Culture-Independent Analysis of Bacterial Community Composition during Bioremediation of Crude Oil-Polluted Soil.
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Date
2012-07
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Abstract
Aim: To use cultivation-independent techniques based on DGGE of PCR-amplified 16S
rRNA gene and to evaluate bacterial community composition during bioremediation of
crude oil-polluted soil.
Study Design: Molecular fingerprints of bacterial populations involved in the active phase
of crude oil biodegradation were generated with DGGE after 16S rRNA gene amplification.
Place and Duration of Study: Department of Microbiology and Plant Pathology, University
of Pretoria, South Africa, between March and August 2008.
Methodology: Crude oil-degrading bacteria in soil microcosms contaminated with 4%
crude oil and then biostimulated with nitrogen-phosphorus-potassium inorganic fertilizer
(NPK: designated PN soil), calcium ammonium nitrate (designated PU soil) and poultry
droppings (designated PP soil) respectively were characterized with PCR of the gene for
the small subunit (SSU) of the bacterial ribosome. Total culturable heterotrophic and
hydrocarbon utilizing bacteria were enumerated using plate count and Bushnell Haas
media. Total organic carbon content was measured throughout the study period to indirectly
determine the effect of microbial activity on carbon content in biostimulated treatments as
against controls. Gas chromatography was used to monitor hydrocarbon degradation with
time while electron microscopy examined community richness during hydrocarbon
degradation. Reamplified dominant DGGE bands (550bp) were cleaned up and sequenced
using an ABI 3130XL genetic analyzer. Electropherograms were inspected with Chromas Lite 2.01. Sequence identification was performed using BLAST.
Results: Dendogram of the DGGE bands constructed using Jaccard coefficient algorithm
revealed that communities from PU and PP-amended soils each formed distinct clades
whereas PN treated soil showed less association when compared with PU and PP
respectively. Fifty distinct bands were excised, reamplified by PCR and sequenced.
Sequence analysis revealed the presence of phylogenetically distinct known hydrocarbon
degrading bacteria like Corynebacterium spp., Dietzia spp., Janibacter sp. low G+C Gram
positive bacterial clones Nocardioides spp., Rhodococcus erythropolis and uncultured
bacterial clones. Forty successful sequence data obtained from the excised DGGE bands
were submitted to GenBank database under accession numbers GU451069 to GU451108.
Chromatograms of the residual hydrocarbons in test treatments and controls showed that
biodegradation occurred markedly in treated soils in this order PN>PU>PP while no
significant loss was observed in the oil-contaminated control on days zero and 42
respectively. Bacterial counts increased significantly in PN, PU and PP treatments and not
in controls PC and OC. Total organic carbon increased appreciably in PN, PU and PP
respectively from day zero to day 28. Electron micrographs of microbial consortia in the
nutrient-amended soils revealed presence of active populations induced by biostimulation
as against the sparsely populated controls.
Conclusion: The results suggest that nutrient amendment stimulates and selects
indigenous soil bacteria that are able to degrade petroleum hydrocarbons.
Description
Keywords
Sequences, DGGE, crude oil, biodegradation, bioremediation, electron micrographs
Citation
Chikere Chioma Blaise. Culture-Independent Analysis of Bacterial Community Composition during Bioremediation of Crude Oil-Polluted Soil. British Microbiology Research Journal. 2012 Jul; 2(3): 187-211.