Biochar-assisted control of antibiotic-resistant bacteria and methane yield optimization in two-stage anaerobic digestion under organic load and antibiotic stress

This study explores the interactions between microbial communities, antibiotic resistance, and biogas production in anaerobic digestion systems, focusing on the acidogenic (AP) and methanogenic (MP) phases under varying organic loads, cefazolin (CEZ) exposure, and biochar supplementation. High organic loading (10 g/L glucose) significantly suppressed CEZ-resistant bacteria (CEZ-r) during the AP phase. However, their abundance markedly rebounded in MP, rising from 0.30 % to 36.28 % in control, indicating phase-specific dynamics. CEZ residues increased CEZ-r by 2.49 % and 9.30 % at 0 and 5 g/L glucose during AP. Although AP suppressed CEZ-r to 0.23 % in the CEZ-added reactor at 10 g/L glucose, MP rebounded CEZ-r to 8.30 %. In addition, CEZ exposure reduced methane yields by up to 28.14 %, likely due to the suppression of Methanosaetaceae and impaired acetic acid conversion. In contrast, biochar addition effectively reduced CEZ-r abundance to below 1.00 % at moderate to high organic loads and alleviated CEZ-induced inhibition on methane production. Biochar also enhanced Methanosaetaceae abundance (up to +6.55 %) compared to the control and promoted more efficient substrate utilization, possibly by facilitating direct interspecies electron transfer. These findings emphasize the role of organic load and digestion phase in shaping antibiotic resistance and system performance. Furthermore, biochar addition effectively mitigates the negative impacts of antibiotic residues, stabilizes microbial communities, and enhances biogas production.