Enzymes for improved hydrolysis of lignocellulosics ...ABSTRACT The considerably low β-glucosidase...

Enzymes for improved

hydrolysis of lignocellulosics

Doctoral dissertation

Tamás Juhász

Supervisor: Dr. Kati Réczey

Department of Agricultural Chemical Technology

Budapest University of Technology and Economics

Hungary

2005

ABSTRACT The considerably low β-glucosidase activity : FPA ratio of 0.5-0.6 represented by the enzyme

complex of Trichoderma reesei RUT C30 results in poor hydrolysis potential. To overcome

this problem culture conditions were developed to enhance β-glucosidase production.

Mandels’ medium supplemented with tris-maleate buffer proved to be efficient for cellulase

production with elevated β-glucosidase activity. β-Glucosidase activity : FPA ratio of the

obtained enzymes were around the optimal on Solka Floc, lactose and glucose carbon

sources. Furthermore, there were no considerable changes in maleic acid concentration of

cultivations throughout the fermentations, therefore it is tempting to speculate that maleic

acid is a kind of β-glucosidase inductor. In addition, in cultures with tris-maleate buffer pH

values varied in a narrow range, thus tris-maleate buffered cultures can be used in shake

flask experiments for modeling fermentations running at constant pH.

Tris-maleate buffer system was applied in shake flask experiments to test steam pretreated

corn stover, spruce and willow for cellulase production. On steam pretreated corn stover

higher cellulase activities could be obtained than on Solka Floc that was used as reference.

However, the achieved β-glucosidase activity was higher on Solka Floc compared to steam

pretreated corn stover. On the other two steam pretreated materials lower enzyme activities

were reached than on Solka Floc. The produced enzymes and two commercial cellulases

(Celluclast 1.5L and Econase CE) were tested in hydrolysis experiments using the three

pretreated materials and Solka Floc as substrates. Generally, the highest yields were

obtained using the enzyme produced on steam pretreated corn stover. Moreover, the

highest sugar yield was reached on steam pretreated corn stover using the enzyme

produced on steam pretreated corn stover. Similar results were obtained in the case of

chemically pretreated corn fiber. Significantly higher sugar yields were obtained in the

hydrolysis of corn fiber using the enzyme produced on the same substrate i.e. on corn fiber

than applying commercial cellulases.

It was shown that mannanase and endoglucanase expressed coordinatedly on the examined

carbon sources, since a good correlation was observed between the two enzyme activities.

This observation might be valuable information to understand the regulation mechanism of

hemicellulase genes in Trichoderma reesei.

Results of mixed cultivation of Aspergillus niger and Trichoderma reesei indicate that

Trichoderma can be used to produce glucose for Aspergillus from lignocellulosic wastes,

which might be advantageous if cheap raw material is needed for large-scale production of β-

glucosidase by Aspergillus species.

1

PREFACE

This work was carried out at Budapest University of Technology and Economics, Department

of Agricultural Chemical Technology between 2001 and 2005. I thank to Prof. Béla Sevella,

head of department, for giving me the opportunity to work at the department.

My most sincere thanks are due to my supervisor associate professor Dr. Kati Réczey for

providing the excellent working facilities at BUTE. I am very grateful for her support,

encouragement, constructive criticism and help during this work. I also express my thanks to

Dr. Zsolt Szengyel who helped me a lot. A great deal of appreciation goes to Professor Liisa

Viikari and Dr. Matti Siika-aho for giving me the opportunity to do part of my research at VTT

Biotechnology (Technical Research Centre of Finland).

I want to express my thanks for the pleasant collaboration to Dr. Béla Simándi, Dr. Edit

Székely, Dr. Erika Vági and Dr. Ildikó Kmetz at BUTE Department of Chemical Engineering;

to Prof. Dr. Guido Zacchi, Dr. Beatriz Palmarola-Adrados, Dr. Mats Galbe, Dr. Malek

Alkasrawi and Karin Öhgren at University of Lund; to Michael J. Bailey, Juha Tähtiharju, Satu

Homan and Jan Sclenar at VTT Biotechnology.

I also wish to thank all the personnel at BUTE Department of Agricultural Chemical

Technology for the positive and creative working atmosphere: Dr. Enikő Varga, Nóra Szijártó,

Zsófia Kádár, Dóra Dienes, Melinda Gáspár, Zsuzsanna Babics, Bálint Kupcsulik, Áron

Németh, Laura Leitgib, Krisztina Kozma, Gergely Kálmán, Ágnes Sárdi, Róbert Fülöp, Judit

Ádám, Eszter Balogh, Norbert Soós, Balázs Kiss, Krisztina Loksa, Veronika Lajtos and

Bernadett Kondor.

Financial supports from the EU "TIME" project (ENK6-CT-2002-00604); Hungarian Ministry

of Education (NKFP-OM-00231/2001); The National Research Fund of Hungary (OTKA

T025234); Varga József Foundation; Rubik Foundation, Hungarian Academy of Engineering;

Pro Renovanda Cultura Hungariae, Student Scientific Foundation and Bizáki Puki Péter

Foundation are gratefully acknowledged.

2

LIST OF PUBLICATIONS

This work is based on the following publications (Appendices I-V.), which are referred to in

the text by their Roman numerals. Additional unpublished data are also presented.

I. Juhász, T., Szengyel, Zs., Szijártó, N., Réczey, K., The effect of pH on the cellulase

production of Trichoderma reesei RUT C30, Appl. Biochem. Biotech., 2004, 113-116, 201-

211.

II. Juhász, T., Egyházi, A., Réczey, K., Towards understanding the β-glucosidase production

by Trichoderma reesei, Appl. Biochem. Biotech., 2005, 121-124, In press.

III. Juhász, T., Szengyel, Zs., Réczey, K. Siika-aho, M., Viikari, L., Characterization of

cellulases and hemicellulases produced by Trichoderma reesei on various carbon sources,

Proc. Biochem., 2005, In press.

IV. Gáspár, M., Juhász, T., Szengyel, Zs., Réczey, K., Fractionation and utilization of corn

fiber carbohydrates, Proc. Biochem., 2005, 40, 1183-1188. V. Juhász, T., Kozma, K., Szengyel, Zs., Réczey, K., Production of beta-glucosidase by

mixed culture of Aspergillus niger BKMF 1305 and Trichoderma reesei RUT C30, Food

Technol. Biotechnol., 2003, 41, 49-53.

Other related papers by the same author:

VI. Juhász, T., Székely, E., Simándi, B., Szengyel, Zs., Réczey, K., Recovery of a

recombinant thermostable endoglucanase from E. coli using supercritical carbondioxide cell

disruption, Chem. Biochem. Eng. Q., 2003, 17, 131-134.

VII. Palmarola-Adrados, B., Juhász, T., Galbe, M., Zacchi, G., Hydrolysis of non-starch

carbohydrates of wheat starch effluent for ethanol production, Biotech. Prog., 2004, 20, 474-

479.

3

ABBREVIATIONS AXE acetyl xylan esterase

β-Glu β-glucosidase

CBHI cellobiohydrolase I

CE Celluclast 1.5 L enzyme (Novozymes, Bagsværd, Denmark)

CO enzyme complex produced on steam pretreated corn stover (SPCS)

CPCF chemically pretreated corn fiber

EC Econase CE enzyme (ROAL Oy, Finland)

EG endoglucanase

FPA filter paper activity

FPU filter paper unit

SPCS steam pretreated corn stover

SF enzyme complex produced on Solka Floc

SP enzyme complex produced on steam pretreated spruce (SPS)

SPS steam pretreated spruce

SPW steam pretreated willow

WI enzyme complex produced on steam pretreated willow (SPW)

4

CONTENTS

1. Introduction.........................................................................................................5 2. Literature background .......................................................................................7 2.1. Lignocelluloses ........................................................................................7 2.2. Pretreatments of lignocelluloses..............................................................8 2.3. Lignocellulolytic enzymes ........................................................................9 2.3.1. Cellulase enzyme complex ..........................................................9 2.3.2. Hemicellulases.............................................................................10 2.3.3. Application of cellulolytic enzymes...............................................11 2.4. Measurement of cellulase and hemicellulase activities ...........................11 2.5. Production of fungal cellulases and hemicellulases ................................12 2.5.1. Why Trichoderma reesei RUT C30?............................................12 2.5.2. Induction and regulation of enzyme production ...........................13 2.5.3. Culture conditions ........................................................................15 2.5.3.1. Carbon sources for cellulase production..........................15 2.5.3.2. Carbon sources for hemicellulase production..................18 2.5.3.3. Nitrogen sources..............................................................18 2.5.3.4. Effect of cultivation pH .....................................................19 2.5.3.5. Effect of cultivation temperature ......................................20 2.5.4. Fermentation methods.................................................................21 2.6. β-Glucosidase production by Aspergillus species and cocultures ...........21 2.7. Hydrolysis of lignocellulosic residues ......................................................22 2.8. Background and outline of the thesis.......................................................25 3. Materials and methods.......................................................................................26 3.1. Substrates and carbon sources...............................................................26 3.2. Inoculum and enzyme production in shake flasks ...................................27 3.2.1. Trichoderma reesei ......................................................................27 3.2.2. Aspergillus niger ..........................................................................27 3.2.3. Aspergillus niger and Trichoderma reesei ...................................28 3.2.4. Comparing the applied media......................................................28 3.3. T. r. and A. n. cofermentation in Biostat CDSU-3 fermentor....................29 3.4. β-Glucosidase production in fermentor flasks by A. n. and T. r. ..............26 3.5. Protein and enzyme assays.....................................................................29 3.6. Hydrolysis ................................................................................................30 3.7. HPLC analysis .........................................................................................31 4. Results and discussion .....................................................................................32 4.1. Production of cellulase complex with high β-glucosidase activity............32 4.1.1. pH effect - selection of buffers .....................................................32 4.1.2. Testing of tris-maleate buffer .......................................................37 4.2. Substrate selection for enzyme production and hydrolysis......................40 4.2.1. Fermentation on various substrates.............................................40 4.2.2. Comparing the spectra of obtained enzymes ..............................42 4.2.3. Hydrolysis test of produced and commercial enzymes................45 4.3. Production of β-glucosidase by cofermentation of T. r. and A. n. ............48

5. Conclusions – New scientific thesis…… .........................................................50 6. References ……..................................................................................................52

5

1. Introduction

Due to the impact of greenhouse effect on the Earth’s climate, biomass originated alternative

fuels have gained remarkable attention during the past decades. Ethanol seems to be a

potential substitute of gasoline in internal combustion engines due to its advantageous

physical properties. Lignocellulosics are ideal raw materials that could support large-scale

fuel ethanol production, since vast quantities of lignocelluloses are produced every year,

constituting the most abundant renewable natural resource available on this planet (1). As it

is presented in Figure 1, ethanol produced from biomass i.e. lignocelluloses do not increase

the carbon-dioxide concentration in the air, since the released CO2 is consumed by

photosynthesizing plants. Furthermore, ethanol production from biomass has several other

advantages. For instance, new fuel ethanol plants can reduce unemployment and

dependency on fuel import. In addition, agricultural overproduction can also be reduced by

growing energy plants instead of food crops.

Figure 1 Closed carbon-dioxide circle

One of the process alternatives suggested for the production of fuel ethanol from

lignocellulosic materials is based on the enzymatic saccharification of cellulose (see Figure

2). For efficient conversion of the cellulose fraction, large enzyme dosage per unit of raw

material has to be applied, which due to the high market price of cellulases significantly

increases the overall production cost of ethanol (1, 2).

6

Figure 2 Ethanol production from lignocelluloses

Research on cellulolytic enzymes began in the 1950s and in the past decades cellulase

based technologies have aroused worldwide research activity (3). To improve cellulase

production many efforts have been made in genetic modification of strains, optimization of

culture conditions, characterization of enzymes in genetic and biochemical levels to produce

effective enzyme system for enzymatic hydrolysis of cellulose (4). In spite of the extensive

research work, the real breakthrough has not yet succeeded (3, 4). Therefore, the production

of enzymes for improved hydrolysis still remains an issue in the next decades. However,

Novozymes claims according to their research program that their cellulase complex will be

available around 2012, which will be cheap and suitable enough for economical cellulose

hydrolysis (5).

In spite of that, it is important to investigate further cellulase production, since its production

on site (“in situ”) can reduce ethanol production costs compared to processes, where

commercial cellulases are applied (1-3). The main aim of present study was to investigate

conditions that enable the efficient hydrolysis of lignocellulosics. On the one hand,

parameters of enzyme production were studied to produce enzymes with high hydrolysis

capacity. On the other hand, pretreated lignocellulosics were examined to select a raw

material that is suitable both for enzyme production and hydrolysis.

Pretreatment of

lignocelluloses

Cellulose

hydrolysis

Ethanol

production

Enzyme

fermentation

Ethanol

destillation

7

2. Literature background

2.1. Lignocelluloses

Main components of lignocelluloses, which are the most common biopolymers in nature, are

cellulose, hemicelluloses and lignin. Lignocelluloses also contain a few percent of mainly

terpenoid compounds that can be extracted by various solvents. Therefore, they are usually

referred as extractives (6).

Cellulose (see Figure 3) is a glucohomopolymer and its linear, unbranched chains contain

up to 10,000 1,4-β-linked β-D-glucopyranose units, which are in chair conformation (6). The

repeating units are cellobiose, since every other unit is rotated 180° around the main axis of

cellulose chain. The parallel chains are held together by hydrogen bonds to form fibrils.

Despite of cellulose is a hydrophilic molecule, it is insoluble in water (6, 7). Cellulose has

crystalline and less ordered i.e. amorphous parts, which exhibit less resistance (7).

O

OO

O H O H

C H 2 O HO H

C H 2 O H

O H

OO

OO

O H O H

C H 2 O HO H

C H 2 O H

O H

ββ

β

Figure 3 Structure of cellulose

Hemicelluloses are more heterogeneous, highly branched polysaccharides and composed

of several different monosaccharides and some other biomolecules (6). There are four types

of hemicelluloses: xylans, mannans, arabinans and galactans regarding to the main sugar

components in their backbones (8, 9).

Xylans have 1,4-linked β-D-xylopyranose backbones. In hard wood 10% of xylose units

contain 4-O-methyl-α-D-glucuronic acid and 60-70% of the xylose residues are acetylated

(10). In contrast, in soft wood xylans backbones are further substituted by α-L-

arabinofuranoside and no acetylated xylan are observed (10). In addition, both in soft and

hard wood some arabinofuranoside units are esterified by ferulic acid and p-coumaric acid

(11).

8

Backbones of mannans consist of β-1,4-linked glucose and mannose units, which are

distributed randomly and substituted with α-D-galactose units by 1,6 linkage. O-acetyl-

galactoglucomannans are the main hemicelluloses in soft wood, while hard wood contains

only a few percent of mannans with no galactose and acetyl groups (10). The main chain

sugars of arabinans and galactans are L-arabinose and D-galactose, respectively (9). These

hemicelluloses are also referred as pectic polysaccharides and they are usually not

significant components of wood lignocelluloses (9, 12).

Lignin is presented in Figure 4. It is a highly branched macromolecule and composed of

several types of aromatic acids such as guajacylpropan and p-hydroxyphenylpropan. Lignin

has great role in preventing lignocellulosic materials against biological attacks (13).

CH2OH

OH

O

O

CH2OH

OH

OH

CH2OH

OH

CH2OH

OHO

CH3O

CH3O

OHO

CH2OH

OHO

O CH3CH2OH

CH3OH

OH

CH2OH

OHO

CH2OH

O

CH2OH

OHOCH3O

O

O

O

HOCH2

CH3OCH3

CH3O

CH3OOH

CH3O

CH3O

OH

O

CH3O

O

CH3O

CH3O

CH3O

OH

CH3O

O

OHCH3O

Figure 4 Structure of lignin

2.2. Pretreatments of lignocelluloses

Usually various pretreatments are applied to break down the intact structure of

lignocellulosics. Lignin is closely associated with hemicellulose, thus preventing enzymes

accessing cellulose. Therefore, the aim of pretreatments is to degrade the lignocellulosic

material to enhance enzymatic degradation (14). In many pretreatment methods most of the

hemicelluloses are removed and the remaining fraction i.e. mixture of degraded lignin and

cellulose is used as substrate either for hydrolysis or for cellulolytic enzyme production (14).

It is important to perform pretreatments in such way to avoid the degradation of hydrolysis

products and the formation of inhibitors. These inhibitors, for instance furfurol, hydroxyl-

methyl-furfurol, acetic acid etc., can hinder the consequent enzyme or ethanol production by

causing toxic environment to fungi or yeasts (14, 15).

9

Pretreatment methods can be classified into physical, chemical, biological and combined

pretreatments regarding to the applied method for the disruption of lignocellulosic matrix.

Physical pretreatments, such as grinding, radiation and pyrolysis are found to be ineffective

(14, 15). Biological pretreatment by lignin-solubilizing filamentous fungi is relatively slow and

the substrate is partly consumed by the microbes. Chemical pretreatments applying acids,

alkalis, peroxides and organosolvents are reported to be effective methods. However, the

consumed chemicals can be expensive and might cause environmental problems (15).

Combined pretreatments are found to be the most suitable methods. Currently, steam

explosion and wet oxidation, which is a reaction involving oxygen and water at high

temperature and pressure, are the most studied methods (15, 16). Detailed results on

pretreatments can be found elsewhere (14-19).

2.3. Lignocellulolytic enzymes 2.3.1. Cellulase enzyme complex

For the complete hydrolysis of cellulose, cellulolytic microorganisms produce a whole set of

enzymes i.e. cellulases, which act synergistically in the degradation process (20). Cellulase

enzyme complex can be classified into of three types of enzymes. Regarding to an older

theory, exoglucanases or cellobiohydrolases (CBH) cleave cellobiose units from the end of

cellulose chain. Endoglucanases (EG) randomly attack cellulose in the middle of the chain,

which results in the release of cello-oligosaccharides and new chain ends that enables

exoglucanases to express their hydrolytic action (21). The mechanism that is most accepted

today is presented in Figure 5 according to Enari et al. (22).

Figure 5 Mechanism of cellulose hydrolysis (22)

Cellulose

Cellodextrines Cellobiose

CBH I CBH II

CBH I CBH II

EG I, EG II

Glucose β− G

10

The difference is that cellulose is first attacked by cellobiohydrolases and the released

cellodextrines are further degraded by endoglucanases. The resultant cellobiose is converted

to glucose by β-glucosidases (21, 22). Strictly speaking, β-glucosidase is not a cellulase

since this enzyme does not act on water insoluble cellulose. However, it shares a common

feature with endo- and exoglucanases, namely the specificity towards β-1,4-glucosidic bonds

(22, 23).

In culture filtrates of Trichoderma reesei, of which cellulase complex is the best

characterized, there are at least four different endoglucanases (EGI, EGII, EGIII, EGV), two

different cellobiohydrolases (CBHI, and CBHII) (24) and at least two, an intra- and an

extracellular β-glucosidase (24, 25). CBHI and CBHII are the major cellulases secreted by T.

reesei, constituting approximately 60-70% and 20% of the total cellulolytic protein,

respectively (24, 26, 27). CBHI is typically active on crystalline cellulose and releases

cellobiose units from the reducing end of cellulose, while CBHII has also activity against

amorphous cellulose and acts from the non-reducing end of the cellulose chain (28, 29).

EGI is an enzyme that exhibits high activity on soluble cellulose derivatives and has relatively

lower activity towards crystalline cellulose (30). Furthermore, EGI also catalyzes

transglycosylation reactions (31, 32) and it can hydrolyze xylan (33). EGII is a specific

endoglucanase that attacks insoluble cellulose and it is the only enzyme that can hydrolyze

cellotriose (34). The other endoglucanases (III-V) constitute around 1% of the total cellulolytic

protein and are usually referred as low molecular weight EGs (35).

2.3.2. Hemicellulases

As hemicelluloses are more heterogeneous polymers than cellulose, therefore several

enzymes play role in their degradation. The main enzymes needed for hemicellulose

hydrolysis are endo-enzymes i.e. endo- xylanase and mannanase. Xylanase enzymes

degrade 1,4-β-D-xylosidic linkages in xylans, while mannanases hydrolyze backbones of

mannan, resulting in the solubilization of short oligosaccharides (36). Accessory enzymes

like β-xylosidases and side group cleaving enzymes like α-arabinosidase and α-

galactosidase, which release arabinose and galactose molecules, respectively, further

degrade these oligosaccharide fragments (36, 37). The acetyl xylan esterase (AXE) cleaves

acetyl groups from sugar units of xylan (37,38).

11

2.3.3. Application of cellulolytic enzymes

Cellulases and hemicellulases can be used in the extraction and clarification process of

vegetable juices (39), for production of fruit nectars (40) and extraction of olive oil (41).

Moreover, the enzymes can improve the quality of bakery products (42). Cellulolytic enzymes

are used in beer brewing (43) and wine production technologies (44). In addition, the

enzymes serve as animal feed (45) and washing powder (46) components. Furthermore,

cellulases and hemicellulases are used in textile (47), pulp and paper industry (48). Last but

not least cellulolytic enzymes can be used in the process of bioethanol production for the

degradation of lignocellulosic residues, which application is hopefully going to be part of our

every day life in the near future (3).

2.4. Measurement of cellulase and hemicellulase activities To determine the activity of various components of cellulase complex, different substrates

are used. However, in most cases not only one but several enzyme components play role in

the degradation of the applied model substrate. In spite of that, enzyme activities depend on

model substrates can be well evaluated and used to characterize enzyme complexes.

Overall cellulase activity is measured against Whatman No I filter paper substrate and the

enzyme activity is expressed in FPU/mL (filter paper unit). The enzyme complex hydrolyzes

cellulose to oligo-, di- and monosaccharides that are determined colorimetrically by dinitro-

salycilic acid (DNS) (49). Mannanase is measured by locust bean gum substrate that mostly

comprises of mannane (50). The released saccharides are measured by DNS method. CMC

(carboxy-methyl-cellulose) and HEC (hydroxy-ethyl-cellulose) are suitable substrates for

endoglucanase measurements (51). Endoglucanases cleave free chain ends that can be

determined by the DNS method. Both endo- and exoglucanases (see Materials and

Methods) can be measured against 4-methylumbelliferyl-β-D-lactoside (52). Enzymes cleave

bonds between lactose and 4-methylumbelliferyl and the released aromatic compound i.e. 4-

methylumbelliferyl can be measured by photometer. α-Galactosidase, β-xylosidase, α-

arabinosidase and β-glucosidase assays have the same principle as endo- and

exoglucanase measurement (53-56). These enzymes release p-nitro-phenyl molecules from

model substrates, which can be measured colorimetrically (see Materials and Methods).

Acetyl xylan esterase is measured against an acetylated xylo-oligo-saccharide and the

released acetic acid is assayed either by HPLC or by test kits (57).

12

2.5. Production of fungal cellulases and hemicellulases

Cellulolytic and hemicellulolytic microbes play a key role in the carbon cycle of Earth by

decomposing lignocellulosic residues. Both bacteria and fungi produce cellulase enzymes

(58). However, for industrial purposes filamentous fungal species are the most suitable

cellulase producers, since most of their enzymes are extracellular (59). Furthermore, fungi

produce a whole set of cellulolytic enzymes, while bacteria usually produce only few

components of cellulases and hemicellulases (58, 59).

2.5.1. Why Trichoderma reesei RUT C30?

The most extensively studied filamentous fungus for cellulase fermentation is Trichoderma

reesei, which was discovered during the II. World War, due to the degradation of tents and

other textiles. In 1951, after the isolation of the wild type strain (T. reesei QM 6a), the

research continued in strain-development (see Figure 6) that led to the isolation of the

hypercellulolytic Trichoderma reesei RUT C30 mutant strain at Rutgers University in 1977

(59, 60).

Figure 6 Strain-development of Trichoderma reesei (60)

In the research T. reesei RUT C30 is the most applied microbe for cellulase fermentation

(59). It is a cre1 mutant strain, which means that monosaccharides do not cause carbon

catabolite repression.

Trichoderma reesei (1951) QM 6a (wild type)

QM 9123

QM 9414

Rut M-7

Rut NG 14

Rut C 30 (1971)

Rut EPI series

L- series

VTT-D series CL- series

MG- series

MCG-77

MHC- series

13

Therefore, cellulases can also be produced in the presence of glucose (61). T. reesei RUT

C30 produces cellulases with approximately three times higher activity than the wild type

strain T. reesei QM 6a (62). It should be noted that the strain-development did not stopped at

T. reesei RUT C30. However, significantly less is known about the new mutant strains

compared to T. r. RUT C30 (63). 2.5.2. Induction of enzyme production

In the early years it was of particular interest to explain how the synthesis of cellulases can

be turned on in the presence of polymeric cellulose. Mandels et al. (64) suggested that T.

reesei produces constitutive level of cellulase that are bound onto the surface of conidia (65).

These bound enzymes enable the primer attack on cellulose. Later the cleaved cello-

oligosaccharides can act as cellulase inducers (65). This theory was later proved by

immunological methods (66). Moreover, it has been reported that the true inducer of

cellulases is sophorose that is a glucose disaccharide with β-1,2 linkage (64, 67). Strongly

supports this view that the constitutive cell-bound β-glucosidase can form sophorose from

cellobiose by transglycolisation activity (68). It should be added that lactose can also induce

cellulase production in smaller extent. However, the induction mechanism is still not clear

(69, 70).

Transcription of cellulase genes starts approximately 20 minutes after induction. Regulation

of genes are under transcription control, which is proved by mRNA probes (66, 68, 71). The

levels of individual enzymes correlate with the steady state levels of respective transcripts

(71). It has been shown that expression of different cellulase genes is coordinated. Thus,

expression levels of cellulases are approximately the same in various conditions (68, 72).

There is usually also a feed back control, which means that the produced glucose repress

cellulase synthesis (73). This inhibition is usually referred as catabolite repression and it

cannot be observed in the case of T. reesei RUT C30 (61, 73).

β-Glucosidase I is induced much lower level than the other cellulases. However, it seems

that the highest β-glucosidase and other cellulase activities can be reached in the same

culture conditions (36). It has been demonstrated that there are at least two types of β-

glucosidases in Trichoderma reesei (74). Recently, Takashima et al. (75) have provided

evidence for β-glucosidase II of Trichoderma reesei, which is a constitutive and mostly

intracellular enzyme that also has transglycolisation activity (19, 75).

14

Both β-glucosidases are expressed on lactose, cellobiose, cellulose and on inert carbon

sources i.e. on sorbitol and glycerin in the presence of sophorose (19). Furthermore, β-

glucosidases are constitutively produced on glucose by Trichoderma reesei RUT C30 (19,

75).

T. reesei produces two types of specific xylanases and an unspecific one i.e. endoglucanase

I (76). All three enzymes are induced by the presence of xylan. Sophorose induces xylanase

and endoglucanase, while induction by xylobiose lead to the formation of only the two

specific xylanases (76-78). On glucose a basal production of xylanase can be observed,

which suggest that xylanase production is partially under carbon catabolit repression in

Trichoderma reesei RUT C30 (36).

There seems to be evidence for low constitutive level of xylanase expression, which is the

responsible for the formation of inducer from xylan i.e. xylobiose. In contrast to cellulase

system, it has been reported that the inactivation of surface bound xylanase activity did not

affect the ability of T. reesei to form xylanase in the presence of xylan (79). Therefore, it can

be argued that the secreted constitutive level of xylanase has great role in formation of

xylanase inducers (79).

Very few data are available on the induction of other hemicellulases. It has been shown (36)

by mRNA analysis that on various carbon sources various enzymes were produced and no

clear correlations were observed. However, it has been demonstrated that arabinose is the

most suitable monosaccharide inducer of α-arabinosidase (80), while α-galactosidase was

induced by galactose and arabinose (81). β-Xylosidase I is produced if xylanase formation

can be observed (36). Biely et al. have found that acetyl xylan esterase is co-expressed with

endoglucanase (82). In addition, it is well known that in T. reesei RUT C30 not only xylanase

but also acety xylan esterase and α-galactosidase are under carbon catabolite repression

caused by cre1 gene (36). Induction of Trichoderma reesei mannanase has been least

studied. However, Margolles-Clark et al. (36) have showed that its was rather poorly

produced except on cellulose.

15

2.5.3. Culture conditions

It is a well-established fact that parameters of cultivation affect significantly the production of

cellulases and hemicellulases (83). The most important factors are the quality and quantity of

carbon source. The applied media should be also supplemented with suitable nitrogen

source, salts and trace elements. Applied pH strategy and temperature also have

considerable effects on enzyme production (83).

2.5.3.1. Carbon sources for cellulase production

Carbon sources can be classified into soluble and insoluble materials. The most studied

soluble carbon sources are glucose and lactose. On glucose low cellulase activities can be

obtained, since only constitutive enzyme synthesis can be observed. Glucose as carbon

source is usually used in scientific experiments, for instance to obtain information on

regulation of cellulase genes or testing inhibitory effect of compounds etc. (84). Lactose can

be applied as carbon source for larger-scale enzyme production. Chaudhuri et al. (85) have

produced 1.72 FPU/mL and 2.84 FPU/mL filter paper activity on 20 g/L and 40 g/L lactose

containing media, respectively. These results illustrate also the concentration effect of carbon

source. The higher is the applied concentration, the higher enzyme activity can be obtained.

However, enzyme yields on the basis of 1 g applied carbon source usually decrease with the

increasing concentration (83, 85, 86).

Carbon source concentration cannot be increased over a certain level, especially in the case

of insoluble carbon sources. Stirring, rheological and mass transfer problems can be

occurred if high concentration insoluble carbon source is used (86, 87). Application of

insoluble carbon sources for enzyme production, for instance Solka Floc (88, see Tables 1-

3), waste paper (89, 90), wheat bran (91), wheat straw (92), corn stover (83), wood (93) and

corn fiber (94) have been extensively studied. The results have shown that pure cellulose

(Solka Floc, Avicel, Sigmacel etc.) is the best carbon source among insoluble materials.

However, pure cellulose is quite expensive. Agricultural and forestry byproducts would be

more economical. However, to get good enzyme yields, lignocellulosics should be pretreated

prior to enzyme fermentation. Therefore, for larger-scale enzyme production usually

pretreated materials are used. Tables 1-3 summarize the most relevant results obtained by

Trichoderma reesei RUT C30 in shake flask experiments.

16

Carbon source FPA β-Glu. a. P Y Ref.

FPU/

mL IU/mL

FPU/

L*h

FPU/

g carb. s

30 g/L glucose 0.90 - 9.5 30.0 95

30 g/L glucose + 15 g/L lactose 2.80 - 30.0 62.2 95

15 g/L lactose 1.50 - 15.0 100.0 95

10 g/L lactose 1.50 - 6.9 150.0 96

10 g/L lactose + 5 g/L Alon* 3.00 - 13.9 300.0 96

Table 1 Soluble carbon sources for cellulase fermentation by Trichoderma reesei RUT C30 in shake flasks (P=productivity, Y= yield, on the basis of 1 g carbon source) * colloid additive Carbon source FPA β-Glu. a. P Y Ref.

FPU/

mL IU/mL

FPU/

L*h

FPU/

g carb. s

10 g/L cellulose (Sigmacell) 1.20 0.60 10.0 120.0 63

50 g/L Solka Floc 14.40 - 74.0 240.0 83

10 g/L cellulose (Whatman CF-11) 2.50 - 11.6 250.0 96

10 g/L cellulose + 5 g/L Alon* 4.50 - 20.8 450.0 96





10 g/L cellulose + 10 g/L starch 3.50 0.70 14.6 175.0 97

60 g/L cellulose + 36 g/L corn steep l. 3.20 - 13.3 53.3 98

20 g/L Solka Floc 5.60 0.30 23.0 280.0 100

20 g/L Solka Floc 4.20 - 29.0 210.0 101

50 g/L Solka Floc 8.00 - 55.0 160.0 101

100 g/L Solka Floc 8.00 - 26.0 80.0 101

40 g/L cellulose (Whatman CF-11) 4.10 0.50 17.0 102.5 102

Table 2 Cellulose carbon sources for cellulase fermentation by Trichoderma reesei RUT C30 in shake flasks (P=productivity, Y= yield, on the basis of 1 g carbon source) * colloid additive

17

Carbon source FPA β-Glu. a. P Y Ref.

FPU/

mL IU/mL

FPU/

L*h

FPU/

g carb. s

20 g/L grain husk 0.37 0.20 3.1 18.5 63

10 g/L hydrolyzed hard wood saw dust 0.93 0.87 7.8 165.0/cel 99

10 g/L hydrolyzed soft wood saw dust 0.29 0.53 2.4 53.0/cel 99

10 g/L hydrolyzed and SO2

impregnated soft wood saw dust 0.68 0.80 5.7 126.0/cel. 99

40 g/L steamed aspen 1.60 0.20 6.8 40.0 102

38 g/L steamed aspen +

2 g/L cellulose 3.10 0.30 12.9 77.5 102


4 g/L cellulose 3.60 0.40 14.9 90.0 102


6 g/L cellulose 3.90 0.40 16.3 97.5 102

10 g/L steam pretreated willow 1.04 0.31 5.4 104.0 103

5 g/L steam pretreated willow +

5 g/L hemicellulose fraction 1.33 0.47 6.9 133.0 103

20 g/l steam pretreated willow 1.58 0.44 8.2 79.0 103

10 g/L steam pretreated willow +

10 g/L hemicellulose fraction 1.79 0.43 9.3 86.0 103

Steam pretreated willow

(10 g/L cellulose) 1.30 0.20 7.4 130.0/cel. 104

Steam pretreated willow

(10 g/L cellulose) 0.66 - 3.9 66.0/cel. 105

20 g/L steam pretreated oak wood 4.25 - 17.7 212.5 106

20 g/L acid percolated oak wood 3.25 - 13.5 162.5 106

Table 3 Pretreated carbon sources for cellulase fermentation by Trichoderma reesei RUT C30 in shake flasks (P=productivity, Y= yield, on the basis of 1 g carbon source)

When comparing the results in Tables 1-3 it should be taken into account that cellulase

researchers all over the world use a large variety of methods for cellulase production and

enzyme activity measurements. Therefore, in most cases enzyme activity values alone do

not definitely cover results of experiments. Thus, comparing results of cellulase

fermentations found in literature needs due foresight.

18

It should be also added that nowadays solid-state fermentation is the most studied method

(2.5.4.) of cellulase production. Recent results published on submerged cultures are rather

focused on genetic and molecular biological aspects of cellulolytic enzyme production (70).

2.5.3.2. Carbon sources for hemicellulase production

Cellulase production is usually coupled with hemicellulase, especially with xylanase

production. T. reesei RUT C30 can produce high levels of xylanase on xylan containing

media (107). Hence, the aforementioned pretreated materials are good carbon sources for

xylanase production (107, 108). Detailed information on xylanase production can be found in

articles presented by Gamerith et al. (107), Bailey et al. (108) and Haapala et al. (109).

In contrast to xylanase production, very little is known about mannanase production by

Trichoderma. Interestingly, it seems that mannanase can be produced better on cellulose

than on locust bean gum, which represents the main substrate of the enzyme, since it mostly

consists of galactoglucomannan (36).

Trichoderma reesei produces low levels of accessory hemicellulases compared to other

species. Therefore, only few data are available on the production of α-arabinosidase and α-

galactosidase. Roche et al. (110) produced α-arabinosidase on sugar beet pulp grown in

solid-state cultures, while Zeilinger et al. (81) carried out submerge fermentation using locust

bean gum for the production and characterization of α-galactosidase. Biely et al. (82) have

studied the carbon source requirements for production of acetyl xylan esterase. The highest

activity was reached when T. reesei RUT C30 was cultivated in a medium containing

deacetylated larchwood that was a mixture of 0.9% xylan and 0.1% cellulose. Almost the

same results were obtained using deacetylated birchwood and pure cellulose (1.0%), while

very low acetyl xylan esterase activities were reached on glucose, xylose and cellobiose

(82).

2.5.3.3. Nitrogen sources Besides carbon sources, nitrogen sources have also considerable effect on the production of

cellulolytic and hemicellulolytic enzymes. It has been demonstrated that higher enzyme

activities could be obtained using elevated nitrogen source concentrations. Tangnu et al. (83)

have reported that higher urea concentration led to higher filter paper activity and shorter

fermentational time.

19

Lu-Kwang et al. (111) have continued Tangnu’s work and demonstrated that the highest

enzyme activities could be achieved by applying carbon and nitrogen sources in the

concentration ratio of 1:1 (g/L). However, carbon source was limiting even in the system of

1:0.25 (C:N) (111). Doppelbauer et al. (112) have studied the effect of peptone

concentration. They obtained 1.0 FPU/mL and 1.7 FPU/mL filter paper activities using 0.5 g/L

and 2.0 g/L peptone, respectively.

Finding a cheap substitute of peptone for larger-scale enzyme production is also of particular

interest. Unfortunately, on most natural nitrogen sources, for instance on rape or soy

extracts, lower enzyme activities could be reached than on peptone (113). However, peanut

extract proved to be suitable for cellulase production. On Avicel carbon source doubled

concentration of peanut extract increased FPA two fold (113). Corn steep liquor is also a

suitable and widespread nitrogen source (98).

2.5.3.4. Effect of cultivation pH

It has been indicated that pH and pH controlling strategies have great effect on the amount of

cellulase produced (62, 83, 101, 112, 114-122). It has been shown that depending on the

nature of the carbon source used to induce cellulase production of Trichoderma strains,

different initial pH of the cultivation may be optimal for maximum cellulase yield. Ryu and

Mandels (62) have reported that pH range 3.0-4.0 was optimal for pure cellulose carbon

source, however higher initial pH (5.0-6.0) was recommended for lignocelluloses. For sugar

cane bagasse containing media (free of water soluble sugars), pH between 5.0 and 6.0 was

observed to be optimal using T. reesei QM 9123 (114). In other studies maximum yield of

cellulases was obtained in the range of pH 3.0 to 5.0 (115-117).

In shake flask cultures the pH control is usually limited to either addition of buffering salts

such as phosphates, or periodic manual pH adjustment, which is obviously tedious and less

effective. The use of ammonium sulfate as the major nitrogen source in the Mandels’

medium (119) requires a more compelling buffering system.

Without buffering, the pH drops quickly during the first stage of the fermentation due to the

depletion of ammonia and liberation of protons (86, 120, 121). Application of KH2PO4 –

K2HPO4 buffer system (4 g/L) for controlling the pH in shake flasks proved to be inefficient in

compensation of the acidification. However, higher cellulase activity was obtained in the

buffered system than in the basal Mandels’ medium (91).

20

Kadam and Keutzer (117) have investigated several organic acid buffer systems, i.e. acetate,

succinate, phthalate and citrate, unfortunately the efficiency of the various buffer systems

was not reported.

Tangnu et al. (83) have studied the influence of pH on cellulase production of T. reesei RUT

C30 in pH-controlled fermentor on cellulose carbon source. In the pH range of 4.0-6.0, no

significant effect on the production rate and final cellulase yield was observed, however the

β-glucosidase production was affected to a large extent. At pH 4.0 and 5.0 the β-glucosidase

activity was gradually increasing until it reached its maximum value by the 8th day of

cultivation. Controlling the pH in the fermentor to 6.0 increased the production rate of β-

glucosidase considerably and approximately 30% higher enzyme yield was achieved by the

4th day than at lower pH values. However, during the final stage of the fermentation the level

of β-glucosidase activity decreased to the same value as measured at lower pH levels.

Hendy et al. (101) have reported that performing the fermentation above pH 5.0 resulted in a

significant loss of cellulase activity on Solka Floc carbon source.

Instead of keeping the pH at a constant value during the whole fermentation process pH

profiling was recommended by Doppelbauer et al. (112). On lignocellulosic waste carbon

sources, for the growth phase of T. reesei, the pH of cultivation was suggested to be

maintained at 4.0, while in the later stage of production and secretion of cellulases, an

elevated pH level of 5.0 was recommended. In another study, pH cycling coupled with

temperature profiling increased the amount of cellulases by 13% compared to the control

case during which the pH was maintained at a constant value (118).

2.5.3.5. Effect of temperature Temperature has also significant effect on enzyme production. Tangnu et al. (83) have

performed experiments to find the optimal temperature for enzyme production on cellulose

carbon source. They have reported that keeping the cultivations in the beginning at 31°C and

after a few days at 28°C resulted in the highest enzyme activities. On lactose carbon source

Merivuori et al. (122) have partly confirmed Tangnu’s findings by proving that the optimal

temperature for cellulase production is 28°C.

21

2.5.4. Fermentation methods The most studied and simplest fermentation method is the batch technique. However,

applying fed-batch cultivation higher activity, productivity and yield could be obtained (see

Table 4). As presented in Table 4 cellulase production in continuous cultivations usually lead

to lower enzyme activities, thus it is very rarely applied (59, 101).

Fermentation method Carbon source FPA

(FPU/mL)

P

(FPU/L*h)

Y

(FPU/g)

Batch 168 h

50 g/L Solka Floc 8.00 55.0 160.0

Batch 336 h

100 g/L Solka Floc 8.00 26.0 80.0

Fed-batch 300 h

Feed: 1.7g/h

100 g/L Solka Floc

(Final conc.) 26.10 109.0 261.0

Continuous (One stage) D=0.046 h-1

Feed: 20 g/L

Solka Floc 2.10 97.0 105.0

Continuous (Two stages) D=0.017 h-1

Feed: 20 g/L

Solka Floc 3.30 56.0 165.0

Table 4 Effect of fermentation method on cellulase production (59, 101)

Solid-state fermentations (SSF) are also applied for cellulase production. However, in SSF

processes to control fermentation parameters and produce enzyme in larger-scale can be

extremely difficult. In spite of that SSF is extensively studied and it is a widespread method,

especially in the Far East. More information on SSF for cellulase production can be found

elsewhere (123-125).

2.6. β-Glucosidase production by Aspergillus species and cocultures

Aspergillus strains are known for their ability to produce β-glucosidase with significantly

higher yields than Trichoderma species. In most cases, where the augmentation of β-

glucosidase activity is needed, Aspergillus β-glucosidase is applied, for instance Novozyme

188 (Novozymes , Bagsværd). Several Aspergillus species produce β-glucosidase, such as

A. niger, A. wentii, A. fumigatus, A. phoenicis, A. foetidus (126, 127). However, A. niger is the

most studied filamentous fungus for β-glucosidase production (128-130).

22

Cofermentations of Aspergillus and Trichoderma species were examined by Duff et al. on

starch (97) and starch-cellulose mixture (121). The highest β-glucosidase activity obtained

was 2.9 IU/mL while the enzyme mixture had a cellulolytic activity of 3.0 FPU/mL.

Maheshwari et al. (131) investigated the cofermentation of the two species on cellulosic

waste. They have found that yields in cellulose hydrolysis were two or three times higher if

enzymes derived from cofermentations were used compared to hydrolysis experiment, where

enzymes form monocultures were applied. Madamwar et al. (132) performed semi-solid state

cofermentations of the two fungal species and obtained 20–40 % higher cellulolytic enzyme

activities with mixed cultures than with monocultures.

In the abovementioned cases the aim of mixed cultures was to produce high β-glucosidase

activity and FPA at the same time. However, Aspergillus strains usually have shorter

adaptation and generation time. Thus, in most submerged fermentations, especially if not

pure cellulose is the carbon source, Aspergillus strains suppress Trichoderma, which lead to

lower levels of FPA values. Therefore, it can be advantageous if cofermentations are

designed such way that Trichoderma produces only low levels of cellulases, just enough for

the breakdown of carbon source to supply Aspergillus with mono-, di- and oligosaccharides.

This method might lead to higher β-glucosidase activity than it can be achieved with the

previous method. The produced β-glucosidase can be added to Trichoderma culture filtrates,

which elevates the hydrolysis potential of the enzyme (see below). It should be worked out

by several experiments if the previous or the latter method gives better enzyme yield. 2.7. Hydrolysis of lignocellulosics and the hydrolysis potential of cellulases

Either acid or enzymes can hydrolyze lignocelluloses (133). Acid hydrolysis can be classified

into one-step concentrated and two-step dilute acid processes. Acid hydrolysis played

significant role during World War II by substituting gasoline (134, 135). However, fuel ethanol

was not competitive with gasoline when the fuel supply chain was rebuilt after the war. Main

drawbacks of acid hydrolysis are sugar degradation that cause low process yields, corrosion

and environmental problems and high cost of acid consumption and recovery (136).

In the enzymatic process much milder conditions are used, due to the specificity of applied

enzymes. However, cellulosic residues should be pretreated prior to hydrolysis, for instance

using conditions similar to acid hydrolysis. Cellulases are very susceptible to end-product

inhibition caused by cellobiose and partly glucose (137).

23

Furthermore, part of endo- and exoglucanases adsorb irreversible to their substrate and to

lignin that causes the loss of cellulase activity (138). Castanon et al. (139) have indicated

that these cellulases are bound to inactive sites of cellulose and they cannot be recycled at

all. On the other hand, cellulases adsorb reversible with the help of their cellulose-binding

domain (CBD) onto the surface of the solid cellulose where they catalyze the hydrolysis.

After several sequential hydrolytic reactions the enzyme desorbs and returns to the bulk fluid

(140). Velkovska et al. (141) have proven that cellulase adsorption can be best characterized

by a Langmuir adsorption isotherm that is presented in Figure 7.

Figure 7 Example of adsorption isotherm of cellulases (141)

Considering the aforementioned phenomena, in order to get high sugar yield high enzyme

dosage should be used and all factors should be optimized that affects the hydrolysis (142).

Generally, the applied cellulase activity varies between 7 FPU/g and 33 FPU/g substrate

depending on the used substrate (133, 142). Usually, it is necessary to augment β-

glucosidase activity to avoid inhibitory effect of formed cellobiose. β-Glucosidase

augmentation is performed by external β-glucosidase addition, which further increases the

cost of hydrolysis (142). Thus, it is highly important to use an enzyme mixture that exhibits

good hydrolysis ability. In contrast, the cellulolytic enzyme produced by most fungi, including

Trichoderma reesei RUT C30, contains inadequate amount of β-glucosidase, which leads to

poor hydrolysis potential (59). Considering cellulases, the optimal hydrolysis potential of the

enzyme complex corresponds to 1.0-1.5 β-glucosidase activity : FPA ratio (143). In contrast,

in Trichoderma culture filtrates β-glucosidase and filter paper activity ratio is only around 0.7

(59). Hence, to develop a method that enables the production of cellulases with adequate

amount of β-glucosidase is of particular interest. On the other hand, end-product inhibition

can be avoided by sugar removal.

0 5

10 15 20 25

0 1 2

Cellulase concentration in liquid (FPU/mL)

Adsorbedcellulase

(FPU/g cellulose)

24

This can be accomplished by applying ultrafiltration or simultaneous saccharification and

alcohol fermentation (SSF) (142). SSF is a comprehensively studied one-step method for

alcohol production using starch or lignocelluloses as raw materials. Detailed results on SSF

can be found elsewhere (144-146).

Hydrolysis is usually carried out at elevated temperature (45-50°C) above the growth

temperature of most microorganism to avoid the loss of carbon substrate. Generally, the

optimal pH of applied enzymes is used that is around pH 4.8 in most cases (142). Substrate

concentration exceeds 10% very rarely, due to rheological problems (87). Hydrolysis is

usually conducted for a few days resulting in 70-80% conversion (142). To improve the yield

and fasten hydrolysis speed, it has been suggested to use surfactants by lowering surface

tension of solid substrate and enhancing enzyme adsorption. However, surfactant-mediated

enhancement in hydrolysis is substrate dependent and its mechanism is still not clear, thus

the optimal application of surfactants for hydrolysis still remains an issue to develop (147).

It is well known that there are synergisms among various cellulase components. However,

several results showed that the synergism is not as simple as it was indicated in earlier

studies (20, 23, 142). On the other hand, it is possible that some interactions exist between

cellulase and hemicellulase enzymes. For instance, as it was mentioned above, EGI has also

xylanase activity (76). Interestingly, it seems that acetyl xylan esterase has particular role in

the degradation of lignocellulosics by cleaving ester-linked non-carbohydrate residues,

thereby increasing biodegradability.

Grohman et al. (148) have shown that due to deacetylation xylan became 5-7 times more

digestible, which in turn made cellulose three times more digestible. Poutanen and Puls

(149) have demonstrated that partial enzymatic deacetylation of birchwood xylooligomers

enhanced xylan degradation three-fold.

25

2.8. Background and outline of the thesis

At the Department of Agricultural Chemical Technology, Budapest University of Technology

and Economics, extensive research is being done since the beginning of the 1990s on the

production of fuel ethanol from lignocellulosic residues. The research work is to be classified

into pretreatment of lignocelluloses, cellulase production and alcohol fermentation. This

thesis represents a small part of the scientific work carried out on cellulase production.

The general aim of the present work was to find alternative methods to improve the

hydrolysis of lignocellulosics. The first part summarizes the experiments carried out to

produce cellulase enzyme complex with high β-glucosidase activity. A buffer system was

developed that enables Trichoderma reesei shake flask experiments at constant pH and at

the same time enhances β-glucosidase production to obtain cellulase enzyme complex with

adequate amount of β-glucosidase. The second part deals with experiments performed to

test steam pretreated corn stover, spruce and willow for enzyme production and hydrolysis,

while the third part studies a new approach to β-glucosidase production on waste paper

carbon source by mixed cultures of Trichoderma reesei and Aspergillus niger.

26

3. Materials and Methods

3.1. Substrates and Carbon sources

Solka Floc (delignified cellulose), glucose, lactose, steam pretreated and willow (SPW),

spruce (SPS), corn stover (SPCS) and chemically pretreated corn fiber (CPCF) were used as

carbon sources for cellulase enzyme production and as substrates for hydrolysis. After

pretreatments most of the hemicellulose fraction was removed by thorough washing and the

remained solid fraction mostly consisted of cellulose was used as substrate or carbon

source.

Pretreated wood and corn stover samples were obtained from Sweden while corn fiber was

obtained from Hungary. Prior to steam pretreatments materials were impregnated with SO2.

Steam pretreatments were performed by loading the samples into a 1 L steam gun. After the

treatment time at a certain temperature (see Table 5), the pretreated materials were released

by rapid depressurization to let the material to explode into a cyclone. Chemical pretreatment

was performed by keeping corn fiber at 120°C for 120 minutes in 2.5% NaOH and 0.006

H2O2 solution. Pretreated materials were collected, filtrated, washed with distilled water and

analyzed using a modified Hägglund´s method (150).

Substartes / carbon sources

Solka Floc SPCS SPS SPW CPCF Paper

Pretreatment conditions

Time (min) - 5 5 12 120 -

Temperature (°C) - 190 215 190 120 -

SO2 (%) - 2 3 2 - -

NaOH (%) - - - - 2.5 -

H2O2 (%) - - - - 0.006 -

Composition (%)

Cellulose 78.8 56.9 50.4 52.9 49.4 52.0

Xylan 14.2 6.8 0.0 3.8 13.8 16.6

Dry matter 97.0 20.2 22.0 25.2 100.0* 100.0*

Table 5 Composition of carbon sources (based on dry matter) and pretreatment conditions

(* carbon sources were kept in a drier until they were used for fermentation or hydrolysis)

27

Waste wrapping paper was used as carbon source for β-glucosidase production by mixed

cultures of Aspergillus niger and Trichoderma reesei (see Table 5). The paper was cut into

about 2X2 cm pieces and put into water. A kitchen blender was used to disintegrate paper

and produce paper slurry that was filtrated on a 150 µm mesh nylon filter via vacuum and

dried at 105°C. The paper was kept in drier until it was used for fermentation.

3.2. Inoculum and enzyme production in shake flasks

3.2.1. Trichoderma reesei

Trichoderma reesei RUT C30 (ATCC#: 56765) stock cultures were maintained on agar slants

containing: 20 g/L malt extract, 5 g/L glucose, 1 g/L proteose peptone and 20 g/L bacto agar.

After few weeks at 28°C conidia were suspended in 5 mL of sterile water and 1 mL of this

spore suspension was transferred aseptically to 750 mL Erlenmeyer flask containing 200 mL

of the sterile medium prepared referred as modified Mandels´ medium (119) in which the

concentrations of nutrients were: 0.40 g/L urea, 1.87 g/L (NH4)2SO4, 2.67 g/L KH2PO4, 0.53

g/L (0.3 g/L) CaCl2 x 2H2O, MgSO4, 0.33 g/L yeast extract, 1.00 g/L proteose peptone

together with 10 g/L Solka Floc cellulose powder. The medium was also supplemented with

the following trace elements: 7 mg/L FeSO4 x 7H2O, 20 mg/L CoCl2, 2 mg/L MnSO4, 2 mg/L

ZnSO4. After 4 days at 28°C and 350 rpm the inoculum was ready.

A total volume of 20 mL mycelium suspension, obtained from the inoculum cultures, was

used to initiate growth in a 750 mL E-flask containing 200 mL of a modified Mandels’ medium

(119, Paper I), which was supplemented with the components of buffers (Paper I, II). The

carbohydrate concentrations of the aforementioned carbon sources were equivalent with 11

g/L glucose. After inoculation flasks were incubated in an orbitary shaker at 28°C and 350

rpm.

3.2.2. Aspergillus niger

Aspergillus niger BKMF-1305 stock cultures were maintained on agar slants containing: 20

g/L malt extract, 5 g/L glucose, 1 g/L proteose peptone and 20 g/L bacto agar. After few

weeks at 30°C conidia were suspended in 5 mL of sterile water and 1 mL of this spore

suspension was transferred aseptically to 750 mL Erlenmeyer flask containing 150 mL of

sterile and pH adjusted (pH=5.5) medium prepared according to Mandels (119).

28

Mass concentrations of nutrients were: urea 0.3 g/L, (NH4)2SO4 1.4 g/L, KH2PO4 2.0 g/L,

CaCl2 0.3 g/L, MgSO4 0.3 g/L, yeast extract 0.25 g/L, proteose peptone 0.75 g/L together

with 10 g/L glucose. The medium was also supplemented with the following trace elements:

FeSO4 x 7H2O 5 mg/L, CoCl2 20 mg/L, MnSO4 1.6 mg/L, ZnSO4 1.4 mg/L. After 3 days at 30

°C and 350 rpm the inoculum was ready.

3.2.3. Aspergillus niger and Trichoderma reesei

10 mL of Trichoderma and 5 mL of Aspergillus mycelium suspensions, obtained from

inoculum cultures, were used to initiate growth in 750 mL Erlenmeyer flasks containing 150

mL of modified Mandels’ medium, as described above, at pH=5.5. The applied carbon source

was 20 g/L waste paper that was equivalent with 11.4 g/L glucose and 3.8 g/L xylose and

arabinose (analyzed by Hägglund’s method (150)). After the inoculation culture flasks were

incubated in an orbitary shaker at 30°C and 350 rpm for 7 days. Samples were withdrawn

every day and at the same time pH was adjusted to 6.0 by addition of either sterile NaOH or

H2SO4 solutions. The samples were centrifuged at 8000 rpm for 5 min and the supernatants

were collected for the analysis of β-glucosidase activities.

3.2.4. Comparing the applied media

For production of Aspergillus niger inoculum original salt and nitrogen source concentrations

were used (119), while carbon source (glucose) concentration was elevated from 7.5 g/L to

10 g/L because Aspergilli grew faster than Trichoderma and it needed more carbon source to

obtain good inocula after 3 days. In contrast, in Trichoderma experiments, where 10 g/L

cellulosic carbon source was used (equivalent with 11 g/L glucose), salt concentrations were

also elevated by 1.33 fold (=10/7.5) to avoid the limitation of nitrogen source and other salts.

In cofermentations the amounts of salts and nitrogen sources were doubled to cover nutrition

demands of both microbes. However, later it was proven (unpublished data) that modification

of salt and nitrogen source concentrations did not have significant effect on cellulase and β-

glucosidase activites in all sorts of fermentation.

29

3.3. Trichoderma reesei and Aspergillus niger cofermentation in Biostat CDSU-3 laboratory fermentor

Upscale enzyme production experiments were performed in a 31-L double-walled stainless

steel laboratory fermentor (Biostat CDCU-3; B Braun Biotech, Germany). Mixed fermentation

of A. niger BKMF-1305 and T. reesei RUT C30 was performed in modified Mandels’ medium

(119) (20 g/L waste wrapping paper as carbon source) as described in the previous section.

Prior to sterilization at 121°C for 20 min, the nutrients, carbon source, and trace elements

required for 20 L of production medium were dissolved in 19.5 L of tap water.

During sterilization, vapor equivalent to about 500 mL of water was bled through the gas

exhaust system in order to achieve sterile conditions. After sterilization, the temperature of

the fermentor was decreased to 30°C and 1 L of starter culture (670 mL of Trichoderma and

330 mL of Aspergillus inoculum) was aseptically added to initiate growth and enzyme

production. The fermentation was carried out with an agitation rate of 250 rpm for 168 h.

Samples were withdrawn regularly and centrifuged at 8000 rpm for 5 min. Supernatants were

collected and enzyme activities were measured. The initial pH was 5.5 and it was let to drop

down to 3.5. Afterwards it was continuously set to pH of 6.0 by automatic addition of sterile,

10 wt% solutions of either H2SO4 or NaOH. The aeration was 0.5 VVm throughout the

fermentation. To avoid the formation of foam, silicon oil–based Sigma-Aldrich Antifoam A

(Munsch, Germany) in 30% ionic emulsion was added manually four times a day at about 6-

hour intervals.

3.4. Protein and enzyme assays

All samples were analyzed in triplicates and the mean values were calculated. The relative

standard deviation of enzyme activity measurements were always below 5%. Protein content

of samples was determined using BSA as standard by either the Coomassie (151) or the

Lowry assay (152). Overall cellulase activity of samples was measured as filter paper activity

(FPA) expressed in filter paper units (FPU) using Mandels’ procedure (49). Part of FPA

measurements were repeated by setting β-glucosidase activity to 1.4 IU/mL by adding

commercial β-glucosidase (Novozyme 188, Bagsværd, Denmark) to avoid the effect of

different β-glucosidase levels on FPA values (153). β-Glucosidase activity was measured

using essays based on Berghem’s method (56). 4-nitrofenil-β-D-glucopyranoside was

applied as substrate in concentrations of 5 mM (pH 4.8) and 1 mM (pH 5.0) in chapters 3.1.

and 3.2., respectively.

30

Endoglucanase activitiy was assayed against HEC (hydroxy-ethyl-cellulose) as substrate

using a method based on IUPAC’s procedure (51). 1 mL substrate (2% HEC, dissolved in

citrate buffer pH 4.8, 0.1 M) and 0.5 mL sample was incubated at 50°C for 10 minutes. The

reaction was terminated by adding 3 mL of 3,5-dinitrosalicylic acid (DNS) reagent and boiling

for 5 minutes. After cooling, 16 mL distilled water was added and the absorbance was

measured at 540 nm. One international unit (IU) was defined as the release of 1 µmol

glucose / minute. EG I and CBH I activities were measured against 4-methylumbelliferyl-β-D-

lactoside (MUmbL) either with or without 1 mM cellobiose (52).

The obtained activity without cellobiose was referred as EG I & CBH I activity, while the

activity in the presence of cellobiose was considered as EG I activity, since cellobiose is a

competitive inhibitor for CBH I. CBH I activity was calculated by subtracting EG I activity from

EG I & CBH I activity.

Xylanase activity was assayed against xylan (Roche Co.) by Bailey´s method (154). β-

Xylosidase, α-galactosidase and α-arabinosidase activities were determined using 4-

nitrophenyl-β-D-xylanopyranoside, 4-nitrophenyl-α-D-galactopyranoside and 4-nitrophenyl-α-

L-arabinofuranoside substrates, respectively (53-55). Acetyl xylan esterase (AXE) activity

was measured against an acetylated xylo-oligomer (kindly supplied by Jürgen Puls) and the

liberated acetic acid was assayed using acetic-acid test kit (Boehringer Co.) (57). Locust

bean gum was used as substrate for mannanase activity determination (50). Enzyme

activities were expressed in both IU and nkat (1 nkat = 0.06 IU).

3.5. Hydrolysis

In the case of Solka Floc, steam pretreated corn stover, spruce and willow the hydrolysis was

performed in 50 mL test tubes with a working volume of 30 mL at 45 °C. The substrate

containing 0.6 g cellulose was suspended in 0.1 M acetate buffer (pH 5.0) and the slurry was

supplemented with either the produced or commercial cellulase enzymes (15 FPU/g

cellulose). The applied commercial enzymes were Celluclast 1.5 L (Novozymes, Bagsværd

Denmark) and Econase CE (ROAL, Finland). Samples were withdrawn after 24 h of

hydrolysis, centrifuged and the supernatants were analyzed by HPLC.

31

In the case of corn fiber substrate the hydrolysis was performed in 100 mL E-flasks with a

working volume of 50 mL at 50 °C. 2.5 g of dried chemically pretreated corn fiber was

suspended in 0.05 M acetate buffer (pH 4.8) and the slurry was supplemented with the

enzyme produced on corn fiber or with industrial cellulase enzymes (30.4 FPU/g cellulose

and 15.2 IU/g cellulose, filter paper and β-glucosidase activities, respectively).

The industrial enzymes were Celluclast 1.5 L and Novozyme 188 (Novozymes, Bagsværd

Denmark). Samples were withdrawn five times, centrifuged and the supernatants were

analyzed by HPLC.

3.6. HPLC analysis For sugar and organic acid determinations an Aminex HPX-87H (Bio-Rad, Hercules, CA,

USA) organic acid column was used at 65°C. The mobile phase was 5 mM sulfuric acid at a

flow rate of 0.5 mL/min. For the detection of various components separated on the analytical

column a refractive index detector (Shimadzu) was used.

32

4. Results and Discussion

4.1. Production of cellulase complex with high β-glucosidase activity

The considerably low β-glucosidase activity : FPA ratio of 0.5-0.6 represented by the enzyme

complex of Trichoderma reesei RUT C30 results in poor hydrolysis potential. To overcome

this problem culture conditions are needed to be developed in order to enhance β-

glucosidase production (59, 142, 143). Experiments are presented here that were carried out

to test buffer systems for production of cellulases with elevated β-glucosidase activity,

particularly considering the effect of pH, which is known as a significant factor that affects β-

glucosidase production.

4.1.1. pH effect - selection of buffers

The effect of pH on β-glucosidase production by Trichoderma reesei RUT C30 has been

comprehensively studied on Solka Floc, which is delignified cellulose powder and the most

suitable and widespread carbon source on which cellulase production has been studied.

When planning shake flask experiments for investigating the effect of pH, it should be taken

into consideration that constant pH cannot be kept in cultivation flasks. pH drops quickly

during the first stage of fermentation owing to the depletion of ammonia and liberation of

protons. Therefore, periodic manual pH adjustment is needed, which is tedious, less

effective, time consuming and results in a saw tooth-like pH profile instead of constant pH

(Paper I, 119).

To overcome this problem media can be supplemented by buffer components. The applied

buffers should have a sufficiently high buffering capacity to compensate the rapid pH drop

and it should be capable of controlling the pH in the range of our interest. Inorganic

phosphate salts were excluded during the selection of potential buffer systems, because they

have already been proved to be insufficient (91). Although, citric acid and acetic acid have

already been examined in a previous study, the pH range in which they were applied was

much lower than our primary interest (107). Thus, these organic acids were also tested in

experimental series along with tris-maleate, maleate, and succinate as shown in Table 6.

33

All buffer systems were applied in 0.1 M concentration at pH 5.0 or 6.0, at which the most of

cellulase fermentations have been performed (83, 101, 114-117). The concentration of

various organic acids in fermentation broth was determined using HPLC.

Buffer System Cellulase β-Glucosidase pH Stability

Citric Acid – Citrate, pH 5.0 Poor Poor Poor

Acetic Acid – Acetate, pH 5.0 — — Very good

Succinic Acid – NaOH, pH 5.0 Very good Very good Poor

Tris-maleate, pH 6.0 Good Very good Very good

Maleate, pH 6.0 Good Very good Very good

Table 6 Application of organic acid buffer systems in shake flask cultures on Solka Floc

Acetic acid : acetate buffer system applied at pH 5.0 kept the pH constant throughout the

fermentation. No changes in acetic acid concentration could be observed in the medium.

However, no enzyme activities were detected after 7 days of cultivation. Inhibition of

microbial growth by acetic acid could be a reasonable explanation for the poor performance

of T. reesei. In contrast, employing succinic acid in the fermentation medium at pH 5.0

resulted in rather high enzyme activities compared to other buffer systems used. However,

the pH fluctuated over a wide range. At the beginning of the cultivation, buffering capacity

was not high enough to prevent acidification caused by ammonia depletion, while in the final

stage, succinic acid was taken up by the fungus and a dramatic pH shift to alkaline region

was observed (Paper I.)

In contrary to previous results reported by Kadam and Kautzer (107), citric acid buffer could

not keep pH at 5.0. Similarly to succinic acid buffer system, citric acid was also consumed by

T. reesei RUT C30, and an extensive basification of the medium was observed. In this case

cellulases and β-glucosidase enzymes were produced in moderate amounts.

Cultivation of T. reesei RUT C30 in tris-maleic acid, maleic acid-NaOH buffer systems were

promising. In the case of these buffers pH 6.0 was applied, since these buffers can be used

at higher pH values than 5.2. Both FPA and β-glucosidase activities were considerably high

in the fermentation broth using these systems. Furthermore, these buffer systems managed

to maintain the pH around the desired value with great stability. In addition, Trichoderma did

not consume maleic acid (Paper I, II). Therefore, these buffer systems were chosen to

quantitatively compare them with four other pH-controlling strategies to investigate which pH

profile gives the highest β-glucosidase activity. The experimental setup is outlined in Table 7.

34

Cultures pH-controlling strategy Average pH

I Daily pH adjustment to 5.0 4.7

II pH adjustment in case pH drops below 4.0, starting pH: 5.0 4.1

III Daily pH adjustment to 6.0 5.7

IV pH adjustment in case pH drops below 4.0, starting pH: 6.0 4.6

V 0.1 M, tris–maleic acid buffer, pH 6.0, no manual adjustment 6.0

VI 0.1 M, Maleic acid–NaOH buffer, pH 6.0, no manual adjustment 6.0

Table 7 pH-Controlling Strategies Followed in Shake-Flask Cultivation of T. reesei RUT C30

Filter paper activities about 1.2 – 1.4 FPU/ml were measured on day 7 of cultivation in each

condition (Paper I). In contrast, β-glucosidase activities varied in the wide range of 0.4 and

1.5 IU/ml. The results are presented on Figure 8. It seems the higher is the average pH, the

higher is the obtained β-glucosidase activity. It is obvious that pH 6.0 is better initial pH than

pH 5.0. In addition, the highest β-glucosidase activities were obtained in buffered cultures.

Moreover, 25% higher activities were reached in cultivations supplied by tris-maleate buffer

than in cultures with maleate. Thus, tris-maleate buffer system was selected to evaluate it on

three carbon sources against two other pH strategies (Table 8, Paper II).

Figure 8 β-Glucosidase activity vs time for shake-flask cultivations using different pH-controlling strategies. (—■—), Condition I; (—□—), condition II; (—▲—), condition III; (—∆—), condition IV; (—●—), condition V; (—○—), condition VI.

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4 5 6 7

Time (day)

IU/mL

35

4.1.2. Testing of tris-maleate buffer All together nine experimental setups were tested against each other as shown in Table 8.

Experiment Carbon source pH control Average pH

A Solka Floc daily 5.7

B Solka Floc - 3.9

C Solka Floc buffer (0.10 M) 5.9

D glucose daily 5.7

E glucose - 3.7

F glucose buffer (0.10 M) 6.0

G lactose daily 5.7

H lactose - 4.9

I lactose buffer (0.10 M) 6.0

Table 8 Experimental setup and average pH values in fermentations on various carbon sources using different pH strategies

To study pH effect in detail, experiments were performed not only on Solka Floc, but on

glucose and lactose, as well (Paper II). Effect of pH might be dependent on the applied

carbon source, since Trichoderma produces cellulase not in the same extent on various

carbon sources. Glucose, for instance does not induce cellulase production, while lactose

induces enzyme formation. However, lactose is not as strong inducer as cellulose i.e. Solka

Floc.

The three applied pH-controlling strategies were as follows. In experiments A, D, G daily

manual pH adjustment was carried out, while in experiments B, E and H pH control was not

performed. Cultivations C, E and I were supplemented by the components of tris-maleate

buffer system (0.1 M, pH 5.8) to keep pH in the range of 5.6 and 6.0.

The results are presented in Figure 9 A and 9 B. The obtained enzyme activities are in good

accordance with results published elsewhere (59). The highest FPA values were around 1.15

FPU/mL that were reached on Solka Floc, which is constituted by cellulose and xylan.

Therefore, it is rather good carbon source for cellulase production. Lactose can also induce

the production of cellulases in smaller extent (69, 95). The obtained filter paper activities on

lactose were 40% less than reached on Solka Floc. Glucose does not repress cellulase

production in Trichoderma reesei RUT C30, since it is cre1 mutant strain (4). However, only

low levels of cellulases can be produced on glucose, because of the lack of inducers (4).

Thus, only around 0.40 FPU/mL cellulase activities were obtained on glucose.

36

Figure 9 A Filter paper activities ( 4th day, 7th day, 7th day with added β-glucosidase)

Figure 9 B Endoglucanase activities (7th day)

0.0

0.5

1.0

1.5

A B C D E F G H I

FPU/mL

0

2.5

5

7.5

10

A B C D E F G H I

IU/mL

37

Filter paper activities measured with additional β-glucosidase were higher than determined

without β-glucosidase and there were minor differences within the experiments where the

same carbon source was used. In addition, there were also minor differences regarding to

endoglucanase activities using different pH strategies on the same carbon source (see

Figure 9 B). The highest endoglucanase activities were obtained on Solka Floc, while on

lactose and glucose 30% and 75% lower activities were reached, respectively.

Figure 10 Extracellular β-glucosidase activities ( 4th day, 7th day)

Figure 10 shows that the highest β-glucosidase activities after 7 days of fermentation were

obtained on each carbon source in experiments with tris-maleate buffer. The highest

activities (1.4 IU/mL) were reached on Solka Floc and lactose with tris-maleate buffer, while

60% lower β-glucosidase activity was obtained on buffered glucose media. In buffered

cultures the reached activities were 140% higher on Solka Floc and glucose, 550% higher on

lactose compared to non-buffered, pH-adjusted cultures, respectively. The obtained β-

glucosidase activities in pH-adjusted cultures were two and three times higher than in

cultures without pH adjustment, which is in good accordance with the observation that higher

pH enhances β-glucosidase production. It can be seen in Table 7 that pH adjusted cultures

had higher average pH (5.6-5.7) than non-pH-adjusted cultivations (3.7-4.9). In buffered

cultures the average pH values varied between 5.9 and 6.0.

0.0

0.5

1.0

1.5

A B C D E F G H I

IU/mL

38

The reached β-glucosidase activities were the highest in buffered cultures. Therefore, it is

tempting to speculate that buffer components provoke β-glucosidase production. Strongly

support this view that in buffered cultures not only β-glucosidase activities, but also

extracellular protein concentrations were considerably higher compared to non-buffered

cultivations. According to Table 8 in experiments with tris-maleate buffer the reached protein

concentrations were almost two times higher than obtained in non-buffered cultures.

Moreover, in buffered cultures major changes were in protein concentrations between the 4th

and 7th days, which were believed to be mainly due to β-glucosidase production.

Protein concentration (mg/mL)

4th day 7th day

A 0.83 0.88

B 0.53 0.87

C 1.01 1.46

D 0.24 0.39

E 0.13 0.31

F 0.41 0.65

G 0.31 0.46

H 0.26 0.39

I 0.72 0.91

Table 9 Protein concentrations (4th and 7th day)

However, the average pH values of buffered cultures were slightly higher than non-buffered

cultivations. Therefore, to prove that not the higher pH, but the buffer components

themselves provoke enhanced β-glucosidase production, tris-maleate buffer was tested in

the concentration range of 0.05 M and 0.15 M using Solka Floc carbon source. Time profiles

of obtained filter paper and β-glucosidase activities are presented in Figures 11 A-B. It

seems that because of buffer salts in buffered cultivations Trichoderma reesei needs longer

adaptation time compared to non-buffered cultures. Therefore, the higher is the

concentration of buffer, the lower is the initial enzyme production rate. However, until the

seventh day of experiments the differences in enzyme activities were equalized. Therefore, it

can be concluded that the concentration of buffer components in the range of 0.05-0.15 M

have no effect on final β-glucosidase and filter paper activities.

39

Figure 11 A Time courses of filter paper activities in experiments with various buffer concentrations (Buffer concentration:♦ 0.05 M, 0.10 M, 0.15 M, x without buffer)

Figure 11 B Time courses of β-glucosidase activities in experiments with various buffer concentrations (Buffer concentration:♦ 0.05 M, 0.10 M, 0.15 M, x without buffer)

0.0

0.5

1.0

1.5

0 1 2 3 4 5 6 7

FPU/mL

Time (day)

0.0

0.5

1.0

1.5

0 1 2 3 4 5 6 7Time (day)

IU/mL

40

Average pH values were in the range of 5.6-6.0, since lower buffer concentrations had

insufficient buffering capacity to compensate pH changes. In cultures with 0.05 M tris-

maleate buffer the average pH was lower than in non-buffered, pH-adjusted cultivations,

which proves that the considerably high β-glucosidase activity is not caused by the high pH.

In addition, previous experiments indicate that maleic acid is the buffer component that

enhances cellulase production, since similar results could be obtained without tris-

hydroxymethyl-amino-methane by applying maleate buffer (Paper I).

4.2. Substrate selection for enzyme production and hydrolysis

It has been proven that Solka Floc is a suitable carbon source (Paper I, II) for cellulase

production. However, it is uneconomical for large-scale enzyme production. Instead of

expensive Solka Floc, pretreated lignocellulosics, derived from agricultural and forestry

wastes or from energy crops should be used.

It seems that considerably higher β-glucosidase activity can be obtained on all three tested

carbon sources if cultivations are supplemented by tris-maleate buffer. The buffer system

was also tested on a few pretreated lignocellulosic carbon sources in experiments as follows.

The produced enzymes were tested against each other by their composition and hydrolysis

potential. It has been reported that hemicellulolytic activities also affect cellulose hydrolysis

(76, 148). Thus, hemicellulolytic activities were also studied.

4.2.1. Fermentation on various substrates

Cellulase enzymes were produced by Trichoderma reesei RUT C30 on steam pretreated

spruce, willow, corn stover and on Solka Floc as a reference. The same cellulose

concentration was applied at each carbon source. The used medium was supplemented with

buffer components according to experiments presented in the previous chapter. The

obtained enzymes were SP (enzyme produced on steam pretreated spruce), WI (enzyme

produced on steam pretreated willow), CO (enzyme produced on steam pretreated corn

stover) and SF (enzyme produced on Solka Floc), respectively. The produced enzymes and

two commercial cellulases, CE (Celluclast 1.5 L) and EC (Econase CE) were characterized

with respect to the various activities and protein content (Paper III).

Figure 12 shows the main enzyme activities and protein concentrations obtained on the

seventh day of fermentations. As revealed by the graph both filter paper and endoglucanase

activities of CO were the highest.

41

In addition, the highest protein concentration was also reached on steam pretreated corn

stover. However, the β-glucosidase activity of CO was lower compared to the reference

enzyme i.e. SF. The activities and protein concentrations of both WI and SP were lower than

that of SF.

Figure 12 Main enzyme activities and protein concentrations (7th day)

It should be noted that in this set of experiments the obtained β-glucosidase activity on Solka

Floc was lower compared to the abovementioned experiments. This is probably due to the

biological variability. In addition, it is certain that the slight differences in β-glucosidase

assays (see Materials and Methods) had also effect on the calculated β-glucosidase activity.

Furthermore, little variations in fermentation conditions, for instance in temperature, agitation,

sampling time etc. also have effect on final enzyme activity. Nevertheless, the reached β-

glucosidase activity : FPA ratio was still considerably higher than the basal 0.5-0.6. However,

on steam pretreated corn stover and willow the ratios of enzyme activities were only around

0.6. To evaluate these results it should be taken into consideration that usually on pretreated

lignocellulosic substrates lower β-glucosidase activity can be reached than on cellulosic

carbon source (see Table 2 and 3). The ratio of 0.5-0.6 is typical only in experiments with

pure cellulose. For instance on 10 g/L cellulose (Sigmacell) (63) 1.20 FPU/mL filter paper

and 0.60 IU/mL β-glucosidase activities were reached, respectively.

0

0.5

1

1.5

2

2.5

FPA (FPU/mL)

Endoglucanase activity (10 IU/mL)

CBH I activity (IU/mL)

β-glucosidase activity(IU/mL)

Protein concentration(mg/mL)

SF CO WI SP

42

While on steam pretreated willow, with cellulose concentration of 10 g/L, 1.30 FPU/mL and

0.20 IU/mL β-glucosidase activity were achieved, respectively (104). Considering the

aforementioned results, it is believed that buffer components still enhanced β-glucosidase

production, however in lower extent. On the other hand, on steam pretreated spruce the

obtained enzyme ratio was higher than 1.0.

4.2.2. Comparing the spectra of obtained enzymes Table 10 shows the obtained specific enzyme activities. There were minor differences among

the overall cellulase activities (FPA) and the activities of main cellulase components i.e.

exoglucanases and endoglucanases. Generally, the activities of cellulase produced on

spruce were slightly lower than those produced on the other carbon sources. Within the

hemicellulases, there were more differences among the produced enzymes. As shown in

Figure 13, the higher was the xylan concentration of substrate, the higher was the obtained

xylanase activity, corresponding to both absolute and specific activities. Thus, it seemed

obvious that xylan induced xylanase production. However, even in the absence of xylan, i.e.

in the case of steam pretreated spruce as carbon source, a low constitutive xylanase

production could be observed. β-Xylosidase activity was also related to xylan content of

carbon source. SP had the lowest, while SF had the highest β-xylosidase activity. These

findings are in good accordance with the results reported by Kubicek and Penttilä (4). They

showed that xylanase and β-xylosidase production is clearly induced by xylans (4).

Figure 13 Xylose content of substrates versus measured xylanase activities (7th day)

Xylanase activity (nkat/mL, nkat/mg)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

SPS (0.0%) SPW (3.8%) SPCS (6.8%) Solka Floc (14.2%) Xylan concentration in carbon

Activity (nkat/mL) Specific activity (nkat/mg)

43

Enzymes Protein Specific enzyme activities (FPU/mg, nkat/mg)

mg/mL FPA Endoglu-canase EG I CBH I

β-glucosidase

Xylanase Manna-nase

Acetyl xylan

esterase

α-galacto-sidase

β-xylosidase

α-arabino-sidase

Produced enzymes (supernatants of fermentation broths)

SF 1.89 0.58 149 4.2 18.3 8.0 1985 75.0 5.5 0.2 12.5 18.4

CO 2.23 0.52 117 3.8 17.5 4.7 1077 40.5 1.5 1.4 3.5 16.2

SP 0.83 0.45 104 3.7 22.5 8.8 203 33.1 0.4 1.1 2.6 23.5

WI 1.05 0.56 136 5.4 23.0 5.8 868 67.0 2.2 1.3 3.5 23.0

Industrial enzymes

CE 125 0.55 130 4.6 17.1 4.2 100 26.1 14.1 0.1 2.3 4.3

EC 110 0.63 153 6.7 16.3 4.5 255 21.9 7.7 6.4 2.9 13.8 Table 10 Specific enzyme activities and protein concentrations (7th day)

(Relative standard deviations of specific activities were below 10%)

44

As it can be seen in Figure 14, acetyl xylan esterase was probably co-produced with

endoglucanase. Biely et al. (82) have reported that acetyl xylan esterase was co-produced

with both endoglucanase and xylanase. In contrast, I did not detect any correlation between

xylanase and acetyl xylan esterase activities.

Figure 14 Specific acetyl xylan esterase, mannanase, endoglucanase activities (7th day)

As it is presented in Figure 14, a good correlation could be observed between specific

mannanase and endoglucanase activities. Thus, there is a good possibility that mannanase

expression is regulated coordinatedly with endoglucanases. However, Margolles-Clark et al.

(36) indicated that mannanases were not co-produced with cellulases on most of the carbon

sources (36). Moreover, it was reported that mannanase is regulated coordinatedly with α-

galactosidase (53). In contrast, I conclude that no correlation could be observed between

mannanase and α-galactosidase activities. There were minor differences among the

produced enzymes regarding specific α-galactosidase and α-arabinosidase activities.

However, SF had very low, almost zero specific α-galactosidase activity. There were major

differences among the produced enzymes in terms of specific β-glucosidase activities. SF

had the highest and CO had the lowest specific β-glucosidase activity.

It is almost certain that differences among β-glucosidase activities were caused by the nature

of carbon sources, since all culture conditions were the same and the effect of pH was

excluded by buffering the cultures with tris-maleate buffer system. As a result of buffering,

the pH was kept in the range of 5.8 – 6.1.

Carbon sources

Specific enzyme activity (nkat/mg)

0

25

50

75

100

125

150

175

SPS SPCS SPW Solka Floc

Specific acetyl xylan esterase activity (0.1 x activity)

Specific mannanase activity

Specific endoglucanase activity

45

These results show that steam pretreated spruce and willow were considerably worse carbon

sources compared to Solka Floc, which was used as a reference. In contrast, on steam

pretreated corn stover higher cellulolytic enzyme activities were reached than on Solka Floc.

Finally, to compare the commercial and produced enzymes, it can be seen that the specific

xylanase, mannanase and β-glucosidase activities of commercial enzymes were generally

lower than those of the produced enzymes. On the other hand, acetyl xylan esterase

activities of the commercial enzymes were the highest. Celluclast and Econase have almost

similar enzyme spectra, especially in terms of cellulases. However, some differences could

be observed concerning hemicellulases. The specific α-galactosidase activity of Celluclast

was almost zero, while Econase had rather high specific α-galactosidase activity. There were

smaller differences between the two enzymes with respect to specific α-arabinosidase,

xylanase and acetyl xylan esterase activities.

4.2.3. Hydrolysis test of produced and commercial enzymes

Solka Floc, steam pretreated spruce, willow and corn stover were hydrolyzed by the four

produced and the two commercial enzymes (Paper III). The enzymes were dosed in such

way that the filter paper activities per gram of cellulose in the substrate were the same in all

cases to ensure constant enzyme/substrate ratio. The obtained sugar yields are presented in

Table 11.

Enzyme Substrate

Glucose yield (%) Xylose yield (%)

Solka F SPCS SPS SPW Solka F SPCS SPW

SF 36 55 23 28 26 33 10

CO 46 59 33 53 25 28 19

SP 31 30 20 25 19 13 6

WI 40 34 21 48 24 13 15

CE 52 41 20 37 32 21 7

EC 47 34 20 44 18 18 17

Table 11 Glucose and xylose yields after 24 h of hydrolysis (expressed in percent of

theoretical) (Relative standard deviations were around 10%)

46

It can be seen that the performance of CO was the best in almost all cases. Table 12

summarizes the enzyme activities used in hydrolysis, it seems that the good hydrolysis ability

of CO cannot be explained by its higher enzyme activities.

Enzymes FPA β-Glu. AXE CBH I Xylanase EG

FPU/g c.S. nkat/g cellulose in substrate

SF 15.0 228 158 523 56843 4269

CO 15.0 144 45 530 32754 3555

SP 15.0 232 11 596 5384 2766

WI 15.0 117 45 466 17583 2745

CE 15.0 113 380 463 2710 3519

EC 15.0 107 184 389 6066 3636

Table 12 Enzyme activities used in the hydrolysis experiments

The yields of xylose varied between 6% and 33% (see Table 11). Generally, the more

xylanase was in the hydrolysate, the higher xylose yield was obtained.

Substrate accessibility was not the same regarding to the used enzyme. Steam pretreated

corn stover was the best hydrolysable substrate in experiments with CO, SF and SP and the

performance of CO was considerably better on steam pretreated corn stover compared to

the other substrates. In contrast, WI degraded steam pretreated willow significantly better

than the other substrates.

These results bear some similarity to experiments where chemically pretreated corn fiber

was used for fermentation and hydrolysis (Paper IV). Cellulase enzyme produced on

pretreated corn fiber was used for hydrolysis of the same substrate. The results of hydrolysis

were compared to the performance of commercial enzymes. Celluclast 1.5 L and Novozyme

188 were applied to set the same filter paper and β-glucosidase activities. Hydrolysis

conversion obtained after 24 h hydrolysis are presented in Figure 15. It seems that the

enzyme produced on pretreated corn fiber could degrade corn fiber substrate approximately

15% more efficiently than commercial enzymes.

47

Figure 15 Glucose yields after 24 h of hydrolysis expressed in percent of theoretical, ─■─

enzyme produced on pretreated corn fiber (CPCF), --●-- commercial enzymes, relative

standard deviation is approximately 4%

According to these observations, it can be argued that it might be advantageous, at least in

the case of some substrates, to use the produced enzyme for the hydrolysis of the same

substrate as it was used as carbon source for fermentation. Obviously, the efficiency of the

enzyme production depends also on other factors than the bare chemical composition of the

raw material, such as accessibility of various components and their chemical or physical

associations.

0 10 20 30 40 50 60 70 80 90

100

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

Cellulose conversion (%)

48

4.3. Production of β-glucosidase by cofermentation of Trichoderma reesei and

Aspergillus niger

The production of β-glucosidase enzyme has also great importance, since it can be used to

elevate the hydrolysis potential of cellulase complex if it contains insufficient amount of β-

glucosidase. β-Glucosidase is usually produced by Aspegillus species on glucose containing

media. However, lignocellulosic materials can replace sugar if a microbial consortium is used

for enzyme production (Paper V).

Enzyme production of a well-known β-glucosidase producer, Aspergillus niger (BKMF 1305),

was examined in a culture, where it was cocultivated with Trichoderma reesei RUT C30. The

experiments were designed in such way that cellulase production of Trichoderma was kept at

low level, just enough for the breakdown of cellulosic carbon source, thereby supporting the

β-glucosidase production of Aspregillus. This type of Aspergillus can produce only low

amount of cellulase. Therefore, without Trichoderma it is hardly able to utilize cellulose.

The fermentation was optimized in shake flask experiments (Paper V.) and the optimized

conditions were used to perform enzyme production in a laboratory scale fermentor.

Mandels’ medium was used with 20 g/L waste paper as carbon source, which was equivalent

with 11.4 g/L glucose and 3.8 g/L xylose and arabinose.

The obtained enzyme activities are summarized and compared with results published by

Brumbauer et al. (128) in Table 13. Brumbauer et al. performed β-glucosidase fermentations

by monocultures of Aspregillus niger using the same strain and almost the same conditions.

The only significant difference was the applied medium. Brumbauer used 10 g/L glucose

carbon source with basal level of Mandels’ salts and nitrogen source, while in mixed

fermentations 20 g/L waste paper was applied as carbon source and the concentration of

other nutrients was doubled compared to the basal level. It was suspected that the elevated

level of salts and nitrogen source might have positive influence on β-glucosidase production

in mixed cultivation. However, later it was proven, that the elevated nutrient concentration did

not cause considerably higher β-glucosidase activity (unpublished data).

49

Shake flask experiments Fermentor

A. n. + T. r. A. niger* A. n. + T. r. A. niger*

Fermentation time (h) 168 168 125 96

Activity (IU/mL) 3.07 2.80 2.87 2.10

Productivity (IU/l*h) 18.3 19.1 22.9 21.8

Yield (IU/g glucose) 269.3 280.0 251.8 210.0

Yield (IU/g carbohydrate) 202.0 280.0 188.8 210.0

Table 13. Comparison of β-glucosidase production by mixed culture of A. niger & T. reesei

and monoculture of A. niger (* Brumbauer et al. (128))

Considering that the price of sugar is about 1 €/kg (155), while waste paper costs

approximately 0.04 €/kg (156), it can be argued that this 25 fold difference between the

prices of the two carbon sources might be advantageous in the case of large-scale enzyme

production.

Furthermore, results of mixed fermentation experiments indicate a new approach to enzyme

production. Trichoderma can be used to produce glucose for another microbe from

lignocellulosic wastes, which might be advantageous if cheap raw material is needed for

large-scale production of a valuable product fermented by Aspergillus species.

50

5. Conclusions – New scientific thesis

1. 0.1 M tris-maleate buffer could keep pH in the range of 5.8 and 6.1 (initial pH: 6.0)

throughout shake flask fermentations by Trichoderma reesei RUT C30 on Solka Floc,

glucose, lactose, steam pretreated corn stover, spruce and willow.

2. In cultivations with tris-maleate buffer β-glucosidase activities increased more than

100% compared to non-buffered cultures on Solka Floc, glucose and lactose.

Moreover, the obtained β-glucosidase activity : FPA ratios were around the optimal in

buffered cultures on all three carbon sources.

3. Steam pretreated corn stover was an excellent carbon source for cellulase

fermentation and ideal substrate for hydrolysis. In tris-maleate buffered cultures of

Trichoderma reesei RUT C30 10-40% higher cellulase activities (FPA, endo- and

exoglucanase) could be obtained on steam pretreated corn stover than on Solka Floc

that was used as reference. However, the reached β-glucosidase activity was higher

on Solka Floc compared to steam pretreated corn stover, which was probably due to

enzyme adsorption onto lignin.

4. Two industrial and the four produced enzymes were tested on the four substrates

that were used as carbon source for enzyme production (steam pretreated corn

stover, spruce and willow and Solka Floc). The highest yields were obtained using the

enzyme produced on steam pretreated corn stover. Moreover, the highest sugar

yield among all experiments was reached on steam pretreated corn stover using the

“own enzyme” i.e. the enzyme produced on steam pretreated corn stover. It might be

advantageous, at least in the case of some substrates (for instance corn stover and

corn fiber), to use the “own enzyme” for hydrolysis. This observation is also supported

by results obtained in the case of chemically pretreated corn fiber. Significantly higher

sugar yields were reached in the hydrolysis of corn fiber using the “own enzyme” than

applying commercial cellulases. This observation shows one benefit of on site

enzyme production.

5. It was found that mannanase and endoglucanase expressed coordinatedly on the

examined carbon sources, since a good correlation was observed between the two

enzyme activities. This observation might be valuable information to understand the

regulation mechanism of hemicellulase genes in Trichoderma reesei. However, to

confirm this observation more experiments are needed.

51

6. Approximately the same amount of β-glucosidase enzyme could be produced by

mixed fermentations of Aspergillus niger and Trichoderma reesei on cheap waste

paper carbon source compared to fermentation of Aspergillus niger on glucose. The

produced β-glucosidase can be added to cellulase complex to elevate its hydrolysis

potential.

52

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Enzymes for improved hydrolysis of lignocellulosics ...ABSTRACT The considerably low β-glucosidase...

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