Aislamiento, selección e identificación de cepas de levadura de elevada acumulación de lípidos en la cuenca baja – media del río Chicama
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Date
2021
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Universidad Nacional de Trujillo
Abstract
En la búsqueda de alternativas menos contaminantes, menos toxicas, biodegradables, renovables, sostenibles y ecológicas para la obtención de petroquímicos, los oleoquímicos han adquirido gran importancia en los diferentes campos de la industria. Sin embargo, su producción ocasiona consumo de agua, áreas de cultivo, alimentos y provocan contaminación al medio ambiente, por ello las levaduras oleaginosas se convierten en una nueva alternativa para la producción de lípidos de manera renovable y sostenible. En este sentido, el objetivo de la presente investigación-tesis, fue aislar, seleccionar e identificar cepas de levadura con elevada acumulación de lípidos. Para ello se realizó un muestreo de los suelos de la cuenca baja – media del río Chicama. Las muestras fueron incubadas en medio de enriquecimiento a 30 °C x 60 h a 150 rpm. De estas se realizó diluciones, las cuales se cultivaron en medio selectivo de xilosa a 30°C x 72 h para obtener cultivos puros de levadura, posteriormente fueron cultivadas utilizando un medio con nitrógeno limitante a base de glucosa y sulfato de amonio a 30 °C x 96 h a 150 rpm. Para la determinación de lípidos, se realizó una dilución de las levaduras, la cual fue sometida a ultrasonido a 45 Khz. por 2 minutos, y luego sumergida en agua con hielo a -4°C por 2 minutos. La muestra, se mezcló con disolventes que se volatilizaron con los lípidos celulares y se calculó el porcentaje de lípidos por gravimetría. Se realizó 3 repeticiones y se seleccionó los 3 cultivos con mayor acumulación de lípidos, codificados como: P21, P11 y P53 con 61.2±5.0%, 67.4±7.4% y 67.3±4.5% respectivamente. Finalmente, las cepas fueron identificadas utilizando el secuenciamiento por el método de Sanger de las regiones ITS. Las cepas fueron identificadas como Rhodotorula mucilaginosa.
ABSTRACT In the search of less toxic, biodegradable, renewable, sustainable and ecological options for obtaining petrochemicals, oleochemicals and other lipid derivatives have developed great importance in different fields of industry. However, due to the fact that its production causes waste of water, cultivation areas, food and causes contamination to the environment, oil yeasts have been proposed as a new renewable and sustainable source for lipid biosynthesis. In this sense, the objective of the present research-thesis was to isolate, select and identify yeast strains with high lipid biosynthesis. For this, a sample of the soils of the lower-middle basin of the Chicama River was carried out. The samples were incubated in enrichment medium at 30 ° C x 60 h at 150 rpm. Of these, dilutions were made, which were cultivated in xylose selective medium at 30 ° C x 72 h to obtain pure yeast cultures, which were subsequently cultivated using a medium with limiting nitrogen based on glucose and ammonium sulfate at 30 ° C x 96 h at 150 rpm. For the determination of lipids, a dilution of the yeasts was carried out, which was subjected to ultrasound at 45 Khz. for 2 minutes, and then immersed in ice water at -4 ° C for 2 minutes. The sample was mixed with solvents that volatilized with the cellular lipids and the percentage of lipids was calculated by gravimetry. 3 repetitions were carried out and the 3 cultures with the highest lipid biosynthesis were selected, coded as: P21, P11 and P53 with 61.2 ± 5.0%, 67.4 ± 7.4% and 67.3 ± 4.5% respectively. Finally, the strains were identified using sequencing by the Sanger method of the ITS regions. The identified strains were Rhodotorula mucilaginosa.
ABSTRACT In the search of less toxic, biodegradable, renewable, sustainable and ecological options for obtaining petrochemicals, oleochemicals and other lipid derivatives have developed great importance in different fields of industry. However, due to the fact that its production causes waste of water, cultivation areas, food and causes contamination to the environment, oil yeasts have been proposed as a new renewable and sustainable source for lipid biosynthesis. In this sense, the objective of the present research-thesis was to isolate, select and identify yeast strains with high lipid biosynthesis. For this, a sample of the soils of the lower-middle basin of the Chicama River was carried out. The samples were incubated in enrichment medium at 30 ° C x 60 h at 150 rpm. Of these, dilutions were made, which were cultivated in xylose selective medium at 30 ° C x 72 h to obtain pure yeast cultures, which were subsequently cultivated using a medium with limiting nitrogen based on glucose and ammonium sulfate at 30 ° C x 96 h at 150 rpm. For the determination of lipids, a dilution of the yeasts was carried out, which was subjected to ultrasound at 45 Khz. for 2 minutes, and then immersed in ice water at -4 ° C for 2 minutes. The sample was mixed with solvents that volatilized with the cellular lipids and the percentage of lipids was calculated by gravimetry. 3 repetitions were carried out and the 3 cultures with the highest lipid biosynthesis were selected, coded as: P21, P11 and P53 with 61.2 ± 5.0%, 67.4 ± 7.4% and 67.3 ± 4.5% respectively. Finally, the strains were identified using sequencing by the Sanger method of the ITS regions. The identified strains were Rhodotorula mucilaginosa.
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Keywords
Rhodotorula mucilaginosa, Estrés metabólico, oleoquímicos, levaduras oleaginosas, Levaduras