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Pseodomonas sp. download (4), Main references:
Farag, S., A. Soliman, N.,…
Pseodomonas sp. 
Characteristics
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A Gram-negative,rod-shaped bacterium
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Source
Oil spill site in Bahary area (Alexandria, Egypt)
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Isolation
- Sample A, from soil contaminated by mechanic oil spill site in the Abo-Quir Alexandria.
- Sample B is water contaminated by mechanic oil spill site in Bahary Alexandria.
- Sample C was collected from soil around gasoline area.
- Sample D is from soil contaminated by oil spill in the Red Sea near the oil refining region.
The samples' pH level was then determined. Their moisture and organic matter content was also analyzed.
For the enrichment phase, the method used was soil enrichment method.
1) 200 ml of sterile mineral salt media, MSM was added to a 500 ml Erlenmeyer flask along with 10 g soil/10 ml water samples that are oily polluted. Next, 2 ml (1%) v/v of crude oil was put into the mixture.
2) The mixture was then incubated for at 30 °C for seven days in an orbital shaker operating at 200 rpm. After that, several subculturings was carried out on the same media with 1% crude oil each for 7 days.
3) The cultures' serial dilutions were prepared. A sample was then spread onto two sets of duplicate MSM oil agar plates with 1% (v/v) crude oil as a substrate. NaCl with concentration of 2% was used for the growth of marine bacteria
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Composition of MSM consistes of these components (g/l)
1.8 K2PO4, 1.2 KH2PO4, 4.0 NH4Cl, 0.2 MgSO4. 7H2O, 0.1 NaCl and 0.01 FeSO4. 7H2O (pH 7.4)
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Screening
- Gravimetric analysis was done in order to find out individual quantitative estimation of oil degradation by collected isolates.
- In each flask, 2% (v/v) of MSM that was supplemented with 1% (v/v) of initially sterilized crude oil was added to pre-culture inoculate suspensions (24 h growth)
- The control experiment was carried out without the addition of isolates and the flasks were then incubated at temperature of 30 °C for six days. The incubation step took place in an orbital shaker that operated at 200 rpm.
- Several isolates was then selected for further investigation. The potent isolates selected were sp29, sp48 and sp50.
Identification
- The cells from overnight culture had been collected for centrifugation.
- Next, the genomic DNA was isolated by a modified method of Sambrook
- Amplification was carried out using prokaryotes 16S rRNA with a specific forward primer: 50-AGAGTTTGATCMTGGCTCAG-30 and reverse primer: 50-TACGGYACCTTGTTACGACTT-30.
- The product from PCR was sequenced using the similar PCR primers.
5.The DNA similarities, multiple sequence alignment and molecular phylogeny were assessed using Blast program such as ncbi.
- The obtained sequence will be submitted into GenBank under accession number (ac: KP202717).
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Biotechnological Importance of microbe
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2,5-Furandicarboxylic acid
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Main references:
Farag, S., A. Soliman, N., & Abdel-Fattah, Y. (2018, January 6). Statistical optimization of crude oil bio-degradation by a local marine bacterium isolate Pseudomonas sp. sp48. Retrieved January 14, 2021, from https://www.sciencedirect.com/science/article/pii/S1687157X18300015?via%3DihubWeimer, A., Kohlstedt, M., Volke, D. C., Nikel, P. I., & Wittmann, C. (2020). Industrial biotechnology of Pseudomonas putida: advances and prospects. Applied Microbiology and Biotechnology, 104(18), 7745–7766. https://doi.org/10.1007/s00253-020-10811-9