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.

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