lignin depolymerization by fungal secretomes is … · ghs and/or esterases might enhance the...
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1
LIGNIN DEPOLYMERIZATION BY FUNGAL SECRETOMES
IS ENHANCED WITH A BACTERIAL SINK
Davinia Salvachúa 11/11/16
Frontiers in Biorefining, Georgia
2
Background - Lignin in a biorefinery, TEA, and LCA.
- Challenges doing biology with lignin
- Production of adipic acid from aromatic compounds
- Biological lignin depolymerization: fungi, bacteria, and enzymes
Lignin depolymerization by fungal secretomes is enhanced with a
bacterial sink
Conclusions
OUTLINE
3
1. Introduction
Abengoa, Babilafuente (Spain)
4
4
TEA and LCA scenarios for lignin valorization
Addition of lignin process
equipment increases MFSP from
$4.99 to $5.43/GGE
Plot is based on % lignin
conversion, of 80% solubilized
upstream
5
Lignin recovery in a biorefinery
Deacetylation
Separations/ Cat.
Upgrading
NaOH
Solid
Liquor
C6/C5-derived
HC FUELS
Mechanical
Refining Enzyme
hydrolysis
Biological
conversion
LIGNIN
LIGNIN
Lignin recovery before carbohydrate conversion
Lignin recovery after carbohydrate conversion
Biomass
DMR-EH lignin Black liquor
Corn stover DMR-EH
(%) (%)
Lignin 14.80 60.00
Ash 2.54 2.18
Glucan 36.48 9.24
Xylan 30.18 9.36
Galactan 1.76 1.04
Arabinan 3.52 1.62
Acetate 2.71 0.72
Proteins NA 6.07
Total mass balance 91.99 90.23 Salvachúa et al, Green Chemistry, 2016
DMR-EH lignin present native-lignin like
properties DMR pretreatment generates a highly digestible substrate, enabling effective sugar recovery.
Lignin content is up to 60 % and its average molecular weight is 9,200 Da.
6
Challenges working with lignin: from a biologist´s point of view
Challenges
1- Biological depolymerization:
by bacteria and fungi
2- Highly heterogeneous
• Monomeric, oligomeric, and polymeric lignin.
• Diverse product slate
• Lignin from different
pretreatments is not the same
1- Highly Recalcitrant
• Difficult to depolymerize • Insolubility
Approaches
3- Toxic High phenolic content
2- Biological funneling enables metabolism of
heterogeneous aromatics
3- Fermentation development Use of very robust bacteria
TCA CYCLE
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MUCONATE Titer: 40 g/L
Yield: 100 %
Productivity: 0.5 g/L/h
BIO-NYLON
Separations
Purification
Fermentation with Production of muconate from benzoic, p-coumaric, ferulic acid
Purity: 99.8%
ADIPIC ACID
What we can do with aromatic compounds
Precursor for nylon-6,6 (85% of its use)
The most commercially important dicarboxylic acid
Market volume: 2.6 million tons/year
engineered P. putida LIGNIN
(aromatic
compounds)
Polymerization Catalysis
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Lignina
Lignina
Lignina
Lignina
Lignina
Lacasas
PeroxidasasLacasas
Peroxidasas
Lacasas
Peroxidasas
AAO
AAO?
Lignina
Lignina
AAD
QR
Repolimerización
Martínez et al. (2005)
Peroxidases (MnP, LiP, VP, DyP…)
Laccases
Oxidases (AAO, Glyoxal oxidase…)
Reductases (AAD, QR…)
Biological lignin depolymerization
• Best organisms depolymerizing lignin • Non-genetically tractable
1- White-rot FUNGI
2- BACTERIA
3- ENZYMES from white-rot fungi
• Poor lignin degraders • Genetically tractable • Production of value added-compounds
from aromatic compounds
4- ENZYMES + BACTERIA
• Fungal enzymes + bacteria • Recombinant bacteria secreting
ligninolytic enzymes
3- ENZYMES from white-rot fungi
4- ENZYMES + BACTERIA
9
Alkyl-aryl Phenolics (20%)
Peroxidases Laccases + mediator
Peroxidases Laccases
C-Cβ
Alkyl-aryl
Non- Phenolics (80%)
Laccases and peroxidases generate radicals that can further initiate alkyl-aryl cleavage, C-Cβ, cross linking, demethoxylation, and ring cleavage of aromatic compounds.
Lignin breakdown by ligninolytic enzymes
COOH COOH ●
Cross linking of low molecular weight
10
2- Lignin depolymerization by fungal enzymes is
enhance with a bacterial sink
11
Repolymerization of LMW lignin
Depolymerization of HMW lignin DMR-EH LIGNIN
VALUE-ADDED COMPOUNDS FROM
LIGNIN
“Microbial sink” for LMW species
Fungal oxidoreductases
DMR pretreatment
EH
Sugar conversion
CORN STOVER
Solids Liquor
OH
OHO
Polymerization vs. repolymerization
- Polymerization of low molecular weight lignin is “unavoidable” when pure laccases and peroxidases are utilized
- Other enzymes present in secretomes might avoid repolymerization (e.g. AAO or VAO reduce aromatic radicals).
- Radical scavenger: bacteria
Salvachúa et al, Green Chemistry, 2016
12
12 secretomes from white-rot fungi were
screened while growing in the presence of DMR-EH
Pleurotus eryngii was the fungi producing the highest laccase levels
Bjerkandera sp. was the fungi producing more peroxidases
9 days- secretomes were used for depolymerization studies
Enzyme screening in fungal cocktails
0
2500
5000
7500
10000
0 3 6 9 12 15
Pleurotus eryngii
0
500
1000
1500
2000
0 3 6 9 12 15
Phellinus robustus
0
500
1000
1500
2000
0 3 6 9 12 15
Bjerkandera sp.
0
500
1000
1500
2000
0 3 6 9 12 15
Panus tigrinus
0
500
1000
1500
2000
0 3 6 9 12 15
Trametes versicolor
0
500
1000
1500
2000
0 3 6 9 12 15
Pleurotus ostreatus
Time (days)
Time (days)
Time (days)
Time (days)
Time (days)
Time (days)
Act
ivit
y (m
U/m
L)A
ctiv
ity
(mU
/mL)
Act
ivit
y (m
U/m
L)
E
DC
F
0
2000
4000
6000
8000
10000
0 3 6 9 12 15
Laccase Peroxidase I Peroxidase II
Mn-Peroxidase Aryl-alcohol oxidase Lignin peroxidase
A BMnP, VP AAO LiP
VP, DyP VP, DyP, MnP
0
50
100
150
200
0 3 6 9 12 15
0
50
100
150
200
0 3 6 9 12 15
0
2000
4000
6000
8000
10000
0 3 6 9 12 15
Pleurotus eryngii
0
500
1000
1500
2000
0 3 6 9 12 15
Bjerkandera sp.
mU
/mL
mU
/mL
Salvachúa et al, Green Chemistry, 2016
13
GPC: Effect of pH and enzyme dosage on lignin MW
Laccases vs. peroxidases
P. eryngii secretome pH 4.5 P. eryngii secretome pH 7
Bjerkandera sp. secretome pH 4.5 P. eryngii + Bjerkandera sp. secretome pH 4.5
Apparent MW (Da) Apparent MW (Da)
0
20
40
60
80
100
100 1000 10000 100000 1000000
Control pH4.5
LA 10 U/g
LA 50 U/g
LA 100 U/g
Lac 3d 100 U/g
0
20
40
60
80
100
100 1000 10000 100000 1000000
Series1
Series2
Series3
Series4
3d
Control pH 4.5
LA 10 U/g
LA 50 U/g
LA 100 U/g
LA 100 U/g*
Control pH 7
LA 3 U/g
LA 15 U/g
LA 30 U/g
LA 30 U/g*
0
20
40
60
80
100
100 1000 10000 100000 1000000
Series1
Series2
Series3
Series4
3d
0
20
40
60
80
100
100 1000 10000 100000 1000000
Series1
Series2
Series3
Series4
3d
Control pH 4.5
PE 8 U/g
PE 40 U/g
PE 80 U/g
PE 80 U/g*
Control pH 4.5
LA 5 U/g + PE 4 U/g
LA 25 U/g + PE 20 U/g
LA 50 U/g + PE 40 U/g
LA 50 U/g + PE 40 U/g*
No
rmal
ize
d r
esp
on
se (
%)
No
rmal
ize
d r
esp
on
se (
%)
Control pH 4.5
LA 10 U/g
LA 50 U/g
LA 100 U/g
LA 100 U/g*
Control pH 7
LA 3 U/g
LA 15 U/g
LA 30 U/g
LA 30 U/g*
Control pH 4.5
PE 8 U/g
PE 40 U/g
PE 80 U/g
PE 80 U/g*
Control pH 4.5
LA 5 U/g + PE 4 U/g
LA 25 U/g + PE 20 U/g
LA 50 U/g + PE 40 U/g
LA 50 U/g + PE 40 U/g*
Mw
12,000
10,000
8,800
3,500
2,000
Mw
12,000
15,000
11,000
5,600
11,000
Mw
12,000
9,600
7,700
5,600
5,400
Mw
6,100
7,400
5,600
3,200
2,300
Salvachúa et al, Green Chemistry, 2016
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Depolymerization with P. eryngii secretome
GPC: Effect of incubation time at pH7 and enzyme dosage on lignin MW
2000
3000
4000
5000
6000
7000
Days
MW
(D
a)
0
Control 3 U
LA/g
10 U LA/g
30 U LA/g
*
0
20
40
60
80
100
100 1000 10000 100000
Log MW (Da)
3 U LA/g CTL
1 d, 3 U/g
2d, 3 U/g
3d, 3 U/g
4 d, 3 U/g
0
20
40
60
80
100
100 1000 10000 100000
Log MW (Da)
30 U LA/g
LMW lignin
HMW
lignin
CTL
1d
2d
3d
4d
No
rma
lize
d r
esp
on
se (
%)
No
rma
lize
d r
esp
on
se (
%) Apparent MW (Da)
Apparent MW (Da)
Repolymerization occurs after 3 days of incubation in the studied conditions and all the enzyme dosages
Dialyzed and non dialyzed secretomes were
compared. No significant differences were found.
Control with/without boiled enzymes presented same trends.
Salvachúa et al, Green Chemistry, 2016
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Salvachúa et al, Green Chemistry, 2016
• The peak intensities of C6 in cellulose slightly decreased for longer treatments as well as for anomeric peaks in cellulose and xylan.
• Carbohydrates in DMR-EH lignin might be slightly depolymerized by the fungal secretome.
• C and Cβ peaks, from the β-O-4 structure, decreased in 2 and 3 days, but the C peak intensity did not change in 4 days.
• The methoxyl peak (OCH3) also decreased, indicating that demethoxylation occurred, likely
catalyzed by laccase.
Control 3 days 4 days
Rui Katahira
Depolymerization with P. eryngii secretome
Lignin peaks
Carbohydrate peaks
16
Salvachúa et al, Green Chemistry, 2016
Expanded HSQC spectra in the aromatic region
Depolymerization with P. eryngii secretome
Control 3 days 4 days
• Peaks of S2/6 and G2/5/6 decreased in 3 days, but slightly increased at 4 days
• The peak for PCA2/6 decreased somewhat at 2 days, not at 3 and 4 days
• FA peak decreased considerably at 2, 3, and 4 days.
Repolymerized portion
17
Evaluating bacterial sink hypothesis N
orm
aliz
ed
re
spo
nse
(%
)
0
20
40
60
80
100
120
100 1000 10000 100000
Apparent MW (Da)
5888-56-1
5888-56-5
5888-56-3
5888-56-8
DMR
DMR+S
DMR+B
DMR+S+B
Insoluble fraction
0
2000
4000
6000
8000
10000
1 2 3 4 5 6 7 8
DMR
DMR
+S+B
DMR
+B
DMR
+S
DMR
DMR
+S+B
DMR
+B
DMR
+S
Soluble fraction Insoluble fraction
DM
R-E
H li
gnin
Mw
(D
a)
Treatments
Bacteria enhances depolymerization of insoluble
lignin Lignin molecular weight increased in the treatment with secretome and bacteria likely due to the consumption of soluble and low molecular weight lignin.
Salvachúa et al, Green Chemistry, 2016
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Bacterial sink: aromatic compounds
0.0
0.5
1.0
1.5
2.0
2.5L
L+S
L+B
L+B+S
Aro
ma
tic
s (g
/L)
DMR
DMR+S
DMR+B
DMR+S+B
Soluble fraction
p-coumarate Ferulate
4-Hydroxybenzoate 4-hydroxybenzaldehyde
Protocatechuate
Vanillin/vanillic acid
Bacteria utilize low molecular weight lignin Vanillin appears duringthe bacterial treatment
4-hydroxybenzaldehyde is not polymerized by the enzymes and utilized by the bacteria.
COOH COOH ●
p-coumarate
Ferulate
4-hydroxybenzaldehyde
4-Hydroxybenzoate
Repolymerization
Salvachúa et al, Green Chemistry, 2016
19
Looking for the “perfect” enzyme cocktail
38%
23%
11%
5%
14%
5% 4% Oxidoreductases
Glycoside hydrolyses
Proteinases
Esterases
Other function*
Hypothetical
intracellular*
41%
23%
14%
6%
5% 4% 4%
2% 1%
GMC oxidoreductases
Laccase
Aryl-alcohol oxidases
Copper radical oxidases
Other oxidases
DyP-type peroxidases
FAD, FMN-linked oxidoreductases
Glyoxal oxidases
Aldo/keto reductases
P. eryngii secretome: Functional classification
P. eryngii secretome: Oxidoreductases
Biorefinery lignin streams not only contain lignin and exhibit different properties
GHs and/or esterases might enhance the utilization of lignin streams by bacteria
GMC oxidoreductases and laccases are the main enzymes involved in DMR-EH lignin depolymerization
by P. eryngii
Lignin depolymerization in Nature is a very complex process… from enzymes to organisms
Salvachúa et al, Green Chemistry, 2016
20
Conclusions
• Fungal enzymes depolymerize solid and high molecular weight lignin
• Bacteria enhance that depolymerization
• Although depolymerization is significant, solubilization of the solid fraction
is not highly improved.
• A lignin “pre-treatment” would be needed in solid lignin to enhance
lignin solubilization yields for further biological upgrading
21
Acknowledgements
Gregg T. Beckham
Rui Katahira
Nicholas S. Cleveland
Payal Khanna
Michael G. Resch
Brenna A. Black
Christopher Johnson
Derek R. Vardon
Eric M. Karp
Samuel O. Purvine
Erika M. Zink
Scott Baker
Alicia Prieto
María J. Martínez
Angel T. Martínez
Blake A. Simmons
John M. Gladden
Salvachúa et al, Green Chemistry, 2016
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I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin I´m not going to burn lignin
THANKS! QUESTIONS?