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Genetic diversity among petroleum hydrocarbon degrading bacteria isolated from crude oil and oily sludge contaminated sites

Student Name: Mr Dhruva Bhattacharya
Guide: Dr Banwari Lal
Year of completion: 2003


Incidences of crude oil spills are increasing at an alarming rate in India. The contamination of soil by crude oil in India is a major concern considering the huge network of oil pipelines that transport crude oil to and from various refineries. The crude oil spills occur due to leakage from the joints of the crude oil pipelines, accidents of the oil tankers during transportation of crude oil, anthropogenic activities and pilferage activities. The oily sludge contamination is also a major environmental concern as it severely damages the surrounding ecosystems. The oily sludge is a hazardous hydrocarbon waste generated by the petroleum refineries during the processing of the crude oil. The storage of the crude oil in the tanks results in the deposition of heavy fractions of crude oil at bottom of the storage tanks. The heavy fractions of crude oil, which constitute the different polycyclic aromatic hydrocarbon compounds, asphaltenes etc. also form the oily sludge. The improper disposal of oily sludge in open pits leads to the contamination of the terrestrial ecosystem since many of its constituents are highly toxic, carcinogenic and are poorly biodegradable in nature.

The petroleum hydrocarbons in nature are degraded by different group of microorganisms, which include bacteria, cyanobacteria, fungi and other eukaryotic organisms. The microbial remediation of crude oil and oily sludge contaminated site is carried out by diverse groups of microorganisms particularly the indigenous bacteria present in the crude oil contaminated soil. These microorganisms are capable of utilising hydrocarbons as sole source of carbon and energy.

Documentation of bacterial diversity in a site contaminated with crude oil and oily sludge is essential. This is because it helps in isolation and identification of novel bacterial strains having capability to degrade wide range of the recalcitrant compounds of crude oil and oily sludge. The evaluation of genetic diversity is necessary to understand the phylogenetic perspective of the bacterial strains. This is also essential to elucidate the diversity at the genus and species level for the conservation of the indigenous microflora.

The present investigation encompassed an approach where the bacterial diversity of seven geoclimatically different crude oil and oily sludge contaminated sites (five oil refineries and two oil exploration sites) was documented. The oil refineries and oil exploration sites were situated in the Northern, North-eastern, Western and Eastern regions of India. The age of the crude oil and oily sludge contamination at the sampling sites also varied. The oil refineries and oil exploration sites were selected for the present study because the soils at these locations get contaminated with the toxic petroleum hydrocarbons in the day to day activities. The diversity was evaluated by cultivation based and gene cloning approaches.

The summary of the above study is as follows:
• The petroleum hydrocarbon degrading bacterial strains were isolated by enrichment cultures techniques from the crude oil spill and oil sludge contaminated soil samples procured from the seven geoclimatically different sites in India. A total of 159 cultivable bacterial strains were isolated from seven different sampling sites. The bacterial strains were purified and preserved as pure culture stocks at –70 C with 25 % glycerol. The bacterial strains were maintained on a minimal salt media with crude oil as the sole source of carbon and energy.

• The phenotypic studies and biochemical characterisation of the bacterial strains were studied based on substrate utilisation profiles generated with Biolog microbial identification system. Of the total bacterial strains isolated, both Gram negative and Gram positive bacteria were obtained but Gram negative bacterial strains were dominant. The identification of the bacterial strains was performed by sequencing the genes encoding 16S rRNA. The partial 16S rDNA sequence based identification and phylogenetic analysis was performed for all the bacterial strains. The sequencing of the complete genes encoding 16S rRNA was performed randomly for representative strains to validate the results of the partial gene sequencing. It was observed that the bacterial strains isolated from different sampling locations were dominantly phylogenetically affiliated to the proteobacteria(93 % of isolates were belonging to the proteobacteria).

• The bacterial strains were predominantly affiliated to subclass of proteobacteria. Of the total bacterial strains, 51 % of the bacterial strains were phylogenetically affiliated to the genus Pseudomonas. Of the total Pseudomonas sp. isolated from the different sites, P. citronellolis and P. aeruginosa were the most dominant strains at all the seven contaminated sites. The second most dominant genus identified at the different sampling sites was the Acinetobacter sp.

• The degradation of total petroleum hydrocarbon (TPH) fraction of crude oil was estimated for all the bacterial strains isolated. The bacterial strains showed varying TPH degradation in a range from 20 % to upto 95 %. Most of the bacterial strains showed more degradation preferentially for the aliphatic fractions of TPH than the aromatic and asphaltene fractions. This could be because the aliphatic fractions are less toxic and are easily degradable constituents of TPH.

• The genetic diversity was studied in the bacterial strains isolated from the different sampling sites. This was evaluated by whole genome analysis with repetitive DNA sequences (ERIC, REP and BOX) based DNA fingerprinting and PCR based ribotyping.

• Complex genomic profiles were obtained for all the strains by whole genome analysis based on rep-PCR. The bacterial strains isolated from different sites showed heterogeneous genomic profiles. This elucidated a distinct genetic diversity among the bacterial strains isolated from different sampling locations. Presence of unique amplicons distinguished the bacterial strains isolated from specific geoclimatic locations. Certain bacterial strains isolated from diverse sampling sites were showing similarities in their rep-PCR profiles, suggesting that strains were regionally endemic. The divergent evolution of the bacterial strains, which could be related by descent from a common ancestor, could also be a probable reason.

• The rep-PCR fingerprints were not specific to species since a species isolated from different sites was also showing variation in the genomic DNA fingerprints. This suggested the existence of intraspecies diversity. The cluster analysis of the combination of the rep-PCR results could deduce phylogenetic relationships between the strains and to evaluate the genetic diversity. Certain genotypic clusters delineated among the bacterial strains were specific to a particular genus. Similarly genotypic clusters specific to proteobacteria were also observed.

• The ribotype profiles discriminated the strains based on the 16S-23S rDNA internally transcribed spacer polymorphism.

• The intraspecies genetic diversity was studied in the 29 strains of Pseudomonas citronellolis isolated from the six different crude oil and oily sludge contaminated sites. The intraspecies diversity was evaluated by repPCR based genomic DNA fingerprinting, fluorescent amplified fragment length polymorphism, amplified 16S ribosomal DNA restriction analysis, PCR based and restriction fragment length polymorphism of the 16S-23S rRNA internally transcribed spacer. The different molecular techniques were targeted to study the intraspecies diversity among the P. citronellolis in details

• Based on the unweighted pair group method clustering analysis (UPGMA) of the rep-PCR fingerprints, the 29 strains of P. citronellolis were delineated into 12 genotypic groups distributed among 7 distinguishable ribotype patterns. The rep-PCR genotypes were specific to the isolation site of the strains. The genotypic distinctions of the 29 different P. citronellolis strains isolated from different sampling sites reflect the difference in location of the sampling sites with respect to geo-climatic regions of India. The fact that the soil at these sites was contaminated with different types of crude oil and oily sludge may also be a contributing factor. The strains of P. citronellolis isolated from Digboi refinery were most genotypically diverse which could be because the soil at Digboi refinery was contaminated with crude oil and oily sludge for nearly 100 years, allowing for genotypic diversification. The REP primer set generated a greater number of distinct genotypic patterns for P. citronellolis. The ribotype patterns of the strains of P. citronellolis showed multiple amplicons that strongly indicated the polymorphism of the rRNA spacer region.

• On basis of the amplified 16S ribosomal DNA restriction analysis (ARDRA), the UPGMA analysis clustered the 29 strains of P. citronellolis into XIV genotypic groups. The ARDRA showed the differential distribution of the strains in the genotypic groups, which was not related to the isolation site of the strain. All the ARDRA genotypic clusters had heterogeneous distribution of the P. citronellolis strain. The distinct ARDRA patterns of the 29 strains of P. citronellolis strains could be due to increased mutation under stress, facilitating adaptation of the strains to stressful environments. This could also be due to the different types of crude oil and oily sludge contamination and varying age of contamination at the sites.

• The fluorescent amplified fragment length polymorphism (FAFLP) was used to differentiate among the microbial strains and closely related species. The FAFLP results segregated the 29 P. citronellolis strains into eight genotypic clusters and the FAFLP clusters were specific to the isolation site of the strains

• The 16S rRNA gene cloning approach detected the presence of different bacterial species and non-culturable bacterial population at a crude oil and oily sludge contaminated site, which were not obtained by the cultivation based studies. Of the fifty 16S rDNA clones randomly analyzed by sequencing, thirty-one unique sequences were identified. Nine of the 16S rDNA sequences of the clones gave the closest sequence match to uncultured bacteria. The uncultured bacteria were mostly belonging to proteobacteria. Six of the 16S rDNA clone sequences were giving homology match with the yet un-identified bacteria. The 16S rRNA gene cloning provided an assessment of the diversity of the soil samples than the plate culture technique.

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