Characterization of Biomass for Understanding Recalcitrance: Approaches from the Bioenergy Science Center

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Characterization of Biomass for Understanding Recalcitrance: Approaches from the Bioenergy Science Center Presented to Frontiers in Biorefining ctober 21 st, 2010 Paul Gilna 1,

1 Characterization of Biomass for Understanding Recalcitrance: Approaches from the Bioenergy Science Center Presented to Frontiers in Biorefining ctober 21 st, 2010 Paul Gilna 1, Suzy Fowler 1, and Mark Davis 2, and Brian H. Davison 1 BioEnergy Science Center and 1 ak Ridge National Laboratory, ak Ridge, TN 2 National Renewable Energy Laboratory, Golden C

The BioEnergy Science Center BESC: A multi-institutional DE-funded center dedicated to understanding and modifying plant biomass recalcitrance Samuel Roberts Noble Foundation

North Carolina State University Virginia Polytechnic Institute University of California Los Angeles 322 People in 20 Institutions ak Ridge National Laboratory University of

2 The BioEnergy Science Center BESC: A multi-institutional DE-funded center dedicated to understanding and modifying plant biomass recalcitrance Samuel Roberts Noble Foundation National Renewable Energy Laboratory Brookhaven National Laboratory Cornell University University of Minnesota Washington State University University of California Riverside North Carolina State University Virginia Polytechnic Institute University of California Los Angeles 322 People in 20 Institutions ak Ridge National Laboratory University of Georgia University of Tennessee Dartmouth College West Virginia University Georgia Institute of Technology ArborGen, LLC Ceres, Incorporated Mascoma Corporation Verenium Corporation 2

Hydrocarbons Synthetic biology Hydrocarbons Recalcitrance vercoming this barrier will cut processing

3 Access to the sugars in lignocellulosic biomass is the current critical barrier Ethanol Butanol Conventional enzyme fermentation Fermentation Alcohols Consolidated bioprocessing Chemical catalysis Hydrocarbons Synthetic biology Hydrocarbons Recalcitrance vercoming this barrier will cut processing costs significantly and be used in most conversion processes This requires an integrated multidisciplinary approach 3

4 4 A two-pronged approach to increase the accessibility of biomass sugars

5 5 BESC Will Revolutionize How Biomass is Processed

Genetic block in lignin biosynthesis in switchgrass increases ethanol yields Phenylalanine PAL Agrobacteriummediated transformation of switchgrass HC C4H HC CoAS H H 4-coumaraldehyde HH2C cinnamate H

6 Genetic block in lignin biosynthesis in switchgrass increases ethanol yields Phenylalanine PAL Agrobacteriummediated transformation of switchgrass HC C4H HC CoAS H H 4-coumaraldehyde HH2C cinnamate H 4-coumaroyl alcohol H lignin H 4-coumaric acid 4CL H 4-coumaroyl CoA CCR CAD HCT R- R- CoAS Lignin pathway H 4-coumaroyl shikimic acid or quinic acid C3H H caffeoyl shikimic acid or quinic acid HCT H caffeoyl CoA H H Ethanol Yield per Weight of Biomass (g/g) Wild-type switchgrass Ethanol yield Noble Foundation transgenic switchgrass 6 The Samuel Roberts NBLE Foundation CCoAMT CoAS feruloyl CoA CCR coniferaldehyde X. Fu and Z. Wang (Noble), J. Mielenz (RNL), support from USDA/DE H CH3 H CH3 H CAD F5H HH2C G lignin CH3 H coniferyl alcohol H CH3 H H F5H 5-hydroxyconiferaldehyde CMT HH2C 5-hydroxyconiferyl alcohol H CH3 H CH3 sinapaldehyde CH3 H H CAD HH2C CMT CH3 H CH3 sinapyl alcohol S lignin

7 Noble Foundation Comt Mutant Switchgrass Requires % Less Enzyme Than Wildtype Switchgrass 400 WILD TYPE TRANSGENIC 350 4X Ethanol (mg/ g cellulose) X WT 5 WT 15 WT 60 TG 5 TG 15 TG 60 Plant Line and Enzyme Loading ' 0.5% H2S4

8 Enzyme titration has been repeated at lower severity yielding identical results: not a pretreatment effect! = wild-type = transgenic Ethanol (mg/g cellulose) ' 0.5% H 2 S 4 WILD-TYPE TRANSGENIC 4X 3X WT 5 WT 15 WT 60 TG 5 TG 15 TG 60 Plant Line and Enzyme Dose 8

9 Lower Enzyme Requirement Significantly Reduces the Price of Ethanol 2008 Minimum Ethanol Selling Price (MESP) for biomass ethanol estimated at $2.43/ US gallon with enzyme 32 /gallon* US DE enzyme cost target is 8 for MESP of $1.33 Comt switchgrass genetic block reduced enzyme requirement by % Lower enzyme doses will likely reduced enzyme costs to 16-8 / gallon Pretreatment conditions impacts the enzyme requirement synergistically Milder pretreatment conditions will reduce pretreatment inhibitors further improving ethanol processing costs with the Comt switchgrass 9 *Andy Aden NREL Technical Report TP MAY 2008

pathway H 4-coumaroyl shikimic acid or quinic acid H caffeoyl shikimic acid or quinic acid H caffeoyl CoA H H Ethanol Yield per Weight of Biomass (g/g) 0.

00 Wild-type switchgrass Ethanol yield Noble Foundation transgenic switchgrass 10 The Samuel Roberts NBLE Foundation CCoAMT CoAS feruloyl CoA CCR

10 Genetic Block in Lignin Biosynthesis in Switchgrass Increases Ethanol Yields Phenylalanine PAL Agrobacteriummediated transformation of switchgrass HC C4H HC 4CL CoAS CCR H CAD H 4-coumaraldehyde HH2C cinnamate H 4-coumaroyl alcohol H lignin H 4-coumaric acid H 4-coumaroyl CoA HCT R- C3H R- HCT CoAS Lignin pathway H 4-coumaroyl shikimic acid or quinic acid H caffeoyl shikimic acid or quinic acid H caffeoyl CoA H H Ethanol Yield per Weight of Biomass (g/g) Wild-type switchgrass Ethanol yield Noble Foundation transgenic switchgrass 10 The Samuel Roberts NBLE Foundation CCoAMT CoAS feruloyl CoA CCR coniferaldehyde X. Fu and Z. Wang (Noble), J. Mielenz (RNL), support from USDA/DE H CH3 H CH3 H CAD F5H HH2C G lignin CH3 H coniferyl alcohol H CH3 H H F5H 5-hydroxyconiferaldehyde CMT HH2C 5-hydroxyconiferyl alcohol H CH3 H CH3 sinapaldehyde CH3 H H CAD HH2C CMT CH3 H CH3 sinapyl alcohol S lignin

11 The Transformation Pipeline (TP) is Fully perational for BESC Plant Wall Mutants 450 genes submitted to TP; 326 genes accepted 477 constructs accepted in TP: (353 Populus, 124 Switchgrass/VIGs) 50 Populus construct lines sent to PIs: (1030 lines; ~10,000-20,000 plants) 79 Virus Induced Gene Silenced (VIGs) (foxtail millet) Constructs nucleotide-sugar GT CWP CWP-R GH Polysac Modify Lignin vesicular transport TF signalling ther 45 stable Switchgrass transformation 11

12 A key part of both strategies: Measure, understand, and model biomass recalcitrance Biomass formation and modification Characterization and modeling Biomass deconstruction and conversion 12

13 This requires a suite of anaytical technologies HTP methods to screen many samples for composition and digestibility Detailed methods to understand plant cell wall structure 13

14 High-throughput characterization pipeline for the recalcitrance phenotype Screening thousands 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 Detailed chemical and structural analyses of specific samples 14

Mining variation to identify key genes in biomass

samples for Populus association and activation-tag

genetic marker map to identify allelic variation

biosynthesis database Skagit (Sedro Woolley) Sugar

(Longview) 100 mi 200 km Puyallup (rting) Establish

populations with thousands of plants Existing

15 Mining variation to identify key genes in biomass composition and sugar release Collected ~1300 samples for Populus association and activation-tag study High-throughput screening pipeline Create genetic marker map to identify allelic variation Identify marker trait association Cell wall biosynthesis database Skagit (Sedro Woolley) Sugar release assay Skykomish (Monroe) Columbia (Longview) 100 mi 200 km Puyallup (rting) Establish common gardens for association and activation-tag populations with thousands of plants Existing collections (N = 500; 1 2 trees/site) New collections (N = 580; trees/site) 15

16 First step in the high throughput pipeline Uses <5 mg/sample Provides Lignin estimate, S/G ratio, and data for PCA ver 3000 samples run Current Campaigns: Poplar: RNL QTL study Foxtail Millet

4 BESCPP001211 20.95 1.6 BESCPP001215 24.13 1.9 BESCPP001219 24.43 1.8 BESCPP001223 23.46 1.2 BESCPP001227 22.01 1.5 BESCPP001231 20.10 1.3 BESCPP001235 19.47 1.6 BESCPP001239 22.52 2.

16 16 First step in the high throughput pipeline Uses <5 mg/sample Provides Lignin estimate, S/G ratio, and data for PCA ver 3000 samples run Current Campaigns: Poplar: RNL QTL study Foxtail Millet VIGS: Noble Arabidopsis: UGA/CCRC Switchgrass: UTK Pine: RNL High Throughput Lignin Estimation Using Molecular Beam Mass Spectrometery Sample Lignin S/G BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP Lignin Composition Prepared Samples BESC0001-BESC00, X-expl: 32%,22% BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP BESCPP 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High-throughput screening to analyze natural

pretreatment only Sugar release varies from

17 High-throughput screening to analyze natural Populus trees Screening of 1200 natural Populus trees shows high natural variability in compostion and digestibility Hot water as pretreatment only Sugar release varies from 25% to >90% of theoretical value Environmental vs genetic? 17

18 The High-Throughput Pretreatment and Hydrolysis (HTPH) System has Analyzed >10,000 Samples in FY2010 for Composition and Digestibility Unique BESC samples submitted and analyzed by HTP pipelines (not including replicates). CRCC RNL UCR Noble U. Tennessee Total Analytical Pyrolysis Recalcitrance Samples from Industrial, International, and external collaborations. ArborGen Purdue U. Copenhagen Edenspace Total Analytical Pyrolysis Recalcitrance

lab facilities with samples 18,000+ samples shipped

19 LIMS Data and Sample Tracking 35,000+ samples 25,000 added in FY10 60 organisms 50 experimental campaigns 35 lab facilities with samples 18,000+ samples shipped between facilities (13,000 in FY10) Metadata and analyses captured 19

20 Detailed analysis of specific samples inform cell-wall chemistry and structure Detailed analysis Detailed analysis Imaging MSAFM: IR spectra of localized cell walls AFM of switchgrass showing cellulose microfibrils Immunolocalization using wall antibodies on switchgrass Chemistry NMR for cellulose crystallinity Fractionation and chromatography Mass spectrometry for key metabolites 2D 1 H-NMR sees altered bonds in polysaccharides and lignin in biomass 20

21 CARS (Coherent Anti-Stokes Raman Scattering) Imaging of Lignin in Interfascicular Fiber Cell Walls in Transgenic Alfalfa 21 S-Y Ding (NREL) and X. S. Xie (Harvard) tools under BER imaging grant; sample analysis under BESC, 2010 Mutant alfalfa from Noble Foundation

Quantitative Coherent Raman Imaging of Plant Cell Wall Polymers shows differential localized lignin kinetics Distribution of different polymers in plant cell wall types directly affects the

22 Quantitative Coherent Raman Imaging of Plant Cell Wall Polymers shows differential localized lignin kinetics Distribution of different polymers in plant cell wall types directly affects the efficiency of chemical pretreatment of enzymatic hydrolysis of biomass. ur findings provide new prospective in rational design strategy for genetic improvement of energy plant, as well as for optimization of pretreatment of enzyme hydrolysis by determination of hydrolysis kinetics of lignin and cellulose in plant cell walls Different rates of delignification reflect the differences in accessibility of catalyst Quantitative imaging of lignin distribution and lignin bleaching kinetics in lignin-down-regulated alfalfa Acid chlorite delignification of plant cell walls using 0.1 N HCl and 10% NaCl2 was monitored by SRS Zeng et al., Bioenergy Research (2010) 22

Mode-Synthesizing Atomic Force Microscope is a new AFM method to characterize lignocellulosic biomass at the nanoscale 23 Mode

Single mode MSAFM revealed variations in mechanical properties and composition within the tissue sample.

23 Mode-Synthesizing Atomic Force Microscope is a new AFM method to characterize lignocellulosic biomass at the nanoscale 23 Mode synthesizing atomic force microscopy (MSAFM) is a generalized technique exploiting the way various materials oscillate at multiple frequencies. Demonstrated nanoscale topography and subsurface imaging for Populus and switchgrass samples at the 50 nm resolution using MSAFM. Single mode MSAFM revealed variations in mechanical properties and composition within the tissue sample. FTIR spectroscopic characterization of similar samples was undertaken to evaluate differential wall chemistries. MSAFM will be used to see plant structure and chemistry within the native and treated cell walls. Populus samples; Tetard et al., Nature Nanotechnol. (2010). Received 2010 R&D 100 award

24 In Depth Characterization 1. High Throughput Pretreatment and Enzymatic Hydrolysis 2. Conventional Washed Solids Pretreatment and Enzymatic Hydrolysis to Determine Sugar Release 3. X-ray micro CT 4. Endoxylanase fingerprinting of glucuronoxylan and arabionxylan using MALDITF-MS( CCRC, UGA) 5. Glycosyl-linkage analyses 6. Neutral and acidic glycosyl residue composition of biomass by high-performance anion-exchange chromatography with pulsed amperometric detection 7. Glycosyl-linkage analyses 8. Per--acetylation of biomass for analysis by NMR spectroscopy 9. Sequencing of xylo-oligosaccharides generated by endoxylanase-treatment of glucuronoxylan and arabionxylan using ESI-MS and NMR spectroscopy 10. Neutral sugar composition by GC-FID 11. Biomass solubilization 12. Ball-milled Biomass 13. Advanced Imaging (AFM, Coherent Raman Microscopy, TIRF-M, TEM) 14. High throughput Pyrolysis Molecular Beam Mass Spectrometry 15. Wet Chemistry 16. Lignin structural characterization of biomass using NMR spectroscopy 17. Imaging Mass Spectrometry using ToF-SIMS and MALDI-MS 18. Investigating cellulase-cellulose interaction using FRET 19. Cryo-microtome biomass 20. FT-IR analysis 21. Scanning electron microscopy of native and processed biomass (SEM) 22. Carbohydrate Biomass Analysis 23. Molecular weight determination by GPC 24. Whole cell wall structural analysis of plant biomass using NMR spectroscopy. 25. Chemical and ultrastructural analysis of biomass (% crystallinity) by solid state NMR 13 C cross polarization magic angle spinning (CP/MAS) NMR spectroscopy 26. Supramolecular structure analysis (pore distributions) via a solvent probe by solid state NMR and magnetic resonance micro-imaging (MRI) 27. Glycome profiling of cell wall and biomass samples 28. Scanning Near-field Acoustic Photothermal Spectroscopy (under development) 24 Institutions: CCRC, UCR, GT, NREL, RNL and UF (non BESC)

25 25 Strategy Part 3: Identify, Understand and Manipulate Biological Catalysts to vercome Recalcitrance

26 verexpression of the Mutant adhe Confers Enhanced Ethanol Tolerance in C. thermocellum Three strains tested in C. thermocellum DSM 1313 wild-type background vector only control additional wt gene adhe via plasmid mutant gene adhe via plasmid Ethanol dose effect observed and only C. thermocellum with mutant gene adhe grow with 5% (v/v) ethanol added D600nm vector only control mutant adhe wt adhe time (h) 10 1 No ethanol added D600nm vector only control mutant adhe wt adhe % (v/v) ethanol added vector only control mutant adhe wt adhe time (h) Whether can we achieve a higher product titer still has to be addressed. D600nm % (v/v) ethanol added time (h)

27 Translating discoveries to the scientific community 187 scientific publications (August 2010) ~33% of publications include external collaborators at non-besc Institutions New Pubs Total BESC publications have already been cited 495+ times in peer-reviewed journals Several publications in top-tier journals 50 0 Mar 2008 Sept 2008 Mar 2009 Sept 2009 Mar 2010 Aug 2010 Nature Biotechnology, 2008, Lynd et al., How biotech can transform biofuels PNAS, 2008, Shaw et al., Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield Nature Nanotechnology, 2010, Tetard et al., New modes of subsurface atomic force microscopy through nanomechanical coupling 22 inventions disclosed (under evaluation by BESC Commercialization Council) 27

28 Thank you SCIENCE RETREAT JUNE BESC is a U.S. Department of Energy Bioenergy Research Center supported by the ffice of Biological and Environmental Research in the DE ffice of Science