Selection of phenol degrading microorganism
The endophytic bacterial strain K. rhizophila 14asp was selected on the bases of its growth rate and survival at elevated initial phenol concentrations on MSM agar plates. The selected bacteria grew by using phenol as the only carbon source.
During the study it was also observed that after 24 hours of phenolic stress, colonies of K. rhizophila 14asp appeared white and lubricious from clear yellow morphology.
Optimization Analysis
The rate of degradation of a pollutant influenced by various factors, but environmental fluctuations is most affecting ones [6]
The effects of different physio-chemical factors in the degradation efficiency of phenol were studied in order to optimize the performance of bacterium for obtaining the highest biomass and phenol degradation rate.
Effect of incubation temperature on growth and degradation
The Exponential growth period was observed at 1500 mg L-1 which was selected for further physio-chemical parameters. The influence of these parameters was evaluated on rate of phenol degradation and growth of K. rhizophila 14asp. This growth period was further confirmed by faster consumption rate of phenol.
The experimental data on the cell biomass (OD600) of K. rhizophila 14asp at four different incubation temperatures (Fig. 1A) showed non-significant differences in biomass (OD600 nm). As well as Fig. 1(B) also expressed non-significant differences among residual phenol degradation profile in wide range of temperature. However, it was recorded from (Fig. 1B) that degradation profile at 35 °C was higher. The amount of phenol degraded was increased at optimum temperature.
Most field and laboratory studies on phenol biodegradation showed that rate of degradation were more affected by temperature rather than microbial growth [14]. In general, most studies on phenol degradation have been carried out at an optimum temperature of 30 °C [15,8,16]. We found that phenol degradation rate was best at 35 °C and showed non-significant change in degradation rate between 22-40 °C. The reason for this consequence will need further investigations.
Effect of different pH on growth and biodegradation
The time course profile of K. rhizophila 14asp growth in 1500 mg L-1 phenol containing MSM at pH 6-8 was shown in Fig. 2(A and B). The cell growth was non-significantly affected by change in tested pH range. However K. rhizophila 14asp showed little retarded growth at acidic pH. While 7 and 7.3 pH showed non-significant differences and lead to significantly increase in cell growth. The remaining substrate concentration (%) profile of K. rhizophila 14asp was shown in Fig. 2(B). Results of this degradation profile showed that K. rhizophila 14asp optimally degraded phenol at 7.3pH.
The optimum pH 7.3 was gradually fluctuated with increase in initial concentration and was in good agreement with the previous reported study [17]. It has also been reported in literature that various strains efficiently degrade phenol at near a neutral pH [18]. A similar observation was made in this study.
Phenol degradation
Effect of substrate concentration on growth rate and biodegradation
In order to investigate the effect of substrate concentration on growth rate of bacterium and rate of biodegradation of phenol, experiment was performed at different initial concentrations of phenol. As seen in (Fig. 3A) bacterium showed maximum growth rate h-1 at 1500 mg L-1 phenol but with increase in initial concentration growth rate inversely effected. Similar results were reported in earlier studies [19].
Fig. 3(B) showed the results of residual phenol degradation (%) profile of a wide range of initial concentration 1500 mg L-1 to 6500 mg L-1 with 24 hrs time intervals. Further evaluation through Tukey's Multiple Comparison Test showed that first initial concentration has comparable a high degradation rate than other initial concentrations. Results illustrated that with the increase in substrate concentration % degradation and degradation rate slows down (Fig. 3B & 6).
Effect of substrate concentration on cell yield
Cell yield (Y) was expressed in terms of substrate concentration to illustrate the relation between cell increase and substrate loss (Eq.1) [20]. Where Xo and Xm are the initial and maximum dry cell concentrations, Co and Cs are initial substrate concentration and substrate concentration at the maximum cell concentration respectively.
Fig. 4 showed effect of substrate concentration on cell yield (Y) which was calculated by linear regression analysis through GraphPad Prism 5 software. Obtained maximum biomass concentration mg L-1 (Xm) for various final substrate concentration was 0.21 (R2 = 0.8087) which was smaller when compared with the values recorded by other researchers [1,21,22]. It might be due to high initial concentration of substrate. However, it ratified the adoptability and resistance of selected bacteria against toxic effects of phenol.
Kinetics and modeling of phenol degradation
It was previously described that microbial growth relates to consumption or degradation rate of pollutant [23], later on it was proved that substrate degradation was the measure of microbial performance [24]. Most studies were available on specific growth rate but limited data was available on kinetic modeling with respect to degradation rate [25,22]. In concern to existing knowledge, we have calculated influence of substrate concentration on degradation rate with kinetic modeling.
So, phenol degradation study was further established by kinetic modeling by considering growth rate as constant. All the q values (degradation rate h-1) calculated for Sₒ values were used in phenol degradation study. Typical plot of various initial concentrations to find the value of q (section. 2.6) was a straight line which indicated that the degradation rate was first order (Fig. 5).
Fig. 6 displayed q variations for different So values. The experimental maximum degradation rate (qmax) was obtained as 0.002 at 1500 mg L-1 initial concentration. As the initial concentration increased, degradation rate was decreased.
Kinetic parameters of three theoretical models (already described in Table 1) were estimated by experimentally derived q values. Table 1 indicated that Edward model was best fit to entire experimental data among three described models.
Evaluation of degradation kinetics by model fitting has shown that all the three models could predict the experimental data (Table 1) [26]. However, in all three models Edward gave the best fit for experimental data (R2 = 0.991, SD = 0.018) as the evaluated degradation rates were very similar of experimental values. The Edward model showed high R2 value which also confirmed its accuracy. The distinctive kinetic characteristic of K. rhizophila 14asp was observed as high Ks (Table 1) better phenol degradation activity. Previous literature has given the fact that high Ki value revealed the higher resistance to substrate inhibition [27]. Ki value (1766.935) for Edward model showed that K. rhizophila 14asp has strong inhibition effect due to increase in initial concentration but proved great tolerance towards phenol biodegradation.