IDENTIFICATION AND CHARACTERIZATION OF POTENTIAL PHENOL DEGRADING BACTERIAL STRAINS ISOLATED FROM MUNICIPAL SEWAGE , BILASPUR , CHHATTISGARH

Phenol and its derivatives are consistently causing harmful effects to an aquatic ecosystem. The present study focused on the isolation and characterization of potential phenol degrading bacterial strains and subsequently optimization of media ingredients for efficient phenol degradation by potential bacterial strains. Bacterial strains were isolated from municipal sewage, Bilaspur (21'47 and 23'8 N 81'14 and 83'15 E). After optimization phenol degradation rate was increased by 1.84 fold for PDB 5 (from 40.37% to 74.67%) and 1.39 fold for PDB 11 (from 58.62% to 81.51%) at 500mg/l initial phenol concentration. PDB 5 and PDB 11 were identified as Streptococcus sp. PDB 5 and Pseudomonas sp. PDB 11 respectively as potential phenol degrading bacterial strains. These strains can further be used in microbially assisted phenol degradation to remove phenol derivatives present in industrial wastewater.


Introduction
Effluents of Petrochemical, coke oven, dye industries, plastic and fiberglass manufacturing unit, pulp and paper production, herbicide manufacturing and oil refineries have been reported for higher amount of phenolic compounds (Paisio et al., 2013, Mohite et al., 2010, El-Ashtoukhy et al., 2013, Veeresh et al., 2005, Jadhav and Vanjara, 2004).These phenolic compounds enter into aquatic food chain when untreated or by improper treated industrial wastewater drained into natural water reservoirs e.g.ponds, lakes, rivers, and oceans.Further phenolic compounds cause harm to aquatic living organisms (e.g.fish) and related organisms which depend on them as a food source (e.g.Eagle and humans).WHO quoted that short-term exposure of excess phenol inside human body causes liver and kidney damage, harmful effects on respiratory system and growth retardation.
Physical methods (such as ultraviolet rays, ozonation), chemical methods (e.g.hydrogen peroxide and Fenton's reaction) and its combination (physio-chemical methods) have been applied to remove phenolic compounds from industrial wastewater (Lin et al., 1994;Zilli et al., 1993).Presently the chemical treatments have been used in most of the industrial wastewater treatments.But it has been found to produce harmful secondary pollutants and costly as well (Agarry et al., 2008).The biological degradation of phenolic compounds is an alternative approach to overcome aforementioned issues.It is an eco-friendly and costeffective method to remove excess phenolic compounds from the industrial wastewater.The pure or mixed cultures of microorganisms which are able to utilize phenolic compounds as an energy source can be used for biological degradation of phenols (Allsop et al., 1993;Wang et al., 1999;Yuan et al. 2000;Sa et al, 2001).Diverse microorganisms like bacteria, yeasts, algae and filamentous fungi have been reported for their metabolic capability to degrade phenol at high concentrations (Sivasubramanian and Namasivayam, 2014).
Microorganisms can metabolize phenolic compounds by either ortho-or meta-cleavage (Dagley, 1971;Agarry et al., 2008).Phenol hydroxylase has known as the first enzyme in both metabolic pathways (ortho-or meta-pathway).The final products of both ortho-and meta-pathways are entering into the Tricarboxylic acid cycle (TCA) for complete mineralization (Shingler, 1996).

Research Article
The present study is focused on the isolation and characterization of phenol-degrading bacteria from municipal sewage, Bilaspur and optimization of different parameters to enhance the rate of phenol degradation by potential bacterial isolates.

Sampling
Samples were collected from Municipal sewage water, Bilaspur (21'47 and 23'8 N latitudes and 81'14 and 83'15 E latitudes) for the isolation of Phenol degrading bacterial strains and stored in sterile plastic containers at 4 o C until used.

Isolation
Serial dilution method was adopted for isolation of bacterial strains.Serially diluted samples were inoculated in Nutrient Agar Media (NAM) plates and incubated at 37 0 C for 24h.These NAM plates served as master plates for the isolation of phenol degrading bacterial strains.After incubation, the pure cultures of bacteria were prepared by streak plate method.

Phenol Degradation
The ability of the acclimatized bacterial strains to degrade phenol was determined.Experiments were carried out in 150 ml Erlenmeyer flask containing 50 ml of MSM with phenol concentration at 500 mg/1.A loop full bacterial culture (McFarland Standard-0.5; 1.5×10 8 cells) was aseptically inoculated in the flasks containing sterilized MSM and incubated at 37 o C in the shaker (150 rpm).Samples were withdrawn at every 24h time interval (till the completion of incubation period), centrifuged and analyzed for phenol concentration for the assessment of the rate of phenol degradation by respective bacterial strains.Initial phenol concentration was subtracted by residual phenol concentration (concentration of phenol remained after the microbial phenol degradation) for the assessment of phenol degrading efficiency of microbial strains.Percentage phenol degradation (g/l) was calculated as per formula is shown below: Initial phenol concentration -initial amount (mg/l) of phenol Residual phenol concentration-amount (mg/l) of phenol remained after biodegradation

Phenol Estimation Assay
Phenol concentration was determined quantitatively by a colorimetric method by using 4-aminoantipyrine (4-AAP) as the chromophore.Protocol was adopted from Standard Methods for the Examination of Water and Wastewater (Greenberg et al., 1992) with some modification.Reaction mixture consisted of 0.9 ml double distilled water, 0.1 ml supernatant (sample), 50 µl of 2N NH4OH (pH-10.0),25µl of 2% 4-AAP and 25 µl 8% of K3Fe(CN)6 at 40 0 C. Optical density was measured at 510 nm.

Estimation of Cell Biomass Concentration
Cell biomass concentration was estimated in terms of CFU/ml as per standard method.

Morphological and Biochemical Characterization
Morphological, Physiological and Biochemical characterization (as per described in Bergey's Manual) were done for the identification of potential phenol degrading bacterial isolates.

Optimization of Bacterial Strains for Maximum Phenol Degradation
Physiochemical parameters and media composition for bacterial strain under un-optimized and optimized condition are shown in Table 2. PDB 5 and PDB 11 showed maximum phenol degradation at 35 0 C and 37 0 C respectively while pH 6.5 and 7.5 respectively (Table 2).Finally after optimization phenol degradation rate was found to increase from 40.37% to 74.67% for PDB 5 and from 58.62% to 81.51% for PDB 11 (Table 3).

Conclusion
The present research was focused on the isolation and media optimization for phenol degrading bacterial strains.Streptococcus sp.PDB 5 and Streptococcus sp.PDB 11 were identified as potential phenol degrading bacterial strains.These bacterial strains would be effectively applied on microbially assisted phenol degradation of industrial wastewater which consisted of excess phenolic compounds.
Further mutagenesis and kinetic study will be applied on Streptococcus sp.PDB 5 and Streptococcus sp.PDB 11 to enhance their phenol degrading efficiency at higher phenol concentration (≤500mg/l).

Table 1 :
Percentage phenol degradation rate by bacterial isolates

Table 2 :
Un-optimized and optimized condition for bacterial isolates

Table 4 :
Morphological characteristics of bacterial isolates

Table 3 .
Streptococcus sp.PDB 5 and Pseudomonas sp.PDB 11 were identified as potential phenol degrading bacterial strains.
Greece whereWang et  al., revealed that in their experiment Streptococcus showed maximum phenol degradation rate at pH 6.5 and temperature 32 o C. Later (2010) Mohite et al., revealed that Streptococcus epidermis isolated from soil was able to degrade 200mg/l of phenol and confirmed by both spectrophotometric and HPLC analysis.In 2015 Mohite again conducted the study on biotransformation of phenol and its derivatives and found that 4-nitrophenol was best transformed by Streptococcus epidermis at pH 7.0 and temperature 32 o C.