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Lignocellulosic enzymes Liisa Viikari University of Helsinki Department of Applied Chemistry and Microbiology US-EC
Lignocellulosic enzymes
Liisa Viikari
University of Helsinki
Department of Applied Chemistry and Microbiology
US-EC Task Force on Biotechnology Research
Biotechnology for the Development of Sustainable Bio-energy
San Francisco, US, 21-22 February 2007
OUTLINE OF THE PRESENTATION 1. Background: from first to second generation fuels 2. Approaches to

OUTLINE OF THE PRESENTATION

1. Background: from first to second generation fuels

2. Approaches to improve bioethanol production processes

3. Potential of thermostable enzymes in cellulose hydrolysis

4. Other enzymes

5. Conclusions

GREENHOUSE GAS REDUCTIONS Doornbosch & Steenblick, OECD, 2007

GREENHOUSE GAS REDUCTIONS

GREENHOUSE GAS REDUCTIONS Doornbosch & Steenblick, OECD, 2007

Doornbosch & Steenblick, OECD, 2007

Raw materials First and second generation biofuels Processes Products 1. Generation: Sugar- Sugar Corn Wheat

Raw materials

First and second generation biofuels

Processes

Products

1. Generation: Sugar- Sugar Corn Wheat cane beet
1. Generation:
Sugar-
Sugar
Corn
Wheat
cane
beet

Sugars C 6 H 12 O 6 , C 12 H 22 O 11 Starch (C 6 H 10 O 5 ) n

1. Generation Bioethanol C 2 H 5 OH (Hydrolysis) Fermentation > 5 % gasoline ~0,5
1. Generation
Bioethanol C 2 H 5 OH
(Hydrolysis)
Fermentation
> 5 % gasoline
~0,5 €/litre
Esterification
1.
Generation
Cracking
Methylester-diesel
> 5 % diesel-mix
~0,7 €/litre
Enzyme/acidhydrolysis, fermentation
(2010… 2015)
2. Generation
Synthetic biodiesel
C n H 2n+2
Gasification
(2010… 2015)
Fisher-
Tropsch
Bioethanol, butanol
etc
1. Generation: Fatty acids (C 18 H 34 O 2 ) Rapeseed Palm oil Jatropha
1. Generation:
Fatty acids
(C 18 H 34 O 2 )
Rapeseed Palm oil Jatropha Algae
2. Generation: cellulose, hemicellulose (C 5 H 10 O 5 ) n , (C 6
2. Generation:
cellulose, hemicellulose
(C 5 H 10 O 5 ) n , (C 6 H 10 O 5 ) n
Straw
Bagasse E-Crops
Wood
Lignocellulose as raw material Because of the resistant structure of cellulose and natural composite structures

Lignocellulose as raw material

Because of the resistant structure of cellulose and natural composite structures of lignocellulosics, efficient pretreatment technologies are needed prior to the enzymatic hydrolysis

Agricultural

residues

Wood residues

Agricultural residues W o o d r e s i d u e s Lignin 22
Lignin 22 % Cellulose 50 % Hemi- cellulose 23 %
Lignin
22 %
Cellulose
50 %
Hemi-
cellulose
23 %

Extractives

Cellulose 38 %

Hemicellulose 32 %

Lignin 17 %

Other 13 %

5 %

Ref. Wyman, 1994

Sorted municipal solid waste

Herbaceous energy crops

1994 Sorted municipal solid waste Herbaceous energy crops Cellulose 45 % Ash 15 % Lignin 10
Cellulose 45 % Ash 15 % Lignin 10 % Hemicellulose 9 % Other carbohydrates 9
Cellulose 45 %
Ash 15 %
Lignin 10 %
Hemicellulose 9 %
Other carbohydrates 9 %
Protein 3 %
Other 9 %

Cellulose 45 % Hemicellulose 30

Lignin 15 %

Other 10 %

THE CHALLENGING RAW MATERIAL Diameter of each tracheid is approximately 30 µm (left), wood cell

THE CHALLENGING RAW MATERIAL

Diameter of each tracheid is approximately 30 µm (left), wood cell wall layers S1-S3:

secondary cell wall layers, P: primary wall, M.L. middle lamella (middle) and lignin- carbohydrate complex of the secondary cell wall (right)

lamella (middle) and lignin- carbohydrate complex of the secondary cell wall (right) Adapted from Kirk and

Adapted from Kirk and Cullen (1998).

GENERAL OUTLINE OF THE LIGNOCELLULOSE-TO- BIOETHANOL PROCESS Simultaneous or separate saccharification and fermentation

GENERAL OUTLINE OF THE LIGNOCELLULOSE-TO- BIOETHANOL PROCESS

Simultaneous or separate saccharification and fermentation

Solid residue

Fermen- Hydrolysis Distillation tation or separation Fuels:
Fermen-
Hydrolysis
Distillation
tation
or
separation
Fuels:

Fermentation of sugars (hexoses and pentoses) to ethanol by yeast or bacteria

Ethanol

Concentration and separation of product

Renewable

lignocellulosic

materials

Pre-

treatment

product Renewable lignocellulosic materials Pre- treatment Physical deconstruction and fractionation by refining,
product Renewable lignocellulosic materials Pre- treatment Physical deconstruction and fractionation by refining,

Physical deconstruction and fractionation by refining, steam explosion or other methods

Hydrolysis of cellulose and hemicellulose by acid or enzymes

Courtecy of K. Reczey

IMPROVEMENT OF THE ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSE Composition and accessibility of substrate n Feedstock

IMPROVEMENT OF THE ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSE

Composition and accessibility of substrate n Feedstock improvement (long term) n Pretreatment and fractionation of
Composition and accessibility of substrate
n
Feedstock improvement (long term)
n
Pretreatment and fractionation of cellulose, hemicellulose and
lignin (short term)
Properties of cellulases
n
Specific activity
n
End-product inhibition
n
Stability
Composition and production of enzyme mixtures
n
Optimal cellulase mixtures
n
Optimal accessory enzymes
n
Efficient production of necessary components
Hydrolysis technologies
n Separate/simultaneous, recycling of enzymes etc.
Main enzymes in lignocellulose hydrolysis n Cellulases n Endo-b-1.4-glucanases, cellobiohydrolases, b-glucosidases n

Main enzymes in lignocellulose hydrolysis

n Cellulases n Endo-b-1.4-glucanases, cellobiohydrolases, b-glucosidases n Fungal cellulases e.g. Trichoderma,
n
Cellulases
n
Endo-b-1.4-glucanases, cellobiohydrolases, b-glucosidases
n
Fungal cellulases e.g. Trichoderma, Humicola, Acremonium
n
Bacterial cellulases e.g. Clostridium thermocellum
n
Hemicellulases
n
Backbone degrading enzymes
Enzymes removing the side groups
n b-xylosidases
n
n
Lignin modifying enzymes?
n
Laccases, peroxidases
n
Enzymes hydrolyzing lignin-carbohydrate complexes?
n
Other helper enzymes/proteins?
n Swollenin
POTENTIAL ADVANTAGES OF THERMOSTABLE ENZYMES IN LIGNOCELLULOSE HYDROLYSIS Higher specific activity, i.e. decreased enzyme

POTENTIAL ADVANTAGES OF THERMOSTABLE

ENZYMES IN LIGNOCELLULOSE HYDROLYSIS

Higher specific activity, i.e. decreased enzyme loading Higher stability; i.e. extended life-time, reuse of enzymes
Higher specific activity, i.e. decreased enzyme loading
Higher stability; i.e. extended life-time, reuse of enzymes
Allow more flexibility for process configuration
Allow process with improved integration in terms of heat recovery
and recycling of process streams
When expressed in plants, allow more flexible processing
Allow increased dry matter content due to lower viscosity at high
temperature
BIOETHANOL PRODUCTION CONCEPTS with various options in relation to process temperature 200— PRETREATMENT 70 —

BIOETHANOL PRODUCTION CONCEPTS with various options in relation to process temperature

200— PRETREATMENT 70 — Total 60 — Liquefaction Liquefaction Bacterial SSF hydrolysis 50 — Total
200—
PRETREATMENT
70
Total
60
Liquefaction
Liquefaction
Bacterial SSF
hydrolysis
50
Total
Sacchari-
hydrolysis
fication
40
SSF
Fermentation
Fermentation
SSF
Fermentation
30
DOWNSTREAM PROCESSING, DISTILLATION
Temperature

Viikari et al. (2007) Advances in Biochemical Engineering Biotechnology 108, 121-145

Thermostable enzymes Methylumbelliferyl lactoside (MULac) used as a substrate At Cel7A ( ) Ct Cel7A

Thermostable enzymes

Methylumbelliferyl lactoside (MULac) used as a substrate

At Cel7A ( ) Ct Cel7A ( ) Ta Cel7A ( ) T. reesei Cel7A
At Cel7A ( )
Ct Cel7A ( )
Ta Cel7A ( )
T. reesei Cel7A ( )

Voutilainen et al. (2008) Biotech. Bioeng. (in press)

Results:

•T opt 65 o C for Ct Cel7A and Ta Cel7A, and 60 o C for At Cel7A and ~ 60 o C for Tr Cel7A •Ct Cel7A clearly the most active cellobiohydrolase on soluble substrate(already at lower temperatures).

Hydrolysis of microcrystalline cellulose at 70 o C

2-module versions of the cellobiohydrolases

at 70 o C 2-module versions of the cellobiohydrolases At Cel7A ( ) Ct Cel7A (
at 70 o C 2-module versions of the cellobiohydrolases At Cel7A ( ) Ct Cel7A (
at 70 o C 2-module versions of the cellobiohydrolases At Cel7A ( ) Ct Cel7A (

At Cel7A ( ) Ct Cel7A ( ) Ta Cel7A + Ct CBM ( ) Ta Cel7A + Tr CBM ( ) T. reesei Cel7A )

Results:

Ta Cel7A + Tr CBM the most efficient enzyme

The time-course of Avicel hydrolysis was followed for 24 hours by

measuring soluble reducing sugars.

Voutilainen et al. (2008) Biotech. Bioeng. (in press)

Kinetic constants and cellobiose inhibition , soluble model substrate, 22 o C Enzyme   CNPLac

Kinetic constants and cellobiose

inhibition, soluble model substrate, 22 o C

Enzyme

 

CNPLac

 
 

k

cat

K

m

k

cat /K m

K i (Glc 2 ) (µM)

Type

of

(min -1 )

(µM)

(min -1 M -

inhibition

1

)

 

Ct Cel7A

19 ±1

2000

±200

9.5

x 10 3

39 ±14

comp.

Ta Cel7A

1.7

±0.1

990

±70

1.7

x 10 3

107

±14

comp.

At Cel7A

2.8

±0.1

2100

±150

1.3x 10 3

141

±25

comp.

Tr Cel7A

2.6

±0.05

520

±30

5.0

x 10 3

19± 4

comp.

Voutilainen et al. (2008) Biotech. Bioeng. (in press)

HYDROLYSIS OF STEAM PRETREATED SPRUCE • Thermostable enzymes (CBH, EG, b -Glu, XYL): 9.8 FPU/g

HYDROLYSIS OF STEAM PRETREATED SPRUCE

Thermostable enzymes (CBH, EG, b-Glu, XYL): 9.8 FPU/g cellulose Reference enzymes (Celluclast + Novozym 188): 11.5 FPU/g

100 90 80 70 60 50 40 30 20 10 0 35°C 45°C 55°C 60°C
100
90
80
70
60
50
40
30
20
10
0
35°C
45°C
55°C
60°C
35°C
45°C
55°C
60°C
T. reesei enzymes
Thermostable
enzymes
Hydrolysis
(% of theor. maximum)

0h24h 48h 72h

24h0h 48h 72h

48h0h 24h 72h

72h0h 24h 48h

Viikari et al. (2007) Advances in Biochemical Engineering Biotechnology 108, 121-145

HEMICELLULOSES AND HEMICELLULASESA Ph MeGlcA Ara Ac Ara Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl

A Ph MeGlcA Ara Ac Ara Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
A
Ph
MeGlcA
Ara
Ac
Ara
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Xyl
Endoxylanase
a-Glucuronidase
Esterase
B--Xylosidase
a-Arabinosidase
Ph
= phenolic groups
Ac
= acetyl
Ara =
arabinose
Xyl = xylose
MeGlcA = methyl
glucuronic acid
B Gal Ac Ac Glc Man Man Glc Man Man Man Man Man Glc Man
B
Gal
Ac
Ac
Glc
Man
Man
Glc
Man
Man
Man
Man
Man
Glc
Man
Endomannanase
b-Mannosidase
Esterase
a -Galactosidase
b-Glucosidase
Gal = Galactose
Man = Mannose
Glc = Glucose
Ac
= Acetyl

Hemicellulases are essential components in efficient LC enzyme mixtures

The need for accessory enzymes depends on the substrate & pretreatment used

CONCLUSIONS: IMPROVED LIGNOCELLULOSE ENZYMES Feed stock improvement n Improved raw materials & pretreatments for

CONCLUSIONS: IMPROVED LIGNOCELLULOSE ENZYMES

Feed stock improvement

n

Improved raw materials & pretreatments for better hydrolyzability

n

Modified carbohydrate/lignin structures

n Expression of LC enzymes in plants Cellulases & other enzymes

n

Short & long term challenges for enzyme development

n

Thermostability a generally useful parameter

n

Integrated hydrolysis technologies

for enzyme development n Thermostability a generally useful parameter n Integrated hydrolysis technologies
Acknowledgements: Financial support European Union, TIME project (ENK6-CT-2002-00604) The Academy of Finland VTT Mati

Acknowledgements:

Financial support European Union, TIME project (ENK6-CT-2002-00604) The Academy of Finland VTT Mati Siika-aho Anu
Financial support
European Union, TIME project (ENK6-CT-2002-00604)
The Academy of Finland
VTT
Mati Siika-aho
Anu Koivula
Sanni Voutilainen
ROAL
Jari Vehmaanperä
Terhi Puranen
Marika Alapuranen
TIME partners, especially
Guido Zacchi, LU, Sweden
Francesco Zimbardi, ENEA, Italy
Kati Reczey, BUTE, Hungary