Factors Affecting Cellulolysis by Ruminococcus Albus W

JouRNAL oF BAcrTOLOGY, May 1973, p. 729-737 Vol. 114, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.S.A.

Factors Affecting Cellulolysis by Ruminococcus albus W. R. SMITH,' IDA YU, AND R. E. HUNGATE Department of Bacteriology, University of California, Davis, California 95616 Received for publication 10 November 1972

The factors influencing the digestion of pebble-milled cellulose by enzymes were studied by using several strains of Ruminococcus albus including a mutant characterized by a more eccentric location of its colony in the clearing produced by digestion of the cellulose in the thin layer lining the wall of a culture tube. Most of the cellulase is extracellular. As much as 65% of the cellulose could be digested by the cell-free enzymes provided the quantity of cellulose was small. Fresh enzyme was repeatedly administered or the digestion experiment was arranged in a dialysis bag through which digestion products could diffuse. Cellobiose and, to a lesser extent, glucose inhibited digestion. Pebble-milled filter paper, moist crystalline cellulose from cotton, and dry crystalline cellulose (Sigmacel) were digested, but in decreasing rapidity, respectively. Carboxymeth- ylcellulose was digested more rapidly than pebble-milled cellulose but to approximately the same final extent as judged by Cu reduction values. Cell walls from alfalfa were digested. The enzyme preparation was active over the pH range 6.0 to 6.8 and showed most rapid cellulose digestion at 45 C. Part of the cellulolytic activity was irreversibly destroyed by exposure to oxygen. Much of the enzyme was absorbed on cellulose. The absorption and desorption character- istics, as well as the partial inhibition by oxygen, indicate that multiple enzymes are involved.

Much cellulose is digested in the rumen. single colony growing in cellulose agar inocu- Cell-free cellulose-digesting extracts of mixed lated with strain 6 (Fig. la). Leatherwood (11) rumen bacteria have been obtained (9), and a has ascribed cleared zones to the combined multiplicity of cellulases has been demon- action of two cellulases and a non-cellulolytic strated (7, 8). molecule. The zones from his strains were Of the numerous cellulolytic bacteria isolated circular, with the colony much nearer the center from the rumen, strains resembling the type than was the case with our strains, although his strain of Ruminococcus albus (4) are among the photographs (11) suggest a slightly eccentric most active. The agar is entirely cleared of location. cellulose around colonies in agar roll tubes, with When the agar medium in which R. albus is a sharp border between digested and undigested growing also contains cellobiose, the cellulose substrate (Fig. la). The separation of the colony around the colony does not clear until most of from the site of digestion shows that the enzyme the cellobiose has been fermented (6), an ob- is extracellular. The colony is located eccentri- servation suggesting involvement of cellobiose cally (its distance from the upper border of in some sort of control mechanism. This phe- cellulose digestion being less than from the nomenon has been observed also by Fusee and lower border) in the oval clearing when the Leatherwood (2) who reported evidence for a colony grows in a thin agar layer lining the cellobiose repression of cellulase production. inner wall of a vertically incubated culture Of the cellulase activity in rumen contents, tube, but is located in the center of a circular 20% was ascribed to R. albus on the basis of the clearing in a horizontally incubated layer. Even decrease in cellulolysis caused by antiserum greater eccentricity of the colony location oc- specific against the cellulase of a strain of R. curred in strain 6L (Fig. lb), noted first as a albus (10). This paper reports on the cellulolytic activity 'Present address: G. D. Searle & Co. Ltd., High Wy- of the mixed enzymes of R. albus and on the combe, Bucks., England. factors affecting it. 729 730 SMITH, YU, AND HUNGATE J. BACTERIOL.

strain was grown in continuous culture (8-h cycle time) on 0.1% cellobiose by previously described methods (5). Yeast extract (0.1%, Difco) added to batch cultures on minimal medium supported more rapid growth initially, but continuous cultures with added yeast extract failed to grow after 7 days. In contrast, a continuous culture containing 10% rumen fluid ran successfully for 3 months on cellobiose without losing the capacity for cellulase production when inoculated into cellulose agar medium. In most experiments, enzyme was from batch cultures grown on rumen fluid plus cellobiose or cellulose. The RAM strain was used unless otherwise indicated. Substrates. Pebble-milled cellulose (PMC) was prepared by tearing up 30 g of Whatman no. 1 filter paper and placing it in a porcelain jar (ca. 4-liter capacity) with one liter of water. Enough flint pebbles were used so that the liquid just covered them. The jar was rolled for 24 h at 74 rpm. The liquid containing the cellulose was drained off, and the pebbles were washed with enough water to bring the milled cellulose to a concentration of 20 mg/ml. Carboxymethyl- cellulose (CMC) was obtained as product 70M from Hercules Powder Co. Powdered crystalline cellulose (Sigmacel) was obtained from Sigma Chemical Co. It was suspended in distilled water and auto- claved at 15 lb/in2 for 20 min. Crystalline cellulose, FIG. 1. Colonies of strain 6 (a) and strain 6L (b) in which was never dried after preparation, was obtained the thin layer of cellulose agar lining the wall of a roll by heating 5 g of absorbent cotton in 220 ml of 2.5 N tube. HCI for 20 min (preparation A) or 45 min (preparation B) at 103 C. The insoluble residue was collected, washed on a Buchner funnel, and suspended in 150 MATERIALS AND METHODS ml of water with the aid of a Waring blend6r. Bacterial strains. Cultures of R. albus strain RAM Alfalfa cell walls were prepared by picking the tops in a were grown in a balanced mineral solution containing of lush prebloom alfalfa and mincing them biotin, aminobenzoic acid, pyridoxin, isobutyrate, Waring blendor which contained water. The blended isovalerate, and 2-methylbutyrate (5). This medium material was transferred to a graduated cylinder, and is designated minimal medium. With addition of the particles were allowed to settle. The suspension in and cellulose or cellobiose and some rumen fluid (2 to 33%, the lower part of the cylinder was again blended water vol/vol) it was designated the stock culture medium. retumed to the cylinder for mixing with wash The mineral portion, including 0.5% sodium bicar- and for settling. The coarsest particles were filtered bonate, 0.03% Na2S-9H2O, and 0.03% cysteine hydro- off with a seive, and the suspension of small particles chloride, equilibrated with O2-free CO2, is designated was washed repeatedly by sedimentation until little mineral medium. chlorophyll could be seen. Sodium hydroxide (1%, At times it was difficult to maintain the strain in wt/vol) was added, the washing process was re- vigorous cellulolytic condition during repeated trans- peated until the pH of the wash water was almost neu- fers in the cellulose rumen fluid agar medium. Occa- tral, and then the pH was adjusted to 7.0 with HCl. sionally, vigor could be at least partially regained by Alfalfa cell walls prepared in this way did not give the subculturing in 30% rumen fluid cellulose liquid blue color characteristic of cellulose treated with 70% medium and then inoculating a 10% rumen fluid H2SO4 plus iodine. cellulose agar dilution series from which an actively For enzyme experiments, the aqueous suspension of cellulolytic colony was picked. Revived cultures grew the cellulosic substrate (except CMC) was centrifuged well in the minimal medium alone (or with 2% rumen to give a pellet, and the supernatant fluid was poured fluid) but were not always identical to the strain off to avoid dilution of the enzyme preparation. The before its revival. By selecting the most actively centrifuge tube was gassed out to exclude 02, the cellulolytic colonies, cellulolysis was preserved, but enzyme solution and toluene were added, and the the activity after 3 years in culture fell to about half tube and contents were treated as desired. that of the original and was less than that of the two Measurement of cellulase activity. Cultures were more recently isolated strains, 6 and N3. These strains usually grown on 0.1 or 0.2% cellobiose or cellulose. in turn showed changes leading to a gradual reduction Exposure of cells and enzyme to atmospheric oxygen in cellulolytic activity. They were routinely cultured was avoided at all steps by using closed containers on clarified rumen fluid (33%, vol/vol) added to gassed out with CO2 freed of oxygen by passage over mineral agar medium containing cellulose. hot Cu. The cells in a spent culture were sedimented During initial enzyme experiments, the RAM by centrifugation at 10,000 x g for 20 min, and the VOL. 114, 1973 CELLULOLYSIS BY R. ALBUS 731 supernatant fluid was used as the source of enzyme. In the entire 800 ml of culture. Since the superna- some experiments the supernatant fluid was first tant fluid showed good activity and its enzymes filtered through a membrane filter (0.45-Am porosity; presumably resembled those in the cells, it was Millipore Corp.). Toluene was added to the supernatant fluid if cells had not been removed by filtration. used as the source of enzyme in all subsequent It did not diminish cellulase activity. experiments. Use of the supernatant fluid The solution to be tested for cellulase was in- avoided the problem of separating the intracel- cubated with the substrate at 39 C for various periods. lular cellulase from other cell components. In some experiments, the supernatant fluid was Effect of kind of substrate. An insoluble mixed with the substrate cellulose at 4 C and in- cellulose substrate was used as more representa- cubated at this temperature for 2 h to adsorb the tive of the material naturally attacked. The cellulase on the cellulose. The latter was sedimented, action of strain N3 was tested on PMC, Sigma- and the supernatant fluid was discarded and replaced cel, and crystalline cellulose preparations A and with mineral solution. When the degree of adsorption was studied, the supernatant fluid from the first B (Table 1). centrifugation was provided with cellulose and in- The time course of digestion of PMC and cubated in parallel with the sediment. In most experi- crystalline B cellulose with the RAM strain is ments, the adsorption procedure was used to partially shown in Fig. 2. The digestion of the crystalline purify the cellulase. Most of it was selectively ad- material is about half as fast but is sustained sorbed, and many impurities, including any soluble satisfactorily. The PMC was used in most carbohydrate in the medium, were discarded with the subsequent experiments. Cell walls prepared supernatant fluid. from fresh alfalfa were significantly more rap- Extent of enzyme digestion was usually measured idly digested than was the PMC, but were not by determining the difference between the initial and final weight of cellulose. In some experiments the as convenient to prepare or handle and showed cellulose or the solubilized carbohydrates, or both, a higher blank value. were measured with the anthrone procedure (13). In Effect of substrate concentration. A 10-ml all experiments with CMC, increase in reducing sample (lower curve, Fig. 3) or a 50-ml sample power was measured by Cu reduction (12). A control (upper curve) of sterile, filtered culture super- for the reducing materials in the medium was neces- sary. Values for weight loss and increase in soluble carbohydrate agreed to within a few percent. Attack TABLE 1. Soluble sugars produced by supernatant on CMC was also followed viscosimetrically with an fluid from N3 acting on various preparations of Ostwald viscometer at 30 C. cellulose Chromatography. Initially, sugars (50 Ag total) Time of Amountof were separated on one-dimensional strips of What- incubation sugar formed man no. 4 filter paper and detected with phthalic acid with enzyme as cellobiose and aniline in n-butanol saturated with water. The (h) (mg) chromatograms were developed with n-butanol- ethanol-water (5:1:4). Later, thin-layer chromatog- PMC 3.5 3.4 raphy with silica gel was used with a 3:2:8 ratio of Crystalline A 3.5 2.24 solvents. The silica gel plates were first tested with PMC 24 9.5 the phthalic acid-aniline reagent, photographed, and Crystalline A 24 4.7 then sprayed with 50% (vol/vol) sulfuric acid. They PMC 2.75 2.4 were heated for 30 min at 100 C to carbonize the Crystalline B 2.75 1.6 organic matter, and again photographed. The acid Sigmacel 2.75 0.5 treatment disclosed higher-molecular-weight com- pounds not detected with aniline phthlate. RESULTS Extracellular nature of the cellulase. The bacterial cells in 800 ml of a culture grown on 0.1% cellobiose were collected by centrifugation and suspended in 25 ml of the culture supernatant fluid. PMC (67 mg) was added to a total volume of 30 ml (25 ml of supernatant fluid), and the mixture was incubated anaerobically with toluene for 48 h. The culture supernatant Time of Incubation (hours) fluid was tested similarly. The supernatant FIG. 2. Time course of digestion of PMC (-) and fluid alone (25 ml out of the 800) showed 21% of crystalline B cellulose (A); 16 ml of supematant fluid the cellulose solubilized as compared to 44% for from a culture of strain N3 grown on 0.2% cellobiose combined supernatant fluid plus the cells from was incubated under toluene with 32 mg ofsubstrate. 732 SMITH, YU, AND HUNGATE J. BACTERIOL. phosphate buffer. Subsequently, the mineral medium, containing both phosphate and bicar- bonate, equilibrated with carbon dioxide, was used. Temperature and pH. Culture supematant a fluid (25 ml) was incubated with 100 mg of Co PMC (each sample at a different temperature) and after 24 h was tested for soluble carbohy- 0 drates (Table 2). U)I The stability with time was tested by incubating replicate 10-ml samples of supernatant fluid anaerobically at 39 C without substrate and by adding substrate at various times to one of them. After 60 days the cellulolytic activity had diminished only slightly. The influence of pH on digestion was ex- Mg Cellulose amined. The results (Fig. 5) show an almost FIG. 3. Effect of quantity of PMC substrate on constant activity between pH 6.0 and 6.6. This amount of soluble carbohydrate formed during 24 h of is a common pH range for rumen contents of incubation in 10 ml of mineral medium. See text for animals on a roughage ration. details. Extent of adsorption. The extent of adsorp- natant fluid was added to each of several tion of enzyme was tested by mixing 100 mg of amounts (5 to 550 mg, respectively) of PMC in PMC with 25 ml of supernatant fluid for 2 h at 4 culture tubes, and the mixture was refrigerated for 2 h with occasional shaking. The cellulose was separated by centrifugation, the supernatant fluid was discarded, and the cellulose was suspended in 10 ml of mineral medium plus 0.5 ml of toluene and incubated at 39 C for 24 h in an atmosphere of CO2. The cellulose was sedimented, and the increment of soluble sugar in the supernatant fluid of each tube was measured (Fig. 3). Enzyme concentration. Various volumes of culture supernatant fluid were added to 5, 40, or 67.4 mg of PMC and adsorbed for 2 h at 4 C. The cellulose was sedimented by centrifugation, and the supernatant fluid was replaced with 10 ml of mineral medium and incubated 20 h (Fig. 4). In the first experiment, 10 ml of supernatant fluid gave the maximal (80%) digestion of 5 mg Ml Supernatont of cellulose. Above this amount, the extent of FIG. 4. Effect of amount of enzyme on amount of digestion decreased and then began to increase cellulose digested. again. With 40 or 67.4 mg of cellulose, the two curves were identical but differed from the 5-mg TABLE 2. Effect of temperature on cellulolytic trial in that digestion increased with increasing activity culture supernatant fluid up to 16 ml. Effect of the inorganic composition of the Increase" in soluble carbohydrate Incubation temp (mg) test medium. When enzyme was adsorbed on (C) 67.4 mg of PMC and sedimented and the a b supernatant fluid was removed and replaced, 18% of the cellulose was digested when 10 ml of 20 -0.7 -0.5 culture supernatant fluid was added, 10% when 30 4.4 4.3 fresh mineral medium (phosphate plus bicar- 39 18.7 18.1 45 23.2 22.2 bonate-CO2 buffer) was added, and 4.7% with 53 3.7 3.5 phosphate buffer. Incubation time was 20 h. In a second experiment the activity in the mineral a Supernatant fluid before incubation contained 5.6 medium was again more than twice that in the mg of soluble carbohydrates. VOL. 114, 1973 CELLULOLYSIS BY R. ALBUS 733 the cellulose. Tubes 5 to 8 were kept anaerobic I0 during adsorption. The enzyme activity was tested by centrifug- 0 ing all tubes after enzyme adsorption and re- placing the supernatant fluid anaerobically X6 / with 10 ml of anaerobic mineral medium. Oa (2 ~0 ml) was then injected into tubes 7 and 8, respectively, and all were incubated at 37 C for 20 h, after which the cellulose was sedimented and the soluble carbohydrate in the supernatant fluid was determined (Table 5). cn Extent of digestion. The extent of digestion a 0 . . . with time was examined by measuring soluble 5.4 56 5.8 6.0 6.2 6A4 6 sugar concentration after various intervals. Cul- PH ture supernatant fluid (15 ml) was adsorbed by FIG. 5. Effect of pH on cellulolytic activity. 60 mg of cellulose for 2 h at 0 C. The cellulose was collected by centrifugation and washed C. The cellulose was sedimented by centrifuga- once with mineral medium before being in- tion. The supernatant fluid was removed and adsorbed with a second batch of cellulose. This TABLE 3. Extent of adsorption on cellulose cellulose, in turn, was sedimented and the Cellobiose was discarded. Mineral medium Cellobiose from fromise supernatant Sample first adsorption from tecond (25 ml) was added to each cellulose sediment (mg) adsorption (with its adsorbed enzyme) and incubated at 39 (mg) C for 21 h (Table 3). The results indicate a ratio A 19.6 2.7 of adsorbed to dissolved enzyme of about 6.5 B 14.1 2.9 under the conditions of the experiment. At 39 C C 18.3 2.5 the degree of adsorption was about the same as at 4 C. Average 17.3 2.7 The adsorption equilibrium was tested by desorption of enzyme previously adsorbed on TABLE 4. Effect of pH on desorption 100 mg of PMC at 4 C. The cellulose plus adsorbed enzyme was collected by centrifuga- pHpH Enzyme (mg/25~~Sugarml) Adsorbed/desorbed tion, and the supernatant fluid was discarded. The sedimented cellulose with adsorbed en- 5.7 Adsorbed 12.8 zyme was equilibrated 2 h at 4 C with 25 ml of 5.7 Deserbed 0 co mineral medium adjusted to a selected The pH. 6.7 Adsorbed 17.5 cellulose was then separated by centrifugation; 6.7 Desorbed 0.4 25 ml of mineral medium was added to the cellulose, and 100 mg of cellulose was added to 7.5 Adsorbed 27.5 10 the separated supematant fluid. They were 7.5 Desorbed 2.8 incubated at 39 C for 21 h. The enzymatic 8.1 Adsorbed 24.6 6 activity was measured at pH 6.7. The ratios of 8.1 Desorbed 3.9 enzyme adsorbed to desorbed at various pHs are shown in Table 4. TABLE 5. Effects of 02 on the enzyme activity Oxygen effect. The effect of oxygen on enzymatic activity was tested by distributing 10 Cellulose ml of sterile culture supernatant fluid anaerobi- digested cally into each of eight stoppered tubes contain- Tube Treatment (mg) ing CO. at atmnospheric pressure. °2 (5 ml each) a b was injected into tubes 1 to 4 and all eight tubes were shaken ovemight at 37 C. The contents of 1 and 2 Exposed throughout to O2 2.5 2.7 tubes 1 and 2, respectively, were added to 40 mg 3 and 4 Exposed overnight to 02 but 2.5 1.9 of sedimented cellulose and incubated for 2 h at reduced before adsorption 4 C under an of CO,, but the 5 and 6 Protected throughout from 02 4.0 4.2 atmosphere 7 and 8 Protected from O til enzy- 2.5 2.3 medium was not reduced. Tubes 3 and 4 were matic activity tested treated similarly except that H,S was added to Blanks No enzyme 0.2 0.0 reduce the contents before they were added to 734 SMITH, YU, AND HUNGATE J. BACTERIOL. cubated at 39 C with mineral medium plus between the seeming stability of the cellulolytic toluene. At intervals, 0.5-ml samples were re- enzymes and the limited digestion from a single moved analerobically and tested for soluble application of supernatant fluid suggested that carbohydrat,es (Fig. 6). hydrolysis was inhibited by its products. This The exteint of cellulose digestion with re- was tested by incubating 67.4 mg of PMC (i) peated applications of fresh culture supernatant with 25 ml of supernatant fluid in a dialysis bag fluid was te:sted. After incubation of 67.4 mg of surrounded by 470 ml of anaerobic minimal PMC at 39 C for 24 h with 25 ml of culture medium, (ii) with 25 ml of supernatant fluid supernatant fluid, the cellulose was sedi- plus 220 ml of added minimal medium, and (iii) mented, and[the supernatant fluid was removed with 25 ml of supernatant fluid with no addi- and replace(d with another 25 ml. This was re- tions. peated at intervals (Table 6). The increment in The cellulose in the undialyzed, undiluted soluble carb4ohydrate amounted to a total of 52.5 preparation decreased by 14.2 mg after 48 h, mg, calculat;ed as cellobiose, equivalent to 49.7 with no additional loss during further incuba- mg of cellul4ose. The weight of residual cellulose tion. The undialyzed, diluted supernatant (ii), was 17.6 mg. Further digestion of the PMC showed 8.1 mg of cellulose digested after 48 h could occur iwith further applications of enzyme, and 16.9 mg after 14 days. The cellulose in the but even in broth cultures some of the cellulose dialysis bag (i) showed 20.2 mg of cellulose never appeared to be as completely digested as digested after 48 h and 43.8 mg (64%) digested it was arourid colonies in agar. after 14 days. The results indicate inhibition of Inhibitioin of digestion. The discrepancy enzymatic activity by a small molecular product of digestion. The effects of several concentrations of cel- u lobiose were examined. To diminish changes in 0 the cellobiose concentration due to cellobiose 4C 6 production during the enzymatic attack, 25-ml i samples of culture supernatant fluid (RAM) j plus 42.5 of in ml 0 mg PMC 5 of water were .jI0 placed in dialysis sacs, each suspended in 270 0, ml of minimal medium containing the test concentration of cellobiose. All solutions and 02 . procedures were designed to avoid exposure to cI oxygen. The samples were incubated for 4 days, O ...a.and the remaining cellulose was then collected, FG 0 4 8 12 16 20 24 dried, and weighed. The percentage of cellulose Time of Incubation ( hours) digested is plotted in Fig. 7 against the percent- FIG. 6. Exttent of cellulose (PMC) digestion with age of cellobiose in the dialyzing solution. Even time. 0.01% cellobiose was inhibitory and a 1% concentration caused a 95% inhibition. TABLE 6. Extent of cellulose digestion obtained with Glucose also inhibited cellulose digestion, successive applications of fresh enzyme during each though not as effectively as cellobiose, 0.5% 24-h interval glucose showing a 61% inhibition. Cellulase from cellobiose and cellulose Timea oforiginal Total Residual cultures. The effect of cellobiose on cellulase (days) celluloseb digested cellulose formation was examined by comparing the digested (mg) (mg) sugar formed by the supernatant fluid of 7-day 1 14.2 9.6 cellulose with that from 22-h cellobiose cul- 2 17.1 2192 tures. These supernatant fluids (10 ml of each) 4 16.1 32.1 were adsorbed with 40 mg of PMC for 2 h at 4 C 5 7.1 36.9 and then centrifuged, the supernatant fluids 7 10.2 43.8 were discarded, 10 ml of mineral medium was 8 3.8 46.4 added, and the solution was incubated at 39 C. 11 2.8 48.3 After 4 h and at 27 h a tube was removed and 12 2.2 49.8 centrifuged, and the soluble sugar was deter- 13 49.8 17.6 mined in the supernatant fluid. Results on total ' Time of irncubation and of replacement superna- amounts are shown in Table 7. The table tant fluid. includes also the results from a second experi-

Original CEellulose = 67.4 mg. ment with a different batch of PMC. The VOL. 114, 1973 CELLULOLYSIS BY R. ALBUS 735 showed more glucose than cellobiose, with both the aniline phthalate and the sulfuric acid 1 \ reagent. Aniline phthalate showed with the 30 _ CMC a smudge of slower-moving material, and the sulfuric acid showed most of the carbon in a very black spot at about the cellotetraose posi- S tion. The aniline phthalate reaction gave simi- 0 20 _ lar results with PMC as enzyme substrate, with e another band due to cellotriose. With sulfuric acid, additional bands of cellotetraose and higher-weight polymers from PMC were demon- 0 .05 .10 J5 20 ' O 1.0 strated. Initial Celloblose Concentrotion The increase in copper reduction values of the FIG. 7. Inhibition of cellulase activity by cellobi- supernatant fluid acting on PMC or CMC was ose. measured in two experiments (Fig. 8). In experiment 2 only 10% as much supematant fluid was TABLE 7. Comparison ofsugars produced by enzymes added to the CMC substrate as to the PMC. In from cultures grown on cellulose and cellobiose both experiments, the Cu reduction values for Soluble sugar (mg/10 ml) the two substrates were similar after long incubation. A parallel test on viscosity changes in Expt Cellulose- Cellobiose- CMC showed that within 2 h the viscosity fell grown cells grown cells rapidly to a level only slightly higher than that Expt 1 at 20 h. Most of the change came within the first 4 h 1.5 1.3 20 min. 20 h 2.7 3.8 Chromatography of the products of enzyme Expt 2 from cellulose-grown cells acting on CMC and 20 h 6.5 7.4 on PMC in experiment 1 gave the results shown in Fig. 9. Higher-weight polymers were obtained with each substrate, but those from the CMC amount of enzymatic activity of the cellobiose contained more glucose residues than did the culture was slightly higher than that of the ones from PMC. The RAM strain was used in cellulose culture, although both substrates were these experiments. It consistently produced used at 0.2% concentration. mannose in concentrations about equal to the The supematant fluids from this second ex- glucose, presumably because the supernatant periment were deionized, concentrated to a fluid contained cellobiose epimerase (1). small volume, and spotted for thin-layer chromatography. For the enzyme from both cellobiose- and cellulose-grown cultures, the cellobiose 0 spot was more intense than the glucose spot, as 0 tested with aniline phthalate. For the cellulose- grown enzyme, an additional three bands, interpreted as cellotriose, cellotetraose and cellopen- taose, were visible with sulfuric acid, and in addition a band 4 mm from the origin was very Co4X,0 dark. With sulfuric acid, the enzyme from cellobiose-grown cells showed only cellotriose and traces of cellotetraose as additional products. The products of action of enzyme from cellulose-grown cells on 40 mg of PMC and on 40 Incubotion Time (hours) mg of CMC were compared by thin-layer chro- FIG. 8. Digestion of CMC and PMC by superna- matography. Cell-free supernatant fluid (10 ml) tant fluid from culture grown on 0.2%o PMC. Expt 1: was used as enzyme source, with incubation for Decrease in viscosity of 40 mg of CMC/10 ml of 40 h at 39 C. Approximately the same amount of supematant fluid, arbitrary units, A; Cu-reducing material formed from 40 mg of CMC, A; Cu-reducing Cu-reducing material was formed from each material formed from 40 mg of PMC, 0. Expt 2: substrate, equivalent to 10 mg of cellobiose. Cu-reducing material formed from 40 mg of CMC by I This concentration of products probably inhib- ml of supernatant fluid plus 9 ml of mineral medium, ited further digestion in both cultures. *; Cu-reducing material formed from 40 mg of PMC Chromatography of the products from CMC by 10 ml ofsupernatant fluid, 0. 736 SMITH, YU. AND HUNGATE J. BACTERIOL. milled Whatman no. 1 filter paper cellulose. Desiccation of the crystalline cellulose (Sigma- a b cel) lessened its digestibility as compared to the material kept continuously in water after the acid treatment. But the effectiveness of the enzyme preparation in also digesting the crystalline material indicates that a true "cellulase" is formed. The more rapid digestion of CMC than of PMC may be due to a more rapid attack on this substrate by the enzyme, but it could also result FIG. 9. Chromatograph (TL) of products of di- from the greater solubility of the CMC. Since gested CMC and PMC, as detected with aniline cellulose is insoluble, its concentration is a phthalate (a) or H~SO, (b). 1, Products from 40 mg of relatively meaningless term and cannot be com- CMC digested in 10 min at 39 C by 10 ml of pared to the concentration of the soluble sub- supernatant fluid. 2, Cellobiose (Rf 0.39), glucose (R, strates. The cellulose surface exposed to the 0.52),(R.and mannose 0.56) standards. The glucose action of the enzyme is the limiting factor, and and mannose spots are not well separated. 3, Products from 40 mg of PMC digested 7 h in 10 ml of this is not easily equated with concentration. supernatant fluid. Because the natural cellulosic substrates of importance are insoluble, studies on the PMC DISCUSSION better reflect factors influencing cellulase activ- Loss in weight of insoluble PMC is a useful ity in nature. A cessation of digestion of both practicable index to the cellulolytic capacities CMC and PMC at about the same concentra- of the cell-free fluid from cultures grown on tion of accumulated Cu-reducing sugars sug- either cellobiose or cellulose. The slightly gests that sugar inhibits the digestion of both greater amount of activity from cellobiose- substrates. grown cultures could be due to adsorption of The experiments on inhibition by oxygen and some cellulase on the small amounts of residual on sorption are consistent with a postulate of cellulose left after growth of the bacteria, or to multiple cellulolytic enzymes in the culture the greater age of the cellulose cultures. The supernatant fluid. The partial inhibition by enzyme from cellulose and cellobiose-grown oxygen can be interpreted as a complete inhibi- cells formed generally similar digestion prod- tion of one or more, but not all, of the enzymes, ucts. and the difference in the adsorption equilibrium No inducation of suppression of cellulase shown for the activity in the supernatant fluid production by cellobiose was noted, in agree- as compared to that in adsorbed enzyme may ment with the results of Hammerstrom et al. (3) also be due to differences in the adsorption that cellulase was constitutive in Clostridium ratios of different enzymes. thermocellum. The effect of cellobiose is to Additional evidence of multiple enzymes was inhibit the action of the enzyme rather than its obtained when a cellulose agar culture tube of production. The effectiveness of low cellobiose strain 6 was observed (Fig. lb) to contain the concentrations suggests that in very limited mutant colony, strain 6L, forming an oval microhabitats, such as the space between cell clearing with an axial ratio of length/width and cellulose, cellobiose may affect cellulolytic much greater than in the parent type. Mutants activity. The advantage to Ruminococcus of forming circular areas of clearing, with central cellobiose inhibition of cellulase may lie in a colony, were observed in the RAM strain after it diminution of the cellobiose supplied to com- had been cultured continuously for several years peting non-cellulolytic species. Also, the total before it was finally lost. Also mutants not cellulose available to Ruminococcus would be digesting cellulose were observed. conserved. An essentiality of close proximity to cellulose has been proposed (6) to explain the ACKNOWLEDGMENTS inability of cellulolytic Ruminococcus and This investigation was supported by Public Health Service grant AI-07406 from the National Institute of Allergy and Butyrivibrio cells to decompose cellulose in the Infectious Diseases. lower dilutions of cellulose agar series inoculated directly from rumen contents. This con- LITERATURE CITED trasts with their rapid dissimilation of cellulose when grown in broth cultures. 1. Amein, M., and J. M. Leatherwood. 1969. Mechanism of cellobiose epimerase. Biochem. Biophys. Res. Com- Crystalline cellulose from absorbent cotton mun. 36:222-227. was less rapidly digested than was pebble- 2. Fusee, M. C., and J. M. Leatherwood. 1972. Regulation of VOL. 114, 1973 CELLULOLYSIS BY R. ALBUS 737 cellulase from Ruminococcus. Can. J. Microbiol. 9. Kitts, W. D., and L. A. Underkofler. 1954. Hydrolytic 18:347-353. products of cellulose and the cellulolytic enzymes. Agr. 3. Hammerstrom, R. A., K. D. Claus, J. W. Coghlan, and R. Food Chem. 2,639-645. H. McBee. 1955. The constitutive nature of bacterial 10. Leatherwood, J. M. 1965. Cellulase from Ruminococcus cellulases. Arch. Biochem. Biophys. 56:123-129. albus and mixed rumen microorganisms. Appl. Micro- 4. Hungate, R. E. 1957. Microorganisms in the rumen of biol. 13:771-775. cattle fed a constant ration. Can. J. Microbiol. 11. Leatherwood, J. M. 1969. Cellulose complex of Rumino- 3:289-311. coccus and a new mechanism for cellulose degradation, 5. Hungate, R. E. 1963. Polysaccharide storage and growth p. 53-59. In G. J. Hajny and E. T. Reese (ed.), Cellu- efficiency in Ruminococcus albus. J. Bacteriol. lases and their applications. Advances in chemistry 86:848-854. series, no. 95. American Chemical Society, Washington, 6. Hungate, R. E. 1966. The rumen and its microbes. D.C. Academic Press Inc., New York. 12. 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