Microorganisms isolated from soil samples using an enrichment culture technique have been in the minimal media where diazinon favoured as a sole carbon ...
Isolation and Identification of Diazinon Degrading bacteria and Residue concentrations of Diazinon by HPLC Arezoo Alipour1, Ali Alizadeh2, Pejman khodaygan3 1, 2 and 3- Graduate student and Assistant Prof. Associate Prof. Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan
Corresponding Author: alializadeh2004@ gmail.com
Abstract Microorganisms isolated from soil samples using an enrichment culture technique have been in the minimal media where diazinon favoured as a sole carbon source. A diazinon biodegradation study was performed in liquid medium with four bacterial strains labeled S1,S2,S3 and S4 that were isolated from lately agricultural soil. All these isolates were able to entirely reduced 50 mg l-1 diazinon in mineral salt medium (MSM) as a only carbon source within 15 days of incubation. Diazinon residues were measured at continuous duration until 15 days after incubation, compared with control samples. Diazinon recovery rate was condacted at 0.1 and 1 mg kg-1, the obtained values were 80.30 and 91.80%, respectively, limit of detection (LOD) was 0.4 mg kg-1 while limit of quantification (LOQ) was 0.2 mg kg1 . Diazinon half- life values (T1/2) were 4.66, 3.76, 3.84 and 3.87 days for S1, S2, S3 and S4, respectively and control value was 6.44 days. No significant effect on diazinon occurred with S1 (Stenotrophomonas. maltophilia), S2 (Pseudomonas. stutzeri), S3 (Alealigen. sp) and S4 (Pantoea. ananatis) treatments showed considerable effect that increased diazinon degradation rate compared with control treatment. These results highlight the potential of these bacteria to be applyed in the purify of polluted pesticides wastage in the environment. Key words: Bacteria , Biodegradation, Diazinon, HPLC
1. Introduction Organophosphorus pesticides (opps) have been used extensively in agricultural practice for more than 40 years. The toxic action of opps is achieved through the inhibition of acetylcholine esterase, an enzyme essential for normal nerve impulse transmission (Abo and Aly,2011). The continued use of these pesticides over the years has led to environmental and ecological problems (Corley.,2003). Excessive and persistent use of pesticides results in environmental harm. The quality of soils, ground, water, surface waters as well as the air, can be compromised by pesticide contamination (Dubey and Fulekar, 2012). Most organophosphorus pesticides have a similar general structure, including three phosphoester linkages, and hydrolysis of one of the phosphoester bonds significantly decreases the toxicity of pesticides by removing acetyl cholinesterase inactivating properties. Pesticides in soil and water can be degraded by biotic pathways; however, biodegradation by microorganisms is the primary mechanism of pesticide breakdown and detoxification in many soils. Thus microbes can have a major effect on the persistence of pesticides in soil (Abo- Amer., 2012). Diazinon is an organophosphate insecticide and is the fifth most common pesticide used by homeowners, with two to four million pounds used annually ( Alhewairini et al., 2016). Diazinon released into water or soil is exposed to volatilization, photolysis, hydrolysis, and biodegradation ( Baishya and Sharma 2014). Diazinon can also be biodegraded under 1
anaerobic conditions. Hydrolysis is an important method for degradation, specifically at low pH. Diazinon has a relatively short half-life in water, ranging from 7 to 12 weeks in the presence of microorganisms ( Barathidasan et al.,2014). Degradation products of diazinon include diazoxon, a toxic metabolite, and 2- isopropyl -6- methyl -4- hydroxypyimidine, a persistent and less toxic product (Briceno et al.,2015). Microbial degradation is the main factor determining the fate of diazinon and other organophosphorus pesticides in the environment. Many authors have indicated that bacterial strains belonging to different taxonomic groups have great potential for degrading organophosphorus pesticides (Cycon et al.,2009). Studies of microbial degradation are useful in the advancement of bioremediation mechanisms for the detoxification of these insecticides by microorganisms. Bioremediation is specified as the process by which organic wastage is biologically treated under controlled conditions and converted to an inoffensive state, or to contaminant levels within concentration limits determined by jurisdiction (Chanika et al.,2011). Bioremediation, which involves the use of microbes to detoxify and degrade contaminants, has received increasing interest as a biotechnological approach for cleaning polluted environments. Generally, this method of bioremediation is achieved through nutrient utilization, and aeration. Isolation of indigenous bacteria able to metabolize organophosphate pesticide compounds have received significant attention, because bacteria provide a method of in situ detoxification (Chen et al.,2011). The goal of this experiment was to isolate bacterial strains, and test them for their potential use in the bioremediation of diazinon- contaminated soil. We used a method based on high performance liquid chromatography (HPLC) in order to ascertain the capability of the strains for utilizing diazinon as the sole carbon source in liquid medium. We also measured the effects of additional carbon sources on the rates of diazinon degradation. Diazinon degrading bacteria may be applied either directly or indirectly in the bioremediation of diazinon contaminated soils. In this study, we isolated diazinon degrading bacteria from soil and assessed their capacity for degrading diazinon.
2. Material and methods 2.1. Chemicals Standard certified of diazinon (99.0% pure analytical grade) was obtained from the SigmaAldrich, chemical company. All other chemicals and solvents were high purity grade reagents. 2.2. Media The mineral salt medium (MSM, pH; 7.0) containing (g L-1) (NH4)2SO4, 2.0g; KH2PO4, 1.5g; FeSO47H2O, 0.001g.MgSO4 7H2O, 0.2g; CaCl2 2H2O0.01g; Na2HPO4, 1.5g was used in this study. Diazinon was added to the mineral salt medium after sterilization. The mineral salt medium together with diazinon was used in the biodegradation studies (Abidi., 1991). 2.3. Sampling Soil samples, were collected from a field at Zarand, Iran for environmental studies. Collected soils were ground, dried in air, and sieved. 2.4. Enrichment of the samples and preparation The aim of this step was to adapt the soil micro flora to the insecticide, diazinon. The mineral salt medium (MSM) was used in this step of the enrichment procedure. Following enrichment of the soil sample, 1ml of contaminated soil suspension was taken and added to 9ml sterile saline water and a serial dilution(10-1 -10-5 ) technique was followed for culturing the sample. 2.5. Isolation and culture conditions
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Five gram of soil was added to an Erlenmeyer flask (250 mL) containing 100 mL MSM with diazinon (50 mg L-1) as the sole carbon source and was incubated at 30°C on a rotary shaker at 200 r.p.m. for 15 days. After 3 days of incubation at 30°C, microbial colonies became visible. We selected and purified these colonies and tested their degrading capability by inoculation in liquid medium. Pesticide residues were measured by high performance liquid chromatography (HPLC) according to the method of Krause et al (1989). 2.6. Identification of diazinon degrading isolates After separating bacteria, according to their morphological characteristics, four individual bacterial strains were selected as diazinon- degrading bacteria and subcultured to obtain pure cultures. These isolates were investigated for their oxidase, catalase, oxidative fermentation and Gram stain. 2.7. Biodegradation of pesticides in liquid medium Soil samples were extracted and cleaned up according to the method of Krause et al (1989) Soil samples (10g) were shaken mechanically with 100 ml of MSM for 15 days in 250 ml glass stopper bottles. This was performed in triplicate. The extract was filtered through a clean pad of cotton. Then, 1ml of filtrate was concentrated using a rotary evaporator on water bath set at 40 to remove acetonitrile and then extracted twice with 50 ml chloroform. The combined chloroform extract was dried using anhydrous sodium sulfate and then evaporated to dryness at 40 using a rotary evaporatory for HPLC analysis (Deng et al., 2015). 2.8. HPLC Systems High performance liquid chromatography conditions: Briefly, column: Hichrom C18 with 25× 14 mm dimension: Mobile phase: Acetonitrile (800/0), water (200/0) flow rate: 1/5 ml min-1; Absorbance254nm. We used a Cecil 1100 HPLC apparatus equipped with power stream software (Waters, UK); all tests were repeated three times. 2.9. Standard curve To prepare the standard solutions, first stock solution with a concentration of 50 mg L-1 was prepared in acetonitrile and stored at -15 C. And then the standard solutions by diluting to draw the standard curve and regression equation was prepared. Five concentration 0.2, 1, 5,10, 15 mg L-1 were produced in aceton and injected into the HPLC. Y= mc + i In this equation Y, C, m, i Device response, concentration. The slope and intercept are shown respectively (Corley., 2003). 2.10. Method authentication studies To examine the effect of extraction and clean up, recovery studies were performed. Three samples for diazinon treatment were spiked at two levels 0.1 and 1 mg/ kg-1 of the pure insecticide standard solution and extraction and clean up were performed as described earlier. The concentration of diazinon in the final extracts was calculated. Recovery values achieved were 80.30 and 91.80%, respectively. Also Limit of determination (LOD) and limit of quantitation (LOQ) of the analytical method used were estimated, values were 0.4 and 0.2 mg L-1, respectively. 2.11. Kinetic studies The diazinon degradation rate constant (K) was determined using the kinetic model Ct= C0×e-kt, where C0 is initial concentration of diazinon at time zero, Ct is the concentration of diazinon at time, t is the degradation period in days, and K is the rate constant (d-1). The halflife (T1/2) of diazinon was determined using the algorithm T1/2= Ln2. K-1. Correlation
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coefficient (R2) and Regression Equation were calculated from liner equation between Ln ( Ct/ C0) of chemical data and time. 2.12. Assay for determination of Diazinon concentration The concentration of diazinon was measured by High Performance Liquid Chromatography (HPLC) using the retention time and peaks corresponding to reference standards. For detecting diazinon, HPLC method was used according to (Romeh and Hendawi 2014). 2.13. Statistical analysis The obtained data were analyzed using analysis of variance and spss software version21 (spss Inc., Chicago, IL, USA).
3. Results 3.1. Isolation, screening and Quantification of Diazinon Degrading Bacteria Based on morphological properties of the bacterial colonies, 10 isolates were selected and repeated sub- culture on diazinon agar (containing50 mgL-1 diazinon) was performed until uniform colonies were found. Finally, through visual observation, four fast growing bacterial isolates were selected and designated as S1, S2, S3 and S4. These isolates were further investigated in more detailed studies. 3.2. Identification of diazinon degrading isolates By means of comparing the results of morphological and various biochemical tests (Table1) with Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1984), and using 'ABIS6' online software (ABIS6, 2012) for bacteria identification. Finally, to complete the process of identifying amplified and sequenced area 16 SrDNA as goldastandard in each of the isolates was performed.
Table1.Morphological and biochemical characteristics of 4 different diazinon degrading isolates grown on nutrient agar at 30 for 24 hours Test S1 S2 S3 S4 Oxidase + + + + Catalase + + + OF + + + Gram Reaction Shape
Cicular
Rod
spreading and irregularly
round, matt and granular
Color
Creamy
Yellow
Yellow
white to cream
OF: Oxidative Fermentative; + for positive reaction, - for negative reaction
3.3. Standard calibration curves The standard calibration was obtained by running three injections of 5 standard concentrations (1, 5, 10, 15 mg L-1) of diazinon through the liquid chromatography apparatus. 4
A curve of the peak area versus the concentration of diazinon is shown in (Figure 1). The calibration plot was linear over the concentration range of 1 to 15 mg L-1 and the correlation coefficient value was 0.9844. 1200
peak area
1000 800
y = 16.206x+50.12 R² = 0.9844
600 400 200 0 0
5
10
concetration mg L-1
15
20
Figure 1. Standard calibration curve for diazinon
3.4. Diazinon residues Data table 2 shows the content of diazinon as mg L-1 found after incubation with the tested bacteria strains and the degradation rate compared to control treatment. The data presented suggests that the most effective bacterial strains for speed of diazinon degradation rate were S2 (Pseudomonas. stutzeri) and S4 (Pantoa. anatis), respectively. Calculated residue half - life values (T1/2) for the S2,S4 treatments were 3.76, and 3.87 days, respectively and were 3.84, and 4.66 days for S3 (Alealigen. Sp) and S1 (Stenotrophomonas. Maltophilia) respectively, compared to the control treatment which was 8.9 days. Results indicated that p values for S2 and S4 were 0.03, and 0.04, respectively less than the null hypothesis H0 (no significant effect) at p
