Sequential Microbial-Photocatalytic process for degradation of neonicotinoid pesticide

Abstract

Imidacloprid (IMI) insecticide was selected from the neonicotinoid family on the basis of its intensive usage throughout the year for wheat and paddy crop in Punjab (India). Soil samples for isolation of bacteria were collected from paddy field and five bacterial strains (T1-T5)were isolated on enrichment media (NB) containing 50 mgL-1 of IMI. Out of these five, strain T1 and T5 exhibited the highest growth and further identified/characterized on the basis of morphological, biochemical, physiological characterization and phylogenetic analysis using 16S rRNA sequencing. Results showed that both of the isolates are a member of Enterobacter sp. and hereafter mentioned as ATA1 and ATA2. Phylogenetic tree were constructed for both isolates by neighbour joining method using GenBank-NCBI database. A close similarity of strain ATA1 was confirmed with Enterobacter asburiae JCM6051 (AB004744) with 98.1% gene sequence whereas strain ATA2 has shown 98% similarity with Enterobacter hormaechei ATCC 49162 strain (AZ508302). Both isolates were referred as Enterobacter sp. ATA1 and Enterobacter sp. ATA2. A significant growth shown by strain ATA1 as compared to strain ATA2 was further selected for degradation studies. Initially, tolerance to IMI at various concentrations (50-150 mgL-1) in minimal media was performed and observed more growth for 50 mgL-1 as compare IMI=100 mgL-1 and 150 mg L-1. Various co-metabolites (Maltose, Sucrose, Fructose, Lactose and Glucose) were used as additional source of carbon for both growth and degradation studies in minimal media broth. Among, these studied co-metabolites, glucose (0.1% w/v) found to show maximum growth, indicated its effective use as carbon source than the others. Co-metabolite degradation studies were performed in minimal media with glucose (MMG 0.1%) under the optimized concentration of IMI (50 mgL-1) and glucose (0.1% w/v) and degradation of ~45% was confirmed at 3rd of incubation. No notable change was observed upto 15 days of degradation time. LC chromatogram analysis supported the results for its degradation and metabolites formation till 15 days of incubation. Microcosm study in soil was performed and various parameters (pH, inoculums size, initial concentration of imidacloprid and flooding of soil) were optimized under laboratory conditions. At variable pH = 1-11, highest degradation (68%) was percieved at pH = 7 after 15 days of incubation for fixed concentration of IMI (50 mgkg-1 of soil). Effect of initial concentration of IMI (25-100 mgkg-1of IMI) at pH = 7 showed that 50 mgkg-1 of IMI lead to better degradation (74%) and hence considered as optimum. These results were further supported by GC-MS chromatograms where peak height/peak area for IMI is least while using its concentartion = 50 mgkg-1, revealing it to be an optimum amount. Highest degradation of IMI (74%) was obtained at inoculum size of 2×107 CFU g-1 soil and confirmation by GC-MS chromatograms.Influence of flooding/nonflodding conditions for degradtion of IMI, was studied and degradation was found to be more in non-flooded (60%) conditions as compared to flooded (50%). Identification of metabolites carrioed out through GC-MS analysis during microbial degradation of IMI. Six metabolites (I-1 to I-6) have been identified through their mass spectra. Photocatalytic degradation studies were performed using TiO2 as photocatalyst in soil.Photolysis and adsorption studies in soil confirmed insignificant change in the concentration of IMI when kept under dark for 18 h. Moreover, photolysis of IMI in soil also showed no notable change in its amount. Optimization of catalyst dose was done by varying TiO2 amount from 0.1gkg-1 to 0.5gkg-1 and it was observed that degradation increases with increase in amount upto 0.3 gkg-1 and thereafter it decreases. This optimum TiO2 dose was further used for optimizing other four experimental parameters namely UV light intensity, initial conc. of IMI, pH, and depth of soil using central composite design (CCD) based on response surface methodology (RSM).Analysis of experimental data was supported by Design-Expert Software (trial version 9.0.3.1, Stat-Ease, Inc., MN, USA). The 30 experimental set up obtained by RSM were experimentally performed and the obtained results have been plotted –ln(C/Co) vs. Time. For all the experiments, plots were straight line with regression values from 0.993 to 0.974, indicating that degradation of IMI followed the L-H kinetics and pseudo first order kinetics. Highest degradation of 83% was achieved (pH = 3, intensity of light = 30 Wm-2 and initial IMI = 10 mgL-1) after 18 h of UV light irradiation and the corresponding apparent rate constant (k) was determined to be 15×10-4 min-1 followed by 9.3×10-3 min-1. Adequacy and significance of the studied model was verified by applying ANNOVA test with very low (<0.0001) probability value (P) for A (pH) and D (concentration of IMI) in comparison to other parameters B (intensity of light) and C (depth of soil). Additionally,adequacy of selected model (response surface reduced quadratic model) with real system was confirmed by analyzing the correlation between observed and predicted values. The response factor of calculated residual values showed that all data points lie close to straight line and within 95% confidence intervals line with mean values near zero confirming the good correlation between experimental and predicted values. Moreover, identification and probable mechanism involved for the formation of various intermediates during the photodegradation of IMI with quantitative estimation of various ions (nitrate, nitrite, and chloride) was also carried out. Based upon the results obtained, a probable mechanism for conversion of one intermediate into another during decomposition of imidacloprid, and a stoichiometric mass balanced equation were being proposed.Sequential studies were performed for microbial-photocatalytic (MP) and photocatalytic-microbial degradation processes (PM). It was found that residual amount of IMI after 15 day + 18 h of degradation in MP process (~2.6 mgL-1 = 96%) is significantly lower than that found in PM process (~7.0 mgL-1 = 87%) confirming betterment of former process. Results were further verified by time course analysis of common metabolite (m/z =128 & 228) after LC-MS analysis. It was found that in case of MP process the metabolites became highest of their concentration in the initial 5 days of incubation and thereafter start degrading or inter-converting into other compounds. However, for PM process an opposite has been observed where the formation of these intermediates continues till 15 d + 18 h of degradation. These results confirmed the superiority of MP process comparative to PM process. In order to find out the optimum degradation time for mineralization of IMI, samples obtained after 5th and 10th day of microbial degradation were further subjected to the photocatalytic degradation under similar conditions and were analyzed for LC-MS spectrum.It was observed that more number of metabolite were formed (reflected by number of peaks)after 15 d + 18 h of degradation in relation with 5 d + 18 h and 10 d + 18 h. This clearly suggested that time of 15 d + 18 h is optimum for mineralization of IMI under the conditions specified in present study. However, not much significant difference in degradation of IMI was observed after 10th and 15th day of incubation under the optimized conditions. Hence, LCMS analysis was performed and results showed that despite of almost same degradation after 10th and 15th day the formation of metabolites is more in later. Therefore, it can be concluded that in terms of mineralization 15th day is better compared to 10th day.