Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments

Abstract

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant (13)C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. (13)C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of delta-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong (13)C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant (13)C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.

FIG. 1

Pore water profiles of SO₄²⁻ (⧫) and CH₄ (●) for select Eel River Basin seep samples. (A) Profile for a seep core used in the generation of the clone libraries Eel-36a and Eel-36e; (B) chemical profile for a seep core in which ANME-2 and Desulfosarcinales aggregates were detected by FISH (depth, 3 to 6 cm) (Fig. 5).

FIG. 2

Phylogenetic tree showing relationships of 16S rDNA archaeal clone sequences from Santa Barbara Basin and Eel River Basin seep sites (in boldface) to selected cultured and environmental euryarchaeotal sequences in the database. Boldface Eel River Basin clones containing accession numbers were previously reported in reference . Environmental sequences included 2MT and 2C from anoxic salt marsh sediments (29), CRA from deep sea sediments (46), pISA from hydrothermal vent sediments (39). The tree was generated by neighbor-joining analysis and corrected with a mask that included only 60% of the conserved regions. One thousand bootstrap analyses were performed, and percentages greater than 50% are reported. The bar represents a 10% estimated sequence divergence. environ., environmental.

FIG. 3

Reconstructed ion chromatogram of the alcohol fraction from sample Eel-pc 36 (6 to 9 cm). Labeled peaks designate compounds with isotopic compositions indicating a partial or exclusive derivation from anaerobic methanotrophic microbes. ∗, C₁₄ to C₁₇ acyclic alcohols from bacteria; #, sn-1-MAGE with ether-linked C₁₄ to C₁₈ acyclic alcohol moieties from bacteria. The numbers beside “DAGE” designate carbon numbers of ether-linked alkyl moieties expressed as numbers, e.g., DAGE-15/15 is diether with two C₁₅-alkyl moieties. phy-gly, sn-1-monophytanylglycerolether (archaea); AR, archaeol (archaea); OH-AR, sn-2-hydroxyarchaeol (archaea); STD, standard.

FIG. 4

Phylogenetic tree showing relationships of 16S rDNA δ-proteobacterial clone sequences from Santa Barbara Basin and Eel River Basin seeps (in boldface) to selected cultured and environmental proteobacterial sequences in the database. Environmental sequences included sva from arctic sediment (47) and SB from a benzene-mineralizing enrichment culture (31). The tree was generated by neighbor-joining analysis and corrected with a mask that included only 60% of the conserved regions. One thousand bootstrap analyses were preformed, and percentages greater than 50% are reported. The bar represents a 10% estimated sequence divergence.

FIG. 5

Whole-cell FISH of putative methane-oxidizing consortia in methane seep sediments. (A) DAPI stain of aggregates from a 3- to 6-cm sediment depth from Eel River Basin cold seeps. Sediment preperations were simultaneously hybridized with a fluoroscein-labeled probe for Desulfosarcina-Desulfococcus members (DSS658) (B) and a Cy-3 labeled archaeal ANME-2-group specific probe (EelMSMX932) (C). (D) Overlay of both Cy-3 ANME-2 group (red) and fluoroscein Dulsulfosarcinales (green) results to show the architecture of the aggregate. Scale bar = 10 μm.