Studies on the Nitrogen Requirements of Some Ruminal Cellulolytic Bacteria.

Knowledge of the nitrogen compounds utilized for growth of ruminal cellulolytic bacteria is of considerable importance to an understanding of the nitrogen economy of ruminants and of factors affecting ruminal cellulose digestion. Work with mixed cultures of bacteria from the rumen has shown that cellulose digestion occurs with ammonia or urea, which rapidly yields ammonia, as the sole source of nitrogen (Burroughs et al., 1951). These workers and others showed that cellulose digestion was stimulated if protein was included and that ammonia or urea stimulated cellulose digestion above that obtained with protein as nitrogen source. Others have shown that certain amino acids can replace the protein in stimulation of cellulose digestion. These included valine, leucine, and isoleucine (MacLeod and Murray, 1956), valine, leucine, isoleucine, and proline (Dehority et al., 1957) and proline, alanine, and methionine (Trenkle, Cheng, and Burroughs, 1957). Hall et al. (1954) obtained stimulation of cellulose digestion above that obtained with ammonia with materials believed to be peptides, whereas Bentley et al. (1954) obtained similar results with xanthine, uracil, guanine, and adenine. In a survey of nitrogen compounds replacing urea as the sole source of nitrogen for cellulose digestion by the mixed population, Belasco (1954) found that amides of certain monocarboxylic acids, guanidine, creatinine, creatine, uric acid, and allantoin were quite active. In work with mixed cultures, it was never certain whether or not the nitrogen compounds stimulating cellulose digestion had a direct effect on cellulolytic organisms. The work of MacLeod and Murray (1956) and Dehority et al. (1957, 1958) indicated that volatile fatty acids such as n-valeric plus isovaleric acids could replace the amino acids when urea was the only nitrogen source. Both groups of workers suggested that the amino acids were stimulatory because they were converted by other ruminal organisms to the active volatile fatty acids. Indeed, work with pure cultures indicates that many cellulolytic strains of the genus Ruminococcus and Bacteroides succinogenes require volatile fatty acids such as isovaleric or isovaleric plus n-valeric acids for growth even when supposedly adequate levels of amino acids are available (Allison, Bryant, and Doetsch, 1958; Bryant and Doetsch, 1955); and it is also known that other ruminal microorganisms produce these acids from amino acids (El-Shazly, 1952; Dehority et al. 1958; Bladen, Bryant, and Doetsch, 1961) and n-valerate can be produced from carbohydrate (Elsden et al., 1956). Although a considerable number of pure cultures of cellulolytic bacteria of undoubted significance in the rumen have been isolated in recent years, relatively little work has been done on their nitrogen requirements for growth. Fletcher (1956) and Ayers (1958) considered amino acids (casein hydrolyzate) to be essential for growth of the type strain of Ruminococcus albus and a strain of Ruminococcus flavefaciens, respectively, when grown in media also containing ammonia. The latter strain also required adenine and guanine. Allison, Bryant, and Doetsch (1959) found that very little UL-C'4 chorella protein hydrolyzate was incorporated into cells of R. flavefaciens strain C94, grown with ammonia as the main source of nitrogen, and suggested that the organism synthesizes most of its amino acids in preference to utilization of exogenous preformed amino acids. Unpublished data of Allison indicate that this strain will grow with ammonia as the sole source of nitrogen. B. succinogenes did not require any amino acid for growth, grew quite well with only cysteine and ammonia present in the medium, and required ammonia for growth even in the presence of 19 amino acids, purines, and pyrimidines (Bryant, Robinson, and Chu, 1959). Gill and King (1958) found no absolute requirement for any single amino acid by a cellulolytic strain of the genus Butyrivibrio but no growth occurred when cysteine was the only amino acid available. Quite good growth was obtained with only histidine, isoleucine, methionine, lysine, cysteine, tyrosine, and valine available. It was of interest that large amounts of ammonia were assimilated and this was not appreciably affected by the amount of casein hydrolyzate in the medium. The purpose of the present work was to explore the nitrogen requirements of strains of R. albus and R. flavefaciens and to obtain further information on the nitrogen compounds utilized for growth of B. succinogenes.