Spatial evidence of cryptic methane cycling and methylotrophic metabolisms along a land-ocean transect in a California coastal wetland

Abstract Methylotrophic methanogenesis in the sulfate reduction zone of wetland and marine sediment has recently been coupled to anaerobic oxidation of methane (AOM), now referred to as the cryptic methane cycle. In this study we present evidence of cryptic methane cycling activity within the sulfate-reducing zone, along a land-ocean transect within the Carpinteria Salt Marsh Reserve (CSMR), Carpinteria, CA, USA consisting of four stations; two brackish, one marine, and one hypersaline. The top 20 cm of sediment collected along the transect was subjected to geochemical and molecular analysis, in vitro methanogenesis batch incubations, and radiotracer incubations using 35 S-SO 2- 4 , 14 C-mono-methylamine, and 14 C-CH 4 to find evidence of cryptic methane cycling activity. Results showed that subsurface porewater salinity increased with increasing sediment depth in the two brackish stations while remaining close to saline or hypersaline in the other stations, suggesting complex subsurface hydrology across the CSMR. Methane concentrations were consistently low (3 to 28 µM) except at the marine station, which showed increasing methane with increasing sediment depth (max 665 µM). In vitro methanogenesis batch incubations showed no linear build-up of methane over time, except with sediment from deeper intervals at the marine station, suggesting a process that is limiting methane production in the sediment. AOM rates from direct measurements with 14 C-CH 4 were low in the two brackish and hypersaline stations (0.03 to 0.66 nmol cm −3 d −1 ) while at the marine station AOM rates increased with increasing sediment depth (Max 19.4 nmol cm −3 d −1 ). Total organic carbon (TOC) and total organic nitrogen (TON) were highest in the sediment intervals near the surface at the two brackish stations and hypersaline station, while at the marine station TOC and TON were higher towards the bottom of the core (<0.5% to 5.4% for TOC and 0.02% to 0.38% for TON). Porewater sulfate concentrations were never limiting (9 to 91 mM) across the transect despite sulfate reduction actvity (1.5 to 2506 nmol cm −3 d −1 ) in sediment intervals near the surface. Porewater sulfide and iron (II) profiles revealed that the sediment transitioned from predominantly iron-reducing to a predominantly sulfate-reducing between the two brackish stations and the marine and hypersaline stations. Metabolomic analysis of porewater revealed that substrates for methanogenesis (i.e., acetate, methanol, and mono-methylamine) were mostly below detection, but some samples from the 0-1.5 cm, 9.5-10.5 cm, and 14.5-15.5 cm depths showed non-quantifiable amounts of mono-methylamine and methanol, indicating rapid turnover of these substrates. Acetate had quantifiable amounts in some depth intervals at most stations ranging between 45 and 72 µM. Estimated methanogenesis from mono-methylamine was detected throughout the sediment at all stations, with the highest rates found in intervals close to the surface, ranging between 0.14 and 3.8 nmol cm −3 d −1 . Differences between the rate constants (k) of methanogenesis from 14 C-mono-methylamine and AOM from either direct injection of 14 C-CH 4 or 14 C-mono-methylamine derived 14 C-CH 4 , point towards a separate metabolic process activity metabolizing mono-methylamine to inorganic carbon. Molecular analysis revealed that microbial communities at the two brackish stations are closer related to each other than the communities at the marine and hypersaline stations. Molecular analysis also revealed the presence and overlap of methanogenic and anaerobic methanotrophic archaea but only in the marine and hypersaline stations, suggesting that the two organisms are involved with cryptic methane cycling either as a couple or by methanogenic archaea capable of both methanogenesis and AOM. Orders within the Desulfobacterota phylum capable of sulfate and iron reduction were detected throughout the sediment and are potentially responsible for sulfate reduction rates and buildup of reduced iron across the transect. We conclude that the results in this work show strong evidence of cryptic methane cycling activity in the top 20 cm of sediment in the CSMR. Based off the evidence the cryptic methane cycle is likely preventing major buildup of methane in the sulfate-reducing zone due to rapid cycling of carbon between methanogenesis and AOM. Our data revealed methanogenic and anaerobic methanotrophs are present in the CSMR which are likely responsible for cryptic methane cycling. Furthermore, our data point towards methylamine utilization by both methanogenic archaea and non-methanogenic microorganisms in the CSMR. We hypothesize that sulfate reduction, by groups of sulfate-reducing bacteria, is actively consuming methylamine alongside methanogenic archaea, but additional work is needed to confirm this metabolic activity and identify who is responsible.