SUMMARYThe in vitro breakdown of degraded and undegraded varieties of cellulose was examined by using pure strains of bacteria isolated from the rumen of cattle. One strain of Bacteroides succinogenes, two strains of Ruminococcus albus and two strains of Ruminococcus jlavefaciens were allowed to ferment ground cellulose powder (prepared from filter paper), cellulose powder (Whatman) and undegraded cotton fibres, the extent of breakdown being followed by loss of weight of the insoluble substrate.All five organisms were highly active on degraded ground cellulose powder and dissolved 72-90 yo, but only one organism, B. succinogenes strain s-85, was equally effective on cellulose powder (Whatman) or on undegraded cotton fibres. R. jlavefaciens strain FD-1 was somewhat less potent on the latter substrates, achieving 40 and 60 yo dissolution, respectively, of cellulose powder (Whatman) and cotton fibres. R. albus strain 7 and R. Jlavefaciens strain c-94 had negligible effects on cotton fibres (10 and 0 Yo solubilization, respectively). R. albus strain D-89, producing 40 yo solubilization of cotton fibres, was intermediate in activity between R. albus strain 7 and R. Jlavefaciens strain FD-'I. Cell-free preparations from culture filtrates of B. succinogenes strain s-85 gave only 4 yo breakdown of ground cellulose powder and up to 9 yo breakdown of cellulose powder (Whatman) in 17 days. Cell-free filtrates from the metabolism fluid of R.flavefaciens strain FD-1 or from the disintegrated organisms brought about 46 and 36 yo solubilization, respectively, of ground cellulose powder, but failed to attack cotton fibres. The results support the view that the capacity of an organism or cell-free enzyme to attack any one particular form of cellulose is no criterion of its ability to attack less degraded or undegraded types of cellulose.

True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum . Such activity depends upon the presence of Ca 2+ 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 carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70°C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity.

1. A purified cellulolytic component C(1) was isolated free from associated activities of the cellulase complex and shown to act as a beta-1,4-glucan cellobiohydrolase on both simple and complex forms of native cellulose. 2. The enzyme releases terminal cellobiose units from cellulose, its extent of action being determined principally by the product and by the nature of the substrate. 3. Component C(x) of the cellulase system is not required for the action of component C(1) (cellobiohydrolase). The enzyme synergizes extensively with cellobiase in extending the hydrolysis of native and of less-complex forms of cellulose to at least 70% with the liberation of glucose. 4. The cellobiohydrolase is relatively unstable, with an optimum at pH5 and a K(m) of 0.05mg/ml. The enzyme is inhibited by its product, from which it is released by cellobiase. 5. Of other compounds tested against the cellobiohydrolase the metal ions Cu(2+), Zn(2+), phenylmercuric and Fe(3+) are increasingly effective inhibitors. Glucose has no action at concentrations found inhibitory with cellobiose. 6. The relationship of the enzyme to the entire cellulase complex is discussed.

1. The catalytic decomposition of undegraded cellulose in the form of cotton fibres is described with hydrogen peroxide at 0.4-0.04% (w/v) concentration in the presence of ferrous salts at pH3-5. 2. Complete solubilization of 5mg. of cotton fibres occurred in about 7 days in the presence of 0.4% hydrogen peroxide and 0.2mm-ferrous sulphate at the optimum pH4.2-4.3. 3. With 0.4% hydrogen peroxide the most rapid decomposition of cellulose was confined to ferrous sulphate concentrations of approx. 2-0.02mm. If the concentrations of the reagents were decreased in proportion extensive breakdown occurred but much more slowly. 4. In the primary stages of breakdown cotton fibres were disintegrated to very short fibres. These were subsequently solubilized, but there was little accumulation of soluble material. Organic matter was lost from solution as the reaction progressed. 5. Other naturally occurring cellulose-containing materials, such as grass, straw, hay and sawdust, were also disintegrated and solubilized by hydrogen peroxide and ferrous sulphate.

Although an extensive literature is available describing cell-free cellulolytic preparations which attack soluble cellulose derivatives, there is a dearth of information on the enzymic breakdown of insoluble cellulose, and virtually nothing is known about the breakdown of undegraded cellulose of the type associated with native cotton fibres [For reviews see Siu (1951), Halliwell (1959)]. Despite the failure of cell-free preparations made from them to attack undegraded cellulose, truly cellulolytic organisms can quantitatively transform all types of cellulose, undegraded or otherwise, to soluble products. The present report concerns an examination of the breakdown of an acid-swollen cellulose by culture filtrates from Myrothecium verrucaria. This substrate is an insoluble although degraded cellulose and was chosen firstly because the relatively mild method of preparation involves only small changes in the degree of polymerization (Walseth, 1952), and secondly only after having shown that our enzyme preparations produoed significant solubilization of this substrate. Conditions governing the enzymic breakdown of insoluble cellulose are described below and compared with the enzymic hydrolysis of the soluble cellulose derivative, carboxymethylcellulose. MATERIATLS AND METHODS Preparation of the culture filtrate Cultures of M. vemrcaria (I.M.I. 45541, Commonwealth Mycological Institute, Kew, Surrey), grown on the salt medium of Saunders, Siu & Genest (1948) with cellulose powder (Whatman) or de-waxed cotton fibres as substrate, were aerated at 280 by incubation in a reciprocal shaking machine operating at 60-90 strokes of 38 mm. horizontal movement per minute. After fermentation for 1-3 weeks most of the organisms and all of the residual cellulose were separated off by filtration through a sintered-glass filter (porosity 3). The filtrate was centrifuged at 54 000g for 30 min. at 10 and subsequently passed through a sinteredglass bacteriological filter (porosity 5/3). In the later work the final filtration was found to be unnecessary by inoculation on cellulose-agar slopes. The cell-free culture supernatant or filtrate fraction was used as the source of cellulolytic enzymes. For enzyme assays the following average volumes were used: for method (a) 0-2 ml. of undiluted filtrate; for method (b) 0.1 ml. of filtrate diluted 50 times.

Cellulolytic enzyme components of culture filtrates of Trichoderma koningii were fractionated on ionic and non-ionic forms of Sephadex and on cellulose powder (Whatman) and examined for their ability to hydrolyse soluble carboxymethyl-cellulose, and to saccharify, solubilize and form short fibres from native undegraded cellulose of the type found in cotton. DEAE-Sephadex provided two CM-cellulase components and a C(1) component; the C(1) component acted weakly and solely on cotton, forming soluble products but not short fibres. The ability to form short fibres was confined almost wholly to one of the CM-cellulase components which completely degraded cotton, minimally to soluble products and extensively to short fibres. The latter action was unaffected by the presence of the other two components. The two CM-cellulase components solubilized cellulose synergistically whereas the short-fibre-forming component and C(1) component were inhibitory.

SUMMARY: The optimal conditions for accurate and reproducible analysis of cellulose hydrolysis by micro-organisms, particularly from the m e n , were determined. Rumen micro-organisms were found to require approximately 50 mg. substrate (cellulose powder) in a p0 hr. incubation at 3 7 ' with 1 ml. of m e n liquor, and a total volume of 8 ml., in static vessels. All types of cellulose or simpler substituted derivatives, from soluble carboxymethylcellulose through insoluble cellulose powder to cotton fibres, are nearly quantitatively hydrolysed by mixed rumen micro-organisms. The effect of the inorganic composition of the medium, of antibacterial agents (thymol, fluoride, toluene) and of concentration procedures on the cellulolytic activity of rumen micro-organisms was examined. Rumen protozoa appear to play a minor role in cellulose degradation; their effective activity is due mainly to closely associated, possibly ingested, bacteria. The validity of the results obtained by various methods of ' cellulase ' assay is discussed.

No abstract