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The cellulolytic activity of cell-free extracts of 15 species of entodiniomorphid protozoa grown in vivo and under two conditions in vitro has been measured using six different assays with substrates varying in complexity from carboxymethylcellulose to microcrystalline cellulose. Although there were differences between the assays, the highest activities were found in Eudiplodinium maggii, Epidinium ecaudatum caudatum and Ostracodinium obtusum bilobutn with little or no activity in five Entodinium spp. There was no consistent effect of growth conditions on cellulase content although with protozoa grown in vitro those grown on grass alone tended to contain more cellulase than those grown on starch and grass.A survey of methods for the release of cellulase from other rumen fractions was made and mild treatment with ultrasound of the fraction suspended in water gave the maximal release of enzyme with the highest specific activity. Using this method 62 % of the total rumen cellulase was found in the protozoal fraction and 27 % was associated with the plant debris in a normal sheep, fed on hay and oats, with an A-type protozoal population.

SUMMARYWashed suspensions of Entodinium caudatum grown in vitro and incubated anaerobically incorporated 14C from [14C]glucose into the cell pool as [ 1 4 C ] g l~~~~e , maltose and hexose phosphate and into protozoal polysaccharide and intracellular bacteria as a glucose polymer. The uptake of sugars into the pool was by an active process (predominant at low sugar concentrations) probably into the protozoal endoplasm, and by a passive process (predominant at high concentrations) into another part of the cell. Uptake of glucose by the active process was not sufficiently rapid to increase the level of glucose in the pool by more than 17% per hour; this was probably insufficient to increase appreciably the rate of glucose utilization. These protozoa hydrolysed starch to maltose and glucose and the level of these sugars in the pool of protozoa metabolizing starch grains was controlled in part by the inhibitory effect of these sugars on the enzymes that formed them. Studies on the enzymes involved in the metabolism of starch showed that the glucose in starch was hydrolysed to free glucose before phosphorylation. In experiments on the effect of carbohydrates on the survival of protozoa, starch was the most effective in prolonging protozoal life, followed by maltose and glucose in that order. The greater effectiveness of maltose was correlated with the finding that the rate of uptake of maltose carbon was always greater than that for glucose when the two sugars were present in equimolar concentrations. However, the rate for both sugars was markedly decreased in the presence of penicillin and neomycin. Evidence is presented that the protozoal ectoplasm may be freely permeable to sugars in the medium and that there is a barrier between the ectoplasm and endoplasm. 14C from r4C]starch grains was incorporated by the protozoa but there was no synthesis of protozoal protein from carbohydrate.