Análisis de la formación de biopelículas de acidithiobacillus ferrooxidans ATCC 53993 sobre soportes inorgánicos y orgánicos y evaluación de su potencial biotecnológico en el tratamiento de aguas contaminadas con arsénico inorgánico.
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Date
2024
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Universidad de Concepción
Abstract
El arsénico (As) es un metaloide altamente tóxico para todas las formas de vida, el cual es liberado al ambiente por fuentes naturales y actividades antrópicas. El consumo de agua contaminada con As es un problema a nivel mundial, lo que representa un problema a la salud pública. Para remediar esta situación, existen tecnologías como la adsorción, método basado en la capacidad de retención de contaminantes en la superficie de adsorbentes. En la búsqueda de nuevos adsorbentes, los óxidos de hierro como Schwertmannita (Sch) y Jarosita (Jt), presentan buenas propiedades de adsorción de As, compuestos que pueden ser biomineralizados por bacterias acidófilas oxidantes de hierro como Acidithiobacillus ferrooxidans. La biomineralización de óxidos de hierro es un proceso mediado por factores como la formación de biopelículas y las interacciones del Fe(III) con diferentes estructuras de la matriz extracelular (ECM, del inglés extracelular matrix), por lo que es necesario caracterizar las biopelículas formadas por Acidithiobacillus ferrooxidans ATCC 53993, incluyendo componentes de su matriz extracelular, y evaluar su utilidad en el tratamiento de aguas contaminadas con As inorgánico mediante biomineralización de óxidos de hierro.
Así, se evaluó la viabilidad de A. ferrooxidans ATCC 53993 a diferentes concentraciones de As inorgánico, mediante análisis espectrofotométricos midiendo la densidad óptica a 430 nm. Se analizó la formación de biopelículas sobre soportes inorgánicos y orgánicos mediante ensayos de adherencia evaluados por microscopia de epifluorescencia y con microscopia electrónica de barrido. La caracterización de las estructuras presentes en la ECM se realizó mediante análisis de distribución de tamaño, microscopia electrónica de transmisión y análisis proteómico. Además, se analizaron los óxidos de hierro biomineralizados por biopelículas de A. ferrooxidans ATCC 53993 sobre un soporte orgánico inerte, los cuales fueron caracterizados mediante microscopia electrónica de barrido asociado a espectroscopia de rayos X de energía dispersiva y difracción de rayos X, junto también con una evaluación de su capacidad de adsorción de As mediante Espectrometría de Masas con Plasma Acoplado Inductivamente.
Los resultados indicaron que A. ferrooxidans ATCC 53993 fue capaz tolerar hasta 2 mM de As(III) y 32 mM de As(V). Además, de adherirse a sustratos inorgánicos (pirita y azufre elemental) y a un soporte orgánico inerte (bioball), formando biopelículas. En cuanto a la caracterización de su ECM, esta cepa es capaz de producir vesículas de membrana, las cuales presentan capacidad de adherencia a pirita y azufre elemental como también una capacidad oxidativa de hierro reducida con respecto a las células. Por último, las biopelículas de A. ferrooxidans ATCC 53993 son capaces de biomineralizar óxidos de hierro, principalmente Sch y Jt. Los cuales presentaron una capacidad para adsorber As(III) de 23.2 mg·g-1. Valores que sugieren la posibilidad de desarrollar un sistema de tratamiento de aguas contaminadas con As basado en la inmovilización de Acidithiobacillus ferrooxidans ATCC 53993 sobre bioballs y que permiten la biomineralización de óxidos de hierro y éstos pueden capturar el As inorgánico. Como proyecciones a este proyecto quedan estudiar el desarrollo de un sistema de tratamiento a mayor escala para evaluar los rendimientos de biomineralización de óxidos de hierro como de la adsorción de As. Por otra parte, surge la necesidad de esclarecer las funciones de las MVs dentro del contexto de la formación de biopelículas de esta bacteria, junto con la interacción con minerales que puede influir en la biomineralización de óxidos de hierro.
Arsenic (As) is a highly toxic metalloid to all forms of life, released into the environment through natural sources and anthropogenic activities. The consumption of water contaminated with As is a global issue, posing a public health problem. To remediate this situation, technologies such as adsorption, a method based on the retention capacity of contaminants on the surface of adsorbents, are employed. In the search for new adsorbents, iron oxides such as Schwertmannite (Sch) and Jarosite (Jt) exhibit good As adsorption properties. These compounds can be biomineralized by iron-oxidizing acidophilic bacteria like Acidithiobacillus ferrooxidans. The biomineralization of iron oxides is mediated by factors such as biofilm formation and the interactions of Fe(III) with different structures of the extracellular matrix (ECM). Thus, it is necessary to characterize the biofilms formed by Acidithiobacillus ferrooxidans ATCC 53993, including components of its extracellular matrix, and evaluate their utility in the treatment of water contaminated with inorganic As through the biomineralization of iron oxides. In this study, the viability of A. ferrooxidans ATCC 53993 at different concentrations of inorganic As was evaluated using spectrophotometric analysis by measuring optical density at 430 nm. Biofilm formation on inorganic and organic supports was analyzed through adherence assays evaluated by epifluorescence microscopy and scanning electron microscopy. The characterization of structures present in the ECM was performed using size distribution analysis, transmission electron microscopy, and proteomic analysis. Additionally, iron oxides biomineralized by biofilms of A. ferrooxidans ATCC 53993 on an inert organic support were characterized using scanning electron microscopy associated with energy dispersive X-ray spectroscopy and X-ray diffraction, along with an evaluation of their As adsorption capacity using Inductively Coupled Plasma Mass Spectrometry. The results indicated that A. ferrooxidans ATCC 53993 was able to tolerate up to 2 mM of As(III) and 32 mM of As(V). Additionally, it adhered to inorganic substrates (pyrite and elemental sulfur) and an inert organic support (bioball), forming biofilms. Regarding the characterization of its ECM, this strain is capable of producing membrane vesicles, which exhibit adhesion to pyrite and elemental sulfur, as well as a reduced iron oxidation capacity compared to the cells. Lastly, the biofilms of A. ferrooxidans ATCC 53993 can biomineralize iron oxides, primarily Sch and Jt, which demonstrated an As(III) adsorption capacity of 23.2 mg·g⁻¹. These values suggest the potential to develop a water treatment system for As contamination based on the immobilization of Acidithiobacillus ferrooxidans ATCC 53993 on bioballs, allowing for the biomineralization of iron oxides capable of capturing inorganic As. Future projections for this project include studying the development of a larger-scale treatment system to evaluate the efficiency of iron oxide biomineralization and As adsorption. Additionally, there is a need to clarify the functions of MVs within the context of biofilm formation by this bacterium, as well as the interaction with minerals, which can influence the biomineralization of iron oxides.
Arsenic (As) is a highly toxic metalloid to all forms of life, released into the environment through natural sources and anthropogenic activities. The consumption of water contaminated with As is a global issue, posing a public health problem. To remediate this situation, technologies such as adsorption, a method based on the retention capacity of contaminants on the surface of adsorbents, are employed. In the search for new adsorbents, iron oxides such as Schwertmannite (Sch) and Jarosite (Jt) exhibit good As adsorption properties. These compounds can be biomineralized by iron-oxidizing acidophilic bacteria like Acidithiobacillus ferrooxidans. The biomineralization of iron oxides is mediated by factors such as biofilm formation and the interactions of Fe(III) with different structures of the extracellular matrix (ECM). Thus, it is necessary to characterize the biofilms formed by Acidithiobacillus ferrooxidans ATCC 53993, including components of its extracellular matrix, and evaluate their utility in the treatment of water contaminated with inorganic As through the biomineralization of iron oxides. In this study, the viability of A. ferrooxidans ATCC 53993 at different concentrations of inorganic As was evaluated using spectrophotometric analysis by measuring optical density at 430 nm. Biofilm formation on inorganic and organic supports was analyzed through adherence assays evaluated by epifluorescence microscopy and scanning electron microscopy. The characterization of structures present in the ECM was performed using size distribution analysis, transmission electron microscopy, and proteomic analysis. Additionally, iron oxides biomineralized by biofilms of A. ferrooxidans ATCC 53993 on an inert organic support were characterized using scanning electron microscopy associated with energy dispersive X-ray spectroscopy and X-ray diffraction, along with an evaluation of their As adsorption capacity using Inductively Coupled Plasma Mass Spectrometry. The results indicated that A. ferrooxidans ATCC 53993 was able to tolerate up to 2 mM of As(III) and 32 mM of As(V). Additionally, it adhered to inorganic substrates (pyrite and elemental sulfur) and an inert organic support (bioball), forming biofilms. Regarding the characterization of its ECM, this strain is capable of producing membrane vesicles, which exhibit adhesion to pyrite and elemental sulfur, as well as a reduced iron oxidation capacity compared to the cells. Lastly, the biofilms of A. ferrooxidans ATCC 53993 can biomineralize iron oxides, primarily Sch and Jt, which demonstrated an As(III) adsorption capacity of 23.2 mg·g⁻¹. These values suggest the potential to develop a water treatment system for As contamination based on the immobilization of Acidithiobacillus ferrooxidans ATCC 53993 on bioballs, allowing for the biomineralization of iron oxides capable of capturing inorganic As. Future projections for this project include studying the development of a larger-scale treatment system to evaluate the efficiency of iron oxide biomineralization and As adsorption. Additionally, there is a need to clarify the functions of MVs within the context of biofilm formation by this bacterium, as well as the interaction with minerals, which can influence the biomineralization of iron oxides.
Description
Tesis presentada para optar al grado académico de Doctora en Ciencias Biológicas Área Biología Celular y Molecular
Keywords
Microbiología, Biotecnología, Metales pesados