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, N McKain, N D Walker, N R McEwan, C J Newbold, R J Wallace

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Abstract

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 muM) 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 mu 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 111, 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 1, 11, 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.

Original languageEnglish
Pages (from-to)4925-4931
Number of pages7
JournalApplied and Environmental Microbiology
Volume68
Issue number10
DOIs
Publication statusPublished - Oct 2002

Keywords

  • 16S ribosomal-RNA
  • bacteroides-ruminicola
  • prevotella-ruminicola
  • protein-degradation
  • nitrogen-metabolism
  • streptococcus-bovis
  • SP-NOV
  • monensin
  • invitro
  • deamination

Cite this

Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen. / Eschenlauer, S C P ; McKain, N ; Walker, N D ; McEwan, N R ; Newbold, C J ; Wallace, R J.

In: Applied and Environmental Microbiology, Vol. 68, No. 10, 10.2002, p. 4925-4931.

Research output: Contribution to journalArticle

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T1 - Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen

AU - Eschenlauer, S C P

AU - McKain, N

AU - Walker, N D

AU - McEwan, N R

AU - Newbold, C J

AU - Wallace, R J

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N2 - 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 muM) 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 mu 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 111, 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 1, 11, 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.

AB - 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 muM) 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 mu 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 111, 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 1, 11, 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.

KW - 16S ribosomal-RNA

KW - bacteroides-ruminicola

KW - prevotella-ruminicola

KW - protein-degradation

KW - nitrogen-metabolism

KW - streptococcus-bovis

KW - SP-NOV

KW - monensin

KW - invitro

KW - deamination

U2 - 10.1128/AEM.68.10.4925-4931.2002

DO - 10.1128/AEM.68.10.4925-4931.2002

M3 - Article

VL - 68

SP - 4925

EP - 4931

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 10

ER -