Identification of novel methane-, ethane-, and propane-oxidizing bacteria at marine hydrocarbon seeps by stable isotope probing

Abstract

Marine hydrocarbon seeps supply oil and gas to microorganisms in sediments and overlying water. We used stable isotope probing (SIP) to identify aerobic bacteria oxidizing gaseous hydrocarbons in surface sediment from the Coal Oil Point seep field located offshore of Santa Barbara, California. After incubating sediment with (13)C-labeled methane, ethane, or propane, we confirmed the incorporation of (13)C into fatty acids and DNA. Terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of the 16S rRNA and particulate methane monooxygenase (pmoA) genes in (13)C-DNA revealed groups of microbes not previously thought to contribute to methane, ethane, or propane oxidation. First, (13)C methane was primarily assimilated by Gammaproteobacteria species from the family Methylococcaceae, Gammaproteobacteria related to Methylophaga, and Betaproteobacteria from the family Methylophilaceae. Species of the latter two genera have not been previously shown to oxidize methane and may have been cross-feeding on methanol, but species of both genera were heavily labeled after just 3 days. pmoA sequences were affiliated with species of Methylococcaceae, but most were not closely related to cultured methanotrophs. Second, (13)C ethane was consumed by members of a novel group of Methylococcaceae. Growth with ethane as the major carbon source has not previously been observed in members of the Methylococcaceae; a highly divergent pmoA-like gene detected in the (13)C-labeled DNA may encode an ethane monooxygenase. Third, (13)C propane was consumed by members of a group of unclassified Gammaproteobacteria species not previously linked to propane oxidation. This study identifies several bacterial lineages as participants in the oxidation of gaseous hydrocarbons in marine seeps and supports the idea of an alternate function for some pmoA-like genes.

FIG. 1.

(A) ¹³C enrichment of phospholipid fatty acids of ¹³C methane (M), ethane (E), and propane (P) incubations and ¹²C controls in initial sediment (t0) and at final time points. The 16:1 and 18:1 isomers were not differentiated. The dashed line indicates natural abundance levels of ¹³C. (B) Proportion of ¹³C incorporated into each fatty acid.

FIG. 2.

Mass spectra for the 16:1 fatty acid molecular ion peak, showing the extent of ¹³C labeling at the final time point of the methane, ethane, and propane incubations. The m/z values for the spectra span the full range from no ¹³C incorporation (m/z = 268) to full ¹³C labeling (m/z = 268 + 16 = 284). The spectra oriented upward correspond to incubations with ¹³C labeling, whereas the inverted spectra correspond to control incubations with substrate lacking ¹³C labeling. Each spectrum is normalized to the height of its tallest peak.

FIG. 3.

Relative abundances of 16S rRNA sequences in clone libraries from heavy DNA (fraction 4 or 6 [noted as Heavy A or Heavy B] or both) from the three time points of the ¹³C methane, ethane, and propane incubations, selected controls (light DNA from the ¹³C incubations and heavy DNA from the ¹²C controls), and the initial sediment. Sequences were grouped using the RDP Classifier tool; “other” combines sequences that, classified at the order level, represented less than 5% of the clones in any individual clone library. Representative sequences from each of the groups indicated in methane, ethane, or propane oxidation are included in the phylogenetic tree in Fig. 4.

FIG. 4.

Neighbor-joining phylogenetic trees of the 16S rRNA gene sequences from groups involved in methane, ethane, or propane oxidation, based on their abundance in both the heavy DNA clone libraries and the heavy T-RFLP fractions, relative to light DNA and ¹²C controls. (A) Gammaproteobacteria; (B) Betaproteobacteria. Sequences from this study are shown in bold, with predicted MspI T-RF lengths in parentheses. Reference sequences from GenBank are shown with accession numbers in parentheses. Filled circles indicate bootstrap values above 90% and open circles bootstrap values above 50% (2,000 replicates).

FIG. 5.

Neighbor-joining phylogenetic tree of pmoA gene sequences from seep sediment (t0) and methane and ethane heavy DNA sequences, plus reference sequences from GenBank (accession numbers in parentheses). Only the divergent sequences from the ethane SIP sample are shown; others were identical to those in the t0 and methane SIP samples. Filled circles indicate bootstrap values above 90% and open circles bootstrap values above 50% (2,000 replicates). The alphaproteobacterial pmoA and gammaproteobacterial amoA sequences are condensed for clarity.

FIG. 6.

T-RFLP fingerprinting of the 16S rRNA gene in density gradient fractions from day 3 of the ¹³C (A) and ¹²C (B) methane, day 6 of the ¹³C (C) and ¹²C (D) ethane, and day 6 of the ¹³C (E) and ¹²C (F) propane incubations. Major T-RFs were identified by in silico digestions of clone library sequences with MspI. Less-abundant T-RFs (<5% of the total peak area in any of the heavy fractions) were combined and are shown as “other.” Fractions with insufficient DNA for analysis are left blank.