UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Degradation of sawdust by Cellulomonas fimi enzymes Vondette, Nancy Anne 1982

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1982_A6_7 V66.pdf [ 5.78MB ]
Metadata
JSON: 831-1.0095441.json
JSON-LD: 831-1.0095441-ld.json
RDF/XML (Pretty): 831-1.0095441-rdf.xml
RDF/JSON: 831-1.0095441-rdf.json
Turtle: 831-1.0095441-turtle.txt
N-Triples: 831-1.0095441-rdf-ntriples.txt
Original Record: 831-1.0095441-source.json
Full Text
831-1.0095441-fulltext.txt
Citation
831-1.0095441.ris

Full Text

DEGRADATION OF SAWDUST BY CELLULOMONAS FIMI ENZYMES by NANCY ANNE VONDETTE B . S c , U n i v e r s i t y of King's C o l l e g e , 1978 Hon. Cert., U n i v e r s i t y of King's C o l l e g e , 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1982 © Nancy Anne Vondette, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 19 tL DE-6 (3/81) 1 ABSTRACT Cellulomonas f i n d was grown on minimal media w i t h casamino acids and yeast e x t r a c t added. A v i c e l was found to be the best c e l l u l o s i c carbon source f o r the production of c e l l u l a s e enzymes. The M i l l i p o r e U l t r a f i l t r a t i o n System was found to be the most e f f i c i e n t method of c o n c e n t r a t i n g the enzyme preparations. Unpretreated sawdust samples of four d i f f e r e n t softwood species were degraded between 12 and 16 percent over a 15-day treatment. Increasing the c o n c e n t r a t i o n of substrate lead to a lower percent degradation but a higher o v e r a l l degradation. Chemical pretreatment d i d not appreciably increase d e g r a d a b i l i t y of the samples. P h y s i c a l pretreatments decrease the degrada-b i l i t y of the sawdust samples. The l o t e c h pretreatment, which i s a combination of chemical and p h y s i c a l pretreatment, gives a substrate that i s degraded by the Cellulomonas f i m i enzyme p r e p a r a t i o n . The pretreatment makes 50% of the sample water s o l u b l e . In 3 days, a f u r t h e r 35% i s degraded from large i n s o l u b l e chunks i n t o i n -s o l u b l e small p a r t i c l e s which remain i n suspension. There i s 6% degraded i n t o s o l u b l e s t a t e . This leaves only 9% of the i n i t i a l sample l e f t i n the p e l l e t . With longer i n c u b a t i o n , one would expect the degradation to continue. Table o f Co n t e n t s A b s t r a c t 1. I n t r o d u c t i o n 1 1.1. H i s t o r i c a l view o f the c e l l u l a s e complex 1 1.1.1. How the system worked 1 1.1.2. C l enzyme 1 1.1.3. Cx, the endoglucanase 1 1.1.4. Ex o g l u c a n a s e 1 1.1.5. I n d u c t i o n o f the C e l l u l a s e Complex 2 1.1.6. R e c o n c i l i a t i o n w i t h P r e s e n t Day t h e o r y 2 1.2. P r e s e n t day v e r s i o n o f c e l l u l a s e complex 2 1.2.1. I n t r o d u c t i o n 2 1.2.2. Endoglucanase 3 1.2.2. C e l l o b i o h y d r o l a s e 3 1.2.4. E x o g l u c o s i d a s e 3 1.2.5. C e l l o b i a s e 4 1.2.6. B - l , 4 - e n d o g l u c a n a s e p r o d u c t i o n as a f u n c t i o n of l i m i t e d carbon s o u r c e 4 1.2.7. I s o l a t i o n o f C e l l o b i o h y d r o l a s e 5 1.2.8. S h i f t i n g system t o e x o g l u c o s i d a s e 5 1.2.9. I s o l a t i o n o f B - g l u c o s i d a s e 6 1.2.10. C e l l , l i n e improvement 6 1.2.11. I n h i b i t i o n o f B - g l u c o s i d a s e i n h i b i t i o n 7 1.3. I n f l u e n c e o f Polyenzyme C e l l u l a s e Complex C o m p o s i t i o n 7 1.3.1. I n t r o d u c t i o n 7 1.3.2. C e l l o b i a s e D e f i c i e n t Group 8 1.3.3. B a l a n c e d Systems 9 1.3.4. E x o g l u c o s i d a s e D e f i c i e n t , and C e l l o b i a s e E x c e s s Group 9 1.3.5. A d d i n g M i s s i n g Enzymes to Make a B a l a n c e d System 10 1.3.6. O v e r a l l Rate L i m i t i n g Step 11 1.4. R e g u l a t i o n o f C e l l u l a s e P r o d u c t i o n 12 1.4.1. C e l l u l a s e P r o d u c t s T e s t e d as I n d u c e r s 12 1.4.2. Glu c o s e P r o d u c t s as I n d u c e r s 13 1.4.3. R e p r e s s i o n o f C e l l u l a s e P r o d u c t i o n 13 1.4.4. S i t e o f C e l l u l a s e P r o d u c t i o n R e p r e s s i o n 14 1.4.5. R e g u l a t i o n o f C e l l u l o l y t i c System by Sophorose 14 1.4.5.1. I n t r o d u c t i o n 14 1.4.5.2. M e t h y l - B - g l u c o s i d e I n t r o d u c t i o n o f B g l u c o s i d a s e 15 1.4,.5.3. C e l l u l a s e I n d u c t i o n a f t e r B g l u c o s i d a s e I n d u c t i o n 15 1.4.5.4. M u l t i p l e Role o f Sophorose 16 1.4.5.5. S i t u a t i o n i n Trichod e r m a r e e s e i 16 1.4.6.. C e l l u l a s e P r o d u c t i o n w i t h Respect to C e l l Growth 16 1.4.7. C e l l l i n e improvements 17 - i i -1.5. B a c t e r i a l Cellulase Enzymes 18 1.5.1. Clostridium acetobutylicum 18 1.5.2. Clostridium therrnoce 11 u 1 um 18 1.5.3. Aleallgenes f a e c a l i s 19 1.5.4. Buedomonas fluoresceins var ce1lulosa 19 1.5.4.1. Introduction 19 1.5.4.2. Cellulase Components and Culture Period Period 21 1.5.4.3. Carbon Supply Control 22 1.5.4.4. C e l l o t r i a s e 22 1.5.4.5. Carbon Source 23 1.5.5. C e l l v l b r i o fulvus 23 1.5.5.1. Cellulases 23 1.5.5.2. Growth on Cellulose " " 24 1.5.6. Cellulomonas sp (Ilbc) 24 1.5.6.1. Diauxic growth 25 1.5.6.2. Cellulase Production Inhibition 26 1.5.6.3. Degradation of C e l l u l o s i c Substrates 26 1.5.6.4. Synergistic E f f e c t with Psuedomonas 27 1.5.7. Cellulomonas CS1-1 27 1.5.7.1. Cellulases 27 1.5.7.2. CS1-7 28 1.5.7.3. Degradation of C r y s t a l l i n e Cellulose 28 1.5.7.4. Product Inhibi t i o n Resistance by CS1-17 29 1.5.7.5. Enzyme Location i n CS-1 and CS1-7 30 1.5.7.6. Enzyme Location i n CS1-1 and CS1-17 30 1.5.7.7. Comparison of B glucosidase A c t i v i t y 31 1.5.7.8. Other Enzymes and Their Locations 31 1.5.7.9. Saturation Point of B xylosidase and xylanase 32 1.5.7.10. Sugar Accumulation from Sugarcane Bagasse 33 1.5.7.11. Synergistic e f f e c t with Trichoderma reesei 33 1.5.8. Cellulomonas fimi 34 1.5.8.1. Cellulases 34 1.5.8.2. Cellulase Production Derepressed 35 1.5.8.3. End Product Inhibi t i o n of Enzyme 35 1.5.8.4. Enhancement of Cellulase Production 35 1.5.9. C e l l v i b r i o gilvus 36 1.5.9.1. Cellulase mode of action 36 1.5.9.2. Energy from glucose and cellobiose 37 1.5.9.3. Enzyme Associated with Ribosomes 38 1.6. C e l l u l u l o s i c Substrate Pretreatments 38 1.6.1. Chemical Pretreatment 38 1-. 6.1.1. Chemical composition of C e l l u l o s i c Substrates 38 1.6.1.2. . Strategy for Hemicellulose Removal 39 1.6.1.3. Strategy for Lignin Removal 39 1.6.1.4. NaOH Treatment of Barley Straw 40 1.6.1.5. NaOH Treatment of Rice Straw 40 1.6.1.7. NaOH and Heat Treatment of Sugarcane Bagasse 41 - i i i -1.6.1.8. Treating Softwoods with NaOH 42 1.6.1.9. H2SO4 Treatment of Sugarcane Bagasse 42 1.6.1.10. Solvent processing 43 1.6.1.11. Comparison of Chemical Pretreatments 43 1.6.1.11.1. Results 43 1.6.1.11.2. Caustic Pretreatment 44 1.6.1.11.3. Sodium Sulphite Pretreatment 44 1.6.1.11.4. Hypochlorite Pretreatment 44 1.6.1.11.5. Peracetic Pretreatment 45 1.6.1.11.6. Butanol Pretreatment 45 1.6.1.11.7. Ethylene Glycol Pretreatment 45 1.6.1.11.8. Dilute H2SO4 Pretreatment 45 1.6.1.11.9. Lignin Concentration in Substrates 45 1.6.2. Physical Pretreatments 46 1.6.2.1. Introduction 46 1.6.2.2. B a l l m i l l i n g 46 1.6.2.3. F i t z m i l l i n g 47 " 1.6.2.4. Roller m i l l i n g 47 1.6.2.5. Comparison of M i l l i n g Types 47 1.6.2.6. Cost of commutation 48 1.6.2.7. Gamma Irrad i a t i o n 48 1.6.2.8. Gamma Irrad i a t i o n of Hemlock Sawdust 48 1.6.2.9. UV l i g h t and Sodium Nitrate Pretreatment 49 1.6.2.10. Size Reduction and Oxidative Heat 49 1.6.2.11. Extreme Temperature Pretreatment 49 1.6.2.12. NaOH and Pressure Cooking as a Pretreatment 5 0 1.6.2.13. Iotech Pretreatment 1.6.3. Approaches to Increase D i g e s t i b i l i t y 5 1 1.6.3.1. Structural features Affecting D i g e s t i b i l i t y 51 1.6.3.2. Increasing Accessible Surface Area 52 1.6.3.3. Decreasing Degree of C r y s t a l l i n i t y 52 1.6.3.4. Decreasing Degree of Substitution 5 3 2. Methods 54 2.1. DNS-CMC Assay 54 2.2. Growth media 54 2.3. Storage 55 2.4. Limiting Amount of Carbon Source 55 2.5. Concentration of Enzyme 56 2.6. Substrates for Enzyme Degradation 56 2.6.1. Sawdust Samples • 56 2.6.2. Autoclaved 56 2.6.3. Acid Treatment 57 2.6.4. Alkaline Treatment 57 2.6.5. Water Treatment 57 2.6.6. Kraft Treatment 57 2.6.7. Iotech; Treatment 57 2.7. Sugar Assays 58 2.7.1. D i n i t r o s a l i c y l i c acid (DNS) Assay 58 - i v -2.7.2. Anthrone Assay 58 2.7.3. Somogyi Assay 58 2.8. Measuring Degradation of Sawdust Samples 59 2.8.1. Measuring S o l u b i l i z a t i o n of a Sample 59 2.8.2. Measuring Reduction i n P a r t i c l e Size of a Sample 60 3. Results and Discussion 62 3.1. Optimum Growth Media 62 3.1.1. Media Used 62 3.1.2. K l e t t Measurements 62 3.1.3. Viable C e l l Count 64 3.1.4. Enzyme A c t i v i t y 64 3.1.5. Discussion 64 3.2. Limiting Amount of CMC 65 3.2.1. Introduction 65 3.2.2. Measuring Growth on Varying Concentrat Concentrations 65 3.2.3. Discussion 67 3.3. E f f e c t of Various C e l l u l o s i c Carbon Sources on Cellulase Production 67 3.4. Concentration of Enzyme 67 3.4.1. Concentrating Small Volumes 67 3.4.2. Concentrating Large Amounts of Supernatant 68 3.5. S t a b i l i t y of the Enzyme 68 3.5.1. S t a b i l i t y of Refrigerated Enzyme 68 3.5.2. S t a b i l i t y of Frozen Enzyme 68 3.6. Enzymic Degradation of Various Untreated Sawdusts 69 3.6.1. Introduction 69 3.6.2. Results 69 3.6.3. Discussion 71 3.7. Degradation of Pretreated Sawdust 71 3.7.1. Degradation of Chemically Pretreated Sawdust 71 3.7.1.1. Introduction 71 3.7.1.2. Results 72 3.7.1.3. Discussion 74 3.7.2. Degradation of Physically Pretreated Sawdust 74 3.7.2.1. Introduction 74 3.7.2.2. Results 75 3.7.3.3.. Discussion 77 3.8. Increasing Percent of Substrate 77 3.8.1. Introduction 77 3.8.2. Results 77 3.8.3. Discussion 78 3.9. Degradation of Iotech Pretreated Sawdust 78 3.9.1. Introduction 78 3.9.2. Results . 7 8 3.9.3. Discussion 79 - V -3.10. Measurement of H60 Degradation by S e t t l i n g 79 3.10.1. Introduction 79 3.10.2. Sugar Assays of Suspension 79 3.10.3. Measuring Optical Density of Suspension 80 3.10.3.1. Introduction 80 3.10.3.2. Optical Density at 550nm of Suspension 80 3.10.3.3. Discussion 80 3.11. Measurement of Freeze-Dried Suspension 80 3.11.1. Introduction 80 3.11.2. Results 81 3.11.3. Discussion ' 82 3.12. Separation of Suspension into Soluble Fraction and Small P a r t i c l e Fraction 82 3.12.1. Introduction 3.12.2. Separation by Centrifugation 82 3.12.2.1. Results 83 3.12.2.2. Discussion 84 3.12.3. Separation by F i l t r a t i o n 84 3.12.3.1. Results 84 3.12.3.2. Discussion 84 3.12.4. F i l t r e Separation over 3 Day Experiment 85 3.12.4.1. Introduction 85 3.12.4.2. Results 86 3.12.4.3. Discussion 86 3.13. E f f e c t of Using Different Concentrations of Enzyme on H60 Degradation 87 3.13.1. Introduction 87 3.13.2. Percent Recovery in Soluble Fraction 88 . 3.13.2.1. Results 88 3.13.2.2. Discussion 89 3.13.3. Percent Recovery in Non Soluble Fraction of the Suspension 91 3.13.3.1. Results 91 3.13.3.2. Discussion 94 3.13.4. Percent Recovery i n P e l l e t 94 3.13.4.1. Results 95 3.13.4.2. Discussion 98 L i s t o f I l l u s t r a t i o n s g.No. 1 C e l l u l a s e d e f i c i e n t systems 2 Balanced systems 3 Exo g l u c o s i d a s e d e f i c i e n t , c e l l o b i a s e excess systems 4 K l e t t measurement of growth on v a r i o u s media 5 Measurement of growth on media w i t h v a r y i n g amounts of CMC 6 Degradation of v a r i o u s sawdust samples 7 Degradation of c h e m i c a l l y p r e t r e a t e d sawdust samples 8 Degradation of p h y s i c a l l y p r e t r e a t e d sawdust samples 9 Recovery i n s o l u b l e f r a c t i o n 1 0 Recovery i n non-soluble p o r t i o n of suspension 1 1 Recovery i n p e l l e t 1 2 Degradation of p e l l e t -x-1. Introduction 1.1. H i s t o r i c a l view of the c e l l u l a s e complex 1.1.1. How the system worked The c e l l u l a s e systems in various organisms have been studied i n great d e t a i l . Norkran's i n i t i a l theory was a quite simple system with just three enzymes involved: / Crystalline^ Cl/amorphous\ Cx /smallerVnoglucanase /glucose^ 1.1.2. Cl enzyme C l , which attacks the c r y s t a l l i n e portion of the ce l l u l o s e , has as i t s chief product cell o b i o s e . In Trichoderma v i r i d e Cl has a molecular weight of 60,000. (Norkrans, B., 1967). 1.1.3. Cx, the endoglucanase Cx, also known as endoglucanase, attacks the amorphous substrate released by C l . This in turn releases smaller cellooligosaccharides. The molecular weight measured varied between 49,000 ( L l , L.H., et a l . , 1965) and 52,000. The optimum substrate length i s 6 glucosyl units. (Norkrans, B., 1967). 1.1.4. Exogluconase The exogluconase removes successive glucosyl moieties from the non-reducing end of the polymer chain since only non-reducing intermediates appear. ( L i , L.H., et a l . , 1965). The molecular weight of the exoglucanase i s 76,000 and completes the hydrolyis to glucose. (Norkrans, B., 1967). osaccharide -2-1.1.5. Induction of the Cellulase Complex. Mandels and her colleagues found that Trichoderma produced c e l l u l a s e when grown on c e l l u l o s e lactose, glucose and cellobiase. (Mandels, M., et a l . , 1957). As i t turns out of these substrates only c e l l u l o s e i s an inducer. Lactose i s an inducer because i t s uptake i s slow. Glucose and cellobiose are inducers because they are contaminated with sophorose. 1.1.6. Reconciliation with Present Day Theory. These cel l u l a s e s f i t into the present day theory as follows Cl,has two functions 1) releasing cellobiose from the ends of c r y s t a l l i n e c e l l u l o s e and 2) cleaving within the c r y s t a l l i n e releasing long cellooligosaccharides. Cx, the endoglucanase, releases smaller c e l l o -oligosaccharides, but i t i s actually the same enzyme as Cl, and i t s substrate i s the c r y s t a l l i n e c e l l u l o s e . The exoglucanase i n the old theory i s the exo-glucosidase i n the present day theory. B glucosidase, which i s missing from this scheme, converts the cellobiose to glucose. 1.2. Present Day Version of Cellulase Complex. 1.2.1. Introduction "The most uptodate theory of the ce l l u l a s e system for a l l c e l l u l o l y t i c organisms, as proposed by Klesov and his colleagues, contains 4 enzymes working on 3 substrates with glucose as the f i n a l product: — * 5 [ E 1 J 1 1 - k 4 [ E j J s i n i t i a l substrate Gn = long cellooligosaccharides G2 = cellobiose G = glucose E l - endoglucanase E2 = cellobiohydrolase E3 = cellobiase E4 = exoglucosidase 1.2.2. Endoglucase. Endoglucanase i s the enzyme that attacks the i n i t i a l insoluble c e l l u l o s e substrate. The product of this attack i s either long cellooligosaccharides that can be attacked by other enzymes in the system, or cellobiose. 1.2.3. Cellobiohydrolase Cellobiohydrolase, also known as B-1,4- glucam cellobiohydrolase, converts the long cellooligosaccharides to cellobiose by removing glucose dimers from the end of the cellooligosaccharide. 1.2.4. Exoglucosidase Exoglucosidase, on the other hand, removes single glucose units o f f of the cellooligosaccharide. -4-1.2.5. Cellobiase Cellobiase, also known as B-glucosidase, s p l i t s cellobiose into two glucose units. (Klesov, A.A., et a l . , 1981). 1.2.6. B-l,4-endoglucanase production as a function of l i m i t e d carbon source. The formation of the B-14, endoglucanase of Trichoderma Tignorum was investigated by Lobanok in the presence of l i m i t e d consumption of readily metabolized carbon sources. The compounds were introduced into the medium at a constant rate by the method of d i f f u s i o n . The data indicated an inverse relationship between the rate of growth of the fungus and enzyme synthesis. The activation of the c e l l u l a s e synthesis, when substrate i s l i m i t i n g , i s not s p e c i f i c a l l y associated with one carbon source or another but depends on the rate of i t s consumption. Limiting the carbon source increased the enzyme y i e l d by 200-600 f o l d with most carbon sources. When lactose was used as a li m i t e d carbon source there was only a twofold increase i n c e l l u l a s e production, but with unlimited lactose the cellu l a s e productose i s already high. Lactose uptake i s very slow so that i t s consumption i s slow even at high concentrations. The use of 2,4 dinitrophenol, which i n h i b i t s the anaerobic pathways of glucose catabolism, or malonic acid, which blocks the TCA cycle, gave a 5-7 f o l d increase in c e l l u l a s e production with a simultaneous slowdown -5-in growth of fungal culture on glucose. From these two results, i t becomes apparent that the production of endoglucanase i s influenced by l i m i t a t i o n of carbon sources and i s dependent on the resultant growth rate. (Labanok, A.G., et a l . , 1975). 1.2.7. Isolation of cellobiohydrolase. Berghem e t : a l . p u r i f i e d an enzyme by chromatography on BioGel P-10, DEAE-Sephadex, i s o l e c t r i c focusing and Biogel P-60 which showed no contamination with B glucosidase or endoglucanase. B glucosidase contamination would result in glucose appearing i n the media. If there was endoglucanase a c t i v i t y there would be CMCase a c t i v i t y . The carboxy methyl substitution on CMC prevents c e l l o -biohydrolase action. Thi,s enzyme degrades Avicel, H 2 P O 4 - swollen Avicel and cellotraose with cellobiose as the p r i n c i p a l reaction product. Therefore, this enzyme i s p u r i f i e d cellobiohydrolase. It has a molecular weight of 46,000 and i t s i s o e l e c t r i c point i s pH 3.79 (10°C) (Berghem, L.E.R., et a l . , 1973). 1.2.8. S h i f t i n g System to exoglucosidase. Nojirimycin i s a potent i n h i b i t o r of B-glucosidase. Sternberg found that when i t was added to low concen-trations of cellulos e (0.1%) and enzyme the t o t a l amount of sugar released was unchanged but most of the sugar released was i n the form of cellobiose not glucose. -6-When high concentrations (10%) of ce l l u l o s e were degraded by enzyme, the addition of nojirimycin made no difference, the end product was glucose. When nojirimycin was added, cellobiose b u i l t up in concentration. The enzyme that produced the glucose under conditions of high c e l l u l o s e and nojirimycin was the exoglucosidase. Since the sub-strate for exoglucosidase i s the long cellooligosaccharides the buildup of cellobiose has no i n h i b i t o r y e f f e c t . The exoglucosidase has a lower a f f i n i t y for the long c e l l o -biohydrolase and B glucosidase, therefore i t requires a higher cellul o s e concentration for the exoglucosidase a c t i v i t y to compensate for the lack of B glucosidase. (Sternberg, D., 1976). 1.2.9. Isolation of B-glucosidase The Trichoderma v i r i d e , now renamed Trichoderma  reesei, B-glucosidase was p u r i f i e d by ammonium sulphate p r e c i p i t a t i o n , ion exchange chromatography, i s o e l e c t r i c focusing and molecular sieve chromatography. Berghem found no enzyme a c t i v i t y toward Avicel or carbonymethyl-cellulos e could be detected i n the p u r i f i e d material under the conditions of the assay. The p u r i f i e d enzyme converted cellobiose into glucose. The molecular weight of the enzyme was 47,000 with an i s o e l e c t r i c point of 5.74 (10°C). (Berghem, L.E.R., et a l . , 1974). 1.2.10. C e l l l i n e improvement Mutants of the parent s t r a i n (QM9414) of Trichoderma  reesei have been reported by Labudova and his colleagues -7-with B-glucosidase levels increased by 3 fo l d . These mutants, MHC15 and MHC22, have additional 8-galactosidase peaks on an i s o e l e c t r i c focusing gel. The two mutants are also more highly branched. Separate B-glucosidases are important in c e l l wall formation. Therefore, t h i s increased B-galactosidase, which formed new peaks on i s o e l e c t r i c focusing gel could be associated with the c e l l wall formation rather than the c e l l u l a s e complex. (Labduova, I., et a l . , 1981). 1.2.11. Inhibition of B-glucosidase i n h i b i t i o n . Woodward found Trichoderma reesei B-glucosidase was i n h i b i t e d by many derivatives and isomers of glucose. These included B-glucose, glucose , D-giucose-L-cy'steine , L-glucose, D-glucose-6-phosphate and D-glucose-1-phosphate. Fructose, on the other hand,was a very poor i n h i b i t o r through the range of concentration of 2-100mM. This suggests that the high rate of cellobiose hydrolysis catalyzed by B-glucosidase might be prolonged by con-verting the reaction product, glucose, to fructose, a fermentable sugar, using glucose isomerase. (Woodward, J., et a l . , 1981). 1.3. Influence of Polyenzyme c e l l u l a s e complex composition. 1.3.1. Introduction. The influence of the composition of the polyenzyme cel l u l a s e complexes on the nature of the steps l i m i t i n g -8-the rate of hydrolysis of isoluble (natural) c e l l u l o s e was studied by Klasov and his colleagues. The approach was to use ce l l u l a s e preparations lacking in various enzymes from Geotrichum, Trichoderma, and Aspergillus. The f i r s t group, including Geotrichum candidum (cellobiase depleted), Trichoderma reesei, and Rapidase, were r e l a t i v e l y d e f i c i e n t i n cellobiase. The cellobiase/ endoglucanase r a t i o was 0.002-0.02. The glucose formed during c e l l u l o s e hydrolysis i s presumably mostly due to enoglucanase (E^) and not cellobiase (Eg). The cellobiose concentration accumulated continuously during the reaction on cotton l i n t e r but didn't reach an equilibrium. The equilibrium rate of glucose formation was provided for by the lower bienzyme pathway of E l & E4 as long as K4>kl. l.'3.2. Cellobiase d e f i c i e n t group. k 5 l l ] S -*GT lacking or very f 1 l i t t l e time product formation The resultant Fig. 1. i s shown in F i g . l Cellobiase d.eficient systems -9-1.3.3. Balanced Systems The second group, including Geotrichum can dldum, Trichoderma kongii and Tricoderma lignorum, were r e l a t i v e l y balanced systems. The r a t i o of c e l l o b i a s e / endoglucanase r a t i o was 0.2 to 0.3. • K 5 I*ll-k l [ E j 2 | E j J kg [Eg] -K}n ^ » G 2 -k 4fE 4]-With a balanced system, one would predict that the system w i l l reach an equilibrium with respect to cellobiose and predict a quicker glucose formation. Experimentation [Product] mM time proved this prediction correct as shown in Fig.2 1.3.4 Fig.2. Balanced Systems Exoglucosidase Deficient and Cellobiase Excess-Group. The t h i r d group, including Aspergillus niger, Aspergillus foetidus, and Trichoderma longibrachiatum, were de f i c i e n t in exoglucosidase (E4),• and had a r e l a t i v e excess of cellobiase (E3) Exoglucosidase a c t i v i t y was less than 3 units/ml. Glucose was formed almost exclu-s i v e l y from the a c t i v i t y of cellobiase. k s [ E i ] L — - ' • " " f c 4 Q ' very l i t t l e , or none The time required to reach equilibrium by the i n t e r -mediate, cellobiose, should be close to the lag period . of glucose formation. At high cellobiase concentrations the equilibrium cellobiose concentration should be low. Fig.3. Exoglucosidase Deficient, Cellobiase Excess Systems Adding Missing Enzymes to Make a Balanced System. Trichoderma v i r i d e c e l l u l a s e containing only 0.5 units/ml B-glucosidase was is o l a t e d by Sternberg and his colleagues. When this Trichoderma v i r i d e c e l l u l a s e was added to Solka Floe the cellobiose concentration reached 12 mg/ml. When added to Avicel the cellobiose concentration reaches 8 mg/ml. Aspergillus phoenicis produced B glucosidase at a concentration of 12 Units/ml. The addition of this Aspergillus phoenicis c e l l u l a s e to the Trichoderma v i r i d e c e l l u l a s e , to a B-glucosidase concentration of 6 Units/ml reduced the cellobiose concen-t r a t i o n to 2 mg/ml in Solka Floe, and 1 mg/ml, in Av i c e l . rime glucose been experimentally proven as shown in This prediction has Fig.3. -11-This, i n ef f e c t , turned a Group 1 enzyme system, the Trichoderma v i r l d e c e l l u l a s e , into a Group 2 balanced system, Trichoderma v i r i d e and Aspergillus phoenicis. (Sternberg, D., et a l . , 1977). In this system of cel l u l a s e s Klesov found the formation of glucose was lim i t e d normally by at least 2 or 3 enzymes. Addition of cellobiase to the ce l l o b i a s e -d e f i c i e n t system lead to an increase in glucose production and cellobiose reaching a low equilibrium concentration. Addition of exoglucosidase to the exoglucosidase-deficient system lead to increased glucose production and a higher equilibrium concentration of cellobiose due to increased o v e r a l l c e l l u l a s e a c t i v i t y . (Klesov, A.A., et a l . , 1981). 1.3.6. Overall Rate Limiting Step Only in Aspergillus foetidus, enriched i n cellobiase, was the l i m i t i n g step the endoglucanase. In other cases the step l i m i t i n g the rate of glucose formation with the p a r t i c i p a t i o n of intermediate cellobiose was cello b i a s e . It was shown by Klesov that, when an excess of cellobiase was added to the reaction system, the step l i m i t i n g microcrystalline c e l l u l o s e hydrolysis by a l l of the cel l u l a s e complexes became the attack' on the i n i t i a l insoluble substrate by endoglucanases. In a l l of the ce l l u l a s e systems studied in the presence of excess cellobiase, 2 l i n e a r correlation was found between Steady state rate of glucose production from micro-c r y s t a l l i n e c e l l u l o s e and the a c t i v i t y of the endoglucanases -12-in these complexes. It was shown that the action of a l l the ce l l u l a s e complexes studied i s described by e s s e n t i a l l y the same k i n e t i c p r i n c i p l e s . This suggests that the same mechanism of hydrolysis of insoluble cellulose holds for c e l l u l a s e preparations of di f f e r e n t o r i g i n s . (Klesov, A.A., et a l . , 1980). 1.4. Regulation of Cellulase Production. 1.4.1. Cellulase Products Tested as Inducers. Five carbon sources were tested by Allen and his colleagues to determine i f they induce c e l l u l a s e pro-duction from Trichoderma reesei. (1) 50% (w/v) glucose, (2) 16% (w/v) cellobiose, (3) a r t i f i c i a l hydrolysis syrup (reagent grade glucose, xylose, cellobiose and gentiobiose), (4) synthetic reversion syrup (glucose incubated with B-galactosidase), (5) a 50% ce l l u l o s e hydrolysate. These five syrups were added under computer control to control C02 evolution. This controlled the growth rate at a set l e v e l . The following c e l l u l a s e concentrations resulted: Substrate Amount consumed 1) - 60 g/1 2) 60 3) 60 . 4) 60 5) 60 5) 115 Cellulase units/ml] 0 0 0 1.8 4.0 10.7 E x t r a c e l l u l a r B glucosidase .37 .94 1.0 .6 .35 1.8 -13-Th. i s indicated cellobiose i s not a natural inducer, since alone or in muxture i t didn't induce. Cellulose hydro-lysates and B-galactosidase reversion syrups are known to contain transfer products (B dimers and trimers of glucose) and i t appears one or more of these i s an inducing compound. (Allen A.L., et a l . , 1981) 1.4.2. Glucose products as Inducers Glycerol, glucose and i t s derivatives, gluco-disaccharides including sophorose and gentiobiose, cellooligosaccharides, c e l l u l o s e and derivatives of sophorose were tested by Nisizawa and his colleagues to see i f they induced c e l l u l a s e formation in washed myceluim. Only sophorose and gentiobiose enhanced e x t r a c e l l u l a r c e l l u l a s e , with gentiobiose inducing one seventh as much as sophorose. Gentiobiose i s quite often contaminated with sophorose due to the process by which i t i s produced, so induction by gentiobiose may actually be due to sophorose. Cellulase induction optimum of _3 sophorose was 10 M. (Nisizawa, T., et a l . , 1971). If a high concentration of glucose i s used induction takes place. This i s because sophorose i s present in glucose preparations at a concentration of 0.0058%. (Mandels, M., et a l . , 1962) . 1.4.3. Repression of Cellulase Production. The production of c e l l u l a s e enzymes are repressed by such things as ATP, glycerol, pyruvate, succinate, -14-malate, gluconate, glutamate, glucose and maltose. This i s in d i c a t i v e of catabolite repression (Nisizawa, T., et a l . , 1971). 1.4.4. Site of Cellulase Production Repression. Nisizawa et a l . showed that glucose i n h i b i t e d the production of c e l l u l a s e , B glucosidase, and xylanase. The i n h i b i t i o n by glucose was compared with those by actinomycin D, which affects transcription, and puromycin, which affects translation, and i t resembled the puromycin and d i f f e r e d considerably from the actinomycin D i n extent and length of time required for complete cessation of c e l l u l a s e production after i t s addition. Glucose was s t i l l i n h i b i t o r y 60 min a f t e r actionomycin D was added, when no more MRNA was produced. Therefore, i t appears that repression of c e l l u l a s e formation occurs mainly at the t r a n s l a t i o n a l l e v e l of c e l l u l a s e production. (Nisizawa, T., et a l . , 1972). 1.4.5. Regulation of the C e l l u l o l y t i c System by Sophorose 1.4.5.1. Introduction. An elegant theory for the regulation of the c e l l u -l o l y t i c system i n Trichoderma reesei by sophorose has been made by Sternberg, et a l . Sophorose has two e f f e c t s , B-glucosidase repression and ce l l u l a s e induction. The repression of B-glucosidase was more sensi t i v e to sophorose concentration than was c e l l u l a s e induction. -15 -Half maximum repression of B-glucosidase occurred at 0. 5JJM with complete repression to c o n s t i t u t i t i v e levels at QyxU. The threshold for c e l l u l a s e induction was at 4uM. Half maximum induction occurred at 160juM. The threshold for sophorose hydrolysis by p-glucosidase was 90uM. and half maximum sophorose hydrolysis occurred at 1400uM. No other mono- or di-saccharide s i g n i f i c a n t l y repressed 0-glucosidase. In a c e l l u l a s e l e s s mutant of Trichoderma reesei B-glucosidase was s t i l l repressed by sophorose. Therefore repression of B-glucosidase by sophorose appeared to be independant of c e l l u l a s e induction. 1.4.5.2. Methyl-B-glucoside Induction of 3-glucosidase. Methyl 3-glucoside induces B-glucosidase without af f e c t i n g c e l l u l a s e induction. Sophorose can repress this methyl B-glucoside-induced B-glucosidase production at any point in i t s induction with the maximum l e v e l of B-glucosidase produced being equivalent to those reached 2 hours l a t e r in the control. 1.4.5.3. Cellulase induction a f t e r ,B-glucosidase Induction. Cellulase induction was the same with or without methyl-J3-glucoside when sophorose was added at 0 time, but was diminished when sophorose was.added afte r p-glucosidase induction was already underway. Therefore ,B-glucosidase induction seems to take precedence over . ce l l u l a s e induction. -16 -1.4.5.4. Multiple Role of Sophorose. The multiple role of a single substance, sophorose, as a repressor and substrate of one enzyme, as well as an inducer of a seperate enzyme system that has no a c t i v i t y against i t , i s unique. Advantages for the regulation of a sequence of enzymatic reactions occur, however, since by repressing B-glucosidase synthesis, the potential for hydrolysis of sophorose i s reduced and that for c e l l u l a s e induction i s increased. If sophorose i s added to mycelia which have induced le v e l s of B-glucosidase, the induction of c e l l u l a s e i s lessened. These findings help explain why B-glucosidase levels are low in c e l l u l o s e fermentations with Trichoderma  reesei. They are produced at constitutive l e v e l s of only .1-.3 Units/mg. protein. The natural inducer of c e l l u l a s e in Trichoderma reesei may be, l i k e sophorose, a soluble compound which represses jB-glucosidase synthesis during growth on c e l l u l o s e . (Sternberg, D., et a l . , 1980). During the c u l t i v a t i o n of wild type Trichoderma  v i r i d e on microcrystalline c e l l u l o s e , the synthesis of c e l l bound ce l l u l a s e s , whose a c t i v i t y against f i l t r e paper was measured by Volfova, preceded c e l l growth. During growth some of these are released into media as e x t r a c e l l u l a r enzymes. The rate of synthesis of extra-c e l l u l a r f i l t r e paper, ce l l u l a s e s increased during c e l l 1.4.5.5. Situation in Trichoderma r e e s i i . 1.4.6. Cellulase Production and C e l l Growth -17 -growth reaching maximum at the t r a n s i t i t i o n to stationary-phase when growth dropped. The rate of synthesis of bound enzyme during active growth was almost constant. In stationary phase the rate of synthesis of both ( f i l t r e paper) cellulases dropped sharply, ceasing well before c e l l l y s i s sets i n . (Volfova, 0., et a l . , 1981). The bound ce l l u l a s e s Kyslikova found were released into the media at the stage of c e l l l y s i s . At th i s time extra-c e l l u l a r c e l l u l a s e s attained maximum a c t i v i t y . (Kyslikova, E., et a l . , 1981). 1.4.7. C e l l l i n e improvements. Many attempts have been made to mutate Trichoderma  reesei to increase the c e l l u l a s e l e v e l s . Two examples of successful mutagenesis are VTT-D-81033 and Rut C-30. VTT-D-80133 was formed, by Bailey, by further mutagenizing VTT-D-78085, a mutant of the parent s t r a i n , ZM9414. It retained the high c e l l u l a s e production of VTT-D-78085 and was more stable. The cel l u l a s e and exocellobiohydrolase were increased by approximately twofold and the B glucosidase by approximately 1.7 f o l d compared to the parent s t r a i n . Other e x t r a c e l l u l a r proteins are also increased. Amyloglucosidase was increased 7.2 fold, xylanase was increased 1.6 fo l d , and mannase was increased 1.7 f o l d . (Bailey, M.J. et a l . , 1981). Rut C-30, i s o l a t e d by Tangnu, produced high c e l l u l a s e a c t i v i t i e s , considerable amounts of xylanase and -18-B-glucosidase. The xylose produced can be converted into alcohol, 1,4 butanediol, or acetic acid by organisms such as Fusaruim oxysporum. (Tangnu, S.K., et a l . , 1981). 1.5. B a c t e r i a l Cellulase Enzymes 1.5.1. Clostridium acetobutylicum Clostridium acetobutylicum according to Allcock, was used in industry to make acetone and butanol from corn starch or molasses before the discovery of synthetic processes. Clostridium acetobutylicum produces a carboxymethyl c e l l u l a s e and a cellobiase, both at quite low levels when induced. The carboxymethel c e l l u l a s e a c t i v i t y in the supernatant i s 0.40 umol.glucose/ml.min. The cellobiase a c t i v i t y i s 0.50 umol.glucose/ml.min. The carboxymethylcellulase i s not repressed by glucose and i s induced by a small molecule present i n molasses that can be dialyzed out. (Allcock, E.R., 1981). 1.5.2. Clostridium thermocellulum Ng showed that Clostridium thermocellulum .released both endoglucanase and exoglucanase into i t s supernatant. The r a t i o of endoglucanase/exoglucanase produced was higher for Clostridium thermocellulum than for Trichoderma reesei; As a result of t h i s , the i n i t i a l (15 min) hydrolytic products from microcrystalline c e l l u l o s e were long chain oligosaccharides instead of short chain oligosaccharides. Clostridium thermocellulum produced -19 -no e x t r a c e l l u l a r c e l l o b i a s e o r B - x y l o s i d a s e . As a r e s u l t o f t h i s , c e l l o b i o s e and x y l o b i o s e were the major l o n g term (24 hour) h y d r o l y s i s p r o d u c t s o f A v i c e l , S o l k a F l o e , o r x y l a n . The enzymes were v e r y heat s t a b l e m a i n t a i n i n g h i g h a c t i v i t y a t 60-70°C. The enzymes were a l s o n o t e a s i l y i n h i b i t e d ; the a d d i t i o n o f 73mM g l u c o s e o r 29mM c e l l o b i o s e caused no i n h i b i t i o n o f the C l o s t r i d i u m  t h e r m o c e l l u l u m enzymes b u t caused 20% i n h i b i t i o n o f Trichoder m a r e e s e i enzymes. (Ng, T.K., 1981). 1.5.3. A l c a l i g e n e s f a e c a l i s A l c a l i g e n e s f a e c a l i s was an a e r o b i c b a c t e r i a i s o l a t e d from sugarcane bagasse by Han which produced an i n d u c i b l e B - g l u c o s i d a s e . I t produced t h i s J 3 - g l u c o s i d a s e even i n t h e presence o f g l u c o s e , and was i n d u c t e d by c e l l o b i o s e , l a c t o s e , and B - m e t h y l - D - g l u c o s i d e . The s p e c i f i c i t y o f t he enzyme was t e s t e d by measuring the c o m p e t i t i v e i n h i b i t i o n o f p a r a n i t r o p h e n y l - B - g l u c o s i d e (PNPG) h y d r o l y s i s b y " B - g l u c o s i d e s . C e l l o b i o s e caused the most i n h i b i t i o n , f o l l o w e d by g l u c o s e and then B-methyl g l u c o s i d e . T h i s showed t h a t the a c t i v i t y i s m a i n l y towards 0 - g l u c o s i d e s . The enzyme was heat i n a c t i v a t e d above 55°C. 90% o f the a c t i v i t y was d e s t r o y e d by h e a t i n g t o 58°C f o r 16 m i n u t e s . (Han. H.W., 1969) . 1.5.4. P'suedomohas f l u o r e s c e n s v a r . e e l l u l o s a 1.5.4.1. I n t r o d u c t i o n Psuedomonas f l u o r e s c e n s var. c e l l u l b s a produced t h r e e c e l l u l a s e A, B, and C. A and B were found e x t r a --2Q-c e l l u l a r l y . They were found by Susuki in the super-natant 6f a centrifuged sample of growth medium. C was found i n an interwall location and was released into the supernatant when the c e l l s were osmotically shocked (Yamane, et al.', 1971). These three enzymes were tested for t h e i r a c t i v i t y towards many c e l l u l o s i c substrates. The number of glucose units released by the enzyme during the test period were as follows: Substrate A B__ C cellobiose 0 0 0 c e l l o t r i o s e 0 0 1010 cellotetraose 1190 230 1850 cellopentaose 2250 240 1370 cellohexaose 3520 410 1810 c e l l o d e x t r i n 80 7 640 swollen c e l l u l o s e 3 7 4 Avicel .7 .4 .2 CMC 1150 90 1860 c e l l o b i o s l s o r b i t o l 0 0 0 c e l l o t r i o s y l s o r b i t o l 0 0 230 c e l l o t e t r a o s y l s o r b i t o l 560 30 850 p Nitrophenyl B-D glucoside 0 0 0 P Nitrophenyl B cellobioside .4 .3 8 From these results (Susuki, H., et a l . , 1969) two main conclusions can be made: 1) No c e l l bound or e x t r a c e l l u l a r c e l l u l a s e can attack a substate with only two glucose units. Only the c e l l bound c e l l u l a s e , (C), can attack a substrate with 3 glucose units in i t , such as c e l l o t r i o s e . The extracellulae c e l l u l a s e s , A and B, require a substrate with at least 4 glucose units, such as cellotetraose. 2) In general, c e l l u l a s e A has at least 5 times the a c t i v i t y of c e l l u l a s e B. -21-1.5.4.2. Cellulase components and Culture Period. The culture medium of Psuedomonas fluorescens var. .cellulos.a was run down a column after various times in the culture period by Yoshikawa et a l . Several e x t r a c e l l u l a r c e l l u l a s e components of d i f f e r e n t molecular weights were found. The number of components increased in p a r a l l e l with the culture period. These c e l l u l a s e components (peaks I, II, III, IV and V) could be e l e c t r o -p h o r e t i c a l l y separated into two fractions, c e l l u l a s e A and Cellulase B, in varying proportions. Peak I was composed almost exclusively of c e l l u l a s e B; peak V was composed almost exclusively of c e l l u l a s e A. The r a t i o of t o t a l c e l l u l a s e A to t o t a l c e l l u l a s e B increased during growth. Peak I, which was composed almost ex-c l u s i v e l y of c e l l u l a s e B, and which has the largest molecular weight among the detectable e x t r a c e l l u l a r c e l l u l a s e s , was found to decrease i n amount during culture, and almost disappeared at the l a t e r stages of culture. Peak I was also the only component found in the intrawall f r a c t i o n during the early stages of culture i n which active secreation of e x t r a c e l l u l a r cellulases was taking place. These facts strongly suggest that the multiple peaks in the medium are formed secondarily from peak I outside the c e l l . (Yohikawa, T., et a l . , 1974). This theory was substanteated by Susuki by the fact that p u r i f i e d c e l l u l a s e B can be converted to -22 -c e l l u l a s e A by some factor present i n Buedomonas fluorescens var c e l l u l o s a spheroplasts.grown on ce l l u l o s e or sophorose. thi s conversion can be followed by zone electrophoresis of the sample. (Susuki, H. et a l . , 1969). When a zone electrophoresis gel was s l i c e d into ten equal sections by Yamane, cel l u l a s e A was found i n section 6 and ce l l u l a s e B i s found in section 3. (Yamane, K., et a l . , 1971). When grown i n cultures in which the rate of c e l l growth was lim i t e d by carbon supply to one t h i r d of the normal rate of 0.5% cellobiose, Yamane found the t o t a l c e l l u l a s e a c t i v i t y increased .from 50 units, mostly cell-bound, to 180 units,mostly c e l l f r e e . When sophorose, gentiobiose, glucose, cellobiose, arabinose, xylose and galactose were tested i n the controlled carbon supply experiments, a l l , except galactose, caused enhancement of cel l u l a s e production even though growth rates were lower. 1.5.4.3. Carbon supply control. Carbon supply control leads to the increase of other e x t r a c e l l u l a r enzymes such as amylase. These increases were not due to autolysis since B-galactosidase, an i n t r a c e l l u l a r enzyme, did not increase in the super-natant. (Yamane, K., et a l . , 1970). The main product of c e l l u l a s e A or cel l u l a s e B degradation of c e l l u l o s i c substrates was c e l l o t r i o s e . (Yamane, K. et a l . , 1971). 1.5.4.4. C e l l o t r i a s e . Cellulase C appeared absorbed into c e l l wall -23-constituents and/or cytoplasmic membrane. Cellulase C converts c e l l o t r i o s e into cellobiose and glucose. Both of these products then pass into the c e l l where i n t r a -c e l l u l a r B-glucosidase can further degrade the c e l l o -biose. The production of ce l l u l a s e C i s independant of the production of cellul a s e A and ce l l u l a s e B (Yamane, K., et a l . , 1971). 1.5.4.5. Carbon Source The rate of c e l l u l a s e production i s affected by the carbon source i n the growth medium. Yamane et a l . showed that the amount of cel l u l a s e produced on varying c e l l u l o s i c compounds was as follows: Cellulose E x t r a c e l l u l a r I n t r a c e l l u l a r A v i c i l CMC DS.7 CMC DS.4 Cellulose Powder E i l t r e Paper *G-25 G-4 G-3 G-2 513. 7 407.9 117. 3 230 143 24.4 15 .6 7.8 2.5 58.1 55 . 3 42.9 21 30 61.5 37.8 34.2 36 .2 *glucose polymers. 1 As can be seen by these results there was a reverse relationship between rate of c e l l u l a s e production and s u s c e p t i b i l i t y of c e l l u l o s i c substances to the action of c e l l u l a s e (Yamane, K. et a l . , 19.70). 1.5.5. C e l l v i b r i o fulvus. 1.5.5.1. Cellulases. C e l l v i b r i o fulvus i s an aerobic c e l l u l o l y t i c bacterium which has been recently r e c l a s s i f i e d as a -24-Psuedomonas. When c e l l s were grown on glucose or c e l l o -biose, Berg found a l l the CMC-hydrolyzing enzyme was cell-bound. Part.of t h i s a c t i v i t y was found on the c e l l surface; part of the a c t i v i t y i s found i n the periplasm and bound to an i n t e r i o r membrane f r a c t i o n . The produc-tion of c e l l u l a s e was repressed by•glucose concentrations higher than 0.04% i n the medium. The enzyme was produced at a constant rate of 5 units/mg. protein on c e l l o b i o s e . As the sugar concentrations became low the location changed from c e l l bound to c e l l free. Growth on c e l l u l o s e gave c e l l free c e l l u l a s e active against CMC at the rate of 40 units/mg. protein. (Berg, B., 1975). 1.5.5.2. Growth on Cellulose No growth was obtained on long cotton f i b r e s . Growth did occur on cotton i f the fibres were cut into small pieces (this opens up the lumen), and on f i l t r e paper and chromatography powders derived from cotton. Reducing sugars were not found i n these c e l l u l o s e cultures showing that the cellulases produce only as much reducing sugar as the bacteria could use. (Berg, B., et a l . , 1972a) C e l l v i b r i o fulvus degraded the very compact fibres of prepared cotton very slowly. By electronmicroscopy i t can be seen that the bacteria penetrated the lumen and degraded the fibres from within l i g n i n - f r e e pulp fi b r e s , which have a very open structure were rapidly degraded from within by the C e l l v i b r i o fulvus. In contrast to this mode of action, Sporocytophaga myxb co ceo1de s -25 -attacked the fibres both from the outside and from within by making close contact with the c e l l u l o s e . This bacteria worked i n this manner because a l l of i t s cellulases are cell-bound. (Berg. B., et a l . , 1972b). 1.5.6. Cellulomonas sp (Ilbc) 1.5.6.1. Diauxi-e growth. Cellulomonas sp (Ilbc) ,' Cellulomonas f 1 avi gen a, has been examined by Enriquez i n fermentation systems using a l k a l i pretreated sugarcane bagasse. During batch operation diauxic growth was found which cannot be explained by catabolite repression since the reducing sugar l e v e l in the medium remained below 40 mg/l. The variation in r a t i o of ce l l u l o s e to hemicellulose during the fermentation suggested the i n i t i a l u t i l i z a t i o n of the ea s i l y degradable substrate, that i s , the hemicellulose and amorphous cell u l o s e , u n t i l t h e i r concentration became l i m i t i n g , followed by the u t i l i z a t i o n of the c r y s t a l l i n e c e l l u l o s e . During the f i r s t log phase the l i q u i d contained pentoses (xylose and arabinose) along with glucose, cellobiose, and galactose. During the second log phase glucose and cellobiose were the major f r a c t i o n . This indicated an i n i t i a l lack of endoglucanase which was brought into e f f e c t in the second log phase Cellulomonas sp (Ilbc) was capable of converting 70% of the a l k a l i pretreated sugarcane bagasse with a resultant 0.355g biomass/g. bagasse feed.(Enriquez, A., 1981). -26-1.5.6.2. Cellulase Production Inh i b i t i o n . The amount of i n h i b i t i o n of cel l u l a s e production was determined by Beguin for varying constituents in the media. The cel l u l a s e (CMCase) active free i n the super-natant and the ce l l u l a s e bound to cel l u l o s e which couldn't be washed o f f by a buffered solution was measured: grown on maximum cel l u l o s e maximum soluble bound a c t i v i t y c e l l u l o s e glucose ce l l u l o s e + cellobiose c e l l u l o s e + glycerol c e l l u l o s e + acetate acetate 250 25 25 155 160 10 30 15 160 10 Substrates that were more e a s i l y digested, and products of c e l l u l o s e degradation repressed the production of ce l l u l a s e . In the absence of cel l u l o s e or i n the presence of non-repressing substrates, such as acitate, no c e l l u l a s e was formed. In a l l cases no ce l l u l a s e a c t i v i t y was found u n t i l the c e l l s were in stationary phase. (Beguin, P., et a l . , 1977). 1.5.6.3. Degradation of C e l l u l o s i c Substrates The enzyme was tested by Han to see how well i t degraded various c e l l u l o s i c substrates-. The % digestion of the substrates follow: - 27-substrate f i l t r e paper ce l l u l o s e paper PAB ce l l u l o s e CM c e l l u l o s e A20 c e l l u l o s e Cotton f i b r e paper towel bagasse p i t h bagasse fibres sorgo bagasse a l k a l i - t r e a t e d sorgo bagasse % digestion 55 .0 34 .0 41.0 28.0 20.5 0 30.0 10 .0 3.4 15 .0 80 .0 (Han, Y.W. e t a l . , 1968) 1.5.6.4. Synergistic E f f e c t with Psuedomonas Srinivasan found a syn e r g i s t i c e f f e c t when Cellulomonas  flavigena and Psuedomonas were grown together on sugar -cane bagasse. Psuedomonas by i t s e l f could not grown on bagasse. Ce1lulomon as flavigena slowly grew to an o p t i c a l density of 0.10. An equal mixture of the two bacteria grew very quickly to an o p t i c a l density of 0.30. (S r i n i v a s i n , V.B., et a l . , 1969). 1.5.7. Cellulomonas CS1-1 1.5.7.1. Cellulases A Cellulomonas s t r a i n (CS1-1) which could readily degrade cottonwool was isola t e d from s o i l by Choi. The amount of enzyme produced varied with the carbon source supplied: Carbons'ource Cellobiose CMC Avicel CMCase i C e l l bound 2 2.5 10 units/ml 1 e x t r a c e l l u l a r undetectable 30 2 Bglucosidase units x 16 at max. Fatmax 18 at maximum 103/ml. reducing sugar mg/ml - .2 in 2 .2 in 7 days days pH 7-5 rapidly 7 stable 7-5 slowly Vaible cells/ml 109in 2 days 109in 2 109in 4 days drops when days pH drops stable stable -28 -1.5.7.2. CS1-7 A.mutant strain,- CS1-7, of CS1-1, made by UV i r r a d i -ation of the parent s t r a i n by Choi et a l . , altered i t s a b i l i t y to degrade cottonwool and had higher lev e l s of enzyme production CS1-5 CS1-7 days to clear Avicel plate 3.5 2 . days to clear Avicel + glucose plate 10 6 % cottonwool digestion in 5 days 16 24 Bglucosidase units/mg protein i n 2 days .01 .02 E x t r a c e l l u l a r CMCase units/ml in 2 days 3 15 Cellbound CMCase units/ml in 2 days 6 15 (Choi, W.Y., et a l . , 1978) 1.5.7.3. Degradation of C r y s t a l l i n e Cellulose Both CS1-7 and CS1-17, another UV mutant of CS1-1 degraded c r y s t a l l i n e c e l l u l o s e (cottonwool) more e f f e c t i v e l y than the parent s t r a i n . The amount of degradation varied with the conditions under which the degradation occurred. Three conditions were tested by Haggett. 1) stationary incubation i n the standard medium, 2) stationary incubation i n the standard medium buffered by the addition of morpholino propane sulphonic acid (MOPS) -at 40mM. 3) shaken at 200 r.p.m. in the standard medium. The resultant degradation under these conditions were as follows: -29 -Strain Stationary standard medium Stationary standard medium •+ MOPS Shaken standard medium CS1-1 26% 35% 63% CS1-7 32 44 CS1-17 36 51 68 pH . 5.3 6.8 7.9 Viable c e l l s / ml at maximum 10 6 10 9 ioi° The mutants achieved higher reducing sugar lev e l s i n the growth medium: Reducing sugar Stationary Stationary Shaken mg/ml standard medium standard medium standard + MOPS medium CS1-1 CS1-7 CS1-17 0.20 0.77 0.68 0 .20 0 .58 0 .61 ,10 10 Under shaken conditions the reducing sugars were u t i l i z e d as quickly as i t was produced. 1.5.7.4. Product Inhibition Resistance by CS1-17 The mutant CS1-17 enzyme was quite resi s t a n t to product i n h i b i t i o n . The amount of reducing sugar released from a l k a l i pretreated bagasse into the media by the enzyme in the presence of various degradation products was measured by Choudhury: Inhibitor (g/1) % A c t i v i t y 24 hrs % A c t i v i t y 48 hrs. none glucose (40) glucose (80) cellobiose (20) cellobiose (40) xylose (40) ethanol (40) 100 88 75 90 77 90 88 100 85 71 88 7.3 87 82 -30-In comparison Trichoderma reesei had only 40% a c t i v i t y in the presence of 4% ethanol. (Choudhury, N., et a l . , 1980a). 1.5.7.5. Enzyme Location in CS1-1 & CS1-7 The location of the various enzymes was then determined i n CS1-1 and CS1-7, measured in a stationary MOPS-buffered incubation: Avicelase Units CMCase Units Bglucosidase units 10 6viable c e l l s 1 0 1 3 v i a b l e c e l l 1 0 1 4 v i a b l e c e l l s Strain Location 3 days 5 days 8 days 3 days 5 days 8 days 3 days 5 days 8 day CS1-1 c e l l bound 5 60 5 5 20 10 10 CS1-7 40 - 270 25 — 20 50 15 15 CS1-1 c e l l debris 5 - 445 55 — 40 30 20 30 CS1-7 " " 5 - 500 55 — 90 50 25 45 CS1-1 e x t r a c e l l u l a r 3100 965 4650 350 175 285 5 - 10 CS1-7 14000 3350 6500 480 250 265 5 - 10 1.5 From these results two main conclusions could be de-termined. F i r s t , that-Avicelase and CMCase were b a s i c a l l y e x t r a c e l l u l a r and Bglucosidase was b a s i c a l l y cellbound. Second, the mutant CS1-7 produced two to three times the amount of enzyme of the parent s t r a i n , CS1-1. 7.6. Enzyme Location in CS1-1 and CS1-17. This type of comparison was made between the mutant s t r a i n CS1-17 and the parent s t r a i n in a shaking incubation with the following r e s u l t s : -31" Avicelase Units 1 0 1 6 v i a b l e c e l l s Strain Location 2 3 4 2 3 4 2 3 4 days days days days days days days days days CS1-1 c e l l bound 60 100 20 5 10 • 5 5 5 CS1-17 It IT 125 200 175 20 15 — 10 5 5 CSl-1 c e l l debris 5 65 — 55 75 - 20 20 20 CS1-17 10 45 - 80 90 — 105 45 25 CSl-1 e x t r a c e l l u l a r • 7400. 5000 1690 1095 875 1085 5. 5 — CS1-17 I I 19000 8145 5940 , 1755 1310 1870 5 5 -CMC Units | Bglucosidase 1 0 1 3 v i a b l e c e l l 1 0 1 4 v i a b l e c e l l s From these results one could s i m i l a r l y conclude that CS1-17 was a superior producer of the c e l l u l a s e s . A conclusion as to whether CS1-7 or CS1-17 was better cannot be made since the two studies were done under d i f f e r -ent growth conditions (Haggett, K.D., et a l . , 1979). 1.5.7.7. Comparison of B-glucosidase A c t i v i t y Haggett compared the B-glucosidase a c t i v i t y of CSl-1 and the two mutants, CS1-7 and CS1-17 when grown on glucose and on cellobiose: Strain glucose grown cellobiose grown units/mg protein units/mg protein 24 hrs. 6 hrs CSl-1 CS1-7 CS1-17 0.015 0 .245 0.390 0.030 0 .070 0 .085 When grown on glucose CS1-17 had a 26 f o l d increase in B-glucosidase production over CSl-1. When grown on cellobiose there was approximately a 3 f o l d increase of B-glucosidase production over CSl-1. (haggett, K.D., et a l . , 1978). 1.5.7.8. Other Enzymes and the i r Locations The CSl-1 and CS1-17 were grown on hemicellulose, spun down and tested for soluble enzyme a c t i v i t y in the -32 -supernatant, and insoluble enzyme a c t i v i t y in the broken p e l l e t . Rickard found the broken p e l l e t contained B-xylosidase, x-L-aribinosidase, x-glucosidase, x-galactosidase, B-glucosidase, B-galactosidase, B-mannosidase, B-glucuronidase, and x-mannosidase. Of these only B-xylosidase, x-L-aribionosidase, x-glucosidase, and B-mannosidase were found in the supernatant. They were not found i n the supernatant u n t i l the seond day and were in lower concentrations than i n the insoluble f r a c t i o n . In a l l cases CS1-17 had higher l e v e l s of soluble enzymes than CS1-1. From the l i s t of enzymes found, Cellulomonas possesses a range of glucosidase a c t i v i t i e s which could be necessary for e f f e c t i v e hydrolysis of the hemicellulose fractio n of the bagasse. (Rickard, P.A.D., et a l . , 1981b). When tested by Rickard et a l . , CS1-17 was found to be improved over CS1-1 in i t s a b i l i t y to hydrolyse xylan containing substrates such as commercial xylan, bagasse holocellulose and hemicellulose (Rickard, P.A.D., et a l . , 1981a) 1.5.7.9. Saturation Point of B xylosidase and xylanase Both B-xylosidase and xylanase were tested to measure their saturation points. Xylanase has an endo action cleaving polymers to oligomers of 2 or more sugar units. Xylobiose i s not hydrolysed by xylanase. Xylanase a c t i v i t y increased with the increase of xylan concentration in the growth media to 0.33%. It then decreased to one -33-t h i r d of the maximum a c t i v i t y when the xylan concentration was increased to 1.65 or 3.3%. B-xylanosidase cleaves xylobiose to xylose. B-xylosidase a c t i v i t y increased l i n e a r l y with increased substrate up to 0.033% at which concentration i t was saturated. Further increase i n xylanopyranoside substrate up to 0.3% caused no change i n a c t i v i t y . (Rickard, P.A.D., et a l . , 1980). 1.5.7.10. Sugar Accumulation from Sugarcane Bagasse. An active c e l l u l o y t i c culture was obtained by Choudhury following growth of CS1-17 for 24 hours at 32°C on a l k a l i treated sugarcane bagasse (10-20g/l). Conditions were then changed to find conditions favouring sugar accumulation from fresh a l k a l i pretreated bagasse added to the 24 hour culture at a concentration of 75g./l. After incubation for 48 hours at 37° under anaerobic, aerobic, and aerobic with .2% sodium azide added, reducing sugar was accumulated at 22.8, 23.7 and 25.6 g/1 respectively. Approximately 83% of thi s was released within the f i r s t 18 hours. The reducing sugar released contained 14% xylose, 35% glucose, and 25% cellobiose. If exogenous cellobiase was added the cellobiose was converted to glucose. (Choudhury, N., et a l . , 1980b). 1.5.7.11. Synergistic e f f e c t with Trichoderma reesei. This may explain the sy n e r g i s t i c e f f e c t when adding both CS1-17 enzyme and Trichoderma reesei enzyme, making a more balanced c e l l u l a s e system, to a l k a l i pretreated sugarcane bagasse, to the reducing sugar l e v e l : -34 -Enzyme preparation Reducing sugar y i e l d (g/1) Cellulomonas 23 T.reesei.at 0.1 FPU/ml 11 T.reesei at 0.25 FPU/ml 25.5 T.reesei at 0.5 FPU/ml 34.5 .1 FPU/ml 42 Cellulomonas + T. reesei Cellulomonas + T. reesei Cellulomonas + . T. reesei Cellulase a c t i v i t y as measured i n f i l t r e paper units (FPU) in mixtures of the enzyme also showed synergism: Preparation measured FPU/ml Cellulomonas ' 0.12 Cellulomonas +0.1 FPU/ml T.reesei 0.37 Cellulomonas +0.25 FPU/ml T. reesei0.62 Cellulomonas +0.5 FPU/ml T.reesei 1.92 (Choudhury, N., et a l . , 1981) 1.5.8. Cellulomonas fimi 1.5.8.1. Cellulases. A Cejlwlomonas sp i s o l a t e d by Stewart et at., from s o i l which had the c h a r a c t e r i s t i c s of Cellulumonas fimi ATCC 484. In this wild type s t r a i n the production of ce l l u l a s e was repressed by high glucose concentrations. A mutant was formed by treatment with N-methyl-N-nitro-N-nitrosoguanadine, and picked by plat i n g on a ce l l u l o s e plate with 0.5% glucose added. Derepressed mutants showed clearing around the colony. The amount of ce l l u l a s e produced when the wild type and mutant s t r a i n were grown on ce l l u l o s e medium with either glucose or cellobiose added was measured in units/ml of culture media: -35-wild type mutant Cellulose + 1% glucose 0 .78 Cellulose + 0.1% glucose .3 .58 Cellulose + 1% cellobiose 0 1.4 Cellulose + 0.1% cellobiose .35 .62 1.5.8.2. Cellulase Production Derepressed From these results i t could be seen that addition of degradation products did not i n h i b i t c e l l u l a s e pro-duction. Indeed, when larger quantities of i n h i b i t o r were added, larger amounts of ce l l u l a s e were produced due to increased growth. 1.5.8.3. End Product Inhibition of Enzyme The enzyme i t s e l f was end product i n h i b i t e d . The enzyme a c t i v i t y against CMC was tested in the presence of. cellobiose by Stewart. Low le v e l s of 0.05% increased CMCase a c t i v i t y over the control without cellobiose added. As more cellobiose was added the CMCase a c t i v i t y was repressed. Another method for testing repression was by placing concentrated enzyme in precut wells in ce l l u l o s e plates. If no i n h i b i t o r was added clearing to a diameter of.5mm occured within 4 days. Equivalent clearing occured with 0.5% glucose added to the plate, showing that the enzyme"was not i n h i b i t e d by glucose. If cellobiose at a concentration of 0.5% was added to the plate no clearing occured, showing that the enzyme i s i n h i b i t e d by cellobiose. 1.5.8.4. Enhancement of Cellulase Production. An enhanced l e v e l of c e l l u l a s e production by the mutant s t r a i n was then sought by varying the grown medium -36-carbon source. The resultant c e l l u l a s e units/ml follow: Grown on +0.03% glucose +0.5% glucose 0.05% cellobiose 0.05% sophorose 0 no growth 0.4 0.2 0.4 0 .85 0 .95 0.01% f i l t r e paper 0.1% f i l t r e paper 0.1% f i l t r e paper + 0.03% 0.2 0.9 glucose 0.1% f i l t r e paper + 0.5% glucose 0.3 1.4 Enzyme a c t i v i t y increased when high glucose concentrations were used, and approximately doubled when cellobiose or sophorose was added. The addition of 0.1% c e l l u l o s e resulted i n a threefold increase in a c t i v i t y . (Stewart, B.J., et a l . , 1976) 1.5.9. C e l l v i b r i o gilvus 1.5.9.1. Cellulase mode of action. C e l l v i b r i o gilvus, recently renamed as a Cellulomonas, i s an aerobic c e l l u l o l y t i c bacterium that has a c o n s t i t u i t i v e c e l l u l a s e system. (Hulcher, F.H., et a l . , 1958). Four d i s t i n c t c e l l u l a s e components have been i s o l a t e d by Storvick et a l . When crude enzyme containing a l l four components was used to degrade ce l l u l o s e , cellobiose was the only product (Storvick, W.O., et a l . , 1960). Analysis of the intermediates and products during hydrolysis of c e l l u l o s e oligosaccharides and chemically reduced cellulo s e oligosaccharides indicated p r e f e r e n t i a l attack at the second glucosyl bond from the non reducing end. -37 -14 K i n e t i c studies of the release of isotope from C cellulose-oligosaccarides confirmed this conclusion. (Storvick, W.O., et a l . , 1963). Hulcher, et a l . found C e l l v i b r i o gilvus grew better on cellobiose than on glucose. (Hulcher, F.H., et a l . , 1958a). 1.5.9.2. Energy from glucose and cellobiose. A cellobiose phosphorylase i n i t i a t e s metabolism of cellobiose. Convergence in the metabolism of glucose and cellobiose occurs at two points. 1) direct forma-tion of glucose from half of the cellobiose as a r e s u l t of phosphorolytic cleavage and 2) formation of fructose-6-phosphate from both. The difference between the sugars as energy sources for growth stems from the direct oxidation of glucose to gluronic acid. Cellobiose i s cleaved phosphorically and further metabolized by reactions leading to more complete oxidation and there-fore greater y i e l d of energy/mole of hexose consumed. (Hulcher, F.H. et a l . , 1958b). Cellobiose was synthesized 14 by Swisher with C l a b e l l i n g on the reducing glucosyl moiety. C e l l s given this l a b e l l e d cellobiose derived-80% of respiratory C02_ from the reducing glucosyl and 20% from the non reducing glucose. The glucose-1-phosphate produced by phosphorolysis of cellobiose was less extensively converted to CO^ than either the glucose released by phosphorolysis of cellobiose or glucose from media. (Swisher, E.J., et a l . , 1964). -38-1.5.9.3. Enzyme Associated with Ribosomes. The amount of c e l l u l a s e and cellobiase that was associated with ribosomes was measured by Carpenter. The c e l l u l a s e a c t i v i t y was contained mostly in the membrane associated ribosomes, indicating that c e l l u l a s e was translated on membrane bound ribosomes to a i d i n export. The cellobiase a c t i v i t y was contained mostly i n the free ribosomes from ruptured c e l l s , i n d i c a t i n g that the cellobiase was translated on free enzymes because i t i s not exported. (Carpenter, S.A., et a l . , 1967). 1.6. C e l l u l o s i c Substrate Pretreatments 1.6.1. Chemical Pretreatment 1.6.1.1. Chemical composition of c e l l u l o s i c substrates. The ce l l u l o s e in wood i s a l i n e a r polymer of B-D-glucopyranose units linked with B(l,4) glucosidic bonds. The degree of polymerization ranges from 15 to 10-14,000 and averages 3,000 glucose units. The '.. hemicellulose in wood consists of r e l a t i v e l y short hetero-polymers of glucose, xylose, galactose, mannose, arabinose and uronic acids of glucose and galactose, linked together by 1-3, 1-6 and 1-4 glucosidic bonds. Most molecules are l i n e a r but some possess short branches. The degree of polymerization seldom exceeds .200. Lignin i s a complex 3-dimensional polymer formed from p-hydronycinnamyl alcohols. The constituents of these substrates follow: -39" % by weight . Cotton Birch Spruce Holocellulose c e l l u l o s e non c e l l u l o s i c polysaccharide l i g n i n protein Extractable extraneous materials Ash (Cowling, E.B., et a l . 1969) The lignin-hemicellulose-cellulose (LHC) complex consists of interpenetrating polymers that are highly organized to r e s i s t chemical attack. This resistance can be attributed to the c.rys t a l l i n i t y of the c e l l u l o s e , the s t e r i c effects of l i g n i n and hemicellulose, and the hydrophobic effects of l i g n i n . There may also be protective resins or simple sugars adhering to the LHC complex that can i n t e r f e r e with hydrolysis. 1.6.1.2. Strategy for Hemicellulose Removal The primary strategies in the removal of hemi-ce l l u l o s e from the s i t e of reaction are breaking of the hydrogen bonds between hemicellulose and c e l l u l o s e and breaking ester cross linkages between hemicellulose and l i g n i n . 1.6.1.3. Strategy for Lignin Removal. The major l i g n i n removal strategies are to break hydrogen bonds with the other two constituents and to eliminate the hydrophobic coating e f f e c t that l i g n i n has in shielding c e l l u l o s e . Swelling agents (water, 94 77 .6 89 44 .9 5 32 .7 0 19 . 3 1 • 3 0 .5 2 .5 2 .3 1 .2 0 . 3 70.7 46 .1 24 .6 26.3 0.2 2.5 0.3 -40-ammonia,amines, a l k a l i s ) are the standard means of breaking hydrogen bonds. Ester cross l i n k s are broken by chemical reactions involving acids or bases (Lipinsky, E.S., 1979) . 1.6.1.4. NaOH treatment of Barley Straw. In one experiment Pietersen et a l . compared un-treated barley straw to two types of NaOH treated straw. The f i r s t treatment consisted of spraying 5.7% NaOH on the straw and then pressing i t in cobs press under high pressure. The second treatment continued from the f i r s t by extracting the straw with water at 115°C for 15 minutes and vacuum drying at 40°C. A l l three preparations were milled to 40 mesh in a Wiley m i l l . The degree of u t i l i z a t i o n of the preparations by a standardized c e l l u l a s e preparation was: (Pietersen, N., 1975) 1.6.1.5. NaOH treatment of Rice Straw. Han found r i c e straw treated with 4% NaOH for 15 minutes at 100°C increased t o t a l s o l u b l i z a t i o n by enzyme treatment from 29.4 to 73.0% This NaOH treatment increased c e l l y i e l d of mixed Cellulomohas and Alcaligenes •faecales cultures by three f o l d . (Han, Y.W., et a l . , 1974). treated % u t i l i z a t i o n untreated NaOH and washed NaOH treated 1% solution NaOH treated 2% solution 30 71 58 56 -41-1.6.1.6. NaOH and Heat Treatment of Sugarcane Bagasse The e f f e c t of a l k a l i and heating on sugarcane bagasse % NaOH digestion by enzyme treatment has been tested: Temperature Time (minutes) % Digestion 2 80 30 44 2 80 15 42 2 100 15 43 10 100 30 43 10 25 90 35 30 80 30 53 30 100 15 59 50 100 90 89 50 25 90 79 High concentrations of a l k a l i and heating lead to the best results (Han, Y.W. et a l . , 1968). 1.6.1.7. NaOH or Ammonia Treatment of Jute Tarkow found the fi b r e saturation point of jute was doubled the amount of water absorbed by a sample, by treatment with 1% NaOH or l i q u i d ammonia. The maximum e f f e c t was reached after 3 hours of treatment. The swelling capacity of the substrate was approximately doubled. This, with the ind i c a t i o n that the upper molecular weight l i m i t for penetration of d i f f u s i n g materials was increased, accounts for the improved d i g e s t i b i l i t y of the material. Untreated jute was only 3.5% digestible whereas treated jute was 27% d i g e s t i b l e . The increase in swelling capacity resulted from the saponification or ammonolysis of esters of 4-0-methyl-glucuronic a c i d attached to xylan chains. In the natural condition these esters act as crosslinks l i m i t i n g the -42-swelling or dispersion of polymer segments in water. (Tarkow, H., et a l . , 1969). 1.6.1.8. Treating softwoods with NaOH M i l l e t t found softwoods as a group exhibited d i g e s t i b i l i t i e s by c e l l u l a s e preparations in the 1-5% range and were unresponsive to a l k a l i treatment. This difference in response appeared related to the l i g n i n content of the wood. The f i r s t 40% of the l i g n i n could be removed from Douglas f i r without any increase i n enzyme d i g e s t i b i l i t y at a l l . ( M i l l e t t , M.A., et a l . , 1975). 1.6.1.9. E^SO^ Treatment of Sugarcane Bagasse Han found treating the sugarcane bagasse with 50% H 2S0 4 at 121°C for 15 minutes and then d i l u t i n g the mix to 1% E^SO^ at 121°C for 15 minutes reduced the sugar y i e l d to 23% enzyme digestion from 28%. Increasing the H2SO^ concentration in the second half reduced the sugar y i e l d further. This probably was due to degradation of the hydrolyzed sugars by the acid. The l i g n i n context of acid treated sugarcane bagasse was higher than that of a l k a l i treated material. Lignin content had a negative co r r e l a t i o n to d i g e s t i b i l i t y , therefore, the low d i g e s t i b i l i t y of acid treated bagasse may be due to i t s high l i g n i n content. Acid may s o l u b i l i z e the e a s i l y degraded (amorphous) part of the ce l l u l o s e leaving the undigestible ( c r y s t a l l i n e ) part of the ce l l u l o s e in the residue. (Han, Y.W., et a l . , 1974). -43-1.6.1.10 Solvent processing. According to Lipinsky solvent processing has been studied by a Perdue research team. Three treatments were determined to be e f f e c t i v e : i 1) Cadoxen method; using a mixture of cadmium s a l t s , ethylenediamine and a l k a l i 2) f e r r i c t a r t r a t e 3) sulphuric acid/methanol method These three techniques were o r i g i n a l l y used in c e l l u l o s e analysis. The challenge of these processes was the recovering of s u f f i c i e n t amounts of solvent and coproduct. in high enough yields to render them commercially a a t t r a c t i v e . Toxicity of cadmium compounds and supply problems with t a r t a r i c acid were s i g n i f i c a n t drawbacks to the f i r s t two methods. However, other metal chelates may be found which overcome these objections. The sulphuric acid/methanol process didn't have th i s problem. These three techniques att a i n increased surface area by destroying c r y s t a l l i n i t y . (Lipinsky, E.S., 1979). 1.6.1.11. Comparison of Chemical Pretreatments 1.6.1.11.1. Results. Chemical pretreatments were compared by Fan et a l . , by trea'ting wheat straw with a large number of pretreat-ments, and then measuring the d i g e s t i b i l i t y by enzyme treatment. The results were: -44-Type of Treatment Maximum D e l i g n i f i c a t i o n Legnin Coversion Content 1. Caustic RT 36 .0% 8 .6 10 .6 2. Caustic AC 62 .0 43 .5 6 .5 3. Sulphite RT 10 .6 16 .9 9 .6 4. Sulphite AC 38 .0 45 .5 6 .4 5 . Hypochlorite RT 36 .4 40 .9 6 .8 6 . Hypochlorite AC 55 .9 16 .6 9 .7 7. Peracetic acid 54 .5 76 .2 2 .8 8. Butanol 10 .6 5 .2 10 .9 9 . Ethylene glycol-B 40 .0 10. Ethylene glycol AC 54 .5 83 .8 1 .9 11. Diluted H2SO4 25 .4 17 .0 12. Standard 5 .5 0 11 .6 RT - room temperature AC - autoclaved B - b o i l e d 1.6.1.11.2. Caustic Pretreatment The caustic pretreatment consisted of a solution of 1% NaOH and 10% wheat straw. Half of the mixture stood at room temperature (R.T.) and half was autoclaved for 2 hours. The straw was washed u n t i l the water became neutral. 1.6.1.11.3. Sodium Sulphite Pretreatment In the sodium sulphite treatment, a mixture of 13.7% sodium sulphite and 14% wheat s t r a t was made. Half of the mixture was l e f t at room temperature; half was autoclaved for 2 hours. Both were then washed u n t i l the wat-er was neutral. 1.6.1.11.4. Hypochorite pretreatment. The hypochlorite pretreatment consisted of a mixture of dilute sodium hypochlorite (4-6% NaOCl) and 10% wheat straw. Half of the mixture stood at room temperature while half was autoclaved for 2 hours. -45-Both were washed u n t i l the odour of hypochlorite was gone . • 1.6.1.11.5. Peracetic Pretreatment. In the peracetic pretreatment, the wheat straw was boiled at 100° for 30 minutes at a concentration of 10% in peracetic acid (a 1:1 mixture of acetic anhydride and 35% hydrogen peroxide). The straw was then washed u n t i l the water became neutral. 1.6.1.11.6. Butanol Pretreatment In the butanol pretreatment the wheat straw was autoclaved for 1§ hours at a concentration of 10% in a 50% butanol solution. 0.005% aluminium chloride was added as a catalyst. The straw was then washed u n t i l the water became neutral. 1.6.1.11.7. Ethylene glycol Pretreatment. In the ethylene glycol pretreatment a mixture of 8% wheat straw, and 2% HCl was made in-ethylene g l y c o l . Half of the mixture was boiled at 170° for 30 minutes, half was autoclaved for 1 hour. The straw was washed u n t i l the water became neutral. 1.6.1.11.8. Dilute H 2S0 4 Pretreatment. The d i l u t e sulphric acid pretreatment consisted of boil-ing a 6% solution of wheat straw in 4.4% H2S04, for 1 hour. The straw was then washed u n t i l the water became neutral. 1.6.1.11.9. Lignin Concentration in substrates. The pretreatments gave substrates with a range -46-of 11.5 to 1.9% lignin. content. The d i g e s t i b i l i t y increased rapidly with d e l i g n i f i c a t i o n up to about 50% d e l i g n i f i c a t i o n . Further d e l i g n i f i c a t i o n gave a smaller increase in d i g e s t i b i l i t y . (Fan, L.T., et a l . , 1981). 1.6.2. Physical Pretreatments 1.6.2.1. Introduction Physical pretreatments f a l l into two groups: the mechanical and the non-mechanical. The mechanical treatments include b a l l m i l l i n g , two r o l l m i l l i n g , hammer m i l l i n g , c o l l o i d m i l l i n g , vibro energy m i l l i n g and extrusion. These pretreatments use shearing and impact-ing forces in order to reduce the c r y s t a l l i n i t y of the substrate, therefore increasing i t s s u s c e p t i b i l i t y to enzyme action. The non-mechanical pretreatments cause decomposition by harsh external forces other than shearing and impacting forces. They include i r r a d i a t i o n , UV i r r a d i a t i o n , p y r o l y s i s , steaming, freezing, autoclaving and explosive depressurization. 1.6.2.2. B a l l M i l l i n g B a l l M i l l i n g employed a 5-L porcelain jar m i l l with a 50 v o l . % charge of 1 inch diameter porcelain spheres. The j a r was rotated at about 52 r.p.m. for 15 or 30 min. The wheat straw was added to f i l l the void volume among the b a l l s . - 4 7 -1.6.2.3. F i t z m i l l i n g . The F i t z m i l l i n g involved a shearing action in the m i l l . The size reduction depends on the screen size on the exit port of the m i l l . 1.6.2.4. Roller M i l l i n g . In r o l l e r m i l l i n g , the extent of size reduction was varied by changing the m i l l i n g time. In extrusion, pressure could be added by an outlet die. 1.6.2.5. Comparison of M i l l i n g types Type of pretreatment Maximum d i g e s t i b i l i t y % B a l l m i l l i n g 4h. 23.6 8h 23.3 16h. 19.1 24h. 19.1 F i t z m i l l i n g coarse 8.2 fine 9.1 Roller m i l l i n g 0.25h. 12.4 0.50h. 14.5 Extrusion under pressure 5.8 no pressure 5.8 Control 5 .5 (Fan, L.T., et a l . , 1981). The p a r t i c l e size was not the only contributing factor when considering a physical or m i l l i n g treatment. The action of the m i l l and the m i l l i n g history (time, temperature p r o f i l e ) both contributed to the change in c r y s t a l l i n i t y or change in s u s c e p t i b i l i t y . For example, SW40, which i s hammer mill e d Solka Floe sized between 400 and 500 mesh, was 50% s a c c a r i f i e d in 48 hours In contrast, b a l l m i l l e d SW40 that was also sized between 400 and 500 mesh was 78% saccharified in 48 hours. (Nystrom J., 1975). -48-1.6.2.6. Cost of Commutation Conventional commutation ( b a l l m i l l i n g , and two r o l l m i l ling) were r e l a t i v e l y e f f e c t i v e i n improving a c c e s i b i l i t y of c e l l u l o s e , but cost in energy i s pro h i b i t i v e . (Lipinsky, E.S., 1979). 1.6.2.7. Gamma Irr a d i a t i o n In i r r a d i a t i o n pretreatment Lipinsky sealed the sample in a jar i n the presence of a i r . The jars were placed in a ^ CO o' c e l l and ir r a d i a t e d at the rate of 500 rad/min u n t i l the dosages of 10 Mrad, 30 Mrad and 50 Mrad were reached. The maximum conversion of the wheat straw was then measured. Pretreatment Maximum d i g e s t i b i l i t y % 10 Mrad 5.5 30 Mrad 13.9 50 Mrad 18.8 Control 5.5 Doses larger than lOMrad were needed for any ef f e c t to to be noticable. (Fan, L.T., et al.,1981). f j - i r r a d i a t i o n causes depolymerization of a l l the molecules. (Lipinsky, E.S 1949). 1.6.2.8. Gamma Irradiation of Hemlock, Sawdust. Hemlock sawdust was subjected by K i t t s to 8 ir r a d i a t i o n up to a maximum of 1.46 x 10 rads. The resu l t i n g substrate was tested by in v i t r o rumen fermen-tation tests. It was found that % dry matter disappearance, and ce l l u l o s e digestion showed a steady increase with increasing i r r a d i a t i o n l e v e l s . -49-Irradiation .% digestion % dry matter Reducing Sugar dosage disappearance released ug/ml 0 4.85 x 10 6 4.0 x 10 7 7.52 x 10 7 1.11 x 10 8 1.46 x 10 8 0 38 25 79 86 199 190 175 256 ( K i t t s , W.D., et a l . , 1969) 1.6.2.9. UV l i g h t and sodium n i t r a t e Pretreatment. Waste c e l l u l o s i c materials from garbage were, pretreated by Rogers in order to increase s u s c e p t i b i l i t y of the cel l u l o s e to biodegradation. The only treatment found to give a s i g n i f i c a n t increase in biodegradation was photochemical treatment, consisting of UV l i g h t and sodium n i t r a t e . (Roger, C.J., et a l . , 1972). 1.6.1.10. Size Reduction and Oxidative Heat Ghose found considerable improvement in susceptib-i l i t y of ce l l u l o s e to hydrolysis was possible by a two step process of size reduction and oxidative heat t r e a t -ment . The optimum heat treatment is for 25 min at 200°C. (Ghose, T.K., 1969). 1.6.2.11. Extreme Temperature Pretreatment. The use of heat alone has also been t r i e d by M i l l e t t . Heating to 200°C for. 32 hours gave only a 35% increase in the maximum acid hydrolysis with a 27% increase in maximum sugar y i e l d s . ( M i l l e t t , M.A., et a l . , 1965). Freezing has also been t r i e d by Bykov. The c e l l u l o s i c material was frozen to -75°C in a water suspension. This reduced both the te n s i l e strength and the degree -50-of polymerization of the substrate. The e f f e c t was augmented by repeated freezing and thawing. The energy requirements for t h i s treatment were too great for i t to be a viable treatment. (Bykov, A.N., et a l . , 1961). 1.6.2.12. NaOH and Pressure Cooking as a Pretreatment. Han/et a l showed that pressure cooking and NaOH treatment could be combined to increase the growth of Cellulomonas on.sugarcane bagasse. If less than 3% NaOH was used no increase in y i e l d i s found by the addition of autoclaving. When 3% NaOH i s used pressure of over 200 p . s . i . for 5 min i s needed for s i g n i f i c a n t differences. Treatment mg protein/ml. c e l l y i e l d 3% NaOH no steam 4.68 (Han, Y.W., et a l . , 1974). 1.6.2.13. Iotech Pretreatment. According to Lipinsky the Iotech pretreatment i s a process that makes use of explosive depressurization applied to the intact plant c e l l materials, such as wood chips. P r i o r to the explosive depressurization, the biomass raw material was subjected to a s p e c i f i c pretreatment with steam and heat under pressure. It is speculated that the i n i t i a l steaming treatment puts substantial quantities of water into the i n t e r c r y s t a l l i n e regions of the cel l u l o s e , while the acetic acid generated 50 p . s . i . 19 min 65 p . s . i . 10 min 230 p . s . i . 5 min 260 p . s . i . 5 min 4.58 5 .24 6.48 7.20 -51-by hydrolysis of acetate groups in the hemicellulose depolymerizes the hemicellulose to a considerable degree. The glass t r a n s i t i o n temperature of l i g n i n was exceeded. When the pressure was released rapidly the weakened lignin-hemicellulose-cellulose complex l i t e r a l l y exploded. The l i g n i n coalesced i n droplets that were soluble in methanol, ethanol or acetone. Mechanical abrasion of the fibres occured during the explosive depressurization. A unique nonfibrous product resulted which contains l i g n i n that no longer coated the carbohydrate f r a c t i o n and was highly soluble; and i n which the hemicellulose had become largely water soluble. The c e l l u l o s e contained in the carbohydrate f r a c t i o n did not lose i t s c r y s t a l l i n i t y but had such an enormous surface area compared with the s t a r t i n g material that i t was e a s i l y hydrolysed enzy-matically. This process was e f f e c t i v e because of the combination of treatments, no one of which was very e f f e c t i v e in i t s e l f . (Lipinsky, E.A., 1979). 1.6.3. Approaches to Increase D i g e s t i b i l i t y . 1.6.3.1. Structural Features Affecting D i g e s t i b i l i t y . Structural features which affect enzymatic de-gradation are: 1) the moisture content, 2) the size and d i f f u s i b i l i t y of the enzyme molecules involved i n r e l a t i o n to the size and surface properties of the gross cappileries and the spaces between m i c r o f i b r i l s and the cellulo s e molecules in the amorphous region, 3) the degree -52-of c r y s t a l l i n i t y of the c e l l u l o s e , 4) i t s unit c e l l dimensions, 5) the conformation and s t e r i c r i g i d i t y of the anhydroglucose units, 6) the degree of polymerization of the c e l l u l o s e , 7) the nature of the substances with which the c e l l u l o s e i s associated, and 8) the nature, concentration and d i s t r i b u t i o n of substituent groups. (Cowling, E.B., et a l . , 1969). 1.6.3.2. Increasing Accessible Surface Area. Cotton l i n t e r s were swollen to an increasing extent by treatment with phosphoric acid of increasing concentration. Using a number of simple sugars plus a series of dextran molecules ranging in molecular weight from 180 to 2.4 x 10 7 and in diameter from 8 to 1600A0. Stone et a l . , measured the pore volume and calculated the surface area within the swollen fibres accessible to a l l the molecules within t h i s range. The substrates were incubated with a commercial c e l l u l a s e preparation and the i n i t i a l rate of reaction was compared with the a c c e s s i b i l i t y of the substrate to molecules of various si z e s . There was a l i n e a r relationship between the i n i t i a l reaction rate and the surface area within the c e l l u l o s e gel accesible to a 40A° diameter molecule. (Stone, J.E., et a l . , 1969). 1.6.3.3. Decreasing Degree of C r y s t a l l i n i t y . Norkrans found there was a correlation between increased degree of c r y s t a l l i n i t y and increased resistance to enzyme attack. (Norkrans, B., 1950). The c r y s t a l l i n e -53-region of c e l l u l o s e could be considered as a source of protection for the amorphous regions, preventing access of the enzyme to the amorphous region. This protection may be r e c i p r o c a l . The amorphous region provides pro-tection by providing s i t e s that are r e l a t i v e l y readily attacked by enzymes. Unless the gross structure has been greatly increased i n a c c e s s i b i l i t y , the glucose formed by enzyme action may diffuse from the reaction s i t e slowly. The presence of a high glucose concentration may i n h i b i t the action of the enzyme on both the amorphous and c r y s t a l l i n e regions of the c e l l u l o s e . Therefore, pretreatments must allow for easy removal of the products. (Lipinsky, E.S., 1979). 1.6.3.4. Decreasing Degree of Substitution. Reese found the degree of substitution of soluble derivates i s a primary factor i n determining the resistance of these materials to microbial attack. Increasing the degree of substitution increases resistance. Immunity i s conferred when every anhydroglucose unit i s sub-s t i t u t e d . (Reese, E.T., et a l . , 1950). -54-2. Methods 2.1. ' DNS-CMC Enzyme Assay The DNS Reagent i s made up i n advance and i s stable when kept under dark, refri g e r a t e d conditions. The reagent i s made by adding l.Og d i n i t r o s a l i c y l i c acid, 0.2g. phenol, 0.05g. sodium sulphite, and 20.Og Rochelle s a l t (sodium potassium tartrate) to 50.0ml of a 2% sodium hydroxide solution. This i s then diluted to 100.0ml. In order to measure enzyme a c t i v i t y , 0.5ml. of the enzyme i s added to 1.0ml of a 4.0% low v i s c o s i t y CMC in 0.1M phosphate buffer (pH7) solution. This i s then incubated at 30°C for 30-60 min. The exact time i s noted. 1.5ml. of DNS Reagent i s added at the solution mixed and then placed i n a b o i l i n g water bath for 15 minutes. The O.D. of the solution i s then measured at ,550 or 640nm. A blank i s made for each'sample in which the DNS Reagent i s added before the CMC i n order that •the enzyme has no time to react with the CMC. The O.D. i s compared to a standard glucose curve. Enzyme a c t i v i t y i s calculated in units/ml. of enzyme preparation, where 1.0 units i s equal to 1.0 mg. glucose equivalents released/minute. 2.2. Growth Media The minimal media for growth of Cellulomonas fimi contains 0.1% NaNOg, 0.1 K 2HP0 4, 0.5% Mg S0 4, and 0.05% KCl. The e f f e c t of adding 0.05% yeast extract and/or 0.1% casamino acids was determined. For carbon sources - 5 5 -glucose, CMC (carboxy methyl c e l l u l a s e ) , acid pre-c i p i t a t e d c e l l u l o s e , acid swollen f i l t r e paper, and av i c e l at a concentration of 1.0% were t r i e d . The glucose, CMC, and a v i c e l are not pretreated. The acid p r e c i p i t a t e d c e l l u l o s e i s prepared by adding fibrous c e l l u l o s e powder to 85% phosphoric acid. This i s then placed i n a b o i l i n g water bath for 4 min and then added to an ice water solution. This i s blended, then rinsed and neutralized. This i s then held as a stock solution. The acid swollen f i l t r e paper i s prepared by soaking the f i l t r e paper in a 2N HCl solution for 48-72 hours with one change of acid. The f i l t r e paper i s then washed for 12-24 hours with several changes in water. The pH i s adjusted to pH7 and then the solution i s blended. The growth of the bacteria i s followed using K l e t t readings, viable place counts, and enzyme assays. 2.3. Storage The enzyme was held at 40°C to check s t a b i l i t y . The e f f e c t on enzyme a c t i v i t y of quick freezing and storage of samples with or without 4% glycerol was determined. 2.4. Limiting Amount of Carbon Source The l i m i t i n g concentration of C-source was determined by adding 0.5% or 0.05% or any of the con-centrations between at 0.05% incremental steps. The -56-growth was followed using K l e t t readings. 2.5. Concentration of Enzyme Three f i l t r i n g systems were used to concentrate the enzyme. The Amicon system was used with a PM 10 (10,000 dalton exclusion pore size) f i l t r e in i t . An immersible CX U l t r a f i l t r e (10,000 dalton exclusion pore size) was used attached to a vacuum pump. The t h i r d system was a M i l l i p o r e U l t r a f i l t r a t i o n System with 2 square feet of 10,000 dalton exclusion pore si z e f i l t r e in i t . The e f f i c i e n c y of the three systems was com-pared by length of time needed to concentrate a sample and by measuring the enzyme a c t i v i t y in the concentrate and in the effluent. 2.6. Substrates for Enzyme degradation 2.6.1. Sawdust samples. Sawdust was prepared from spruce, douglas f i r and hemlock by sanding with a b e l t sander. P a r t i c l e s of a s u f f i c i e n t l y reduced size were selected by passing them through a 200 mesh sieve. These sawdust preparations were further pretreated i n several manners. 2.6.2. Autoclaved. The sawdust sample i s autoclaved for 30 minutes and the pressure i s released quickly. -57-2.6.3. Steamed. The sawdust i s placed i n a thin layer i n a p e t r i plate and placed i n a steamer where i t i s steamed for one hour. 2.6.4. Acid treatment. The sample i s soaked i n 2N HCl for 24 hours. It i s then rinsed and the pH i s adjusted to 7. The sample i s then dried. 2.6.5. Alkaline treatment. The sample i s soaked i n 2N NaOH for 24 hours. It i s then rinsed and the pH i s adjusted to 7. The sample i s dried. 2.6.6. Water treatment. The sample i s soaked i n d i s t i l l e d water for 24 hours, and then dried. 2.6.7. Kraft treatment. The sample i s autoclaved for 30 minutes in a 30% suphidity solution (IN NaOH, .428 M Nz 2S). The sample i s then rinsed and the pH i s adjusted to 7. The sample i s then dried. 2.6.8. Iotech treatment. The sample was received already pretreated. This consists of a pretreatment of steam and heat under pressure.and then an explosive release of the pressure. -58-2.7. Sugar Assays 2.7.1. D i n t r o s a l i c y l i c acid (DNS) assay Using the DNS reagent mentioned previously, equal amounts of the sugar solution or an appropriate d i l u t i o n and the DNS reagent are mixed, and then placed i n a b o i l i n g water bath for 15 minutes. The OD i s measured at 550 or 640 nm. and then compared to a standard curve for the same wavelength. 2.7.2. Anthrone Assay. Reagent i s made by adding 50 mg anthrone and l.Og thiourea to a s t i l l warm solution of 28 ml. d i s t i l l e d water and 72ml. concentrated H^SO^. The reagent must be aged at 4°C for at least 4 hours before using. 5.0 ml of Anthrone reagent i s added to 1.0 ml. of sugar sample or an appropriate d i l u t i o n . This i s then mixed well, capped and placed i n a b o i l i n g water bath for 15 minutes. The samples are c o l l e d for 20 minutes i n a room temperature water bath before measuring the O.D. at 620 nm. The value obtained i s compared to a standard curve which must be prepared for each set of samples. 2.7.3. Somogyi Assay. Reagent i s made by dissolving 12.Og Rochelle s a l t , 20.Og anhydrous sodium carbonate, and 25.Og -59-anhydrous sodium bicarbonate in 500 ml of d i s t i l l e d water. This solution i s poured into a second solution made of 6.5g of CuSO^ H^^ O in 100 ml of d i s t i l l e d water. Into t h i s lOg potassium iodate, and 18.g potassium oxalate are added. This i s then diluted to 1000 ml. This solution i s stable at room temperature. 5.0 ml of Reagent i s added to 5.0 ml of sugar solution or an appropriate d i l u t i o n . It i s mixed and placed i n a b o i l i n g water bath for 15 min, and then cooled to 35-40°. 1.0 ml of 5N H 2 S 0 4 i s added. When sample has dissolved the sample i s t i t r a t e d with 0.005N Sodium thiosulphate. The value of the blank, obtained by b o i l i n g 5 ml of water with 5.0 ml of reagent and then t i t r a t i n g t h i s , i s subtracted from the samples value and the result i s compared to a standard curve. 2.8. Measuring Degradation of Sawdust Samples. 2.8.1. Measuring solubization of a sample. A sample weighed to 5 decimal points accuracy i s incubated with an enzyme sample i n an erylemeyer flask, on a rotary shaker, at 30°C for a set length of time. The sample i s then f i l t r e d through a tared GHF f i l t r e . The f i l t r e i s dried and then weighed. The percent recovery i s found by the equation: -60-% Recovery = (wt of f i l t r e + sample) -(wt of f i l t r e ) i n i t i a l wt. of sample The percent degradation can be obtained by running a blank sample in which the sawdust i s incubated with water. Percent recovery i s then found by the equation: % Degradation = 100%-(% Recovery(blank)-% Recovery(Sample)) 2.8.2. Measuring Reduction i n P a r t i c l e Size of a Sample A sample weighed to 5 decimal points accuracy i s incubated with an enzyme sample in a stoppered, tared test tube, on a rotary shaker, at 30°C for a set length of time. The test tube with the sample in i t i s then set upright in a test tube rack and allowed to s e t t l e for 30 minutes. After this period the contents of the test tube has separated into a p e l l e t and a cloudy supernatant. The supernatant i s c a r e f u l l y pipetted o f f of the p e l l e t . Water i s then added to the p e l l e t to the same l e v e l that the enzyme was. The same i s shaken and then allowed to s e t t l e for 30 min. The supernatant is removed and added to the f i r s t supernatant. This proce-ss i s repeated u n t i l the supernatant, after 30 minutes s e t t l i n g , i s clear. The t o t a l supernatant i s then passed through a tared GFC f i l t r e . T h e f i l t r a t e i s c o l l e c t e d and then freeze-dried i n a tared flask. The f i l t r e with the fine p a r t i c l e s (remain i n suspension for 30 minutes) on i t i s dried. The p e l l e t containing large p a r t i c l e s incapable of remaining in suspension for 30 minutes, i s freeze dried i n the tared test tube. The percent recovery of the sample i n each portion can be determined by the equations: % Recovery=(wt of freeze-dried test tube-wt of empty test tube) wt of i n i t i a l sawdust sample % Recovery=(wt of dried f i l t r e - i n i t i a l wt of f i l t r e ) (fine p a r t i c l e s )  wt of i n i t i a l sawdust sample % Recovery=(wt of freeze dried f l a s k - i n i t i a l wt of flask) (soluble portion)  wt of i n i t i a l sawdust sample In each case two control samples must be run. The f i r s t is a sample of sawdust with water in order to determine the percentage of each p a r t i c l e size in the o r i g i n a l sample. The second control i s a sample of sawdust with boiled enzyme. This determines in which portion the weight of the enzyme i t s e l f w i l l be found. -62-3. Results & Discussion 3.1. Optimum Growth Media 3.1.1. Media Used. The minimal media was composed of 0.1% NaNO^, 0.1% K 2HP0 4, 0.5% MgS04 and 0.5% KC1. The media tested for growth and c e l l u l a s e production were compared as follows: Media nutrient minimal CMC . Yeast casamino glucose broth media 0.5% extract acids 1% 0.05% 0 .1% 1 + - - - -2 - + + + 3 - + + + - + 4 - + + _ . + + 5 - + + + + 6 - + + - + -7 _ ' • + + + + + 3.1.2. Kle t t measurements The growth upon these media was followed by Kle t t measurements u n t i l they reached stationary phase. The Kl e t t value for the uninnoculated media was subtracted from the time readings and the resultant values are graphed in Fig.4. Graph of following measurements.' time 0 18hr. 23hr. 42hr. 48hr. 6 7hr. 73hr. 97hr. 1 0 4 22 76 86 -2 0 7 . 83 203 230 279 284 3 0 11 97 514 -4 0 2 26 50 92 550 550 5 0 3.5 72 266 303 388 393 6 0 0 2 9 13 41 51 7 0 122 495 --63-600 *1 time (hours) F i g . 4 K l e t t measurement of growth on various media -64-3.1.3. Viable C e l l Count At the f i n a l point for each media a sample was taken and the number of viable c e l l s was measured by 2 standard plate count: Media Time of sample # viable c e l l s / m l . 3.1.4, 1 2 3 4 5 6 7 48 hrs 73 42 73 73 73 23 2.10 x 10 7 1.38 x 10 6 3.7 x 108 2.1 x 10 8 6 . 3 x 10 7 1.8 x 10 5 3.9 x 10 8 Enzyme a c t i v i t y The enzyme a c t i v i t y of these f i n a l points was also measured using the DNS-assay. Separate controls had to be taken for each media since the background l e v e l of reducing sugar varied. Media Time of sample Enzyme A c t i v i t y (Units/ml) 1. 2 3 4 5 6 7 48 hrs. 73 42 73 73 73 23 7 16 .4 0 2 .5 20 .5 16 . 0 3.1.5. Discussion From these three results several conclusions can be made: 1) The addition of glucose, while allowing good growth of the bacteria, i n h i b i t s the production of c e l l u l a s e . In each case where glucose was added, medias 3, 4 and 7, the growth was very good but the ce l l u l a s e production -65-was very low or immeasurable. 2) When grown on CMC as the carbon source enzyme a c t i v i t y was good. When both casamino acids and yeast extract were added to the media there was an increase in growth and an increase in c e l l u l a s e production. Due to these results i t was decided that the best media would consist of a c e l l u l o s i c carbon source i n minimal media with casamino acids and yeast extract added. 3.2. Limiting amount of CMC 3.2.1. Introduction The l i m i t i n g amount of CMC added i n the media was sought. This amount of CMC would be used up before the c e l l s entered stationary phase. Since CMC i s a soluble carbon source, centrifugation of the media w i l l remove the c e l l s but w i l l not remove any remaining CMC. If any CMC i s present i n an enzyme preparation for sawdust degradation, the CMC w i l l be p r e f e r e n t i a l l y attacked and the r e s u l t i n g reducing sugars produced would not be from the degradation of sawdust. 3.2.2. Measuring growth on varying concentrations. The amount of CMC i n each K l e t t flask varied from a low of 0.05% CMC, r i s i n g by increments of 0.05 to a maximum of 0.5%. The growth in each flask was measured by K l e t t readings. These measurements are graphed in Fig.5. - 6 6 -F i g . 5 . Measurement of amounts of CMC growth on media wi t h v a r y i n g - 6 7 -3.2.3. Discussion From the graphed res u l t s , one. can see three basic groups. The f i r s t group with high CMC concentrations, 0.35 - 0.50% CMC, reach stationary phase without using up a l l the available CMC. The second group with moderate CMC concentrations, 0.15 - 0.30% CMC, did not reach stationary phase and did not appear to run out of carbon source. The t h i r d group with low CMC concentrations, 0.05 - 0.10%, reach a stationary phase. They appear to run out of usable carbon source. 3.3. E f f e c t of Various C e l l u l o s i c Carbon Sources on Cellulase Production. The l e v e l of enzyme production i s quite low when C.fimi i s grown on CMC. Acid Precipitated Cellulose and Avicel were tested to see i f the ce l l u l a s e production was higher on them. Whereas growth on CMC for 3 days results in only about 20 units/ml., growth on acid p r e c i p i t a t e d c e l l u l o s e produces 240 units/ml in 4 days. The best substrate was A v i c e l . The resultant growth got up to 9 2.9 x 10 viable cells/ml i n 3 days and the cel l u l a s e was up to 500 units/ml. 3.4. Concentration of Enzyme 3.4.1. Concentrating small volumes. Starting with 200 ml. samples of supernatant, the samples were concentrated by Amicon concentration and by U l t r a f i l t r e concentration. The Amicon concentrated - 6 8 -th e 200ml sample to 20 ml. When the Amicon effluent was tested for enzyme a c t i v i t y none was found. When the U l t r a f i l t r e effluent was tested enzyme was found at one quarter the a c t i v i t y of the untreated supernatant. The U l t r a f i l t r e membrane probably contains c e l l u l o s e and i s being degraded allowing the enzyme through the membrane. 3.4.2. Concentrating large amounts of Supernatant. While the Amicon i s good enough for concentrating small volumes when large volumes of enzyme need to be concentrated the Amicon would be too slow. The M i l l i p o r e system i s capable of handling the large volumes required for larger scale experiments. The M i l l i p o r e system can reduce 20 l i t r e s of supernatant to 100 ml in about 4 hours. In doing so there i s no enzyme a c t i v i t y allowed through into the ef f l u e n t . Only about 5% of the a c t i v i t y i s l o s t during the concentration process. 3.5. S t a b i l i t y of the enzyme. 3.5.1. S t a b i l i t y of Refrigerated Enzyme. If enzyme i s stored at 4°C with sodium azide added, the enzyme i s stable for at least a month. 95% of the o r i g i n a l a c t i v i t y was s t i l l present in a sample stored for a month. 3.5.2. S t a b i l i t y of Frozen Enzyme. Samples of enzyme were quick frozen "as i s " or with 40% glycerol added. When the sample was frozen "as i s " 20% of the a c t i v i t y was l o s t within 24 hours, -69-but the remaining 80% was then stable for a month. Therefore the glycerol i s an unnecessary addition. This i s just as well since i t adds so much reducing sugar to the enzyme that the background becomes larger than the a c t i v i t y when measured by the DNS-assay. 3.6. Enzyme Degradation by Various Untreated Sawdusts. 3.6.1. Introduction Four types of sawdust, 2 di f f e r e n t samples of hemlock, spruce and Douglas f i r were incubated with enzyme with 2800 units/ml of a c t i v i t y . Samples were removed immediately or at 3, 9 or 15 days of incubation. The samples were f i l t e r e d and the % Recovery was measured. 3.6.2. Results Type of wood Days of i n i t i a l wt wt. of f i l t r e % Recovery Incubation of sawdust f i l t r e + sample of sawdust _' sample (mg) (mg) (mg) Hemlock 1 0 100 .63 27.06 122 46 94 8 3 100 .24 26 .06 116 89 90 6 9 100 .94 27 .12 110 33 82 4 15 100 .93 27.21 108 51 80 6 Hemlock 2 0 100 .52 26 .46 118 57 91 6 3 100 .16 26 .02 113 .56 87 4 9 100 .56 27 .22 107 59 79 9 15 100 .09 27.01 104 85 77 8 Spruce 0 100 . 74 27.87 122 .02 93 5 3 100 .69 27.28 115 .00 87 1 9 100 .69 27.17 108 .73 81 0 15 100 .03 28.04 .105 .51 77 .4 Douglas f i r 0 100 .54 26 .34 123 .47 96 .6 3 100 .79 27.27 118 .84 90 .9 9 100 .17 27.88 112 .88 84 .9 15 100 .51 27 .24 118 .84 84 .2 The results are graphed in Fig.6. -70-3 9 days of incubation Douglas f ir Hem 71 Hem/< Spruce /ock I ock2 15 Fig. Degradation, of Various Sawdust Samples -71-The amount of degradation i s the difference between 0 days of incubation and 15 days of incubation: Type of wood % of Degradation hemlock 1 hemlock 2 spruce douglas f i r 14.2 13 .8 16 .1 12 .4 3.6.3. Discussion Each of the four d i f f e r e n t sawdust samples appear to be degraded to about the same amount. In each of these softwoods the percentages of lignin. hemicellulose and c e l l u l o s e are roughly the same. Therefore since they have been treated the same, one would expect them to be degraded to the same degree. 3.7. Degradation of Pretreated Sawdust 3.7.1. Degradation of Chemically Pretreated Sawdust 3.7.1.1. Introduction. Alkaline pretreated, acid pretreated, Kraft pretreated, water pretreated, and untreated sawdust were incubated with enzyme with 2913 units/ml. The samples were removed immediately or in 1 or 4 days. The samples were f i l t e r e d and the % recovery measured. -72-3.7.1.2. Results Pretreatment Enzyme Days of i n i t i a l wt. of f i l t r e % Recovery incubation wt of f i l t r e + of sawdust (mg) sample Sawdust sample (mg) ; . (mg)  Alkaline active 0 100 .18 27 .67 123 .79 95 .9 ! 1 1 100 .60 27 .21 120 .74 93 .0 1 1 4 100 .14 27 .62 117 .82 90 .1 boiled 4 100 .43 28 .30 125 .65 96 .9 Acid active 0 100 .23 27 .93 124 .77 96 .6 1! 1 100 .00 26 .81 120 .90 94 .1 11 4 100 .49 26 .92 120 .80 93 .4 boi l e d 4 100 .32 27 .63 125 .49 97 .5 Kraft active 0 100 .59 27 .86 126 .73 98 .4 ii 1 100 .68 27 .91 122 .99 94 .4 4 100 .53 27 .44 121 .34 93 .4 boi l e d 4 100 .89 27 .72 128 .44 99 .8 water active 0 100 .43 27 .51 125 .02 97 .1 11 1 100 .56 26 .83 120 .12 92 .8 11 4 100 .70 27 .83 121 .28 92 .8 boi l e d 4 100 .20 28 .00 126 .36 98 .2 untreated active 0 100 .57 27 .94 123 .38 94 .9 11 1 100 .67 26 .85 117 .74 90 .3 11 4 100 .37 27 .64 117 .24 89 .3 boi l e d 4 100 .34 27 .47 123 .38 95 .6 The results of th i s table are graphed i n Fig.7. -73-r A c i d ,and Kraft -$\lkalme Untreated days of mcufoat/on F i g . 7.. Degradation, of Chemically Pretreated Sawdust Samples -74-The amount of degradation i s the difference between the fourth day's recovery and the recovery for the boiled enzyme: Pretreatment % of Degradation Alkaline 6.8 Acid 4.1 Kraft 6.4 Water 5.4 Untreated 6.3 3.7.1.3. Discussion The alkaline pretreatment appears to increase d e g r a d i b i l i t y s l i g h t l y . Acid pretreatment makes the sample less degradable. This i s probably due to the acid clearing out the more eas i l y degraded portions of the sawdust. The Kraft treatment does not s i g n i f i c a n t l y improve degradability. The water treatment appears to remove some of the smaller more e a s i l y degraded sawdust p a r t i c l e s making the remaining sawdust s l i g h t l y less degradable. 3.7.2. Degradation of Physically Pretreated Sawdust Samples. 3.7.2.1. Introduction Autoclaved, steamed and untreated samples of sawdust were incubated with enzyme with 5400 units/ml. of a c t i v i t y . For comparison a sample of acid pretreated sawdust was also incubated. The samples were removed immediately of aft e r 1 or 4 days. The samples were f i l t r e d and the % Recovery were measured: -75-3.7.2.2. Results Pretreatment Days of I n i t i a l wt ..Wt of Incubation of sawdust f i l t r e sample mg mg F i l t r e + sample mg % Recovery Autoclaved 0 100.31 27.47 126.38 98.6 1 100.56 27.75 115.09 86 .9 4 100.02 27.86 114 .98 87.1 Steamed 0 100.44 27.50 126.50 99 .0 1 100 .53 27.82 118.29 90 .0 4 100.41 27.22 117.20 89 .6 Untreated 0 100.75 27.53 126.97 98.7 1 100.34 27.34 112.96 85 .3 4 100 .05 27.66 113.03 85 .3 Acid 0 88.00 27.00 114.50 99 .0 1 87.86 27.78 110.25 93.9 4 87.84 27.04 110.13 94.6 The results of this table are graphed i n Fig.8 . -76-w 80.0\ T3 § 70-0-CO O60.0-I 50.01 o o a> 40.01 OC 0 30.0 ^ Ac/d Steamed Autoclaved Untreated 20.0-10.0-o days ofincubaf/on F i g . 8. Degradation of physically pretreated sawdust samples. -77-Th e amount of degradation i s the difference between 0 days of incubation and 4 days of incubation: Pretreatment % Degradation Autoclaved 11.5 Steamed 8.4 Untreated 13.4 Acid 4.4 3.7.2.3. Discussion Both of the physical pretreatments decrease the degradability of the sawdust. Heat and pressure are supposed to nonspecifically increase the surface area of the p a r t i c l e s . This does not appear to have happened. Instead the treatment has proven counter productive. 3.8. Increasing percent of substrate 3.8.1. Introduction Untreated sawdust was added to enzyme with 5400 units/ml of a c t i v i t y at 1, 3, 5 and 7% concentration. These samples were incubated for 4 days and then removed. The samples were f i l t r e d and the % Recovery measured. Controls with boiled enzyme were also run for 4 days. 3.8.2. Results % of Sawdust Enzyme I n i t i a l wt wt of f i l t r e % Recovery of sample f i l t r e + sample of Sample mg. mg mg . 1 'active 100.30 27.45 110.95 83.5 boiled 100.19 27 .38 123.82 96 .3 3 active 300.00 27.03 278.24 83.7 boiled 300.87 26 . 71 313.36 95 .3 5 active 500.18 27.83 450.36 84.5 boiled 500.32 31.44 502.71 94.2 7 active 700.06 30.48 642.50 87.4 boiled 700.14 31.69 693.93 94.6 -78-Th e % Degradation i s the difference between the sample with boiled enzyme. % of S a w d u s t % of Degradation 1 3 5 7 12.8 11.6 9.7 7.2 3.8.3. Discussion The addition of higher concentrations of substrate leads to lower % degradation, but the t o t a l amount degraded does increase. The enzyme may be i n h i b i t e d by the end product of degradation. 3.9. Degradation of Iotech Pretreated Sawdust Iotech pretreated sawdust was added to enzyme with 9600 units/ml of a c t i v i t y at a concentration of 2% by weight. Two types of Iotech pretreated sawdust were used, 56V and H60. Of the two, H60 has been more exten-s i v e l y pretreated. The samples were incubated for 4 days and then removed. A control with boiled enzyme was also run. The samples were f i l t r e d and the % recovery The H60 does not f i l t r e well through a 25mm. G.F.C. f i l t r e . " It had to be f i l t r e d through a 55 mm. G.F.C. f i l t r e . 3.9.2. Results Pretreatment Enzyme I n i t i a l wt of f i l t r e f i l t r e + % Recovery Sample mg. wt. mg. sample mg'.' ' 56V H60 active boiled active boiled 200.35 200.42 200.45 200.26 31 .19 30.19 156.79 156.31 175 .9 177 .3 200 . 3 247.8 72 .2 73.4 31.7 61.3 -79-3.9.3. Discussion With 56V the difference between active and boiled enzyme incubation i s minimal. The r a t i o of % Recovery of active to bo i l e d enzyme i s 72.2/73.4 = .98. On the * other hand, with H60 there i s a large difference between active and bo i l e d enzyme samples. The ra t i o of % Recovery of active to bo i l e d enzyme i s 31.7/61.3 = .52. There i s s i g n i f i c a n t degradation of the H60 pretreated sawdust. 3.10. Measurement of H60 Degradation by S e t t l i n g . 3.10.1. Introduction. The f i l t e r i n g of H60 doesn't give r e l i a b l e results because the f i l t r e clogs up and f i l t r a t i o n becomes too slow. Because of th i s , the s e t t l i n g techniques was used. The enzyme v i s i b l y worked on the H60 degrading the large p a r t i c l e s into much smaller p a r t i c l e s that e a s i l y stayed in suspension. A p a r t i c l e that stayed in the super-natant for 30 minutes was considered to be part of the suspension. 3.10.2. Sugar Assays of Suspension. When the suspension was tested for reducing sugars by the DNS, Anthrone or Somogyi assays, some component in the H60 digest supernatant interferes with the assays so that the assays don't work. This condition i s apparent in both acid degraded and untreated suspensions. Acid degradation, composed of adding 72% H 2S0 4 for 1 hour, then d i l u t i n g to 3% -80-H 2S0 4 and b o i l i n g for 4 hours, and then neutralizing, reduces cellooligosaccharides to monomers. The un-treated suspension i s composed of the larger c e l l o -oligomers in suspension. 3.10.3 Measuring Optical Density of Suspension. 3.10.3.1. Introduction. In this experiment enzyme, bo i l e d enzyme and water were incubated with H60 pretreated sawdust for 5 days. On day 1, 2, 4 and 5 a sample i s removed and allowed to s e t t l e for 30 minutes. The o p t i c a l density of the samples over the range of 250-850 nm. was measured. The OD at 550 was selected as giving a representative value. 3.10.3.2. Optical Density of 550 nm. of Suspension. Sample Day 1 Day 2 Day 4 Day enzyme .221 .225 .232 .275 boiled enzyme .180 .203 .222 .245 water .10 .007 .012 .005 3.10.3. Discussion. The values of the b o i l e d enzyme samples are not consistent enough for the enzyme sample to be s i g n i f i c a n t . Even the water samples are not very consistent. 3.11. Measurement of Freeze dried Suspension. 3.11.1. Introduction. In this experiment enzyme, b o i l e d enzyme and water were incubated with H60 pretreated sawdust for 5 days. On day 1 through day 5 a sample was taken o f f and allowed to s e t t l e for 30 minutes. One suspension was removed -81-and freeze dried. The weight for water on H60 w i l l give the value for the amount of small p a r t i c l e s and soluble f r a c t i o n present in the sample before enzyme treatment. The weight for bo i l e d enzyme on H60 w i l l r give the value for the small p a r t i c l e s and soluble f r a c t i o n present in the sample before enzyme treatment plus the weight of the enzyme. The weight for enzyme on H60 w i l l give the value of the small p a r t i c l e s and soluble f r a c t i o n present in the sample before enzyme treatment plus the weight of the enzyme plus any new small p a r t i c l e s and soluble p a r t i c l e s which have been released due to enzyme action on the i n i t i a l l y large p a r t i c l e s . 3.11.2. Results Day Sample I n i t i a l Wt of flask(g) Wt Suspension % Reocovery wt of + flask(g) of sample Sample mg. in sus-' . pension  1 enzyme boiled 100. 39 60 .30247 60 39304 90 .22 enzyme 100 41 77 .07321 77 .16868 95 .08 water 100 . 40 76 .29523 76 .3402 47 .60 2 enzyme boiled . 100. 08 62 .57268 62 .66175 94 .99 enzyme 102 23 78 .51443 78 .59372 77 .56 3 enzyme boiled 100. 26 77 .55995 77 .66604 105 .81 enzyme 102 26 72 .14661 72 24216 93 .43 4 enzyme boiled 100'. 51 74 .96281 75 06700 103 .66 enzyme . 100 81 75 .11888 75 .21073 91 .11 5 enzyme boiled 101 59 77 .45454 77 .57742 120 .96 enzyme 101 80 81 .03550 81 13239 95 .18 -82-Average v a l u e f o r b o i l e d enzyme =93.7 Weight o f enzyme - b o i l e d enzyme - water = ( p a r t i c l e s + enzyme) - ( p a r t i c l e s ) = 9 3 . 7 - 4 7 . 6 = 46.1 Day Wt o f p a r t i c l e s i n enzyme t r e a t e d s u s p e n s i o n 1 44.12 2 48.89 3 59.71 4 57.56 5 74.86 P a r t i c l e s r e l e a s e d by enzyme = enzyme - b o i l e d enzyme. Day Wt o f p a r t i c l e s r e l e a s e d by enzyme 25% of i n i t i a l w e i g h t 1 0 2 1.29 3 12.11 4 9.96 5 27.26 3.11.3. D i s c u s s i o n A s i g n i f i c a n t q u a n t i t y o f the l a r g e p a r t i c l e s a re r e l e a s e d by the a c t i o n o f the enzyme t o form s m a l l p a r t i c l e s t h a t s t a y i n s u s p e n s i o n . 3.12. S e p a r a t i o n o f Suspension i n t o S o l u b l e F r a c t i o n and S m a l l P a r t i c l e F r a c t i o n 3.12.1. I n t r o d u c t i o n Samples o f H60 were i n c u b a t e d w i t h enzyme w i t h 3400 u n i t s / m l a c t i v i t y , b o i l e d enzyme o r w a t e r . The samples were removed a f t e r 2 through 6 days and a f i n a l sample was removed a f t e r 14 days. In a l l c ases the s u s -p e n s i o n was removed. F o r the samples taken o f f on days 2 t h r o u g h 6 the s m a l l p a r t i c l e s were removed from the -83-soluble f r a c t i o n by centrifugation. For the f i n a l sample they were separated by f i l t r a t i o n using a GSC f i l t r e with a 0.12 um pore s i z e . 3.12.2.1. Results. Separation by centrifugation had to be done quickly and c a r e f u l l y as prolonged s i t t i n g or any rough handling caused the p i l l e t to resuspend. Day Treatment Soluble Flask Wt(g) Flask + I n i t i a l % or sample(g) sample Recovery Insoluble mg. 2 H 90 S 103.31624 103.36339 100.20 47.1 A I 83.15407 83.16038 6.3 boiled S 94.41016 94.51723 100.13 106 .9 enzyme I 52 .45594 52 .46734 11.3 enzyme s 95 .92880 96.04461 100 .12 115.7 I 83.85328 83.86472 11.4 3 H 90 s 95 .00818 95 .05850 100.42 50 .1 A I 58.72883 58.73339 4.5 boiled s 94 .25479 94.37130 100.37 116 .1 enzyme I 75 .54391 75.54963 5.7 enzyme s 97.71643 97.83405 100.32 117.2 I 66.28200 66 .29421 12 . 4 H 90 s 96.45734 96 .50636 100.52 48 .8 A I 78 .85197 78.85991 10.0 boiled s 71.41480 71.53173 100 .50 116.3 enzyme I 102.64234 102 .64972 7.3 enzyme s 108.95566 109.07017 100.47 114 .0 I 75.14106 75 .15890 17.8 5 H90 s 103.61419 103.66891 101.76 53.8 A I 80.04074 ^ . 80.04486 4.0 boiled s 96.30487 96 .61626 101.00 110.3 enzyme I 64 .30627 64.31750 11.1 enzyme s 100.62600 100.71240* 100.55 85 .9* I 71 .54357 71.5887 15 .2 6 H90 s 93.97483 94.01614 101.76 40.6 I 76 .40183 76.41774 15.6 boiled s 97.13944 97.23426 101.00 93.9 enzyme I 76 .93221 76.95940 26 .9 enzyme s 94 .42958 94.55065 100.00 121.1 I 79.68147 79 .69300 11.5 *cracked and leaking. -84-3.12.2.2. Discussion These results are indi c a t i v e of enzyme action on the H60 pretreated sawdust which increases both the amount of small p a r t i c l e s which w i l l stay in suspension and the soluble f r a c t i o n . Because of trouble with resuspension of the p e l l e t in some cases the soluble f r a c t i o n measure higher than i t should. 3.12.3. Separation by F i l t r a t i o n 3.12.3.1. Results. The f i n a l sample taken o f f afte r 2 weeks gives these re s u l t s : Treatment Soluble or Flask(g) or Flask + , O r i g i n a l % Recovery Insoluble filtre(mg) sample Sample Ho0 S 71 .87851 71 .93172 102 .68 51.8 2 I 125 .41 127 .66 2.2 bo i l e d S 81 .67618 81 .76952 102 .39 91.2 enzyme I 122 .68 125 .18 2 .4 enzyme S 32 .02878 32 .15262 102 .33 120 .9 I 121 .89 151 .80 29 .2 3.12.3.2. Discussion This technique i s more r e l i a b l e than the c e n t r i -fugation method, since such care does not need to be taken to prevent resuspension. After 2 weeks of incu-bation a considerable amount of degradation has taken place. The amount released by enzyme action i s the difference between the value for enzyme and the value for b o i l e d enzyme. According to these calculations 54% of the sample i s previously in solution i . e . released by water; 30% of the sample i s released into solution and -85-26% of the sample i s released into suspension. Un-fortunately this adds up to 110%. This indicates that growth has managed to overcome the sodium azide i n -h i b i t i o n . This growth probably accounts for about 15% of the t o t a l since the p e l l e t , consisting of the large p a r t i c l e s , i s almost non existant after 2 weeks. 3.12.4. F i l t r e Separation over 3 day experiment 3.12.4.1. Introduction Samples of H60 were incubated with enzyme with 2000 units/ml a c t i v i t y , b oiled enzyme or water. They were taken o f f after 6, 25, 58 or 72 hours. They were allowed to s e t t l e for 30 minutes and the suspension was removed. The soluble and insoluble fractions were separated by f i l t r a t i o n . -86-3.12.4.2. Results Incubation Treatment Soluble Wt of Flask + I n i t i a l % Time or filtre(mg) sample Sample Recor Insoluble or flask(g) (mg)or(g) mg very 6 hr H 2 0 S I 59.95872 122.03 60.00575 122 .63 100.10 47.0 0.6 b o i l e d enzyme S I 64 .42597 126.76 64.54112 128 .89 100.17 115 .0 2.1 enzyme S I 34.02833 127.60 34.14493 154 .54 100 .25 116 .3 26 .9 25 hrs H 2 0 S I 77.03135 125.21 77.07966 126.26 100.41 48.1 1 .0 b o i l e d enzyme s I 74.67109 129.55 74.78783 131.90 100.51 116.1 2.3 enzyme s I 33.36282 125.94 33.48493 156 .57 100 .62 121.4 30 .4 58 hrs H 2 0 s I 76.86429 124.70 76.91160 126.00 100.65 47.0 1.3 b o i l e d enzyme s I 80.80970 127.09 80.92732 131.86 100 .69 116 .8 4.7 enzyme s I 35.72486 122.51 35.85985 151 .60 100.73 124 .1 28 .8 72 hrs. H 2 0 s I 78.63215 124.31 78.68203 125 .53 100.74 49 .5 1.2 b o i l e d enzyme s I 77.19100 124.50 77.30861 129.00 100.82 116 .7 4.5 enzyme s I 33 .28048 122 .80 33.40406 155.72 100 .93 122.4 35 .4 3.12.4.3. Discussion In t h i s short experiment no growth has a chance to overcome the e f f e c t of the sodium azide. In this case 50% of the sample i s previously released i . e . can be released by water. The amount released by enzyme action i s the difference between bo i l e d enzyme and active 'enzyme. About 35% of the i n i t i a l sample i s released into the insoluble small p a r t i c l e s in suspension f r a c t i o n . About 6% of the i n i t i a l sample i s released into the soluble f r a c t i o n . This leads to a t o t a l of r -87-91% being in suspension. The p e l l e t , composed of large p a r t i c l e s , has been reduced to a very small s i z e . 3.13. E f f e c t of Using Different Concentration of Enzyme on H60 Degradation 3.13.1. Introduction Sample of H60 at 1% concentration were incubated with enzyme at 2000, 1000, and 500 units/ml a c t i v i t y . Controls of b o i l e d enzyme were run for each of the enzyme a c t i v i t y l e v e l s , since the weight of enzyme w i l l be reduced i n the d i l u t e d enzyme a c t i v i t y l e v e l s . Water controls were also run. The samples were removed after 0.5, 2, 4, 23, 46, and 71 hours of incubation. The sample was allowed to s e t t l e for 30 minutes and the .. • suspension was removed. The suspension was separated into a soluble f r a c t i o n and a small p a r t i c l e non-soluble suspension f r a c t i o n . The % Recovery for these two fractions was determined. The p e l l e t was also weighed and the % Recovery of large unaltered p a r t i c l e s was measured. -88-3.13.2. Percent Recovery in Soluble Fraction 3.13.2.1. Results. This i s the portion of the suspension which can pass through the f i l t r e . This includes most of the enzyme. Time Treatment I n i t i a l Flask Wt(g) Wt flask + % (Hr) Sample(mg) sample(g) Recovery 0.5 2000 units 100 . 71 74. 04904 74. 15634 106. 5 bo i l e d 2000 100 . 83 80 . 06673 80 . 17007 102 . 5 1000 105. 15 76 . 59516 76 . 67027 71 . 4 bo i l e d 1000 102 . 14 77. 90017 77. 97281 71. 1 400 101. 15 74. 38346 74 43560 51 . 5 boi l e d 400 102 . 86 83. 05730 83. 11068 51 8 H20 101. 05 75 . 00778 75 04897 40 . 8 2. 2000 102 . 96 81. 32351 81 . 43588 109 . 1 boiled 2000 103. 73 79. 15779 79. 25988 98. 4 1000 102. 71 75 . 12118 75 19888 75 . 6 boi l e d 1000 100. 65 80. 78337 80. 85563 71. 8 400 101 83 76 . 00361 76 05866 54. 1 boiled 400 102 66 73. 74043 73 79299 51 2 H2O 100. 83 82. 23317 82 27458 41. 1 4. 2000 100 50 83. 08456 83 19597 110 9 boiled 2000 101 23 77 40421 77 50645 101 0 1000 101 12 71 20220 71 28190 78 8 boiled 1000 107 39 75 36960 75 44647 71 6 400 103 66 78 16642 78 .22426 55 8 boi l e d 400 100 72 75 90284 75 .95401 50 8 H2O 100 83 74 17280 74 .21545 42 3 23. 2000 101 41 109 56851 109 56851 111 6 boi l e d 2000 101 86 79 03006 79 .03006 101 .6 1000 104 .48 93 .59130 93 .59130 83 .0 boiled 1000 102 .52 59 .91526 59 .91526 72 .3 400 103 .58 98 .27783 98 .27783 57 .0 boiled 400 100 .29 69 .15574 69 .15574 50 .3 H20 103 .16 77 .09292 77 .09292 41 .3 46 . 2000 104 .18 102 .64071 102 .75 749 112 .1 boiled 2000 101 .96 95 .18958 95 .29450 102 .9 1000 100 .79 102 .81523 102 .89971 83 .8 boiled 1000 103 .53 97 .18208 97 .25704 72 .4 400 100 .28 111 .74346 111 .80318 59 .6 boiled .400 103 .42 73 .91700 73 .97119 52 .4 H20 100 .60 80 .34802 80 .39039 42 .1 71. 2000 101 .58 85 .55551 85 .67055 113 .3 boiled 2000 100 .99 95 .57282 95 .67837 104 .5 1000 100 .05 98 .46042 98 .54696 86 .5 boiled 1000 101 .21 71 .07883 71 .15159 71 .9 400 100 . 76 108 .66734 108 .73164 63 .8 boiled 400 103 .29 79 .20600 79 .25929 51 .6 H20 102 .89 83 .83469 83 .88039 44 .4 -89-Th e amount that i s soluble i n the o r i g i n a l sample i s the percent that i s released into the water. This averages about 42% of the sample. The difference between the recovery from the enzyme sample and the bo i l e d enzyme sample i s the amount above the 42% that i s released due to the action of the enzyme. Enzyme a c t i v i t y 0.5 2 4 23 . 46 71 hrs 2000 units/ml 4% 10 .7 .9.9 10.0 9.2 8.8 1000 0 3 .8 7.2 10.7 10.4 14 .6 400 0.3 2 .9 5.0 6.7 7.2 12.2 This i s graphed i n Fig.9. 3.13.2.2. Discussion. The amount that i s converted into a soluble form seems to be l i m i t e d to around 10%. The enzyme with the highest a c t i v i t y , 2000 units/ml. very quickly reaches this l i m i t i n g . The two samples with lower a c t i v i t i e s take longer to reach this l i m i t . Probably the soluble f r a c t i o n , which i s composed of oligomers of 3 or less, i s i n h i b i t i n g the release of more into the soluble f r a c t i o n . -90 F i g . 9. R e c o v e r y i n S o l u b l e F r a c t i o n -91-3.13.3. Percent Recovery in Non Soluble Fraction of the Suspension. 3.13.3.1. Results. This i s the portion of the suspension which can not pass through the f i l t r e . This includes very l i t t l e enzyme. Time Treatment I n i t i a l f i l t r e wt. f i l t r e + % Recovery (Hr) Sample (mg) sample (mg) i _ u . ( m g ) 2000 units 101. 71 120 64 122. 18 1 5 boi l e d 2000 100 . 83 121 52 121. 74 0 .2 1000 105 . 15 123 77 124. 81 1 .0 boiled 1000 102. 14 123.43 124 44 1 .0 400 101. 15 124 46 124 58 0 .1 boi l e d 400 102. 86 126 .66 126 76 0 .1 H20 101. 05 126 .42 126 48 0 .1 2000 102. 96 124 .70 127 14 2 .4 boi l e d 2000 103 . 73 124 .24 124 64 0 .4 1000 102 . 71 124 .24 125 00 0 .7 boi l e d 1000 100 . 65 125 .88 126 11 0 .2 400 101. 83 124 .00 125 05 1 .0 bo i l e d 400 102 . 66 125 .41 125 77 0 .4 H 20 100 . 83 126 .29 126 .62 0 .3 2000 100. 50 124 .57 127 15 2 .6 boiled 2000 101. 23 123 .82 124 .48 0 .7 1000 101 . 12 123 .86 125 .33 1 .5 boiled 1000 107 39 125 .07 125 .62 0 .5 400 103 66 124 .80 126 .00 1 .2 boiled 400 100 72 122 .16 122 .56 0 .4 H20 100 83 128 .55 128 .74 0 .2 2000 101 41 124 .07 144 .55 20 .2 boi l e d 2000 101 86 125 .04 126 .40 1 .3 1000 104 46 126 .34 142 .00 15 .0 boiled 2000 102 52 125 .00 125 .51 0 .5 400 103 58 121 .94 128 .49 6 .3 boiled 400 100 29 127 .18 127 .72 0 .5 H20 103 16 124 .25 124 .90 0 .6 2000 104 .18 123 .58 145 .76 21 . 3 boiled 2000 101 .96 119 .61 120 .53 0 .9 1000 100 .79 125 .85 142 .36 16 .4 boi l e d 1000 103 .53 123 .37 124 .30 0 .9 400 100 .28 124 .62 133 .84 9 .2 boiled 400 103 .42 124 .95 125 .62 0 .6 H 20 100 .60 122 .83 123 .51 0 .7 -92-Time Treatment I n i t i a l f i l t r e wt. f i l t r e + % Recovery (Hr) Sample (mg) sample (mg) (mg) 71. 2000 101.58 121.69 148.27 26 .2 boiled 2000 100 .99 124.23 126.06 1.7 1000 100.05 122 .42 141.73 19.3 boiled 1000 101.21 121.23 122 .05 0.8 400 100.76 119.66 130.34 10.7 boiled 400 103.29 123.41 124.61 1.2 H20 102.89 122.68 123.31 0.6 The amount that i s i n the non-soluble portion of the suspension in the o r i g i n a l sample i s the percent that i s released into the water. This averages about 0.4% of the sample. The difference between the recovery from the enzyme sample and the b o i l e d enzyme sample i s the amount about the 0.5% that i s released due to the action of the enzyme. Enzyme a c t i v i t y 0.5 2 4 23 46 71 h o u r s 2000 units/ml 1.3 2.0 1.9 18.9 20.2 24.5 1000 0 0.5 1.0 14.5 15.5 18.5 400 0 0.6 0.8 5.8 8.6 9.5 These results are graphed in Fig.10. -93-F i g . 1 0 Recovery in Non-Soluble Portion of Suspension -94-3.13.3.2. Discussion. These results show no l e v e l i n g o f f of the release of these small p a r t i c l e s . The enzyme with the highest enzyme a c t i v i t y , 2000 units/ml., produces these small p a r t i c l e s at a faster rate than the enzyme solutions with lower enzyme a c t i v i t i e s . 31.13.4. Percent Recovery in P e l l e t 3.13.4.1. Results. This i s the portion of the sample l e f t when the suspension i s removed. This includes very l i t t l e enzyme. Time Treatment I n i t i a l Wt.tube (g) tube + sample. .% Recovery ml. Sample (mg) (g) 2000 units 100 .71 17 .25742 17. 31019 52 .4 boiled 2000 100 .83 17 .37867 17. 43535 56 .2 1000 105 .15 17 .24510 17. 30420 56 .2 boiled 1000 102 .14 17 .51070 17. 56751 55 .6 400 101 .15 16 .85715 16 . 91380 56 .0 boiled 400 102 .86 17 .19263 17. 25047 56 .2 H20 101 .05 16 .87640 16 . 93329 56 .3 2000 102 .96 17 .90706 17. 95339 45 .0 boiled 2000 103 .73 17 .32561 17. 38329 55 .6 1000 102 .71 16 .01562 16 . 07057 53 .5 boi l e d 1000 100 .65 15 .78383 15. 84013 55 .9 400 101 .83 16 .02242 16. 07780 54 .4 boiled 400 102 .66 15 .73511 15. 79257 56 .0 H 20 100 .83 15 .75266 15. 80913 56 .0 2000 100 .50 17 .26648 17. 31281 46 .1 boiled 2000 101 .23 17 .20063 17. 25568 54 .4 1000 101 .12 16 .88316 16 . 93342 49 .7 boiled 1000 107 .39 15 .75591 15 . 81352 53 .6 400 103 .66 16 .87342 16 . 92732 52 .0 boiled 400 100 .72 17 .05835 17. 11434 55 .6 2000 101 .41 17 .34571 17. 37390 27 .8 boiled 2000 101 .86 16 .04445 16 . 09936 53 .9 1000 104 .48 16 .71882 16 . 75246 32 .2 boiled 1000 102 .52 16 .87668 16 . 93243 54 .4 400 103 .58 17 .18221 17. 22882 45 .0 boiled 400 100 .29 17 .27580 17. 33075 54 .8 H 20 103 .16 16 .81247 16 86896 54 .8 -95-Time Treatment I n i t i a l Wt.tube (g) tube + sample % Recovery (Hr) ml. Sample (g) (mg)  2000 units 104 .18 15 .18564 15 .21377 27 .0 boiled 2000 101 .96 17 .81821 17 .87508 55 .8 1000 100 .79 17 .07242 17 .10256 29 .9 boiled 1000 103 .53 15 .88861 15 .94463 54 .1 400 100 .28 17 .26720 17 .30882 41 .5 boiled 400 103 .42 17 .40914 17 .46475 53 .8 H 20 100 .60 17 .33556 17 .38981 53 .9 2000 101 .58 17 .70727 17 .73206 24 .4 boiled 2000 100 .99 17 .26969 17 .32278 52 .6 1000 100 .05 17 .41142 17 .43633 24 .9 boiled 1000 101 .21 17 .04341 17 .09707 53 .0 400 100 .76 15 .63403 15 .67010 35 .8 boiled 400 103 .29 17 .41444 17 .47032 54 .1 H20 102 .89 17 .44157 17 .49744 54 .3 The percent of the sample that i s degraded into either the soluble f r a c t i o n or the non-soluble fr a c t i o n of the suspension, i s the difference between the enzyme sample and the boi l e d enzyme sample. The amount that i s l e f t in the water p e l l e t i s the portion of the sample that starts out insoluble, about 56%. % Recovery Enzyme 0 .5 2 4 23 46 71 hours 2000 units/ml 52 .4 45 .0 46 .1 27. 8 27 .0 24 .4 1000 56 .2 53 .5 49 .7 32. 2 29 9 24 .9 400 56 .0 54 .4 52 .0 45. 0 41 5 35 .8 These, figures are graphed in Fig.11. % Degradation Enzyme 0 5 2 4 23 46 71 hours 2000 units/ml 3 8 10 .6 8 3 26 1 28 .8 28 .2 1000 0 2 .4 3 9 22 2 24 .2 28 .1 400 0 2 1 .6 3 6 9 8 12 .3 18 .3 These figures are graphed i n Fig.12. 60.0J F i g . 1 1 . R e c o v e r y i n P e l l e t -9 7-200Gunits/ml. looo units/ml. 4 0 0 units/ml. 0524 23 46 71 hours of incubation F i g . 1 2 . D e g r a d a t i o n o f P e l l e t -98-3.13.4.2. Discussion. These results seem to indicate that the degradation plateaus at about 29% of the i n i t i a l sample. Since 42% of the i n i t i a l sample i s already soluble this means a t o t a l of 71% of the sample has been degraded by the combination of Iotech pretreatment and enzyme degrada-t i o n . In previous experiments with either higher enzyme a c t i v i t i e s or longer incubation periods, the plateau with 29% of the sample l e f t in the p e l l e t does not e x i s t . The p e l l e t i s almost completely released into suspension. -99-REFERENCES Allcock, E.R., and Woods, J.R.: Appl. Environ. Microbiol. 41, 539-541 (1981) Allen, A. L. , and Mortensen, R .E..: Biotech. 23, 2641-2645 (1981) Bailey, M.J. and Nevalainen, M.H.: Enzyme microb. Technol. 3, 153-157 (1981) Beguin, P., Eisen, H. and Koupas, A.: J. Gen. Microbiol. 101, 191-196 (1977) Berg, B., Hofsten, B.v. and Pettersson, G.: J. appl. Bact. 35, 201-214 (1972a) Berg. B., Hofsten, B.v. and Petterson, G.: J. appl. Bact. 35, 215-219 (1972b) Berg. B.: Can. J. Microbio. 21, 51-57 (1975) Berghem, L.E.R. and Pettersson, L.G.: Eur. J. Biochem. 37, 21-30 (1973) Berghem, L.E.R. and Pettersson, L.G.: Eur. J. Biochem. 46, 295-305 (1974) Bykov, A.N., and Frolov, S.S.: Khim Volokna 2, 33 (1961) Carpenter, S.A., and Barnett, L.B.: Arch. Biochem. Biophys. 122, 1-7 (1967) Choi, W.Y., Haggett, K.D., and Dunn, N.W.; Aust. J. B i o l . S c i . 31,553-564 (1978) Choudhury, N. Gray, P.P. and Dunn, N.W.; Biotech. Let. 2, 427-428 (1980a) Choudhury, N. Gray, P.P., and Dunn, N.W.; European J. Appl. Microbiol. Biotechnol. 11, 50-54 (1980b) Choudhury, N., Dunn, N.W. and Gray, P.P.: Biotech. Let. 3_ 493-496.(1981) Cowling, E.B., and Brown, W.; Adv. Chem. Ser. 95, 152-186 (1969) de Menezes, H.C, de Menezes, T.J.B. and Boas, H.V.Jr.: Biotechnol. Bioeng. 15, 1123-1129 (1973) Donefer, E., Adeleye, I.O.A., and Jones, T.O.A.C.: Adv. Chem. Ser. 95, 328- (1969) -100-Enriquez, A.: Biotechnol. Boieng. 23, 1423-1429 (1981) Fan, L.T., Gharpuray, M.M., and Lee, Y.A.: Biotechnol. Bioeng. Symp. No.n, 29-45 (1981) Ghose, T.K.:. Biotechnol. Bioeng. 11, 239-261 (1969) Ghose, T.K. andKostick, J.A.: Biotechnol. Bioeng. 12, 921-946 (1970) Haggett, K.D., Choi, W.Y., and Dunn, N.W.: European J. Appl. Microbiol. Biotechnol. 6, 189-191 (1978) Haggett, K.D., Gray, P.R., and Dunn, N.W.: European J. Appl. Microbiol. Biotechnol. 8, 183-190 (1979) Han, Y.W., and Srinivasan, V.R.: Appl. Microbiol. 16, 1140-1145 (1968) Han, Y.W. and Srinivasan, V.R.: J. B a c t e r i o l . 100, 1355-1353 (1969) Han, Y.W., a n d C a l l i h a n , CD.: Appl. Microbiol. 27, 159-165 (1974) Harmsen, L., and Nissen, T.V.: Nature 206, 319 (1965) Huang, A.A.: Biotechnol. Bioeng. 17, 1421-1433 (1975) Hulcher, F.H. and King, K.W.: J. B a c t e r i o l . 76, 265-570 (1958a) Hulcher, F.H. and King, K.W.: J. B a c t e r i o l . 76, 571-577 (1958b) Hulme, M.A. and Stranks, D.W.: Nature 226,469-470 (1970) Kirk, T.K: Biotech. Bioeng. Symp. 5, 139-150 (1975) K i t t s , W.D., Kirshnamurti, C.R., Shelford, J.A. and Huffman, J.G.: Adv. Chem. Ser. 95, 279-286 (1969) Klesov, A.A., and Churilova, I.V.: Biochemistry (Biokhimiya) 45, 1282-1290 (1980) Klesov, A.A. 'and Grigorash, S.Y.: Biochemistry (Biokhimiya) 46, 86-94 (1981) Kyslikova, E., and Volfova, 0.: F o l i a Microbiol. 26_, 303-308 (1981) Labudova, I., Farkas, V., Bauer, S., Kolarova, N. and Branyik, A.: Duropean J. Appl. Microbio. Biotechnol. 12, 16-21 (1981) -101-L i , L.H., Flora, R.M. and King, K.W.: Arch. Biochem. Biophys. I l l , 439-447 (1965) Lipinsky, E.S.: Adv. Chem. Ser. 181, 1-23 (1979) Lobanok, A.G. and Pavolvskaya, Z.I.: Microbiology (Mikrobiologiya) 44, 25-28 (1975) Mandels, M. and Reese, E.T.: J. B a c t e r i o l . 73, 269-278 (1957) Mandels, M. Parrish, F.W., and Reese, E.T.: J. B a c t e r i o l . 83, 400-408 (1962) Mandels, M., Medeiros, J.E., Andreotti, R.E., Bi s s e t t , F.H.: Biotech. Bioeng. 23, 2009-2026 (1981) M i l l e t t . M.A. and Goedken, Y.L.: Tappi 48, 367 (1965) M i l l e t t , M.A., Baker, A.H. andSatter, L.D.: Biotech. Bioeng. Symp. 5, 193-219 (1975) Ng, T.K. and Zeikus, J.G.: Appl. Environ. Microbio. 42, 231-240 (1981) Nisizawa, T., Suzuki, H., Nakayama, M. and Nisizawa, K.: J. Biochem. 70, 375-385 (1971) Nisizawa, T., Suzuki, H., and Nisizawa, K.: J. Biochem. 71, 999-1007 (1972) Norkrans, B.: Physiol. Plant. 3, 75-87 (1950) Norkrans, B.: Adv. Appl. Microbio. 9, 91-130 (1967) Nummi, M., Niku-Paavola, M.L., Enari, T.M., and Raunio, V.: Analyt. Bio. 116, 137-141 (1981) Nystrom, J.: Biotech. Bioeng. Symp. 5, 221-224 (1975) Okada, G., Nisikawa, K, and Suzuki, H. : J. Biochem. 6_3, 591-607 (1968) Petersen, N.: Biotechnol. Bioeng. 17, 361-374 (1975) Reese, E.T., Siu, R.G.H., and Levinson, H.S.: J. B a c t e r i o l . 59, 485-497 (1950) Reese, E.T. and Levinson, H.S.: Physiol. Plantarum 5, 345-366 (1952) -102-Rickard, P.A.D., and Laughlin, T.A.: Biotech. Let. 2, 363-368 (1980) Rickard, P.A.D. a n d P e i r i s , S.P.: Biotech. Let. 3, 39-44 (1981a) Rickard, P.A.D., Rajoka, M.I., and Ide, J..A.: Biotech. Let.3, 487-492 (1981b) Rogers, C.J., Coleman, E., Spino, D.F., and P u r c e l l , T.C.: Environ. S c i . Techno. 6, 715-719 (1972) Srinivasan, V.R. and Han, Y.W.: Adv. Chem. Ser.' 95, 447-460 (1969) Stenberg, D.: Appl. Environ. Microbiol. 31, 648-654 (1976) Sternberg, D., Vijayakumar, P, and Reese, E.T.: Can. J. Microbiol. 23, 139-147 (1977) Sternberg, D. and Mandels, G.R.: J. B a c t e r i d . ' 144, 1197-1199 (1980) Stewart, B.J., Leatherwood, J.M.: J. B a c t e r i o l . 128, 609-615 (1976) Stone, J.E., Scallan, A.M., Donefer, E. and Ahlgren, E.: Adv. Chem. Ser. 9_5, 219-238 (1969) Storvick, W.O., and King, K.W.: J. B i o l . Chem. 235(2), 303-307 (1960) Storvick, W.O., Cole, F.E., and King, K.W.: Biochemistry, 2, 1106-1110 (1963) Summers, R.J. and Srinivasan, V.R.: Appl. Environ. Micro. 37, 1079-1084 (1979) Summers, R.J., Boudreaux, D.P. and Srinivasan, V.R.: Appl. Envir. Micro. 38, 66-71 (1979) Suzuki, H., Yamane, K. and Nisizawa, K.: Adv. Chem. Ser. 95, 60-82 (1969) . Swisher, E.J:, Storvick, W.O. and King, K.W.: J. B a c t e r i o l . 88, 817-820 (1964) Tangnu, S .K .,. Blanch , H.W., Wilke, C.R.: Biotech. Bioeng. 23, 1837-1849 (1981) Tarkow, H. and Fei s t , W.C: Adv. Chem. Ser. 95, 197-217 (1969) -103-Updegraff, D.M.: Biotechnol. Bioeng. 13, 77-97 (1971) Volfova, 0. and Kyslikova, E.: F o l i a Microbio. 26, 309-313 (1981) Wood, T.M.: Biochem. J. 115, 457-464 (1969) Woodward, J. and Arnold, S.L.: Biotechnol. Bioeng. 23, 1553-1562 (1981) Yamane, K., Suzuki, H., Hirotani, M., Ozawa, H., Nisizawa, K.: J. Biochem,(Japan) 67, 9-18 (1970) Yamane, K., Yoshikawa, T., Suzuki, H. and Nisizawa, K.: J. Biochem. (Japan) 69, 771-780 (1971 Yoshikawa, T., Suzuki, H. and Nisizawa, K.: J. Biochem. (Japan) 75, 531-540 (1974) 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095441/manifest

Comment

Related Items