Effects of brackish water inflow on methane-cycling microbial communities in a freshwater rewetted coastal fen

Rewetted peatlands can be a significant source of methane (CH4), but in coastal ecosystems, input of sulfate-rich seawater could potentially mitigate these emissions. The presence of sulfate as an electron acceptor during organic matter decomposition is known to suppress methanogenesis by favoring the growth of sulfate reducers, which outcompete methanogens for substrate. We investigated the effects of a brackish water inflow on the microbial communities relative to CH4 production-consumption dynamics in a freshwater rewetted fen at the southern Baltic Sea coast after a storm surge in January 2019 and analyzed our data in context with the previous freshwater rewetted state (2014 serves as our baseline) and the conditions after a severe drought in 2018 (Fig. 1). We took peat cores at four previously sampled locations along a brackishness gradient to compare soil and pore water geochemistry as well as the microbial methane- and sulfate-cycling communities with the previous conditions. We used high-throughput sequencing and quantitative polymerase chain reaction (qPCR) to characterize pools of DNA and RNA targeting total and putatively active bacteria and archaea. Furthermore, we measured CH4 fluxes along the gradient and determined the concentrations and isotopic signatures of trace gases in the peat. We found that both the inflow effect of brackish water and the preceding drought increased the sulfate availability in the surface and pore water. Nevertheless, peat soil CH4 concentrations and the C-13 compositions of CH4 and total dissolved inorganic carbon (DIC) indicated ongoing methanogenesis and little methane oxidation. Accordingly, we did not observe a decrease in absolute methanogenic archaea abundance or a substantial change in methanogenic community composition following the inflow but found that the methanogenic community had mainly changed during the preceding drought. In contrast, absolute abundances of aerobic methanotrophic bacteria decreased back to their predrought level after the inflow, while they had increased during the drought year. In line with the higher sulfate concen trations, the absolute abundances of sulfate-reducing bacteria (SRB) increased - as expected - by almost 3 orders of magnitude compared to the freshwater state and also exceeded abundances recorded during the drought by over 2 orders of magnitude. Against our expectations, methanotrophic archaea (ANME), capable of sulfate-mediated anaerobic methane oxidation, did not increase in abundance after the brackish water inflow. Altogether, we could find no microbial evidence for hampered methane production or increased methane consumption in the peat soil after the brackish water inflow. Because Koebsch et al. (2020) reported a new minimum in CH4 fluxes at this site since rewetting of the site in 2009, methane oxidation may, however, take place in the water column above the peat soil or in the loose organic litter on the ground. This highlights the importance of considering all compartments across the peat-water-atmosphere continuum to develop an in-depth understanding of inflow events in rewetted peatlands. We propose that the changes in microbial communities and greenhouse gas (GHG) fluxes relative to the previous freshwater rewetting state cannot be explained with the brackish water inflow alone but were potentially reinforced by a biogeochemical legacy effect of the preceding drought.