AVALIAÇÃO GÊNICA E PROTEICA DE CONSÓRCIOS MICROBIANOS NA DEGRADAÇÃO DO DIFENOCONAZOL.

Abstract

The extensive use of pesticides is crucial to meet agricultural productivity in Brazil, but it has negative impacts on the environment and human health. Among pesticides, Difenoconazole (DFZ) stands out for inhibiting the fungal growth of various phytopathogens. However, it is classified as highly dangerous and has low biodegradability, environmental persistence, and bioaccumulation in human and animal tissues. Given these characteristics, bioremediation is seen as a viable alternative that utilizes microorganisms to convert pollutants into less toxic compounds. It focuses on the production of microbial enzymes, especially oxidoreductases, becoming an economical and promising strategy with potential for degrading recalcitrant compounds like DFZ. This project aims to explore the use of microbial consortia to degrade DFZ. It involves evaluating bacterial growth in media supplemented with DFZ, constructing bacterial consortia, analyzing the toxicity of media containing DFZ before and after biological treatment, and investigating the proteomic and genomic profiles of microbial consortia. Bacteria from the molecular biology laboratory library – ITP/SE were activated in LB (Luria Bertani) and minimal salt medium (MSM), and MSM supplemented with commercial DFZ at 0.05%. Six consortia consisting of bacterial trios and one consortium consisting of all isolates used in this study were constructed. Microbial growth with supplemented media was assessed, and antagonism tests, as well as ecotoxicity tests on lettuce seeds, were conducted. The gene profile was verified by PCR for catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Finally, the protein profile was examined using 12% SDS-polyacrylamide gel electrophoresis. Results indicated that the isolates have the capacity for growth in these media, indicating their potential for pesticide growth and degradation. Antagonism tests did not show inhibitory halos, indicating that microorganisms do not inhibit each other's growth and do not exhibit antagonistic activity. Regarding consortium growth, C2, C4, C6, and C7 showed the highest growth at 48 h and 72 h, indicating adaptability and degradation capacity to the pesticide-containing medium. Ecotoxicity tests showed that treatment with DFZ by consortia C2 and C7 reduced pesticide toxicity by 50%. Protein profile analysis revealed four common bands around 30 kDa among isolates MI1 (Bacillus cereus.), MI3 (Bacillus sp.), P.sp (Pseudomonas sp.), and consortium C7, likely corresponding to peroxidase enzymes associated with the DFZ degradation process. The gene profile showed that microorganism MI29 (Bacillus sp.) has the catechol 2,3-dioxygenase gene, and microorganism 8BR has the catechol 1,2-dioxygenase gene. In conclusion, this work led to the formation of an effective consortium in DFZ degradation, reducing its toxicity and indicating the involvement of oxidoreductase enzymes in the pesticide degradation process through proteomic-enzymatic analysis.