Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.Methane is a potent greenhouse gas that is implicated in global warming (35). Of the 600 Tg of methane released into the atmosphere each year, 55 to 70% is anthropogenic (48). Enteric fermentation of ruminant livestock is the largest source of anthropogenic methane, contributing between 20 and 25% (48). During enteric fermentation, archaea in the rumen (methanogens) produce methane mainly through the stepwise reduction of CO 2 (4H 2 ϩ CO 2 3 CH 4 ϩ 2H 2 O) (47). As well as contributing to greenhouse gas emissions, methanogenesis is energetically wasteful representing a loss of between 2 and 12% ingested feed energy (23). Reductive acetogenesis is a hydrogenotrophic pathway (4H 2 ϩ 2CO 2 3 CH 3 COOH ϩ 2H 2 O) that results in an energy gain for ruminant livestock through the production of acetate (22) and could be an alternative hydrogen sink to methanogenesis if methanogenesis is suppressed (16).The bacteria capable of reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway (acetogens) exist in a range of environments, including sediments, wastewater treatment systems, soils, and animal gut systems, and they are likely to be natural microbiota of all ruminants (11,22). Naturally, however, reductive acetog...

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Belmont Red Composite rumen-cannulated steers (n = 8, 364 ± 8.4 kg liveweight, LW LW; least squares means ± s.e.m.) were used to assess effects of feeding tea seed (Camellia sinensis L.) saponin (TSS) supplementation on performance, methanogenesis, fermentation pattern and rumen microbial communities. The expectation was to use TSS to potentially modulate the rumen microbial population and decrease enteric methane (CH 4) production. The steers were fed twice a day with a basal diet (BD) that contained a mixture of 0.15 Rhodes grass (Chloris gayana) hay plus 0.85 of a commercial concentrate before CH 4 emissions were measured in open-circuit respiratory chambers for 48 h. Steers were then adapted progressively to doses of 20 and 30 g/day of TSS for 10 and 6 days, respectively before new CH 4 measurements were recorded. Final placement in chambers was conducted after 13 days of TSS removal (BDP). Rumen fluid samples from each steer were collected for the treatments BD, BD + 20 g TSS, BD + 30 g TSS and

Molecular information suggests that there is a broad diversity of acetogens in the rumen, distinct from any currently isolated acetogens. We combined molecular analysis with enrichment culture techniques to investigate this diversity further. Methane-inhibited, hydrogenotrophic enrichment cultures produced acetate as the dominant end product. Acetyl-CoA synthase gene analysis revealed putative acetogens in the cultures affiliated with the Lachnospiraceae and Ruminococcaceae as has been found in other rumen studies. No formyltetrahydrofolate synthetase genes affiliating with acetogens or with 'homoacetogen similarity' scores >90% were identified. To further investigate the hydrogenotrophic populations in these cultures and link functional gene information with 16S rRNA gene identity, cultures were subcultured quickly, twice, through medium without exogenous hydrogen, followed by incubation without exogenous hydrogen. Comparison of cultures lacking hydrogen and their parent cultures revealed novel Lachnospiraceae and Ruminococcaceae that diminished in the absence of hydrogen, supporting the hypothesis that they were likely the predominant acetogens in the enrichments. Interestingly, a range of Bacteroidetes rrs sequences that demonstrated <86% identity to any named isolate also diminished in cultures lacking hydrogen. Acetogens or sulphate reducers from the Bacteroidetes have not been reported previously; therefore this observation requires further investigation.