Differential protein expression during growth on model and commercial mixtures of naphthenic acids in Pseudomonas fluorescens Pf-5.

Boyd A Mckew,Matthew S Ross,Michael A Hough,Corinne Whitby,Lisa M Gieg,Richard Johnson,Metodi Metodiev,Lindsay Clothier,Karl Skeels,Jonathan W Martin,Max Frenzel

doi:10.1002/mbo3.1196

Abstract

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.

Highlights

In the Athabasca region of Northern Alberta, Canada, surface mining and caustic hot water extraction of bitumen have resulted in the accumulation of vast quantities of wastewaters known as oil sands process-affected water (OSPW) (Quagraine et al, 2005; Siddique et al, 2011)
After a short lag phase, growth was observed after 6 h on pyruvate and after 4 days on n- BPBA and the commercial naphthenic acids (NAs) mixture (Acros) (Figure A1), which is similar to previous NA biodegradation studies (Johnson et al, 2011, 2013)
Additional unidentified peaks were observed that had longer retention times, suggesting they are less polar or less water-soluble compounds. These findings confirmed that P. fluorescens Pf-5 degraded n-BPBA and that the initial degradation steps involved the removal of two carbons from the carboxyl side chain indicative of beta-oxidation as previously shown (Johnson et al, 2011, 2012, 2013)

Summary

Introduction

In the Athabasca region of Northern Alberta, Canada, surface mining and caustic hot water extraction of bitumen have resulted in the accumulation of vast quantities of wastewaters known as oil sands process-affected water (OSPW) (Quagraine et al, 2005; Siddique et al, 2011). For effective OSPW remediation and site reclamation, effective modes of NA degradation or removal will be crucial Despite their recalcitrance and toxicity, very little is known about the mechanisms of NA biodegradation and the enzymes involved (Whitby, 2010). Was found to degrade aromatic NAs by both the beta-and omega-oxidation pathways (Johnson et al, 2012) Such studies have made some headway, the proteins involved in NA biodegradation (e.g., transport and metabolic pathways) are still unknown. Their identification has in part been hampered by the chemical complexity of environmental NAs and the identification of the individual components

Objectives

Results

Conclusion