Abstract
The use of conductive materials, such as granular activated carbon (GAC), for optimization of the anaerobic digestion (AD) process has garnered attention in recent years; however, its impact on the dynamics of the microbiome and resistome in continuous AD systems remains unclear, especially under temperature variation. This study combined culture-based bacterial enumeration and shotgun metagenomics to investigate the impact of two GAC application strategies, suspended and packed, on the fate of pathogens (viable Escherichia coli) and ARGs during the AD of cattle manure at 40 and 45 degrees C. The results show that GAC mitigated the process imbalance and shock induced by temperature transition. The microbial community in the AD sludge was highly impacted by temperature but not GAC, while GAC biofilms showed notably higher archaeal abundance. All AD reactors reduced viable E. coli, with the highest reduction occurring in the packed GAC reactors (95.70-96.24%), followed by the suspended GAC (94.53-95.69%), and then the non-GAC (92.77-94.24%). Culturable tetracycline-resistant bacteria were reduced below the quantification limit in all reactors. Reduction of ampicillin-resistant bacteria showed stochastic trends at 40 degrees C but improved at 45 degrees C, indicating limited impact by GAC. ARGs and mobile genetic elements (MGEs) were reduced in all reactors at comparable levels, regardless of GAC addition. Temperature transition exerted a mixed effect, with higher reduction of some resistance classes (MLS, tetracycline, and multidrug) and lower reduction of others (bacitracin, aminoglycoside, beta-lactam, and streptothricin). Mantel test and Procrustes analysis revealed a significant correlation between the resistome and the bacterial community, inferring that shifts in the ARG host population were a major determinant of the fate of ARGs. Overall, GAC was beneficial to reactor stability but had a minimal influence on the reduction of E. coli, ARGs, and MGEs. It is highly recommended to monitor antimicrobial resistance using both culture-based and culture-independent methods.