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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
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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
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3 | Bioenergy Technologies Office
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
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4 | Bioenergy Technologies Office
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
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5 | Bioenergy Technologies Office
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
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6 | Bioenergy Technologies Office
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
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7 | Bioenergy Technologies Office
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
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8 | Bioenergy Technologies Office
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%
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9 | Bioenergy Technologies Office
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
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10 | Bioenergy Technologies Office
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
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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
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12 | Bioenergy Technologies Office
• 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
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13 | Bioenergy Technologies Office
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)
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14 | Bioenergy Technologies Office
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
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15 | Bioenergy Technologies Office
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
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16 | Bioenergy Technologies Office
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
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17 | Bioenergy Technologies Office
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
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18 | Bioenergy Technologies Office
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
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19 | Bioenergy Technologies Office
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
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20 | Bioenergy Technologies Office
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
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21 | Bioenergy Technologies Office
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
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22 | Bioenergy Technologies Office
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
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Additional slides
• Publications • Acronyms • Additional Technical Accomplishment Slides
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24 | Bioenergy Technologies Office
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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25 | Bioenergy Technologies Office
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
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26 | Bioenergy Technologies Office
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 + Mn
2+ 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
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27 | Bioenergy Technologies Office
• 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
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28 | Bioenergy Technologies Office
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
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29 | Bioenergy Technologies Office
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
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30 | Bioenergy Technologies Office
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
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31 | Bioenergy Technologies Office
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
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32 | Bioenergy Technologies Office
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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33 | Bioenergy Technologies Office
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
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34 | Bioenergy Technologies Office
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 DepolymerizationProject GoalQuad ChartProject OverviewTechnical ApproachManagement ApproachTechnical Results – OutlineRelevanceFuture WorkSummaryAcknowledgementsAdditional slides