Phylogenetic analysis of methanogens from the bovine rumen

Abstract

Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified. , an organism not previously described in the rumen. The third cluster contained rDNA sequences that may form a novel group of rumen methanogens. The current set of 16S rRNA hybridization probes targeting methanogenic Archaea does not cover the phylogenetic diversity present in the rumen and possibly other gastro-intestinal tract environments. New probes and quantitative PCR assays are needed to determine the distribution of the newly identified methanogen clusters in rumen microbial communities.

Background

Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified.

Results

, an organism not previously described in the rumen. The third cluster contained rDNA sequences that may form a novel group of rumen methanogens.

Conclusions

The current set of 16S rRNA hybridization probes targeting methanogenic Archaea does not cover the phylogenetic diversity present in the rumen and possibly other gastro-intestinal tract environments. New probes and quantitative PCR assays are needed to determine the distribution of the newly identified methanogen clusters in rumen microbial communities.

Background

]. We have undertaken this study to examine the free living community of methanogens within bovine rumen fluid using comparative sequence analysis of cloned 16S rRNA genes (rDNA) amplified from total DNA extracted from clarified rumen fluid.

Results and discussion

).

used in this study were greater than 98 % similar, yet these organisms are considered distinct species.

), were associated with subclusters Mbr I and Mbr II, indicating that these clusters are not strictly associated with planktonic methanogens.

.

] speculated that the presence of an archaeal group not covered by archaeal group specific probes may be responsible for the lack of additivity of these probes when compared to signals from the general archaeal probe. From 20 to 60 % of the general archaeal probe signal they detected in rumen samples was not accounted for when probe signals specific for Methanococcaceae, Methanobacteriaceae, Methanosarcinales, and Methanomicrobiales were summed. The latter three probes currently cover all rumen methanogens that have been successfully cultured.

] to the control of rumen methanogenesis difficult.

].

. This may be due to differences in sample preparation, animal diet or geographic region. Clearly more studies are needed on the effects of diet and animal species on the diversity of methanogens in the rumen. Of particular benefit would be studies relating the phylogenetic diversity of methanogens in the rumen, using techniques such as Denaturing Gradient Gel Electrophoresis (DGGE), to the amount of methane produced by the animal.

Conclusions

] would not hybridize to a potentially important group of rumen methanogen sequences identified in this study. Additional studies need to be conducted to determine the quantitative significance of the newly identified groups, as well as how the diversity of rumen methanogens varies with animal diet.

Rumen sampling and animal diets

Rumen samples were obtained after manual mixing of rumen contents from five rumen-fistulated mature Holstein dairy cows. The contents were squeezed through two layers of cheesecloth and then subsampled for extraction of DNA. The cattle were mid-lactation cows fed a total mixed ration consisting of 9 % hay, 26 % alfalfa haylage, 30 % corn silage, and 35 % concentrate (13 % barley, 50.8 % corn, 28.1 % roasted soybeans, plus vitamins/minerals). Samples from the cattle were obtained prior to the first morning feed.

DNA extraction, PCR amplification, cloning and sequencing

].

Phylogenetic analysis

.

Reference strains and cloned sequences used in phylogenetic analysis

).

Acknowledgments

This research was supported by the Dairy Farmers of Canada and Agriculture and Agri-Food Canada through the Matching Investment Initiative. Critical reading of the manuscript by Dr. Athol Klieve is gratefully acknowledged. This paper represents LRC contribution number 3870005.

Figures and Tables

was used as the out-group. The bar represents a sequence divergence of 10 %.