Uncovering the mechanisms of ethanol stimulation on magnetite-enhanced anaerobic process treating oxytetracycline contained wastewater.

Magnetite has been proved to facilitate direct interspecies electron transfer (DIET)-based syntrophys and might alleviate inhibitory effects of antibiotics in anaerobic digestion (AD), while feeding ethanol was an effective approach to enrich the DIET partners. However, most of the existing studies were conducted at fixed ethanol concentration, few attentions were paid on the effects of differential ethanol proportion on AD, the underlying roles and mechanisms of ethanol stimulation remains unclear. This study systematically investigated the impact of ethanol stimulation on anaerobic processes treating oxytetracycline (OTC)-contaminated wastewater at varying proportions (20%, 50%, and 80%, based on equivalent COD value). In the presence of magnetite, ethanol stimulation promoted the methane production from 244.9 mL/g COD to a maximum 434.2 mL/g COD, with the most pronounced enhancement observed at high ethanol proportions. In particular, the average methane production obtained at 50% and 80% ethanol was 328.5 and 297.7 mL/g COD, respectively, whereas the enhancement of 20% ethanol stimulation was relatively limited. Concurrently, more stable COD removal and OTC reduction was noted in the existence of both magnetite and high ethanol proportions. Microbial analysis revealed the pivotal roles of Methanosaeta, alongside the predominance of Methanobacterium, in regulating COD conversion and driving methanogenesis through the CO ₂ reduction pathway. Notably, high ethanol proportions fostered the enrichment of exoelectrogens (Geobacter, Desulfovibrio) in the magnetite-amended system, accompanied by the up-regulation of genes involved in organic metabolism pathways. Further investigation of functional genes highlighted the prevalence of pilA enrichment in the magnetite-amended system at low ethanol proportions, whereas omcS became more abundant at high ethanol proportions.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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