Abstract
Ibuprofen (IBU) is a significant contaminant frequently found in wastewater treatment plants due to its widespread use and limited removal during treatment processes. This leads to its discharge into the environment, causing considerable environmental concerns. The use of microorganisms has recently been recognized as a sustainable method for mitigating IBU contamination in wastewater. In this study, new bacteria capable of growing in a solid medium with IBU as the only carbon source and removing IBU from a liquid medium were isolated from environmental samples, including soil, marine, mine, and olive mill wastewater. Four bacterial strains, namely Klebsiella pneumoniae TIBU2.1, Klebsiella variicola LOIBU1.1, Pseudomonas aeruginosa LOIBU1.2, and Mycolicibacterium aubagnense HPB1.1, were identified through 16S rRNA gene sequencing. These strains demonstrated significant IBU removal efficiencies, ranging from 60 to 100% within 14 days, starting from an initial IBU concentration of 5 mg per litre. These bacteria have not been previously reported in the literature as IBU degraders, making this work a valuable contribution to further studies in the field of bioremediation in environments contaminated by IBU. Based on the IBU removal results, the most promising bacteria, K. pneumoniae TIBU2.1 and M. aubagnense HPB1.1, were selected for an in silico analysis to identify genes potentially involved in IBU biodegradation. Interestingly, in the tests with TIBU2.1, a peak of IBU transformation product(s) was detected by high-performance liquid chromatography, while in the tests with HPB1.1, it was not detected. The emerging peak was analysed by liquid chromatography-mass spectrometry, indicating the presence of possible conjugates between intermediates of IBU biodegradation. The proteins encoded on their whole-genome sequences were aligned with proteins involved in an IBU-degrading pathway reported in bacteria with respective catabolic genes. The analysis indicated that strain HPB1.1 possesses genes encoding proteins similar to most enzymes reported associated with the IBU metabolic pathways used as reference bacteria, while strain TIBU2.1 has genes encoding proteins similar to enzymes involved in both the upper and the lower part of that pathway. Notably, in the tests with the strain having more candidate genes encoding IBU-catabolic enzymes, no IBU transformation products were detected, while in the tests with the strain having fewer of these genes, detection occurred.