Particle size of zero-valent iron affects the risks from antibiotic resistance genes in waste activated sludge during anaerobic digestion

Zero-valent iron (ZVI) is the promising enhancer for sludge anaerobic digestion (AD) performance and for mitigating the proliferation of antibiotic resistance genes (ARGs). However, concerns about its size effects in shifting the behavior and risk of ARGs in sludge, during the AD process. Here, the metagenomics-based profile of ARGs, along with their potential (pathogenic) hosts in sludge were investigated, during mesophilic AD enhanced by ZVI with three different sizes. Results showed that the size of ZVI affected the profiles of ARGs, with nano-ZVI (nZVI, 50 nm) demonstrating the most significant reduction in abundance (by 45.0 %) and diversity (by 8.6 %) of total ARGs, followed by micron-ZVI (150 mu m) and iron scrap (1 mm). Similar trends were also observed for highrisk ARGs, pathogens, and potential pathogenic hosts for ARGs. Notably, nZVI achieved the greatest reductions in the abundance of risk ARGs and potential pathogenic hosts (superbugs) by 58.8 % and 53.9 %, respectively. Correlation and redundancy analyses revealed that, the size of ZVI induced concentration differences in ammonium nitrogen, pH, carbonaceous matters, iron, and potential microbial hosts were the main reasons for the variation in the risk of ARGs. Moreover, the down-regulation of genes involved in oxidative stress contributed to the lower risk of ARGs in the three ZVI groups, especially in nZVI. This study provides insights into AD processes of solid wastes using ZVI enhancers.