Bacterial bioaugmentation for paracetamol removal from water and sewage sludge. Genomic approaches to elucidate biodegradation pathway

Wastewater treatment plants (WWTPs) are recognized as significant contributors of paracetamol (APAP) into the environment due to their limited ability to degrade it. This study used a bioaugmentation strategy with Pseudomonas extremaustralis CSW01 and Stutzerimonas stutzeri CSW02 to achieve APAP biodegradation in solution in wide ranges of temperature (10-40 °C) and pH (5-9), reaching DT₅₀ values < 1.5 h to degrade 500 mg L^-1 APAP. Bacterial strains also mineralized APAP in solution (<30 %), but when forming consortia with Mycolicibacterium aubagnense HPB1.1, mineralization significantly increased (up to 74 % and 58 % for CSW01 +HPB1.1 and CSW02 +HPB1.1, respectively), decreasing DT₅₀ values to only 1 and 9 days. Despite the complete degradation of APAP and its high mineralization, residual toxicity throughout the process was observed. Three APAP metabolites were identified (4-aminophenol, hydroquinone and trans-2-hexenoic acid) that quickly disappeared, but residual toxicity remained, indicating the presence of other non-detected intermediates. CSW01 and CSW02 degraded also 100 % APAP (50 mg kg^-1) adsorbed on sewage sludge, with DT₅₀ values of only 0.7 and 0.3 days, respectively, but < 15 % APAP was mineralized. A genome-based analysis of CSW01 and CSW02 revealed that amidases, deaminases, hydroxylases, and dioxygenases enzymes were involved in APAP biodegradation, and a possible metabolic pathway was proposed.