Utilização de xilose para a produção de ácido lático por isolados de Weizmannia sp. e abordagem da evolução adaptativa laboratorial
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Data
2023-07-20
Autores
Santos, Larissa Provasi [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O ácido lático é um ácido orgânico de grande ocorrência, utilizado em diferentes setores industriais, como farmacêutico, alimentício, cosmético e têxtil, ganhando destaque também por atuar como monômero na síntese de biopolímeros. Há duas formas de se obter o ácido lático, através da utilização de produtos de origem petrolífera, ou por meio de bioprocessos. Esse último já ocupa a maior parte do mercado produtor, mas ainda há obstáculos a serem superados, como a produção da biomolécula a partir de substratos de baixo custo, utilizando materiais lignocelulósicos que é um dos maiores desafios. Outro obstáculo é a purificação do ácido lático, pois os microrganismos produtores desse composto não toleram faixas muito baixas de pH, tornando-se indispensável o uso de agentes neutralizantes, como por exemplo, o carbonato de cálcio que podem gerar resíduos como o gesso. Com isso, o objetivo desse estudo foi isolar Weizmannia sp. produtora de ácido lático a partir de bagaço de cana-de-açúcar, e utilizar o planejamento experimental para otimizar a produção do metabólito. Adicionalmente realizar uma evolução adaptativa laboratorial (ALE) para obtenção de cepas resistentes a concentração do ácido no meio durante seu crescimento e produção da biomolécula. Os experimentos foram conduzidos em frascos Erlenmeyers e os isolados foram cultivados em meio xilose- extrato de levedura- peptona (XYP), contendo 20 g/L da fonte de carbono. A identificação dos isolados foi dada através da técnica de PCR do 16S rDNA, seguido de sequenciamento. O delineamento composto central rotacional (DCCR) foi realizado com os componentes do meio de cultivo, como fonte de carbono (xilose), fonte de nitrogênio (peptona e extrato de levedura) e acetato de sódio. A quantificação do ácido lático e o consumo da fonte de carbono foi realizada em HPLC. Dos isolados selecionados dois foram considerados promissores para a produção de ácido lático, sendo que Weizmannia coagulans BLMI apresentou maior produção de ácido lático em anaerobiose, chegando a 21,93±0,9 g/L e rendimento de 69,18 %, e a cepa Weizmannia ginsengihumi BMI foi capaz de produzir 19,79±0,8 g/L com um rendimento de 70,46 %. Por W. ginsengihumi ser uma espécie sem registros na literatura até o momento para a produção de ácido lático, o DCCR foi realizado com essa cepa. O DDCR mostrou que as variáveis fonte de carbono e nitrogênio tanto na forma linear quanto quadrática apresentaram influência na resposta, mas as interações das variáveis não tiveram significância (p < 0,05). E a variável que apresentou maior influência foi a xilose em nível linear. A análise de superfície de resposta indicou que as concentrações ótimas de fonte de carbono e fonte de nitrogênio foram 32,5 e 3,5 g/L, respectivamente, sem a necessidade de adicionar acetato de sódio ao meio de cultivo, levando à produção de 20,02±0,19 g/L e produtividade de 0,55 g/L/h após 36 horas de fermentação, e uma concentração de açúcar residual de 12,59±0,51 g/L. A fermentação utilizando os hidrolisados da semente de goiaba e bagaço de cana-de-açúcar apresentaram uma produção de ácido lático de 0,18±0,02 e 1,26±0,37 g/L, respectivamente, uma produção extremamente baixa quando comparado com a literatura. Nos ensaios da ALE, a bactéria W. coagulans BLMI passou por 71 transferências, totalizando 280 gerações, sendo capaz de suportar uma concentração de 90 mM de ácido lático com um pH do meio em 4,65. Nas fermentações com a cepa selvagem e a cepa evoluída, W. coagulans BLMI 171, sem a presença de agente neutralizante produziram 7,70±0,43 e 6,47±0,9 g/L, respectivamente após 48 horas de fermentação. Quanto a produção de ácido acético foi maior mesmo os ensaios sendo conduzidos em anaerobiose, BLMI produziu 7,96±0,35 g/L e BLMI 171 produziu 8,11±0,08 g/L após 48 horas de fermentação. Dessa forma, Weizmannia sp. foi capaz de fermentar a xilose para a produção de ácido lático em anaerobiose, apesar da cepa BMI não ser capaz de crescer e nem produzir o ácido lático a partir dos resíduos. BLMI demonstrou boa resistência cultivada em meio com pH de 4,65, mas para a produção de ácido lático são necessárias intervenções moleculares.
Lactic acid is an organic acid of great occurrence, used in different industrial sectors, such as pharmaceuticals, food, cosmetics, and textiles, and also gaining prominence for acting as a monomer in the synthesis of biopolymers. There are two ways to obtain lactic acid, through the use of products of petroleum origin, or bioprocesses. The latter already occupies most of the producing market, but there are still obstacles to be overcome, such as the production of the biomolecule from low-cost substrates, using lignocellulosic materials, which is one of the biggest challenges. Another obstacle is the purification of lactic acid, as the microorganisms that produce this compound do not tolerate very low pH ranges, making it essential to use neutralizing agents, such as calcium carbonate, which can generate residues such as gypsum. Thus, this study aimed to isolate Weizmannia sp. producer of lactic acid from sugarcane bagasse, and to use the experimental design to optimize the production of the metabolite. Additionally, carry out an adaptive laboratory evolution (ALE) to obtain strains resistant to acid concentration in the medium during their growth and production of the biomolecule. The experiments were carried out in Erlenmeyers flasks and the isolates were cultivated in xylose-yeast extract-peptone (XYP) medium, containing 20 g/L of the carbon source. The identification of the isolates was given through the 16S rDNA PCR technique, followed by sequencing. The rotational central composite design (DCCR) was performed with the components of the culture medium as carbon source (xylose), nitrogen source (peptone and yeast extract), and sodium acetate. The quantification of lactic acid and the consumption of the carbon source was performed in HPLC. Of the selected isolates, two were considered promising for the production of lactic acid, with Weizmannia coagulans BLMI showing the highest production of lactic acid in anaerobic conditions, reaching 21.93±0.9 g/L and a yield of 69.18%, and the strain Weizmannia ginsengihumi BMI was able to produce 19.79±0.8 g/L with a yield of 70.46%. Because W. ginsengihumi is a species with no records in the literature so far for the production of lactic acid, the DCCR was performed with this strain. The DDCR showed that the carbon and nitrogen source variables in both linear and quadratic forms influenced the response, but the interactions of the variables were not significant (p < 0.05). And the variable that showed the greatest influence was xylose at a linear level. Response surface analysis indicated that the optimal concentrations of carbon source and nitrogen source were 32.5 and 3.5 g/L, respectively, without the need to add sodium acetate to the culture medium, leading to the production of 20.02±0.19 g/L and productivity of 0.55 g/L/h after 36 hours of fermentation, and a residual sugar concentration of 12.59±0.51 g/L. Fermentation using hydrolysates of guava seed and sugarcane bagasse showed a lactic acid production of 0.18±0.02 and 1.26±0.37 g/L, respectively, an extremely low production when compared to the literature. In the ALE assays, the bacteria W. coagulans BLMI underwent 71 transfers, totaling 280 generations, being able to support a concentration of 90 mM of lactic acid with a medium pH of 4.65. Fermentations with the wild strain and the evolved strain, W. coagulans BLMI 171, without the presence of a neutralizing agent produced 7.70±0.43 and 6.47±0.9 g/L, respectively, after 48 hours of fermentation. As for the production of acetic acid, it was higher even in the tests being conducted in anaerobic conditions, BLMI produced 7.96±0.35 g/L and BLMI 171 produced 8.11±0.08 g/L after 48 hours of fermentation. Thus, Weizmannia sp. was able to ferment xylose to produce lactic acid in anaerobiosis, despite the BMI strain not being able to grow or produce lactic acid from waste. BLMI showed good resistance grown in a medium with a pH of 4.65, but molecular interventions are required for lactic acid production.
Lactic acid is an organic acid of great occurrence, used in different industrial sectors, such as pharmaceuticals, food, cosmetics, and textiles, and also gaining prominence for acting as a monomer in the synthesis of biopolymers. There are two ways to obtain lactic acid, through the use of products of petroleum origin, or bioprocesses. The latter already occupies most of the producing market, but there are still obstacles to be overcome, such as the production of the biomolecule from low-cost substrates, using lignocellulosic materials, which is one of the biggest challenges. Another obstacle is the purification of lactic acid, as the microorganisms that produce this compound do not tolerate very low pH ranges, making it essential to use neutralizing agents, such as calcium carbonate, which can generate residues such as gypsum. Thus, this study aimed to isolate Weizmannia sp. producer of lactic acid from sugarcane bagasse, and to use the experimental design to optimize the production of the metabolite. Additionally, carry out an adaptive laboratory evolution (ALE) to obtain strains resistant to acid concentration in the medium during their growth and production of the biomolecule. The experiments were carried out in Erlenmeyers flasks and the isolates were cultivated in xylose-yeast extract-peptone (XYP) medium, containing 20 g/L of the carbon source. The identification of the isolates was given through the 16S rDNA PCR technique, followed by sequencing. The rotational central composite design (DCCR) was performed with the components of the culture medium as carbon source (xylose), nitrogen source (peptone and yeast extract), and sodium acetate. The quantification of lactic acid and the consumption of the carbon source was performed in HPLC. Of the selected isolates, two were considered promising for the production of lactic acid, with Weizmannia coagulans BLMI showing the highest production of lactic acid in anaerobic conditions, reaching 21.93±0.9 g/L and a yield of 69.18%, and the strain Weizmannia ginsengihumi BMI was able to produce 19.79±0.8 g/L with a yield of 70.46%. Because W. ginsengihumi is a species with no records in the literature so far for the production of lactic acid, the DCCR was performed with this strain. The DDCR showed that the carbon and nitrogen source variables in both linear and quadratic forms influenced the response, but the interactions of the variables were not significant (p < 0.05). And the variable that showed the greatest influence was xylose at a linear level. Response surface analysis indicated that the optimal concentrations of carbon source and nitrogen source were 32.5 and 3.5 g/L, respectively, without the need to add sodium acetate to the culture medium, leading to the production of 20.02±0.19 g/L and productivity of 0.55 g/L/h after 36 hours of fermentation, and a residual sugar concentration of 12.59±0.51 g/L. Fermentation using hydrolysates of guava seed and sugarcane bagasse showed a lactic acid production of 0.18±0.02 and 1.26±0.37 g/L, respectively, an extremely low production when compared to the literature. In the ALE assays, the bacteria W. coagulans BLMI underwent 71 transfers, totaling 280 generations, being able to support a concentration of 90 mM of lactic acid with a medium pH of 4.65. Fermentations with the wild strain and the evolved strain, W. coagulans BLMI 171, without the presence of a neutralizing agent produced 7.70±0.43 and 6.47±0.9 g/L, respectively, after 48 hours of fermentation. As for the production of acetic acid, it was higher even in the tests being conducted in anaerobic conditions, BLMI produced 7.96±0.35 g/L and BLMI 171 produced 8.11±0.08 g/L after 48 hours of fermentation. Thus, Weizmannia sp. was able to ferment xylose to produce lactic acid in anaerobiosis, despite the BMI strain not being able to grow or produce lactic acid from waste. BLMI showed good resistance grown in a medium with a pH of 4.65, but molecular interventions are required for lactic acid production.
Descrição
Palavras-chave
Pentose, Resíduo frutorefinaria, Ácidos orgânicos, Weizmannia sp., Planejamento experimental, Fruit refinery residue, Organic acids, Experimental planning