Integrated 13C-DNA Stable Isotope Probing and Metagenomics Approaches to Identify Bisphenol A Assimilating Microorganisms and Metabolic Pathways in Biofilms

Bisphenol A (BPA) is a persistent environmental contaminant requiring effective removal strategies. Biofilms offer advantages over conventional activated sludge for refractory compound degradation, yet the specific microorganisms and mechanisms driving BPA removal in biofilms remain poorly understood. This study employed an integrated approach, combining ¹³C-DNA stable isotope probing (SIP) and metagenomics to identify BPA-assimilating microorganisms and elucidate their metabolic pathways in biofilms. Two moving bed biofilm reactors (MBBRs) were operated at contrasting BPA concentrations (500 μg/L and 10 mg/L) to enrich distinct microbial communities. Using DNA-SIP, we revealed differences in assimilating bacteria across diverse concentrations of BPA-enriched biofilms. Simultaneously, we reconstructed the genomes of these assimilating bacteria, dissecting the functional genes essential to the degradation process and identifying significant gene variations among different assimilating bacteria. By integrating these gene functions, we constructed the BPA metabolic pathway, which surprisingly comprised genes from various assimilating bacteria. This research significantly advances our understanding of BPA-assimilating bacteria within biofilms and provides valuable insights for refining biofilm technologies aimed at BPA removal from wastewater.