Vol. 43, No. 5 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1982, p. 1125-1132 0099-2240/82/051125-08$02.00/0 Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum ERIC A. JOHNSON, MITSUJI SAKAJOH, GEOFFREY HALLIWELL, ASHWIN MADIA, AND ARNOLD L. DEMAIN* Fermentation Microbiology Laboratory, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 13 October 1981/Accepted 13 December 1981 True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca2' and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70°C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellu- lose, respectively. In the absence of substrate at temperatures up to 70°C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity. The rate-limiting step in the conversion of native forms of cellulose to valuable fermenta- tion products such as ethanol (3) is its depolym- erization to sugars, a limitation imposed to vari- ous extents by the proportion of crystalline cellulose which is present. However, even in native cotton, the crystalline portion can be completely solubilized by a true cellulase; also degraded are simpler amorphous and derived forms of cellulose. Since the discovery of such activity in cell-free filtrates from Trichoderma species (T. reesei, formerly T. viride [18], and T. koningii [7]), a few other fungi have been found to secrete cellulase (for review see 10), but none appears to be as effective as the mutants of T. reesei (10, 23). Although some aerobic bacteria (2) and especially anaerobic cellulolytic bacteria such as Bacteroides succinogenes (6, 11), Clos- tridium thermocellum (19, 21, 26), and Acetivi- brio cellulolyticus (16) may grow more rapidly than the fungi on derived or native forms of cellulose, they have so far failed to show extra- cellular cellulolytic activities comparable with that from Trichoderma. The anaerobic, cellulolytic, and thermophilic bacterium, C. thermocellum, has received some attention in the past for its ability to carry out a limited attack on derived forms of cellulose (21, 22). The presence of carboxymethyl-cellulase (CM-cellulase), or possibly a weak ability to degrade native cellulose, has been detected in the extracellular fluid of this species (21, 22). The activity is said to be oxygen stable, low in "exoglucanase" (21), and unaffected by Ca21 (22) in experiments where cellulolysis was mea- sured on crystalline cellulose (Avicel) or its Remazol-dyed derivative during periods up to 2 h. The use of derived celluloses and short incu- bation periods, however, does not clarify wheth- er such enzyme preparations are true cellulases. In short incubation periods, even up to 24 h, initial rates of cellulolysis by bacterial and fungal enzyme preparations may equal or even exceed that of the cellulase from Trichoderma. Howev- er, such activity does not indicate the prepara- tion's ability to achieve extensive cellulolysis, particularly of native cellulose, over longer peri- ods, in the manner that characterizes the Tricho- derma enzyme. The inability of extracellular bacterial cellulases to show significant break- down of native and crystalline forms of cellulose may well be due to the inefficiency or instability of one or more components of the cellulase complex found to be essential in the Tricho- derma system. Relatively little is known about the enzyme complex of anaerobic cellulolytic microbes. The present report concerns the cellu- lase of C. thermocellum, its main components, and its efficacy as compared with the Tricho- derma complex when acting on native and de- rived forms of cellulose. MATERIALS AND METHODS Sources of enzymes. A crude cellulase preparation was obtained from B. Faison who used C. thermocel- lum ATCC 27405 grown in a 12-liter Microferm fer- mentor (New Brunswick Scientific Co., New Bruns- wick, N.J.) on CM-4 cellobiose medium (4) at 60°C and 60 rpm for 68 h. In her work, the broth was chilled to 4°C and then centrifuged at 18,000 x g for 20 min to remove cells. The supernatant fluid was treated with solid (NH4)2SO4 to 80% saturation and stored over- night at 4°C. The precipitate was harvested by centrif- 1125 on November 27, 2020 by guest http://aem.asm.org/ Downloaded from
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Vol. 43, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1982, p. 1125-11320099-2240/82/051125-08$02.00/0
Saccharification of Complex Cellulosic Substrates by theCellulase System from Clostridium thermocellum
ERIC A. JOHNSON, MITSUJI SAKAJOH, GEOFFREY HALLIWELL, ASHWIN MADIA, AND
ARNOLD L. DEMAIN*Fermentation Microbiology Laboratory, Department of Nutrition and Food Science, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139
Received 13 October 1981/Accepted 13 December 1981
True cellulase activity has been demonstrated in cell-free preparations from thethermophilic anaerobe Clostridium thermocellum. Such activity depends upon thepresence of Ca2' and a thiol-reducing agent of which dithiothreitol is the mostpromising. Under these conditions, native (cotton) and derived forms of cellulose(Avicel and filter paper) were all extensively solubilized at rates comparable withcellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulasewas displayed at 70°C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellu-lose, respectively. In the absence of substrate at temperatures up to 70°C,carboxymethylcellulase was much more unstable than the Avicel-hydrolyzingactivity.
The rate-limiting step in the conversion ofnative forms of cellulose to valuable fermenta-tion products such as ethanol (3) is its depolym-erization to sugars, a limitation imposed to vari-ous extents by the proportion of crystallinecellulose which is present. However, even innative cotton, the crystalline portion can becompletely solubilized by a true cellulase; alsodegraded are simpler amorphous and derivedforms of cellulose. Since the discovery of suchactivity in cell-free filtrates from Trichodermaspecies (T. reesei, formerly T. viride [18], and T.koningii [7]), a few other fungi have been foundto secrete cellulase (for review see 10), but noneappears to be as effective as the mutants of T.reesei (10, 23). Although some aerobic bacteria(2) and especially anaerobic cellulolytic bacteriasuch as Bacteroides succinogenes (6, 11), Clos-tridium thermocellum (19, 21, 26), and Acetivi-brio cellulolyticus (16) may grow more rapidlythan the fungi on derived or native forms ofcellulose, they have so far failed to show extra-cellular cellulolytic activities comparable withthat from Trichoderma.The anaerobic, cellulolytic, and thermophilic
bacterium, C. thermocellum, has received someattention in the past for its ability to carry out alimited attack on derived forms of cellulose (21,22). The presence of carboxymethyl-cellulase(CM-cellulase), or possibly a weak ability todegrade native cellulose, has been detected inthe extracellular fluid of this species (21, 22).The activity is said to be oxygen stable, low in"exoglucanase" (21), and unaffected by Ca21(22) in experiments where cellulolysis was mea-sured on crystalline cellulose (Avicel) or its
Remazol-dyed derivative during periods up to 2h. The use of derived celluloses and short incu-bation periods, however, does not clarify wheth-er such enzyme preparations are true cellulases.In short incubation periods, even up to 24 h,initial rates of cellulolysis by bacterial and fungalenzyme preparations may equal or even exceedthat of the cellulase from Trichoderma. Howev-er, such activity does not indicate the prepara-tion's ability to achieve extensive cellulolysis,particularly of native cellulose, over longer peri-ods, in the manner that characterizes the Tricho-derma enzyme. The inability of extracellularbacterial cellulases to show significant break-down of native and crystalline forms of cellulosemay well be due to the inefficiency or instabilityof one or more components of the cellulasecomplex found to be essential in the Tricho-derma system. Relatively little is known aboutthe enzyme complex of anaerobic cellulolyticmicrobes. The present report concerns the cellu-lase of C. thermocellum, its main components,and its efficacy as compared with the Tricho-derma complex when acting on native and de-rived forms of cellulose.
MATERIALS AND METHODSSources of enzymes. A crude cellulase preparation
was obtained from B. Faison who used C. thermocel-lum ATCC 27405 grown in a 12-liter Microferm fer-mentor (New Brunswick Scientific Co., New Bruns-wick, N.J.) on CM-4 cellobiose medium (4) at 60°C and60 rpm for 68 h. In her work, the broth was chilled to4°C and then centrifuged at 18,000 x g for 20 min toremove cells. The supernatant fluid was treated withsolid (NH4)2SO4 to 80% saturation and stored over-night at 4°C. The precipitate was harvested by centrif-
ugation, dissolved in 50 mM sodium citrate buffer (pH5.7), reprecipitated by the addition of 4 volumes ofsaturated (NH4)2SO4, and again stored overnight be-fore dissolution in citrate buffer. This preparation wasdesalted by passage through Biogel P-2 (Bio-Rad Lab-oratories, Richmond, Calif.), diluted to 1 mg of proteinper ml, and stored frozen (B. Faison, S.M. thesis,Massachusetts Institute of Technology, Cambridge,Mass., 1981).A second batch of enzyme was prepared similarly
but with C. thermocellum grown in a 100-liter fermen-tor containing 0.5% Solka-Floc SW-40 (Brown Co.,Berlin, N.H.) as carbon source. The fermentor wasstirred slowly at 50 rpm and gassed with N2 for the first19 h of the 60 h fermentation. At 60 h, cysteine-hydrochloride was added to a final concentration of0.1% and the cells were removed in a Sharples centri-fuge (type M47-16Y, Sharples Corp., Philadelphia,Pa.). This extracellular preparation has retained itscellulolytic activity for at least a month at 4°C, and forat least 4 months at -20°C. When required for enzy-matic work, it was centrifuged at 18,000 x g for 15 minat room temperature, and the supernatant fluid (-200,ug of protein per ml by the Coomassie blue method[1]) was either used as such or precipitated with(NH4)2SO4 and redissolved in acetic or succinatebuffer.
Dried powders from T. reesei were prepared at theNatick Laboratories (T. reesei QM 9414 cellulase 0.61mg of protein per ml, 0.32 filter paper units [FPU] permg, and T. reesei RUT C-30, 0.53 FPU/mg). Thesewere prepared in solution at 5 FPU/ml and 9 FPU/ml,respectively, which is equivalent to their broth con-centrations.Methods of enzymatic analysis. Cellulase activity of
the Clostridium cellulase was followed in most casesby the decrease in turbidity (measured at 660 nm in amodel 330 spectrophotometer, G. K. Turner Asso-ciates, Palo Alto, Calif.) of an Avicel suspension (typePH 105, 20-,um particles; FMC Corp., Marcus Hook,Pa.). Three milligrams of this substrate was incubatedfor about 18 h in Hungate tubes at 60°C with 3 ml ofbuffer (0.1 M succinic acid-NaOH, pH 5.5; 0.2 Msodium acetate-acetic acid, pH 5.5; or as indicated intext), 0.5 ml of 1% CaCl2 2H20, 0.5 ml of 0.1 Mdithiothreitol (DTT; Sigma Chemical Co., St. Louis,Mo.), various amounts of enzyme, and water to 5 ml.This concentration of cellulose (0.6 mg/ml) was usedso that cellulolysis would proceed to completion (7, 8,12, 14) within a relatively short time with minimuminterference from products (12).
Cellulase activity was also measured on Avicel andnonpowdered celluloses by loss in dry weight. Threemilligrams of filter paper (6-mm diameter diskspunched from Whatman no. 1 filter paper circles) ornonabsorbent cotton (SP cotton coil, C-8355-3, Ameri-can Scientific Products, Bedford, Mass.) were incu-bated with the Clostridium cellulase under the sameconditions as with Avicel. Such cellulosic substratescannot be measured turbidimetrically (see below).The Trichoderma enzyme (1 ml), 3 ml of buffer (pH
4.8; 0.2 M acetate-acetic acid), and 3 mg of Avicel,filter paper, or cotton, in a total volume of 5 ml withwater, was incubated in Hungate tubes at 37 or 50°Cbut otherwise as for the clostridial enzyme above. Allenzymatic assays were unshaken.Measurement of residual cellulose. Since reducing
agents such as cysteine (in the culture medium) andDTT (in the enzyme assay) interfere in the determina-tion of reducing sugars (the products of cellulolysis),and filter paper and cotton cannot be measured turbi-dimetrically, solubilization of these cellulose sources(and occasionally, Avicel) was followed by the deter-mination of residual cellulose, thus avoiding any as-sumptions about the nature of the saccharificationproducts. After incubation of the enzyme with eachsubstrate, residual cellulose was washed repeatedly bycentrifugation and determined colorimetrically withK2Cr2O7 (9) by a modified procedure with threefoldgreater volumes of each reagent.
CM-celiulase activity (endo-1,4-0-D-glucanase, E.C.3.2.1.4.). CM-cellulase activity was determined in theabsence of reducing agents with ferricyanide reagent(13) as reducing sugars liberated (60°C, pH 5.8, 45 min)from carboxymethylcellulose (CM-cellulose, 0.61% fi-nal concentration). The reaction was terminated with0.2 ml of 2% anhydrous Na2CO3 followed by 1 ml of0.064% KCN-0.52% Na2CO3 mixture to raise the pHto 10.6.The CM-cellulose chosen as enzymatic substrate
was type 9M31, lot No. 80954 (Hercules, Inc., Wil-mington, Dela.); other types were either more resist-ant to enzymatic hydrolysis (type 12M31; Hercules),too viscous (type 7H4; Hercules), or showed too largea reducing sugar "blank value" (CM-cellulose type C-8758, Sigma).
Phosphoric acid-swollen Avicel was prepared bytreating Avicel for 2 h at 4°C according to Tansey (25).
Buffer solutions. The effect of pH on enzymaticactivity was measured in the following buffers: pH 2 to3 with 0.2 M sodium acetate-HCl; pH 4 to 6.5 with 0.2M sodium acetate-acetic acid or with 0.1 M succinicacid-NaOH; pH 7 to 9 with 0.05 M Tris-hydrochloride;pH 8 to 10 with 0.05 M boric acid-0.05 M KCl-NaOH.All buffers contained 0.025% sodium azide to preventgrowth of microorganisms.Growth of C. thermocellum on agar plates. We grew
C. thermocellum on agar plates as described previous-ly (24) but with 1% compression-milled corn stover (20,um diameter, provided by L. Spano), Avicel, oramorphous cellulose (Sigmacel 100) as growth sub-strates. Clearing zones were photographed after 8 daysof incubation at 60°C in an anaerobic jar.
RESULTSCa2' and sulfhydryl reducing compounds as
requirements of the cellulase system of C. thermo-cellum. C. thermocellum, plated on agar mediumcontaining Avicel, Sigmacel 100, or compres-sion-milled corn stover, gave rise to coloniessurrounded by clear zones in 5 to 7 days (Fig. 1),suggesting the presence of an extracellular cellu-lase. However, extracellular broth from the or-ganism, prepared on cellobiose medium as inMaterials and Methods, showed only weak cel-lulolytic activity (0.2 to 0.4 mg of reducingsugars per ml of broth per h) in the filter paperassay (18), confirming earlier findings by Gor-don et al. (5). Similar results were obtained withthe Bio-Gel-treated ammonium sulfate enzymepreparation. Considerable improvements in ac-tivity as measured by the turbidimetric assay
FIG. 1. Clear zones produced by C. thermocellumgrown on compression-milled corn stover (A), micro-crystalline Avicel (B), and amorphous cellulose (C).
with Avicel as substrate were achieved whensuccinate replaced citrate as the buffer and a
sulfhydryl reducing compound was added. Suchchanges had little effect on the initial rate ofAvicel hydrolysis, but greatly increased the ulti-mate extent of breakdown (Fig. 2). In the pres-ence of 5 mM DTT, Avicel was completelysolubilized; in the absence of DTT, solubiliza-tion stopped after 24 h. Other effective reducingagents included cysteine, sodium dithionite, glu-tathione, and mercaptoethanol. DTT (5 mM) hadno effect on the solubilization of phosphoricacid-swollen Avicel or trinitrophenylcarboxy-methyl-cellulose prepared by the method ofHuang and Tang (15).The stimulatory effect of DTT on the break-
down of Avicel does not agree with the observa-tion by Ng et al. (21) that Avicel hydrolysis by
the culture filtrate of C. thermocellum strainswas unaffected by oxygen and did not appear torequire a reducing agent. However, Ng andZeikus (22) later found that filtrates of C. ther-mocellum LQRI were strongly inhibited by Ag+and Hg2' and that this inhibition could be re-versed by cysteine or DTT. These discrepanciescould be the result of the "low yield of exoglu-canase" reported by Ng et al. (21) in theirbroths, the use of dyed rather than untreatedAvicel in their later paper (22), or the use of C.thermocellum strains different from ours.The cellulase activity of our C. thermocellum
enzyme preparation was also stimulated byCa2+. EDTA inhibited completely the celluloly-sis of Avicel by the Bio-Gel-treated preparationeven in the presence ofDTT (Fig. 3). Addition ofMgCl2 (7 mM) improved cellulolysis slightly,whereas 7 mM CaCl2 was far more effective andenabled the enzyme preparation to achieve com-plete solubilization of the substrate. These re-sults differ somewhat from those of Ng andZeikus (22) with cellulase from C. thermocellumLQRI. They reported no inhibition by Ca2+ orEDTA and did not mention any stimulatoryeffect of Ca2+. They did observe inhibition byethylene glycol-bis-(P-aminoethyl ether)-N,N-tetraacetic acid trisodium salt, i.e., EGTA; how-ever, this was not reversed by Ca2+.The effect of DTT was most marked in the
desalted, Bio-Gel-treated enzyme preparation, atreatment which simultaneously removed sulf-hydryl-reducing compounds (cysteine) from thegrowth medium. The same stimulatory effect
FIG. 2. Influence of DTT concentration on Avicelhydrolysis by C. thermocellum cellulase. Fifty micro-grams of Biogel-treated extracellular protein wereincubated with 3 mg of Avicel, and the absorbance(660 nm) of the suspension was determined with time.Succinate buffer with no Ca2+ was used.
could be observed (but to a lesser extent) withextracellular broth and with ammonium sulfate-precipitated enzyme.
Action of Clostridium and Trichoderma cellu-lases on native and derived forms of cellulose. Inthis section of the work, we used an extracellu-lar broth from C. thermocellum grown on Solka-Floc. Equal broth volumes (1 ml) of the Clostrid-ium (0.2 mg of protein per ml) and T. reeseiQM9414 (9.5 mg of protein per ml) cellulasewere examined quantitatively for their ability tosolubilize cotton, filter paper, and Avicel asmeasured by loss in weight. The crude Tricho-derma cellulase acted rapidly on filter paper,less so on Avicel, and slowest on cotton (Fig. 4),whereas the Clostridium enzyme showed thereverse pattern, cotton being saccharified mostrapidly and faster than the Trichoderma cellu-lase. Filter paper, however, showed greaterresistance to hydrolysis by the clostridial cellu-lase, particularly in the early stages, possiblybecause of limiting amounts of a cellulase com-ponent. Of note was the Clostridium enzyme'sability to achieve essentially complete hydroly-sis of all three forms of cellulose in the mannerexpected of a true cellulase. Solubilization ofcellulose in Fig. 4 was measured not only fromits loss in weight but, in the case of Tricho-derma, also as (i) total soluble carbohydrate by
0.3 .
0.2
0
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0mgo4 0.1
NO ADDITIONS
cc2
20 40 60 80
HOURS OF HYDROLYSIS
100
FIG. 3. Influence of Ca2" on Avicel hydrolysis byC. thermocellum cellulase. Reaction conditions werethe same as in Fig. 2, but the incubation mixturescontained 5 mM DTT and either 10 mM EDTA, 7 mMCaCl2, or 7 mM MgCl2.
the phenol method (14) and (ii) as glucose andcellobiose using a glucose oxidase procedure (8).Determinations (i) and (ii) gave values similar tothose obtained from loss in weight, indicatingthat the products were essentially all glucose.The DTT present in the Clostridium enzymehydrolysates interfered with similar determina-tions. However, quantitative high-pressure liq-uid chromatography (Waters Associates, Mil-ford, Mass.) of 50-h hydrolysates of Avicel (96%solubilization, Fig. 4) on a column of HPX-87-H(Bio-Rad) showed the sample to consist of 86%cellobiose and 14% glucose. DTT did not inter-fere with the turbidimetric assay of Avicel hy-drolysis. In contrast to its stimulation of theClostridium cellulase, DTT had no effect on theTrichoderma enzyme.
Effect of pH and temperature on the hydrolysisof Avicel and CM-cellulose. One of the mostreadily recognizable components of the cellulasecomplex is its endo-3-glucanase, i.e., CM-cellu-lase. The activity of this component in theammonium-sulfate-precipitated enzyme prepa-ration from C. thermocellum was compared un-der standard conditions with the action of the
FIG. 4. Solubilization of native and derived cellu-loses by cellulase of T. reesei QM 9414 and C. thermo-cellum. One milliliter of culture broth (fresh or recon-stituted) was incubated at 37°C and pH 4.8 acetatebuffer (Trichoderma) or 600C and pH 5.5 succinatebuffer (Clostridium) with cotton, filter paper, or Avi-cel. The clostridial enzyme incubation mixture con-tained 7 mM CaCl2 and 10 mM DTT.
same preparation on Avicel. The preparationshowed an optimum of about pH 6.1 on CM-cellulose and of 5.7 on Avicel (Fig. 5). Theenzyme showed similar behavior in both acetateand succinate buffers between pH 4.0 and 6.5.The pH optima, 5.7 and 6.1, for the Avicelaseand CM-cellulase activities, respectively, con-trast with those found for C. thermocellum Nibroth (21) where the optimum pH values forAvicelase and CM-cellulase were 5.4 and 5.2,respectively. Furthermore, strain Ni brothshowed 80 to 100% of its maximum activitythroughout the pH range 4 to 8.Subsequent investigations employed buffers
ofpH 5.8; under this condition, the same ammo-nium-sulfate fraction was most active at 700C onAvicel and on CM-cellulose. The Avicelase ac-tivity was almost nonexistent at 80°C (Fig. 5). Incontrast, at 80°C, CM-cellulase retained 40% ofits maximum potency.The effect of temperature (30, 37, 50, 60, 70,
and 80°C) on enzymatic stability during a 5-hincubation at pH 5.8 in the absence of substratewas followed by adding Avicel and incubating
0:E
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0.
t:F4
.-
Da
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pH
30 40 50 60 70 80
TEMPERATURE (°C)
FIG. 5. Effect of (A) pH and (B) temperature onthe activity of C. thermocellum cellulase on Avicel andCM-cellulose. The ammonium sulfate enzyme prepa-ration at broth strength was incubated with buffersolutions described in the text. Temperature studieswere done at pH 5.8 in acetate buffer. Avicel hydroly-sis was measured turbidimetrically. CM-cellulase ac-tivity was measured by estimation of reducing sugars.
TABLE 1. Stability of enzymatic activities' in theclostridial preparation against CM-cellulose and
Avicel in the absence of substratesResidual activity (%)
CMCaseb Avicelase
30 31 8537 56 8550 38 8560 25 8570 13 8580 13 0
aResidual activities were measured by the standardmethods on CM-cellulose and turbidimetrically onAvicel.
b CMCase, CM-cellulase.
for a further 16 h at 60°C. The Clostridiumcellulase lost all activity in 5 h at 80°C but lostonly 15% of its activity between 37 and 70°C. Incontrast, the same cellulase preparation, incu-bated at the same temperatures and pH 5.8 for45 min without CM-cellulose, revealed markedloss of enzymatic activity when tested for resid-ual endoglucanase, even at 30°C (69% loss),followed by increasing losses in activity (to 87%)up to 80°C (Table 1). This suggests the presenceof more than one enzymatic component, possi-bly an exoglucanase or cellobiohydrolase.
Cellulolytic activity of the celiulases of T. reeseiand C. thermocellum under optimal conditions.The previous experiment comparing T. reeseiand C. thermocellum cellulases (Fig. 4) em-ployed a temperature of 37°C for Trichoderma,this temperature being optimal for the cellobio-hydrolase (C1) component of T. koningii overlong periods (12). However, in the short-term (1-h) assay employed by Mandels et al. (17), 50°C isused, being optimal for the CM-cellulase compo-nent in the culture filtrate. Therefore, it was ofinterest to compare the cellulase of T. reesei at50°C with the clostridial cellulase (at 60°C). Forthis experiment, 1 ml of the best available T.reesei preparation (RUT C-30; Ryu and Mandels[23]; Montenecourt and Eveleigh [20]) was usedat culture broth strength (9 FPU/lml). The resultsmeasured turbidimetrically on Avicel (Fig. 6A).and as loss in weight of Avicel and cotton (Fig.6B) confirm those found in Fig. 4. In addition,the Trichoderma enzyme showed slightly im-proved activity on both substrates at 50°C com-pared with 37°C, rates that were equalled orexceeded by the Clostridium cellulase.
DISCUSSIONThe present report shows clearly the presence
of true cellulase activity in a bacterial extracellu-lar preparation. The bacterium C. thermocellum
produces an extracellular enzyme which, in thepresence of Ca2' and DTT, has the ability tosolubilize native and derived forms of cellulose(cotton, filter paper, and Avicel) at a rate and toan extent comparable with T. reesei cellulase.Filter paper was the preferred substrate for theTrichoderma cellulase but was a poor substratefor the bacterial cellulase in the initial stages ofhydrolysis. In contrast, cotton presented less ofa problem to the Clostridium cellulase than tothe Trichoderma enzyme. In the presence ofCa2' and DTT, the cell-free Clostridium cellu-lase solubilized Avicel at the rate of 0.11 mg/h.This is only slightly less than the rate observedwith growing cells, i.e., 0.14 mg/h.
Care is needed in choosing a suitable cellulos-ic substrate for cellulase preparations and also ininterpreting results from such activities. Filterpaper may be a good substrate for fungal cellu-lases (if those from T. viride and T. koningii aretypical), but it may not be a good substrate forbacterial cellulases (if C. thermocellum cellulaseis typical). Cotton, however, is a suitable sub-strate for cellulase preparations from both mi-
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ul
J
ui
<i
a
0 21 42 0 21 42
HOURS OF HYDROLYSIS
FIG. 6. Hydrolysis of cotton and Avicel under op-timal conditions by the cellulases of T. reesei RUT C-30 (Tr) and C. thermocellum (Ct). The Trichodermaenzyme incubation was done in pH 4.8 acetate buffer,and the Clostridium enzyme incubation was done inpH 5.8 acetate buffer. (A) Turbidimetric measurementof Avicel hydrolysis. (B) Colorimetric determinationof residual cellulose using Avicel or cotton. For eachorganism, 1 ml of culture broth (fresh or reconstituted)was used.
crobial sources. Such fibers are representativeof the most resistant cellulose fraction found innatural polysaccharides, material that cellulasepreparations must hydrolyze if microbial proc-essing of cellulosic material is to be effectiveindustrially in providing new sources of energy,chemicals, and single-cell protein.Whatever cellulosic substrate is chosen (but
particularly if these are derived or treatedforms), the results of cellulolytic assays havelittle meaning if such substrates are not exten-sively attacked, as is common in short incuba-tion periods. This applies whether or not theinitial hydrolysis rate appears similar to that ofthe Trichoderma cellulase. In these circum-stances, the extent of hydrolysis is a far morerealistic assessment of the potency of a cellulasepreparation than are "units" of specific activity.This precaution is of even greater importancewith derived or treated celluloses which cancontain different amounts of more susceptiblecellulose fractions. A comprehensive estimate ofthe smaller molecular chains of cellulose is givenby the 1 and y cellulose contents, fractionsdefined as material soluble in 17.5% (wt/wt)NaOH at 20°C and consisting of molecules ofdegree of polymerization of 10 to 200. a-Cellu-lose is insoluble in the same concentration ofalkali and has a degree of polymerization of>200. The a + -y cellulose was determined byloss in weight of 50 mg of the substrate, correct-ed for moisture content (11); the cotton fibers,filter paper, and Avicel used above had 3%, 3%,and 29% 13 + -y cellulose content, respectively.Thus, microcrystalline Avicel consists of almost30% of readily available, relatively small mole-cules which may well be hydrolyzed by one oranother component of an incomplete and ineffi-cient cellulase system at an initial rate (especial-ly in short-term assays) comparable with thatshown by a true cellulase as from Trichodermaspecies. In extended assays, however, such par-tial cellulase preparations fail to achieve theefficiency of solubilization shown by the cellu-lase of Trichoderma or of the C. thermocellumof the present report. Only when a preparationhas been shown to contain true cellulase activityis it possible to consider short-term assays witha suitable substrate.
ACKNOWLEDGMENTSWe thank B. Montenecourt and M. Mandels for gifts of T.
reesei cellulase, L. Spano for a gift of compression-milled cornstover, B. Faison for a preparation of clostridial cellulase, andHerve Cellard for the HPLC analyses.G.H. acknowledges with thanks the award of a NATO
Fellowship. E.A.J. acknowledges the fellowship provided byNational Distillers and Chemical Co. and a research grantfrom Eastman Kodak Co.
This research was supported by contract EC-77-5-02.4198from the U.S. Department of Energy.
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