Enhanced Diclofenac Biodegradation by Bacterial Strains and a Microbial Consortium from Activated Sludge: Toxicity Assessment and Insights into Microbial Community Dynamics

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug whose presence in environmental matrices has led to its classification as an emerging contaminant. Developing effective and sustainable removal strategies is therefore essential. In this study, <i>Pseudomonas aeruginosa</i> CSWD.1, <i>Pseudomonas</i> sp. CSWD.2, and a microbial consortium (MC) were isolated from activated sludge through enrichment cultures with DCF and employed as laboratory models to investigate DCF biodegradation capacity under a biosafety-aware framework. Biodegradation assays supplemented with glucose showed limited removal (45.5%) by CSWD.1, whereas CSWD.2 and the MC achieved complete elimination (100%) of 10 mg L^-1 DCF in 21 and 5 days, respectively. Three extracellular metabolites, 4'-hydroxy-diclofenac (4'-OH-DCF), 5-hydroxy-diclofenac (5-OH-DCF), and putative NO₂-DCF, were detected, with concentrations varying during degradation. Persistence of 4'-OH-DCF and tentatively identified NO₂-DCF after 28 days was potentially associated with increased toxicity relative to the abiotic control. Overall, the results suggest that evaluating metabolites and their toxicity is essential, requiring isolation of additional microorganisms able to degrade 4'-OH-DCF and NO₂-DCF to combine with the microorganisms isolated in this study. Metabarcoding analysis of the microbial consortium after bioremediation revealed the dominant bacterial population of <i>Burkholderia</i> (88.9% relative abundance) and a predominant fungal genus <i>Talaromyces</i> (80.1%), indicating that both bacteria and fungi may be associated with DCF transformation. These results provide insights into microbial community dynamics and their potential application in designing effective consortia for DCF bioremediation.