Occurrence of unusual steroids and hopanoids derived from aerobic methanotrophs at an active marine mud volcano

Surface sediment samples from two microbial habitats (centre and adjacent Beggiatoa mats) at an active CH4-emitting mud volcano on the Norwegian margin of the Barents Sea (Haakon Mosby Mud Volcano – HMMV) were analyzed for their steroid and hopanoid biomarker distributions and associated stable carbon isotopic composition. Substantial abundances of a suite of six steroids (four 4α-methyl sterols and two 4α-methyl steroid diols) and three hopanoids (diploptene, diplopterol and Δ2-diplopterol) were found, together with biomarkers from anaerobic methanotrophic consortia (ANME-3/Desulfobulbus aggregates) at the surface sediment of the mats. All of the steroid and hopanoid biomarkers, some not been described before, were substantially depleted in 13C (δ13C between −77‰ and −68‰), proving biosynthesis by an organism involved in methane consumption. Molecular analysis (16S rRNA sequencing and fluorescence in situ hybridisation) performed in a parallel study showed that, besides ANME-3/Desulfobulbus consortia, aerobic methanotrophs of the order Methylococcales are also abundant with high cell numbers. These independent findings suggest a spatially close vicinity of active aerobic and anaerobic methanotrophic communities on the order of millimetres, not reported before for the marine environment. At the centre location, where methane oxidation is performed solely by Methylococcales species, predominantly 4α-methyl-3β-sterols, together with high abundances of specific fatty acids (i.e. C16:1ω9c and C16:1ω8c), were detected. The additional presence of new and unusual biomarkers for aerobic methanotrophy at the Beggiatoa mats seems to indicate either varying strains of Methylococcales types or an energy stress adaptation of the dominant species at the latter sampling site. Moreover, consideration is given to the new biomarkers being intermediates in the biosynthetic formation of steroids and hopanoids in methanotrophic bacteria.