5% of the total archaea. To date, the RCC has been found in many ruminants, including cattle [1, 4, 6–8, 11], sheep [2, 5, 11], goats [9, 12], water buffalo [10], and red deer [11]. Further the proportion of RCC within the total methanogen populations is high (up to 80%) [11, 13]. However, most of these studies have been conducted using sequencing-based culture-independent molecular

methods. The role of RCC in the rumen remains unclear in the absence of cultivated isolates. Further, although RCC has been labeled as a group of methanogens, there is little evidence to support that the RCC is methanogen [13]. Recently, Poulsen et al. see more [8] investigated the impact of rapeseed oil on the abundance of rumen microorganisms and their gene expression by metatranscriptomics, and found that methylamines might be the substrates for RCC. They further verified this by in vitro experiment which was composed of adding trimethylamine (TMA) to

bovine rumen fluids and incubating for 24 hours. The results showed that methane production increased 22%, accompanied by a three fold increase for the abundance TAM Receptor inhibitor of RCC. Moreover, the recently reported Methanomassiliicoccus luminyensis from human feces, which was clustered within RCC clade in our present study, could use hydrogen to reduce methanol to methane [14]. Borrel et al. [15] published the genome sequence of another RCC related isolate (Candidatus Methanomethylophilus alvus) from human gut and reported Thiamet G this isolate contains genes needed for methylotrophic methanogenesis from methanol

and methylamines. Padmanabha et al. [16] reported that a chicken gut isolate (Methanoplasma gallocaecorum strain DOK-1) belonging to RCC clade could strictly use hydrogen to reduce both methylamines and methanol to methane. In agreement with Wright et al. [2] suggesting a new order, Paul et al. [17] strongly proposed that these unclassified Thermoplasmatales sequences (as referred as RCC and its phylogenetic relatives) represents the seventh order of methanogenic archaea, based on the comparative phylogenetic analysis of the 16S rRNA genes and mcrA gene sequences, together with the enriched cultures from the higher termites and millipedes and the recently reported isolate M. luminyensis. Thus, the methanogenic archaeon in this order are widely distributed in marine habitat, soil, and in the intestinal tracts of termites and mammals. Although the exact contribution of RCC to rumen methane production still remains unclear, they possibly play an important role in the methanogenesis, due to their high percentage in the rumen methanogen population [11, 13]. Therefore, the cultivation and isolation of these unique RCCs from rumen has become increasingly important for understanding the role of RCC in the rumen. However, many attempts have been made, but the isolation of anoxic pure RCC from the rumen still remains unsuccessful.