Identification of Novel Methane-, Ethane-, and Propane-Oxidizing Bacteria at Marine Hydrocarbon Seeps by Stable Isotope Probing

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 ¹³C-labeled methane, ethane, or propane, we confirmed the incorporation of ¹³C 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 ¹³C-DNA revealed groups of microbes not previously thought to contribute to methane, ethane, or propane oxidation. First, ¹³C 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, ¹³C 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 ¹³C-labeled DNA may encode an ethane monooxygenase. Third, ¹³C 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.Hydrocarbon seeps are widespread along continental margins and emit large amounts of oil and gas into the surrounding environment. This gas is primarily composed of methane, a powerful greenhouse gas, and marine hydrocarbon seeps are estimated to contribute 20 Tg year⁻¹ methane to the atmosphere, representing about 5% of the total atmospheric flux (21, 39). Seeps of thermogenic gas also release an estimated 0.45 Tg year⁻¹ ethane and 0.09 Tg year⁻¹ propane to the atmosphere (20). Each of these three fluxes would be substantially larger if not for microbial oxidation in the sediments and water column (68). Methane, ethane, and propane are subject to anaerobic oxidation in anoxic sediments and water columns (44, 53, 68) or to aerobic oxidation in oxic and suboxic water columns and oxygenated surface sediment (10, 47, 53, 80). We focus here on aerobic oxidation.The majority of known aerobic methane-oxidizing bacteria are members of either Gammaproteobacteria (type I) or Alphaproteobacteria (type II) (29), though several strains of highly acidophilic methanotrophic Verrucomicrobia have also been recently isolated (63). Most methanotrophs are capable of growth only on methane or other one-carbon compounds (17, 29), using a methane monooxygenase (MMO) enzyme to oxidize methane to methanol. There are two known forms of this enzyme: soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO). sMMO is a soluble, di-iron-containing monooxygenase found only in certain methanotrophs and typically expressed only under low-copper conditions (57). In contrast, pMMO is a membrane-bound enzyme believed to contain copper and iron (26). It is found in all known methanotrophs, with the exception of species of the genus Methylocella (16). pmoA, the gene encoding the α subunit of pMMO, is often used to identify methanotrophic bacteria (54). Very few methanotrophs from marine environments have been cultured (22, 49, 72, 74), but several previous studies of marine methanotrophs (35, 62, 77, 82, 85) have been performed with culture-independent methods and have almost exclusively detected type I methanotrophs. Many of the pmoA sequences from methane seep sites are quite different from those of cultured organisms, suggesting that these environments may contain many novel methanotrophs (77, 82, 85).Even less is known about the organisms that oxidize ethane or propane in marine environments. The number of such isolates, which primarily represent high G+C Gram-positive bacteria (Nocardia, Pseudonocardia, Gordonia, Mycobacterium, and Rhodococcus) or Pseudomonas species, is limited (70). Nearly all of these strains were isolated from soil and selected for their ability to grow on propane or n-butane as the sole carbon source. Most propane-oxidizing strains can oxidize butane, as well as a range of longer chain n-alkanes, but differ in the ability to oxidize ethane. These strains show little, if any, ability to oxidize methane, and none have been shown to grow with methane as the sole carbon source (13, 27, 38, 45, 65). As with methane metabolism, the first step in aerobic ethane and propane metabolism is the oxidation of the alkane to an alcohol (70). Several different enzymes are known to catalyze this step. Thauera butanivorans uses a soluble di-iron butane monooxygenase related to sMMO to oxidize C₂ through C₉ n-alkanes (18, 73). Gordonia sp. strain TY-5, Mycobacterium sp. strain TY-6, and Pseudonocardia sp. strain TY-7 contain soluble di-iron propane monooxygenases that are capable of both terminal and subterminal propane oxidation and differ in their substrate ranges (45, 46). Nocardioides sp. strain CF8 is believed to possess a copper-containing monooxygenase similar to pMMO and ammonia monooxygenase (27, 28). An alkane hydroxylase typically used to oxidize longer-chain n-alkanes has also shown some ability to oxidize propane and butane but not ethane (38). The variety of enzymes and their substrate ranges make it difficult to identify ethane or propane oxidizers with a single functional gene.In order to identify the organisms responsible for methane, ethane, and propane oxidation at hydrocarbon seeps, we used stable isotope probing (SIP). SIP allows the identification of organisms actively consuming a ¹³C-labeled substrate of interest, based on the incorporation of ¹³C into biomass, including DNA and lipids (67). We collected sediment from the Coal Oil Point seep field and incubated sediment-seawater slurries with ¹³C methane, ethane, or propane. Samples were removed at three time points, chosen to ensure sufficient ¹³C incorporation into DNA while minimizing the spread of ¹³C through the community as a result of cross-feeding on metabolic byproducts. ¹³C-DNA was separated from ¹²C-DNA by CsCl density gradient ultracentrifugation, and we used the fractionated DNA for terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis. We also measured ¹³C incorporation into fatty acids in order to confirm significant ¹³C enrichment in membrane lipids, to determine the carbon labeling pattern for each substrate and lipid, and to further characterize the composition of the microbial community.