Evaluación de la producción de poli(3-hidroxialcanoato) de las cepas 3228, 32, 1804, 321, 2894, 3641 y 1517 de bacillus megaterium utilizando glicerol crudo como única fuente de carbono
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2019
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es
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Universidad Andrés Bello
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Licencia CC
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Los plásticos convencionales son polímeros sintéticos, derivados a partir de materias primas no renovables como el petróleo. Se caracterizan por tener una gran resistencia a la temperatura y ser maleables, haciéndolos muy versátiles por lo que han formado parte indispensable de nuestra vida diaria. El principal problema de los plásticos es su largo tiempo de degradación, entre 100 a 1000 años, lo que ha generado una amenazada importante al medio ambiente y la salud humana. Se han buscado opciones para poder subsanar este problema, donde los biopolímeros o polímeros naturales comienzan a ganar interés. Estos se caracterizan por tener propiedades termoplásticas y ser completamente biodegradables. Dentro de este tipo de polímeros naturales, se encuentran los polihidroxialcanoatos (PHAs), los cuales son sinterizados naturalmente por una gran variedad de bacterias Gram positivo y Gram negativo. Uno de los microrganismos modelo para la producción de biopolímeros es Bacillus megaterium, la cual tiene la capacidad de adaptarse a fuentes de carbono no convencionales para sintetizar PHAs. Dentro de los PHAs más conocidos se encuentra el poli(3-hidroxibutirato) (PHB) el cual fue el primer tipo de biopolímero en ser aislado y caracterizado en la bacteria B. megaterium, con propiedades similares al polietileno y polipropileno. Este microrganismo fue uno de los primeros estudiados para la producción de biopolímeros, ha sido utilizado por muchos años en la industria de la biotecnología, debido a su gran tamaño celular y su versatilidad metabólica. Como se mencionó anteriormente, los PHAs tienen propiedades similares a los plásticos derivados del petróleo, esto hace considerar a los polihidroxialcanoatos como posibles sustitutos. Sin embargo, tienen elevado costos de producción, por lo que se buscan estrategias para economizar el proceso, una de ellas la utilización de residuos industriales como el glicerol crudo. En un intento para sintetizar PHAs a partir de desechos industriales, en este trabajo se realizó la caracterización y producción de estos con cepas de Bacillus megaterium DSM319, 3228, 32, 1804, 321, 2894 y 1517 utilizand o glicerol puro (GP) y crudo (GC) como fuente de carbono a 30 y 3T por 72 y 96 horas. Las cepas con los mejores rendimientos de PHA (DSM319, 3228 y 2894) fueron seleccionadas para los análisis de cinéticas de crecimiento y producción de PHA en glicerol crudo (GC). Para los cultivos con GP se obtuvo valores de la tasa específica de crecimiento de 0,5 h'', 0,46 h'' y 0,47 h"1, para DSM319, 3229 y 2894, respectivamente; mientras que al utilizar GC como fuente de carbono las tasas de crecimiento disminuyeron 10 veces para la cepa DSM319 y para las cepas 3228 y 2904, disminuyó a 0,4 h"1. En cuanto a la producción de PHA, la cepa DSM319 mostró una acumulación de 15% de su peso seco en biopolímero con GP, mientras que al utilizar GC esta disminuye a un 1% de acumulación. La cepa 3228 alcanza una acumulación del 41% y 24% con glicerol puro y crudo respectivamente. La cepa 2894 presentó una acumulación de PHA del 36% con GP, mientras que al utilizar GC la acumulación disminuye a 2%. Finalmente, se determinó que los mejores rendimientos obtenidos de producción de PHA con glicerol crudo fueron de la cepa Bacillus megaterium 3228, en condiciones de crecimiento a 30°C durante 72 horas de incubación.
Conventional plastics are synthetic polymers produced from non-renewable raw materials such as petroleum. They are characterized for having high temperature resistance and being moldable, making them very versatile and part of our everyday life. The most important problem with plastics is the long period of degradation around 100 and 1000 years, what has generated a big treat to the environment and human health. Biopolymers or natural polymers are important alternatives to avoid the use of petroleum-based plastics because they have similar properties as conventional plastics, such as thermostability, being moldable and biodegradable. Polyhydroxyalkanoates (PHAs) are a type of biopolymer, they are synthesized by Gram positive and Gram negative bacteria. Bacillus megaterium is one of the model microorganisms used to synthesize PHAs, which has the ability to adapt to nonconventional carbon sources. The poly(3-hydroxybutyrate) is the most common biopolymer, was first isolated and characterized by Bacillus megaterium and it has similar properties to polyethylene and polypropylene. B. megaterium is one of the most studied microorganisms for biopolymer production, it has been used for many years in biotechnological processes because of its big cellular size and metabolic versatility. As mentioned before PHAs has similar properties to petroleum-based plastics, which implies a possible replacement for conventional plastics. However, the production processes associated to PHA production are very expensive, so in aim to compete with petrochemical plastic is necessary to search for strategies that reduce production costs, one of them is to use industrial waste such as raw glycerol. In this work to attempt synthesize polyhydroxyalkanoates from an industrial waste, we characterized the produced PHA using Bacillus megaterium DSM319, 3228 32, 1804, 321, 2894 and 1517 strains, using pure and raw glycerol as a carbon source, at 30 and 3TC and for 72 and 96 hrs. The strains with the better yields (DSM319, 3228 and 2894) were selected for further experiments using raw glycerol. For the cultures with pure glycerol the specific growth rate (p) was 0,5 h'', 0,46 h'' and 0,47 h'' for DSM318, 3228 and 2894, respectively, whereas in the raw glycerol cultures the Amax decreased ten times for the DSM319 strain, and for the 3228 and 2894 strain the specific growth rate reduce to 0,4 h'1. Through PHA extraction we determined that about 15% of the cell dry mass was amassed as PHA by the DSM319 strain using pure glycerol as a carbon source, whereas in the raw glycerol cultures the accumulation of PHA reduce to a 1%. The 3228 strain reached a PHA accumulation of 41 % and 24% in pure and raw glycerol, respectively. Finally, the 2894 strain reached a 36% PHA accumulation in pure glycerol, whereas in raw glycerol the PHA content decreased to a 2%. We determined that the best yield for PHA production in raw glycerol was obtained by Bacillus megaterium 3228 strain at 30°C and 72 hrs of incubation.
Conventional plastics are synthetic polymers produced from non-renewable raw materials such as petroleum. They are characterized for having high temperature resistance and being moldable, making them very versatile and part of our everyday life. The most important problem with plastics is the long period of degradation around 100 and 1000 years, what has generated a big treat to the environment and human health. Biopolymers or natural polymers are important alternatives to avoid the use of petroleum-based plastics because they have similar properties as conventional plastics, such as thermostability, being moldable and biodegradable. Polyhydroxyalkanoates (PHAs) are a type of biopolymer, they are synthesized by Gram positive and Gram negative bacteria. Bacillus megaterium is one of the model microorganisms used to synthesize PHAs, which has the ability to adapt to nonconventional carbon sources. The poly(3-hydroxybutyrate) is the most common biopolymer, was first isolated and characterized by Bacillus megaterium and it has similar properties to polyethylene and polypropylene. B. megaterium is one of the most studied microorganisms for biopolymer production, it has been used for many years in biotechnological processes because of its big cellular size and metabolic versatility. As mentioned before PHAs has similar properties to petroleum-based plastics, which implies a possible replacement for conventional plastics. However, the production processes associated to PHA production are very expensive, so in aim to compete with petrochemical plastic is necessary to search for strategies that reduce production costs, one of them is to use industrial waste such as raw glycerol. In this work to attempt synthesize polyhydroxyalkanoates from an industrial waste, we characterized the produced PHA using Bacillus megaterium DSM319, 3228 32, 1804, 321, 2894 and 1517 strains, using pure and raw glycerol as a carbon source, at 30 and 3TC and for 72 and 96 hrs. The strains with the better yields (DSM319, 3228 and 2894) were selected for further experiments using raw glycerol. For the cultures with pure glycerol the specific growth rate (p) was 0,5 h'', 0,46 h'' and 0,47 h'' for DSM318, 3228 and 2894, respectively, whereas in the raw glycerol cultures the Amax decreased ten times for the DSM319 strain, and for the 3228 and 2894 strain the specific growth rate reduce to 0,4 h'1. Through PHA extraction we determined that about 15% of the cell dry mass was amassed as PHA by the DSM319 strain using pure glycerol as a carbon source, whereas in the raw glycerol cultures the accumulation of PHA reduce to a 1%. The 3228 strain reached a PHA accumulation of 41 % and 24% in pure and raw glycerol, respectively. Finally, the 2894 strain reached a 36% PHA accumulation in pure glycerol, whereas in raw glycerol the PHA content decreased to a 2%. We determined that the best yield for PHA production in raw glycerol was obtained by Bacillus megaterium 3228 strain at 30°C and 72 hrs of incubation.
Notas
Memoria de Titulo (Ingeniera en Biotecnología)
Palabras clave
Biopolímeros, Bacterias Anaeróbicas, Análisis.