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

7

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

8

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

14

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

15

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

18

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

22

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?