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Appl Environ Microbiol. Oct 2002; 68(10): 4925–4931.
PMCID: PMC126416

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 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.

Deamination of amino acids in the rumen, which leads to the loss of NH3 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 NH3 formation was carried out by some of the numerically predominant species of ruminal bacteria that had been identified as producing NH3 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 NH3 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 NH3 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 sensitive to monensin. Since ruminal NH3 concentrations are lower when ruminants receive this dietary ionophore, it was deduced that the new species must consist of significant NH3 producers in vivo. The species isolated, Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum (29), were able to grow rapidly on Trypticase as the sole energy source and were atypical of the most numerous ruminal species, but even at a low population size they had deaminative activity sufficient to contribute significantly to NH3 production by the mixed population in vivo.

Subsequently, similar bacteria—so-called ammonia-hyperproducing (HAP) bacteria—have been isolated in New Zealand (2) and Australia (26). In the former study, Attwood et al. (2) isolated 14 morphologically different species from pasture-grazed cows, sheep, and deer. The isolates were similar in function to the Russell et al. HAP species, but all were genotypically different. A greater diversity of HAP species was also indicated in the isolates made by McSweeney et al. (26) from goats receiving tannin-rich Calliandra calothyrsus. Furthermore, some of the isolates were saccharolytic and/or proteolytic, suggesting that the HAP niche is not occupied only by asaccharolytic organisms but also by organisms with wider biochemical functions.

Rates of NH3 production in ruminal fluid appear to vary greatly depending on diet, and it is not always necessary to invoke the activity of the high-activity NH3 producers to explain observed rates of NH3 production by the mixed ruminal population (42). The aims of the present experiments were to measure NH3 production by different methods and, using different substrates, to enumerate and characterize the high-activity Trypticase fermenters in sheep with apparently lower rates of NH3 production, and, from the effects of monensin, to assess their role in NH3 production in the mixed ruminal population. The results explain previous contradictions in measurements of NH3 production rates, both in pure and mixed cultures, and extend significantly the range and knowledge of metabolic functions of HAP bacteria isolated from ruminants, in this case from sheep.

(S.C.P.E. undertook some of these experiments as part of an exchange agreement between the Institut Universitaire de Technologie, Université d'Auvergne, Clermont-Ferrand, and the Robert Gordon University, Aberdeen.)

MATERIALS AND METHODS

Animals and sampling.

Four rumen-fistulated sheep received a maintenance diet of hay, barley, molasses, fishmeal, and vitamins and minerals (500, 299.5, 100, 91, and 9.5 g kg of dry matter−1) twice daily. Samples of ruminal fluid were removed 2 to 3 h after feeding, kept warm and under CO2, and strained through four layers of muslin.

Measurement of ammonia production in ruminal fluid in vitro.

Strained ruminal fluid from the sheep was incubated in vitro under CO2 in a shaking water bath at 39°C with or without added substrate at 2 or 20 mg ml−1, with or without 5 μM monensin. This concentration was based on that used by Chen and Russell (10), which is similar to the estimated monensin concentration in vivo (46). Substrates included casein (Sigma, Poole, Dorset, United Kingdom), Trypticase Peptone (Becton Dickinson Microbiology Systems, Cockeysville, Md.), soluble soybean protein, Soya Peptone (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom), and an amino acid mixture based on the composition of casein. Soluble soybean protein was prepared by dissolving 100 g of soybean flour (type I; Sigma) in 500 ml of distilled water at room temperature for 2 h, filtering through Whatman no. 1 filter paper and centrifuging the filtrate at 10,000 × g for 15 min. The amino acid mixture comprised Gibco casein hydrolysate no. 5 (Life Technologies Ltd., Paisley, United Kingdom) plus added l-tryptophan (0.87%), l-methionine (0.17%), and l-cysteine (0.14%). Monensin (5 μM; Sigma) was added as 0.32 ml of a 10-mg ml−1 solution in ethanol per liter of strained ruminal fluid at zero time. Ammonia production was linear during the incubations, and rates of NH3 production were determined by linear regression. Samples (8 ml) were removed at 0, 0.5, 1, 2, 4, 6, and 8 h into 2 ml of 25% perchloric acid; chilled on ice for 15 min; and then centrifuged at 27,000 × g for 20 min. The supernatant was neutralized with KOH, and the precipitate of potassium perchlorate was sedimented by repeating the centrifugation. Ammonia was determined in the supernatant fluid by an automated phenol-hypochlorite method (50), and protein was determined on the perchloric acid precipitate using the Folin reagent as described before (47).

A second method, used by Russell et al. (36), in which ruminal fluid is diluted fourfold in basal growth medium and incubated for 6 h, was carried out for comparison. We referred to this method as the batch culture type of incubation. Two samples were removed from each sheep on separate days. Each sample was incubated in triplicate. Ammonia and protein concentrations were measured at 0 and 6 h. The specific rate of NH3 production was calculated from these values in two ways, using either the protein concentration at zero time or the average of the 0- and 6-h samples.

Bacterial counts.

Samples of strained ruminal fluid were diluted serially 10-fold under CO2 in a vitamin and mineral medium with no carbohydrate source, based on that described by Chen and Russell (12). The basal medium contained, per liter, 292 mg of K2HPO4, 292 mg of KH2PO4, 480 mg of Na2SO4, 480 mg of NaCl, 100 mg of MgSO4 · 7H2O, 55 mg of anhydrous CaCl2, 1.0 ml of 0.1% resazurin, 600 mg of cysteine hydrochloride, and vitamin and mineral solutions (12). The medium was adjusted to pH 7.0 before autoclaving. These dilutions were used to inoculate (1%, vol/vol) Hungate tubes containing four different liquid media: medium A, complete medium M2 (20); medium B, basal medium plus Trypticase Peptone (15 g/liter; Becton Dickinson Microbiology Systems); medium C, medium B plus 5 μM monensin; medium D, basal medium plus Casamino Acids (15 g/liter; Difco, Becton Dickinson Europe, Meylan, France). Five tubes were inoculated for each dilution, the gas phase was 100% CO2, and tubes were incubated at 39°C. The optical density at 650 nm was determined periodically using an LKB Novaspec spectrophotometer. The population size exceeding a given optical density was determined using most-probable-numbers tables (1).

Plate counts.

The same dilutions of ruminal fluid were used to inoculate petri dishes of medium B in agar (2%). Plates were incubated at 39°C for 7 days. All discernible colonies were counted. All dilutions, inoculations, and incubations were done in an anaerobic chamber with a CO2-N2-H2 (10/85/5) atmosphere.

Isolation and identification of Trypticase utilizers.

Dilutions of ruminal fluid were made as before and inoculated into medium B. The cultures were incubated at 39°C for 7 days, and then 20 μl of the 10−6 and 10−7 dilutions were streaked crosswise onto plates of medium B and incubated again for 7 days. Colonies of different morphology were picked and moved into liquid medium M2 (20). After overnight growth, cultures were used to inoculate tubes containing medium B and medium B to which had been added (1% each) glucose, maltose, xylose, and cellobiose. Growth was assessed turbidimetrically as before. Fermentation products were determined by capillary gas-liquid chromatography of supernatants from cultures grown in liquid medium M2 (31). Morphological, physiological, and biochemical characteristics were determined by standard methods (21), and API 20A strips (bioMérieux, Lyon, France) were used to determine sugar utilization, indole production, and gelatin liquefaction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with SDS-treated whole cells using 8 to 18% acrylamide gradient gels (Amersham Biosciences UK Ltd., Little Chalfont, Bucks, United Kingdom).

Deaminase and peptidase activities of isolates.

Ammonia production was determined using overnight cultures of bacteria grown in liquid M2 medium (20) or in medium B plus 1% sugars. Deaminase activity was estimated from the rate of NH3 production in pure batch cultures incubated with Trypticase (15 mg ml−1) as the sole energy source, using culture medium (medium B) and methods described by Russell et al. (36). Peptidase activities were measured using overnight M2-grown cultures only. Cells were harvested by centrifuging at 27,500 × g for 15 min at 4°C. Pellets were washed once with anaerobic 25 mM potassium phosphate (pH 7.0), resuspended to half the original volume in the same buffer, and used in peptidase assays in multiwell plates. All operations except the incubation of the plates were carried out under CO2. The assays contained 100 μl of 0.5 mM Ala2-p-nitroanilide (Ala2-pNA) or GlyArg-4-methoxynaphthylamide (GlyArg-MNA) dissolved in anaerobic 25 mM potassium phosphate buffer (pH 7.0), 50 μl of 4 mM dithiothreitol, and 50 μl of cell suspension. Peptidase activities were estimated from the release of p-nitroaniline or 4-methoxynaphthylamine (49).

Deaminase and peptidase results are the means of determinations carried out with three different cultures. Deaminase was calculated from NH3 concentrations at the beginning and end of 6-h incubations, as described by Russell et al. (36). Peptidase activity was calculated by linear regression of product formation measured hourly for 3 h.

rDNA analysis.

DNA from isolates was either extracted by methods described by McEwan et al. (25) or by using Qiagen Ltd. (Crawley, West Sussex, United Kingdom) genomic 100/G tips, following the manufacturer's guidelines. Partial sequences of 16S ribosomal DNA (rDNA) were amplified using universal primers (18) forward, P0124, 5′-CACGGATCCGGACGGGTGAGTAACACG, and reverse, P1394, 5′-TGGTGTGACGGGCGGTGTGT, in a mixture which contained a 1 μM concentration of each of the primers, a 200 μM concentration of each of the deoxynucleoside triphosphates, 50 mM KCl, and 1.5 M MgCl2 in 10 mM Tris-HCl (pH 8.3) buffer. Genomic DNA (0.1 μg) and 2.5 U of Taq DNA polymerase were added to 100 μl of this mixture, and amplification was carried out using 35 cycles of 94°C for 1 min followed by 55°C for 1 min and 72°C for 3 min. Amplification was confirmed by agarose gel electrophoresis. On one occasion (isolate A2) these primers failed to produce a product and PCR was repeated using the above conditions, but replacing the reverse primer, P1394, with an alternate reverse primer, P1115, 5′-GTGAAGCTTAGGGTTGCGCTCGTTG. This primer change allowed a fragment of the A2 16S rDNA to be amplified and used for identification analysis.

Amplified DNA was cloned into the PCR2.1-TOPO vector (Invitrogen BV, Leek, The Netherlands) following the manufacturer's instructions. Plasmids were isolated from recombinant colonies using WizardPlus SV Minipreps (Promega), following the manufacturer's instructions, and the nucleotide sequence of the insert was determined using an ABI Prism 377XL DNA sequencer.

DNA similarity searches were performed using the BLAST server at http://genome.eerie.fr/bin/blast-guess.cgi

Bacteria.

Prevotella albensis M384 was originally isolated from a sheep rumen at the Rowett Research Institute (43). C. aminophilum (ATCC 49906), C. sticklandii (ATCC 12662), and P. anaerobius (ATCC 27337) were isolated from enrichments inoculated with bovine ruminal fluid (12, 36).

Nucleotide sequence accession numbers.

The 16S rDNA sequence of strain 6 has been deposited in the GenBank data library under accession number AJ310135, with the provisional name Eubacterium pyruvivorans. Isolate A2 was deposited as AJ251324, provisionally called Atopobium oviles. The Desulfomonas isolate B1 is AJ318100. Isolate 4 was not named, and its partial 16S rDNA sequence was deposited as AJ318205.

RESULTS

Ammonia production in ruminal fluid in vitro.

The apparent rate of NH3 production by mixed ruminal microorganisms depended on the substrate, the concentration of substrate, and the method used (Table (Table1).1). NH3 was formed more than twice as rapidly from casein compared to soybean protein, while NH3 was formed from Trypticase, a pancreatic casein hydrolysate containing predominantly peptides, as rapidly as from casein. Soya Peptone was degraded more slowly than Trypticase, and free amino acids corresponding to the amino acid content of casein were degraded more slowly than the corresponding peptides. The rate of NH3 production was on average 56% lower with substrate added at 2 mg/ml than with that at 20-mg/ml substrates. The average protein content of strained ruminal fluid from these sheep was 7.6 mg ml−1; therefore, the specific activity of NH3 production from Trypticase (20 mg/ml) can be calculated to be 9.5 nmol mg of protein−1 min−1. At a substrate concentration of 2 mg ml−1, the apparent rate of NH3 production was lower, particularly for amino acids, with values of 4.8, 3.9, and 2.3 nmol mg of protein−1 min−1 for casein, Trypticase, and amino acids, respectively (calculated from Table Table11).

TABLE 1.
Influence of monensin on NH3 production by mixed ruminal microorganisms in vitro

In contrast, when NH3 formation was determined by diluting ruminal fluid in growth medium with Trypticase as the energy source and incubating for 6 h as described by Russell et al. (36), the mean specific activity was calculated to be 19.7 ± 11.0 nmol mg of protein−1 min−1, calculated using the protein concentration at zero time. The protein concentration increased during the course of the incubation, however, and if the average of the 0- and 6-h concentrations was used in the calculation, the values were 16.4 ± 8.7 nmol mg of protein−1 min−1.

Between 40 and 61% (average, 48%) of NH3 formation from proteins, peptides, or amino acids at 20 mg/ml was inhibited by 5 μM monensin. The range was 23 to 36% and the average was 28% with substrates at 2 mg/ml.

Assessment of population size of bacteria capable of growth on peptides and amino acids.

When dilutions of strained ruminal fluid were inoculated into liquid media in Hungate tubes, both the number of tubes showing growth and the cell density achieved increased with the time of incubation, although the relative proportions changed little with time (results not shown). Maximum growth occurred after 7 days. Most-probable-number calculations were carried out based on two criteria, namely, an optical density (OD) of >0.1 and an OD of >0.2. Approximately twice as many tubes grew to an OD of >0.1 as grew to an OD of >0.2 in all media (Table (Table2).2). Total numbers were about 109 ml−1, while numbers capable of growing on Trypticase and amino acids after 7 days were about 107 and 105 ml−1, respectively. Monensin decreased Trypticase fermenters by a factor of about 10.

TABLE 2.
Most-probable-number counts of Trypticase- and amino acid-utilizing bacteria in ruminal fluid from the sheep rumena

Plate counts on medium with Trypticase as the sole energy source gave colony numbers very similar to the most-probable-number counts, at (3.2 ± 0.70) × 107 ml−1 (mean ± standard deviation). The colonies were very small and took a week to develop.

Properties of isolates.

In order to determine if the bacteria capable of growth on peptides and amino acids were the same as those isolated elsewhere, 19 isolates were obtained from the highest dilutions of medium B from the four sheep and were characterized genetically and physiologically. They all failed to grow on nutrient agar in air. The growth of all the isolates was inhibited by 5 μM monensin. None of the isolates hydrolyzed gelatin. The bacteria were allocated to six groups based on morphology and fermentation products (Table (Table33).

TABLE 3.
Properties of Trypticase-utilizing bacterial isolates

Group I consisted of four isolates from sheep A. 16S rDNA analysis of isolate 4 indicated that the bacteria were most closely related to a Clostridium sp., isolated from an anaerobic digester, which uses gelatin as the sole source of carbon and energy (G. N. Jarvis and C. Stroempl, nucleotide sequence number AJ002593 [http://www.ncbi.nlm.nih.gov]). They did not ferment sugars; indeed adding sugars to medium B decreased the cell density obtained. Group I isolates produced a range of fermentation products, and two of the strains appeared to utilize a little of the lactate present in M2. Rates of NH3 production were 128 to 200 nmol mg of protein−1 min−1 in medium 2, increasing to 342 to 754 nmol mg of protein−1 min−1 in medium B.

Group II comprised two isolates from sheep B. They were also asaccharolytic, and their fermentation products were valerate, isovalerate, and caproate. A little lactate was utilized, as with group I, but this group was characterized by decreases in the concentrations of propionate and butyrate during growth on medium 2. Isolate 6 was most closely related to another ruminal Eubacterium sp. also isolated as a HAP species (2) and was also closely related to oral Eubacterium spp. (6). Rates of NH3 production from Trypticase were similar to those in group I, and they were also induced by growth in medium B.

Group III, isolates from three sheep, had much lower rates of NH3 production than the other groups and probably do not qualify as HAP. 16S rDNA analysis indicated that they were most closely related to Atopobium minutum. They grew on sugars added to medium B, and API strips indicated that they fermented glucose, sucrose, and maltose. The genus Atopobium contains organisms within the low-G+C-content gram-positive bacteria which were formerly classified as Lactobacillus or Streptococcus (14). Group III isolates similarly were lactate producers.

Group IV isolates were all from one sheep. SDS-PAGE patterns of their whole-cell protein indicated that there were a few bands slightly different. Their fermentation patterns were similar to those from group II, except that they produced acetate and the caproate produced was about half of the amount produced by group II. 16S rDNA analysis showed that isolate E2 was most closely related to isolate 6, but only at 96% identity. Propionate and butyrate were utilized. Their ammonia-producing activity was high, and was induced three- to fivefold by growth in medium B.

Group V was a single isolate, B1, most closely related to the sulfate reducer D. piger, formerly Desulfovibrio pigra. Isolate B1 was only a weak sulfate reducer, based on cultivation in SIM medium (21). It was ureolytic and fermented glucose, sucrose, and maltose. Its NH3 production activity was highest of all of the isolates on M2. More than 20 mM lactate was formed on M2.

Group VI isolates were from three sheep. The five members of the group were asaccharolytic. The isolates' NH3-producing activity was high but not induced by growth on medium B to the same degree as that of the other isolates. Two of the isolates were sequenced. The closest relative was Acidaminococcus fermentans.

Peptidase and deaminase activities of isolates.

Dipeptidyl peptidase (DPP) activity against GlyArg-MNA was absent in all of the isolates except group III, the Atopobium-like isolates. Activity against Ala2-pNA was present in all isolates, at values ranging from 0.5 to 4.6 nmol min−1 mg of protein−1. The same DPP activities were also measured in the original HAP species, P. anaerobius, C. aminophilum, and C. sticklandii. Activities against GlyArg-MNA were zero, and those against Ala2-pNA were 0.60, 0.43, and 3.76 nmol min−1 mg of protein−1, respectively. With P. albensis, rates of hydrolysis of GlyArg-MNA and Ala2-pNA were 5.04 and 6.06 nmol min−1 mg of protein−1, respectively.

DISCUSSION

The measurements made here of rates of NH3 production from different substrates, the influence of monensin on these rates, and the properties of bacteria isolated on the basis of being able to grow on Trypticase have important implications for understanding the biochemistry and microbial ecology of NH3 production in the sheep rumen.

NH3 production rates from the different substrates at 2 mg/ml in mixed ruminal digesta were consistent with previous observations that casein is more rapidly degraded than most other proteins (4) and that the uptake and deamination of amino acids is the rate-limiting step in casein catabolism (30, 53). This is the first time differences in rates of breakdown have been seen with different peptide mixtures, although numerous instances have been observed with pure peptides (5, 48, 53). Since peptide concentrations in the rumen reach a maximum of about 200 mg of N liter−1 (4, 11, 45, 52), or 1.25 mg of peptides ml−1, and since amino acids rarely accumulate (42), one would surmise that results with higher substrate concentrations (20 mg ml−1) and corresponding rates of NH3 production have less relevance to the in vivo situation.

It has been pointed out elsewhere (42) that published values for rates of NH3 production vary considerably, because of possible dietary, species, and methodological differences. This is the first direct comparison of different methods. A rate of 3.9 nmol mg of protein−1 min−1 was observed when strained ruminal digesta were incubated with Trypticase (2 mg ml−1), in contrast to a value five times higher when a batch culture type of incubation of diluted ruminal fluid with 15-mg ml−1 Trypticase incubation was carried out in fresh medium (36). Likely maximum in vivo rates of NH3 production can be calculated from experiments in which casein (4) or Trypticase (54) was added to the rumen of sheep and cows, respectively. In the former study, NH3 concentration rose from approximately 12 to 25 mM in 2 h, a rate of 6.5 μmol ml−1 h−1; in the latter study, NH3 concentration rose from 3 to 18 mM in 3 h, a rate of 5.0 μmol ml−1 h−1. Thus, in vivo rates of NH3 production are higher than the in vitro values shown in Table Table11 but considerably lower than those indicated from the batch culture type of incubation. NH3 production rates from more typical, less rapidly degraded protein sources will be lower still (27, 42, 47).

The effects of monensin give an impression of the roles of different types of bacterial populations involved in NH3 production. There tended to be a greater effect of monensin on protein breakdown than on peptide breakdown, presumably reflecting effects on bacteria such as Streptococcus bovis and Butyrivibrio fibrisolvens, which are among the many species of ruminal microbes which are involved in protein digestion (33, 34, 40, 43). HAP bacteria are generally sensitive to monensin; thus, the maximum contribution they make to NH3 production must be in the region 23 to 36% similar to that seen by Yang and Russell with Trypticase breakdown (54). A similar conclusion was drawn from kinetic modeling in hay-fed cattle by Rychlik and Russell (37), although the importance of HAP bacteria in grain-fed animals was at least fourfold lower. The present data would indicate that the importance of monensin-sensitive bacteria is less when available protein is lower. The monensin effect might be a little misleading, however, because not all HAP bacteria are sensitive to monensin under ruminal conditions (22) and because non-HAP species like Prevotella spp. may adapt to break down peptides more slowly upon exposure to monensin (28), similarly causing decreased rates of NH3 formation. It must be remembered too that there are strong synergies between bacteria possessing different functions. Madeira et al. (24) demonstrated that HAP bacteria alone probably play a minor part in peptide breakdown—as shown here, they do not possess DPP activity, the main peptidase activity in ruminal digesta (16, 17, 44)—instead acting synergistically with Prevotella spp. to convert peptides to NH3.

The numbers of HAP bacteria, as a proportion of the total population, were not very different from the estimates made in cattle by Chen and Russell (7) and Russell et al. (35). Very few bacteria grew on amino acids alone, suggesting that bacteria which grow on amino acids as a source of energy also have a biosynthetic requirement for peptides. Benefits of peptides as N source in comparison to amino acids have been seen frequently before with ruminal bacteria (9, 12, 15).

The bacteria isolated here as being capable of growth on Trypticase were, like those isolated before (7, 8, 12, 29, 35, 36), monensin-sensitive anaerobes. They were predominantly asaccharolytic and belonged to the low-G+C-content gram-positive bacteria, which form the majority of the rumen bacterial population (41). They were, however, taxonomically different from the P. anaerobius, C. aminophilum, and C. sticklandii originally isolated by Russell's group (8, 36). They were also different from HAP species isolated by Attwood et al. (2) and by McSweeney et al. (26). Groups I, II, and IV were to a major extent functionally similar to the previous Eubacterium and Clostridium isolates. The utilization of butyrate by groups II and IV to form caproate and the utilization of propionate by group II to form valerate are reminiscent of the metabolism of Clostridium kluyveri in converting ethanol and acetate to butyrate and caproate, in order to generate ATP and enable the NADH formed as a result to be reoxidized (38).

Groups V and VI comprised D. piger and A. fermentans, neither recognized previously to be HAP species. D. piger is grouped in the delta group of mesophilic nonsporing sulfate-reducing bacteria (51). The main means of energy generation in these bacteria is by coupling the oxidation of electron donors to sulfate reduction. Some use various organic molecules, such as lactate and pyruvate, as electron donors, and it has been observed that the growth of many species is enhanced by peptone. The weak sulfate reduction, the finding that lactate was a net product of fermentation, and the finding that adding sugars to the medium stimulated growth would suggest that the physiology of this isolate is significantly different from that of D. piger (21). Sulfate-reducing bacteria are usually present in the rumen (39), although a link with amino acid metabolism and sulfate reduction has not been made before. Gibson et al. (19) estimated that 3 to 10% of human fecal sulfate-reducing bacteria were Desulfomonas spp. A. fermentans utilizes glutamate as a carbon source and has been isolated previously from the rumen on the basis of its ability to produce tricarballylic acid from trans-aconitic acid (32). Attwood et al. (2) obtained a Peptostreptococcus sp. as a HAP bacterium, as had Chen and Russell (7), and noted that its ability to grow on amino acids might occur via its ability, like A. fermentans, to metabolize glutamate via the hydroxyglutarate pathway.

The bacteria isolated here are taxonomically and functionally similar to nonsaccharolytic isolates from other habitats, such as gingival fluid and anaerobic digesters. Protein is consistently more available than sugars as a source of energy in these environments, and to some extent also in the rumen. The strategies the bacteria use to survive in the rumen are therefore relevant to other ecosystems, including the large intestine of ruminants and nonruminants. The ability of other isolates to reduce sulfate and also to utilize sugars may reflect the metabolic versatility necessary to survive under anaerobic conditions where nutrient availability is highly variable.

Acknowledgments

This work was supported by the Scottish Executive Environment and Rural Affairs Department.

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