Anchoring-decomplexation-mineralization: A biofilm-mediated pathway for EDTA-Cu transformation and recovery

Complexed heavy metals are emerging wastewater contaminants. Conventional coagulation-precipitation efficiently removes free metal ions but not chelated species such as EDTA-Cu, which persist through biological treatments and impair performance, while their impacts on microbial function and fate remain poorly understood. Here, using electroactive biofilms exposed to an EDTA-Cu gradient (5-80 mg/L) in bioelectrochemical systems, we identified an anchoring-decomplexation-mineralization pathway governing EDTA-Cu transformation. EDTA-Cu was primarily retained within loosely bound extracellular polymeric substances (EPS, ∼53 %), but excessive accumulation (>20 mg/L) formed a coverage layer that induced intracellular retention and functional collapse. Microbial decomplexation by EDTA-Cu-responsive genera (Sphaerochaeta, Pseudomonas, Stenotrophomonas, and Achromobacter) released Cu^2 + , which nucleated with phosphate to form Cu₃(PO₄)₂ nanoflowers accounting for 32-50 % of biofilm-associated copper. Transcriptomic profiling revealed a stress-dependent shift, with two-component regulatory networks sustaining electron transfer, efflux, and EPS synthesis under moderate stress (5 mg/L) but collapsing when EDTA-Cu exceeded 20 mg/L. Overall, this study elucidates the mechanistic basis of complexed heavy metal-microbe interactions, offering a foundation for the sustainable optimization of next-generation biological treatment technologies.