"The BioEnergy Science Center is
focused specifically on the challenge
of overcoming biomass
recalcitrance. If we can manage to
understand and solve this challenge,
we will not only improve our ability
to generate cellulosic ethanol, but
we also will open doors to a vast
array of renewable and sustainable
product pipelines that can serve
many different missions. To address
these important issues, we have
assembled a team of world leaders
in their fields from 20 different institutions and have built a
culture of integration and collaboration that has significantly
accelerated our rate of scientific discovery and application."
– Paul Gilna
Paul Gilna, BESC director, also is deputy director of the Biosciences Division at Oak Ridge National Laboratory. An expert in computational biology, Gilna previously served as executive director of CAMERA (Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis), a project providing researchers with bioinformatic tools and services.
The DOE BioEnergy Science Center (BESC), led by Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee, focuses on the fundamental understanding and elimination of biomass recalcitrance— the resistance of cellulosic biomass to enzymatic breakdown into sugars. BESC approaches the problem of biomass recalcitrance from two directions by closely linking (1) plant research to make cell walls easier to deconstruct and (2) microbial research to develop multitalented biocatalysts tailor-made to produce biofuels from this modified plant material in a single step.
Scientists at national laboratories, universities, and private companies that make up the BESC team have extensive experience with studying biomass recalcitrance, and they have made fundamental advances in a wide range of related sciences. The Joint Institute for Biological Sciences systems biology research facility at ORNL serves as the central hub for coordinating research among all BESC partners. BESC's research is organized into three focus areas: (1) Biomass Formation and Modification, (2) Biomass Deconstruction and Conversion, and (3) Characterization and Modeling.
Some recent highlights of BESC research are featured below. By understanding the myriad factors that collectively determine biomass recalcitrance, BESC researchers are providing foundational knowledge that will streamline processing and reduce costs for many different approaches to plant feedstock and cellulosic biofuel production.
1. Biomass Formation and Modification
BESC biomass formation and modification research involves understanding the genetics and biochemistry of plant cell-wall biosynthesis and working directly with two potential bioenergy crops—switchgrass and poplar—to develop varieties that are easier to break down into fermentable sugars. Computational models are being developed to help BESC researchers identify target genes and successful strategies for modifying biosynthetic pathways to generate cell walls that can be readily deconstructed into sugars for biofuel production. Target genes are turned on or off in thousands of poplar and switchgrass samples generated and studied by BESC, and then these samples are characterized to assess how these modifications affect plant cell walls. These modified plants are growing in greenhouses and at several sites to help differentiate genetic versus environmental influences on plant trait development (see figure, Bioenergy Crop Research at BESC, this page). By understanding the synthesis and assembly of the polysaccharides and lignin in plant biomass, BESC researchers are reducing cell-wall recalcitrance using methods that can be applied to a wide range of woody and herbaceous plants.
2. Biomass Deconstruction and Conversion
Two key hypotheses drive biomass deconstruction and conversion research at BESC: (1) microorganisms can be engineered to enable consolidated bioprocessing (CBP)— a game-changing, one-step, microbe-mediated strategy for directly converting plant biomass into ethanol and (2) enzymes and microbial biocatalysts can be understood and engineered to synergize with recalcitrance-reducing plant modifications to achieve better biomass deconstruction.
Model organisms for CBP development include species of Clostridia bacteria that rapidly degrade pure cellulose and then ferment the resulting sugars into ethanol (see figure, BESC Research on Biomass-Deconstructing Microbes). These microbes deconstruct biomass using cellulosomes— multifunctional enzyme complexes that specialize in degrading the mix of complex carbohydrates in cell walls. BESC is studying the structures and activities of these poorly understood multienzyme complexes to design new variants that are more efficient at deconstructing cell walls.
BESC researchers also are investigating microbes that can rapidly degrade biomass at near-boiling temperatures, such as species of Caldicellulosiruptor isolated from hot springs at Yellowstone National Park. By developing genetic tools for these difficult-to-manipulate microbes, BESC is working to engineer novel microbes that add biofuel production capabilities to their native cellulose-degrading abilities. These microbes and their enzymes could provide new biomass-degrading capabilities resistant to the heat and stresses of industrial processing.
Bioenergy Crop Research at BESC. Transformed Populus shoots grow in a greenhouse. These plants are altered in targeted cell-wall pathway genes. [Image courtesy of Oak Ridge National Laboratory]
BESC Research on Biomass-Deconstructing Microbes. Isolated from decaying grass compost, Clostridium cellulolyticum degrades cellulosic biomass using multienzyme complexes called cellulosomes. This scanning electron micrograph shows C. cellulolyticum cells growing on switchgrass biomass. [Photo by Thomas Hass and Shi-You Ding, National Renewable Energy Laboratory]
3. Characterization and Modeling
Advancing BESC goals to develop improved plant materials and CBP methods that facilitate cost-effective conversion of biomass to fermentable sugars will require detailed knowledge of (1) the chemical and physical properties of biomass that influence recalcitrance, (2) how these properties can be altered by engineering plant biosynthetic pathways, and (3) how biomass properties change during pretreatment and how such changes affect biomass-biocatalyst interactions during deconstruction by enzymes and microorganisms.
To examine chemical and structural changes that occur in the modified plant cell walls of switchgrass and poplar, BESC analyzes thousands of native and modified biomass samples in a high-throughput screening pipeline that can perform compositional analysis, pretreatment, and enzyme digestibility studies. This screen already has revealed that a few native poplar samples can release most of their sugars using just a mild (hot water) pretreatment. Promising candidates selected from this screening pipeline are passed along to other partner institutions for a variety of detailed chemical, physical, and imaging analyses. The resulting data are incorporated into computational models and simulations used to predict relationships between biomass structure and recalcitrance.
Modeling and simulation tools are part of a knowledgebase BESC is establishing to maintain and share data, materials, experimental processes, and scientific insights across the distributed BESC community. One component of this knowledgebase is a comprehensive set of tools for discovering biomass recalcitrance genes in plant genomes and building pathways for cell-wall synthesis. By extracting and combining results from isolated experiments, this knowledgebase serves as a biological discovery platform for integrating diverse experimental, theoretical, and computational approaches that will help define the genomic and physical bases of plant cell-wall recalcitrance. A public version of the knowledgebase is at http://besckb.ornl.gov.
Translation of BESC Science into
Translating BESC research results into the testing of applications and potential commercial deployment is an important step toward reaching DOE's bioenergy objectives. BESC has formed a "commercialization council" of technology-transfer and intellectual property (IP) management professionals from partner institutions to evaluate the commercial potential of new inventions arising from BESC research and to promote and facilitate the licensing of BESC IP. Inventions are posted on the center's website (http://bioenergycenter.org/). Some early inventions address techniques for plant and microbial genetic transformation, special microscopy methods, and innovations in biomass sample handling. To build external relationships that can promote commercialization of new technologies, BESC provides opportunities for companies to become BESC Industry Affiliates. Several BESC research partners and affiliates have pilot facilities for testing BESC improvements. The University of Tennessee–Genera Energy and Dupont Danisco Cellulosic Ethanol have established a demonstration plant for turning corn cobs and switchgrass into ethanol in Vonore, Tennessee. Mascoma, Verenium, Ceres, and ArborGen also have pilot and field sites.
Education and Outreach
To prepare the next generation of bioenergy scientists, BESC provides interdisciplinary research opportunities to graduate students, postdocs, and visiting scientists. In addition to these activities in higher education, BESC is teaming with the Creative Discovery Museum in Chattanooga, Tennessee, to raise awareness of cellulosic biofuels, carbon emissions from energy use, and obstacles to a successful biofuel economy. Targeting fifth-graders, BESC education and outreach efforts make information accessible to the general public and reach students when they still are excited about science. Lessons piloted to thousands of students at schools in Georgia and Tennessee are available to schools nationwide. BESC also began "Science Night" programs that build on these classroom lessons and are offered to students and their families. The National Geographic Jason Project featured BESC science in a program developed for high school students. The BESC website features announcements about BESC outreach and educational programs, seminars and presentations describing BESC research, and other resources.
Location of Center: ORNL Campus, Oak Ridge, Tennessee
Key Targets from a Complex Family of Lignin
Biosynthesis Genes Identified in Switchgrass
Although lignin content and composition have been manipulated in several plant species by targeting the monolignol biosynthesis pathway, little is known about the genes and enzymes associated with this pathway in switchgrass. Cinnamoyl CoA reductase (CCR) catalyzes the first step in this pathway dedicated to monolignol synthesis. However, switchgrass contains numerous copies of CCR-like genes, complicating the selection of the best gene targets for altering lignin to reduce cell-wall recalcitrance. By analyzing the RNA of expressed CCR genes, BESC researchers show that one of the expressed genes (PvCCR1) encodes an enzyme actively involved in lignification and thus is a prime target for down-regulation to improve the degradability and sugar yield from switchgrass. Ongoing research is investigating how reducing the expression of the PvCCR1 gene impacts lignin composition and plant structure. This research was reported in Escamilla-Treviño, L. L., et al. 2009. "Switchgrass (Panicum virgatum) Possesses a Divergent Family of Cinnamoyl CoA Reductases with Distinct Biochemical Properties," New Phytologist 185, 143–55.
Amount of Cellulose Synthase Proteins in Different Subcellular Fractions. The pellet fraction of extracted xylem proteins is enriched in membrane proteins. Significantly higher amounts of a key membrane protein, cellulose synthase, are associated with the pellet fraction relative to the other fractions. [Image from Kalluri et al. 2009]
Thousands of Proteins from Developing Xylem
Cells in Poplar Are Identified
Woody biomass in trees primarily consists of the secondary cell walls of dead xylem tissue, so developing xylem cells are useful models for investigating secondary cell-wall formation. To provide subcellular context for identified protein functions and to enhance the detection of low-abundance proteins, subcellular fractionation techniques were used to obtain crude (soluble protein), pellet (insoluble protein), and nuclear protein fractions for analysis. Applying an automated approach known as MudPIT (Multidimensional Protein Identification Technology), BESC researchers successfully isolated and identified 6,000 different proteins from developing xylem cells in the stems of poplar plants. Results from this project greatly expanded the number of proteins that had been identified in previous poplar proteome studies. The protein products of several cell-wall synthesis genes (e.g., cellulose synthase, sucrose synthase, and polygalacturonase) were found to be associated with cellular membranes (see figure), and numerous new candidate genes for cell-wall synthesis were discovered—many are promising targets for further functional genomic analysis. Measuring differences in the whole proteomes of different poplar variations will increase understanding of the fundamental properties that underlie the recalcitrance of woody biomass to degradation. This research was reported in Kalluri, U. C., et al. 2009. "Shotgun Proteome Profile of Populus Developing Xylem," Proteomics 9, 4871–80.
Caldicellulosiruptor bescii on Birchwood Xylan. [Image courtesy of Mike Adams, University of Georgia]
Heat-Tolerant Bacteria Efficiently Degrade
Presenting the possibility of eliminating the pretreatment step from cellulosic biofuel production, a hot springs bacterium known as Caldicellulosiruptor bescii has shown that it can efficiently degrade crystalline cellulose, xylan (a hemicellulose), and various types of non-pretreated biomass including hardwoods such as poplar, high-lignin grasses such as switchgrass, and low-lignin grasses such as Bermuda grass (see figure). With an optimal growth temperature of 75°C, C. bescii was able to break down 65% of switchgrass biomass without pretreatment. This bacterium is the most heat-tolerant biomass degrader known (withstanding temperatures up to 90°C), and it primarily produces hydrogen as an end product when grown on plant biomass. BESC researchers have discovered another hot springs bacterium (Caldicellulosiruptor obsidiansis), isolated from Yellowstone National Park, that thrives at 78°C and can ferment all the simple sugars in cell-wall polysaccharides into diverse products including ethanol. Combining the functional capabilities of C. bescii and C. obsidiansis theoretically could yield organisms that both deconstruct and ferment plant biomass at temperatures above the boiling point of ethanol (78.4°C). Producing ethanol in the vapor phase could greatly reduce the inhibitory effects of ethanol on cell growth. C. bescii (formerly called Anaerocellum thermophilum DSM 6725) findings are from Yang, S. J., et al. 2009. "Efficient Degradation of Lignocellulosic Plant Biomass, Without Pretreatment, by the Thermophilic Anaerobe 'Anaerocellum thermophilum' DSM 6725," Applied and Environmental Microbiology 75(14), 4762– 69. The discovery of C. obsidiansis was reported in Hamilton- Brehm, S. D., et al. 2010. "Caldicellulosiruptor obsidiansis sp. nov., an Anaerobic, Extremely Thermophilic, Cellulolytic Bacterium Isolated from Obsidian Pool, Yellowstone National Park," Applied and Environmental Microbiology 76(4), 1014–20.
Validation of Gene Expression in Vascular Tissues. To confirm the expression of identified genes in vascular tissues, probes that specifically bind the mRNA of a small number of the identified genes were applied to switchgrass tissue. The darker regions indicate the vascular bundles where the probes bound the abundant mRNA from the active expression of targeted genes in these regions. [Image courtesy of Elison Blancaflor, The Samuel Roberts Noble Foundation]
Researchers Target Expressed Genes in Vascular
Tissues of Switchgrass
Using a laser-based technique for microdissecting plant tissues, BESC researchers have targeted and analyzed DNA that is actively expressed in switchgrass vascular tissues where secondary cell walls are synthesized and reinforced with lignin. A total of 2,766 unique genes were identified from 5,734 expressed DNA segments (known as expressed sequence tags or ESTs). A significant number of these expressed sequences are novel with no significant hits to existing EST data. A small subset of the identified genes was targeted with labeled probes to visualize the expression of these genes in live plant tissue (see figure), and researchers found that several genes have much higher expression in the vascular bundles. The gene list generated from this study provides an important genomic resource for narrowing the range of molecular targets that could play key roles in modifying the lignin content of switchgrass and other related bioenergy crops. This research was reported in Srivastava, A. C., et al. 2010. "Collection and Analysis of Expressed Sequence Tags Derived from Laser Capture Microdissected Switchgrass (Panicum virgatum L. Alamo) Vascular Tissues," BioEnergy Research, DOI: 10.1007/s12155-010-9080-8.
New Strategy Enhances Microbial Resistance
to Inhibitory Pretreatment Chemicals
The chemical and physical processes for pretreating biomass help unravel the complex matrix of cell-wall components and enhance enzyme accessibility to these materials, but pretreatments also generate chemicals such as acetate that inhibit sugar fermentation to biofuels. Using a combination of adaptation, genetic engineering, and systems biology tools, BESC researchers have developed acetate-resistant strains of two industrial ethanol producers (the bacterium Zymomonas mobilis and the yeast Saccharomyces cerevisiae) by changing the expression of genes encoding transport proteins that move substances across the cell membrane. These proteins (called antiporters) transport proton and sodium ions and form gradients that are adversely impacted by the presence of acetate. By resequencing a Z. mobilis strain that had been adapted to withstand high acetate concentrations, BESC researchers discovered specific mutations in antiporter genes that enable acetate resistance. The specific antiporter mutations were validated using genetically engineered Z. mobilis and yeast showing the broad impact of these changes. This research is reported in Yang, S., et al. 2010. "Paradigm for Industrial Strain Improvement Identifies Sodium Acetate Tolerance Loci in Zymomonas mobilis and Saccharomyces cerevisiae," Proceedings of the National Academy of Sciences 107(23), 10395-400.
Genomic Science-Related BER Research Highlights