- Email: [email protected]
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides
Saudi Journal of Biological Sciences (2013) 20, 257–264
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides Seema Jilani
*
Department of Environmental Studies, Sindh Madressatul Islam University, Aiwan-e-Tijarat Road, Karachi, Pakistan Received 10 November 2012; accepted 15 February 2013 Available online 14 March 2013
KEYWORDS Toxic pesticides; Industrial wastewater; Bacterial isolate; Activated sludge; COD; HPLC analysis
Abstract In Pakistan, to increase agricultural production, higher amounts of fertilizers and pesticides are being used. The residues of the applied pesticides stay in the environment and therefore causing contamination of air, water and land. Moreover, agricultural industries are also contributing relatively high quantities of toxic pesticides into the environment. Since most of them have no treatment facilities. These pesticides may be toxic, mutagenic or carcinogenic. They may be bioaccumulated or biomagnified by the biota. Therefore its removal from environmental systems needs special attention. In this study, bacterial isolate, Pseudomonas, designated as IES-Ps-1, was used to assess its potential for pesticide removal from industrial wastewater using the biosimulator (activated sludge process). During experimental studies conducted in the flask as well as in biosimulator, it was observed that IES-Ps-1 grows normally at low concentrations of added insecticides when compared with the control test (without pesticide). However, at high concentrations the microbial count decreased but no death occurred and the culture remained in lag phase. In many cases, the growth of organisms in the presence of the particular substrate serves as an indication about its metabolic potential. However, to confirm these results, chemical oxygen demand (COD) and HPLC analysis were performed. Under aerobic culture conditions using mechanical aerators in biosimulator, almost complete removal of Cypermethrin at 20 mg/L dose occurred during 48 h. The study findings indicate that IES-Ps-1 strain, can be used for the treatment of the pesticide contaminated environment. Such study may be valuable to scientist and engineers, who are trying to develop methods for the treatment of toxic organic waste using the biological treatment process. ª 2013 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
* Tel.: +92 21 99217501–3; fax: +92 21 99217504. E-mail address: [email protected]. URL: http://www.smiu.edu.pk. Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
1. Introduction In Pakistan, insecticides, particularly Cypermethrin is mainly used for cotton crop protection and for forestry and public health management. Because of low water solubility and relatively high lipoaffinity, its presence indicates a strong bioconcentration potential in aquatic organisms (Sapiets et al.,
1319-562X ª 2013 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sjbs.2013.02.007
258 1984; Kollman and Segawa, 1995). It is reported that Cypermethrin acts on the nervous system and is toxic to bees, other beneficial insects, earthworms, fish and shrimps (Stepheson, 1982). At present, to meet the needs of food requirements for growing population in the country, farmers are using higher amounts of fertilizers and pesticides but at the expense of environment and health. At present, besides pesticide contamination from agricultural field, the agricultural industries are also contributing relatively high quantities of toxic pesticides into the environment. Since most of them have no treatment facilities or have a grossly inadequate arrangement. The Karachi coastal region has now become the dumping ground of hazardous waste, receiving huge quantities of untreated industrial and agricultural wastes. In order to prevent water pollution and to protect human health, an appropriate wastewater treatment system, easy to operate and suitable for environmental conditions is needed in the country. Among various treatment technologies, the bioremediation technology has been found to be very effective and economical in the treatment of hazardous waste (Grady, 1986; Ritmann et al., 1988; Enrica, 1994). It is well known that specific bacterial culture is capable to degrade the hazardous organic compounds if provided the right environmental capabilities (Mandelbaum et al., 1995; Ramos et al., 1995; Smith-Greeier and Adkins, 1996; Hashmi, 2001). It has been observed that these microorganisms perform their activity efficiently in the activated sludge system (Baker and Dold, 1995; Quan et al., 2004). Although, most synthetic organic compounds are biodegraded easily, making the biological treatment a technically feasible alternative for many environmental problems (Grady, 1990; Kelley et al., 1993; Chaudhry, 1994; Zacharias et al., 1995; Giraud et al., 2001; Bharati et al., 2002; Kao et al., 2004). However, in some cases, specific compounds have either resisted the biodegradation, or their degradation occurs very slowly thus make biological treatment ineffective. Therefore, it is essential for the biological treatment processes, to promote and maintain a microbial population that can metabolize the target wastes. The objective of the present research study was to assess the growth and biodegradation potential of bacterial isolate in the presence of Cypermethrin pesticide using biosimulator (activated sludge system). The study findings would be valuable to scientists and engineers who are trying to develop methods for the treatment of hydrophobic compounds like Cypermethrin pesticide which are resistant otherwise to conventional biological wastewater treatment. 2. Materials and methods 2.1. Chemicals, media and bacterial culture Commercial grade Organophosphate (Malathion, Methamidophos), carbamate (Cartap) and pyrethroid (Cypermethrin) pesticides were purchased from local markets and used in the research study. Because of low aqueous solubility of pesticides, a stock solution of Malathion (5.7 mg/ml), Methamidophos (10 mg/ ml) and Cypermethrin (1 mg/ml) was prepared in GR grade methanol (Merck). However, Cartap solution (9.7 mg/ml) was prepared in sterile distilled water. The bacterial culture, Pseudomonas (IES-Ps-1) was available for this research study and was isolated by Hashmi
S. Jilani (2001) from agricultural soil using enrichment culture technique. However, Cypermethrin adapted bacterial culture was obtained by acclimatization of IES-Ps-1 strain in a gradually increased concentration of Cypermethrin (5–120 mg/L) in a nutrient medium. Adapted IES-Ps-1 was stored at 4 C on nutrient agar slopes containing 0.1 mg/L Cypermethrin. When a new batch of test was performed with different doses of pesticides, the stock culture was first subcultured into 10 ml nutrient broth, aerobically grown and subsequently utilized for characterization, growth and biodegradation studies. Characterizations of IES-Ps-1 were performed using morphological, cultural and biochemical tests using methods described by Collins and Lyne (1985) up to the stage of genus. 2.2. Growth kinetics and biodegradation studies For growth study, 2.5 ml of 24 h old culture prepared in nutrient broth was inoculated into a 25 ml nutrient broth flask and flasks containing different concentrations of Malathion, Methamidophos, Cartap and Cypermethrin. Non shaken condition was used as it actually represents the environmental conditions where microorganisms are usually exposed. A control experiment without insecticide in nutrient broth was used for comparison. Samples from each flask were drawn, tested and counted. Miles and Misra technique was used for bacterial growth study. The growth of IES-Ps-1 in biosimulator was determined by viable cell enumeration immediately after inoculation and at 24, 48, 72, and 96 h later. However, to determine the IES-Ps1 capability for Cypermethrin degradation, batch experiments were performed using the biosimulator (activated sludge) at different temperatures, dissolved oxygen and Cypermethrin concentration. Approximately, 350 ml bacterial culture inoculated into wastewater sample (8.5 L) containing an appropriate quantity of Cypermethrin was transferred into the borosilicate glass jar of a compact bench scale stainless steel biosimulator (Fig. 1). The sample was strongly agitated by impeller with flat stirring paddles and by four vertical baffles. The required temperature was maintained by the built in thermostat and the DO concentration was achieved by diffused aeration using pressure pump and mechanical aeration regulated through continuous agitation. 2.3. Analytical method The water samples were analyzed for temperature, pH, dissolved oxygen and COD as per standard procedures (APHA, 1998). However, for HPLC analysis, the water samples extracted in n-hexane reagent were allowed to dry at 70 C using a vacuum rotary evaporator. The dried residue was then dissolved in 10 ml methanol (GR grade) and filtered through a 0.2 lm membrane. An aliquot 20 lL, was taken from the organic phase and the quantification of Cypermethrin was carried out using HPLC method described earlier (Jilani and Khan, 2006). Each sample was analyzed three times to obtain mean value. 2.4. High pressure liquid chromatography (HPLC) HPLC (Shimadzu, Japan) chromatography system consisted of a solvent delivery pump LC-10 AS, connected with an
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides
Figure 1
259
General layout of a biosimulator (activated sludge treatment system).
autoinjector model SIL-6A and a rheodyne injection valve fitted with a sample loop (20 ll). The chromatographic separation was achieved on a reverse phase C18 column with a guard column and monitored by UV-detector (visible spectrophotometer detector SPD-10A) set at 220 nm. The output of the detector was connected to a chromatopack (CR6A). The mobile phase consisted of methanol (Merck HPLC grade) since Cypermethrin is miscible with alcohol. The filtered methanol was degassed prior to use by sonication. The flow rate was adjusted at 2 ml/min with total elution time of 10 min for each run. 3. Results and discussion 3.1. Characterization and adaptation of IES-PS-1 Bacterial characterization, based on the morphological, cultural and biochemical tests indicates that the IES-Ps-1 strain belongs to the genus Pseudomonas according to ‘‘Bergey’s Manual of Systematic Bacteriology’’ (1994). Further, the experimental results of the present study, as well as of other researchers, indicate that bacteria belonging to the genus Pseudomonas are gram-negative, rod-shaped, highly oxidative, aerobic and metabolically versatile and have been reported to degrade phenolic compounds (Hughes and Cooper, 1996) and other aromatic substances (Christodoulatos et al., 1997; Lee et al., 1998; Ramanathan and Lailithakumari, 1999; Martin et al., 2000; Hashmi, 2001; Maria et al., 2002). During the period of Cypermethrin adaptation, it was observed that the bacteria undergo stress and its growth consequently slowed down. Further, the bacteria changed its normal rod-shaped morphology to coccus. However, this change was temporary, as the cells recovered to original rod form after a few days.
3.2. Bacterial growth in flask The comparative growth response of IES-Ps-1 in the presence of different concentrations of Malathion, Methamidophos, Cartap and Cypermethrin as shown in Fig. 2 revealed the degree of bacterial sensitivity or resistance and the amount of growth stimulation when exposed to different concentrations and type of pesticides. Similar growth studies have been conducted and reported by other researchers as they provide information about the biodegradation potential of microorganisms (Smith-Greeier and Adkins, 1996; Lee et al., 1998; Karpouzas and Walker, 2000; Martin et al., 2000; Hashmi, 2001; Maria et al., 2002). Comparison of the viable count data with an earlier study conducted on shaking water bath by Hashmi (2001), indicates that the growth performance of IES-Ps-1 in control experiments with no pesticides was much better. The observed generation time was 45 min with specific growth rate of 0.0219 min 1. Whereas, without shaking condition, used in the present study, the generation time increased to 69 min with specific growth rate of 0.014 min 1. These results proved that as an IES-Ps-1 culture is aerobic, it performed better in a shaking water bath where greater quantity of oxygen might enhance the oxidation capability of these organisms. The overall findings of this research study suggest that IESPs-1 can grow in the presence of added pesticides. However, the toxicity pattern observed is as follows: Cartap > Cypermethrin > Methamidophos Malathion. From these results it appears that Cartap and Cypermethrin pesticides are more toxic than Methamidophos and Malathion. It is worth mentioning here that since the culture was Malathion adapted, therefore IES-Ps-1 growth in the presence of organophosphorus pesticides increased its tolerance and hence more growth
260
S. Jilani
Figure 2 Comparative growth response of IES-Ps-1 in nutrient broth and broth containing different concentrations of pesticides. = IES-Ps-1 growth in nutrient broth containing no pesticide (Control). Graph 1 = IES-Ps-1 growth in the presence of Malathion (n 35, m 50 & · 110 mg/L). Graph 2 = IES-Ps-1 growth in the presence of Methamidophos (n 80, m160 & · 300 mg/L). Graph 3 = IES-Ps-1 growth in the presence of Cartap (n 60, m 80 & · 120 mg/L). Graph 4 = IES-Ps-1 growth in the presence of Cypermethrin (n 40, m 60 & · 80 mg/L).
observed even at high concentrations of Methamidophos and Malathion. During growth kinetic studies as explained above using different concentrations of pesticides, the death phase was not observed even at high concentrations; however the viable count gradually decreased. The possible explanation of these may be the non adaptation of IES-Ps-1 in the presence of added pesticide as an acclimation period is necessary for microorganisms in order to activate the production of degradative enzymes. This may account for the prolonged lag phase which was observed at high concentrations of all the added pesticides. Another reason may be the less availability of dissolved oxygen (DO), as it is reported that increased organic load might decrease the DO concentration (Corbitt, 1998). Based on the present study findings as well as by Hashmi (2001), it can be concluded that IES-Ps-1 strain could be used for remediation of pesticide contamination.
Figure 3 Total cell count of bacteria in biosimulator containing 40 mg/L Cypermethrin.
3.3. Bacterial growth in biosimulator (activated sludge system) The growth of IES-Ps-1 in the presence of Cypermethrin (Figs. 3 and 4) indicates that bacteria can grow fast in biosimulator as a higher number of cells were observed compared to the shaken flask condition. The maximum count at 24 h with 40 mg/L Cypermethrin was 13 ± 1.73 · 107 CFU/ml and with 80 mg/L, it was 19 ± 2.65 · 107 CFU/ml. However, the generation time at these concentrations (40 and 80 mg/L) was 57 and 53 min. On the other hand in control experiments, the cell count at 24 h was relatively low (7 ± 1.73 · 107 CFU/ml) with a marked increase in generation time (98 min). It was further noted that the growth at 40 mg/L dose significantly increased after 48 h incubation. But at 80 mg/L dose, the growth was slightly less but continued till 96 h incubation and a count of 7 · 107 CFU/ml was recorded. The observed growth may be due to the availability of nutrients and
Figure 4 Total cell count of bacteria in biosimulator containing 80 mg/L Cypermethrin.
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides favorable environmental conditions in biosimulator which allow the cells to survive till 96 h. In contrast, the population density in the control experiment (no Cypermethrin) was comparatively less (0.1 · 107 CFU/ml). Since 78–88% Cypermethrin degradation observed after 48 h, it appears that biodegradation actually occurred by the acclimated IES-Ps-1 culture bioaugmented in wastewater samples. Further the presence of bacterial cells in log phase during biodegradation indicates that substrate conversion would be at its maximum as described by Gray (1989) and similarly observed in this study. 3.4. Influence of physicochemical conditions on biodegradation rates 3.4.1. pH The data as reported in Table 1, indicate that IES-Ps-1 can retain their degradation ability in a wide range of pH with optimum degradation at 30 C temperature where the pH of the water sample was found near neutral range (pH 7.33). This finding is supported by Ashok and Seth (1989), who reported that isolated Pseudomonas strain can grow between pH 5.5 and 9.5 with optimum growth at pH around 7.0. Similarly, Mandelbaum et al. (1995) found that atrazine degradation in the pH range of 5.5–8.5 by Pseudomonas strain was not affected. Moreover it is reported that the tolerable limits for pH in activated sludge ranged between pH 6.0 and 9.0 (Hanel, 1988). 3.4.2. Temperature Temperature is among the important environmental parameters that can influence the microbial growth as well as treatment efficiency (Comeau et al., 1993). During Cypermethrin degradation, the direct correlation was found between temperature and microbial activity. Significant removal occurred when biosimulator was operated at 28–30 C temperature using 8–9 mg/L DO, whereas moderate degradation was observed at ambient (18–25 C) and 38 C temperature (Table 1). Similar optimal temperature (28–30 C) for Pseudomonas
Table 1
261
growth in activated sludge was also reported by Schlegel (1969). 3.4.3. Dissolved oxygen Comparative performance evaluation at different dissolved oxygen concentrations for Cypermethrin degradation is presented in Table 1. It was noted that the initial dissolved oxygen concentration in the wastewater sample ranged between 1.5 and 2.6 mg/L. In order to maintain aerobic conditions in biosimulator, the mechanical aeration in the reactor not only provided sufficient dissolved oxygen but also kept Cypermethrin in suspension, such that the bacterial growth and COD removal were greatly enhanced. During experiment it was observed that the biodegradation performance of IES-Ps-1 significantly improved at 8–9 mg/L dissolved oxygen using mechanical aeration (250 rpm) in the temperature range of 28–30 C. 3.4.4. Chemical oxygen demand Results as summarized in Table 1, explain the lower degradation at high Cypermethrin concentrations and a good agreement between COD removal and Cypermethrin degradation rates especially at low concentrations. These results are in accordance to previous findings reported by Berchtold et al. (1995), who noticed the same correlation between the COD removal and biodegradation of 2,4-DAT and 2,4 and 2,6 diamino toluene degradation by acclimated bacteria (Pesce and Wunderlin, 1997). Similar correlations were also observed by Ramanathan and Lailithakumari (1999) during biodegradation of hazardous chemicals. Moreover, during treatment, it was also observed that even at a high organic load, the biosimulator function is satisfactory when operated under controlled temperature and dissolved oxygen with retention time of 48 h. These findings are in agreement with Toprak [38] who reported that COD removal during treatment mainly depends on temperature and influent COD concentration.
Comparative performance evaluation of IES-Ps-1 for Cypermethrin degradation after 48 h.
Parameters
pH
COD removal
Cypermethrin degradation
Conc. (mg/l)
% Removal
Conc. (mg/l)
% Degradation
Effect of Cypermethrin concentration (mg/l) Cypermethrin conc. (mg/l) 20 8.60 40 8.30 80 7.81 125 7.83
80 1080 4500 13767
97 82 54 24
– 8.2 42 118
No peak 81 51 18
Effect of temperature (C) at 80 mg/L dose Temperature (C) Ambient temp. (18–25) 7.80 28–30 7.33 38–40 7.50
4500 867 4000
54 89 52
42 9.0 42
51 88 48
Effect of dissolved oxygen (mg/L) at 80 mg/L dose Dissolved oxygen (mg/L) 5–6 mg/L 7.87 5000 8–9 mg/L 7.81 4500 8–9 mg/L (30 C temp.) 7.33 867 11–12 mg/L 8.20 1300
31 54 89 83
50 42 9.0 17
32 51 88 78
Data reported in table indicate average values of three experiments.
262
S. Jilani
3.5. Biodegradation of Cypermethrin The data reported in Table 1 and HPLC chromatogram shown in Figs. 5–7 clearly indicate that increased concentration of Cypermethrin (40–125 mg/L) in biosimulator decreased the degradation rates and the complete removal occurred at 20 mg/L dose (Jilani and Khan, 2006). However, optimizing the treatment conditions like pH, temperature, and dissolved oxygen in activated sludge, IES-Ps-1 could effectively remove higher concentrations of Cypermethrin. Similar results were also reported by other researchers (Ashok and Seth, 1989; Smith-Greeier and Adkins, 1996; Collins and Daugulis, 1997; Pesce and Wunderlin, 1997; Lee et al., 1998; Goudar and Strevett, 2000). During the experimental work it was observed that the optimum temperature for growth and degradation of Cypermethrin by IES-Ps-1 culture remained between 28 and 30 C. Using a similar concentration of Cypermethrin (80 mg/L), when the temperature of the reactor decreased or increased (38 C), the degradation rates were significantly reduced. At ambient temperature, 51% Cypermethrin degradation occurred whereas at 38 C temperature, it was 48% (Fig. 5). In contrast, when biosimulator temperature maintained between 28 and 30 C using mechanical aeration (8–9 mg/L), keeping Cypermethrin concentration constant (80 mg/L), >88% removal was achieved after 48 h of aerobic treatment (Fig. 6). Similar optimal temperature (28–30 C) for growth of Pseudomonas in activated sludge was also reported by Schlegel (1969) and Chaterjee et al. (1982). Moreover, Karpouzas and Walker (2000) reported that unlike high temperatures, low growth temperatures for Pseudomonas putida strain are usually not lethal. Overall study findings described that this may be the first instance in which high concentrations of Cypermethrin degradation were achieved in short retention time of 48 h. Although the transformation of permethrin (50 mg/L) by pure culture of Pseudomonas fluorescence under aerobic conditions with a half-life of less than 5 days was reported by Maloney et al. (1998) and the removal of technical grade Cypermethrin from
Figure 6 HPLC chromatograms showing comparative effect of temperature on Cypermethrin (80 mg/L) degradation at 9 mg/L DO. a: ambient temp.; b: 30 C; c: 38 C.
Figure 7 HPLC chromatograms showing comparative effect of DO on Cypermethrin (80 mg/L) degradation. a: 6 mg/LDO; b: 9 mg/L DO & 9 mg/L DO at 30 C; c: 12 mg/L.
60 to 6 mg/L by Pseudomonas species in 20 days was reported by Grant and Betts (2003). From the study findings, it can be concluded that biodegradation performance mainly depends on Cypermethrin concentration. In addition, during the treatment optimal residence time needs to be assessed while taking into account the Cypermethrin concentration but it appeared that 2 days would be a convenient time to reach satisfactory biodegradation at low Cypermethrin concentrations (<20 mg/L). 4. Conclusions Following conclusions may be drawn from this study: Figure 5 HPLC chromatograms showing comparative biodegradation rates at different Cypermethrin concentrations. a: 45 mg/ L; b: 80 mg/L; c: 120 mg/L.
Malathion degrading bacterial isolate, IES-Ps-1, can be used for biodegradation of pesticide wastes, as IES-Ps-1
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides showed potential to grow in the presence of Cypermethrin. Cypermethrin concentration and the temperature were found to be a principal governing factor in removing Cypermethrin. Because of the low aqueous solubility of Cypermethrin, mechanical aeration in biosimulator proved to be very effective in reducing the concentration of Cypermethrin. As mechanical aeration not only provided the maximum dispersion of Cypermethrin in wastewater but also maintained the sufficient dissolved oxygen required for the growth of IES-Ps-1. Complete removal of Cypermethrin (20 mg/L) would only be possible if an appropriate organism (IES-Ps-1) and optimum operating conditions (temperature, dissolved oxygen, and mechanical aeration) be maintained in biosimulator.
Acknowledgement The partial financial support of the National Drainage Program of WAPDA, Pakistan is greatly acknowledged. References APHA (American Public Health Association), 1998. Standard methods for the examination of water and wastewater. American Public Health Association, American Water Works Association, Water Pollution Control Federation, 20th ed. Washington DC, USA. Ashok, K.S., Seth, P.K., 1989. Degradation of Malathion by microorganism isolated from industrial effluents. Bull. Environ. Contam. Toxicol. 43, 28–35. Baker, P.S., Dold, P.L., 1995. COD and nitrogen mass balances in activated sludge systems. Water Res. 29, 633–643. Berchtold, S.R., Vanderloop, S.L., Suidan, M.T., Maloney, S.W., 1995. Treatment of 2,4-dinitrotoluene using a two-stage system: fluidized-bed anaerobic granular activated carbon reactors and aerobic activated sludge reactors. Water Environ. Res. 67, 1081– 1091. Hensyl, W.R. (Ed.), 1994Bergey’s Manual of Determinative Bacteriology, ninth ed. Williams & Wilkins Company, Baltimore. Bharati, J.B., Seema, S.S., Pradnya, P.K., 2002. Bioremediation of an industrial effluent containing Monocrotophos. Curr. Microbiol. 45, 346–349. Chaterjee, D.K., Kilbane, J.J., Chakrabarty, A.M., 1982. Biodegradation of 2,4,5-trichlorophenoxy acetic acid in soil by a pure culture of Pseudomonas cepacia. Appl. Environ. Microbiol. 44, 514–516. Chaudhry, G.R., 1994. Biological Degradation and Bioremediation of Toxic Chemicals. Dioscorides Press, Portland, OR, USA. Christodoulatos, C., Koutsospyros, A.D., Brodman, B.W., Korfiatis, G.P., 1997. Biodegradation of diphenylamine by selected microbial cultures. J. Environ. Sci. Health 32 (1), 15–30. Collins, L.D., Daugulis, A.J., 1997. Biodegradation of phenol at high initial concentrations in two phase partitioning batch and fed-batch bioreactors. Biotechnol. Bioeng. 55, 155–162. Collins, C.H., Lyne, P.M., 1985. Microbiological Methods. 5thEdition. Butterworth and Co (Publishers) Ltd. Environmental Engineering. 116 (5), 805–828. Comeau, Y.C., Greer, W., Samson, R., 1993. Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation. Appl. Microbiol. Technol. 38, 681– 687. Corbitt, R.A., 1998. Standard Handbook of Environmental Engineering, second ed. McGraw Hill, New York, Chap 6.
263
Enrica, G., 1994. The role of microorganism in environmental decontamination. In: Aristeo, Renzoni (Ed.), Contaminants in The Environment – A Multidisciplinary Assessment of Risk to Man and Other Organisms. Lewis Publisher, pp. 235–246, 25. Giraud, F., Guiraud, P., Kadri, M., Blake, G., Steiman, R., 2001. Biodegradation of anthracene and fluoranthene by fungi isolated from an experimental constructed wetland for wastewater treatment. Water Res. 35, 4126–4136. Goudar, C.T., Strevett, K.A., 2000. Substrate inhibition kinetics of phenol biodegradation. Water Environ. Res. 72, 50–55. Grady, C.P.L., 1986. Biodegradation of hazardous waste by conventional biological treatment. Hazard. Waste Hazard. Material 3, 333–365. Grady, C.P.L., 1990. Biodegradation of toxic organics. Status and potential. J. Environ. Eng. 116, 805–828. Grant, R.J., Betts, W.B., 2003. Biodegradation of the synthetic pyrethroid cypermethrin in used sheep dip. J. Appl. Microbiol. 36, 173–176. Gray, N.F., 1989. The effect of small changes in incubation temperature on the five day biochemical oxygen demand test. Environ. Technol. Lett. 10, 253–258. Hanel, K., 1988. Biological Treatment of Sewage by the Activated Sludge Process. Ellis Horwood, Chichester, Wiley, New York. Hashmi, I., 2001. Microbiological transformation of hazardous waste during biological treatment. Ph.D. Thesis. Institute of Environmental Studies, University of Karachi, Pakistan. Hughes, S., Cooper, D., 1996. Biodegradation of phenol using the selfcycling fermentation process. Biotechnol. Bioeng. 51, 112–119. Jilani, S., Khan, M.A., 2006. Biodegradation of Cypermethrin by Pseudomonas in a batch activated sludge process. Int. J. Environ. Sci. Technol. 3, 371–380. Kao, C.M., Chai, C.T., Liu, J.K., Yeh, T.Y., Chen, K.F., Chen, S.C., 2004. Evaluation of natural and enhanced PCP biodegradation at a former pesticide manufacturing plant. Water Res. 38, 663–672. Karpouzas, D.G., Walker, A., 2000. Factors influencing the ability of Pseudomonas putida strains epI and II to degrade the organophosphate ethoprophos. J. Appl. Microbiol. 89, 40–48. Kelley, L., Freeman, J.P., Evans, F.E., Cerniglia, C.E., 1993. Identification of metabolites from the degradation of fluoranthene by Mycobacterium sp. strain PYR-1. Appl. Environ. Microbiol. 59, 800–806. Kollman, W., Segawa, R., 1995. Interim Report of the Pesticide Chemistry Database Environmental Hazards Assessment Program. Department of Pesticide Regulation. Lee, S.G., Yoon, B.D., Park, Y.H., Oh, H.M., 1998. Isolation of a novel pentacholorophenol-degrading bacterium, Pseudomonas sp. Bu 34. J. Appl. Microbiol. 85, 1–8. Maloney, S.E., Maule, A., Smith, A.R.W., 1998. Microbial transformation of the pyrethroid insecticides. Permethrin, Deltamethrin, Fastac, Fenvalerate and Fluvalinate. Appl. Environ. Microbiol. 54, 2874–2876. Mandelbaum, R.T., Allen, D.L., Wackett, L.P., 1995. Isolation and characterization of Pseudomonas sp. that mineralized the s-trazine herbicide atrazine. Appl. Environ. Microbiol. 61 (4), 1451–1457. Maria, K., Graciela, C., Zauscher, F., 2002. Biodegradation of two commercial herbicides (Gramoxone and Matancha) by the bacteria Pseudomonas putida. Electronic J. Environ. Biotechnol. 5, 182–195. Martin, M., Mengs, G., Plaza, E., Garbi, C., Sanchez, M., Gibello, A., Gutierrez, F., Ferrer, E., 2000. Propachlor removal by Pseudomonas strain GCH 1 in an immobilized cell system. Appl. Environ. Microbiol. 66, 1190–1194. Miles, A.A., Misra, S.S., 1938. The estimation of bacterial power of the blood. J. Hyg. Camb. 38, 732–749. Pesce, S.F., Wunderlin, D.A., 1997. Biodegradation of 2,4- and 2,6diaminotoluene by acclimated bacteria. Water Res. 31, 1601–1608. Quan, X., Hanchang, S., Hong, L., Pingping, Lv., YI, Q., 2004. Enhancement of 2,4-dichlorophenol degradation in conventional
264 activated sludge systems bioaugmented with mixed special culture. Water Res. 38, 245–253. Ramanathan, M.P., Lailithakumari, D., 1999. Complete mineralization of methyl parathion by Pseudomonas sp. A3. Appl. Biochem. Biotechnol. 80, 1–12. Ramos, T.L., Duque, E., Huertas, M.J., Haidour, A., 1995. Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentration of aromatic hydrocarbons. J. Bacteriol. 177, 3911–3916. Ritmann, B.E., Jacson, D.E., Storck, S.L., 1988. Potential for treatment of hazardous organic chemical with biological process. Biotreatment systems. In: Wis, D.L. (Ed.). CRC Press, Boca Raton, FL, pp. 15–64, 3. Sapiets, A., Swaine, H., Tandy, M.J., 1984. Cypermethrin. In: Zweig, G., Sherma, J. (Eds.), Analytical Methods for Pesticides and Plants Growth Regulators. Academic Press, New York, p. 33, XIII.
S. Jilani Schlegel, H.G., 1969. Allgemeine Mikrobiologie Thieme Stuttgart. In: Hanel, K. (Ed.), Biological Treatment of Sewage by the Activated Sludge Process. Ellis Horwood, Chichester, Wiley, New York. Smith-Greeier, L.L., Adkins, A., 1996. Isolation and characterization of soil microorganisms capable of utilizing the herbicide dichloro-p-methyl as a sole source of carbon and energy. Can. J. Microbiol. 42, 221–226. Stepheson, R.R, 1982. Aquatic toxicology of Cypermethrin. In: Acute toxicity to some freshwater fish and invertebrates in laboratory tests. Aquatic. Toxicol. 2, 253–270. Toprak, H., 1995. Removal of soluble chemical oxygen demand from domestic waste waters in a laboratory scale anaerobic waste stabilization ponds. Water Res. 29, 923–932. Zacharias, B., Lang, E., Hanert, H.H., 1995. Biodegradation of chlorinated aromatic hydro-carbons in slow sand filters simulating conditions in contaminated soil – Pilot study for in situ cleaning of an industrial site. Water Res. 29 (7), 1663–1671.
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides Seema Jilani
*
Department of Environmental Studies, Sindh Madressatul Islam University, Aiwan-e-Tijarat Road, Karachi, Pakistan Received 10 November 2012; accepted 15 February 2013 Available online 14 March 2013
KEYWORDS Toxic pesticides; Industrial wastewater; Bacterial isolate; Activated sludge; COD; HPLC analysis
Abstract In Pakistan, to increase agricultural production, higher amounts of fertilizers and pesticides are being used. The residues of the applied pesticides stay in the environment and therefore causing contamination of air, water and land. Moreover, agricultural industries are also contributing relatively high quantities of toxic pesticides into the environment. Since most of them have no treatment facilities. These pesticides may be toxic, mutagenic or carcinogenic. They may be bioaccumulated or biomagnified by the biota. Therefore its removal from environmental systems needs special attention. In this study, bacterial isolate, Pseudomonas, designated as IES-Ps-1, was used to assess its potential for pesticide removal from industrial wastewater using the biosimulator (activated sludge process). During experimental studies conducted in the flask as well as in biosimulator, it was observed that IES-Ps-1 grows normally at low concentrations of added insecticides when compared with the control test (without pesticide). However, at high concentrations the microbial count decreased but no death occurred and the culture remained in lag phase. In many cases, the growth of organisms in the presence of the particular substrate serves as an indication about its metabolic potential. However, to confirm these results, chemical oxygen demand (COD) and HPLC analysis were performed. Under aerobic culture conditions using mechanical aerators in biosimulator, almost complete removal of Cypermethrin at 20 mg/L dose occurred during 48 h. The study findings indicate that IES-Ps-1 strain, can be used for the treatment of the pesticide contaminated environment. Such study may be valuable to scientist and engineers, who are trying to develop methods for the treatment of toxic organic waste using the biological treatment process. ª 2013 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
* Tel.: +92 21 99217501–3; fax: +92 21 99217504. E-mail address: [email protected]. URL: http://www.smiu.edu.pk. Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
1. Introduction In Pakistan, insecticides, particularly Cypermethrin is mainly used for cotton crop protection and for forestry and public health management. Because of low water solubility and relatively high lipoaffinity, its presence indicates a strong bioconcentration potential in aquatic organisms (Sapiets et al.,
1319-562X ª 2013 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sjbs.2013.02.007
258 1984; Kollman and Segawa, 1995). It is reported that Cypermethrin acts on the nervous system and is toxic to bees, other beneficial insects, earthworms, fish and shrimps (Stepheson, 1982). At present, to meet the needs of food requirements for growing population in the country, farmers are using higher amounts of fertilizers and pesticides but at the expense of environment and health. At present, besides pesticide contamination from agricultural field, the agricultural industries are also contributing relatively high quantities of toxic pesticides into the environment. Since most of them have no treatment facilities or have a grossly inadequate arrangement. The Karachi coastal region has now become the dumping ground of hazardous waste, receiving huge quantities of untreated industrial and agricultural wastes. In order to prevent water pollution and to protect human health, an appropriate wastewater treatment system, easy to operate and suitable for environmental conditions is needed in the country. Among various treatment technologies, the bioremediation technology has been found to be very effective and economical in the treatment of hazardous waste (Grady, 1986; Ritmann et al., 1988; Enrica, 1994). It is well known that specific bacterial culture is capable to degrade the hazardous organic compounds if provided the right environmental capabilities (Mandelbaum et al., 1995; Ramos et al., 1995; Smith-Greeier and Adkins, 1996; Hashmi, 2001). It has been observed that these microorganisms perform their activity efficiently in the activated sludge system (Baker and Dold, 1995; Quan et al., 2004). Although, most synthetic organic compounds are biodegraded easily, making the biological treatment a technically feasible alternative for many environmental problems (Grady, 1990; Kelley et al., 1993; Chaudhry, 1994; Zacharias et al., 1995; Giraud et al., 2001; Bharati et al., 2002; Kao et al., 2004). However, in some cases, specific compounds have either resisted the biodegradation, or their degradation occurs very slowly thus make biological treatment ineffective. Therefore, it is essential for the biological treatment processes, to promote and maintain a microbial population that can metabolize the target wastes. The objective of the present research study was to assess the growth and biodegradation potential of bacterial isolate in the presence of Cypermethrin pesticide using biosimulator (activated sludge system). The study findings would be valuable to scientists and engineers who are trying to develop methods for the treatment of hydrophobic compounds like Cypermethrin pesticide which are resistant otherwise to conventional biological wastewater treatment. 2. Materials and methods 2.1. Chemicals, media and bacterial culture Commercial grade Organophosphate (Malathion, Methamidophos), carbamate (Cartap) and pyrethroid (Cypermethrin) pesticides were purchased from local markets and used in the research study. Because of low aqueous solubility of pesticides, a stock solution of Malathion (5.7 mg/ml), Methamidophos (10 mg/ ml) and Cypermethrin (1 mg/ml) was prepared in GR grade methanol (Merck). However, Cartap solution (9.7 mg/ml) was prepared in sterile distilled water. The bacterial culture, Pseudomonas (IES-Ps-1) was available for this research study and was isolated by Hashmi
S. Jilani (2001) from agricultural soil using enrichment culture technique. However, Cypermethrin adapted bacterial culture was obtained by acclimatization of IES-Ps-1 strain in a gradually increased concentration of Cypermethrin (5–120 mg/L) in a nutrient medium. Adapted IES-Ps-1 was stored at 4 C on nutrient agar slopes containing 0.1 mg/L Cypermethrin. When a new batch of test was performed with different doses of pesticides, the stock culture was first subcultured into 10 ml nutrient broth, aerobically grown and subsequently utilized for characterization, growth and biodegradation studies. Characterizations of IES-Ps-1 were performed using morphological, cultural and biochemical tests using methods described by Collins and Lyne (1985) up to the stage of genus. 2.2. Growth kinetics and biodegradation studies For growth study, 2.5 ml of 24 h old culture prepared in nutrient broth was inoculated into a 25 ml nutrient broth flask and flasks containing different concentrations of Malathion, Methamidophos, Cartap and Cypermethrin. Non shaken condition was used as it actually represents the environmental conditions where microorganisms are usually exposed. A control experiment without insecticide in nutrient broth was used for comparison. Samples from each flask were drawn, tested and counted. Miles and Misra technique was used for bacterial growth study. The growth of IES-Ps-1 in biosimulator was determined by viable cell enumeration immediately after inoculation and at 24, 48, 72, and 96 h later. However, to determine the IES-Ps1 capability for Cypermethrin degradation, batch experiments were performed using the biosimulator (activated sludge) at different temperatures, dissolved oxygen and Cypermethrin concentration. Approximately, 350 ml bacterial culture inoculated into wastewater sample (8.5 L) containing an appropriate quantity of Cypermethrin was transferred into the borosilicate glass jar of a compact bench scale stainless steel biosimulator (Fig. 1). The sample was strongly agitated by impeller with flat stirring paddles and by four vertical baffles. The required temperature was maintained by the built in thermostat and the DO concentration was achieved by diffused aeration using pressure pump and mechanical aeration regulated through continuous agitation. 2.3. Analytical method The water samples were analyzed for temperature, pH, dissolved oxygen and COD as per standard procedures (APHA, 1998). However, for HPLC analysis, the water samples extracted in n-hexane reagent were allowed to dry at 70 C using a vacuum rotary evaporator. The dried residue was then dissolved in 10 ml methanol (GR grade) and filtered through a 0.2 lm membrane. An aliquot 20 lL, was taken from the organic phase and the quantification of Cypermethrin was carried out using HPLC method described earlier (Jilani and Khan, 2006). Each sample was analyzed three times to obtain mean value. 2.4. High pressure liquid chromatography (HPLC) HPLC (Shimadzu, Japan) chromatography system consisted of a solvent delivery pump LC-10 AS, connected with an
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides
Figure 1
259
General layout of a biosimulator (activated sludge treatment system).
autoinjector model SIL-6A and a rheodyne injection valve fitted with a sample loop (20 ll). The chromatographic separation was achieved on a reverse phase C18 column with a guard column and monitored by UV-detector (visible spectrophotometer detector SPD-10A) set at 220 nm. The output of the detector was connected to a chromatopack (CR6A). The mobile phase consisted of methanol (Merck HPLC grade) since Cypermethrin is miscible with alcohol. The filtered methanol was degassed prior to use by sonication. The flow rate was adjusted at 2 ml/min with total elution time of 10 min for each run. 3. Results and discussion 3.1. Characterization and adaptation of IES-PS-1 Bacterial characterization, based on the morphological, cultural and biochemical tests indicates that the IES-Ps-1 strain belongs to the genus Pseudomonas according to ‘‘Bergey’s Manual of Systematic Bacteriology’’ (1994). Further, the experimental results of the present study, as well as of other researchers, indicate that bacteria belonging to the genus Pseudomonas are gram-negative, rod-shaped, highly oxidative, aerobic and metabolically versatile and have been reported to degrade phenolic compounds (Hughes and Cooper, 1996) and other aromatic substances (Christodoulatos et al., 1997; Lee et al., 1998; Ramanathan and Lailithakumari, 1999; Martin et al., 2000; Hashmi, 2001; Maria et al., 2002). During the period of Cypermethrin adaptation, it was observed that the bacteria undergo stress and its growth consequently slowed down. Further, the bacteria changed its normal rod-shaped morphology to coccus. However, this change was temporary, as the cells recovered to original rod form after a few days.
3.2. Bacterial growth in flask The comparative growth response of IES-Ps-1 in the presence of different concentrations of Malathion, Methamidophos, Cartap and Cypermethrin as shown in Fig. 2 revealed the degree of bacterial sensitivity or resistance and the amount of growth stimulation when exposed to different concentrations and type of pesticides. Similar growth studies have been conducted and reported by other researchers as they provide information about the biodegradation potential of microorganisms (Smith-Greeier and Adkins, 1996; Lee et al., 1998; Karpouzas and Walker, 2000; Martin et al., 2000; Hashmi, 2001; Maria et al., 2002). Comparison of the viable count data with an earlier study conducted on shaking water bath by Hashmi (2001), indicates that the growth performance of IES-Ps-1 in control experiments with no pesticides was much better. The observed generation time was 45 min with specific growth rate of 0.0219 min 1. Whereas, without shaking condition, used in the present study, the generation time increased to 69 min with specific growth rate of 0.014 min 1. These results proved that as an IES-Ps-1 culture is aerobic, it performed better in a shaking water bath where greater quantity of oxygen might enhance the oxidation capability of these organisms. The overall findings of this research study suggest that IESPs-1 can grow in the presence of added pesticides. However, the toxicity pattern observed is as follows: Cartap > Cypermethrin > Methamidophos Malathion. From these results it appears that Cartap and Cypermethrin pesticides are more toxic than Methamidophos and Malathion. It is worth mentioning here that since the culture was Malathion adapted, therefore IES-Ps-1 growth in the presence of organophosphorus pesticides increased its tolerance and hence more growth
260
S. Jilani
Figure 2 Comparative growth response of IES-Ps-1 in nutrient broth and broth containing different concentrations of pesticides. = IES-Ps-1 growth in nutrient broth containing no pesticide (Control). Graph 1 = IES-Ps-1 growth in the presence of Malathion (n 35, m 50 & · 110 mg/L). Graph 2 = IES-Ps-1 growth in the presence of Methamidophos (n 80, m160 & · 300 mg/L). Graph 3 = IES-Ps-1 growth in the presence of Cartap (n 60, m 80 & · 120 mg/L). Graph 4 = IES-Ps-1 growth in the presence of Cypermethrin (n 40, m 60 & · 80 mg/L).
observed even at high concentrations of Methamidophos and Malathion. During growth kinetic studies as explained above using different concentrations of pesticides, the death phase was not observed even at high concentrations; however the viable count gradually decreased. The possible explanation of these may be the non adaptation of IES-Ps-1 in the presence of added pesticide as an acclimation period is necessary for microorganisms in order to activate the production of degradative enzymes. This may account for the prolonged lag phase which was observed at high concentrations of all the added pesticides. Another reason may be the less availability of dissolved oxygen (DO), as it is reported that increased organic load might decrease the DO concentration (Corbitt, 1998). Based on the present study findings as well as by Hashmi (2001), it can be concluded that IES-Ps-1 strain could be used for remediation of pesticide contamination.
Figure 3 Total cell count of bacteria in biosimulator containing 40 mg/L Cypermethrin.
3.3. Bacterial growth in biosimulator (activated sludge system) The growth of IES-Ps-1 in the presence of Cypermethrin (Figs. 3 and 4) indicates that bacteria can grow fast in biosimulator as a higher number of cells were observed compared to the shaken flask condition. The maximum count at 24 h with 40 mg/L Cypermethrin was 13 ± 1.73 · 107 CFU/ml and with 80 mg/L, it was 19 ± 2.65 · 107 CFU/ml. However, the generation time at these concentrations (40 and 80 mg/L) was 57 and 53 min. On the other hand in control experiments, the cell count at 24 h was relatively low (7 ± 1.73 · 107 CFU/ml) with a marked increase in generation time (98 min). It was further noted that the growth at 40 mg/L dose significantly increased after 48 h incubation. But at 80 mg/L dose, the growth was slightly less but continued till 96 h incubation and a count of 7 · 107 CFU/ml was recorded. The observed growth may be due to the availability of nutrients and
Figure 4 Total cell count of bacteria in biosimulator containing 80 mg/L Cypermethrin.
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides favorable environmental conditions in biosimulator which allow the cells to survive till 96 h. In contrast, the population density in the control experiment (no Cypermethrin) was comparatively less (0.1 · 107 CFU/ml). Since 78–88% Cypermethrin degradation observed after 48 h, it appears that biodegradation actually occurred by the acclimated IES-Ps-1 culture bioaugmented in wastewater samples. Further the presence of bacterial cells in log phase during biodegradation indicates that substrate conversion would be at its maximum as described by Gray (1989) and similarly observed in this study. 3.4. Influence of physicochemical conditions on biodegradation rates 3.4.1. pH The data as reported in Table 1, indicate that IES-Ps-1 can retain their degradation ability in a wide range of pH with optimum degradation at 30 C temperature where the pH of the water sample was found near neutral range (pH 7.33). This finding is supported by Ashok and Seth (1989), who reported that isolated Pseudomonas strain can grow between pH 5.5 and 9.5 with optimum growth at pH around 7.0. Similarly, Mandelbaum et al. (1995) found that atrazine degradation in the pH range of 5.5–8.5 by Pseudomonas strain was not affected. Moreover it is reported that the tolerable limits for pH in activated sludge ranged between pH 6.0 and 9.0 (Hanel, 1988). 3.4.2. Temperature Temperature is among the important environmental parameters that can influence the microbial growth as well as treatment efficiency (Comeau et al., 1993). During Cypermethrin degradation, the direct correlation was found between temperature and microbial activity. Significant removal occurred when biosimulator was operated at 28–30 C temperature using 8–9 mg/L DO, whereas moderate degradation was observed at ambient (18–25 C) and 38 C temperature (Table 1). Similar optimal temperature (28–30 C) for Pseudomonas
Table 1
261
growth in activated sludge was also reported by Schlegel (1969). 3.4.3. Dissolved oxygen Comparative performance evaluation at different dissolved oxygen concentrations for Cypermethrin degradation is presented in Table 1. It was noted that the initial dissolved oxygen concentration in the wastewater sample ranged between 1.5 and 2.6 mg/L. In order to maintain aerobic conditions in biosimulator, the mechanical aeration in the reactor not only provided sufficient dissolved oxygen but also kept Cypermethrin in suspension, such that the bacterial growth and COD removal were greatly enhanced. During experiment it was observed that the biodegradation performance of IES-Ps-1 significantly improved at 8–9 mg/L dissolved oxygen using mechanical aeration (250 rpm) in the temperature range of 28–30 C. 3.4.4. Chemical oxygen demand Results as summarized in Table 1, explain the lower degradation at high Cypermethrin concentrations and a good agreement between COD removal and Cypermethrin degradation rates especially at low concentrations. These results are in accordance to previous findings reported by Berchtold et al. (1995), who noticed the same correlation between the COD removal and biodegradation of 2,4-DAT and 2,4 and 2,6 diamino toluene degradation by acclimated bacteria (Pesce and Wunderlin, 1997). Similar correlations were also observed by Ramanathan and Lailithakumari (1999) during biodegradation of hazardous chemicals. Moreover, during treatment, it was also observed that even at a high organic load, the biosimulator function is satisfactory when operated under controlled temperature and dissolved oxygen with retention time of 48 h. These findings are in agreement with Toprak [38] who reported that COD removal during treatment mainly depends on temperature and influent COD concentration.
Comparative performance evaluation of IES-Ps-1 for Cypermethrin degradation after 48 h.
Parameters
pH
COD removal
Cypermethrin degradation
Conc. (mg/l)
% Removal
Conc. (mg/l)
% Degradation
Effect of Cypermethrin concentration (mg/l) Cypermethrin conc. (mg/l) 20 8.60 40 8.30 80 7.81 125 7.83
80 1080 4500 13767
97 82 54 24
– 8.2 42 118
No peak 81 51 18
Effect of temperature (C) at 80 mg/L dose Temperature (C) Ambient temp. (18–25) 7.80 28–30 7.33 38–40 7.50
4500 867 4000
54 89 52
42 9.0 42
51 88 48
Effect of dissolved oxygen (mg/L) at 80 mg/L dose Dissolved oxygen (mg/L) 5–6 mg/L 7.87 5000 8–9 mg/L 7.81 4500 8–9 mg/L (30 C temp.) 7.33 867 11–12 mg/L 8.20 1300
31 54 89 83
50 42 9.0 17
32 51 88 78
Data reported in table indicate average values of three experiments.
262
S. Jilani
3.5. Biodegradation of Cypermethrin The data reported in Table 1 and HPLC chromatogram shown in Figs. 5–7 clearly indicate that increased concentration of Cypermethrin (40–125 mg/L) in biosimulator decreased the degradation rates and the complete removal occurred at 20 mg/L dose (Jilani and Khan, 2006). However, optimizing the treatment conditions like pH, temperature, and dissolved oxygen in activated sludge, IES-Ps-1 could effectively remove higher concentrations of Cypermethrin. Similar results were also reported by other researchers (Ashok and Seth, 1989; Smith-Greeier and Adkins, 1996; Collins and Daugulis, 1997; Pesce and Wunderlin, 1997; Lee et al., 1998; Goudar and Strevett, 2000). During the experimental work it was observed that the optimum temperature for growth and degradation of Cypermethrin by IES-Ps-1 culture remained between 28 and 30 C. Using a similar concentration of Cypermethrin (80 mg/L), when the temperature of the reactor decreased or increased (38 C), the degradation rates were significantly reduced. At ambient temperature, 51% Cypermethrin degradation occurred whereas at 38 C temperature, it was 48% (Fig. 5). In contrast, when biosimulator temperature maintained between 28 and 30 C using mechanical aeration (8–9 mg/L), keeping Cypermethrin concentration constant (80 mg/L), >88% removal was achieved after 48 h of aerobic treatment (Fig. 6). Similar optimal temperature (28–30 C) for growth of Pseudomonas in activated sludge was also reported by Schlegel (1969) and Chaterjee et al. (1982). Moreover, Karpouzas and Walker (2000) reported that unlike high temperatures, low growth temperatures for Pseudomonas putida strain are usually not lethal. Overall study findings described that this may be the first instance in which high concentrations of Cypermethrin degradation were achieved in short retention time of 48 h. Although the transformation of permethrin (50 mg/L) by pure culture of Pseudomonas fluorescence under aerobic conditions with a half-life of less than 5 days was reported by Maloney et al. (1998) and the removal of technical grade Cypermethrin from
Figure 6 HPLC chromatograms showing comparative effect of temperature on Cypermethrin (80 mg/L) degradation at 9 mg/L DO. a: ambient temp.; b: 30 C; c: 38 C.
Figure 7 HPLC chromatograms showing comparative effect of DO on Cypermethrin (80 mg/L) degradation. a: 6 mg/LDO; b: 9 mg/L DO & 9 mg/L DO at 30 C; c: 12 mg/L.
60 to 6 mg/L by Pseudomonas species in 20 days was reported by Grant and Betts (2003). From the study findings, it can be concluded that biodegradation performance mainly depends on Cypermethrin concentration. In addition, during the treatment optimal residence time needs to be assessed while taking into account the Cypermethrin concentration but it appeared that 2 days would be a convenient time to reach satisfactory biodegradation at low Cypermethrin concentrations (<20 mg/L). 4. Conclusions Following conclusions may be drawn from this study: Figure 5 HPLC chromatograms showing comparative biodegradation rates at different Cypermethrin concentrations. a: 45 mg/ L; b: 80 mg/L; c: 120 mg/L.
Malathion degrading bacterial isolate, IES-Ps-1, can be used for biodegradation of pesticide wastes, as IES-Ps-1
Comparative assessment of growth and biodegradation potential of soil isolate in the presence of pesticides showed potential to grow in the presence of Cypermethrin. Cypermethrin concentration and the temperature were found to be a principal governing factor in removing Cypermethrin. Because of the low aqueous solubility of Cypermethrin, mechanical aeration in biosimulator proved to be very effective in reducing the concentration of Cypermethrin. As mechanical aeration not only provided the maximum dispersion of Cypermethrin in wastewater but also maintained the sufficient dissolved oxygen required for the growth of IES-Ps-1. Complete removal of Cypermethrin (20 mg/L) would only be possible if an appropriate organism (IES-Ps-1) and optimum operating conditions (temperature, dissolved oxygen, and mechanical aeration) be maintained in biosimulator.
Acknowledgement The partial financial support of the National Drainage Program of WAPDA, Pakistan is greatly acknowledged. References APHA (American Public Health Association), 1998. Standard methods for the examination of water and wastewater. American Public Health Association, American Water Works Association, Water Pollution Control Federation, 20th ed. Washington DC, USA. Ashok, K.S., Seth, P.K., 1989. Degradation of Malathion by microorganism isolated from industrial effluents. Bull. Environ. Contam. Toxicol. 43, 28–35. Baker, P.S., Dold, P.L., 1995. COD and nitrogen mass balances in activated sludge systems. Water Res. 29, 633–643. Berchtold, S.R., Vanderloop, S.L., Suidan, M.T., Maloney, S.W., 1995. Treatment of 2,4-dinitrotoluene using a two-stage system: fluidized-bed anaerobic granular activated carbon reactors and aerobic activated sludge reactors. Water Environ. Res. 67, 1081– 1091. Hensyl, W.R. (Ed.), 1994Bergey’s Manual of Determinative Bacteriology, ninth ed. Williams & Wilkins Company, Baltimore. Bharati, J.B., Seema, S.S., Pradnya, P.K., 2002. Bioremediation of an industrial effluent containing Monocrotophos. Curr. Microbiol. 45, 346–349. Chaterjee, D.K., Kilbane, J.J., Chakrabarty, A.M., 1982. Biodegradation of 2,4,5-trichlorophenoxy acetic acid in soil by a pure culture of Pseudomonas cepacia. Appl. Environ. Microbiol. 44, 514–516. Chaudhry, G.R., 1994. Biological Degradation and Bioremediation of Toxic Chemicals. Dioscorides Press, Portland, OR, USA. Christodoulatos, C., Koutsospyros, A.D., Brodman, B.W., Korfiatis, G.P., 1997. Biodegradation of diphenylamine by selected microbial cultures. J. Environ. Sci. Health 32 (1), 15–30. Collins, L.D., Daugulis, A.J., 1997. Biodegradation of phenol at high initial concentrations in two phase partitioning batch and fed-batch bioreactors. Biotechnol. Bioeng. 55, 155–162. Collins, C.H., Lyne, P.M., 1985. Microbiological Methods. 5thEdition. Butterworth and Co (Publishers) Ltd. Environmental Engineering. 116 (5), 805–828. Comeau, Y.C., Greer, W., Samson, R., 1993. Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation. Appl. Microbiol. Technol. 38, 681– 687. Corbitt, R.A., 1998. Standard Handbook of Environmental Engineering, second ed. McGraw Hill, New York, Chap 6.
263
Enrica, G., 1994. The role of microorganism in environmental decontamination. In: Aristeo, Renzoni (Ed.), Contaminants in The Environment – A Multidisciplinary Assessment of Risk to Man and Other Organisms. Lewis Publisher, pp. 235–246, 25. Giraud, F., Guiraud, P., Kadri, M., Blake, G., Steiman, R., 2001. Biodegradation of anthracene and fluoranthene by fungi isolated from an experimental constructed wetland for wastewater treatment. Water Res. 35, 4126–4136. Goudar, C.T., Strevett, K.A., 2000. Substrate inhibition kinetics of phenol biodegradation. Water Environ. Res. 72, 50–55. Grady, C.P.L., 1986. Biodegradation of hazardous waste by conventional biological treatment. Hazard. Waste Hazard. Material 3, 333–365. Grady, C.P.L., 1990. Biodegradation of toxic organics. Status and potential. J. Environ. Eng. 116, 805–828. Grant, R.J., Betts, W.B., 2003. Biodegradation of the synthetic pyrethroid cypermethrin in used sheep dip. J. Appl. Microbiol. 36, 173–176. Gray, N.F., 1989. The effect of small changes in incubation temperature on the five day biochemical oxygen demand test. Environ. Technol. Lett. 10, 253–258. Hanel, K., 1988. Biological Treatment of Sewage by the Activated Sludge Process. Ellis Horwood, Chichester, Wiley, New York. Hashmi, I., 2001. Microbiological transformation of hazardous waste during biological treatment. Ph.D. Thesis. Institute of Environmental Studies, University of Karachi, Pakistan. Hughes, S., Cooper, D., 1996. Biodegradation of phenol using the selfcycling fermentation process. Biotechnol. Bioeng. 51, 112–119. Jilani, S., Khan, M.A., 2006. Biodegradation of Cypermethrin by Pseudomonas in a batch activated sludge process. Int. J. Environ. Sci. Technol. 3, 371–380. Kao, C.M., Chai, C.T., Liu, J.K., Yeh, T.Y., Chen, K.F., Chen, S.C., 2004. Evaluation of natural and enhanced PCP biodegradation at a former pesticide manufacturing plant. Water Res. 38, 663–672. Karpouzas, D.G., Walker, A., 2000. Factors influencing the ability of Pseudomonas putida strains epI and II to degrade the organophosphate ethoprophos. J. Appl. Microbiol. 89, 40–48. Kelley, L., Freeman, J.P., Evans, F.E., Cerniglia, C.E., 1993. Identification of metabolites from the degradation of fluoranthene by Mycobacterium sp. strain PYR-1. Appl. Environ. Microbiol. 59, 800–806. Kollman, W., Segawa, R., 1995. Interim Report of the Pesticide Chemistry Database Environmental Hazards Assessment Program. Department of Pesticide Regulation. Lee, S.G., Yoon, B.D., Park, Y.H., Oh, H.M., 1998. Isolation of a novel pentacholorophenol-degrading bacterium, Pseudomonas sp. Bu 34. J. Appl. Microbiol. 85, 1–8. Maloney, S.E., Maule, A., Smith, A.R.W., 1998. Microbial transformation of the pyrethroid insecticides. Permethrin, Deltamethrin, Fastac, Fenvalerate and Fluvalinate. Appl. Environ. Microbiol. 54, 2874–2876. Mandelbaum, R.T., Allen, D.L., Wackett, L.P., 1995. Isolation and characterization of Pseudomonas sp. that mineralized the s-trazine herbicide atrazine. Appl. Environ. Microbiol. 61 (4), 1451–1457. Maria, K., Graciela, C., Zauscher, F., 2002. Biodegradation of two commercial herbicides (Gramoxone and Matancha) by the bacteria Pseudomonas putida. Electronic J. Environ. Biotechnol. 5, 182–195. Martin, M., Mengs, G., Plaza, E., Garbi, C., Sanchez, M., Gibello, A., Gutierrez, F., Ferrer, E., 2000. Propachlor removal by Pseudomonas strain GCH 1 in an immobilized cell system. Appl. Environ. Microbiol. 66, 1190–1194. Miles, A.A., Misra, S.S., 1938. The estimation of bacterial power of the blood. J. Hyg. Camb. 38, 732–749. Pesce, S.F., Wunderlin, D.A., 1997. Biodegradation of 2,4- and 2,6diaminotoluene by acclimated bacteria. Water Res. 31, 1601–1608. Quan, X., Hanchang, S., Hong, L., Pingping, Lv., YI, Q., 2004. Enhancement of 2,4-dichlorophenol degradation in conventional
264 activated sludge systems bioaugmented with mixed special culture. Water Res. 38, 245–253. Ramanathan, M.P., Lailithakumari, D., 1999. Complete mineralization of methyl parathion by Pseudomonas sp. A3. Appl. Biochem. Biotechnol. 80, 1–12. Ramos, T.L., Duque, E., Huertas, M.J., Haidour, A., 1995. Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentration of aromatic hydrocarbons. J. Bacteriol. 177, 3911–3916. Ritmann, B.E., Jacson, D.E., Storck, S.L., 1988. Potential for treatment of hazardous organic chemical with biological process. Biotreatment systems. In: Wis, D.L. (Ed.). CRC Press, Boca Raton, FL, pp. 15–64, 3. Sapiets, A., Swaine, H., Tandy, M.J., 1984. Cypermethrin. In: Zweig, G., Sherma, J. (Eds.), Analytical Methods for Pesticides and Plants Growth Regulators. Academic Press, New York, p. 33, XIII.
S. Jilani Schlegel, H.G., 1969. Allgemeine Mikrobiologie Thieme Stuttgart. In: Hanel, K. (Ed.), Biological Treatment of Sewage by the Activated Sludge Process. Ellis Horwood, Chichester, Wiley, New York. Smith-Greeier, L.L., Adkins, A., 1996. Isolation and characterization of soil microorganisms capable of utilizing the herbicide dichloro-p-methyl as a sole source of carbon and energy. Can. J. Microbiol. 42, 221–226. Stepheson, R.R, 1982. Aquatic toxicology of Cypermethrin. In: Acute toxicity to some freshwater fish and invertebrates in laboratory tests. Aquatic. Toxicol. 2, 253–270. Toprak, H., 1995. Removal of soluble chemical oxygen demand from domestic waste waters in a laboratory scale anaerobic waste stabilization ponds. Water Res. 29, 923–932. Zacharias, B., Lang, E., Hanert, H.H., 1995. Biodegradation of chlorinated aromatic hydro-carbons in slow sand filters simulating conditions in contaminated soil – Pilot study for in situ cleaning of an industrial site. Water Res. 29 (7), 1663–1671.