2015 DOE BioEnergy Technologies Office Principal Investigators: (BETO) Project Peer Review Gregg Beckham (NREL) Date: March 25th, 2015 John Gladden (SNL) Technology Review Area: Biochemical Conversion Organizations: National Biological Depolymerization (WBS Renewable Energy 2.3.2.100) Laboratory and Sandia National Laboratory

1 | Bioenergy Technologies Office eere.energy.gov Project Goal

Goal and Outcome: develop a biological approach to depolymerize solid lignin for upgrading of low MW aromatic compounds to co-products

Relevance to BC Platform, BETO, industry: lignin valorization is key for meeting HC fuel cost and sustainability targets in integrated biorefineries

Residual Ligninolytic Aromatic- Biorefinery Lignin catabolizing bacteria

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Quad Chart

Timeline Barriers • Start date: October 2014 • Bt-F Hydrolytic Production • End date: September 2017 • Bt-I Catalyst Efficiency • Percent complete: ~30% • Bt-J Biochemical Conversion Process Integration

Budget Partners and Collaborators • BETO Projects: Pretreatment and Process Total Planned Hydrolysis, Lignin Utilization, Biochemical Funding FY14 Costs Platform Analysis, Pilot Scale Integration, (FY15-Project Enzyme Engineering and Optimization End Date) • BETO-funded National Lab Projects: Oak Ridge National Laboratory (A. Guss) DOE $222,410 $1,752,590 • Office of Science funded efforts: Joint funded BioEnergy Institute, Environmental Molecular Sciences Laboratory, Pacific Northwest National Funds are split 50/50 between NREL Laboratory (R. Robinson, E. Zink) and SNL • Academic collaborators: Swedish University of Agricultural Sciences, University of Portsmouth

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Project Overview

Project History: • Began as a BETO seed project in FY14 between NREL and SNL • Achieved major milestone at end of FY14 • Complementary effort to other BETO-funded lignin projects

Project Context: • Lignin valorization is a primary challenge in biochemical conversion processes • Leverage studies in biological lignin depolymerization with new synthetic biology techniques and new process concepts in biological funneling

High-Level Objective: • Employ biology for depolymerization and aromatic catabolism of residual lignin

Lignin Depolymerization Lignin Upgrading • Focus on residual solids from • Develop aromatic metabolic map Deacetylation-Mechanical Refining Prt. • Elucidate aromatic transport mechanisms • Examine fungal and bacterial ligninolytic • Understand rate limitations in aromatic enzymes for solid lignin depolymerization catabolism • Develop effective enzyme secretion • Develop optimized modules for production systems of value-added products 4 | Bioenergy Technologies Office

Technical Approach

DMR Pretreatment Biomass Degraded lignin Sugars for fuels

DDR-EH lignin Lignin-degrading Enzymes: Laccases & Enzymatic Hydrolysis : residual lignin from deacetylation and mechnical-refining pretreatment and enzymatic hydrolysis (DMR-EH) Approach: • Characterize enzymatic degradation of DMR-EH lignin • Identify parameters for optimal biological lignin degradation • Engineer organisms convert depolymerized lignin into valuable bioproducts Primary challenges: • Effective analytical tools to quantify lignin depolymerization • Minimizing repolymerization of high MW lignin during depolymerization • Identifying and overcoming aromatic transport and catabolic limitations Use microbial cell factories to upgrade lignin Success factors: • Achieving high yields of low MW lignin species • Generating aromatics that can be metabolized by microbes • Producing products with high market value from lignin 5 | Bioenergy Technologies Office

Management Approach

Critical Success Factors: Management Approach: • Employ tools of synthetic biology and • Use quarterly milestones to track lignin analytical chemistry progress and down-select options

• Leverage experience from two • Milestone priority dictated by biomass conversion centers depolymerization and overcoming key risks in yields • Strategic hire in biological lignin depolymerization • Phone calls every 3 weeks, site visits every 6 months • Work with Biochemical Platform Analysis Project to identify optimal co- • Contributions from both partners on targets lignin analytical chemistry leveraging respective expertise • Leverage input and collaborations from other BETO-funded projects in lignin • Divide research in a manner that leverages each partners strengths

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Technical Results – Outline

• Examined purified enzymes and fungal- derived secretomes on several lignin substrates for ability to depolymerize lignin

Lignin • Determined that reaction conditions and enzyme composition are key factors in Repolymerization ENZYMES Depolymerization promoting lignin depolymerization

• Found evidence that lignin repolymerization is an issue that must be addressed to Co- achieve maximum lignin depolymerization. products Solution: microbial “sink” Aromatic Catabolism

• For FY15, we have assembled a list of Strain Consumes Growth on Lignin Aromatics microbial “sinks” and initiated a detailed Amycolatopsis sp. Y Y Pseudomonas fluorescens Y Y characterization of DMR-EH lignin Pseudomonas putida KT2440 Y Y Etc… Y Y 7 | Bioenergy Technologies Office

FY15 Milestone: Characterization of solid DMR-EH

Compositional analysis Molecular weight distribution

Sample ID Content (%) Ash 2.18 Mn Mw PD Lignin 66.0 2,000 10,000 5.0 Glucan 9.24 Xylan 9.36 Galactan 1.04 Arabinan 1.62 Fructan 0.00 Acetate 0.72 Total sugar 21.26 Total 90.2

2D-NMR for lignin chemical composition (HSQC)

Moiety Abundance 1 1 6 2 6 H 2 5 S 3 5 3 H3 C O 4 OC H 3 4 S 17% O O R R O O G 45% b a 1 O O H 2% 6 FA 2 b 5 3 1 a 4 PCA 27% 6 G 2 1 O 5 3 6 2 R 4 OC H PCA 3 FA 9% O 5 R 4 O C H 3 O R

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Microbial Lignin Depolymerization

Enzymatic cocktail

Laccases Peroxidases

Manganese (MnP) Lignin (LiP) Dye-decolorizing (DyP) Versatile (VP)

Enzymatic assays

O2 H2O2 Aryl-alcohol (AAO) H O 2 Free radicals

LIGNIN DEPOLYMERIZATION

FY14 Research: Start with Fungi • Use purified fungal enzymes to optimize conditions for lignin depolymerization • Examine ligninolytic enzyme cocktails produced by basidiomycete fungi to optimize conditions and identify the enzymes present in natural secretomes 9 | Bioenergy Technologies Office

Optimizing Enzymatic Lignin Depolymerization

Ligninolytic enzyme activity is a strong function of temperature and pH Top Enzymes • Screened 9 commercial purified fungal laccases Enzyme Function Source Organism Name and peroxidases for ability to solubilize lignin Laccase Trametes versicolor Tv • Used active enzymes to optimize the reaction Laccase Agaricus bisporus Ab Laccase Rhus vernicifera Rv conditions and create cocktail Laccase Pleurotus ostreatus Po • pH and Temp are critical factors for Lignin depolymerization Unknown LiP Green enzymes used in ligninolytic cocktails • Optimal enzyme loads depend on pH, T

Ligninolytic Enzymes Depolymerize or Solubilization of DMR-EH Lignin by Repolymerize Lignin Depending on Reaction Ligninolytic Cocktail 60

Conditions 0.8 50

0.6 40 30 0.4 20 Solubility(%) 0.2

Depolymerize Depolymerize 10 0

Repolymerize 0

ugE/mgL 0.4 4 0.4 4 inLignin 1x 0.5x 1x 0.5x -0.2 -10 Temp 30°C 30°C Tv Lac -20 20°C 30°C -0.4 pH Po Lac Change Change -30 DDR-EH Lignin Solubilization (ABS 280nm) Solubilization Lignin -0.6 LiP Enzyme Loadings are also very important! 10 | Bioenergy Technologies Office

Screening of natural fungal secretomes

Screen with 12 white-rot fungi Ligninolytic Enzyme induction on DDR-EH lignin • Pleurotus ostreatus • Irpex lacteus • Panus tigrinus • Bjerkandera adusta • Bjerkandera sp. • Cerioporopsis subvermispora • Pleurotus eryngii • Phellinus robustus • Polyporus alveolaris • Stereum hirsutum Production of fungal secretomes in the presence of • Trametes versicolor lignocellulose can induce and/or accelerate the • Phanerochaete chrysosporium production of ligninolytic and cellulolytic enzymes

Profile ligninolytic enzyme activities Production of ligninolytic cocktails ABTS assay

Reactive Blue 19 assay

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Characterization of ligninolytic cocktails

Pleurotus eryngii Phellinus robustus Bjerkandera sp. ABTSLaccase     (MnPs) ( laccase, AAO, VP, MnP ) ( laccase, MnP, DyP, AAO) 2000 ABT 2000 ABTS + H2O2 2000S 2000 Peroxidase

1500 ABTS + H2O2+ MnP I 1500 1500 Mn 1500 9 days

9 days

MnSO4 ABT 7 days mU/mL 1000 MnP II S + 1000 1000H2O 1000 mU/mL mU/mL Laccase x 10 2 mU/mL VeratrylAAO alcohol 500 500 500 500 VeratrylLiP alcohol+ ABT H2O2 S + 0 H2O 0 RBDyP 19 0 2+0 0 5 10 15 0 5 10 15 Mn 0 5 10 15 0 5 10 15 Days Days DaysDays

Control Secretomes from: • Identified organisms that Pleurotus eryngii Bjerkandera sp. Phellinus robustus 9000 produce the most activity of 8000 pH4.5 7000 specific ligninolytic enyzmes pH7 6000 60% decrease P. eryngii 50% decrease MW • shows highest 5000 MW 4000 lignin depolymerization, 3000 lowest MW pH 7 +2 +2 2000 H2O2 + H2O2 + Mn H2O2 + Mn Mn+2 • Surpassed FY14 Go/No-Go 1000

Molecular weight average (kDa) weight Molecular average 0 • pH plays a major role in lignin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 12 | Bioenergy Technologies Office depolymerization extents

P. eryngii secretome depolymerizes DMR-EH Lignin

Number-averaged Molecular Weight Distribution upon ligninolytic enzyme treatment at pH 7

2500 3 U laccase/g DDR Lignin 11 U laccase/g DDR 2000 30 U laccase/g DDR Repolymerization Depolymerization 1500 (kDa) n 1000 M

500  Need to create conditions

0 that promote Depolymerization No1 21 23 34 54 enzymes day days days days

P. eryngii is able to depolymerize DDR-EH lignin to low MW species

• 50% of lignin species are smaller than the control (Mn) in a single day of treatment • Apparent repolymerization by 4 days of incubation • Repolymerization may be prevented if we incorporate a low MW aromatic compound “sink” • Work in Pseudomonas putida suggests microbes are an ideal “sink”” as they can be used to convert the lignin degradation products into value-added compounds

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One primary strategy going forward: microbial “sink”

High MW Lignin Microbial “Sink” Co- products Fungal Aromatic Laccases & Catabolism Peroxidases

Re-polymerization

Utilize microbial sink to uptake aromatic species upon depolymerization • Similar to SSF or CBP concepts in polysaccharide valorization approaches • Requires understanding of aromatic transport and catabolism: “aromatic metabolic map” • Need a promiscuous microbe with broad substrate specificity, genetic tractability • Co-product selection will require consideration of separations and other process variables including oxygen demands in solid media

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Conditioning DMR-EH lignin for biological treatments

• DMR-EH lignin is a challenging substrate to obtain high conversion yields to monomeric species. • pH will be crucial for obtaining high lignin solubilization as well as performing enzyme treatments and growing the organisms

Effect of pH on Growth on DMR-EH Lignin 0.60 Organism

) 0.50 5 600 0.40 8 0.30 14

0.20 22 24 Growth (OD Growth 0.10 31 0.00 33 7 8 9 10 11 12 pH

• FY15 Milestone: Identified 27 organisms (bacteria and fungi) that can metabolize lignin subunits and can grow on lignin • Remaining FY15 Milestones: Down-select to top microbial “sinks” and begin detailed characterization of aromatic metabolism

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Salvachua et L-CBP: Bacteria also depolymerize lignin al., in review

L-CBP ALKALINE PRETREATED LIQUOR FROM CORN STOVER Lignin Consolidated DDR-EH Bioprocessing lignin

O O O O O O HO

HO High Mw Aromatic Lignin Monomers Laccases & Peroxidases

Intracellular Or Extracellular Metabolites Production Lignin Depolymerization Depolymerization Lignin Monomers for Catabolism Catabolism for Monomers

For fuels, materials, & chemicals

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Proteomics to fingerprint ligninolytic enzymes

• Bacteria also depolymerize lignin with laccases and peroxidases, but produce much smaller amounts of enzymes than fungi • Proteomics studies will provide detailed information about fungal and bacterial enzymes implicated in lignin depolymerization and catabolism • Awarded competitive allocation for proteomics at EMSL, analysis ongoing

Control P. putida

BACTERIA: Pseudomonas putida KT2440 Acinetobacter ADP1 Amycolatopsis sp. Rhodococcus jostii RHA1 FUNGI Pleurotus eryngii

Analysis of the secretome at 9 EXTRACELLULAR INTRACELLULAR and 15 days

1, 3, 5 days of bacterial culture

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Evaluating optimal depolymerization and sink

Cellulosic Bioprocessing

Pretreatment Biofuels& Bioproducts Sugars Cellulase Biomass Microbes Enzymes

Lignin Ligninolytic Enzymes or Higher pH or Low MW Temperature Aromatics

• Develop an effective lignin depolymerization approach that enables near complete conversion to co-products • Determine the optimal configuration of microbes and enzymes: multiple enzymes, multiple microbes, L-CBP 18 | Bioenergy Technologies Office

Relevance

Lignin valorization will be essential to achieve 2022 HC fuel cost targets Highlighted in 2011 Review/CTAB as a key gap in BC Platform Key MYPP areas for process improvement via lignin utilization: Hydrolytic enzyme production Key Stakeholders and Impacts: • Evaluating ligninolytic enzymes • Lignocellulosic biorefineries: TEA has shown that • Demonstrated effectiveness of refineries must valorize lignin to be competitive with fungal secretomes at neutral pH petroleum on DMR-EH substrate • Lignin-derived aromatics could be useful for the Catalyst efficiency fuels, chemical, and material science industries • Combined depolymerization and • Efficient lignin depolymerization is a major barrier to sink concept for lignin lignin utilization • Selective transformation of lignin to advanced fuels and chemicals • A successful outcome of this project will lead to technologies that can efficiently depolymerize lignin and simultaneously convert it into value-added Biochemical Conversion Process bioproducts via microbial cell factories Integration • Examining ability of biological catalysts to function on a process-relevant substrate

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Future Work

Lignin Depolymerization • Use omic analysis to understand microbial lignin depolymerization • Engineer microbial sinks to depolymerize lignin by expression of heterologous enzymes • Examine ability of microbial “sinks” to depolymerize DMR-EH alone or with fungal enzymes

Lignin Transport and Catabolism • Mine genomes of microbial “sinks” to identify catabolic pathways to leverage for products • Conduct metabolomics analysis with 13C labeled aromatics to identify intermediates • Identify genetic “parts” that can be used from metabolic engineering of microbes to produce bioproducts from lignin

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Summary

1) Approach: • Develop a biological approach to depolymerize solid lignin for upgrading of low MW aromatic compounds to co-products

2) Technical accomplishments • Examined purified enzymes and fungal-derived secretomes on several lignin substrates for ability to depolymerize lignin • Determined that reaction conditions and enzyme composition are key factors in promoting lignin depolymerization • Found microbial aromatic “sinks” may be critical for extensive lignin depolymerization

3) Relevance • TEA shows lignin valorization is critical for lignocellulosic biorefineries • Low molecular weigh aromatics have value in several industries

4) Critical success factors and challenges • Achieving high yields of low MW, upgradeable species • Minimizing lignin monomer repolymerization • Overcoming aromatic transport and catabolic rate limitations

5) Future work: • Increase efficiency of biological lignin depolymerization • Implement a microbial aromatic “sink” to prevent lignin repolymerization and produce valuable bioproducts

6) Technology transfer: • Generate IP around lignin depolymerization methodologies • Generate novel microbial lignin conversion strategies

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Acknowledgements

• Edward Baidoo • Eric Karp • Adam Bratis • Payal Khanna • Xiaowen Chen • Bill Michener • Tanmoy Dutta • Michael Resch External Collaborators • Rick Elander • Davinia Salvachua • Adam Guss, Oak Ridge National Laboratory • Mary Ann Franden • Blake Simmons • R. Robinson, E. Zink, EMSL Pacific Northwest • Chris Johnson • Melvin Tucker National Laboratory • David Johnson • Derek Vardon • John McGeehan, University of Portsmouth • Rui Katahira • Jerry Ståhlberg, Mats Sandgren, Swedish University of Agricultural Sciences

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Additional slides

• Publications • Acronyms • Additional Technical Accomplishment Slides

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Publications

Publications in review: 1. D. Salvachua et al., “Lignin Consolidated Bio-Processing: Simultaneous lignin depolymerization and co-product generation by bacteria”, in review.

Publications in print: 2. A. Ragauskas, G.T. Beckham, M.J. Biddy, R. Chandra, F. Chen, M.F. Davis, B.H. Davison, R.A. Dixon, P. Gilna, M. Keller, P. Langan, A.K. Naskar, J.N. Saddler, T.J. Tschaplinski, G.A. Tuskan, C.E. Wyman, “Lignin Valorization: Improving Lignin Processing in the Biorefinery”, Science (2014), 344, 1246843.

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Acronyms

• DDR-EH Lignin: Deacetylated, Disk-Refined, Enzymatically Hydrolyzed Lignin • DyP: Dye-decolorizing Peroxidase • LiP: Lignin Peroxidase • MnP: • MW: Molecular Weight • VP: Versatile Peroxidase

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Characterization of ligninolytic cocktails

To measure enzyme activities, we utilized UV- and colorimetric assays to follow substrate oxidation

Laccases: ABTS at pH 5 Reactive blue 19 at pH 3

Manganese Peroxidase (MnP):

ABTS + H2O2 at pH5 (also for DyP and VP) ABTS assay 2+ ABTS + H2O2 + Mn at pH 5 MnSO4 + H2O2 at pH 5

Lignin Peroxidase (LiP):

Veratryl alcohol + H2O2 at pH 3

Dye-decolorizing peroxidase (DyP):

Reactive Blue 19 + H2O2 at pH 3

Versatile peroxidase (VP): Reactive black 5 + H2O2 at pH 5 Reactive Blue 19 assay

Aryl-alcohol oxidase (AAO): Veratryl alcohol at pH 5

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Detailed characterization of lignin depolymerization

• In order to further develop depolymerization strategies and microbial aromatic “sinks” we must first have a detailed understanding of the DMR-EH lignin macromolecule and degradation products • Will employ a variety of analytical techniques to characterize the lignin, including mass spectrometry (MS), size exclusion chromatography (SEC), and nuclear magnetic resonance (NMR)

NMR- linkages MS- identification SEC- size

Green Chem. Nature Protocols 7, 1579–1589 (2012) , 2014, 16, 2713-2727 27 | Bioenergy Technologies Office

Biological Lignin Depolymerization: Fungi

• Laccases and peroxidases Enzymatic cocktail are the major enzymes Laccases Peroxidases involved in microbial lignin depolymerization Manganese Lignin Dye- Versatile O 2 (MnP) (LiP) decolorizing (VP) Other • Require O2 and H2O2 (DyP) oxidases respectively for activity Aryl- H O H2O2 2 alcohol (AAO) Approach: Free radicals • Characterized microbial degradation of DDR-EH lignin

• Identify parameters for optimal LIGNIN DEPOLYMERIZATION biological lignin degradation

FY14 Research: Start with Fungi • Use purified fungal enzymes to optimize conditions for lignin depolymerization • Examine the lignolytic enzyme cocktails produced by basidiomycete fungi to optimize conditions and identify the enzymes present in natural secretomes

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DMR-EH lignin depolymerization

Control Secretomes from:

Pleurotus eryngii Bjerkandera sp. Phellinus robustus 9000

8000 pH4.5

7000 pH7 60% decrease MW 6000 50% decrease MW

5000

4000 AS AS 3000 +2 +2 +2 H2O2 + Mn Mp= Mp= H2O2 + Mn H2O2 + Mn 2000 300 260

Molecular weight average (kDa) average weight Molecular 1000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Laccase* (U/g DMR) 0 - 0- 0- 0 100- 100- 28- 28- 400- 400 30- 30- 8- 8 Peroxidase* (U/g DMR) 0 - 0- 0- 0 0- 90 - 0- 0.1 0 - 7 -0.01 - 0.01

P. eryngii secretome is an effective lignin degrading enzyme cocktail • Obtained major difference between pH 4.5 and 7 in both initial MW and MW change • P. eryngii samples seem able to produce a substantial amount of low MW species • Selected P. eryngii secretome at pH 7 for more in-depth experimental studies 29 | Bioenergy Technologies Office

Treatment of DMR-EH lignin with the P. eryngii secretome

GPC chromatogram

Fungal ligninolytic enzymes are clearly depolymerizing lignin, however, as we can see at 4 days of incubation no more depolymerization is observed although the enzymes are still active. Is it due to repolymerization?

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Treatment of DMR-EH lignin with the P. eryngii secretome

Different enzyme dosages at pH 7 DDR-EH lignin (2%) P. eryngii secretome Time-course analysis (3, 11, 30 U laccase/g substrate) Autoclaved

GPC results: Mw and Mn are the values at which there are equal masses or number of molecules on each side, respectively.

0d 4d 4d - Inactivated enzymes 7000 2500 0d 4d 4d - Inactivated enzymes 6000 2000 5000 3 3U/g U/g ND 4000 1500 11 U7g ND 3000 11 U/g KDa KDa KDa KDa 1000 2000 3030 U/g U/g ND 500 1000

0 0 Control1 1 day2 2 day3 3 day4 4 day5 Control1 1 day2 2 day3 3 day4 4 day5

P. eryngii secretome shows rapid DDR-EH lignin depolymerization

• 50% of lignin species are smaller than the control (Mn) in a single day of treatment • Control experiments with boiled enzymes and without enzymes show no depolymerization • Analyzed detailed molecular weight distributions (next slide)

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Current Key Milestones and Findings

• Examined purified enzymes and fungal-derived secretomes on several lignin substrates for ability to depolymerize lignin • Determined that reaction conditions and enzyme composition are key factors in promoting lignin depolymerization • Found evidence that lignin repolymerization is an issue that must be addressed to achieve maximum lignin depolymerization • Repolymerization may be prevented using a microbial “sink” to remove low molecular weight lignin products form the reaction • Superseded our FY14 Go/No-Go of attaining 20% lignin depolymerization • For FY15, we have assembled a list of microbial “sinks” and initiated a detailed characterization of DMR-EH lignin

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Workflow

Down-select and Choose optimal method characterize lignin- to quantify lignin enriched substrate depolymerization

Challenges Screen Sufficient isolated Depolymerization enzymes lignin ? substrate

Sufficient Develop and Transport and incorporate Catabolism? microbial sink

Endogenous Exogenous (Secreted) Ligninolytic Ligninolytic Enzymes? Enzymes?

Co-Product Engineering 33 | Bioenergy Technologies Office

FY15 Milestone: Characterization of solid DMR-EH

Compositional analysis 2D-NMR (HSQC) Sample ID Content (%) Lignin side chain Carbohydrate region Ash 2.18 1 1 2 Lignin 66.0 6 2 6 5 3 5 3 O C H Glucan 9.24 4 4 3 O O Saturated Xylan 9.36 R R aliphatic region H G Galactan 1.04 1 Arabinan 1.62 6 2 5 3 Fructan 0.00 H 3C O 4 O C H 3 O Acetate 0.72 R Total sugar 21.26 S O O Total 90.2 O O Aromatic region b b a a Molecular weight distribution 1 1 6 2 6 2 5 3 5 3 O C H S 4 4 3 2/6 Mn Mw PD O O OCH3 R R G2 PCAb 2,000 10,000 5.0 FA PCA G5 + G6 (H3/5) FA ( H 3C O ) b Aa H O g (Pyridine) (b-O-4)

H O H Sugar a b O 4 2/6 1 O C H PCA 6 2 3 2/6 3 (Pyridine) S 17% Ab ( H 3C O ) 5 O C H 3 4 (b-O-4) O G 45%

PCAa+FAa H 2% A (b-O-4) (Pyridine) PCA 27% FA 9% C1 in sugar

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