Lignocellulosic enzymes

Liisa ViikariUniversity of HelsinkiDepartment of Applied Chemistry and Microbiology

US­EC Task Force on Biotechnology ResearchBiotechnology for the Development of Sustainable Bio­energySan Francisco, US, 21­22 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

Processes ProductsRaw materials

1. Generation

Methylester­diesel

• > 5 % diesel­mix• ~0,7 €/litre

(Hydrolysis)Fermentation

Esterification

Bioethanol C2H5OH

• > 5 % gasoline• ~0,5 €/litre

1. Generation

Synthetic biodieselCnH2n+2

Bioethanol, butanoletc..

2. Generation

Cracking

Enzyme/acidhydrolysis, fermentation(2010… 2015)

Gasification(2010… 2015)

Fisher­Tropsch

2. Generation:cellulose, hemicellulose

(C5H10O5)n, (C6H10O5)n

BagasseStraw WoodE­Crops

1. Generation:Fatty acids

(C18H34O2)Palm oilRapeseed Jatropha

1. Generation:Sugars C6H12O6, C12H22O11

Starch (C6H10O5)n

Sugarbeet

Sugar­cane

WheatCorn

First and second generation biofuels

Algae

Lignocellulose as raw materialBecause of the resistant structure of cellulose and natural

composite structures of lignocellulosics, efficientpretreatment technologies are needed prior to the

enzymatic hydrolysis

Cellulose 38 %Hemicellulose 32 %

Lignin 17 %

Other 13 %

Cellulose50 %Hemi­

cellulose23 %

Lignin22 %

Extractives5 %

Agriculturalresidues

Wood residues Sorted municipalsolid waste

Herbaceousenergy crops

Cellulose 45 %

Hemicellulose 9 %Lignin 10 %

Other carbohydrates 9 %

Ash 15 %

Protein 3 %Other 9 %

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

Ref. Wyman, 1994

Adapted from Kirk and Cullen (1998).

THE CHALLENGING RAW MATERIALDiameter of each tracheid is approximately 30 µm (left), wood cell wall layers S1­S3:secondary cell wall layers, P: primary wall, M.L. middle lamella (middle) and lignin­carbohydrate complex of the secondary cell wall (right)

Simultaneous or separatesaccharificationand fermentation

Fermen­tation Distillation

orseparation Fuels:

Ethanol..

Renewablelignocellulosicmaterials

Hydrolysis

Pre­treatment

Solid residue

GENERAL OUTLINE OF THE LIGNOCELLULOSE­TO­

BIOETHANOL PROCESS

Physical deconstructionand fractionation byrefining, steam explosionor other methods

Hydrolysis of celluloseand hemicelluloseby acid or enzymes

Fermentation of sugars(hexoses andpentoses) to ethanol byyeast or bacteria

Concentration andseparation ofproduct

Courtecy of K. Reczey

IMPROVEMENT OF THE ENZYMATICHYDROLYSIS OF LIGNOCELLULOSE

Composition and accessibility of substraten Feedstock improvement (long term)n Pretreatment and fractionation of cellulose, hemicellulose and

lignin (short term)Properties of cellulases

n Specific activityn End­product inhibitionn Stability

Composition and production of enzyme mixturesn Optimal cellulase mixturesn Optimal accessory enzymesn Efficient production of necessary components

Hydrolysis technologiesn Separate/simultaneous, recycling of enzymes etc.

Main enzymes in lignocellulose hydrolysis

n Cellulasesn Endo­β­1.4­glucanases, cellobiohydrolases, β­glucosidasesn Fungal cellulases e.g. Trichoderma, Humicola, Acremoniumn Bacterial cellulases e.g. Clostridium thermocellum

n Hemicellulasesn Backbone degrading enzymesn Enzymes removing the side groupsn β­xylosidases

n Lignin modifying enzymes?n Laccases, peroxidasesn Enzymes hydrolyzing lignin­carbohydrate 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 life­time, reuse of enzymes

• Allow more flexibility for process configuration

• Allow process with improved integration in terms of heat  recoveryand recycling of process streams

• When expressed in plants, allow more flexible processing

• Allow increased dry matter content due to lower viscosity at hightemperature

BIOETHANOL PRODUCTION CONCEPTSwith various options in relation to process temperature

30 —

40 —

50 —

70 —

Tem

pera

ture

200 —

60 —

Sacchari­fication

DOWNSTREAM PROCESSING, DISTILLATION

SSF FermentationSSFFermentation

Bacterial SSFLiquefactionTotal

hydrolysis Liquefaction

Fermentation

PRETREATMENT

Totalhydrolysis

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

Thermostable enzymes

Methylumbelliferyl lactoside (MULac) used as a substrate

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

•Topt 65 oC for Ct Cel7Aand Ta Cel7A, and  60 oCfor At Cel7A and ~ 60 oC forTr Cel7A•Ct Cel7A clearly the mostactive cellobiohydrolase onsoluble substrate(already atlower temperatures).

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

Hydrolysis of microcrystalline cellulose at 70 oC2­module versions of the cellobiohydrolases

The time­course of Avicel hydrolysis was followed for 24 hours bymeasuring soluble reducing sugars.

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

Results:•Ta Cel7A + Tr CBM  themost efficient enzyme

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

Kinetic constants and cellobioseinhibition, soluble model substrate, 22oC

comp.19± 45.0 x 103520 ±302.6 ±0.05Tr Cel7A

comp.141 ±251.3x  1032100 ±1502.8 ±0.1At Cel7A

comp.107 ±141.7 x 103990 ±701.7 ±0.1Ta Cel7A

comp.39 ±149.5 x 1032000 ±20019 ±1Ct Cel7A

Type  of

inhibition

Ki (Glc2)

(µM)

kcat/Km

(min­1M­

1)

Km

(µM)

kcat

(min­1)

CNPLacEnzyme

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

HYDROLYSIS OF STEAM PRETREATED SPRUCE

0

10

20

30

40

50

60

70

80

90

100

35°C 45°C 55°C 60°C 35°C 45°C 55°C 60°C

T. reesei enzymes Thermostableenzymes

Hyd

roly

sis

(% o

f the

or. m

axim

um)

0h24h48h72h

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

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

HEMICELLULOSES AND HEMICELLULASES

Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl

MeGlcA Ara Ara

Ph

Ac

Endoxylanase α­Glucuronidaseα­ArabinosidaseΒ−­Xylosidase

Esterase

Xyl Xyl

Xyl

PhAraXyl

===

phenolic groupsarabinosexylose

AcMeGlcA

==

acetylmethyl

A

glucuronic acid

Glc Man Man Glc Man Man Man

Gal Ac Ac

Man Man Glc Man

B

Endomannanase β­Mannosidaseβ­Glucosidaseα ­Galactosidase

Esterase

GalMan

= Galactose= Mannose

GlcAc

= Glucose= Acetyl

Hemicellulases are essential components in efficient LC enzyme mixtures

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

CONCLUSIONS: IMPROVEDLIGNOCELLULOSE ENZYMES

Feed stock improvementn Improved raw materials & pretreatments

for better hydrolyzabilityn Modified carbohydrate/lignin structuresn Expression of LC enzymes in plantsCellulases & other enzymesn Short & long term challenges for enzyme

developmentn Thermostability a generally useful

parametern Integrated hydrolysis technologies

Acknowledgements:Financial supportEuropean Union, TIME project (ENK6­CT­2002­00604)The Academy of Finland

VTTMati Siika­ahoAnu KoivulaSanni Voutilainen

ROALJari VehmaanperäTerhi PuranenMarika Alapuranen

TIME partners, especially

Guido Zacchi, LU, SwedenFrancesco Zimbardi, ENEA, ItalyKati Reczey, BUTE, Hungary