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Isolation of anaerobic cellulolytic thermophiles and production and purification of the cellulase from… Lee, Byong Hoon 1972

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ISOLATION OF ANAEROBIC CELLULOLYTIC THERMOPHILES AND PRODUCTION AND PURIFICATION OF THE CELLULASE FROM CLOSTRIDIUM THERMOCELLULASEUM M-7 by BYONG HOON LEE B.Agr., Chun Chon National Agr icu l tu ra l College, Korea, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Microbiology We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1972 In presenting th is thesis in pa r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the Universi ty of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lable fo r reference and study. I fu r ther agree that permission fo r extensive copying of th i s thesis fo r scholar ly purposes may be granted by the Head of my Department or by his representat ives. I t i s understood that copying or publ icat ion of th is thesis fo r f inanc ia l gain shal l not be allowed without my wr i t ten permission. Department of Microbiology The Universi ty of B r i t i s h Columbia Vancouver 8, Canada Date March 13, 1972. ABSTRACT An enrichment procedure led to the i s o l a t i o n , by the cel lu lose r o l l tube method, of a number of ac t i ve ly c e l l u l o l y t i c anaerobic thermo-p h i l i c bac ter ia . Two isolates were terminal ly sporing rods and were ten ta t i ve ly i d e n t i f i e d as Clostr idium thermocellulaseum (Enebo, 1951). Strain M-7 (0.6 ym x 4.0 urn) from manure grew opt imal ly at 58°C to 63°C, pH 6.0 to 6.5 and did not require organic n i t rogen. St ra in C-19 (0.3 ym x 4.5 ym) from compost was s imi la r but grew opt imal ly at 50°C to 68°C, pH 7.5. Both u t i l i z e d cel lobiose and a wide range of other sugars. Stra in C-19 did not u t i l i z e glucose, ra f f inose and inos i to l but did use i n u l i n . The mean generation times in a r i ch nu t r ien t medium containing cel lobiose were 35 min f o r s t ra in M-7 and 25 min f o r s t ra in C-19. St ra in M-7 had a mean generation time of 2 rjr when grown on ce l lu lose . Yeast ext ract (0.5%) stimulated growth and cel lu lase production by s t ra in M-7 but was i n h i b i t o r y at higher concentrat ions. Other organic nitrogen sources acted s i m i l a r l y . Cellulose at 1.0% gave maxi-mum cel lu lase production a f te r 72 hr incubation of s t ra in M-7. Higher concentrations of cel lu lose were not completely degraded in 72 hr . Strain M-7 did not produce cel lu lase when grown on any carbon source other than cel lu lose substrates. The addi t ion of cel lobiose (0.3%) and glucose (0.4%) prevented cel lu lose hydrolysis in ce l lu lose medium. This may have been repression of synthesis but cel lu lase was inh ib i ted by both sugars. i i i i i Both C-, cel lu lase (degrades native cel lu lose) and C cel lu lase (g-1,4-glucanase) a c t i v i t i e s in s t ra in M-7 cultures were assayed by measuring the l i be ra t i on of reducing sugars, using d i n i t r o s a l i c y l i c ac id . Both a c t i v i t i e s had optima at pH 6.5 and 67°C. C x ce l lu lase could conveniently be assayed by a new automated procedure. Stra in M-7 was very ac t ive ly c e l l u l o l y t i c when compared to previously microbial species. The 48 hr cul ture contained C a c t i v i t y (56 yg glucose/min/ml A from carboxymethyl cel lu lose) and C-| a c t i v i t y (8 yg glucose/min/ml from cotton f i b r e s ) ; the r a t i o of C,:C was 1:7. The ce l lu lase(s) from I A s t ra in M-7 were e x t r a - c e l l u l a r , produced during exponential growth but were not free in the growth medium u n t i l 50% of the cel lu lose was hydrolyzed. Glucose and cel lobiose were the only soluble products l iberated by the cel lu lase from ce l lu lose ; ZnCl^ p rec ip i ta t ion appeared to be a good method f o r the concen-t r a t i o n of cel lu lase a c t i v i t y but subsequent p u r i f i c a t i o n was not successful. I soe lec t r i c focusing indicated the presence of four C A cel lulases (pi 4 .5 , 6.3, 6 .8 , and 8 . 7 ) . DEAE-Sephadex chromatography indicated three C components. A I t is concluded that C_. thermocellulaseum M-7 produces ce l lu lase(s) capable of rap id ly hydrolyzing native ce l lu lose. The rapid production and high a c t i v i t y of cel lulases from th is organism strongly support the basic premise that increased hydrolysis of cel lu lose is possible at elevated temperature. TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW ' . . 3 MATERIALS AND METHODS 13 I . Chemicals 13 I I . Culture media 13 1 . Basal enrichment medium . • 13 2. Solid media 14 3. Growth medium • 14 4. F i l t e r paper medium 14 5. Cellulose medium 15 I I I . Sources of s t ra ins and procedures of i so la t i on of anaerobic c e l l u l o l y t i c thermophil ic bacteria . . . 15 IV. C r i t e r i a of pur i ty and maintenance 16 V. Cellulase assay 16 V I . Paper chromatography • 17 V I I . ATP assay 19 V I I I . Determination of protein . . 19 IX. Determination of to ta l cel lu lose . 19 X. Cell f rac t iona t ion 19 iv Page RESULTS 20 I . I so la t ion and character izat ion of anaerobic c e l l u l o l y t i c thermophil ic bacter ia 20 1 . I so la t ion of pure cultures 20 2. C r i t e r i a of pur i t y and maintenance 20 3. Character ist ics of isolates 23 a. Morphological character is t ics 23 b. Cultural character is t ics 23 c. Growth character is t ics 23 d. Nu t r i t i on 23 I I . Cultural condit ions fo r cel lu lase production by C_. thermocel lulaseum M-7 26 1. Local izat ion of cel lu lase in C_. thermo-cenulaseum M-7 26 2. The e f fec t of complex nitrogenous compounds on growth and cel lu lase (C x ) production by C_. thermocenulaseum M-7 26 3. The e f fec t of cel lu lose concentration on growth and cel lu lase (C x ) production by C_. thermocel lulaseum M-7 28 4. The e f fec t of inoculum size on growth and cel lu lase (C x ) production by C_. thermo- cel lulaseum M-7 28 5. The e f fec t of carbohydrates on growth and • cel lu lase (C x ) production by C_. thermo- cenulaseum M-7 31 6. The e f fec t of various concentrations of cel lobiose and glucose on C x production by C_. thermocenulaseum M-7 31 vi Page 7. The effect of Tween 80 on the production of cellulase (Cx) and growth by C_. thermo-cellaseum M-7 31 8. The kinetics of cellulase production by C_. thermocellulaseum M-7 35 III. Cellulase assay methods 37 IV. Concentration and purification of cellulase from culture supernatant of C_. thermocellulaseum M-7 . . 43 1. Concentration of cellulase 43 a. Ammonium sulphate precipitation 43 b. ZnC^ precipitation 47 c. Adsorption on cellulose 47 2. Purification of cellulase 47 DISCUSSION 54 BIBLIOGRAPHY 60 LIST OF TABLES Table Page I . Substrates and methods of cel lu lase assay 11 I I . Comparison of carbohydrate fermentation 27 I I I . Local izat ion of cel lu lase in C_. thermo-cellulaseum M-7 28 IV. The e f fec t of yeast ext ract on growth and cel lu lase (C x ) production by C_. thermo- cellulaseum M-7 29 V. The e f fec t of cel lu lose concentration on growth and cel lu lase (C x ) production by C_. thermocellulaseum M-7 30 V I . The e f fec t of sugars on growth of C. thermo-cellulaseum M-7 and cel lu lase (C^) production . . . . 33 V I I . The e f fec t of various concentrations of c e l l o -biose and growth on cel lu lase (C x ) production and growth of C_. thermocellulaseum M-7 34 V I I I . The e f fec t of the addi t ion of Tween 80 on the y i e l d of cel lu lase (C x ) and growth by C_. thermocellulaseum M-7 35 IX. The e f fec t of glucose and cel lobiose concen-t r a t i o n on cel lu lase a c t i v i t y 44 X. Zinc chlor ide p rec ip i ta t ion of cel lu lase from the cul ture supernatant of C_. thermocellulaseum M-7 . . 48 X I . The e f fec t of ZnCl 2 on cel lu lase a c t i v i t y 49 X I I . Adsortion and desortion of cel lu lase on cel lu lose column 50 X I I I . Pu r i f i ca t i on of cel lu lase from the cu l ture supernatant of C_. thermocel lulaseum M-7 51 v i i LIST OF FIGURES Figure Page 1 . Flow diagram in Technicon autoanalyzer 18 2. I so la t ion of anaerobic c e l l u l o l y t i c thermophil ic bacteria 21 3. Pure c e l l u l o l y t i c colonies of s t ra in M-7 on cel lu lose agar plates a f te r 5 days 22 4. Phase contrast photomicrograph of s t ra in M-7(a) and s t r a i n C-19(b) . ' 24 5. Growth curve fo r s t ra in M-7 and s t ra in C-19 25 6. The e f fec t of inoculum size on growth and ce l lu lase production of C_. thermocenulaseum M-7 32 7. The k inet ics of cel lu lase production by C_. thermo-cenulaseum M-7 ; 36 8. The e f fec t of incubation time on cel lu lase a c t i v i t y 38 9. The e f fec t of enzyme concentration on cel lu lase a c t i v i t y 39 10. The e f fec t of temperature on cel lu lase a c t i v i t y 40 11. The e f fec t of pH on cel lu lase a c t i v i t y 41 12. The e f fec t of CMC concentration on C x cel lu lase a c t i v i t y 42 13a. Typical ca l i b ra t ion curve (a) in automated C x ce l lu lase assay 45 13b. Typical ca l ib ra t ion curve (b) in automated C x ce l lu lase assay 46 14. DEAE-Sephadex chromatography 52 15. Separation of cel lu lase (C x ) of cul ture supernatant of C_. thermocellulaseum M-7 by i soe lec t r i c focusing . . 53 v i i i ACKNOWLEDGEMENTS The author wishes to express his grat i tude to Dr. T. H. Blackburn, f o r his encouragement, advice and c r i t i c i s m throughout the course of th is work. Thanks are also extended to the other committee members: Dr. D. G. K i lburn , Dr. R. A. J . Warren, and Dr. B. C. McBride fo r t he i r cooperative cont r ibu t ions . The author would l i k e to extend a very special thanks to Dr. J . J . R. Campbell, Head of the Department of Microbiology and other facu l ty members, fo r granting th is opportunity fo r study and research at The Universi ty of B r i t i s h Columbia. The valuable technical assistance and advice of Mr. B. Walsh, Mr. V. Singh, Mr. W. Ramey, and Miss W. Charlotte are great ly appreciated. This work was supported in part by Department of the Environment, Water Resources Research grant (#65 -1651), Canada. ix ISOLATION OF ANAEROBIC CELLULOLYTIC THERMOPHILES AND PRODUCTION AND PURIFICATION OF THE CELLULASE FROM CLOSTRIDIUM THERMOCELLULASEUM M-7 x INTRODUCTION The microbial degradation of cel lu lose is one of the most important processes in nature. I t contr ibutes one of the major steps in maintaining the balance between the opposing synthet ic and degrada-t i ve reactions in the carbon cycle. With the continued increase in urban populations the problem of disposing of so l id wastes such as garbage (40-60% cel lu lose) has become of major importance (Feldman, 1969). The most e f fec t i ve disposal would be a recycl ing process whereby waste cel lu lose is converted in to a new food source such as sugar. The conversion of cel lu lose to glucose can be accomplished chem-i c a l l y or enzymatical ly. Chemical hydro lys is , however, has l im i ted value because of the high rate of destruct ion of the glucose monomer (Walseth, 1952). Consequently, the success attained by enzymatic con-version of starch into sugars ( S i n c l a i r , 1965; Underkofler, 1969) motivated cel lu lase workers to consider what might be s i m i l a r l y achieved in the case of ce l lu lose. Yet, despite the i r great importance, re la -t i v e l y l i t t l e work has been done on cel lulases as they are d i f f i c u l t to measure and p u r i f y . There are c o n f l i c t i n g opinions concerning the number of com-ponents in cel lu lase systems. Reese et a l . (1950) established that at least two components were present in Trichoderma v i r i de cel lu lase preparations. The C-i enzyme is believed to be required f o r the 1 2 hydrolysis of nat ive , highly ordered forms of ce l lu lose (cotton f ib res ) before C enzymes can cleave them. The C enzymes, consist of exo- and A A endo-e-1,4-glucanases, l iberated reducing sugars from chemically modified cel lu lose (carboxy methyl ce l lu lose) or C-| - modified ce l lu lose . As y e t , the mechanism of C-| enzyme is s t i l l obscure (S iu , 1951 , 1963; Gascoigne and Gascoigne, 1960; Selby and Mait land, 1963, 1967; Norkrans, 1967). L i t t l e is known regarding the propert ies of the exo-8-1,4-glucanase < (Reese, 1969). L i t t l e information is avai lable on the p o s s i b i l i t y of obtaining large amounts of sugar by enzymatic degradation of ce l lu lose . Katz and Reese (1968) showed that a 30% glucose solut ion could be obtained by hydrolysis of ground and heat treated ce l lu lose . Ghose and Kostick (1969) showed that while heat treated cel lu lose was a poor subst i tu te from Trichoderma v i r i d e ce l lu lase , the combination of heat treatment and pa r t i c le size reduction increased s u s c e p t i b i l i t y to hydro lys is . Ghose and Kostick (1970 also demonstrated the p o s s i b i l i t y of continuous con-version of the substrate in to a product containing 50% glucose, over an incubation period of 40 hours, i f the products were removed as fas t as they were formed. In 30 hours a 14% glucose solut ion was obtained from a 30% cel lu lose suspension by using a cel lu lase from Trichoderma v i r i d e mutant QM 9123 (Mandels, Weber and Parizek, 1971). The glucose was separated from substrate and enzyme by u l t r a f i l t r a t i o n . Commercial u t i l i z a t i o n of cel lu lase remains unat t ract ive due to 1) the low level of a c t i v i t y of the cel lulases ava i lab le , 2) the i n -soluble and res is tan t nature of substrate, and 3) the presence of impuri t ies in waste ce l l u los i c mater ia ls . 3 Since increased conversion rate is of importance, the possible advantage of using thermophil ic microorganisms has been studied (McBee, 1948, 1950; Enebo, 1949, 1951). Although the resul ts to date have been disappointing in f a i l i n g to provide an i n d u s t r i a l l y pract ica l process, the basic premise that increased degradation rates are possible at elevated temperature remains unchallenged. The object of these studies was to : a) iso la te anaerobic thermo-p h i l i c c e l l u l o l y t i c microorganisms, b) determine the cu l tu ra l conditions fo r optimal cel lu lase product ion, c) develop assay methods fo r cel lu lase a c t i v i t y , and d) pur i f y the ce l lu lase . LITERATURE REVIEW C e l l u l o l y t i c enzyme production has been reported from a broad range of microorganisms. Since cel lu lose is insoluble and cannot pass through the ce l l membrane, cel lulases are thought to be ex t race l lu la r enzymes. Most c e l l u l o l y t i c microorganisms secrete cel lu lase in to the cul ture medium and digest the high polymeric cel lu lose in to soluble products that can be assimilated by the organism. Cellulases are also often released by lysing the bacteria wi th agents such as toluene (Norkrans, 1967) i n d i -cating that at least cer ta in cel lu lase components ex is t bound to the c e l l s . Although few studies have been made on the loca l i za t ion of ce l lu lases, Suzuki et a l . (1969) and Yamane et a l . (1970, 1971 ) working with Pseudomonas fluorescens var. cel lu losa found three cel lu lase com-ponents, one of which was local ized in the " i n t r a w a l l " or "periplasmic" 4 f rac t ion of the c e l l , and the other two in the ex t race l lu la r f rac t i on or cul ture medium. Cultural factors inf luencing cel lu lase production include com-posi t ion of the cul ture medium such as qua l i t y and quant i ty of cel lu lose used, the amounts of metal sa l ts present, the pH, the temperature, and adequacy of the oxygen (Whitaker and Thomas, 1963; Lyr , 1964). C e l l u l o l y t i c enzymes are generally considered to be formed only in the presence of cel lu lose but the soluble products of the ce l lu lose , especial ly ce l lob iose, are thought to induce cel lu lase (Mandels and Reese, 1960). Other disaccharides wi th B-glycoside linkage such as lactose and sophorose also induce cel lu lase (Mandels, Parrish and Reese, 1962). Recently, Nisizawa et a l . (1971) found that cel lu lase was induced in mycelia of Trichoderma washed with a small amount of sophorose and was inh ib i ted in vivo by glucose, g l y c e r o l , and ATP. In an invest igat ion with V e r t i c i l l i u m alboatrum, Talboys (1958) reported that cel lobiose or cel lu lose would induce Cx whereas glucose, sucrose, lactose, and starch a l l inh ib i ted the cel lu lase formation. Gupta and Heale (1970) presented evidence that of a large number of sugars and polysaccharides, only cel lobiose was the spec i f ic inducer of Cx while many of the sugars repressed Cx induction in the presence of carboxy-methyl -cel lu lose. Horton and Keen (1966) working with Pyrenochaeta t e r r e s t r i s , a fungus in infected onion roo ts , showed that cel lu lase -4 synthesis was repressed by glucose concentrations of 5 x 10 M and they presented evidence f o r the regulat ion of the cel lu lase synthesis by c e l l u l o l y t i c products through a repressor-inducer mechanism. There i s , however, evidence that cel lu lase produced by some micro organisms is c o n s t i t u t i v e . C e l l u l o l y t i c f i l t r a t e s have been obtained from cultures of Aspergi l lus luchuensis grown on glucpse, g l y c e r o l , ce l lob iose, and starch (Reese and Levinson, 1952) and from Clostr idium  thermocellulaseum grown on xylose (Enebo, 1954) and C_. thermocel 1 urn grown on xylose and hemicellulose (Hammerstrom et a l . , 1955). Myers and Eberhart (1966) invest igated mutations of Neurospora  crassa which involved a regulatory gene fo r the simultaneous production of cel lobiase and ce l lu lase . This gene; was d i s t i n c t from a regulatory gene f o r e - a r y l glycosidase a c t i v i t y although cel lobiose induced a l l three a c t i v i t i e s in wi ld- type mutants. More recent ly , a mutant s t r a i n , designated as Trichoderma v i r i d e QM 9123 which secretes twice as much cellulase; as i t s parent was obtained by i r r a d i a t i n g conidia of the parent Trichoderma v i r i d e QM 6a wi th high energy electrons (Mandels, Weber, and Parizek, 1971). Increases in ce l lu lase production have been achieved in mixed cu l tu re . An anaerobic thermophil ic bacterium Clostr idium thermo- cellulaseum degraded cel lu lose much more e f f e c t i v e l y in the presence of other nonce l lu lo ly t i c bacter ia (Enebo, 1949). Hungate (1964) also found that d i g e s t i b i l i t y was bet ter with a mixture of c e l l u l o l y t i c bacter ia and protozoan. S imi la r ly adding the aerobic bacterium Alcaligenes to cul ture of a c e l l u l o l y t i c Cellulomonas sp. resulted in a marked increase in growth and ce l lu lose degradation (Han and Srinivasan 1968). Most recent ly Hofsten et a l . (1971) discovered a synergis t ic e f fec t of ce l lu lose degradation between the c e l l u l o l y t i c Sporocytophaga 6 bacterium and the non-ce l lu lo l y t i c gram negative eubacterim. Although much is known of the biology of many of the aerobic c e l l u l o l y t i c micro-organisms, the avai lable information on the anaerobic bacter ia is less complete because so few have been obtained in pure cu l tu res . Anaerobic c e l l u l o l y t i c bacter ia have been isolated from many sources and are responsible f o r a c t i v i t i e s such as the digest ion of cel lu lose in animal digest ive t r a c t s , the se l f heating of hay, peat format ion, and decompo-s i t i o n in composte. Few reports are avai lable on the c e l l u l o l y t i c a c t i v i t y of cul ture f i t r a t e s of pure cultures of rumen organisms (Hal l iwe l l and Bryant, 1963; Leatherwood, 1965; Tyler and Leatherwood, 1967). The f a i l u r e of early attempts to cu l t i va te rumen c e l l u l o l y t i c bacter ia has been a t t i -buted by Hungate (1966) to : a) lack of nu t r i t i ona l factors present in rumen f l u i d (Bryant and Doetsch, 1954), b) presence of oxygen, c) use of too high concentration of cel lu lose and types of cel lu lose not readi ly attacked, and d) the confusion resu l t ing from the general lack of success on obtaining pure cultures of anaerobic c e l l u l o l y t i c bacteria from any hab i ta ts . I t has been maintained that anaerobic c e l l u l o l y t i c thermophiles cannot be isolated by the techniques used f o r other anaerobic bacter ia . Their existence was deduced from the disappearance of cel lu lose in manure p i les at temperatures above 50°C (Macfadyen and B l a x a l l , 1896). Pochon (1942) characterized them as extremely var iable while Enebo (1949) indicated that they could not be grown in pure cu l tu res , but re -quired the presence of symbionts. V i l joen et a l . (1926) described the 7 i so la t ion of an anaerobic c e l l u l o l y t i c thermophile which they named as Clostr idium thermocellum. This organism los t the a b i l i t y to ferment cel lu lose a f t e r having been cul tured on glucose media. Further studies by Imseneski (1940) demonstrated that th is apparently var iable behavior was due to contamination of the cu l tu re . Subsequently, McBee (1948) isolated two cultures by the technique developed by Hungate (1944) and proposed the name, Clostr idium thermocellum as used e a r l i e r by V i l joen et a l . (1926). These were the f i r s t cultures of anaerobic c e l l u l o l y t i c thermophiles which could be used fo r morphological and physiological s tudies. A l l these cul tures degraded cel lg lose even a f te r repeated sub-cul ture on sugar media and McBee (1948) concluded that e a r l i e r studies were suspect due to contaminated cu l tu res . The rate of ce l lu lose degra-dation by C_. thermocellum was low, y ie ld ing CO^, Hg, ethanol , formic a c i d , acetic ac id , l a c t i c ac id , succinic a c i d , and possibly butyr ic acid and methane. End product i n h i b i t i o n of degradation could occur in C_. thermocellum and the degradation of cel lu lose to glucose was thought to be the a c t i v i t y of two d i f f e r e n t enzymes: a) a cel lu lase which hydro-lyzed cel lu lose to glucose and act ive at 68°C, and b) a cel lobiase which hydrolyzed cel lobiose to glupose and inact ive at 68°C. S i u , Nelson and McBee (1955) confirmed that C_. thermocellum could grow on cel lu lose and cel lobiose but not on glucose and demonstrated the presence of a c e l l o -biose phosphorylase in c e l l - f r e e extracts of the organisms. Subsequently, cel lobiose phosphorylase was shown in Ruminococcus f l av i fac iens (Ayers, 1958), Ce l l v ib r io g i lvus (Hulcher & King, 1958) and Cellulomonas f imi (Sato & Takahashi, 1967). Enebo (1951) isolated a pure cul ture morphologically 8 ident ica l to C_. thermocel!um (McBee, 1948) which he named Clostr idium  thermocellulaseum. C_. thermocellulaseum hydrolyzed cel lu lose mainly to cel lobiose and glucose and produced CC^, h^, ethanol , formic, ace t i c , l a c t i c and succinic acids and was capable of CC^ f i x a t i o n . McBee (1948) observed that cultures f a i l e d to grow a f te r 2 or 3 transfers on cel lu lose medium lacking growth factors but Enebo could make up to 10 transfers on mineral medium without loss of v i a b i l i t y . More s t r i k i n g dif ferences occurred in the fermentation products: C_. thermocellulaseum gave very good carbon recovery (102%) in the fermen-ta t ion which indicated C0£ f i x a t i o n but C_. thermocellum gave only 70% recovery. Recently, an act ive cul ture of an obl igate c e l l u l o l y t i c thermophil ic bacteria has been isolated in Russia (Loginova et a l . , 1962, 1966). I t s main metabolic products were ethanol , l a c t i c , ace t i c , formic acids as well as propionic and butyr ic acids which were found in small quant i t ies from growth in 1% to 3% cel lu lose media (Shcherbakov, 1968). No other thermophil ic c e l l u l o l y t i c isolates are known to produce butyr ic ac id . Knowledge of the s t ruc tura l features of cel lu lose is relevant to any discussion of i t s enzymatic degradation. The chemical consti tuents of wood and cotton f ibers include ce l lu lose , several hemicelluloses, l i g n i n , a wide var ie ty of other materials including cer ta in nitrogeneous substances, and a small amount of inorganic matter. The cel luloses of wood and cotton are both l inear polymers of 3-D-glucopyranose units l inked by g-1,4-glycosidic bonds having a degree of polymerization (the number of glucose uni ts per molecule) as few as 15 to as high as 14,000 9 with an average of about 3,000. The most abundant hemicelluloses in cotton are pect ic substances and those in wood are r e l a t i v e l y short heteropolymers of glucose, xylose, galactose, mannose and arabinose, as well as uronic acids of glucose and galactose l inked together by S-1,3, 6-1,6, and 6-1,4 glucpsidic bonds. Lignin is a complex three-dimentional polymer formed from p-hydroxy cinnamyl a lcohols. A port ion of the l i g n i n is believed to be l inked by covalent bonds to cer ta in of the hemi-cel luloses (Freudenberg, 1965). Cotton consists of nearly pure c r y s t a l l i n e ce l lu lose . Almost a l l the hemicelluloses and extraneous materials are contained in the cu t i c l e and primary wall layers . In wood, hemicelluloses and l i g n i n form a matrix surrounding the ce l lu lose . The l i g n i n apparently prevents the cel lulases and hemicellulases of many microorganisms from contacting a s u f f i c i e n t number of glyCosidic bonds to permit s i g n i f i c a n t hydrolysis (Cowling and Brown, 1969), Other features of ce l l u los i c materials that might determine the i r s u s c e p t i b i l i t y to enzyme degradation include: 1) the moisture contents of the f i b e r (Stone and Scal len, 1967); 2) the size and d i f fus i - * b i l i t y of the enzyme molecules (Stone, Scal lan, Donefer, and Ahlgren, 1969); 3) the degree of c r y s t a l l i t y of the cel lu lose (Norkrans, 1950; Walseth, 1952; Reese et a l . , 1957); 4) the un i t ce l l dimensions of the c r y s t a l l i t e s present--ri . e . , the repeating three-dimensional un i t w i th in the c r y s t a l l i n e regions (Honeyman, 1959; Rautela and King, 1968); 5) the conformation and s t r i c t r i g i d i t y of the glucose uni ts (Thoma and Koshland, 1960); 6) the degree of polymerization of the cel lu lose 10 (Norkrans, 1950; King, 1963; G i l l i gan and Reese, 1954); 7) the nature, concentrat ion, and d i s t r i b u t i o n of subst i tuent groups (Reese et a l . , 1950; Reese, 1957). Contradictory statements regarding cel lu lase a c t i v i t i e s have arisen as a resu l t of the wide range of ce l l u los i c substances. Although factors in addi t ion to chain length, such as c r y s t a l l i t y or cross-l inkage, also goyern the rate and extent of attack of an enzyme on ce l lu lose , the mean degree of polymerization is the most useful guide in assessing the p robab i l i t y of enzyme at tack. Cellulase invest igators have t r i e d to overcome some of these d i f f i c u l t i e s by using modified ce l lu loses . Some of the common substances used, including the water soluble ol igoglucosides, and some cel lu lose der ivat ives are presented in Table I , which also gives the methods of measuring the a c t i v i t y . Since native cel lu lose is highly res is tan t to hydrolysis by most "cq l lu lases , " soluble der ivat ives of ce l lu lose , p a r t i c u l a r l y sodium carboxymethylcellulose (CMC) have been widely used as tes t substrates. The c e l l u l o l y t i c a c t i v i t y against soluble substrates has been measured from the change in v iscos i ty or the production of reducing sugars. When a c t i v i t y is assayed v iscomet r i ca l l y , uni ts of a c t i v i t y are based on the i n i t i a l rates of change of the reciprocal of the spec i f i c v i s c o s i t y , i . e . , (d l / n sp ) /d t . The production of reducing sugars has been assayed by a number of methods., including the co lor imetr ic fer r icyanide (Park and Johnson, 1949), arsenomolybdate reagent (Festenstein, 1958; Hash & King, 1958; Myers & Northcote, 1959; Thomas, 1956), oxidat ion wi th iodine in Table I . Substrates and methods of c e l l u l a s e assay. Subs t ra tes Methods References 1) I n so l ub l e : Nat ive c e l l u l o s e : c o t t o n , undried c o t t o n , d r i ed c o t t o n , dewaxed 2) P h y s i c a l l y modi f ied c e l l u l o s e : a -Ce l l u l o s e from wheat straw Hyd ro ce l l u l o se c o t t o n , swo l len (H 3 P0 4 ) C o l l o i d a l c e l l u l o s e so l (DP. 200-300) Cel lophane C e l l o d e x t r i n Morpholog ica l changes by: M i c roscop i c observat ion E l e c t r on mic roscop ic observat ions Tens i l e s t rength and a l k a l i - s w e l l i n g M i c roscop i c observat ions c e l l u l o s e res idue ( g r a v i m e t r i c a l l y or c o l o r i m e t r i c a l l y ) Reducing sugars T u r b i d i t y and reducing sugars , c e l l u l o s e res idue DP and reducing sugars T u r b i d i t y and DP reduc ing sugars Retardat ion Reducing sugars: r a t i o moles o l i gosaccha r i des Marsh (1957) Po r t e r e t a l . (1960) Reese & G i l l i g a n (1954) Blum and Stah l (1952) H a l l i w e l l (1963) Selby (1961) Grimes et a l . (1957) Norkrans (1950) L i e t a l . (1963) Walseth (1952) G i l l i a a n & Reese (1954) Myers & Northcote (1959) Whitaker (1953) Norkrans (1950) Norkrans & Ranby (1956) Thomas (1956) Whitaker (1956) Table I (continued) Substrates Methods References 3) Soluble: Carboxymettiyl ee l lu lose Methyl cel lu lose Ethyl hydroxy ethyl ce l lu lose Hydroxy ethyl c e l l -ulose 4 ) 01igoglucosides: £-1 -,4-01 i gogl ucos i des MethyJ fl-1,4-gluco-sides 5) Resorufin acetate: Viscosi ty broken bonds/second (viscometrvcal ly) Reducing sugars Viscosi ty Viscosi ty Viscosi ty and reducing sugars Reducing sugars and chromatographic separation Fluorometric Reese et a l . (1950) Whitney e t a l . (1969) Hulme & Strauks (1970) Almin & Eriksson (1967, 1968) Hulme (T971) Toyama & Stiibata (1961) Aitken et a l . (1956) Reese et a l . (1950) Lyr (1959J Joos et a l . (1969) Blum & Stahl (1952) Whitaker e t a l . (1954) Hanstein & Whitaker (1963) Gui lbaul t & Heyn (1967) ro 13 an a lka l ine medium (Festenstein, 1958), and the co lor imet r ic d in i t ro - ! s a l i c y l i c acid (M i l l e r e t a l . ,1960; Reese et a l . ,1950; Mandels & Weber, 1969; Gascoigne & Gascoigne» 1960). Enzyme a c t i v i t y against insoluble substrates has also been estimated from the loss in weight of residual ce l lu lose (Saunders et a l . . , 1948; Blum & S tah l , 1952; Walseth, 1952; H a l l i w e l l , 196] ) , the formation of to ta l soluble carbohydrates ( H a l l i w e l l , 1961), the change in v iscos i ty pf residual ce l lu lose (Walseth, 1952; Reese et a l . , 1957; Selby, 1961), or the increase in reducing sugars (Whitaker, 1953; Thomas, 1956; Myers & Northcote, 1959; Sison et a l . , 1963). MATERIALS AND METHODS I. Chemicals The media const i tuents and other chemicals were of reagent grade. Microcrystal ce l lu lose (Cellex-MX), Biogel P-l50 were purchased from Bio-Rad Laborator ies, carboxymethylcellulose (CMC,7H) from Hercules Incorporated, and.DEAE-Sephadex A-50 was obtained from Pharmacia Uppsala. I I . Culture Media 1 . Basal enrichment medium The basal enrichment medium which is given below was according to Blackburn (1968). Solut ion A, 4.5% K 2HP0 4; Solution B, 4.5% KH 2P0 4; 9.0% (NH 4 ) 2 S0 4 and 9.0% NaCl; Solution C, 0.9% MgS04 and 0.9% CaCl 2 . One ml of each of these solut ions and 0.1 ml of 0.1% resazurin so lu t ion were made up to 90 ml wi th d i s t i l l e d water and autoclaved at 15 lb fo r 14 20 min in screw-capped b o t t l e s . Immediately a f t e r s t e r i l i z a t i o n , the caps were screwed t i g h t and the bot t les were brought to the room temper-ature. Five ml of 1% (W/V) cysteine-HCl ( s t e r i l i z e d by autoclaving) and 5 ml of 10% (W/V) NaHC03 ( f i l t e r e d through a M i l l i po re f i l t e r , 0.45 y size) were added to the basal medium j u s t before inocu la t ion . 2. Sol id media The semi-sol id media, r o l l tubes and plates contained 0.7%, 1.5% and 2.5% agar, respect ive ly . 3. Growth medium, 1 The medium f o r the growth studies consisted of 0.5% (W/V) yeast ext ract in 5 ml of basa,! medium in 16 x 125 mm rubber stoppered tubes. The carbon sources were autoclaved separately at a concentration of 10% (W/V) before addi t ion to the s t e r i l i z e d basal medium at a concen-t r a t i o n of 0.5%. The growth at various temperatures was determined by using the basal medium which was supplemented with 0.5% (W/V) ce l lob iose. The pH range fo r growth was determined by adjust ing the pH of basal medium wi th 3.ON HC1 or NaOH pr io r to the inocu la t ion . The growth e f fec t of 0.01% (W/V) b i o t i n , 0.01% (W/V) casamino acid hydrolyzate, 0.01% (W/V) thiamine and 10% (W/V) glutamine was tested on basal agar plates containing 0.5% (W/V) ce l lob iose. Discs of Whatman No. 1 paper were spaked in the solut ions of b i o t i n , casamino acids, thiamin or glutamine were placed on the surface of plates which were spread with tes t organism. 4. F i l t e r paper medium The f i l t e r paper medium contained 0.5% (W/V) yeast extract in basal 15 medium (5 ml) in which was placed a piece of Whatman No. 1 paper ( 1 x 5 cm) in 18 x 160 mm rubber stoppered tubes. 5. Cellulose medium The cel lu lose medium in a l l experiments, unless otherwise de-f i n e d , consisted of 1% microcrystal cel lu lose and 0.5% (W/V) yeast extract added to the basal medium. These cultures were grown e i ther in magnetically s t i r r e d water-jacketed vessels (100 ml or 500 ml) or in a Fermentation Design Fermentor (14 l i t r e capaci ty, FD, I n c . , Allentown, Pa. ) . The cultures were incubated anaerobically under CO2 at 60°C. The inocula used were from the logari thmic phase cu l tu res , grown in 0.5% (W/V) glucose or 0.5% (W/V) cel lobiose basal medium supplemented with 0.5% yeast ex t rac t . The cultures were examined by a l i g h t microscope fo r contamination before any study was made. I I I . Source of s t ra ins and procedures of i so la t ion of anaerobic c e l l u l o l y t i c thermophil ic bacteria Samples of decayed manure, hay, grass, wood ch ips, sewage sludge and compost were col lected from the campus of The Universi ty of B r i t i s h Columbia and several areas of Vancouver. Each sample was wrapped by Whatman No. 1 f i l t e r paper and put in to a series of tes t tubes (18 x 150 mm) anaerobically at 60°C. A piece of d is integrated paper was inoculated in to 500 ml of basal medium containing 1% cel lu lose in a s t i r r e d water-jacketed vessel at 60°C. Cultures were examined da i ly fo r growth by measuring the i n -crease of ATP, protein and v isua l l y fo r cel lu lose disappearance. Samples 16 taken from the ac t ive ly c e l l u l o l y t i c cul tures were inoculated in to a series of semi-sol id ce l lu lose tubes which were incubated in tempera-ture gradient between 45°C to 75°C. The c e l l u l o l y t i c colonies from the semi-sol id tubes were d i lu ted in to r o l l tubes (Hungate, 1966). IV. C r i t e r i a of pur i t y and maintenance C e l l u l o l y t i c colonies from r o l l tubes were fu r the r pu r i f i ed by repeated a l ternate t ransfer on 0.5% cel lobiose and on 0.5% cel lu lose agar p la tes, This process was repeated twice. The pure isolates were preserved e i ther by freeze^drying in serum or by storage at low temper-ature (-20°C) in ce l lu lose medium plus 10% (W/V) g l yce ro l . Cultures were subcultured monthly from the frozen stock cu l tu res . V. . Cellulase assay The cel lu lase assay was based on the determination of reducing sugar l iberated from cotton or carboxymethylcellulose as the substrates. The estimation of reducing sugar was carr ied out by d i n i t r o s a l i c y l i c acid (DNS) reagent (Fisher and S te in , 1961). One un i t of cel lu lase was defined as the amount of enzyme which would release 1 yg of glucose from cotton or CMC per minute at 60°C. The react ion mixture f o r the cel lu lase assay contained 1 ml of enzyme solut ion and the substrates in 1 ml of 0.1M sodium phosphate bu f fe r , pH 6.5. C x ce l lu lase (e-1,4-glucanase) was assayed using 10 mg CMC and C-| ce l lu lase (required f p r C x to act on native cel lu lose) was assayed using 50 mg of cot ton. Af ter incubation at 60°C (10 min fo r C x 17 and 60 min fo r ) , the reaction was stopped by the addi t ion of 2 ml of DNS reagent. The tubes were placed in a bo i l i ng water bath f o r 5 min and then brought to room temperature. The samples of C-| cel lu lase assays were f i l t e r e d . The absorbance of the color produced was measured at 570 nm using a Bausch and Lomb Spectonic 20 color imeter. The absorbance readings were d i r e c t l y proport ional to the enzyme a c t i v i t y over a l im i ted range and therefore the amount of glucose was calculated from a standard curve. The C v ce l lu lase a c t i v i t y assayed by using an autoanalyzer (Technicon Instruments, Corporat ion). The automated method involved only s l i g h t modi f icat ion of the established DNS method. The volume de-l ivered by each size of tubing f o r each time in terva l was maintained as shown in flow diagram (Figure 1 ) . The experimental samples contain-ing substrate (CMC) were run with appropriate controls (0.IM sodium phosphate buf fer ) which did not contain CMC. A ca l ib ra t ion curve was prepared by p l o t t i n g the peak height {% transmittance) against cel lu lase concentration (units/ml cul ture supernatant). The cel lu lase concentration of unknown samples was determined from the peak height (di f ference between tes t and cont ro l ) by d i rep t comparison with the ca l ib ra t ion curve, VI. Paper chromatography Paper chromatography of enzymatic products of cel lu lase hydro-l ys i s was performed on Whatman No. 1 f i l t e r paper using a solvent system (n-Butanol: pyr id ine: water = 6 : 4 : 3 ) and spray reagent (a lka l ine CELLULASE(Cv ) TIMING END/ 9 m i n . SINGLE MIXER WATER JACKETED DIGESTOR,1x40ft(1-0mm.) m m HEATING BATH 4 0 ' C O l L i 4 m i n . (1-6 mm.) SINGLE MIXER TUBE SlZE(inches) 0-045 S A M P L E Q 0-051 AIR n 0 0 4 5 SUBSTRATE ^ OR CONTROL Q 0 0 5 1 RFAGENT Q 0061 BUFFER , r , ^ W A S T E 4 ^ 0 ° - Q 9 Q F ^ Q W C » PROPORTIONING PUMP E L L COLORIMETER RECORDER 5875 mu 15 mm Tubular f/c Fig. 1 . Flow diagram in Technicon autoanalyzer. 19 AgNOg). The enzyme products were prepared by reacting cul ture super-natant and cel lu lose fo r 24 hr at 60°C. The degradation products from cel lu lose cul ture were obtained at 2 hr in terva ls and i d e n t i f i e d by using D-glucose and D-cellobiose as the standard. V I I . ATP assay ATP was assayed by the rmethod of Stanley and Williams (1969). V I I I . Determination of protein Protein was determined by the method of Lowry et a l . (1951). Protein from ce l l s was extracted in 1.0% sodium dodecyl sulphate at 100°C fo r 15 min. IX. Determination of to ta l cel lu lose The to ta l cel lu lose content in the cul ture residue was deter-mined by the anthrone method (Viles and Silverman, 1949) or by the gravimetric method ( H a l l i w e l l , 1958). X. Cell f rac t iona t ion Cultures were centr i fuged at 10,000 x g fo r 15 min and the supernatant was assayed fo r ex t race l lu la r ce l lu lase. The pe l l e t obtained from the above cent r i fugat ion was washed once with 0.85% NaCl, twice with 0.05M sodium phosphate bu f fe r , pH 7.0 and f i n a l l y the pe l l e t was sonicated at 10,000 cycles fo r 90 sees at maximum power in a Biosonik (Bronwil l S c i e n t i f i c , Rochester, N.Y.). The sonicated mixture was 20 centr i fuged at 10,000 x g fo r 15 min. The supernatant was assayed fo r i n t r a c e l l u l a r ce l lu lase . RESULTS I . I so la t ion and character izat ion of anaerobic c e l l u l o l y t i c thermophil ic bacteria T. I so la t ion of pure cultures The i so la t i on procedure is described in methods. Zones of c e l l u -lose c lear ing were observed w i th in a week when enrichments from the manure or compost were inoculated in to semi-sol id cel lu lose agar medium incubated at 55°C to 60°C (Figure 2 a and b ) . The c e l l u l o l y t i c zones were seen in the low d i l u t i o n r o l l tubes a f te r 3 days (Figure 2 c ) , and by the 7th day these zones became v i s i b l e in the high d i l u t i o n tubes. When these colonies were inoculated onto f i l t e r paper medium, the f i l t e r paper was dis integrated considerably in less than 2 days. 2. C r i t e r i a of pur i t y and maintenance The method of mul t ip le t rans fe rs , as described in methods, should have eliminated nonce l lu lo l y t i c bacter ia l contaminants. The isolates s t i l l retained the i r a b i l i t y to degrade ce l lu lose . Two c e l l u l o l y t i c s t ra ins were i so la ted , s t r a i n M-7 from manure (Figure 3) and s t r a i n C-19 from compost. Ei ther iso la te when inoculated in to f i l t e r paper medium dis integrated the paper in 24 hours. Cultures were v iable a f te r freeze-drying or by storage at -20°C, without any Toss of c e l l u l o l y t i c a c t i v i t y over a period of 2 years. a b c F ig . 2. Iso la t ion of anaerobic c e l l u l o l y t i c thermophil ic bacter ia . a) Semi-solid cel lu lose agar tube showing cel lu lose degradation w i th in a week at 55°C to 60°C. b) Complete cel lu lose disappearance wi th in 4 weeks between 51°C to 64°C. c) C e l l u l o l y t i c colonies developed in low d i l u t i o n tube by 3rd day. Fig. 3. Pure c e l l u l o l y t i c colonies of s t ra in M-7 on cel lu lose agar plate a f te r 5 days. 23 3. Character ist ics of isolates a) Morphological character is t ics Both s t ra ins were gram-negative, s t ra igh t or s l i g h t l y curved rods. Stra in M-7 and C-19 ce l l s were about 0.6 ym (wide) by 4.0 ym (long) and 0.3 ym by 4.5 ym, respect ively (Figure 4 a and b ) . The iso-lates produced endospores which were oval and occupied a terminal posi -t i o n . However, spores are rare ly produced in young cul ture on l i q u i d media. M o t i l i t y was not observed. b) Cultural character is t ics Both isolates formed minute, co lo r less , c i r cu la r subsurface colonies. The colonies were surrounded by a clear zone of cel lu lose degradation and often by yel lowish pigments on ce l lu lose . c) Growth character is t ics Stra in M-7 grew opt imal ly from 58°C to 63°C and s t r a i n C-19 from 50°C to 68°C. The optimum pH was 6.0-6.5 fo r s t r a i n M-7 and 7.5 fo r s t ra in C-19. Neither s t ra in grew under aerobic condi t ions. The mean generation t imes, in a r i ch medium, f o r s t ra in M-7 and s t ra in C-19 were 35 and 25 minutes, respect ively (Figure 5 ) . d) Nu t r i t i on Both isolates grew well on cel lu lose or cel lobiose media con-ta in ing 0.5% yeast ex t rac t . Stra in M-7 grew well on ce l lob iose, glucose, starch and s t ra in C-19 grew only on cel lobiose and s tarch. Rumen f l u i d was not required and did not st imulate growth. B i o t i n , thiamine, 24 (a) (b) Fig. 4. Phase contrast photomicrograph of s t ra in M-7(a) and s t ra in C-19(b). The ce l l s in the photo were from log phase cul ture (0.3 0D660) grown on 0.5% cel lobiose supplemented with 0.5% yeast extract and 0.5% t ryptose. Mag x 3000. G r o wth(hours ) Fig . 5. Growth curve fo r s t ra in M-7 and s t ra in C-19. Both strains were grown in 0.5% cel lobiose basal medium containing 0.5% yeast extract and 0.5% tryptose at 60°C. . 2 6 casamino acids and glutamine stimulated growth of s t ra in M-7. B i o t i n , thiamine and glutamine stimulated growth of s t r a i n C-19. Stra in M-7 resembled s t ra in C-19 in the range of carbon u t i l i z a t i o n except that s t ra in M-7 used glucose, ra f f i nose , and inos i to l but did not use i n u l i n (Table I I ) . There is a marked s i m i l a r i t y between s t ra in M-7 and Clostridium thermocenulaseum (Enebo, 1951). Stra in M-7 is ten ta t i ve ly assigned th is name. I I . Cultural conditions fo r cel lu lase pro-duction by C_. thermocellulaseum M-7 1 . Local izat ion of cel lu lase in C_. thermocellulaseum M-7 As shown in Table I I I , most of the C, and C cel lu lase a c t i v i t i e s were found in the ex t race l lu la r f r a c t i o n . However, a small f rac t i on of C a c t i v i t y was also detected in the i n t r a c e l l u l a r f r a c t i o n , x J 2. The e f fec t of complex nitrogenous compounds on growth and cel lu lase (C ) production by C_. thermocellulaseum M-7 C_. thermocellulaseum M-7 produced ex t race l lu la r cel lu lase in the basal medium containing cel lu lose as the sole carbon source. The addi-t ion of yeast extract stimulated the growth and C x ce l lu lase a c t i v i t y . The optimum concentration of yeast extract was 0.5%. Over th is concen-t ra t i on there was an i n h i b i t i o n of growth and enzyme production (Table IV ) . Some other complex nitrogenous compounds such as t ryp tose, proteose peptone, and casamino acids, were also t r i e d at various concen-t ra t ions replacing yeast ex t rac t . Their e f fec t was the same as yeast ex t rac t . 27 Table I I . Comparison of carbohydrate fermentat ion. Stra in M-7 and s t ra in C-19 were grown fo r 2 days in basal medium containing a single carbon source (0.5%) as described in methods. The resul ts are the mean of three separate experiments. (NT, not tested; - , negative growth; +, pos i t ive growth with gas). Carbohydrates Stra in Stra in C. thermo- C. thermo-M-7 C-19 cellulaseum cel lum b Monosaccharides Arabinose + + + + Fructose + + + -Galactose + + - -Glucose + - + -Mannose + + + -Rhamnose + + - NT Ribose + + NT NT Xylose + + + + Disaccharides Cellobiose + + + + Lactose + + - -Maitose + + + -Melibiose - - - -Sucrose + + - -Trisaccharides Trehalose + + - -Raffinose + - - NT Melezitose - - NT NT Polysacchrides Starch + + - -Glycogen + + NT NT Inu l in - + NT -Dextrin + + NT -Sugar alcohol Dulc i to l - - - -Glycerol - - - -Inoc i to l + - NT -Mannitol + + - -Sorbi tol + + - -Substituted sugar Sa l ic in + + NT -aCarbon u t i l i z a t i o n by Enebo (1951). ^Carbon u t i l i z a t i o n by McBee (1948). 28 Table I I I . Local izat ion of cel lu lase in C_. thermocellulaseum M-7. C_. thermocellulaseum M-7 was grown on cel lu lose medium and was f ract ionated as described in methods. Culture Ext race l lu lar I n t r a c e l l u l a r day Cellulase units/ml cel lu lase units/ml C l C x C l C x 0 0 0 0 0 1 0.7 1 .8 0 0 2 3.0 29.0 0 2.4 3 5.5 49.8 0 2.0 4 5.0 48.5 0 0 5 5.0 48.0 0 0 3. The e f fec t of cel lu lose concentration on growth and cel lu lase (C x ) production by C_. thermocellulaseum M-7 Various concentrations of cel lu lose added to the growth medium showed a marked e f fec t on the growth (Table V) . C cel lu lase a c t i v i t y A was maximum at 1% cel lu lose concentration a f te r 72 hr of incubation and the ce l l growth reached to maximum at about 3% cel lu lose concentration a f te r 72 hr . There was a s ign i f i can t drop in the pH of the cul ture medium at higher cel lu lose concentrations. 4 . The e f fec t of inoculum size on growth and cel lu lase production by C_. thermocellulaseum M-7 The production of cel lu lase increased with time of incubation up to 3 days. A 0.5% inoculum was s u f f i c i e n t to give maximum ce l l 29 Table IV. The e f fec t of yeast extract on growth and cel lu lase (C x ) production of C. thermocellulaseum M-7. C_. thermocel!uTaseum M-7 was grown on f i l t e r paper medium as described in methods, supplemented with various con-centrations of yeast ex t rac t . Incubation was fo r 3 days at 60°C and the cul ture supernatant was used f o r C x cel lu lase assay. The resul ts are the mean of three sep-arate experiments. Yeast (%) C x a c t i v i t y Cell protein Specif ic a c t i v i t y extract (uni ts /ml) (mg/ml) (units/mg ce l l protein) 0 6.2 0.34 18.0 0.1 9.5 0.38 25.0 0.2 14.6 0.39 37.4 0.3 16.2 0.47 35.5 0.4 19.6 0.81 24.2 0.5 22.4 0.92 24.3 0.6 12.3 0.70 17.6 0.7 11.2 0.64 18.0 0.8 9.0 0.51 18.0 30 Table V. The e f fec t of cel lu lose concentration on growth and cel lu lase (Cx) production by C_. thermocellulaseum M-7. C_. thermocellulaseum M-7 was grown on ce l lu lose , as described in methods, fo r 3 days at 60°C and samples were analyzed d a i l y . Culture supernatant was assayed fo r C x a c t i v i t y , the ce l l pe l l e t fo r p ro te in . Concentration of cel lu lose {%) Hours Cx a c t i v i t y (un i ts /ml) Cell protein (mg/ml) pH of the supernatant 0.5 0 0.04 6.7 1.0 0 0 0.05 6.7 2.0 0 0.05 6.6 3.0 0 0.06 6.7 0.5 5.4 0.2 6.5 1.0 24 4.5 1 .8 6.2 2.0 3.3 1 .8 6.2 3.0 3.0 0.4 6.4 0.5 30.3 2.2 5.9 1.0 48 27.0 2.8 5.3 2.0 11.7 2.4 5.2 3.0 14.4 1 .9 5.4 0.5 29.4 1 .6 5.8 1 .0 72 33.0 2.9 5.2 2.0 22.5 2.9 4.7 3.0 21 .3 3.6 4.9 31 growth and cel lu lase in 72 hr (Figure 6 ) . The lag in growth and c e l l u -lase production where a 2.0% inoculum was used, cannot be explained. 5. The e f fec t of carbohydrates on growth and cel lu lase (C x ) production by C_. thermo- cellulaseum M-7 C_. thermocellulaseum M-7 grew readi ly on many carbohydrates as the carbon source with some exceptions (Table V I ) . The data in Table VI demonstrates that C cel lu lase was produced only in the presence of A cel lu lose and CMC. C cel lu lase was not produced when s t r a i n M-7 was A grown on other carbohydrates. 6. The e f fec t of various concentrations of cel lobiose and glucose on C x production by C_. thermocellulaseum M-7 The addi t ion of increasing concentrations of e i ther cel lobiose or glucose to the cel lu lose cul ture medium of C_. thermocellulaseum M-7 resulted in increased i n h i b i t i o n of cel lu lase production and par t i a l i n h i b i t i o n of growth. As shown in Table V I I , there was no cel lu lase a c t i v i t y detected in cul ture supernatants from cultures containing e i ther 0.3% cel lobiose or 0.4% glucose. 7. The e f fec t of Tween 80 on the production of cel lu lase (C x ) and growth by C_. thermo- cellulaseum M-7 Surfactants have been observed to increase y ie lds of cel lu lase (Reese and Maguire, 1971). The addi t ion of Tween 80 to the cel lu lose cultures resulted in marked decrease in cel lu lase production and growth of C. thermocellulaseum M-7 (Table V I I I ) . 0 2 4 4 8 7 2 9 6 0 2 4 4 8 7 2 9 6 i g . 6. The e f fec t of inoculum size on growth and cel lu lase production of C_. thermocellulaseum M-7. C_. thermocellulaseum M-7 was grown on cel lu lose medium, as described in methods, fo r 4 days at 60°C and samples were assayed d a i l y . Culture supernatant was the source of C x a c t i v i t y and the ce l l p e l l e t , of p ro te in . When the absorbance of the cul ture at 660 nm was 0.3 (4x10^ c e l l s / m l ) , the d i f f e ren t inocula were prepared. ( O — O 1 l o o p , # — # 0 . 2 5 % , A — 0.50%,O • 1.0%, • • 2.0%). CulturcChr.) Table V I . The e f fec t of sugars on growth of C_. thermocel lulaseum M-7 and cel lu lase (C x ) production. C_. thermocellulaseum was grown in basal medium contain-in g"TJT5T3?e^sT^eTtTact and 0.5% of one of the fo l lowing sugars. A f te r 2 days of incubation at 60°C, the cul ture supernatant was assayed fo r C x a c t i v i t y . Growth was determined by fo l lowing the opt ical density at 660 nm. The resul ts are the mean of three separate experiments. - indicates no growth. Sugars Cx a c t i v i t y Cell growth (0.5%) (un i ts /ml) (0D660) Monosaccharides Arabinose 0 0.73 Fructose 0 0.44 Galactose 0 0.70 Glucose 0 0.52 Mannose 0 0.05 Rhamnose 0 0.67 Ribose 0 0.45 Xylose 0 0.53 Disaccharides Cellobiose 0 0.63 Lactose 0 0.64 Maltose 0 0.53 Melibiose , -Sucrose 0 0.80 Trisaccharides Trehalose 0 0.32 Raffinose 0 0.04 Melezitose - -Polysaccharides Starch 0 0.49 Glycogen 0 0.67 Inu l in - -Dextrin 0 0.31 Carboxymethyl cel lu lose 10 0.20 Cellulose 34 * Sugar alcohols Dulc i to l - -Glycerol -0.21 Inoc i to l 0 Mannitol 0 0.11 Sorbitol 0 0.63 Glycosides and subst i tuted sugar 0.43 Glucosamine 0 Sal ic in 0 0.50 2.3 mg ce l l p ro te in /ml . 34 Table V I I . The e f fec t of various concentrations of cel lobiose and glucose on cel lu lase (Cx) production and growth by C_. thermocellulaseum M-7. C. thermocellulaseum M-7 was grown on basal f i l t e r paper medium, as described in methods, wi th various concentra-t ions of cel lobiose and glucose. A f te r 6 days, the cul ture supernatant was assayed fo r C x a c t i v i t y and the ce l l p e l l e t , fo r p ro te in . Concentration Cell Specif ic of cel lobiose C x a c t i v i t y protein a c t i v i t y or glucose [%) (un i ts /ml) (mg/ml) (units/mg) Cellobiose 0 0.05 0.10 0.20 0.30 0.40 0.50 Glucose 0 0.05 0.10 0.20 0.30 0.40 0.50 32.0 2.88 11 .0 26.0 2.81 9.2 21.6 2.38 9.1 17.0 1.98 8.6 0 0.74 0 0 1 .49 0 0 1 .63 0 32.5 2.36 13.7 30.6 2.46 12.4 20.0 2.30 8.7 19.6 2.57 7.6 21 .0 2.30 8.2 0 1 .25 0 0 1 .86 0 35 Table V I I I . The e f fec t of the addi t ion of Tween 80 on the y i e l d of ce l lu lase (C%) and growth by C_. thermocellulaseum M-7. C_. thermocellulaseum M-7 was grown on f i l t e r paper medium as described in methods with various concen-t ra t ions of Tween 80 f o r 3 days at 60°C. The c u l -ture supernatant was the source of C x a c t i v i t y and the ce l l p e l l e t , f o r p ro te in . Concentration of Tween.80.(%) Cx a c t i v i t y (un i ts /ml) Cell prote in (mg/ml) 0 25.2 0.78 0.1 6.8 0.29 0.2 3.6 0.23 0.4 2.4 0.18 0.6 0 0 0.8 0 0 1.0 0 0 8. The k inet ics of cel lu lase production by C_. thermocellulaseum Mr7 The resul ts in Figure 7 showed that maximal a c t i v i t i e s of C-| and C were detected a f te r 48 hr incubation and tend to decrease s l i g h t l y A during fu r ther incubat ion. The C-j:Cx r a t i o at 48 hr of c u l t i v a t i o n was 1:7. The pattern of cel lu lase production was coincident wi th the bacter ia l growth except that i t lagged s l i g h t l y behind i t . Cellulose disappearance began immediately a f te r inoculat ion of C_. thermocel lulaseum M-7 and approximately 50% of the cel lu lose was hydrolyzed before c e l l u -lase became detectable in the growth medium. The mean generation time was about 2 hr in ce l lu lose cu l tu re . Cu l tu rc (hours ) Fig. 7 . The kinet ics of cel lu lase production by C_. thermocellulaseum M - 7 . C_. thermocellulaseum M - 7 was grown in 5 0 0 ml of ce l lu lose medium as des-cribed in methods for 6 0 hrs at 6 0 ° C . Samples were taken at 2 hr in terva ls and were assayed. ( O — O ce l l p r o t e i n , 0 0 ce l l u lose , p — H | C I ^ act iv i ty ,A—-A Cx a c t i v i t y ) . <=n 37 I I I . Cellulase assay methods As shown in Figure 8, r e l a t i v e l y short times of incubation were necessary as the assay was not l inear fo r e i ther C-j or Cx a c t i v i t i e s when an ODgyg greater than 0.15 was produced. This could have been avoided by using higher concentration of CMC than the f i n a l concentration of 0.5% that was used in the standard assay. Both C-j and C x a c t i v i t i e s were found to be d i r e c t l y proport ional to the enzyme concentrations in the assays up to 0.5 ml of the cul ture supernatant which usually contained 0.45 mg of protein per ml (Figure 9 ) . For opt ica l density increments of greater than 0.15 (28 C un i ts ) a c t i v i t y was read from a standard A curve. Both C-j and C x ce l lu lase a c t i v i t i e s were maximum at 67°C as shown in Figure 10. C x a c t i v i t y decreased more rapid ly than C-j at tem-peratures in excess of 67°C and below 60°C. Both enzymes demonstrated very l i t t l e a c t i v i t y at 45°C. In Figure 1 1 , cel lu lase a c t i v i t y was seen to vary wi th pH. Both C-| and C x a c t i v i t i e s showed optima near pH 6.5 and have almost the same pH spectrum. C-j a c t i v i t y decreased more rapid ly than C at pH 8.0. The e f fec t of increasing the CMC concentra-A t ion in the C cel lu lase assay is shown in Figure 12. There was a A sharp increase in C cel lu lase a c t i v i t y wi th increasing concentrations A of CMC and the enzyme was not saturated with substrate w i th in the range used. There was d i f f i c u l t y in handling the CMC solut ions at high con-centrat ions as they were very viscous. Glucose and cel lobiose were the only soluble products l iberated by the cel lu lase from ce l lu lose , as de-tected by paper chromatography. Three un ident i f ied spots were detected on chromatograms of cu l ture supernatants. Both these sugars are 38 0 5 0 20 4 0 60 8 0 100 120 I n c u b a t i o n t i m e (min.) Fig. 8. The e f fec t of incubation, time on cel lu lase a c t i v i t y . Cellulase a c t i v i t i e s were assayed as described in methods. 1 ml of cul ture supernatant of C_. thermocellulaseum M-7 contained 0.45 mg pro te in . ( # — % , C] a c t i v i t y ; 0 — O > C x a c t i v i t y ) . 39 E c O N If) d d 0-25 r 0-20 -0-15 -0 - 1 0 -0 0 5 0 0 1 1 1 1 0-2 0-4 0-6 0-8 1-0 I F i l t r a t c ( m l ) i i i i i i 0 14 2 8 42 5 6 I C x u n i t s i i i i i i 0 2 0 4 0 6 0 8 0 C| u n i t s F i g . 9. The e f f e c t o f enzyme concen t ra t i on on c e l l u l a s e a c t i v i t y . Ce l l u lase a c t i v i t i e s were assayed as descr ibed i n methods. Vary ing q u a n t i t i e s o f c u l t u r e supernatant o f C_. thermo-ce l lu laseum M-7 were added to assay m i x t u r e s . ( # # , C] a c t i v i t y ; 0 — 0 > C x a c t i v i t y ) . TGmperaturG°C Fig. 10. The e f fec t of temperature on cel lu lase a c t i v i t y . 0.1 ml (0.045 mg protein) of cul ture supernatant of C_. thermocel lulaseum M-7 was added to assay mixtures which were preincubated in a tempera-ture gradient. Incubation was fo r 30 min and 120 min fo r C] and C x a c t i v i t i e s , respect ive ly . ( # — # , C-j a c t i v i t y ; 0 O ' C x a c t i v i t y ) . 4 0 5 0 6 0 7 0 8 0 P H Fig. 11. The e f fec t of pH on cel lu lase a c t i v i t y . Cellulase a c t i v i t i e s were assayed as described in methods. 1 ml of cul ture supernatant of C_. thermocellulaseum M-7 contained 0.45 mg pro te in . 0.1 M sodium c i t r a t e buf fer replaced 0.1 M sodium phosphate at pH values below 6.0. ( • — 0 , C] a c t i v i t y ; Q — O ' a c t i v i t y ) . 42 0 - 8 0 1 0 2 0 3 0 4 0 Final CMC(%>) in assay digest Fig. 12. The e f fec t of CMC concentration of C x ce l lu lase a c t i v i t y . Cellulase a c t i v i t i e s were assayed as described in methods. 1 ml (0.45 mg protein) of cul ture supernatant of C_. thermocel 1ulaseum M-7 was used to assay C x a c t i v i t y in the presence of d i f f e r e n t amounts of CMC. ( O — O • C x a c t i v i t y ) . u t i l i z e d by s t r a i n M-7 and 0.4% glucose or 0.3% cel lobiose completely inh ib i ted the act ion of cel lu lase on f i l t e r paper (Table I X ) . The automated assay proved to be a convenient method fo r the determination of C cel lu lase a c t i v i t y in a large number of samples, A as when f ract ions were col lected in the p u r i f i c a t i o n studies. Figure 13 a shows the re la t ionsh ip between peak heights (% transmission) fo r d i l u t i o n of cul ture supernatant of known cel lu lase a c t i v i t y . The c a l i -brat ion curve from these peak heights is shown in Figure 13 b. IV. Concentration and p u r i f i c a t i o n of cel lu lase from cul ture supernatant of C. thermo- cellulaseum M-7 1 . Concentration of cel lu lase The fo l lowing methods fo r cel lu lase concentration were examined before a p u r i f i c a t i o n was attempted: a) Ammonium sulphate p rec ip i ta t i on Ammonium sulphate was added to 40 ml of cul ture supernatant (36.4 C cel lu lase u n i t s / m l ) . Precipi tates were col lected a f t e r 60%, A 70%, 80%, and 90% saturat ion by ammonium sulphate. Those f ract ions were dissolved in 5.0 ml of 0.1M sodium phosphate bu f fe r , pH 6.5. No cel lu lase a c t i v i t y was detected in these f ract ions because the presence of ammonium sulphate was la te r found to in te r fe re wi th the measurement of reducing sugars by d i n i t r o s a l i c y l i c acid method. This e f fec t became clear when the ammonium sulphate was removed by overnight d ia lys is at 4°C against 0.05M sodium phosphate bu f fe r , pH 6.5 and about 20% of the 44 Table IX. The e f fec t of glucose and cel lobiose concentration on ce l lu lase a c t i v i t y . Cellulase a c t i v i t y was assayed on 1% f i l t e r paper f o r 60 minutes. 1 ml of cul ture supernatant con-ta in ing 0.45 mg protein was added to the reaction mixture in varying concentrat ion. Sugar Glucose Cellobiose {%) O D ( A ) % of control O D ( A ) % of control 0 0.30 100 0.29 100 0.05 0.32 103 0.28 95 0.10 0.29 99 0.27 96 0.20 0.31 101 0.15 51 0.30 0.19 62 0 0 0.40 0 0 0 0 0.50 0 0 0 0 0 0-2 04 0-6 08 10 0-2 0 4 0 6 0 8 10 Di lu t ion of culture supernatant Test B lank Fig. 13a. Typical ca l ib ra t ion curve in automated C x ce l lu lase assay. The automated C x cel lu lase a c t i v i t y was assayed as described in methods. Di lut ions of cul ture supernatant (un i ts /ml) containing CMC were run against. the cont ro ls . 46 I -o o O) a o CL 20 15 -10 -0 0 0-2 0 4 0-6 0-8 F i l t r a t e ( m l ) 10 0 14 28 C x u n i t s 42 5 6 Fig. 13b. Typical ca l ib ra t ion curve in automated C x ce l lu lase assay. The enzyme units were determined from the peak height (di f ference between test and cont ro l ) by d i rec t comparison * with the ca l ib ra t ion curve. 47 or ig ina l C a c t i v i t y could be recovered. No cel lu lase could be detected A in the dialysed supernatants. b) ZnCl 2 p rec ip i ta t i on (Ensign and Wolfe, 1966) The resul ts of a typ ica l experiment are shown in Table X. The cel lu lase a c t i v i t y of the cul ture supernatant was pu r i f i ed 3.5 f o l d , the recovery was only 20%. Since ZnC^ was found to i n h i b i t ce l lu lase a c t i v i t y completely at 0.4% (Table X I ) , i t was necessary to remove ZnCl2 by d ia lys is before assaying the enzyme. c) Adsorption on cel lu lose There was complete adsorption of C x and C-| cel lu lase on cel lu lose columns but unfortunately i t could not a l l be recovered (Table X I I ) . The C-j cel lu lase was not released by any of the treatments. Only 34% of C a c t i v i t y was recovered in the water and buf fer e f f l u e n t s . There A was also problems in obtaining cel lu lase on cel lu lose as reducing sugars were l iberated even at 4°C. This gave high blank values in the cel lu lase assay. 2. Pu r i f i ca t i on of cel lu lase The Tris-EDTA and c i t r a t e extracts by a ZnCl 2 p rec ip i ta te from 5 l i t r e s of cul ture supernatant were pooled (Table X I I I ) . This pooled material had a low 260-absorbance which indicated a low nucleic acid content. Concentration of the ext ract using a Diaf lo XM-50 membrane (exclusion 50,000 molecular weight) resulted in some loss of C A a c t i v i t y , none of which could be detected in the f i l t r a t e . The 48 Table X. Zinc chlor ide p rec ip i ta t ion of cel lu lase from the cul ture supernatant of C_. thermocellulaseum M-7. 2 ml of a 10% ZnCl2 solut ion was added dropwise to the cul ture supernatant (50 m l , 37 Cx uni ts /ml) with vigorous s t i r r i n g . The prec ip i ta te was col lected by cent r i fuga-t ion at 10,000 xg fo r 15 min and was sonicated fo r 20 sec at 10,000 cycles in 10 ml of 0.05 M Tr is buf fer (pH 8.0) containing 0.01 M sodium EDTA. This suspension was centr i fuged as above and the prec ip i ta te was sonicated again in 10 ml of 0.1 M c i t r a t e buf fer (pH 5 .0 ) . Cel lu-lase a c t i v i t y was assayed as described in methods. Fraction Total a c t i v i t y (C x un i ts ) Total protein (mg) Specif ic a c t i v i t y (units/mg) Fold p u r i f i -cat ion Yield of a c t i v i t y (%) Culture supernatant 1850 21.0 88 1 100 ZnCl2 supernatant 420 3.75 112 1.3 23 Tris-EDTA extract 263 1.63 162 1.7 14 Ci t ra te extract 378 1 .25 302 3.5 20 concentrated ext ract was chromatographed on DEAE-Sephandex A-50 (Figure 14). C cel lu lase a c t i v i t y was detected in three peaks whose combined X a c t i v i t y represented 9.7% of that in s ta r t i ng mater ia l . No C-| cel lu lase was detected in any f r a c t i o n . A l l C cel lu lase a c t i v i t y in the pooled A peaks was los t when run through Biogel P-150. The p u r i f i c a t i o n was not successful as the recovery was low (9.7%) and the p u r i f i c a t i o n i n s i g -n i f i c a n t (1.5X) up to the point at which C cel lu lase was f i n a l l y l o s t . A In a separate experiment the Cv cel lulases in the cul ture A 49 Table X I . The e f fec t of ZnCl 2 on cel lu lase a c t i v i t y . ZnCl2 solut ion was added to react ion mix-tures in varying concentrations. Cellulase a c t i v i t y was assayed as described in methods. Cone, of ZnCl 2 (%) C units/ml A % of Control 0.00 41.0 100 0.05 33.8 82.4 0.10 33.6 82.0 0.20 23.2 57.6 0.40 0 0 0.60 0 0 supernatant were resolved by i soe lec t r i c focusing (Figure 15). The C X cel lu lase a c t i v i t y was found to have four d i f f e r e n t i soe lec t r i c points at pH 4 .5 , 6.3, 6 .8 , and 8.7. Most cel lu lase had a pi of 6.8 and was not located in a f rac t i on high in p ro te in . The to ta l recovery of C X cel lu lase in a l l the f ract ions was 128% with a 1.5-fold p u r i f i c a t i o n . 50 Table X I I . Adsortion and desort ion of ce l lu lase on cel lu lose column. A column (3.0 by 60 cm) containing 50 g of c r y s t a l -l i ne cel lu lose was equi l ibrated wi th 0.05 M sodium phosphate bu f fe r , pH 7.0 and 100 ml of cu l ture supernatant was passed through i t . The adsorbed enzyme was eluted with 200 ml of mineral s a l t medium (pH 6 . 7 ) , 200 ml of d i s t i l l e d water and then 1 M NaCl in 0.05 M sodium phosphate bu f fe r . Fractions (5 ml) was assayed f o r Ci and C x a c t i v i -t ies as described in methods. Fraction Cn to ta l units C to ta l uni ts A Recovery of C x a c t i v i t y (%) Culture supernatant 600 4300 100 mi neral sa l t 0 0 0 water 0 664 15 buf fer 0 800 19 51 Table X I I I . Pu r i f i ca t i on of ce l lu lase from the cul ture supernatant of C_. thermocellulaseum M-7. 10 l i t r e s of 1.0% c rys ta l l i ne cel lu lose medium was inoculated with 100 ml of a log phase cul ture of C. thermocellulaseum M-7 and cu l t i va ted anaero-F i c a l l y fo r 48 hr at 60°C. Af te r c e n t r i f i g a t i o n in Sharpies, 5 l i t e r batches of cu l ture super-natant were adjusted to pH 6.7 with 3N NaOH and cooled to 4°C. 100 ml amounts of a 10% solut ion of ZnCl2 was added. The prec ip i ta te was col lected by cen t r i f uga t i on , homogenized fo r 30 sec in 0.05 M Tr is (pH 8.0) containing 0.01 M sodium EDTA and secondly in 0.1 M c i t r a t e buf fer (pH 5.0) in a Waring Blender. The combined extracts were concen-t rated by Diaf lo u l t r a f i l t r a t i o n using a XM-50 membrane and were applied to a column of DEAE-Sephadex A-50 (3 x 60cm). The f ract ions containing cel lu lase were pooled, concentrated and run through Biogel P-150 (3 x 60cm). Step assay to ta l uni ts (xl03) to ta l protein (mg) spec i f ic a c t i v i ty (units/mg) puri -f i cat ion recovery of a c t i v i t y (%) 1.Culture supernatant C l C x 23.0 235.0 1890 12.0 124.0 1 1 100 100 2.Discard sup. of Zn ppt. C l C x 3.2 59.5 500 6.4 119.0 - 14.0 25.3 3. Tris-EDTA and c i t r a t e wash of Zn ppt C l C X 3.1 100.0 1735 1 .7 62.0 -13.5 42.6 4.XM-50 d ia f l o 6.3 77.5 1170 5.3 66.2 -27.0 33.0 5.DEAE-Sephadex 0 22.8 122.5 0 186.0 1 .5 0 9.7 6.Biogel P-150 0 0 -- -0 0 0 20 4 0 6 0 80 100 120 140 160 F r a c t i o n no. Fig. 14. DEAE-Sephadex chromatography. A 10 ml sample (step 4 , Table X I I I ) was applied to a column (3.0 by 60 cm) of DEAE-Sephadex which had been equi l ibrated with 0.05 M sodium phosphate bu f fe r , pH 7.0. The cel lu lase was eluted by a l inear NaCl gradient from 0.0 to 1.0 M and the f ract ions of 6.5 ml were co l lec ted . The cel lu lase a c t i v i t y ( • ) , E28O (so l id l i ne ) and NaCl concentration ( ) were measured. F r a c t i o n no. Fig. 15. Separation of cel lu lase (C x) of cul ture supernatant of C_. thermocel!ulaseum M-7 by i soe lec t r i c focusing. 30 ml of dialysed cul ture supernatant containing 35 Cx uni ts and 0.35 mg protein/m! was added to an LKB-8100 electrofocusing column with LKB Ampho-l ine car r ie r ampholytes to give a pH-gradient from 3.0 to 10.0 a f te r 1,000 V fo r 48 hr . 2 ml f ract ions were col lected and C x ce l lu lase a c t i v i t y ( o ) , the OD28O (•) a n d the pH (so l id l i ne ) were measured. DISCUSSION 54 Though only two isolates were selected fo r study, they were representative of s imi lar thermophil ic c e l l u l o l y t i c bacter ia which were isolated by enrichment cu l ture from a wide var ie ty of sources. The r o l l tube method of Hungate (1966) was best fo r the i so la t i on of pure cultures as c e l l u l o l y t i c colonies developed rap id ly and could be easi ly recognized. I t was more d i f f i c u l t to maintain anaerobic conditions using plates in anaerobic j a r s , some exposure to oxygen was inev i tab le . I t was more d i f f i c u l t to recognize c e l l u l o l y t i c colonies on plates and longer incubation was necessary. Neither iso la te was, however, pa r t i cu -l a r l y sensi t ive to exposure to the atmosphere and did not require a very low redox potent ia l fo r growth. A l l s t ra ins were isolated in the basal medium plus ce l lu lose. No organic nitrogen source was required although these great ly stimu-lated growth. Aerobic thermophil ic c e l l u l o l y t i c bacter ia appear to have an obl igate requirement fo r organic nitrogen (Stutzenberger, 1971; Bellamy, 1969). I t was important to ensure that isolates were uncontaminated as other workers found d i f f i c u l t y in removing contaminating bacter ia l species (McBee, 1948; Enebo, 1951; Hungate, 1966). There has been a h is tory of ce l lu lose hydrolysis being more e f f i c i e n t l y performed by mixed populations (Enebo, 1951; Hofstein et a l . , 1971). In order to ensure that non-ce l lu lo l y t i c contaminants be e l iminated, the cultures were plated a l te rna te ly on cel lobiose and ce l lu lose. No contaminants 55 were observed and the st ra ins l os t none of the i r c e l l u l o l y t i c a b i l i t y . The most rapid growth was attained in enriched media. Mean generation times of 35 and 25 minutes were calculated fo r s t ra ins M-7 and C-19 respect ive ly , in yeast , tryptose cel lobiose medium. I t was d i f f i c u l t to accurately measure bacter ia l growth in cel lu lose-contain ing media, due to the opacity of the ce l lu lose . Growth was measured by ATP production or by bacter ia l protein production. These two methods gave s imi la r resul ts the former being more sens i t i ve , the l a t t e r being more convenient f o r denser cu l tu res . Measurement of growth in the cel lu lose medium, by protein production, gave a mean generation time of 2 hr fo r s t ra in M-7. F i l t e r paper was observed to completely disappear in 2 days, a contrast with 7 to 14 days noted fo r anaerobic thermophi1es, by McBee (1948) and Enebo (1951). Stra in M-7 resembled s t r a i n C-19 in the range of substrates which i t fermented except that i t used glucose, ra f f i nose , and inos i to l but did not use i n u l i n . Both s t ra ins appeared to possess wider fermentative powers than e i ther C_. thermocellulaseum (Enebo, 1951) or C_. thermocel 1 urn (McBee, 1948). Stra in M-7, however, is s u f f i c i e n t l y s imi la r to C_. thermocel lulaseum to be assigned to that species. Clostr idium thermocellulaseum M-7 produced an ex t race l lu la r cel lu lase when grown on ce l lu lose . Almost no C a c t i v i t y was l iberated on the d is in tegra t ion of the c e l l s . The growth requirements of C_. thermo- cellulaseum M-7 are simple, growth and C x ce l lu lase were produced in the basal cel lu lose medium from a small inoculum (Table I V ) . Both growth and cel lu lase production were stimulated by the addi t ion of 56 yeast extract up to 0.5% although the highest d i f f e r e n t i a l rate of pro-duction was at 0.2% yeast ex t rac t . High concentrations of ce l lu lose gave high y ie lds of bacteria but apparently lower quant i t ies of ce l lu lase. The l a t t e r observation may have been due to adsorption of cel lu lase to undegraded ce l lu lose . Other c e l l u l o l y t i c microorganisms have been observed to be inh ib i ted by 1.0% cel lu lose (McBee, 1948; Hungate, 1966; Stutzenberger, 1971) which was optimum fo r s t ra in M-7. Cellulose was required in the growth medium fo r cel lu lase production although good growth was obtained on other carbohydrates. The role of cel lu lose as an inducer of fungal cel lulases has been demonstrated by many workers but in addi t ion some oligosaccharides such as cel lobiose (Mandels and Reese, 1960), sophorose (Mandels, Parrish and Reese, 1962; Nisizawa et a l . , 1971) and lactose (Mandels and Reese, 1957) allow fo r some cel lu lase production. I t has been suggested (Hulme and Strauks, 1970) that ce l lu lase is not produced on easi ly metabolized carbon sources and that i t is only produced on these carbon sources as i t is slowly metabolized and does not cause catabol i te repression. Cellulase was not detected in cel lu lose media to which c e l l o -biose or glucose was added. I t i s assumed that th is was a repression of cel lu lase synthesis rather than an i n h i b i t i o n of ce l lu lase funct ion although such an i n h i b i t i o n can be demonstrated by s imi la r concentrations of glucose (0.3%) and cel lobiose (0.2%). Gupta and Heale (1970) found that the C cel lu lase production by V e r t i c i l l i u m albo-atrum in CMC media was inh ib i ted by a large number of sugars. They observed that low growth rates were related to increased cel lu lase production. 57 The addi t ion of Tween 80 (0.1%) inh ib i ted the growth of s t r a i n M-7 and decreased the y i e l d of ce l lu lase . C_. thermocellulaseum M-7 d i f f e r s from Trichoderma v i r i d e in th is respect as Tween 80 (0.2%) increased cel lu lase production in a cel lobiose medium (Reese and Maguire, 1971) or a cel lu lose medium (Reese and Maguire, 1969). Cellulose disappearance from cul ture media (Figure 7) showed that ce l lu lase was produced e a r l i e r than i t could be detected in the cul ture medium. Free cel lu lase was not found u n t i l 50% of the cel lu lose had been hydrolysed. I t is assumed that the cel lu lase was bound to undegraded cel lu lose although i t is possible that i t may have been c e l l -bound. The adsorption of ce l lu lase to cel lu lose has been observed (Hungate, 1966; Norkrans, 1967). The C-| and C x ce l lu lase a c t i v i t i e s were produced at the same rate and the ra t ios remained at approximately 1:7. This could indicate that one enzyme was responsible fo r both a c t i v i t i e s . Subsequent data from a c t i v i t y spectra and p u r i f i c a t i o n to some extent substantiated th is observation. The C-| and C x ce l lu lase a c t i v i t i e s were 8 yg and 56 yg glucose/ min/ml of 2 day cul ture supernatant. This represents a large and rapid production of ce l lu lase when one compares i t wi th Trichoderma v i r i de C-| and C x cel lulases (0.04 yg and 1.7 yg glucose/mi n/ml of 18 day c u l -ture) as calculated from Mandels and Reese (1964). The rapid produc-t ion and high a c t i v i t y of the s t ra in M-7 cel lu lase is probably a re f l ec t i on of the high temperature optimal fo r growth and assay. How-ever, Stutzenberger (1971) showed that the maximal C 1 /C x r a t i o was approximately 1:450 a f te r 6 days of c u l t i v a t i o n at 55°C and the C, 58 a c t i v i t y was i n s i g n i f i c a n t , probably due to the i r reve rs ib le binding of the enzyme on insoluble ce l l u los i c mater ia ls . Most cel lulases operate maximally at low pH values; Trichoderma v i r i d e , pH 4.5 to 5.5 (Mandels & Weber, 1969), Myrotheciurn Verrucar ia, pH 5.0 to 6.0 ( H a l l i w e l l , 1961), Thermomonospora curvata (Stutzenberger, 1971), pH 6.0 , Poria v a i l l a n t i ; pH 3.5 (Sison et a l . , 1963), and at low temperature, but a few such as the 3-1,6-glucanases of SporotriChum pruinosum (Reese & Mandels, 1963) and Thermomonospora curvata (Stutzenberger, 1971) have optima at 60°C to 70°C. Zinc chlor ide p rec ip i ta t i on appeared to o f f e r a convenient method fo r the concentration and pa r t i a l p u r i f i c a t i o n of the cel lu lase (Table X) but on a large scale experiment (Table X I I I ) no p u r i f i c a t i o n was obtained. In subsequent manipulations a l l ce l lu lase a c t i v i t y was l o s t . Pu r i f i ca t i on of cel lulases has been attempted and abandoned by more workers than i t is possible to quote. They appear to be extremely d i f f i c u l t enzymes with which to work.. The high y ie lds obtainable by the convenient method of concen-t r a t i o n leads us to hope that the cel lulases of C_. thermocellulaseum may be pur i f i ed to homogeneity. The number of components wi th c e l l u -l o l y t i c a c t i v i t y is unknown, some evidence suggests that C-| and C x may be ident ica l but the data from i soe lec t r i c focusing indicated four cel lulases and DEAE-Sephadex chromatography resolved three components. 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