Isolation of Pesticide-Degrading Microorganisms Using Winogradsky Columns: A Sustainable Approach to Bioremediation

In India, pesticides play a significant role in modern agriculture by enhancing crop yields and protecting plants from pests. However, their toxic and persistent nature poses a serious threat to the environment and human health. Among them, chlorpyrifos, an organophosphate pesticide, is of particular concern due to its wide use and long-term residual presence in soil and water. This calls for urgent and eco-friendly solutions such as biodegradation through microbial action. In the present study, Winogradsky columns were employed as a natural, cost-effective enrichment system to isolate native bacterial strains capable of degrading chlorpyrifos. Soil samples were collected from a farm, nursery, and garden in pesticide-contaminated regions and were used to construct Winogradsky columns. These columns allowed for the development of nutrient and oxygen gradients, facilitating the selective enrichment of pesticide-degrading microbial communities over 8 weeks. Following enrichment, five bacterial isolates—designated as G1, F2, F3, F4, and F5—were obtained. These isolates underwent both qualitative (clear zone formation) and quantitative (UV spectrophotometric) assays to evaluate their pesticide degradation potential. Further studies assessed the effect of environmental conditions such as temperature, pH, and NaCl concentration on bacterial growth to determine the robustness of the isolates. Morphological and biochemical characterization, along with reference to Bergey’s Manual of Systematic Bacteriology, identified the isolates as Staphylococcus aureus (G1), Pseudomonas aeruginosa (F2 and F5), and Micrococcus luteus (F3), while isolate F4 remained unidentified. Antibiotic susceptibility testing using the Kirby-Bauer disc diffusion method revealed sensitivity to most common antibiotics, ensuring their biosafety for environmental use. To investigate the genetic basis of pesticide degradation, plasmid DNA isolation and plasmid curing were performed. Four isolates (F2, F3, F4, F5) were found to possess plasmids, but after curing with ethidium bromide, all strains retained their ability to degrade chlorpyrifos, suggesting that the responsible genes are located on the bacterial chromosome, ensuring genetic stability. This study demonstrates the effectiveness of using Winogradsky columns to enrich and isolate indigenous chlorpyrifos-degrading bacteria, highlighting their potential application in sustainable bioremediation of pesticide-contaminated soils.