2009 Annual Report 1a.Objectives (from AD-416) 1). To isolate and identify ruminal bacteria responsible for ruminal lysine degradation. 2). To study the effect of the feed additive, monensin, and bacteriocins on lysine degradation. 1b.Approach (from AD-416) Lysine is often an amino acid that limits milk production, but bacteria responsible for wasteful ruminal lysine degradation have not yet been identified. Mixed ruminal bacteria will be enriched with lysine in vitro, and bacteria capable of utilizing lysine will be isolated, characterized physiologically, tested for their susceptibility to monensin and bacteriocins, and identified via 16 S rDNA sequencing. Cattle will be supplemented with lysine to see if these same bacteria increase in vitro. The experiments with live cattle have been completed and cow is no longer needed. The manuscript entitled "Cattle will be supplemented with lysine to see if these same bacteria increase in vitro," was submitted one year ago and has been accepted. 3.Progress Report Because of their very rapid rate of amino acid fermentation, ruminal hyperammonia producing bacteria (HAB) can reduce the efficiency of protein utilization by ruminant animals. Quantitative data on the distribution of HAB among animals, the population sizes of HAB in the rumen, and the metabolic diversity of these bacteria are essential for including their activities in nutritional models. We have established enrichment cultures of HAB from the rumens of hay-fed and silage fed dairy cattle, and have demonstrated that amino acid degradation and the production of ammonia (0.15 M concentration, specific activity 0.4-0.5 micromoles ammonia per minute per mg cell protein) and expected volatile fatty acid products reach stability by the fourth enrichment culture transfer. Analysis of bulk DNA from the enrichments by the community fingerprinting method ARISA revealed that enrichments from hay-fed and silage fed cows developed similar bacterial community compositions. Sixteen strains of apparent HAB have been isolated from these enrichments. All display slower growth in pure culture than do the enrichments themselves, suggesting that other ruminal bacteria may be important in cross-feeding nutrients to the HAB. Research conducted under this agreement was monitored through phone calls between the ADODR and the Cooperator.