Bacteria and Archaea physically associated with Gulf of Mexico gas hydrates

Abstract

Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The delta(13)C of hydrate-bound methane varied from -45.1 per thousand Peedee belemnite (PDB) to -74.7 per thousand PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 x 10(9) cells g(-1)) and gas hydrate (mean = 1.0 x 10(6) cells ml(-1)). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.

FIG. 1

Map of sampling sites. Locations of known shallow gas hydrates, major gas and oil seeps, and chemosynthetic communities across the northen continental slope of the GOM are also indicated (38).

FIG. 2

Phylogenetic analysis of bacterial SSU rRNA gene clones from sample AT425. This phylogenetic tree was rooted with Sulfolobus acidocaldarius. A mask of 989 nucleotides, including all nonambiguously aligned positions, spanning nearly the full length of the SSU rRNA gene, was included. Bootstrap values (100 replications) generated by the neighbor-joining method are shown above relevant nodes, and those generated by maximum-parsimony analysis are shown below. Only bootstrap values above 70 are shown. Sequences from isolates are in italics, sequences from environmental gene clones are in plain text, and sequences from the AT425 sample are in boldface. GenBank accession numbers of the sequences from other studies are included. CFB, Cytophaga/Bacteroides/Flavobacterium. Th, Thermus.

FIG. 3

Phylogenetic analysis of archaeal SSU rRNA gene clones from sample AT425. This unrooted phylogenetic tree was obtained by methods described in the legend to Fig. 2. A mask of 695 nucleotides, including all nonambiguously aligned positions, spanning approximately 900 nucleotides of the 5′ end of the SSU rRNA gene, was included in the analysis. Sequences from isolates are in italics, sequences from environmental gene clones are in plain text, and sequences from the AT425 sample are in boldface. Sequences labeled “Eel” are from a study of the Eel River area off the coast of northern California (15), and sequences labeled “2C” or “2M” are from a study of a coastal salt marsh (31). GenBank accession numbers of the sequences from other studies are included.