More than sulfidation: Roles of biogenic sulfide in attenuating the impacts of CuO nanoparticle on antibiotic resistance genes during sludge anaerobic digestion.

Biogenic sulfide (BS) in anaerobic digesters was previously suggested to mitigate the potential impacts of metallic nanoparticles (M-NPs) on antibiotic resistance genes (ARGs) propagation by sulfidation of the M-NPs. In this study, a new role of BS in regulating ARGs responses to M-NPs is reported. It was observed that CuO NPs at environmentally relevant level had no significant effects on the spread of ARGs. However, higher dosage (50 mg/gTSS) contributed to the propagation of ARGs, whose abundances would be effectively reduced by 74-115% if BS production was stimulated. Instead, introduction of EDTA, a metal ion chelator, resulted in much lower attenuation efficiencies (12-40%), indicating that restriction of the bioavailability of CuO NPs might not be the only reason for the buffering of ARG responses in the presence of BS. Further investigation showed that the presence of BS together with activation of key enzymes (O-acetyl serine sulfhydrylase and γ-glutamylcysteine synthetase) supplied and favored the biosynthesis and transformation of cysteine, which mitigated the oxidative stress induced by CuO NPs. Moreover, the amounts of cysteine and its metabolite glutathione in sludge were associated with the abundances of ARGs negatively, implying that in situ generated cysteine was the important ARGs regulator. Exploration of possible mechanisms revealed that the biosynthesized cysteine might limit gene transfer potential via mobile genetic elements, as cysteine restricted the abundances of intI 1, Tn916/1545 and ISCR 1. In addition, the cysteine remarkably alleviated the copper stress and copper resistance, which in turn blocked possible co-selection between copper and antibiotic resistance. This work provides new insight into attenuation of the bio-effects of NPs in digesters.