Isolation of a Most Potent Bacterial Strain from Soil …
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2.2 Reagents
Phenol-(analytical grade), 4-amino antipyrene, potassium ferricyanide, ammonium
chloride and ammonium hydroxide solution. Our targeted concentration of phenol
was prepared by mixing the properly weighed phenol to distilled water.
2.3 Bacterial Media
The nutrient agar medium with following composition was used for cultivation of
bacteria.
Peptone—0.5%, beef extract—0.3%, agar—3.0%.
2.4 Isolation and Screening of Most Potent Bacterial Strain
The bacterial strain capable of removing phenol was isolated by soil enrichment and
serial dilution plate count method. The soil was enriched with 500 ppm phenol for
10 days. After serial dilution and plate count isolated colonies were transferred in
individual slant. A total of 29 isolated colonies were transferred to slant.
Screening was done to select most potent strain. Each of the isolated colonies
was transferred to liquid medium and incubated 37 °C for 24 h in the presence
of 500 ppm phenol. After fermentation, the fermented broth was centrifuged and
the clear supernatant was used for spectrophotometric estimation of residual phenol
content.
2.5 Estimation of Residual Phenol Content
Residual phenol concentration was measured in spectrophotometer at 510 nm wavelength followed by APHA method. Residual phenol content was calculated from the
standard curve made with the known concentrations of phenol.
2.6 Morphological, Biochemical, and Phylogenetic
Characterization of the Isolated Bacterial Strain
Morphological and biochemical characterization of the isolated bacterial strain was
done as per Bergey’s Manual of Determinative Bacteriology (Holt et al. 1993).
Phylogenetic assay was made by 16s rDNA method.
289
2.2 Reagents
Phenol-(analytical grade), 4-amino antipyrene, potassium ferricyanide, ammonium
chloride and ammonium hydroxide solution. Our targeted concentration of phenol
was prepared by mixing the properly weighed phenol to distilled water.
2.3 Bacterial Media
The nutrient agar medium with following composition was used for cultivation of
bacteria.
Peptone—0.5%, beef extract—0.3%, agar—3.0%.
2.4 Isolation and Screening of Most Potent Bacterial Strain
The bacterial strain capable of removing phenol was isolated by soil enrichment and
serial dilution plate count method. The soil was enriched with 500 ppm phenol for
10 days. After serial dilution and plate count isolated colonies were transferred in
individual slant. A total of 29 isolated colonies were transferred to slant.
Screening was done to select most potent strain. Each of the isolated colonies
was transferred to liquid medium and incubated 37 °C for 24 h in the presence
of 500 ppm phenol. After fermentation, the fermented broth was centrifuged and
the clear supernatant was used for spectrophotometric estimation of residual phenol
content.
2.5 Estimation of Residual Phenol Content
Residual phenol concentration was measured in spectrophotometer at 510 nm wavelength followed by APHA method. Residual phenol content was calculated from the
standard curve made with the known concentrations of phenol.
2.6 Morphological, Biochemical, and Phylogenetic
Characterization of the Isolated Bacterial Strain
Morphological and biochemical characterization of the isolated bacterial strain was
done as per Bergey’s Manual of Determinative Bacteriology (Holt et al. 1993).
Phylogenetic assay was made by 16s rDNA method.