctic regions, frozen soils (permafrost) sequester an estimated 1.6 trillion metric tons of carbon, more than 250 times the amount of greenhouse gas emissions attributed to the United States in 2009. Concerns are growing about the potential impact on the global carbon cycle when rising temperatures thaw the permafrost and release the trapped carbon. Microbes may significantly influence the eventual outcome through their involvement in carbon cycling. New research on permafrost microbes has discovered a previously unknown, yet abundant microbe that produces methane, a far more potent greenhouse gas than carbon dioxide. A draft of this microbe’s genome was determined by assembling DNA fragments isolated from permafrost. The DOE Joint Genome Institute (JGI) had previously identified several microbes that produced methane ("methanogens") as a metabolic byproduct, and used this knowledge to identify enough fragments of the new microbe’s DNA to assemble a draft of its genome. The abundance of this novel methanogen implies that it could be an important factor in methane production under permafrost thawing conditions. The research, published in Nature, was carried out by scientists at JGI, Lawrence Berkeley National Laboratory, and U.S. Geological Survey.

Reference: Mackelprang, R., et al. 2011. “Metagenomic Analysis of a Permafrost Microbial Community Reveals a Rapid Response to Thaw,” Nature 480, 368-71. DOI: 10.1038/nature10576. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 19, 2011

DOE User Facilities Help Explain Workings of Key Metabolic Enzyme

Carbonic anhydrase (CA) converts bicarbonate ion to carbon dioxide and back. It is a key part of the metabolism of humans, animals, plants, and microbes that involves carbon dioxide. Engineered and stabilized forms of CA are being studied for use to capture CO2 from flue gas at coal-fired power plants and as part of algal biofuel production. Three recent publications improve our understanding of how CA works using the unique capabilities of DOE's National Synchrotron Light Source (NSLS) and Los Alamos Neutron Science Center (LANSCE). X-ray crystallography at the NSLS was used to show how human CA recognizes molecules to which it might bind. These data support the authors' hypothesis from thermodynamic considerations that "the shape of the water in the (HA) binding cavity may be as important as the shape of the cavity." The second study, used neutron diffraction of human CA at LANSCE to show that the catalytic site CA changes when the pH of the water around it decreases from 10.0 to 7.8. This observation, the first of its kind, enabled the authors to define more clearly the proton transfer that occurs when CA catalyzes the carbon dioxide—bicarbonate conversion. These studies will help scientists re-engineering CA designs for CO2 capture, biofuel production, and other applications.

The NSLS studies were carried out by scientists at Brookhaven’s Macromolecular Crystallography Research Resource jointly with scientists from Harvard University, while the LANSCE experiments were carried out by scientists at Los Alamos’ Protein Crystallography Station in collaboration with scientists from the University of Florida.

Reference: Snyder, P.W., et al. 2011. "Mechanism of the Hydrophobic Effect in the Biomolecular Recognition of Arylsulfonamides by Carbonic Anhydrase," Proceedings of the National Academy of Sciences (USA), DOI: 10.1073/pnas.1114107108. (Reference link)
[Discussed in Ball, P. 2011. "Biophysics: More Than a Bystander," Nature 478, 467-68. (DOI:10.1038/478467a) (Reference link)]

Mecinovic, J., et al. 2011. "Fluoroalkyl and Alkyl Chains Have Similar Hydrophobicities in Binding to the 'Hydrophobic Wall' of Carbonic Anhydrase," Journal of the American Chemical Society 133, 14017. DOI: 10.1021/ja2045293. (Reference link)

Fisher, Z., et al. 2011 "Neutron Structure of Human Carbonic Anhydrase II: A Hydrogen-Bonded Water Network "Switch" Is Observed Between pH 7.8 and 10.0," Biochemistry 50, 9421-23. DOI: 10.1021/bi201487b. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 18, 2011

Genomic Science Program Scientist To Become Editor of Analytical Chemistry

Jonathan Sweedler of the University of Illinois, Urbana-Champaign, has been named the new editor of the American Chemical Society journal Analytical Chemistry, the most widely read journal in this field. His research has focused on bioanalytical chemistry, specifically on small volume peptidomics and metabolomics. Sweedler has been funded since the late 1990s for the development and application to DOE missions of a variety of analytical techniques, initially for genome sequencing and currently for genomic applications of mass spectrometry. He has a current Office of Biological and Environmental Research award with his collaborator Paul Bohn of Notre Dame to study the molecular interactions among microbes, and between microbes and plants. The objective of this work is to develop correlated chemical and spatial information, employing mass spectrometry and confocal Raman imaging to characterize key molecular species and events in multicellular processes. Sweedler is a chemistry professor and director of the Roy J. Carver Biotechnology Center at the University of Illinois.

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 12, 2011

Mapping Sensory Systems in Sulfate-Reducing Bacteria

Sulfate-reducing bacteria (SRBs) play important roles in the decomposition of organic matter, cycling of nutrients, and transformation of heavy metals in subsurface environments. Sensing and responding to minute shifts in nutrient levels, potentially damaging or toxic conditions, and the presence of other microbes is critical to their lifestyle. Systems involving two components, paired sets of sensor and regulator proteins that control gene expression, are an important sense/response mechanism in bacteria, but it remains extremely difficult to establish relationships between the systems and larger networks of regulated genes. Researchers at Lawrence Berkeley National Laboratory have now completed the first-ever map of two-component regulatory systems for the model microbe SRB Desulfovibrio vulgaris using a cell-free approach based on direct binding of purified regulator proteins to genome fragments. Genes involved in nutrient acquisition, growth, stress response, and community assembly were mapped onto specific response regulators, providing a greatly enhanced understanding of how SRBs react to changing environmental conditions and mediate key processes in the subsurface.

Reference: Rajeev, L., E. G. Luning, P. S. Dehal, M. N. Price, A. P. Arkin, and A. Mukhopadhyay. 2011. “Systematic Mapping of Two Component Response Regulators to Gene Targets in a Model Sulfate Reducing Bacterium,” Genome Biology 12:R99. DOI:10.1186/gb-2011-12-10-r99. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 10, 2011

Maize Juvenility Gene Enhances Biofuel Production from Bioenergy Crops

The sugars in plant cell walls have the potential to be converted on a large scale to biofuels; however, these sugars are locked in a rigid lignin matrix, inhibiting their extraction and conversion into biofuels. Researchers have now discovered a potential way around this obstacle through studies of the maize Corngrass1 (Cg1) gene, which promotes maintenance of juvenility in maize plants. Since juvenile plant material contains less lignin, they hypothesized that this mutant might produce plants whose sugars would be more easily extracted and converted into biofuels. When the Cg1 gene was transferred into other plants, including the potential bioenergy crop switchgrass, the amount of starch and subsequent glucose release was significantly higher than from the wild type plants even without expensive pretreatment. These results offer a promising new approach for the improvement of dedicated bioenergy crops. The research was carried out at the USDA-ARS, University of California, Berkeley, DOE's Joint BioEnergy Institute, and the Energy Biosciences Institute, and supported in part by the joint USDA-DOE Plant Feedstocks Genomics for Bioenergy program. It is published in the Proceedings of the National Academy of Sciences.

Reference: Chuck, G. S., C. Tobias, L. Sun, F. Kraemer, C. Li, D. Dibble, R. Arora, J. N. Bragg, J. P. Vogel, S. Singh, B. A. Simmons, M. Pauly, and S. Hake. 2011. "Overexpression of the Maize Corngrass1 MicroRNA Prevents Flowering, Improves Digestibility, and Increases Starch Content of Switchgrass," Proceedings of the National Academy of Sciences (USA) 108(42), 17550-55. DOI:10.1073/pnas.1113971108. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 27, 2011

Microbial Production of Bisabolane, a New Terpene-Based Biofuel

Development of next-generation biofuels will require economical production of high-energy compounds that are compatible with existing vehicle engines and fuel distribution infrastructures. To this end, researchers at the DOE Joint Bioenergy Institute (JBEI) have been exploring potential fuel properties of molecules in the terpene family. Many terpene molecules possess properties similar to petroleum-derived fuel compounds, and industrial microbes such as yeast and E. coli have been previously engineered for terpene compound synthesis for pharmaceutical production. In a new study published in Nature Communications, JBEI scientists describe production of the terpene bisabolane, a molecule with fuel properties similar to D2 diesel. After identifying bisabolane as a promising biofuel, the team embarked on a series of targeted genetic modifications to terpene synthesizing E. coli and yeast strains, resulting in microbial production of the compound using simple sugars as the starting material. Unlike other biofuels such as ethanol and isobutanol, bisabolane was found to be relatively nontoxic to the microbes and thus could potentially be produced at higher yields. Efforts are currently underway to screen the fuel properties of biologically produced bisabolane and develop improved fermentation strategies that would enable scaling of production to commercial levels.

Reference: Peralta-Yahya, P. P., M. Ouellet, R. Chan, A. Mukhopadhyay, J. D. Keasling, and T. S. Lee. 2011. "Identification and Microbial Production of a Terpene-Based Advanced Biofuel," Nature Communications 2:483. (DOI: 10.1038/ncomms1494) (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 16, 2011

Engineering Microbes to Produce Biodiesel Precursors

Biodiesel production typically starts with oil-rich energy crops such as soybean, palm, or rapeseed, which are harvested and converted into fatty acids from which biodiesels or other fuels are derived. The cost of expanding crop production is a limiting factor in allowing biodiesel to compete with fossil fuel sources. One alternative is to avoid the plant entirely and directly synthesize the precursor fatty acids in bacteria, bypassing several upstream steps, reducing production costs, and raising final yields. A team of researchers, including members of the DOE Joint Genome Institute, now has developed a process to engineer bacteria to produce biodiesel with the help of a novel fatty acid synthesis enzyme. The enzyme, identified and characterized from several bacterial sequences, was inserted into the commonly used model microbe E. coli to prove that it was involved in fatty acids synthesis. The fatty acid pathway was further engineered to improve the generation of biodiesel precursors. This new work provides an alternative route for the synthesis of biofuel molecules. The pathway they describe is a first step in the generation of biodiesel and, with further optimization, may lead to the production of a cost-efficient, next-generation biofuel. The results have just been published in Applied and Environmental Microbiology.

Reference: Nawabi, P., S. Bauer, N. Kyrpides, and A. Lykidis. 2011. "Engineering Escherichia coli for Biodiesel Production Utilizing a Bacterial Fatty Acid Methyltransferase," Applied and Environmental Microbiology 77(22), 8052-61. DOI:10.1128/AEM.05046-11

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 12, 2011

Understanding How Environmental Microbes Make Uranium Less Soluble

Uranium is one of the major contaminants at DOE cleanup sites. It was usually released into the environment as the highly soluble uranyl ion (uranium (VI)). This ion interacts with bacteria and minerals in the ground to form reduced uranium (IV), notably in the mineral uraninite, a form that is much less soluble than uranium (VI). Less soluble uranium (IV) species are less likely to be moved out of the initially contaminated zone and into nearby rivers or aquifers by groundwater. New research has shown that biologically produced uraninite in a natural underground environment dissolves much more slowly than uraninite prepared in the laboratory. Researchers have developed a model showing that the slower dissolution is due to the presence of biomass that limits the reoxidation rate of the uranium (IV) in uraninite and diffusion of oxidized uranium into the groundwater. This understanding will be used in developing improved models of uranium transport in contaminated environments. Field studies were carried out at the Old Rifle, Colorado, Integrated Field Research Challenge site, while experiments to determine the forms of uranium present were conducted at the Stanford Synchrotron Radiation Lightsource.

Reference: Campbell, K. M., et al. 2011. "Oxidative Dissolution of Biogenic Uraninite in Groundwater at Old Rifle, CO," Environmental Science and Technology 45, 8748–54. DOI: 10.1021/es200482f. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 02, 2011

Capturing Carbon in the Dark Ocean

Contributions to the carbon cycle in the ocean's water column below the penetration of sunlight have not yet been explained either mechanistically or quantitatively, although a significant part of ocean carbon fixation is known to be due to microbial activities. Current oceanographic models suggest that archaea, the prevalent microbial domain in the oceans, do not adequately account for the carbon that is being fixed in the dark ocean. New research using sequencing technology has identified microbes involved in capturing carbon in the twilight zone, the region of the ocean that lies between 200 meters and 1,000 meters beneath the surface. This study discovered specific types of bacteria (the other domain of prokaryotic microbes besides the archaea) that may be responsible for this major, previously unrecognized component of the dark ocean carbon cycle. The report's authors isolated and identified bacteria from water samples collected in the South Atlantic and North Pacific oceans. They found that "...previously unrecognized metabolic types of dark ocean bacteria may play an important role in global biogeochemical cycles, and their activities may in part reconcile current discrepancies in the dark ocean's carbon budget." A better model of carbon cycling in the oceans will help experts predict future CO2 concentrations in the atmosphere and oceans and impacts of altered CO2 fluxes on ocean biogeochemistry. This work involved researchers from the DOE Joint Genome Institute.

Reference: Swan, B., et al. 2011. "Potential for Chemolithoautotrophy Among Ubiquitous Bacteria Lineages in the Dark Ocean," Science 333, 1296-1300. DOI: 10.1126/science.1203690. (Reference link)

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 01, 2011

Direct Mass Spectrometric Imaging of Cellulose and Hemicellulose in Populus Tissue

Pretreatment of bioenergy feedstocks produces complex chemical changes that need to be understood to evaluate the effectiveness of different pretreatment regimens. Feedstock imaging can provide useful information, but high molecular specificity is required to identify components such as cellulose and hemicellulose and to produce useful spatial images. Simple mass spectrometry (MS) is limited by the complexity of the plant tissue. University of Florida researchers have successfully overcome this difficulty by applying matrix-assisted laser desorption/ionization mass spectrometry (MALDI) linear ion trap tandem MS technology. In tandem MS, the material goes through two consecutive rounds of MS instead of one. While single MALDI MS images of young Populus wood stems show an even distribution of both cellulose and hemicellulose, tandem MS produces very different images of the distribution of the two plant components. The new strategy offers the high molecular specificity needed for analyzing complex lignocellulosic biomass and will be applicable to many plant species that are potential bioenergy resources.

Reference: Lunsford, K. A., G. Peter, and R. Yost. 2011. "Direct Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Imaging of Cellulose and Hemicellulose in Populus Tissue," Analytical Chemistry 83(17), 6722-30. (DOI: 10.1021/ac2013527) (Reference link)

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 01, 2011

Engineering Microbes for Optimized Biofuel Production

Redirecting a microbe's metabolic pathways to make desired products frequently results in slower growth, lower yield, and other negative impacts that reduce production efficiency. This is often related to the accumulation of toxic intermediates at metabolic "bottlenecks" in microbes lacking natural pathways to use, redirect, or dispose of these compounds. Researchers at the DOE Joint Bioenergy Institute (JBEI) have observed this phenomenon in E. coli strains expressing an engineered pathway for the synthesis of terpene, a precursor of several different hydrocarbon biofuels. To alleviate this toxicity, the team screened genome databases to identify variants of the enzyme in other organisms that are able to process the problematic compound. The enzymes were expressed in vitro and assayed for activity, and genes encoding the most promising candidates were engineered into E. coli. This produced a set of strains with varying synthesis properties under different growth conditions. Subsequent manipulation of gene expression levels, cofactor pools, and redox conditions resulted in a 120% improvement in terpene production over the initial strain. These results further improve an already promising industrial microbe and demonstrate the potential of coupled systems biology and targeted metabolic engineering for enhancing biofuel production.

Reference: Maa, S. M., D. E. Garcia, A. M. Redding-Johanson, G. D. Friedland, R. Chan, T. S. Batth, J. R. Haliburton, D. Chivian, J. D. Keasling, C. J. Petzold, T. Lee, and S. R. Chhabra. 2011. "Optimization of a Heterologous Mevalonate Pathway Through the Use of Variant HMG-CoA Reductases," Metabolic Engineering 13(5), 588-97. (DOI: 10.1016/j.ymben.2011.07.001) (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 01, 2011

Improving Understanding of Microbial Interactions with the Environment

Transporter proteins control the flow of large and small molecules in and out of the cell and are a primary means for organisms to interface with the environment. Transporters affect cellular metabolic capabilities and influence signaling pathways and regulatory networks that are key to the cell’s behavior. DOE researchers have confirmed the efficacy of a high-throughput methodology to rapidly and specifically identify the molecules transported by these proteins. The new technique measures the change in the melting temperature of proteins. Using Rhodopseudomonas palustris as a test case, they found a variety of compounds bound to the transporters studied that were not predicted using standard computational methods. These findings illustrate the potential of this method to expand our ability to predict the response of microbes and cells to environmental changes, such as the utilization of environmental nutrients and the ejection of toxic compounds.

Reference: Giuliani, S. E., A. M. Frank, D. M. Corgliano, C. Seifert, L. Hauser, and F. R. Collart. 2011. "Environment Sensing and Response Mediated by ABC Transporters," BMC Genomics 12(Supplement 1), S8. (DOI: 10.1186/1471-2164-12-S1-S8) (Reference link)

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 01, 2011

Poplar Roots Influence Microbial Community Composition

Poplar, a model organism for woody perennials, is a promising bioenergy feedstock for producing cellulosic biofuels. Poplar roots establish intimate associations with various microorganisms, both bacterial and fungal, that are beneficial to both plant and microbe. However, these associations are still poorly understood. Researchers at Oak Ridge National Laboratory have published the first results of a comprehensive study of the poplar rhizosphere (soil in direct contact with plant roots) and endophytic (living within plant tissues without causing harm) microbial communities from mature, natural poplar stands. They investigated microbial diversity among root endophyte and associated rhizosphere communities from two poplar populations differing in soil and stand characteristics near the Caney Fork River in central Tennessee. Although soil was not a major determinant of microbial distribution and diversity, the rhizosphere and endophyte communities of both bacteria and fungi were distinct. The results suggest that tissues within naturally occurring poplar roots provide a unique niche for these microorganisms. The research has implications for the growth and management of poplar plantations established for biofuel production.

Reference: Gottel, N. R., H. F. Castro, M. Kerley, Z. Yang, D. A. Pelletier, M. Podar, T. Karpinets, E. Uberbacher, G. A. Tuskan, R. Vilgalys, M. J. Doktycz, and C. W. Schadt. 2011. "Distinct Microbial Communities Within the Endosphere and Rhizosphere of Populus deltoides Roots Across Contrasting Soil Types," Applied and Environmental Microbiology 77(17), 5934-44. (DOI:10.1128/AEM.05255-11) (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 21, 2011

New Microfluidic Device Enables Characterization of Environmental Microbes

Microbes play critical roles in global scale environmental processes such as carbon cycling and the movement and degradation of environmental contaminants at waste sites. Understanding and predicting the roles of particular types of microbes in these processes remains extremely challenging, since over 90% of environmental microbes cannot be grown in the lab and existing approaches do not allow identification of specific cell types or quantification of their abundance. Researchers at Lawrence Berkeley National Laboratory and Sandia National Laboratories have now developed a new microfluidic device called µFlowFISH that enables the high-throughput identification of the types and abundance of microbes from environmental samples. Microbial cells are moved through the chip-mounted device using electrical currents, fluorescently labeled using diagnostic probes, and counted in a flow cytometry chamber. After initial testing with microbes that could be cultured, µFlowFISH was used to analyze microbes in groundwater samples from the DOE Hanford 100H cleanup site, targeting organisms known to be involved in uranium immobilization. Results from the device were in good agreement with more cumbersome and time-intensive techniques, requiring 100-fold less sample and far less time. Coupled to "omics" methods for comprehensive microbial community analysis, µFlowFISH presents a powerful new tool for dissecting microbial community structure and function in a variety of environments. Reference: Peng, L., R. J. Meagher, Y. K. Light, S. Yilmaz, R. Chakraborty, A. P. Arkin, T. C. Hazen, and A. K. Singh. 2011. "Microfluidic Fluorescence In Situ Hybridization and Flow Cytometry (µFlowFISH)," Lab on a Chip 11, 2673-79. DOI: 10.1039/c1lc20151d.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 18, 2011

A "Meraculous" Algorithm for Whole-Genome Assemblies

DNA sequencing technologies generate a tremendous amount of genomic data compared to just a few years ago. Today, however, most genomic data is for small DNA fragments that need to be assembled back into a whole genome to elucidate the biological function of the parent organism. This represents a computational challenge for the sequencing community, in particular when the amount of genomic data reaches more than a hundred million fragments. DOE Joint Genome Institute researchers have now developed an efficient algorithm, Meraculous, to assemble the short genomic fragments into whole genome sequences. Meraculous can quickly and accurately assemble microbial genomes with a fraction of the computer memory required for more traditional methods, thanks to the use of novel techniques in graph theory and in memory-efficient hashing schemes. JGI staff have tested this method on Pichia stipiti, a microbe that efficiently produces ethanol from the five-carbon sugar xylose and found that they were able to quickly reconstruct 95% of the genome, error free. Research at JGI continues to advance this algorithm with applications to more complex plant genomes planned.

Reference: Chapman, J. A., I. Ho, S. Sunkara, S. Luo, G. P. Schroth, and D. S. Rokhsar. 2011. "Meraculous: De Novo Genome Assembly with Short Paired-End Reads," PLoS ONE 6(8), e23501. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 16, 2011

Assessing Carbon Impacts of Land-Use Choices for Bioenergy Crops

The Conservation Reserve Program (CRP) contains over 13 million hectares of former croplands now in grasslands, providing a reservoir of biodiversity, water quality, and carbon sequestration benefits. However, these benefits could be lost if the land is converted back to agricultural use for biofuel production. Scientists from the DOE Great Lakes Bioenergy Research Center analyzed the effects that converting CRP lands to annual crops for biofuel production (continuous corn and corn-soybean rotation, each either tilled or permanent no-till) would have on greenhouse gas (GHG) emissions as compared with directly harvesting perennial grasses on these lands for cellulosic ethanol. They report that although a no-till management regime of an annual bioenergy crop would reduce the carbon debt significantly compared with tilling, harvesting perennial grasses would result in virtually no GHGs lost, because the disruption required when converting to annual crops would be avoided. This is the first time field trials have been used instead of model predictions. The trials show that carbon debt can be avoided and climate change mitigated by directly using unconverted CRP grasslands for cellulosic feedstock production. The results will be helpful in developing strategies for producing bioenergy crop systems.

Reference: Gelfand, I., T. Zenone, P. Jasrotia, J. Chen, S. K. Hamilton, and G. P. Robertson. 2011. "Carbon Debt of Conservation Reserve Program (CRP) Grasslands Converted to Bioenergy Production," Proceedings of the National Academy of Sciences of the United States of America 108(33), 13864-69. (DOI: 10.1073/pnas.1017277108) ( (Reference link)

Contact: John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 08, 2011

Key Ethanol Tolerance Gene Identified in Biomass-Degrading Bacteria

If a single organism could breakdown cellulosic biomass and synthesize biofuels, a process known as consolidated bioprocessing, it could significantly increase the efficiency and reduce the costs of biofuel production. Some biomass-degrading microbes such as Clostridium thermocellum can also synthesize ethanol, but they are poisoned by relatively low ethanol concentrations compared to sugar fermenters such as yeast or E. coli. Researchers at the DOE Bioenergy Science Center (BESC) have now identified a key gene in C. thermocellum that is related to enhanced ethanol tolerance. The team analyzed genomes of C. thermocellum mutants that could tolerate higher than normal ethanol concentrations, and found a consistently modified gene involved in alcohol metabolism. By analyzing the structure of the encoded protein, it was determined that the mutation causes significant alterations to central ethanol metabolism. The identification of this gene will enable more targeted metabolic engineering approaches to improve production of ethanol and other biofuels in C. thermocellum and other biomass-degrading microbes useful for consolidated bioprocessing.

Reference: Brown, S. D., A. M. Guss, T. V. Karpinets, J. M. Parks, N. Smolin, S. Yang, M. L. Land, D. M. Klingeman, A. Bhandiwad, M. Rodriguez, Jr., B. Raman, X. Shao, J. R. Mielenz, J. C. Smith, M. Keller, and L. R. Lynd. 2011. "Mutant Alcohol Dehydrogenase Leads to Improved Ethanol Tolerance in Clostridium thermocellum," Proceedings of the National Academy of Sciences of the United States of America 108, 13752-57. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 05, 2011

Conifer-Rotting Fungus Offers Potential New Strategy for Lignocellulose Degradation

Due to its abundance and high cellulose content, wood has great potential as raw material for the production of biofuels. However, wood also contains lignin, a hard-to-degrade polymer that poses a major obstacle to converting its cellulose into liquid fuels. White rot fungi have evolved mechanisms to digest lignin and cellulose, and scientists are trying to take advantage of these capabilities. Now, new research using genome sequencing and comparative analysis of the brown rot fungus Serpula lacrymans has discovered a different strategy used by this boreal forest fungus to extract the energy-rich cellulose from conifer wood. A comparison of the gene content in white and brown rot fungi indicates that the enzymatic machinery to degrade lignin has been eliminated in brown rot fungi, enabling it to specifically target cellulose, separating it from the recalcitrant lignin. The researchers also discovered that in the presence of wood, S. lacrymans produces variegatic acid, a phenolate compound that helps in reducing iron ions to Fe+2, which are required for the initial non-enzymatic steps in cellulose degradation upon wood colonization by the fungus. These insights provide researchers with new strategies to potentially bypass the problem of eliminating lignin from renewable woody feedstocks for transportation fuel production. The research has just been published in Science and was carried out by an international consortium including researchers at DOE's Joint Genome Institute in Walnut Creek, CA, and its partners HudsonAlpha Institute for Biotechnology (Huntsville, AL) and Pacific Northwest National Lab (Richland, WA).

Reference: D. C. Eastwood, et al. 2011. "The Plant Cell Wall-Decomposing Machinery Underlies the Functional Diversity of Forest Fungi", Science, 333, 762-65. DOI:10.1126/science.1205411. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 02, 2011

New Insights on Algal Metabolism

Photosynthetic algae are a potential bioenergy source; however, significant unknowns about their basic metabolic properties have hindered development of algae for biofuel production. DOE researchers now present a new metabolic network reconstruction and a genome-scale model of light-driven metabolism for the alga Chlamydomonas reinhardtii. This approach represents a significant advance over previous metabolic models for this organism since it incorporates greatly improved functional gene annotations, experimental validation of gene expression, and quantitative reaction measurements under different light conditions. This model allows enhanced understanding and prediction of photosynthetic growth properties (including lipid synthesis) under varying conditions and provides a broad knowledgebase of potential targets for directed metabolic engineering. This publication was featured in the Editor’s Choice section of the August 12th issue of Science.

Reference: Chang, R. L., L. Ghamsari, A. Manichaikul, E. F. Hom, S. Balaji, W. Fu, Y. Shen, T. Hao, B. Palsson, K. Salehi-Ashtiani, and J. A. Papin. 2011. "Metabolic Network Reconstruction of Chlamydomonas Offers Insight into Light-Driven Algal Metabolism," Molecular Systems Biology 7:518. DOI:10.1038/msb.2011.52. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 26, 2011

Symbiotic Relationship with Fungi Benefits Bioenergy Feedstock Poplar

The forest soil environment is teeming with microbial communities, including a group of mutualistic fungi known as the ectomycorrhizae. These organisms develop a close association with tree roots, establishing an exchange of nutrients and sugars essential for the health of both plant and microbe. While this phenomenon has been known for a long time, the signaling and regulatory mechanisms of this exchange are poorly understood. Researchers at the DOE Oak Ridge National Laboratory, as part of an international collaboration, have identified and characterized a protein called Mycorrhizal Induced Small Secreted Protein 7 (MiSSP7) that is secreted from the ectomycorrhizal fungus Laccaria bicolor in response to signals diffused from the roots of poplar trees, a promising bioenergy feedstock. They found that this very small protein is imported into the nucleus of the host plant cell where it alters the expression of certain plant genes, similar to the manner in which fungal pathogens work. The result is a "reprogram-ming" of plant cells, through which a beneficial, symbiotic relationship between fungus and plant is established. This relationship enhances growth and productivity of the tree. Understanding the underlying mechanism will help address diverse DOE missions, including bioenergy production, environmental remediation, and carbon cycling and sequestration.

Reference: Plett, J. M., M. Kemppainen, S. D. Kale, A. Kohler, V. Legué, A. Brun, B. M. Tyler, A. G. Pardo, and F. Martin. 2011. "A Secreted Effector Protein of Laccaria bicolor Is Required for Symbiosis Development," Current Biology 21, 1197&ndash1203. DOI:10.1016/j.cub.2011.05.033. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 12, 2011

Impact of Bioenergy Feedstocks on Agricultural Landscapes

Simplification of the agricultural landscape due to expansive monocultures of individual crops reduces habitat diversity and has long been believed to increase insect pest pressure with a resulting need for more insecticides. This assumption seems logical, but has lacked supporting scientific evidence, evidence needed to establish a science-based land-use policy that includes dedicated bioenergy crops. Now, researchers at the DOE Great Lakes Bioenergy Research Center (GLBRC) have reported an analysis of cropping systems across 562 counties in seven Midwestern states. They found a significant correlation between insecticide use and land simplification (i.e., less natural habitat). The results suggest that plantings of more minimally managed perennial bioenergy crops requiring less insecticide use may mitigate some of the negative effects associated with continued simplification. This study provides a scientific basis for understanding the impact that the greater demand for bioenergy feedstocks will have on the agricultural landscape.

Reference: Meehan, T. D., B. P. Werling, D. A. Landis, and C. Gratton. 2011. "Agricultural Landscape Simplification and Insecticide Use in the Midwestern United States," Proceedings of the National Academy of Sciences of the United States of America 108, 11500–505. DOI: 10.1073/pnas.1100751108.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 11, 2011

Engineering a Better Switchgrass

Perennial grasses such as switchgrass are considered prime candidates for bioenergy feedstocks because of their potential for substantial biomass yields on marginal lands. An approach that promises further improvement in this species is genetic transformation, the introduction and expression of desirable genes from other sources to increase yields and reduce recalcitrance. Current transformation technology, however, uses promoters (segments of DNA that control the expression of desired genes) from other plants making them inefficient for use in switchgrass. Researchers from the DOE BioEnergy Science Center (BESC) now report the identification of novel promoter regions from a specific switchgrass gene that is found in all eukaryotes and that can be used for efficient genetic transformation in switchgrass. A variety of transgenic plants constructed with these promoters exhibited significantly higher gene expression levels than observed using the non-switchgrass promoters, showing great potential for driving transgenic expression in switchgrass and other plants. This is the first characterization of native switchgrass promoter sequences for transgene expression. The results will facilitate improvement of switchgrass and other bioenergy feedstocks through engineering of key bioenergy-relevant traits.

Reference: Mann, D. G. J., Z. R. King, W. Liu, B. L. Joyce, R. J. Percifield, J. S. Hawkins, P. R. LaFayette, B. J. Artelt, J. N. Burris, M. Mazarei, J. L. Benentzen, W. A. Parrott, and C. N. Stewart. 2011. "Switchgrass (Panicum virgatum L.) Ubiquitin Gene (PvUbi1 and PvUbi2) Promoters for Use in Plant Transformation," BMC Biotechnology 11, DOI: 10.1186/1472-6750-11-74. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 30, 2011

In Search of Enzymes for Biofuel Production

Some microbes contain enzymes that can break down lignocellulosic biomass, such as that found in switchgrass or Miscanthus. But there are few suitable methods for finding these enzymes in complex microbial communities. Researchers at the DOE Joint BioEnergy Institute (JBEI) have developed a new method that uses nanostructure initiator mass spectroscopy (NIMS). It enables rapid and accurate characterization of enzymes in complex microbial and environmental samples (e.g., microbial compost). Using this new technology, JBEI researchers have characterized a broad range of environmental and purified microbial samples, further optimizing selected samples for enzymatic activity and stability in the presence of ionic liquids, which are being tested by JBEI for use in biofuel production. This new NIMS-based approach may aid in finding more efficient ways to convert biomass into lignocellulosic biofuels.

Reference: Reindl, W., K. Deng, J. M. Gladden, G. Cheng, A. Wong, S. W. Singer, S. Singh, J.-C. Lee, C.-H. Yao, T. C. Hazen, A. K. Singh, B. A. Simmons, P. D. Adams, and T. R. Northen. 2011. "Colloid-Based Multiplexed Screening for Plant Biomass-Degrading Glycoside Hydrolase Activities in Microbial Communities," Energy and Environmental Science 4, 2884–93. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 23, 2011

Special "Biofuels Outlook" Section in Nature

The June 23, 2011, issue of the journal Nature features a special supplementary section that outlines the current state of biofuels research, highlights recent advances, and discusses potential issues associated with the expanded use of biomass-derived transportation fuels. Articles in the supplement discuss development of advanced drop-in biofuels compatible with existing engines, new approaches to economically break down tough lignocellulosic plant material into fermentable sugars, the potential of dedicated biomass feedstocks to reduce “food vs fuel” and water demand issues, and a variety of other topics relevant to biofuels development. Researchers involved in all three DOE Bioenergy Research Centers, as well as numerous scientists pursuing independent biofuels research supported by DOE’s Genomic Science program, discuss their work in the supplement. The collected articles provide a valuable resource for communicating the current state of biofuels R&D to the broader scientific community and the general public.

Reference: Grayson, M., et al. 2011. "Nature Outlook: Biofuels," Nature 474 (7352), supp S1–S43.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 17, 2011

What Makes a Plant a Plant?

DNA sequencing has generated vast collections of genes for all types of organisms; however, determining the roles of the proteins coded within those genes is a difficult task and the functions of many of those proteins are still unknown. Researchers at the UCLA-DOE Institute for Genomics and Proteomics and at the DOE Joint Genome Institute in Walnut Creek, California, have now provided new information on the function of genes that are uniquely found in plants and green algae. Comparing the genes present in the genomes of 20 photosynthetic organisms with those of non-photosynthetic organisms, the investigators compiled GreenCut2, an inventory of nearly 600 plant-specific genes. As the function of more than half of those 600 genes is not known, this work sheds new light on genes needed for plant-specific processes, including those related to the chloroplast (the photosynthetic organelle of plant cells). Further analysis of those proteins of unknown function showed that many of them are likely involved in protein modification, gene regulation, and transport of molecules to the chloroplast. This new knowledge provides insights on plant evolution and will help researchers better understand how plants work, enabling them to harness their potential to provide alternative energy sources.

Reference: Karpowicz, S., S. E. Prochnik, A. R. Grossman, and S. S. Merchant. 2011. "The GreenCut2 Resource: A Phylogenomically-Derived Inventory of Proteins Specific to the Plant Lineage," Journal of Biological Chemistry 286, 21427–439.

Contact: Pablo Rabinowicz, SC-23.2, (301) 903-0379; Susan Gregurick, SC-23.2, (301) 903- 7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 16, 2011

A Step Closer to the "Greening" of Commercial Biofuel Production

Ionic liquids, a relatively new class of "green" solvents, can break down a wide range of feedstocks for biofuels, producing high yields of sugar and relatively pure lignin with short treatment times. However, even after scale up, ionic liquids are expensive compared with other pretreatment options. To determine which biofuel production parameters have the greatest impact on total cost, DOE's Joint BioEnergy Institute (JBEI) conducted a techno-economic analysis. A publically available techno-economic model of a biofuel refinery was developed, using ionic liquid pretreatment, to show a prioritized research path for fundamental understanding, process engineering, and operational improvements that would enable the use of ionic liquids in a commercial setting. The model results indicate, in decreasing order of significance, the importance of high prices for lignin byproducts, reducing the cost of the ionic liquid solvents and the concentration of the solvent used, and increasing the rate of solvent recovery. This analysis will lead to improvements in the cost effectiveness of biofuel production using ionic liquid-based processes.

Reference: Klein-Marcuschamer, D., B. A. Simmons, and H. W. Blanch. 2011. "Techno-Economic Analysis of a Lignocellulosic Ethanol Biorefinery with Ionic Liquid Pretreatment," Biofuels, Bioproducts and Biorefining, DOI: 10.1002/bbb.303.

Contact: John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 15, 2011

Exploring the Cellulose Degradation Machinery of Hot Springs Bacteria

Members of the Caldicellulosiruptor genus of bacteria, originally discovered in terrestrial hot springs, are unique in their ability to efficiently degrade cellulosic plant biomass at temperatures over 70°C. Researchers at the DOE Bioenergy Science Center (BESC) at Oak Ridge National Laboratory previously sequenced the genomes of several Caldicellulosiruptor species and characterized their abilities to degrade corn stover, switchgrass, and other biomass feedstocks. In a new study, BESC scientists used mass spectrometry-based proteomics to compare the complex mixture of enzymes secreted by two Caldicellulosiruptor species during cellulose degradation. Both of the organisms deployed carefully regulated configurations of multifunctional cellulase modules, tethered cellulose binding elements, and proteins that bind released sugars and return them to the cell. All of these elements were traced back to encoding genes on sequenced genomes. The secreted cellulase fractions from the Caldicellulosiruptors were found to work optimally at 85°C and pH 5, indicating significantly higher thermal stability and acid tolerance than current commercially available cellulase cocktails. These results present a promising source of novel cellulase enzymes for industrial development and provide new insights into the diversity of tools that microbes have at their disposal for biomass breakdown.

Reference: Lochner, A., R. J. Giannone, M. Rodriguez, Jr., M. B. Shah, J. R. Mielenz, M. Keller, G. Antranikian, D. E. Graham, and R. L. Hettich. 2011. “Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis,” Applied and Environmental Microbiology 77, 4042–54.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 09, 2011

Circadian-Controlled Pathways Facilitate Adaptation to a Changing Environment

Plants and other organisms synchronize their internal processes with the environment through circadian clocks to cope with natural cycles of light and temperature. These temporal rhythms coordinate physiological and metabolic processes with daily and seasonal changes by helping coordinate gene expression that enable organisms to adapt. Researchers at Oregon State University and collaborators used a combination of genomics and bioinformatics technologies to investigate daily rhythms in gene expression in the monocot plant rice and the dicot plant poplar. They compared their findings to work previously performed in the model plant Arabidopsis. They found a high degree of conservation across the three species among the cycling patterns of many circadian clock genes. This new research indicates that a core regulatory network is conserved across higher plants, although some cases of species-specific diurnal/circadian-associated regulatory circuits were observed. The findings have implications for engineering plants with enhanced vigor, fitness, and adaptation to changing environments. The research was supported in part by the joint USDA-DOE Plant Feedstocks Genomics for Bioenergy program.

Reference: Filichkin, S. A., G. Breton, H. D. Priest, P. Dharmawardhana, P. Jaiswal, S. E. Fox, T. P. Michael, J. Chory, S. A. Kay, and T. C. Mockler. 2011. "Global Profiling of Rice and Poplar Transcriptomes Highlights Key Conserved Circadian-Controlled Pathways and cis-Regulatory Modules," PLoS ONE 6(6):e16907. (DOI: 10.1371/journal.pone.0016907) (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 02, 2011

Solving the Mysteries of Cellobiose Stability Using High-Performance Computing

Cellobiose, a two glucose basic repeat unit of cellulose, is formed in enzymatic or acidic hydrolysis of plant biomass and is the precursor compound that microbes digest to produce cellulosic biofuels. Because this process happens outside of the microbial cell, understanding the structure and stability of cellobiose in solution provides a framework for improving microbial biofuel production. Interestingly, the low-temperature, gas-phase stable, preferred structure of cellobiose is cis, while the high temperature structure is trans. However, in cellulose itself, cellobiose is always in the trans state. Researchers believe that the stability of trans-cellobiose could be due to the water environment that surrounds it. Now, an international collaborative study has found that water molecules hydrate cellobiose collectively instead of binding to cellobiose separately and sequentially as was previously assumed. The team used DOE’s National Energy Research Scientific Computing Center, a high-performance computing facility, to simulate cellobiose dynamics together with vibrational spectroscopy experiments. Their results suggest that water dynamics could play a critical role in determining the most stable structure of cellobiose. The next step in this research will be to produce a simulation of cellobiose that includes the quantum and dynamically polar nature of water. It is anticipated that this new research will provide insight into how to optimize the hydrolysis of plant-derived cellulose, a key step in the production of biofuels. The computational aspects of the research were funded by DOE's SciDAC program.

Reference: Pincu, M., et al. 2011. "Isotopic Hydration of Cellobiose: Vibrational Spectroscopy and Dynamical Simulations," Journal of Physical Chemistry A, DOI: 10.1021/jp112109p. (Reference link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 25, 2011

Biological Impacts of Climate Change on Coral Reefs

Over the past two decades, scientists have linked the decrease in the pH levels of the global oceans and the corresponding slowdown in coral growth to the increasing levels of carbon dioxide trapped in the atmosphere and which, in turn, are being absorbed in the ocean. As coral reefs are the primary habitat for several marine organisms, their decline has significant impacts on the health of the marine ecosystems and ocean productivity. To better understand how corals contribute to the global carbon cycle, the DOE Joint Genome Institute (JGI) generated a dataset of expressed sequence tags or ESTs, small portions of a genome that can be used to help identify unknown genes and chart their locations along the sequence, from the reef-building coral Acropora palmate. In a study published online May 25, 2011, in PLoS ONE, a team of researchers including DOE JGI’s Erika Lindquist compared the A. palmate EST dataset to an EST dataset of another reef-building coral to identify the proteins involved in helping corals adapt to global climate change. The comparative analysis identified several proteins evolving at an accelerated rate, such as those involved in immunity, reproduction and sensory perception. “The category that was the most enriched with rapidly evolving proteins —cell adhesion—may also be related to symbiosis,” noted the study authors in their paper. These proteins are expected to evolve under positive selection due to the need for readjustments, e.g., due to the “arms race” between the coral and the bacterial symbionts. This research provides insights into the impacts of climate change at the biological level.

Reference: Voolstra, C. R., S. Sunagawa, M. V. Matz, T. Bayer, M. Aranda, et al. 2011. “Rapid Evolution of Coral Proteins Responsible for Interaction with the Environment,” PLoS ONE 6(5),e20392. DOE:10.1371/journal.pone.0020392.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Acropora table coral on the Great Barrier Reef (Image (c) Pete Faulkner, Mission:awareness/Marine Photobank).



May 19, 2011

Using New Computational Methods To Improve Biofuel Production

Lignin gives plants their strength and helps make them resistant to diseases, but it also complicates the use of plant material for biofuel production because of its recalcitrance to deconstruction. Researchers have successfully manipulated the lignin biosynthetic pathway in biofuel-producing plant species; however, the modified plants often have unexplained or undesirable biological features. It is difficult to predict, given our current ability to model plant metabolic processes, how individual biosynthetic pathways connect together, influence each other, and are controlled. To address this challenge, Yun Lee and co-workers at DOE’s BioEnergy Research Center (BESC) have developed a new computational method that combines metabolic modeling with Monte Carlo (random sampling) simulations to enable the analysis of many biological pathways simultaneously. When this method was applied to the prediction of lignin biosynthesis in alfalfa, BESC researchers found that lignin generation was not due to a single process but involved many pathways. In addition, the researchers predicted, and later confirmed, that a possible control for lignin biosynthesis was the signaling molecule salicylic acid. This work addresses the complexity of plant biosynthetic pathways and provides a computational method that can help researchers decipher them, providing new tools that can be used to improve biofuel production.

Reference: Lee, Y., F. Chen, L. Gallego-Giraldo, R. A. Dixon, and E. O. Voit. 2011. "Integrative Analysis of Transgenic Alfalfa (Medicao sativa L.) Suggests New Metabolic Control Mechanisms for Monolignol Biosynthesis," PLoS Computational Biology 7(5), e1002047.

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 16, 2011

Fungal Lesson in Improving Large-Scale Chemical Production

The chemical compound citric acid has been produced on a large-scale basis for decades with the help of the filamentous fungus Aspergillus niger. The fungus also has enzymes that can be used to help break down plant cell walls for biofuel production, and it plays a key role in the carbon cycle.

For biofuels, A. niger is a highly relevant organism since it has already been scaled up, shown to be safe, and used for enzyme production. An A. niger strain was selected for sequencing by the DOE Joint Genome Institute (JGI) in 2005.

In a recent paper, an international team of collaborators including JGI compared the genome of the citric-acid producing A. niger strain with another strain that had undergone mutagenesis for enzyme production. The fungal genomes are expected to help industry generate green chemicals and fuels from sustainable sources. The comparative analysis allowed the team to identify the key genes to each strain’s predominant characteristics. This information, along with genomic data from additional Aspergillus strains being sequenced at the DOE JGI should facilitate further optimization of these strains for different bio-products.

Reference: Andersen, M. R., et al. 2011. “Comparative Genomics of Citric-Acid-Producing Aspergillus niger ATCC 1015 Versus Enzyme-Producing CBS 513.88,” Genome Research. Published online May 4, 2011, DOI:10.1101/gr.112169.110.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Aspergillus niger. (Image: Sue Karagiosis, PNNL)



May 16, 2011

Quantum Dot Thermometers Measure Temperatures Inside Single Living Cells

Small temperature differences inside individual cells affect kinetics and shift chemical equilibria, but also alter the physical state of biomaterials such as DNA and proteins. Technology for detecting such temperature variations could lead to insights into biological mechanisms related to a wide range of metabolic processes in bioenergy-relevant systems. New research has shown that quantum dots can serve as nano thermometers to measure local temperature responses inside single living cells following exposure to external chemical and physical stimuli. Quantum dots are semiconductors in the form of crystals that fluoresce with colors determined by crystal size and chemical composition. The spectral shifts in the photoluminescence produced by the quantum dots were used to map intracellular heat generation from different organelles and compartments in cells following exposure to stress from high calcium levels and cold shock. These results are the first experimental evidence for inhomogeneous intracellular temperature progression in cells. The research was carried out at the Berkeley Lab and Princeton University and published in ACS Nano.

Reference: Yang, J.-M., H. Yang, and L. Lin. 2011. "Quantum Dot Nano Thermometers Reveal Heterogeneous Local Thermogenesis in Living Cells," ACS Nano 5(6), 5067-71. (DOI: 10.1021/nn201142f) (Reference link)

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 09, 2011

Spikemoss Genome Aids Biofuels Researchers

The genome of a small plant is providing biofuels researchers with information that could influence the development of candidate biofuel feedstock plants and offering botanists long-awaited insights into plant evolution. A team of researchers, including from DOE’s Joint Genome Institute (JGI), used a comparative genomics approach on Selaginella moellendorffii and 14 other plants up and down the phylogenetic tree to identify the core genes likely to be present in a common ancestor to land plants.

“When you burn coal, you’re burning Selaginella’s ancestors,” said Purdue University botanist Jody Banks, who led the 2005 DOE JGI Community Sequencing Program project. The Selaginella research community has grown up around the availability of the genome since 2009 through the DOE JGI’s plant portal Phytozome. The spikemoss genome has revealed the transition from mosses to plants with vascular systems involving fewer genes than going from a non flower-producing vascular plant to one that does.

The spikemoss genome is already proving useful for biofuels researchers. For example, Banks’ colleague Clint Chapple, a coauthor on the paper and a Purdue colleague, has been using the Selaginella genome to study the pathways by which the three different types of lignin are synthesized in plants. He and his team have used enzymes from the lignin-synthesizing pathway in Selaginella to modify the canonical lignin-producing pathway in Arabidopsis to produce the polymer.

Reference: Banks, J. A., et al. 2011. “The Selaginella Genome Identifies Genetic Changes Associated with the Evolution of Vascular Plants,” Science 332, 960–63. Published online May 5, 2011, DOI:10.1126/science.1203810.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Selaginella moelledorffii. (Image by Jing-Ke Weng, Salk Institute)



May 06, 2011

Wood Degrading Fungi Use Specialized Systems for Degrading Different Plant Types

“Brown rot” and “white rot” fungi from forest floors are among the few organisms on Earth that can fully degrade both the long, repeated sugar chains (cellulose and hemicellulose) and the complex, interlinked network of aromatic compounds (lignin) that make up woody plant material. The two classes of fungi use distinct (but poorly understood) enzyme systems to break down biomass and show strong preferences for particular types of wood. A collaborative team of researchers at the DOE Great Lakes Bioenergy Research Center and the DOE Joint Genome Institute have examined representative species of brown and white fungi to determine which specific genes involved in biomass deconstruction are deployed to attack aspen or pine wood. These studies revealed that the two types of fungi used distinct deconstruction systems, and the expression of these systems was heavily influenced by the type of wood being degraded. Many genes identified in the study correspond to known biomass degradation enzymes, but a significant fraction have no currently known catalytic function and will be the subject of further investigation. The results of this study increase our understanding of molecular mechanisms that allow degradation of biomass and could lead to the identification of new systems for plant deconstruction and biofuels production.

Reference: Wymelenberg, A. V., et al. 2011 “Gene Expression of Wood Decay Fungi Postia placenta and Phanerochaete chrysosporium is Significantly Altered by Plant Species,” Applied and Environmental Microbiology, doi:10.1128/AEM.00508-11.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 05, 2011

Poplar Rust Fungus Is First Tree Pathogen Sequenced

Rust plant pathogens make up a large fungal group that cannot survive on their own, so they use crops as hosts, leading to reduced yields and potentially hindering efforts to grow biomass for fuel. To learn more about these pathogens, a 2006 Community Sequencing Program project at the DOE Joint Genome Institute (JGI) generated the 101-million base pair genome of the poplar leaf rust fungus Melampsora larici-populina, the first tree pathogen sequenced.

The fungal project complements work as poplar leaf rust outbreaks weaken poplar trees, a candidate bioenergy feedstock whose genome sequence was published by JGI in 2007. A new study that involved a JGI researcher compares the genomes of poplar leaf rust and wheat stem rust fungi, the latter sequenced by the Broad Institute, in order to develop better biocontrol methods. In combination with the genome sequence of Populus, published in 2006, researchers will be able to compare and dissect the molecular interactions that lead to symbiotic versus pathogenic responses in the host plant.

Reference: Duplessis, S., et al. 2011. "Obligate Biotrophy Features Unraveled by the Genomic Analysis of Rust Fungi," PNAS Early Edition, www.pnas.org/content/108/22/9166

Contact: SC-23.2, (301) 903-4742, Dan Drell
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Original confocal microscopy image of infected leaves from poplar cv. (Image by Stéphane Hacquard, INRA Nancy)



April 25, 2011

Comparative Genomics of Social Amoebae

Found in soils worldwide, slime molds such as Dictyostelium discoideum are perhaps best known by their behavior in the presence or absence of food. When food is plentiful, the social amoeba behave as individuals, but when food is scarce, they come together to form multicellular “fruiting bodies” that look like a flower bud atop a single stalk or foot composed of a fifth of the amoebae that have sacrificed themselves for the group.

Studying social amoebae allows researchers to learn more about multicellularity because these amoebae can exist in both single-cell and multicellular states. From a bioremediation perspective however, slime molds are important candidates in cleaning up sites contaminated with chemicals and radioactive materials.

In a recent paper, researchers from DOE’s Joint Genome Institute and Baylor College offer a second Dictyostelium genome, and compare the 33-million base draft sequence produced using the Sanger platform with the finished genome of the model organism D. discoideum.

Separated by 400 million years of evolution, Dictyostelium purpureum is a close relative of D. discoideum and shares many of the same characteristics. Aside from their food-related behaviors, they also have a highly sophisticated recognition system that allows them to distinguish same-species Dictyostelium from others. The researchers found that the genes involved in sociality evolve more rapidly, probably due to continuous adaptation and counter-adaptation.

Reference: Sucgang, R., et al. 2011. “Comparative Genomics of the Social Amoebae Dictyostelium discoideum and Dictyostelium purpureum,” Genome Biology 12:R20, DOI:10/1186/gb-2011-12-2-r20, reference link

Contact: Dan Drell, SC-23.2, (301) 903-4742, Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



D. purpureum. (Photo by Chandra Jack, Rice University)



April 14, 2011

Understanding the Role of Microbes in Greenhouse Gas Production in Agricultural Soils

It is critical to understand the role of agricultural practices on soil greenhouse gas (GHG) emissions as expanded collections of agricultural residues are considered for bioenergy production and shifts are made to farming dedicated bioenergy crops. Production and consumption of carbon dioxide, methane, and other GHGs are predominantly mediated by soil microbes, yet the relationship between functional processes and microbial diversity in these systems is poorly understood. Researchers at the DOE Great Lakes Bioenergy Research Center (GLBRC) have examined agricultural GHG production, linking these processes to microbial community activities. The study included agricultural soils under various management practices, both successional grasslands on abandoned agricultural land and mature forests or grasslands that had never been farmed. GHG production and consumption rates were correlated to soil microbial community composition. Rates of methane consumption were found to be highest in non-agricultural forests and grasslands, which also showed the greatest diversity of methane-consuming microbes (i.e., methanotrophs). Successional sites were intermediate in terms of both methane consumption and methanotroph diversity, suggesting a gradual recovery process following disruption by traditional tillage agriculture. These results have important implications in considering sustainable establishment and long-term management of bioenergy landscapes and predictive modeling of GHG emissions.

Reference: Levine, U. Y., T. K. Teal, G. P. Robertson, and T. M. Schmidt. 2011. "Agriculture's Impact on Microbial Diversity and Associated Fluxes of Carbon Dioxide and Methane," The ISME Journal, DOI:10.1038/ismej.2011.40. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 07, 2011

Probing the Natural Variation in Poplar Trees to Increase the Yield of Sugars for Biofuels

A promising source of renewable “next generation” fuels is from the lignocellulosic biomass of poplar trees, from which sugars can be extracted and fermented to produce biofuels. These sugars, in the form of cellulose and hemicellulose, are embedded within lignin, a complex polymer composed of varying ratios of phenylpropanoid subunits. The rigid structure of lignin is a critical component of the plant cell wall, but this same trait impedes extraction of the sugars. Researchers at the DOE BioEnergy Research Center (BESC) at Oak Ridge National Laboratory measured lignin content and composition in a large (1100 individual) sample of undomesticated poplar trees and found that variation between individuals was large and significant. Using a high-throughput screening method, samples were tested for total sugar release with or without various pretreatments. The total amounts of sugars released varied widely among samples, and, as expected, a strong negative correlation between sugar release and lignin content was observed. However, the large data set allowed the researchers to discover critical exceptions to the overall correlation. The negative correlation did not apply to trees with a certain composition of lignin, and, for some trees with typical lignin content and composition, a very high volume of sugars were released. These results indicate that although recalcitrance to sugar release is partly determined by lignin content, lignin composition and other factors are also critical, and underscores the need for further research on cell wall structure in order to rationally design high-yielding bioenergy feedstocks for large-scale industrial use. The research has just been published in the Proceedings of the National Academy of Sciences (USA).

Reference: Studer, M.H., M.F. Davis, R.W. Sykes, B.H. Davison, M. Keller , G.A. Tuskan, and C.E. Wyman. 2011. Lignin Content in Natural Populus Variants Affects Sugar Release." PNAS doi:10.1073/pnas.1009252108.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 21, 2011

Correlating Biomolecular Experimental Measurements with Computational Simulations

Understanding the structural changes a biomolecule undergoes during processing is important in the design of, for example, new routes to convert biomass to biofuels. However, when studying these processes it often is difficult to correlate kinetic experiments with computer simulations. Both the experiments and the simulations provide a time-ordered understanding of the biological process at hand, but the results are often hard to compare. Research by an international consortium that includes Jeremy Smith of Oak Ridge National Laboratory has developed a new mathematical method, “Dynamical Fingerprints,” that allows researchers to visualize the essential kinetic features of an experiment and compare these features directly to computational simulation results. Structural changes present in the simulation can be assigned to experimentally observed processes. The new method enables enhanced interpretation of experiments ranging from neutron scattering to fluorescence correlation spectroscopy and Förster resonance energy transfer efficiency. Combining simulations and experiments will enable progress in areas such as biofuel production and design of advanced materials, which require a clear understanding of how molecules move and interact. The research was supported by DOE SciDAC funding and was just published online in the Proceedings of the National Academy of Sciences (USA).

Reference: Noe, F., S. Doose, I. Daidone, M. Löllmann, M. Sauer, J. Chodera and J. Smith. 2011. “Dynamical Fingerprints for Probing Individual Relaxation Processes in Biomolecular Dynamics with Simulations and Kinetic Experiment,” Proceedings of the National Academy of Sciences (USA), Early Edition March 2, 2011 (DOI: 10.1073/pnas.1004646108).

Contact: Christine Chalk, SC-21.1, (301) 903-5152, Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 21, 2011

Finding “Small” Proteins and Discovering How They Affect Plant Biomass Growth

Proteins less than 200 amino acids in length are commonly called “small proteins.” They have recently been found to have important roles in regulating biological processes such as stress response, flowering, and cell-to-cell communication in plants. However, identification of short open reading frames (sORFs), the genes that encode small proteins, has been a problem because their small size makes accurate prediction difficult. Researchers at Oak Ridge National Laboratory, working with scientists at the DOE BioEnergy Research Center, have applied computational biology to gene expression and protein data to discover sORFs encoding small proteins in the promising bioenergy feedstock Populus deltoids (poplar). Using the capacity of the DOE Joint Genome Institute for deep RNA sequencing, they reconstructed high-quality, full-length genes directly from the set of genes expressed in poplar (transcriptome), thus avoiding the uncertainty of prediction from genome sequence. The team then applied three computational filters to enrich for protein-encoding sORFs: prediction based on known protein sequences, evolutionary conservation between poplar and other plants, and protein family clustering. The results demonstrated the efficacy of this strategy in discovering candidate sORFs in sequenced as well as yet unannotated genomes. This method will greatly enhance understanding of the regulatory mechanisms underlying processes such as growth and stress response, features important to the development of high-yielding, sustainable bioenergy feedstocks.

Reference: Yang, X., T. J. Tschaplinski, G. B. Hurst, S. Jawdy, P. E. Abraham, P. K. Lankford, R. M. Adams, M. B. Shah, R. L. Hettich, E. Lindquist, U. C. Kalluri, L. E. Gunter, C. Pennacchio, and G. A. Tuskan. 2011. “Discovery and Annotation of Small Proteins Using Genomics, Proteomics, and Computational Approaches,” Genome Research doi:10.1101/gr.109280.110. Published online March 2, 2011.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 21, 2011

Improved Pretreatment of Biomass by Ionic Liquids Pretreatment

Pretreatment is a critical yet expensive stage in the biomass to biofuels pathway. However, pretreatment can reduce the overall biofuel production cost by facilitating conversion of the raw lignocellulosic biomass material into fermentable sugars and other valuable components. Pretreatment is thought to disrupt the lignin-carbohydrate complex in the cellulose microfibrils. Researchers at the DOE Joint BioEnergy Institute (JBEI) applied X-ray diffraction and small-angle neutron scattering to better understand ionic liquid pretreatment of these materials. The techniques were used to determine structural and surface changes in the biomass as a function of pretreatment conditions. Compared with other biomass samples studied, the ionic liquid pretreatment of switchgrass facilitated a more rapid expansion and conversion of the crystalline cellulose structure into a form more susceptible to enzymatic hydrolysis. The researchers also found that the degree to which lignin is intermixed within the cellulose microfibrils influences the required temperature and duration of an effective ionic liquid pretreatment.

Reference: Cheng, G., P. Varanasi, C. Li, H. Liu, Y. B. Melnichenko, B. A. Simmons, M. S. Kent, and S. Singh. 2011. “Transition of Cellulose Crystalline Structure and Surface Morphology of Biomass as a Function of Ionic Liquid Pretreatment, and Its Relation to Enzymatic Hydrolysis,” Biomacromolecules, dx.doi.org/10.1021/bm101240z.

Contact: John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 07, 2011

Complex Sugar Diet Makes Microbe a Good Candidate for Producing Biofuels

Many current biofuel production scenarios involve breaking down biomass into its component sugars and using microbes to convert these sugars into liquid biofuels. However, plant biomass contains long chains of both six- and five-carbon sugars (cellulose and hemicelluloses, respectively) and the commonly used biofuel-producing microbes such as the yeast Saccharomyces cerevisiae or the bacterium Escherichia coli cannot use both sugars simultaneously. Thus, substantial effort and expense is required to separate the sugars prior to conversion to fuels, resulting in reduced overall process efficiency. Now, researchers at the DOE Joint Bioenergy Institute (JBEI) have demonstrated that the microbe Sulfolobus acidocaldarius can simultaneously consume both types of sugars, efficiently consuming even complex substrate mixtures. S. acidocaldarius is an extremophile capable of growing at high temperatures in acidic conditions with an unusually high degree of genome stability. Altogether, these traits make this organism an attractive candidate for metabolic engineering and further development as industrial biofuel producer.

Reference: Joshua, C. J., R. Dahl, P. I. Benke, and J. D. Keasling. 2011. “Absence of Diauxie During Simultanteous Utilization of Glucose and Xylose by Sulfolobus acidocaldarius,” Journal of Bacteriology 193, 1293–1301.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 07, 2011

Engineering Production of Biofuels From Proteins

Biofuels currently are produced either from carbohydrates (e.g., ethanol from starch or sugar) or lipids (e.g., biodiesel from oils or fats). Both have serious shortcomings, requiring processes that have limited net energy efficiency and yielding byproducts such as nitrous oxide, a potent greenhouse gas. Research has now shown that the third major component of living organisms, proteins, could provide a large-scale source of biofuels without these limitations. Scientists at the UCLA-DOE Institute for Genomics and Proteomics have demonstrated that proteins produced in yeasts, bacteria, and algae can be converted efficiently into long-chain alcohols that are readily used in liquid fuels. The critical step in this research was to engineer metabolic processes into cells that convert the amino acids making up proteins into fuel molecules. These processes enable efficient deamination, or removal of the nitrogen-containing group from the amino acids and conversion of the resulting molecules into fuel alcohols. The nitrogen-containing byproducts are readily captured and recycled to fertilize growth of more of the photosynthetic cells, such as algae. The process can make effective use of sunlight as an energy source and CO2 as a carbon source, as proteins are the principal product of rapid growth in photosynthetic microorganisms. The research was led by James C. Liao and was just published in Nature Biotechnology.

Reference: Huo, Y.-X., K. M. Cho, J. G. Lafontaine Rivera, E. Monte, C. R. Shen, Y. Yan, and J. C. Liao. 2011. “Conversion of Proteins into Biofuels by Engineering Nitrogen Flux,” Nature Biotechnology, published online March 6, 2011, (doi:10.1038/nbt.1789).

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 07, 2011

Making Better Feedstocks for Bioenergy by “Cracking” Switchgrass’s “Backbone”

The perennial grass switchgrass is considered one of the most promising biofuel feedstocks because of its high yield potential and ability to thrive on lands poorly suited for conventional agriculture. However, the presence of lignin within the cell walls, which provides rigidity and pathogen resistance to the plant, also confers resistance to breakdown into constituent sugars. This recalcitrance to cell wall deconstruction limits current efforts to convert these sugars into biofuels. Now researchers at the U.S. Department of Agriculture’s Agricultural Research Service (USDA-ARS), with funding from the joint USDA-DOE Plant Feedstock Genomics for Bioenergy Program, have re-engineered switchgrass to produce a modified lignin that, when subjected to alkaline pretreatment, released a modest but significant increase in glucose compared to control plants. These modified plants have a reduced function of the gene catalyzing the last step in the lignin biosynthetic pathway (cinnamyl-alcohol dehydrogenase, or CAD). These results demonstrate the promise of this approach in developing high-yielding switchgrass lines for biofuel production.

Reference: Saathoff, A. J., G. Sarath, E. K. Chow, B. S. Dien, and C. M. Tobias. 2011. “Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment,” PLoS ONE 6(1): 16416.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 07, 2011

New Metabolic Mapping Capabilities May Lead to Design of More Useful Microbes

The recent development of metabolic flux analysis has enabled better understanding of the physiological state of microbes by tracing the molecules involved in cellular metabolism. Typically, however, metabolic flux analysis requires that molecular reactions be lumped together because it is too difficult to map all of the atoms involved in cellular processes. DOE researchers at Penn State University have tackled this problem head on by using techniques from pattern recognition and graph theory combined with conventional metabolic flux analysis and high-performance computing. They can now automatically trace the path of all atoms (C, O, N, P, S, metals and their ions) as these atoms move through metabolic reactions in E. coli. Thanks to the database they developed as part of this project, this process can be applied to other organisms so that researchers can quickly design and analyze isotopic labeling experiments. This new approach will allow researchers to better understand the physiological state of a microbe and then to design or enhance metabolic processes, such as for bioenergy production or carbon sequestration.

Reference: Ravikirthi, P., P. Suthers, and C. Maranas. 2011."Construction of an E. Coli Genome-Scale Atom Mapping Model for MFA Calculations," Biotechnology and Bioengineering, available online February 2011 (DOI: 10.1002/bit.23070). PubMed.

Contact: (301) 903-7672, SC-23.2, Susan Gregurick
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 07, 2011

One-Stop “Shopping” for Biofuels: A Breakthrough in Consolidated Bioprocessing

In most current biomass-to-biofuel strategies, plant material must be first broken down into its component sugars and then converted to ethanol in a separate step, resulting in a costly and inefficient process. Researchers at the DOE Bioenergy Science Center (BESC) and the University of California, Los Angeles, have now successfully engineered the cellulose-degrading bacterium Clostridium cellulolyticum to convert cellulose directly to isobutanol, a liquid fuel with much higher energy density than ethanol and, unlike ethanol, with the potential to be directly used in current engines. This consolidated bioprocessing (CBP) approach, in which a single organism both deconstructs plant cellulose and converts it to a biofuel in one step, significantly improves overall process efficiency. Until now no single microbe was known to possess the necessary combination of biomass degradation and fuel synthesis properties, and the most promising organisms are extremely challenging to genetically manipulate. This breakthrough thus provides a promising new avenue to engineer similar organisms for single-step conversion of plant biomass to fuels.

Reference: Higashide, W., Y. Li, Y. Yang, and J. C. Liao. 2011. “Metabolic Engineering of Clostridium cellulolyticumfor Isobutanol Production from Cellulose,” Applied and Environmental Microbiology, published online March 4, 2011 (doi:10.1128/AEM.02454-10).

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 03, 2011

New Insight into the Mechanism of Plutonium Transport in the Environment

The potential migration of plutonium in the environment is a concern at DOE sites such as the Hanford Nuclear Reservation and the Nevada Test Site, as well as an issue in nuclear waste disposal for nuclear energy development. Using a number of transmission electron microscopy techniques Lawrence Livermore National Laboratory researchers and collaborating Clemson University scientists have provided important new understanding of the formation and the biogeochemical mechanisms controlling plutonium migration. Once thought immobile in the subsurface, it has been recently recognized that plutonium is capable of being transported with the colloidal faction of groundwater. The researchers examined the interaction of plutonium nanocolloids with environmentally relevant minerals such as iron-containing goethite and silicon-containing quartz. The studies revealed the molecular basis of potential binding through epitaxial growth between the plutonium nanocolloids and colloid goethite that may be a possible mechanism for enhanced plutonium transport. The results improve our understanding of how molecular-scale behavior at the mineral-water interface may facilitate transport of plutonium at the field scale, providing important molecular-level input to improve contaminant transport models and the prediction of plutonium behavior.

Reference: Powell, B. A., Z. Dai, M. Zavarin, P. Zhao, and A. B. Kersting. 2011. "Stabilization of Plutonium Nano-Colloids by Epitaxial Distortion on Mineral Surfaces," Environmental Science and Technology 45, 2698–2703. DOI:dx.doi.org/10.1021/es1033487. (Reference link)

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


March 01, 2011

Assembly Path of Multi-Metal Catalysis Clusters in [FeFe]-Hydrogenases Revealed

Complex enzymes containing iron-sulfur (Fe-S) clusters are ubiquitous in nature where they are involved in a number of reactions fundamental for life, including carbon dioxide and nitrogen fixation and hydrogen metabolism. Because these enzymes have high catalytic rates of hydrogen production, their potential for improving hydrogen–fuel cell technologies is the focus of much interest. One type of such enzymes, the [FeFe]-hydrogenases, is being investigated as an alternative biological catalyst to enzymes containing precious metals such as platinum. The active site of this hydrogenase, the H-cluster, has a [4Fe-4S] subcluster bridged to a 2Fe subcluster. Advancements in understanding how this H-cluster is synthesized in nature could contribute significantly to both the genetic engineering of hydrogen-producing microorganisms and the synthesis of biomimetic hydrogen-production catalysts. X-ray crystallography data from an intermediate, not-yet-mature form of [FeFe]-hydrogenase present insights into how the H-cluster (bio)synthesis occurs. This research was conducted at the Stanford Synchrotron Radiation Lightsource.

Reference: Mulder, D. W., et al. 2010. “Stepwise [FeFe]-Hydrogenase H-Cluster Assembly Revealed in the Structure of HydAΔEFG,” Nature 465, 248–51.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 01, 2011

Improving Access to Cellulose in Biomass for Biofuel Production

The conversion of cellulosic biomass to fermentable sugars usually requires costly, time-consuming pretreatment to increase the material’s porosity, decrease its crystallinity, and reduce the amount of structural lignin in the cell wall. Researchers used small-angle neutron scattering at the High-Flux Isotope Reactor to probe the morphological changes of switchgrass cell walls during dilute acid pretreatment, elucidating the interplay of different biomolecular components in the breakdown process. The results are important for the development of efficient strategies to convert biomass to biofuel.

Reference: Pingali, S. V., et al. 2010. “Breakdown of Cell Wall Nanostructure in Dilute Acid Pretreated Biomass,” Biomacromolecules 11, 2329–35.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 01, 2011

Key Plant Receptors Discovered

The phytohormone abscisic acid (ABA) plays important regulatory roles in physiological pathways for plant growth and development and enables adaptation to environmental stresses, yet the protein recognition mechanisms for this hormone have eluded plant biologists. Crystallographic and small-angle X-ray scattering capabilities at the Advanced Light Source enabled researchers to determine the atomic resolution of the ABA receptor and identify conformational changes on the ABA binding site. Elucidating the structural mechanisms mediating ABA receptor recognition and signaling is essential for understanding and manipulating abiotic stress resistance. These results were listed as one of the top 10 scientific breakthroughs of the year in 2009 by Science.

Reference: Nishimura, N., et al. 2009. “Structural Mechanism of Abscisic Acid Binding and Signaling by Dimeric PYR1,” Science 326, 1373–79.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 01, 2011

Measuring Chemical Changes Inside Living Cells

Understanding how microbes adapt to changing chemical environments is a critical aspect of using them to solve DOE challenges. With synchrotron radiation-based Fourier transform infrared microscopy at the Advanced Light Source, researchers tracked the chemistry of living Desulfovibrio vulgaris cells in real time. The ability to make these dynamic measurements continuously inside selected living cells dramatically increases the usefulness and reliability of information traditionally derived from cells that have been killed and broken apart.

Reference: Holman, H.-Y., et al. 2009. “Real-Time Molecular Monitoring of Chemical Environment in Obligate Anaerobes During Oxygen Adaptive Response,” Proceedings of the National Academy of Sciences (USA) 106, 12599–604.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 01, 2011

Neutron Crystallography Reveals How Carbonic Anhydrases (CAs) Work

CAs are a family of enzymes that play an essential role in the metabolism of carbon dioxide by converting it into a carbonate ion and a proton. Because they are very stable and inexpensive, CAs could be used in significant large-scale applications such as carbon sequestration processes and biofuel production. However, little is known about the arrangement of the active site of CAs while they carry out their function, a gap that has impeded design of optimized CAs for these applications. Neutron crystallography experiments at the Los Alamos Neutron Science Center to determine the structure of human carbonic anhydrase II have revealed the orientation of amino acids around the zinc ion in the active site, as well as the unexpected presence of a water molecule bound to the metal ion. This structural information has enabled development of a mechanism to explain the proton transfer process and is being used to re-engineer the enzyme to be pH insensitive and thermally stable for carbon sequestration or biodiesel production.

Reference: Fisher, S. Z., et al. 2010. “Neutron Structure of Human Carbonic Anhydrase II: Implications for Proton Transfer,” Biochemistry 49, 415–21.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 01, 2011

New Insights into D-Xylose Isomerase (XI)

XI is an important enzyme because it can convert sugars that resist bioconversion to fuel into those readily fermented by, for example, yeasts. Through neutron diffraction experiments at the Los Alamos Neutron Science Center, researchers were able to map the positioning of individual hydrogen atoms as XI moves them from one carbon to another on a sugar molecule. They were able to model how specific amino acids in the XI structure are involved in proton movement. Results may enable new approaches for modifying the enzyme to improve its performance for biofuel and other applications. This research was featured on the June 9, 2010, cover of Structure.

Reference: Kovalevsky, A. Y., et al. 2010. “Metal Ion Roles and the Movement of Hydrogen During Reaction Catalyzed by D-Xylose Isomerase: A Joint X-Ray and Neutron Diffraction Study,” Structure 18, 688–99.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 01, 2011

Understanding Enzymes That Process Sugars and Carbohydrates

In two separate studies at the Advanced Photon Source, researchers used high-resolution synchrotron protein crystallography to determine the crystal structures of ROK (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) fructokinase from Bacillus subtilis and a recombinant a-glucosidase from the human gut bacterium Ruminococcus obeum. The results provided new information about how enzymes bind, recognize, and process carbohydrate substrates and how variations in enzyme structure impact enzyme function. These findings are expected to improve the conversion of biomass to fuels by using structural information to optimize enzymes for bioprocessing.

References: Nocek, B., et al. 2011. “Structural Studies of ROK Fructokinase YdhR from Bacillus subtilis: Insights into Substrate Binding and Fructose Specificity,” Journal of Molecular Biology 406, 325–42.

Tan, K., et al. 2010. “Novel a-Glucosidase from Human Gut Microbiome: Substrate Specificities and Their Switch,” The FASEB Journal 24, 3939–49.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 14, 2011

Learning New Tricks from Fungi to Improve Biomass Processing

Knowing how biomass is degraded in nature will advance understanding in how to process biomass for conversion to biofuels. The biodegradation of plant material generally involves removal of the resistant lignin barrier that prevents enzymes from reaching cellulose and degrading it to sugar. However, brown rot fungi, natural biomass recycler in coniferous forests, degrade biomass without removing much of the lignin. DOE researchers at the University of Wisconsin, Madison, and the Great Lakes Bioenergy Research Center (GLBRC) in Madison, Wisconsin, report that these fungi can disrupt the lignin in wood even though it remains in place. They discovered that key chemical linkages (ethers) in lignin’s complex molecular structure are broken, likely using reactive oxygen species such as hydroxyl radicals. They applied newly developed nuclear magnetic resonance (NMR) technology to look at the chemistry of wood attacked by a brown rot fungus. These results will enable development of new routes to access cellulose in biomass as part of the large-scale production of biofuels and will also improve understanding of natural carbon cycling from wood.

Reference: Yelle, D., D. Wei, J. Ralph, and K. E. Hammel. 2011. “Multidimensional NMR Analysis Reveals Truncated Lignin Structures in Wood Decayed by the Brown Rot Basidiomycete Postia placenta,” Environmental Microbiology doi:10.1111/j.1462-2920.2010.02417.x.

Contact: Arthur Katz, SC-23.2, (301) 903-4932
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 07, 2011

Diversity Among Rice Varieties Indicates Multiple Targets for Biomass Improvement

Breeding cellulosic feedstock crops with enough biomass for sustainable liquid fuel production is a major challenge. We can exploit natural variation in bioenergy-relevant traits, but many of the most promising feedstock crops, such as perennial grasses, have large genomes and limited genetic resources, making breeding for such traits difficult. However, such tools are readily available for rice, a well-studied crop plant that shares many developmental and physiological processes as well as gene content with other grasses. These shared characteristics make rice useful as a model for modifying other newly emerging bioenergy crops. Researchers at Colorado State University, in collaboration with the International Rice Research Institute (IRRI) in the Philippines, assessed variation in traits such as biomass, height, tiller number, plant girth, cell-wall composition, and water-use efficiency among a diverse set of 20 rice varieties at different stages of development. Significant variation was found for all traits, and this variation was determined to be heritable. Additionally, high yields exhibited by different varieties were achieved through different combinations of traits, indicating the contribution of multiple genetic loci to overall biomass productivity and suggesting that multiple targets can be utilized in traditional breeding programs to develop other energy feedstocks with enhanced yield.

Reference: Jahn, C. E., J. Mckay, R. Mauleon, J. Stephens, K. L. McNally, D. R. Bush, H. Leung, and J. E. Leach. 2011. “Genetic Variation in Biomass Traits Among 20 Diverse Rice Varieties,” Plant Physiology 155, 157–68.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 07, 2011

Water Flea Genome Sequenced: Sentinel of Environmental Change

The water flea Daphnia pulex is a keystone species of freshwater ecosystems, a principal grazer of algae, a primary food source for fish, a sentinel of still water inland ecosystems, and a sentinel species used to assess the ecological impact of environmental change. The genome of this species has just been sequenced by DOE’s Joint Genome Institute (JGI). They find that the Daphnia genome is only 200 megabases in size, but contains at least 30,000 genes, which is thought to be about 25% more than in the human genome. More than a third of Daphnia’s genes have no detectable homologs in any other available proteome, and the largest gene families are specific to the Daphnia lineage. These Daphnia-specific genes, including many additional sequenced genes that have not been assigned any functions, are the most responsive genes to ecological challenges. These results will enable better understanding of real-world environmental changes through knowledge of how a genome responds to gene-environment interactions. The study is published in the February 4, 2011, issue of Science magazine.

Reference: Colbourne, J. K., et al. 2011. “The Ecoresponsive Genome of Daphnia pulex,” Science 331, 555–61.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 31, 2011

"Mining" Cows for New Enzymes to Degrade Biomass

Successful development of biofuels depends on being able to break down cellulose-rich feedstocks such as switchgrass. In nature enzymes called cellulases break down plant material into simple sugars that can be converted into biofuels. Cattle and other plant eating animals have microbes that carry out this breakdown in the rumen portion of their stomachs. Now scientists at the DOE’s Joint Genome Institute (JGI) report on a metagenomics study of the microbes in the cow rumen. The JGI team was able to obtain and sequence 270 billion DNA bases from the resident microbes feeding on switchgrass in the rumen of a fistulated cow. The researchers developed a candidate set of 30,000 genes that encoded biomass degrading enzymes. They tested a sample of 90 of the proteins encoded by these genes and found that more than 50% had cellulose degrading activity. The JGI researchers were also able to assemble complete genomes of 15 novel microbial species from the cow rumen sample. The research demonstrates that large scale sequencing and data analysis capabilities are enabling researchers to accurately identify genes of biological interest and to provide draft genomes of uncultured novel organisms in the environment. It also defines a powerful strategy for finding new enzymes with significance for DOE missions. The research was led by Matthias Hess of the JGI and is published in the January 28, 2011, issue of Science.

Reference: Hess, M., A. Sczybra, R. Egan, T.-W. Kim, H. Chokhawala, G. Schroth, S. Luo, D. Clark, F. Chen, T. Zhang, R. Mackie, L. Pennacchio, S. Tringe, A. Visel, T. Woyke, Z. Wang, and E. Rubin. 2011. “Metagenomic Discovery of Biomass-Degrading Genes and Genomes from Cow Rumen,” Science 331, 463–67.

Contact: Dan Drell, SC-23.2, (301) 903-4742, Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 18, 2011

Genomic Analysis Provides New Clues on the Origins of Metabolic Pathways in Earth’s Biosphere

For the first three billion years of life’s history on Earth, microbes were the original and predominant form of life, but evolution during this period remains a mystery due to the lack of significant fossil evidence. Analysis of microbial gene sequences across the tree of life has yielded clues on the development of fundamental biological processes; however, horizontal gene transfer (HGT), the exchange of genetic material across species, has confounded efforts to map out deep evolutionary processes operating over geological time periods. In new results published in the January 6th issue of Nature, researchers at the Massachusetts Institute of Technology describe a new comparative genomics approach for analyzing molecular evolution while accounting for HGT. The authors identified a period of rapid gene innovation between 3.3 and 2.8 billion years ago that gave rise to 27% of modern gene families. This evolutionary burst coincided with a period when oxygen concentrations in the atmosphere rapidly increased. The genes originating during this period include many involved in expanded energy production and metabolic reactions associated with an oxidizing environment. These results shed new light on fundamental processes that have shaped the metabolic potential of life on Earth and that continue to govern adaptation of the biosphere to changing conditions. This research was funded as part of a DOE Science Focus Area at Lawrence Berkeley Lab.

Reference: David, L. A., and E. J. Alm. 2011. “Rapid Evolutionary Innovation During an Archaean Genetic Expansion,” Nature 469, 93–96.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 03, 2011

Making Trees More Bioenergy Friendly

Wood is a heterogeneous compound composed of the polysaccharides cellulose and hemicellulose, from which bioethanol can be derived, and the polymer lignin, which encloses the cellulosic material, provides rigidity and durability to the plant and makes it difficult to convert the cellulosic material to bioethanol. The content and composition of lignin varies by species of tree and by tissue and organ within a tree. A tree with reduced lignin content in the stems but with higher lignin in the roots would provide for more efficient and higher yielding ethanol production while at the same time enhancing carbon sequestration in the non-harvested below-ground tissues. Researchers at the DOE BioEnergy Research Center at Oak Ridge National Lab used pyrolysis molecular beam mass spectroscopy to characterize the lignin content in stems and roots from progeny of a three-generation pedigree of poplar, a tree species widely regarded as a potential biofuel crop. Several genetic regions associated with lignin content were identified that were root- and/or stem-specific, indicating the existence of gene(s) that differentially regulate lignin biosynthesis above and below ground. These results suggest that it may be possible to decrease stem lignin content through conventional or molecular breeding methods without impacting lignin in the roots.

Reference: Yin, T., X. Zhang, L. Gunter, R. Priya, R. Sykes, M. Davis, S.D. Wullschleger, and G.A. Tuskan. 2010. "Differential Detection of Genetic Loci Underlying Stem and Root Lignin Content in Populus," PLoS ONE 5(11):e14021.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 03, 2011

Systems Biology Analysis of Cellulose Degradation by Clostridium thermocellum

The bacterium Clostridium thermocellum is highly specialized to degrade cellulosic plant material through the use of cellulosomes, complex multi-component molecular machines tethered to the bacteria’s surface. The microbe can adjust the modular composition of its cellulosomes in response to various types of substrates and environmental conditions, but the mechanisms regulating this process remain poorly understand. Researchers at the DOE Great Lakes Bioenergy Research Center at the University of Wisconsin, Madison, have completed a global analysis of gene expression in C. thermocellum during controlled growth on cellulose and cellobiose (a simpler two sugar compound). Over 350 genes involved in cellulosome assembly, cellulose chain deconstruction, product uptake, and downstream synthesis of ethanol and hydrogen were observed to be differentially expressed depending on substrate and growth rate. In addition, the study provided new clues on the roles of numerous C. thermocellum genes that are currently categorized as having unknown functions. These results reveal the complex control that C. thermocellum exerts over its cellulose degrading machinery and provides new routes for development of this organism for bioenergy production.

Reference: Riederer, A., T. E. Takasuka, S. Makino, D. M. Stevenson, Y.V. Bukhman, N. L. Elsen, and B. G. Fox. 2010. “Global Gene Expression Patterns in Clostridium thermocellum from Microarray Analysis of Chemostat Culture on Cellulose or Cellobiose,” Applied and Environmental Microbiology, DOE:10.1128/AEM.02008-10

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 06, 2010

A New Mechanism for Microbial Community Metabolism

Outside of laboratories, microbial species rarely exist in isolation. Many important environmental processes are actually mediated by complex communities of microbes. In many cases, two or more species have evolved to perform a cooperative metabolic activity that would be energetically unfavorable for either organism acting independently. Research published in the December 3 issue of Science and led by DOE scientist Derek Lovley of the University of Massachusetts, Amherst, describes a new mechanism by which the bacterium Geobacter metallireducens consumes ethanol, an important intermediate compound in oxygen free soils and sediments, in cooperation with a second organism Geobacter sulfureducens. For this reaction to yield energy for either partner, electrons produced from ethanol oxidation must be rapidly consumed. Although it was previously assumed that the first organism uses a hydrogen production mechanism to pass electrons to its partner, the authors have discovered that electrons are instead directly fed to G. sulfureducens via conductive "nanowires" called pili on the cell surface, resulting in much more efficient collaborative growth. These results provide important new clues on the fundamentals used by microbes to mediate important environmental processes such as carbon cycling and contaminant transformation and suggest intriguing new approaches to direct generation of electricity in microbial fuel cell systems.

Reference: Summers, Z.M., H. E. Fogarty, C. Leang, A. E. Franks, N. S. Malvankar, and D. R. Lovley. 2010. "Direct Electron Exchange Within Aggregates of an Evolved Syntrophic Coculture of Anaerobic Bacteria," Science 330:1413-15.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 06, 2010

Genome of Methane-Oxidizing Microbe Sequenced

Methane is a more potent greenhouse gas than CO2 on a per molecule basis although far more CO2 than methane is released into the atmosphere. Methane production and oxidation (usually conversion to methanol) is a common property of many bacteria. To better understand the basis for bacterial methane processing and its potential role in the global greenhouse gas cycle, the genome sequence of a methane-oxidizing microbe, Methylosinus trichosporium, has now been published. This microbe has been used to elucidate the structure and function of several key enzymes that oxidize methane. In particular, the catalytic properties of a soluble methane monooxygenase enzyme from this bacterium have been studied extensively as it is also involved in biodegradation of recalcitrant hydrocarbons, such as trichloroethylene. The sequence of this bacterium's genome should provide insights into both methane processing and organic contaminant degradation. The sequencing was carried out by the DOE Joint Genome Institute as part of its Community Sequencing user Program.

Reference: Stein, L.Y., et.al. December 2010. "Genome Sequence of the Obligate Methanotroph Methylosinus trichosporium Strain OB3b," Journal of Bacteriology, 192, 6497-98.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 06, 2010

Progress and Prospects for Metabolic Engineering of Microbes for Biofuels Production

In a review article in the December 3, 2010, issue of Science, DOE Joint Bioenergy Institute director Jay Keasling discusses advances in metabolic engineering and outlines current efforts to develop economical production of biofuel compounds by microbes. Keasling points to recent improvements in DNA sequencing, bioinformatics, and systems biology approaches as key elements enabling recent breakthroughs in microbial production of high value products such as pharmaceuticals. As petroleum prices continue to rise, engineering microbes to synthesize next generation biofuels compatible with existing engines and infrastructure has become more feasible economically. However, more work is needed to provide low cost starting materials from cellulosic biomass, improve genetic tools that allow introduction of metabolic pathways and control elements into microbial genomes, and develop a broader range of host microbes that can produce tailored biofuel compounds and withstand stresses associated with industrial fuel production. Given the rapid pace of recent progress in these areas, Keasling considers the prospects for economical microbial production of biofuels from renewable resources to be very strong.

Reference: Keasling, J.D. 2010. “Manufacturing Molecules Through Metabolic Engineering,” Science 330:1355-1358.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 29, 2010

Computational Approaches to Simulate Microbial Ecosystems

A basic challenge in microbial ecology is to understand and to predict the growth and behavior of complex microbial communities, in fact most isolated microbes cannot be readily grown in culture. These communities are important for biogeochemical processes such as nitrification, hydrogen production, and methanogensis. They also show promise for the degradation of complex oligosaccharides in biomass to fermentable sugars for biofuel production. A new method for genome-scale metabolic simulation has been developed by DOE scientists Niels Klitgord and Daniel Segrè of Boston University that will predict the optimal media for promoting the growth of microbes in a community. The method has been successfully tested on a community consisting of hydrogen producing and methane producing microbes as well as the model co-culture Escherichia coli and Saccharomyces cerevisiae. Research is now underway to extend this method to simulating microbial community growth involving more than two species. The new method has just been published in PLoS Computational Biology. This new predictive capability may expand our ability to take advantage of the vast and diverse capabilities found in the microbial world.

Reference: Klitgord, N., and D. Segrè. November 2010. "Environments that Induce Synthetic Microbial Ecosystems," PLoS Computational Biology 6.(Reference Link)

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 22, 2010

DOE Scientists to Receive Franklin Institute Medals

Jillian F. Banfield, a University of California, Berkeley, biogeochemist and geomicrobiologist, will receive the Benjamin Franklin Medal in Earth and Environmental Science, “for discovering the underlying principles of mineral formation and alteration by microbes, which are critical to understanding the form, composition, and distribution of minerals in the presence of living organisms.” Using cutting-edge technology, Banfield has fully characterized this unique microbial ecosystem by sequencing the genomes of the different species of bacteria and cataloguing the proteins they produce. Banfield has been supported by DOE for the past decade. Banfield also is one of five recipients of the 2011 For Women in Science awards from the L'Oréal Foundation and United Nations Educational, Scientific and Cultural Organization (UNESCO) and will received this award on March 3, 2011, at UNESCO headquarters in Paris.

George Church of the Harvard Medical School is recipient of the Franklin Institute’s Bower Award for “innovative and creative contributions to genomic science, including the development of DNA sequencing technologies, as well as for his subsequent efforts to promote personal genomics and synthetic biology.” Church’s research has been supported by DOE since 1988. During this time he has been a leader in bringing improvements in cost and speed to bioanalytical technologies and their applications across the life sciences. Many technologies flowing from his projects have been commercialized.

Banfield and Church are two of seven recipients of the 2011 Franklin Medal, presented every year to “preeminent trailblazers in science, business and technology.”

References:

Bower Award: George Church

Banfield: News article

Contact: Dan Drell, SC-23.2, (301) 903-4742, Marvin Stodolsky, SC-23.2, (301) 903-4475
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 22, 2010

Interactions of Bacteria with Uranium in the Environment

Uranium in the 6+ oxidation state is quite soluble and can thus move rapidly in uranium-contaminated subsurface environments. In contrast, uranium in the 4+ state is highly insoluble, and is therefore less likely to move the subsurface environment. New research has identified important aspects of how bacteria reduce uranium 6+ to uranium 4+, showing that the latter is produced in a variety of forms, not just in the expected, simple form of uraninite (UO2). The authors of the new study used a variety of techniques at the Stanford Synchrotron Radiation Lightsource (SSRL) to characterize the products of uranium reduction in various microbial cultures, including x-ray absorption spectroscopy (XAS). The XAS experiments showed that many of the uranium 4+ products lacked the spectral peak characteristic of uraninite. Instead, a variety of complex solids involving uranium and phosphate, and in some cases also calcium were identified, as well as solids in which uranium 4+ is bound to the surface of the bacterial biomass. These results will be helpful in modeling the mobility of uranium species at contaminated DOE sites. The research was led by Rizlan Bernier-Latmani of the École Polytechnique Fédérale de Lausanne in Switzerland, and involved scientists at SSRL. It is just published online in Environmental Science & Technology.

Reference: Bernier-Latmani, R., et al. 2010. "Non-uraninite Products of Microbial U(VI) Reduction," Environmental Science & Technology, online November 11, 2010. DOI: 10.1021/es101675a

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


November 22, 2010

Predicting Function of Unknown Genes

Recent advances in plant genomics have identified many new genes, but many are of unknown function. Experimental determination of the function of individual genes is difficult because gene duplication occurs frequently among plants so large, functionally redundant gene families are common. Researchers at the DOE Joint BioEnergy Institute have used a phylogenetic (evolutionary relatedness) approach to computationally predict the biological function of individual genes within the very large (1,508-member) rice kinase gene family by combining gene expression data from various rice tissues and different experimental conditions with protein interaction data and looking for similarities. Function could be inferred for genes showing similar patterns in diverse tissues and conditions. Certain members of the kinase gene family regulate the responses of plants to a range of stresses such as drought and pathogens, as well as being involved in other signaling cascades. Rice can be used as a model for bioenergy grass crops such as sorghum and switchgrass, thus integration of gene data from these plants could facilitate functional predictions of genes important for bioenergy-relevant traits.

Reference: Jung, K-H., P. Cao, Y-S. Seo, C. Dardick, and P.C. Ronald. 2010. "The Rice Kinase Phylogenomics Database: A Guide for Systematic Analysis of the Rice Kinase Super-family," Trends in Plant Science 15(11), 595-99.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 22, 2010

The Challenge of Redesigning Lignin for Biofuel Applications

Secondary cell walls of plants contain lignins that provide rigidity and pathogen resistance to the plant, but hinder breakdown of cell walls during biomass processing. This limits the efficient use of plants as bioenergy feedstocks. Lignins are polymers formed from several different chemical monomers and the nature of these monomers determines the properties of the lignin polymer. Modifying the lignin composition could significantly improve the ease of conversion of biomass to biofuel products, while retaining the critical functions of lignins for the plants growing in the field. Researchers at the DOE Great Lakes Bioenergy Center (GLBRC) have found that by altering two genes in Arabidopsis, a plant often used as a research model, a unique lignin is produced that contains a non-traditional monomer. The altered plant exhibits reduced lignin content, a trait desirable for increasing efficiency of deconstruction, but also shows aberrant growth and development and large metabolic shifts. The GLBRC researchers found evidence for genetic interactions between two lignin biosynthetic pathways. These results are an example of the type of unanticipated effects that will need to be taken into account when designing strategies for genetically engineering plant cell walls for bioenergy applications.

Reference: Vanholme, R., J. Ralph, T. Akiyama, F. Lu, J.R. Pazo, H. Kim, J.H. Christensen, B. Van Reusel, V. Storme, R. De Rycke, A. Rohde, K. Morreel, and W. Boerjan. 2010. "Engineering Traditional Monolignols Out of Lignin by Concomitant F5H1-up- and COMT-down-regulation in Arabidopsis," Plant Journal. doe:10.1111/j.1365-313X.2010.04353.x.

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 15, 2010

Jill Banfield to Receive Franklin Medal and L'Oréal-UNESCO Award

DOE-funded scientist Jillian F. Banfield, a University of California, Berkeley, biogeochemist and geomicrobiologist, will receive two prestigious awards - the Benjamin Franklin Medal in Earth and Environmental Science and the L'Oréal-UNESCO 'For Women in Science' award – for her groundbreaking work on how microbes alter rocks and interact with the natural world. She has used cutting-edge techniques to sequence the genomes of the different species of bacteria and to catalogue the proteins they produce, fully characterizing this unique microbial ecosystem. Banfield has been at UC Berkeley since 2001, where she is a professor of earth and planetary science, of environmental science, policy and management, and of materials science and engineering, and a faculty scientist at Lawrence Berkeley National Laboratory. She is one of seven recipients of the 2011 Franklin Medal, presented every year to "preeminent trailblazers in science, business and technology." Banfield is one of five recipients of the 2011 For Women in Science awards from the L'Oréal Foundation and United Nations Educational, Scientific and Cultural Organization (UNESCO). The awards ceremony will take place on March 3, 2011, at UNESCO headquarters in Paris. Each laureate will receive $100,000 in recognition of her contributions to science.

Reference: News Article

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 01, 2010

Methane-Oxidizing Bacterium Sequenced at DOE-JGI

Methane is one of the most important greenhouse gases, 21 times more potent molecule-for-molecule than carbon dioxide. Methane-oxidizing bacteria (methanotrophs) that are common in terrestrial and marine environments help reduce levels of atmospheric methane. To better understand the bacteria involved in the global methane cycle, the DOE JGI sequenced and annotated the genome of Methylosinus trichosporium OB3b. This microbe has been studied extensively to identify and characterize several key enzymes involved in methane oxidation. For example, one crucial enzyme uses copper to efficiently oxidize methane. Aside from genes involved in methane oxidation, genes involved in nitrogen fixation and ammonia transport were also identified. An improved understanding of microbial methane biochemistry will help characterize the biological components of global climate models. The new results were just published online ahead of print in the Journal of Bacteriology.

Reference: Stein, L.Y., S. Yoon, J.D. Semrau, A.A. DiSpirito, J.C. Murrell, S. Vuilleumier, M.G. Kalyuzhnaya, H.J.M. Op den Camp, F. Bringel, D. Bruce, J.-F Cheng, A. Copeland, L. Goodwin, S. Han, L Hauser, M.S.M. Jetten, A. Lajus, M.L. Land, A. Lapidus, S. Lucas, C. Médigue, S. Pitluck, T. Woyke, A. Zeytun, and M.G. Klotzl. "Genome sequence of the obligate methanotroph, Methylosinus trichosporium strain OB3b," Journal of Bacteriology doi:10.1128/JB.01144-10. Published online ahead of print on 15 October 2010.

Contact: Dan Drell, SC-23.2, (301) 903-4742
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 01, 2010

New Microfluidic Chips for Large-Scale Screening of Biomass Hydrolysis

Large numbers of cellulose enzymes, the enzymes used to break down cellulosic biomass to produce fermentable sugars, need to be screened to identify the best enzymes and the most effective processing conditions for biofuel production from cellulosic biomass. Researchers at DOE's Joint BioEnergy Institute (JBEI) have developed a new microfluidic chip-based assay to rapidly and precisely characterize biomass hydrolysis products, especially glycan and xylan sugars. They solved the difficult challenge of separating and identifying these closely related sugars by modeling and optimizing the process in the microfluidic system. They describe the use of this new system, demonstrating its ability to rapidly screen for hydrolysis products on the order of one minute. These results suggest that that this new system could be adapted to large-scale, rapid characterization of cellulase enzyme cocktails. This study was featured on the November 15, 2010 cover of Analytical Chemistry.

Reference: Bharadwaj, R., Z. Chen, S. Data, B.M. Holmes, R. Sapra, B.A. Simmons, P.D. Adams, and A.K. Singh. 2010. "Microfluidic Glycosyl Hydrolase Screening for Biomass-to-Biofuel Conversion," Analytical Chemistry. Released online on October 22, 2010, DOI: 10.1021/ac102243f/

Contact: Susan Gregurick, SC-23.2, (301) 903-7672
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 01, 2010

New Modeling Tool for Optimizing Biofuels Production

Many feedstocks and conversion options are available to produce biofuels. DOE's Joint BioEnergy Institute (JBEI) has developed a new publicly available model to evaluate the relative advantages of various biofuel production approaches. The model includes the flow of materials from feedstocks leaving the farm through finished products leaving the biorefinery. It tracks the use of heat, power, and raw materials and predicts costs as well as energy and material balances. A preliminary, traditional scenario involving corn stover as feedstock, acid pretreatment, and conversion to ethanol using yeast engineered to ferment five and six carbon sugars is the basis of comparison. The model facilitates input from the user community to provide suggestions and modify assumptions. JBEI is using the model to guide its research emphasis. For example, the model predicts that acetate produced during fermentation could limit ethanol production more than the accumulation of ethanol suggesting that feedstocks and feedstock processing should be optimized to reduce acetylation. The model is also highlighted in the October 22 issue of Science Magazine.

Reference: Klein-Marcuschamer, et al. 2010. "Technoeconomic Analysis of Biofuels: A Wiki-Based Platform for Lignocellulosic Biorefineries," Biomass and Bioenergy, doi:10:1016/j.biombioe.2010.07.033.

Contact: John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 01, 2010

Sneak Peak at How Stressed Plants Mobilize the Resources

The ability of plants to withstand stresses depends on a coordinated chain of events from the molecular level to the whole plant. Our ability to effectively develop plants as sustainable feedstocks for biofuels requires that we understand the impacts of these stresses. DOE-funded researchers at Brookhaven National Laboratory and Tufts University have shown that plants re-allocate a significant portion of their below-ground nitrogen resources when defense mechanisms are triggered in response to herbivory (being eaten or under attack). Using a combination of short-lived PET (positron emission tomography) radioisotopes, including carbon-11 and nitrogen-13, administered to leaves of intact tomato plants, they were able to "see" the movement of sugars and amino acids away from the simulated attack sites. The results argue for strong physiological adaptive responses by plants as a tolerance defense mechanism. This research has important implications for bioenergy feedstock development since the next generation of plant feedstocks will need to withstand many environmental challenges including drought, limited nutrients and disease. Modifying plants with the right defense traits could improve the robustness of future feedstocks. The research is reported in the November issue of New Phytologist, along with a commentary on the significance of the new findings.

References:

Gómez, S., R.A. Ferrieri, M. Schueller, and C. M. Orians. 2010. "Methyl Jasmonate Elicits Rapid Changes in Carbon and Nitrogen Dynamics in Tomato" New Phytologist 188, 835-44.

Anten, N.P.R., and R. Pierik. 2010. "Moving Resources Away From the Herbivore: Regulation and Adaptive Significance," New Phytologist 188, 643-45.

Contact: Prem Srivastava, SC-23.2, (301) 903-4071
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Division: SC-23.2 Biological Systems Science Division, BER


October 25, 2010

Could Biofuels Replace a Large Fraction of the U.S. Petroleum Demand?

Sustainability of large-scale biofuel domestic production is a serious concern. A new model has been developed at the DOE Great Lakes BioEnergy Research Center to assess the potential impact of existing and emerging technologies for the production of biofuels and animal feed. The model assumes that all land used for human food, forests, rangeland, and most other uses will not be affected by the production of bioenergy and animal feed. The only land considered for these technologies is currently allocated to animal feed and corn ethanol. The technologies considered in this study include separating and concentrating leaf protein, pretreating forage, and double cropping where possible. These results outlined in a recent article in Environmental Science & Technology indicate the potential for annual production of about 100 billion gallons of ethanol with no impact on domestic food production or indirect land use change, while significantly reducing U.S. greenhouse gas emissions, increasing soil fertility, and promoting biodiversity.

Reference: Dale B., Bals B., Kim S., and Eranki P., "Biofuels Done Right: Land Efficient Animal Feeds Enable Large Environmental and Energy Benefits," Environ. Sci. Technol, 10.1021/es101864b, October 7, 2010

Contact: John Houghton, SC-23.2, (301) 903-8288
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 25, 2010

Human Metabolic Disease Leads to New Understanding of Oil Accumulation in Plants

A major challenge in developing plants for biofuels production is the difficulty involved in breaking down lignocellulosic material, the main constituent of plant biomass. An alternate approach to biofuel production in plants would involve engineering plants to accumulate larger amounts of lipids (the precursors of oils normally found in seeds) in vegetative tissues such as leaves. Lipids and oils could then be directly harvested for biodiesel or converted to other biofuels. DOE researchers at the University of North Texas recently identified a gene in the model plant Arabidopsis thaliana that is surprisingly similar to a gene known to be involved in Chanarin-Dorfman syndrome, a human metabolic disorder that results in excessive production of lipids in non-fatty tissues. When this gene was disrupted in Arabidopsis, plants had a 10-fold increase in total lipid content in vegetative plant tissue although the plants appeared to grow normally. Lipid levels in seeds were unchanged. These results suggest a surprising degree of similarity of lipid metabolism between plants and animals. Although Arabidopsis is unlikely to be developed as a bioenergy feedstock, this represents a major advance in understanding of oil synthesis in plants and presents promising new targets for metabolic engineering of biomass crops.

Reference: James, C.N., P. J. Horn, C. R. Case, S. K. Gidd, D. Zhang, R. T. Mullen, J. M. Dyer, R. G. W. Anderson, and K. D. Chapman. 2010 "Disruption of the Arabidopsis CGI-58 homologue produces Chanarin–Dorfman-like lipid droplet accumulation in plants," Proc. of the Natl. Acad. Sci. 107:17833-17838.

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 12, 2010

New Route to Lignin Biosynthesis Offers New Opportunity to Improve Biofuels Production

The biosynthetic pathway of lignin, the compound that confers stren