Aislamiento preliminar de consorcios microbianos degradadores de pentolita a partir de muestras de suelos, lodos y agua con presencia de explosivos
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2011
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Universidad de La Salle. Facultad de Ingeniería. Ingeniería Ambiental y Sanitaria
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La presente investigación está adscrita a un macro proyecto inter-institucional conformado por la Universidad de la Salle (Centro de Investigaciones de Medio ambiente y salud) CIMAS y la Pontificia Universidad Javeriana (Unidad de Saneamiento y Biotecnología Ambiental) USBA. El macro proyecto lleva como título “Diseño de un Sistema de microorganismos degradadores de PENT y TNT incorporados en el explosivo de Pentolita®”. Este proyecto de investigación hace parte de seis proyectos de tesis realizados en la Universidad de la Salle, el cual tuvo como objetivo evaluar la capacidad biodegradadora del explosivo Pentolita (PENT+TNT) por consorcios microbianos aislados. Para esto se tomaron muestras de suelo, agua y lodos en siete puntos diferentes con presencia de explosivos, las cuales fueron transportadas y almacenadas en el laboratorio de microbiología de la Universidad de La Salle; cada muestra fue agregada a un medio mineral líquido con fuente de carbono (T2) y 100 mg/L de Pentolita (5 g de muestra en 45 ml de medio para las muestras de suelo y lodo; 5 ml de muestra en 45 ml de medio para las muestras de agua) durante un periodo de 59 días a 120 rpm, 21 °C +/- 2 y ausencia de luz. A su vez se realizó monitoreo cualitativo del crecimiento poblacional de los microorganismos (observación de placas in vitro y tinciones de Gram) que conformaron cada uno de los consorcios aislados, encontrando que a los 42 días de incubación se presentó crecimiento bacteriano en 5 puntos de muestreo y en uno se observó la presencia de hongos. Posteriormente se realizaron dos resiembras. La primera resiembra se realizó transfiriendo 5 ml de cada consorcio aislado a un nuevo erlenmeyer con 45 ml de T2 y 100 mg/L de Pentolita, bajo las mismas condiciones de temperatura y agitación trabajadas inicialmente. La segunda resiembra se realizó a los 72 días de incubación de la primera resiembra, transfiriendo 10 ml de cada consorcio en 90 ml de T2 con explosivo. Durante cada resiembra se hizo siembra en agar T2, en donde se cuantificó el número de unidades formadoras de colonia (UFC) por mililitro y se determinaron los morfotipos de las cepas que conforman los consorcios aislados, teniendo en cuenta características como su elevación, color y borde. Finalmente se evaluó la capacidad biodegradadora de los consorcios aislados mediante bioensayos, los cuales fueron preparados en tubos de ensayo agregando 6 ml de un pre- inoculo (pellet de los consorcios microbianos + 70 ml de T2 sin explosivo) y 6 ml de T2 con explosivo. Para los bioensayos se manejaron 4 concentraciones diferentes: 50, 150, 250 y 350 mg/L de Pentolita y se inocularon durante una semana, tiempo en el cual se realizaron extractos al tiempo 0 (cero días de incubación, tiempo 1(cuatro días de incubación) y tiempo 2 (8 días de incubación) para su posterior lectura en la Cromatografía Líquida de Alto Rendimiento (HPLC), utilizando el método EPA 8330B para nitroaromáticos, nitraminas, y ésteres de nitrato
This research is attached to a draft inter-institutional Macro made by La Salle University (Research Center for Environment and Health) CIMAS and La Pontificia Universidad Javeriana (Sanitation Unit and Environmental Biotechnology) USBA. The Macro project is entitled "Design of a System of PENT degraders and incorporated in the explosive TNT to Pentolite ®." This research project is part of six thesis projects conducted at the University of La Salle, which aimed to assess the ability biodegradable Pentolite explosive (PETN + TNT) by microbial consortia isolated. For this, samples of soil, water and sludge at seven different points in the presence of explosives, which were transported and stored in the laboratory of microbiology at the University of La Salle, each sample was added to a liquid mineral medium with power carbon (T2) and 100 mg / L of Pentolite (5 g sample in 45 ml of medium for samples of soil and mud sample 5 ml in 45 ml medium for water samples) over a period of 59 days to 120 rpm , 21 ° C + / - 2 and no light. At the same time were monitored qualitatively the population growth of microorganisms (in vitro observation plates and Gram stains) that make up each of the consortium, finding that at 42 days of incubation, bacterial growth in 5 sampling points and one showed the presence of fungi. Subsequently there were two replant. The first seeding was done transferring 5ml of each consortium isolated a new flask with 45 ml of T2 and 100 mg / l of Pentolite, under the same conditions of temperature and agitation worked initially. The second passage was performed at 72 days of incubation of the first seeding, transferring 10 ml of each consortium in 90 ml of T2 with explosive. During each reseeding plating agar was T2, where we quantified the number of colony forming units (CFU) per milliliter and were determined morphotypes of the strains that make up the consortia, taking into account characteristics such as elevation, color and edge. In the evaluation of the biodegradable capacity consortia isolated by bioassay, which were prepared in test tubes by adding 6 ml of a pre-inoculum (pellet of microbial consortia + 70 ml of T2 without explosive) and 6ml of T2 with explosive. Cultures were handled for 4 different concentrations: 50, 150, 250 and 350 mg / L of Pentolite and inoculated for a week, at which time extracts were made at time 0 (zero days of incubation, time 1 (4 days incubation) and time 2 (8 days incubation) for further reading on High Permormance Liquid Chomatography (HPLC) using EPA method 8330 B for nitroaromatics, nitamines, and nitrate esters
This research is attached to a draft inter-institutional Macro made by La Salle University (Research Center for Environment and Health) CIMAS and La Pontificia Universidad Javeriana (Sanitation Unit and Environmental Biotechnology) USBA. The Macro project is entitled "Design of a System of PENT degraders and incorporated in the explosive TNT to Pentolite ®." This research project is part of six thesis projects conducted at the University of La Salle, which aimed to assess the ability biodegradable Pentolite explosive (PETN + TNT) by microbial consortia isolated. For this, samples of soil, water and sludge at seven different points in the presence of explosives, which were transported and stored in the laboratory of microbiology at the University of La Salle, each sample was added to a liquid mineral medium with power carbon (T2) and 100 mg / L of Pentolite (5 g sample in 45 ml of medium for samples of soil and mud sample 5 ml in 45 ml medium for water samples) over a period of 59 days to 120 rpm , 21 ° C + / - 2 and no light. At the same time were monitored qualitatively the population growth of microorganisms (in vitro observation plates and Gram stains) that make up each of the consortium, finding that at 42 days of incubation, bacterial growth in 5 sampling points and one showed the presence of fungi. Subsequently there were two replant. The first seeding was done transferring 5ml of each consortium isolated a new flask with 45 ml of T2 and 100 mg / l of Pentolite, under the same conditions of temperature and agitation worked initially. The second passage was performed at 72 days of incubation of the first seeding, transferring 10 ml of each consortium in 90 ml of T2 with explosive. During each reseeding plating agar was T2, where we quantified the number of colony forming units (CFU) per milliliter and were determined morphotypes of the strains that make up the consortia, taking into account characteristics such as elevation, color and edge. In the evaluation of the biodegradable capacity consortia isolated by bioassay, which were prepared in test tubes by adding 6 ml of a pre-inoculum (pellet of microbial consortia + 70 ml of T2 without explosive) and 6ml of T2 with explosive. Cultures were handled for 4 different concentrations: 50, 150, 250 and 350 mg / L of Pentolite and inoculated for a week, at which time extracts were made at time 0 (zero days of incubation, time 1 (4 days incubation) and time 2 (8 days incubation) for further reading on High Permormance Liquid Chomatography (HPLC) using EPA method 8330 B for nitroaromatics, nitamines, and nitrate esters
Palabras clave
Muestras de suelos, Explosivos