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 improve bioethanol production processes 3. Potential of thermostable enzymes in cellulose hydrolysis 4. Other enzymes 5. Conclusions

GREENHOUSE GAS REDUCTIONS

Doornbosch & Steenblick, OECD, 2007

First and second generation biofuels

Raw materials
1. Generation: Sugars C6H12O6, C12H22O11 Starch (C6H10O5)n
Sugarcane Sugar beet Corn Wheat

Processes
(Hydrolysis) Fermentation Esterification

Products
1. Generation Bioethanol C2H5OH > 5 % gasoline ~0,5 /litre

1.

Generation: Fatty acids (C18H34O2)

1. Cracking

Generation

Methylester-diesel > 5 % diesel-mix ~0,7 /litre

Rapeseed Palm oil Jatropha Algae

2. Generation: cellulose, hemicellulose (C5H10O5)n, (C6H10O5)n

Straw Bagasse E-Crops Wood

Enzyme/acidhydrolysis, fermentation (2010 2015) Gasification (2010 2015) FisherTropsch

2. Generation Synthetic biodiesel CnH2n+2 Bioethanol, butanol etc..

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 Sorted municipal solid waste Herbaceous energy crops

Lignin 22 % Cellulose Hemicellulose 32 % 50 % HemiLignin 17 % cellulose Other 13 % 23 % Extractives 5%

Cellulose 38 %

Cellulose 45 % Ash 15 % Lignin 10 % Hemicellulose 9 % Other carbohydrates 9 % Protein 3 % Other 9 %

Cellulose 45 % Hemicellulose 30 % Lignin 15 % Other 10 %

Ref. Wyman, 1994

THE CHALLENGING RAW MATERIAL

Diameter of each tracheid is approximately 30 m (left), wood cell wall layers S1-S3: secondary cell wall layers, P: primary wall, M.L. middle lamella (middle) and lignincarbohydrate complex of the secondary cell wall (right)

Adapted from Kirk and Cullen (1998).

GENERAL OUTLINE OF THE LIGNOCELLULOSE-TOBIOETHANOL PROCESS

Simultaneous or separate saccharification and fermentation

Solid residue

Renewable lignocellulosic materials

Pretreatment Hydrolysis

Fermentation

Physical deconstruction and fractionation by refining, steam explosion Hydrolysis of cellulose Fermentation of sugars and hemicellulose (hexoses and or other methods by acid or enzymes pentoses) to ethanol by yeast or bacteria

Distillation or separation Fuels: Ethanol..

Concentration and separation of product

Courtecy of K. Reczey

IMPROVEMENT OF THE ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSE

Composition and accessibility of substrate
n n

Feedstock improvement (long term) Pretreatment and fractionation of cellulose, hemicellulose and lignin (short term)

Properties of cellulases
n n n

Specific activity End-product inhibition Stability Optimal cellulase mixtures Optimal accessory enzymes Efficient production of necessary components Separate/simultaneous, recycling of enzymes etc.

Composition and production of enzyme mixtures

n n n

Hydrolysis technologies
n

Main enzymes in lignocellulose hydrolysis

n Cellulases
n Endo--1.4-glucanases, cellobiohydrolases, -glucosidases n Fungal cellulases e.g. Trichoderma, Humicola, Acremonium n Bacterial cellulases e.g. Clostridium thermocellum

n Hemicellulases
n Backbone degrading enzymes n Enzymes removing the side groups n -xylosidases

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 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

Temperature

60

Total hydrolysis

Liquefaction

Liquefaction Bacterial SSF

50

Total hydrolysis

Sacchari fication SSF Fermentation

40

SSF
30

Fermentation Fermentation

DOWNSTREAM PROCESSING, DISTILLATION

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 ( ) Ta Cel7A ( ) T. reesei Cel7A ( ) Results:

Topt 65 oC for Ct Cel7A and Ta Cel7A, and 60 oC for At Cel7A and ~ 60 oC for Tr Cel7A Ct Cel7A clearly the most active cellobiohydrolase on soluble substrate(already at lower temperatures).

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

Hydrolysis of microcrystalline cellulose at 70 oC

2-module versions of the cellobiohydrolases
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, 22oC

Enzyme kcat (min-1) Km (M) CNPLac kcat/Km (min-1M1)

Ki (Glc2) (M)

Type

of

inhibition

Ct Cel7A Ta Cel7A At Cel7A Tr Cel7A

19 1 1.7 0.1 2.8 0.1 2.6 0.05

2000 200 990 70 2100 150 520 30

9.5 x 103 1.7 x 103 1.3x 103 5.0 x 103

39 14 107 14 141 25 19 4

comp. comp. comp. comp.

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

HYDROLYSIS OF STEAM PRETREATED SPRUCE

Thermostable enzymes (CBH, EG, -Glu, XYL): 9.8 FPU/g cellulose Reference enzymes (Celluclast + Novozym 188): 11.5 FPU/g
100

Hydrolysis (% of theor. maximum)

90 80 70 60 50 40 30 20 10 0 35C 45C 55C 60C 35C 45C 55C 60C

0h 24h 48h 72h

T. reesei enzymes

Thermostable enzymes

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

HEMICELLULOSES AND HEMICELLULASES

A MeGlcA Xyl Xyl Xyl

Ph Ara Xyl Xyl Xyl Ac Xyl Xyl Ara

B Gal Glc Xyl Xyl Man Man Glc Man Ac Ac

Man Man Glc Man Man Man

Xyl

Endoxylanase -Xylosidase

-Glucuronidase -Arabinosidase

Esterase

Endomannanase -Galactosidase

-Mannosidase -Glucosidase

Esterase

Ph = phenolic groups Ara = arabinose Xyl = xylose

Ac = acetyl MeGlcA = methyl glucuronic acid

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 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

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