Dark-light cycle driven metabolic H₂/O₂ switching for benzothiazole removal and sulfate transformation in a nano-Fe₃O₄-assisted anoxic bacterial-microalgal biofilm

Benzothiazole (BTH) and sulfate coexist in thiazole-containing pharmaceutical wastewater, but their biological removal is constrained by conflicting redox requirements for oxidative ring cleavage and reductive sulfate transformation. Here, a nano-Fe₃O₄-assisted anoxic bacterial-microalgal biofilm (ABMB) was constructed to couple dark-light cycle driven metabolic H₂/O₂ switching with pollutant conversion. During long-term operation (60 days) at a hydraulic retention time of 24 h, the nano-Fe₃O₄-assisted ABMB achieved 99.3 ± 0.7% BTH removal, 92.9 ± 2.1% sulfate removal, and 49.8 ± 8.7% total organic carbon removal, outperforming suspended and unmodified biofilm systems. Metabolism analysis indicated that BTH was transformed through hydroxylation and thiazole-ring cleavage to 2-mercaptophenyl-carbamate and further degradable intermediates, whereas sulfate was converted mainly into recoverable elemental sulfur. The 6 h dark/6 h light cycle was optimal for coordinating the sulfate reduction and the BTH oxidation degradation. Metagenomic and physiological analyses further validated that nano-Fe₃O₄ enhanced extracellular electron transfer, regulated photosynthetic activity and optimized biofilm structure, as well as enriched key genes related to BTH oxidation, sulfate reduction, and sulfide oxidation. This system breaks the conventional reliance on microalgae solely for O₂ supply by harnessing a dark-light cycle driven metabolic H₂/O₂ switching mechanism. It provides a paradigm shift in bacterial-microalgal symbiosis with a sustainable, zero-aeration, and resource-oriented strategy for treating thiazole-containing wastewater.