Methanogens are the unique and only group of microorganisms responsible for the generation of more than 90% of Earth`s methane. In terms of the necessary of the biological processes, these archaea use very small range of substrates but they are so adoptable that can be found in various anoxic habitats, even in extreme environments. Research of Arctic permafrost revealed the presence of viable methanogenic archaea, what may lead to the increase of methane flux in permafrost in the future. Several investigations on the CH4 emissions and the methanogenic community of Arctic permanently frozen grounds of different ages have been conducted previously (Rivkina et al., 2007; Wagner et al., 2013; Shcherbakova et al., 2016). The use of cultivated methods allowed to identify and describe new methanogenic species of the genera Methanosarcina and Methanobacterium in the Pliocene and Pleistocene permafrost, which are responsible for the formation of methane under extreme subzero conditions (Rivkina et al., 2004; Rivkina et al., 2007; Krivushin et al., 2010; Shcherbakova et al., 2011).
Tied methane-producing binary culture JL01 was isolated from 2.0-m-deep Holocene permafrost (Kolyma lowland, Russia) after long-term cultivation at 15°C. Using molecular techniques and repeated transfers in the presence of various antibiotics the binary culture was divided into archaeal (JL01) and bacterial (GLS2) strains. Later the bacterium was described as a novel spherical spirochaete Sphaerochaeta associata GLS2T (Troshina et al., 2015).
Preliminary studies of strain JL01 have shown that it was strictly anaerobic, nonmotile methanogenic аrchaeon which cells were organized into aggregates and had a typical appearance for Methanosarcina species. Strain JL01 cell wall corresponded to the Gram-positive type. Methane production was observed under the temperature from 10 to 37oC, pH 5.5-9.0 and NaCl concentration from 0.9 to 10 g L-1. The most suitable substrates for strain JL01 growth were methanol and trimethylamine. The genome sequence was determined in the Centre for Genomic Regulation (Barcelona, Spain). Genome was assembled from 323 contigs. The sequence total and ungapped lengths were 4,186,733 bp and 4,127,022 bp, respectively. GC content was 41.59%. A comparison of the genome sequences of our strain and Methanosarcina mazei S-6T (ANI 98.5%) showed that the Arctic isolate belonged to the species M. mazei despite significant phenotypic differences between strain JL01 and its closest relative M. mazei S-6T.
This study comprises the characterization of methanogenic archaeon strain JL01 and the data of the long-term cooperation with saccharolytic bacterial partner S. associata GLS2T. Supported by genomic data this finding confirm a possibility of methane production in this cooperation without any additional carbon and energy sources.
The genome analysis of S. associata GLS2T shows a particular adaptation of this bacterium towards methanochondroitin matrix of M. mazei JL01 due to the presence of necessary genes responsible for various carbohydrates. It is possible that such cooperation between the methanogen and the bacterium, that we observed in our experiments, allows the participants to carry out a continuous process of decomposition of methanochondroitin of dead methanogen cells to form acetate (Troshina et al., 2015), the substrate for methanogenesis. The analysis of genomes of Sphaerochaeta spp. showed the absence of several genes coding for vitamin B12 synthesis. Methane producing archaea can produce its precursors (Zhang et al., 2008, Caro-Quintero et al., 2012) and can supply them to the bacterium.
Thus, we hypothesize that such interaction allows partners to stay viable and active in the permafrost condition.