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Applied and Environmental Microbiology, October 2002, p. 4925-4931, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4925-4931.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

S. C. P. Eschenlauer,{dagger} N. McKain, N. D. Walker, N. R. McEwan, C. J. Newbold, and R. J. Wallace*

Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom

Received 22 March 2002/ Accepted 9 July 2002

Excessive NH3 production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH3 production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH3 production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH3 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 µM) inhibited NH3 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 µM 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 NH3 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 NH3 production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.

* Corresponding author. Mailing address: Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, United Kingdom. Phone: 44 1224 716656. Fax: 44 1224 716687. E-mail: rjw{at}rri.sari.ac.uk.

{dagger} Present address: Parasitology Laboratory, University of Glasgow, Glasgow G12 8QQ, United Kingdom.

Applied and Environmental Microbiology, October 2002, p. 4925-4931, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4925-4931.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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