Metagenomic and Clumped Isotopologue Evidence for Microbial Methanogenesis in the Zambales Ophiolite

Active serpentinization occurring in the Zambales Ophiolite Range in the Philippines results in ultrabasic (pH > 10) fluid springs high in dissolved hydrogen and methane. Concentrations of dissolved gases varies between the springs; H2 ranges from 16-20% and CH4 by 8-12% by volume. Hydrogen gas is generated by serpentinization, but the provenance of methane is unknown and thought to be thermogenically derived based on a previously-reported δDCH4 values from the Los Fuegos Eternos gas seep1. Here, we present metagenomic and 13CH3D clumped isotopologue evidence for hydrogenotropic and acetoclastic methanogenesis in fluid springs and sediments collected from Manleluag Spring Protected Landscape, Mangatarem, Pangasinan, the Philippines. Methane gas collected from two springs was analyzed on a tunable infrared laser direct absorption spectroscopy (TILDAS) at the Massachusetts Institute of Technology to determine the equilibration temperature of methane. Stable isotope analysis of methane C and H indicate δ13C vs. PDB ratios near -14‰ and δD vs. SMOW ratios near -367‰. The Δ13CH3D approximately -1.0‰ vs. stochastic distribution, which is "anticlumped" (i.e. values <0‰, at which temperature cannot be expressed) and indicative of microbial methanogenesis2. Shotgun metagenomic sequencing analysis of fluids and sediments from Manleluag reveals an abundance of methanogenesis-related genes. Universal methanogenesis genes encoding methyl-coenzyme M reductase (mcr) and heterodisulfide reductase (hdr) are detected in spring fluids and sediments. Genes encoding key steps of both hydrogenotrophic and acetoclastic methanogenesis are present. Universal methane oxidation genes methanol dehydrogenase (mdh) and methane monooxygenase (mmo) are present but less abundant than methanogenesis genes, and not found in all sampling locations. Carbon assimilation genes detected in fluid and sediment metagenomes indicate that the ribulose monophosphate pathway is the predominant methane oxidation mechanism utilized by methanotrophs. This work indicates that the source of microbial methane in Zambales may be very complex and likely involves multiple metabolic pathways. 1. Abrajano et al. (1988). Chem. Geol. 71: 211-222. 2. Wang et al. (2015). Science. 348(6233): 428-431.