Turk J Biol 36 (2012) 561-567 © TÜBİTAK doi:10.3906/biy-1112-44
Bioremediation of endosulfan-contaminated soil by using bioaugmentation treatment of fungal inoculant Aspergillus niger
Tejomyee Sadashiv BHALERAO School of Environmental and Earth Sciences, North Maharashtra University, P.B. No. 80, Jalgaon 425 001, Maharashtra - INDIA
Received: 29.12.2011
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Accepted: 27.04.2012
Abstract: The persistence of endosulfan and intermediate metabolite endosulfan sulfate in the environment and their toxic effects on biota necessitate their removal. This study investigated the bioaugmentation of endosulfan-contaminated soil by fungal inoculant Aspergillus niger ARIFCC 1053. The influence of bioaugmentation by A. niger on endosulfancontaminated soil was evaluated with the help of change in pH and released chloride, and by thin layer chromatography and gas chromatography analysis. Its effects on soil functionality were monitored by estimating dehydrogenase and arylsulfatase enzyme activities. The endosulfan degradation reached an undetectable level on day 15. The pH of the medium was nearly neutral (6.9) at the time of inoculation and it decreased to 3.6 on day 15. The amount of chloride released at particular intervals in the endosulfan degradation ranged from 28 μg mL–1 to 104 μg mL–1. Change in pH and the increase in released chloride correlated with metabolic activities involved in the simultaneous degradation of endosulfan. Endosulfan sulfate, an intermediate metabolite, was detected and had disappeared on day 11 of the process. The increase in enzyme activities is an indicator of soil fertility and suggests possible involvement of these enzymes in endosulfan degradation. These results demonstrate that bioaugmentation by A. niger may be a viable tool for the remediation of soil contaminated with endosulfan. Key words: Endosulfan, bioaugmentation, bioremediation
Introduction The extensive utilization of synthetic pesticides and/ or agrochemicals results in the accumulation of the residues of these toxic chemicals in soil. These accumulated residues deteriorate the overall quality of the soil and that of the surrounding environment, as well. Aside from this, pesticide-formulating industries are also contaminating the environment through various activities (1). Pesticide exposure inflicts chronic and acute threats to human health, and long-term low-dose exposure to pesticides causes immune suppression, hormonal disruption, diminished intelligence, reproductive abnormalities, and carcinoma (2).
Soil bioremediation is an option that offers the possibility to degrade or render various contaminants harmless using natural biological activity. For detoxification of pollutants such as pentachlorophenol (3), diesel oil (4), herbicides (5,6), and polyaromatic hydrocarbons (7,8), bioremediation was employed. Two important approaches of bioremediation are bioaugmentation and biostimulation. Bioaugmentation involves the addition of highly concentrated and specialized populations of specific microbes to a contaminated site to enhance the rate of contaminant biodegradation in the affected soil or water. Bioremediation through bioaugmentation is a promising, innovative, and cost-effective technology 561
Bioremediation of endosulfan-contaminated soil by using bioaugmentation treatment of fungal inoculant Aspergillus niger
for use in the cleanup of hazardous wastes. In this process, microorganisms transform environmental contaminants into harmless end products. The chlorinated organic pesticide endosulfan is the major agrochemical responsible for environmental pollution. Endosulfan is a mixture of 2 stereoisomers, α- and β-endosulfan, at a ratio of 7:3. It is registered under many trade names, including Thiofor, Thiodan, Cyclodan, Thimol, and Malix. It is extremely toxic to fish and aquatic invertebrates and has been reported for mammalian gonadal toxicity (9), genotoxicity (10), and neurotoxicity (11). It enters air, water, and soil environments during its use and manufacturing. Due to its hydrophobic nature, it is also associated with soil. It is reported that the half-life of soil-bound endosulfan is much higher than that of aqueous forms. Several researchers have described endosulfan degradation by bacteria and fungi including Corynebacterium sp., Nocardia sp., Mycobacterium sp., Pseudomonas fluorescens, Penicillium sp., Aspergillus sp., and Phanerochaete chrysosporium (12). However, generation of endosulfan sulfate is the major concern of endosulfan degradation research, as this metabolite is more toxic, persists longer in soils, and has bioaccumulation potential; therefore, detoxification of endosulfan is receiving serious attention (13). Among different groups of microorganisms, fungi have their own advantages in cultivation, maintenance, and tolerance to pesticides over bacteria, especially for chlorinated pesticides. All of these applications attract scientists to use fungi in the biodegradation process. As per the earlier report of Aspergillus niger ARIFCC 1053 showing good results at broth condition (14) in endosulfan degradation, endosulfan was degraded in just 120 h. On the basis of these results, A. niger ARIFCC 1053 was selected for the present biodegradation study of endosulfan contaminated soil. Keeping this view in the present study, the bioaugmentation process was applied for endosulfan degradation in soil. The previously isolated endosulfan degrader fungal culture A. niger ARIFCC 1053 was applied to endosulfan-contaminated soil to study its potential to be used for bioremediation. The degradation study was evaluated in accordance with pH monitoring, chloride release, and thin layer 562
chromatography (TLC)/gas chromatography (GC) analysis. Effects on soil functionality were evaluated by monitoring dehydrogenase and arylsulfatase (ARS) activities. Materials and methods Chemicals Technical grade endosulfan, a 35% emulsified preparation (Excel Industries Ltd., Mumbai, India), was used for the experiments. Standard endosulfan sulfate was obtained from Sigma-Aldrich, USA. All of the other reagents were of high purity and analytical grade. Microorganisms Fungal culture Aspergillus niger ARIFCC 1053, capable of degrading 350 mg L–1 endosulfan and isolated from soil during previous studies, was used in the present study (14). The culture was maintained on agar slopes of modified Czapek Dox medium containing sucrose, 30 g; NaNO3, 2 g; KCl, 0.5 g; MgSO4.7H2O, 0.5 g; glucose, 10 g; FeCl3, 10 mg; endosulfan, 0.5 g; and agar, 12 g in 1 L of distilled water at pH 6.8. Experimental design Locally collected black cotton soil (Jalgaon district, Maharashtra, India), with an undetectable level of endosulfan, was used for the study. The physicochemical characteristics of the soil were as follows: texture, clay loam; pH, 6.9; electrical conductivity, 0.5 mS m–1; organic matter, 2.56%; total nitrogen, 0.08%; and maximum water holding capacity, 0.42 mL g–1. Finely sieved (24 mesh size) soil (200 g) was moistened and placed in a pot for further experiment. The pot was amended with 350 mg (w v–1) of endosulfan and 20 mL of spore suspension (10–8 spores mL–1) of fungal culture A. niger. All of the experiments in this study were carried out in triplicate and the results are the means of the 3. Controls 1 and 2 were run with and without endosulfan for comparison. All of the pots were watered daily to maintain moisture. Endosulfan degradation studies Samples from the pot containing soil with 350 mg L–1 endosulfan and fungal culture A. niger were taken every alternate day up to the complete degradation of
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Analytical methods In order to analyze the residual endosulfan and the metabolites formed, cell-free culture broth was acidified to pH 2.0 with 6.0 M hydrochloric acid and extracted 3 times with ethyl acetate (16). Ethyl acetate fractions from each flask were pooled and aliquots were analyzed using TLC and gas chromatography GC. Samples (20-25 μL) were inoculated on silica gel (60 F 254) TLC plates (E. Merck, India) using an applicator system. The plates were developed in a solvent system comprising hexane, chloroform, and acetone (9:3:1). Spots were visualized by silver nitrate chromogenic reagent (14). GC analysis of the samples was performed with a flame ionization detector on a SE-30 column. The linear temperature gradient applied was from 75 °C to 25 °C at a rate of 3 °C/min. Nitrogen was used as the carrier gas. Soil enzyme activities during endosulfan degradation Dehydrogenase activity Dehydrogenase activity was measured using the triphenyl formazan (TPF) method (17). One gram of soil was taken in a screw cap vial, and added to it was 3.0 mL of 2,3,5-triphenyl tetrazolium chloride solution (3% w v–1) and 1.0 mL of distilled water, forming a thin layer of water above the surface of the soil. The tubes were incubated in the dark at 30 ± 1 °C for 24 h. After appropriate incubation (24 h), 25 mL of methanol was added to each tube, mixed on a vortex, and left to stand for some time. The mixture was then filtered through Whatman filter paper (No. 42) and the optical density of the filtrate was measured spectrophotometrically (Shimadzu UVMini 1240) at 485 nm. Dehydrogenase activity was expressed in terms of TPF produced day–1 g–1 of soil with reference to a standard curve of TPF.
Arylsulfatase activity Assay of ARS involved the colorimetric estimation of the p-nitrophenol (PNP) released by sulfatase activity when soil was incubated with buffered (pH 5.8) sodium-p-nitrophenyl sulfate (PNS) solution and toluene at 37 °C for 1 h (18). Added to 1 g of soil in a 50-mL Erlenmeyer flask were 4 mL of modified universal buffer (pH 5.8), 0.25 mL of toluene, and 1 mL of PNS solution. After mixing the contents, the flasks were stoppered and incubated at 37 °C. After 1 h of incubation, 1 mL of 0.5 M CaCl2 and 4 mL of 0.5 M NaOH were added to the reaction mixture. The contents of the flasks were swirled for few seconds and filtered through Whatman filter paper No. 42. The color intensity of the filtrate was spectrophotometrically measured at 420 nm. The standard curve was drawn at a range of 0-50 μg of PNS. Sulfatase activity was expressed in terms of μg PNP released h–1 g–1 of soil. All of the experiments were carried out in triplicate and the results are the means of the 3. Results and discussion Change in pH during endosulfan degradation in the soil The pH of the soil was nearly neutral at the time of inoculation. It decreased to 3.6 on day 15, at which time the endosulfan was at an undetectable level in the soil (Figure 1). The decrease in pH from nearly neutral to acidic was because of the metabolic activities involved in the simultaneous degradation of the endosulfan. These results show similarity with our previous broth study (14). The observations
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the endosulfan, and they were processed for analysis of pH and estimation of released chloride. All of the experiments were performed in triplicate. The released chloride was estimated using the mercuric thiocyanate method as determined by Jing and Huyop (15). The standard graph of sodium chloride stock solution (0.014 N) was prepared using a series of standards to plot the concentration of released chloride during endosulfan degradation by A. niger.
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Figure 1. Change in pH during endosulfan degradation in the bioaugmentation process.
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of Siddique et al. (19) also suggest that fungal and bacterial strains significantly decreased the pH of culture media during endosulfan degradation. Hussain et al. (20) demonstrated that endosulfan degradation by fungal strain resulted from a decrease in pH; the substantial drop in pH observed in the case of efficient strains could be due to the formation of acidic substances in greater amounts. All of these observations confirm that the pH of the soil decreased according to the simultaneous degradation of the endosulfan. However, in contradiction, Miles and Moy (21) reported an increase in the pH of broth culture with the biodegradation of endosulfan. In this study, after the complete degradation of the endosulfan, the pH of the soil recovered slowly. In both of the controls, there was no significant change in the pH up to day 15. Released chloride during endosulfan degradation The chloride content increased with endosulfan degradation by the bioaugmentation of A. niger in the soil. The amount of chloride released at particular intervals in the endosulfan degradation ranged from 28 μg mL–1 to 104 μg mL–1 (Figure 2). The significant increase of free chloride from the soil amended with endosulfan clearly indicates the degradation of the endosulfan. Verma et al. (22) also demonstrated the same results. Siddique et al. (19) noted that the release of the chloride ions produced probably led to the formation of hydrochloric acid, which reduced the
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pH of the culture medium. This observation correlates with the present bioaugmentation study of A. niger in the soil. In control 1, there was no significant change in the chloride content up to day 15. Endosulfan degradation in the soil as a function of the bioaugmentation process The biodegradation of technical grade endosulfan (containing 70:30 α- and β-endosulfan) was assessed using A. niger in soil. TLC was used to monitor the disappearance of the endosulfan and the formation of endosulfan sulfate. Endosulfan sulfate, an intermediate metabolite, was detected on day 5 in soil treated by bioaugmentation of A. niger, and it disappeared during the degradation process on day 11. The spots of both isomers disappeared on day 15. However, the color intensity of these spots decreased over time. In order to confirm the TLC results, GC analysis of the same samples was carried out. The GC analysis was in accordance with the TLC analysis (Figure 3). Chromatogram A represents the control sample containing endosulfan. Chromatograms B and C represent samples from the bioaugmentation pot that were taken on days 10 and 15. On day 10, a small amount of endosulfan still remained. The number of peaks was seen to decrease with an increase in incubation time and all of the peaks completely disappeared on day 15 (Figure 3). Both of these analyses strongly support the complete degradation of endosulfan and endosulfan sulfate
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Figure 2. Amount of released chloride (μg mL–1) during endosulfan degradation in the bioaugmentation process.
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Figure 3. Gas chromatogram showing metabolites of endosulfan degradation by Aspergillus niger. Chromatograms A-C represent samples drawn at different time intervals: A) day 1, B) day 10, and C) day 15.
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in the bioaugmentation process by A. niger. These results corroborate our previous study of endosulfan degradation (14) and studies on Chlorococcum sp. (23). This suggests that either direct desulfurization of endosulfan sulfate (13) or a novel pathway could be the mechanism of degradation of endosulfan and endosulfan sulfate in A. niger (14). In the present study, the GC analysis showed slower degradation of soil-bound endosulfan than in the culture medium when compared with our earlier study (14). The complete degradation of endosulfan in the soil was achieved with the bioaugmentation process within 15 days. While our earlier study reported that complete degradation of endosulfan (350 mg L–1) occurred within 120 h in broth (14), Awasthi et al. (24) also suggested that the degradation of soil-bound endosulfan was nearly 4-fold slower than that in culture medium. Soil enzyme activities during endosulfan degradation Change in soil dehydrogenase activity (SDA) as a function of endosulfan degradation in the bioaugmentation treatment and 2 controls was investigated every alternate day up to day 15. The SDA ranged from 49 μg g–1 of dry soil on day 1 to 78 μg g–1 of dry soil on day 15 (Figure 4). The rate of increase was steady during the entire bioaugmentation process. The same trend was observed in the ARS activity. The ARS activity was 1.1 μg PNP g–1 on day 1, and it was 3.7 μg PNP g–1 on day 15, at the end of the endosulfan degradation process (Figure 5). In control 1, the enzyme activities showed no further differences within the 15 days; in control 2, both activities were primarily suppressed and did not recover during the 15 days.
The SDA and the ARS activity followed the increasing rate with endosulfan degradation in the bioaugmentation process. These enzyme activities were used to evaluate the efficiency of A. niger, along with the microbial population, in utilizing organic matter in the soil. The increase in ARS activity suggests the mineralization of ester sulfate in the soil (18). The SDA showed the possibility of active utilization (metabolism) of the compound by microbes, either as a nutrient source or for the detoxification of the compound. The increase in SDA during the endosulfan degradation process by bioaugmentation showed similarity with the previous findings of Kalyani et al. (18), and it is a useful indicator of overall microbial activity in soil (25,26). The increase in both activities suggested their possible involvement in endosulfan degradation. Decontamination of toxic pollutants takes time. Biodegradation is becoming the method of choice for the remediation of polluted sites. This investigation analyzed the efficiency of A. niger for the bioremediation of endosulfan-contaminated soil. The performance of the bioaugmentation treatment was examined by monitoring the change in pH, released chloride, and the formation of endosulfan sulfate, and by a direct degradation study by TLC and GC. Its effects on soil functionality were evaluated by monitoring dehydrogenase and ARS activities. The complete degradation of soil-bound endosulfan occurred on day 15 by A. niger. The change in pH and the increase in released chloride correlated with metabolic activities involved in the simultaneous degradation of endosulfan. Endosulfan sulfate, an intermediate metabolite, was detected
80 μg of nitrophenol released hr-1 g-1 of soil
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Figure 4. Dehydrogenase activity (SDA) during endosulfan degradation in the bioaugmentation process.
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Figure 5. Arylsulfatase activity during endosulfan degradation in the bioaugmentation process.
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Bioremediation of endosulfan-contaminated soil by using bioaugmentation treatment of fungal inoculant Aspergillus niger
on day 5 in soil treated by the bioaugmentation of A. niger, and it disappeared during the degradation process on day 11. In this bioaugmentation process, endosulfan degradation suggests either the direct desulfurization of endosulfan sulfate or a novel pathway of degradation by A. niger. The increase in enzyme activities was an indicator of soil fertility and suggests the possible involvement of these enzymes in endosulfan degradation. The strategy of in situ bioremediation by bioaugmentation was applied for soil contaminated with organochlorine pesticide endosulfan in an open pot. It showed that previously isolated fungal culture A. niger can be effectively used for endosulfan degradation. All of these results demonstrate that the bioaugmentation process under optimized conditions may provide a model of bioremediation of pesticide-contaminated soil. However, this particular observation needs further study for confirmation.
Acknowledgments The author gratefully acknowledges DST, New Delhi, for providing financial assistance and also Prof S.T. Ingle, Director of EES, NMU, and Dr Pravin R. Puranik, Assistant Professor, SLS, NMU, for providing the necessary facilities for this study.
Corresponding author: Tejomyee Sadashiv BHALERAO School of Environmental and Earth Sciences, North Maharashtra University, P.B. No. 80, Jalgaon 425 001, Maharashtra - INDIA E-mail:
[email protected]
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