Methane-Oxidizing Bacteria Shunt Carbon to Microbial Mats at a Marine Hydrocarbon Seep

Abstract

The marine subsurface is a reservoir of the greenhouse gas methane. While microorganisms living in water column and seafloor ecosystems are known to be a major sink limiting net methane transport from the marine subsurface to the atmosphere, few studies have assessed the flow of methane-derived carbon through the benthic mat communities that line the seafloor on the continental shelf where methane is emitted. We analyzed the abundance and isotope composition of fatty acids in microbial mats grown in the shallow Coal Oil Point seep field off Santa Barbara, CA, USA, where seep gas is a mixture of methane and CO₂. We further used stable isotope probing (SIP) to track methane incorporation into mat biomass. We found evidence that multiple allochthonous substrates supported the rich growth of these mats, with notable contributions from bacterial methanotrophs and sulfur-oxidizers as well as eukaryotic phototrophs. Fatty acids characteristic of methanotrophs were shown to be abundant and ¹³C-enriched in SIP samples, and DNA-SIP identified members of the methanotrophic family Methylococcaceae as major ¹³CH₄ consumers. Members of Sulfuricurvaceae, Sulfurospirillaceae, and Sulfurovumaceae are implicated in fixation of seep CO₂. The mats' autotrophs support a diverse assemblage of co-occurring bacteria and protozoa, with Methylophaga as key consumers of methane-derived organic matter. This study identifies the taxa contributing to the flow of seep-derived carbon through microbial mat biomass, revealing the bacterial and eukaryotic diversity of these remarkable ecosystems.

Keywords: 16S rRNA gene; intact polar lipids (IPL); methanotrophs; microbial mats; stable isotope probing; sulfide-oxidizing bacteria.

FIGURE 1

Summary of growth and enrichment experiments: (A) Benthic growth device. Rough PVC growth plates (white) were affixed to a large mounting board facing down when deployed, and secured in situ with rebar driven into the seafloor. Individual growth plates were removed by SCUBA divers, leaving the remaining plates undisturbed. For scale, a metric measuring stick is shown at the top of the mounting board. (B) Representative growth plates recovered from Shane Seep during in situ sampling. (C) Overview of sampling and analyses performed on environmental (SEEP) and experimental (SIP) mats.

FIGURE 2

Comparative IPL distribution and FA ¹³C enrichment: (A) Relative IPL (%) abundances in methane enrichment samples. (B) Proportion of ¹³C assimilated to specific fatty acids, determined from relative (%) abundances and δ¹³C values for each lipid.

FIGURE 3

Methanotroph abundance variability across DNA density gradients: (A) Schematic of theoretical density gradients of total community DNA in centrifuge tubes from SIP-labeling (left) and nonSIP (right) experiments. (B) Heatmap of ranked abundances from OTUs (OTU # indicated on Y-axis), belonging to Methylococcaceae subfamily-A in the DNA-SIP (left) and INIT (right) density gradients. Fractions 1–7 from each gradient are labeled with their buoyant densities (g/mL).

FIGURE 4

Correlation analysis of fatty acid ¹³C signatures vs. taxa abundances: (A) Spearman’s rho analysis of ranked OTU abundances versus fatty acid δ¹³C values in SEEP samples. OTUs are ordered by hierarchical clustering on Euclidean distances from Spearman’s rho values. Taxonomic affiliations (lineage, subfamily, and PhyloChip OTU number) are displayed at left for each row. OTUs that have a significant association with at least one fatty acid’s δ¹³C values are highlighted in bold and with an asterisk mark. (B) Selected examples of OTUs with ranked abundances significantly (p ≤ 0.05) associated with fatty acid δ¹³C values in SEEP samples.

FIGURE 5

Microbial food web for the seep mat community. Bacterial carbon transfer under (A) ex situ SIP and (B) in situ conditions. Ex situ, the CO₂ available to autotrophs like Sulfurovum is the product of methane oxidation; in situ, more CO₂ is expected to be supplied directly by the gas seep.