Ammonia Production by Ruminal Microorganisms and Enumeration, Isolation, and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids from the Sheep Rumen

Abstract

Excessive NH 3 production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH 3 production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH 3 production. Ruminal fluid was taken from four rumenfistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH 3 production from Trypticase varied from 1.8 to 19.7 nmol mg of protein ؊1 min ؊1 depending on the substrate, its concentration, and the method used. Monensin (5 M) inhibited NH 3 production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P ‫؍‬ 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 M monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH 3 at >250 nmol min ؊1 mg of protein ؊1 , depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium-and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH 3 production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.Deamination of amino acids in the rumen, which leads to the loss of NH 3 across the ruminal wall, is one of the main causes of inefficient N retention by ruminants (23). For many years, it had been assumed that NH 3 formation was carried out by some of the numerically predominant species of ruminal bacteria that had been identified as producing NH 3 weakly from protein or protein hydrolysates (3). However, Russell and his colleagues (7,8,12,35,36) calculated that these bacteria did not have sufficient activity to account for observed in vitro rates of NH 3 production by the mixed population in their cattle, and they isolated bacteria which were much less numerous than the others but which possessed a specific activity of NH 3 production from Trypticase which was an order of magnitude greater than that of the other species. Moreover, these bacteria, unlike the others, were gram positive and highly sen...

Nutrient digestion, microbial protein synthesis, and blood metabolites of Jersey heifers fed chitosan and whole raw soybeans

“…The decrease in ruminal ammonia production when monensin is supplied can be attributed to inhibitory effects on hyper-ammonia-producing bacteria (Eschenlauer et al, 2002) which have peptidase and deaminase activities (Wallace et al, 1997). Chitosan may have the same effect of monensin in ruminal protein degradation.…”

Cloacibacillus evryensis gen. nov., sp. nov., a novel asaccharolytic, mesophilic, amino-acid-degrading bacterium within the phylum 'Synergistetes', isolated from an anaerobic sludge digester

“…the rumen (Debroas & Blanchart, 1993), and has been restricted to molecular inventories in protein-containing wastewaters (Tang et al, 2005). The major difference between protein degradation in the rumen and in anaerobic digesters is that, in the rumen, the ruminal bacteria (both saccharolytic and asaccharolytic) grow on amino acids and peptides as major energy substrates (Eschenlauer et al, 2002). In anaerobic reactors, however, proteolytic bacteria predominantly mediate protein degradation and the process involved is energy yielding (Ramsay & Pullammanappallil, 2001).…”

Olsenella umbonata sp. nov., a microaerotolerant anaerobic lactic acid bacterium from the sheep rumen and pig jejunum, and emended descriptions of Olsenella, Olsenella uli and Olsenella profusa

“…These species were based on strains from gingival and subgingival sites in humans with periodontitis. A third species with one strain (A2) from the rumen of a sheep had been informally named 'Atopobium oviles' (Eschenlauer et al, 2002) and was renamed 'Olsenella oviles' by Dewhirst et al (2001).…”

“…The methods and results of isolation, 16S rRNA gene sequencebased identification and initial phenotypic characterization were described in detail by Eschenlauer et al (2002). Strains lac15, lac16 and lac31 T were isolated from the mucosal jejunum of a pig that also harboured lac18 T , the type strain of Veillonella magna, at the Institute of Animal Nutrition in 2007.…”