Biodegradation of Chlorpyrifos, Malathion, and Dimethoate by Three

Article pubs.acs.org/JAFC

Biodegradation of Chlorpyrifos, Malathion, and Dimethoate by Three Strains of Bacteria Isolated from Pesticide-Polluted Soils in Sudan Abd Elaziz S. A. Ishag,*,† Azhari O. Abdelbagi,† Ahmed M. A. Hammad,†,⊥ Elsiddig A. E. Elsheikh,‡ Osama E. Elsaid,§ J.-H. Hur,⊗ and Mark D. Laing⊥ †

Department of Crop Protection, Faculty of Agriculture, University of Khartoum, Shambat, Sudan Department of Soil and Environment, Faculty of Agriculture, University of Khartoum, Shambat, Sudan § Faculty of Agricultural Technology and Fish Sciences, Al Neelain University, Khartoum, Sudan ⊗ Department of Biological Environment, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon-do, Republic of Korea ⊥ Discipline of Plant Pathology, School of Agriculture Earth and Environmental Sciences, University of Kwazulu, Natal, South Africa ‡

S Supporting Information *

ABSTRACT: This study was done to identify pesticide-biodegrading microorganisms and to characterize degradation rates. Bacillus safensis strain FO-36bT, Bacillus subtilis subsp. inaquosorum strain KCTC13429T, and Bacillus cereus strain ATCC14579T were isolated from pesticide-polluted soil in Sudan, separately incubated with each pesticide with periodic samples drawn for GC and GC-MS. Pesticide biodegradation followed a biphasic model. α and β half-lives (days) of chlorpyrifos, malathion, and dimethoate in B. safensis culture were 2.13, 4.76; 2.59, 5.66; and 9.5, 11.0, respectively. Values in B. subtilis and B. cereus cultures were 4.09, 9.45 and 4.33, 9.99 for chlorpyrifos; 2.99, 5.36 and 2.43, 4.71 for malathion; and 9.53, 15.11 and 4.16, 9.27 for dimethoate. No metabolite was detected in B. subtilis cultures, whereas a few were detected from B. safensis and B. cereus cultures. Bacterial efficiency can be ordered as B. safensis > B. subtilis > B. cereus for chlorpyrifos and B. cereus > B. subtilis > B. safensis for malathion and dimethoate. KEYWORDS: biodegradation, bacteria, chlorpyrifos, dimethoate, malathion, Sudan

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names, and annual imports exceed 50 tons.9 Dimethoate is the most heavily used insecticides used in Sudan.9 It has been registered under more than 10 trade names, and annual imports exceed 370 tons.9 Recent inventories indicated that these compounds constitute a major fraction of the obsolete pesticide stocks, and therefore they represent a major contributor to soil contamination in hot spots. The conventional methods employed for the remediation of organophosphate-contaminated sites have relied mainly on chemical treatments, recycling, pyrolysis, incineration, and landfills. However, these are inefficient and costly and can lead to the formation of toxic products.10−12 On the other hand, the use of microbes for effective detoxification, degradation, and removal of toxic compounds from contaminated soil and water has emerged as an efficient technique to clean up polluted environments.13 Many bacteria capable of degrading organophosphate pesticides have been isolated from soils around the world.12,14−17 In Sudan, Abdelbagi et al.1,2 and Elmahi18 proposed the potential use of indigenous soil microorganisms to clean highly polluted soils and dump sites. The subsequent preliminary investigation indicated a promising potential of indigenous microorganism in the degradation of endosulfan and γHCH.19

INTRODUCTION Sudan started to use pesticides in the late 1940s. Irrigated cotton schemes are the major sectors using pesticides in the country. The annual consumption of pesticides in Sudan has diminished from a mean of 5000 MT before the 1990s to a range of 2000−3000 MT for several reasons: changes in agriculture policies, a reduction in the area allotted to cotton production, adoption of integrated pest management (IPM).1,2 Poor or substandard storage facilities and inventory management practices led to huge amounts of stored pesticides becoming obsolete. According to the last audit, the amount of obsolete pesticides in Sudan was estimated at 666 tons, with about 6459 m3 of contaminated soil spread in 43 major and minor sites in the country.3 Horizontal and vertical movements of contaminants were reported.4,5 Corroded containers release significant quantities into the soil below. Most of the stores are close to residential areas or fresh drinking water bodies, both surface and groundwater. Chlorpyrifos, malathion, and dimethoate are organophosphorous insecticides widely used for public health purposes, in recreational and residential landscaping as well in the protection of field and horticultural crops.6−8 In Sudan, chlorpyrifos, malathion, and dimethoate have been used for plant protection, termite control in houses, sugar cane plantations, and vector control.9 Malathion is the third most heavily used insecticide in Sudan.9 It has been registered under many trade names, and annual imports exceed 280 tons.9 Chlorpyrifos has also been registered under different trade © 2016 American Chemical Society

Received: Revised: Accepted: Published: 8491

August 7, 2016 October 17, 2016 October 22, 2016 October 22, 2016 DOI: 10.1021/acs.jafc.6b03334 J. Agric. Food Chem. 2016, 64, 8491−8498

Article

Journal of Agricultural and Food Chemistry

ces of 16S rRNA gene were analyzed in the EZBIOCLOUD database (http://www.ezbiocloud.net/eztaxon). Sequencing alignment was performed using the CLUSTALW program;25 the sequences were verified and edited using BioEdit 7.2.5. Phylogenetic Analysis. The phylogenetic trees were constructed using the neighbor-joining algorithm with Mega 6.0. The strengths of internal branches of resulting trees were statistically evaluated by bootstrap analysis with 1000 bootstrap replications. The results of 16S rRNA sequence analysis used to reconfirm the identification of bacterial isolates recognized the isolated strains as follows: strain 1, Bacillus cereus strain ATCC14579T with accession AE016877; strain 2, Bacillus subtilis subsp. inaquosorum strain KCTC 13429T with accession AMXN01000021; and strain 3, Bacillus safensis strain FO36bT with accession ASJD01000027. The identified bacterial strains were subcultured in meat peptone agar (MPA) for 24 h prior to their use in biodegradation studies in mineral salt medium (MSM) (3% carbon content). Preparation of Media. MPA. The MPA was prepared by adding 5 g of meat extract, 7.5 g of peptone, 5 g of NaCl, and 20 g of agar to 1.0 L of distilled water according to the method of Tepper et al.26 MSM. The MSM was prepared following the method described by Tepper et al.26 1 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.001 g of FeSO4·7H2O, 0.01 g of MnSO4·4H2O, and 0.05 g of CaCO3 were added to a conical flask (1500 mL) and, then, the suspension was topped up to 1 L by adding distilled water. The suspensions were autoclaved for 20 min, at 121 °C, allowed to cool at room temperature, and kept in a refrigerator at 4 °C until used. Preparation of the Microbial Inocula. Three aliquots of MPA, 200 mL each, were taken, and each was placed separately in a 250 mL conical flask. Each flask was inoculated with one bacterial isolate using sterilized loops. Inoculated flasks were then sealed with sterilized cotton and placed in an incubator shaker (thermostatic cabinet) at 25 °C for 24 h prior to their use in the biodegradation experiment. Microbial Degradation of Chlorpyrifos, Malathion, and Dimethoate in Mineral Salt Medium. The aim of this experiment was to evaluate the capability of the isolated and identified bacterial types in the degradation of chlorpyrifos, malathion, and dimethoate in carbon-free media (CFM). A total of 60 clean test tubes were sterilized in an oven for 3 h at 180 °C. Ten milliliters of MSM was taken from the stock flasks into each test tube. One milliliter of inoculum was added to each test tube. The cultured test tubes were incubated at 25 °C with 400 mg L−1 chlorpyrifos, malathion, and dimethoate for 0, 3, 7, 14, and 30 days. The experimental units were arranged in a completely randomized design (CRD) with three replicates. Control sets without bacterial inoculum were incubated under the same conditions. All treatments were applied on the recovery test. The recovery sets were immediately extracted and kept in a refrigerator for the gas chromatographic (GC) analysis. Extraction of Chlorpyrifos, Malathion, and Dimethoate from the Culture. Treated cultures were centrifuged at 800 rpm for 10 min to separate the microorganisms from the media. The supernatant was removed by careful decanting and placed in a 100 mL separating funnel. Ten milliliters of dichloromethane was added followed by 5.0 mL of saturated sodium chloride solution and 1.0 mL of methanol. The contents were vigorously shaken for 5 min, and the cork was frequently opened to release the pressure. Contents were allowed to stand for 1 min until separation of layers. The dichloromethane layer was collected in a clean test tube, and the aqueous layer was reextracted twice with 10.0 mL of dichloromethane. Dichloromethane fractions were recombined in a clean test tube, and water was removed by passing the sample through anhydrous sodium sulfate to the filter paper. The solvent was evaporated by a rotary evaporator at 68 °C until dryness, and the residues were reconstituted in 10 mL of hexane and stored in the refrigerator at 4 °C for GC analysis. Gas Chromatographic Analysis. A gas chromatograph equipped with a flame ionization detector (FID) and a DB-5 fused silica capillary column 30 m long with a 0.25 μm i.d. was used for analysis of the extracts. The stationary phase (0.25 mm thickness) was 5% phenyl, methylpolysiloxane. The detector and injector temperatures were 280

The potential of some indigenous soil microorganisms to degrade endosulfan was studied by Elsaid et al.,20 who found that most indigenous microorganisms are capable of degrading endosulfan. Degradation rates can be enhanced by many activators,21 and wild types may be more potent than mutant (tolerant) strains.22 The degradation of pendimethalin by three bacterial strains (Pseudomonas aeruginosa, Bacillus mycoides, and Bacillus cereus) isolated from pesticide-contaminated soil was studied by Shaer et al.,23 who reported that strains of P. aeruginosa are capable of complete mineralization of this compound. On the basis of their promising results, and the hazards caused by pesticide-contaminated soils, this work was initiated to identify and evaluate the potential of indigenous bacterial isolates (Bacillus straina 1, 2, and 3) to degrade chlorpyrifos, malathion, and dimethoate in a mineral salt medium and to identify major degradation products, especially those of toxicological concern, as well as to characterize the degradation rates of the parent compounds.

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MATERIALS AND METHODS

Chemicals and Reagents. Analytical standards of the insecticides chlorpyrifos (96.5%), dimethoate (96%), and malathion (96.5%) were obtained from the Agricultural Research Corp., Sudan. Dichloromethane (99.8%), hexane (99.8%), and methanol (99.8%) were obtained from Fisher Co., U.K. Sodium chloride (99.5%) and anhydrous sodium sulfate (99.0%) were purchased from Lab. Line Co., Sudan. Isolation and Identification of Microorganisms from Pesticide-Polluted Soils. Surface soil samples were randomly collected from pesticide-polluted storage soil in Hasahesa (Gezira irrigation scheme), using a soil auger of 10 cm length and 5 cm diameter. Five augers were taken and mixed thoroughly to make a composite sample (1 kg). The collected sample was placed in a paper bag, labeled, and immediately transported to the Microbiology Laboratory, University of Khartoum. One gram of soil sample was weighed and placed in a sterilized test tube containing 10 mL of distilled water. The contents were well shaken to give the first dilution (10−1). The other five dilutions (10−2, 10−3, 10−4, 10−5, and 10−6) of each soil sample were prepared by serial dilution from the respective preceding concentration. From each of the six serial dilutions of the soil, 1 mL was transferred to the nutrient agar surfaces in the plate. The plates were then incubated in an incubator at 37 °C for 48 h. Purification and Identification of Microorganisms. Predominant microorganisms from morphologically different colony types were selected from plate count agar. The isolates were purified by subculturing. Typical colonies were then streaked onto sterile nutrient agar plates. The plates were incubated at 37 °C for 24 h. The representative colonies of various microorganisms were subcultured in nutrient agar media (on a slope), and then the cultures were kept in the refrigerator at 4 °C until used for the further test. The identification of purified isolates was carried out according to the procedure in Cowan and Steel’s Manual for Identif ication of Medical Bacteria.24 The bacterial isolates were identified by the biochemical test. The identified isolates have been reconfirmed by molecular biotechnology. Extraction of DNA and PCR Amplification and Sequencing of 16S rDNA. Extraction of DNAs was carried out at Macrogen Ltd., Korea. PCR amplification and sequencing of the 16S rRNA gene were performed. The 16S rRNA genes were PCR-amplified from the genomic DNA using the bacterial Macrogen universal primer sets 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3′ and 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′. Analysis of nucleotide sequences, confirmation of microorganism, and homogenic data using rRNA database (NCBI) after amplification of bacteria rRNA were done. All sequences generated in this study have been deposited in GenBank with accession numbers in the Entrez retrieval system (http://www.ncbi.nlm.nih.gov/sites/entrez). The determined sequen8492

DOI: 10.1021/acs.jafc.6b03334 J. Agric. Food Chem. 2016, 64, 8491−8498

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Table 1. Concentrations of Chlorpyrifos, Malathion, and Dimethoate (mM L−1) following Incubation with the Three Bacterial Strains in Mineral Salt Mediuma B. safensis strain FO-36bT

B. cereus strain ATCC14579T

B. subtilis subsp. inaquosorum strain KCTC 13429T

insecticide type

time (days)

chlorpyrifos

0 3 7 14 30

1.14 0.43 0.28 0.15 0.12

± ± ± ± ±

0.0 0.0105C 0.0666C 0.0077B 0.0045C

1.14 0.71 0.43 0.18 0.14

± ± ± ± ±

0.0 0.042A 0.0294B 0.0099B 0.0031A

1.14 0.69 0.49 0.40 0.13

± ± ± ± ±

0.0 0.0028A 0.0235A 0.0037A 0.0028B

0.0 0.0505 0.0441 0.0152 0.0072

LSD

malathion

0 3 7 14 30

1.21 0.54 0.51 0.43 0.14

± ± ± ± ±

0.0 0.0139B 0.0205A 0.0064A 1.62B

1.21 0.52 0.27 0.15 0.12

± ± ± ± ±

0.0 0.0108C 0.0050A 0.0011C 0.565C

1.21 0.61 0.49 0.39 0.24

± ± ± ± ±

0.0 0.0084A 0.0228B 0.0052B 1.065A

0.0 0.0226 0.0358 0.0096 2.351

dimethoate

0 3 7 14 30

1.74 1.40 1.12 0.88 0.53

± ± ± ± ±

0.0 0.0171A 0.0221B 0.0403C 0.0205A

1.75 1.06 1.03 1.01 0.41

± ± ± ± ±

0.0 0.0284A 0.0049C 0.0150B 0.0109C

1.75 1.40 1.35 1.27 0.46

± ± ± ± ±

0.0 0.0131B 0.220A 0.061A 0.0124B

0.0 0.0411 0.255 0.0681 0.0305

a Means followed by the same letter in the same row are not significantly different at p = 0.05 according to least significant difference (LSD). Means ± SD = standard deviation, mM L−1 = milliters mole/liter.

Figure 1. Removal (%) of chlorpyrifos after incubation with the three strains of bacteria in mineral salt medium. and 270 °C, respectively. Nitrogen was used as a carrier gas at a flow rate of 4.23 mL min−1. The oven temperature was programmed as follows: initial temperature was 100 °C, held for 2 min, then increased at 16 °C min−1 to 180 °C, held for 3 min, and subsequently increased by 16 °C min−1 until the final temperature of 240 °C, at which it was held for 3 min. Flow rates of the makeup gas (nitrogen), hydrogen, and air were 30, 40, and 400 mL min−1, respectively. Analysis of the samples was done by duplicate injection of 2.0 μL. Three concentrations (1.142, 0.57, and 0.114 mM L−1 for chlorpyrifos; 1.2121, 0.606, and 0.12 mM L−1 for malathion; and 1.74, 0.87, and 0.17 mM L−1 for dimethoate) of the analytical standard of chlorpyrifos (96.5%), malathion (96.5%), dimethoate (96.0%) were injected under the same conditions, and the response was used for the construction of the standard curve. Reanalysis of the analytical standard was repeated every morning to check for the performance of the machine. The injection septum was replaced when necessary. The limits of detection (LODs) for chlorpyrifos, malathion, and dimethoate were 0.0447, 0.0201, and 0.004 mM L−1, respectively. The recovery of the method ranged from 93 to 95% for chlorpyrifos, from 91 to 94% for malathion, and from 95 to 96% for dimethoate. Gas Chromatography with Mass Spectroscopy (GC-MS) Analysis. Three representative samples were reanalyzed using a Shimadzu GC-Ms Qp2010 system with an AOC-5000 autosampler. The gas chromatograph was fitted with a Rtx5-Ms capillary column, 30

m × 0.25 mm i.d., 0.25 μm film thickness, sourced from Restek. Helium (purity ≥ 99.999%) was used as a carrier gas at a flow rate of 1.24 mL min−1. The splitless injection temperature was 200 °C. The oven temperature was programmed from an initial temperature of 100 °C, held for 2 min, then increased by 16 °C min−1 to 180 °C, held for 3 min, and finally increased by 16 °C min−1 to 240 °C, at which it was held for 3 min. The mass spectrometer was operated with an electron impact (EI) source in the scan mode. The electron energy was 70 eV, and the interface temperature was maintained at 200 °C as well as the ion source. The solvent delay was set to 2 min. Statistical Analysis. The data were analyzed using the analysis of variance (ANOVA). A probability of 0.05 or less was considered significant (SAS 2004 and MINITAB software version 13.20). Data are expressed as means ± SD (standard deviation) from at least three independent trials. A biphasic model was assumed to calculate the loss of chlorpyrifos, malathion, and dimethoate from the media. Calculations were done according to the equation

R = A 0e−αt + B0 e−βt

(1)

where R = the amount of chlorpyrifos, malathion, or dimethoate at t days, A0 and B0 are concentrations of chlorpyrifos, malathion, or dimethoate at t = 0, and α and β are the degradation rate constants for 8493


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Figure 2. Removal (%) of malathion after incubation with the three strains of bacteria in mineral salt medium.

Figure 3. Removal (%) of dimethoate after incubation with the three strains of bacteria in mineral salt medium. first- and second-phase models, respectively. The half-life of the exponential decay was calculated according to eq 2:

t1/2 = (2.303log 2 )/rate constant

B. cereus strain ATCC14579T were 38−87% for chlorpyrifos, 57−90% for malathion, and 39−76% for dimethoate (Figures 1−3). The recovery of the method ranged from 93 to 95% for chlorpyrifos, from 91 to 94% for malathion, and from 95 to 96% for dimethoate. Biodegradation Kinetics. The data in Table 2 indicated that there was a faster rate of degradation in the first phase than in the second. The half-lives (t1/2α) of the first phase of chlorpyrifos biodegradation in media inoculated with B. safensis strain FO-36bT, B. subtilis subsp. inaquosorum strain KCTC 13429T, and B. cereus strain ATCC14579T were estimated at 2.13, 4.33, and 4.03 days, respectively, whereas corresponding values for malathion were 2.59, 2.43, and 2.99 days and the values for dimethoate, 9.5, 4.17, and 9.53 days. On the other hand, the respective values for the second phase (t1/2β) were 4.76, 9.99, and 9.45 days for chlorpyrifos; 5.61, 4.71, and 5.36 days for malathion; and 11, 9.27, and 15.11 days for dimethoate. The first-phase degradation constants for chlorpyrifos, malathion, and dimethoate in media incubated with B. safensis strain FO-36bT, B. subtilis subsp. inaquosorum strain KCTC 13429T, and B. cereus strain ATCC14579T were 0.32, 0.169, and 0.16 day−1; 0.266, 0.284, and 0.227 day−1; 0.07, 0.166, and 0.06 day−1 respectively. The corresponding values for the second phase were 0.14, 0.07, and 0.06 day−1; 0.123, 0.147, and 0.129 day−1; 0.063, 0.074, and 0.036 day−1 (Table 3). Biodegradation Products. Despite the significant decreases in the starting material of chlorpyrifos, no metabolites were detected in the B. subtilis subsp. inaquosorum strain KCTC

(2)

The rate constant of pesticide degradation was calculated by exponential analysis (Microsoft Excel 2010).

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RESULTS Biodegradation of Chlorpyrifos, Malathion, and Dimethoate in Mineral Salt Medium. The results indicated that all three bacterial isolates were able to degrade chlorpyrifos, malathion, and dimethoate in MSM (Table 2). Reductions in levels of the starting pesticides were quite significant and increased with an increase in the incubation period. Generally, degradation rates by B. cereus strain ATCC14579T and B. subtilis subsp. inaquosorum strain KCTC 13429T were low from days 14, 7, and 3 on for chlorpyrifos, malathion, and dimethoate, respectively. However, in B. safensis strain FO-36bT cultures, the rate was low from day 3 for chlorpyrifos and malathion and from day 14 for dimethoate (Table 1). The percentage removal of chlorpyrifos from the medium inoculated with B. safensis strain FO-36bT ranged from 62 to 89%, whereas the corresponding values for malathion and dimethoate ranged from 55 to 88% and from 19 to 70% (Figures 1−3), respectively. On the other hand, removal of chlorpyrifos by B. subtilis subsp. inaquosorum strain KCTC 13429T ranged from 39 to 89%, whereas removal of malathion from the medium ranged from 49 to 80% and that of dimethoate from 19 to 73%. The respective removal values for 8494


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Table 2. Statistical Parameters of Chlorpyrifos, Malathion, and Dimethoate (mM L−1) after Bacterial Biodegradation in Mineral Salt Mediuma insecticide type

statistical parameter

B. safensis strain FO-36bT

B. cereus strain ATCC14579T

B. subtilis subsp. inaquosorum strain KCTC 13429T

LSD

chlorpyrifos

A0 B0 α (day−1) β (day−1) t1/2α (days) t1/2β (days) regression coefficient

0.60 0.32 0.32 0.15 2.13 4.76 0.96

± ± ± ± ± ±

0.0036C 0.0049C 0.0049A 0.00467A 0.0778C 0.273C

0.83 0.50 0.16 0.07 4.33 9.99 0.97

± ± ± ± ± ±

0.0053A 0.00413A 0.004C 0.00502B 0.0153A 0.0644B

0.81 0.47 0.17 0.07 4.09 9.45 0.91

± ± ± ± ± ±

0.00189B 0.00186B 0.001B 0.011C 0.0 551B 0.372A

0.0078 0.0077 0.0036 0.015 0.1113 0.5373

malathion


0.71 0.58 0.27 0.12 2.59 5.62 0.87

± ± ± ± ± ±

0.01B 0.0025A 0.00106B 0.002B 0.01B 0087A

0.77 0.33 0.28 0.15 2.44 4.71 0.97

± ± ± ± ± ±

0072A 0.01C 0.00416A 0.0045A 0.0035C 0.0456C

0.77 0.56 0.23 0.13 2.99 5.36 0.95

± ± ± ± ± ±

0.0052A 0.004B 0.00361C 0.002B 0.0503A 0.0306B

0.0155 0.013 0.0065 0.0067 0.0593 0.0641

dimethoate


1.51 1.16 0.07 0.04 9.50 11.00 0.996

± ± ± ± ± ±

0.0004A 0.0001B 0.0001B 0.001B 0.0006A 0.06B

1.25 1.11 0.17 0.07 4.17 9.27 0.84

± ± ± ± ± ±

0.001C 0.0025C 0.003A 0.004A 0.0036B 0.0021C

1.50 1.40 0.07 0.05 9.53 15.11 0.88

± ± ± ± ± ±

0.0077B 0.015A 0.0002B 0.00458C 0.003A 0.067A

0.009 0.0179 0.0035 0.0071 0.0545 0.1.036

A0 and B0 are the concentrations of chlorpyrifos, malathion, and dimethoate at t = 0, and α and β are the disappearance rate constants for the firstand second-phase models, respectively. Means in the same row followed by the same letter are not significantly different (P = 0.05 according to the LSD). Means ± SD a

Table 3. Mean Lifetimes and Decay Constants of Chlorpyrifos, Malathion, and Dimethoate following Incubation with the Three Isolates of Bacteria mean lifetime

decay constant

insecticide

micro-organism

3 days

7 days

14 days

30 days

3 days

7 days

14 days

30 days

chlorpyrifos

B. safensis strain FO-36bT B. cereus strain ATCC14579T B. subtilis subsp. inaquosorum strain KCTC 13429T

3.08 6.24 5.90

5.02 7.21 8.43

6.89 7.46 13.40

13.28 14.42 13.64

0.32 0.16 0.17

0.20 0.14 0.12

0.15 0.13 0.07

0.08 0.07 0.07

malathion


3.75 3.52 4.38

8.10 4.65 7.65

13.55 6.79 12.35

14.41 12.85 18.35

0.28 0.28 0.23

0.12 0.15 0.13

0.07 0.15 0.08

0.07 0.08 0.05

dimethoate


13.71 6.02 15.86

15.87 13.40 27.33

20.41 25.66 43.60

24.98 22.67 20.94

0.07 0.17 0.06

0.06 0.07 0.04

0.05 0.03 0.02

0.04 0.04 0.05

13429T and B. cereus strain ATCC14579T cultures, whereas a major metabolite (hydroxy O-ethyl O-3,5,6-trichloropyridin-2yl phosphorothioate) was detected in the B. safensis strain FO36bT culture, whereas two metabolites of malathion, malathion monoacid and diacid, were detected after 14 days of incubation with the B. cereus strain ATCC14579T. No malathion metabolites were detected in B. subtilis subsp. inaquosorum strain KCTC 13429T and B. safensis strain FO-36bT cultures. On the other hand, no metabolites of dimethoate were detected in B. safensis strain FO-36bT and B. subtilis subsp. inaquosorum strain KCTC 13429T cultures, whereas major dimethoate metabolites (2-(hydroxy(methoxy)phosphorylthio)acetic acid and tetramethoxy pyrophosphate methylene dithioate) were detected in the B. cereus strain ATCC14579T culture.

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DISCUSSION

Biodegradation is a sustainable, cost-effective, and practical technique for the degradation of pesticides in polluted environments. However, this approach may have some disadvantages, such as the formation of undesirable degradation products that may require desorption or further treatment. The biological removal of chemical pollutants becomes the method of choice because microorganisms can metabolize a variety of xenobiotic compounds, including pesticides, and consequently detoxify or mineralize them. In the present study, the isolates of pesticide-degrading bacteria isolated from the pesticidecontaminated soil were identified by biochemical tests according to the method in Cowan and Steel’s Manual for 8495


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Journal of Agricultural and Food Chemistry Identif ication of Medical Bacteria (Barrow and Feltham)24 and reconfirmed the identification by molecular biotechnology. Three strains of bacteria (B. safensis strain FO-36bT, B. subtilis subsp. inaquosorum strain KCTC 13429T, and B. cereus strain ATCC14579T) are able to degrade chlorpyrifos, malathion, and dimethoate in a MSM. Reduction levels of pesticides were substantial and increased with the increase in the length of the incubation period. The removal of chlorpyrifos from the media inoculated with all three organisms exceeds 87%, whereas the corresponding values for malathion and dimethoate removals exceeded 80 and 76%. Chlorpyrifos, malathion, and dimethoate were reported to be degraded in soil by many microorganisms including the bacteria Bacillus pseudomycoides, Bacillus licheniformis, P. aeruginosa, Pseudomonas fluorescens, B. subtilis, Klebsiella sp., Pseudomonas putida, and Bacillus pumulu27−29 and the fungi Aspergillus niger and Azospirillum lipoferum.30,31 Bacterial degradations of chlorpyrifos, malathion, and dimethoate are dependent not only on culture conditions but also on the isolates and species of bacteria.6 B. cereus, Bacillus mycoides, and P. aeruginosa have been reported as degraders of many organic compounds such as pesticides and petroleum products.23,32,33 The current study agrees with that of Abdelbagi et al.1,2 in that indigenous soil microorganisms could be used to reduce the level of contamination by pesticides in highly polluted storage soils in Sudan. Their suggestion is in line with that of Elzorgani,34 who found that although large quantities of DDT and other pesticides have been applied to crops in the Gezira scheme, Sudan, the soil level was relatively low, which indicates that biological and physical degradation process must be taking place in these soils. This inspection was later confirmed by Ali,19 Elsaid et al.,21,22 Shaer et al.,23 and Abdurruhman et al.35 They demonstrated the ability of indigenous soil microorganisms, especially those isolated from pesticide-polluted soils, to degrade pesticidal pollutants present in storage soils and other hot spots. These pesticides included endosulfan, lindane, pendimethalin, and atrazine screened under conditions of selective and/or mineral salt media or soil. Elsaid and Abdelbagi22 reported that isolated indigenous strains of bacteria and fungi tolerated up to 1000 ppm of endosulfan without significant reduction in their degradation capabilities. Various types of synthetic and natural fertilizers enhanced their degradation rates.21 P. aeruginosa is a versatile microorganism, and previous studies suggested that it can degrade a number of chemicals including many pesticides such as carbaryl,36 malathion,37 chlorpyrifos,38 and p-nitrophenol and parathion.39 P. aeruginosa is a widely distributed Gram-negative soil bacterium, and isolates can be used to implement biodegradation of xenobiotic compounds.40 Environmental factors such as pH and temperature have been found to affect the rates of biodegradation of chlorpyrifos by test microorganisms.40 B. cereus has been reported to degrade malathion and malaxon in a soil slurry system.41 Madhuri29 noted that dimethoate can be highly degraded by Pseudomonas putida and Bacillus pumulu in supplemented medium. Generally, the degradation rates by the three strains of bacteria followed a biphasic model with an initial faster rate in the first phase of degradation followed by a second phase at a slower rate. This phenomenon of biphasic biodegradation in soil is common with many pesticides.23,33,42 The relative importance of the phases depends upon the availability of the pollutants, hydrophobicity, and affinity for organic matter.43

Despite the significant decreases in the starting levels of chlorpyrifos, no metabolites were detected after biodegradation by B. subtilis subsp. inaquosorum strain KCTC 13429T and B. cereus strain ATCC14579T cultures, whereas a major metabolite (hydroxy O-ethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) was detected after biodegradation by a B. safensis strain FO-36bT culture. This metabolite can be formed by O-dealkylation of the ethoxy group by microsomal mixed-function oxidases,44,45 whereas two metabolites of malathion, malathion monoacid and diacid, were detected after 14 days of incubation with B. cereus strain ATCC14579T. These two metabolites can be formed by hydrolysis of the ester linkage of the succinate portion of malathion, possibly by the carboxyl esterase.46 No malathion metabolites were detected after biodegradation by B. subtilis subsp. inaquosorum strain KCTC 13429T and B. safensis strain FO-36bT cultures. On the other hand, two dimethoate metabolites, 2-(hydroxy(methoxy)phosphorylthio)acetic acid and tetramethoxy pyrophosphate methylene dithioate, were detected after biodegradation by a B. cereus strain ATCC14579T culture after 14 days of the incubation. These two metabolites can be formed by an enzyme that could degrade the P−S linkage of dimethoate, which is different from parathion hydrolases, which attack the P−O bond in Gram-negative bacterial strains and produce the metabolites of the compound.47,48 Also, oxidation of the PS bond of dimethoate carboxylic acid can result in the formation of the oxon derivative, which can be hydrolyzed at the CO bond to release a CH3 group to form 2-(hydroxy(methoxy)phosphorylthio)acetic acid.48 The absence of detectable levels of breakdown products in pesticide biodegradation studies involving bacteria and fungi has been reported by many authors.42 The results have indicated the great potential of indigenous microorganisms isolated from pesticide-polluted soils to degrade chlorpyrifos, malathion, and dimethoate under conditions of the mineral salt medium. In light of this study and the previous results,21−23,33 the use of such microorganisms in cleaning pesticide-polluted soils through bioremediation techniques has the potential to be applied as a practical resolution to pollution problems. Results of the current work may provide a foundation for bioremediation of organophosphate-contaminated soils, reducing risks to human health and the environment.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b03334. Figures 1−15. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.E.S.A.I.) Present address: Department of Biological Environment, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon-do, Republic of Korea. E-mail: [email protected]. Cell phone: +821059440339 (Republic of Korea) and +249915378530 (Sudan). Funding

The financial support made available by the Ministry of Higher Education and Scientific Research, Sudan is highly acknowledged. 8496


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Journal of Agricultural and Food Chemistry Notes

(18) Elmahi, A. Distribution of Chlorinated Hydrocarbon Pesticides Residues in Sudan Soil. M.Sc. thesis, University of Khartoum, Sudan, 1996. (19) Ali, M. Naturally Occurring Soil Microorganism in Qurashi Pesticides Store and the surrounding Gezira Soil and their Potential in Degrading Endosulfan α, β and Lindane. M.Sc. thesis, University of Khartoum, Sudan, 2005. (20) Elsaid, G.; Abdelbagi, A.; Elmustafa, A. Microbial degradation of endosulfan in carbon free media and selective media. Res. J. Agric. Biol. Sci. 2010, 6 (3), 257−562. (21) Elsaid, G.; Abdelbagi, A.; Elsheikh, A. Effects of fertilizers (activators) in enhancing microbial degradation of endosulfan in soil. Res. J. Environ. Toxicol. 2009, 3 (2), 76−85. (22) Elsaid, G.; Abdelbagi, A. Comparative biodegradation of endosulfan by mutants and their native microorganisms. Res. J. Agric. Biol. Sci. 2010, 6 (6), 953−961. (23) Shaer, I.; Abdelbagi, A.; Elmustafa, A.; Ahmed, A.; Osama, G. Biodegradation of pendimethalin by three strains of bacteria isolated from pesticides polluted soils. U. K. J. Agric. Sci. 2013, 21 (2), 233− 252. (24) Barrow, I.; Feltham, A. Cowan and Steel’s Manual for Identification of Medical Bacteria, 3rd ed.; Press Syndicate of the University of Cambridge, UK, 2003; 317 pp. (25) Thompson, R.; Marcelino, A.; Polz, F. Heteroduplexes in mixedtemplate amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Res. 2002, 30 (9), 2083−2088. (26) Tepper, Z.; Shilnikova, K.; Pereverzeva, I. A Manual of Microbiology, 4th ed. (in Russian); Kolas Publishers: Moscow, 1994; 170 pp. (27) Thabit, A.; El-Naggar, H. Biodegradation of malathion insecticide by bacteria isolated from agricultural soil and detection of main degradation products by GC-MS. World Rural Observ. 2012, 4 (3), 57−65. (28) Kavi, S.; Reetha, D. Efficiency of bacterial isolates in the degradation of malathion and parathion. Int. J. Pharm. Biol. Arch. 2012, 3 (3), 659−665. (29) Madhuri, J. Degradation of dimethoate by cellulolytic bacteria in cotton soils. Global J. Med. Res. Microbiol. Pathol. 2014, 14 (1), 9−12. (30) Chalamala, R.; Mitta, N.; Muppala, P. Mycodegradation of malathion by a soil fungal isolate, Aspergillus niger. Int. J. Basic Appl. Chem. Sci. 2012, 2 (1), 108−115. (31) Kanade, N.; Ade, B.; Khilare, C. Malathion degradation by Azospirillum lipoferum Beijerinck. Sci. Res. Rep. 2012, 2 (1), 94−103. (32) Okerentugba, O.; Ezeronye, U. Petroleum degrading potentials of single and mixed microbial cultures isolated from rivers and refinery effluent in Nigeria. Afr. J. Biotechnol. 2003, 2 (9), 288−292. (33) Abdurruhman, A.; Abdelbagi, A.; Ahmed, A. Biodegradation of pendimethalin and atrazine by Pseudomonas pickettii isolated from pesticides polluted soil under laboratory conditions. J. Biotechnol. Sci. Res. 2015, 2 (3), 94−102. (34) El Zorgani, A. The Status of DDT Residues in Sudan, progress report; Agricultural Research Corporation: Wad Medani, Sudan, 1982. (35) Vandana, S.; Phale, S. Metabolism of carbaryl via 1,2dihydroxynaphthalene by soil isolates Pseudomonas sp. strains c4, c5, and c6. Appl. Environ. Microbiol. 2005, 71 (10), 5951−5956. (36) Hashmi, I.; Khan, A.; Kim, G. Malathion degradation by Pseudomonas aeruginosa used activated sludge treatment system (biosimulator). Biotechnology 2004, 3 (1), 82−89. (37) Geetha, M.; Fulekar, H. Bioremediation of chlorpyrifos by Pseudomonas aeruginosa using scale up technique. J. Appl. Biosci. 2008, 12, 657−660. (38) Douglas, M.; Dennis, H. Microbial decontamination of parathion and p-nitrophenol in aqueous media. Appl. Microbiol. 1974, 28 (2), 212−217. (39) Fulekar, H. Bioremediation technologies for environment. Indian J. Environ. Prot. 2005, 25 (4), 358−364. (40) Singh, B.; Sharma, S.; Saini, S.; Chadha, S. Biosurfactant production by Pseudomonas sp. and its role in aqueous phase

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Adam Ali Mohamed and Salah Abbass of the National Chemical Laboratories, Federal Ministry of Health, Sudan, for their help with GC-MS analysis. We also thank Kangwon National University (KNU), Republic of Korea, for their help in identification of bacterial isolates by Molecular Biotechnology done in Korean Macrogen Co.

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Biodegradation of Chlorpyrifos, Malathion, and Dimethoate by Three

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