Influence of environmental factors on biodegradation of quinalphos by Bacillus thuringiensis
© The Author(s) 2017
Received: 7 November 2016
Accepted: 18 February 2017
Published: 6 March 2017
The extensive and intensive uses of organophosphorus insecticide—quinalphos in agriculture, pose a health hazard to animals, humans, and environment because of its persistence in the soil and crops. However, there is no much information available on the biodegradation of quinalphos by the soil micro-organisms, which play a significant role in detoxifying pesticides in the environment; so research is initiated in biodegradation of quinalphos.
A soil bacterium strain, capable of utilizing quinalphos as its sole source of carbon and energy, was isolated from soil via the enrichment method on minimal salts medium (MSM). On the basis of morphological, biochemical and 16S rRNA gene sequence analysis, the bacterium was identified as to be Bacillus thuringiensis. Bacillus thuringiensis grew on quinalphos with a generation time of 28.38 min or 0.473 h in logarithmic phase. Maximum degradation of quinalphos was observed with an inoculum of 1.0 OD, an optimum pH (6.5–7.5), and an optimum temperature of 35–37 °C. Among the additional carbon and nitrogen sources, the carbon source—sodium acetate and nitrogen source—a yeast extract marginally improved the rate of degradation of quinalphos.
Display of degradation of quinalphos by B. thuringiensis in liquid culture in the present study indicates the potential of the culture for decontamination of quinalphos in polluted environment sites.
Organophosphate (OP) compounds are part of the most common chemical classes used in the protection of crop and livestock and in the control of diseases transmitted through vectors and account for an estimated 34% of worldwide insecticide sales . In India, usage of OPs has also gradually been increasing with a consistent decline in application of organochlorines and currently makes up 27% of the total sales of pesticides [2–4]. Andhra Pradesh is the biggest user of crop protection chemicals in India and uses 20% of the total pesticides in the country . Among OPs, quinalphos (QP: O,O-diethyl O-quinoxalin-2-yl phospharothioate) is used widely in agriculture in Andhra Pradesh because of its effective control of all pests over different crops, which is reflected by the 5% of total sales of pesticides registered against quinalphos . Quinalphos is a synthetic OP, non-systemic, broad spectrum insecticide, and acaricide extensively used in India owing to its action on inhibition of acetylcholinesterase in target pests [6, 7]. Being ranked as moderately hazardous by the World Health Organization (WHO) and classified as a yellow label (highly toxic) pesticide in India, quinalphos is either banned or restricted in its usage in most of the nations . Nevertheless, quinalphos is still being used to treat the following crops: wheat, rice, groundnut, cotton, sugarcane, coffee and other ornamental crops. Only 1% of the pesticides applied make contact with the target pest, while the remaining 99% of the pesticide drifts into the environment contaminating soil, water and biota [9, 10]. Accidental spills/leaks occurring during transport and storage of industrial materials and agricultural chemicals have polluted areas that were never intended as sites for waste disposal. Thus, soil and water bodies serve as the ultimate receptacle/reservoir for all kinds of pesticides regardless of whether they are applied intentionally or unintentionally.
The extensive usage of quinalphos in agriculture poses a health hazard because of severe inhibition of acetylcholinesterase (AChE) in non-target organisms by quinalphos [11–15], an adverse influence on blood and brain esterase activity in chickens  and fertility efficiency in adult male rats  by quinalphos. Exposure of non-target organisms to quinalphos in the environment depends on the extent of persistence of quinalphos in natural resources which is, in turn, controlled by factors—abiotic and biotic. Influence of abiotic factors such as sun light, pH and TiO2 on degradation of quinalphos in natural resources such as soil and water was examined [18–22]. Relatively, less attention was paid on biotic factors involved in the fate of quinalphos in natural resources . A definite participation of factors, in particular biotic factors, in the degradation of quinalphos in natural resources such as soil and water can only be demonstrated with the isolation of biotic agents with degradation traits from natural resources. Isolation of Ochrobactrum sp. strain HZM with biodegradation of quinalphos from pesticide-contaminated samples has been recently reported . This organism degraded quinalphos by hydrolysis. Strains of Bacillus thuringiensis appeared to be biotic agents for degradation of fipronil and a wide range of pyrethroids in sugarcane fields  and an activated sludge . In view of less understanding of biotic factors in quinalphos degradation, the current study is aimed at isolating bacterial species capable of degrading quinalphos from soil samples collected from horticultural fields and hitting the potential bacterium for assessment of various environmental factors on biodegradation of quinalphos in liquid culture conditions.
Soil samples [organic matter (%)—0.45; nitrogen (%)—1.62; pH—7.86] were collected from a horticultural field in a semi-arid zone at Honegal close to Chikkaballapur, Karnataka, India.
Quinalphos of a technical grade was purchased from Sigma-Aldrich (99.2% purity). This quinalphos was used for bacterial growth as a sole source of carbon and energy. All other chemicals and solvents used in the study were of an analytical reagent grade/HPLC grade and purchased from Sigma-Aldrich.
Culture medium and selective enrichment method
The composition of the mineral salt medium (MSM) was as follows (g L−1): 1.5 NH4NO3, 1.5 K2HPO4·3H2O, 0.2 MgSO4·7H2O, 1.0 NaCl and 1 mL of trace element stock solution. The trace element stock solution contained the following (g L−1): 2.0 CaCl2·2H2O, 0.2 MnSO4·4H2O, 0.1 CuSO4·2H2O, 0.2 ZnSO4·H2O, 0.02 FeSO4·7H2O, 0.09 CoCl2·6H2O, 0.12 Na2MoO4·2H2O and 0.006 H3BO3.
For selective enrichment, 5-g samples of soil were incubated in MSM spiked with quinalphos of the technical grade at 20 µg mL−1 of MSM in a 250-mL Erlenmeyer flask in an orbital shaker (Orbitek LE-IL Model) at 37 °C and 175 rpm. After 10 days of incubation, a 5-mL portion of the culture was transferred to a fresh medium fortified with increasing concentrations of quinalphos up to 200 µg mL−1 in Erlenmeyer flasks and the flasks were incubated for an additional 10 days. After five more transfers, the culture was purified by serial dilution and streak plating onto solidified MSM containing 20 µg mL−1 of quinalphos. Finally, a pure bacterial strain was obtained and designated as OP1.
Identification and characterization of the bacterial isolate
Morphological, physiological and biochemical characterization
Morphological observations of bacterial isolate were made with an optical compound microscope. Physiological and biochemical properties of the isolate were determined by the procedures as described in Bergey’s Manual of Determinative Bacteriology .
16S rRNA gene sequencing and phylogenetic tree analysis
Amplification of the 16S rRNA gene in genomic DNA, extracted from the potential bacterial isolate (OP1) in a standard phenolic extraction procedure , was performed with the universal conserved sequence as primers—16 forward primer sequence, 5′-AGACTCAGGTTTGATCCTGG-3′, and 16 reverse primer sequence, 5′-ACGGCTACCTTGTTACGACTT-3′. The phylogenetic analysis was based on a 16S rRNA gene sequence as described by Qin et al. . Comparison of the determined sequence with those in the GenBank/EMBL database was made using the online tool BLAST programme . Sequences of the OP1 and closely related bacterial spp. were collected and aligned. A neighbour-joining and maximum-likelihood tree was constructed using the Robust Phylogenetic tree online tool [30, 31] to establish the phylogenetic relationship.
Measurement of bacterial growth kinetics on quinalphos
For the preparation of the inoculum, the bacterial isolate OP1 was grown overnight in 50 mL of MSM amended with 20 ppm of quinalphos per mL of MSM and yeast extract (0.1%) on an orbital shaker at 175 rpm at 37 °C. Bacterial cells in the overnight grown culture were harvested aseptically (8000×g, 15 min, 4 °C) and thoroughly washed with MSM and suspended in sterile MSM to get a suspension with the desired OD. For the growth of the bacterial isolate on quinalphos, 50 mL of sterile MSM, spiked with quinalphos at a concentration of 20 µg mL−1, was dispensed into sterile 250-mL Erlenmeyer flasks. After inoculation with the bacterial culture to the final OD of 1.0/mL of MSM, the flasks were incubated on an orbital shaker at 175 rpm at 37 °C. Uninoculated flasks with the fortified medium served as the control. Five-millilitre aliquots from the growing culture broth were withdrawn at 6-h intervals for measurement of turbidity/growth at wavelength of 600 nm in a UV–visible spectrophotometer (Chemito-UV-2600). The total number of viable bacterial colony-forming units in the culture broth was determined by a serial dilution method on nutrient agar medium plates. The specific growth rate of bacterial sp. OP1 was calculated in the logarithmic phase.
Biodegradation of quinalphos
Experiments on biodegradation of quinalphos by the bacterial isolate was undertaken in 250-mL Erlenmeyer flasks in the same manner as done for the growth experiments as mentioned earlier “Measurement of bacterial growth kinetics on quinalphos” section. Flasks containing quinalphos in MSM without inoculum served as controls. At regular intervals of 48 h, 10 mL of culture broth was aseptically withdrawn from the flasks for growth measurements and residue analysis. The culture broth from both uninoculated and inoculated flasks was processed for residue analysis and spun at 8000×g for 15 min in a refrigerated centrifuge (REMI, C24 BL, Hyderabad). The supernatants collected were extracted with dichloromethane with an equal volume of supernatant; this was repeated three times with fresh lots of dichloromethane. The extracts were pooled together, dried over anhydrous sodium sulphate, filtered and allowed to dry at room temperature. The dried residue was dissolved in methanol for UFLC analysis.
Factors influencing biodegradation of quinalphos
In order to assess the effect of various factors on the degradation of quinalphos by OP1, appropriate modifications in the supplementation of additional nutrients to MSM and the growth conditions of the bacterial culture on quinalphos were made. For this purpose, MSM was spiked with 20 mg L−1 of quinalphos and distributed into 250-mL flasks (100 mL per flask). The flasks were supplemented with an additional carbon source, (glucose or sodium acetate), or additional nitrogen sources, NH4Cl, (NH4)2SO4, urea or yeast extract to a final concentration of 0.01% (w/v). Flasks were inoculated with the bacterial suspension to get an initial OD of 1.0, and flasks devoid of inoculum were maintained as controls. These flasks were incubated at 37 °C and shaken at 175 rpm in an orbital shaker; samples were collected at 48-h intervals; and the culture broth was extracted with dichloromethane solvent for residue analysis. The influence of the concentration of quinalphos on its degradation was assessed by growing the bacterial isolate on quinalphos in MSM at different concentrations (20–200 ppm) of quinalphos. In another experiment, flasks containing MSM (pH 7.5) were supplemented with 20 mg L−1 of quinalphos inoculated with the bacterial cell suspension to an initial OD of 1.0 and incubated in a shaker at 175 rpm at different temperatures of 30–45 °C to study the influence of temperature on the degradation of quinalphos. In order to study the effect of pH on quinalphos degradation, OP1 was cultured as described above and only the pH was varied from a pH of 5.5–8.5.
Quinalphos residue analysis by ultra-fast liquid chromatography (UFLC)
The residue of quinalphos extracted from the different experiments was dissolved in methanol and analysed by UFLC-LC 20 AD (Shimadzu, Japan) equipped with a ternary gradient pump, programmable variable-wavelength PDA detector, column oven, and electric sample valve and ODS-2, C18, reverse-phase column (4.6 × 250 mm × 5 μm). The quinalphos residue analysis was conducted using an isocratic mobile phase of methanol. The sample injection volume was 20 µL; the mobile phase was programmed at a flow rate of 1 mL min−1; and quinalphos was detected at 254 nm wavelength under these operating conditions with a retention time of 1.859 min.
All parameters (carbon source, nitrogen source, size of inoculum, concentration of quinalphos, pH and temperature) were compared using a one-way ANOVA analysis. All were tested at P < 0.05 significance level and the Duncan multiple range test was used for separation between treatment means. Statistica v.10, StatSoft (USA) was used for all statistical analysis.
Results and discussion
Identification and characterization of the bacterial isolate
The bacterial strain (OP1) was isolated from a horticultural field by the selective enrichment method. The OP was identified according to the classification scheme outlined by Bergey’s Manual of Determinative Bacteriology . The cell morphology for OP1 was analysed by compound microscopy and observed to display morphological characteristics consistent with the Gram-positive reaction; the colonies were rod-shaped, circular with a crenate margin. In addition, various biochemical tests were performed and recorded as Indole test—negative; Methyl red test—negative; VP test—negative; Citrate utilization test—positive; glucose and lactose fermentation tests—positive; Urease activity—positive; Catalase activity—positive; Nitrate-reductase activity—positive; Starch hydrolysis—positive; Casein hydrolysis—negative; Gelatin liquefaction—negative. Based on these morphological and biochemical characteristics, the strain OP1 is homologous with Bacillus sp.
As the 16S rRNA gene sequence is a proven molecular and taxonomic tool used for the identificat'ion of bacteria isolated from the environment [23, 32–36], the same approach was adopted for the identification of bacterial isolate in the current study. Similarly, strains of Bacillus thuringiensis with capacity to degrade insecticides—cyhalothrin (http://www.nature.com/articles/srep0874) and fipronil [24, 25] were isolated from an activated sludge and sugarcane growing fields, respectively.
Growth rate of Bacillus thuringiensis on quinalphos
Growth of Bacillus thuringiensis on quinalphos
Incubation time in h
Bacillus thuringiensis CFU/mL
26 × 108
190 × 108
570 × 108
600 × 108
95 × 108
Quinalphos was included in MSM as the sole source of carbon and energy for the cultivation of a bacterial culture in the current study. The proliferation of bacterial cells on quinalphos occurred in MSM up to 18 h as reflected by an increase in the viable cell count (Table 1). This indicates the use of quinalphos by bacterial culture as a sole source of carbon.
Under the conditions used in the current study, B. thuringiensis grew more rapidly with increase in cell number and a shorter generation time. Proliferation of organisms is only possible with utilization of OP insecticides as carbon and energy source. Similarly, utilization of quinalphos at 2 mmol L−1 as a sole source of carbon and energy by Ochrobactrum sp. strain HZM and attainment of maximum growth (OD600—0.8) within 6 days of incubation was reported . The growth of bacteria—Pseudomans sp. and Serratia sp.—on diazinon at 50 mg L−1 in MSM was the most effective, attaining a maximum OD660 within 6–10 days . The growth of Pseudomonas putida epI on ethoprophos appeared to be a logarithmic mode between 5 and 35 h of incubation . During this period, the viable cell count in the culture increased from 104 to 106 CFU mL−1. There was a rapid increase in the OD600 value up to 0.9 of the culture of Paracoccus sp. for 2 days with the utilization of 50 µg mL−1 of chlorpyrifos . Alcaligenes sp. JAS1 could grow rapidly on chlorpyrifos at 300 mg L−1 for 5 days and exhibited a high growth rate .
Biodegradation of quinalphos by Bacillus thuringiensis
Variable factors such as additional carbon and nitrogen sources, the size of inoculum density, medium pH, temperature and concentration of quinalphos were examined to optimize the biodegradation of quinalphos by B. thuringiensis.
Influence of additional carbon source on biodegradation of quinalphos
Disappearance of quinalphos occurred to the extent of 84–86% with the culture of B. thuringiensis grown in the presence of sodium acetate or absence of any additional carbon within 2 days of incubation. However, degradation of quinalphos was slower in the cultures of the same bacterium grown with the amendment of glucose as reflected by the recovery of 24% initially added quinalphos at the end of 2-day incubation.
Influence of additional carbon source on degradation of OP compounds varies from one organism to another. For instance, a provision of additional carbon source to Paracoccus strain TRP  and Enterobacter strain B-14  led to a lag phase followed by an accelerated degradation of chlorpyrifos. The degradation of ethoprophos by Pseudomonas putida epI was not markedly influenced by the presence of a supplementary carbon source . Ethoprophos degradation by P. putida epII was slower in the presence of glucose or succinate. On the other hand, glucose enhanced the degradation rate of Bacillus pumilus C2A1 as it completely degraded the chlorpyrifos within 3 days of incubation. However, results of the current study revealed that supplementation of carbon had a different response to the degradation of quinalphos by the B. thuringiensis. The disappearance of quinalphos by B. thuringiensis in the presence of sodium acetate was marginally improved. This might be due to the effect of sodium acetate on the growth of the bacterium.
Influence of an additional nitrogen source on the biodegradation of quinalphos
Quinalphos contains nitrogen atoms in its structure. A provision of another nitrogen source in the medium may cause a reduction in the degradation and utilization of nitrogenous organophosphates as a nitrogen source. The addition of yeast extract and nutrient broth to MSM enhanced the degradation of non-nitrogenous OP pesticide, chlorpyrifos, by the Bacillus pumilus strain . In the current study, supplementation of nitrogen, except for the yeast extract, did not improve degradation of quinalphos.
Influence of inoculum density on the biodegradation of quinalphos
Influence of concentration of quinalphos on its biodegradation
Influence of initial concentration of quinalphos degradation by Bacillus thuringiensis under submerged culture conditions
Residual concentration of quinalphos in MSM
3.37 ± 0.06a
4.54 ± 0.11a
5.50 ± 0.06a
6.12 ± 0.06a
2.30 ± 0.06b
3.22 ± 0.06b
3.00 ± 0.03b
4.75 ± 0.03b
2.04 ± 0.05c
2.37 ± 0.06c
2.76 ± 0.06c
3.68 ± 0.06c
0.77 ± 0.09d
1.56 ± 0.06d
1.55 ± 0.06d
2.13 ± 0.04d
0.53 ± 0.06e
1.07 ± 0.04e
1.25 ± 0.08e
1.64 ± 0.03e
The concentration of quinalphos had no influence on the degradation of quinalphos by B. thuringiensis in this study (Table 2). Degradation of quinalphos occurred to the extent of 75% in the culture of B. thuringiensis grown on MSM with the concentration of quinalphos at 20 mg L−1 as against 98% in the same culture with 200 mg L−1 of quinalphos concentration. No appreciable degradation of quinalphos (1%) was observed in respective inoculated controls in the corresponding period. This observation of the present study is in agreement with the result of degradation of chlorpyrifos at concentration from 10 to 500 mg L−1 by Alcaligenes faecalis strain DSP3 . Anwar et al.  reported degradation of chlorpyrifos by B. pumilus C2A1 at even relatively higher concentrations, i.e. 500 and 1000 mg L−1, to the extent of 90% after 2 weeks. The prolonged lag phase in the degradation of chlorpyrifos at higher concentration could be due to the time taken for the adaptation of microorganisms to produce the necessary enzymes .
Influence of the medium pH on the biodegradation of quinalphos
Influence of temperature on biodegradation of quinalphos
The bacterial isolate from soil samples of a grape vine garden was identified as B. thuringiensis through the selective culture enrichment method, morphological, biochemical and 16S rRNA gene sequence analysis. The optimum environmental conditions for growth and degradation of quinalphos were analysed in shaking conditions and recorded as the inoculum density of (1.0. OD), pH (6.5–7.5), 35–37 °C temperature and high concentration of quinalphos (200 ppm). Additional carbon and nitrogen sources (carbon source—sodium acetate) (nitrogen source—yeast extract) marginally improved the rate of degradation of quinalphos. It thus appears that B. thuringiensis is the best microbial source to be used in a quinalphos/pesticide-contaminated environment.
GSR, KPK and BRR were substantially involved in the conception and design of the study. GSR and BM were responsible for the acquisition and analysis of the data. GSR, BRR and NK drafted the manuscript and correction. ADT helped in the analysis of the data. All authors read and approved the final manuscript.
This work was funded by UGC (F. No. F. 33-205/2007 (SR) and CSIR-SRF sanctioned (Lr. No. 09/383(0048)/2012-EMR-I), New Delhi.
The authors declare that they have no competing interests
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