Produção de ácido gama-poliglutâmico em cultivo submerso em biorreator airlift a partir de bactérias do gênero Bacillus
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Data
2022-03-03
Autores
Kaspary, Rosane Maria
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Universidade Estadual Paulista (Unesp)
Resumo
A biotecnologia está numa posição privilegiada para substituir materiais poluentes e processos químicos por alternativas mais sustentáveis, como por exemplo, o ácido gama-poliglutâmico (y-PGA), um biopolímero de aplicações multifuncionais, que pode ser obtido a partir de materiais considerados subprodutos da indústria, como o farelo de soja, cascas de arroz, glicerol, entre outros compostos ricos em carbono e nitrogênio. Dessa forma, a produção de y-PGA pode ser uma importante aliada das questões ambientais, principalmente para o tratamento de água potável, considerando que não apresenta toxicidade ao ser humano e ao ambiente. Esse biopolímero é um composto natural, solúvel em água e comestível, podendo ser utilizado em diversos tratamentos medicinais incluindo o tratamento de câncer e regeneração de tecidos. É produzido a partir de processos fermentativos por microrganismos do gênero Bacillus, seja por cultivo submerso, semissólido ou em estado sólido. No entanto, para que esse produto seja utilizado em formulações comerciais, é fundamental o melhoramento dos processos de produção bem como de purificação. Apesar da possibilidade de utilizar subprodutos da indústria, o elevado custo da produção de y-PGA ainda restringe sua utilização, principalmente no tratamento de água potável, considerando os métodos tradicionais de tratamento de água utilizados atualmente. Assim, este projeto voltou-se para estudar a aplicação de um biorreator não convencional do tipo airlift e do modo de operação contínua para produção de PGA, avaliando as grandezas de transformação (velocidade específica de crescimento, conversão de substrato em biomassa e produtividade) para duas espécies de Bacillus, B. licheniformis e B. subtilis, com diferentes condições nutricionais. A cinética de crescimento dos bacilos foi estudada em fermentação em batelada com o objetivo de verificar o tempo de conversão dos substratos em biomassa, além de verificar o comportamento geral dos microrganismos. Nos experimentos realizados pode-se observar um tempo de duplicação menor para B. licheniformis. Das fontes de nitrogênio inorgânico utilizadas nesse estudo para obtenção de y-PGA, o composto com melhor aproveitamento no processo foi o sulfato de amônio, que é mais barato tanto em relação a outras fontes inorgânicas como também fontes orgânicas de nitrogênio. Nos experimentos conduzidos para analisar as diferentes fontes de carbono para a produção de γ PGA observou-se em especial a importância em disponibilizar os nutrientes essenciais para produção do biopolímero, os meios suplementados com glicerol apresentaram melhores resultados (P= 1,033 g.L-1.h-1 e YP/S= 0,266 gP.g.subs-1), porém quando em concentrações inferiores à 40 g L-1. A glicose como fonte de carbono também foi importante na produção de γ-PGA, no entanto, observou-se produção de γ-PGA em meio sem glicose, enquanto houve baixa produção quando uma concentração de glicose superior a 15 g L -1 foi adicionada ao meio de cultivo. A adição de 0,3% de n-heptano no início das fermentações como vetor para aumentar a solubilidade de oxigênio no meio de cultivo, foi favorável para o crescimento da biomassa, principalmente para o B. licheniformis, houve aumento também na produção de γ-PGA em comparação aos experimentos sem a adição do solvente orgânico. Os resultados permitiram definir uma composição apropriada para o meio de cultivo, bem como das condições de processo, de modo a obter um rendimento e produtividade maior, com menor custo de operação, considerando ainda a menor geração de resíduos. O rendimento na produção de y-PGA foi ainda melhor no biorreator airlift com alimentação contínua dos nutrientes, utilizando baixas concentrações de carbono e nitrogênio. Dessa forma, a produtividade nessas condições foi de 2,31 g L -1 h -1 com um rendimento de 0,678 (g de biopolímero/g de substrato de carbono), além de baixa produção de resíduos, atendendo com isso os conceitos de sustentabilidade, onde é possível com base em processos biotecnológicos, a obtenção de um produto com alto valor agregado a partir de subprodutos, em processo com custo reduzido e baixo impacto ambiental.
Biotechnology is in a privileged position to replace polluting materials and chemical processes with more sustainable alternatives, such as gamma-polyglutamic acid (γ-PGA), a biopolymer with multifunctional applications, which can be obtained from materials considered by-products of industry, such as soybean meal, rice husks, glycerol, among other compounds rich in carbon and nitrogen. Thus, the production of γ-PGA can be an important ally of environmental issues, especially for the treatment of drinking water, considering that it does not present toxicity to humans and the environment. This biopolymer is a natural, water-soluble and edible compound, which can be used in several medicinal treatments, including the treatment of cancer and tissue regeneration. It is produced from fermentation processes by microorganisms of Bacillus genus, either by submerged, semi-solid or solid state cultivation. However, for this product to be used in commercial formulations, it is essential to improve production processes as well as purification. Despite the possibility of using by-products from the industry, the high cost of producing γ-PGA still restricts its use, especially in the treatment of drinking water, considering the traditional methods of water treatment currently used. Thus, this project focused on studying the application of an unconventional airlift bioreactor and continuous operation mode for the production of γ-PGA, evaluated as transformation quantities (specific growth rate, substrate-to-biomass conversion and productivity) for two Bacillus species, licheniformis and subtilis, with different nutritional conditions. The kinetics of growth of bacillus was studied in fermentation in batch with the objective of verifying the time of conversion of substrates into biomass, in addition to verifying the general behavior of microorganisms. In the experiments carried out, it was possible to observe a shorter doubling time for B. licheniformis. Of the inorganic nitrogen sources used in this study to obtain γ-PGA, the compound with the best use in the process was ammonium sulfate, which is cheaper compared to other inorganic sources as well as organic sources of nitrogen. In the experiments conducted to analyze the different carbon sources for the production of γ-PGA, the importance of making essential nutrients available for the production of the biopolymer was observed, the medium supplemented with glycerol showed better results (P= 1.033 g L-1 h-1 and YP/S= 0.266 gP g.subs-1), but when in concentrations below 40 g L-1. Glucose as a carbon source was also important in the production of γ-PGA, however, γ PGA production was observed in a medium without glucose, while there was low production when a glucose concentration greater than 15 g L-1 was added to the medium. cultivation medium. The addition of 0.3% of n-heptane at the beginning of the fermentations as a vector to increase the solubility of oxygen in the culture medium was favorable for the growth of biomass, especially for B. licheniformis, there was also an increase in the production of γ-PGA compared to experiments without the addition of organic solvent. The results made it possible to define an appropriate composition for the culture medium, as well as the process conditions, in order to obtain a higher yield and productivity, with lower operating costs, considering the lower generation of waste. The yield in the production of γ-PGA was even better in the airlift bioreactor with continuous feeding of nutrients, using Low concentrations of carbon and nitrogen. Thus, the productivity under these conditions was 2.31 g L-1 h-1 with a yield of 0.678 (g of biopolymer/g of carbon substrate), in addition to low waste production, thus meeting the concepts of sustainability, where it is possible, based on biotechnological processes, to obtain a product with high added value from by-products, in a process with reduced cost and low environmental impact.
Biotechnology is in a privileged position to replace polluting materials and chemical processes with more sustainable alternatives, such as gamma-polyglutamic acid (γ-PGA), a biopolymer with multifunctional applications, which can be obtained from materials considered by-products of industry, such as soybean meal, rice husks, glycerol, among other compounds rich in carbon and nitrogen. Thus, the production of γ-PGA can be an important ally of environmental issues, especially for the treatment of drinking water, considering that it does not present toxicity to humans and the environment. This biopolymer is a natural, water-soluble and edible compound, which can be used in several medicinal treatments, including the treatment of cancer and tissue regeneration. It is produced from fermentation processes by microorganisms of Bacillus genus, either by submerged, semi-solid or solid state cultivation. However, for this product to be used in commercial formulations, it is essential to improve production processes as well as purification. Despite the possibility of using by-products from the industry, the high cost of producing γ-PGA still restricts its use, especially in the treatment of drinking water, considering the traditional methods of water treatment currently used. Thus, this project focused on studying the application of an unconventional airlift bioreactor and continuous operation mode for the production of γ-PGA, evaluated as transformation quantities (specific growth rate, substrate-to-biomass conversion and productivity) for two Bacillus species, licheniformis and subtilis, with different nutritional conditions. The kinetics of growth of bacillus was studied in fermentation in batch with the objective of verifying the time of conversion of substrates into biomass, in addition to verifying the general behavior of microorganisms. In the experiments carried out, it was possible to observe a shorter doubling time for B. licheniformis. Of the inorganic nitrogen sources used in this study to obtain γ-PGA, the compound with the best use in the process was ammonium sulfate, which is cheaper compared to other inorganic sources as well as organic sources of nitrogen. In the experiments conducted to analyze the different carbon sources for the production of γ-PGA, the importance of making essential nutrients available for the production of the biopolymer was observed, the medium supplemented with glycerol showed better results (P= 1.033 g L-1 h-1 and YP/S= 0.266 gP g.subs-1), but when in concentrations below 40 g L-1. Glucose as a carbon source was also important in the production of γ-PGA, however, γ PGA production was observed in a medium without glucose, while there was low production when a glucose concentration greater than 15 g L-1 was added to the medium. cultivation medium. The addition of 0.3% of n-heptane at the beginning of the fermentations as a vector to increase the solubility of oxygen in the culture medium was favorable for the growth of biomass, especially for B. licheniformis, there was also an increase in the production of γ-PGA compared to experiments without the addition of organic solvent. The results made it possible to define an appropriate composition for the culture medium, as well as the process conditions, in order to obtain a higher yield and productivity, with lower operating costs, considering the lower generation of waste. The yield in the production of γ-PGA was even better in the airlift bioreactor with continuous feeding of nutrients, using Low concentrations of carbon and nitrogen. Thus, the productivity under these conditions was 2.31 g L-1 h-1 with a yield of 0.678 (g of biopolymer/g of carbon substrate), in addition to low waste production, thus meeting the concepts of sustainability, where it is possible, based on biotechnological processes, to obtain a product with high added value from by-products, in a process with reduced cost and low environmental impact.
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Palavras-chave
Biopolímeros, y-PGA, Biorreatores, Fermentação, Microorganismos, Biopolymers, Bioreactors, Fermentation, Microorganisms