A Morphological and Molecular Study of Bacterial Strains First Reported Locally in the City of Samarra and Isolated from Hydrocarbons Contaminated Soil

• Marwah Luay Muhammed Hafedh1 Afrah Tohma Khalaf 2

  Author

Soil is a fundamental foundation for life on Earth; it is not merely a medium for plant growth and spread, but rather represents a complex biosystem with a diverse array of living organisms. Soil health and the absence of contamination particularly by hydrocarbons and their derivatives reflect the health of the entire ecosystem.  Certain bacteria play a key biochemical role in the functioning of the ecosystem and in maintaining soil structure. Therefore, The aim of this study is to isolate and identify bacterial strains from hydrocarbon-contaminated soil in the city of Samarra at the morphological, microscopic, and molecular levels, and to identify any mutations in them, if present.

Methods: Soil samples were collected from two sites in the city of Samarra: the first site consisted of soil samples from power generators, and the second site consisted of soil samples from fuel filling stations during the period of November 2025, The bacterial culture media were prepared, diluted in a 10-fold series, and inoculated onto the media. The samples were then identified morphologically, microscopically, and using the D2MINI device, followed by molecular identification and sequencing of the 16S rRNA gene, and the results were analyzed.

Results: The results of bacterial identification from both soil types, following cultivation on Blood Agar and MacConkey Agar media, and based on colony morphology, cultural characteristics, microscopic examination, and identification using the D2 MINI Microbial ID/AST system, revealed the presence of Bacillus licheniformis, Bacillus subtilis, and Pseudomonas aeruginosa in both soil types.Molecular identification of these isolates was subsequently performed using the Polymerase Chain Reaction [PCR] technique targeting the 16S rRNA gene. Nucleotide sequencing results were compared with reference sequences available in the NCBI GenBank database. The findings demonstrated that the Bacillus licheniformis and Bacillus subtilis isolates investigated in this study represent novel strains recorded for the first time at the local level in Iraq due to the presence of nucleotide variations and mutations. The Bacillus licheniformis isolate exhibited six mutations with a sequence similarity of 99%, including five transversion mutations and one transition mutation. In contrast, the Bacillus subtilis isolate showed six mutations with a sequence similarity of 98%, comprising five transversion mutations and one insertion mutation. To document these novel strains, they were deposited in the NCBI GenBank database and assigned unique accession numbers for their nucleotide sequences as follows: accession number PZ694937 for Bacillus licheniformis strain Marwa1, and accession number PZ694938 for Bacillus subtilis strain Marwa2 and accession number PZ694939 for Pseudomonas aeruginosa strain Marwa 3.

Conclusion:Our study found that soil samples from power plants and fuel filling stations contaminated with hydrocarbons contained bacterial species that had acquired adaptive and acclimatory mutations to resist these pollutants, leading to the emergence of new strains for the first time locally in the city of Samarra. This was confirmed through DNA-based molecular diagnostic methods, which confirmed the presence of mutations in these strains, identified as Bacillus licheniformis, Bacillus subtilis, and Pseudomonas aeruginosa.

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