Isolation and characterization of phenol degrading organism, optimization using Doehlert design

Phenol degrading organisms were effectively isolated from petroleum contaminated soil. Based on their resistivity to phenol up to 1500 mg l(-1), the bacterial strains were isolated as R1, R2, R3, R4, and R5. Morphological studies showed that most of these organisms as gram-positive, rod-shaped and aerobic. These five isolated organisms showed maximum similarity with Pseudomonas stutzeri NCG1, Bacillus flexus strain MS14-1, Bacillus thuringiensis strain 2PR56-10, Bacillus anthracis strain IHB B 18197, and Bacillus thuringiensis strain Bt 2 based on 16S rRNA sequencing. Preliminary experimental conditions such as pH, temperature, aeration, inoculum age and inoculum volume were studied for maximum phenol degrading organisms along with mixed culture. Under optimized conditions, gram-negative P. stutzeri showed the maximum capability to degrade phenol up to 81.8% at 500 mg l(-1) initial concentration within 84 h. Also, the phenol degradation by Pseudomonas stutzeri was optimized using response surface methodology (RSM) for various parameters viz. pH, temperature, phenol concentration and yeast extract concentration through batch experiments according to Doehlert design. The optimized conditions of pH, temperature and yeast extract concentration were used to determine the kinetic parameters of phenol degradation using various models. The analysis of kinetic data suggests that the Haldane substrate inhibitory model can be used to fit experimental data. Additionally, phenol tolerant Pseudomonas stutzeri produced cis, cis-muconic acid which is a metabolic intermediate specifying the organism followed ortho pathway.