biological lignin depolymerization presentation for beto ......pleurotus ostreatus po lignin...

1 | Bioenergy Technologies Office eere.energy.gov

2015 DOE BioEnergy Technologies Office (BETO) Project Peer Review Date: March 25th, 2015 Technology Review Area: Biochemical Conversion Biological Lignin Depolymerization (WBS 2.3.2.100)

Principal Investigators: Gregg Beckham (NREL) John Gladden (SNL) Organizations: National Renewable Energy Laboratory and Sandia National Laboratory

2 | Bioenergy Technologies Office

Project Goal

Residual Biorefinery Lignin

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

Ligninolytic Enzymes

Aromatic-catabolizing bacteria


Quad Chart

• Start date: October 2014 • End date: September 2017 • Percent complete: ~30%

• Bt-F Hydrolytic Enzyme Production • Bt-I Catalyst Efficiency • Bt-J Biochemical Conversion Process

Integration

Timeline

Budget

Barriers

• BETO Projects: Pretreatment and Process Hydrolysis, Lignin Utilization, Biochemical Platform Analysis, Pilot Scale Integration, Enzyme Engineering and Optimization

• BETO-funded National Lab Projects: Oak Ridge National Laboratory (A. Guss)

• Office of Science funded efforts: Joint BioEnergy Institute, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory (R. Robinson, E. Zink)

• Academic collaborators: Swedish University of Agricultural Sciences, University of Portsmouth

Partners and Collaborators

FY14 Costs

Total Planned Funding

(FY15-Project End Date)

DOE funded $222,410 $1,752,590

Funds are split 50/50 between NREL and SNL


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

• Focus on residual solids from Deacetylation-Mechanical Refining Prt.

• Examine fungal and bacterial ligninolytic enzymes for solid lignin depolymerization

• Develop effective enzyme secretion systems

Lignin Upgrading • Develop aromatic metabolic map • Elucidate aromatic transport mechanisms • Understand rate limitations in aromatic

catabolism • Develop optimized modules for production

of value-added products


Technical Approach

Substrate: 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

Lignin-degrading Enzymes: Laccases & Peroxidases

Degraded lignin

Biomass DMR Pretreatment

Enzymatic Hydrolysis

Sugars for fuels

DDR-EH lignin

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


Management Approach

Management Approach: • Use quarterly milestones to track

progress and down-select options

• Milestone priority dictated by depolymerization and overcoming key risks in yields

• Phone calls every 3 weeks, site visits every 6 months

• Contributions from both partners on lignin analytical chemistry leveraging respective expertise

• Divide research in a manner that leverages each partners strengths

Critical Success Factors: • Employ tools of synthetic biology and

lignin analytical chemistry

• Leverage experience from two biomass conversion centers

• Strategic hire in biological lignin depolymerization

• Work with Biochemical Platform Analysis Project to identify optimal co-product targets

• Leverage input and collaborations from other BETO-funded projects in lignin


Technical Results – Outline

• 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. Solution: microbial “sink”

• For FY15, we have assembled a list of microbial “sinks” and initiated a detailed characterization of DMR-EH lignin

Depolymerization Repolymerization

Lignin

Co-products

Aromatic Catabolism

Strain Consumes Aromatics

Growth on Lignin

Amycolatopsis sp. Y Y

Pseudomonas fluorescens Y Y

Pseudomonas putida KT2440 Y Y

Etc… Y Y

ENZYMES


FY15 Milestone: Characterization of solid DMR-EH

Compositional analysis Molecular weight distribution

Sample ID Content (%) Ash 2.18

Lignin 66.0 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

Mn Mw PD

2,000 10,000 5.0

2D-NMR for lignin chemical composition (HSQC)

O O C H 3 H 3 C O

R

1 2 3

4 5 6

O O C H

R

1 2 3

4 5 6

O R

1 2 3

4 5 6

1 2 3

4 5 6

O O C H 3

R

O O

b a

O R

O O

1 2 3

4 5 6

b a

H

G

S

FA

PCA

Moiety Abundance S 17% G 45% H 2%

PCA 27% FA 9%


Microbial 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

Dye-decolorizing (DyP)

Peroxidases Laccases

Lignin (LiP) Manganese (MnP)

Aryl-alcohol (AAO)

Enzymatic cocktail

LIGNIN DEPOLYMERIZATION

H2O Free radicals

H2O2 O2

Versatile (VP)

Enzymatic assays


Enzyme Function Source Organism Name

Laccase Trametes versicolor Tv

Laccase Agaricus bisporus Ab

Laccase Rhus vernicifera Rv

Laccase Pleurotus ostreatus Po

Lignin

Peroxidase Unknown LiP

Top Enzymes

Green enzymes used in ligninolytic cocktails

Ligninolytic enzyme activity is a strong function

of temperature and pH

• Screened 9 commercial purified fungal laccases and peroxidases for ability to solubilize lignin

• Used active enzymes to optimize the reaction conditions and create cocktail

• pH and Temp are critical factors for depolymerization

• Optimal enzyme loads depend on pH, T

Optimizing Enzymatic Lignin Depolymerization

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

ugE/mgL 0.4 4 0.4 4

Temp 30°C 30°C

pH

Lign

in S

olu

bili

zati

on

(AB

S 2

80

nm

)

Ligninolytic Enzymes Depolymerize or Repolymerize Lignin Depending on Reaction

Conditions

Tv Lac

Po Lac

LiP

-30

-20

-10

0

10

20

30

40

50

60

1x 0.5x 1x 0.5x

20°C 30°C

DDR-EH Ch

ange

in L

ign

in S

olu

bili

ty (

%)

Solubilization of DMR-EH Lignin by Ligninolytic Cocktail

Enzyme Loadings are also very important!

Dep

oly

mer

ize

Rep

olym

erize


Screening of natural fungal secretomes

Screen with 12 white-rot fungi • Pleurotus ostreatus

• Irpex lacteus

• Panus tigrinus

• Bjerkandera adusta

• Bjerkandera sp. • Cerioporopsis subvermispora

• Pleurotus eryngii

• Phellinus robustus

• Polyporus alveolaris

• Stereum hirsutum

• Trametes versicolor

• Phanerochaete chrysosporium

Production of fungal secretomes in the presence of lignocellulose can induce and/or accelerate the production of ligninolytic and cellulolytic enzymes

Ligninolytic Enzyme induction on DDR-EH lignin

Production of ligninolytic cocktails ABTS assay

Reactive Blue 19 assay

Profile ligninolytic enzyme activities


• Identified organisms that produce the most activity of specific ligninolytic enyzmes

• P. eryngii shows highest lignin depolymerization, lowest MW pH 7

• Surpassed FY14 Go/No-Go • pH plays a major role in lignin

depolymerization extents

0

500

1000

1500

2000

0 5 10 15

mU

/mL

Days

ABTS

ABTS + H2O2

ABTS + H2O2+ Mn

9 days

0

500

1000

1500

2000

0 5 10 15

mU

/mL

Days

7 days

0

500

1000

1500

2000

0 5 10 15

mU

/mL

ABTS

ABTS + H2O2

ABTS + H2O2+ Mn

MnSO4

Veratryl alcohol

Veratryl alcohol+ H2O2

RB 19

Laccase x 10

Laccase

Peroxidase

MnP I

MnP II

AAO

LiP

DyP

Phellinus robustus

(laccase, MnP, DyP, AAO)

Pleurotus eryngii

(laccase, AAO, VP, MnP )

Days

Bjerkandera sp.

(MnPs)

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

pH4.5

pH7

H2O2 + Mn+2 H2O2 + Mn+2

Pleurotus eryngii Bjerkandera sp. Phellinus robustus

Secretomes from:

H2O2 + Mn+2

Control

Mo

lecu

lar

wei

ght

aver

age

(kD

a)

60% decrease MW 50% decrease

MW

Characterization of ligninolytic cocktails

0

500

1000

1500

2000

0 5 10 15

mU

/mL

Days

9 days


P. eryngii secretome depolymerizes DMR-EH Lignin

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

Depolymerization Repolymerization

Lignin

Need to create conditions

that promote Depolymerization

Number-averaged Molecular Weight Distribution upon

ligninolytic enzyme treatment at pH 7

0

500

1000

1500

2000

2500

1 2 3 4 5

3 U laccase/g DDR

11 U laccase/g DDR

30 U laccase/g DDR

No enzymes

1 day

2 days

3 days

4 days

Mn (

kDa)


One primary strategy going forward: microbial “sink”

Fungal Laccases & Peroxidases

Microbial “Sink”

Re-polymerization

Co-products

High MW Lignin

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

Aromatic Catabolism


Conditioning DMR-EH lignin for biological treatments

• 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

• 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

0.00

0.10

0.20

0.30

0.40

0.50

0.60

7 8 9 10 11 12

Gro

wth

(O

D600)

pH

Effect of pH on Growth on DMR-EH Lignin

5 8 14 22 24 31 33

Organism


L-CBP: Bacteria also depolymerize lignin Salvachua et

al., in review

ALKALINE PRETREATED LIQUOR FROM CORN STOVER L-CBP Lignin Consolidated

Bioprocessing

OO

HO

HO

OO

OO

High Mw Lignin

Lig

nin

De

po

lym

eriza

tio

n

Mo

no

me

rs fo

r C

ata

bo

lism

DDR-EH

lignin

For fuels, materials,

& chemicals

Aromatic Monomers

Laccases & Peroxidases

Intracellular Or

Extracellular Metabolites Production


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

EXTRACELLULAR INTRACELLULAR

BACTERIA: Pseudomonas putida KT2440

Acinetobacter ADP1 Amycolatopsis sp.

Rhodococcus jostii RHA1

1, 3, 5 days of bacterial culture

FUNGI Pleurotus eryngii

Analysis of the secretome at 9 and 15 days

Control P. putida


Biomass Cellulase Enzymes Microbes

Pretreatment

Cellulosic Bioprocessing

Sugars

Lignin Ligninolytic Enzymes

Low MW Aromatics

Biofuels& Bioproducts

Higher pH or Temperature

or

• 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

Evaluating optimal depolymerization and sink


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 • Evaluating ligninolytic enzymes • Demonstrated effectiveness of

fungal secretomes at neutral pH on DMR-EH substrate

Biochemical Conversion Process Integration • Examining ability of biological

catalysts to function on a process-relevant substrate

Key Stakeholders and Impacts:

• Lignocellulosic biorefineries: TEA has shown that refineries must valorize lignin to be competitive with petroleum

• Lignin-derived aromatics could be useful for the fuels, chemical, and material science industries

• Efficient lignin depolymerization is a major barrier to lignin utilization

• A successful outcome of this project will lead to technologies that can efficiently depolymerize lignin and simultaneously convert it into value-added bioproducts via microbial cell factories

Catalyst efficiency • Combined depolymerization and

sink concept for lignin • Selective transformation of lignin

to advanced fuels and chemicals

Relevance


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

Future Work


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

Summary


Acknowledgements

• Edward Baidoo • Adam Bratis • Xiaowen Chen • Tanmoy Dutta • Rick Elander • Mary Ann Franden • Chris Johnson • David Johnson • Rui Katahira

• Eric Karp • Payal Khanna • Bill Michener • Michael Resch • Davinia Salvachua • Blake Simmons • Melvin Tucker • Derek Vardon

External Collaborators • Adam Guss, Oak Ridge National Laboratory • R. Robinson, E. Zink, EMSL Pacific Northwest

National Laboratory • John McGeehan, University of Portsmouth • Jerry Ståhlberg, Mats Sandgren, Swedish

University of Agricultural Sciences


Additional slides

• Publications • Acronyms • Additional Technical Accomplishment Slides


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.


Acronyms

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


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 + H2O2 + Mn2+ 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

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

ABTS assay

Reactive Blue 19 assay


• 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

Nature Protocols 7, 1579–1589 (2012) Green Chem., 2014, 16, 2713-2727

MS- identification SEC- size

Detailed characterization of lignin depolymerization


Biological Lignin Depolymerization: Fungi

Dye-decolorizing (DyP)

Peroxidases Laccases

Lignin (LiP)

Manganese (MnP)

Aryl-alcohol (AAO)

Enzymatic cocktail

LIGNIN DEPOLYMERIZATION

H2O

Free radicals

H2O2

O2

Versatile (VP) Other

oxidases

• Laccases and peroxidases are the major enzymes involved in microbial lignin depolymerization

• Require O2 and H2O2 respectively for activity

Approach: • Characterized microbial degradation

of DDR-EH lignin • Identify parameters for optimal

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


Peroxidase* (U/g DMR)

DMR-EH lignin depolymerization

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

pH4.5

pH7

H2O2 + Mn+2 H2O2 + Mn+2

Pleurotus eryngii Bjerkandera sp. Phellinus robustus

Mp=300

Mp=260

AS

Secretomes from:

0 - 0- 0- 0 100- 100- 28- 28- 400- 400

0- 90 - 0- 0.1

30- 30- 8- 8

0 - 7 -0.01 - 0.01 0 - 0- 0- 0

H2O2 + Mn+2

Control M

ole

cula

r w

eig

ht

ave

rage

(kD

a)

AS

60% decrease MW

50% decrease MW

Laccase* (U/g DMR)

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


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?

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


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

P. eryngii secretome Different enzyme dosages at pH 7 (3, 11, 30 U laccase/g substrate)

DDR-EH lignin (2%) Autoclaved

Time-course analysis

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

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)

0

500

1000

1500

2000

2500

1 2 3 4 5

3 U/g ND

11 U7g ND

30 U/g ND

Control 1 day 2 day 3 day 4 day

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 Control 1 day 2 day 3 day 4 day

KD

a

KD

a

3 U/g

11 U/g

30 U/g

0d 4d 4d - Inactivated enzymes

0d 4d 4d - Inactivated enzymes


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


Workflow

Down-select and characterize lignin-enriched substrate

Choose optimal method to quantify lignin depolymerization

Screen isolated

enzymes lignin substrate

Develop and incorporate

microbial sink

Sufficient Depolymerization

?

Sufficient Transport and Catabolism?

Co-Product Engineering

Endogenous (Secreted)

Ligninolytic Enzymes?

Exogenous Ligninolytic Enzymes?

Challenges


FY15 Milestone: Characterization of solid DMR-EH

Compositional analysis

Molecular weight distribution

Sample ID Content (%) Ash 2.18

Lignin 66.0 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

Mn Mw PD

2,000 10,000 5.0

2D-NMR (HSQC)

Aromatic region

Lignin side chain Carbohydrate region

Saturated aliphatic region

C1 in sugar

PCAa+FAa

G2

S2/6

(H3/5) G5 + G6

(Pyridine)

(Pyridine)

(Pyridine)

PCAb

PCA2/6

H2/6

FAb Aa (b-O-4)

Ab (b-O-4)

OCH3

Suga

r

O O C H 3 H 3 C O

R

1 2 3

4 5 6

O O C H

R

1 2 3

4 5 6

O R

1 2 3

4 5 6

1 2 3

4 5

6

O O C H 3

R

O O

b a

O R

O O

1 2

3 4

5 6

b a

H O

O H O

O C H 3

O O C H 3

b 4 1

2 3

4 5

6

( H 3 C O )

( H 3 C O )

g

a

H G

S

FA PCA

A (b-O-4)

S 17%

G 45%

H 2%

PCA 27%

FA 9%

3

biological lignin depolymerization presentation for beto ......pleurotus ostreatus po lignin...

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