STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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Chapter 1
Introduction
1.1. Introduction
Urbanization and extensive industrialization leads to the accumulation of a wide variety of pollutants and new technologies implanted to eradicate contaminants from the
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environment. In agricultural field uses of pesticides has become necessary and their indiscriminate routine has inflicted serious environmental implication related to human health (Gavrilescu, 2005; Hussain et al., 2009). Pesticides intentionally made to be injurious and directly introduced into the environment from that only 5 percent reach to the target species and remaining enters into the environment.
Presently, there are multiple possible methods for the clean-up of pesticides among them biodegradation proved to be most promising remedy for the treatment and detoxification of pesticides, particularly by the use of biocatalysts like enzymes, or by using micro-organisms as whole. Ghadiri, 2001; Bavcon et al., 2003; Nawab et al., 2003; Kodaka, et al., 2003; Sassman et al., 2004; Finley et al., 2010 discussed about the
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pesticides degradation by the process of bioremediation as an substitute treatment approach that is effective, economical, , negligibly hazardous and environment-friendly.
Schroll et al., (2004) stated that the degradation ability of microorganisms is directly proportional to their continuing adaptation to these compounds from the environment. Singh and Walker, (2006) further reported that indigenous microorganisms have the potential to be used for the bioremediation provides a cheap and efficient solution.
According to Andreu and Pico, (2004) discussed about the pesticides are studied more than any other environmental contaminant. There is also increasing interest in their metabolites as transformation products can be present at higher levels with lower toxicity to biota in the environment than the parent pesticide itself (Nawab, et al.,
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2003). However, metabolites also have the potential to produce an adverse impact on the environment (Sinclair and Boxall, 2003; Papadakis, and Papadopoulou-Mourkidou, 2002; Pozo et al., 2001). As a result, there is a need to identify metabolites during the process of pesticide detoxification.
Potter and Wadkins, (2006); Redinbo and Potter (2005) stated that microbial enzymes advantageous for the detoxification of many pesticides. Specifically, carboxylesterases degrade many pesticides including pyrethroids (Wheelock et al., 2004; Stok et al., 2004a; Abernathy and Casida, 1973) and organophosphates (Casida and Quistad 2004; Kao et al., 1985)
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Bass and Field, (2011) discussed about the enzyme families responsible for the degradation of pesticides that are Phosphotriesterases (Sogorb et al., 2004), Glutathione S-transferases (Enayati et al., 2005; Fournier et al., 1992) and Cytochrome P450 (Kulkarni and Hodgson 1984).
1.2. Objectives of the Study
The present work is focused towards the use of efficient indigenous microbial strains and their extracellular enzyme for the degradation of the Malathion and Cypermethrin as a pilot study that can be used for commercial explications.
Following are the main objectives which have been focused:
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1. To assess the physicochemical characteristics of wastewater of Lyari River that lead to the contamination of coastal ecosystem. The qualitative and quantitative analysis of wastewater samples have focused on parameters including pH, Temperature, DO
-3 (Dissolve Oxygen), NH3 (Ammonia), TKN (Total Kjeldahl Nitrogen), PO4
(Phosphate), B0D5 (Biological Oxygen Demand), COD (Chemical Oxygen Demand), Total Coliform Count (TCC), Total Fecal Coliform Count(TFCC) and pesticide residues (malathion and cypermethrin) in order to compare range of concentration in accordance to the permissible limit provided by National Environmental Quality Standards (NEQS) for municipal and industrial effluent in Pakistan. (see in Chapter 2)
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2. Isolation, characterization and Polymerize chain reaction (PCR) based identification of indigenous bacteria capable for degrading malathion and cypermethrin. PCR was done for the amplification of the fragment of 16S rDNA gene of the DNA of isolated bacterial strain. The sequences were compared with the nucleotide database in GenBank and then aligned to construct a phylogenetic tree. This work as to describe the potential of pesticide degrading bacteria and to demonstrate eco- friendly technology suitable for wastewater containing pesticide. (see in Chapter 3)
3. Evaluation of Growth kinetics of metabolically versatile bacterial species namely Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa by using
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varying concentration of malathion and cypermethrin in minimal salt media. (see in Chapter 4)
4. Determination of the biodegradation kinetics of isolated metabolically versatile indigenous bacterial species namely Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa under varying concentration of malathion and cypermethrin in minimal salt media. Gas chromatography was performed by using electron capture detector for the confirmation of biodegradation. (see in Chapter 5)
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5. To study degradation of malathion and cypermethrin by carboxylesterase of Escherichia coli IES-02 (KU593482) and analysis of novel product appearance over time by (GC- MS) gas chromatography mass spectrometry analysis. (see in Chapter 6)
1.3. Justification and Likely Benefits
Around the world with the growth of agricultural activities malathion and cypermethrin are extensively used and discharged without any treatment into the environment caused seriously environmental implications because of their high toxicity. The problem is of special concern because of the development of resistance among bacterial strains to pesticides and the presence of pesticide metabolites into environment. Pesticide
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resistance is mainly attributed by excessive and indiscriminate use of pesticides (Jayaraj, 1989).
Gavrilescu, (2005) reported that the environmental pollution caused by pesticides spread over larger area. Therefore, the fate of pesticides pollution is often undefined. In fact, the damages are practically irreparable. To eliminate the antagonistic effect, it is important to treat them efficiently. Therefore, biodegradation is the best possible solution by transform organic matter into nutrients.
Enzymes of microbial species break up complex compounds into metabolites which used by the microbial cells for reproduction and growth. Mixtures of species offer mineralization of particular pollutants completely because the metabolite of one species often serve as substrate to another species.
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Enzymatic degradation of pesticides has two main advantages. Firstly, the degradation continues under relatively mild reaction conditions and secondly, the rate of reaction is faster than that by micro-organisms. Bacterial enzymatic detoxification of organophosphorus and pyrethroids pesticide has studied by many researchers, because it is economical, environment friendly and effective. Desaint et al., (2000); Pieper and Reineke, (2000) suggested that biodegradation are desirable to conventional methods because, microorganisms detoxify complex pollutants into less toxic metabolites. Carboxylesterases are major enzymes responsible for the catalysis of malathion and cypermethrin by the hydrolysis of carboxyl esters and also an important enzyme that deserve further study for applications in environmental monitoring.
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Estimation of metabolites is also advantageous because transformation products can be present at higher levels with lower toxicity to biota in the environment than the parent pesticide itself. As a result, there is a need to identify metabolites during the process of pesticide detoxification. Furthermore kinetics of metabolites correctly describe the degradation of the parent compound. When the pesticide pollution is alarming remediation is obligatory to avoid relocation of pesticides to a more vulnerable areas. The identification of degradation products should facilitate an evaluation of the relative significance of chemical and microbial degradation in the environment.
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1.4. References
Abernathy, C. O., & Casida, J. E. (1973). Pyrethroid insecticides: esterase cleavage in relation to selective toxicity. Science, 179(4079), 1235-1236.
Andreu, V., & Picó, Y. (2004). Determination of pesticides and their degradation products in soil: critical review and comparison of methods. TrAC Trends in Analytical Chemistry, 23(10), 772-789.
Bass, C., & Field, L. M. (2011). Gene amplification and insecticide resistance. Pest management science, 67(8), 886-890.
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Bavcon, M., Trebše, P., & Zupančič-Kralj, L. (2003). Investigations of the determination and transformations of diazinon and malathion under environmental conditions using gas chromatography coupled with a flame ionisation detector. Chemosphere, 50(5), 595-601.
Casida, J. E., & Quistad, G. B. (2004). Organophosphate toxicology: safety aspects of nonacetylcholinesterase secondary targets. Chemical research in toxicology, 17(8), 983-998.
Desaint, S., Hartmann, A., Parekh, N. R., & Fournier, J. C. (2000). Genetic diversity of carbofuran-degrading soil bacteria. FEMS Microbiology Ecology, 34(2), 173-180.
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Enayati, A. A., Ranson, H., & Hemingway, J. (2005). Insect glutathione transferases and insecticide resistance. Insect molecular biology, 14(1), 3-8.
Finley, S. D., Broadbelt, L. J., & Hatzimanikatis, V. (2010). In silico feasibility of novel biodegradation pathways for 1, 2, 4-trichlorobenzene.BMC systems biology, 4(1), 1. Fournier, D., Bride, J. M., Poirie, M., Berge, J. B., & Plapp, F. W. (1992). Insect glutathione S-transferases. Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides. Journal of Biological Chemistry, 267(3), 1840-1845.
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Gavrilescu, M. (2005). Fate of pesticides in the environment and its bioremediation. Engineering in Life Sciences, 5(6), 497.
Gavrilescu, M., & Chisti, Y. (2005). Biotechnology—a sustainable alternative for chemical industry. Biotechnology advances, 23(7), 471-499.
Ghadiri, H. (2001). Degradation of endosulfan in a clay soil from cotton farms of western Queensland. Journal of Environmental Management, 62(2), 155-169.
Hussain, S., Siddique, T., Arshad, M., & Saleem, M. (2009). Bioremediation and phytoremediation of pesticides: recent advances. Critical Reviews in Environmental Science and Technology, 39(10), 843-907.
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Jain, R. K., Kapur, M., Labana, S., Sarma, P. M., Lal, B., Bhattacharya, D., & Thakur, I. S. (2005). Microbial diversity: application on micro- organisms for the biodegradation of xenobiotics. Current science, 89, 101-112.
Jayraj, S. (1989). Advances in biological means of pest control. The Hindu Survey of Indian Agriculture, 181-187.
Kao, L. R., Motoyama, N., & Dauterman, W. C. (1985). Multiple forms of esterases in mouse, rat, and rabbit liver, and their role in hydrolysis of organophosphorus and pyrethroid insecticides. Pesticide biochemistry and physiology, 23(1), 66-73.
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Kodaka, R., Sugano, T., Katagi, T., & Takimoto, Y. (2003). Clay-catalyzed nitration of a carbamate fungicide diethofencarb. Journal of agricultural and food chemistry, 51(26), 7730-7737.
Kulkarni, A. P., & Hodgson, E. (1984). The metabolism of insecticides: the role of monooxygenase enzymes. Annual review of pharmacology and toxicology, 24(1), 19-42.
Nawab, A., Aleem, A., & Malik, A. (2003). Determination of organochlorine pesticides in agricultural soil with special reference to γ-HCH degradation by Pseudomonas strains. Bioresource technology, 88(1), 41-46.
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Papadakis, E. N., & Papadopoulou-Mourkidou, E. (2002). Determination of metribuzin and major conversion products in soils by microwave-assisted water extraction followed by liquid chromatographic analysis of extracts. Journal of chromatography A, 962(1), 9- 20.
Paul, D., Pandey, G., Pandey, J., & Jain, R. K. (2005). Accessing microbial diversity for bioremediation and environmental restoration. TRENDS in Biotechnology, 23(3), 135-142.
Pieper, D. H., & Reineke, W. (2000). Engineering bacteria for bioremediation. Current opinion in biotechnology, 11(3), 262-270.
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Potter, P. M., & Wadkins, R. M. (2006). Carboxylesterases- detoxifying enzymes and targets for drug therapy. Current medicinal chemistry, 13(9), 1045-1054.
Pozo, O., Pitarch, E., Sancho, J. V., & Hernandez, F. (2001). Determination of the herbicide 4-chloro-2-methylphenoxyacetic acid and its main metabolite, 4-chloro-2- methylphenol in water and soil by liquid chromatography–electrospray tandem mass spectrometry. Journal of Chromatography A, 923(1), 75-85. Redinbo, M. R., & Potter, P. M. (2005). Keynote review: Mammalian carboxylesterases: From drug targets to protein therapeutics. Drug discovery today, 10(5), 313-325.
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Sassman, S. A., Lee, L. S., Bischoff, M., & Turco, R. F. (2004). Assessing N, N'-Dibutylurea (DBU) Formation in Soils after Application of n-Butylisocyanate and Benlate Fungicides. Journal of agricultural and food chemistry, 52(4), 747-754.
Schroll, R., Brahushi, F., Dörfler, U., Kühn, S., Fekete, J., & Munch, J. C. (2004). Biomineralisation of 1, 2, 4-trichlorobenzene in soils by an adapted microbial population. Environmental Pollution, 127(3), 395-401.
Sinclair, C. J., & Boxall, A. B. (2003). Assessing the ecotoxicity of pesticide transformation products. Environmental science & technology, 37(20), 4617-4625.
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Singh, B. K., & Walker, A. (2006). Microbial degradation of organophosphorus compounds. FEMS microbiology reviews, 30(3), 428-471.
Sogorb, M. A., Vilanova, E., & Carrera, V. (2004). Future applications of phosphotriesterases in the prophylaxis and treatment of organophosporus insecticide and nerve agent poisonings. Toxicology letters, 151(1), 219-233.
Stok, J. E., Huang, H., Jones, P. D., Wheelock, C. E., Morisseau, C., & Hammock, B. D. (2004). Identification, expression, and purification of a pyrethroidhydrolyzing carboxylesterase from mouse liver microsomes. Journal of Biological Chemistry, 279(28), 29863-29869.
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Wheelock, C. E., Miller, J. L., Miller, M. J., Gee, S. J., Shan, G., & Hammock, B. D. (2004). Development of toxicity identification evaluation procedures for pyrethroid detection using esterase activity. Environmental Toxicology and Chemistry, 23(11), 2699-2708.
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Chapter 2
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Physicochemical and Pesticide Residues Analysis of Wastewater of Lyari River
2.1. Introduction
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Pakistan has agri-based economy where 70% of the human resource is living in villages and are involved directly or indirectly to the agriculture sector (Azmi et al., 2006). The rate of pesticide usage is keep on accelerating in the current face of world food problem (Pimentel, 1995). Pakistan has no exception (Tariq et al., 2007). According to PPSGDP, 2002 presently in Pakistan above 108 types of insecticides are being used for many purposes. In the last 20 years the pesticide utilization trend remarkably increased up to 1169 percent (Technical bulletin, 2000). The predicament is that the farmers are indiscriminately using pesticides without considering their toxic effects. Even the pesticides that are banned by the developed countries are still in used by the developing or under developed countries (Sankararamakrishnan et al., 2005).
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Karachi 24°51′ N 67°02′ E is the commercial hub and prime city of Pakistan which ranked 6th world largest metropolitan area. Agriculture in Karachi city is mostly in the out skirts. Karachi basin is drained by two major rivers namely Malir and Lyari with catchments areas of 2051 and 7045 Km2 respectively (ACE, 1993).
As there is no more fresh water in these rivers, they are merely used for dumping of liquid and solid wastes of both domestic and industrial origin (Beg, 1997). Lyari River is the largest watercourse flowing through urban Karachi, which was predominantly a seasonal river and presently served as drainage system for the connecting industries and vicinities. (Mansoor et al., 2007). Lyari River has very mild slope in the outfall reaches touching almost at zero level downstream of Mauripur Road Bridge. The flow in the river is thus very much influenced by the diurnal rise and fall of the tides. The river
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carries highly polluted wastewater from the north and west of the city and finally dumped it into the Arabian Sea.
Lyari River is about 50 km long, which up till 1950’s had clean water (Asif, 2002). Lyari River drain into the Arabian Sea at Manora channel where it remain stagnant during low tide (Mansoor et al., 2007). At present more than 50 squatter settlements are located along both the banks of Lyari River accommodating approximately 0.8 million persons. These unauthorized encroachments create obstruction in the waterway of Lyari River. Ultimately, the water level increases and storm water drains discharging in Lyari River become ineffective.
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In many places, Lyari River effluent is used for unrestricted irrigation. The people of the nearby communities used to cultivate vegetables mostly through the untreated effluent of Lyari River. These vegetables are supplied to the Karachites. The farmers mostly used two common and commercially available pesticides namely Malathion and Cypermethrin owing to their efficacy and affordability. Malathion (organophosphate) and Cypermethrin (pyrethroid) are also extensively used all over the country. Their application is mostly through ground and aerial sprays (Ware, 2000).
Malathion targets mainly the nervous system of organisms by selectively inhibiting acetylcholinesterase (Bjorling-Poulsen et al., 2008). It is commonly used to control
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insects of a number of crops and stored grain. Malathion classified as a toxicity class III of pesticide (USEPA, 1988).
Cypermethrin is also widely used in agriculture, public health and homes, (Wang and Yan, 2011; Tallur et al., 2008). Cypermethrin as low as 10 µg/L is toxic to the aquatic environment (Pearce, 1997; Vinodhini and Narayanan, 2008; Virtue et al., 2008). Kakko et al., (2004); Cabral and Galendo, (1990) reported about the carcinogenic effects of cypermethrin.
Physicochemical characteristics of wastewater of Lyari River was estimated that lead to the contamination of coastal ecosystem. The qualitative and quantitative analysis of
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wastewater samples focused on parameters including pH, Temperature, DO (Dissolve Oxygen), NH3 (Ammonia),
-3 TKN (Total Kjeldahl Nitrogen), PO4 (Phosphate), B0D5 (Biological Oxygen Demand), COD (Chemical Oxygen Demand), Total Coliform Count, Total Fecal Coliform Count and pesticide residues (malathion and cypermethrin) in order to compare with permissible level provided by National Environmental Quality Standards (NEQS) for municipal and industrial effluent in Pakistan.
2.2. Material and Methods
2.2.1. Chemicals and pesticides standards
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Sodium chloride, Acetone, n-hexane, dichloromethane, anhydrous sodium sulfate were purchased from Merck (Germany) analytical grade. Malathion and cypermethrin standards were purchased from AccuStandard, Inc. (USA) 99.9% pure.
2.2.2. Study Area
Samples were collected from Lyari River outfall Fig. 2.1. (24°51'59.48"N 66°58'20.16"E). These samples were collected twice a month during 2014. In all 24 samples were collected.
2.2.3. Collection of samples
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Wastewater samples were collected using Niskin bottle from the surface (approx. 10 cm) at the area approachable through feet. For the collection of samples pre-sterilized amber glass bottles of 2-litre capacity were used. The samples were collected in a way to avoid floating materials. All samples were grab collection, taken from the pre- designated locations as mentioned in Fig.2.2.
2.2.4. Physicochemical parameters
The physical characteristics of wastewater such as pH was determined using HACH sensation 156 multi parameter dissolved oxygen meter. Dissolved oxygen was
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determined using Jenway 630i dissolved oxygen meter. The DO probe was immersed in the sample stream to an adequate depth and in a manner to ensure sufficient sample movement across the probe-sensing element. The above mentioned parameters were determined onsite.
BOD5 (Biological oxygen demand), COD (Chemical oxygen demand), TKN, NH3, and PO4 were analyzed as per method described in Standard Methods for Water and Wastewater examination (APHA, 2005) Fig.2.3 and compared with National Environmental Quality Standards (NEQS, 2000).
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For measuring total coliform count (TCC) and total fecal coliforms (TFCC), the multiple-tube fermentation technique was employed by the Examination of Water and Wastewater Standard Methods (APHA, 1995) Fig.2.4
2.2.5. Liquid–liquid extraction method
For the extraction of pesticide residues in wastewaters, liquid liquid extraction (LLE) method was used according to method of Alawi et al., (1995). One liter wastewater sample was homogenized with 2 g anhydrous NaCl in separating funnel and extracted twice with 50ml of dichloromethane (2 x 50 ml). The lower layer was separated as
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shown in Fig. 2.5 and dry by anhydrous sodium sulfate. The eluent evaporated by using a rotary evaporator (BUCHI Rotavapor B-740) at 40°C under vacuum. The dried out residue was further dissolved in 10 mL n-hexane for analysis on GC.
2.2.6. GC Analysis
A Gas Chromatograph (Shimadzu's versatile GC-2014), equipped with a WBI injector, Optima 5 column (6.0m 0.32mm capillary column coated with a 0.22µm film) was used for pesticide analysis. Temperature of column at 180°C for 1 min was maintained and raised at the rate of 240°C/min to 360°C and hold for 1min. The injection port
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temperature 260°C was retained. Carrier gas (nitrogen) at a flow rate of 2.11 ml min-1 and the make-up gas with 7.2 ml/min of total flow rate. Electron Capture Detector (ECD) temperature 300°C was used for analysis of cypermethrin and Flame Ionization Detector (FID) was used for the estimation of malathion residue. Individual sample was monitored three times and standard deviation and standard error were calculated.
2.2.7. GC –MS Analysis
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Malathion and Cypermethrin were monitored and confirmed through (GC/MS) gas chromatography system Agilent 7890A (G3440A) prepared with mass spectroscopy detector (MS Agilent 7000 GC/MS triple quadrupole).
2.3. Results and Discussion
Physicochemical analysis of Lyari River was performed to determine the extent of pollution load in the river. Results of all these parameters were compared with National Environmental Quality Standards for municipal and industrial effluents in Pakistan. All these parameters are discussed below and presented in Table 2.1, 2.2 and 2.3.
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2.3.1 Physical Characteristics
Results of pH and DO are given in Table 2.1. The minimum and maximum pH values of Lyari River effluent ranged from 7.3 to 7.8. Whereas, the mean pH value was 7.51. The minimum pH value was found in January and October while maximum pH value was obtained in February and May. In general, the pH of Lyari River effluent is towards alkaline side. Lyari River contains effluent from textile industries located at Sindh Industrial Trading Estate (SITE) and Federal B area industrial area. These are the two industrial areas where most of the textile and hosiery industries are located. Alkaline pH of Lyari River is mainly due to textile effluent which generally has a very high pH values. The mean concentration of DO in Lyari River effluent was 1.95. Whereas minimum and
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maximum DO concentration were 1.6 (May and October) and 2.9 mg/L (August) respectively. High DO concentration was found in August that might be due to high wind velocity, which is responsible for atmospheric dissolution of dissolved oxygen. The typical sewage like smell all along the Lyari River particularly at its outfall is an indication of high organic load that is responsible for creating anoxic conditions. No significant variations pertain to DO concentration was observed during entire study.
Dhage et al., 2006 and Allan et al., 1990 described that low DO concentrations is due to high BOD values which indicate the stress of ecosystem. In general, DO concentration during each month remained less than 3.0 mg/L. It can be claimed that the river is facing hypoxic condition which normally occurs when DO concentration is < 2.0 mg/L (Diaz et
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al., 1995). Lower DO (<4.0 mg/L) followed in both the estuary and adjacent coastal waters largely in summer (Yin et al., 2004). Khan et al., (1999) reported parallel values of DO in Lyari River effluent.
2.3.2 Nutrients
Phosphate ammonia, and TKN were selected as nutrient parameters to report their concentration in the Lyari River effluent. Concentrations of phosphate ion higher to 3.46mg/L. In all the samples, the concentration of ammonia were in noticeable variation (85mg/L to 180mg/L). However the NEQS value (i.e. 40mg/L) is even less than the minimum detected concentration of ammonia in wastewater samples. Whereas, TKN
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concentration in all samples was found to be closer to each other. However, these concentrations are at least 20% higher than the NEQS limit (100mg/L).
Ahn et al., (2007) described that the measurements of TKN and ammonia are necessary to assess the potential of eutrophication in the river. A high content of inorganic nitrogen and phosphorus may give rise to the production of algal blooms eventually causing turbidity of river.
2.3.3. Organic and Inorganic Constituents
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The BOD level of the samples ranged from 1099 to 2457 mg/L, while that of COD from 2009 to 4430 mg/L. Higher BOD and COD lead to eutrophication in the river. Mean values of BOD and COD are 1742 and 3242 mg/L respectively. These values are relatively higher than NEQS (80mg/L and 150mg/L respective).
Prashanth et al., (2006) and Iran et al., (2007) stated that untreated effluent with high concentration of organic matters is responsible for the low DO. Very high BOD5 and COD values represents poor environmental conditions and high pollution level.
2.3.4. Microbiological analysis
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The fate of microbial activities in wastewater is predominant. Bacterial parameters, such as TCC and TFCC was performed. Mean values of TCC and TFCC were 584.4 and 995.1 /100ml respectively. These values are relatively higher than NEQS (<200/100ml and <400/100ml).
2.3.5. Pesticide analysis
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This study focused on the residual concentration of two pesticides (Malathion and Cypermethrin). The study revealed that concentrations of these two pesticides in the wastewater throughout the Lyari River were alarming.
Outcomes of pesticides analysis are presented in Table. 2.3 and Fig. 2.7 and 2.8. Fig. 2.6 represents the standard chromatogram of malathion and cypermethrin. Malathion peak was detected after 15.20 minutes and cypermethrin peak was detected after 32.79 minutes. Gas chromatography mass spectrometry were confirmed the malathion and cypermethrin peaks by evaluation of their molecular ions and fragments with parallel authentic compounds by using library database of NIST (National Institute of Standards
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and Technology). The mass spectrum of malathion and cypermethrin is shown in Fig. 2.9 and 2.10 with molecular weight of malathion 284 and cypermethrin 415.
The mean concentration of malathion was 21.35 ppm while that of cypermethrin was 14.73 ppm. Minimum and maximum concentration of malathion ranged between 42.5 to 59.8ppm. Minimum concentration of malathion was found in March while maximum was obtained in October. It is interesting to note that malathion was only available in the effluent during January, March, April, September and October. The concentration of cypermethrin was relatively low as compared to malathion. The minimum and maximum values of cypermethrin was 19.5 (February) and 50 ppm (June). The average
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concentration of both these pesticides in wastewater samples were higher than the NEQS (0.15mg/L).
No significant trend was observed in the concentration of both the pesticides during the entire study. The main source of these pesticides could be the industrial effluents originated from the four industrial zones of Karachi that are LITE (Landhi Industrial Estate) in the east, SITE (Sindh Industrial Trading Estate) in the north, , HITE (Hub Trading Estate) between Karachi and Gadani in the west and KIA (Korangi Industrial area) in the south. Karachi city generates more than 350 MGD of domestic and industrial waste, of which only 90 MGD (less than 30% of the total waste generated) is partially treated daily at three waste water treatment plants (Hussain, 2007; Khan and Khan, 2007). These treatment plants are located in SITE town called TP-1 (Sher Shah) in
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Jamshaid Town called TP-2 (Mahmoodabad) and in Mauripur called TP-3. TP-1 and TP-2 were built in 1960.
Unrestricted irrigation is common at some places along the course of Lyari River. In particular, the people used to cultivate spinach, corn, tomatoes and lettuce. The unrestricted irrigation fields were found at Hasan square bridge, Lasbella and Teen Hatti bridge and at Lyrai River outfall. The families consumed their own produce and also sell into the nearby markets. It has been observed during the field surveys that the farmers /families indiscriminately using malathion and cypermethrin in particular to save their crops from the attack of insects without considering their harmful effects. This unrestricted use of pesticide could be one of the potential sources of pesticides in Lyari River effluent. During the recent epidemic of Dengue fever (2014-2015) in Karachi
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malathion was also used to kill mosquitoes by Karachi Municipal Corporation. This involves spray in large outdoor areas. Lemon, 1967 reported that malathion also used to kill beetles found in stored grain particularly wheat. Malathion from wheat granaries located in the city may also enter in the Lyari effluent through runoff from areas.
Koirala et al., (2007) stated that haphazard and misuse of pesticides may pollute the water reservoir and eventually enter into the food chain, which cause severe harm to human health, fishes and many other animals, leading to death.
Jing et al., (2010) detected maximum concentration of cypermethrin (0.969 µg/l) in industrial wastewater samples of Beijing. Cypermethrin is potentially toxic to the aquatic life forms even at a concentration as low as 10 µg/l (Vinodhini and Narayanan, 2008). It
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may also have carcinogenic effects (Kako et al., 2004). Stephenson, 1982 determined Cypermethrin toxicity to some species of fish (Cyprinus curpio. Scurdinius etytheophthalmus, Salnro goirdneri, Salmo trutta and Tilapia nilotko). He found 96 hour LC 50 values were within the range 0.4-2.2 µg/l.
Nuzhat et al., (2010) has reported that Karachi harbor is contaminated with organochlorine pesticide and the residual concentration is considerably higher in the vicinity of the discharge point of Lyari River and adjoining areas. This study correlate the present findings. As such desirable aquatic life forms do not exist in Lyari River, however, the continuous discharge of Lyari River effluent into the Arabian Sea is potentially hazardous to marine biodiversity.
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From this preliminary study no selective point source of pesticide was detectable however, pesticide misuse, spillage, and/or inappropriate storage, handling and disposal could be the potential sources of pesticides in Lyari River effluent. Accordingly these sources are responsible, in many cases, for pesticide contamination of water bodies at high concentration levels (Affam et al., 2012).
2.4. Conclusion
This study will provide a groundwork for establishing a monitoring program for commercially available pesticides. This preliminary study will be helpful for the municipal authorities in setting up relevant management policies.
Figures
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Figure. 2.1 Lyari River outfall (24°51'59.48"N66°58'20.16"E)
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Figure. 2.2 Wastewater samples collected from Lyari River
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Figure. 2.3. Physicochemical analysis of wastewater samples (A) Biological oxygen demand (B) Chemical oxygen demand (C) Ammonia and TKN
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Figure. 2.4. Microbiological analysis of wastewater samples
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Figure. 2.5. Liquid–liquid extraction (LLE) for the extraction of pesticide residues
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Figure. 2.6 (A) GC-chromatogram of Malathion standard (10ppm) (B) GC-chromatogram of Cypermethrin standard (10ppm)
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Figure. 2.7 GC- chromatogram of Malathion residues in wastewater
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Figure. 2.8 GC- chromatogram of Cypermethrin residues in wastewater
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Figure. 2.9 Mass spectrum of Malathion
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Figure. 2.10 Mass spectrum of Cypermethrin
Tables
S. No Months pH Dissolved oxygen (ppm) 1 January 7.3 1.8 2 February 7.8 1.7 3 March 7.6 1.9 4 April 7.5 2.1
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5 May 7.8 1.6 Table. 6 June 7.4 2.3 2.1 pH 7 July 7.5 2.5 and 8 August 7.7 2.9 Dissolved 9 September 7.4 1.9 oxygen 10 October 7.3 1.6 values of 11 November 7.4 1.5 12 December 7.5 1.7 Lyari Min.-Max 7.3-7.8 1.6-2.9 River Mean 7.51 1.95 effluent Standard error 0.051 0.122 Standard deviation 0.174 0.42
NEQS 4.0-6.0 6.5-8.5
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STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER -3* S. No Months NH3* TKN* PO4 BOD5* COD* TFCC/100ml TCC/100ml 1 January 85 124.3 2.43 1978 2999 240 120 2 FebruaryTHROUGH 96 134.6 ITS3.65 INDIGENOUS2012 3807 389 654 3 March 112 116.2 3.21 1947 2976 190 210 4 April 127 121.6 2.67 1400 3408 675 1200 5 May 146 114.7 2.74 2184 3992 1100 2000 6 June MICROBIAL112 102 2.1 1343FLORA 2567 746 1200
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Table. 2.2 Descriptive statistics of Physico-chemical and Microbiological analysis of wastewater
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7 July 160 120.7 4.6 1120 2130 334 678 8 August 165 132.1 3.99 1009 2009 444 1400 9 September 152 130 4.34 1943 2876 546 1000 10 October 180 135 5.09 2305 4208 1100 1900 11 November 99 110 4.1 2457 4430 789 1340 12 December 154 139.5 2.7 1210 3510 460 240 Min.- 120- 1099- 2009- 85-180 2.1-5.09 190-1100 120- 2000 Max. 139.5 2457 4430 Mean 132.33 123.39 3.46 1742.2 3242.6 584.4 995.1
Standard 8.99 3.27 error 0.27 143.4 227 87.8 181.8 Standard 31.1 11.34 deviation 0.96 497 786.3 304.4 629.9 NEQS** 40 100 1 80 150 <200 <400 * mg/L ** National Environmental Quality Standards for municipal and industrial effluents in Pakistan
Table. 2.3. Residual concentration of Malathion and Cypermethrin in Lyari River effluent S. No Months Malathion (ppm) Cypermethrin (ppm) 1. January 51 BDL 2. February BDL 19.5
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3. March 42.5 BDL 4. April 55 BDL 5. May BDL 31 6. June BDL 50 7. July BDL 30.7 8. August BDL 30.9 9. September 48 BDL 10. October 59.8 BDL 11. November BDL BDL 12 December BDL BDL Min-Max. 42.5-59.8 19.5-50 Mean 21.35 14.73 Standard Error 6.60 10.99 Standard deviation 26.69 18.30 NEQS 0.15 0.15
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2.5. References
ACE. (1993). Lyari and Malir River pollution study. Associated consulting engineers, Karachi 1-24.
Affam, Augustine Chioma, Shamsul Rahman M. Kutty, and Malay Chaudhuri. (2012). Solar Photo-Fenton Induced Degradation of Combined Chlorpyrifos, Cypermethrin and Chlorothalonil Pesticides in Aqueous Solution. In Proceedings of World Academy of Science, Engineering and Technology, no. 62. World Academy of Science, Engineering and Technology.
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Ahn, C. H., Park, H. D., & Park, J. K. (2007). Enhanced biological phosphorus removal performance and microbial population changes at high organic loading rates. Journal of Environmental Engineering, 133(10), 962-969.
Alawi, M., Khalili, F., & Da'as, K. (1995). Interaction behavior of organochlorine pesticides with dissolved Jordanian humic acid. Archives of Environmental Contamination and Toxicology, 28(4), 513-518.
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Allan, G. L., Maguire, G. B., & Hopkins, S. J. (1990). Acute and chronic toxicity of ammonia to juvenile Metapenaeus macleayi and Penaeus monodon and the influence of low dissolved-oxygen levels. Aquaculture, 91(3), 265-280.
APHA, (2005). Standard Methods for Examination of water and wastewater, 21st Edition.
Azmi, M. A., Naqvi, S. N. H., Azmi, M. A., & Aslam, M. (2006). Effect of pesticide residues on health and different enzyme levels in the blood of farm workers from Gadap (rural area) Karachi— Pakistan. Chemosphere, 64(10), 1739-1744.
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Beg, M. A. A. (1997). Pollution of the Karachi coastal environment. Wildlife and Environment, 5(4), 20-22.
Bjørling-Poulsen, M., Andersen, H. R., & Grandjean, P. (2008). Potential developmental neurotoxicity of pesticides used in Europe. Environmental Health, 7(1), 50.
Zhang, C., Wang, S., & Yan, Y. (2011). Isomerization and biodegradation of beta- cypermethrin by Pseudomonas aeruginosa CH7 with biosurfactant production. Bioresource technology, 102(14), 7139-7146.
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Cabral, J. R. P., & Galendo, D. (1990). Carcinogenicity study of the pesticide fenvalerate in mice. Cancer letters, 49(1), 13-18.
Dhage, S. S., Chandorkar, A. A., Kumar, R., Srivastava, A., & Gupta, I. (2006). Marine water quality assessment at Mumbai West Coast. Environment international, 32(2), 149 -158.
Diaz, R. J., & Rosenberg, R. (1995). Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanography and marine biology. An annual review, 33, 245-03.
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Ge, J., Cong, J., Sun, Y., Li, G., Zhou, Z., Qian, C., & Liu, F. (2010). Determination of endocrine disrupting chemicals in surface water and industrial wastewater from Beijing, China. Bulletin of environmental contamination and toxicology, 84(4), 401-405.
Hussain, M. (2008). Competitive performance evaluation of waste water treatment plants of karachi and impact of untreated waste water on some edible fishes of Arabian sea.
Lima Neto, I. E., Zhu, D. Z., Rajaratnam, N., Yu, T., Spafford, M., & McEachern, P. (2007). Dissolved oxygen downstream of an effluent outfall in an ice-covered river:
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Natural and artificial aeration. Journal of Environmental Engineering, 133(11), 1051-1060.
Kakko, I., Toimela, T., & Tähti, H. (2004). Oestradiol potentiates the effects of certain pyrethroid compounds in the MCF7 human breast carcinoma cell line. Alternatives to laboratory animals: ATLA, 32(4), 383-390.
Khan, M. A., & Khan, M. A. (2007). The potential of waste stabilization ponds effluent as a liquid fertilizer. Pak. J. Bot, 39(3), 817-829.
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Khan, M. A., Shaukat, S. S., Hashmi, I., & Khan, M. A. (1999). A quantitative study of pollution profile of Karachi coast. In Proceeding Seventh Statistics, Seminar, KU. ISBN-969-8397-06X (pp. 105-119).
Khan, N., Khan, S. H., Amjad, S., Muller, J., Nizamani, S., & Bhanger, M. I. (2010). Organo chlorine pesticides (OCPs) contaminants in sediments from Karachi harbour, Pakistan. Journal of the Chemical Society of Pakistan, 32(4), 542-549.
Koirala, P., Khadka, D. B., & Mishra, A. (2007). Pesticide residues as environmental contaminants in foods in Nepal. The Journal of Agriculture and Environment, 8, 96-100.
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Lemon, R. W. (1967). Laboratory evaluation of malathion, bromophos and fenitrothion for use against beetles infesting stored products. Journal of Stored Products Research, 2(3), 197-210.
Mansoor, A., & Mirza, S. (2007). Waste Disposal and Stream Flow Quantity And Quality of Lyari River. Indus Journal of Management & Social Science (IJMSS), 1(1), 70- 82.
NEQS, National Environmental Quality Standards, S.R.O, 549 (I). (2000). Ministry of Environment, Local Government, and Rural Development, Islamabad, Pakistan.
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Pearce, F. (1997). Sheep dips poison river life.
Pimentel, D. (1995). Amounts of pesticides reaching target pests: environmental impacts and ethics. Journal of Agricultural and environmental Ethics, 8(1), 17-29.
Prashanth, S., Kumar, P., & Mehrotra, I. (2006). Anaerobic degradability: effect of particulate COD. Journal of environmental engineering, 132(4), 488-496. Punjab Private Sector Groundwater Development Project PPSGDP. (2002). Environmental assessment and water quality monitoring program. Irrigation and Power Department, Government of the Punjab, Pakistan Technical Report 54.
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R Asif. (2002). Lyari Expressway: woes of displaced families. Dawn (newspaper). 8 August.
Sankararamakrishnan, N., Sharma, A. K., & Sanghi, R. (2005). Organochlorine and organophosphorous pesticide residues in ground water and surface waters of Kanpur, Uttar Pradesh, India. Environment International, 31(1), 113-120.
Stephenson, R. R. (1982). Aquatic toxicology of cypermethrin. I. Acute toxicity to some freshwater fish and invertebrates in laboratory tests. Aquatic Toxicology, 2(3), 175-185.
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Tallur, P. N., Megadi, V. B., & Ninnekar, H. Z. (2008). Biodegradation of cypermethrin by Micrococcus sp. strain CPN 1. Biodegradation, 19(1), 77-82.
Tariq, M. I., Afzal, S., Hussain, I., & Sultana, N. (2007). Pesticides exposure in Pakistan: a review. Environment international, 33(8), 1107-1122.
Technical bulletin.(2000). Directorate of pest warning and quality control of pesticides, Punjab;
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U.S. EPA. Office of Pesticide Programs. (1988). Pesticides in ground water data base: interim report. Washington, DC.
Vinodhini, R., & Narayanan, M. (2008). Bioaccumulation of heavy metals in organs of fresh water fish Cyprinus carpio (Common carp). International Journal of Environmental Science & Technology, 5(2), 179-182.
Virtue, W. A., & Clayton, J. W. (1997). Sheep dip chemicals and water pollution. Science of the total environment, 194, 207-217.
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Ware, G. W. (2000). The pesticide book. Fresno CA. Thomson Publications. 178- 183.
Yin, K., Lin, Z., & Ke, Z. (2004). Temporal and spatial distribution of dissolved oxygen in the Pearl River Estuary and adjacent coastal waters. Continental Shelf Research, 24(16), 1935-1948.
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Chapter 3
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Isolation and characterization of indigenous bacterial strains of wastewater and PCR based identification through 16S rRNA
3.1. Introduction
The impact of industrialization on the quality of the environment is evident and there is a dire need to have eco-friendly strategies. In recent years biodegradation of pollutants by using microbial strains has increased as humanity strives to find possible ways to sustain contaminated environments (Damalas C. A, 2009; FAO, 2002).
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Jamaluddin et al., (2012) studied that many microorganisms isolated from wastewater are helpful in bioremediation and detoxification of toxic xenobiotics. Bacteria maintain ecological balance for their survival by participating in the carbon, oxygen, and nitrogen cycles.
Shanahan, (2004) reported that bacterial identification is significant in order to determine their interaction in an ecosystem, microbial metabolism, mechanisms of gene/enzyme evolution and efficiency for the detoxification of polluted environments.
Shannon and Unterman, (1993); Galli, (1994); Schmid et al., (2001); Bhadhade et al., (2002); Nwuche and Ugoji, (2008); Pazos et al., (2003); Dua et al., (2002) described that microorganisms detoxify the harmful chemicals by mineralization, transformation, or
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organophosphorus alteration. The first strain and its degradative compounds degrading bacterial enzymes have been reported in 1973 (Siddaramappa et al., 1973).
Microbial population exist with a combination of many other type of cells and separated by pure culture. There are several practical applications available for the identification of unknown bacteria. Molecular practices are current and fastest technology for characterization and identification of microbial flora.
Ogram et al., (1987) stated that it is necessary to study the microbial flora by using the procedures of nucleic acid analysis. Rahmani et al., (2006) suggested that PCR (polymerize chain reaction) is a very useful and rapid method, for detection and identification of bacteria.
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In prokaryotic ribosomes16S ribosomal RNA is a section of the 30S small subunit and are used in reconstructing phylogenic tree. Woese, (1987) proposed that the comparison of 16S rRNA sequence of different microorganisms can show evolutionary connection. Bacterial strains have a conserved portion in the 16S rRNA gene which can be amplified by the universal PCR method. Patel, (2001) reported that sequence of 16S rRNA gene (1,500 bp) is sufficient to transport genetic information and has not altered over time and existing as a operons or multigene family.
The present study deals with isolation, characterization and PCR based identification of bacteria. Five indigenous bacterial strains (Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa, Micrococcus luteus, and Staphylococcus aureus) were
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identified by microscopic examination, biochemical tests and 16S rRNA sequence analysis. 16S rDNA gene fragment of the isolated strain DNA was amplified by PCR. 16sF forward and 16sR reverse primers were used. The sequences were compared in the nucleotide database in GenBank and then aligned to construct a phylogenetic tree. This study suggests that the use of pesticide degrading bacteria which is an eco-friendly technology is suitable for the control of wastewater pollution.
3.2. Material and Methods
Identification of unknown bacteria will be carried out by morphological and biochemical and molecular biological techniques can be seen in Fig. 3.2.
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3.2.1. Sample Collection
For the isolation of water born bacterial strains the wastewater samples were collected aseptically from Lyari River outfall (24°51'59.48"N 66°58'20.16"E) Karachi, Pakistan. The sample was collected in a sterile falcon tube and transferred to the laboratory of Institute of Environmental Studies University of Karachi for further processing.
3.2.2. Bacterial Culture Isolation
Bacterial cultures were isolated by incorporating 1.0 ml wastewater sample in 9.0 ml (10-1) normal saline (8.5 gL-1 NaCl). Further serial dilutions were prepared by transferring
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1.0 ml solution from 10-1 dilution tube to attain dilutions up to 10-6. From the dilutions, 0.1 ml culture sample was transferred onto sterilized nutrient agar plate (Fig. 3.1) and lawn was prepared by spreading with sterilized glass rod and incubated for 24 hours at 37°C. Next day, several colonies were obtained having diverse morphological characteristics. All morphological contrasting colonies were further purified by repeated streaking technique.
3.2.3. Morphological Analysis
Different characteristics of bacterial colony, cell morphology were examined in order to identify the bacterial isolates.
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3.2.4. Colony Morphology
Morphological properties of culture colonies were examined after growing the overnight cultures at 37°C on nutrient agar plates. Color, shape, surface, margin and elevation of culture colonies were analyzed by the method of Holt et al., (1994).
3.2.5. Cell Morphology
Bacterial cell type, shape and arrangement were examined by Gram’s staining method (Gephart et al., 1981). For this purpose, a bacterial culture was mixed with a drop of normal saline (8.5 gL-1 NaCl) on glass slide and spread at the center of the slide. It was
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allowed to air dry and heat fixed. Gram’s staining solutions were used to stain the culture smear (Appendix-1). The slide was observed under microscope.
3.2.6. Culture Identification
Characterization and identification of isolated bacterial strains was performed by the analysis of colonial morphology, biochemical test and 16S r DNA sequence.
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3.2.7. Biochemical Analysis
Different biochemical tests were performed for further identification of bacterial isolates including catalase, oxidase, MR-VP, nitrate reduction, starch hydrolysis and citrate utilization test. Besides all of these tests, selective sugars (glucose, sucrose, lactose, mannitol, maltose and fructose) were also tested for sugar fermentation reaction and gas production. Bacterial growth was also monitored by inoculating the culture in NaCl (7.5%) containing medium and on sabouraud dextrose agar plate. Biochemical tests were carried out in the late-logarithmic phase of the bacterial isolates. (Appendix-2).
3.2.8. Screening of Bacterial strain for pesticide degradation
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In a shaking water bath at 37°C five isolated bacterial strains were grown for 24 hour in MSM (Minimal salt medium) spiked with 50 ppm concentration of malathion and cypermethrin. Malathion and cypermethrin left over in the broth of bacterial cultures were quantified by gas chromatography and percentage of degradation of malathion and cypermethrin by bacterial cultures was monitored. Only the culture Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa showed maximum biodegradation of pesticides were selected for further processing by using molecular technique.
3.2.9. Scanning Electron Microscopy
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The bacterial cells morphology was observed by scanning electron microscope (SEM) images. The micrographs were taken using SEM (JSM 6380A Jeol, Japan).
3.2.10. 16S rDNA Gene Sequence Analysis
Isolated Bacterial strains (Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa) were further identified by molecular analysis.
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3.2.11. Extraction of genomic DNA
Genomic DNA Purification Kit Wizard® was used for the extraction of isolated strains DNA. Quantification of DNA was done by Nano drop spectrophotometer (Nanophotometer P-360 Implen Germany). Confirmation of the presence of the isolated DNA was carried out by the agarose gel 1.0% holding 10.0 mg ml-1 ethidium bromide (2.0 µl) electrophoresis and visualized under Gel Doc system (GelDoc 2000, Bio-Rad Laboratories, USA). Gel electrophoresis was done by using 1x TAB buffer (Appendix-3). Purified DNA was stored at −20°C.
3.2.12. Amplification of bacterial 16S rDNA by PCR (Polymerase Chain Reaction)
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Amplification of DNA was performed by PCR using universal bacterial primer set [16SF: 5′-GAGTTTGATCCTGGCTCAG-3′ and 16SR: 5′-AGAAAGGAGGTATCCAGCC-3′]. Total 50.0 µl PCR reaction mixture containing genomic DNA template 50 ng, Taq polymerase 1.0 U, dNTPs mix 200 µM , PCR reaction buffer 5X, MgCl2 1.5 mM, Primers (2.0 µM forward ; 2.0 μM reverse ) and distilled water were used for DNA amplification. The reaction was carried out in Applied BioSystem GenAmp PCR system-2700 by following three steps of thermal cycling.
The PCR settings were 94°C for 5.0 minutes as initial denaturation temperature, 40 cycles of 94°C for 1.0 minute, 55°C for 1.0 minute and 72°C for 10 minutes as an extension temperature.
3.2.13. Agarose Gel Electrophoresis
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After amplification PCR product was visualized on agarose gel (1.0%) containing 2.0 µl ethidium bromide (10.0 mg ml-1). Gel electrophoresis was performed at 8.0 volts cm-1 for 40.0 minutes and the bands were visualized under UV light in a gel documentation system. 1.0 kb DNA ladder (Fermentas) was used.
3.2.14. Purification and Sequencing of PCR Product
Purification of PCR product was done by purifying kit of Promega Wizard SV Gel and PCR Cleanup System. And then sequenced in forward and reverse directions from Eurofins MWG Operon (USA). UV-trans illuminators (MWR LM-20E) were used for sizing a PCR product from agarose gel.
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3.2.15. Sequence analysis and accession number
The Sequences 16S rDNA of the isolates were BLAST and matched in NCBI Genbank database with the available other species 16S rDNA sequences (www.ncbi.nlm.nih.gov/BLAST/). In this research reported Sequences were submitted in Genbank database (www.ncbi.nlm.nih.gov/Genbank).
3.2.16. Construction of Phylogenetic Tree
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Multiple sequence alignment were performed by Mega 6.0 Clustal-W program were used for the reconstruction of phylogenetic tree using likelihood method with 1000 bootstrap values (Tamura et al., 2013). All sequences were retrieved from NCBI Genbank database.
3.3. Results and Discussion
The strains used for the pesticide degradation were isolated from wastewater of Lyari River Karachi, Pakistan. Initially, serial dilution method was used for the isolation and purification of indigenous bacteria after the pure culture study. Only the predominant microbial flora were selected for further identification and processing. The isolated strains were identified by morphological and biochemical characteristics. Further identification was performed by molecular analysis.
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3.3.1. Culture Identification
The colonial characteristics, growth pattern of isolated strain are presented in Table.3.1 and Fig. 3.3, 3.4, 3.5, 3.6 and 3.7). Colonial morphology of Isolate 1 was observed as circular shape, medium size, entire margin, raised elevation, smooth texture, shiny appearance, non-pigmented. Colonial morphology of Isolate 2 was variable and gave the apperenve of mixed culture, coloines are round, whitish and irregular in shape with margins varying from fimbriate to undulate, Colonial morphology of Isolate 3 was small, rough, wrinkled with radient crests, produce diffusible green pigments, Isolate 4 gave the appearance of circular colonies, convex with entire margins, produces a bright yellow, non-diffusible pigment and Isolate 5 gave Short Opaque, smooth, circular – Gray-white colonies.
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Isolate 1, Isolate 2 and Isolate 5 were identified as facultative aerobic bacteria as they survived anywhere in the broth. Isolate 3 and Isolate 4 were obligate aerobic bacteria, it is shown in Fig. 3.3, 3.4, 3.5, 3.6 and3 .7.
Microscopic illustration of bacterial isolated are presented in Fig.3.8 Isolate 1 and 3 show its characteristic as gram negative mean while isolate 2, 4 and 5 were gram positive. According to microscopic characteristics, the isolates were rods and coccus with scattered and clusters arrangements.
Bacterial cells morphology was also observed by scanning electron microscope (SEM) and images presented in Fig. 3.9.
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3.3.2. Biochemical Analysis
Five predominant culture were collected and subjected to biochemical tests. The results of biochemical tests are given in Table 3.1 and Fig. 3.3, 3.4, 3.5, 3.6 and 3.7. Isolated strains confirmation were done by following bergy’s manual of bacteriology.
Five bacterial strains were identified by morphological, colonial and biochemical characteristics. They were presented in Table 3.2 and were Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa, Micrococcus luteus, and Staphylococcus aureus.
3.3.3. Screening of Bacterial strain for pesticide degradation
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All five isolates Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa, Micrococcus luteus, and Staphylococcus aureus were selected for screening process of pesticide degradation. Among them Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa achieved 90- 99% malathion and cypermethrin degradation within 24 hour (see in Chapter 5). Efficient bacterial cultures (Escherichia coli IES-01, Bacillus licheniformis IES-02, Pseudomonas aeruginosa IES-03) with the highest degradation rate were selected for subsequent studies on biodegradation of malathion and cypermethrin.
3.3.4. Molecular characterization of isolated bacterial strains
The identification of pesticide degrading bacteria Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa was done by molecular phylogeny.
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Sacchi et al., (2002) studied that the 16S rRNA gene sequence has been extensively use to identify an unknown bacterium to the genus or species level. Amann et al., (1995) reported that l 16S rRNA gene sequence served as an ultimate genetic technique.
3.3.4.1. DNA Extraction and Quantification
DNA yield determined by two techniques including agarose gel electrophoresis and optical density (absorbance). The extracted purified genomic bacteria isolates were shown in Fig. 3.10. DNA sample concentration and purity was determined presented in
Table 3.4. DNA having A260/A280 ratio of 1.7–2.0 regarded as good-quality. The extracted
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purified genomic DNA of bacterial isolates were load on 1% agarose gel shows the band of isolated DNA which where visualized under UV illumination with a gel documentation system (BioRad, USA).
3.3.4.2. 16S rDNA PCR
For the 16S rDNA approximately 1500bp gene fragment were amplified by using 16SrDNA universal primer it can be seen in (Fig. 3.11).
3.3.4.3. GenBank Deposition
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Isolates obtained GenBank accession numbers presented in (Table. 3.5), Bacillus licheniformis strain were regarded as IES-01 KU593481, Escherichia coli strain was given the name IES-02 KU593482 and Pseudomonas aeruginosa strain IES-03 represented as KU593483. The sequences were aligned to construct a phylogenetic tree. Sequence available in (Appendix-4).
3.3.4.4. Phylogenic dendogram
The phylogenetic tree of Bacillus licheniformis strain IES-01 KU593481 can be seen in Fig. 3.12. The isolated strains are closely related and belong to the family of Bacillaceae. The phylogenetic tree of Escherichia coli strain IES-02 KU593482 can be seen in Fig. 3.13. The isolated strain was closely related and belong to the family of Enterobacteriaceae.
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The phylogenetic tree of Pseudomonas aeruginosa strain IES-03 KU593483can be seen in Fig. 3.14 and it was belong to the family of Pseudomonadaceae.
The dendogram analysis were based on the partially sequence of 16S rDNA. The multiple sequence alignment showed that the grouping in isolated sequence was due to the nucleotide substitutions in the DNA sequences, and these substitutions evenly distributed in the DNA sequences. Taxonomy of bacterial isolates presented n Table 3.3.
3.4. Conclusion
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Five bacterial isolates Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa, Micrococcus luteus, and Staphylococcus aureus were purified and identified in this this study. Screening of bacterial strain for pesticide degradation ability was monitored by gas chromatography analysis. Five of three isolates Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa were more efficient and possessed high degradation ability to degrade almost 99% malathion and cypermethrin and selected for molecular analysis and further study.
Isolates obtained GenBank accession numbers, Bacillus licheniformis strain IES-01 KU593481, Escherichia coli strain IES-02 KU593482and Pseudomonas aeruginosa strain IES-03 KU593483. The sequences were aligned to construct a phylogenetic tree.
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Pesticide degrading bacterial strains was successfully isolated and identified up to Species level and suggested to use for suitable eco-friendly technologies of wastewater pollution controlling programs.
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Figures
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Figure. 3.1. Bacterial cultures isolated by serial dilutions
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Figure. 3.2. I MICROBIAL FLORA identification of unknown bacteria by morphological, biochemical and molecular techniques
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Figure. 3.3 Morphological and biochemical identification of Escherichia coli
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Figure. 3.4 Morphological and biochemical identification of Bacillus licheniformis
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Figure. 3.5 Morphological and biochemical identification of Pseudomonas aeruginosa
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Figure. 3.6 Morphological and biochemical identification of Micrococcus luteus
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Figure. 3.7 Morphological and biochemical identification of Staphylococcus aureus
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Figure. 3.8 Gram staining under microscope A. Escherichia coli, B. Bacillus licheniformis, C. Pseudomonas aeruginosa, D. Micrococcus luteus, E. Staphylococcus aureu
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Figure. 3.9 Scanning Electron Microscopy images of isolated bacteria
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Figure. 3.10. Ethidium bromide stained agarose gel electrophoresis of extracted DNA M: Marker 1.0 kb, 1500bp Lane 1: Escherichia coli Lane 2: Bacillus licheniformis Lane 3: Pseudomonas aeruginosa Lane 4: Negative
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Figure. 3.11 Ethidium bromide stained agarose gel electrophoresis of purified PCR product for 16S rDNA analysis M: Marker 1.0 kb, 1500bp Lane 1: Escherichia coli Lane 2: Bacillus licheniformis Lane 3: Pseudomonas aeruginosa Lane 4: Positive Lane 5: Negative
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Figure. 3.12 The evolutionary tree was reconstructed by neighbour-joining with bootstrap (1000 replications) based on the 16s rDNA sequence of Bacillus licheniformis (Purple and Bold). The sequences were compared with the other homologous region of 16s rDNA of Bacillus licheniformis were retrieved from NCBI data base
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Figure. 3.13 The evolutionary tree was reconstructed by neighbour-joining with bootstrap (1000 replications) based on the 16s rDNA sequence of Escherichia coli (Blue and Bold). The sequences were compared with the other homologous region of 16s rDNA of Escherichia coli were retrieved from NCBI data base
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Identification Tests Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Gram reaction - + - + +
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Figure. 3.14 The evolutionary tree was reconstructed by neighbour-joining with bootstrap (1000 replications) based on the 16s rDNA sequence of Pseudomonas aeruginosa (Green and Bold). The sequences were compared with the other homologous region of 16s rDNA of Pseudomonas aeruginosa were retrieved from NCBI data base
Tables
Table. 3.1 General growth characteristics and biochemical properties of isolated bacterial strain
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Colony morphology Rods Rods Rods Coccus Coccus Tetrads, Single Pair, chains Single Clusters Arrangement clusters Pigmentation - - Blue, green Yellow - Oxygen requirement FA** FA** OA*** OA*** FA** Temperature 20-45°C 20-45°C 20-45°C 20-45°C 20-45°C requirement Motility + + + - - Catalase + + + + + Oxidase - + + + - Citrate - + + + + Nitrate + + + - + MR + + - - + VP - - - - + Starch hydrolysis - + - - -v Growth on 7.5% NaCl - + + + + Growth on SDA* N/A + N/A N/A N/A Glucose AG A A A AG
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Succrose AG A - - A Lactose AG A - - A Manitol AG A A A A Maltose AG A A A A Fructose AG A - A A Sabouraud dextrose agar ** Facultative anaerobe *** obligate aerobe A: acid production; AG: acid gas production
Table. 3.2 Isolated bacterial strains Isolates Identified cultures
Isolate 1 Escherichia coli
Isolate 2 Bacillus licheniformis
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Isolate 3 Pseudomonas aeruginosa
Isolate 4 Micrococcus luteus
Isolate 5 Staphylococcus aureus
Table. 3. 3 Taxonomy of bacterial isolates
Bacillus Pseudomonas Micrococcus Staphylococcus Classification E.coli licheniformis aeruginosa luteus aureus Kingdom Bacteria Bacteria Bacteria Eubacteria Eubacteria Phylum Proteobacteria Firmicutes Proteobacteria Actinobacteria Firmicutes Gamma Gamma Class Bacilli Actinomycetales Coccus Proteobacteria Proteobacteria
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Order Enterobacteriales Bacillales Pseudomonadales Actinomycetales Bacillales Family Enterobacteriaceae Bacillaceae Pseudomonadaceae Micrococcaceae Staphylococcaceae Genus Escherichia Bacillus Pseudomonas Micrococcus Staphylococcus B. Species E. coli P. aeruginosa M. luteus S. aureus licheniformis
Table. 3.4 Quantification of DNA Sample ID Gram DNA Other DNA Staining Purity Contaminants Concentration 260/280 260/230 ng/µl Bacillus licheniformis Positive 1.9 2.206 185
Escherichia coli Negative 1.9 2.568 369 Pseudomonas aeruginosa Negative 1.9 2.128 400
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Table. 3. 5 Strains ID, isolation sources and GenBank accession numbers of isolated bacteria
Genbank Sequence Accession Isolation- Strain ID number Organism source ID
SEQ1 KU593481 Bacillus licheniformis Wastewater IES-01
SEQ2 KU593482 Escherichia coli Wastewater IES-02
SEQ3 KU593483 Pseudomonas aeruginosa Wastewater IES-03 3.5. R
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Shannon, M. J., & Unterman, R. (1993). Evaluating bioremediation: distinguishing fact from fiction. Annual Reviews in Microbiology, 47(1), 715-736.
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Siddaramappa, R., Rajaram, K. P., & Sethunathan, N. (1973). Degradation of parathion by bacteria isolated from flooded soil. Applied microbiology, 26(6), 846-849.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution, 30(12), 2725-2729.
Woese, C. R. (1987). Bacterial evolution. Microbiological reviews, 51(2), 221.
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3.6. APPENDIX
3.6.1. Appendix-1
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Crystal Violet Crystal violet (1.0 g), dissolved in 10.0 ml ethanol (95%)
Ammonium oxalate (0.4 g), dissolved in 40.0 ml double deionized water
Both chemical solutions mixed together.
Gram’s Iodine Iodine crystal (1.0 g)
Potassium iodide (2.0 g)
Both chemical solutions were incorporated in 300.0 ml double deionized water and left for overnight to prepare homogenous solution.
Safranin Solution Stock Solution (10×)
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Safranin (2.5 g) was dissolved in 100.0 ml ethanol (95%).
Working Solution (1×)
10.0 ml Stock solution was mixed with 90.0 ml double deionized water.
Decolorizing Solution Ethanol (95%) was employed as a decolorizing solution.
3.6.2. Appendix-2
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Catalase Hydrogen peroxide 3%
Oxidase Strip method
Sugar fermentation Yeast extract (0.3gm)
Peptone (0.5 gm)
Sugar (1.0gm)
Phenol red (200 µl)
Deionized water (volume makeup 100ml)
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pH + 7.2
Phenol red dye Dissolve (0.1gm) of Phenol red in 95% ethyl alcohol (400 ml
Deionized water (volume makeup 500ml)
MR-VP test CLARK MEDIUM
Peptone (0.5gm)
Glucose (0.5 gm)
Dipotassium hydrogen phosphate (0.5gm)
Deionized water (volume makeup 100ml)
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pH + 7.2
Methyl red pH indicator Methyl red (0.1 gm)
95 % Ethanol (300 ml)
Deionized water (volume makeup 500ml)
VP Barritt’s reagents Solution A
α - nephtol (0.5gm)
95 % Ethanol (10 ml)
Solution B
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Potassium hydroxide (4gm)
Deionized water (volume makeup 10ml)
Nitrate broth Peptone (0.5 gm)
Yeast extract (0.3 gm)
Potassium nitrate (0.1gm)
Deionized water (volume makeup 10oml)
Nitrate test solution solution A
Sufanilic acid (8.0gm)
Acetic acid (1L)
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Nitrate test solution solution B
Alpha naphtyleamine (5.0gm)
Acetic acid (1L)
Citrate agar 23gm/L
Add 1 gm agar agar in (50ml) citrate agar
Starch agar Nutrient broth (1.3 gm)
Starch (0.5 gm)
Agar agar (2.4 gm)
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Deionized water (volume makeup 100ml) pH + 7.2
NaCl broth 1.3 gm nutrient broth
7.5 gm NaCl
Deionized water (volume makeup 100ml)
Sabouraud Dextrose Agar SDA Peptone (1.0 gm)
Glucose (2gm)
Agar Agar (2.4 gm)
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Deionized water (volume makeup 100ml) pH + 5.6
Nutrient broth Nutrient broth (1.3 gm)
Deionized water (volume makeup 100ml) pH + 7.2
Nutrient agar Nutrient broth (1.3 gm)
Agar Agar (2.4 gm)
Deionized water (volume makeup 100ml)
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pH + 7.2
Luria Bertani Broth (LB Broth) Yeast extract (5gm)
Tryptone (10gm)
NaCl (10gm)
Distilled H2O (1L)
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3.6.3. Appendix-3
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50x TAE BUFFER STOCK 1Litre (pH + 8.0)
Tris (hydroxymethyl) – aminomethane 121.1 MW (242gm)
Glacial acetic acid (57.1 ml)
EDTA 0.5 M (100 ml)
1x TAE BUFFER STOCK 1Litre (pH + 8.0)
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50x TAE BUFFER STOCK 1Litre (20ml)
Deionized water (980ml)
1% AGAROSE GEL/1x TBE (100ml)
Agarose 1g)
1x TBE buffer (100ml)
Microwave on high for ~2min. or until clear and slightly bubbling. Cool slightly.
10mg/ml Ethidium Bromide (5µ)l
Pour into a gel tray and remove any bubbles. Let solidify 20min.
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3.6.4. Appendix-4
>IES-01 Bacillus licheniformis strain IES-01 16S ribosomal RNA gene Sirajuddin,S., Khan,M.A. and Siddiqui,N.
Sequence ID Accession number Organism Isolation-source
SEQ1 KU593481 Bacillus. licheniformis Wastewater
TCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGT AAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGT GGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGA TGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG TAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAG CTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCT
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AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAG GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGACTGC
>IES-02 Escherichia coli strain IES-02 16S ribosomal RNA gene Sirajuddin,S., Khan,M.A. and Siddiqui,N.
Sequence ID Accession number Organism Isolation-source
SEQ2 KU593482 Escherichia. coli Wastewater
ATGCAAGTCGAATGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGA AACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGG
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ACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGAC GATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCA GTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAA GTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCT AACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCA GGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTC GTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGG CCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGAT
>IES-03 Pseudomonas aeruginosa strain IES-03 16S ribosomal RNA gene
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Sirajuddin,S., Khan,M.A. and Siddiqui,N.
Sequence ID Accession number Organism Isolation-source
SEQ3 KU593483 Pseudomonas. aeruginosa Wastewater
GCCGATGGCAACAGCCTACACATGCAGTCGAGCGGATGAAGGGAGCTTGCTCCTGGATTCAGCGGCGGACGGGTG AGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTGAGG GAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGG CCTACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGG TCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAG AATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCG TAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTAC TGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCA GTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACC CTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCGATAA GTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGC
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ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGT GCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTA ACGAGCGCAACCCTTGTCCTTAGTAACTAGCACTTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAG GAAGGTGGGGATGAC
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Chapter 4
Growth Kinetics Studies of Isolated Bacterial Culture
4.1. Introduction
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Microbial growth is the dynamic expressions of bacterial life and a relationship between the microbial population and the substrate. The principle of growth kinetics was established in the 1940s and several microbial growth and biodegradation kinetic models have been developed, proposed and used in bioremediation processes to establish the relationships between growth and environmental factors. (Monod 1942, 1949, 1950; Hinshelwood 1946; van Niel 1949; Novick and Szilard 1950; Herbert et al., 1956; Ma´lek 1958; Pfenning and Jannasch 1962; Fencl 1963; Pirt 1965, 1975; Powell et al., 1967; Tempest 1969).
Mateles, (1969); Baltiz, (1996); Ordaz, (2009); Arthur, (2011) reported that study of microbial growth kinetics has been successfully used in optimizing wastewater treatment practices. Different direct and indirect methods are available to predict microbial cell growth. The most well-known methods are observing and counting the
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bacteria, measuring wet or dry mass, and measuring turbidity. Monon, (2012) and Nassif, (2004) reported that several factors affecting bacterial growth rate including, growth medium, temperature, pH and so on. Bacteria are usually grown in batch samples that represent a controlled environment. The growth curve plotted between the logarithms of the number of cells and the time characterized by a succession of growth phases including Lag, Logarithmic, stationary and decline phase. The time required during the exponential growth phase for doubling the initial bacterial population known as generation time (g). (Delignette-Muller, M. L., 1998). Previous reports revealed that the microbial degradation process detoxifies the pesticide contamination and successfully applied to overcome the pollution (Bhadhade et al., 2002).
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Studies have shown that pesticides are being degraded by a wide variety of microorganisms i.e. E.coli (Richins et al., 1997; Langlois et al., 1970; Chen and Mulchandani 1998; Sing and Seth 1989), Pseudomonas( Atit et al., 2013; Muragesan et al. 2010; Cycoń et al., 2009; Jilani and Altat Khan 2006; Hashmi, 2004; Jilani and Althaf Khan, 2004; Deshpande et al., 2001; Nawab et al., 2003; Maria et al., 2002), Bacillus (Yang et al., 2007; Anwar et al., 2009; Langlois et al., 1970; Awasthi et al., 2003; Rivera 1999; Munnecke, 1976; ANG et al.,2008; Zhu and Qiu, 2010; Mandal et al., 2005;; Langlois et al., 1970).
The present study aims at demonstrating the biodegradation potential of metabolically versatile bacterial species namely Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis isolated from Lyari River their growth kinetics studies were performed under varying concentration of malathion and cypermethrin in minimal salt media.
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4.1.1 Pesticides used in the study
The pesticide selected the current study belongs to the class Organophosphate and Pyrethroid, commercially available as malathion and cypermethrin Fig. 4.1.
Malathion, is an organophosphate extensively used to control a wide range of insect. (Bjørling-Poulsen, 2008). Cypermethrin, is a pyrethroid widely used in public health, homes and agriculture (Lin QSet al., 2011). These two pesticides upon inhalation, ingestion and through skin and eyes are highly toxic for human. These compounds are toxic and carcinogenic in nature even at low concentrations. (Anonymous, 1987). These
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pesticides also used as bioresistant compounds, which are nondegradeable during water and wastewater treatment (Bending and Cruz, 2007).
4.2. Material and methods
4.2.1. Chemicals and reagents
Analytical grade chemicals and reagents were used in the study. Malathion - 98% and Cypermethrin - 95.8% (AccuStandard, USA) were obtained from FQSRI, Pakistan
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Agricultural Research Council, University of Karachi. Analytical grade n-hexane was purchased from Sigma-Aldrich, USA.
4.2.2. Preparation of stock and working standards from analytical grade pesticides
Because of low aqueous solubility of pesticides, a stock solution of malathion (2940ppm) and cypermethrin (2874ppm) were prepared by using 0.1500 gm of malathion (98%) and 0.1500 gm of cypermethrin (95.8%) in 30 ml analytical grade n- hexane sonicated for 2 minutes and the volume was made up to 50ml. working standards was prepared form the stock solution. Table. 4.1
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4.2.3. Method validation
The stock solution was used to prepare different concentrations of each pesticides. These dilutions (2, 10, 50, 100, 200 and 300ppm) were then used to plot standard calibration curve (Fig. 4.5) by Agilent 6890N GC system with an ECD (electron capture detector), with an capillary column HP-5MS (0.25mm, 30m, 0.25µm) Fig. 4.2 .The temperatures applied were 280°C and 320°C for the injector and detector. The injector mode was splitless and injector volume was 0. 5µl. The temperature of oven was held at
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80°C for 1min and then raised at 5°C/min until it reaches to 280°C. Nitrogen gas flow rate was 0.8 ml/min.
4.2.4. Minimal Salt Medium (MSM)
The minimal salt medium was prepared by the protocol of Sambrook et al., (1989). The medium contained 5 x M9 salts autoclaved (1L) containing KH2PO4 15g, Na2HPO4.7H2O
64g, NaCl 2.5g, NH4Cl 5g, pH 7.2. Minimal growth medium (1L) consists of 5 x M9 salts (200 ml), 20 ml of 20% carbon source 0.2 m filter sterilized (D-Glucose for control,
Pesticide for test), 2 ml autoclaved 1 M MgSO4 and 0.1 ml autoclaved 1 M CaCl2. The medium were used for the growth kinetics studies.
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4.2.5. Bacterial Culture
Bacterial culture (Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis) were isolated from wastewater of Lyari River capable for degrading malathion and cypermethrin and were used for growth kinetics studies. Fig. 4.3.
4.2.6. Preparation of Inoculum
Inoculum was prepared in three steps:
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1. Inoculum was prepared by loopful of bacterial culture inoculated into 2.5 ml nutrient broth tube (Merck) and incubated on shaker at 160 rpm at 37 °C for 24 hours. After incubation period optical density at 600nm was recorded. 2. 100 µl (6 x 108 CFU/ml McFarland’s Index) of 24 hour grown culture was inoculated into 2.5 ml minimal salt media tubes fortified with different concentration of cypermethrin and malathion (0.1, 0.5, 2.0, 50 ppm) and one tube was used as blank in which glucose was added and incubated on an orbital shaker at 160 rpm for 24hr at 37 °C. Table 4.2 3. 2.5 ml of 24 h grown culture was inoculated into 22.5 ml of minimal salt media flask supplemented with different concentration of malathion and cypermethrin (0.1, 0.5, 2.0, 50 ppm) and one served as blank in which glucose added and incubated for 24 hours at 37 °C. Table 4.3
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4.2.7. Growth kinetic studies of pesticide degrading bacteria
25ml of inoculum was transferred in 225ml of minimal salt medium flasks with different concentrations of malathion and cypermethrin (0.1, 0.5, 2.0, 50 ppm) and one served as blank in which glucose was added and incubated in a shaking water bath at 37 °C (Thermo electron corporation model no. 2873, USA) Fig. 4.4 with the speed of 120 rpm for 24hours. Samples were collected after one hour interval, and bacterial growth was assessed by using UV – spectrophotometer (Kanza Max Biochemis Try, Biolebo, France) at 600nm. Generation time (g) and specific growth were calculated from growth curve. Table 4.4.
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4.3. Results and Discussion
4.3.1. Validation of analytical procedure
Analytical procedure was validated by linearity to obtain test results of peak area as mentioned in (Table 4.5.) which are equivalent to the concentration of analyte in the sample. Concentration of each pesticide shows a peak-to-peak noise ≥ 3 with respect to the baseline, was considered as LOD and the concentration giving a peak-to-peak noise ≥ 10 with respect to the baseline was considered as LOQ.
4.3.2. Growth kinetics Studies
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In growth kinetics study, 25ml of 24 h grown culture were inoculated on to the 225ml of minimal salt medium flasks and incubated under optimal growth conditions for the measurement of turbidity by using the spectrophotometer. The degree of turbidity directly associated to the number of cells, either viable or dead cells. Bacterial cell mass and cell size increased with increasing the turbidity during the growth cycle and affected by physical factors include the pH, temperature, and Nutritional factors. The bacterial growth studied by plotting the sigmoid curve between log of cell number and the incubation time.
The potential pesticide degrader (Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis) were selected to evaluate its growth kinetics under varying concentration of malathion and cypermethrin (0.1, 0.5, 2.0, 50 ppm) in minimal salt media using
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shaking water bath. Such studies have been conducted and reported by several other researcher (Karpouzas and Walker, 2000; Haugland et al., 1990; Maria et al., 2002; Jilani and Khan, 2004; Hashmi, 2004).
4.3.3 Growth kinetics of Bacterial cultures in plain minimal salt medium (Control)
A control test was performed in order to determine the degradation efficiency of isolated strain when exposed to altered concentration of malathion and cypermethrin. Bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis were grown in minimal salt medium without adding pesticide at 37°C in shaking water bath and optical density at 600nm of cultures were measured at regular interval of hour. Results of optical density and log of O.D were measured and represented in (Table 4.6, 4.7, 4.8). At zero time (immediately after inoculation) the optical density of MSM was (-
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1.966, -1.921 and -0.903) in respect of (Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis). Growth of bacterial cultures occurred in MSM up to 6 hours from Escherichia coli, 8 hours for Pseudomonas aeruginosa and 10 hours for Bacillus licheniformis as reflected by increase in log of O.D (Fig 4.9). This indicates that glucose was used as a carbon source.
Bacterial strain first, entered into the Lag phase required some time for adaptation. In this phase breakdown of pesticide was accelerated with increasing cells size and decreasing cell mass. The duration of the lag phase is dependent on the initial inoculum size and the type of medium (Maier, R et al., 2009).
After the lag phase, the bacterial strain were in a rapidly growing and dividing state entered into the Log phase. In this phase bacterial metabolic activity increased and
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replication of DNA was occurred at a constant rate. The growth medium was also consumed and the number of bacteria increased logarithmically. During the log phase bacteria specific growth rate and generation time were calculated by this equation; g=t/n. Generation times of Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis in log phase were 40.9 min, 57.86 min, 117.4 min respectively and presented in (Table 4.9). Growth of bacterial population was continuous until all of the nutrient were depleted with the accumulation of metabolic waste materials that makes unfavorable environment leads to the Death phase.
Yates and Smotzer, (2007) explain that the lag phase is the transition to the exponential phase after the initial population has doubled. It can be understood from (Table. 4.6 and Fig. 4.6) the phase of adaptation of Escherichia coli continued up to 1h, However a
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significant increase in the optical density occurred during 6 hours of incubation, demonstrating that the bacterial culture remains in lag phase for the duration of 1 hour and then moved into Log phase and then achieved stationary phase after 6 hours. Generation time 40.90 min and specific growth rate of 0.024 was calculated. From Table. 4.7 and Fig. 4.7 it may be seen that the phase of acclimatization of Pseudomonas aeruginosa significantly increased up to 8 hours of incubation period. Mean generation time and specific growth rate at 8 h were calculated as 57.86 min and 0.017 respectively. From Table.4.8 and Fig. 4.8 it is seen that the phase of acclimatization of Bacillus licheniformis significantly increased up to 10 hours of incubation period. Mean generation time and specific growth rate at 10 h were calculated as 117.4 min and 0.009respectively.
4.3.4. Growth kinetics studies supplemented with different concentration of Malathion
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Analytical grade Malathion in the range (0.1. 0.5, 2.0, 50 ppm) was used to determine the growth response of Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis.
4.3.4.1. Escherichia coli
The growth response of E.coli were observed when exposed to different concentration (0.1. 0.5, 2.0, 50 ppm) of malathion in mineral salt medium that can be seen in Table 4.4. Results of optical density and log of O.D were measured and represented in (Table 4.10, 4.11, 4.12, 4.13). The growth pattern is shown in Fig. 4.10, 4.11, 4.12, 4.13 and
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cumulative results are presented in Fig. 4.14. Generation time and specific growth rate are recorded in Table 4.14.
After inoculation of 25 ml culture into 225 fresh minimal medium with different concentration of malathion (0.1. 0.5, 2.0, 50 ppm) E.coli entered into adaptability phase without increase in O.D600nm because bacteria did not immediately reproduce in a new medium. During the process of adaptation, the bacteria entered into stressed phase and the growth slowed down. E. coli changed rod shaped morphology into coccus because of increased in malathion concentration (Scanning electron microscopy images presented in Fig. 4.15). However, this change was interim, and cells changed into an original short rod form after adapting time period.
An inverse relationship between pesticide concentration and the bacterial growth was recorded. At low concentrations of malathion (0.1, 0.5 ppm), the adaptation time period
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of bacteria was about 1 hour as the concentration increased (2.0,50 ppm) the bacterial adaptation time period was also increased about 2 hours. When compared with the control test (grown without pesticide), the growth pattern was different as in control sample bacteria required 0.5 hour of adaptation time for growth.
Near the end of the lag phase, the cells double in size in preparation for division. After lag phase bacterial cells entered into the log phase of growth, balanced growth pattern was seen in this phase. Primary metabolites (like amino acids, nucleotides, nucleic acids, lipids, carbohydrates, were produced in this phase. A significant reduction in log of
O.D600nm was observed after 6h of incubation at 0.1, 0.5, 2.0 ppm malathion dose proved to be inhibitory for E.coli. When compared to the control test the log of O.D600nm at 6 hours was 0.698 and the log of O.D600nm at 6 hours with different concentration
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(0.1. 0.5, 2.0, ppm) of malathion was (0.719, 0.529, 0.399) the generation time calculated was 60 min with 0.1ppm malathion dose, 68 min with 0.5ppm of malathion dose, 74.5 min with 2.0ppm of malathion dose and 91.37 min with 50ppm of malathion dose after 8 hours of inoculation.
As the malathion concentration (0.1. 0.5, 2.0, 50 ppm) increased the generation time (g) was also increased and the specific growth rate decreased. The result of the study indicated that malathion concentration in the range of 50 ppm stimulate the growth of
E.coli, however a marked reduction in log of O.D600nm at 8 h was noted with generation time 74.5 min. This indicates that the bacterial enzymes at high concentration suppressed and the growth rate thus decreased see Table 4.14.
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The result of the analysis indicated that malathion concentration in the range of 0.1, 0.5 and 2.0ppm stimulate the growth of E. coli however, a marked reduction in optical density noted when 50ppm malathion dose was used with generation time of 91.37 min. This indicates that bacterial enzyme at high concentration suppressed with decrease in growth rate. It was further noted that E.coli grows faster at low concentration of malathion. However, at higher concentration, the growth of E. coli significantly decreased or increased very slightly during 24 hours of incubation, when compared with control tests. This implies that the suitable degrading enzymes may be suppressed at high concentration and bacteria required an acclimation period to induce the necessary degradative enzymes because of this reason the prolonged lag phase observed at high concentration of pesticide. Sing and Seth, (1989) reported similar
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findings and found significant decreased in the rate of degradation and growth of microorganisms at 200ppm malathion concentration.
4.3.4.2. Pseudomonas aeruginosa
The growth response of Pseudomonas aeruginosa were observed when exposed to different concentration (0.1. 0.5, 2.0, 50 ppm) of malathion and can be seen in Table 4.4. Results of optical density and log of O.D were measured and represented in (Table 4.15, 4.16, 4.17, 4.18). The growth pattern are shown in Fig. 4.16, 4.17, 4.18, 4.19 and cumulative results presented in Fig. 4.20. Generation time and specific growth rate are recorded in Table 4.19.
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After inoculation into fresh minimal medium Pseudomonas aeruginosa entered into adaptability phase with slight increase in O.D600nm because the organism did not immediately reproduce in a new medium entered into stressed phase and the growth slower down. Pseudomonas aeruginosa changed rod shaped morphology into coccus probably of increased in malathion concentration (Scanning electron microscopy images presented in Fig. 4.21). However, this change was interim, and cells changed into an original rod shaped form after adapting time period. An inverse relationship between pesticide concentration and the bacterial growth was recorded. At low concentrations of malathion (0.1, 0.5, 2.0ppm), the adaptation time period of bacteria was about 0.5 hour as the concentration increased (50 ppm) the adaptation time period was increased about 2 hours where Pseudomonas aeruginosa cells grew slowly. When compared with the
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control test (grown without pesticide), the growth pattern was different as in control sample bacteria does not required extra adaptation time for growth.
Cellular metabolism was accelerated near the end of lag phase. After lag phase bacterial cells entered into the log phase of growth. Balanced growth pattern was seen in this phase. Primary metabolites (like amino acids, nucleotides, nucleic acids, lipids, carbohydrates, were produced in this phase. A significant reduction in log of O.D600nm was observed after 8hours of incubation at 0.1, 0.5, 2.0, 50 ppm malathion concentration that may be bacterial inhibitory for Pseudomonas aeruginosa. When compared to the control test the log of O.D600nm at 8 hours was 0.576 and the log of
O.D600nm at 8 hours with different concentration (0.1. 0.5, 2.0, 50ppm) of malathion was (0.468, 0.308, 0.328, 0.398).
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Generation time was calculated to be 64.69 min with 0.1ppm malathion dose, 69.00 min with 0.5ppm of malathion dose 77.41 min with 2.0ppm of malathion dose and 90.56min with 50ppm of malathion dose after 8 hours of inoculation. As the malathion concentration (0.1. 0.5, 2.0, 50 ppm) increased the generation time (g) was also increased and the specific growth rate decreased see Table 4.19.
4.3.4.3. Bacillus licheniformis
The growth response of Bacillus licheniformis were observed when exposed to different concentration (0.1. 0.5, 2.0, 50 ppm) of malathion Table 4.4. Results of optical density
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and log of O.D were measured and represented in (Table 4.20, 4.21, 4.22, 4.23). The growth pattern are shown in Fig. 4.22, 4.23, 4.24, 4.25 and cumulative results presented in Fig. 4.26. Generation time and specific growth rate are recorded in Table 4.24.
After inoculation into fresh minimal medium Bacillus licheniformis cells entered into adaptability phase with slight increase in O.D600nm because bacteria did not immediately reproduce in a new medium entered into stressed phase and the growth slower down. Bacillus licheniformis cells changed rod shaped morphology into coccus probably because of increased in malathion concentration (Scanning electron microscopy images presented in Fig. 4.27. However, this change was temporary, and cells changed into an original rod shaped form after adapting time period. An inverse relationship between pesticide concentration and the bacterial growth was recorded. During this time, the culture used to the new medium and synthesized new enzymes and co-factors for
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metabolic activities (Atlas, 2006b). At low concentrations of malathion (0.1, 0.5, 2.0ppm), the adaptation time period of bacteria was about 0.5 hour as the concentration increased (50 ppm) the bacterial adaptation time period was also increased about 1 hours. In this time period Bacillus licheniformis cells grew slowly. When compared with the control test (grown without pesticide), the growth pattern was different as in control sample bacteria does not required extra adaptation time for growth.
Immediately after the end of the lag phase, the cells double in size in preparation for division. Cellular metabolism was accelerated near the end of lag phase after lag phase bacterial cells entered into the log phase of growth, balanced growth pattern was seen in this phase. Primary metabolites (like amino acids, nucleotides, nucleic acids, lipids, carbohydrates, were produced in this phase. A significant reduction in log of O.D600nm
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was observed after 10 hours of incubation at 0.1, 0.5, 2.0, 50 ppm malathion dose proved to be bacterial inhibitory for
Bacillus licheniformis. When compared to the control test the log of O.D600nm at 10 hours was 0.64 and the log of O.D600nm at 10 hours with different concentration (0.1. 0.5, 2.0, 50ppm) of malathion was 0.32, 0.281, 0.18, 0.059. Generation time calculated was to be 124.7min with 0.1ppm malathion dose, 128.2min with 0.5ppm of malathion dose 137.9min with 2.0ppm of malathion dose and 150.7min with 50ppm of malathion dose after 10 hours of inoculation. As the malathion concentration (0.1. 0.5, 2.0, 50 ppm) increased the generation time (g) was also increased and the specific growth rate decreased as can be seen from Table 4.24.
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Mulla et al., (1981) states that bacteria have the tendency to accumulate, detoxify, or metabolize malathion completely. Rohr Sager et al., (2006) also reported that pesticides exposure may prove directly toxic to microbes such as an initial decrease in a population and certain mutations carry over to successive generations.
Many researchers Williams Robbins et al., (1963); Pratt Bowers et al., (1993); Staley Rohr et al., (2010); Staley, Senkbeil et al., (2012); Staley, Rohr et al., (2011) found that organophosphate pesticides are less toxic to bacterial species as appeared in the present study Malathion was less toxic.
Stanlake and Clark, (1975) were reported that no significant effects on bacteria were observed when exposed to 0.44 µl/g concentration of malathion similarly, Staley, Rohr et al., (2010) demonstrated that malathion at 101 and 202 µg/L did not have any
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significant effect on Escherichia coli and Enterococcus spp. However, malathion 0.5 mg/L inhibited bacterial growth in sludge (Pai, Wang et al., 2009). And also inhibited cyanobacteria permanently at concentrations of 200 mg/L (Torres and O'Flaherty, 1976).
Christie, (1969) detected the non-significant effect of 100 mg/L malathion on the green alga Chlorella pyrenoidosa. Lal R, Lal S, (1988) found partial inhibitory effect on growth of the bluegreen alga Chlorogloea fritschii, and permanently suppressed growth at 200 mg/L concentration of malathion .
4.3.5. Growth kinetics studies supplemented with different concentration of Cypermethrin
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Analytical grade cypermethrin in the range (0.1. 0.5, 2.0, 50 ppm) was used to determine the growth response of Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis.
4.3.5.1. Escherichia coli
E.coli growth response was determined by using different concentration Table 4. 4 (0.1, 0.5, 2.0, 50 ppm) of cypermethrin (Table 4.25, 4.26, 4.27, 4.28). Data in Fig. 4.28, 4.29,
4.30, 4.31 and cumulative results presented in Fig. 4.32 demonstrated the effect of cypermethrin concentrations on the growth of E.coli. An inverse relationship between pesticide concentration and the bacterial growth was recorded. As the cypermethrin
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concentration increased the generation time (g) was also increased and the specific growth rate decreased Table 4.29.
After inoculation E.coli cells entered into the phase of adaptation without increase in
O.D600nm and changed rod shaped morphology into coccus probably because of increased in cypermethrin concentration (Scanning electron microscopy images presented in Fig. 4.15. However, this change was interim, and cells changed into an original rod shaped form after adapting time period. An inverse relationship between pesticide concentration and the bacterial growth was recorded. During this time, the culture get used to the new medium and synthesized new enzymes and co-factors for metabolic activities.
At low concentrations of cypermethrin (0.1, 0.5 ppm), the adaptation time period of E.coli was about 1 hour as the concentration increased (2.0, 50 ppm) the bacterial
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adaptation time period was also increased to about 2 hours in this time period E.coli cells grew slowly. When associated with the control test (grown without pesticide), the growth pattern was different as in control sample where bacteria required 0.5 hour of adaptation time for growth.
It is clear from Table. 4.40 that with malathion, bacteria grows faster than when grows with cypermethrin. Result indicates that E.coli with cypermethrin required more adaptation time as compared to malathion. At higher concentration of cypermethrin (50ppm) the optical density and rate of substrate utilization decreased which prolonged the lag phase. After lag phase bacterial cells entered into the log phase of growth, balanced growth where, pattern was seen A significant reduction in log of O.D600nm was observed after 6hours of incubation at 0.1, 0.5, 2.0, 50 ppm cypermethrin concentration proved to be bacterial inhibitory for E.coli. When compared to the control test the log of
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O.D600nm at 6 hours was 0.698 and the log of O.D600nm at 6 hours with different concentration (0.1. 0.5, 2.0, 50ppm) of cypermethrin was (0.567, 0.4, 0.219, 0.316). The generation time with 0.1ppm of cypermethrin dose was 66min, 0.5ppm of cypermethrin dose was 73.17min, 2.0ppm of cypermethrin dose was 83.91min and 50ppm of cypermethrin dose was 97.95min after 6 hours of inoculation.
The results showed that cypermethrin even at high concentration is relatively harmless to microorganism, however the rate of substrate utilization declined which extended the lag phase. It was further noted that E.coli grow faster at low concentration of pesticide. However at higher concentration, the growth of E.coli significantly decreased or very slightly increased during 24 hours of incubation, when compared with control tests. This implies that the suitable degradative enzymes may be repress at high
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concentration and bacteria required an acclimation period to make the degradative enzymes because of this reason the extended lag phase observed at high concentration of pesticide. Therefore, it can be concluded that the E.coli may play a significant role in pesticide detoxification.
4.3.5.2. Pseudomonas aeruginosa
Pseudomonas aeruginosa growth response was determined by using different concentration (0.1, 0.5, 2.0, 50 ppm) Table 4.4 of cypermethrin (Table 4.30, 4.31, 4.32,
4.33). Data in Fig. 4.33, 4.34, 4.35, 4.36 and cumulative results presented in Fig. 4.37 demonstrated the effect of cypermethrin concentrations on the growth of Pseudomonas aeruginosa. In this case an inverse relationship between pesticide
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concentration and the bacterial growth was recorded. As the cypermethrin concentration increased the generation time (g) was also increased and the specific growth rate decreased Table 4.34.
After inoculation Pseudomonas aeruginosa culture entered into the phase of adaptation without increase in O.D600nm and changed rod shaped morphology into coccus (Scanning electron microscopy images presented in Fig. 4.21) because of increased in cypermethrin concentration. However, this change was interim, and cells changed into an original rod shaped form after adapting time period. An inverse relationship between pesticide concentration and the bacterial growth was recorded. During this
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time, the culture used to the new medium and synthesized new enzymes and co- factors for metabolic activities
(Winn, 2006).
At low concentrations of cypermethrin (0.1, ppm) Pseudomonas aeruginosa does not required extra adaptation time period but where the concentration increased to 0.5, 2.0ppm the adaptation time period was increased about 0.5 hours and with 50 ppm about 1 hour. At this time Pseudomonas aeruginosa cells grew slowly. When correlate with the control test (grown without pesticide) the growth pattern was different as in control sample bacteria does not required extra adaptation time for growth.
Results indicates that Pseudomonas aeruginosa with cypermethrin required more adaptation time as compared to malathion. At higher concentration of cypermethrin
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(50ppm) the optical density and rate of substrate utilization decreased which prolonged the lag phase. After lag phase bacterial cells entered into the log phase of growth, balanced growth pattern was seen in this phase. A significant reduction in log of O.D600nm was observed after 8hours of incubation at 0.1, 0.5, 2.0, 50 ppm cypermethrin dose proved to be bacterial inhibitory for Pseudomonas aeruginosa. When compared to the control test the log of O.D600nm at
8 hours was 0.576 and the log of O.D600nm at 8 hours with different concentration (0.1. 0.5, 2.0, 50ppm) of cypermethrin was (0.388, 0.296, 0.094, 0.218). The generation time with 0.1ppm of cypermethrin dose was 67min, 0.5ppm of cypermethrin dose was 72.3min, 2.0ppm of cypermethrin dose was 81.7min and 50ppm of cypermethrin dose was 96.57min after 8 hours of inoculation.
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The results proved that cypermethrin even at high concentration was relatively less toxic to microorganism, however the rate of substrate utilization decreased which prolonged the lag phase. It was further noted that Pseudomonas aeruginosa grow faster at low concentration of pesticide. However at higher concentration, the growth of Pseudomonas aeruginosa significantly decreased or very slightly increased during 24 hours of incubation, when compared with control tests. This implies that the appropriate catabolic enzymes repress at high concentration and bacteria may need an acclimation period to make the suitable degradative enzymes because of this reason the extended lag phase observed at high concentration of pesticide. Therefore, it can be concluded that the Pseudomonas aeruginosa play a significant role in pesticide detoxification.
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Muragesan et al., (2010) state that P. aeruginosa grow with cypermethrin at 0.1 to 1% concentration. Jilani and Khan, (2006) reported that P. aeruginosa was responsible for the degradation of cypermethrin and lag phase increased with the increase of cypermethrin concentration.
4.3.5.3. Bacillus licheniformis
Bacillus licheniformis growth response was determined by using different concentration (0.1, 0.5, 2.0, 50 ppm) Table 4.4 of cypermethrin (Table 4.35, 4.36, 4.37, 4.38). Data in
Fig. 4.38, 4.39, 4.40, 4.41 and cumulative results presented in Fig.4.42 demonstrated the effect of cypermethrin concentrations on the growth of Bacillus licheniformis. An opposite relationship between pesticide concentration and the bacterial growth was
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recorded. As the cypermethrin concentration increased the generation time (g) was also increased and the specific growth rate decreased Table 4.39.
After inoculation Bacillus licheniformis entered into the phase of adaptation without increase in O.D600nm and changed rod shaped morphology into coccus (Scanning electron microscopy images presented in Fig. 4.27) because of increased in cypermethrin concentration. However, this change was interim, and cells changed into an original rod shaped form after adapting time period. An inverse relationship between pesticide concentration and the bacterial growth was recorded. During this time, the culture used to the new medium and synthesized new enzymes and co-factors for metabolic activities.
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At low concentrations of cypermethrin (0.1, ppm) Bacillus licheniformis does not required extra adaptation time period as the concentration increased (0.5, 2.0ppm) the adaptation time period was increased about 0.5 hours and with (50 ppm) about 1 hour in this time period Bacillus licheniformis cells grew slowly. When associated with the control test (grown without pesticide), the growth pattern was different as in control sample bacteria does not required extra adaptation time for growth.
Results indicates that Bacillus licheniformis with cypermethrin required more adaptation time as compared to malathion. At higher concentration of cypermethrin (50ppm) the optical density and rate of substrate utilization decreased which prolonged the lag phase. After lag phase bacterial cells entered into the log phase of growth, balanced growth pattern was seen in this phase. A significant reduction in log of O.D600nm was
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observed after 10 hours of incubation at 0.1, 0.5, 2.0, 50 ppm cypermethrin dose proved to be bacterial inhibitory for Bacillus licheniformis.
When compared to the control test the log of O.D600nm at 10 hours was 0.64and the log of O.D600nm at 10 hours with different concentration (0.1. 0.5, 2.0, 50ppm) of cypermethrin was (0.271, 0.241, 0.165, 0.042). The generation time with 0.1ppm of cypermethrin dose was 129.0min, 0.5ppm of cypermethrin dose was 132.7min, 2.0ppm of cypermethrin dose was 139.5min and 50ppm of cypermethrin dose was 153 min after 10 hours of inoculation. The results proved that cypermethrin even at high concentration was relatively less toxic to microorganism, however the rate of substrate utilization decreased which prolonged the lag phase.
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It was further noted that for Bacillus licheniformis grow faster at low concentration of pesticide. However at higher concentration, the growth of for Bacillus licheniformis significantly decreased or very slightly increased during 24 hours of incubation, when compared with control tests. This implies that the efficient enzymes repress at high concentration and bacteria may need an acclimation period to induce the necessary degradative enzymes.
Trpton et al., (2003) reported that the bacterial survival and adaptation can offer an effective, economical and eco-friendly clarification for bioremediation. Therefore, it can be concluded that Bacillus licheniformis play a significant role in pesticide detoxification.
Similarly, Binner et al., (1999) reported that cypermethrin had no adverse effect on soil microbes and Murugesan, A. G, et al. (2010) states that Klebsiella Sp, P. aeruginosa and
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E. coli were found dynamic in utilizing 1%cypermethrin whereas, Corynebacterium and Bacillus Sp. were discreetly active in utilizing 0.1% cypermethrin.
Jilani and Altaf Khan, (2004); Ashok and Seth, (1989) reported that the optimum concentration supporting normal bacterial growth was found to be 120 ppm malathion, 60 ppm cypermethrin. Medium with 35mg/L to 50 mg/L concentration of malathion facilitate the bacterial growth while high dose of malathion 200 mg/L inhibited the growth. Medium with 40mg/L to 60 mg/L cypermethrin increased bacterial growth while 80mg/L to 125mg/L significantly decreased the bacterial growth and lag phase increased but no zone of inhibition was observed.
4.4. Conclusion
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It is concluded that malathion and cypermethrin concentration in the range of 0.1, 0.5 and 2.0 ppm stimulate the growth of Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis however, a marked reduction in optical density noted when 50ppm malathion dose was used, this indicates that bacterial enzyme at high concentration suppressed with decrease in growth rate.
The overall findings of this research study suggest that Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis can grow with different concentration of malathion and cypermethrin. However, the toxicity pattern observed is as follows: Cypermethrin >Malathion. From these results it appears that cypermethrin is more toxic than malathion. When comparing the growth of Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis with cypermethrin and malathion, it is cleared from
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results that with malathion, bacteria grows rapidly and with cypermethrin required more adaptation time.
The results proved that malathion and cypermethrin even at high concentration is relatively less toxic to microorganism, however the rate of substrate utilization decreased which prolonged the lag phase.
SEM (Scanning electron microscopy) was used to study the cultural morphology of bacterial isolates. Scanned images reveal that the isolates were rod-shaped cells (Fig. 4.15, 4.21, 4.27). It was also observed by Scanning Electron Microscopy (SEM) that the cells growing in minimal salt medium containing malathion and cypermethrin were changed rod shaped morphology into coccus and changed scattered arrangement into clusters when compare with cells grow in plain minimal salt medium.
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Our studies also corroborating with the findings of Clements and Rohr, (2009); Verro et al., (2009) who found beneficial or adverse effects when microbes exposed to pesticides.
Growth kinetics parameters determined that the complete removal of malathion and cypermethrin would only be possible if an appropriate organism and optimum operating condition be maintained. Bacterial isolate Escherichia coli, Pseudomonas aeruginosa, Bacillus licheniformis are efficient and useful for the treatment of pesticide contaminants in industrial effluent, agriculture waste. Figures
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Figure. 4.1 Chemical structure of Malathion and Cypermethrin
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Figure. 4.2 Preparation of stock and working standards of pesticides
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Figure. 4.3. A. Escherichia coli, B. Pseudomonas aeruginosa, C. Bacillus licheniformis
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Figure. 4.4. Growth kinetics studies of pesticide degrading bacterial culture
1E+10 1.8E+09 B A 9E+09 1.6E+09 R² = 0.9996 8E+09 R² = 0.9992 1.4E+09 7E+09 1.2E+09 6E+09 1.0E+09 5E+09 8.0E+08 4E+09 6.0E+08 3E+09 2E+09 Chromatogrophic Chromatogrophic Area 4.0E+08 1E+09 2.0E+08 0 0.0E+00 0 100 200 300 400 0 100 200 300 400 Cypermethrin Concentration (ppm) Malathion Concentration (ppm)
Figure. 4. 5. Standard calibration curve (a) Malathion (b) Cypermethrin
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1
0.5
0
-0.5 600nm
-1 Log O.D Log -1.5
-2
-2.5
Time (hrs) Figure.4.6. Growth kinetics of E.coli in minimal salt medium (Control)
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1.0
0.5
0.0
-0.5 600nm
-1.0 Log O.D Log -1.5
-2.0
-2.5 Time (hrs)
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Figure. 4.7. Growth kinetics of Pseudomonas aeruginosa in minimal salt medium (Control)
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0.8
0.6
0.4
0.2
600nm 0
-0.2
Log O.D Log -0.4
-0.6
-0.8
-1 Time (hrs)
Figure. 4.8. Growth kinetics of Bacillus licheniformis in minimal salt medium (Control)
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E.coli Pseudomonas. aeruginosa Bacillus. Licheniformis
1
0.5
0
-0.5 Log O.D600nm LogO.D600nm -1
-1.5
-2
-2.5 Time (hrs)
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Figure. 4.9. Cumulative results of growth kinetics studies (Control)
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1 0.8 0.6 0.4 0.2
600nm 0 -0.2
-0.4 Log O.D Log -0.6 -0.8 -1 -1.2 Time (hrs)
Figure. 4.10 Growth kinetics of E.coli in minimal salt medium supplemented with 0.1ppm Malathion
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0.80 0.60 0.40 0.20
0.00 600nm -0.20
-0.40 Log O.D -0.60 -0.80 -1.00 -1.20 Time (hrs)
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Figure. 4.11 Growth kinetics of E.coli in minimal salt medium supplemented with 0.5ppm Malathion
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0.6
0.4
0.2
0
600nm -0.2
-0.4
Log O.D Log -0.6
-0.8
-1
-1.2 Time (hrs)
Figure. 4.12 Growth kinetics of E.coli in minimal salt medium supplemented with 2.0 ppm Malathion
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0.2
0
-0.2
-0.4 600nm
-0.6
-0.8 Log O.D Log
-1
-1.2
-1.4 Time (hrs)
Figure. 4.13 Growth kinetics of E.coli in minimal salt medium supplemented with 50 ppm Malathion
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1 0.1 ppm 0.5 ppm 2.0 ppm 50 ppm
0.5
0
-0.5 Log O.D600nm LogO.D600nm
-1
-1.5 Time (hrs)
Figure. 4.14 Cumulative results of growth kinetics studies of E.coli supplemented with different concentration of Malathion
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Figure. 4.15 A. Scanning electron micrographs of E.coli colonial growth. B. E.coli with malathion C. E.coli with cypermethrin during the phase of adaptation
1
0.5
0 600nm -0.5
Log O.D Log -1
-1.5
-2 Time (hrs)
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Figure. 4.16 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.1ppm Malathion
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0.5
0
-0.5 600nm
-1 Log O.D Log
-1.5
-2 Time (hrs)
Figure. 4.17 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.5ppm Malathion
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0
-0.5 600nm
-1 Log O.D Log
-1.5
-2 Time (hrs)
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Figure. 4.18 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 2.0 ppm Malathion
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0.6
0.4
0.2
0
-0.2 600nm -0.4
-0.6 Log O.D Log -0.8
-1
-1.2
-1.4 Time (hrs)
Figure. 4.19 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 50 ppm Malathion
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1 0.1 ppm 0.5 ppm 2.0 ppm 50 ppm
0.5
0
-0.5 Log O.D600nm LogO.D600nm -1
-1.5
-2 Time (hrs)
Figure. 4.20 Cumulative results of growth kinetics studies of Pseudomonas aeruginosa supplemented with different concentration of Malathion
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Figure. 4.21 A. Scanning electron micrographs of Pseudomonas aeruginosa colonial growth. B. Pseudomonas aeruginosa with malathion C. Pseudomonas aeruginosa with cypermethrin during the phase of adaptation
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0.4
0.2
0
-0.2 600nm -0.4
-0.6 Log O.D Log -0.8
-1
-1.2
-1.4 Time (hrs)
Figure. 4.22 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.1ppm Malathion
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0.2
0
-0.2
600nm -0.4
-0.6
Log O.D Log -0.8
-1
-1.2
-1.4 Time (hrs)
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Figure. 4.23 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.5ppm Malathion
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0.4
0.2
0
-0.2
-0.4 600nm
-0.6
Log O.D Log -0.8
-1
-1.2
-1.4 Time (hrs)
Figure. 4.24 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 2.0 ppm Malathion
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0
-0.2
-0.4
600nm -0.6
-0.8 Log O.D Log
-1
-1.2
-1.4 Time (hrs)
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Figure 4.25 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 50 ppm Malathion
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0.6 0.1 ppm 0.5 ppm 2.0 ppm 50 ppm 0.4
0.2
0
-0.2
-0.4
-0.6
Log O.D600nm L Log O.D600nm -0.8
-1
-1.2
-1.4 Time (hrs)
Figure. 4.26 Cumulative results of growth kinetics studies of Bacillus licheniformis supplemented with different concentration of Malathion
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Figure. 4.27 A. Scanning electron micrographs of Bacillus licheniformis colonial growth. B. Bacillus licheniformis with malathion C. Bacillus licheniformis with cypermethrin during the phase of adaptation
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0.8
0.6
0.4
0.2
0
Log Log -0.2
600nm -0.4
O.D -0.6
-0.8
-1
-1.2 Time (hrs) Figure. 4.28 Growth kinetics of E. coli supplemented with 0.1 ppm Cypermethrin
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0.6
0.4
0.2
0
Log Log -0.2
-0.4 600nm
O.D -0.6
-0.8
-1
-1.2 Time (hrs)
Figure. 4.29 Growth kinetics of E. coli supplemented with 0.5 ppm Cypermethrin
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0.4
0.2
0
-0.2
Log Log -0.4
-0.6600nm
O.D -0.8
-1
-1.2 Time (hrs)
Figure. 4.30 Growth kinetics of E. coli supplemented with 2.0 ppm Cypermethrin
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1
0.5
0 Log Log
-0.5 600nm
O.D -1
-1.5
-2 Time (hrs)
Figure. 4.31 Growth kinetics of E. coli supplemented with 50 ppm Cypermethrin
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1 0.1 ppm 0.5 ppm 2.0 ppm 50 ppm
0.5
0
-0.5 O.D600nm Log O.D600nm -1
-1.5
-2 Time (hrs)
Figure. 4.32 Cumulative results of growth kinetics studies of E. coli supplemented with different concentration of Cypermethrin
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1
0.5
0 Log Log
-0.5 600nm
O.D -1
-1.5
-2 Time (hrs)
Figure. 4.33 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.1ppm Cypermethrin
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0.5
0
-0.5
Log Log 600nm
-1 O.D
-1.5
-2 Time (hrs)
Figure. 4.34 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.5ppm Cypermethrin
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0.2
0
-0.2
-0.4
-0.6 Log Log -0.8
600nm -1
O.D -1.2
-1.4
-1.6
-1.8 Time (hrs)
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Figure. 4.35 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 2.0 ppm Cypermethrin
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0.4
0.2
0
-0.2
-0.4 Log Log -0.6
600nm -0.8 O.D -1
-1.2
-1.4
-1.6 Time (hrs) Figure. 4.36 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 50 ppm Cypermethrin
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1 0.1 ppm 0.5 ppm 2.0 ppm Series1
0.5
0
-0.5 O.D600nm Log O.D600nm -1
-1.5
-2 Time (hrs)
Figure. 4.37 Cumulative results of growth kinetics studies of Pseudomonas aeruginosa supplemented with different concentration of Cypermethrin
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0.4
0.2
0
-0.2
Log Log -0.4
-0.6 600nm
O.D -0.8
-1
-1.2
-1.4 Time (hrs)
Figure. 4.38 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.1ppm Cypermethrin
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0.4
0.2
0
-0.2 Log Log
-0.4 600nm
-0.6 O.D
-0.8
-1
-1.2 Time (hrs)
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Figure. 4.39 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.5 ppm Cypermethrin
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0.4
0.2
0
-0.2
Log Log -0.4
600nm -0.6
O.D -0.8
-1
-1.2
-1.4 Time (hrs)
Figure. 4.40 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 2.0 ppm Cypermethrin
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0.2
0
-0.2 Log Log
-0.4 600nm
-0.6 O.D
-0.8
-1
-1.2
-1.4 Time (hrs)
Figure. 4.41 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 50 ppm Cypermethrin
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50 ppm 2.0 ppm 0.5 ppm 0.1 ppm
0.4 0.2 0 -0.2 -0.4
-0.6 O.D600nm Log O.D600nm -0.8 -1 -1.2 -1.4 Time (hrs)
Figure. 4.42 Cumulative results of growth kinetics studies of Bacillus licheniformis supplemented with different concentration of Cypermethrin
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Tables
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Table. 4.1 Pesticides working standards was prepared in 50 ml n-hexane Working Malathion Cypermethrin standards (ppm) Volume used from Volume used from stock stock 300 5.102 ml 5ml +220 µl 200 3.4 ml 3.5 ml 100 1.70 ml 1.74 ml 50 0.850 ml 0.87 ml 10 170µl 174 µl 5 85 µl 87 µl 2 34 µl 34.8 µl
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Table. 4.2 2.5 ml media volume with culture and different concentration of pesticide Culture Pesticide volume (µl) Minimal salt Concentration volume 125ppm working medium (ppm) (µl) standard volume (ml)
Blank 100 0 2.5
50 100 1000 1.5
2 100 400 2.46
5 100 100 2.49
0.1 100 20 2.498
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Table. 4.3 25 ml media volume with culture and different concentration of pesticide Culture Pesticide volume (µl) Minimal salt Concentration volume 125ppm working medium volume (ppm) (µl) standard (ml) Blank 2.5 0 22.5 50 2.5 10 12.5 2 2.5 0.4 22.1 5 2.5 0.2 22.3 0.1 2.5 0.04 22.46
Table. 4.4 250 ml media volume with culture and different concentration of pesticide Culture Minimal salt Concentration volume Pesticide medium volume (ppm) (µl) volume (µl) (ml)
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Blank 25 0 250 50 25 4.25* 220.75
2 25 4** 221
5 25 1* 224
0.1 25 0.2* 224.8 *Volume taken from stock **Volume taken from 125 ppm working standar
Table. 4.5 Peak area of different concentration of Malathion and Cypermethrin
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Pesticide concentration (ppm) Malathion Cypermethrin Peak area Peak area 2 26358649 117232159 10 50192535 262090522 50 250843214 1291833495 100 551340767 2767193230 200 1104606490 5900545979 300 1643687860 8694358875 2 26358649 117232159 Table. 4.6 Growth kinetics of E.coli in MSM (Control)
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm hours 1 2 3 dev Mean Log 0 0.002 0.018 0.013 0.008 1.019 -1.966 1 0.026 0.013 0.021 0.007 1.015 -1.689 2 0.3 0.298 0.308 0.005 1.012 -0.519 3 1.19 1.182 1.186 0.004 1.009 0.074
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4 2.611 2.579 2.556 0.028 1.066 0.412 5 3.943 3.953 3.993 0.026 1.063 0.598
6 4.981 4.987 4.999 0.009 1.021 0.698 7 4.523 4.549 4.515 0.018 1.042 0.656 8 4.041 4.038 4.029 0.006 1.014 0.606
9 3.891 3.874 3.854 0.019 1.044 0.588 10 3.373 3.391 3.379 0.009 1.021 0.529 11 1.451 1.428 1.417 0.017 1.041 0.156
12 0.491 0.538 0.552 0.032 1.076 -0.2786 13 0.268 0.261 0.266 0.004 1.008 -0.577 14 0.233 0.238 0.246 0.007 1.015 -0.621
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Table. 4.7 Growth kinetics of Pseudomonas aeruginosa in MSM (Control)
Time O.D600nm O.D600nm O.D600nm O.D600nm Std. dev Mean hours 1 2 3 Log 0 0.01 0.011 0.015 0.002646 1.006 -1.921 1 0.021 0.028 0.038 0.008544 1.020 -1.538 2 0.059 0.067 0.062 0.004041 1.009 -1.204 3 0.192 0.201 0.204 0.006245 1.014 -0.701 4 0.614 0.62 0.617 0.003 1.007 -0.210 5 1.261 1.256 1.26 0.002646 1.006 0.100 6 1.919 1.926 1.921 0.003606 1.008 0.284 7 3.048 3.056 3.058 0.005292 1.012 0.485 8 3.761 3.772 3.768 0.005568 1.013 0.576 9 3.157 3.168 3.162 0.005508 1.013 0.500 10 2.691 2.698 2.686 0.006028 1.014 0.430 11 1.989 1.998 1.999 0.005508 1.013 0.300 12 1.618 1.624 1.623 0.003215 1.007 0.210 13 1.379 1.389 1.383 0.005033 1.012 0.141
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14 1.185 1.198 1.191 0.006506 1.015 0.076
Time O.D600nm O.D600nm O.D600nm O.D600nm Std. dev Mean hours 1 2 3 Log 0 0.126 0.122 0.127 0.003 0.125 -0.903 1 0.227 0.235 0.249 0.011 0.237 -0.625 2 0.587 0.594 0.606 0.010 0.596 -0.225 3 1.258 1.263 1.264 0.003 1.262 0.101 4 1.889 1.896 1.905 0.008 1.897 0.278 5 2.515 2.5 2.503 0.008 2.506 0.399 6 3.084 3.069 3.075 0.008 3.076 0.488 7 3.458 3.462 3.482 0.013 3.467 0.54 8 3.881 3.892 3.898 0.009 3.890 0.59
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9 4.068 4.071 4.082 0.007 4.074 0.61 10 4.358 4.369 4.368 0.006 4.365 0.64 11 4.062 4.079 4.08 0.010 4.074 0.61 12 3.881 3.894 3.896 0.008 3.890 0.59 13 3.306 3.315 3.313 0.005 3.311 0.52 14 3.154 3.159 3.174 0.010408 3.162 0.5
Table. 4.8 Growth kinetics of Bacillus licheniformis in minimal salt medium (Control)
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Table. 4.9 Cumulative result of Generation time and specific growth rate (Control)
Isolated strains Generation Time Growth rate constant (min) (min-1) Escherichia coli 40.9 0.024 Pseudomonas aeruginosa 57.86 0.017 Bacillus licheniformis 117.4 0.009
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Table 4.10 Growth kinetics of E.coli in minimal salt medium supplemented with 0.1ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.081 0.079 0.092 0.007 0.084 -1.075 1 0.076 0.083 0.082 0.00379 0.080 -1.095
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2 0.223 0.225 0.224 0.001 0.224 -0.65 3 1.209 1.203 1.203 0.00346 1.205 0.081 4 2.932 2.939 2.942 0.00513 2.938 0.468
5 3.977 3.969 3.97 0.00436 3.972 0.599 6 5.239 5.232 5.237 0.00361 5.236 0.719 7 3.967 3.963 3.958 0.00451 3.963 0.598 8 3.451 3.462 3.465 0.00737 3.459 0.539
9 3.137 3.134 3.129 0.00404 3.133 0.496 10 1.693 1.692 1.698 0.00321 1.694 0.229 11 1.492 1.499 1.498 0.00379 1.496 0.175 12 0.942 0.947 0.95 0.00404 0.946 -0.024 13 0.702 0.698 0.7 0.002 0.700 -0.155 14 0.637 0.632 0.633 0.00265 0.634 -0.198
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Table. 4.11 Growth kinetics of E.coli in minimal salt medium supplemented with 0.5ppm Malathion Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.083 0.087 0.088 0.003 0.086 -1.065 1 0.093 0.091 0.108 0.009 0.097 -1.012 2 0.226 0.223 0.230 0.004 0.226 -0.645 3 1.154 1.148 1.150 0.003 1.151 0.061 4 2.912 2.921 2.919 0.005 2.917 0.465 5 3.084 3.081 3.085 0.002 3.083 0.489 6 3.384 3.378 3.380 0.003 3.381 0.529 7 2.282 2.289 2.286 0.004 2.286 0.359 8 2.037 2.031 2.029 0.004 2.032 0.308 9 1.882 1.887 1.895 0.007 1.888 0.276 10 1.693 1.702 1.700 0.005 1.698 0.23 11 1.549 1.541 1.546 0.004 1.545 0.189 12 0.926 0.922 0.920 0.003 0.923 -0.035 13 0.797 0.791 0.790 0.004 0.793 -0.101
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14 0.548 0.537 0.534 0.007 0.540 -0.268 Table. 4.12 Growth kinetics of E.coli in minimal salt medium supplemented with 2.0 ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.085 0.089 0.090 0.003 0.088 -1.055 1 0.078 0.085 0.081 0.004 0.081 -1.089 2 0.127 0.118 0.117 0.006 0.121 -0.918 3 1.025 1.032 1.027 0.004 1.028 0.012 4 1.711 1.724 1.719 0.007 1.718 0.235 5 1.963 1.971 1.970 0.004 1.968 0.294 6 2.502 2.507 2.509 0.004 2.506 0.399 7 1.995 1.989 1.988 0.004 1.991 0.299 8 1.375 1.367 1.371 0.004 1.371 0.137 9 1.062 1.070 1.068 0.004 1.067 0.028 10 1.047 1.041 1.039 0.004 1.042 0.018
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11 1.024 1.019 1.020 0.003 1.021 0.009 12 0.963 0.958 0.957 0.003 0.959 -0.018 13 0.891 0.899 0.889 0.005 0.893 -0.0491 14 0.791 0.799 0.793 0.004 0.794 -0.1
Table. 4.13 Growth kinetics of E.coli in minimal salt medium supplemented with 50 ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.095 0.103 0.102 0.0044 0.100 -1 1 0.062 0.067 0.063 0.0026 0.064 -1.193 2 0.093 0.089 0.088 0.0026 0.090 -1.045 3 0.791 0.798 0.799 0.0044 0.796 -0.099 4 1.162 1.17 1.176 0.0070 1.169 0.068 5 1.298 1.291 1.294 0.0035 1.294 0.112
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6 1.531 1.537 1.546 0.0075 1.538 0.187 7 1.592 1.586 1.588 0.0031 1.589 0.201 8 1.843 1.848 1.857 0.0071 1.849 0.267 9 1.601 1.609 1.611 0.0053 1.607 0.206 10 1.251 1.259 1.258 0.0044 1.256 0.099 11 0.927 0.921 0.92 0.0038 0.923 -0.035 12 0.772 0.78 0.782 0.0053 0.778 -0.109 13 0.671 0.664 0.67 0.0038 0.668 -0.175 14 0.501 0.509 0.507 0.0042 0.506 -0.296
Table. 4.14 Cumulative result of Generation time and specific growth rate of E.coli supplemented with different concentration of Malathion Malathion Concentration Generation Growth rate constant of time E.coli (minute) (minute-1)
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Control 40.90 0.024 0.1ppm 60.40 0.016 0.5ppm 68.05 0.014 2ppm 74.5 0.013 50ppm 91.37 0.010
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.012 0.019 0.02 0.004 0.017 -1.77 1 0.021 0.028 0.023 0.004 0.024 -1.62 2 0.059 0.067 0.066 0.004 0.064 -1.194 3 0.161 0.169 0.171 0.005 0.167 -0.777 4 0.389 0.399 0.401 0.006 0.396 -0.402
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5 0.941 0.959 0.945 0.009 0.948 -0.023 6 1.631 1.642 1.637 0.006 1.637 0.214 7 2.529 2.539 2.537 0.005 2.535 0.404 8 2.931 2.943 2.939 0.006 2.938 0.468 9 2.737 2.746 2.742 0.005 2.742 0.438 10 2.51 2.522 2.521 0.007 2.518 0.401 11 2.416 2.426 2.421 0.005 2.421 0.384 12 1.881 1.891 1.892 0.006 1.888 0.276 13 1.595 1.61 1.594 0.009 1.600 0.204 14 1.257 1.265 1.263 0.004 1.262 0.101 Table. 4.15 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.1ppm Malathion
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Table. 4.16 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.5ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.011 0.021 0.019 0.005 0.017 -1.77 1 0.021 0.028 0.023 0.004 0.024 -1.62 2 0.059 0.067 0.07 0.006 0.065 -1.184 3 0.173 0.178 0.174 0.003 0.175 -0.757 4 0.389 0.399 0.401 0.006 0.396 -0.402 5 0.978 0.965 0.969 0.007 0.971 -0.013 6 1.319 1.311 1.316 0.004 1.315 0.119 7 1.631 1.641 1.638 0.005 1.637 0.214 8 2.027 2.036 2.034 0.005 2.032 0.308 9 1.919 1.928 1.922 0.005 1.923 0.284 10 1.881 1.891 1.892 0.006 1.888 0.276 11 1.587 1.583 1.574 0.007 1.581 0.199 12 1.255 1.259 1.254 0.003 1.256 0.099
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13 0.789 0.796 0.798 0.005 0.794 -0.1 14 0.639 0.628 0.63 0.006 0.632 -0.199
Table. 4.17 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 2.0ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.014 0.015 0.022 0.004 0.017 -1.77 1 0.021 0.028 0.023 0.004 0.024 -1.62 2 0.059 0.066 0.067 0.004 0.064 -1.194 3 0.173 0.168 0.16 0.007 0.167 -0.777 4 0.388 0.401 0.4 0.007 0.396 -0.402 5 0.942 0.948 0.955 0.007 0.948 -0.023 6 1.374 1.383 1.384 0.006 1.380 0.14 7 1.632 1.643 1.635 0.006 1.637 0.214
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8 2.123 2.133 2.128 0.005 2.128 0.328 9 2.013 2.005 2.009 0.004 2.009 0.303 10 1.883 1.887 1.894 0.006 1.888 0.276 11 1.435 1.431 1.431 0.002 1.432 0.156 12 0.529 0.528 0.525 0.002 0.527 -0.278 13 0.269 0.258 0.268 0.006 0.265 -0.577 14 0.231 0.246 0.241 0.008 0.239 -0.621
Table. 4.18 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 50 ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.061 0.07 0.072 0.006 0.068 -1.17 1 0.081 0.089 0.085 0.004 0.085 -1.07 2 0.109 0.1 0.099 0.006 0.103 -0.989
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3 0.217 0.209 0.205 0.006 0.210 -0.677 4 0.491 0.501 0.505 0.007 0.499 -0.302 5 0.952 0.951 0.942 0.006 0.948 -0.023 6 1.373 1.382 1.386 0.007 1.380 0.14 7 1.927 1.919 1.923 0.004 1.923 0.284 8 2.508 2.502 2.491 0.009 2.500 0.398 9 2.365 2.359 2.357 0.004 2.360 0.373 10 2.281 2.289 2.287 0.004 2.286 0.359 11 2.029 2.021 2.019 0.005 2.023 0.306 12 1.979 1.992 1.987 0.007 1.986 0.298 13 1.931 1.941 1.937 0.005 1.936 0.287 14 1.829 1.821 1.822 0.004 1.824 0.261
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Table. 4.19 Cumulative result of Generation time and specific growth rate of Pseudomonas aeruginosa supplemented with different concentration of Malathion Malathion Generation time Growth rate constant of Concentration (minute) Pseudomonas aeruginosa (minute-1) Control 57.86 0.017 0.1ppm 64.69 0.015 0.5ppm 69.00 0.014 2ppm 77.41 0.013 50ppm 90.56 0.011
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Table. 4.20 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.1ppm Malathion
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Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log
0 0.077 0.076 0.066 0.006 0.073 -1.1366 1 0.127 0.121 0.121 0.003 0.123 -0.91 2 0.307 0.301 0.298 0.005 0.302 -0.5199 3 0.671 0.674 0.664 0.005 0.670 -0.1741
4 1.293 1.297 1.298 0.003 1.296 0.1126 5 1.542 1.547 1.557 0.008 1.549 0.19 6 1.732 1.739 1.742 0.005 1.738 0.24 7 1.867 1.861 1.858 0.005 1.862 0.27 8 1.954 1.947 1.949 0.004 1.950 0.29 9 1.993 1.991 2.002 0.006 1.995 0.3 10 2.083 2.087 2.098 0.008 2.089 0.32
11 1.908 1.901 1.907 0.004 1.905 0.28 12 1.643 1.647 1.654 0.006 1.648 0.217 13 1.371 1.378 1.383 0.006 1.377 0.139
14 1.011 1.019 1.019 0.005 1.016 0.007
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Table. 4.21 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.5 ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.075 0.078 0.069 0.005 0.074 -1.13 1 0.098 0.099 0.103 0.003 0.100 -1 2 0.247 0.238 0.235 0.006 0.240 -0.6199 3 0.579 0.568 0.575 0.006 0.574 -0.241 4 1.025 1.031 1.032 0.004 1.029 0.0126 5 1.217 1.209 1.206 0.006 1.211 0.083 6 1.327 1.319 1.318 0.005 1.321 0.121 7 1.447 1.441 1.438 0.005 1.442 0.159 8 1.574 1.565 1.572 0.005 1.570 0.196 9 1.708 1.697 1.701 0.006 1.702 0.231 10 1.916 1.911 1.903 0.007 1.910 0.281 11 1.845 1.839 1.838 0.004 1.841 0.265
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12 1.674 1.669 1.67 0.003 1.671 0.223 13 1.583 1.59 1.593 0.005 1.589 0.201 14 1.571 1.579 1.572 0.004 1.574 0.197 Table. 4.22 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 2.0 ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.071 0.076 0.072 0.003 0.073 -1.1366 1 0.094 0.096 0.082 0.008 0.091 -1.043 2 0.194 0.193 0.185 0.005 0.191 -0.7199 3 0.536 0.534 0.526 0.005 0.532 -0.2741 4 1.026 1.032 1.03 0.003 1.029 0.0126 5 1.379 1.372 1.374 0.004 1.375 0.1383 6 1.391 1.399 1.397 0.004 1.396 0.1448 7 1.438 1.43 1.429 0.005 1.432 0.156
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8 1.471 1.476 1.48 0.005 1.476 0.169 9 1.48 1.474 1.483 0.005 1.479 0.17 10 1.517 1.511 1.513 0.003 1.514 0.18 11 1.467 1.461 1.459 0.004 1.462 0.165 12 1.329 1.324 1.329 0.003 1.327 0.123 13 1.297 1.292 1.285 0.006 1.291 0.111 14 1.046 1.039 1.035 0.006 1.040 0.017 Table. 4.23 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 50 ppm Malathion
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.073 0.077 0.067 0.005 0.072 -1.14 1 0.067 0.064 0.077 0.007 0.069 -1.16 2 0.101 0.105 0.101 0.002 0.102 -0.99 3 0.201 0.205 0.207 0.003 0.204 -0.69 4 0.472 0.478 0.486 0.007 0.479 -0.32
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5 0.791 0.795 0.797 0.003 0.794 -0.1 6 1.006 1.001 1 0.003 1.002 0.001 7 1.025 1.023 1.036 0.007 1.028 0.012 8 1.063 1.066 1.078 0.008 1.069 0.029 9 1.118 1.109 1.108 0.006 1.112 0.046 10 1.142 1.149 1.146 0.004 1.146 0.059 11 1.072 1.078 1.079 0.004 1.076 0.032 12 1.043 1.047 1.044 0.002 1.045 0.019 13 1.024 1.029 1.024 0.003 1.026 0.011 14 1.021 1.028 1.021 0.004 1.023 0.01
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Table. 4.24 Cumulative result of Generation time and specific growth rate of Bacillus licheniformis supplemented with different concentration of Malathion Malathion Concentration Generation time Growth rate constant of (minute) Bacillus licheniformis (minute-1) Control 117.4 0.009 0.1ppm 124.7 0.008 0.5ppm 128.2 0.008 2ppm 137.9 0.007 50ppm 150.7 0.007
Table. 4.25 Growth kinetics of E. coli supplemented with 0.1 ppm Cypermethrin
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Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.081 0.083 0.088 0.004 0.084 -1.075 1 0.083 0.085 0.09 0.004 0.086 -1.065 2 0.451 0.459 0.461 0.005 0.457 -0.34 3 1.142 1.143 1.152 0.006 1.146 0.059 4 1.611 1.619 1.624 0.007 1.618 0.209 5 2.503 2.497 2.501 0.003 2.500 0.398 6 3.694 3.687 3.688 0.004 3.690 0.567 7 3.143 3.147 3.153 0.005 3.148 0.498 8 2.554 2.56 2.562 0.004 2.559 0.408 9 2.423 2.429 2.428 0.003 2.427 0.385 10 1.591 1.597 1.589 0.004 1.592 0.202 11 1.005 1.011 1.012 0.004 1.009 0.004 12 0.791 0.795 0.792 0.002 0.793 -0.101 13 0.649 0.641 0.643 0.004 0.644 -0.191
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14 0.571 0.578 0.577 0.004 0.575 -0.24
Table. 4.26 Growth kinetics of E. coli supplemented with 0.5 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.082 0.081 0.086 0.003 0.083 -1.081 1 0.085 0.082 0.075 0.005 0.081 -1.093 2 0.487 0.496 0.486 0.006 0.490 -0.31 3 1.259 1.251 1.249 0.005 1.253 0.098 4 1.805 1.795 1.809 0.007 1.803 0.256 5 2.452 2.445 2.45 0.004 2.449 0.389 6 2.511 2.518 2.507 0.006 2.512 0.4 7 2.477 2.468 2.47 0.005 2.472 0.393
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8 2.004 2.01 2.013 0.005 2.009 0.303 9 1.846 1.84 1.836 0.005 1.841 0.265 10 1.601 1.609 1.611 0.005 1.607 0.206 11 1.531 1.54 1.543 0.006 1.538 0.187 12 1.162 1.168 1.17 0.004 1.167 0.067 13 0.793 0.799 0.791 0.004 0.794 -0.1 14 0.572 0.576 0.578 0.003 0.575 -0.24
Table. 4.27 Growth kinetics of E. coli supplemented with 2.0 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.083 0.082 0.087 0.003 0.084 -1.075 1 0.093 0.104 0.103 0.006 0.100 -1 2 0.107 0.101 0.1 0.004 0.103 -0.989 3 1.002 1.009 1.01 0.004 1.007 0.003
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4 1.287 1.279 1.281 0.004 1.282 0.108 5 1.529 1.531 1.544 0.008 1.535 0.186 6 1.651 1.659 1.657 0.004 1.656 0.219 7 1.569 1.559 1.561 0.005 1.563 0.194 8 1.309 1.302 1.308 0.004 1.306 0.116 9 1.221 1.231 1.23 0.006 1.227 0.089 10 1.114 1.12 1.124 0.005 1.119 0.049 11 0.834 0.845 0.839 0.006 0.839 -0.076 12 0.681 0.689 0.682 0.004 0.684 -0.165 13 0.561 0.571 0.575 0.007 0.569 -0.245 14 0.461 0.469 0.467 0.004 0.466 -0.332
Table. 4.28 Growth kinetics of E. coli supplemented with 50 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. Mean O.D600nm
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hours 1 2 3 dev Log 0 0.084 0.081 0.085 0.002 0.083 -1.08 1 0.021 0.029 0.026 0.004 0.025 -1.595 2 0.012 0.02 0.018 0.004 0.017 -1.776 3 0.429 0.419 0.417 0.006 0.422 -0.375 4 1.502 1.508 1.51 0.004 1.507 0.178 5 1.929 1.922 1.918 0.006 1.923 0.284 6 2.076 2.069 2.065 0.006 2.070 0.316 7 2.449 2.44 2.441 0.005 2.443 0.388 8 2.613 2.622 2.62 0.005 2.618 0.418 9 1.994 1.987 1.991 0.004 1.991 0.299 10 1.339 1.332 1.32 0.010 1.330 0.124 11 0.387 0.379 0.377 0.005 0.381 -0.419 12 0.269 0.262 0.267 0.004 0.266 -0.575 13 0.209 0.201 0.204 0.004 0.205 -0.689
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14 0.197 0.191 0.19 0.004 0.193 -0.715
Table. 4.29 Cumulative result of Generation time and specific growth rate of E. coli supplemented with different concentration of Cypermethrin Cypermethrin Generation Growth rate constant of Concentration time E. coli (minute) (minute-1) Control 40.90 0.024 0.1ppm 66 0.015 0.5ppm 73.17 0.0136 2ppm 83.91 0.011 50ppm 97.95 0.010
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Table. 4.30 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.1ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.014 0.021 0.016 0.004 0.017 -1.77 1 0.028 0.031 0.032 0.002 0.030 -1.52 2 0.059 0.066 0.071 0.006 0.065 -1.184 3 0.173 0.179 0.173 0.003 0.175 -0.757 4 0.389 0.399 0.39 0.006 0.393 -0.406 5 0.978 0.969 0.965 0.007 0.971 -0.013
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6 1.631 1.642 1.637 0.006 1.637 0.214 7 2.339 2.348 2.313 0.018 2.333 0.368 8 2.448 2.439 2.443 0.005 2.443 0.388 9 2.111 2.119 2.096 0.012 2.109 0.324 10 1.881 1.889 1.894 0.007 1.888 0.276 11 1.578 1.584 1.582 0.003 1.581 0.199 12 1.231 1.261 1.276 0.023 1.256 0.099 13 0.789 0.795 0.799 0.005 0.794 -0.1 14 0.638 0.637 0.622 0.009 0.632 -0.199
Table 4.31 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 0.5 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.024 0.021 0.015 0.005 0.020 -1.7
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1 0.029 0.032 0.03 0.002 0.030 -1.52 2 0.087 0.079 0.076 0.006 0.081 -1.094 3 0.172 0.168 0.161 0.006 0.167 -0.777 4 0.502 0.495 0.5 0.004 0.499 -0.302 5 0.974 0.967 0.964 0.005 0.968 -0.014 6 1.318 1.311 1.308 0.005 1.312 0.118 7 1.924 1.917 1.915 0.005 1.919 0.283 8 1.971 1.981 1.979 0.005 1.977 0.296 9 1.439 1.428 1.43 0.006 1.432 0.156 10 0.841 0.832 0.834 0.005 0.836 -0.078 11 0.672 0.661 0.663 0.006 0.665 -0.177 12 0.507 0.513 0.515 0.004 0.512 -0.291 13 0.497 0.512 0.491 0.011 0.500 -0.301 14 0.497 0.486 0.49 0.006 0.491 -0.309
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Table. 4.32 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 2.0 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.023 0.021 0.019 0.002 0.021 -1.675 1 0.029 0.034 0.022 0.006 0.028 -1.55 2 0.056 0.048 0.05 0.004 0.051 -1.289 3 0.159 0.151 0.155 0.004 0.155 -0.81 4 0.462 0.456 0.453 0.005 0.457 -0.34 5 0.799 0.789 0.795 0.005 0.794 -0.1 6 1.041 1.049 1.044 0.004 1.045 0.019 7 1.152 1.161 1.163 0.006 1.159 0.064 8 1.248 1.239 1.238 0.006 1.242 0.094 9 1.142 1.149 1.146 0.004 1.146 0.059 10 1.039 1.049 1.046 0.005 1.045 0.019 11 0.831 0.84 0.847 0.008 0.839 -0.076 12 0.689 0.679 0.684 0.005 0.684 -0.165
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13 0.562 0.571 0.574 0.006 0.569 -0.245 14 0.461 0.469 0.467 0.004 0.466 -0.332 Table. 4.33 Growth kinetics of Pseudomonas aeruginosa in minimal salt medium supplemented with 50 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.054 0.049 0.054 0.003 0.052 -1.28 1 0.047 0.038 0.036 0.006 0.040 -1.395 2 0.042 0.037 0.039 0.003 0.039 -1.406 3 0.102 0.111 0.105 0.005 0.106 -0.975 4 0.219 0.208 0.214 0.006 0.214 -0.67 5 0.529 0.521 0.524 0.004 0.525 -0.28 6 1.043 1.032 1.038 0.006 1.038 0.016 7 1.431 1.442 1.443 0.007 1.439 0.158 8 1.658 1.647 1.651 0.006 1.652 0.218 9 1.589 1.578 1.588 0.006 1.585 0.2
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10 1.569 1.559 1.561 0.005 1.563 0.194 11 1.432 1.441 1.443 0.006 1.439 0.158 12 1.339 1.328 1.334 0.006 1.334 0.125 13 1.279 1.267 1.275 0.006 1.274 0.105 14 0.991 0.983 0.992 0.005 0.989 -0.005 Table. 4.34 Cumulative result of Generation time and specific growth rate of Pseudomonas aeruginosa supplemented with different concentration of Cypermethrin Cypermethrin Generation time Growth rate constant of Concentration (minute) Pseudomonas aeruginosa (minute-1) Control 57.86 0.017 0.1ppm 67 0.015 0.5ppm 72.3 0.014 2ppm 81.7 0.012 50ppm 96.57 0.010
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Table. 4.35 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.1ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.079 0.071 0.072 0.004 0.074 -1.13 1 0.122 0.131 0.133 0.006 0.129 -0.89 2 0.248 0.237 0.235 0.007 0.240 -0.6199 3 0.471 0.482 0.48 0.006 0.478 -0.321 4 0.791 0.787 0.805 0.009 0.794 -0.1 5 1.233 1.241 1.242 0.005 1.239 0.093 6 1.389 1.382 1.38 0.005 1.384 0.141 7 1.541 1.549 1.546 0.004 1.545 0.189
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8 1.629 1.619 1.617 0.006 1.622 0.21 9 1.749 1.736 1.74 0.007 1.742 0.241 10 1.861 1.871 1.867 0.005 1.866 0.271 11 1.837 1.843 1.842 0.003 1.841 0.265 12 1.678 1.669 1.666 0.006 1.671 0.223 13 1.582 1.587 1.597 0.008 1.589 0.201 14 1.571 1.579 1.572 0.004 1.574 0.197
Table. 4.36 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 0.5 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. Mean O.D600nm
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hours 1 2 3 dev Log 0 0.079 0.071 0.078 0.004 0.076 -1.12 1 0.107 0.101 0.092 0.008 0.100 -1 2 0.197 0.188 0.187 0.006 0.191 -0.7199 3 0.368 0.359 0.36 0.005 0.362 -0.441 4 0.771 0.781 0.777 0.005 0.776 -0.11 5 1.151 1.159 1.158 0.004 1.156 0.063 6 1.359 1.349 1.348 0.006 1.352 0.131 7 1.449 1.439 1.438 0.006 1.442 0.159 8 1.577 1.569 1.565 0.006 1.570 0.196 9 1.668 1.661 1.661 0.004 1.663 0.221 10 1.749 1.739 1.737 0.006 1.742 0.241 11 1.711 1.782 1.661 0.061 1.718 0.235 12 1.679 1.668 1.666 0.007 1.671 0.223 13 1.582 1.587 1.597 0.008 1.589 0.201
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14 1.571 1.577 1.574 0.003 1.574 0.197 Table. 4.37 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 2.0 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.079 0.073 0.067 0.006 0.073 -1.137 1 0.097 0.089 0.086 0.006 0.091 -1.043 2 0.193 0.187 0.192 0.003 0.191 -0.720 3 0.429 0.421 0.418 0.006 0.423 -0.374 4 0.797 0.789 0.792 0.004 0.793 -0.101 5 1.027 1.024 1.012 0.008 1.021 0.009 6 1.291 1.299 1.302 0.006 1.297 0.113 7 1.371 1.379 1.375 0.004 1.375 0.138 8 1.391 1.399 1.397 0.004 1.396 0.145 9 1.438 1.429 1.43 0.005 1.432 0.156
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10 1.465 1.467 1.455 0.006 1.462 0.165 11 1.323 1.329 1.33 0.004 1.327 0.123 12 1.296 1.287 1.291 0.005 1.291 0.111 13 1.046 1.039 1.035 0.006 1.040 0.017 14 1.004 1.008 1.016 0.006 1.009 0.004
Table. 4.38 Growth kinetics of Bacillus licheniformis in minimal salt medium supplemented with 50 ppm Cypermethrin
Time O.D600nm O.D600nm O.D600nm Std. O.D600nm Mean hours 1 2 3 dev Log 0 0.078 0.074 0.065 0.007 0.072 -1.140 1 0.062 0.071 0.07 0.005 0.068 -1.170 2 0.106 0.101 0.1 0.003 0.102 -0.990 3 0.167 0.159 0.161 0.004 0.162 -0.790 4 0.328 0.32 0.323 0.004 0.324 -0.490
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5 0.635 0.628 0.63 0.004 0.631 -0.200 6 1.026 1.029 1.015 0.007 1.023 0.010 7 1.047 1.038 1.035 0.006 1.040 0.017 8 1.052 1.054 1.064 0.006 1.057 0.024 9 1.079 1.072 1.071 0.004 1.074 0.031 10 1.101 1.098 1.106 0.004 1.102 0.042 11 1.096 1.089 1.097 0.004 1.094 0.039 12 1.055 1.048 1.046 0.005 1.050 0.021 13 1.038 1.039 1.028 0.006 1.035 0.015 14 1.028 1.019 1.016 0.006 1.021 0.009
Table. 4.39 Cumulative result of Generation time and specific growth rate of Bacillus licheniformis supplemented with different concentration of Cypermethrin Cypermethrin Generation Growth rate constant of Concentration time Bacillus licheniformis
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(minute) (minute-1)
Control 117.4 0.009 0.1ppm 129.0 0.008 0.5ppm 132.7 0.008 2ppm 139.5 0.007 50ppm 153 0.007
Table. 4.40 Cumulative result of Generation time and specific growth rate of E. coli Malathion Cypermethrin
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Pesticide Concentration Generation Growth rate Generation time Growth rate time constant of (minute) constant of (minute) E.coli E.coli (minute-1) (minute-1) Control 40.90 0.024 40.90 0.024 0.1ppm 60.40 0.016 66 0.015 0.5ppm 68.05 0.014 73.17 0.0136 2ppm 74.5 0.013 83.91 0.011 50ppm 91.37 0.010 97.95 0.010
Table. 4.41 Cumulative result of Generation time and specific growth rate of Pseudomonas aeruginosa Malathion Cypermethrin Pesticide Concentration Generation Growth rate Generation time Growth rate time constant of (minute) constant of (minute) Pseudomonas Pseudomonas aeruginosa aeruginosa
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(minute-1) (minute-1) Control 57.86 0.017 57.86 0.017 0.1ppm 64.69 0.015 67 0.015 0.5ppm 69.00 0.014 72.3 0.014 2ppm 77.41 0.013 81.7 0.012 50ppm 90.56 0.011 96.57 0.010
Table. 4.42 Cumulative result of Generation time and specific growth rate of Bacillus licheniformis Malathion Cypermethrin Pesticide Concentration Generation Growth rate Generation time Growth rate time constant of (minute) constant of (minute) Bacillus Bacillus licheniformis licheniformis (minute-1) (minute-1) Control 117.4 0.009 117.4 0.009 0.1ppm 124.7 0.008 129.0 0.008
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0.5ppm 128.2 0.008 132.7 0.008 2ppm 137.9 0.007 139.5 0.007 50ppm 150.7 0.007 153 0.007
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Tempest, D. W. (1969). The place of continuous culture in microbiological research. Advances in microbial physiology, 4, 223-250.
Torres, A. M. R., & O'Flaherty, L. M. (1976). Influence of pesticides on Chlorella, Chlorococcum, Stigeoclonium (Chlorophyceae), Tribonema, Vaucheria (Xanthophyceae) and Oscillatoria (Cyanophyceae)*. Phycologia, 15(1), 25-36.
Trpton, D. K.; Roiston, D. E and Scow, K. M. (2003). Bioremedation and Biodegradation. J. Environment. Qual, 32: 40-46.
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Van Niel, C. B. (1949). The kinetics of growth of microorganisms. Parpart (ed.), The chemistry and physiology of growth. Princeton University Press, Princeton, NJ, 91- 102.
Verro, R., Finizio, A., Otto, S., & Vighi, M. (2008). Predicting pesticide environmental risk in intensive agricultural areas. II: Screening level risk assessment of complex mixtures in surface waters. Environmental science & technology, 43(2), 530-537.
Wang, C. H., & Koch, A. L. (1978). Constancy of growth on simple and complex media. Journal of bacteriology, 136(3), 969-975.
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Williams, P. P., Robbins, J. D., Gutierrez, J., & Davis, R. E. (1963). Rumen bacterial and protozoal responses to insecticide substrates. Applied microbiology, 11(6), 517- 522.
Winn, W. C. (2006). Koneman's color atlas and textbook of diagnostic microbiology. E. W. Koneman (Ed.). Lippincott williams & wilkins.
Yang, Y., Chen, H., Wu, Y., Yang, Y., & Wu, S. (2007). Mutated cadherin alleles from a field population of Helicoverpa armigera confer resistance to Bacillus thuringiensis toxin Cry1Ac. Applied and Environmental Microbiology, 73(21), 6939-6944.
Yates, G. T., & Smotzer, T. (2007). On the lag phase and initial decline of microbial growth curves. Journal of theoretical biology, 244(3), 511-517.
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Zhu, J., Zhao, Y., & Qiu, J. (2010). Isolation and application of a chlorpyrifos-degrading Bacillus licheniformis ZHU-1. African Journal of Microbiology Research, 4(22), 2410-2413.
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Chapter 5
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Biodegradation Studies on pesticides (Malathion and Cypermethrin)
5.1. Introduction
Biodegradation is the reduction in complexity of chemical compounds (Alexander, 1994). Biodegradation is the process by which xenobiotics are broken down by living organisms into metabolites (Marinescu et al., 2009). According to the Bennet et al., 2002 biodegradation is used to describe any biologically mediated alteration in a substrate.
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Fritsche and Hofrichter, (2008) studied the microbial organisms convert complex substance through enzymatic processes. Wolt et al., (2001) described that biodegradation studies conducted for the purpose of regulatory decision making with respect to pesticide use. Pesticides although beneficial for agricultural growth, but their excessive use causes serious environmental damage (Diez, 2010). Some of the pesticides used in agricultural remain in the environment for several months. Only 10 percent of pesticides reach to the target specie and remaining amount spread in the environment through wind, rain, fog, leaching, improperly cleaning or disposing of empty containers, equipment washings as well as mixing (Tariq et al., 2006; Ahad et al., 2001) and deposited on environment and effects to other organism such wildlife and public health. Therefore, remediation of contaminated sites is currently underway in order to develop economical and convenient methods for the detoxification of pesticide.
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Malathion and cypermethrin are commonly used pesticides all over the world. S-(1,2-dicarbethoxyethyl) -O, O-dimethyldi-thiophosphate (Malathion) is an organophosphate pesticide extensively used as a substitute to the DDT for the control of wide range of field crop pests, household insects, flies and animal parasites (Bjorling, 2008; Barlas, 1996). Despite its high toxicity, malathion is still extensively used throughout the world (Kumar et al., 1996). Cyclopropanecarboxylic acid, 3-(2, 2 dochoroethenyl)-2, 2 dimethyl-,cyano(3-phenoxyphenyl) methyl ester (Cypermethrin) is a fourth generation pyrethroid pesticide most common among the available synthetic chemical pesticides. It was first synthesized in 1974 and widely used in agriculture and public health (Lin et al., 2011). It works by quickly affecting the insect’s nervous system (NPIC, 1998).
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These pesticides upon inhalation, ingestion and through skin and eyes contact may be highly toxic for human. These compounds are toxic and carcinogenic in nature even at low concentrations (U.S. Public Health Service. 1995; Pham et al., 2004; Kaur et al., 1997). Prolonged exposure associates with teratogenic and mutagenic effects (Sharara et al., 1998; Getenga et al., 2000).
Various conventional, biological and chemical methods used for the degradation of malathion and cypermethrin including advanced oxidation processes (Shang et al., 2003; Bhatkhande et al., 2004), Fenton oxidation (Canizares et al., 2006; Sedlak and Andren, 1991) and electrochemical oxidation (Asmussen et al., 2009; Brillas et al., 2003).
Chen et al., (2012) and Lin et al., (2011) reported that conventional methods for
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pesticide disposal need high operational cost and are low in efficiency. Similarly, Ibrahim et al., (2014) demonstrated that methods are not only expensive and time-consuming, but also they do not provide a complete degradation. Mulla et al., (1981) reported that the amount of microbial degradation is far better than chemical degradation in natural systems.
Microbial degradation is the best means of detoxification of pesticides (Bhatnagar et al., 1989; Kudo, 2003) because of its operational flexibility, low operational costs, easy handling, and degradation of a wide variety of organic compounds (Schmit et al., 2009). Similarly, Contreras et al., (2003), Saaty and Booth, (1994) and Horne, (2002) reported that bioremediation is an eco-friendly methodology and suitable economical substitute to other conventional practices.
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Sethunathan, (1973); Sethunathan and Yoshida, (1973); Siddaramappa et al., (1973) reported that the organophosphorus compounds degrading bacterial strain were identified first in 1973 and their degradative enzymes have been extensively studied. Indigenous microbial flora for successful bioremediation of polluted sites have been reported by many researchers (El-Deeb et al., 2000; Bhadbhade et al., 2002 and Mohamed et al., 2010).
Microbial biodegradation commonly used for pollution control (Galli, 1994; Bhadbhade et al., 2002) of environmental pollutants (Nwuche and Ugoji, 2008; Schmid et al., 2001; Dua et al., 2002; Nwuche and Ugoji, 2010; Pazos et al., 2003).
The present study aims at demonstrating the biodegradation kinetics of metabolically versatile indigenous bacterial species namely Escherichia coli, Bacillus licheniformis,
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Pseudomonas aeruginosa under varying concentration of malathion and cypermethrin in minimal salt media. The isolation and characterization of these microorganisms are already described in Chapter 3. This work involves GC-ECD (Gas Chromatography Electron Capture Detector) analysis for the confirmation of biodegradation and the study suggests that the use of pesticide degrading bacteria is an eco-friendly technology suitable for wastewater pollution.
5.2. MATERIALS AND METHODS
5.2.1. Chemicals and reagents
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In the study analytical grade chemicals and reagents were used. Malathion - 98% and Cypermethrin - 95.8% (AccuStandard, USA) were obtained from FQSRI, Pakistan Agricultural Research Council, University of Karachi. Analytical grade n- hexane was purchased from Sigma-Aldrich, USA.
5.2.2. Growth medium
MSM (Mineral salt medium) were prepared according the method of Sambrook et al., (1989). The composition of the media in given in the Table. 5.1 and Fig. 5.1.
5.2.3. Biodegradation studies
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The biodegrading ability of selected strains Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa were tested by using five flasks of 500 mL of minimal salt medium were prepared. Each flask containing 225mL of minimal salt medium with different concentration of malathion and cypermethrin (0.1, 0.5, 2.0, 50 ppm) and 25mL of individual bacterial inoculum then placed in shaking water bath for 24 hours at 37°C and 120rpm. While control flask was taken without adding bacterial culture.
Fig. 5.2. Shows the Sample extraction method for pesticide residues analysis. 25mL of sample was taken after 0, 8 and 24 hours of inoculation and residues of malathion and cypermethrin were extracted twice with n-hexane (2 x 25mL) by the equal amount of n- hexane to each aliquot was added. After vigorous shaking of separatory funnel the organic layer was separated and dried out by sodium sulphate then evaporated by rotary
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evaporator (BUCHI Rotavapor B-740). 10mL analytical grade n-hexane was used to dissolve the dried residues and subjected to GC examination in Food Quality and Safety Research Institute, Pakistan agricultural research council (PARC), Karachi, Pakistan. Each sample was estimated three times and the mean and standard deviation were calculated.
5.2.4. Determination of Malathion and Cypermethrin by GC analysis
Residual malathion and cypermethrin were monitored by Agilent 6890N GC system with (ECD) Electron Capture Detector, with a capillary column HP-5MS (0.25mm, 30m, 0.25µm).The functional temperatures were 280°C and 320°C for the injector and detector. The injector mode was splitless and injector volume was 0. 5µl. The temperature of oven was held for 1min at 80°C and then raised @ 5°C/min until it
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reaches to 280°C. Nitrogen gas flow rate was 0.8 ml/min.
5.3. Results and discussion
The study was conducted to produce reliable data of biodegrading capability of isolated microorganisms and to develop a better understanding of the biological treatment process.
5.3.1. GC analysis of Malathion and Cypermethrin degradation
For the determination of malathion and cypermethrin degradation by the strains of Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa. Gas chromatography technique were used. Malathion peak was detected after the 7.81 minutes Fig. 5.3 and
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cypermethrin peak after 12.68 minutes shown in Fig. 5.4. Chromatogram showing Malathion and Cypermethrin degradation peaks after 4, 8 and 24 hours as shown in Fig. 5.5.
Biodegradation was quantified by comparison of pesticide concentration in inoculated and un-inoculated (control flask). The concentration of pesticide, which supported the bacterial growth during growth kinetic study, was selected for quantitative evaluation of biodegradation. Concentration of malathion and cypermethrin left were calculated by multiplication of peak corrected area (peak area-control area) with standard concentration (12.5ppm) and divided by standard peak area.
Result of percentage degradation of Malathion by Escherichia coli after 4, 8 and 24 hours are shown in Table 5.2, 5.3 and 5.4 and Fig. 5.6, 5.7, 5.8 and 5.9. The obtained
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results showed significant removal of malathion inoculated with Escherichia coli and almost 61% (0.1ppm), 88 % (0.5ppm), 98.5% (2.0 ppm) and 99.1% (50 ppm) was degraded during the first four hours afterwards, malathion was further degraded to 92% (0.1ppm), 96 % (0.5ppm), 99.1% (2.0 ppm) and 99.3% (50 ppm) in 8 hours and almost 99.9% removal of malathion was attained within 24hours. Malathion concentration decreased after 24 hours from 25 to 0.91 µg/250ml (0.1ppm) , 125 to 5.45 µg/250ml (0.5ppm), 500 to 1.80 µg/250ml (2.0 ppm) and 12500 to 51.36µg/250ml (50 ppm).
Percentage degradation of Malathion by Bacillus licheniformis after 4, 8 and 24 hours are shown in Table 5.6, 5.7 and 5.8 and Fig. 5.10, 5.11, 5.12 and 5.13 and nearly 58% (0.1ppm), 96.5 % (0.5ppm), 58% (2.0 ppm) and 96.5% (50 ppm) was degraded in first four hours. After 8 hours duration 93% (0.1ppm), 99.1 % (0.5ppm), 93% (2.0 ppm) and
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99.1% (50 ppm) was removed and almost 99% malathion disappeared form the medium within 24 hours of incubation period. Malathion concentration decreased after 24 hours from 25 to 0.91 µg/250ml (0.1ppm) , 125 to 5.45 µg/250ml (0.5ppm), 500 to 1.80 µg/250ml (2.0 ppm) and 12500 to 51.36µg/250ml (50 ppm).
Results of percentage degradation by Pseudomonas aeruginosa are presented in Table 5.10, 5.11 and 5.12 and Fig. 5.14, 5.15, 5.16 and 5.17. The results indicated that almost 61% (0.1ppm) , 96 % (0.5ppm), 60% (2.0 ppm) and 96% (50 ppm) was degraded during the first four hours afterwards, malathion was further degraded to 79% (0.1ppm) , 97 % (0.5ppm), 79% (2.0 ppm) and 98% (50 ppm) in 8 hours and 82 % (0.1, 2.0ppm) 99.9% (0.1, 50 ppm) removal of malathion was attained within 24hours. Malathion concentration decreased after 24 hours from 25 to 4.45 µg/250ml (0.1ppm) , 125 to
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1.733 µg/250ml (0.5ppm), 500 to 2.32 µg/250ml (2.0 ppm) and 12500 to 12.55 µg/250ml (50 ppm).
Table 5.5, 5.9 and 5.13 and Fig. 5.30, 5.32 and 5.34 shows cumulative result of percentage of Malathion degradation by Escherichia coli, Bacillus licheniformis, and Pseudomonas aeruginosa. Result of percentage degradation of Cypermethrin by Escherichia coli after 4, 8 and 24 hours is shown in Table. 5.14, 5.15 and 5.16 and Fig. 5.18, 5.19, 5.20 and 5.21. Removal of cypermethrin inoculated with Escherichia coli was slower initially as compared to malathion degraded by Escherichia coli and almost 59% (0.1ppm) , 85 % (0.5ppm), 96% (2.0 ppm) and 99.5% (50 ppm) cypermethrin removal was achieved within 4 hours. After 8 hours degradation rate was increased with 91% (0.1ppm), 92 % (0.5ppm), 99.6% (2.0 ppm) and 99.9% (50 ppm) and after 24 hours 99.9% complete removal of
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cypermethrin were seen by gas chromatography. Cypermethrin concentration decreased within 24 hours from 25 to 0.00 µg/250ml (0.1ppm) , 125 to 0.33 µg/250ml (0.5ppm), 500 to 0.26 µg/250ml (2.0 ppm) and 12500 to 0.98 µg/250ml (50 ppm).
Percentage degradation of Cypermethrin by Bacillus licheniformis after 4, 8 and 24 hours is shown in Table. 5.18, 5.19 and 5.20 and Fig. 5.22, 5.23, 5.24 and 5.25. Degradation rate was observed slower in this case and almost 30% (0.1ppm), 91% (0.5ppm), 29.9% (2.0 ppm) and 91% (50 ppm) was degraded in first four hours. After 8 hours duration 30% (0.1ppm), 94.7 % (0.5ppm), 31% (2.0 ppm) and 95% (50 ppm) were degraded. Degradation rate was observed slower till 24 hour of incubation 33.2% (0.1ppm), 95.6% (0.5ppm), 33.2% (2.0 ppm) and 95.6% (50 ppm). Cypermethrin concentration decreased
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within 24 hours from 25 to 17 µg/250ml (0.1ppm) , 125 to 4.0 µg/250ml (0.5ppm), 500 to 22.05 µg/250ml (2.0 ppm) and 12500 to 13.70 µg/250ml (50 ppm).
Results of percentage degradation by Pseudomonas aeruginosa are presented in Table 5.22, 5.23 and 5.24 and Fig. 5.26, 5.27, 5.28 and 5.29. Removal of cypermethrin inoculated with Pseudomonas aeruginosa was slower initially as compared to malathion degraded by Pseudomonas aeruginosa and almost 34 (0.1ppm) , 90 % (0.5ppm), 34% (2.0 ppm) and 91% (50 ppm) cypermethrin removal was achieved within 4 hours. After 8 hours degradation rate was increased with 94% (0.1ppm), 92 % (0.5ppm), 94% (2.0 ppm) and 91.6% (50 ppm) and after 24 hours 98% removal of cypermethrin were seen by gas chromatography. Cypermethrin concentration decreased within 24 hours from 25
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to 0.30 µg/250ml (0.1ppm) , 125 to 12.53 µg/250ml (0.5ppm), 500 to 14.43 µg/250ml (2.0 ppm) and 12500 to 4.85 µg/250ml (50 ppm).
Table 5.17, 5.21, 5.25 and Fig. 5.31, 5.33 and 5.35 shows cumulative result of percentage of Cypermethrin degradation by Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa. The results also demonstrating that the reduction in peak height and peak area as compared with control is due to reduction in pesticide concentration the can be seen in Fig. 5.5. Degradation of malathion and cypermethrin (50ppm) by Escherichia coli was found to be high and achieved complete removal within 4 hours of incubation. Jilani and Khan, (2006) reported that as the concentration of cypermethrin increased it will directly effect on the degradation efficiency of Pseudomonas sp. It can be concluded
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that such isolated strain can be useful for biodegradation of industrial effluent and can detoxify agricultural waste containing pesticides. Similar findings were also described by Kim et al., (2005) who stated that the malathion degradation rate was very high and almost 60% of initial 500 ppm malathion was removed within half hour by fungal cutinase.
Many researcher reported that the bacterial species capable of complete mineralization of malathion with less toxic product are as Pseudomonas (Goda et al., 2010; Matsumura and Boush, 1966; Abo-Amer, 2007; Paris et al., 1975; Andleeb et al., 2013; Imran et al., 2004; Pankaj et al., 2013) Bacillus (Kamal et al., 2008; Singh et al., 2012; Kumari et al., 2012; Thabit and El-Naggar, 2013; Laveglia and Dahm, 1977; Mohamed et al., 2010; Sarnaik, 2004; Adhikari, 2010) Escherichia coli (Zhang, et al., 2004; Mulchandani, et al., 1999).
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Microorganisms degrading Pyrethroid, are Pseudomonas (Chen et al., 2011; Grant et al., 2002; Zhang et al., 2011; Majid et al., 2012) Bacillus ( Chen et al., 2012; Murugesan et al 2010; Majid, et al., 2012). E.coli (Nishraj et al., 2012; Murugesan et al 2010; Majid, et al., 2012)
5.4. Conclusion
Microorganisms are beneficious for the regeneration of our environment. The scope of this work demonstrates the elimination of malathion and cypermethrin from the polluted environment by isolated bacterial strains can be considered as innocuous and efficient technology and used for treating agricultural wastes contaminated with pesticides. The microbial floral continuously exposed to toxic compounds naturally
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therefore, these microorganisms can survive in polluted environments by having high bioremediation potential.
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Figures
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Figure. 5.1 Minimal salt medium
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Figure.5.2 Sample extraction for pesticide residues analysis
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Figure.5.3 GC chromatogram of 12.5ppm of Malathion standard at 7.81 minutes
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Figure.5.4 GC chromatogram of 12.5ppm of Cypermethrin standard at 12.68
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0
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(A) (B) Figure. 5.5 Chromatogram showing Malathion and Cypermethrin degradation peaks. (A) GC chromatogram of Malathion degradation peaks. (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour (B) GC chromatogram of Cypermethrin degradation peaks (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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Figure. 5.6 0.1 ppm Malathion degraded by E. coli in minimal salt media
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Figure. 5.7 0.5 ppm Malathion degraded by E. coli in minimal salt media
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Figure. 5.8 2.0 ppm Malathion degraded by E. coli in minimal salt media
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Figure. 5.9 50 ppm Malathion degraded by E. coli in minimal salt media
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Figure. 5.10 0.1 ppm Malathion degraded by Bacillus licheniformis in minimal salt media
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Figure. 5.11 0.5 ppm Malathion degraded by Bacillus licheniformis in minimal salt media
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Figure. 5.12 2.0 ppm Malathion degraded by Bacillus licheniformis in minimal salt media
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Figure. 5.13 50 ppm Malathion degraded by Bacillus licheniformis in minimal salt media
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Figure. 5.14 0.1 ppm Malathion degraded by Pseudomonas aeruginosa in minimal salt media
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Figure. 5.15 0.5 ppm Malathion degraded by Pseudomonas aeruginosa in minimal salt media
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STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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423
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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120
100
80
60 remaining 40
20 2 ppm 2 Malathion concentration % 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.16 2.0 ppm Malathion degraded by Pseudomonas aeruginosa in minimal salt media
424
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60 remaining 40
20 50 ppm 50 Malathion concentration % 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.17 50 ppm Malathion degraded by Pseudomonas aeruginosa in minimal salt media
425
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
426
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60
remaining 40
20
0.1 ppm 0.1 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.18 0.1 ppm Cypermethrin degraded by E. coli in minimal salt media
427
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60
remaining 40
20
0.5 ppm 0.5 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.19 0.5 ppm Cypermethrin degraded by E. coli in minimal salt media
428
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
429
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60 remaining 40
20 2 ppm 2 Cypermethrin concentration % 0 0 5 10 15 20 25 30 Time (hours)
Figure.5.20 2.0 ppm Cypermethrin degraded by E. coli in minimal salt media
430
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60 remaining 40
20
50 ppm 50 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure.5.21 50 ppm Cypermethrin degraded by E. coli in minimal salt media
431
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
432
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60
remaining 40
20
0.1 ppm 0.1 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.22 0.1 ppm Cypermethrin degraded by Bacillus licheniformis in minimal salt media
433
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60
remaining 40
20
0.5 ppm 0.5 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.23 0.5 ppm Cypermethrin degraded by Bacillus licheniformis in minimal salt media
434
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
435
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60 remaining 40
20
2 ppm 2 Cypermethrin concentration % 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.24 2.0 ppm Cypermethrin degraded by Bacillus licheniformis in minimal salt media
436
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60 remaining 40
20
50 ppm 50 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.25 50 ppm Cypermethrin degraded by Bacillus licheniformis in minimal salt media
437
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
438
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60
remaining 40
20
0.1 ppm 0.1 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.26 0.1 ppm Cypermethrin degraded by Pseudomonas aeruginosa in minimal salt media
439
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60
remaining 40
20
0.5 ppm Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
Figure. 5.27 0.5 ppm Cypermethrin degraded by Pseudomonas aeruginosa in minimal salt media
440
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
120
100
80
60 remaining 40
20
2 ppm 2 Cypermethrin concentration % 0 0 5 10 15 20 25 30 Time (hours)
441
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Figure. 5.28 2.0 ppm Cypermethrin degraded by Pseudomonas aeruginosa in minimal salt media
120
100
80
60 remaining 40
20
50 ppm 50 Cypermethrin % concentration 0 0 5 10 15 20 25 30 Time (hours)
442
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Figure. 5.29 50 ppm Cypermethrin degraded by Pseudomonas aeruginosa in minimal salt media
443
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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Figure. 5.30 Percentage of residual Malathion in minimal salt media inoculated with E.
444
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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coli
445
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
446
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Figure. 5.31 Percentage of residual Cypermethrin in minimal salt media inoculated with E. coli
447
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
448
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Figure. 5.32 Percentage of residual Malathion in minimal salt media inoculated with Bacillus licheniformis
449
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
450
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Figure. 5.33 Percentage of residual Cypermethrin in minimal salt media inoculated with Bacillus licheniformis
451
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
452
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Figure. 5.34 Percentage of residual Malathion in minimal salt media inoculated with Pseudomonas aeruginosa
453
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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454
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Figure. 5.35 Percentage of residual Cypermethrin in minimal salt media inoculated with Pseudomonas aeruginosa
Tables
Table. 5.1. Recipe for 1 L of 5 x M9 salt (autoclaved) and 1 L of supplemented Minimal growth medium
5 x M9 salts (autoclaved)
KH2PO4 15.0 g
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STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Na2HPO4.7H2O 64.0 g
NaCl 2.5 g
NH4Cl2 (pH adjusted to 7.2 with NaOH) 5.0 g
Minimal growth medium
5 x M9 salt (autoclaved) 200.0 ml
D-glucose stock (20 g100 ml) (0.2 _m filter sterilized) 20.0 ml
Basal Vitamins Eagle Media 10.0 ml
456
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
1 M MgSO4 (autoclaved) 2.0 ml
1 M CaCl2 (autoclaved) 0.1 ml
457
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.2. Percentage of Malathion degraded by E. coli in minimal salt media after 4 hours Initial Retenti Peak Initial Malathion Malathion Malathion Malathion on corrected concentration concentration degradation after concentration Time area (µg/250ml) After 4hrs 4hrs of incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 64052897 12.5 - - 0.1 7.81 4985842 25 9.73 61.06 0.5 7.81 7500758 125 14.6 88.32 2 7.81 3843894 500 7.5 98.55 50 7.81 53990801 12500 105 99.16
Table. 5.3. Percentage of Malathion degraded by E. coli in minimal salt media after 8 hours Initial Retenti Peak Initial Malathion Malathion Malathion Malathion on corrected concentration concentration degradation after concentration Time area (µg/250ml) After 8 hrs 8hrs of incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 64052897 12.5 - - 0.1 7.81 909,538 25 1.77 92.9
458
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
0.5 7.81 2811758 125 5.623 95.61 2 7.81 2108920 500 4.11 99.17 50 7.81 45962851 12500 89.69 99.28
Table. 5.4. Percentage of Malathion degraded by E. coli in minimal salt media after 24 hours Initial Retenti Peak Initial Malathion Malathion Malathion Malathion on corrected concentration concentration degradation after concentration Time area (µg/250ml) After 24 hrs 24 hrs of incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 64052897 12.5 - - 0.1 7.81 458933 25 0.917 99.63 0.5 7.81 2790717 125 5.45 95.64 2 7.81 901246 500 1.802 99.63 50 7.81 25682144 12500 51.364 99.58
459
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
Concentration (ppm) % degradation
Concentration (ppm) 4 hours SADIA SIRAJUDDIN 8 hours 24 hours
Average STDEV Average STDEV Average STDEV
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STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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0.1 61.32 60.39 61.47 61.06 0.58 92.9 91.67 92.45 92.34 0.62 99.63 99.45 99.22 99.43 0.21
0.5 87.16 89.38 88.49 88.34 1.11 95.64 94.68 95.33 95.61 0.49 95.5 94.76 95.94 95.64 0.6
2 98.4 98.21 99.05 98.55 0.44 99.17 99.04 99.24 99.15 0.1 99.63 99.45 99.35 99.48 0.14
461
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
50 99.14 99.32 99.03 99.16 0.14 99.28 99.43 99.19 99.3 0.12 99.8 99.83 99.83 99.75 0.14
Table. 5.5. Cumulative result of percentage Malathion degradation by E. coli in minimal salt media
462
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.6. Percentage of Malathion degraded by Bacillus licheniformis in minimal salt media after 4 hours Initial Retenti Peak Initial Malathion Malathion Malathion remains Malathion on corrected concentration concentration after 4 hrs of concentration Time area (µg/250ml) After 4 hrs incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 7075604.5 12.5 0.1 7.81 595317 25 10.51 58 0.5 7.81 3044750 125 53.78 56.96 2 7.81 1045069 500 17.14 96.57 50 7.81 2317837 12500 38.76 99.68
463
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.7. Percentage of Malathion degraded by Bacillus licheniformis in minimal salt media after 8 hours Initial Retenti Peak Initial Malathion Malathion Malathion remains Malathion on corrected concentration concentration after 8hrs of concentration Time area (µg/250ml) After 8 hrs incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 7075604.5 12.5 0.1 7.81 909538 25 1.77 92.9 0.5 7.81 2790717 125 5.45 95.64 2 7.81 2108920 500 4.11 99.17 50 7.81 45962851 12500 89.69 99.28
Table. 5.8. Percentage of Malathion degraded by Bacillus licheniformis in minimal salt media after 24 hours Initial Retenti Peak Initial Malathion Malathion Malathion remains Malathion on corrected concentration concentration after 24 hrs of concentration Time area (µg/250ml) After 24 hrs incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 7075604.5 12.5
464
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
0.1 7.81 458933 25 0.917 99.63 0.5 7.81 2,811,758 125 5.623 95.50 2 7.81 901246 500 1.802 99.63 50 7.81 25682144 12500 51.364 99.58
465
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Malathion
Concentration % degradation (ppm) 4 hours 8 hours 24 hours
466
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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Average STDEV Average STDEV Average STDEV
0.1 57.96 57.97 58.07 58 0.06 92.89 92.94 92.87 92.9 0.04 99.69 99.61 99.59 99.63 0.05
0.5 96.6 96.59 96.52 96.57 0.04 99.11 99.19 99.21 99.17 0.05 99.69 99.61 99.59 99.63 0.05
2 57.96 57.97 58.07 58 0.06 92.89 92.94 92.87 92.9 0.04 99.69 99.61 99.59 99.63 0.05
467
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
50 96.6 96.59 96.52 96.57 0.04 99.11 99.19 99.21 99.17 0.05 99.69 99.61 99.59 99.63 0.05
Table. 5.9. Cumulative result of percentage Malathion degradation by Bacillus licheniformis in minimal salt media
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STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.10. Percentage of Malathion degraded by Pseudomonas aeruginosa in minimal salt media after 4 hours Initial Retenti Peak Initial Malathion Malathion Malathion remains Malathion on Time corrected concentration concentration after 4 hrs of concentration (min) area (µg/250ml) After 4 hrs incubation (µg/ml) (µg/250ml) (%) Standard 7.81 7075604.5 12.5 0.1 7.81 553415 25 9.776 60.89 0.5 7.81 3034004 125 53.59 57.13 2 7.81 1035090 500 18.28 96.34 50 7.81 1708783 12500 30.18 99.75
469
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.11. Percentage of Malathion degraded by Pseudomonas aeruginosa in minimal salt media after 8 hours Initial Retenti Peak Initial Malathion Malathion Malathion remains Malathion on corrected concentration concentration after 8hrs of concentration Time area (µg/250ml) After 8 hrs incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 7075604.5 12.5 - - 0.1 7.81 291200 25 5.144 79.42 0.5 7.81 569132 125 10.05 91.95 2 7.81 721997 500 12.75 97.44 50 7.81 867108 12500 12.31 99.00
Table. 5.12. Percentage of Malathion degraded by Pseudomonas aeruginosa in minimal salt media after 24 hours Initial Retenti Peak Initial Malathion Malathion Malathion remains Malathion on corrected concentration concentration after 24 hrs of concentration Time area (µg/250ml) After 24 hrs incubation (µg/ml) (min) (µg/250ml) (%) Standard 7.81 7075604.5 12.5 - -
470
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
0.1 7.81 252197 25 4.45 82.17 0.5 7.81 98102 125 1.733 98.61 2 7.81 131591 500 2.32 99.53 50 7.81 2419538 12500 12.55 99.96
471
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Malathion
Concentration % degradation (ppm) 4 hours 8 hours 24 hours
472
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Average STDEV Average STDEV Average STDEV
0.1 79.4 79.4 79.3 82.1 82.1 61.0 60.9 60.9 60.98 0.07 4 3 9 79.42 0.03 82.2 9 2 82.17 0.04
0.5 96.3 97.4 97.4 97.4 99.5 99.5 99.4 96.3 96.3 9 96.34 0.04 5 6 1 97.44 0.03 3 7 9 99.53 0.04 2 61.0 60.9 60.9 60.98 0.07 79.4 79.4 79.3 79.42 0.03 82.2 82.1 82.1 82.17 0.04
473
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
2 4 3 9 9 2
50 96.3 97.4 97.4 97.4 99.5 99.5 99.4 96.3 96.3 9 96.34 0.04 5 6 1 97.44 0.03 3 7 9 99.53 0.04
Table. 5.13. Cumulative result of percentage Malathion degradation by Pseudomonas aeruginosa in minimal salt media
474
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.14. Percentage of Cypermethrin degraded by E. coli in minimal salt after 4 hours Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 4 hrs concentration (min) area concentration After 4 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 22350446 12.5 - - 0.1 12.68 1861671 25 10.41 58.36 0.5 12.68 4578093 125 20.0 84 2 12.68 4195598 500 18.39 96.322 50 12.68 5409254 12500 23.71 99.811
Table. 5.15. Percentage of Cypermethrin degraded by E. coli in minimal salt after 8 hours
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STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
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Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 8 hrs concentration (min) area concentration After 8 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 22350446 12.5 - - 0.1 12.68 40611 25 2.27 90.91 0.5 12.68 1851043 125 10.35 91.72 2 12.68 261064 500 1.46 99.700 50 12.68 1583414 12500 8.855 99.93
Table. 5.16. Percentage of Cypermethrin degraded by E. coli in minimal salt after 24 hours Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 24 hrs concentration (min) area concentration After 24 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 22350446 12.5 - - 0.1 12.68 - 25 - - 0.5 12.68 6062086 125 0.33 99.73 2 12.68 4847776 500 0.26 99.99 50 12.68 17715385 12500 0.98 99.99
476
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
477
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Cypermethrin
Concentration % degradation (ppm) 4 hours 8 hours 24 hours
478
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Average STDEV Average STDEV Average STDEV
0.1 58.3 58.7 59 58.71 0.32 90.91 90.87 90.85 90.87 0.03 100 99.96 99.99 99.98 0.02
0.5 84 84.4 85.5 84.6433 0.77 91.72 91.32 92 91.68 0.34 99.73 98.9 99.56 99.3 0.43
2 96.3 96.3 95.9 96.2 0.26 99.7 99.5 99.67 99.62 0.1 99.99 99.32 99.12 99.47 0.45
479
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
50 99.8 99.7 99.2 99.59 0.33 99.93 99.97 99.95 99.95 0.02 99.99 99.89 99.71 99.86 0.14
Table. 5.17. Cumulative result of percentage Cypermethrin degradation by E. coli in minimal salt after
480
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.18. Percentage of Cypermethrin degraded by Bacillus licheniformis in minimal salt after 4 hours Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 4 hrs concentration (min) area concentration After 4 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 5378617 12.5 - - 0.1 12.68 754569 25 17.53 29.85 0.5 12.68 26167479 125 6.08 95.13 2 12.68 1913481 500 44.46 91.11 50 12.68 664213 12500 15.43 99.87
Table. 5.19. Percentage of Cypermethrin degraded by Bacillus licheniformis in minimal salt after 8 hours
481
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 8 hrs concentration (min) area concentration After 8 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 5378617 12.5 - - 0.1 12.68 743867 25 17.2 30.84 0.5 12.68 255592 125 5.94 95.24 2 12.68 1141339 500 26.52 94.69 50 12.68 589401 12500 13.69 99.89
Table. 5.20. Percentage of Cypermethrin degraded by Bacillus licheniformis in minimal salt after 24 hours Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 24 hrs concentration (min) area concentration After 24 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 5378617 12.5 - - 0.1 12.68 718684 25 16.70 33.19 0.5 12.68 173336 125 4.03 96.77 2 12.68 948903 500 22.05 95.58 50 12.68 589693 12500 13.70 99.89
482
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
483
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Table. 5.21 Cumulative result of percentage Cypermethrin degradation Bacillus licheniformis in minimal salt
484
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
Cypermethrin
% degradation
Concentration (ppm) 4 hours 8 hours 24 hours
Average STDEV Average STDEV Average STDEV
0.1 29.9 29.9 29.8 29.9 0.0 30.9 30.9 30.8 30.8 0.0 33.2 33.2 33.1 33.2 0.1
485
STUDIES ON BIODEGRADATION OF PESTICIDE FROM WASTEWATER THROUGH ITS INDIGENOUS MICROBIAL FLORA
SADIA SIRAJUDDIN
0.5 91.1 91.2 91.1 91.1 0.0 94.8 94.6 94.7 94.7 0.1 95.7 95.6 95.5 95.6 0.1
2 29.9 29.9 29.8 29.9 0.0 30.9 30.9 30.8 30.8 0.0 33.2 33.2 33.1 33.2 0.1
50 91.1 91.2 91.1 91.1 0.0 94.8 94.6 94.7 94.7 0.1 95.7 95.6 95.5 95.6 0.1
486
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Table. 5.22. Percentage of Cypermethrin degraded by Pseudomonas aeruginosa in minimal salt after 4 hours Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 4 hrs concentration (min) area concentration After 4 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 69,775,288 12.5 - - 0.1 12.68 9,206,154 25 16.49250 34.03 0.5 12.68 14,468,404 125 25.9196 79.27 2 12.68 25818655 500 46.253221 90.75 50 12.68 9,256,397 12500 16.5825 99.86
Table. 5.23. Percentage of Cypermethrin degraded by Pseudomonas aeruginosa in minimal salt after 8 hours Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 8 hrs concentration (min) area concentration After 8 hrs of incubation
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(µg/ml) (µg/250ml) (µg/250ml) (%)
Standard 12.68 69,775,288 12.5 - - 0.1 12.68 852,799 25 1.5277 93.89 0.5 12.68 10,320,780 125 18.48931 85.21 2 12.68 23,395,392 500 41.9120 91.62 50 12.68 8,410,812 12500 15.0676 99.88
Table. 5.24. Percentage of Cypermethrin degraded by Pseudomonas aeruginosa in minimal salt after 24 hours Initial Retention Peak Initial Cypermethrin Cypermethrin Cypermethrin Time corrected Cypermethrin concentration remains after 24 hrs concentration (min) area concentration After 24 hrs of incubation (µg/ml) (µg/250ml) (µg/250ml) (%) Standard 12.68 69,775,288 12.5 - - 0.1 12.68 171739 25 0.30 98.77 0.5 12.68 1.253244 125 12.53 90.00 2 12.68 8,057,843 500 14.43 97.12 50 12.68 2707861 12500 4.851 99.962
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Cypermethrin % degradation Concentration (ppm) 4 hours 8 hours 24 hours
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Average STDEV Average STDEV Average STDEV
0.1 34.0 34.1 34.0 34.0 0.1 93.9 93.9 93.8 93.9 0.0 98.8 98.7 98.8 98.8 0.1
0.5 90.8 90.8 90.7 90.8 0.0 91.7 91.6 91.6 91.6 0.0 97.1 97.1 97.2 97.1 0.1 2 34.0 34.1 34.0 34.0 0.0 93.9 93.9 93.8 93.9 0.0 98.8 98.7 98.8 98.8 0.1
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50 90.8 90.8 90.7 90.8 0.0 91.7 91.6 91.6 91.6 0.0 97.1 97.1 97.2 97.1 0.1
Table. 5.25. Cumulative result of percentage Cypermethrin degradation by Pseudomonas aeruginosa in minimal
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coli. Biotechnology progress, 20(5), 1567-1571.
5.6. Appendix 5.6.1. Appendix-1
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GC chromatogram of 0.1 ppm Malathion degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 0.5 ppm Malathion degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 2.0 ppm Malathion degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 50ppm Malathion degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
5.6.2. Appendix-2
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GC chromatogram of 0.1ppm Malathion degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 0.5ppm Malathion degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 2.0ppm Malathion degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 50ppm Malathion degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
5.6.3. Appendix-3
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GC chromatogram of 0.1ppm Malathion degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 0.5ppm Malathion degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 2.0ppm Malathion degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 50ppm Malathion degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
5.6.4. Appendix-4
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GC chromatogram of 0.1 ppm Cypermethrin degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 0.5 ppm Cypermethrin degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24h
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GC chromatogram of 2.0 ppm Cypermethrin degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 50 ppm Cypermethrin degradation by Escherichia coli (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
5.6.5. Appendix-5
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GC chromatogram of 0.1ppm Cypermethrin degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 0.5ppm Cypermethrin degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 2.0ppm Cypermethrin degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 50ppm Cypermethrin degradation by Bacillus licheniformis (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
5.6.6. Appendix-6
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GC chromatogram of 0.1ppm Cypermethrin degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 0.5ppm Cypermethrin degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 2.0ppm Cypermethrin degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour
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GC chromatogram of 50ppm Cypermethrin degradation by Pseudomonas aeruginosa (a) high peak at 4hour, (b) medium peak at 8hour, (c) small peak at 24hour Chapter 6
Degradation studies on Malathion and Cypermethrin by Carboxylesterase enzyme of Escherichia coli IES-02 (KU593482) and analysis of novel product appearance by GC-MS
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6.1. Introduction
The use of pesticides in controlling agricultural pests continue to increase and their usage possibly cannot be discontinued in future. Thus environments will be a continuous sink for the millions of pounds of the world's pesticides. At present, environmental impact of the residual organophosphorus pesticides (e.g. Malathion) and pyrethroids pesticides (e.g. Cypermethrin) draw more attention (Liu et al., 2005; Lee et al., 2002).
Riser-Roberts, (1998) and Marttinen et al., (2002) stated that for the detoxification of pesticides many procedures are reported but many of these do not involve in complete
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elimination rather modified them or transferred from one phase to another. Current disposal methods of pesticides includes, incineration, soil injection, photodecomposition, chemical degradation, biodegradation by disposal pits, landfills and enzymatic treatment. However, enzymatic degradation offer an innovative “green” solution for the elimination of xenobiotics (Phale et al., 2007).
Bollag, (1974) while studying enzymatic treatments suggested that it is necessary to investigate the metabolic reactions, enzyme systems and identify the resulting products because biochemical reactions involved in pesticide metabolism contribute to the clarification of the relationship between chemical structure and susceptibility to
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probable microbial transformations and also give a basis for the understanding of pesticide short or long persistence in a natural environment.
Munnecke, (1980) suggested that soluble or immobilized enzymes can be useful for detoxification of contaminated areas and pesticide-industrial effluents. Esterase used for the ester bonds formation. Esterase do not require cofactors and this property makes them attractive biocatalysts (Godinho et al., 2011). Esterase play a key role in the degradation of pollutants (Bornscheuer 2002; Cheng et al., 1996; Liu et al., 2005). Carboxylesterases used as elimination of toxic compounds by hydrolyzing the ester bond are more effective than existing chemical methods (Satoh, 1987; Jakoby and Ziegler 1990; Liu et al., 2005; Tallur et al., 2008; Zhang et al., 2010; Guo et al., 2009).
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Naveen et al., (2011) reported that biological treatment involves the transformation of complex compound into non-hazardous. In addition, the metabolites may be more easily degraded by other microorganisms. Another advantage of enzyme treatment is that the bio specificity of the parent molecule is destroyed. Reclamation of pesticides by appropriate enzymes rather than by whole microbial cells is advantageous since enzymes have tendency to tolerate extreme environmental conditions like: pH, temperature high salt and solvent concentrations.
Enzymatic detoxification of organophosphorus insecticides reported by many researchers (Kim et al, 2005; Cheng et al., 1993; Gilbert et al., 2003; Richins et al., 1997; Chen-Goodspeed; 2001; Di Sloudi et al., 1999).
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Kasai, (2004); Liang, (2005) and Maloney (1993) stated that pyrethroid-degraded by microorganisms by the oxidation of cytochrome P450s and hydrolysis of ester bonds by the action of carboxylesterases.
Five pesticide degrading bacterial strains (Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa, Micrococcus luteus, and Staphylococcus aureus) were isolated in this study (see in Chapter 2) from wastewater of Lyari River. In which three bacterial strains (Escherichia coli, Bacillus licheniformis, Pseudomonas aeruginosa) were proved to be efficient in malathion and cypermethrin degradation (see in Chapter 5). Results in chapter 5 shows that among these three bacterial species Escherichia coli found more efficacious than other species. Gas chromatography analysis confirmed that
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50 ppm malathion and cypermethrin residues almost completely disappears within 4 hours of incubation.
In the present study Carboxylesterase was purified from Escherichia coli capable for the hydrolysis of malathion and cypermethrin. Carboxylesterase degradation activity of malathion and cypermethrin by using `crude, partially purified and purified enzyme was monitored and gas chromatography analysis confirmed that 50 ppm Malathion and Cypermethrin residues completely degrade within 1 hour of incubation. Gas chromatography mass spectrometry analysis confirmed the presence of degradation metabolites formed by the action carboxylesterase activity.
Carboxylesterase assayed by Gas chromatography and Mass spectrometry confirmed the
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transformation products formed during the enzymatic biodegradation. Previously Gas chromatography used to determine lipase activity (Plank and Lorbeer, E, 1995) and High performance liquid chromatography used for the estimation of several enzymes activities (Gilham and Lehner, 2005; Brown and Krstulovic, 1979; Soeda et al., 1981; Zakaria and Brown, 1981; Pace et al., 1989; Pietta et al., 1987).
For the present study the carboxylesterase enzyme were extracted from Escherichia coli strain and were used for the biodegradation of malathion and cypermethrin.
6.2. Material and methods
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6.2.1. Extraction of extracellular protein of Escherichia coli capable for degrading Malathion and Cypermethrin
For enzyme production, fresh minimal salt media (see in 5 chapter 5.2.2) supplemented with 50 ppm of malathion and cypermethrin was inoculated with Escherichia coli (for preparation of inoculum see in 4 chapter 4.2.6). The bacterial culture was incubated for 24 hours at 37°C on a rotary shaker at 150 rpm (Thermo electron corporation model no. 2873, USA) and centrifuge (5810 R, Eppendorf AG, Hamburg, Germany) at 40006x g for 30 min at 4°C. Cell pallets were removed and the resulting cell free filtrate were used for additional purification as an enzyme source and stored at -20° C until further use. Total protein content was estimated in the cell free filtrate using BSA (Bovine serum albumin) as a standard. Presented in Fig.6.1.
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6.2.2. Estimation of Total Protein
Total protein concentration was determined by Lowry’s method (Lowry et al., 1951)
Reagents Preparation
Reagent-A Sodium hydroxide (4.0 g) Sodium carbonate (20.0 g) Sodium hydroxide was dissolved in 300.0 ml double deionized water. Sodium carbonate was also added. Double deionized water were used to volume makeup up to 1 L and
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stored at 4°C.
Reagent-B1 Copper sulfate (1.0 g) Double deionized water (100.0 ml) were used to dissolve coper sulfate and kept at room temperature.
Reagent-B2 Sodium potassium tartrate (2.0 g) Double deionized water (100.0 ml) were used to dissolve Sodium potassium tartrate and kept at room temperature.
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Reagent-C
Reagent A, B1 and B2 were mixed together (100:1:1). This reagent was freshly prepared before every use.
Reagent-E
This reagent was freshly diluted prior to every use prepared by diluting Folin-Ciocalteu reagent in 1:1 ratio with double deionized water.
Standard (Stock Solution: 1.0 mg ml-1)
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0.1 g BSA (Bovine serum albumin) was used as a stock standard, dissolved in 100.0 ml double deionized water. Stock solution kept at 4°C.
Working Solution (0.250 mg ml-1)
Stock solution (1.0 ml) was diluted by adding double deionized water (3.0 ml) that prepared 0.250 mg ml-1 final concentration of the working solution.
Total Protein Assay
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Test tubes were labelled as 1, 2, 3, 4, 5, 6, 7, 8, 9 and S10 that contained 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 ml of working standard solution, respectively. Separate tube was marked as test (T) that contained the sample (0.025 ml). Final volume of all standard tubes and test (T) were made up to 1.0 ml by incorporating double deionized water. Another tube was marked as blank that contained 1.0 ml double deionized water only. Freshly prepared reagent-C (5.0 ml) was incorporated in all tubes, vortex thoroughly and incubated for 15 minutes at room temperature. Reagent-E (0.5 ml) was added in all tubes and re-incubated for 15.0 minutes in dark. Optical density was recorded against reagent blank at 650 nm.
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Absorbance values of all standard tubes were used to plot the standard curve. Total protein (mg ml-1) was estimated by the formula given below: 푻풐풕풂풍 푷풓풐풕풆풊풏 (풎품 풎풍-1) 푶푫 풐풇 푻풆풔풕 = × 푪풐풏풄풆풏풕풓풂풕풊풐풏 풐풇 푺풕풂풏풅풂풓풅 푶푫 풐풇 푺풕풂풏풅풂풓풅 ퟏ. ퟎ × 푽풐풍풖풎풆 풐풇 푺풂풎풑풍풆
6.2.3. Purification of pesticide degrading protein
The Cell free filtrate (CFF) containing the enzyme was used for purification and all of the experiments were performed at 4°C presented in Fig. 6.2.
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6.2.3.1. Partial purification of pesticide degrading protein
Escherichia coli pesticide degrading protein was further purified by gradient precipitation technique ranging from 20 % to 60 % saturation of ammonium sulfate. Ammonium sulfate was gradually added in CFF (cell free filtrate) with nonstop stirring at 4° C and kept overnight for complete equilibration. The precipitate formed and collected by centrifugation at 4,000 x g for 30 min and the then obtained pellet dissolved in the 50 mM (pH 7.5) phosphate buffer saline. Enzyme activity and total protein was determined after ammonium sulfate precipitation of the samples.
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6.2.3.2. Purification of pesticide degrading protein
Partially purified pesticide degrading protein were desalted by desalting column PD-10 (GE Healthcare UK Ltd. Little Chalfont, Buckinghamshire, UK). Enzyme activity and total protein was determined after de-salting the samples. Centricon® centrifugal filter device (Ultracel YM-10. Millipore corporation USA) of 10.0 kDa membrane was used for concentration of partially purified desalted sample. Enzyme activity and total protein was determined after filtration of the samples.
6.2.4. Enzyme Assays for Carboxylesterase
Catalytic activity of malathion and cypermethrin was assayed by addition of 100µl
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enzyme (crude, partially purified, purified) into 5ml Phosphate Buffer Saline 50 mM (pH 7.5) containing 5 ppm of malathion and cypermethrin and incubated for 1 hour at 37° C. After that, samples were extracted with 5 ml n-hexane and the pesticide residues were monitored by gas chromatography (Agilent 6890N) with an electron capture detector by the method of Chen et al., (2011). A control experiment without malathion and cypermethrin in Minimal salt media was used for comparison.
Unit Definition
“One unit of enzyme activity (U) is defined as the amount of enzyme required to liberate
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1.0 µmol of product per minute under standard assay conditions”.
Unit Calculation
푼 푻풐풕풂풍 풂풄풕풊풗풊풕풚 (푼) = ( ) 푴 µ 풎풊풏 푷풆풂풌 풄풐풓풓풆풄풕풆풅 풂풓풆풂 풐풇 풔풂풎풑풍풆 = 푷풆풂풌 풂풓풆풂 풐풇 푺풕풂풏풅풂풓풅 ퟏ. ퟎ × 푪풐풏풄풆풏풕풓풂풕풊풐풏 풐풇 푺풕풂풏풅풂풓풅 × 푽풐풍풖풎풆 풐풇 푺풂풎풑풍풆 ퟏ. ퟎ ퟏ. ퟎ × × 푴풐풍풆풄풖풍풂풓 풘풆풊품풉풕 풐풇 풑풆풔풕풊풄풊풅풆 푹풆풂풄풕풊풐풏 푻풊풎풆 (풎풊풏)
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푻풐풕풂풍 푼풏풊풕풔 푺풑풆풄풊풇풊풄 푨풄풕풊풗풊풕풚 = 푻풐풕풂풍 푷풓풐풕풆풊풏
푻풐풕풂풍 푼풏풊풕풔 (풎풍 풎풊풏-1) 풀풊풆풍풅 (%) = × ퟏퟎퟎ 푯풊품풉풆풔풕 풗풂풍풖풆 풐풇 푻풐풕풂풍 푼풏풊풕풔 (풎풍 풎풊풏-1)
푷풖풓풊풇풊풆풅 풔풕풆풑 풔풑풆풄풊풇풊풄 풂풄풕풊풗풊풕풚 푷풖풓풊풇풊풄풂풕풊풐풏 풇풐풍풅 = 풄풓풖풅풆 풔풕풆풑 풔풑풆풄풊풇풊풄 풂풄풕풊풗풊풕풚
6.2.5. Molecular Weight Estimation of Carboxylesterase
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Fig. 6.3. Presented Native polyacrylamide gel electrophoresis (PAGE), was performed with 12.5% resolving gel and 4.0% stacking gel for the assessment of molecular weight by comparing its migration rates with known molecular weight standard markers. The technique was performed according to the method described by Ornstein (1964) and Davis (1964) with some modification.
Preparation of Reagents
Acrylamide Bis-acrylamide: (Solution A) 30.0 g Acrylamide 0.8 g Bis-acrylamide
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Both the polymer and cross linker were dissolved in double deionized water (80.0 ml) and the volume was made up to 100.0 ml. Filtered by Whatman® filter paper and stored at 4°C.
1.5 M Tris-HCl: (Solution B) pH-8.8 36.3 g Tris-HCl The salt was dissolved in double deionized water (200.0 ml) to attain 1.5 M concentration and pH-8.8 was achieved by gradually incorporating 1.0 N hydrochloric acid (HCl).
0.5 M Tris-HCl : (Solution C) pH-6.8 6.05 g Tris-HCl
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The salt was dissolved in double deionized water (100.0 ml) to achieve 0.5 M concentration and the pH was adjusted up to 6.8 by gradually incorporating 1.0 N hydrochloric acid (HCl). The buffer was stored at 4°C.
Ammonium persulfate (APS, 2.0%): (Solution E) Ammonium persulfate (10.0 g) freshly prepared The salt was dissolved in double deionized water 100.0 ml and placed on ice bath before use.
TEMED (N, N, N', N'-Tetramethylethylenediamine)
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Stored at 4°C.
0.025 M Tris-glycine Reservoir Buffer (pH-8.5) 3.025 g Tris 14.4 g Glycine Tris and Glycine were dissolved in 1000.0 ml double deionized water pH 8.5 maintained and kept at 4°C.
2.0 × Tris-HCl Sample Diluting Buffer (pH-6.8) 10%Glycerol 0.5 M Tris-HCl (Solution C) Bromophenol blue (Tracking dye)
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Glycerol (2.0 ml) was mixed thoroughly with solution C (2.5 ml) and few crystals of bromophenol blue dye was added. The volume was made up to 10.0 ml using double deionized water. Aliquots (1.0 ml) were prepared and stored at −20°C.
Tracking Dye Few crystals of Bromophenol blue were dissolved in sample diluting buffer. Molecular Weight Marker Molecular weight marker SDS7, Sigma-Aldrich, USA (14, 000 – 66,000 Dalton). Specification of marker can be seen in Table 6.1 Marker was prepared and used according to manufactured instruction. Preparation of Gel
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Composition of the gel prepared for Native-PAGE electrophoresis is presented in Table 6.2.
Staining of Gel At the end of the electrophoresis, protein bands were stained by the silver staining procedure of Morrissey (1981). Gel documentation system (Gel Doc 2000, Universal Hood, BioRad Laboratories Inc., USA) was used to estimate the approximate molecular mass of carboxylesterase with Quantity One Quantitative Software.
Silver Staining Preparation of Reagents Fixative 1
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Methanol (80 ml) Acetic Acid (20 ml) Fixative 2 Ethanol (50 ml) Acetic Acid (25 ml) 10x Oxidant 0.32 M Potassium dichromate (2.5 gm) 0.032 M Nitric acid (500 µl) 250 ml volume makeup with double deionized water and stored at 4° C. 1x oxidant freshly prepared from 10 x stock. 10x Silver Nitrate 0.12 M Silver Nitrate (5.10 gm)
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250 ml volume makeup with double Deionized water and stored at 4° C. 1x oxidant freshly prepared from 10 x stock.
Developer (Freshly Prepared) Sodium Carbonate (29.68 gm) Formaldehyde (0.5ml) 1 L volume makeup with double Deionized water
Stop Solution 5% Acetic acid
Time intervals for Staining Gel
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After Electrophoresis Gel immediately transferred to Fixative 1 and Table 6.3 represents the time intervals are appropriate for Polyacrylamide Gel.
6.2.6. Identification of metabolites produced by Malathion and Cypermethrin degradation
The non-degraded residues of malathion and cypermethrin were monitored through GC analysis and metabolites of malathion were monitored by GC MS Agilent 7890A (G3440A) with mass spectroscopy detector (MS Agilent 7000 GC/MS triple quadrupole) with auto-sampler, the injector mode was splitless and injector volume was 2.0 µL at an inlet temperature of 250°C. The analyses were done in electron ionization mode (70eV).
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An Agilent USB 393752HHP- 5MS column (30m × 250 µm × 0.25 µm) was used with a temperature program of 1 min at 70°C raised to 280°C for 20 min at 5°C/min. Helium gas at a flow rate of 3mL/min. The identified metabolites were matched with standard authentic compounds from the library database of NIST, USA (National Institute of Standards and Technology) available at HEJ, Research Institute of Chemistry, University of Karachi.
6.3. Results and Discussion
6.3.1. Malathion and Cypermethrin degrading enzyme Purification
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Malathion and cypermethrin degrading enzyme was partially purified by ammonium sulfate precipitation, desalted by PD-10 desalting column and concentrated by Centricon® centrifugal filter device. The purification results shown in Table 6.4 and 6.5. As a result of fractional precipitation of malathion degrading enzyme it was observed that the fold purification increased up to 1.19 times as compared to crude. The enzyme activity was detected in the retainant of filtration device indicating that the molecular weight of the enzyme is greater than 10 kDa. Finally malathion degrading protein was purified with a yield of 7.34% by 1.35-fold to a specific activity of 54.52 U/mg. Similarly in the case of cypermethrin degrading enzyme as a result of fractional precipitation fold purification increased up to 1.71 times as compared to crude. The enzyme activity was detected in the retainant of filtration device indicating that the molecular weight of the enzyme is
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greater than 10 kDa. Finally cypermethrin degrading protein was purified with a yield of 17.36% by 2.46-fold to a specific activity of 35.25 U/mg.
6.3.2. Estimation of Molecular weight of Malathion and Cypermethrin degrading enzyme by Native PAGE
For the identification of approximate molecular weight of malathion and cypermethrin degrading carboxylesterase native polyacrylamide gel electrophoresis was performed.
The results represented that malathion degrading carboxylesterase shows three sharp bands of 33.0, 30.0, 28.0 kDa (Fig. 6.4 Lane. 1). Approximate molecular weight of
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carboxylesterase was determined by corresponding it with the relative mobility of standard molecular weight marker (Fig. 6.4 Lane. M). Similar findings were also described by Yoshii et al.,(2008) who stated that the molecular weight of carboxylesterase band I (33kDa), II (30kDa) and III (28kDa) of the wheat kernel extract. Generally carboxylesterase are low molecular weight protein and different researcher reported their molecular weight in the range of 25-60 kDa. In earlier studies Barber et al.,(1968) also performed native PAGE and reported that there are slow moving and fast moving esterase with hydrolyzing activity of malathion. In another study Torres et al., (2008) reported that Bacillus licheniformis is able to produce 38.4 kDa esterase. Xie et al., (2013) reported that malathion hydrolyzing carboxylesterase appeared as a single band of 57.82 kDa.
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Molecular weight of cypermethrin degrading purified protein was approximately 31.0 KDa. The purified enzyme gave a single bands of cypermethrin hydrolyzing carboxylesterase (Fig. 6.5 Lane. 1). Approximate molecular weight of carboxylesterase was determined by corresponding it with the relative mobility of standard molecular weight marker (Fig. 6.5 Lane. M). Similar findings were also studied by Wang et al., (2009) and Li et al., (2008) that carboxylesterase molecular weight was approximately about 31 kDa. Fan et al., (2012) reported that the molecular weight of pyrethroid degrading esterases was found between the range of 29.0-44.3 kDa. Chen et al., (2013) reported molecular mass 41 kDa of beta-cypermethrin degrading enzyme.
Zhai et al., (2013) isolated Ochrobactrum anthropi from activated sludge capable of
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degrading pyrethroids pesticides by degrading enzymes appeared with a single band of 25 kDa. Many scientist described pyrethroid-hydrolyzing enzymes. Kasai, (2004) reported permethrinase with molecular mass of approximately 61 kDa from Bacillus cereus, Miguel and Eugenio, (2002) reported molecular weight 56 kDa of pyrethroid hydrolase.
Goullet, (1984) studied carboxylesterase B and Regnier et al., (1974) studied esterase with molecular mass of approximately 32 kDa produced by Escherichia coli. Zhang et al., (2008) reported that the E.coli carboxylesterase provides easier and safer remedy to use for the degradation of organophosphorus compounds. Carboxylesterase with different molecular weights have been reported, such as 53 kDa of carboxylesterase type-B from Bacillus sp. (Prim et al., 2001), 65 kDa of carboxylesterase B1 from Culexpipiens (Lan et al., 2006) and 31 kDa from Sphingobium sp. 597
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Sayali et al., (2013) reported that esterase are less explored enzymes having low literature survey as compared to the lipases so the future scope of research is highly valuable. In the present study malathion degrading carboxylesterase appeared with three sharp bands of 33.0, 30.0, 28.0 kDa and cypermethrin degrading carboxylesterase appeared with single band of 31.0 kDa.
6.3.3. Residual quantification analysis of Malathion and Cypermethrin by Gas Chromatography
GC was used for the determination of malathion and cypermethrin residual product degraded by the enzyme produced by E.coli. Malathion peak was detected at 7.81 minute and cypermethrin peak was detected at 12.68 minute can be seen in Fig. 6.6 598
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At each step of carboxylesterase purification, malathion and cypermethrin degradation was observed (Table. 6.6, 6.7 and Fig. 6.7, 6.8). The results identified that the purified enzyme strongly degraded malathion and cypermethrin (50ppm) within 1 hour, percentage degradation can be seen in Fig. 6.9, 6.10. Karns et al., (1987) stated that uses of cell free enzyme was successful in degradation process because whole microbial cells required fresh inoculum and medium nutrient.
6.3.4. Detection of Malathion Cypermethrin metabolites by GC-MS
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Different analytical techniques were used to determine pesticide residues in wastewater samples. Among those methods Gas chromatography mass spectrometry elected as the preferred method. Van Eerd et al., (2003) studied that In Pesticide metabolism firstly, parent compound changed by hydrolysis, reduction and oxidation reactions to produce less lethal product than the parent. Secondly metabolites of pesticide conjugate to amino acid or sugar, which reduces toxicity. Finally conjugated metabolite converted into non- toxic secondary conjugates. In these processes bacteria produces hydrolase, oxygenase and peroxidase.
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The degradation products of malathion and cypermethrin were extracted and analyzed gas chromatography mass spectrometry. The peaks of metabolite were confirmed by library database of national institute of standards and technology. The GC analysis peaks revealed that there were six malathion metabolites peaks occurring around 8–27 minutes (Table 6.8) including, monocarboxylic acid, dicarboxylic acid, succinic acid mercapto diethyle ester S-dimethyl phosphorodithioate, Oxalic acid isobutyl nonyl, Ethyl hydrogen fumarate and Diethyl maleate which appeared after 8hours of incubation. The mass spectrum of malathion metabolites are shown in Fig. 6.11, 16.2, 6.13.
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The carboxylesterase represent ideal enzymes for malathion detoxification. These results are consistent with previous reports which suggest the formation of these metabolites mainly via carboxylesterase activity (Paris et al., 1975; Lewis et al., 1975; Walker et al., 1976; Singh et al., 1989; Kim et al., 2005). In recent years, a number of carboxylesterase have been characterized in microorganisms that showed esterase activity (Morana et al., 2002; Mnisi et al., 2005).
Kim et al., (2005) reported that Malathion is toxic compound, and enzymatic decomposition processes by carboxylesterase rapidly converted malathion into less toxic compounds like malathion monoacid (MMA), malathion diacid (MDA), alcohols and carboxylic acids by ester hydrolysis. The result has been reported previously by
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Yoshii et al., (2008) that the carboxylesterase band I (33kDa), II (30kDa) tended to produce comparatively a large amount of malathion monocarboxylic acid and lowest molecular weight carboxylesterase band III (28kDa) have a tendency to produce a relatively small amount of malathion dicarboxylic acid.
6.3.5 Proposed Malathion degradation pathway
In the enzymatic degradation malathion metabolites were analyzed through GCMS. The metabolites retention time can be seen in (Table 6.8). It is significant that the
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transformation products formation is depended on the enzyme used in catalytic activity. Therefore, it is confirmed that the purified enzyme was carboxylesterase with three sharp bands of 33.0, 30.0, 28.0 kDa. The transformation products were identified and confirmed by the similarity of molecular ions of authentic compounds.
On the basis of the metabolites a novel malathion degradation pathway was offered (Fig. 6.14) The obtained data indicated that malathion was detected as a parent compound at 26.53 min, M.W 285 (Fig. 6.11 A B) and (Fig. 6.11 C) shows malathion degradation product including, malathion monocarboxylicacid 26.25 min, M.W 302 (Fig. 6.12 A), malathion dicarboxylic acid 23.48 min, M.W 274 (Fig. 6.12 B) with parallel formation of diethyl malate 8.19 min, M.W 172 (Fig. 6.13 C) were detected. Small
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amount of other metabolite was also detected, including ethyl hydrogen fumarate 8.58 min, M.W 143 (Fig. 6.13 B). Laveglia et al., (1997) reported similar malathion degradation pathway in which mono and diacid metabolites formed by the activity of carboxylesterase. Kamal et al., (2008) also reported that strain of Bacillus thuringiensis degrade malathion into monocarboxylic acid and dicarboxylic acid.
Carboxylesterase activity, in which malathion transform into monoacid and diacid, is the principal degradative pathway (Matsumura and Boush, 1966). Our and previous studies might also be different in terms of the malathion degradation efficiency of E. coli and formation of new minor metabolites.
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6.3.6 Proposed Cypermethrin degradation pathway
The GC analysis peaks revealed that there were Five cypermethrin metabolites peaks occurring around 16–41 minutes (Table 6.9) including, Benzyl butyl phthalate, Trichloroacetic acid hexadecyl ester, Phthalic acid isobutyl octadecyl ester, Cyclopentanecarboxylic acid and dodecyl ester and Phenol 2,4- bis 1,1, dimethyl ethyl which appeared after 8hours of incubation. The mass spectrum of cypermethrin metabolites are shown in Fig. 6.15, 6.16, 6.17. It has been confirmed by Miyamoto et al., (1968) that carboxylesterase hydrolyzed ester bond and responsible for the pyrethroids detoxification. Many other strains capable for degrading pyrethroids have been isolated and degradation studies were also studied (Guo et al., 2009; Zhang et al.,
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2010; Tallur et al., 2008; Maloeny et al., 2013). Cypermethrin degradation pathway was reported by (Roberts and Standen, 1977; Chen et al., 2011). Chen et al., (2012) and Liang et al., (2007) stated that for the detoxification of cypermethrin the hydrolysis of its carboxyl ester linkage was the first step.
Cypermethrin-derived degradation products were analyzed through GCMS and the RT (retention time) of metabolites can be seen in (Table 6.9). Transformation products significantly depended on the activity of used enzyme. Therefore, it is confirmed that the purified enzyme was carboxylesterase with single sharp bands of 31.0 kDa. The transformation products were identified and confirmed based on the similarity of their molecular ions with accurate compounds. On the basis of the metabolites, a novel
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cypermethrin degradation pathway was proposed (Fig. 6.18). The obtained data indicated that cypermethrin was detected as a parent compound at 40.56 min, M.W 281 (Fig. 6.15 A B) and (Fig. 6.15 C) shows cypermethrin degradation product including, Benzyl butyl phthalate 33.05 min, M.W 281 (Fig. 6.16 A), Trichloroacetic acid hexadecyl ester 26.84 minute, M.W 239 (Fig. 6.16 B) with parallel formation of Phthalic acid isobutyl octadecyl ester 24.41 min, M.W 223 (Fig. 6.16 C) were detected. Small amount of other metabolite was also detected, including Cyclopentanecarboxylic acid, dodecyl ester 23.45 min, M.W 194 (Fig. 6.17 A) and Phenol 2,4- bis 1,1, dimethyl ethyl 16.64 min, M.W 206 (Fig. 6.17 B).
The traditional methodologies of detoxification of pesticides like recycling, incineration landfilling and pyrolysis are destructive for environment. Therefore, enzymatic processes
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offers an economical and innocuous strategy for detoxification of toxic compounds. (Sogorb and Vilanova, 2002).
6.4. Conclusion
The outcomes of present research provided the advantageous knowledge for the remediation of malathion and cypermethrin by Escherichia coli isolated from polluted wastewater. It can be regarded as effective remediation technology and the proposed results should be operative for treating pesticide contaminated wastes. In this study malathion and cypermethrin degrading carboxylesterases was purified that shows the maximum malathion degradation (5ppm) within 1hour.
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Malathion and cypermethrin transformation products detected by GC/MS and the proposed degradation pathway was established. Malathion and cypermethrin metabolites confirmed the occurrence of carboxylesterase in Escherichia coli. Applications of cell free enzyme was successful in degradation process because whole microbial cells required fresh inoculum and medium nutrient.
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Figures
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Figure. 6.1 Production of pesticide degrading protein
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Figure. 6.2. Purification of pesticide degrading protein
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Figure. 6.3 Native polyacrylamide gel electrophoresis (PAGE)
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Figure. 6.4 Purification of Malathion degrading carboxylesterase from E.coli (28, 30, 33 KDa)
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(M) Low molecular weight markers were used purchased from (SDS7, Sigma-Aldrich, USA) (1) Proteins recovered during various purification steps were separated by Native (PAGE) gel electrophoresis, and stained with silver staining
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Figure. 6.5 Purification of Cypermethrin degrading carboxylesterase from E.coli (31 KDa) (M) Low molecular weight markers were used purchased from (SDS7, Sigma-Aldrich, USA) (1) Proteins recovered during various purification steps were separated by Native (PAGE) gel electrophoresis, and stained with silver staining
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(A)
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(B) Figure. 6.6 (A) Malathion standard 12.5 ppm (B) Cypermethrin standard 12.5 ppm
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Figure. 6.7 GC chromatogram of 5 ppm Malathion degradation by Carboxylesterase
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Figure. 6.8 GC chromatogram of 5 ppm Cypermethrin degradation by Carboxylesterase
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Figure. 6.9 Malathion % degradation
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Figure. 6.10 Cypermethrin % degradation
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(A) (B)
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(C) Figure. 6.11 (A) GC-chromatogram of Malathion standard (12.5ppm) (B) Accurate Malathion mass spectrum of the peak at 26.54 (C) GC chromatogram of Malathion main degradation products after 8hours of incubation
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(A)
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(B)
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(C) Figure. 6.12 Accurate mass spectrum of proposed malathion transformation products (A) malathion monocarboxylic acid with m/z 302at 26.253 min (B) malathion dicarboxylic acid with m/z 274 at 23.48 min (C) succinic acid , Mercapto, diethyle ester, S-ester with O, S-dimethyl phosphorodithioate with m/z 283 at 23.24 min
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(A)
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(B)
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(C) Figure. 6.13 Accurate mass spectrum of proposed malathion transformation products (A) Oxalic acid isobutyl nonyl ester with m/z 113 at 13.89 min (B) Ethyl Hydrogen Fumarate with m/z 143 at .8.58 min (C) Diethyl maleate with m/z 1172 at 8.19 min
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Figure. 6.14 The proposed pathway for detoxification of Malathion on the basis of the transformation products by the enzyme from E. coli
(A)
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(B)
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(C) Figure. 6.15 (A) GC-chromatogram of Cypermethrin standard (12.5ppm) (B) Accurate Cypermethrin mass spectrum of the peak at 40.57 (C) GC chromatogram of Cypermethrin main degradation products after 8hours of incubation 649
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(B)
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(C) Figure. 6.16 Accurate mass spectrum of proposed malathion transformation products (A) Benzyl butyl phthalate with m/z 281 at 26.84 min (B) Trichloroacetic acid hexadecyl ester with m/z 239 at 23.48 min (C) Phthalic acid isobutyl octadecyl ester with m/z 223 at 24.41min
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(A)
(B) Figure. 6.17 Accurate mass spectrum of proposed malathion transformation products (A) Cyclopentanecarboxylic acid, dodecyl ester with m/z 194 at 23.45 min (B) Phenol 2,4- bis (1,1, dimethyl ethyl)with m/z 206 at 16.64 min
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ProteinMICROBIAL Appropriate FLORA Molecular Weight (kDa) Albumin (Bovine) 66.0 Albumin (Egg) 45.0 Glyceraldehyde 3- phosphate Dehydrogenase 36.0
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FFigure 6.18. The proposed pathway for detoxification of Cypermethrin on the basis of the transformation products by the enzyme from E. coli
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(Rabbit Muscles Carbonic anhydrous (Bovine) 29.0 Trypsinogen (Bovine Pancreas) 24.0 Trypsin Inhibitor (Soybean) 20.0 α – Lactalbumin (Bovine albumin) 14.2 Tables
Table. 6.1 Specification of Molecular weight marker SDS7, Sigma-Aldrich, USA (14, 000 – 66,000 Dalton)
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Table. 6.2 Composition of electrophoresis gel for Native-PAGE
Gel Components Stacking Gel (4 %) Resolving Gel (12.5%) Solution A 1.5 ml 6.25 ml Solution B ----- 3.75 ml Solution C 1.2 ml ----- Solution E 0.25 ml 0.5 ml Double deionized water 7.04 ml 7.865 ml TEMED 0.006 ml 0.01 ml
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Table. 6.3 Silver staining procedure time intervals for Polyacrylamide Gel Reagents Time Fixative 1 30 minute Fixative 2 15 minute Fixative 2 15 minute Oxidizer 5 minute Double Deionized Water 5 minute Double Deionized Water 5 minute (until all yellow color removed) 1x Silver Reagent 20 minute Double Deionized Water 1 minute Developer 30 second Developer 5 minute Developer (optional) 5 minute Stop Solution 5 minute or over night
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Table. 6.4 Purification steps of Malathion degrading enzyme from E. coli Total Total Specific Purification Percent Purification steps Protein activity activity Fold yield (mg) (U**) (U/mg) Crude 1.848 74.159 40.12 1 100 20%* 0.335 14.709 43.9 1.09 19.83 40%* 0.375 17.27 46.05 1.14 23.28 60%* 0.287 13.81 48.1 1.19 18.622 Desalting 0.188 9.346 49.71 1.23 12.596 Filtration 0.1 5.45 54.52 1.35 7.34 * Ammonium sulfate precipitation
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** One unit of enzyme activity (U) was defined as the amount required to catalyze formation of 1 mmol of product per minute
Table. 6.5 Purification of Cypermethrin degrading protein from E.coli Total Specific Total Protein Purification Percent Purification steps activity activity (mg) Fold yield (U*) (U/mg) Crude 2.83 40.61 14.3 1 100 20%* 0.83 12.61 15.19 1.06 31.05 40%* 0.74 11.98 16.2 1.13 29.5 60%* 0.42 10.28 24.49 1.71 25.31 Desalting 0.322 9.07 28.16 1.96 22.33 Filtration 0.2 7.05 35.25 2.46 17.36 * Ammonium sulfate precipitation 662
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** One unit of enzyme activity (U) was defined as the amount required to catalyze formation of 1 mmol of product per minute
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Table. 6.6 Malathion degradation by purified enzyme Corrected Malathion % remaining After After 60 Purification steps Area After 20 min 40 min min Crude 9521527 45.30 70.46 88.12 20%* 1888539 20.56 35.62 41.30 40%* 2217356 50.90 80 85.97 60%* 1773200 70.73 94.56 95.10 Desalting 1200000 78.72 96.82 97.04 Filtration 700000 81.24 98.02 98.27 * Ammonium sulfate precipitation
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Table. 6.7 Cypermethrin degradation by purified enzyme Cypermethrin % Corrected After After 60 Purification steps remaining Area 40 min min After 20 min Crude 1024562 59.92 80.53 94.68 20%* 318125 57.74 91.2 94.96 40%* 302402 80.10 92.85 95.21 60%* 259451 90.33 94.76 95.89 Desalting 228790 91.41 96 96.37 Filtration 177840 95.56 96.99 97.18
* Ammonium sulfate precipitation
Table. 6.8 Metabolites produced by E. coli with 50 ppm of malathion Peak Mass to charge Retention ratio (m/z) Chemical name in NIST library time
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26.53 285 Malathion 26.25 302 Malathion Monocarboxylic Acid 23.48 274 Malathion Dicarboxylic Acid 23.24 283 Succinic acid , Mercapto, diethyle ester, S- ester with O, S-dimethyl phosphorodithioate 13.89 113 Oxalic acid isobutyl nonyl ester 8.58 143 Ethyl hydrogen fumarate 8.19 172 Diethyl maleate
Table. 6.9 Metabolites produced by E.coli with 50 ppm of Cypermethrin Peak Mass to charge Retention Chemical name in NIST library ratio (m/z) time 40.56 281 Cyclopropanecarboxylic acid, 3-(2,2 dochoroethenyl)-2,2 dimethyl-,cyano(3- phenoxyphenyl) methyl ester 33.05 281 Benzyl butyl phthalate
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26.84 239 Trichloroacetic acid hexadecyl ester 24.41 223 Phthalic acid isobutyl octadecyl ester 23.45 194 Cyclopentanecarboxylic acid, dodecyl ester 16.64 206 Phenol 2,4- bis (1,1, dimethyl ethyl)
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