Water Soluble Fraction of Crude Oil Uncouples Protease Biosynthesis and Activity in Hydrocarbonoclastic Bacteria; Implications for Natural Attenuation

Author(s): Atim David Asitok, Sylvester Peter Antai, Maurice George Ekpenyong

Three hydrocarbonoclastic bacteria isolated from crude oil-impacted mesotidal waters of the Niger Delta area of Nigeria, and identified, by 16S rRNA sequencing, as Vibrio fluvialis OWPB63, Serratia sp. MWPB18 and Pseudomonas fluorescens OWPB17 demonstrated commendable protease production ability in skimmed milk-minimal medium. Protease biosynthesis commenced in all three bacteria before the first 2 h of fermentation with peak productivities of 4.12, 0.73 and 1.12 μg/mL/h reached at 12, 36 and 48 h for Vibrio fluvialis, Serratia sp. and Pseudomonas fluorescens respectively. Activities of the enzymes assessed with azocasein as standard substrate were respectively 116.61, 174.56 and 145.84μg/mL/min. Median inhibition concentration (IC50) for Vibrio fluvialis enzyme biosynthesis, significantly (P<0.05, 0.01) influenced by exposure time, was <10% (<0.91 mg/L) with a corresponding IC50 for protease activity of <50% (<4.55 mg/L). This suggests that protease biosynthesis was more amenable to toxicity by WSF than its activity. Supplementation of respective crude bacterial proteases in pure-culture biodegradation medium containing casein-N significantly (P = 0.005; R2 = 0.9914) enhanced hydrocarbonoclasis, however only 13.8 mg C-CO2 was evolved in the mixed-culture study as against 98.4 mg in the control containing NH4Cl. These findings imply that inhibition of bacterial protease biosynthesis by WSF of crude oil reduces nitrogen availability thus limiting natural attenuation opportunities oil-spilled aquatic ecosystems.

Crude oil, Water soluble fraction, Protease, biosynthesis, Toxicity, Vibrio fluvialis OWPB63

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