Kinetics of high concentrated phenol biodegradation by Acinetobacter baumannii.
Phenol is an organic, aromatic compound that occurs naturally in the environment  and is water-soluble and highly mobile . Phenol is the basic structural unit in a wide variety of synthetic organic compounds . Now a day's phenol is used by many industries in the manufacturing of product like insulation materials, adhesives, lacquers, paint, rubber, ink, dyes, illuminating gases, perfumes, soaps and toys . The main use of phenol is as a feedstock for phenol resins, bisphenol A and caprolactam. The microorganisms that are normally used in phenol degradation include Pseudomonas species , Candida tropicalis , Azotobacter species , Rhodococcus species , and Acinetobacter species . In the present study Phenol-degrading microorganism was isolated and its performance in treating the compound was investigated.
Materials and Methods Isolation of Bacterial Strains
Soil samples from cotton fields of Kanchikacherla, Krishna district, was collected and mixed thoroughly, grinded and made into a fine powder. From this 1gm of soil was taken and serial dilutions was made. From these dilutions, 10-6 dilution sample was taken and inoculated into minimal medium containing 125mg/l phenol. This culture was incubated for a week from this a Loop full is transferred into fresh minimal medium with 125mg/L phenol. This culture was again incubated for 48 hrs and growth of bacteria and phenol degradation was tested. The initial phenol concentration and the final phenol concentration after incubation period were observed, from this we concluded the bacteria we isolated can degrade phenol successfully.
Minimal medium is used for the growth of microorganism. The components are Potassium di hydrogen phosphate 3 g/l, Disodium hydrogen phosphate 6 g/l, NaCl 5g/l, Ammonium Chloride 2 g/l, these components were dissolved in water and autoclaved then Magnesium Sulfate 0.1 g/l is added to the medium, plates were prepared using the same compounds and in addition to them Agar15 g/l is added.
Bacterial culture growing in Minimal medium with phenol was taken and centrifuged at 5000 rpm for 20 min, for 2ml of bacterial supernatant 0.1ml of folincalcitue reagent and 0.3 ml sodium carbonate was added, the mixture was incubated for 60min.Deep blue color was observed and is estimated at 750nm.
Immobilization of Microorganism
The phenol degrading bacteria was harvested after 30h of growth from 1 L of culture medium. The cell pallet obtained by centrifugation at 5000 rpm for 10 min at 4 [degrees]C, obtained was used for immobilization in alginate matrices. Alginate (4% w/v) was solubilised in boiling water and autoclaved at 121 [degrees]C for 15 min. A 10 ml of bacterial cell suspension was added to 100 ml of sterilized alginate solution and mixed by stirring on a magnetic stirrer. This alginate-cell mixture was extruded drop by drop into a cold, sterile 0.1 M calcium chloride solution through a burette. The gel beads formed were left in the solution for one hour before being filtered off. The beads were washed with distilled water and used for experiments. These beads were washed successively with distilled water and saline and were packed tightly in a glass column, from the top of the column 1000mg/L phenol is introduced by using a peristaltic pump (2Lh-1) whose initial OD reading is taken at 760 nm and noted. The phenol poured into column is left for sometime in order to allow the immobilized cells for uptake of the phenol. From the bottom of the column phenol is collected at regular time interval of 2 hrs and its OD is measured at 760 nm.
Results and Discussion
The isolated bacterium was identified as Acinetobacter baumanniisp using BIOLOG. Ten batch cultivation experiments were carried out using phenol as single limiting substrate for Acinetobacter baumannii. Different initial phenol concentrations of 125 mg/L to 1000 mg/L were used. The extent of phenol degradation using these different initial phenol concentrations was investigated for several batch residence times by intermittent sampling. Figure1 also depicts the typical cell growth curve. The cell growth curve has typical exponential and stationary phases with increasing lag phase as increasing phenol concentrations. 10 different initial phenol concentrations ranging from (125-1000) mg/L were completely degraded (consumed) at different residence times. Maximum growth was observed for lower concentration and growth maximum is decreasing with increasing concentrations of phenol. As shown in Figs.2, which indicates the comparison of the time course for phenol substrate consumption of all the ten batches, it is evident that the rate of degradation decreased with increase in the initial phenol concentration. The bacteria entered into log phase 20hr to 30hr depend upon initial phenol concentration and complete degradation is varied from 36hr to 48hr .It observed that the encapsulated bacteria in sodium alginate has more degrading ability than free cell batch cultivation experiments. The immobilized cells showed quicker degradation rates and complete degradation were observed within 25hrs (Fig.No.3).
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It is observed that as the initial phenol concentration increased the duration of the lag phase increased; and thereby, prolonging the biodegradation time as a result of decrease in the rate of degradation. This observation is supported by the earlier works 9-11. According to Prpich and Daugulis 12, the rate of substrate consumption was suggested to be the most important measure of microbe performance. Zilli et al. 14 gave a similar report. As seen from Fig. 3, the specific phenol consumption rate (rs) increased with the increase in the initial phenol concentrations. Therefore, it seems that there is also an influence of the initial phenol concentration on the specific phenol consumption rate. According to Layokun et al 15 the growth of microorganisms corresponds to the degradation of the substrate. Hence, the growth of microorganisms on phenol can be described by the most commonly used kinetic models that can be based on specific substrate consumption rate as proposed by Posten13 and which have been used by Zilli et al.14. In this work, phenol was completely degraded and kinetic model of Monod based on specific substrate consumption rate was used to evaluate the degradation of phenol at different initial concentration by this microbe. The classical method of obtaining kinetic parameters is to linearize kinetic models. Recently, non-linear least squares computer fitting of data to model equations has been used 16. The non-linear least square fitting routine of MATLAB was used to fit the Monod kinetic model to the different batch experimental data. The parameters of Monod (Ks and rsmax) were found using the curve fitting tool of MATLAB and then fitted to the experimental calculated specific phenol consumption rate and the corresponding phenol concentration. The result revealed that, Monod has a correlation coefficient (R2) greater than 0.90 and the least root mean square error (RMSE = 0.00165), and thus, indicating a very good fit to the batch experimental data and the MONOD's graph is as shown below(x-axis = Substrate concentration (mg/lit) ; y-axis = Phenol consumption rate (mg/mg/hr)).
[FIGURE 4 OMITTED]
The observation of substrate inhibition due to phenol can be modeled using substrate inhibition models described in literature16. The experimental results of specific phenol consumption rate variation with initial phenol concentration was fitted to five inhibition models of Haldane9, Yano and Koga17, Aiba et al. 18, Teissier and Webb19 . The model with the best fit was selected on the basis of highest correlation coefficient (R2) and the least root mean square error (RMSE).
The experimental batch biodegradation data was fitted to Haldane, Yano and Koga, Aiba et al., Teissier, and Webb, substrate inhibition models. It was shown that of all the models, the Aiba et al. model represented the biodegradation data reasonably very well, and hence, may be proposed as the best model to describe the phenol biodegradation behaviour of this microbe and the AIBA's graph is as shown below(x-axis = Substrate concentration (mg/lit); y-axis = Phenol consumption rate (mg/mg/hr)).
[FIGURE 5 OMITTED]
The kinetic parameters of the models (rsmax, Ks and Ki) were estimated using the non--linear regression routine of MATLAB, and the results along with the R2 and RMSE are given in Table 1.
Conclusions and Future Work
The kinetic constants evaluated using the models showed good tolerance and growth of the indigenous organism, and therefore, a complete degradation of the phenol substrate. The performance of the indigenous strain in biodegradation of phenol in the nutrient medium is good.
From experimental results we can conclude that Acinetobacter baumannii can degrade the phenol effectively even at high concentrations of phenol. We observed that majority of biodegradation takes place during exponential phase. Further research can be done by continuous fermentation and we can study the kinetics in continuous fermentation process.
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S.B.C. Prasad, R. Satish Babu, R. Chakrapani, C.S.V. Ramachandra Rao *
Department of Biotechnology, DVR & HS MIC College of Technology, Kanchikacherla, Andhra Pradesh, India.
* Corresponding Author E-mail: email@example.com
Table 1 Model rmax (mg/mg/h) Ks K1 K2 Monod 0.04103 268.6 -- -- Aiba 0.1793 268.5 -- -- Haldane 2.351 -6.683 -- -- Yano & Koga 17.12 68.73 .1897. 8.764 Tessier 0.8121 0.3345 -- -- Webb .02559 -14.63 -- -- Model Ki R2 RMSE Monod -- .9453 .00165 Aiba .7772 .9453 .001905 Haldane 1.67 -10.44 .0255 Yano & Koga -- -13.18 .03066 Tessier .2132 -16.36 .03141 Webb -8.578 .02408 .007448