Structures of free-living and protozoa-associated methanogen communities in the bovine rumen differ according to comparative analysis of 16S rRNA and mcrA genes

Abstract

Structures of free-living and protozoa-associated methanogen communities in the bovine rumen differ according to comparative analysis of 16S rRNA and mcrA genes Structures of free-living and protozoa-associated methanogen (PAM) communities from foragefed cattle were investigated by comparative sequence analysis of 16S rRNA and methyl coenzyme M reductase (mcrA) gene clone libraries. The free-living and protozoa-associated communities were composed of the same three genera [namely Methanobrevibacter, Methanomicrobium and rumen cluster C (RCC), which is distantly related to Thermoplasma]; however, the distribution of the methanogen genera differed between the two communities. Despite previous reports of potential bias for the degenerate mcrA primer set, the 16S rRNA (n5100 clones) and mcrA (n592 clones) gene libraries exhibited a similar distribution pattern for the three methanogenic genera. RCC was more abundant in the free-living community and represented 72 and 42 % of the 16S rRNA and mrcA gene sequences, respectively, versus 54 and 13 % of the 16S rRNA and mrcA gene sequences from the PAM community, respectively. The majority of RCC sequences from the free-living and protozoa-associated communities belonged to different species-level operational taxonomic units. Methanobrevibacter species were more abundant in the PAM community and represented 42 and 79 % of clones for the 16S rRNA and mrcA gene libraries, respectively, versus 9 and 27 % of 16S rRNA and mrcA gene clones from the free-living community, respectively. Methanomicrobium species were predominantly free-living. Primers for quantitative PCR were designed to target specific methanogen groups and used to assess the effect of a high-grain diet on methanogen species composition. Switching the ruminant diet from forage to high-grain resulted in reduced protozoal diversity, along with a profound overall reduction in the relative abundance of RCC and an increase in the relative abundance of free-living Methanobrevibacter spp. It was unclear whether the reduced abundance of RCC in grain-fed animals was due to the loss of symbiotic protozoa species or due to broader changes in the rumen environment that affected both RCC and protozoa. Importantly, results from this study emphasize the need to consider the different methanogen communities when developing strategies for mitigating methane emissions in ruminants. INTRODUCTIONRuminants are recognized as a major contributor to methane (CH 4 ) emissions within the agricultural sector. Methane, which is an end product of fermentation, is of concern because of its detrimental environmental effects (IPCC, 2007) and it represents a substantial loss of dietary energy (up to 12 %) to the animal (Johnson & Johnson, 1995). Practical and efficacious methods for decreasing ruminant enteric CH 4 emissions have yet to be developed, but are critical to ensuring environmentally and economically sustainable ruminant production.Methane is produced by methanogenic archaea ('methanogens'), which typically comprise ,3 % of the rumen...

Buccal Swabbing as a Noninvasive Method To Determine Bacterial, Archaeal, and Eukaryotic Microbial Community Structures in the Rumen

“…ISO3F5 followed the reverse trend, with the lowest relative abundances in sheep on 100MS and 65MG. Methanogens (among them species of the order Methanomassiliicoccales) and ciliate protozoa have been reported to live in close proximity in the rumen (49,50), and the advantages of a symbiotic relationship for both the archaeal and ciliate protozoal partners are well recognized (51). Our study confirms that high-grain diets are generally characterized by lower ciliate protozoal diversity.…”

“…In contrast to the 100LS, 100MS, and 25MG diets, protozoal communities were composed of only two major genera, namely, Entodinium and Epidinium, in samples from animals feeding on 65MG. Comparable trends from the bovine rumen have led to the hypothesis that a decreased relative abundance of Methanomassiliicoccales in animals on high-graincontaining diets could be due to the loss of the symbiotic protozoal partner (50). However, the similar responses of Methanomassiliicoccales and some protozoa to dietary changes could also be due to physiological or metabolic changes in the rumen environment that adversely affect both Methanomassiliicoccales and certain protozoal genera independently of each other (50).…”

“…Comparable trends from the bovine rumen have led to the hypothesis that a decreased relative abundance of Methanomassiliicoccales in animals on high-graincontaining diets could be due to the loss of the symbiotic protozoal partner (50). However, the similar responses of Methanomassiliicoccales and some protozoa to dietary changes could also be due to physiological or metabolic changes in the rumen environment that adversely affect both Methanomassiliicoccales and certain protozoal genera independently of each other (50). In congruence with the observations by Petri et al (48), but in contrast to early reports by Hungate et al (52), we found significantly lower relative abundances of members of the phylum Firmicutes (which contains mostly Gram-positive bacteria) in animals feeding on medium (25MG)-and high (65MG)-grain diets, while Bacteroidetes and Proteobacteria were present at significantly higher relative abundances.…”

Analysis of the rumen bacteria and methanogenic archaea of yak (Bos grunniens) steers grazing on the Qinghai-Tibetan Plateau

“…After excluding chimeras and invalid sequences, we obtained 110 valid sequences. These sequences were grouped into 14 OTUs based on 95% threshold of mcrA sequence identity (Tymensen et al, 2012). The 14 representative OTUs, 36 reference sequences downloaded from GenBank are included in phylogenetic tree (Fig.…”

Extracellular Quinones Affecting Methane Production and Methanogenic Community in Paddy Soil

“…All mcrA sequences were aligned, and the evolutionary distance matrices were calculated using MEGA 5.0 [48]. The sequences were assigned to individual operational taxonomic units (OTUs) based on a sequence similarity of at least 95 % using the furthest-neighbor algorithm as implemented in mothur v.1.26.0.…”