SUBSTRATE INHIBITION KINETICS OF PHENOL DEGRADATION BY PSEUDOMONAS AERUGINOSA (NCIM 2074)

Transcription

1 International Journal of Environmental Pollution Control & Management Vol. 3, No. 1, July-December 2011; pp SUBSTRATE INHIBITION KINETICS OF PHENOL DEGRADATION BY PSEUDOMONAS AERUGINOSA (NCIM 2074) M. V. V. Chandana Lakshmi 1, V. Sridevi 1, M. Narasimha Rao 2 & A. V. N. Swamy 3 1 Associate Professor, Centre for Biotechnology, Department of Chemical Engineering, A.U, Visakhapatnam-03, Andhra Pradesh, India. 2 Principal, Al-Ameer College of Engineering and Information Technology, College of Engineering, Gudilova, Anandapuram, Visakhapatnam, Andhra Pradesh, India. 3 Professor, Department of Biotechnology, J.N.T.U, Pulivendula, Anantapur District Andhra Pradesh, India. Abstract: The biodegradation of phenol by Pseudomonas aeruginosa (NCIM 2074) was carried out in a batch system and the optimum conditions were determined. At an optimum ph 7, temperature 32 C, agitation speed 160 rpm, carbon source (glucose) 0.5 g/l, nitrogen source (ammonium chloride) 0.2 g/l, metal ion concentration (Zn 2+ ) 0.02 g/l the percentage of phenol degradation was 80.45%. Phenol had a strong inhibitor effect on the microbial growth and Haldane model was used to describe the substrate inhibition. Model parameters were estimated by non-linear regression analysis using Force 2.0 software. The values of the parameters obtained were µ max = h -1 ; K S = mg/l and K I = mg/l. Keywords: Phenol; Biodegradation; Pseudomonas aeruginosa; Substrate inhibition; Kinetics. 1. INTRODUCTION Environmental pollution, especially with the hazardous and recalcitrant chemicals, is one of the major problems faced by the developing countries like India (Saravanan et al. 2008). Phenol, is one such toxic pollutant discharged in to the environment by various industrial operations, which include petroleum refineries, chemical and pharmaceutical industries, wood processing (Xabier Sevillano et al. 2008), plastic/coke oven industries, phenol resin manufacturing processes, textiles, dying, varnish industries, smelting, organic chemicals with high levels (up to several grams per liter). Phenol is a troublesome contaminant in surface waters and gives an objectionable taste to municipal drinking waters at even lower concentrations (Seker et al. 1997). Phenol is toxic by ingestion, contact or inhalation, and harmful to aquatic organisms (George P. Prpich and Andrews J. Daugulis, 2005). Hence, these wastewater streams require proper treatment before being discharged (Banerjee et al. 2001) to preserve the environment and the health of human beings (Liu et al. 2009). Efficient treatment methods are available for the degradation of phenol, and when compared with physico-chemical methods (Chandana and Sridevi, 2009), the biodegradation method of phenol reduction is universally preferred, because of lower costs and the possibility of complete mineralization (Khaled M. Khleifat, 2006). In this sense biological treatment of phenol contamination has therefore gained an increasing attention in pollution prevention. However, phenol is not readily biodegradable and it is very toxic to most types of microorganisms at

2 104 / INTERNATIONAL JOURNAL OF ENVIRONMENTAL POLLUTION CONTROL & MANAGEMENT sufficiently high concentrations (Prieto et al. 2002). Phenol can inhibit the growth rate even of those species that have the metabolic capability of using it as a substrate for growth (Agarry et al. 2008). The knowledge of aerobic biodegradation kinetics of toxic substances is a very important issue for the design of wastewater plants (Ziagova and Liakopoulou-Kyriakides, 2007) hence, the present work was carried out to study the growth kinetics of P. aeruginosa (NCIM 2074) in the biodegradation of phenol. The Haldane model was used to describe the phenol inhibition. 2. KINETICS 2.1. Estimation of Kinetic Parameters from Batch Experiments The mass balances for bacterial cells and phenol in a batch system with constant volume can be written as follows dx dt = µ X (1) ds 1 dx = dt Y dt (2) where X is the biomass concentration, S phenol concentration, t time, Y X/S yield coefficient and µ specific growth rate of bacterial biomass. Monod s kinetic expression for the specific growth rate may be used only if phenol is assumed to be non-inhibitory at high concentrations (Arutchelvan et al. 2006). X S µ = K µ max S S + S (3) where µ max is the maximum specific growth rate and K S half saturation constant. On the other hand, if phenol inhibits microbial growth at high concentrations, there is a critical concentration of phenol above which the specific growth rate of the microorganism decreases with increasing phenol concentration. Haldane (Haldane, 1930) suggested the following expression for this case, where K I inhibition constant. µ = K S µ maxs S + S + K At higher substrate concentrations, S» K S, the above equation reduces to the following equation: 2 I

3 SUBSTRATE INHIBITION KINETICS OF PHENOL DEGRADATION BY / 105 or This is the linearized Haldane s equation. µ maxs µ = 2 S S + K 1 1 S = + µ µ K I µ max I max (4) (5) 3.1. Microorganisms and Culture Medium 3. EXPERIMENTAL P. aeruginosa used in these experiments was obtained from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (NCL) Pune, India. In order to create a sufficient stock, the bacteria was first grown in a minimal salt medium (MSM) consisting of dihydrogen potassium phosphate: 1.5 g l -1 ; potassium dihydrogen phosphate: 0.5 g l -1 ; ammonium sulfate: 0.5 g l -1 ; sodium chloride: 0.5 g l -1 ; sodium sulfate: 3.0 g l -1 ; yeast extract: 2.0 g l -1 ; glucose: 0.5 g l -1 ; ferrous sulfate: g l -1 and calcium chloride: g l -1 at constant g l -1 phenol concentration. The bacterial culture was kept in solid form with the addition of agar, at 4 C as a stock of bacterial inoculum Acclimation of the Bacteria The acclimatization of the culture was performed in MSM. Initially, the culture was grown in MSM with 20 mg/l phenol. Later, cell mass was centrifuged and inoculated to MSM with 40 mg/l phenol and incubated. The third, fourth and fifth incremental addition of phenol i.e up to 100 mg/l, respectively, were made in MSM and the cultures were grown. The samples were analyzed regularly for phenol and growth Batch Experiments Batch experiments were conducted in Erlenmeyer flasks to study the effects of initial ph, temperature, agitation speed, carbon, nitrogen sources, metal ion concentration and the initial phenol concentration on the biodegradation of phenol. The samples were analyzed regularly for phenol and growth Estimation of Phenol Concentration The undegraded phenol was estimated quantitatively by the spectrophotometric method using 4-aminoantipyrene as color indicator (APHA, 1989) at an absorbance of 510 nm Optimization of Physical Parameters Effect of ph 4. RESULTS AND DISCUSSION Most organisms cannot tolerate ph values below 4.0 or above 9.0 (Kim and Armstrong, 1981). At low (4.0) or high (9.0) ph values, acids or bases can penetrate into cells more easily, because

4 106 / INTERNATIONAL JOURNAL OF ENVIRONMENTAL POLLUTION CONTROL & MANAGEMENT they tend to exist in undissociated form, under these conditions and electrostatic force cannot prevent them from entering cells (Robertson and Alexander 1992; Chitra, 1995; Annadurai et al. 1999). For optimum microbial activity in the environment, the preferred range of ph is between 6 to 8 (McLelland, 1996). Therefore, it is not surprising to find that most microorganisms have evolved with ph tolerances within this range (Suthersen, 1999). In the present investigation, the ph range selected was from 4.0 to 10.0 and the results are shown in Figure 1. It is evident from Figure 1 that phenol degradation increased up to neutral ph and then started decreasing as the medium ph deviates from neutral condition. Therefore a ph of 7 was considered to be the optimum value for the phenol degradation. Figure 1: Effect of ph on the Biodegradation of Phenol by P.aeruginosa Effect of Temperature Temperature is an important factor affecting the performance of cells, regulating the influence on the rate of metabolism. The influence of temperature on enzyme activity could be rationalized since the rate of enzyme reaction increases with increase in temperature and the rate of movement of molecules is slower at lower temperature than at higher temperature. Experiments were carried out at temperatures ranging from C to study its effect on phenol degradation. From Figure 2 it is evident that the phenol degradation increased up to a temperature of 32 C and further increase in temperature decreased the phenol degradation. Hence, phenol degradation capability of P.aeruginosa was optimum at 32 C Effect of Agitation Speed Agitation provides uniform mixing of the fermentation medium and the cells will be uniformly exposed to the medium components. The effect of shaking on the phenol-degrading ability of P.aeruginosa was investigated by varying the speed in the range from rpm and the results

5 SUBSTRATE INHIBITION KINETICS OF PHENOL DEGRADATION BY / 107 Figure 2: Effect of Temperature on the Biodegradation of Phenol by P.aeruginosa were shown in Figure 3. From Figure 3 it was observed that the phenol degradation increased up to an agitation speed of 160 rpm and above that, there was a decrease in phenol degradation. The increase in biodegradation rate may be due to adequate mass transfer thus allowing more oxygen to be dissolved and made available for the degradation. While the decrease may be due to higher shear stress effect, thus leading to cell loss or lower biomass concentration. Hence, the optimum agitation rate was considered to be 160 rpm. Figure 3: Effect of Agitation Speed on the Biodegradation of Phenol by P.aeruginosa

6 108 / INTERNATIONAL JOURNAL OF ENVIRONMENTAL POLLUTION CONTROL & MANAGEMENT 4.2. Optimization of Nutritional Parameters Effect of Carbon Source Different carbon sources were selected for two reasons. First, phenol is a toxic compound representing wastes of industrial origin. Second, these conventional carbon sources are non-toxic, a common substrate which can represent wastes of urban or agricultural origin. In this study, the effect of six different carbon sources namely glucose, galactose, maltose, xylose, fructose and sucrose in the range of g/l on the degradation of phenol were studied and the results were shown in Figure 4. From Figure 4, it can be observed that glucose is the best carbon source among all for phenol degradation. Phenol degradation increased up to a glucose concentration of 0.5 g/l and thereafter it decreased and the degradation was almost inhibited at a concentration of 2.5 g/l. This may be due to catabolite repression by glucose (Papanastasiou and Maiwer, 1982) i.e the presence of glucose could inhibit utilization of the target substrate. Figure 4: Effect of Carbon Source on the Biodegradation of Phenol by P.aeruginosa Effect of Nitrogen Source The effect of four nitrogen sources namely ammonium chloride, ammonium nitrate, ammonium tartrate and ammonium acetate in the range of g/l on the degradation of phenol were studied and the results were shown in Figure 5. From Figure 5 it was observed that at a concentration of 0.2 g/l of ammonium chloride the phenol degradation was increased and further increase in the ammonium chloride concentration, a detrimental effect was observed. Hence, the optimum concentration of ammonium chloride was 0.2 g/l. The enhanced rate of phenol degradation at less than 0.2 g/l ammonium chloride can be attributed to the attenuation of phenol toxicity by ammonium chloride and the increase in cell mass formed as a result of the additional nitrogen source.

7 SUBSTRATE INHIBITION KINETICS OF PHENOL DEGRADATION BY / 109 Figure 5: Effect of Inorganic Nitrogen Source on the Biodegradation of Phenol by P.aeruginosa Effect of Metal Ion Concentration The tolerance of different strains to metals varies widely and it is necessary to determine the optimum concentration to avoid the inhibitory effects caused when these cations are present in toxic concentrations. The present study investigates the effect of four different metal sources viz iron, cadmium, copper and zinc in the range of 0.01 to 0.05 g/l on phenol degradation by P.aeruginosa and the results were shown in Figure 6. It was observed that zinc degraded maximum phenol when compared to iron, cadmium and copper. Figure 6: Effect of Metal Ion Concentration on the Biodegradation of Phenol by P.aeruginosa

8 110 / INTERNATIONAL JOURNAL OF ENVIRONMENTAL POLLUTION CONTROL & MANAGEMENT It may be observed from Figure 6 that phenol degradation increased up to a concentration of 0.02 g/l of zinc and further increase in zinc concentration had a detrimental effect on phenol degradation. Hence, the optimum concentration of zinc for phenol degradation by P.aeruginosa was found to be 0.02 g/l Phenol Degradation Kinetics Shake flask experiments were conducted to examine the effects of various initial concentrations of phenol on the degradation of P.aeruginosa. The initial phenol concentrations used were from mg/l (Figure 7). Results of these studies show that higher the initial concentration of phenol the more time it takes to be degraded completely. Figure 8 represents the growth curve of P.aeruginosa at different concentrations namely mg/l and at optimum physical and nutritional parameters (ph 7, temperature 32 C, agitation speed 160 rpm, carbon source (glucose) 0.5 g/l. nitrogen source (ammonium chloride) 0.2 g/l, metal ion concentration (Zn 2+ ) 0.02 g/l). It was observed that the lag phase increases with increase in the initial phenol concentration. The increase in lag time depends on the inoculum size and on the inhibition constant. In this study, the optimum inoculum size 7 (%v/v) was chosen for the study of kinetics. Since the inoculum was previously acclimatized to phenol, substrate inhibition at low concentrations most probably was absent. This may be due to changes in the transport mechanism of the substrate across the cell membrane at a low concentration (Shishido and Toda, 1996). Hence, at low phenol concentrations an inhibition effect is negligible. But at high phenol concentrations, the microbial flora, although adapted to phenol, seemed to be inhibited as substrate inhibition effects became important. Figure 7: Phenol Degradation by P.aeruginosa in Batch Reactor

9 SUBSTRATE INHIBITION KINETICS OF PHENOL DEGRADATION BY / 111 Figure 8: Growth Curve of P.aeruginosa at various Substrate Concentrations Evaluation of Growth Kinetics (µ max, K S & K I ) for the Experimental Batch System with Optimized Parameters Haldane model, is a good model for the determination of biokinetic parameters viz., maximum specific growth rate µ max, the half-saturation constant K S and the inhibition constant K I. A semi-logarithmic graph is used to calculate µ from the curves of optical density as a function of time. The specific growth rate (µ) for each concentration of phenol was calculated and plotted as shown in the Figure 9. Figure 9: Specific Growth Rates for Phenol Concentrations from 0 to 600 mg/l

10 112 / INTERNATIONAL JOURNAL OF ENVIRONMENTAL POLLUTION CONTROL & MANAGEMENT Maximum specific growth rate µ max, the inhibition constant K I, the half-saturation constant K S were obtained by using the non-linear regression analysis and fitting the experimental data for the specific growth rate as a function of phenol concentration with Force 2.0 software and the values obtained are µ max = h -1 ; K S = mg/l and K I = mg/l. The curve obtained (Figure 9) had a high correlation coefficient (R 2 ) of It indicated that the maximal growth rate of P.aeruginosa was obtained at 200 mg/l phenol. Below this concentration, growth seemed to be suboptimal due to substrate limitation, and above this concentration growth was inhibited increasingly due to substrate inhibition. At high phenol concentrations, some cells were damaged, leading to a decreased metabolic activity. In addition, death of a part of the cells seemed to contribute further to apparent inhibition of growth at high concentrations. The biokinetic parameters obtained in this study were supported by the researchers as given in the Table 1. Table 1 Comparison of Growth Kinetic Parameters Observed by Various Researchers with the Present Study Haldane s model Authors Bacterial System Concentration µ max K s K I Other strain range (mg/l) (h -1 ) (mg/l) (mg/l) conditions: temperature ( C)/pH Pawlowsky & Mixed culture I Batch ± 0.5/ 6.6 Howell (1973) Mixed Culture II Batch ± 0.5/ 6.6 (filamentous organisms) Kumaran & Pseudomonas fluoroscens Batch Paruchuri (1997) 2218 (phenol only) pooled culture (phenols) (P.fluoroscens, P.putida, P.cepacia, A. calcoaceticus, Candida tropicalis) Saravanan et al., Mixed culture ± 1/ b Buitron et al., Mixed culture / Mini Bajaj et al., Mixed culture ± 2/ This study P.aeruginosa NCIM 2074 Batch /7 5. CONCLUSION Biodegradation of phenol by P.aeruginosa was carried out in a batch reactor. Phenol can be utilized as a sole source of carbon and energy. Phenol had a strong inhibitory effect on the growth of cells and it was well described by the Haldane model. In this study it was found that the specific growth rates decreased as the initial phenol concentration increased, suggesting that substrate inhibition may play some role in the rate of phenol biodegradation. Model parameters were determined as µ max = h -1 ; K S = mg/l and K I = mg/l by non-linear regression analysis using Force 2.0 software. References Saravanan, P., Pakshirajan, K., Prabikumar Saha (2008), Biodegradation of Phenol and m-cresol in a Batch Operates Internal Loop Airlift Bioreactor by Indigenous Mixed Microbial Culture Predominantly Pseudomonas Species. Bioresource Technology, 99,

11 SUBSTRATE INHIBITION KINETICS OF PHENOL DEGRADATION BY / 113 Xabier Sevillano, Jose R. Isasi, Francisco J. Penas (2008), Feasibility Study of Degradation of Phenol in a Fluidized Bed Bioreactor with a Cyclodextrin Polymer as Biofilm Carrier. Biodegradation, 19, Seker, S., Beyenal, H., Salih, B., Tanyolac, A. (1997), Multi-substrate Growth Kinetics of Pseudomonas putida for Phenol Removal. Applied Microbiology and Biotechnology, 47, George P. Prpich, Andrews J. Daugulis (2005), Enhanced Biodegradation of Phenol by a Microbial Consortium in a Solid-liquid Two Phase Partitioning Bioreactor. Biodegradation, 16, Banerjee, I., Jayant M. Modak, Bandopadhyay, K., Das, D., Maiti, B. R. (2001), Mathematical Model for Evaluation of Mass Transfer for Evaluation Mass Transfer Limitations in Phenol Biodegradation by Immobilized Pseudomonas putida. Journal of Biotechnology, 87, Liu, Y. J., Zhang, A. N., Wang, X. C. (2009), Biodegradation of Phenol by Using Free and Immobilized Cells of Acinetobacter sp. XA05 and Sphingomonas sp. FG03. Biochemical Engineering Journal, 44, Chandana, M. V. V., Sridevi, V. (2009), A Review on Biodegradation of Phenol from Industrial Effluents. Journal of Industrial Pollution Control, 24, 1. Khaled M. Khleifat (2006), Biodegradation of Phenol by Ewingella Americana: Effect of Carbon Starvation and Some Growth Conditions. Process Biochemistry, 41, Prieto, M.B., Hidalgo, A., Rodriguez, F. C., Serra, J. L., Llama, M. J. (2002), Biodegradation of Phenol in Synthetic and Industrial Wastewater by Rhodococcus erythropitics. UPV-1 Immobilized in Air-stirred Reactor with Clarifier. Applied Microbiology and Biotechnology, 58, Agarry, S. E., Solomon, B. O., Layokun, S. K. (2008), Substrate Inhibition Kinetics of Phenol Degradation by Binary Mixed Culture of Pseudomonas aeruginosa and Pseudomonas fluorescence from Steady State and Wash-out Data. African Journal of Biotechnology, 7, Ziagova, M., Liakopoulou-Kyriakides, M. (2007), Kinetics of 2, 4-dichlorophenol and 4-Cl- m-cresol Degradation by Pseudomonas sp. Cultures in the Presence of Glucose. Chemosphere, 68, Arutchelvan, V., Kanakasabai, V., Nagarajan, S., Muralikrishnan, V. (2006), Kinetics of High Strength Phenol Degradation Using Bacillus brevis. Journal of Hazardous Materials, B129, Haldane, J.B.S., Enzymes, Longman Publishers, London (1930). American Public Health Association (APHA) American Water Work Association, Water Pollution Control Federation, Standards Methods for the Examination of Water and Wastewater, 17th Edition, Washington DC, 55 (1989) Kim, J.W., Armstrong, N.E.A. (1981), Comprehensive Study on the Biological Treatabilites of Phenol and Methanol-II the Effects of Temperature, ph, Salinity and Nutrients. Water Research, 15, Robertson, B. K., Alexander, M. (1992), Influence of Calcium, Iron and ph on Phosphate Availability for Microbial Mineralization of Organic Chemicals. Applied and Environmental Microbiology, 58, Chitra, S. (1995), Studies on Biodegradation of Phenolic Compounds by Pseudomonas pictorum. Ph.D. Thesis CLRI, University of Madras, Chennai-25, Indian. Annadurai, G., Mathalai Balan, S., Murugesan, T. (1999), Box-Behnken Design in the Development of Optimized Complex Medium for Phenol Degradation Using Pseudomonas putida (NICM 2174). Bioprocess Engineering, 21, McLelland, S. P. (1996), Supporting a Ground Water and Soil Natural Remediation Proposal. Site Remediation News Letters Archieves, 8, Suthersen, S. S. (1999), In situ Bioremediation. Chap.5. Boca Raton, CRC Press. Papanastasiou, A.C., Maiwer, W. J. (1982), Kinetics of Biodegradation of 2, 4-dichlorophenoxyacetate in the Presence of Glucose, Biotechnology and Bioengineering, 24, Shishido, M., Toda, M. (1996), Apparent Zero-order Kinetics of Phenol Biodegradation by Substrate-inhibited Microbes at Low Substrate Concentrations. Biotechnology and Bioengineering, 50,