Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities

Abstract

The methane-rich, hydrothermally heated sediments of the Guaymas Basin are inhabited by thermophilic microorganisms, including anaerobic methane-oxidizing archaea (mainly ANME-1) and sulfate-reducing bacteria (e.g., HotSeep-1 cluster). We studied the microbial carbon flow in ANME-1/ HotSeep-1 enrichments in stable-isotope-probing experiments with and without methane. The relative incorporation of (13)C from either dissolved inorganic carbon or methane into lipids revealed that methane-oxidizing archaea assimilated primarily inorganic carbon. This assimilation is strongly accelerated in the presence of methane. Experiments with simultaneous amendments of both (13)C-labeled dissolved inorganic carbon and deuterated water provided further insights into production rates of individual lipids derived from members of the methane-oxidizing community as well as their carbon sources used for lipid biosynthesis. In the presence of methane, all prominent lipids carried a dual isotopic signal indicative of their origin from primarily autotrophic microbes. In the absence of methane, archaeal lipid production ceased and bacterial lipid production dropped by 90%; the lipids produced by the residual fraction of the metabolically active bacterial community predominantly carried a heterotrophic signal. Collectively our results strongly suggest that the studied ANME-1 archaea oxidize methane but assimilate inorganic carbon and should thus be classified as methane-oxidizing chemoorganoautotrophs.

Fig. 1.

Results of lipid stable-isotope probing. (A and B) Isotopic shifts of (A) hydrogen and (B) carbon, expressed as weighted-average Δδ of total fatty acids (FA, light gray bars), archaeols as phytanes (Phy, dark gray bars), and glycerol dibiphytanyl glycerol tetraethers (GDGTs) as biphytanes (BPs, black bars) during the experiment. (C) Production of lipids (prod_lipid, green bars) and assimilation of inorganic carbon (assim_IC, blue bars) into bacterial (Left) and archaeal (Right) lipids. Time series experiments in C are presented as average values (n = 3, error bar = SD). The ratio of assim_IC to prod_lipid (R_a/p) gives a measure for the dominance of a heterotrophic (R_a/p < 0.3) or autotrophic (R_a/p ~ 1) mode of carbon fixation. In the absence of methane, R_a/p values are not presented because of low values detected for archaeal inorganic carbon lipid assimilation. The symbol “+” indicates incubations with ¹³C_DIC ∼ 9.6% ¹³C, D ∼ 3%, and unlabeled methane. Incubations with¹³CH₄ contained ∼15.9% ¹³C.

Fig. 2.

Carbon assimilation and lipid production of microbial lipids. Compared are carbon assimilation (assim_IC) and production (prod_lipid) of bacterial fatty acids (diamonds) and archaeal ether lipid-derived isoprenoids (circles) in the (A) presence and (B) absence of methane after 24 d of incubation (see Fig. S4 for more details).

Fig. 3.

Relative increase in individual lipid production in incubations with methane compared with incubations without methane. Increases are plotted against the ratio of carbon assimilation to lipid production (R_a/p) as an indicator for auto- or heterotrophic source organisms of the lipid. Archaeal ether lipid-derived hydrocarbons (circles) and bacterial fatty acids (diamonds) were produced in either the presence (red symbols) or the absence (white symbols) of methane. R_a/p values in the absence of methane were not determined for iC_16:1ω6, C_16:1ω7, and iC_18:1ω6 and for BP(0) and BP(1) due to assim_IC values close to the detection limit. Shaded bars indicate ranges primarily interpreted to reflect heterotrophy (brown) or autotrophy (blue).