AN ADVANCED STUDY ON IN-SITU MYCOREMEDIATION OF PESTICIDE CONTAMINATED AGRICULTURAL SOIL AND ITS SUSTAINABILITY

Abstract

The dawn of the 21st century has marked the boom of pesticide industries across the world that ameliorated not only the agricultural business but also the public health sector. However, the increasing global consumption of pesticides generates several toxic compounds posing severe effect to environment and public health. Many recalcitrant pesticides accumulate in the soil and, reach various environmental components, such as air and surface water. Generally, the recalcitrant pesticides are adsorbed to the soil and, thus, are inadequately accessible to bacteria due to their intracellular degradation processes. However, the filamentous nature of fungi offers a great advantage over bacteria because it helps the fungi to permeate through the soil environment effectively. Of the various physicochemical methods proposed, bio-based removal of pesticides has been suggested as sustainable and an attractive method of treating contaminated environmental matrices due to their economic and non-toxic nature. Bioremediation has also been recommended as a potentially attractive, ecofriendly, and economical solution for a variety of contaminants. Given that, the study has been undertaken to effectively restore the pesticide-contaminated soils, exploiting the potent isolated fungi. The study objectives take in: isolation and identification of pesticide-resistant fungi, assessing its tolerance level to pesticides in liquid culture and soil systems, bioaugmentation studies, dissipation kinetics and statistical analyses, furthermore the residual quantification and metabolite identification. A comparative bioaugmentation studies were carried out on mixed carbamate (Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-ylmethylcarbamate) and Carbaryl (1-Naphthylmethylcarbamate)) contaminated soil using newly isolated Acremonium sp. (MK514615). The experiments were performed in the range of biomass (0.2~0.6 g kg-1), temperature (23~33 oC), pH (6~9), and moisture (10~30%). Towards the development of the bioaugmentation strategy, the process parameters were optimized employing Central Composite Design-Rotatable Method. To confirm the model appropriacy and sustainability of the optimization procedure, bioremediation experiments were conducted onto carbamate-contaminated soils.About 97.65%/99.06% removal was achieved for carbofuran and carbaryl, respectively. The kinetic data showed the degradation process was characterized by the rate constants (k) of 0.4758, 0.325 and 0.0277 d-1, with the corresponding half-lives (t1/2) of 5.3903, 2.1327 and 8.3610 d for zero, first and second-order respectively. GC-MS analysis of fungal-treated carbofuran samples detected the metabolites generated during the process; identified as carbofuran-7-phenol (tR 6.42 min; m/z 165); 2-Hydroxy-3-(2-hydroxy-2-methylpropyl) phenyl-N-methylcarbamate (tR 5.73 min; m/z 240); 3-(2-hydroxy-2-methylpropyl) benzene-1,2-diol (tR 3.60 min; m/z 183). Similarly, α-naphthol (tR 7.42 min; 144 m/z); 1,4-naphthoquinone (tR 6.84; m/z 158) and benzoic acid (tR 5.26 min; m/z 104) were identified in treated carbaryl samples. Similarly biodegradation studies were conducted for carbamates mixture using Carb.1b strain isolated from soil. Based on morphology and 18S rRNA sequencing, the Carb.1b strain was identified as an Ascochyta sp. CBS 237.37 (MG786925). The isolate was employed in two growth mediums (added carbon/carbon-free) enriched with carbamates (25 to 85 mg L-1) to assess its degradation efficacy. Response Surface Methodology was exploited to determine the optimum conditions for studied parameters. Over 86.7%/90.15% of carbamates (85 mg L-1) were removed in added carbon/carbon-free medium, respectively, with half-life of 26 d and R2 ranging 0.982-0.999; indicating high tolerance of carb.1b strain towards carbamates. The widespread use of type-II pyrethroids in agricultural and residential practices has raised serious public-health and environmental concerns. With this in view, continuous biodegradation experiments were carried out using the fungal consortium to effectively degrade Deltamethrin (α-(Cyano-3-phenoxybenzyl)-3-(2,2-dibromovinyl-2,2-dimethylcyclopropane carboxylate) and 3-phenoxybenzaldehyde. The biodegradation of DMT followed the single first-order kinetic model, with R2 ranged 0.986-0.995. The degradation of 3-PBA with same strain proves to be laudable since it is not only quite persistent; however, it also limits pyrethroids biodegradation due to its antimicrobial activities. The values of degradation rate constant (k) ranged 0.01672 to 0.2717 d−1 with half-life values (t1/2) of 2.55 to 4.14 d; which has been significantly reduced compared to the reported studies. DMT/3-PBA (100 mg Kg-1) dissipation rates were considerably higher in treated soil system (T3) than in non-sterile (T4) and sterile soil without fungi (T5).The continuous mycoremediation experiments coupled with bioaugmentation studies were performed using newly isolated Aspergillus sp. The transforming ability of the strain PYR-P2 was evaluated in minimal salt media, where fungus utilized up to 500 mg L-1 of pyrethroid mixture (Cypermethrin (CYP), Cyfluthrin (CYF), Cyhalothrin (CYH)). Under the optimized conditions, strain was implemented for the bioaugmentation studies in natural and sterile soil (NS/SS) systems spiked with pyrethroids (100 mg Kg-1). The highest pyrethroid removal percentages were observed in the fungally-augmented natural soil system where CYP (99.94%) was degraded at a higher rate than CYF (96.52%), CYH (94.48%), and pyrethroid mixture (86.49%) accompanied by a decrease in pyrethroid half-life (t1/2). The pesticide residues were quantified, and metabolites generated were identified using chromatographic-techniques. Overall, this study provides novel insights on the interaction between pesticides and fungi at a structural and functional levels; and should be considered in developing new bioremediation technologies. These advanced research efforts exploiting new fungal species can be practiced to promote the use of mycoremediation for the pesticide-contaminated milieu and to develop field-worthy remediation systems.