Activity of Ancillary Heterotrophic Community Members in Anaerobic Methane-Oxidizing Cultures

Abstract

Consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria mediate the anaerobic oxidation of methane (AOM) in marine sediments. However, even sediment-free cultures contain a substantial number of additional microorganisms not directly related to AOM. To track the heterotrophic activity of these community members and their possible relationship with AOM, we amended meso- (37°C) and thermophilic (50°C) AOM cultures (dominated by ANME-1 archaea and their partner bacteria of the Seep-SRB2 clade or Candidatus Desulfofervidus auxilii) with L-leucine-3-¹³C (¹³C-leu). Various microbial lipids incorporated the labeled carbon from this amino acid, independent of the presence of methane as an energy source, specifically bacterial fatty acids, such as iso and anteiso-branched C_15:0 and C_17:0, as well as unsaturated C_18:1ω9 and C_18:1ω7. In natural methane-rich environments, these bacterial fatty acids are strongly ¹³C-depleted. We, therefore, suggest that those fatty acids are produced by ancillary bacteria that grow on ¹³C-depleted necromass or cell exudates/lysates of the AOM core communities. Candidates that likely benefit from AOM biomass are heterotrophic bacterial members of the Spirochetes and Anaerolineae-known to produce abundant branched fatty acids and present in all the AOM enrichment cultures. For archaeal lipids, we observed minor ¹³C-incorporation, but still suggesting some ¹³C-leu anabolism. Based on their relatively high abundance in the culture, the most probable archaeal candidates are Bathyarchaeota, Thermoplasmatales, and Lokiarchaeota. The identified heterotrophic bacterial and archaeal ancillary members are likely key players in organic carbon recycling in anoxic marine sediments.

Keywords: anaerobic oxidation of methane; archaea; bacteria; heterotrophy; lipid biomarkers; stable isotope probing.

FIGURE 1

Bacterial and archaeal lipid patterns (A,B) and their δ¹³C values (C,D) in the original cultures AOM37 and AOM50 and the isolated bacterium Ca. D. auxilii. Phy and BP data are derived from total lipid extracts.

FIGURE 2

Development of ΔHS^– (mM, A) and Δδ¹³C_DIC (‰, B) relative to T₀ in the incubation experiments with the AOM37 and AOM50 cultures over 28 days and the Ca. D. auxilii culture over 40 days.

FIGURE 3

Development of δ¹³C values (in ‰ relative to T₀) of bacterial FAs during ¹³C-leu incubation of AOM37 and AOM50 with and without (w/o) CH₄ over 28 days (experiments 1 and 3, respectively).

FIGURE 4

Pattern of ¹³C-incorporation into bacterial FAs in the AOM37 and AOM50 cultures during ¹³C-leu incubation with and without (w/o) CH₄ after 28 days (experiments 1 and 3, respectively).

FIGURE 5

Development of δ¹³C values (in ‰ relative to T₀) of archaeal lipid-derived isoprenoid hydrocarbons during ¹³C-leu incubation of AOM37 and AOM50 cultures with and without (w/o) CH₄ over 28 days (experiments 1 and 3, respectively). In the AOM50 culture, the amounts of IPL-Phy and IPL-BP0 were too low to obtain δ¹³C values.