Effects of nanozyme on environmental fate and dissemination of antibiotic resistance genes in anaerobically digested sludge

While nanozymes have been shown to promote organics hydrolysis and methane yield in sludge anaerobic digestion (AD), their impact on the fate of antibiotic resistance genes (ARGs) remains a critical knowledge gap. This study presents a comprehensive investigation into how nanozymes influence the environmental behavior of ARGs in AD systems. Nanozyme exposure increased total ARG abundance in a concentration-dependent manner, while simultaneously decreasing the abundance of mobile genetic elements. Specific ARGs, such as adeF, sul1, bla_CTX-M−123, tetW/N/W, sul2, and rmtA, showed increased relative abundances, while rpsL and aadA3 levels decreased. Furthermore, nanozyme exposure led to the enrichment of putative antibiotic-resistant bacteria such as Nitrospira, Dechloromonas, Longilinea, Methylibium, and Candidatus Contendobacter, but decreased the abundance of Acidothermus, Mycobacterium, and Candidatus Microthrix. The conjugation transfer frequency was increased by nanozyme, suggesting enhanced horizontal gene transfer potential. Despite a distinct reduction in adenosine triphosphate level (65.3–87.8% lower than the control), the reactive oxygen species production rate increased markedly, particularly at the highest nanozyme concentration. A noticeable increase in the protein-to-polysaccharide ratio and the upregulation of the key functional pathway of extracellular polymeric substance secretion further supported the potential role of this nanozyme in promoting ARG dissemination. These findings underscore the need for careful consideration of the long-term environmental impacts of nanozyme exposure, particularly regarding the potential for ARG dissemination when nanozyme-treated sludge is applied to natural environments.