In vitro screening for oil degrading bacteria and evaluation of their biodegradation potential for hydrocarbon

Twenty seven potential hydrocarbon degrader bacterial isolates were isolated from soil that has been exposed to crude petroleum oil. The hydrocarbon degradation potentialities of these isolates were assessed against n-tetradecane. Four isolates (AT3, AT5, AT11 and AT15) were the most potent isolates. These isolates were identified via morphological, biochemical and 16S-rRNA gene sequencing techniques as (Pseudomonas stutzeri, Bacillus thuringiensis, Bacillus pumilus and Bacillus cereus), respectively. AT3 isolate was the highest n-tetradecane degrader. This isolate exhibited specific growth rate in Bushnell-Hass medium amended with hydrocarbon, n-tetradecane of 1.213 h -1 and growth yield of 1.311 g cells g hydrocarbon -1 . An efficient biodegradation up to 90% was performed by AT3 bacterial isolate. Genetic fingerprinting was carried out using RAPD-PCR and SDS-PAGE methods to characterize and conduct phylogenetic relationship among the four most potent isolates. Thirty six specific markers were obtained and used to characterize the four studied isolates. Different specific markers for AT3 isolate were generated. The closest genetic distance was found between isolate AT11 and AT15 (0.55), while the lowest genetic similarity was between AT3 and AT5 (0.41). The introduced markers could be used for tracking the most potent isolates.


INTRODUCTION
Saudi Arabia is the world's largest producer and exporter of oil, and has one quarter of the world's known oil reserves.The global transport of oil from production centers to the world market has been established through road, rail, pipeline and shipping infrastructures.As a consequence, there is a constant risk of oil spills in almost every environment worldwide.The oil spillage poses a serious environmental problem, due to the possibility of air, water and soil contamination.There is an increased interest in promoting environmental friendly methods in the process of cleaning oil-polluted sites.Biological treatment of environmental pollutants is preferred over physicochemical as the former is cost effective, efficient and environmentally friendly (Ojo, 2006).Microbial remediation of hydrocarbon compounds was found to be an available alternative method over the conventional methods.Microbial treatment can control hydrocarbons pollution by reducing the length of the hydrocarbon molecules and by producing products that act as biosurfactants and solvents (Banat, 1995).Microorganisms isolation from oil polluted sites is commonly taken as evidence that these microorganisms are active degraders of that pollutants (Okerentugba and Ezeronye, 2003).Several studies reported that mixed cultures carry out more extensive biodegradation of hydrocarbons than pure cultures (Foght et al., 1999;Komukai et al., 1996;Malik and Ahmad, 2012).There are several advantages relying on indigenous microorganisms rather than adding microorganisms to degrade hydrocarbons.So, it is very useful to screening for new indigenous microorganism degrading hydrocarbon.Isolation, identification and characterization of potential indigenous biodegrading isolates are helpful in the manipulation, utilization and tracking these potent isolates.Various methodologies were adopted to identify the immense complex bacterial community potential problems in culture ability of many of the members (Abou-Shanab, 2007).
Serological and bacteriological methods are not sensitive enough to differentiate all bacterial isolates (Taghi et al., 2008).Therefore, several molecular approaches now provide powerful adjuncts to the culturedependent techniques.Now Combination of colonial morphological, physiological, biochemical, serological and molecular markers is essential for successful identification either to the genus level or more frequently to the species level (Millar et al., 2007).Total cellular proteins as well as DNA-based analyses can contribute significantly to the characterization of bacteria that have been successfully isolated from different environments (Leisner et al., 1994;Vos et al., 1995;Durrani et al., 2008).SDS-PAGE is an important molecular technique used for the identification at species level of whole cell proteins and it has the advantage of being fairly simple and rapid to perform.However, this technique requires extensive data to cover all known target species (Leisner et al., 1994;Durrani et al., 2008).Genomic DNA fingerprinting using random amplified polymorphic DNApolymerase chain reaction (RAPD-PCR) was found to be useful in differentiating among closely related bacteria (Williams et al., 1990).Besides being easier and cheaper, these methods are as effective as the more labor intensive restriction fragment length polymorphism (RFLP) techniques for establishing genetic relationships and identifying bacterial genomes (Selenska-Pabell et al., 1996).Also, RAPD-PCR is an effective technique for typing microbial isolates such as Brucella spp.(Behroozikhah et al., 2005), Pseudomonas stutzeri, Enterobacter aerogenes, Pseudomonas pseudoalcaligenes, Pseudomonas maltophila and Pseudomonas vesicularis (El -Tarras et al., 2010;Awad et al., 2011).Bacterial 16S-rRNA is a common target for taxonomic purposes and identification, largely due to the mosaic composition of phylogenetically conserved and variable regions within the gene (Gurtler and Sanisich, 1996;Bayoumi et al., 2010).
The objectives of the present study are to isolate and characterize bacterial strains capable to remediate crude  oil and similar hydrocarbons.Api profiles, total protein profile, RAPD-PCR as well as 16S-rRNA sequencing procedures were employed for characterization and identification of bacterial isolates.

Enumeration of microorganisms
Soil samples were enumerated by making tenfold serial dilution of samples using physiological saline.From the diluted sample, using a dropper pipette, 1 ml of each dilution was dropped onto Petri dish then the plate count agar, Actinomycetes agar media and Sabourauds media for fungi amended with n-tetradecane were poured (Gerdhardt et al., 1984).Duplicates of plates were used for each dilution.Plates were incubated for 48 to 72 h at 30°C in an incubator.Each inoculum of microorganism developed into a discreet colony.All plates yielding 30 to 300 colonies were counted.
The number of viable microorganisms in the sample was calculated from the number of colonies formed, the volume of inoculum used by dropper pipette and the dilution factor expressed in colony forming unit (CFU) (Krieg, 1984).

Isolation of crude oil degrading bacteria
The microorganisms used in this study were obtained by the enrichment culture technique by inoculating Bushnell-Haas enrichment mineral media with fertile garden soil and crude oil.Samples were grown for two months in a rotary shaker for 25 cycles.The culture media used were an enrichment medium for isolation of bacterial degrading organism and nutrient agar.Bushnell-Haas broth medium containing (g/l): MgSO 4 .7H 2 O, 0.2; K 2 HPO 4 , 1.0; KH 2 PO 4 , 1.0; FeCl 3 , 0.05; NH 4 NO 3 , 1.0; CaCl 2 , 0.02; pH to 7.2 and sterilize at 121°C for 15 min.Inoculation was done using Bushnell-Haas enrichment medium of 10 ml in 250 ml flasks into which ten grams of the contaminated soil was added and incubated at 30°C for 4 weeks.Samples that were turbid were subcultured into nutrient agar using nutrient broth medium, as diluents to observe the morphological characteristics of the isolates.
Colonies showing a good growth and characters were picked and streaked on new Bushnell-Haas minimal agar plates amended with n-tetradecane.A rapidly growing, visually distinct colony and a separate, morphologically unique isolate were selected for further analysis and purified by repeated plating.

Microorganisms identification
The morphological characteristics of the isolates were identified by gram stain and biochemical reactions.The biochemical reactions include glucose fermentation, oxidase test, catalase production reaction, cell motility; egg yolk reaction and reaction in tryptose soya broth were performed.Bacteria were isolated, identified and named based on morphological, physiological and biochemical characteristics presented in Bergey's Manual of Determinative Bacteriology (Holt et al., 1994) and the APi Kit profiles (Api, bioMerieux, France, 2009).

Culture growth conditions
Nutrient broth medium was used.Its composition was the following ((g/l): pepton, 10; yeast extract, 3; NaCl, 8; distilled water, 1000 ml) to grow mother cultures in 125 ml Erlenmeyer flasks with 10 ml of medium and incubated with shaking (150 xg) at 30°C overnight.
The mother cultures of the isolates were cultivated in Bushnell-Haas enrichment mineral medium containing 3 ml of n-tetradecane as a sole carbon and energy source.Bacteria were cultivated in 250 ml Erlenmeyer flasks with 100 ml of medium and incubated in rotary shaker (150 xg) at 30°C.

Optical density and biomass measurement
The turbidity of the cultures was determined by measuring the Optical Density (OD) at a wavelength of 595 nm in 2 ml cuvettes using a spectrophotometer (Biophotometer plus, Eppendorf).The net dry weight for the biomass was determined simultaneously.A 1 mL of culture was centrifuged at 1500 xg for 10 min, washed twice with distilled water, poured into a pre-weighed container, dried overnight at 90°C to constant weight and cooled for reweighing.The linear relation between O.D 595 and dry mass was obtained.Cultures were usually harvested at absorbency 0.660.Cells were harvested by centrifugation for 5 min at 3,000 x g at room temperature.

Growth rate measurement
The growth rates of cultures in exponential phase were determined from linear regressions of log10 absorbency vs. time, calculating a least squares fit of data from the exponential growth phase, and determining the slope of this line.The instantaneous growth rate (μ) was determined from the slope of this line x ln10; μ had the dimensions h -1 (Koch, 1981).

Hydrocarbon measurements
Residual n-tetradecane was determined using the method used by Martinez-Checa et al. (2002).The analyses were carried out in triplicate in a TechComp D-7900 ver.1.30 Gas Chromatograph equipped with a flame ionization detector using a 30 m x 0.32 mm, 0.25 μm internal diameter, polar DB1 fused silica capillary column (Supelco).The carrier gas was nitrogen at a flow rate of 20 ml/min.The temperature of the injector was 230°C and that of the flame ionization detector was maintained at 320°C.The oven temperature after sample injection (2 μl) was 1 min at 50°C, increasing to 200°C at 15°C/min and held at this temperature for 2 min then raised to 280°C with an increasing rate of 15°C/min and held for 2 min. 1 μl of pure n-tetradecane dissolved in 10 ml hexane was used as standard.

Sample preparation and SDS-PAGE analysis
Culture samples (withdrawn from growing cultures, when they reached an OD 420 of 0.5,) were centrifuged (4,000 × g for 20 min at 4°C) in Eppendorf tubes.Supernatant was discarded and the pellet was suspended in 0.5 ml PBS (Phosphate Buffer Saline).After centrifugation the supernatant was discarded and the pellet was resuspended in 1 ml of distilled water.The cell pellets as well as sediments from fractionated cells were resuspended in SDS-PAGE sample buffer (Bradford, 1976).The proteins were separated by electrophoresis on 12% (w/v) SDS-polyacrylamide gels (Laemmli, 1970) using a BioRad Mini-Protean apparatus.The molecular weight of separated bands was determined in reference to the (SDS marker, Sigma-7H and Fermentas SM 0661 protein ladder).The separated proteins were stained with coomassie brilliant blue (CBR) R-250 and de-stained by overnight shaking incubation in methanol, acetic acid and water de staining buffer.The gels were visualized under the white light and documented using a GeneSnap 4.00-Gene Genius Bio Imaging System (Syngene; Frederick, Maryland, USA).

DNA extraction
The cell pellets from all isolates were used to extract genomic DNA using (Jena Bioscience, Germany) extraction kit by following the manufacturer's instructions.

Random amplified polymorphic DNA (RAPD)
Nine different primers were used in PCR reaction which consists of 10 pmol of each different arbitrary 10-mer primers and 25 to 50 ng of genomic DNA and 12.5 μl of 2x SuperHot PCR Master Mix (Bioron, Ludwigshafen, Germany).The names and sequences of these oligoprimers are listed in Table 2.The RAPD-PCR amplification reactions were performed in Eppendorf® thermal cycler using the following PCR program: 1cycle at 94°C, 4 min; 35 additional cycles consisting of 94°C 5 s, 37°C 20 s and 72°C 20 s.After the amplification, the PCR reaction products were electrophoresed with 100 bp ladder marker (Fermentas, Germany) on 10 x 14 cm 1.5%-agarose gel (Bioshop; Canada) for 30 min using Tris-borate-EDTA Buffer.The gel was stained with 0.5 μg/ml of ethidium bromide (Bioshop; Canada).

PCR amplification of 16SrRNA gene
Primer sequences used to amplify the 16S-rRNA gene fragment were: U1 [5CCA GCA GCC GCG GTA ATA CG3] and U2 [5ATC GG(C/T)TAC CTT GTT ACG ACT TC3] according to (Kumara et al., 2006).The PCR master mix contained10 Pmol of each primer and 12.5 μl of 2x SuperHot PCR Master Mix (Bioron, Ludwigshafen, Germany) mixed with 50 to 100 ng of DNA template.Sterile d.H 2 O was added to a final volume of 25 μl.Thermal cycler (Uno II , Biometra, Germany) program was 94°C for 4 min., 94°C for 1 min., 55°C for 1 min., 72°C for 1.5 min, the number of cycles was 35 cycle and the post PCR reaction time was 72°C for 5 min.

Analysis of the PCR products
After the amplification, the PCR reaction products were electrophoresed with 100 bp ladder marker (Fermentas, Germany) on 10 x 14 cm 1.5%-agarose gel (Bioshop; Canada) for 30 min using Tris-borate-EDTA Buffer.The gels were stained with 0.5 ug/ml of ethidium bromide (Bioshop; Canada), visualized under the UV light and documented using a GeneSnap 4.00-Gene Genius Bio Imaging System (Syngene; Frederick, Maryland, USA).

Gels analysis
The SDS polyacrylamide gels and agarose digital image files were analyzed using Gene Tools software from Syngene.The densitometric scanning of each based on its three characteristic dimensions was carried out.Each band was recognized by its length, width and intensity.Accordingly, the relative amount of each band was measured and scored.

Sequencing of 16S-rRNA gene
The 990 bp PCR-products of each isolate were purified from excess primers and nucleotides by the use of AxyPrep PCR Clean-up kit (AXYGEN Biosciences, Union City, California, USA) and directly sequenced using the same primers as described for the

Determination of genetic relationship
In order to determine the genetic relationship among studied bacteria SDS-PAGE and RAPD data were scored for presence (1) or absence (0) of the bands.The data were transferred to a statistical software program, Statistical Package for Social Science, version 10.00 (SPSS Inc, Chicago, Illinois, USA) to obtain analytical statistics in the form of Jaccard's similarity coefficient (S) showing the genetic similarity among different examined bacterial isolates based on pair-wise comparison.The dendrogram was constructed using the Average Linkage between groups.

Nucleotide sequence accession numbers
Nucleotide sequences have been deposited in the GenBank database under accession nos.JQ342094 for P. stutzeri AT3, JQ621963 for Bacillus thuringiensis AT5, JQ638269 for Bacillus pumilus AT 11 , JQ303325 for Bacillus cereus AT15.

Degrading bacteria isolation
To isolate bacteria that degrade crude oil, Bushnell-Hass medium was supplied with various concentrations of ntetradecane as the sole carbon and energy source.The medium inoculated with fresh garden soil and incubated at 30°C and pH 7.0 on a rotary shaker (150 xg) for 25 cycles.This set was last for 3 months after which serial dilutions of the obtained culture were prepared and plated on Bushnell-Hass medium agar plates with crude oil at 10, 20, 50 ml.-1 as the sole carbon and energy source (Lal and Khanna, 1996).The microbial colonies thus developed were further tested, purified and sub-cultured on the same medium.Twenty seven microbial isolates representing the different colony morphologies observed with the ability to grow on n-tetradecane as sole carbon and energy source were obtained (Table 1).These include 27 isolates as short rods and long rod bacilli bacteria.All isolates were identified morphologically and physiologically.Phenotypic examination of the recovered bacteria isolate revealed that they belong mainly to the genera of Bacillus, Pseudomonas, Actenomycetes, Enterobacter, and Acinetobacter.Isolates were identified on the basis of their cultural and biochemical characteristics according to Bergey's Manual of Determinative Bacteriology (9 th edition) as well as API kit profiles.Four isolates were selected for further studies (Table 1).The study was limited to degradation of n-tetradecane by four isolates.This selection was based on growth rate, biomass, and macroscopic differences in colony morphologies and microscopic examination of bacterial cells as well.

Biodegradation of hydrocarbon
The isolates were grown in Bushnell-Hass minimal medium amended with n-tetradecane as sole carbon and energy source to determine their growth rate, biomass yield, and biodegradation activity.Biodegradation activity of bacterial isolates revealed a varying hydrocarbon biodegradation rates of 50.6 to 90% with isolate P. stutzeri AT3 being the superior (Figure 1).Most growth occurred in the first 3 days of cultivation period for all isolates followed by rapid growth reaching a maximum biomass concentration of 2.9 g l -1 after 5 days.Isolates might degrade 3 g/100 ml of n-tetradecane completely in 5 days only (Figure 1).All isolates showed highest  4).Strain AT3 and AT 11 produced the highest biomass on used hydrocarbon being 2.810 and 2.870 g l -1 , respectively (Table 4).Lazar et al. (1999) achieved 95.3% petroleum hydrocarbon degradation from paraffinic crude oil at 28°C in 10 to 12 days by using a consortium of 15 bacterial isolates.The reduction in wax appearance temperature and heavy hydrocarbon fractions by biodegradation of paraffinic hydrocarbons using Pseudomonas and Actinomyces species was noticed (Etoumi, 2007).It was mentioned that the lower the concentration of hydrocarbons the higher was the utilization.With the other three isolates, AT 5, AT11 and AT15 slow growth with much longer lag phases, being 40 h with the three isolates (Figure 1, B, C and D).Also, low n-tetradecane utilization was recorded.This resulted in lower biomass being 1.9 and 1.6 g.l -1 , respectively, compared to isolate AT3.Residual undegraded n-tetradecane was recovered at the end of cultivation period with the two isolates.
The maximum specific growth rate (μ m ) of 1.213 h -1 was recorded for isolate AT3 compared with only 0.073, 0.044 and 0.919 h -1 for isolates AT5, AT11 and AT15, respectively (Table 2).Moreover, isolate AT3 produced more biomass from hydrocarbons being 1.311 g cells l -1 n-tetradecane.The growth yield values recorded for the three isolates were 0.78, 0.63 and 1.101g cell/g ntetradecane, respectively (Table 3).
Isolation of alkane degrading microorganisms from oil contaminated soil has been reported by several researchers.Nazina et al. (2005) have obtained hydrocarbon oxidizing Geobacilli strains from formation waters of oil fields.Hydrocarbon degrading members of the family Bacillaceae were found to dominate the oil contaminated soil of Kuwait (Mohamed et al., 2006).In addition, a bacterial isolate Geobacillus thermodenitrificans that shows selective degradation of long chain alkanes, similar to the degradation pattern of isolate AT3, was also isolated (Wang et al., 2006).

Identification of isolates
The bacterial isolates were subjected to morphological and biochemical tests (Table 1).The isolates AT5, AT11 and AT15 possessed typical cellular and colonial morphologies, physiological, biochemical, and nutritional features that resembled it to Bacillus genus.

Molecular and biochemical characterization of isolates
According to the alignment at the National Center for Biotechnology Information (NCBI), the studied isolates AT3, AT5, AT11 and AT15 (Figure 5) were identified as P. stutzeri, B. pumilus, B. thuringiensis and B. cereus, respectively (DeSantis et al., 2006) .
Based on the SDS-PAGE data there are 49 produced bands from four examined bacterial isolate (Figure 2).Out of which 13 were monomorphic bands with homogeneity (26.5%).AT15 isolate was the highest band producer and followed by AT11, AT5 and AT3 being 35, 32 and 30 bands, respectively (Table 5).
The total number of amplified bands through all primers and isolates was 237.Variations in the size and number of amplified fragments from each primer were detected.The size of amplified fragments ranged from approximately 100 bp in primer D7 (AT3 and AT15) to approximately 1500 bp in primers (C9, D7 and D8) in (AT3, AT11 and AT15 isolates), respectively.The maximum number (20 fragments) was amplified with primer D8.Table 7 shows the specific markers obtained across RAPD-PCR analysis.Primers D7 generated the highest number (7) of specific markers.In contrast, C6 and D4 produced the least number of markers, two per primer.
As regards to genetic relationships, data presented in (Table 6) showed that, the highest genetic similarity was between isolate AT15 and AT11 ( 55%), while the genetic similarity between isolate AT3 and AT5 was the lowest (41%).

DISCUSSION
High molecular weight hydrocarbons are highly difficult to degrade.High molecular weight biosurfactants are highly efficient emulsifiers that work at low concentrations and exhibit considerable substrate specificity, they are produced by a large number of bacteria and they are composed of polysaccharides, proteins, lipopolysaccharides, lipoproteins etc. (Banat et al., 2000;Zhang et al., 2012).In general microorganisms produce biosurfactants to increase their interfacial area for contact to give improved uptake of hydrophobic substrates.However, it has been observed that the exopolymers synthesized by these strains in media with glucose as carbon and energy source, had a remarkable capacity of emulsifying hydrocarbon compounds (Martinez-Checa et  , 2002;Zhang et al., 2012).Taking into the account the existence of the heterotrophic bacteria that ranged from 4.2 x10 5 to 9.8 x 10 6 cell ml -1 in the fresh soils, the indigenous microbial communities are likely to contain microbial populations of different taxonomic characteristics, which might be capable of degrading the contaminating chemicals (Barth and Atlas, 1977).This observation is in line with reports of Shahaby and EL-Tarras (2011), that there was an increase in heterotrophic bacterial population in the presence of dispersant agents.
The microbial density of crude oil contaminated soil was decreased compared to non-contaminated samples being 71.3 to 81% for bacteria, 50 to 72.1% for fungi and 31.2 to 65.6% for Actniomycetes.However, microbial density of oil degrading bacteria was increased in the enrichment culture than non-treated soils being 53 to 77.2% for bacteria, 54.7 to 57.3% for fungi and 33.3 to 58.8%) for Actinomycetes (data not shown).
Isolation of alkane degrading microorganisms from oil contaminated soil has been reported by several researchers.Nazina et al. (2005) have obtained hydrocarbon oxidizing Geobacilli strains from formation waters of oil fields.Hydrocarbon degrading members of the family Bacillaceae were found to dominate the oil contaminated soil of Kuwait (Mohamed et al., 2006).Moreover, the bacterial strain G. thermodenitrificans that shows selective degradation of long chain alkanes, similar to the degradation pattern of isolate AT3, was also isolated (Wang et al., 2006).
The local isolates would have great application in bioremediation of crude oil contaminated sites.Bioaugmentation treatment with such a wide spectrum of degraders would be the preferable choice of treatment over many others.The biodegradation increased fast and the metabolism was high enough to maintain the bacterial activity stable.It was also observed that the first day of incubation was the most important and critical stage for the biodegradation of the hydrocarbon.Results obtained in this study are similar with results obtained from soil samples at Hokaiddo and Japanese Costal water (Tanaka et al., 1993;Shahaby and El-Tarras, 2011).The behavioral patterns of the hydrocarbon-utilizing bacteria in enrichment media containing different concentrations of hydrocarbon (n-tetradecane) present an interesting observation (data not shown).
In order to determine the optimum hydrocarbon concentration for microbial growth, further growth rate experiments at different concentrations of n-tetradecane hydrocarbon were carried out.Growth of bacterial strains was evaluated by measuring culture optical density (OD) at 595 nm.The strains showed different growth rates in different media (data not shown).B. pumilus AT3 showed maximum growth rate values.Most growth occurred in the initial 24 h.The concentration of 3% hydrocarbon was the optimum and had a μ max of 1.213 h −1 .
A reduction in wax appearance temperature and heavy hydrocarbon fractions by biodegradation of paraffinic hydrocarbons using Pseudomonas and Actinomyces species was recorded (Etoumi, 2007;Zhang et al., 2012).It was reported that the lower the concentration of hydrocarbons the higher was the utilization (Wang et al., 2010).Obtained molecular and biochemical genetic data documented that the studied isolates belongs to two different genera as showed in Figure 4.The dendrogram showed phylogenetic tree which divided into two main groups.The first main group consisted of three isolates AT11, AT15 and AT5.The closest genetic distance was found between isolate AT11 and AT15 which were first clustered together and then with isolate AT5.The second group includes isolate AT3 (Figure 4).The 16S-rRNA gene sequencing identified studied bacterial isolates as P. stutzeri, B. pumilus, B. thuringiensis and B. cereus, this results is in agreement with previous studies reported that such bacteria species capable to biodegrade hydrocarbons (Singh and Lin, 2008;Shahaby and El-Tarras, 2011;Awad et a., 2011;Hassanshahian et al., 2012).These two genera have focused on its wide range of diverse degrading capabilities and potential application in bioremediation.This might be due to the great genetic diversity among these two genera which confirmed by different pattern of amplification and restriction enzyme digestion based on RAPD and SDS PAGE.This diversity supports the utilization of these isolates as bacterial consortium to perform more bio derivative potentialities (Wang et al., 2010).Thirty six specific markers across RAPD primers were identified and characterized four studied isolates.Such markers considered as specific markers for tracking these bacterial isolates Knowledge obtained from this study could help in understanding the biodegradation of petroleum  hydrocarbon in contaminated sites, as well as to design efficient biocatalyst allowing transformation of oil fractions into valuable compounds.The isolation of pure strains from such a consortium has also been achieved, its hydrocarbon degradation ability confirmed, and the different effects of hydrocarbon on their degrading capacity have been shown.Complete identification of these isolates has been carried out and further work continues on their characterization and abilities to produce surfactants.More research is necessary to understand the fundamental mechanisms of enhancement and inhibition in the microbial degradation of super high concentration of toxic compounds.However, these micro-organisms could be used very effectively for in situ bioremediation in an environment which is highly contaminated with petroleum hydrocarbon.However, further research could be carried out on these isolates, on genetic manipulation for improvement and exploitation as bioremediation vehicles.

Figure 4 .
Figure 4. Phylogenetic tree of the four tested bacterial strains using RAPD-PCR and SDS-PAGE analysis.

Figure 5 .
Figure 5. Agarose gel electrophoresis of 990bp PCR fragment of 16S rRNA gene among four studied bacterial isolates with 100bp DNA ladder.

Table 1 .
Morphology, physiology, growth, and biodegradation of n-tetradecane by four cultures in Busnell-Hass medium amended with n-tetradecane as the sole energy and carbon source.

Table 2 .
Growth kinetics of four isolates grown in Bushnel-Hass medium amended with n-tetradecane as carbon and energy source.
amplification process.The products were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (ABI Applied Biosystems, Foster City, California, USA) on a 3130XL Genetic Analyzer (Applied Biosystems).The bacterial 16S rDNA sequences obtained were then aligned with known 16S rDNA sequences in Genbank using the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information, and percent homology scores were generated to identify bacteria.

Table 3 .
List of primers, their nucleotide sequences and total number of bands for each isolate are produced by nine primers.

Table 4 .
Biodegradation rates (%) of four isolates in Bushnell-Hass medium amended with n-tetradecane as energy and carbon source.

Table 5 .
Number of bands for each isolate produced by SDS-PAGE.

Table 6 .
Similarity coefficient values among the four studied bacterial isolates.

Table 7 .
Specific markers for studied bacterial isolates across RAPD-PCR analysis.