Effects of higher temperature on antibiotic resistance genes for in-situ biogas upgrading reactors with H₂ addition

In-situ biogas upgrading by H₂ injection is a promising method for bio-natural gas production, yet the effect of H₂ addition on antibiotic resistance genes during the in-situ biogas upgrading process remains unknown. We analyzed mesophilic and thermophilic in-situ biogas upgrading digesters with intermittent or continuous mixing models using metagenomic and metatranscriptomic methods to evaluate the effects of H₂ addition on antibiotic resistance profiles. We found that H₂ addition had less impact in the mesophilic reactor. In the thermophilic reactor, the influenced antibiotic resistance ontology (AROs) was mostly bound to the integral membrane transporters of the ATP-binding cassette and major facilitator superfamily. The annotated gene numbers of four drug classes, including macrolide, glycopeptide, lincosamide, and fluoroquinolone, increased distinctly after H₂ addition. Acetate concentration is a vital indicator for distinguishing the abundance of different antibiotic efflux pumps. Most of the AROs influenced by Ruminiclostridium replaced the original dominant species Clostridium, and the versatile genus Methanosarcina was the sole methanogen correlated with the altered AROs of efflux pumps conferring antibiotic resistance. The introduced H₂ was synthesized to CH₄via the hydrogenotrophic pathway of Methanosarcina flavescens, and part of the consumed H₂ was used for cell growth.