Assessing the Impact of Site Legacy on Methanogen Community Composition and Activity in the Soil of a Restored Freshwater Mineral Wetland Through the Use of 13C-DNA Stable Isotope Probing

Wetlands are the largest natural source of atmospheric methane (CH₄), a potent greenhouse gas produced through microbial methanogenesis in the soil. Methanogen community composition and activity are shaped by soil habitat conditions. How methanogen communities respond to long term shifts in those conditions, for example brought on by climate change, are still widely debated. We examined the effect of previous land use and long-term soil oxygenation on methanogen community composition and function by comparing a restored wetland site previously converted for agriculture to a relatively undisturbed natural wetland site. Intact soil cores from both wetland sites were incubated in either an oxic or anoxic redox state with the addition of ¹³C labelled acetate. The incubations were performed under two different redox conditions to test community tolerance and activity. During the incubation headspace gas samples were collected and at the end soil cores were destructively sampled for DNA extraction. Extracted DNA was analyzed using ¹³C-DNA stable isotope probing, allowing us to isolate community members actively metabolizing acetate for CH₄ production. The active community composition will be determined by sequencing 16S rRNA genes for bacteria and archaea. Headspace gas was analyzed for CH₄ and CO₂ concentration as well as ¹³C-content, allowing verification of the connection between the active methanogens and the CH₄ fluxed from the soil. We predict that active methanogen community composition in the restored site incubations will include oxygen tolerant methanogens and will produce more CH₄ compared to natural wetland cores. Restored site anoxic redox incubations will also produce CH₄, indicating a broader range of redox conditions under which methanogenesis occurs. These findings will inform how the effects of past site disturbance dictate response to future disturbances, such as those brought on by climate change.