2.3. Isolation, purification, and identification of phenol degrader bacteria

The pour-plate method was used to isolate phenol-degrading bacteria on P-NA using enrichment media containing phenol, P-NB, and P-NA (Usman et al., 2021). Only the single colonies grown on the P-NA medium were transferred independently to a fresh medium in a Petri plate and subcultured in several slants of maintenance medium (Mueller Hinton Agar), labeled with the initial designations, and stored for future investigation.

Morphological examinations and biochemical analysis using the API 20NE kit (bioMerieux, Marcy Etoile, France) have been evaluated for the identification of phenol degraders. Confirmation of the identification was evaluated using 16S rDNA sequencing. Genomic DNA was extracted according to Hassen et al. (2018) from an overnight-grown culture, and PCR amplification was performed using a forward primer (5′-AGAGTTTGATCMTGGCTCAG-3′) and a reverse primer (3′-TACCTTG TTACGACTT-5′) for 16S rRNA. The obtained 16S rRNA sequences were integrated into the database using the automatic alignment tool, and a phylogenetic tree was generated through distance matrix analysis using the NT system. Database searches and comparisons were performed using the BLAST search tool with the National Centre for Biotechnology Information (NCBI) database, and accession numbers were obtained.