Produção e caracterização de ramnolipídios a partir de substratos alternativos
Carregando...
Data
2022-06-14
Autores
Yoshimura, Ingrid
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Biossurfactantes são substâncias sintetizadas por microrganismos e que possuem a capacidade de reduzir a tensão superficial e interfacial. Os ramnolipídios (RLs) são biossurfactantes da classe dos glicolipídios, que apresentam atividade de superfície elevada e maior rendimento de produção. Eles atuam em diferentes setores da indústria, mas não são amplamente utilizados devido ao seu alto custo de obtenção. Ramnolipídios produzidos por Pseudomonas aeruginosa são constituídos por uma mistura de espécies homólogas, onde os mono-ramnolipídios e os di-ramnolipídios são predominantes. A razão entre esses homólogos resulta em diferentes propriedades e aplicabilidades para o composto, fato que pode ser influenciado pelas fontes de carbono utilizadas. Para aprofundar os estudos nesse âmbito, é interessante levar em consideração a contribuição individual de cada homólogo nas propriedades finais do ramnolipídio. Entretanto, tais análises são dependentes do desenvolvimento de metodologias de separação desses compostos. Dessa maneira, nessa pesquisa procurou-se estudar a produção de ramnolipídios a partir de dois substratos alternativos, o óleo da semente da goiaba e o d-limoneno, subprodutos provenientes da frutorrefinaria. Os objetivos foram obter maior rendimento desse metabólito, otimizar a produção através de planejamento experimental, caracterizar o RL produzido e desenvolver metodologias de separação das principais espécies homólogas. Os experimentos foram conduzidos em laboratório a partir de fermentações em frascos utilizando P. aeruginosa LBI 2A1. A caracterização do tensoativo ocorreu através de espectrometria de massas, testes de turbidez e tensão superficial e avaliação da atividade de emulsificação. A separação dos homólogos foi realizada através de cromatografia em coluna e complexos de inclusão. As fermentações realizadas com d-limoneno demonstraram baixa viabilidade para crescimento insipiente do microrganismo e baixa produção do metabólito. O óleo de semente de goiaba mostrou-se como uma fonte de carbono mais favorável para o crescimento microbiano e produção do biossurfactante, e por isso foi utilizado para realização de um delineamento central composto rotacional. As variáveis independentes foram: concentração da fonte de carbono e de nitrogênio. O delineamento mostrou que as variáveis nas formas quadráticas influenciaram na resposta, assim como a interação entre as mesmas (p<0,05). A variável mais significativa foi concentração de N. A análise de superfície de resposta indicou que as concentrações ótimas de C e N foram de 100 e 10 g/L, respectivamente e levaram à produção de 39,97 g/L de ramnolipídios, com predominância de mono-RL. A composição de espécies homólogas variou em relação à fonte de carbono utilizada e uma espécie homóloga de RL, de m/z 555 (ainda não descrita na literatura) foi encontrada com a fonte óleo de semente de goiaba. Em relação aos testes de atividadede superfície, a concentração micelar crítica do RL diminuiu em soluções ácidas; a adição do propilenoglicol favoreceu a formação de agregados e o índice de emulsificação do RL foi favorecido por meios mais alcalinos. Por fim, na separação das espécies homólogas, os complexos de inclusão com a ciclodextrina mostram-se como uma metodologia potencial, mas ainda demonstraram a necessidade de um número maior de experimentos para sua confirmação. E, a separação em coluna cromatográfica possibilitou a eluição de mono-ramnolipídios com 99% de pureza.
Biosurfactants are substances synthesized by microorganisms that have the ability to reduce surface and interfacial tension. Rhamnolipids (RLs) are biosurfactants of the glycolipid class, which have high surface activity and higher production yield. They operate in different sectors of the industry, but they are not widely used due to their high cost of obtaining. Rhamnolipids produced by Pseudomonas aeruginosa are composed of a mixture of homologous species, where mono-rhamnolipids and di-rhamnolipids are predominant. The ratio between these homologs results in different properties and applicability for the compound, a fact that can be influenced by the carbon sources used. To deepen the studies in this area, it is interesting to consider the individual contribution of each homologue to the final properties of rhamnolipid. However, such analyzes are dependent on the development of methodologies for the separation of these compounds. Thus, this research sought to study the production of rhamnolipids from two alternative substrates, guava seed oil and d-limonene, by-products from the fructo-refinery. The aims were to obtain higher yields of this metabolite, optimize production through experimental planning, characterize the RL produced and develop methodologies for separating the main homologous species. The experiments were carried out in the laboratory using flask fermentations with P. aeruginosa LBI 2A1. The characterization of the surfactant occurred through mass spectrometry, turbidity and surface tension tests and evaluation of the emulsification activity. Separation of homologs was performed by column chromatography and inclusion complexes. Fermentations carried out with d-limonene showed low viability for incipient growth of the microorganism and low production of the metabolite. Guava seed oil proved to be a more favorable carbon source for microbial growth and biosurfactant production, and therefore it was used to perform a central composite rotational design. The independent variables were: carbon and nitrogen source concentration. The design showed that the variables in the quadratic forms influenced the response, as well as the interaction between them (p<0.05). The most significant variable was N concentration. The response surface analysis indicated that the optimal concentrations of C and N were 100 and 10 g/L, respectively, and led to the production of 39.97 g/L of rhamnolipids, predominantly of mono-RL. The composition of homologous species varied in relation to the carbon source used and a homologous species of RL, of m/z 555 (not yet described in the literature) was found with the source guava seed oil. Regarding the surface activity tests, the RL critical micellar concentration decreased in acidic solutions; the addition of propylene glycol favored the formation of aggregates and the RL emulsification index was favored by more alkaline media. Finally, in the separation of homologous species, the cyclodextrin inclusion complexes shows up as a potential methodology, but it still demonstrated the need for a greater number of experiments to confirm it. And, the chromatographic column separation allowed the elution of mono-rhamnolipids with 99% purity.
Biosurfactants are substances synthesized by microorganisms that have the ability to reduce surface and interfacial tension. Rhamnolipids (RLs) are biosurfactants of the glycolipid class, which have high surface activity and higher production yield. They operate in different sectors of the industry, but they are not widely used due to their high cost of obtaining. Rhamnolipids produced by Pseudomonas aeruginosa are composed of a mixture of homologous species, where mono-rhamnolipids and di-rhamnolipids are predominant. The ratio between these homologs results in different properties and applicability for the compound, a fact that can be influenced by the carbon sources used. To deepen the studies in this area, it is interesting to consider the individual contribution of each homologue to the final properties of rhamnolipid. However, such analyzes are dependent on the development of methodologies for the separation of these compounds. Thus, this research sought to study the production of rhamnolipids from two alternative substrates, guava seed oil and d-limonene, by-products from the fructo-refinery. The aims were to obtain higher yields of this metabolite, optimize production through experimental planning, characterize the RL produced and develop methodologies for separating the main homologous species. The experiments were carried out in the laboratory using flask fermentations with P. aeruginosa LBI 2A1. The characterization of the surfactant occurred through mass spectrometry, turbidity and surface tension tests and evaluation of the emulsification activity. Separation of homologs was performed by column chromatography and inclusion complexes. Fermentations carried out with d-limonene showed low viability for incipient growth of the microorganism and low production of the metabolite. Guava seed oil proved to be a more favorable carbon source for microbial growth and biosurfactant production, and therefore it was used to perform a central composite rotational design. The independent variables were: carbon and nitrogen source concentration. The design showed that the variables in the quadratic forms influenced the response, as well as the interaction between them (p<0.05). The most significant variable was N concentration. The response surface analysis indicated that the optimal concentrations of C and N were 100 and 10 g/L, respectively, and led to the production of 39.97 g/L of rhamnolipids, predominantly of mono-RL. The composition of homologous species varied in relation to the carbon source used and a homologous species of RL, of m/z 555 (not yet described in the literature) was found with the source guava seed oil. Regarding the surface activity tests, the RL critical micellar concentration decreased in acidic solutions; the addition of propylene glycol favored the formation of aggregates and the RL emulsification index was favored by more alkaline media. Finally, in the separation of homologous species, the cyclodextrin inclusion complexes shows up as a potential methodology, but it still demonstrated the need for a greater number of experiments to confirm it. And, the chromatographic column separation allowed the elution of mono-rhamnolipids with 99% purity.
Descrição
Palavras-chave
Caracterização de homólogos, d-limoneno, Óleo de semente de goiaba, Pseudomonas aeruginosa, Purificação, d-limonene, Guava seed oil, Homologs characterization, Purification