Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps

Abstract

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.

FIG 1

Sediment depth profiles of methane concentrations and methanogenesis rates. (A) White Mat 14. (B) Edge of White Mat 14. The relative proportions of acetate, bicarbonate, and dimethylamine methanogenesis rates are represented in pie charts for each sediment section. The size of the pie chart is proportional to the total methanogenesis rate. The methane concentrations are from Vigneron et al. (23).

FIG 2

DGGE analysis of archaeal 16S rRNA diversity. (A) DGGE profiles for 20 samples represented by letters (A to T) in the dendrogram in panel B. The different media are represented by symbols: dots, acetate/H₂-CO₂; stars, H₂-CO₂; diamonds, trimethylamines. (B) Dendrograms from cluster analysis of DGGE profiles. The underlined samples were selected for analysis of the phylogenetic diversity of methanogens. Act, acetate; WM14a, WM14 PC3; WM14b, WM14 PC4; EWM14a, EWM14 PC8; EWM14b, EWM14 PC11; Z1, 0 to 6 cmbsf; Z2, 6 to 10 cmbsf; Z3, 10 to 15 cmbsf.

FIG 3

Neighbor-joining (NJ) phylogenetic tree of the archaeal 16S cDNA sequences amplified from selected enrichment cultures. Maximum-likelihood (ML) topologies were similar. Bootstrap support values obtained for NJ/ML analyses are reported at the nodes (1,000 replicates). Sequences from this study are in boldface. Highly similar sequences (>97% identical) from the same sample were clustered, but only one representative sequence is shown. The scale bar indicates five substitutions per 100 nucleotides.

FIG 4

Hypothetical model (not to scale) of microbial methane cycling in the Sonora Margin cold seep sediments. Each microbial group is characterized by a specific function: group 1, methylotrophic methanogenesis by Methanococcoides lineages directly from surface organism inputs or after primary degradation by various bacteria (Desulfovibrio, Desulfobacterium, and Desulfuromonas) previously detected in environmental samples (60); group 2, hydrogenotrophic methanogenesis by Methanogenium lineages after organic-matter degradation by fermentative bacteria (Firmicutes) previously detected in environmental samples (24, 60); group 3, methane production by the deepest methanogenic communities detected in the deepest (1 to 9 mbsf) sediments of the Sonora Margin cold seeps (46) and other, potentially unidentified methanogens; group 4, potential methanogenesis activity of ANME communities (68, 70). SRB, sulfate-reducing bacterium.