Isolation and characterization of proteolytic ruminal bacteria from sheep and goats fed the tannin-containing shrub legume Calliandra calothyrsus

Tannins in forages complex with protein and reduce the availability of nitr ogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from th e rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% conde nsed tannin) were isolated on proteinaceous agar medium overlaid with eithe r condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin, Fifte en genotypes were identified, based on 16S ribosomal DNA-restriction fragme nt length polymorphism analysis, and all were proteolytic and fermented pep tides to ammonia, Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), wh ile the other isolates did not utilize or had low growth on these substrate s. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin, One isola te, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone, Phylogenetic analysis of the 16S rib osomal DNA sequence showed that Lp1284 was related (97.6%) to Clostridium b otulinum NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a pro teolytic, ammonia-hyperproducing bacterium from the rumen, In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with c ondensed tannin.