The BioEnergy Science Center: Initial Results in Overcoming Biomass Recalcitrance

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1 The BioEnergy Science Center: Initial Results in Overcoming Biomass Recalcitrance Brian H. Davison, Ph.D. Interim Deputy Director, BioEnergy Science Center August 12, 2009

Major DOE Biofuels Project Locations (DOE 2008) Geographic, Feedstock, and Technology Diversity Pacific Ethanol (Boardman, OR) DOE Joint Bioenergy Institute

Verenium Corp (San Diego, CA) Lignol Innovations (Commerce City, CO) Cargill Inc (Minneapolis, MN) Abengoa (Hugoton, KS) NewPage (Wisconsin Rapids, WI) POET

Joseph, MO) Gas Technology Institute (Des Plaines, IL) DOE Great Lakes Bioenergy Research Center (Madison, WI) Mascoma (Monroe County, TN) Southern Research

IN) Ecofin, LLC (Washington County, KY) Range Fuels (Soperton, GA) RSE Pulp & Chemical, LLC (Old Town, ME) GE Global Research (Niskayuna, NY) DSM Innovation Center

Biorefinery Projects 2 Four Improved Enzyme Projects Five Projects for Advanced Organisms Five Thermochemical Biofuels Projects Three Bioenergy Centers DOE Joint

2 Major DOE Biofuels Project Locations (DOE 2008) Geographic, Feedstock, and Technology Diversity Pacific Ethanol (Boardman, OR) DOE Joint Bioenergy Institute (Berkeley, CA) Novozymes (Davis, CA) Ceres, Inc (Thousand Oaks, CA) Genencor (Palo Alto, CA) Emery Energy (Salt Lake City, UT) BlueFire Ethanol (Corona, CA) Verenium Corp (San Diego, CA) Lignol Innovations (Commerce City, CO) Cargill Inc (Minneapolis, MN) Abengoa (Hugoton, KS) NewPage (Wisconsin Rapids, WI) POET (Emmetsburg, IA) Iowa State University (Ames, IA) Regents of the University of Minnesota (Minneapolis, MN) ICM (St. Joseph, MO) Gas Technology Institute (Des Plaines, IL) DOE Great Lakes Bioenergy Research Center (Madison, WI) Mascoma (Monroe County, TN) Southern Research Institute (Birmingham, AL) DOE Bioenergy Science Center (Oak Ridge, TN) Mascoma (Lebanon, NH) Cornell University (Ithaca, NY) Purdue University (West Lafayette, IN) Ecofin, LLC (Washington County, KY) Range Fuels (Soperton, GA) RSE Pulp & Chemical, LLC (Old Town, ME) GE Global Research (Niskayuna, NY) DSM Innovation Center (Parsippany, NJ) Dupont (Wilmington, DE) Research Triangle Institute (Research Triangle Park, NC) Seven Small-Scale Biorefinery Projects Four Commercial-Scale Biorefinery Projects 2 Four Improved Enzyme Projects Five Projects for Advanced Organisms Five Thermochemical Biofuels Projects Three Bioenergy Centers DOE Joint Solicitation Biomass Projects Regional Partnerships South Dakota State University, Brookings, SD Cornell University, Ithaca, NY University of Tennessee, Knoxville, TN Oklahoma State University, Stillwater, OK Oregon State University, Corvallis, OR

3 3 GTL Core Science Goals at All Scales

4 4 DOE-GTL Mission Challenges for Biology

5 5 DOE Bioenergy Research Centers: Multi-Institutional Partnerships

The BESC Team: A multi-institutional DOEfunded center dedicated to understanding and modifying plant biomass

Laboratory University of Georgia National Renewable Energy Laboratory University of Tennessee Georgia Institute of

6 The BESC Team: A multi-institutional DOEfunded center dedicated to understanding and modifying plant biomass recalcitrance Joint Institute for Biological Sciences (JIBS) Alternative Fuels User Facility Oak Ridge National Laboratory University of Georgia National Renewable Energy Laboratory University of Tennessee Georgia Institute of Technology Samuel Roberts Noble Foundation Dartmouth College ArborGen, LLC Verenium Corporation Mascoma Corporation Ceres, Inc. Individuals from University of California-Riverside, Cornell University, Washington State University, University of Minnesota, North Carolina State University, Brookhaven National Laboratory, Virginia Polytechnic Institute 6 Complex Carbohydrate Research Center (CCRC)

7 The Joint Institute for Biological Sciences (JIBS) JIBS will house a complete suite of tools for state-of-art systems biology Eleven MS devices to measure hundreds of proteins and chemicals of life at once HTP Growth Screen Controlled Cultivation Bioanalyzer and Nano-Drop High-density, multi-plex arrays Hybridization Station Microarray scanner 2 mm resolution scanner 7 Gary Sayler Director, Joint Institute for Biological Sciences 14 N/ 15 N Quantitative proteomics Metabolomics by GC, GC-MS, HPLC 454 Pyrosequencer

Access to the Sugars in Lignocellulosic Biomass is the

Conventional Enzyme Fermentation Fermentation

Catalysis Hydrocarbons Synthetic Biology Hydrocarbons

and be used in most conversion processes This requires

Modified plants to field trials: Year 5 New or improved

8 Access to the Sugars in Lignocellulosic Biomass is the Current Critical Barrier Alcohols Ethanol Butanol Conventional Enzyme Fermentation Fermentation Consolidated Bioprocessing Recalcitrance Chemical Catalysis Hydrocarbons Synthetic Biology Hydrocarbons Solving this will cut processing costs significantly and be used in most conversion processes This requires an integrated multidisciplinary approach Timeframe Modified plants to field trials: Year 5 New or improved microbes to development: Years 4 5 Analysis and screening technologies: Year 3 on 8

9 9 A Two-pronged Approach to Increase the Accessibility of Biomass Sugars

10 10 BESC Will Revolutionize How Biomass is Processed

11 11 Strategy Part 1: Identify, Understand and Manipulate the Plant Cell Wall Genes Responsible for Recalcitrance

Functional modifications of both 1 and 2 walls may decrease recalcitrance Primary Wall 90% polysaccharide Dividing and growing cells Pectin Hemicellulose Cellulose

12 Functional modifications of both 1 and 2 walls may decrease recalcitrance Primary Wall 90% polysaccharide Dividing and growing cells Pectin Hemicellulose Cellulose (proteins) Secondary Walls 70 80% polysaccharide Some cells with structural roles 12 Malcolm O Neill, CCRC Mohnen et al. (2008) Pectin Hemicellulose Cellulose Lignin (proteins)

13 13 Establishment of Plant Biomass Recalcitrance Gene KnowledgeBase (KB)

14 Reference Genomes * BESC Curation * Gene Discovery Tools * Pathway building *Links to BESC data Links to LIMS system Parang, Uberbacher et al., 14

Example: Targeted Cell Wall Synthesis: Tension Stress Study:

2006 Tension wood is formed on upper side of bent stems and

wall thickness Special layer of wall: Gelatinous G-layer Increased

15 Example: Targeted Cell Wall Synthesis: Tension Stress Study: Background Patten et al Tension wood is formed on upper side of bent stems and characterized by: Increased number of xylem cells Increased cell wall thickness Special layer of wall: Gelatinous G-layer Increased cellulose content Decreased lignin content Parallel orientation of microfibrils 15 Tension stress study is a cross-project effort.

Harvested, pooled and processed (where needed) developing xylem and phloem

16 Tension Stress Study: Experiment Two genotypes of Populus plants used in the bending experiment. Two weeks of mechanical bending. Harvested, pooled and processed (where needed) developing xylem and phloem tissues from tension, opposite and normal wood types. 16 Control erect plants Mechanically bent plants

17 Discovery-based Approach to Identify Recalcitrance-Associated Genes via Analysis of Natural Variation 17

Mining Genetic Variation in Switchgrass Create

upland SG VS-16 into 385 pseudo F1 clones HTS

385 genotypes Sugar Release Assay Analytical

18 Mining Genetic Variation in Switchgrass Create diverse population by cross lowland SG AP-13 and upland SG VS-16 into 385 pseudo F1 clones HTS Pipeline AP- 13 VS- 16 Pseudo F 1 population of 385 genotypes Sugar Release Assay Analytical Pyrolysis Create Genetic Marker Map to identify allelic variation Identify Marker Trait Association Cell Wall Biosynthesis Database Clones ready for field planting 18

Mining Variation to Identify Key Genes in Biomass Composition and Sugar

Study HTS Pipeline Skagit (Sedro-Woolley) Skykomish (Monroe) Columbia

200 km Existing collections (N = 500; 1-12 trees/site) New collections (N =

variation Identify Marker Trait Association Cell Wall Biosynthesis Database

19 Mining Variation to Identify Key Genes in Biomass Composition and Sugar Release Collected ~1300 samples for Populus Association and Activation-tag Study HTS Pipeline Skagit (Sedro-Woolley) Skykomish (Monroe) Columbia (Longview) Puyallup (Orting) Sugar Release Assay Analytical Pyrolysis 100 mi 200 km Existing collections (N = 500; 1-12 trees/site) New collections (N = 580; trees/site) Create Genetic Marker Map to identify allelic variation Identify Marker Trait Association Cell Wall Biosynthesis Database Establish common gardens for association and activation tag populations with 1000s of plants 19

20 20 Strategy Part 2: Biomass Recalcitrance Measure, Understand, and Model

21 HTP Characterization Pipeline for the Recalcitrance Phenotype Screening of 1000 s of samples Composition analytical pyrolysis, IR, confirmed by wet chemistry Pre-treatment new method with dilute acid and steam Enzyme digestibility sugar release with enzyme cocktail 21 Detailed chemical and structural analyses of specific samples

22 22 LIMS Reports are Used for Science and for MTA

23 Detailed Analysis of Specific Samples Inform Cell-wall Chemistry and Structure Detailed analysis Imaging AFM of switchgrass showing cellulose microfibrils Detailed analysis Bio-ultraCAT for 3-D density of Populus cell walls Immunolocalization using wall antibodies on Populus protoplasts Chemistry Fractionation and chromatography Mass Spectrometry for key metabolites 23 NMR for cellulose crystallinity 2D 1 H-NMR sees altered bonds in polysaccharides and lignin in biomass

24 24 Strategy Part 3: Identify, Understand and Manipulate Biological Catalysts to Overcome Recalcitrance

Microbial Hydrolysis and Enzymatic Hydrolysis: A Fundamentally Different Relationship Between Microbes and Cellulose Enzymatic hydrolysis (classical approach)

Hydrolysis mediated by CE complexes Enzymes (several) both bound and free Cells may or may not be present Hydrolysis mediated mainly by CEM complexes Enzymes

25 Microbial Hydrolysis and Enzymatic Hydrolysis: A Fundamentally Different Relationship Between Microbes and Cellulose Enzymatic hydrolysis (classical approach) Microbial hydrolysis (CBP) Cellulase enzyme(s), E Cellulase enzyme(s), E Cellulose, C Microbes, M (non-cellulolytic) Cellulose, C Microbes, M (cellulolytic) Hydrolysis mediated by CE complexes Enzymes (several) both bound and free Cells may or may not be present Hydrolysis mediated mainly by CEM complexes Enzymes both bound and free Cells both bound and free Yeast, enzymes with biomass, Dumitrache and Wolfaardt C. thermocellum on poplar, Morrell-Falvey and Raman, ORNL 25

State-of-the-art cultivation techniques to isolate novel high-temperature microbes with powerful

26 Biodiversity Access for New Biocatalysts Hypothesis: Will higher temperature anaerobic microbes be more effective? State-of-the-art cultivation techniques to isolate novel high-temperature microbes with powerful lignocellulolytic enzymes Collect samples from thermal biotopes Establish primary enrichment cultures at relevant temperatures and conditions Sampling at Yellowstone National Park October 2007 and July

27 High-Throughput Isolation Using Flow Cytometry Establish consortium Identify members Flow-cytometer Select different gates Anaerobic 75 o C ΔpH indicates growth 27 27

28 Caldicellulosiruptor sp. OB47 OB 47 on Switchgrass Particle Life-Dead Stain & Confocal Microscopy Image by Jennifer Morell-Falvey Gram-positive bacterium Rod Shaped ~0.5 x 1-2µm T opt C Fermentative heterotroph Produces H 2, Acetate, Lactate, CO 2 and Ethanol 28

29 Schematic of Cellulosome OlpA Cthe_0452 CELL SdbA Cthe_0735?? Type I Cohesin Type II Cohesin CELLULOSOME Scaffoldin (CipA) Cellulose Dockerin Dockerin Catalytic Units Cellulose Binding Domain OlpB Orf2p???? Cthe_1806? Cthe_0736? 29 (adapted from Carlos Fontes, 2007 Gordon Research Conference on Cellulases and Cellulosomes )

30 C. thermocellum Cellulosome was Analyzed under Several Conditions Protein (mg/l) Cultivation on Avicel shown Cellulosome Cell protein Cellulosome protein Time (hr) kda Run 1 Run 2 Quantitative Proteomics: Shot-gun Grown on cellubiose, Avicel,, and pretreated switchgrass at ~1L Cellulosome is released when growth slows Cellulosome isolation via affinity digestion method In-solution trypsin digestion, following by shot-gun proteomics (LC-MS/MS) Quantitative proteomics with 15 N labeled substrates 30

31 Characterization of a C. thermocellum Mutant that Utilizes Cellulose Rapidly Genome position Speedy 454 contigs Speedy SNPs CDS 454 Resequencing identified 78 mutated loci in Speedy mutant 25 mutant loci common with ORNL wild-type strain Transcriptomics and further analysis underway ORNL wild-type 454 contigs 31 ORNL wild-type SNPs

32 32 BESC: Bioenergy Related Microbes kb

Integrating Expression, Proteomics, SNPs,

thermocellum ATCC 27405 proteomics Pathway

pathways Metabolite tracing Locus Tag: SO_1631

kinase, PyrHOrganism: Clostridium thermocellum

.1713915 Function: This protein, also called

33 Integrating Expression, Proteomics, SNPs, Metabolites on Cellular Systems Clostridium thermocellum ATCC proteomics Pathway Component Details SNP mapping Expression on pathways Metabolite tracing Locus Tag: SO_1631 Gene Symbol: pyrh Gene Description: uridylate kinase, PyrHOrganism: Clostridium thermocellum Location: Function: This protein, also called UMP kinase, converts UMP to UDP by adding a phosphate from ATP. It is the first step in pyrimidine biosynthesis. Notes: Citation: Annotation Date: Domain(s): COG MIST INTERPRO: Pathway(s): BeoCyc KEGG: Information: ORNL GenBank IMG SEED Expression on genome map 33

34 Computational Microscope Assay and Analysis Framework Knowledgebase Metabolic pathways Ontologies LIMS Integrate experimental data with external reference sets Assays Phenotypes Microarray Experimental conditions Gene expression Treatments Mass spec Imagining Protein expression Protein interaction 2D gels Genotypes Mutants DNA sequence SNPs Orthologs Proteins Metabolites & concentrations Genome sequence Gene structure Infer significant processes Operons Pathways Navigate pathways 34

35 BESC Website New website has been deployed Elements include General information Educational and professional level components area for controlled access by BESC members Inventions 35

36 Highlights 2008 until June Scientific presentations at meetings and conferences worldwide 82 Scientific publications 17 Workshops and seminars for BESC researchers and graduate students 16 Inventions disclosed which are under evaluation by the BESC Commercialization Council Scientific collaboration with the University of British Columbia has contributed over 250 additional Populus samples at no cost to BESC 85+ Presentations to Stakeholders (Secretary, Under Secretaries, Congressmen and Staff Members, Businessmen, etc.) 70+ Television, Print, and Radio Interviews Education program with the Creative Discovery Museum in Chattanooga, Tennessee to develop a Biofuels Outreach Lesson Co-sponsored Global Venture Challenge 2008 in April at ORNL 36

37 Lessons Learned A coherent vision, goal and theme is critical to create a team, mission and excitement This vision must be reinforced by lots of communication (early and on-going) Have your arguments early and honest and based on strong scientific judgment and respect developed with input from the whole team After vision is defined let the science lead Make sure success is defined through clear milestones and deliverables Management needs to strongly encourage collaborations By intentional scientific plans such as our pipelines where samples are exchanged and must be coordinated and discussed By natural means such as providing time at retreats where people find common interests and start working together By directive such as our insistence that all projects in the Computational Biology and Data Analysis activity must find and align with a BESC experimental team. A strong integrated operations team is essential for start-up (contracts, ops, tech transfer, support) Well developed plans and processes provide the necessary framework to address and adapt to project challenges expeditiously 37

38 Lessons Learned Leveraging unique strengths between Universities, National Laboratories and Industries creates synergy Universities: strong innovation National Laboratories: high degree of consistency and breadth of resources/expertise Industry: agility and market understanding Pretreatment Pipeline Example UC Riverside led the development of a novel approach to high throughput pretreatment and digestion which saved 900k over existing technology NREL adapted, fine-tuned and deployed this innovative system in a manner that significantly reduced method uncertainty and increase throughput 10X ArborGen has begun propagating plant transformants based on information gleaned from the resulting tests 38

39 A Dispersed Center: the Good, the Bad, the Reality You can be highly integrated without being co-located Allowing scientists to participate from home institutions enables the assembly of a team with a much higher proportion of the most accomplished experts Capital costs are lowered by utilizing existing infrastructure/ equipment available at partner facilities Travel costs are higher due to need for periodic center meetings and workshops Establishing and maintaining trust between partners is imperative Organizational discipline is critical to success Communication, communication, communication.. 39

40 40 BESC is organized to provide clear operations and science accountability

41 Bio-Energy at ORNL 41 Managed by UT-Battelle for the Department of Energy www. bioenergycenter.org Tech. Transfer Nat l Transport. Res. Center Bioproducts, etc.

Tennesse Biofuels Initiative January 31 2007: Governor Bredesen

feedstock production, R&D, pilot production, fuel distribution $40M

and UT Leverages industrial partners Builds on TN biomass

Managed by UT-Battelle for the Department of Energy Conceptual

42 Tennesse Biofuels Initiative January : Governor Bredesen began a significant investment in biofuels More than $70M for feedstock production, R&D, pilot production, fuel distribution $40M for a pilot plant (groundbreaking fall 2008) Leverages R&D at ORNL and UT Leverages industrial partners Builds on TN biomass resources: Corn stover Switchgrass Soybeans Tree residue Poplar 42 Managed by UT-Battelle for the Department of Energy Conceptual drawing: Genera, LLC Pilot Biorefinery in Collaboration with UT and DuPont/Danisco, LLC 42

43 SAFER (Southeast Agriculture Forestry and Energy Research) Has three pilot working groups Feedstocks and infrastructure Linked with SunGrant and Regional Partnerships Feedstock biology Common garden working group BioPiloting sharable resources 43 Managed by UT-Battelle for the Department of Energy

44 Thank you Retreat December BESC is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science

Wheat Straw Corn Stover 0 20 40 60 80 100 Million dry

45 Nearly 200 million dry tons of cellulosic feedstock required Cellulosic feedstock requirements Energy Crops Wheat Straw Corn Stover Million dry tons 45 Managed by UT-Battelle for the Department of Energy

Sustainable Biofuel Production

Sustainable Biofuel Production A U.S. Department of Energy Perspective Alison Goss Eng Office of the Biomass Program May 28, 2008 A Sustainable Biofuels Industry Social Promotes social goals Rural livelihoods