The effect of substrate concentration on the methane-driven interaction network

Methane, the primary substrate for aerobic methanotrophs, regulates the rate of methanotrophic activity and shapes the composition of the methane-oxidizing community. Given that methane-derived carbon may fuel the food web in the soil, methane availability can potentially be a key determinant, structuring the network of the interacting methane-oxidizing community. Here, we determined the response of the methane-driven interaction network to different methane concentrations (∼1.5 %_v/v, 3 %_v/v, and 7 %_v/v), indicative of different levels of energy flow through the soil food web, using a stable isotope probing approach with ¹³C-methane coupled to a co-occurrence network analysis in a microcosm study. The accumulated ¹³C-atom fraction in the total carbon content increased from 1.08 % (background level) to an average of 7.2 % in the incubation under 7 %_v/v methane, indicating that the carbon-flow via the methanotrophs can significantly contribute to the total carbon in the rice paddy soil. The ¹³C-enriched 16 S rRNA gene sequencing analysis revealed the predominance of gammaproteobacterial methanotrophs and Methylocystis. The composition of the actively growing (¹³C-labelled) bacterial community was dissimilar in the incubation under ∼3 %_v/v than under 1.5 %_v/v and 7 %_v/v methane. This was also reflected in the co-occurrence network analysis, where the topological properties indicated a more complex and connected network in the incubation under 3 %_v/v methane. It thus appears that moderate methane concentrations fostered closer associations among members of the methane-oxidizing community. Overall, our research findings showed that the methanotrophs can contribute to the total soil carbon, and methane concentrations not only shifted the bacterial community, including the methanotrophic composition, but also affected bacterial interactions.