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Anaerobic fungi are key players in the breakdown of fibrous plant material in the rumen, but not much is known about the composition and stability of fungal communities in ruminants. We analyzed anaerobic fungi in 53 rumen samples from farmed sheep (4 different flocks), cattle, and deer feeding on a variety of diets. Denaturing gradient gel electrophoresis fingerprinting of the internal transcribed spacer 1 (ITS1) region of the rrn operon revealed a high diversity of anaerobic fungal phylotypes across all samples. Clone libraries of the ITS1 region were constructed from DNA from 11 rumen samples that had distinctly different fungal communities. A total of 417 new sequences were generated to expand the number and diversity of ITS1 sequences available. Major phylogenetic groups of anaerobic fungi in New Zealand ruminants belonged to the genera Piromyces, Neocallimastix, Caecomyces and Orpinomyces. In addition, sequences forming four novel clades were obtained, which may represent so far undetected genera or species of anaerobic fungi. We propose a revised phylogeny and pragmatic taxonomy for anaerobic fungi, which was tested and proved suitable for analysis of datasets stemming from high-throughput next-generation sequencing methods. Comparing our revised taxonomy to the taxonomic assignment of sequences deposited in the GenBank database, we believe that >29% of ITS1 sequences derived from anaerobic fungal isolates or clones are misnamed at the genus level.

Homoacetogens produce acetate from H 2 and CO 2 via the Wood-Ljungdahl pathway. Some homoacetogens have been isolated from the rumen, but these organisms are expected to be only part of the full diversity present. To survey the presence of rumen homoacetogens, we analyzed sequences of formyltetrahydrofolate synthetase (FTHFS), a key enzyme of the Wood-Ljungdahl pathway. A total of 275 partial sequences of genes encoding FTHFS were PCR amplified from rumen contents of a cow, two sheep, and a deer. Phylogenetic trees were constructed using these FTHFS gene sequences and the translated amino acid sequences, together with other sequences from public databases and from novel nonhomoacetogenic bacteria isolated from the rumen. Over 90% of the FTHFS sequences fell into 34 clusters defined with good bootstrap support. Few rumen-derived FTHFS sequences clustered with sequences of known homoacetogens. Conserved residues were identified in the deduced FTHFS amino acid sequences from known homoacetogens, and their presence in the other sequences was used to determine a "homoacetogen similarity" (HS) score. A homoacetogen FTHFS profile hidden Markov model (HoF-HMM) was used to assess the homology of rumen and homoacetogen FTHFS sequences. Many clusters had low HS scores and HoF-HMM matches, raising doubts about whether the sequences originated from homoacetogens. In keeping with these findings, FTHFS sequences from nonhomoacetogenic bacterial isolates grouped in these clusters with low scores. However, sequences that formed 10 clusters containing no known isolates but representing 15% of our FTHFS sequences from rumen samples had high HS scores and HoF-HMM matches and so could represent novel homoacetogens.Feed ingested by ruminant animals is fermented in the rumen by a complex community of microbes. This community produces, among other products, the volatile fatty acids acetate, propionate, and butyrate, which are absorbed across the rumen wall and satisfy a large part of the animals' carbon and energy requirements. Hydrogen gas (H 2 ) is also formed and is the major precursor of the methane (CH 4 ) formed in ruminant animals. This ruminant-derived CH 4 is a contributor to global greenhouse gas emissions (46) and also represents an energy loss for the animals (34). Proposed ruminant greenhouse gas mitigation strategies include using feeds that produce less CH 4 and more volatile fatty acids (31). Alternative strategies include interventions that slow or halt methanogenesis by vaccination, using natural inhibitors found in plants, and supplementing feed with fats and oils or small-molecule inhibitors (31, 32). In the absence of methanogenesis, accumulation of H 2 could lead to a decrease in the rate of feed fermentation (31, 53) and hence a decrease in animal productivity. Other microbes that use H 2 without producing methane could be valuable in conjunction with intervention strategies that inhibit methanogens. This possibility has sparked interest in possible inoculation of ruminants with alternative H 2 users.Bacteria tha...

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10 7 to 10 8 cells g ؊1 ) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate-and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H 2 utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H 2 utilization was similar to that in conventional lambs. H 2 utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H 2 utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H 2 in the rumen.Methane (CH 4 ) eructated from ruminants represents a loss of 8 to 13% of the digestible energy ingested by the animal (71) and contributes to global warming. The amount of methane produced by ruminants varies with the farming system, the nature of the feed, the feeding level, the feed digestibility, and the animal species (6,67,71). The annual production of methane by ruminants, estimated to be 80 to 120 ϫ 10 6 tons or approximately 15% of total anthropogenic methane emissions (16,63), is the second largest biogenic source of methane after rice paddy fields. Decreasing methane emissions from ruminant livestock is desirable in order to both reduce greenhouse gases in the atmosphere and improve energy capture during digestion.Nutritionists have been trying for a long time to mitigate rumen methane emissions in order to enhance animal performance but so far have not been successful. The methods most commonly attempted involve utilization of antibiotics and ionophores (58), halogenated methane analogues (20,37,59), heavy metals (70), lipid-rich materials such as coconut oil (21,26,54,55,56), probiotics (58), bacteriocin (47), and numerous chemicals (1, 4). Immunization against methanogens (79), elimination of ciliate protozoans which support methanogen populations (64), and addition of acetogenic bacteria to rume...

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