Phylogenetic diversity of dominant bacterial communities during bioremediation of crude oil-polluted soil

Abstract

Bioremediation of hydrocarbon pollutants is advantageous owing to the cost-effectiveness of the technology and the ubiquity of hydrocarbon degrading microorganisms in the soil. Soil microbial diversity is affected by hydrocarbon perturbation thus selective enrichment of hydrocarbon utilizers occurs. Hydrocarbons interact with the soil matrix and soil microorganisms determining the fate of the contaminants relative to their chemical nature and microbial degradative capabilities respectively. Bacterial dynamics in crude oil-polluted soil microcosms undergoing bioremediation were investigated over a 42-day period. Four out of the five microcosms containing 4kg of pristine soil each were contaminated with 4% Arabian light crude oil. Three microcosms were amended with either 25g of NPK fertilizer, calcium ammonium nitrate or poultry droppings respectively while the fourth designated oil-contaminated control was unamended. The fifth microcosm had only pristine soil and was set up to ascertain indigenous bacterial community structure pre-contamination. Biostimulated soils were periodically tilled and watered. Hydrocarbon degradation was measured throughout the experimental period by gas chromatography. Gas chromatographic tracing of residual hydrocarbons in biostimulated soils showed marked attenuation of contaminants starting from the second (day 14) till the sixth (day 42) week after contamination whereas no significant reduction in hydrocarbon peaks was seen in the oil contaminated control soil throughout the 6-week experimental period. Molecular fingerprints of bacterial communities involved in aerobic biodegradation of crude oil hydrocarbons in biostimulated soils and controls were generated with DGGE using PCR-amplification of 16S rRNA gene obtained from extracted total soil community DNA. DGGE fingerprints demonstrated that NPK, calcium ammonium nitrate and poultry droppings selected different bacterial populations during the active phase of oil degradation. Cluster analysis of DGGE bands using simple matching group average setting revealed that poultry droppings-amended soils and calcium ammonium nitrate-amended soils formed distinct clades meaning that the treatment selected similar bacterial populations for each of the treatments whereas NPK soils showed less association. Excision, reamplification and sequencing of dominant DGGE bands in biostimulated soils revealed the presence of distinct hydrocarbon degraders like Corynebacterium spp., Dietzia spp., low G+C Gram positive bacteria and some uncultured bacterial clones. Phylogenetic analysis of the 16S rRNA gene sequences of these dominant bacterial communities was conducted using the neighbour joining method of PHYLIP. Two distinct clades appeared in the tree clustered members of the Actinobacteria and Firmicutes separately. The overall data suggested that Gram positive bacteria especially members of the Actinobacteria may have a key role in bioremediation of crude oil-polluted soil.

Keywords

• Bacterial dynamics
• Arabian light crude oil
• bioremediation
• phylogenetic analysis
• PHYLIP