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Naphthenic acids (NAs) are carboxylic acids with the formula (C n H 2n + Z O2) and are among the most toxic, persistent constituents of oil sands process‐affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC‐MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf‐5 on a model NA (4′‐n‐butylphenyl)‐4‐butanoic acid (n‐BPBA) and commercial NA mixture (Acros). By day 11, >95% of n‐BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10–18 carbon compounds of the Z family −2 to −14 (major NA species in this mixture). A total of 554 proteins (n‐BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl‐CoA dehydrogenase and acyl‐CoA thioesterase II, which catalyze part of the fatty acid beta‐oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl‐carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf‐5 was using existing fatty acid catabolic pathways (among others) during NA degradation., Oil sands mining operations produce vast amounts of oil sands process‐affected waters (OSPW) that contain recalcitrant and toxic naphthenic acids (NAs), yet little is known about the mechanisms of NA biodegradation and the enzymes involved. Here, we identified several proteins overexpressed in Pseudomonas fluorescens Pf‐5 during growth on NAs and included multiple transporters (e.g. ABC transporters), suggesting a cellular protective response from NA toxicity, alongside proteins associated with the fatty acid, lipid, and amino acid metabolism. We postulate that P. fluorescens Pf‐5 is using existing fatty acid catabolic pathways (among others) during NA degradation and these protein targets could be exploited in future biotechnological efforts for OSPW reclamation.

Supplementary Table S1

Naphthenic acids (NAs) are carboxylic acids with the formula (CnH2n+ZO2) and are the toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf5 on a model NA (4-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs; including several transporters (e.g. ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid and amino acid metabolism were also overexpressed; including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.