Biodegradación de la atrazina mediante hongos del Parque Nacional do Iguaçu
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2018-12-12
Autores
Esparza Naranjo, Samantha Beatríz
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A lo largo de las últimas décadas, el uso indiscriminado de agrotóxicos en actividades agrícolas ha provocado que una gran porción de las cantidades aplicadas permanezca o se disemine en el suelo, agua superficial y subterránea de los ecosistemas asociados a los campos de cultivo, comprometiendo la salud de microorganismos, plantas, animales y el ser humano. La atrazina, un herbicida ampliamente utilizado para el control de maleza, presenta moderada persistencia ambiental y alta toxicidad, siendo necesaria la búsqueda de linajes microbianos para la biorremediación de ambientes contaminados por este agrotóxico. A pesar de que el alto contenido de hierro (Fe) presente en los suelos del Parque Nacional do Iguaçu (PNI) puede haber seleccionado microorganismos con potencial de biodegradación de agrotóxicos diferentes a los ya descritos, la diversidad microbiana del PNI es prácticamente desconocida. Así, este trabajo tiene como objetivo evaluar el potencial de biodegradación de atrazina por hongos del PNI, al definir un método de selección eficiente de hongos en base a su producción de biomasa y de lacasa en presencia de la atrazina, además de su capacidad para degradar el herbicida. Además, se buscó seleccionar a los aislados con el mayor potencial de degradación del herbicida y caracterizarlos usando técnicas morfológicas y moleculares. Para definir el método de selección, se determinó el peso seco del micelio, la cantidad de lacasa producida, midiendo el cambio de coloración provocado por la oxidación del sustrato enzimático ABTS en el espectrofotómetro y se cuantificó la atrazina (ATZ) y los metabolitos desisopropilatrazina (DIA) y desetilatrazina (DEA). Como método de preparo de muestra y análisis se utilizó la Microextracción Líquido-Líquido Dispersiva (DLLME), en conjunto con Cromatografía Gaseosa acoplada al Espectrómetro de Masas (GC - MS). No se pudo establecer un método de selección de hongos con potencial de degradar atrazina al no existir una relación entre degradación de la ATZ, producción de biomasa y producción de lacasa. Por tanto, se sometió a análisis cromatográficas a todos los hongos. En este estudio, el potencial de degradación de ATZ fue analizado en términos de formación de DEA, al conseguir diferenciar aislados con y sin esta capacidad. No obstante, se determinó que los hongos tienen capacidad para degradar ATZ y producir DIA, con un porcentaje que no difiere significativamente entre los aislados. Tampoco existe diferencia significativa entre los productores de DEA (aislados DF, IB, GW y GU). Estos cuatro fueron identificados usando características morfológicas, por la observación de estructuras reproductivas, y moleculares, al realizar el análisis filogenético con las secuencias de ADN ribosomal, de la región ITS-1 e ITS-4. GU y DF pertenecen al género Fusarium, GW presenta proximidad filogenética con el género Fusarium y Aschersonia, e IB no fue considerado para el análisis filogenético por la baja calidad de la secuenciación. Los organismos del género Fusarium se destacan por ser fitopatógenos y catalizan la ATZ a través de la retirada de los terminales n-alquilo, en este caso evidenciado por la formación de DEA. Con base en estos resultados, nuevos análisis cromatográficos serán realizadas para obtener el perfil metabólico, así como análisis de toxicidad de los productos generados, ya que los aislados son candidatos para consorcios microbianos que permitan alcanzar el objetivo de la biorremediación.
Over the last decades, the indiscriminate use of pesticides in agricultural activities has caused a large accumulation of these components in soil, surface water and groundwater of the ecosystems associated to crop fields, compromising the health of microorganisms, plants, animals and human beings. Atrazine, an herbicide widely used for weed control, has moderate environmental persistence and high toxicity, making it necessary to search for microbial strains for bioremediation of environments contaminated by this herbicide. Although the high iron (Fe) content associated with Iguaçu National Park (PNI) soils may have selected microorganisms with herbicide biodegradation potential different from those already described, PNI ́s microbial diversity is practically unknown. Thus, this work aimed to evaluate the potential of atrazine biodegradation by fungi from PNI, by using an efficient method of selection of fungi based on its biomass and laccase production in presence of atrazine, as well as, their herbicide degradation potential. Also, the isolates with the greatest herbicide degradation potential were selected, and they were characterized by morphological and molecular approaches. In the selection method, mycelium dry weight and laccase production amount were determined. The enzymatic production was measured by change in coloration caused by the oxidation of the ABTS enzyme substrate in a spectrophotometer. To quantify atrazine (ATZ) and its metabolites (deisopropylatrazine (DIA) and deethylatrazine (DEA)), Dispersive Liquid-Liquid Microextraction (DLLME), associated to Gas Chromatography coupled to the Mass Spectrometer (GC-MS), was used as sample preparation and analysis method. It was not possible to establish a selection method of fungi with the potential to degrade atrazine since there is no relationship between ATZ degradation, biomass production, and laccase production. Therefore, chromatographic analyses were carried out in all fungi. In this study, the degradation potential of ATZ was analyzed by DEA formation, since it was possible to differentiate isolates with and without this capacity. However, fungi have the capacity to degrade ATZ and produce DIA, in a quantity that does not differ significantly among isolates. Also, there is no significant difference between DEA producers (isolates DF, IB, GW, and GU). These four were identified at the genus level using morphological (observation of reproductive structures) and molecular characteristics (phylogenetic analysis using ribosomal DNA sequences of ITS-1 and ITS-4 region). GU and DF belong to genus Fusarium, GW presents phylogenetic proximity to genus Fusarium and Aschersonia, and IB was not considered in the phylogenetic analysis due to low-quality sequence. The genus Fusarium strains stand out for being phytopathogens, and they catalyze ATZ through the removal of N-alkyl terminals, in this case, evidenced by DEA formation. Based on these results, new chromatographic analyzes will be carried out aiming at obtaining the metabolic profile, as well as toxic analyses of generated products. The isolates are candidates for microbial consortia that allow achieving the goal of bioremediation
Over the last decades, the indiscriminate use of pesticides in agricultural activities has caused a large accumulation of these components in soil, surface water and groundwater of the ecosystems associated to crop fields, compromising the health of microorganisms, plants, animals and human beings. Atrazine, an herbicide widely used for weed control, has moderate environmental persistence and high toxicity, making it necessary to search for microbial strains for bioremediation of environments contaminated by this herbicide. Although the high iron (Fe) content associated with Iguaçu National Park (PNI) soils may have selected microorganisms with herbicide biodegradation potential different from those already described, PNI ́s microbial diversity is practically unknown. Thus, this work aimed to evaluate the potential of atrazine biodegradation by fungi from PNI, by using an efficient method of selection of fungi based on its biomass and laccase production in presence of atrazine, as well as, their herbicide degradation potential. Also, the isolates with the greatest herbicide degradation potential were selected, and they were characterized by morphological and molecular approaches. In the selection method, mycelium dry weight and laccase production amount were determined. The enzymatic production was measured by change in coloration caused by the oxidation of the ABTS enzyme substrate in a spectrophotometer. To quantify atrazine (ATZ) and its metabolites (deisopropylatrazine (DIA) and deethylatrazine (DEA)), Dispersive Liquid-Liquid Microextraction (DLLME), associated to Gas Chromatography coupled to the Mass Spectrometer (GC-MS), was used as sample preparation and analysis method. It was not possible to establish a selection method of fungi with the potential to degrade atrazine since there is no relationship between ATZ degradation, biomass production, and laccase production. Therefore, chromatographic analyses were carried out in all fungi. In this study, the degradation potential of ATZ was analyzed by DEA formation, since it was possible to differentiate isolates with and without this capacity. However, fungi have the capacity to degrade ATZ and produce DIA, in a quantity that does not differ significantly among isolates. Also, there is no significant difference between DEA producers (isolates DF, IB, GW, and GU). These four were identified at the genus level using morphological (observation of reproductive structures) and molecular characteristics (phylogenetic analysis using ribosomal DNA sequences of ITS-1 and ITS-4 region). GU and DF belong to genus Fusarium, GW presents phylogenetic proximity to genus Fusarium and Aschersonia, and IB was not considered in the phylogenetic analysis due to low-quality sequence. The genus Fusarium strains stand out for being phytopathogens, and they catalyze ATZ through the removal of N-alkyl terminals, in this case, evidenced by DEA formation. Based on these results, new chromatographic analyzes will be carried out aiming at obtaining the metabolic profile, as well as toxic analyses of generated products. The isolates are candidates for microbial consortia that allow achieving the goal of bioremediation
Abstract
Descrição
Trabajo de Conclusión de Curso II presentado al
Instituto Latinoamericano de Ciencias de la Vida y de la
Naturaleza de la Universidad Federal de la Integración
Latinoamericana como requisito parcial para la
obtención del título de grado en Ciencias Biológicas –
Ecología y Biodiversidad.
Orientadora: Profa. Dra. Rafaella Costa Bonugli Santos
Co-orientadora: Profa. Dra. Marcela Boroski
Palavras-chave
Biodegradación de pesticidas, Parque Nacional do Iguaçu (PNI), Atrazina (ATZ), Desisopropilatrazina (DIA), Desetilatrazina (DEA)
Citação
ESPARZA NARANJO, Samantha Beatríz. Biodegradación de la atrazina mediante hongos del Parque Nacional do Iguaçu. 2018. 65 f. Trabajo de Conclusión de Carrera (Ciencias Biológicas – Ecología y Biodiversidad) – Universidad Federal de la Integración Latinoamericana, Foz do Iguaçu,
2018