Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence

525

406

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.

Section: Rumen Methanogensmentioning

confidence: 99%

Microbial ecosystem and methanogenesis in ruminants

Morgavi

Forano²,

Martin

et al. 2010

525

406

“…millerae (50.4% to 54.1% and 65.2%), unidentified (12.4% to 23.7% and 25.4%) and A. boonei-like (6.0% to 15.5% and 19.9%) clones was lower in the LM library. Up to now, only Shin et al (2004) reported the methanogenic molecular diversity in the rumen fluid, solid and associated to the epithelium. They showed a relatively similar methanogenic diversity among the different fractions.…”

Section: Discussionmentioning

confidence: 99%

“…This may be due to the sample collection practice. In the Shin et al (2004) study, the rumen fluid and solid were separated by filtration, and the microorganisms on the rumen epithelium were collected by scraping with a spatula. In present study, after filtration, the rumen liquid was further centrifuged by low speed in order to remove feed micro-particles remaining in the rumen fluid.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

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...

“…are members of the order Methanomicrobiales that appear to be abundant in certain rumens (Yanagita et al, 2000;Tajima et al, 2001;Shin et al, 2004;Chaudhary et al, 2012), although the basis of their apparent dominance over Methanobrevibacter spp. in those cases is not known.…”

Section: Overview Of Rumen Ecology/methanogensmentioning

confidence: 99%

Progress in the development of vaccines against rumen methanogens

Wedlock

Janssen

Leahy

et al. 2013

Vaccination against rumen methanogens offers a practical approach to reduce methane emissions in livestock, particularly ruminants grazing on pasture. Although successful vaccination strategies have been reported for reducing the activity of the rumen-dwelling organism Streptococcus bovis in sheep and S. bovis and Lactobacillus spp. in cattle, earlier approaches using vaccines based on whole methanogen cells to reduce methane production in sheep have produced less promising results. An anti-methanogen vaccine will need to have broad specificity against methanogens commonly found in the rumen and induce antibody in saliva resulting in delivery of sufficiently high levels of antibodies to the rumen to reduce methanogen activity. Our approach has focussed on identifying surface and membrane-associated proteins that are conserved across a range of rumen methanogens. The identification of potential vaccine antigens has been assisted by recent advances in the knowledge of rumen methanogen genomes. Methanogen surface proteins have been shown to be immunogenic in ruminants and vaccination of sheep with these proteins induced specific antibody responses in saliva and rumen contents. Current studies are directed towards identifying key candidate antigens and investigating the level and types of salivary antibodies produced in sheep and cattle vaccinated with methanogen proteins, stability of antibodies in the rumen and their impact on rumen microbial populations. In addition, there is a need to identify adjuvants that stimulate high levels of salivary antibody and are suitable for formulating with protein antigens to produce a low-cost and effective vaccine.Keywords: vaccine, antigen, antibody, methane, methanogens, Methanobrevibacter ruminantium Implications A number of different mitigation strategies are being investigated for reducing methane emissions from livestock, including the development of vaccines that specifically target the methane-producing methanogens in the rumen. Previous studies on vaccinating ruminants against the rumen-dwelling organisms Streptococcus bovis and Lactobacillus spp. have demonstrated the feasibility of vaccinating animals to produce salivary antibodies that neutralise the activity of these microbes in the rumen (Shu et al., 1999(Shu et al., , 2000a(Shu et al., , 2000b(Shu et al., and 2001Gill et al., 2000). Owing to the complexity of the rumen microbiota and diversity of methanogens in the rumen, an effective vaccine for reducing methane emissions from ruminants will need to target a wide range of methanogens in the rumen among an even more diverse assemblage of bacteria, fungi and protozoa. This paper reviews the progress towards developing an anti-methanogen vaccine. IntroductionA range of different strategies are being investigated to reduce methane emissions from farmed ruminants (reviewed by Martin et al., 2010;Buddle et al., 2011;Clark, 2013). Vaccination of ruminants against rumen methanogens has the potential to reduce methane emissions by decreasing the number or activity of meth...