Crude oil biodegradation by a mixed bacterial culture

Abstract

Biological methods to remediate petroleum hydrocarbon-contaminated wastes typically rely on mixed cultures with broad substrate specifically. In this study, the overall objective was to gain an appreciation for microbe-microbe and microbe-substrate interactions in bath fermentation systems using Bow River crude oil as a model substrate. Due to hydrocarbon insolubility the mixed-bacterial culture was limited by substrate availability. Chemical surfactants were tested as a means to improve biodegradation. A detailed study with nonylphenol ethoxylates revealed that a hydrophile-lipophile balance of 13 was optimal for degradation enhancement at supra-critical micellization concentrations not exceeding a critical level.

The culture contained a wide variety of trypticase soy agar-culturable bacteria, and fermentations with various hydrocarbon mixtures were initially dominated y Pseudomonas/Flavimonas and Stenotrophomonas spp. A chemical surfactant increased the lag time of the Stenotrophomonas sp. and exposure to Bow River saturates selected for an Acinetobacter calcoaceticus strain. In any case, a greater variety of mainly non-hydrocarbon degrading bacteria were isolated following prolonged incubation. The culture exhibited greatest activity against the saturate and aromatic fractions, and low molecular weight volatile hydrocarbons, normally ignored in such biodegradation studies, were degraded in closed systems.

A rapid and sensitive solid phase microextraction methodology was developed for monitoring volatile hydrocarbon degradation in live cultures at 30oC. A 30 um polydimethylsiloxane fibre extracted C5 and C16 alkanes and aromatics, and C5 to C11 could be quantified. Volatile hydrocarbon-degrading capabilities were retained only in cultures grown on crude oil in sealed flasks, or in open flasks amended with yeast extract. Otherwise, metabolic capacity decreased with inoculum age and correlated with reduced proportions of hydrocarbon-degrading bacteria in biodegradation flasks.

Pure and co-culture studies confirmed both the degradation hierarchy and chemical surfactant effects. Crude oil biodegradation by a non-adherent, non-emulsifying, accession-limited Pseudomonas sp. (strain JA5-B45) was greatly enhanced by chemical surfactant. In the absence of surfactant, degradation by this strain was slightly enhanced by an adherent, mycolic acid-capsule producing Rhodococcus sp. (strain F9-D79) able to emulsify oil. The Rhodococcus sp. efficiently degraded alkanes up to C32 but was inactive against aromatics. In co-culture, it was shown that chemical surfactant enhanced biodegradation by directly increasing substrate solubility and by removing Rhodococcus sp. strain Fp-D79 from the hydrocarbon-water interface.