AN ABSTRACT OF THE THESIS OF

Tor Soren Nordmark for the degree of Master of Science in Food Science and Technology presented on November 24. 1993 .

Title: A Filter Paper Assay for Low Activities and the Cultivation of reesei on Acid Whev and Sweet Whev Permeate

Abstract approved: Michael H. Penner

The traditional filter paper assay for saccharifying cellulase

originally described by M. Mandels et al (1976) has been modified to

make possible low activity determinations of Trichoderma

. The enzymatic activity appears to decline during a

prolonged incubation period if no precautions have been taken. By

means of adding bovine serum albumin and potassium chloride as

protein stabilizers and sodium azide as an antimicrobial agent filter

paper activities in the range from 0.02 to 0.37 (IUPAC assay, 1987)

can be estimated by extending the incubation time up to 20 hours. Filter paper activity values obtained by this method may be compared to those obtained by the IUPAC assay by using a conversion factor from 1.4 to 1.7.

Acid whey and sweet whey permeate have been investigated as media for growth and metabolite production by

QM 9414 using shake flask cultures and spore inocula. In the case of acid whey the mycelial growth after 2 weeks is 13 mg dry weight

/ml substrate. The specific growth rate is 0.29 / day. The appears to metabolize the whey protein the first 2 weeks. The alkalinity of acid whey rises continuously over a three week period up to a pH of 8.5. In the case of whey permeate the maximal mycelial weight gain is 4.4 mg/ml which appears after 8 days. A rise in net soluble protein level comes after 3-5 days and reaches a maximum value of 0.23 mg/ml after 2 weeks. The pH of whey permeate rises continuously to 7.5 after 3 days and then slowly declines. The net production of cellulases is low on both media. Dilution 1:6 of the acid whey, supplementation with ammonium sulfate and pH- adjustments did not enhance the production of cellulases. Acid whey supports a significant growth and sweet whey permeate shows potential for extracellular protein production. A literature review surveys the composition and uses of acid whey, environmental aspects of whey wastes, the fungus

Trichoderma reesei, the mode of action of the Trichoderma reesei cellulase system and the structure of in cotton and wood. A Filter Paper Assay for Low Cellulase Activities and the Cultivation of Trichoderma reesei on Acid Whey and Sweet Whey Permeate

by Tor Soren Nordmark

A THESIS submitted to Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Completed November 24, 1993 Commencement June 1994 APPROVED:

Professor of Food Science and Technology in charge of major

y" 1r \ >lead of the Department of Food Science and Technology

,ry in v — | i »ii \y t . i Dean of GrackJaie Schoo} \

Date thesis is presented November 24. 1993

Typed by Tor Soren Nordmark Acknowledgement

I wish to thank Dr. Michael Penner for encouragement and supportive ideas throughout the experimental work as well as assistance during the preparation of this thesis. His ability to give me freedom in determining research approaches and topics also stimulated the progress. Dr. Alan Bakalinsky offered me laboratory space and added valuable views to my microbiological perspectives. This work has been supported by funds from the Western Dairy Foods Research Center and the Oregon State University Agricultural Experiment Station to which I wish to express my gratitude. Finally I would like to express my gratefulness to Ean-Tun Liaw and other colleagues in the department who generously made arrangements in their own work in order to accommodate for my experiments and with whom I had many helpful discussions. I I

Table of Contents

1. General Introduction and Literature Review

1.1 Project goals 1

1.2 Background

1.2.1 Environmental aspects on whey wastes 2

1.2.2 Whey and whey permeate 4

1.2.3 Industrial processing of whey media 9

1.2.4 The microfungus Trichoderma reesei 11

1.2.5 The Trichoderma reesei cellulase system 14

1.2.6 Cellulose structure and cellulase action 16

1.2.7 The measurement of cellulolytic 2 2 activity

1.3 Research outline 24

2. The Application of the Filter Paper Assay for Cellulase at Low Activity Levels

2.1 Introduction 26

2.2 Materials and methods

2.2.1 Materials 30

2.2.2 Enzyme preparation 3 0 11 I

2.2.3 Enzymatic hydrolysis 31

2.3 Results and discussion

2.3.1 Principles of an improved assay 3 3

2.3.2 Activity development studies 3 8

2.3.3 Stabilization studies 44

2.4 Conclusion 50

3. Cultivation of Trichoderma reesei on Acid Whey and Sweet Whey Permeate

3.1 Introduction 51

3.2 Materials and methods

3.2.1 Substrates 54

3.2.2 Microbe 55

3.2.3 Incubation experiments 55

3.2.4 Reagents and analytical procedures 56

3.3 Results and discussion

3.3.1 The development of mycelial biomass 58

3.3.2 Metabolite production and effects on 61 substrate composition.

3.4 Final comments 80

4. Bibliography 82 Appendices

A. A protocol for the maintenance and handling of 9 7 Trichoderma reesei cultures on potato dextrose agar

B. Spore counting on PDA plates by using Rose Bengal 102

C. A protocol for the modified IUPAC filter paper 103 assay

D. An experiment demonstrating the modified IUPAC 109 filter paper assay

E The pigment production by Trichoderma reesei 113 on liquid media

F. A protocol for preincubation and postincubation 114 processing when recovering proteins on whey media List of Figures

Fig. 1. The of T. longibrachiatum. 1 3

Fig. 2. The Trichoderma reesei cellulase system. 15

Fig. 3. Cellulose structure. 17

Fig. 4. Attachment of cellulases to the cellulose. 19

Fig. 5. Modes of cellulase action. 21

Fig. 6. The relation between the amount of glucose 36 released in a filter paper assay and the logarithm of (A) the enzyme concentration and (B) the incubation time.

Fig. 7. The level of reducing sugar as glucose produced by 4 0 Trichoderma reesei QM 9414 (A) and viride (B) cellulase during a time extended filter paper assay in the absence of stabilizers plotted against the product of enzyme concentration (E as dilution level) and time (t in hours).

Fig. 8. The amount of reducing sugar as glucose released 41 by cellulase at different concentrations of non-macerated Whatman #1 filter paper and cellulase activities.

Fig. 9. Incubation of Trichoderma reesei QM 9414 cellulase 4 3 when the substrate (Whatman #1 filter paper) is either (A) always present or (B) present only during an assay hour ending by the times shown. In case A four groups of different dilutions each cover the points where E x t is equal to the original value, whereas in case B all enzyme is undiluted. V I

Fig. 10. The level of reducing sugar as glucose produced by 4 7 Trichoderma reesei (A) and viride (B) cellulase during time extended filter paper assays when stabilizers are used plotted against the product of enzyme concentration (E as dilution factor) and time (t in hours). All standard deviation bars are covered by the symbols.

Fig. 11. The result of filter paper assays (triplicate 48 samples) using stabilized (S) and non-stabilized (NS) Trichoderma reesei (A) and viride (B) cellulase. The conversion factors (f) between the apparent activities are 1.4 and 1.7 respectively.

Fig. 12. The growth of T. reesei on filter-sterilized acid 59 whey.

Fig. 13. The growth of T. reesei on filter-sterilized whey 6 2 permeate.

Fig. 14. The development of pH when T. reesei is cultivated 6 3 on thermally sterilized acid whey.

Fig. 15. The development of soluble protein as BSA when T. 6 5 reesei is grown on thermally sterilized acid whey.

Fig. 16. The development of soluble protein as BSA when T. 6 6 reesei is grown on filter-sterilized acid whey.

Fig. 17. The amount of glucose released in a filter paper 6 8 assay (IUPAC) when T. reesei is cultivated on filter-sterilized acid whey.

Fig. 18. The time course of pH when T. reesei is grown on 6 9 filter-sterilized acid whey that has been diluted 1:6. VI I

Fig. 19. The development of pH when T. reesei is grown on 71 filter-sterilized sweet whey permeate.

Fig. 20. The development of soluble protein as BSA when T. 7 2 reesei is grown on filter-sterilized sweet whey permeate.

Fig. 21. The amount of glucose released in a filter paper 74 assay (IUPAC) when T. reesei is cultivated on filter-sterilized sweet whey permeate.

Fig. 22. The effect of 0.1 % (w/v) Solkafloc addition on the 76 level of soluble protein as BSA when T. reesei is grown on filter-sterilized sweet whey permeate

Fig. 23. The change in lactose level with and without added 78 0.1 % (w/v) Solkafloc when T. reesei is grown on filter-sterilized sweet whey permeate.

Fig. 24. If 0.001 % Rose Bengal is added to the PDA then the 102 7". reesei colonies will appear more distinct and limited as can be seen from the photos above. Higher concentrations can kill the fungus.

Fig. 25. The amount of reducing sugar as glucose released 111 in a modified filter paper assay for different incubation times and for 2 dilutions. T. viride cellulase is used. VIII

List of Tables

Table 1. The chemical composition of acid whey. 7

Table 2. Relative filter paper activities obtained 112 from modified assay data for different dilutions compared with expected values based on modified assay data for a stock solution of T.viride cellulase.

Table 3. The pigment production by Trichoderma 113 reesei on liquid media. A FILTER PAPER ASSAY FOR LOW CELLULASE ACTIVITIES AND THE CULTIVATION OF TRICHODERMA REESEION ACID WHEY AND SWEET WHEY PERMEATE

1 . GENERAL INTRODUCTION AND LITERATURE REVIEW

1.1 PROJECT GOALS

The present study was undertaken as a part of the general efforts to find useful applications for acid whey and other whey products. One of the foci was to explore the properties of acid whey as a substrate for the cultivation of the microfungus Trichoderma reesei. The growth of the fungus as well as its excretion of metabolic products into the whey were of interest. Little information in this matter is available and thus few processes or projects have been started where acid whey is employed for the production of useful commodities.

At the same time there was a need to develop and refine analytical tools that could be utilized for whey products. Among the metabolites produced by Trichoderma reesei is the cellulase system

- a group of that degrade the cellulosic part of such materials as vegetable and fruit peel, cotton and wood. Thus the applications of an assay designed for the often occurring low cellulolytic activities could be widespread. Obviously, access to such an assay would be of value when investigating cellulase production by Trichoderma reesei growing on acid whey.

Finally spin-off effects of this study would include extended know-how about the handling and processing of whey related entities under cultivation conditions.

1.2 BACKGROUND

1.2.1 Environmental aspects on whey wastes

For every kg of milk used in cheese manufacture about 90 % ends up as fluid whey. The U. S. dairy plants produce 20 billion kg of whey annually (Abril and Stull 1989, Sell 1992) including 10 % acid whey (Hui 1992). One third of this amount is directly utilized in human foods or animal feeds. The remaining two thirds of the whey is, for instance, disposed of as landfill (irrigation) by pipeline or truck transfer (Sell 1992). In areas with abundant rainfall and vegetation some fertilization benefits can be obtained. Even more purposefully research has demonstrated that acid whey can be used effectively in reclaiming sodic soil (Jones et al 1993). However the high levels of nitrogen, phosphorus and minerals in whey can kill vegetation and the spread has thus to be managed carefully (Zall

1979).

The disposal of whey remains the greatest waste problem for the dairy industry because of the great volume, the high biological oxygen demand (BOD) and the often acidic properties.

Nearly 90 % of U. S. dairy plants are connected to sewer systems

(Sell 1992) and most of the whey waste is still transported to them or directly into streams. A dairy plant may discharge 10-100,000 kg whey daily. The BOD for whey is 30-60,000 mg/liter (the lower range applies to acid whey). The lactose accounts for 90 % of this value. Every 1000 kg whey requires 4.5 million liters of fresh aerated water for its biological oxidation. This corresponds to the daily BOD load from 470 people. Obviously concern is raised about the damage to aquatic life support systems and the overload on municipal sewage treatment facilities. Thus the load from one dairy plant is equal to that from a medium size town (Zall 1979).

Acid whey has currently very limited applications and thus relatively more of such whey is being disposed of. Its acidic character causes corrosion to metal tanks and pipelines thereby increasing the cost of its handling. The use of ultrafiltration through

2 nm membranes for the purposes of protein removal and concentration has in recent years emerged as a promising whey treatment alternative. Lactose can then be removed by means of reverse osmosis and the product has a BOD of only 3 % of its original value (Sell 1992).

Agricultural waste, domestic refuse and industrial waste are the three major waste sources in the human society. As indicated above some management alternatives are to make use of the so- called waste (minimization [Snider 1992]) and to perform waste treatment before disposal (Kharbanda and Stallworthy 1990). As with all changes, values and costs are involved which may slow down the progress towards a more harmonic relation to natural resources. This thesis project should be seen as a contribution in that direction.

1.2.2 Whev and whev permeate

Whey is the liquid drained from the curd when making cheese or manufacturing casein. If the goal is to produce swiss or cheddar type cheeses, pasteurized milk is first inoculated with lactic acid culture and then the enzyme rennet is added to coagulate the caseins. The coagulum is cut into small blocks and heat treated to expel whey, whereafter the whey is drained out. The cheese is then generally left for ripening. The whey obtained from this procedure is sweet whey that has a maximum titratable acidity of 0.16 % as lactic acid (Code of Federal Regulations, April 1993) and usually a pH within the range 5.8-6.6 (Zadow 1986). When cottage cheese or quarg type cheeses are produced, skim milk is the common raw material. It is pasteurized at 72 0C for 15 sec. (High Temperature

Short Time) and a high amount of starter culture - usually

Lactococcus lactis and cremoris together with a flavor giving strain such as Leuconostoc citrovorum (Scott 1981) or Lactococcus diacetylactis - are then added. Nowadays a small amount of rennet is also often supplied to improve the firmness of the coagulum. Part or all of the lactic culture may be replaced by naturally formed lactic acid or by hydrochloric acid. The cottage cheese curd is heat treated at 50-55 0C for 1-2 hours (Fox 1987) and the whey is then removed. The quarg curd is traditionally not heat treated and the final cheese product has a smoother consistency. Acid whey has a pH in the range 4.0-5.0 and a titratable acidity of 0.4-0.6 % as lactic acid (Zall 1979). If the acid present is due to the action of the starter culture, a significant amount of lactose - approximately 0.7

% - is used up. Other cheesemaking procedures may result in titratable acidity and pH values between those for sweet and acid whey (Zadow 1986).

The whey composition varies with farm, breed, season, type of cheese and aging of the whey. About half of the milk solids are present in whey. Average data for acid whey are listed in Table 1.

Minerals are listed in order of descending concentration. From an operational standpoint, however, proteins that are present in the pH

4.6 supernatant of milk are referred to as whey proteins. That includes the proteose peptone fraction which is essentially a set of heat stable fragments from betacasein (Hui 1993). Whey products have GRAS status (Code of Federal Regulations, April 1993) and are considered of good nutritional value for human use (Zadow 1986).

The whey proteins have a balanced amino acid composition and a high digestibility (Hui 1992). Whey and in particular acid whey is rich in Table 1. The chemical composition of acid whey.

Lactose 4.3 %

Lactic acid 0.7 %

Protein 0.7 %

Betalactoglobulins 50% Major aminoacids Alfalactalbumins 15% Glutamic acid Bovine serum albumin 5% Aspartic acid Immunoglobulins (IgG) 10% Leucine Proteose peptones, 20% Lysine residual casein and enzymes

Fat (Sat'd : Monounsat'd Polyunsat'd = 0.04 % (skim milk) = 20 : 10 : 1) 0.3-0.9 % (whole milk)

Ash 0.5 % (0.4 - 0.8 %)

Potassium 1.4 g/kg Calcium 1.0 g/kg P, Cl, Na, S, Mg Zn, Fe, Cu, Mn, Se

Vitamins

B2 1.2 ppm Pantothenic acid 3.3 ppm B12 1.5 ppb

Water 93.7 % 8

minerals such as calcium and phosphorus. The contents of water soluble vitamins such as riboflavin are also of importance. The lactose has low sweetness and that is considered an advantage in some food applications. Whey cheeses - gjeitost or mysost type - are popular in some countries. They are produced by cooking the whey until the desired concentration of solids is achieved. People who do not have sufficient betagalactosidase activity in their digestive tract will have problems with these products due to lactose intolerance. Available composition data for whey also indicate that all of the nutrients generally required for fungal growth are present. Unpasteurized whey has a high count of molds and yeasts.

Ultrafiltration of whey results in whey permeate that can be sweet or acid depending on the raw material. Common membranes have cut-off values around 15-20 kD and thus most proteinaceous material is removed. The whey may first have been clarified from phospholipoproteins by an initial heat treatment in the presence of calcium and a following microfiltration using ceramic membranes

(Daufin et al 1992). 1.2.3 Industrial processing of whey media

Sweet whey aimed for use in foods must be processed within hours of its removal from the curd. Acid whey at a pH of 4.7 or lower is more stable because of the inhibited bacterial growth. The whey is first pasteurized and concentrated by means of vacuum evaporation or reverse osmosis in the plant and then traditionally spray-dried to yield whey powder. Caking and lumping in whey based products may be due to lactose hygroscopicity. In order to avoid this phenomenon, or to get delactosed whey powder, crystallization should be performed before the spray-drying step (Hui 1992, Zadow

1986). Demineralized whey can be obtained by using electrodialysis.

Acid whey from a cottage cheese plant has reportedly been clarified and treated with cationic and anionic columns to remove most of the salt and lactose before it is used in vegetable oil based dips and other products (Schexnayder 1991). Efforts to use acid whey in fermentation processes have been made (Stewart and Gilliland 1982) as well as to use it in sweet syrups after ultrafiltration and direct enzymatic treatment (Abril and Stull 1989). Clarified and ultrafiltrated whey leaves a retentate, i. e. whey protein 10

concentrate (WPC). This product can be utilized in baking to improve foaming properties. Upon heating dispersions of WPC can act as gelling agents mainly due to the content of betalactoglobulin. It may be further processed to yield individual whey proteins or hydrolyzed to yield peptides for specific dietetic purposes. A main application of WPC's is in the production of baby formulas (Hui 1992).

Ultrafiltration of unpretreated whey and particularly acid whey causes extensive membrane fouling and flux reduction. This varies with membrane type but is generally due to the phospholipoproteins and is markedly increased by the mineral contents, i. e. calcium.

Cottage cheese whey is rich in colloidal calcium phosphate (Heng and

Glatz 1991, Morr 1989, Pierre et al 1992). Sugars as lactose tend to precipitate in the membrane pores (Kuo and Cheryan 1983). Removal or modification of the phospholipoproteins or chelation of the calcium will improve the flux rate. It has also been observed that increased transmembrane pressure results in increased retention of calcium and lactose when cottage cheese whey is ultrafiltrated

(Roehl 1990). 11

1.2.4 The microfungus Trichoderma reesei

Trichoderma sp. constitute up to 3 % of the total fungal population in many forest soils especially those with an acidic character. They are likely to be found in abundance in the rhizosphere in coniferous forests as well as in the litter of humid mixed hardwood forests (Eveleigh 1985, Kendrick and Parkinson

1990). Pasture soils and a wide range of other habitats also contain

Trichoderma sp. As saprobes they are important in the natural recycling of nutrients. They are metabolically versatile and degrade a variety of carbonic substances such as cellulose, starch, pectin, xylan, laminaran and chitin. Nitrogen sources like proteins and ammonium compounds are all readily utilized. Potassium is a major mineral requirement and it is also a major wood ash and whey component. Trichoderma sp. can degrade hydrocarbons and are thus found in oil-polluted soils and even in jet fuel, rubber and polyethylenes (Eveleigh 1985). They are aerobic and have a septate mycelium with the cell wall made up of layers of betaglucans and chitin. The wild type ancestor to T. reesei QM 9414 is T. longibrachiatum (Montenecourt 1983), which is a mesophilic soft- 12

rot fungus. Its general placement among living organisms can be seen in Figure 1. There are still considerable problems with the taxonomic classification of species (Herrera-Estrella 1993), however, and the morphological differences between Trichoderma sp. is a task for expert microbiologists (Eveleigh 1985). The colonies of

T. reesei QM 9414 often appear yellow due to pigment production.

Under restrained conditions and provided some short exposure to blue light has occurred (see section 1.2.5), green rough-walled conidia (asexual spores) are produced from tip cells in the branched mycelium. Hyphal tips are also the centra for protein synthesis. The presence of sterile mycelium makes colonies appear white.

The T. reesei QM 9414 genome has a size of at least 33 million basepairs that are located on seven different chromosomes

(Maentylae et al 1992). Cellulase genes are found on two of them.

Thus both exoglucanases (CBH) and one endoglucanase (EG) gene are located on one single chromosome whereas another endoglucanase gene is found on a different chromosome. These genes have been expressed in hosts to obtain more pure cellulases for the study of cellulase action. The QM 9414 strain produces relatively small 13

Kingdom FUNGI

Class ASCOMYCETES

Subclass EUASCOMYCOTIDAE

Order

Family

Teleomorph: Anamorph: Genus TRICHODERMA

Species RUFA LONGIBRACHIATUM

Strain: First called T. viride Renamed T. reeseiQM 6A

Fig. 1. The taxonomy of T. longibrachiatum. 14

amounts of betaglucosidase (BG). The cellulases display high

intrinsic stability at room temperature, but their activity is

affected by periods of drying or freezing and more so in the first

case than in the second (Sinsabaugh and Linkins 1989).

Although not generally considered as an invasive human

pathogen, T.viride has been able to cause fungal peritonitis in a weakened individual (Loeppky et al 1983). Another patient was

proven to have a pulmonary fungus ball consisting solely of T. viride

mycelia (Escudero et al 1976). People with specific sensitivity or who are already afflicted by a fungal skin disease should protect themselves from direct contact with the mycelium.

1.2.5 The Trichoderma reesei cellulase system

Although an inducer of cellulase in Trichoderma reesei is still to be found, much information has been collected regarding the

regulatory mechanisms in this fungus (see Fig. 2). Cellulose,

sophorose, maltose, lactose, glucose and many others are all able to

provoke synthesis of cellulases. A conidial cellulase system is

known to exist with a composition that differs from the mycelial cellulase system. Access to some light is of importance in the 15

CONIDIA: inhibits CBH & EG Constitutive cellulases: synthesis & activity cell wall bound: 50 % CBH II exo-exo synergistic ^^^ cellobiose (major product) 25 % CBH I attack on crystalline ""™ cello-oligosaccharides EG III cellulose d-cellobiono-1,5-lactone ' plasma membrane bound: (all complete cellulases (from oxidized cellulose) betaglucosldase (alkaline) known contain CBHs)

MYCELIUM: Sporulation extracellular within 24 hours betalinked disaccharide betaglucosldase 4 permease (pH>S) CBH II expression glucose enhanced transglycosylation ' repression of via intracellular betaglucosldase betaglucosldase responding nucleotide (provokable by sophorose) synthesis palindrome in the CBH II or EG I promotor other intermediate or sophorose (pH 3 optimum) t provoker(s) cyclic AMP elevated \ / "true" inducer adenyl cyclase elevated

gene transcription within 20 min. and with Light at 3B5 nm and 445 nm constant ratio between CBH I, CBH II and EG I peaks starts induction

translation on ribosomes/ER (more ER present in hypersecreting mutants) Light also stimulates: * hyphal branching (on solid media) vacuoles (with EG I) & Golgi bodies; * the TCA cycle whereby the ATP enzymatic regulation of cellulases level rises

plasma membrane

Attack on cellulose: -^ Release ot induced cellulases:

CBHs and most EGs contain cellulose up to 70% of soluble protein is cellulases, binding domains; cell wall heteroglycans all of which are N-glycosylated for heal may have affinity for cellulose. and proteolysis stability; CBHs and EGs EGs rapidly hydrolyse amorphous may be O-glycosylated to ease secretion. cellulose. Different enzyme systems are exhibited Synergism exists between CBH I, CBH II on different substrates. One example: and EGs. 60% CBH I 20% CBH II 10% EG I EG III and betaolucosidase

Fig. 2. The Trichoderma reesei cellulase system. 16

lifecycle of Trichoderma reesei primarily because it is required for sporulation in this asexual fungus. Constitutive cellulases on the spore surface start the breakdown of cellulose to smaller molecules that can be recognized by the fungus as the sugars just mentioned are recognized. Transport mechanisms carry the information to the genetic material where transcription occurs. Following translation enzymatic modification of the cellulases takes place, whereafter they are released from the cell surface (Farkas et al 1990, Kubicek

1987, Kubicek et al 1990, Kubicek et al 1993, Messner et al 1991,

Messner and Kubicek 1990 and Szakmary et al 1991).

1.2.6 Cellulose structure and cellulase action

Cellulose as a macromolecular substance with many origins has been a hard object to get grasp of in terms of its many variants and how the functional properties are related to the structure. Recent work has cast new light on this issue (see Fig. 3-4). It is apparent that efforts to quantify the interaction between cellulose and its environment must be a complicated task. Although its smallest repeating unit is cellobiose that in turn consists of 2 glucose

(glucopyranosyl) units, there are numerous options for the design of 17

The structures considered are primarily mature native cotton and wood. The chemical reactions only take place at the submicrofibhl level or lower.

Cellulose fiber Macrofibril

Macrofibril (parallel) or Up to a few hundreds microfibril (parallel or of microfibrils spaced randomly woven) layers at 20 nm (but depends wound around a central on the degree of swelling). axis; reversal of direction Diameter less than 15 occurs. microns. Cavities and pores may be present.

Submicrofibril Microfibril

Consists of stacked unit crystal 9-12 submicrofibrils (elementary cells of parallel cellulose chains fibrils) together with amorphous lined up in distinct planar layers. regions every 100 nm of length. Diameter 2-6 nm.The cellulose Diameter 3-10 nm, length 0.1- chains - as well as the submicro- 1000 microns. Small 60 nm fibrils themselves - may be long crystallites (micelles) of twisted together in some species. stacked glucan sheets connected by weak Van der Waal's forces can be found.

Unit crystal cell Cellulose molecule

Ribbon-like approx. 2-fold Repeated 1.0 nm long helix of 2 cellulose chains beta-1,4-linked anhydro- (1 staggered central chain cellobiose units. The D.P. and 4 quarter chains) in all is 3,500-36,000 gluco- polymorphs. The native pyranosyl units, that are monoclinic unit crystal cell in chair conformation and dimensions are 0.8x0.8x1.0 alternatingly rotated half nm depending on cellulose a turn along the chain source and polymorph. length. Non-reducing ends appear to be stereochemi- cally identical.

Fig. 3. Cellulose structure. 18

The six known polymorphs result from a set of binding options; they thereby differ in density and surface area. Most native cellulose is of type I where k1, l-beta and parallell arrangement dominate.

Secondary structure Tertiary structure: of intrachain H-bonds: k1 = C6-0 and ring-0 bind to A) Different lattice parameters. C3-0 on the adjacent B) Different interchain hydrogen glucopyranosyl residue bonding pattern (types l-alpha and k2 type bonding from and the more stable l-beta). these 2 residues to the C) Parallel or antiparallel (that connecting residues on provides for intersheet H-bonds) other cellobiose units. chain polarity. K2= Ring-0 binds to C3-0 on the adjacent residue.

Fig. 3 cont'd. Cellulose structure. 19

Accessibility Dimensions and shape vary with cellulose morphology, type of enzyme and stage of hydrolysis. The CBH's are Binding to 5x20 nm in size. Competitive crystalline adsorption may occur. surface

Tightness of Increased crystallinity results binding in increased affinity for both CBH's and EG's. The glyco- sylation in their binding domains may provide for hydrophobic interactions that are enhanced by decreased pH. Synergism requires good binding.

Binding to The surface area is Water can be H-bonded amorphous 4 times larger than monomolecularly to the region in crystalline regions. hydroxylgroups (and on The binding domain in crystalline surfaces); it the cellulase is of can also be loosely adsorbed less importance. polymolecularly. Swelling will reduce the crystallinity left and increase accessibility while water breaks H-bonds and rearranges the higher order structure of cellulose.

Fig. 4. Attachment of cellulases to the cellulose. 20

the whole cellulosic material. Characteristics as degree of polymerization (D. P.) and crystallinity index (C. I.) must be complemented with many other data such as the adsorption properties of other substances like cellulases to accessible cellulose surfaces over time. Some cellulosic materials appear more inert than others. Thus filter paper and microcrystalline cellulose

(Avicel) are relatively inert substrates, whereas mechanically treated, amorphous cellulose is much easier to degrade when using cellulolytic enzymes. (Atalla and Vanderhart 1989, Coughlan 1992,

Dinwoodie 1989, Kirk-Othmer 1979, Nevell and Zeronian 1985,

Seymour and Carraher 1988, Sarko 1985, Turbak and Sakthivel 1990,

Weimer et al 1991). The mechanisms by which cellulase systems degrade cellulose are slowly being revealed. The access to more pure crystalline cellulose as well as the possibility of expressing cellulase genes in hosts to obtain more pure or modified enzymes have contributed considerably to this progress. The cellulase system of T. reesei is the best studied and the features of its degradation of cellulose are outlined in Figure 5. Endoglucanases (EG) make cuts in the crystalline cellulose surface thereby leaving fragments that can 21

Synergism occurs primarily in the degradation of crystalline regions but not lor all types of substrate and the extent varies considerably with the concentrations and conditions involved. The hydrolytic mechanisms employed by the cellulase active sites have been described as lysozyme like and beta-amylase like. The numbers 1-8 below refer to a time sequence of events. The T. reesei system is featured.

(1) Diffusion by CBH's and EG's break the weak H-bonds between the (nicrofibrils. Microfibril (2a) Endoglucanases (EG III) (4) Possible water uptake after addition EG's help prepare crystalline of CBH's results in increased acces- regions for attack by CBH's but sible surface area. easily degrade amorphous regions, easier if the cellulose (2b) Endoglucanases (EG III) is in a staggered array. They Apart from amorphous regions, EG's attach readily along the may make superficial cuts in the microfibril surface and crystalline surface. This activity is cleave internal glycosidic facilitated if the area is weakened bonds in the amorphous by crystal faults, skewing of adjacent cellulose, whereby free sheets or binding of other enzymes, chain ends become exposed intersheet hydrogen bonds would for action by CBH's. have an opposite effect. Whiskerlike submicrofibrils, that remain attached (3a) CBH I to the lattice, are splayed off from Binds along crystal edges and the surface. The EG binding domain cuts reducing ends. It coopera- may help unzip the microfibril by tes synergisticaliy with EG's y disrupting the (H-) bonding pattern in detachment and digestion V in the crystal structure. When acting of surface submicrofibrils. It I (at the common pi) in a complex with facilitates EG's disruptive / CBH, maximal synergism is obtained. work upon binding and cuts Only crystal faces with accessible the outermost chains in 2,3- and 2,3,6- hydroxyl groups are the submicrofibril whiskers. attacked. The CBH activity creates EG I has minimal binding to clusters and plaques of crystalline cellulose and appears not digestion products. to act synergisticaliy with CBH's.

Formation of Clusters (S) and (3b) CBH II Plaques (6), composed of swollen This enzyme is more substrate but water insoluble cellulose inter- specific than CBH I and binds mediates together with crystal and preferentially to crystal tips. chain fragments in various propor- Cooperates synergisticaliy with tions. EG's in amorphous parts and with CBH I in crystalline parts when Crossfracture (7) caused by the cutting liberated submicrofibril action of an EG-CBH complex. chains, since CBH I prepares new sites for attack by CBH II. Beta-Glucosidase CBH II removes cellobiose Cleaves cellobiose to glucose units, units from the non-reducing end whereby product inhibition of EG and It degrades amorphous CBH activities is decreased. Weak cellulose less efficiently, although inhibition by glucose of the synergism synergism between CBH I and between EG and CBH has been reported. CBH II has been reported on Some EG's may convert cellobiose amorphous substrate. Complex to glucose by transglycosylation formation between CBH's occur. if cellulose is present.

Fig. 5. Modes of cellulase action. 22

be further degraded by the exoglucanases (CBH). The CBH's are not required for the degradation of the amorphous regions of the cellulose. Cellobiases (betaglucosidases) will cleave cellobiose to glucose. (Beguin and Aubert 1992, Hoshino et al 1992, Klyosov 1990,

Sprey and Bochem 1991, 1992 and 1993, Tomme et al 1990, Walker and Wilson 1991, White 1982).

1.2.7 The measurement of cellulolvtic enzyme activity

There is widespread interest in the application of cellulase enzymes to technologies associated with forest products utilization, textile processing, food processing and biomass conversion. These technologies are dependent on inexpensive sources of cellulase enzymes with high specific activities.

Consequently, considerable research activity is directed at identifying novel cellulase enzyme systems with properties which may be exploited for industrial purposes. An obvious prerequisite for any of these studies is an appropriate method for the measurement of cellulase activity. Microorganisms which readily degrade native cellulose secrete a family of cellulolytic enzymes which act in concert to solubilize/saccharify crystalline cellulose. 23

In many cases it is of primary interest to measure the specific activity of the complete enzyme mixture produced by a given microbe. This is in contrast to assays designed to measure the activity of a specific component of the complete enzyme system.

The need for an assay of complete cellulase activity is particularly true for the cellulases due to the well documented synergism between individual cellulase enzymes. Substrates that are particularly good for measuring complete cellulase systems are those which contain an appreciable fraction of crystalline cellulose, such as filter paper, Avicel and cotton. Thus the water soluble carboxymethyl cellulose (CMC) is no longer used for the estimation of total cellulase activity. Viscosimetric methods were designed for evaluation of the degree of hydrolysis in the produced slurry. At the present time the most widely used assay for total cellulase activity is the IUPAC filter paper assay (Ghose 1987), the origin of which can be traced back 25 years (Mandels and Weber 1969). The experimental work described in section 2 considerably extends the range of detectable activities while building on the principles of the IUPAC assay. 24

1.3 RESEARCH OUTLINE

Trichoderma reesei QM 9414 was used to evaluate the

properties of acid whey as a growth substrate for Trichoderma

microfungi. Acid whey batches were obtained from two different

regional dairy plants in order to work with commercial type

substrates. Ultrafiltrated whey was used as a complementary whey

based substrate. It is easier to process whey permeate than acid whey. The employment of whey permeate also offered an opportunity

to determine the effects of the whey proteins on the outcome of the

experiments. Efforts were made to keep the whey media as close to

unprocessed characteristics as possible despite the necessity of

sterilization and pretreatment. Laboratory protocols for the

processing of the culture samples and in some cases improved assay

methods had to be developed before comparative studies could be

carried out. In most cases, the parameters monitored included fungal growth, protein production, pH of the medium and production of cellulase enzyme activity. Experiments were designed to evaluate the major period of fungal growth, i. e. prior to extensive mycelial 25

deterioration. Efforts were made to boost the yield of cellulase enzymes in some cultures.

A major part of this thesis involved the development of a cellulase enzyme assay appropriate for enzyme solutions of relatively low activity. The first part of this phase of the research was to evaluate the fate of cellulase enzymes during prolonged incubation under IUPAC filter paper assay conditions. The IUPAC assay conditions were then modified to accommodate enzyme preparations of low activity. This modification involved the identification of appropriate enzyme stabilizers. To ensure a broad applicability, a commercial cellulase preparation from Trichoderma viride was tested in addition to Trichoderma reesei QM 9414. 26

2. THE APPLICATION OF THE FILTER PAPER ASSAY FOR CELLULASE AT LOW ACTIVITY LEVELS

Key words: Cellulase activity, cellulase stability, filter paper assay, incubation time, Trichoderma.

2.1 INTRODUCTION

Cellulases are produced by a large number of aerobic and anaerobic fungi and bacteria. Trichoderma sp. are known for their potent ability to enzymatically degrade cellulosic materials.

Cellulolytic enzymes have found applications in the fields of forestry, biomass conversion, food processing and cellulosic waste treatment. The amount, type and cooperative pattern of the molecules active in any given cellulase system depend on the source of the enzyme, the substrate and the environmental conditions

(Cochet 1991). Three classes of cellulase enzymes cellobiohydrolases (CBH), endoglucanases (EG) and betaglucosidases

(BG) - are now well recognized. Only a few organisms produce complete cellulase systems, i.e. they possess a pronounced ability

(Stahlberg et al 1993) to break down crystalline cellulose. 27

In actual biomass material cellulose is generally embedded in other structures containing hemicelluloses, lignin, proteins, lipids etc. It is difficult to assess cellulase enzyme preparations of lignocellulosic raw materials. Several cellulase assays exist; however, it has been difficult to correlate the different assays due to the complexity of the substrates and the synergism between the enzymes. Currently, cellulase assays based on the degradation of filter paper are widely accepted. The early filter paper assay from 1969 (Mandels and Weber 1969) was modified a few years later in order to obtain good linearity over a wide range of activities and to better account for a combined activity on amorphous and crystalline substrates (Griffin 1973). Mandels et al (1976) suggested that international enzyme units should be calculated on the basis of 2 mg glucose released from a 50 mg piece of filter paper.

The assay recommended by the IUPAC Commission on

Biotechnology is the filter paper assay described by Ghose (Ghose

1987). If appropriate dilutions have been made, a graph displaying how the amount of reducing sugar as glucose varies with the 28

logarithm of the enzyme concentration will provide the concentration corresponding to exactly 2 mg glucose. International

Units (IU) are defined as the number of (imoles/min of substrate converted. If each complete hydrolytic event is considered to produce 1 molecule of reducing sugar (glucose), then 0.37 lU/ml only need to be divided by the concentration (i.e. the inverse of the dilution factor) in order to get the filter paper activity (FPA). Low cellulase activities as defined by this assay are encountered in many applications. A low level can be due to type of strain, growth medium, time of harvest and application conditions. The measurement of low activities is difficult since the traditional plot of reducing sugar versus the logarithm of the enzyme concentration becomes less steep and is non-linear. Thus extrapolation of low activity values is not appropriate. To assay low activity solutions one must concentrate samples by laborious ultrafiltration or freezedrying, with a risk of some loss of activity (Roseiro et al

1993). No other method is currently available for the determination of lUPAC-FPA's for low activity samples. 29

Forintek Canada Corp., Ottawa has described a filter paper assay resembling lUPAC's except that lower amounts of glucose/ml are targeted, the activity values thereby being larger than the lUPAC-FPA's (Chan et al 1989). Another approach for low activity samples involves the addition of a small level of BG to cellulases

(Breuil et al 1992) of low BG activity as is the case for Trichoderma reesei. Apart from BG addition the ratio between FPA and cellobiase

(BG) activity has been suggested as a measure of the hydrolytic potential of cellulase enzyme preparations (Cochet 1991). The significance of any of these filter paper based assays obviously depends on how the enzyme is used and the substrate employed.

The method described in the following permits the estimation of lUPAC-FPA's at levels up to 20 times lower than those previously assessable. No additional sample processing and no expensive reagents are required. 30

2.2 MATERIALS AND METHODS

2.2.1 Materials

Reagent components were dinitrosalicylic acid (Sigma Chemical

Co., MO) and sodium hydroxide, potassium sodium tartrate, phenol and sodium metabisulfite all of which were obtained from Mallinckrodt Specialty Chemicals Co., KY. Potential cellulase stabilizing agents tested were sorbitol, D-mannitol, inositol, glycerol, L-ascorbic acid, bovine serum albumin (fraction

V), heavy mineral oil, potassium chloride, calcium chloride and magnesium sulfate. The antibacterial agent sodium azide was added at 0.02 M concentration to the citrate buffer.

2.2.2 Enzvme preparation

Trichoderma reesei QM 9414, from which a complete enzyme system is obtained, was grown in our laboratory using shake-flask cultures for 7 days at 27 - 28.5 0C in dim light. The hammer-milled spruce cellulose, Solkafloc SW 40 (James River Inc.,

Berlin, NH), was used at the concentration level of 8 g/l for cellulase production together with Mandel's medium [ 1.5 g/l ammonium sulfate, 2 g/l monobasic potassium phosphate, 0.32 g/l 31

calcium chloride, 0.32 g/l magnesium sulfate heptahydrate, 1 ml/I trace metal solution (containing ferrous and manganese sulfate, zinc chloride and cobalt chloride), 0.31 g/l urea, 0.8 g/l proteose peptone

(DIFCO Laboratories, Ml), 1 m/l Tween 80]. The flask contents were pH-adjusted to 4.8, filtered through 1.2 fim glass microfiber filters and subsequently concentrated 20-fold by using a

Minitan ultrafiltration unit (Millipore Corp., MA). The enzyme fraction was first precipitated by addition of cold acetone (70 % of the total volume). Following centrifugation, the enzyme precipitate was washed three times with 100 % acetone, then vacuum dried at +

2 "C using a Rotovapor equipment (Buchi Co.). The result was a crude enzyme powder, which was stored at + 4 0C. The commercial preparation Cellulysin (Calbiochem Corp., CA) containing

Trichoderma viride cellulase was employed without further processing.

2.2.3 Enzymatic hydrolysis

IUPAC filter paper assay conditions were used if not otherwise stated. All filter papers (Whatman #1) were weighed to 1 mg accuracy. Enzyme stock solutions were made up by dissolving crude 32

cellulase powder in double distilled water immediately before use.

The reducing properties of the hydrolysates were evaluated by the dinitrosalicylic acid assay in all cases (Breuil and Saddler 1985,

Miller 1959). 33

2.3 RESULTS AND DISCUSSION

2.3.1 Principles of an improved assay

The classical Michaelis-Menten theory based on the initial action of a single enzyme species (E) on a well defined substrate (S) states that when So » Eo the initial velocity is:

Vo = (Vmax x So)/(KM + So) = (kcat X EQ X SO)/(KM + So)

In case So » KM then VQ is proportional to Eo and the rate of product

(P) formed is:

dP/dt = - dS/dt = VQ = kca, x Eo

Integration of the Michaelis-Menten equation in the same case shows that P is proportional to time (t) (Kuchel and Ralston 1988).

However, for several reasons these relations cannot be taken for granted under the condition used in the filter paper assay (Schwald et al 1988, Mandels et al 1976, Mandels et al 1978). The cellulase system consists of multiple enzyme species acting 34

synergistically, activators and inhibitors might be present and the substrate is not of a single type. Steady-state conditions are mostly lacking (Tomme et al 1990). In particular, an extension of the incubation time could cause a decline in enzymatic activity due to denaturation processes. Laboratories utilizing IUPAC filter paper assay conditions have presented data for 0.5 - 2 hour incubations employing cellulase activities in the range 0.2 - 0.7 FPU which suggests that a change in hydrolysis time may substitute for an equal change in enzyme activity (concentration) provided that the same level of substrate conversion is achieved (Ghose 1987, Mandels et al 1976). This means that E and t may appear symmetrically as

Ext in an integrated rate equation, it does not mean that P is proportional to E and t or that the enzymatic hydrolysis follows

Michaelis-Menten kinetics in the filter paper assay. It is recognized on empirical basis that, at enzyme concentrations releasing close to

2 mg reducing sugar as glucose in the IUPAC one hour assay, the glucose level is related to the logarithm of the enzyme concentration by a proportionality factor (k), that represents type of substrate, type of cellulase and other assay conditions: 35

A P = k x A log E

Thus a plot of P vs. log E for different dilutions of an enzyme sample is a straight line (Reese and Mandels 1971). The same plot for the same dilutions of an enzyme sample of another activity is also a straight line parallel to the former line since the k-value is the same. If Eactive is independent of time, then time can substitute for enzyme concentration (see below) and a plot of P vs. log t for different incubation times is also a straight line with the same k-value as above. An enzyme acting at a m-fold lower concentration but over a m-fold longer incubation time would result in the same line as the original enzyme solution would give. Figure 6 is a theoretical plot of P against log E and log t for conditions when E does not change during the incubation period.

The reaction rate in general in the assay is a function of So and the time dependent variables E, S, A (activator concentration) and I (inhibitor concentration). If variations in So, A and I between assays can be considered insignificant and assuming that the effect of substrate recalcitration etc. is time independent if the final 36

mg glucose

X-cellulase: sample (A) # 1 and dilution 1:1

X-cellulase: sample # 2 and dilution 1:1 (alt sample # 1 and dilution 1:2)

log E

mg glucose X-cellulase: sample # 1 and incubation time 1 hour (B)

X-cellulase: sample # 2 and incubation time 1 hour (alt. sample # 1 and incubation time 0.5 hour)

log t

Fig. 6. The relation between the amount of glucose released in a filter paper assay and the logarithm of (A) the enzyme concentration and (B) the incubation time. 37

degree of substrate conversion is the same in the assays, then integration of the rate equation yields the amount of product formed:

t P = J f [E(t)] dt 0

If as suggested above P is a function of Eo x t only and thus no change in cellulolytic activity takes place over time, then a m-fold decrease in the original enzyme concentration together with a m-fold increase in the incubation time will always result in the same P value. Thus if P is plotted against Eo x t for a set of different enzyme concentrations, then this set of curves will cover each other. If on the other hand E varies with time, then the plots for different enzyme concentrations will appear separated from each other since, for instance, E may be lowered over a prolonged time owing to denaturation .

Stabilization of the enzymatic activity could thus allow the application of an extended time assay when only small amounts of cellulase are available. Assuming the validity of the model the FPA of the original sample in the low activity range could be calculated 38

from

FPA = [f x 0.37]/[conc. x #hours] (filter paper units)

where f is a factor that accounts for any difference in hydrolysis

rate due to any necessary assay modification. The experiments were

designed to demonstrate the validity of this hypothesis by

investigating the development of cellulase activity over several

hours and trying to find means for maintaining the original activity.

2.3.2 Activity development studies

Any diagram of reducing sugar vs. the product of enzyme

concentration and incubation time for a set of different dilutions would reveal if there is any time dependent decline in enzymatic

activity (Selwyn 1965). Four different dilutions (1:1, 1:3, 1:9 and

1:27) in duplicates of Trichoderma reesei cellulase were each

incubated with filter paper during periods of four different lengths

(1, 3, 9 and 27 hours). The subsequent DNS assay resulted in the data

plot in Figure 7:A. Since different dilutions appear to give rise to different graphs the enzyme activity seems to vary with time, i.e. 39

consistent with a loss of enzyme activity. Multistep thermal denaturation (Dominguez et al 1992, Lencki et al 1992, Baker et al

1992), proteolytic activity (Haab et al 1990) and substrate inhibition (Huang and Penner 1991, Liaw and Penner 1990, Mandels et al 1981) may be causative agents separately or in combination.

Trichoderma viride cellulase was employed in a study (Fig. 7:B) that was otherwise identical to the previous experiment. The same conclusions as before can be drawn in this case and thus an activity decline is expected to occur in case of other Trichoderma cellulase preparations as well.

The possibility exists that substrate inhibition takes place with filter paper as a substrate under the assay conditions. Therefore duplicates of 3 different concentrations of Trichoderma viride cellulase were each incubated together with 4 different concentrations of filter paper. No significance for substrate inhibition can be extracted from the result shown in Figure 8. It may be noted that the glucose level is still relatively dependent on the substrate concentration at the 50 mg level. Since the Whatman

#1 filter papers have a weight variation range of ±10 % (Whatman 40

mg glucose (A)

Dilution 1:27 -o— Dilution 1:9 Ext -o— Dilution 1:3

-A Dilution 1:1

mg glucose (B)

Ext

Fig. 7. The level of reducing sugar as glucose produced by Trichoderma reesei QM 9414 (A) and viride (B) cellulase during a time extended filter paper assay in the absence of stabilizers plotted against the product of enzyme concentration (E as dilution level) and time (t in hours). 41

...••■•0

3 -

a mg glucose * * » 0 2- 0 6

1 - ^••-•o 0 0

0- —u—T 1 1 1 50 100 150 200 250 mg filterpaper

—□— 0.005 FPU —O— 0.02 FPU

—■ o—• 0.08 FPU

Fig. 8. The amount of reducing sugar as glucose released by Trichoderma viride cellulase at different concentrations of non-macerated Whatman # 1 filter paper and cellulase activities.

Co. 1993), weighing is advisable when the filter paper assay is carried out.

The next experiments (Fig. 9) were conducted in order to further explore the role of the substrate in the hydrolytic process when the amount of product formed is kept close to the required 2 mg in the 42

filter paper assay. The reducing sugar time course for Trichoderma reesei cellulase was followed when substrate was either continuously present or present only during an assay hour ending by the times indicated and the enzyme thus first preincubated. In the first case duplicates of 4 different concentrations were incubated for periods of different lengths so that the product E x t equal to the original value should be contained in each group of dilutions. As the figure shows dilution and time are not interchangeable with regard to the amount of glucose obtained. Rather there is a continuous decline in activity, thus prohibiting the direct employment of a time extended filter paper activity concept. This is apparent also from the trend in the second case with an undiluted sample that always has the original E x t value. These data points lie closely to the data points from the diluted samples with the same E x t value. Thus substrate inhibition is not likely to be a major factor whereas protease activity, thermal denaturation of grace period or biphasic type (Lencki et al 1992) and lack of steady-state conditions (Selwyn

1965) remain as possible reasons for the observed time courses.

Measures must be taken in order to prevent an activity change. 43

mg glucose (A)

i—i—i—i—i—r 9 10 11 12 13 14 15 hours

mg glucose (B)

i—i—i—i—r 10 11 12 13 14 15 hours

Fig. 9. Incubation of Trichoderma reesei QM 9414 cellulase when the substrate (Whatman # 1 filter paper) is either (A) always present or (B) present only during an assay hour ending by the times shown. In case A four groups of different dilutions each cover the points where E x t is equal to the original value, whereas in case B all enzyme is undiluted. 44

2.3.3 Stabilization studies

Several conditions can be expected to influence the determined cellulase activity. Commonly used protein stabilizers include various sugars, polyhydroxyalcohols, aminoacids and kosmotropic salts (Kristjansson and Kinsella 1991, Timasheff 1992, Timasheff and Arakawa 1989). Both cellulose and cellulase show some susceptibility to oxygen attack (Rodriguez et al 1991, Scott 1965).

Metal ions can alter the rate of the reactions (Forouhi and Gunn

1983). Fibers and other macromolecules have a general promoting and stabilizing effect on the cellulase (Volkin and Klibanov 1989).

Proteins such as BSA can serve as artificial substrates for protein degrading agents and thus act to preserve cellulase molecules

(Reese and Mandels 1980, Whitaker 1972). Shaking can cause significant denaturation with loss of apparent activity due mainly to surface tension forces but also to shear forces (Tjerneld et al

1991). I have continued the work on stabilization of cellulases that was conducted by Reese and Mandels in 1980. My pilot screening studies (data not shown) revealed that sorbitol and L-ascorbic acid increased the detected level of reducing sugar in the DNS assay. 45

The L-ascorbic acid also appeared to cause a continuous decline in absorbance readings when the DNS assay was carried out. The use of degassed buffer and pure heavy mineral oil covering the incubated volume did not result in any significant increase in filter paper activity. Bovine serum albumin appeared to have a good stabilizing effect and even to increase the apparent FPA. Potassium chloride also showed good stabilizing properties (Manonmani and Joseph

1993) but lowered the apparent FPA. Glycerol and mannitol decreased the activities considerably. Calcium chloride and magnesium sulfate both caused formation of a precipitate when the

DNS reagent was added. Sodium azide at the concentration employed did not significantly influence the activity values (Reese and

Mandels 1980). Shaking of the enzyme until foam formation significantly lowered the activity values. The most promising stabilizers were bovine serum albumin and potassium chloride in combination. Accordingly we then employed potassium chloride

(1 M) together with bovine serum albumin (1 mg/ml) as stabilizers in the next study where Trichoderma reesei cellulase and

Trichoderma viride cellulase were utilized. The experiments were 46

conducted in the same way as the first experiments in order to see if a time independent enzymatic activity could be achieved. Figure

10 shows that the graphs for different dilutions cover each other within a 5 % margin. That is not more than the variation in FPA that can be found when a crude sample is analyzed for cellulolytic activity. Thus the original stabilized cellulolytic activity appears to have been maintained. The results from these two enzyme preparations suggest that enzyme concentration and incubation time can be considered as interchangeable entities when the stabilizers are used. I hypothesize that the presence of a large amount of added protein and the enhanced water structure due to potassium chloride can be expected to slow down the cellulolytic activity.

Triplicates of cellulase from Trichoderma reesei and Trichoderma viride were incubated with and without added stabilizers at dilutions suitable for obtaining a filter paper activity in each case

(i. e. production of 2 mg glucose in a one hour assay). The result is shown in Figure 11 where the lines are parallel as expected and from which the conversion factors (f), i.e. the ratio between the dilution factors required to reach the 2 mg glucose level, were 47

mg glucose (A)

Dilution 1:27

■o Dilution 1:9 Ext -o— Dilution 1:3 Dilution 1:1

mg glucose (B)

Ext

Fig. 10. The level of reducing sugar as glucose produced by Trichoderma reesei (A) and viride (B) cellulase during time extended filter paper assays when stabilizers are used plotted against the product of enzyme concentration (E as dilution factor) and time (t in hours). All standard deviation bars are covered by the symbols. 48

mg glucose (A)

dilution (log scale)

mg glucose (B)

0.10 1.00 dilution (log scale)

Fig. 11. The result of filter paper assays (triplicate samples) using stabilized (S) and non-stabilized (NS) Trichoderma reesei (A) and viride (B) cellulase. The conversion factors (f) between the apparent activities are 1.4 and 1.7 respectively. 49

determined to 1.4 and 1.7 respectively. Thus some minor variation in this factor should be expected between different

Trichoderma cellulase preparations. 50

2.4 CONCLUSION

Low filter paper activities from various Trichoderma sp. can be determined from a modified filter paper assay, where bovine serum albumin, potassium chloride and sodium azide are added and the incubation time is prolonged up to 20 hours. The slower reaction rate necessitates the employment of a conversion factor between the unit defined by this modified assay and the regular FPU. The equation presented here provides the number of filter paper units

(IUPAC) within a minor error margin. 51

3. CULTIVATION OF TRICHODERMA REESEI QM 9414 ON ACID WHEY AND SWEET WHEY PERMEATE

Key words: Acid whey, growth, soluble protein, Trichoderma, whey permeate.

3.1 INTRODUCTION

Acid whey as a fairly rich medium could be expected to support considerable fungal growth (Smith and Berry 1975). T. reesei grows on Solkafloc and glucose media despite pH values as low as 3 (Brown et al 1990) and has thus proven to be acid tolerant enough for use on acid whey that has a normal pH range from 4 to 5. Acid whey permeate has proven to yield high ethanol concentrations when fermented by Saccharomyces fragilis (Chen 1991). Sweet whey permeate is used as a growth substrate for the production of ethanol, lactic acid, citric acid, single cell protein, bakers' yeast and antibiotics (Hui 1992 and Champagne et al 1990) by other microbes such as Lactococcus lactis (Christopherson and Zottola

1989), Lactobacillus bulgaricus, Kluyveromyces fragilis,

Kluyveromyces bulgaricus (Zadow 1986) and Aspergillus niger

(Maddox 1983). Earlier studies have shown that deproteinated whey is more promising than sweet whey as a medium for cellulase 52

production by Trichoderma reesei MCG 80 (Allen and Andreotti

1982). Some drawbacks are that whey is deficient in inorganic

nitrogen and has a high content of mineral salts that may interfere with growth (Marth 1970) and processing (Moore-Landecker 1990,

Zadow 1986). Certain agents in whey media as well as autoclaving of these media have been found to inhibit the growth of or the

metabolite production in some bacteria and viruses (Marth 1970,

Anderson et al 1986). Environmental pollution by whey wastes has

long been a problem for the food industry (Sell 1992). By using acid whey or whey permeate as a growth medium for fungi, a twofold goal could be accomplished: reduction of whey wastes and a facilitated production of a wide range of utilizable products. Since a significant decrease in the cost of cellulase production could be expected if the cultivation of Trichoderma reesei on whey media

results in a fair yield of cellulase, an objective of the study was to explore the feasibility of such a technique. Other applications of

Trichoderma sp. are the production of mycelial protein for feedstock purposes and their ability to transform many complex natural materials as well as xenobiotics (Eveleigh 1985). They are 53

considered to possess disease biocontrol potential (Jackson et al

1991). Trichoderma species are known to produce mycotoxins such as trichotoxin A (a cyclic polypeptide with some antimicrobial activity) (Taber and Taber 1978), trichodermin (a trichothecene) and trichoviridin (an isocyanide) (Eveleigh 1985). The small amounts of these compounds have motivated the Food Additives and

Contaminants Committee of the Ministry of Agriculture, Fisheries and Food in the United Kingdom to classify Trichoderma reesei in group B, i. e. substances that on the available evidence may be regarded provisionally acceptable for use in food but about which further information should be gathered (Chaplin and Bucke 1990). In the U. S. Trichoderma metabolites do not yet appear in the GRAS list

(Code of Federal Regulations april 1993). Trichoderma strains also produce yellow pigments such as anthroquinones (Eveleigh 1985).

However, no data have been presented in the literature on the growth of T. reesei on acid whey. The purpose of the present study was to fill this information gap. 54

3.2 MATERIALS AND METHODS

3.2.1 Substrates

Fresh acid whey (pH 4.6 and 4.4 respectively) was obtained from

Sunny Brooks Dairy, Corvallis, OR and from Echo Springs Dairy,

Eugene, OR. Whey permeate (pH 6.3) was provided by the Department of Food Science at Utah State University, Logan, UT. These media were vacuum-filtered in several steps using Celite powder (D3877 followed by D 5384, both from Sigma Chemical Co., MO) and 1.2 jim glass fiber filters. Sterilization was normally carried out by filtration through 0.2 jim Nalgene cellulose nitrate membranes (VWR

Scientific). In the initial study on acid whey (Fig. 14 and 15) thermal sterilization was conducted at 121 0C for 15 min. The hammer-milled spruce cellulose, Solkafloc SW 40 (James River Inc.,

Berlin, NH), at 8 g/l was normally used as the control together with

Mandels' medium [1.5 g/l ammonium sulfate, 2 g/l monobasic potassium phosphate, 0.32 g/l calcium chloride, 0.32 g/l magnesium sulfate heptahydrate, 1 ml/I trace metal solution (containing ferrous and manganese sulfate, zinc chloride and cobalt chloride),

0.31 g/l urea, 0.8 g/l proteose peptone (DIFCO Laboratories, Ml), 55

1 ml/l Tween 80]. This control was always adjusted to pH 6.5 and

thermally sterilized at 121 0C for 15 min. In the growth study on whey permeate (Fig. 13) a control consisting of 8 g/l maltose

monohydrate together with Mandels' medium was employed and

filter-sterilization was conducted as described above.

3.2.2 Microbe

Trichoderma reesei QM 9414 was obtained as a freeze-dried

preparation from ATCC and grown on potato dextrose agar (DIFCO

Laboratories, Detroit, Ml) slants or plates at 30 0C for 1-2 weeks in

the laboratory (Smith and Onions 1983). An aqueous spore suspension

(3-125 million CFU/ml) was used as inoculum (0.1-0.4 ml/100 ml

medium).

3.2.3 Incubation experiments

The inoculated media were contained in Erlenmeyer flasks

(duplicates or triplicates) with glasswool plugs and aluminum foil

caps. The maximum liquid to volume ratio was 100 ml in a 250 mL

flask. The incubations were performed at 28 0C in an Orbit

Environshaker (Lab.-Line Instruments, Inc., Melrose Park, IL) with a

speed setting of 100 r. p. m. 56

3.2.4 Reagents and analytical procedures

Spore counting was carried out on potato dextrose agar (PDA) and on PDA with added Rose Bengal at 0.001 %. Soluble protein was determined according to the Bradford method (reagent provided by

Bio-Rad Laboratories, Richmond, CA) with bovine serum albumin as standard. Samples aimed for determination of cellulolytic activity were centrifuged at 5000 g for 1 hour using Centricon polysulfone ultrafiltration membranes with 10 kD M. W. cut-off (Amicon, Inc.,

Beverly, MA), then subjected to size exclusion chromatography by using Econopac 10 DG desalting columns with 6 kD M. W. cut-off polyacrylamide gel (Bio-Rad Laboratories, Richmond, CA) and subsequently freeze-dried and diluted as appropriate for the following assay. Glucose levels were assayed in accordance with the

IUPAC method (Ghose 1987) for filter paper activity determination employing the dinitrosalicylic acid method. Cellulolytic activity was determined by a low-activity variant of the IUPAC method developed in our laboratory and using potassium chloride (1 M) and bovine serum albumin (1 mg/ml) as protein stabilizers and sodium azide

(0.02 M) as an antimicrobial. Lactose was assayed by using a 57

kit from Boehringer Mannheim Biochemicals, Indianapolis, IN, employing lactase, hexokinase and glucose-6-phosphate dehydrogenase. All reagent chemicals were of analytical grade.

Mycelial growth was assayed by vacuum filtering the sample and washing it with at least 3 x 10 ml cold filtered tap water on a 1.2

|im glass fiber disk until no foam appeared, drying the retentate at

102 0C for 16 hours, cooling it in a desiccator and weighing. 58

3.3 RESULTS AND DISCUSSION

3.3.1 The development of mvcelial biomass

When active growth starts it involves only apical and subapical hyphal cells (Nielsen 1993) and then the degree of branching and the pellet size will determine much of the growth characteristics

(Garraway and Evans 1984). T. reesei QM 9414 grows well on acid whey as can be seen in Figure 12. The lag phase may parallel the finding that the lag on cellulosic media using a spore inoculum is long and unpredictable (Sternberg 1976b). The doubling time was found to be 2.4 days which corresponds to a specific growth rate of 0.29 (= In 2 / 2.4 ) per day. After 2 weeks and a mycelial dry weight gain of 13 mg/ml the growth has not reached the stationary phase. As a comparison T. viride QM 9123 growing on glucose (23 mg/ml) supplemented with Mandels' medium reached a maximum of

11 mg/ml (Brown and Halsted 1975). The mold Aspergillus niger has been shown to have a specific growth rate of 0.62/day on grape waste, which was considered a good growth (Kuzmanova et al 1991).

Microfungi in general contain 19-47 % protein (Smith and Berry

1975). Protein data are sometimes based on Kjeldahl nitrogen 59

100

mg mycelial dry weight/ml (log scale) 10

days

□ Acid whey # 1 O Acid whey # 2 O Dil. acid whey # 1 A Dil. acid whey # 2 S Dil. acid whey (adjusted) # 1 ♦ Dil. acid whey (adjusted) # 2

Fig. 12. The growth of 7. reeseion filter-sterilized acid whey. 60

values multiplied by 6.25. However, the high amount of non-protein nitrogen in microfungi means that the corresponding amount of real protein is lower. Figure 12 also shows the effect of dilution 1:6 of acid whey. The low level of nutrients causes the time scale to shrink

(Megee et al 1970) and the maximum mycelial mass to decrease. The maximum mycelial mass in the case of undiluted acid whey is predicted to be approximately 22 mg/ml based on the dilution factor above. A duplicate set of the diluted acid whey flasks were supplemented with ammonium sulfate to 14 mM (Garraway and Evans 1984). This adjustment does not appear to have influenced growth, indicating that ammonium nitrogen is not growth limiting. The diluted acid whey graphs in Figure 12 are consistent with cell death occurring after 7 days. The death phase may involve cell autolysis or turnover (Sikyta 1983, Freitag 1989,

Sternberg and Dorval 1979), in which case a declining growth curve appears. No stationary phase was observed in any of the experiments reported in Figure 12. The absence of a true stationary phase often occurs among filamentous fungi (Garraway and Evans

1984, Schaffner and Toledo 1991, Wilke et al 1983). 61

On sweet whey permeate (Fig. 13) the maximum mycelial yield is 4.4 mg/ml. The 0.8 % maltose control reached 3.3 mg/ml. This can be compared with a 0.5 % maltose culture which yielded a maximum

2.2 mg/ml mycelial weight using T. viride (Mandels and Reese 1957).

The growth curves on diluted acid whey and whey permeate are similar. This suggests that neither lactose nor mineral constituents are growth limiting, since the peptide content is about the same in both media. The higher final biomass yield in the case of undiluted acid whey is thus likely to result from its higher protein content relative to the diluted and permeate media. Since the carbon and mineral contents vary considerably between the media it is reasonable to assume that peptide nitrogen is growth limiting.

The findings above support that acid whey is superior to sweet whey permeate as a growth medium.

3.3.2 Metabolite production and effects on substrate composition

Figure 14 shows how pH changes when thermally sterilized, pH- adjusted and non-supplemented acid wheys are used as substrates.

The first pH-changes are likely to be a result of the thermal treatment since no such variations occur if the acid whey is filter- 62

mg mycelial dry weight/ml

□ Whey permeate # 1 O Whey permeate # 2 O 0.8 % Maltose # 1 A 0.8 % Maltose # 2

Fig. 13. The growth of T. reeseion filter-sterilized whey permeate. 63

PH

days

—o Acid whey pH 4.4 • o Acid whey pH 5.0 —-o—- Acid whey pH 5.5

—A— Acid whey pH 6.0 ---ffl--- Acid whey pH 6.5

--♦-- Solkafloc pH 6.5

Fig. 14. The change in pH when T. reesei is cultivated on thermally sterilized acid whey. 64

sterilized (data not shown). The following one unit rise in pH parallels a decline in soluble protein level (Fig. 15). Also in the case of filter-sterilized acid whey there is a considerable decline in soluble protein (Fig. 16). These two findings appear to support the hypothesis that whey proteins are digested and ammonia released into the medium. At any particular time point metabolite production is generally inversely related to growth (Megee et al 1970). There is a 50-60 % difference in the initial protein values for the thermally sterilized (Fig. 15) and the filter-sterilized (Fig. 16) acid whey preparations. This difference is similar to the percentage of whey proteins that can be heat coagulated (Hui 1992). It is also apparent from the figures that the net specific protein production rate is similar on acid whey and Solkafloc medium (Fig. 15). The maximal net soluble protein production is 0.17 mg/ml for thermally sterilized and 0.19 mg/ml for filter-sterilized acid whey. These values are likely to reflect actual metabolite production since there was no indication of cell lysis and the rise comes immediately after the digestion phase. As mentioned below the development of enzymatic activity parallels the rise in soluble protein level in the 65

Cold Incubation storage

mg protein/ml

Acid whey pH 4.4 -O Acid whey pH 5.5 -O-— Acid whey pH 6.5 -A— Solkafloc pH 6.5

Fig. 15. Change in soluble protein as BSA when T. reesei is grown on thermally sterilized acid whey. 66

mg protein/ml

days

Acid whey # 1 -o Acid whey # 2 -o-— Solkafloc # 1

-A— Solkafloc # 2

Fig. 16. Change in soluble protein as BSA when T. reesei is grown on filter-sterilized acid whey. 67

experiment employing filter-sterilized whey permeate and also in the case of the filter-sterilized acid whey (Fig. 16 and 17).

I have found that acid whey which has been filter-sterilized, diluted and supplemented with ammonium sulfate, shows a similar initial pH dip as the Solkafloc medium (data not shown). The development of pH when Trichoderma reesei is cultivated using a cellulosic carbon source such as Solkafloc together with Mandels' medium is well known. There is a rapid decrease initially to pH 3 or less (Garraway and Evans 1984) due to consumption by the mold of ammonia from the ammonium sulfate (Sternberg 1976b). Such low pH values can inactivate produced enzymes (Sternberg 1976a) and favor protease formation on peptone media. Peptones can also substitute for carbohydrates as a carbon source. When the ammonium ions are used up the pH starts to rise due to release of ammonia when peptones are digested (Moore-Landecker 1990).

Simultaneously the fungus starts production of digestive enzymes, for instance cellulases if a suitable provoker (Kubicek et al 1993) is present such as cellulose. The filter-sterilized and diluted but unsupplemented acid whey displays a strong rise in pH (Fig. 18). The 68

1.5- mg glucose

0.5-

days

Acid whey # 1 •o Acid whey # 2 -o— Solkafloc # 1 Solkafloc # 2

Fig. 17. The amount of glucose released in a filter paper assay (IUPAC) when T. reesei is cultivated on filter-sterilized acid whey. 69

PH

□ Dil. acid whey # 1 O Dil. acid whey # 2 O Solkafloc # 1 A Solkafloc # 2

Fig. 18. The time course of pH when T. reesei is grown on filter-sterilized acid whey that has been diluted 1:6. 70

rise is the same as on undiluted acid whey (data not shown). The pH profiles for growth on the diluted and undiluted acid wheys do not correlate with those for growth on whey permeate. This observation may be a result of the differences in protein and peptide composition and/or mineral contents for the acid wheys and whey permeate. The buffering capacities of the two media could also differ. The increased risk for irreversible denaturation of proteins and enzymes when pH rises above 8-9 (Townend 1978), especially at elevated temperatures, could harm the yield of desired metabolites.

When sweet whey permeate is used as a substrate there is also a pH increase initially (Fig. 19). The low amount of peptides in whey permeate will limit the rise in pH and favor an earlier start of net protein production (Fig. 20). Thus a marked increase in soluble protein takes place after 3-5 days and a maximum value of 0.23 mg protein/ml is attained after 2 weeks. The long term trends with declining pH can be explained as an effect of organic acid production under conditions of oxygen access and ongoing autolysis.

T. reesei produces pigment compounds that are released into liquid and solid growth media. Visible spectra of these pigments in 71

PH

days

Whey permeate # 1 Whey permeate # 2 -o- Solkafloc # 1 Solkafloc # 2

Fig. 19. The development of pH when T. reesei is grown on filter-sterilized sweet whey permeate. 72

0.5

0.4- mg 0.3 protein/ml 0.2

0.1-

days

Whey permeate # 1

■o- •■••• Whey permeate # 2

■o— Solkafloc # 1

-A— Solkafloc # 2

Fig. 20. Change in soluble protein as BSA when T. reesei is grown on filter-sterilized sweet whey permeate. 73

cone, sulfuric acid reveals a marked increase in absorption of wavelengths shorter than 470 nm. The apparent pigment concentration is correlated with the pH of the media. Visually, the color of the growth media changes with incubation time. This color change appears related to the pH of the culture broth. I have compared the pigmentation during growth on acid whey, lactose and

Solkafloc media at initial pH values of 4.4 and 6.5. Acid whey gives the strongest color development reaching an apparent maximum after 1-2 weeks. Lactose and Solkafloc media give less pigmentation consistent with a smaller final mycelial weight and lower pH values due to the lower peptide contents. See appendix E for further information on culture pigmentation.

Figure 21 shows the cellulolytic filter paper activity measured as glucose after incubation on whey permeate. The development is consistent with the development of extracellular protein (Sternberg

1976b) (Fig. 20) and demonstrates that a small cellulase activity is produced on unsupplemented whey permeate. In terms of IUPAC filter paper activities the difference between the Solkafloc and the whey permeate is much bigger due to the nonlinear relationship between 74

mg glucose

days

Whey permeate # 1 •o- Whey permeate # 2 -o- Solkafloc # 1 Solkafloc # 2

Fig. 21. The amount of glucose released in a filter paper assay (IUPAC) when T. reesei is cultivated on filter-sterilized sweet whey permeate. 75

the amount of glucose released in the assay and the cellulase concentration. Supplementation of whey permeate with 0.1 %

Solkafloc appeared to increase the soluble protein level by 25 % (Fig.

22). ANOVA analysis (Fischer's protected LSD) shows a 90 % significance for the differences on day 3. The result is consistent with a substitution by Solkafloc for some lactose as carbon source in the case of the supplemented whey permeate.

Addition of 0.2-0.5 % cellulose to 4-6 % lactose media has been found to enhance the soluble protein level and the cellulase activity using the strains T. reesei CL-847 (Warzywoda et al 1983) and C-5

(Chaudhuri and Sahai 1993a). Figure 17 illustrates the development of cellulase activity on acid whey. It is apparent that the net cellulase production is low. This is probably in part due to extracellular proteases emitted into the medium because of the protein and peptide contents in acid whey and whey permeate (Haab et al 1990). No protease inhibitors are known that could prevent digestion of cellulases to more than 50 % (Haab et al 1990). Dilution of the acid whey 1:6 resulted in even lower cellulase enzyme activities (data not shown) and the IUPAC filter paper activity after 76

mg protein/ml

Whey permeate

•■••O Whey permeate + Solkafloc -O—- Solkafloc

Fig. 22. The effect of 0.1 % (w/v) Solkafloc addition on the level of soluble protein as BSA when T. reesei is grown on filter-sterilized whey permeate. 77

7 days was determined to 0.02 FPU. This finding suggests that some whey proteins present after the dilution are inhibitory to cellulase production and/or that the medium was deficient in growth factors.

Cellulase is assumed to originate in the apical hyphal wall (Gow et al 1993) where, for instance, lactose is known to provoke cellulase synthesis (Ryu et al 1979). When cellulase is produced industrially on lactose media the pH is generally kept close to 5.

Cultivation on different media results in different pH profiles and cellulase production is not tied to a- particular profile (Esterbauer et al 1991). Consumption of lactose by the mold when growing on whey permeate is demonstrated in Figure 23. It is clear that after 10 days growth there was only limited reduction in the lactose content of the media. The high lactose content (4.5-5 %) in whey (Hui 1993), appears to favor growth (Chaudhuri and Sahai 1993b) instead of metabolite production (Mandels and Reese 1957, Farkas et al 1987).

High concentrations of rapidly metabolized carbon sources strongly repress cellulase formation (Mandels and Weber 1969).

High peptone concentrations in cellulosic media are strongly inhibitory to cellulase production (Mandels and Weber 1969). The 78

g lactose/I E3 DayO H Day 10

Whey Whey permeate permeate + Solkafloc

Fig. 23. The change in lactose level with and without added 0.1 % (w/v) Solkafloc when T. reesei is grown on filter-sterilized sweet whey permeate. 79

experiments above have all resulted in low net yield of cellulase. It thus seems unlikely that acid whey or whey permeate without further processing could be used as media for cellulase production by Trichoderma reesei QM 9414. 80

3.4 FINAL COMMENTS

It appears of interest to conduct further studies on acid whey which showed potential for mycelial mass production. The focus when molds are grown is perhaps more often on the secondary metabolites, i. e. products that appear when there is a nutrient deficiency and which do not have any obvious function for the fungus.

Primary metabolites and digestive enzymes are also of interest in

many applications. As far as proteinaceous metabolites are of concern sweet whey permeate appears to be a better alternative than acid whey. If a precultured broth had been used instead of a spore inoculum the lag period could have been shorter (Sternberg

1976b) than in the previous experiments. This can be of economical

importance in an industrial setting. Acid whey permeate could be worth a separate study due to its low protein content and the

economical benefits of acid whey utilization. The productivity of

other T. reesei enzymes than cellulase such as glucanases,

(Peberdy and Ferenczy 1985), pectinases (Haltmeier et al

1983) and xylanases (Poutanen and Puls 1989) on whey media need

to be addressed. T. reesei has also been called a promising 81

alternative host for protein production (Penttilae et al 1991).

Optimal conditions for the production of extracellular proteins will

be dependent on nutritional and temperature requirements of the fungus, handling of the substrate, ranges for denaturation,

precipitation and aggregation of the protein, ease of harvesting and of the costs involved in each case. 82

4. BIBLIOGRAPHY

Abril, J. R. and Stull, J. W. 1989. Lactose hydrolysis in acid whey with subsequent glucose isomerisation. J. Sci Food Agric, 48, 511- 514.

Allen, A. L. and Andreotti, R. E. 1982. Cellulase Production in Continuous and Fed-batch Culture by Trichoderma reesei MCG80. Biotechnol. Bioeng. Symp., 12, 451-459.

Anderson, T. M., Bodie, E. A., Goodman, N. and Schwartz, R. D. 1986. Inhibitory effect of autoclaving whey-based medium on propionic acid production by Propionibacterium shermanii. Appl. Environ. Microbiol., 51:2, 427-428.

Atalla, R. H. and Vanderhart, D. L. 1989. Studies on the structure of cellulose using raman spectroscopy and solid state 13C NMR. In: C. Schuerch (ed), Cellulose and wood - chemistry and technology. Proceedings of the tenth cellulose conference. State Univ. of New York, Syracuse, NY.

ATCC Catalogue of Filamentous Fungi. 1991. American Type Culture Collection, Rockville, MD.

Baker, J. O., Tatsumoto, K., Grohmann, K., Woodward, J., Wichert, J. M., Shoemaker, S. P. and Himmel, M. E. 1992. Thermal denaturation of Trichoderma reesei cellulases studied by differential scanning calorimetry and tryptophan fluorescence. Proceedings of the thirteenth symposium on biotechnology for fuels and chemicals. Appl. Biochem. Biotechnol., 34-35, pp. 217-232.

Beguin, P. and Aubert, J-P. 1992. Cellulases. In: Lederberg, J. (ed), Encyclopedia of microbiology. Vol. 1. Academic Press, Inc., New York, NY. 83

Breuil, C, Chan, M., Gilbert, M and Saddler, J. N. 1992. Influence of beta-glucosidase on the filter paper activity and hydrolysis of lignocellulosic substrates. Bioresour. Technol., 39, 139-142.

Breuil, C. and Saddler, J. N. 1985. Comparison of the 3,5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying for reducing sugars and determining cellulase activity. Enzyme Microb. Technol., 7, 327-332.

Brown, D. E. and Halsted, D. J. 1975. The effect of acid pH on the growth kinetics of Trichoderma viride. Biotechnol. Bioeng.M, 1199- 1210.

Brown, D. E., Hasan, M., Lepe-Casillas, M. and Thornton, A. J. 1990. Effect of temperature and pH on lipid accumulation by Trichoderma reesei. Appl. Microbiol. Biotechnol., 34:3, 335-339.

Champagne, C P., Goulet, J. and Lachance, R. A. 1990. Production of bakers' yeast in cheese whey ultrafiltrate. Appl. Environ. Microbiol., 56:2, 425-430.

Chan, M., Breuil, C, Schwald, W. and Saddler, J. N.I989. Comparison of methods for quantifying the hydrolytic potential of cellulase enzymes. Appl. Microbiol. Biotechnol., 31, 413-418.

Chaplin, M. F. and Bucke, C. 1990. Enzyme technology. Cambridge University Press, Cambridge, p.77.

Chaudhuri, B. K. and Sahai, V. 1993a. Production of cellulase enzyme from lactose in batch and continuous cultures by a partially constitutive strain of Trichoderma reesei. Enzyme Microb. Technol., 15:6, 513-518.

Chaudhuri, B. K. and Sahai, V. 1993b. Production of cellulase using a mutant strain of Trichoderma reesei growing on lactose in batch culture. Appl. Microbiol. Biotechnol., 39, 194-196. 84

Chen, Hung-Chang. 1991. Up-flow attached-bed bioreactor for continuous ethanol fermentation. Biotechnol. Prog., 7:4, 311-314.

Christopherson, A. T. and Zottola, E. A. 1989. Whey permeate as a medium for mesophilic lactic acid streptococci. J. Dairy Sci. 72, 1701-1706.

Cochet, N. 1991. Cellulases of Trichoderma reesei: influence of culture conditions upon the enzymatic profile. Enzyme Microb. Techno!., 13, 104-109.

Code of Federal Regulations. April 1, 1993. Vol. 21. Office of the Federal Register, National Archives and Records Administration.

Coughlan, M. P. 1992. Enzymic hydrolysis of cellulose: An overview. Bioresour. Technol., 39, 107-115.

Dade, H. A. 1960. Methods in use at the Commonwealth Mycological Institute. In: Herb. I. M. I. Handbook, Commonwealth Agricultural Burreaux, Soman-Wherry Press, Norwich, England.

Daufin, G., Michel, F. and Merin, U. 1992. Ultrafiltration of defatted whey: influence of some physicochemical characteristics. The Australian Journal of Dairy Technology, 47, 7-13.

Dinwoodie, J. M. 1989. Wood: Nature's cellular, polymeric fibre- composite. The Institute of Metals, London.

Dominguez, J. M., Acebal, C, Jimenez, J., Delamata, I., Macarron, R. and Castillon, M. P. 1992. Mechanisms of thermoinactivation of endoglucanase I from Trichoderma reesei QM 9414. Biochem. J., 287:2, 583-588.

Esterbauer, H., Steiner, W., Labudova, I., Hermann, A. and Hayn, M. 1991. Production of Trichoderma cellulase in laboratory and pilot scale. Bioresource Technol. 36, 51-65. 85

Eveleigh, D. E. 1985.Trichoderma. In: Demain, A. L. and Solomon, N. A. (ed.), Biology of Industrial Microorganisms. The Benjamin / Cummings Publishing Company, Inc. Menlo Park, CA, pp. 487-509.

Farkas, V., Gresik, M., Kolarova, N., Sulova, Z. and Sestak, S. 1990. Biochemical and physiological changes during photoinduced conidiation and derepression of cellulase synthesis in Trichoderma. In: C. P. Kubicek, D. E. Eveleigh, H. Esterbauer, W. Steiner and E. M. Kubicek-Pranz (eds), Trichoderma reesei cellulases: , genetics, physiology and application. The Royal Society of Chemistry, Cambridge, U.K.

Farkas, V., Sestak, S., Gresik, M., Kolarova, N., Labudova, I. and Bauer, S. 1987. Induction of Cellulase in Trichoderma reesei grown on lactose. Acta Biotechnol. 7:5, 425-429.

Forouhi, E. and Gunn, D. J. 1983. Some effects of metal ions on the estimation of reducing sugars in biological media. Biotechnol. Bioeng.,25, 1905-1911.

Fox, P. F. 1987. Cheese: An overview. In: P. F. Fox (ed), Cheese: chemistry, physics and microbiology. Vol. 1. General aspects. Elsevier Applied Science, New York, NY, p. 18-21.

Freitag, M. 1989. Measuring extracellular enzymes in pure and mixed cultures of Trametes versicolor Pilat and Trichoderma harzanium Rifai. Thesis. Oregon State University, Corvallis, OR, p. 92.

Garraway, M. O. and Evans, R. C. 1984. Fungal nutrition and physiology. John Wiley and Sons, New York, NY, pp. 103, 110, 235.

Ghose, T. K. 1987. Measurement of cellulase activities. Pure Appl. Chem., 59:2, 257-268.

Gow, N. A. R., Miller, P. F. P. and Gooday, G. W. 1993. Life at the apex: growth of the hyphal tip. J. Chem. Tech. Biotechnol., 56, 217-219. 86

Griffin, H. L. 1973. Filter paper assay - Effect of time and substrate concentration on cellulase activity. Anal. Biochem., 56, 621-625.

Haab, D., Hagspiel, K. and Szakmary, K. 1990. Formation of the extracellular proteases from Trichoderma reesei QM 9414 involved in cellulase degradation. J. Biotechnol., 16, 187-198.

Haltmeier, T., Leisola, M., Ulmer, D., Waldner, R. and Fiechter, A. 1983. Pectinase from Trichoderma reesei QM 9414. Biotechnol. Bioeng. 25, 1685-1690.

Heng, M. H. and Glatz, C. E. 1991. Chemical pretreatments and fouling in acid cheese whey ultrafiltration. J. Dairy Sci.,74, 11-19.

Herrera-Estrella, A., Goldman, G. H., Van Montagu, M. and Geremia, R. A. 1993. Electrophoretic karyotype and gene assignment to resolved chromosomes of Trichoderma spp. Molecular Microbiology, 7:4, 515- 521.

Hoshino, E., Kanda, T., Sasaki, Y. and Nisizawa, K. 1992. Adsorbtion mode of exo- and endo-cellulases from Irpex lacteus {Polyporus tulipiferae) on cellulose with different crystallinities. J. Biochem., 111, 600-605.

Huang, X. and Penner, M. 1991. Apparent substrate inhibition of the Trichoderma reesei cellulase system. J. Agric. Food Chem., 39:11, 2096-2100.

Hui, Y. H. 1993. Dairy Science and Technology Handbook, Vol.1: Principles and properties. VCH Publishers Inc., New York, NY, p. 9, 14, 26.

Hui, Y. H. 1992. Encyclopedia of food science and technology. Vol. 4. John Wiley and Sons, Inc., New York, NY, pp. 2836, 2842. 87

Jackson, A. M., Whipps, J. M. and Lynch, J. M. 1991. Effects of temperature, pH and water potential on growth of four fungi with disease biocontrol potential. World J. Microbiol. Biotechnol., 7:4, 494-501.

Jones, S. B., Robbins, C. W. and Hansen, C. L. 1993. Sodic soil reclamation using cottage cheese (acid) whey. Arid Soil Research and Rehabilitation, 7:1, 51 -61.

Jong, S. C. 1981. Isolation, cultivation and maintenance of conidial fungi. In: C. T. Cole and B. Kendrick (eds.), Biology of conidial fungi. Vol. 2. Academic Press, New York, NY, pp. 551-577.

Kendrick, W. B. and Parkinson, D. 1990. Soil fungi. In: D. L. Dindal (ed.), Soil biology guide. John Wiley & Sons, New York, NY, pp. 49-52.

Kharbanda, O. P. and Stallworthy, E. A. 1990. Waste management. Towards a sustainable society. Gower Publishing Co. Ltd., U. K.

Kirk-Othmer. 1979. Encyclopedia of chemical technology. Vol. 5 and 7. John Wiley and Sons, New York, NY.

Klyosov, A. A. 1990. Trends in biochemistry and enzymology of cellulose degradation. Biochemistry, 29:47, 10577-10585.

Kolev, D., Witte, K. and Wartenberg, A. 1991. Synergistic cooperation of cellulases of Trichoderma reesei as distinguished by a new filter-paper destruction assay. Ada Biotechnol., 11:4, 359-365.

Kristjansson, M. M. and Kinsella, J. E. 1991. Protein and enzyme stability: structural, thermodynamic and experimental aspects. In: J. E. Kinsella (ed.), Advances in food and nutrition research. Vol. 35. Academic Press, Inc., San Diego, CA, pp. 237-316. 88

Kubicek, C. P. 1987. Involvment of a conidial endoglucanase and a plasma-membrane-bound betaglucosidase in the induction of endoglucanase synthesis by cellulose in Trichoderma reesei. J. General Microbiol., 133, 1481-1487.

Kubicek, C. P., Messner, R., Fritscher, C, Strauss, S. and Kubicek- Pranz, E. M. 1990. Regulatory aspects of formation and secretion of cellulases by Trichoderma reesei. In: C. P. Kubicek, D. E. Eveleigh, H. Esterbauer, W. Steiner and E. M. Kubicek-Pranz (eds), Trichoderma reesei cellulases: biochemistry, genetics, physiology and application. The Royal Society of Chemistry, Cambridge, U.K.

Kubicek, C. P., Messner, R., Gruber, F., Mach, R. L. and Kubicek-Pranz, E. M. 1993. The Trichoderma cellulase regulatory puzzle: From the interior life of a secretory fungus. Enzyme Microb. Technol., 15, 90- 99.

Kuchel, P. W. and Ralston, G. B. 1988. Theory and problems of biochemistry. McGraw Hill, Inc., New York, NY, pp. 266.

Kuo, K., and Cheryan, M. 1983. Ultrafiltration of acid whey in a spiral-wound unit: effect of operating parameters on membrane fouling. J. Food Sci., 48, 1113-1118.

Kuzmanova, S., Vandeska, E. and Dimitrovski, Aco. 1991. Production of mycelial protein and cellulolytic enzymes from food wastes. J. Industrial Microbiol., 7, 257-262.

Kyriacou, A., Neufeld, R. J. and Mackenzie, C. R. 1989. Reversibility and competition in the adsorbtion of Trichoderma reesei cellulase components. Biotechnol. Bioeng., 33, 631-637.

Lencki, R., Arul, J. and Neufeld, R. 1992. Effect of subunit dissociation, denaturation, aggregation, coagulation and decomposition on enzyme inactivation kinetics: 1. First-order behavior. Biotechnol. Bioeng., 40, 1421-1426. 89

Liaw, E-T and Penner, M. 1990. Substrate-velocity relationships for the Trichoderma viride cellulase - catalyzed hydrolysis of cellulose. Appl. Environ. Microbiol., 56:8, 2311-2318.

Loeppky, C. B., Sprouse, R. F., Carlson, J. V. and Everett, E. D. 1983. Trichoderma viride peritonitis. Southern Med. J., 76:6, 798-799.

Maddox, I. S. 1983. Production of citric acid from whey permeate by fermentation using Aspergillus niger. New Zealand J. Dairy Sci Technol., 18:2, 161-168.

Maentylae, A. L, Rossi, K. H., Vanhanen, S. A., Penttilae, M. E., Suominen, P. L. and Nevalainen, K. M. H. 1992. Electrophoretic karyotyping of wild-type and mutant Trichoderma longibrachiatum (reesei) strains. Curr. Genet, 21, 471-477.

Mandels, M., Andreotti, R. and Roche, C. 1976. Measurement of saccharifying cellulase. Biotechnol. Bioeng. Symp., 6, 21-33.

Mandels, M., Dorval, S. and Medeiros, J. 1978. Saccharification of cellulase with Trichoderma cellulase. In: W. W. Schuster (ed.), Proceedings. Second annual fuels from biomass symposium. Rensselaer Polytechnic Institute, Troy, New York, NY, pp. 632-635.

Mandels, M., Medeiros, J., Andreotti, R. and Bissett, F. 1981. Enzymatic hydrolysis of cellulose: evaluation of cellulase culture filtrates under use conditions. Biotechnol. Bioeng., 23, 2009-2026.

Mandels, M. and Reese, E. 1957. Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J. Bacteriol., 73:2, 269-278.

Mandels, M. and Weber, J. 1969. The production of cellulases. In: Hajny, G. J. and Reese, E. T. (ed.), Cellulases and their applications. Adv. Chem. Ser. 95, A. C. S., Washington D.C., pp. 391-414. 90

Manonmani, H. K. and Joseph, R. 1993. Purification and properties of an extracellular proteinase of Trichoderma koningii. Enzyme Microb. Technol.^S:?, 624-628.

Marth, E. H. 1970. Fermentation products from whey. In: B. H. Webb and Whittier, E. O. (ed.), Byproducts from milk. Second edition. The Avi Publishing Company, Inc., Westport, CT, p. 43.

Megee III, R. D., Kinoshita, S., Fredrickson, A. G. and Tsuchiya, H. M. 1970. Differentiation and product formation in molds. Biotechnol. Bioeng., 12, 771-801.

Messner, R. and Kubicek, C. P. 1990. Evidence for a single specific betaglucosidase in cell walls from Trichoderma reesei QM 9414. Enzyme Microb. Techno!., 12, 685-690.

Messner, R., Kubicek-Pranz, E. M., Gsur, A. and Kubicek, C. P. 1991. Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains. Arch. Microbiol., 155, 601-606.

Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31:3, 426-428.

Montenecourt, B. 1983. Trichoderma reesei cellulases. Trends in Biotechnology, 1:5, 156-161.

Montenecourt, B., Eveleigh, D., Elmund, K. and Parcells, J. 1977. Antibiotic disks - an improvement in the filter paper assay for cellulase. Biotecnol. Bioeng., 20, 297-300.

Moore-Landecker, E. 1990. Fundamentals of the fungi. Prentice Hall, Englewood Cliffs, NJ, p. 300.

Morr, C. V. 1989. Whey proteins: Manufacture. In: P. F. Fox (ed.), Developments in dairy chemistry - 4. Functional milk proteins. Elsevier Applied Science, London and New York, p. 262. 91

Nevell, T. P. and Zeronian, S. H. 1985. Cellulose chenistry fundamentals. In: T. P. Nevell and S. H. Zeronian (eds), Cellulose chemistry and its applications. Halsted Press division of John Wiley and Sons, New York, NY.

Nielsen, Jens. 1993. A simple morphologically structured model describing the growth of filamentous microorganisms. Biotechnol. Bioeng.,A^, 715-727.

Peberdy, J. F. and Ferenczy, L. 1985. Fungal protoplasts. Marcel Dekker, Inc., New York, NY, p. 36.

Penttilae, M., Teeri, T., Nevalainen, H. and Knowles, J. 1991. The molecular biology of Trichoderma reesei and its application to biotechnology. In: Peberdy, J. F., Caten, C. E., Ogden, J. E. and Bennett, J. W. (eds.), Applied molecular genetics of fungi. Symposium Series of the British Mycological Society, Cambridge University Press, UK,18, pp. 85-102.

Pierre, A., Legraet, Y., Faucuant, J., Piot, M., Durier, C. and Kobilinsky, A. 1992. Optimization of physico-chemical factors for whey clarification. Lait, 72:4, 405-420.

Poutanen, K. and Puls, J. 1989. The xylanolytic enzyme system of Trichoderma reesei. A. C. S. Symp. Ser. 399, 630-639.

Reese, E. T. and Mandels, M. 1971. Enzymatic degradation in cellulose and cellulose derivatives. Ed. N. B. Kales and L. Segal. John Wiley and Sons. Vol. 5. Part 5, pp. 1088-1094.

Reese, E. and Mandels, M. 1980. Stability of the cellulase of Trichoderma reesei under use conditions. Biotechnol. Bioeng., 22, 323-335. 92

Rodriguez, H., Ponce, T., de la Torre, M. and Enriquez, A. 1991. Effect of oxygen and air flow on xylanolytic and cellulolytic activity from bacterial cultures grown on bagasse pith. Biotechnol. Lett., 13:8, 563-566.

Roehl, D. A. 1992. Studies of the rejections of minerals during the ultrafiltration of cottage cheese whey. Mai, 30/02, 261.

Roseiro, J. C, Conceicau, A. C, Amaral-Collaco, M. T. 1993. Membrane concentration of fungal cellulases. Bioresour. Technol. , 43, 155- 160.

Ryu, D., Andreotti, R., Mandels, M., Gallo, B. and Reese, E. T. 1979. Studies on quantitative physiology of Trichoderma reesei with Two- Stage continuous culture for cellulase production. Biotechnol. Bioeng., 21, 1887-1903.

Sarko, S. 1985. Cellulose and amylose: crystal structures and properties. In: Crescenzi, V., Dea, I. C. M. and Stivala, S. S. (eds), New developments in industrial polysaccharides. Gordon and Breach Science Publishers, New York, NY.

Schaffner, D. W. and Toledo, R. T. 1991. Cellulase production by Trichoderma reesei when cultured on xylose-based media supplemented with sorbose. Biotechnol. Bioeng., 37, 12-16.

Schexnayder, K. 1991. 'Whey' ahead of the game. Dairy Foods, 92:3, 58-61.

Schwald, W., Chan, M., Breuil, C. and Saddler, J. N. 1988. Comparison of HPLC and colorimetric methods for measuring cellulolytic activity. Appl. Microbiol. Biotechnol., 28, 398-403.

Scott, G. 1965. Atmospheric oxidation and antioxidants. Elsevier Publ. Co., Amsterdam, pp. 320-321. 93

Scott, R. 1981. Cheesemaking practice. Applied Science Publishers Ltd., London, U. K., pp. 342-347.

Sell, N. 1992. Industrial pollution control: Issues and techniques. Second edition. Van Nostrand Reinhold, New York, NY, pp. 315-323.

Selwyn, M. J. 1965. A simple test for inactivation of an enzyme during assay. Biochim. Biophys. Ada, 105, 193-195.

Seymour, R. B. and Carraher, C. E. 1988. Polymer chemistry - an introduction. Marcel Dekker, Inc., New York, NY. p. 180.

Sikyta, B. 1983. Methods in industrial microbiology. Halsted Press division of J. Wiley and Sons, New York, NY, p.186.

Sinsabaugh, R. L. and Linkins, A. E. 1989. So/7 Biol. Biochem., 21:6, 835-839.

Smith, J. E. and Berry, D. R. 1975. The filamentous fungi. Vol. 1. Industrial mycology. John Wiley and Sons, New York, NY, p. 18, 81, 261.

Smith, D. and Onions, A. H. S. 1983. The preservation and maintenance of living fungi. Commonwealth Mycological Institute, Surrey, U. K..

Snider, E. H. 1992. Waste minimization. In: L. K. Wang and M. H. S. Wang (eds), Handbook of industrial waste treatment. Vol. 1. Marcel Dekker, Inc., New York, NY, pp. 1-60.

Sprey, B. and Bochem, H. P. 1991. Electron microscopic observations of cellulose microfibril degradation by endocellulase from Trichoderma reesei. FEMS Microbiol. Letters, 78, 183-188.

Sprey, B. and Bochem, H. P. 1992. Effect of endoglucanase and cellobiohydrolase from Trichoderma reesei on cellulose microfibril structure. FEMS Microbiol. Letters, 97, 113-118. 94

Sprey, B. and Bochem, H. P. 1993. Formation of cross-fractures in cellulose microfibril structure by an endoglucanase- cellobiohydrolase complex from Trichoderma reesei. FEMS Microbiol. Letters, 106, 239-244.

Stahlberg, J., Johansson, G. and Petterson, G. 1993. Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose. Biochim. Biophys. Ada, 1157, 107-113.

Sternberg, D. 1976a. A method for increasing cellulase production by Trichoderma viride. Biotechnol. Bioeng., 18, 1751-1760.

Sternberg, D. 1976b. Production of cellulase by Trichoderma. Biotechnol. Bioeng. Symp., 6, 35-53.

Sternberg, D. and Dorval, S. 1979. Cellulase production and ammonia metabolism in Trichoderma reesei on high levels of cellulose. Biotechnol. Bioeng., 21, 181-191.

Stewart, C. F. and Gilliland, S. E. 1982. Processing cottage cheese whey. U. S. Dep. Agric. Washington. The Service, Oct., 1129:31.

Szakmary, K., Wotawa, A. and Kubicek, C. P. 1991. Origin of oxidized cellulose degradation products and mechanism of their promotion of cellobiohydrolase I biosynthesis in Trichoderma reesei. J. General Microbiol., 137, 2873-2878.

Taber, W. A. and Taber, R. A. 1978. Ascomycetes. In: Laskin, A. I. and Lechevalier, H. A. (ed.), CRC Handbook of Microbiology. Vol. II. Fungi, protozoa and viruses. CRC Press, Inc., West Palm Beach, FL, p.140.

Tamada, M., Kasai, N. and Kaetsu, I. 1988. Estimation of cellulase activity based on glucose productivity. Biotechnol. Bioeng., 32, 920-922. 95

Timasheff, S. 1992. Stabilization of protein structure by solvent additives. In : T. Ahern and M. Manning (ed.), Stability of protein pharmaceuticals. Part B. In vivo pathways of degradation and strategies for rotein stabilization. Plenum Press, New York, NY, pp. 265-285.

Timasheff, S. and Arakawa, T. 1989. Stabilization of protein structure by solvents. In: T. E. Creighton (ed.), Protein structure a practical approach. IRL Press at Oxford University Press, Oxford, pp. 331-345.

Tjerneld, F., Persson, I. and Lee, J. 1991. Enzymatic cellulose hydrolysis in an attrition bioreactor combined with an aqueous two-phase system. Biotechnol. Bioeng., 37, 876-882.

Tomme, P., Heriban,. V. and Claeyssens, M. 1990. Adsorption of two cellobiohydrolases from Trichoderma reesei to Avicel: Evidence for "exo-exo" synergism and posssible "loose complex" formation. Biotechnol. Letters, 12:7, 525-530.

Townend, R. E. 1978. Coprecipitation of acid cheese whey and tannery waste and recovery of protein. Biotechnol. Bioeng. 20, 589-599.

Turbak, A. F. and Sakthivel, A. 1990. Solving the cellulose enigma. Chemtech, July, 444-446.

Volkin, D. and Klibanov, A. 1989. Minimizing protein inactivation. In: T. E. Creighton (ed.), Protein function a practical approach. IRL Press at Oxford University Press, Oxford, pp. 1-24.

Walker, L. P. and Wilson, D. B. 1991. Enzymatic hydrolysis of cellulose: An overview. Bioresour. Technol., 36, 3-14.

Warzywoda, M., Ferre, V. and Pourquie, J. 1983. Development of a culture medium for large scale production of cellulolytic enzymes by Trichoderma reesei. Biotechnol. Bioeng., 25, 3005-3010. 96

Weimer, P. J., French, A. D. and Calamari, T. A. 1991. Differential fermentation of cellulose allomorphs by ruminal cellulolytic bacteria. Appl. Environ. Microbiol., 57:11, 3101-3106.

Whatman Co. by oral communication 1993.

Whitaker, J. 1972. Principles of enzymology for the food sciences. Marcel Dekker, Inc., New York and Basel, p. 321.

White, A. R. 1982. Visualization of cellulases and cellulose degradation. In: R. M. Brown Jr. (ed), Cellulose and other natural polymer systems. Plenum Press, New York, NY.

Wilke, C. R., Maiorella, B., Sciamanna, A., Tangnu, K., Wiley, D. and Wong, H. 1983. Enzymatic hydrolysis of cellulose. Theory and applications. Noyes Data Corporation, Park Ridge, NJ, pp. 16-24.

Wood, T. and Bhat, K. M. 1988. Methods for measuring cellulase activities. In: W. Wood and S. Kellogg (ed.), Methods in Enzymology. Vol. 160. Biomass Part A. Cellulose and Hemicellulose. Academic Press, Inc., New York, NY, pp. 87-112.

Zadow, J. G. 1986. Utilization of milk components: Whey. In: R. K. Robinson (ed.), Modern dairy technology. Vol. 1. Advances in milk processing. Elsevier Applied Science Publishers. London and New York, pp. 273, 292, 296.

Zall, R. R. 1979. Whey treatment and utilization. In: J. H. Green and A. Kramer (eds), Food processing waste management. Avi Publishing Company, Inc., Westport, CT. pp. 175-180. APPENDICES 97

A. A PROTOCOL FOR THE MAINTENANCE AND HANDLING OF TRICHODERMA REESEI CULTURES ON POTATO DEXTROSE AGAR

Media preparation and inoculum

1) Trichoderma reesei grows well on potato dextrose agar (PDA).

Since it is a rich medium it tends to give rise to excessive

mycelial growth. Weaker media, such as potato carrot agar

(PCA) and malt extract agar (MA), are also suitable growth

media (Smith and Onions 1983). Addition of leaf litter or a

piece of filter paper can improve the growth. One third of the

volume of a screw capped test tube should be PDA, i. e. 6 ml

agar in a 17 ml test tube. Plates often offer better sporulation

conditions than tubes; fill them with 16-20 ml PDA.

Media formulation

Potato dextrose agar (DIFCO) 39 mg/ ml distilled water

or

Diced potatoes 300 g Glucose 20 g Agar 15 g Dist. water 1.0 L 98

Boil finely diced potatoes in 500 ml of water until thoroughly cooked; filter through cheesecloth and add water to filtrate to 1.0 L. The agar is dissolved in the filtrate by heating, and the glucose is added prior to sterilization (ATCC Catalogue of Filamentous Fungi 1991).

2) Use a revived freeze-dried fungal preparation from ATCC or a

contaminant free sample from an old plate or slant with good

sporulation as inoculum.

3) Transfers should be made during night or morning hours that are

reasonably dust free.

4) Minimize the necessary heat treatment of the PDA. Preheat the

PDA in a waterbath past 90 0C but do not boil the agar before

sterilization at 121 0C for 15 min. This should give an agar with

a pH in the range from 5.5 to 5.7.

5) The filling of the agar and the spore transfer should be done in a

sterile hood. Wash shelf and table areas with ethanol (>70 %)

before use. Wear gloves.

6) The agar should be close to solidification (40-50 0C) when it is

poured into the tubes by using a disposable sterile pipette. 99

The tubes should then be tilted so that the agar barely

reaches the neck of the tube. Allow the water vapor to

completely leave the plate or tube so that enough oxygen is

present for the growth and no condensation of water will occur.

Inoculation

7) Inoculate the agar within hours after solidification. Surface

streaks are better than inoculation with a spore suspension.

Sterilize the inoculation needle in a flame and move it into the

hood.

8) Smear the tube area close to the screwcap with a ring of

petroleum jelly. Thereby the risk of infection by dust mites is

decreased (Dade 1960).

Incubation

9) Incubate under dark conditions immediately after inoculation.

The best temperature for sporulation is 30 0C. Keep the tube

caps unscrewed the first 24 hours and then tighten them but not

completely. 100

10) The agar should turn yellow after 2 days.

11) After 3-4 days expose the mycelium to daylight or blue light for

at least a few seconds.

12) Use the cultures after 7-10 days if the spores will be utilized

for inoculation of liquid media.

Culture preservation

13) At least one tube should be stored at -20 0C for 24 hours before

placement in regular cold storage since adults, nymphal

instars and eggs of mites will then be killed (Jong 1981).

14) Store well sporulated cultures in a dark area at 4 0C.

Mites are mostly immobile and the fungal growth, variation and

metabolism is retarded at that temperature (Smith and Onions

1983).

15) The agar tends to dry out and curl after several months at 4 0C.

Transfers can be done after 4-6 months. Avoid

frequent transfers in order to decrease the risk of

contamination with Rhizopus stolonifer, many Penicillium

species and others. 101

16) Liquid nitrogen storage and freeze-drying of cultures are

superior techniques for long-term preservation. 102

B. SPORE COUNTING ON PDA PLATES BY USING ROSE BENGAL

PDA + 0.001 % Rose Bengal PDA Dilution 10-8 Dilution 10-8

PDA + 0.001 % Rose Bengal PDA Dilution 10-7 Dilution 10-7

Fig. 24. If 0.001% Rose Bengal is added to the PDA then the T. reesei colonies will appear more distinct and limited as can be seen from the photos above. Higher concentrations can kill the fungus. 103

C. A PROTOCOL FOR THE MODIFIED lUPAC FILTER PAPER ASSAY

Introduction

This assay permits the estimation of total cellulase activities

in accordance with the lUPAC assay (Ghose 1987) in the range from

0.37 FPU to 0.02 FPU by employing certain enzyme stabilizing agent

and extending the incubation time up to one day. The assay has the

advantages and disadvantages that accompany the regular lUPAC

assay. Cellulosic substrates other than filter paper may result in

different rankings of the cellulase activity.

If the intention is to get lUPAC filter paper activities and not

only relative activities, a new Trichoderma cellulase preparation

should be assayed by this method for determination of the

conversion factor (f) between the regular lUPAC filter paper unit and

the unit defined by the modified assay. This is done by carrying out a

one hour assay under lUPAC conditions but with stabilizers added to

half the number of incubated enzyme tubes. 104

Ingredient preparation

1) Dinitrosalicylic acid (DNS) reagent:

Sodium hydroxide 19.8 g Distilled water 1416 ml

Dissolve the above and add:

3,5-Dinitrosalicylic acid 10.6 g

When all the above has been dissolved add the following:

Rochelle salt 306 g (=Potassium sodium tartrate) Phenol (melt in a 50 0C water bath) 7.6 m I Sodium metabisulfite 8.3 g

Titrate 3 ml of the DNS reagent with phenolphtalein with 0.1 N HCI. Some addition of NaOH to the reagent is normally necessary to get the HCI volume into the required range from 5 to 6 ml (1 ml 0.1 N HCI = 2 g NaOH). The final pH is 13-14.

Store this solution at room temperature for shorter times and at + 4 0C for longer periods.

2) Substrate:

Whatman #1 50 mg filter paper (dry) 105

This amount corresponds to approximately 50 x 12 mm. Trim it to ± 1 mg. Curl the papers (around a thin stainless steel rod).

3) Sodium citrate buffer:

Sodium hydroxide 13.75 g Citric acid monohydrate 52.50 g

Dissolve the above in distilled water so that the final volume is 250 ml. This stock solution should have a pH close to 3.9 and can be stored at + 4 0C until used.

Then take 100 ml solution and add distilled water until the volume is 1950 ml. Add 5 N sodium hydroxide solution until the pH is almost 4.80. Then add distilled water until the total volume is 2000 ml. Use this solution fresh or store at + 1 0C for up to a week.

4) Glucose standards:

Glucose 1.00 g (dry in vacuum or at 60 0C for 5 hours)

Add distilled water until the total volume is 100 ml and mix thoroughly. Dispense small quantities into plastic vials and store them at - 20 0C until used.

Then make 5 dilutions of the above 10 mg/ml stock as follows: 106

Concentratioi i Stock Dist. water (mg/ml) (ml) (ml)

6.67 2 1 5.00 2 2 3.33 1 2 2.00 1 4 1.00 1 9

Mix each solution thoroughly and store at + 4 "C until used the same day.

5) Sodium citrate buffer with stabilizers:

Sodium citrate buffer 100 ml Bovine serum albumin 150 mg Potassium chloride 11.18g Sodium azide 30 mg

Use this solution fresh.

Place the amounts under 6) to 8) below in screw-capped test tubes of 25 ml volume or more. Store them at + 4 0C until used in the assay.

6) Substrate blank solutions: Reagent blank solutions:

citrate buffer 1.5 ml

7) Assay standards:

citrate buffer 1.0 ml glucose standard (dilution) 0.5 ml 107

8) Enzyme solutions: Enzyme blank solutions:

citrate buffer 1.0 ml (w. stabilizers) enzyme 0.5 m I

Prepare for 3 data points in duplicates (see 14) below) by taking 6 x 2 x 0.5 ml (enzyme solutions and enzyme blank solutions) from the original enzyme preparation. The incubation times must be chosen so that both data points above and below 2 mg glucose are obtained in the assay. Label the tubes according to the incubation times which should be in a geometric series. Transfer the 0.5 ml solutions slowly and take care not to leave any remnants of solution.

Performing the assay

9) Preincubate the solutions under 6) to 8) for 10 min. at + 50 0C. Then start the regular incubation by adding one filter paper to each tube. Stab the papers gently with a glass rod so that they stay under the liquid surface. Cover the tubes.

10) After the desired incubation period stop the reaction by adding 3 ml DNS. Boil the tubes for exactly 5 minutes in a vigorously boiling water bath. Keep screw caps on. Immediately cool in an ice bath.

11) Add 20 ml distilled water to each test tube and invert the tubes several times so that the pulp moves from end to end. Let the tubes stand for 20 min.

12) Calibrate the spectrophotometer by using the reagent blanks. Remove samples from the top third of the test tube. Read the absorbances at 540 nm. Make water dilutions as 108

required to get the absorbances between 0.1 and 0.7. Be sure that the pH is still highly alkaline.

Calculations

13) Get a regression line for the assay standards. Translate the blank corrected absorbances to mg reducing sugar as glucose released in the assay (i. e. in 0.5 ml).

14) Plot grams of glucose vs. the logarithm of the number of hours on a semilogarithmic paper. Find the number of hours that corresponds to 2 mg glucose.

15) Calculate the IUPAC filter paper activity from:

FPA = f x 0.37 / [(dilution)-i x # hours]

If only relative activities are of interest, the factor f above can be excluded. 109

D. AN EXPERIMENT DEMONSTRATING THE MODIFIED IUPAC FILTER PAPER ASSAY

Enzyme

A 6.18 mg/ml stock solution of a commercial T. viride cellulase

(Cellulysin) was made.

Materials

Bovine serum albumin (1 mg/ml) and potassium chloride (1 M) were present in the incubated volume. Sodium azide was utilized as an antimicrobial agent at 0.087 % and 0.013 % concentration in test runs 1 and 2 respectively.

Test runs

In both runs 1:1, 1:1.5 and 1:2 dilutions of the stock solution were incubated in duplicates with stabilizers for one hour in order to determine the apparent filter paper activities. Duplicates of a

1:7 dilution were incubated for 5 and 10 hours in test run 1 and for

3.5, 5 and 7 hours in test run 2. Duplicates of a 1:14 dilution were 110

incubated for 5, 10 and 20 hours in test run 1 and for 7, 10 and 14 hours in test run 2.

Result

Figure 25 shows how the amount of reducing sugar varies with the incubation time for the two dilutions in the second test run. The intersection with the 2 mg line gives the number of hours to be used in the calculation of the filter paper activity. Table 2 shows relative activity data. Apparently, similar activities are obtained independent of the incubation times. This talks in favor of the modified assay as a useful tool when testing samples of low cellulase activity. 111

mg glucose

hours (log scale)

D Dilution 1:7

O Dilution 1:7 o Dilution 1:14

A Dilution 1:14

Fig. 25. The amount of reducing sugar as glucose released in a modified filter paper assay for different incubation times and for 2 dilutions. T. viride cellulase is used. 112

Table 2. Relative filter paper activities obtained from modified assay data for different dilutions compared with expected values based on modified assay data for a stock solution of 7". viride cellulase.

TEST RUN Apparent FPA Average Av./7 jAv./14 SE(Mean) # 1, undiluted 0.57 # 2, undiluted 0.55 0.56 0.08! 0.04 0.01 |# 1, dil. 1:7 0.069 : #2, dil. 1:7 0.087 0.078 0.009 # 1, dil. 1:14 0.038 # 2, dil. 1;14 0.04 0.039 0.001 1 13

E. THE PIGMENT PRODUCTION BY TRICHODERMA REESEI ON LIQUID MEDIA

Table 3. The pigment production by Trichoderma reesei on liquid media.

DAYS ACID WHEY LACTOSE iSOLCAFUX pH4.4 pH6.5 pH 4.4 jpH 6.5 pH4.4 pH6.5

whiteish 0 whiteish whiteish colorless jcolorless i whiteish

3 milky yellow yellowish lyellowish yellowish white

6 yellowish egg yolk yellowish ilemon yellow pale yellowish yellowish gray (pH=5.4) yellow !(pH=4.4) (pH=3.2)

9 egg yolk brownish lemon yellow ibrownish lemorM^ellow^ brownish yellow yellow (pH=4.4) lyellow m=m yellow (pH=5.8) (pH=6.9) !(PH=6.1) i (PH-4.1) 14 yellow dark yellow grayish yellow : f i S 20 brownish brownish i i gray 1 14

F. A PROTOCOL FOR PREINCUBATION AND POSTINCUBATION PROCESSING WHEN RECOVERING PROTEINS ON WHEY MEDIA

Acid whey or whey permeate

PREINCUBATION STEPS

FILTRATION

1. 1.2 [im glass fiber / vacuum

2. 1.2 urn glass fiber with a 0.5 " layer of Celite D 3877 / vacuum

3. 1.2 |im glass fiber with a 0.25" layer of Celite D 5384 / vacuum

4. 1.2 (im glass fiber / vacuum

FILTERSTERILIZATION

Nalgene 0.2 jim cellulose nitrate filter cups / vacuum 115

POSTINCUBATION TREATMENT FOR PROTEIN RECOVERY;

FILTRATION

1.2 |xm glass fiber / vacuum

ULTRAFILTRATION

Amicon's Centricon 10 YM membrane filters (10 kD cut-off)

Centrifuge at 5000 g/1 h 15 min/4 "C

Turn filter unit and centrifuge at 800 g/2 min/4 0C

Volume adjustment w. dist. water

SIZE EXCLUSION CHROMATOGRAPHY

BioRad's Econopac 10 DG columns with polyacrylamide gel (6 kD cut-off) Gravity flow / 4 0C Preserve the gel after use in 0.02 % aq. NaNa

LYOPHILIZATION

-60 0C/7 urn Hg/60 h I Store in vacuum desiccator at 4 0C (or store at -20 0C)