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Petroleum as crude oil while drilling, as refinery effluents, as slop oil emulsion solids and as water from ballast tanks may find their way into the adjoining land and surface or ground water through accidental or deliberate disposal. The storage of the crude oil in tanks results in 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. form the oily sludge. The improper disposal of oily sludge by dumping it in open pits leads to the contamination of the terrestrial ecosystem. There have been huge loss of agricultural land because of the crude oil spills and dumping of oily sludge. Petroleum hydrocarbon contamination is a major environmental concern since many of its constituents are highly toxic, carcinogenic and are poorly biodegradable in nature. Among the various methods used for reclamation and remediation of environments contaminated with petroleum hydrocarbons, the use of biological methods have been well reviewed and acknowledged. With the new biotechnological innovations in pollution control, the documentation of microbial diversity at such sites will help in formulating novel strategies for efficient and effective reclamation of contaminated sites.
A polyphasic approach compromising of both conventional methods and nucleic acid techniques were used in the present study for proper understanding bacterial microflora associated with the sites contaminated with total petroleum hydrocarbons. The present investigation emphasized the aspects of functional and genomic diversity of the total petroleum hydrocarbon degrading bacterial strains prevalent in soil contaminated with TPH. These sites were situated in the different geo-climatic regions of India and history of the petroleum hydrocarbon contamination at these sites varied.
The summary of the above study is as follows:
1. A total of eight sites were selected for this study. These sites were situated in different geoclimatic locations of India. The sites of Digboi refinery, Guwahati refinery and BRPL refinery, Bongaigoan are in the north-eastern part of India. HPCL refinery, Visakhapatnam is situated in the south-eastern region of India. Jamnagar refinery and Vadinar oill off loading site are in the western region of India and Mumbai is situated in the south-western India. While the site Cochin refinery is located in the southern part of the country.
2. There was a variation observed in the type, levels, and history of total petroleum hydrocarbon contamination prevalent at these sites. the sampling location at Digboi refinery had the longest contamination history of over 100 years while contamination history at sampling points in Jamnagar refinery five years. The soil samples collected from sites Digboi refinery, BRPL refinery, Bongaigoan, and Cochin refinery had higher level contamination of aromatic fractions of total petroleum hydrocarbons (TPH). Whereas soil samples from Jamnagar refinery, HPCL refinery, Visakhapatnam and BPCL refinery, Mumbai had high level contamination of aliphatic fractions of TPH.
3. A total of 165 culturable hydrocarbon degrading bacterial strains were isolated from soil samples contaminated with TPH procured from eight sampling sites located at different geoclimatic regions of India locations. Preliminary characterisation of the culturable bacterial strains was based on Gram reaction, colony morphology and substrate utilisation profiles generated by Biolog system. Sequencing the genes encoding 16S rRNA was performed for identification of bacterial strains. Phylogenetic affiliation of the culturable bacterial cells was confirmed after validation of the phylogenetic tree of the individual bacterial isolates.
4. Among the total culturable bacterial strains isolated, Gram negative-rod shaped bacterial strains were dominant. The culturable bacterial strains were predominantly affiliated to the -subclass of proteobacteria. A total of 165 bacterial strains isolated in the present study were affiliated to eighteen different bacterial genera. The most dominant species identified from different sites was Acinetobacter baumannii. The bacterial strains isolated from TPH contaminated soil samples from Digboi refinery had the maximum bacterial diversity with eleven species. The diversity of culturable bacterial strains was least in TPH contaminated soil samples procured from Vadinar oil off loading site and Jamnagar refinery site with three bacterial species respectively.
5. Genetic diversity was evaluated among the 165 bacterial strains by whole genome analysis with repetitive DNA sequences (Enteric Repetitive Intergenic Consensus, Repetitive Entergenic Palindromes and BOXAIR) based DNA fingerprinting and tRNA intergenic length polymorphism. Bacterial strains from different sites showed heterogeneous genomic profiles elucidating a distinct genetic diversity among them. Maximum genomic diversity was observed among the bacterial strains isolated from Digboi refinery. Presence of unique amplicons differentiated bacterial strains isolated from specific geoclimatic locations. Bacteria isolated from diverse sampling sites were showing similarities in their genomic fingerprinting profiles suggesting that strains were regionally endemic. Genomic fingerprints were not species specific since a species isolated from different sites was also showing variation in the genomic DNA fingerprints. This suggested the existence of intraspecies diversity.
6. The functional diversity of the 165 culturable bacterial strains was assessed by estimating the degradation potential of different fractions of TPH and four different PAH compounds. The culturable bacterial strains showed degradation potential of TPH in a range from 30 % - 90 %. Gas chromatography analysis indicated the majority of the bacterial strains showed more degradation preferentially for the aliphatic fractions of TPH than the aromatic fractions.
7. The bacterial strains isolated from the Digboi refinery, Cochin refinery and BRPL refinery, Bongaigoan could effectively degrade the aromatic fractions of TPH compounds. These sites had comparatively longer TPH contamination history and were contaminated with aromatic fractions of TPH. Among the 165 bacterial strains, 59 strains showed effective degradation of PAH compounds. The bacterial strains from Digboi refinery, Cochin refinery and BRPL refinery, Bongaigoan could degrade high molecular weight PAH compounds taken for the study. While the bacterial strains from Jamnagar refinery, Vadinar off loading site and HPCL refinery, Visakhapatnam could degrade only the low molecular weight PAH compounds.
8. A rare bacterial strain identified as Leclercia adecarboxylata TERI 4040 was isolated from soil samples collected from total petroleum hydrocarbon contaminated soil procured from Digboi refinery. This bacterial strain could efficiently degrade pyrene along with differential capability to degrade other polycyclic aromatic hydrocarbons. This enteric bacterial strain showed a novel metabolic pathway for pyrene degradation. An intermediate 1- hydroxypyrene, previously not described for pyrene metabolism was identified by gas chromatography mass spectrometry method.
9. Catechol 2, 3 dioxygenase (C23DO) is a key enzyme in PAH degradation and catechol is a central intermediate in PAH degradation pathway. Thus, detection and screening of catabolic genes encoding C23DO enzyme was done by a PCR based approach with four different degenerate primer sets to screen the aromatic degrading bacterial. All the bacterial strains that showed positive amplification with any of the primer sets could effectively degrade PAH compounds. The PCR based screening by four primer sets indicated a positive correlation between the distribution of catabolic genes among the bacterial strains and degradative ability of PAH compounds by these bacterial strains.
10. Despite the taxonomic diversity observed within the bacterial strains, the distribution of catabolic genes revealed a distinct homogeneity in their presence among the bacterial strains. The bacterial strains that showed positive amplicon with 23DO primers variegated over nine different species Similarly, tol primer set gave positive amplification with seven different bacterial species, PI primer set showed amplification with eight different bacterial species and ndo primers gave amplification with six different bacterial species respectively. The study indicated that the characteristics of the total petroleum hydrocarbon contaminants that are prevalent in the sites influenced the distribution of catabolic genes in the isolated bacterial strains.
11. Among the four primer sets used for this study, degenerate primer set of 23DO showed a broader detection of catabolic genes encoding catechol 2 3 dioxygenase (C23DO). Thus phylogenetic analysis of 900-bp fragment amplified with 23DO primer for 26 bacterial strains isolated from seven different geographical sites was done. The phylogenetic analysis of the C23DO sequences indicated that the sequences obtained from bacterial strains of specific site were grouped together. It was also observed that the bacterial strains whose C23DO sequences are grouped together showed similar ability to degrade specific PAH. As observed with the screening of catabolic genes, the sequence analysis of C23DO fragment reiterates the importance of selective pressure by the contaminants on bacterial microflora.
12. Among the eight selected sites for the present study, Digboi had the longest history of TPH contamination and the strains isolated from this site indicated the maximum diversity. Thus this site was selected to study the total bacterial community by a culture independent approach. The total community analysis of the contaminated soil sample from Digboi was studied by DGGE analysis of community 16S rRNA gene and analysis of 16S DNA clone library. These approaches detected the presence of few bacterial species that were not obtained by the cultivation-based studies. However, there were few bacterial species that were identified with the culture dependent techniques but were not detected by culture independent technique. The community profile of the bacterial groups that were detected among the different operational taxonomic units (OTUs) by culture independent methods were found to be similar (not identical), when compared with bacterial groups detected by the culture dependent methods. The analysis of DGGE profiles and 16S rDNA gene clone library indicated the predominance of - proteobacterial group among the OTUs as also indicated by the culture dependent studies.
13. The ecological parameters prevalent in the contaminated sites are influential in obtaining a genomic variation among the bacterial strains thriving in these contaminated sites. This genomic diversity in turn effects the functional diversity of the bacterial strains. The shifts in microbial community composition could be induced by changes in environmental factors such as temperature, moisture content and substrate levels. In strenuous environment such as TPH contaminated soil, bacterial strain tries to exploit different opportunities for its adaptation. The long-term exposure of contaminants at a particular site and higher levels of contaminants induces selective pressures for survival of indigenous microflora.
14. The process of natural selection might have favoured bacterial strains thriving in these sites to have an access for the limiting substrate resources in TPH contaminated sites, allowing functional diversification of the strains. The genomic diversity among the bacterial strains isolated from these contaminated sites can me postulated to happen due to high frequency transfer of mobile genetic elements in environments saturated with recalcitrant pollutants. In stressed environments such as oil contaminated sites, the possibilities of horizontal and lateral gene transfers are quite evident and the homology of catabolic gene observed in this study suggests that gene flux mediated by conjugative plasmids have an important role to play.
15. In this study it was thus observed long-term exposure of contaminants at a particular site and higher levels of contaminants induces selective pressures for survival of indigenous microflora. This possibly lead to a series of genetic recombination for successful adaptation of the bacterial flora in such contaminated sites This in turn have lead to observe a degradative variability among the bacterial flora from contaminated sites. Further studies on the expression level catabolic genes among the bacterial strains are warranted that will give a better insight on the role of contaminants at the genomic level.
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