In Vitro Study of Lipid Biosynthesis in an Anaerobically Methane-Oxidizing Microbial Mat

The anaerobic oxidation of methane (AOM) is a key process in the global methane cycle, and the majority of methane formed in marine sediments is oxidized in this way. Here we present results of an in vitro ¹³CH₄ labeling study ([delta]¹³CH₄, [approximately]5,400[per mille]) in which microorganisms that perform AOM in a microbial mat from the Black Sea were used. During 316 days of incubation, the ¹³C uptake into the mat biomass increased steadily, and there were remarkable differences for individual bacterial and archaeal lipid compounds. The greatest shifts were observed for bacterial fatty acids (e.g., hexadec-11-enoic acid [16:1[Delta]11]; difference between the [delta]¹³C at the start and the end of the experiment [[Delta][delta]¹³C[subscript start-end]], [approximately]160[per mille]). In contrast, bacterial glycerol diethers exhibited only slight changes in [delta]¹³C ([Delta][delta]¹³C[subscript start-end], [approximately]10[per mille]). Differences were also found for individual archaeal lipids. Relatively high uptake of methane-derived carbon was observed for archaeol ([Delta][delta]¹³C[subscript start-end], [approximately]25[per mille]), a monounsaturated archaeol, and biphytanes, whereas for sn-2-hydroxyarchaeol there was considerably less change in the [delta]¹³C ([Delta][delta]¹³C[subscript start-end], [approximately]2[per mille]). Moreover, an increase in the uptake of ¹³C for compounds with a higher number of double bonds within a suite of polyunsaturated 2,6,10,15,19-pentamethyleicosenes indicated that in methanotrophic archaea there is a biosynthetic pathway similar to that proposed for methanogenic archaea. The presence of group-specific biomarkers (for ANME-1 and ANME-2 associations) and the observation that there were differences in ¹³C uptake into specific lipid compounds confirmed that multiple phylogenetically distinct microorganisms participate to various extents in biomass formation linked to AOM. However, the greater ¹³C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.