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INTRODUCTION
Petroleum-based products are the major source of energy for industry and daily life.Used motor oil is the brown-to-black oily liquid removed from a motor vehicle, with increasing demands of fossil fuel energy, extensive exploration of natural sources has caused number of large scale accidental spills of crude oil and resulted in environmental disasters (Darsa et al., 2014), Lubricant oil from pump motors, automobile engines and engines of boats and ships constitute an important pollutant of water and soil ecosystems, leading to devastating damage to the aquatic ecosystem at times of accidents.There are wide varieties of microorganisms in water and soil known to utilize petroleum hydrocarbons as an energy source and degrade them (Gopinath et al., 2015).Bacteria are the most active agents in petroleum degradation, and they work as primary degraders of spilled oil in environment (Rahman et al., 2003;Brooijmans et al., 2009).It has long been a popular bioremediation process in case of oil or petroleum contaminated soil (Beaudin et al., 1996;Van Gestel et al., 2003).Compost is rich in carbon, organic nitrogen, phosphorous and mineral compounds required for growth of microorganisms helping to degrade the oil (Joo et al., 2007).Several bacterial species have been identified as having the ability for oil degradation.In general, Bacillus sp. has been identified as petroleum hydrocarbon degrader Ghazali et al., 2004) and is known as naphthalene and pyrene degrader (Ron and Rosenberg, 2001;Zhuang et al., 2002).B. axarquiensis, B. thuringiensis and B. cereus isolates able to degrade the aromatic fraction of the crude petroleum oil under both non-saline and saline conditions (Lobna, et al., 2017).
Petroleum oil is the main pollutant specially in petroleum countries producers.In This research study looked for bacterial strains have ability to degrade petroleum oily pollutants.Two bacterial isolates from 23 isolates have high ability to degrade engine oil were characterized and identified.The two bacterial isolates have been identified phenotypically and genotypically methods, as Bacillus subtilis strain BS1 and Bacillus axarquiensis strain BA1, these deposited in national center for biotechnology information (NCBI Gen Bank) under the Accession numbers MF375213.1 and MF375110.1 respectively.Two isolated strains have ability to grow in minimal medium containing petroleum oil with maximum bacterial growth was determined by spectrophotometry.Bacillus subtilis growth exhibited 1.7 OD at 600nm after 2 days of incubation time whereas Bacillus axarquiensis exhibited 1.14 OD at 600 nm after 3 days of incubation time with minimal medium containing 2% petroleum oil.

Collection of samples
Used motor oil was obtained from automobile workshops.The used oils were collected in well washed and airdried plastic bottles.

Isolation of petrol degrading bacteria
The collected oil samples were serially diluted up to 10 7 and 0.1 mL from the dilutions 10 5 and 10 6 were spread plated on mineral salt medium (magnesium sulphate 0.2 g, calcium chloride 0.02 g, mono potassium phosphate 1 g, di potassium phosphate 1 g, ammonium nitrate 1 g and ferric chloride 0.05 g) containing 2.5% of petrol (Bushnell, and Haas, 1941), the plates were incubated at 37°C for 24 hrs.(cited in Darsa et al., 2014), and from the developed colonies, one was selected for further experiments.

Biodegradation studies
One hundred mL of minimal medium (dextrose 1 g, ammonium sulphate 1 g, dipotassium phosphate 7 g, monopotassium phosphate 2 g, sodium citrate 0.5 g and magnesium sulphate 0.1 g) with petrol and one mL inoculum from the overnight culture maintained in nutrient broth during the logarithmic phase were added to 250 mL Erlenmeyer flasks which were subjected to a period of four days.Flasks were incubated in a shaker at 30 °C at 100 rpm.After specified time, flasks were taken out.The ability of the isolated strain to degrade petrol was studied by determining the parameter optical density (OD).

Identification of petrol selected bacterial degrading strains Phenotyping Identification
Gram staining and biochemical tests such as catalase, oxidase, Voges proskaeur, and indole production, sporulation test were carried out for strain identification (Holt et al., 1994).

Genotyping identification by 16S rRNA gene DNA Extraction
The genomic DNA of Bacillus strains was isolated using modification method from Sambrook (Sambrook, et al., 1989).

Identification by 16S Ribosomal RNA gene
PCR Amplification according to Sambrook (Sambrook, et al., 1989) The PCR amplification reactions were performed in a total volume of 50 L.Each reaction mixture contained the following solutions will prepared: 2 L of DNA (40 ng), 1 L of 10 pmol forwarded 16S-rRNA primer (5′-AGAGTTTGATCMTGGCTCAG -3′ ); 1 L of 10 pmol reverse 16S rRNA primer (5′-AACTGGAGGAAGGTGGGAT -3′ ); 0.8 L of 12.5 mM (dNTP's); 5 L of PCR buffer included MgCl2, and 0.2 L Taq polymerase (1 Unit) and water-free DNAse and RNAse were added up to 50 L.The PCR apparatus was programmed as follows: 3 min denaturation at 95 C, followed by 35 cycles that consisted of 1 min at 95 o C, 1 min at 58 C, and 1 min at 72 o C, and a final extension was 10 min at 72 o C. The products of the PCR amplification were analyzed by agarose gel electrophoresis (2%).2.6.3.PCR Cleanup and 16S rRNA Sequencing.The PCR products were cleaned up for DNA sequencing following the method described by Sambrook et al. 1989.Automated DNA sequencing based on enzymatic chain terminator technique, developed by Sanger (Sanger et al., 1977).

Phylogenetic Analysis:
Similarity analysis of the nucleotides was performed by BLAST searches against sequences available in GenBank.For phylogenetic tree construction, multiple sequences were obtained from GenBank and the alignments were performed using clustal W program.A phylogenetic tree was built using MEGA 5 software (Tamura et al., 2011).The morphological and physiological characteristics of the isolated strains were compared with the data from Bergey's Manual of Determinative Bacteriology (Holt et al., 1994)

Identification by 16S Ribosomal RNA (rRNA)
The DNA was purified and analyzed by gel electrophoresis, the quality of the DNA for each sample has been identified for further investigation.The amplified 16S rRNA gene from the DNA of Bacillus sp.BS1 and Bacillus sp.BA1 was determined using 2% agarose gel.The size of the amplified fragments was determined by using size standard (Gene ruler 100bp-3000bp DNA ladder).The PCR products were visualized under UV light and photographed using gel documentation system.Approximately 1200bp of 16S rRNA gene was amplified.The PCR products were purified and sequenced using 16S forward primer.The sequences of Bacillus sp.BS1 and Bacillus sp.BA1 were deposited in national center for biotechnology information (NCBI GenBank) under the Accession numbers (MF375213, MF375110), respectively.The basic local alignment search tool (BLAST) algorithm was used to retrieve for homologous sequences in GenBank.The Bacillus sp.BS1 revealed 99% identity to Bacillus subtilis while Bacillus sp.BA1 revealed 99% identity to Bacillus axarquiensis.Based on the morphological, biochemical, and molecular characteristics, the Bacillus sp.BS1 and Bacillus sp.BA1 were designated as Bacillus subtilis BS1 and Bacillus axarquiensis BA1, respectively.A phylogenetic tree based on the comparison of 16S rRNA sequences of reference strains was constructed.The phylogenetic analysis was performed with 1200bp sequences using the software MEGA 5 (Tamura et al., 2010).DNA nucleotide sequence of 16S rRNA gene of BA1 strain that was amplified by PCR and partially sequenced for Bacillus subtilis (MF375213.1)and nucleotide sequence was the following:

Biodegradation of petroleum oils
Changes in the optical density of medium during the four days of treatment of petrol by Bacillus subtilis and Bacillus axarquiensis strain are illustrated in Figs. 3,  4. Optical density values exhibited an increase during the initial period of treatment up to 3 days and afterwards showed a decline.Maximum bacterial growth of Bacillus subtilis and Bacillus axarquiensis in medium with petrol oil were 1.7 OD at 600 nm with 2% petroleum oil after 2 days incubation time and 1.14 OD at 600 nm with 2% petroleum oil after 3 days of incubation time respectively.Bacteria can degrade carbon compounds in petroleum oil as a necessary source of energy (AMSA, 2004).There are many different varieties of hydrocarbons and over millions of years' bacteria have evolved catalytic enzymes that are specific for degradation reactions.Some of the simpler compounds can be degraded by a very wide variety of bacteria but the ability to degrade hydrocarbons is found in fewer species.No single bacterium can make all the different enzymes, instead; each kind of bacterium specializes in a few hydrocarbons as preferred food sources (Owaid, 2008).

CONCLUSION
According to data of this research study, two bacterial strains isolated and identified, have ability of biodegradation of petroleum oil, the two given strains identified as Bacillus subtilis , Bacillus axarquiensis and they can be used as active agents in petroleum degradation process.Further study needed to imply of the identified strains in field of contaminant water by oil.

Fig. 1 :
Fig. 1: Phylogenetic dendrogram based on 16S rRNA gene sequences showing the position of strain BS1 among members of the genus Bacillus species.

Fig. 3 :
Fig. 3: Changes in the optical density of the culture medium during the degradation of petrol by the bacterium Bacillus subtilis.

Fig. 4 :
Fig. 4: Changes in the optical density of the culture medium during the degradation of petrol by the bacterium Bacillus axarquiensis

Strains Isolation and Identification Identification by Morphological and Biochemical Tests Isolation and Characterization of B. subtilis MF375213.1 and B. axarquiensis 33
A total of 23 pure cultures of sporeforming bacteria were isolated and purified which obtained from different samples collected from Assiut city, Egypt.All isolates shown the ability to degrade petroleum oil.Two Bacillus isolates have high ability to degrade petroleum oil purified and gained the names Bacillus sp.BS1, and Bacillus sp.BA1.
revealed that Bacillus sp.BS1 and Bacillus sp.BA1 are matching with those of B. subtilis group.In previous articles on taxonomy, species included in the B. subtilis group.The two strains are aerobic, motile, Gram-positive rods, and spore forming.The results of biochemical tests of the Bacillus sp.BS1 and Bacillus sp.BA1 indicated that they are related to B. subtilis, and B. axarquiensis which summarized in Table 1.Bacillus axarquiensis (a.xar.qui.en9sis.N.L. 1. physiological and biochemical tests for the selected strains. Tab Phylogenetic dendrogram based on 16S rRNA gene sequences showing the position of strain BA1 among members of the genus Bacillus species. Ron, E.Z. and Rosenberg E. (2001).Natural roles of biosurfactants.Environ.Microbiol., 3: 229-236.Sambrook J., Fritsch E. F. and Maniatis T. (1989).Owaid, Y. H. (2008).The common and solitary action of some bacterial isolates on biodegradation of average Kirkuk crude oil.J. Sci.Rafidain, 19: 101-115.