Anaerobic digestion at hyper-mesophilic temperatures: Microbiome and antibiotic resistome in full-scale agricultural...

Temperatures between 40°C and 50°C are increasingly implemented in full-scale agricultural anaerobic digestion (AD), yet the microbial diversity and antibiotic resistome dynamics within this temperature range remain poorly understood. Here, we defined this range as "hyper-mesophilic" and surveyed five full-scale sites. Significant differences were found in the bacterial community structure, potentially stemming from feedstock combination (high vs low/non-manure) and operating temperature. Sites operating at 44°C exhibited superior attenuation efficiency (81-92 %) for antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) than sites operating at 41°C (41-83 %). High-risk clinically important ARGs such as sul1, lnuA, tet(O), and tet(L) persisted in sites blending livestock manure. Potential hosts of ARGs were identified and included opportunistic human pathogens like Enterococcus faecalis, Staphylococcus aureus, and Clostridioides difficile. The tnpA transposon accounted for > 50 % of the total MGEs and frequently co-localised with ARGs, while the class 1 integrase, intI1, was only detected in manure-blended AD. Based on prevalence in plasmids, ARGs showed higher mobility potential in sites blending chicken manure. The results obtained here provided initial insights into hyper-mesophilic AD and reinforced the importance of conducting surveillance for crop AD, with or without manure, as part of wider efforts to mitigate antimicrobial resistance in agroecosystems.