INTRODUCTION:
Therefore, it is a necessity to mitigate their negative consequences. Many researchers have focused on the environmental fate of different groups of pesticides. In our study, we focused on a semi-volatile organophosphorous (OPs) insecticide, chlorpyrifos (Odyethyl O-3,5,6- trichloropyridin-2-yl phosphorothioate), (CAS No.2921-88-2). Chlorpyrifos is one of the world's most used insecticides, but due to many observed negative effects on different organisms, it needs careful management3. Usually, it affects both the neuronal synapsis and plasma of the target insects by blocking acetylcholinesterase production by phosphorylation, acetylcholine is then stored in the neuron synapse, which provoke death of the target insect. The half-life of chlorpyrifos in soil is normally between 10 and 120 days, but depending on the type of soil, the environment and other factors, it can vary from two months to over one year4. Different studies showed that CP has a negative effect on different organisms such pollinators and humans. In the public health sector, several investigations have shown that autism, low birth weight and other developmental disabilities may result from prenatal exposure to chlorpyrifos3.
Due to the frequent usage of organophosphate pesticides in very large amounts worldwide and its possible neurotoxicity to humans, effective remediation strategies have been established to reduce its diverse dispersal. New efforts have been made in biotechnological fields which highlight the use of microorganisms for pollutant degradation instead of their disposal5. Microbial chlorpyrifos degradation has been the key target of many studies because other chlorpyrifos removal approaches are inefficient or expensive or harmful to the environment. To date, several microbial species have been isolated from chlorpyrifos polluted soils and their biodegradation efficiency has been investigated6-7. Chlorpyriphos was previously reported as resistant to biodegradation, but subsequent studies identified genera of Enterobacter, Pseudomonas, Bacillus and Klebsiella, which were capable of efficiently degrading CP8-10. In more recent research, several bacterial strains from the Bacillus genera, Alcaligenes, Paracoccus, Gordonia, Sphingobacterium, Mesorhizobium, Streptomyces, Cupriavidus and Ochrobactrum have been found to be able to use CP as a primary carbon source10,7.
CP biodegradation produces 3,5,6-Trichloro-2-pyridinol (TCP) which is considered as the main and the most important degradation component. It is water soluble compared to chlorpyrifos and causes extensive soil and marine contamination4. It has stated that a mixture of CP and TCP can have more toxic effects than either. However, most of bacteria can only degrade chlorpyrifos but not TCP and just a few bacteria can destroy chlorpyrifos and TCP at the same time. The selection of a strain that can degrade chlorpyrifos and TCP efficiently is therefore of a significant importance7.
Hence, the present study was undertaken to isolate and to characterize strains that were able to degrade CP in a liquid culture under different nutritional conditions, to provide a more comprehensive knowledge for the future use of microbial isolates in bioremediation.
MATERIALS AND METHODS:
Chemicals and Media:
Chlorpyrifos standard [O,Odiethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate] with a purity of 99% was received from the U.S. EPA. All other organic solvents and chemicals were of analytical grade and provided from standard commercial suppliers.
MMSM containing (gram per liter): FeSO4.7H2O (0.013g), CaCl2.2H2O (0.013g), MgSO4.7H2O (0.25g), KH2PO4 (7.5g), Na2HPO4 (5g), NH4NO3 (5g), Yeast extract (0.25g) pH 7.0 was used for the screening and for biodegradation tests of bacterial strains.
Bacterial strains and inoculum preparation:
In this study a total of 23 bacterial strains isolated from different origins (agricultural soil, household compost and activated sludge) at the laboratory of Molecular Toxicology were used. For each strain, overnight cultures were prepared in 50ml nutrient broth. After 18h incubation at 37°C, the cell pellet was recovered by centrifugation (6000rpm for15 min), the pellet was then resuspended in 2ml normal saline for a second centrifugation (6000rpm/5min), the supernatant was removed and the pellet was dissolved in 1ml normal saline11.
Tolerance of bacterial strains to chlorpyrifos:
To study the effect of the pesticide on the growth of the bacterial strains, inoculation was carried out on solid MMSM medium supplemented with 25mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 600mg/L, 800mg/L or 1000mg/L CP. The plates were incubated at 37°C for 24h, bacterial strains showing good growth were selected 12.
Screening of CP degraders on liquid medium:
This test deals with the selection of the most efficient bacterial strains able to grow in MMSM containing CP (50mg/l) as sole carbon source and energy. Four ml of the tested bacterial cells were inoculated in 40ml MMSM. The samples were incubated for 4 days in a rotary shaker at 37°C under agitation. Microbial growth was monitored by a UV-Visible spectrophotometer (Amercham) at 600nm every 24h (the test was performed in triplicate)13.
Effect of different concentrations of CP on bacterial growth:
Different concentrations of CP were used in order to determine the maximum CP concentration that is supported by the selected strain Pseudomonas sp. B5-2 without having a negative impact on its optimal growth. A set of Erlenmeyer flasks containing 40ml of MMSM inoculated with different concentrations of CP (100mg/L, 200mg/L, 400mg/L, 600mg/L) were seeded with an overnight culture of the selected strain. Samples without CP were used as control. The flasks were incubated under shaking at 37°C for 48h. The assay was conducted in duplicates13.
CP Biodegradation:
Shake flask studies have been conducted to evaluate the CP degradation capacity of the selected strain B5-2. Ten percent (10%) inoculum of the bacterial cultures were inoculated in MMSM (40mL) containing 200mg/L chlorpyrifos as sole carbon and energy source. Flasks were incubated at 37°C and 150rpm in a rotary shaker. Flasks without bacterial culture were used as control. The capacity of the selected bacterial strain to degrade CP in presence of an extra-carbon source was also tested; in this case, 25g/L of glucose was added to the culture medium. Bacterial growth was measured through the optical density measurement at 600nm. To estimate CP concentration, samples of 2mL were taken after 48h of incubation, centrifuged for 20 min at 4300g, filtered directly into amber vials for HPLC analysis using a Millipore filter (0.22μm). The test was performed in duplicate, along with uninoculated flasks as a control14.
HPLC analysis:
Following sample clean-up, the aliquots from the filtered samples have been quantified using the HPLC method (Shimadzu LC10 Avp-Series LC Solution Software LabSolutions (LC 1.11SP1) fitted with a photodiode array (PDA) PDA detector, SPD-M 10 Avp. The analytical column C18 Reverse Phase Alltech was used (5µm, 250 94.6mm). The composition of the mobile phase was a combination of 90% acetonitrile and 10% water. The flow rate and detection wave length were 0.80mL/min and 290nm, respectively. The samples were passed through syringe filters 0.45µm nylon (Alltech Assoc) prior to HPLC analysis, and were manually injected (20µl) into the HPLC system. The suspected pesticide was identified by comparing peak retention time in samples with peaks in the pure analytical standard 15. Peak area was used to estimate the percentage of degradation using the Eq. (1)16,7.
EI (%) is the CP removal rate (%).
C0 is the peak area of CP at the beginning of the experiment and Ci at the end.
RESULTS AND DISCUSSION:
Tolerance of bacterial strains to chlorpyrifos:
Cultivation of the different strains on MMSM agar showed that the majority of the strains were able to grow on this medium to a concentration of 800 mg/L, however only seven strains (B2-3, B3-1, B5-2, S2, S4 and I11) resisted and grew on the concentration of 1000mg/L. B2-3, B3-1 and B5-2 were isolated from an agricultural soil where pesticides were used continuously to control pests. However, strain S2 was isolated from household compost and S4 and I11 were isolated from activated sludge. Rani et al17 reported that the strain of Providencia stuartii MS09 isolated from soils can tolerate up to 700mg/L of chlorpyrifos and the optimal concentration that has supported the bacterial growth during 24h was between 50 and 200mg/L of chlorpyrifos and a low growth was observed at higher concentrations of the pesticide (300-700mg/L). In this work, strains B2-3, B3-1, B5-2, S2, S4 and I11 resisted up to 1000mg/L of CP, with poor growth at concentrations of 800mg/L and 1000mg/L. These strains were selected to perform the biodegradation tests.
Screening of CP degraders on liquid medium:
The ability of the selected strains to grow on MMSM containing 50mg/L of CP as sole source of carbon and energy was tested in this experiment. The growth of the bacterial cells was determined at 37°C each 24 hours of incubation and the results are shown in Figure1. According to the results, for most strains, cell growth started in the first hours of incubation and go up over time, reaching its maximum level after 48hours of incubation, except for B5-2 strain, where the maximum growth level was obtained after 72h of incubation (OD: 0.709).
The growth of strains Pseudomonas sp. B 3-1, Pseudomonas sp. B 5-2 and P. aeruginosa S4 were better than the growth of the other strains. After 72 h, a sharp decline in cell growth was noticed for almost all the isolates. The growth of the isolates in the presence of CP in MMSM means that they can use CP as a substrate and therefore could degrade it. This may be explained by their good acclimation to pesticide. As a result, they may have the desired enzymes for the breakdown of CP. Similar results were obtained by5, they observed that the bacterial strain Bacillus isolated from agricultural soil treated with pesticide in India can grow and degrade CP as sole carbon source. The strain Pseudomonas sp. B5-2 presented the highest growth rate (OD 600nm =0.709 after 72 h) in presence of CP as sole carbon source it was therefore selected for further analysis.
Figure 1. Screening of bacterial growth in presence of CP (50 mg/L) as sole carbon source in MMSM at 37°Cfor 96h.
Effect of different concentrations of CP on bacterial growth:
Different concentrations of CP were tested to determine the optimum CP concentration that stimulates growth of the selected strain (Figure 2), the results indicated that the CP at concentration between 50-200mg/L promotes bacterial development, with a maximal growth obtained at a concentration of 200mg/L (OD 0.418). At CP concentrations between 400 and 600mg/L, a substantial decrease in bacterial growth was observed. The toxic impact of high CP concentrations may explain these findings. Our results revealed a tolerance of Pseudomonas sp. B5-2 to CP.
Similarly, Rani et al17 have previously used a similar strategy to isolate pesticides-degrading microorganisms. Verma et al18 tested the effect of CP on the growth of several bacterial strains IESDV5, IESDV10, IESDV12, IESDV13 and IESDV28 at different concentrations (2, 4, 6, and 20μl/ml). Only two strains IESDV4 and IESDV27 displayed growth inhibition zone at 4, 6 and 20μl/ml concentrations. In addition, Duraisamy et al5 reported that, generally, microorganisms have a significant capacity to metabolize many organophosphorpus pesticides (OPs). In fact, since the bacteria do not contain acetylcholinesterase (AChE), they are not affected by OPs. At the same time, some of the microorganisms utilize OPs as an energy source. This may explain the resistance of our tested bacterium B5-2 to CP. According to the literature, the genus Pseudomonas is recognized by its ability to mineralize OP pesticides such as CP and to use it as a source of carbon and phosphorus19-20.
Figure 2. Effect of different concentration of CP on Pseudomonas sp. B5-2 growth on MMSM broth at 37°C for 24h.
Biodegradation experiment:
During the biodegradation experiment, the growth of the bacterium in presence of CP (200mg/L) used as single substrate and in presence of glucose with an initial concentration of (25g/L) was monitored (Figure 3). As seen in Figure 3, the strain B5-2 was able to survive in the presence of CP with an efficient growth noticed in presence of glucose (OD600 after 48h was 1.38). HPLC analysis of CP concentration revealed that in metabolic sample (CP as sole carbon source), CP concentration was significantly decreased (67.06%) (Figure 4) while in the presence of glucose as an external carbon source, a slightly decline was observed (14.61%). This result gives evidence that the bacterium B5-2 was able to remove CP better when it was used as a unique carbone source.
Figure 3. Pseudomonas sp. B5-2 growth in presence and in absence of glucose in MMSM broth at 37°C for 96 h under shaking (150rpm)
Figure 4. Biodegradation of CP by Pseudomonas sp. B5-2 in presence and in absence of Glucose in MMSM at 37°C under shaking.
Kumar et al21 reported the ability of different isolated bacteria from different origins namely agricultural soil, activated sludge and effluents to degrade CP co-metabolically and catabolically like different species of Pseudomonas, which include P. putida, P. stutzeri, P. aeruginosa, P. nitroreducens, and P. fluorescence. Biodegradation of chlorpyrifos and its major metabolite 3,5,6-trichloro-2-pyridinol (TCP) were also assessed by Abraham and Silambarasan4 using a novel bacterial strain JAS2 isolated from paddy rhizosphere soil identified as Ochrobactrum sp. JAS2. This strain was able to degrade 300mg/L of chlorpyrifos within 12h of incubation in the aqueous medium and it produced the TCP metabolite. Nevertheless, after 72h of incubation TCP was also completely removed by the JAS2 strain. The effectiveness of three isolated strains: Xanthomonas sp. 4R3-M1, Pseudomonas sp. 4H1-M3 and Rhizobium sp. 4H1-M1 on CP and its primary metabolic product, TCP biodegradation, were investigated by Rayu et al10. The findings indicated that, all of the three bacterial strains almost entirely metabolized CP (10mg/L) and TCP in mineral salt medium as primary source of carbon and nitrogen. In addition, Xanthomonas sp.4R3-M1 and Pseudomonas sp. 4R3-M1 may also eliminate TCP (10 mg/L) as primary carbon and nitrogen source when externally supplied. The co-metabolic degradation of chlorpyrifos in liquid media by Flavobacterium sp. and P. diminuta, which were initially isolated by enrichment with diazinone and parathione has been reported by Singh et al8. However, chlorpyrifos is not used as a carbon source by these microbes. Literature shows that the simultaneous application of additional carbon source with a less energetic compound will sustain cell growth and serve as electron donor, allowing this compound to be biodegraded23-24. This hypothesis is not in line with the present evidence since the removal rate of CP as the only carbon source is greater than that of the alternative carbon source (glucose). These findings may be attributed to catabolic repression, which prevents the oxidation of complex compounds through the existence of an additional simple carbon source. In this case, glucose is considered as a repressor to the synthesis of the adequate enzymes involved in different metabolic pathways25. In other recent research, biodegradation of CP as sole carbon source has been recorded by different bacterial genera as Enterobacter, Pseudomonas, Bacillus, Klebsiella, Alcaligenes, Paracoccus, Gordonia, Sphingobacterium and Mesorhizobium10. According to the literature the ability of microorganisms to breakdown OPs is due to the presence of phosphotriesterases (PTEs) enzymes group. There are three types of well-known bacterial PTEs, namely OPH, methyl parathion hydrolase (MPH) and OP acid anhydralase (OPAA)5.
The obtained results in this study showed that the bacterium Pseudomonas sp. B5-2 has a considerable potential for the elimination of pesticide waste in the environment. Further analyses are needed to explain the degradation mechanism and to determine genes and enzymes that are involved in this process.
ACKNOWLEDGMENT:
Authors are grateful to the Algerian Ministry of Higher Education and Scientific Research and DGRSDT for financial support.
CONFLICT OF INTEREST:
Authors declare that they have no conflict of interest.
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Received on 27.09.2021 Modified on 23.12.2021
Accepted on 25.02.2022 ©AJRC All right reserved
Asian J. Research Chem. 2022; 15(2):115-120.
