Chemicals and culture medium
The tested compounds CHL (99.5% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents (dimethyl sulfoxide DMSO and dichloromethane) used in this study were of high purity and analytical grade. Organic free water was prepared with a Milli-Q/Milli-Ro system (Millipore Corp., Bedford, MA, USA). Stock solutions of chlorpyrifos were prepared at 5000 mg L-1 in dimethyl sulfoxide (DMSO). For each experiment, 1 mL of CHL was added in the medium. For the microbial assays, mineral salt medium (MSM) and Luria-liquid broth (LB) were used. The medium (MSM) contained 20 mL of K2HPO4, 10 mL of MgSO4 7H2O, 10 mL of NaCl, 1 mL of CaCl2, 2 g of boric acid, 0,2 g of ZnSO4, 0,8g of CuSO4, 0,25 g of NaNO, and 0,05g of CO(NO2)2, 1mL of FeSO4 6H2O was added per liter of distilled water. The medium (LB) contained 10 g of peptone, 5 g of yeast extract and 10 g of NaCl per 1 liter of distilled water (pH 7.0). After autoclaving (121°C, 20 min) and cooling the medium was supplemented with a suitable CHL solution prepared as described above.
Sampling
The soil sample used in this study was collected from field site located in Mornag approximately 20 km away from Tunis (36° 67′ 70.39 N, 10° 27′ 53.78 E), Tunisia. The sampling site has been in use for intensive agricultural practices since long and this soil has received leachates after application of chlorpyrifos for several years. Soil sample was collected from a depth of 20 cm. Soil was mixed thoroughly and plant debris were removed. Then soil was sieved at 2mm and kept at 4°C until its use (for less than 3 weeks).
Mineralization of 14C-CHL in soil microcosms
The potential of the soil microbial community to mineralize chlorpyriphos (CHL) was evaluated using 14C-labelled CHL (Izotop, specific activity = 118 µCi/mg) as described by El-Sebai et al. (2007). Four individual replicates of 20 g soil microcosms were studied. For each replicate 1 mL of a methanol solution comprising 60 mg.L-1 of 14C-CHL and 0.068 µ Ci of 14C-CHL was applied on an aliquot of 1g of dried soil. After evaporation CHL contaminated soil was thoroughly mixed with the rest of soil. Soil humidity was then adjusted to 40 % of the water holding capacity (WHC) and kept constant all along the 70 days of incubation in the radiorespirometer. 14CO2 evolved from 14C-CHL was quantified by liquid scintillation counting (LS 6500 Multi-Purpose Scintillation Counter, Beckman) using ACSII scintillation fluid (Amersham) (Storck et al. 2017).
Enrichment procedure, isolation and characterization of chlorpyrifos degrading strain
Preliminary screening experiments were performed to obtain strains that were tolerant to CHL. The study was conducted as described previously by Ben Salem et al. 2016.
Only one soil with three replicates was used in this study. Fresh soil sample was divided in six subsamples of 50 g dry weight equivalent. Aqueous CHL solution were prepared at 4.8g. L-1 (which corresponds to 10 times the recommended dose agronomic purpose) the day of its application starting from Robust ® formulated solution. The duration of experiment was two weeks.
Soil samples were incubated at room temperature under laboratory conditions. They were watered every two days to keep soil humidity at 40% of water holding capacity (WHC). Every two days, they were irrigated with 10mL of commercial chlorpyrifos solutions as 10X concentrated solutions of pesticides to exert a selective pressure favorable to the emergence of degrading bacterial populations. Three samples were not treated with CHL but with equivalent amount of pure water (control). Two-days after CHL application, soil was sampled to immediately carry bacterial isolation. Briefly, one g of soil was added to 10 mL of physiological water (NaCl 9‰) and serially ten-times diluted. 100 µL of 10, 10-2 and 10-4 dilutions were streaked on PCA plates that were incubated for 16 hours at 37°C. Each colony growing on the plate was purified using the Z streak technique.
Primary distinction between all the isolates was based on the size, color and morphology of their colonies on the PCA plates. Bacterial colonies showing different morphologies were selected and further characterized using API 20E (Biomérieux, Lyon, France) following the recommendations of the Bergey’s manual of systematic bacteriology (Krieg et al. 1984). In addition, 16S rRNA amplicon generated by PCR using the 27f and 1492r universal primer pair (Gürtler and Stanisich 1996) from DNA extracted from the bacterial isolate was sequenced as previously described (Devers et al. 2008). 16S rRNA sequence was compared to other sequences available in GenBank database (http: //www. ncbi.nlm.nih.gov/genbank) using the BlastN search analysis (http://www.blast.ncbi.nlm.nih.gov/). Sequence was deposited in the Genbank database (SUB8916609 S10 MW494965).
Bacterial growth kinetics in different liquid media
To investigate the growth of Serratia rubidaea strain ABS 10 with chlorpyrifos, 200 µl of strain (OD 600 = 0.8) were inoculated into 20 mL of either MSM or nutrient broth medium added with CHL at a final concentration of 25 mg.L-1. The culture was incubated at 30±2°C on a rotary shaker at 120 rpm. The bacterial growth was regularly monitored for 5 days by measuring the turbidity of the culture using a spectrophotometer at 600 nm.
Inoculum preparation
A bacterial colony was inoculated in LB medium and grown to reach the exponential phase. It was then collected by centrifugation at 5000 g for 5 min. The cell pellet was washed twice with 0.9% of sterile NaCl and then re-suspended in NaCl to obtain the bacterial suspension at a concentration of approximately 3.106 CFU /mL. The cell density (OD 600 nm) was measured using UV-Visible spectrophotometer (Lu et al. 2013)
Dissipation of chlorpyrifos by S. rubidaea strain ABS 10 in aqueous medium
CHL dissipation studies were performed in 250 mL Erlenmeyer flasks containing 100 mL of sterile MSM supplemented with CHL at 25 mg L1 and inoculated with 1 mL of bacterial strain (approximately 3×106 cells.mL-1). Un-inoculated media comprising with the same concentration of CHL were used as control. All the samples were incubated at 30 ± 2°C on a rotary shaker at 120 rpm for 5 days. Samples were periodically taken from the culture under aseptically conditions to measure the remaining pesticide concentration the culture medium.
Dissipation of chlorpyrifos by S.rubidaea strain ABS 10 in soil
To study the dissipation of chlorpyrifos in sterilized (SS) or natural (NS) soil inoculated with S .rubidaea strain ABS 10 (B) or not S. rubidaea strain ABS 10. Briefly, 100 g of sterilized soil (SS) or natural soil (NS) was placed in a 250 ml Erlenmeyer flask, inoculated or not with 30 ml of a S. rubidaea strain ABS 10 suspension (B) containing 3 × 106 cells.mL−1 and treated with CHL at 25 mg kg−1. The amount of carbon, nitrogen and phosphorous were calculated using the relationship C/N/P 100:10:1. The sources of carbon, nitrogen and phosphorous were glucose, (NH4)2SO4 and K2HPO4 respectively (Martin et al. 2007). Sterilized soil not inoculated with S. rubidaea strain ABS 10 was used to estimate abiotic dissipation of CHL (Pino et al. 2011). All flasks were incubated in an incubator at 30 ± 2 ◦C. Samples were periodically removed aseptically to determine the pesticide concentration. Each treatment was performed in triplicate (ntot=12).
Analytical methods
At regular intervals, 5–10 mL cultures were withdrawn from aqueous medium and centrifuged at 7200 × g for 10 min to obtain a cell-free medium. CHL was extracted twice from the supernatant with an equal volume of dichloromethane (DCM). Organic layers of DCM were pooled and evaporated at 28 ± 2 °C. For the analysis of CHL in soil, 5 g of soil samples was mixed with 10 ml of dichloromethane. The samples were ultra-sonicated for 30 min at 30°C. After that, the mixture was centrifuged for 30 min on a rotary shaker at 120 rpm. Then the samples were allowed to stand until the soil had settled, and the clear supernatant was used to determine the pesticide concentration by GC-MS. Levels of CHL were measured by GC-MS using an Agilent 6850N gas chromatograph (Agilent Technologies, USA), equipped with an Agilent6973 MS detector. A capillary column HP-5MS (30 m, 0.25 mm, 0.50 mm) was used while chromatographic separation was achieved with the following method: the GC oven temperature was initially set at 70°C, for 2 min, and raised to 270°C at a rate of 20°C/min and held for 10 min. The injector and detector were set at 250 and 280°C, respectively. The carrier gas Helium was used as at a constant flow rate of 1 mL/min. Electron impact (70ev) mass spectra were recorded from 100 to 550 amu (atomic mass unit).
Data analysis
The four kinetic models proposed by the FOCUS working group on pesticide degradation kinetics (FOCUS, 2006) were used to calculate pesticide dissipation kinetic parameters. The four kinetic models proposed by the FOCUS working group on pesticide degradation kinetics (FOCUS, 2006) were used to calculate pesticide dissipation kinetic parameters: the single first order kinetic model (SFO), and the biphasic models hockey stick (HS), first order multi-compartment model (FOMC) and double first order in parallel model (DFOP).
Data obtained from the dissipation experiments were fit to the exponential decay model. The first-order model and the DT50 was calculated as follows:
Where Ct is the concentration of pesticide remaining in MSM or soil after t days, C0 is the initial concentration of pesticide in MSM or soil. k and t are the rate constant (d-1) and degradation time in days respectively (Focus, 2006)
Statistical analysis
All the experiments were performed in triplicates. The data were statistically analyzed using two-way analysis of variance (ANOVA). When significant differences test (P≤0.05) were observed, the means were separated using Graphpad Prism, v7.00.