Structure of the archaeal community of the rumen.

Members of the domain Archaea contribute about 0.3 to 3.3% of the microbial small subunit (16S and 18S) rRNA in the rumen (22, 39, 60). Archaea have a range of different metabolisms and are found in many habitats (6), but those known to exist in the rumen are strictly anaerobic methanogens. Yanagita et al. (59) observed that 2.8 to 4.0% of ruminal microorganisms displayed autofluorescence characteristic of F420, a methanogen cofactor, able to be seen under UV illumination during microscopy. Taken together with the small subunit rRNA abundance data, this suggests that a large part of the archaeal population is made up of methanogens. Most species of methanogens can grow using H2 and often formate as their energy sources and use the electrons derived from H2 (or formate) to reduce CO2 to CH4. Some species can grow with methyl groups, oxidizing some to CO2 to produce electrons that are used to reduce further methyl groups to methane. A few species can grow with acetate, effectively dissimilating acetate to CH4 and CO2. However, acetate is not metabolized to CH4 to any significant extent in the rumen (13). This is probably because the rate of passage of rumen contents through the rumen is greater than the growth rate of acetateutilizing methanogens (53). In a normally functioning rumen, proteins and polymeric carbohydrates, which usually make up the largest part of the incoming feed, are fermented by a mixed microbial community to volatile fatty acids (VFAs), NH4 , CO2, and H2. The hydrogen is metabolized by the methanogens. The VFAs are taken up by the animal across the rumen wall and serve as major carbon and energy sources for the ruminant. A part of the VFAs, undigested feed components, and microbial cells leave the rumen and enter the rest of the animal’s digestive tract. The central role of H2 in the rumen fermentation (12) means that, although methanogenic archaea make up only a small part of the rumen microbial biomass, they play an important role in rumen function and animal nutrition. Efficient H2 removal leads to a nutritionally more favorable pattern of VFA formation and to an increased rate of fermentation by eliminating the inhibitory effect of H2 on the microbial fermentation (26, 53). The rumen can be simplistically described as an open system with discontinuous solid (feed) and liquid (saliva and drinking water) inputs and multiple fractions that have different turnover rates (53). The methanogens in the rumen are found free in the rumen fluid, attached to particulate material and rumen protozoa, associated as endosymbionts within rumen protozoa, and attached to the rumen epithelium. The methanogens associated with these different fractions can be expected to have different growth rates since they will be removed from the rumen at different rates. In addition, the animal itself and the feed also influence the rate of passage of digesta through the rumen system (25). These different habitats may allow niche division among the methanogens and may explain some of the observed phylogenetic diversity of rumen archaea. Cultured methanogens from the rumen. Methanogens have been classified into 28 genera and 113 species (11), but many more species can be expected to occur in nature (6). Surprisingly few methanogens have been isolated from the rumen. Those that have been cultured are assigned to only seven species. These are Methanobacterium formicicum (33), Methanobacterium bryantii (16), Methanobrevibacter ruminantium (43), Methanobrevibacter millerae (36), Methanobrevibacter olleyae (36), Methanomicrobium mobile (35), and Methanoculleus olentangyi (16). Methanosarcina spp. have also been cultured from the rumen (1, 34) but are not normally a major part of the archaeal community. In addition, Methanobrevibacter smithii (16) has been reported as being isolated from the rumen, but this is likely to be a strain more closely related to M. millerae (M. Kirs and P. H. Janssen, unpublished data). Cultivation-based studies usually fail to uncover the full extent of microbial diversity. This is because some species are more readily culturable than others and because the size of any single cultivation-based survey is usually too small to give good insight into the community structure (15). The random isolation of methanogens in small-scale cultivation-based studies is useful for obtaining isolates for detailed investigation and also indicates the presence of a species. However, the abundance of different methanogens in different systems cannot be assessed comprehensively using data from the cultivation-based studies that have been made over the past 50 years. Cultivation-independent surveys of ruminal methanogens. Surveys of methanogens and total archaea in the rumen have been made by using PCR to amplify the 16S rRNA genes of archaea, followed by a cloning step in Escherichia coli to separate the different gene variants within the mixed amplicon. An assessment of the dominant groups of archaea in the rumen can be made by comparative sequence analysis and phylogenetic placement of the 16S rRNA genes recovered in such surveys. In some of the studies, not all of the 16S rRNA genes * Corresponding author. Mailing address: AgResearch Ltd., Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand. Phone: 64 6 351 8300. Fax: 64 6 351 8003. E-mail: [email protected]. † Present address: Cawthron Institute, 98 Halifax Street East, Nelson, New Zealand. Published ahead of print on 18 April 2008.