16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs

Skillman, Lucy; Evans, Paul N.; Naylor, Graham E.; Morvan, B.; Jarvis, Graeme N.; Joblin, K. N.

doi:10.1016/j.anaerobe.2004.05.003

Cited by 141 publications

(127 citation statements)

References 37 publications

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“…The most predominant species of methanogens in the three groups were related to the genus Methanobrevibacter. This is in agreement with Pei, Mao, Cheng and Zhu previous studies indicating species belonging to the genus Methanobrevibacter as the major methanogens in the rumen of sheep (Wright et al, 2004 andSkillman et al, 2004;Nicholson et al, 2007) and cattle (Whitford et al, 2001;Skillman et al, 2006;Nicholson et al, 2007;Ouwerkerk et al, 2008), of which some were associated with the rumen protozoa (Tokura et al, 1999a;Sharp et al, 1998;Chargan and Ushida, 2004). The predominance of this genus has also been suggested before based on cultivation studies (e.g.…”

Section: Discussionsupporting

confidence: 92%

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Pei

Mao

Cheng

et al. 2010

Animal

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Three methanogen 16S rRNA gene clone libraries were constructed from liquid (LM), solid (SM) and epithelium (EM) fractions taken from the rumen of Jinnan cattle in China. After the amplification by PCR using methanogen-specific primers Met86F and Met1340R, equal quantities of PCR products from the same fractions from each of the four cattle were mixed together and used to construct the three libraries. Sequence analysis showed that the 268 LM clones were divided into 35 phylotypes with 18 sequences of phylotypes affiliated with the genus Methanobrevibacter (84.3% of clones). The 135 SM clones were divided into 19 phylotypes with 11 phylotypes affiliated with the genus Methanobrevibacter (77.8%). The 267 EM clones were divided into 33 phylotypes with 15 phylotypes affiliated with the genus Methanobrevibacter (77.2%). Clones closely related to Methanomicrobium mobile and Methanobrevibacter wolinii were only found in the LM library, and those to Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii only in the SM library. LM library comprised 12.4% unidentified euryarchaeal clones, SM library 23.7% and EM library 25.5%, respectively. Five phylotypes (accession number: EF055528 and EF055531-EF055534) did not belong to the Euryarchaeota sequences we had known. One possible new genus (represented by phylotype E17, accession number EF055528) belonging to Methanobacteriaceae was identified from EM library. Quantitative real-time PCR for the first time revealed that epithelium fraction had significantly higher density of methanogens, with methanogenic mcrA gene copies (9.95 log 10 (copies per gram of wet weight)) than solid (9.26, P , 0.01) and the liquid (8.44, P , 0.001). The three clone libraries also appeared different in Shannon index (EM library 2.12, LM library 2.05 and SM library 1.73). Our results showed that there were apparent differences in the methanogenic diversity and abundance in the three different fractions within the rumen of Jinnan cattle, with Methanobrevibacter species predominant in all the three libraries and with epithelium fraction having more unknown species and higher density of methanogens.Keywords: rumen methanogen, molecular diversity, different fractions ImplicationsInterest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 2% to 12% of ingested energy as methane (Johnson and Johnson, 1995). However, little is known about their distribution in the rumen of Chinese ruminants. To understand their diversity distribution and their abundance, three methanogenic 16S rRNA gene libraries were constructed from different fractions of rumen of Jinnan cattle and their density determined by quantitative real-time PCR. Our results showed that Methanobrevibacter species were predominant in all three libraries, and that there were differences in the methanogenic diversity and abundance in the three different fractions within the rumen. The study for the first time demonstrated that many unidentified sequences wer...

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Section: Discussionsupporting

confidence: 92%

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Pei

Mao

Cheng

et al. 2010

Animal

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“…This finding is consistent with other studies where Methanobrevibacter strains accounted for the vast majority of methanogens in chicken ceca (Saengkerdsub et al, 2007), the lower termite Reticulitermes speratus (Shinzato et al, 1999) and the rumen Sharp et al, 1998;Tokura et al, 1999;Whitford et al, 2001;Irbis and Ushida, 2004;Skillman et al, 2004Skillman et al, , 2006Wright et al, 2004Wright et al, , 2008. Moreover, this finding supports our hypothesis that the genus Methanobrevibacter is the most dominant constituent of foregut fermentation systems.…”

Section: Discussionsupporting

confidence: 82%

Rumen-like methanogens identified from the crop of the folivorous South American bird, the hoatzin (Opisthocomus hoazin)

2009

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The hoatzin is the only known avian species with foregut fermentation. It is a primarily folivorous feeder and has a distended crop and lower/distal esophagus, which has evolved for the microbial fermentation of ingested feed. Crop samples collected from 10 individual animals from the Apure River area, Apure State, Venezuela were examined for the presence and density of methanogens using 16S rRNA gene clone libraries and real-time PCR prepared from pooled and individual PCR products. A total of 197 clones were examined, revealing 24 different methanogen 16S rRNA sequences, or phylotypes. Of the 24 unique phylotypes, 16 (171 of 197 clones) formed five unique clades within the genus Methanobrevibacter with the largest group of clones (118 clones) 98.7% similar to Methanobrevibacter ruminantium. The remaining eight phylotypes (26 clones) formed four unique clades that had only 94.0-96.7% identity to Methanosphaera stadtmanae. Based upon 98% sequence identity, we identified 17 of the 24 methanogen phylotypes from the hoatzin as possible new species and strains, with three phylotypes representing possible new genera (o94.5% sequence identity). Although none of the hoatzin methanogen phylotypes had 100% sequence identity to any other archaeal sequences in the GenBank database, the hoatzin crop methanogen sequences formed sister groups with known rumen methanogens. Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using real-time PCR, were 5.80 Â 10 9 , 7.93 Â 10 12 and 3.31 Â 10 5 , respectively. The crop microbial data presented here provide an excellent example of convergent evolution of foregut fermentation in the hoatzin, similar to that of ruminants.

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“…Active homoacetogens become established in the rumen of newborn animals as early as 20 h after birth (Morvan et al, 1994). Methanogens became established soon after, as they are detected within 1 to 3 days after birth, but when methanogens' numbers increase, before reaching adult concentrations at 3 weeks of age in lambs, the concentration of homoacetogens decrease (Morvan et al, 1994;Skillman et al, 2004). In adult animals, densities of homoacetogens range from 10 3 to 10 7 /g (Le Van et al, 1998;Fonty et al, 2007).…”

Section: Reductive Acetogenesismentioning

confidence: 99%

Microbial ecosystem and methanogenesis in ruminants

Morgavi

Forano²,

Martin

et al. 2010

Animal

538

406

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Ruminant production is under increased public scrutiny in terms of the importance of cattle and other ruminants as major producers of the greenhouse gas methane. Methanogenesis is performed by methanogenic archaea, a specialised group of microbes present in several anaerobic environments including the rumen. In the rumen, methanogens utilise predominantly H 2 and CO 2 as substrates to produce methane, filling an important functional niche in the ecosystem. However, in addition to methanogens, other microbes also have an influence on methane production either because they are involved in hydrogen (H 2 ) metabolism or because they affect the numbers of methanogens or other members of the microbiota. This study explores the relationship between some of these microbes and methanogenesis and highlights some functional groups that could play a role in decreasing methane emissions. Dihydrogen ('H 2 ' from this point on) is the key element that drives methane production in the rumen. Among H 2 producers, protozoa have a prominent position, which is strengthened by their close physical association with methanogens, which favours H 2 transfer from one to the other. A strong positive interaction was found between protozoal numbers and methane emissions, and because this group is possibly not essential for rumen function, protozoa might be a target for methane mitigation. An important function that is associated with production of H 2 is the degradation of fibrous plant material. However, not all members of the rumen fibrolytic community produce H 2 . Increasing the proportion of non-H 2 producing fibrolytic microorganisms might decrease methane production without affecting forage degradability. Alternative pathways that use electron acceptors other than CO 2 to oxidise H 2 also exist in the rumen. Bacteria with this type of metabolism normally occupy a distinct ecological niche and are not dominant members of the microbiota; however, their numbers can increase if the right potential electron acceptor is present in the diet. Nitrate is an alternative electron sinks that can promote the growth of particular bacteria able to compete with methanogens. Because of the toxicity of the intermediate product, nitrite, the use of nitrate has not been fully explored, but in adapted animals, nitrite does not accumulate and nitrate supplementation may be an alternative under some dietary conditions that deserves to be further studied. In conclusion, methanogens in the rumen co-exist with other microbes, which have contrasting activities. A better understanding of these populations and the pathways that compete with methanogenesis may provide novel targets for emissions abatement in ruminant production.

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16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs

Cited by 141 publications

References 37 publications

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Rumen-like methanogens identified from the crop of the folivorous South American bird, the hoatzin (Opisthocomus hoazin)

Microbial ecosystem and methanogenesis in ruminants

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