Recuperação enzimática na produção de etanol 2g: um estudo sobre capacidade de adsorção entre lignocresol e celulases.
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Data
2019-05-07
Autores
Ferreira, Mariana Teresa Barduco
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Universidade Estadual Paulista (Unesp)
Resumo
A busca por fontes de energias renováveis vêm ganhando cada vez mais importância, pois, com a eminente escassez dos combustíveis fósseis, que são as principais fontes energéticas utilizadas mundialmente e ao mesmo tempo com os problemas ambientais causados por esses combustíveis, o etanol de segunda geração (2G) surge como uma alternativa de energia renovável já que é obtido a partir de biomassa lignocelulósica. Entretanto, as tecnologias para a conversão desta biomassa em açúcares fermentáveis ainda possuem fatores limitantes. Um dos grandes problemas do processo de obtenção do etanol 2G está relacionado ao custo das enzimas celulolíticas. Sendo assim, desenvolver estratégias para a recuperação dessas enzimas é necessário para assegurar a viabilidade econômica do processo de conversão de biomassa. Diversas técnicas estão sendo estudadas e colocadas em prática para promover a recuperação, reciclagem e imobilização enzimática. Entretanto, nenhuma das metodologias utilizadas demonstram, de maneira geral, alta eficiência, custo baixo, rapidez e manutenção da atividade enzimática. A partir da lignina, subproduto da indústria de papel e celulose, pode-se sintetizar o lignocresol. Este, apresenta grandes expectativas de interação enzimática uma vez que apresenta características físico-químicas favoráveis à adsorção. Frente a isto, propusemos a produção deste composto para promover a interação com enzimas para promover recuperação enzimática. A síntese de lignocresol, oriundo da lignina Kraft de Pinus spp., foi feita pela adaptação à metodologia do sistema de separação de fases. Nesse processo, mais especificamente na primeira fase, a madeira é solvatada com o p-cresol (derivado do fenol) para evitar o ataque do ácido concentrado a lignina e assim ela não se dissolver. Desse modo, apenas os carboidratos são dissolvidos pelo ácido concentrado na segunda fase. Para obtenção do complexo, foram realizadas duas suspensões de lignocresol em tampão citrato de sódio (50 mM), ambas com concentração fixa 10 mg/mL. Ensaios de adsorção foram realizados entre o lignocresol e β-glicosidases (TpBgl1 e TpBgl3), e o coquetel enzimático Cellic Ctec 3 (Novozymes). Em relação a adsorção das β-glicosidases, os resultados encontrados demonstram grande adsorção (100-90%) do lignocresol para com TpBgl1 e TpBgl3, em concentrações inferiores à 0,25 e 0,40 mg/mL, respectivamente. Ensaios de adsorção utilizando somente a lignina apresentaram resultados muito inferiores aos de lignocresol, comparados também em concentrações inferiores como demonstradas anteriormente, sendo ausente adsorção de lignina à TpBgl1 e adsorção de lignina à TpBgl3 de 70-50%. Os ensaios de ELS, demonstraram que lignocresol e lignina apresentam potencial-zeta (mV) negativo entre a faixa de pH 4-7. Vale ressaltar que, entre o pH 5-7, o lignocresol apresenta valores maiores negativamente se comparado à lignina. A adsorção do lignocresol proveniente da lignina Kraft com o coquetel Cellic Ctec 3 foi eficiente, a celulase foi efetivamente imobilizada pelo lignocresol. O desempenho da hidrólise enzimática de celulases imobilizadas em lignocresol é de aproximadamente 33% em relação ao da celulase livre na conversão de açúcares. A celulase manteve uma atividade residual após a adsorção em lignocresol. A estabilidade do complexo celulase de lignocresol também foi testada e a atividade foi mantida a partir da formação do complexo. Portanto, todos estes resultados mostram boa adsorção do lignocresol às enzimas, demonstrando que o lignocresol pode ser utilizado como suporte para recuperação enzimática, sendo uma alternativa para redução dos custos na produção de etanol 2G.
The search for renewable energy sources is gaining increasing importance because, with the imminent shortage of fossil fuels, which are the main energy sources used worldwide and at the same time with the environmental problems caused by these fuels, second-generation ethanol (2G) arises as a renewable energy alternative since it is obtained from lignocellulosic biomass. However, the technologies for the conversion of this biomass to fermentable sugars still have limiting factors. One of the major problems in the process of obtaining 2G ethanol is related to the cost of cellulolytic enzymes. Thus, developing strategies for the recovery of these enzymes is necessary to ensure the economic viability of the biomass conversion process. Several techniques are being studied and put into practice to promote recovery, recycling, and enzymatic immobilization. However, none of the methodologies used shows, in general, high efficiency, low cost, fastness and maintenance of the enzymatic activity. From lignin, a byproduct of the pulp and paper industry, lignocresol can be synthesized. This one presents high expectations of enzymatic interaction since it presents physicochemical characteristics favorable to the adsorption. In view of this, we proposed the production of this compound to promote interaction with enzymes to promote enzymatic recovery. The lignocresol synthesis from Pinus spp. Kraft lignin was made by adapting to the phase separation system methodology. In this process, more specifically in the first step, the wood is solvated with the p-cresol (phenol derivative) to avoid the attack of the concentrated acid on the lignin and so it does not dissolve. Thus, only the carbohydrates are dissolved by the acid concentration in the second phase. To obtain the complex, two lignocresol suspensions were made in sodium citrate buffer (50 mM), both with fixed concentration 10 mg / mL. Adsorption assays were performed between lignocresol and β-glycosidases (TpBgl1 and TpBgl3), and the enzymatic cocktail Cellic Ctec 3 (Novozymes). Regarding the adsorption of the β-glycosidases, the results showed high adsorption (100-90%) of lignocresol to TpBgl1 and TpBgl3, at concentrations below 0.25 and 0.40 mg / mL, respectively. Adsorption tests using lignin alone showed much lower results than lignocresol, also compared at lower concentrations as demonstrated previously, absent adsorption of lignin to TpBgl1 and adsorption of lignin to TpBgl3 of 70-50%. The ELS tests showed that lignocresol and lignin present negative zeta potential (mV) between pH range 4-7. It is worth mentioning that, between pH 5-7, lignocresol presents higher values negatively when compared to lignin. The adsorption of lignocresol from Kraft lignin with Cellic Ctec 3 cocktail was efficient, cellulase was effectively immobilized by lignocresol. The performance of the enzymatic hydrolysis of immobilized cellulases in lignocresol is approximately 33% in relation to that of free cellulase in the conversion of sugars. Cellulase was active even after adsorption on lignocresol. The stability of the lignocresol cellulase complex was also tested and activity was maintained from the complex formation. Therefore, all these results show good adsorption of lignocresol to the enzymes, demonstrating that lignocresol can be used as support for enzymatic recovery, being an alternative to reduce costs in the production of 2G ethanol
The search for renewable energy sources is gaining increasing importance because, with the imminent shortage of fossil fuels, which are the main energy sources used worldwide and at the same time with the environmental problems caused by these fuels, second-generation ethanol (2G) arises as a renewable energy alternative since it is obtained from lignocellulosic biomass. However, the technologies for the conversion of this biomass to fermentable sugars still have limiting factors. One of the major problems in the process of obtaining 2G ethanol is related to the cost of cellulolytic enzymes. Thus, developing strategies for the recovery of these enzymes is necessary to ensure the economic viability of the biomass conversion process. Several techniques are being studied and put into practice to promote recovery, recycling, and enzymatic immobilization. However, none of the methodologies used shows, in general, high efficiency, low cost, fastness and maintenance of the enzymatic activity. From lignin, a byproduct of the pulp and paper industry, lignocresol can be synthesized. This one presents high expectations of enzymatic interaction since it presents physicochemical characteristics favorable to the adsorption. In view of this, we proposed the production of this compound to promote interaction with enzymes to promote enzymatic recovery. The lignocresol synthesis from Pinus spp. Kraft lignin was made by adapting to the phase separation system methodology. In this process, more specifically in the first step, the wood is solvated with the p-cresol (phenol derivative) to avoid the attack of the concentrated acid on the lignin and so it does not dissolve. Thus, only the carbohydrates are dissolved by the acid concentration in the second phase. To obtain the complex, two lignocresol suspensions were made in sodium citrate buffer (50 mM), both with fixed concentration 10 mg / mL. Adsorption assays were performed between lignocresol and β-glycosidases (TpBgl1 and TpBgl3), and the enzymatic cocktail Cellic Ctec 3 (Novozymes). Regarding the adsorption of the β-glycosidases, the results showed high adsorption (100-90%) of lignocresol to TpBgl1 and TpBgl3, at concentrations below 0.25 and 0.40 mg / mL, respectively. Adsorption tests using lignin alone showed much lower results than lignocresol, also compared at lower concentrations as demonstrated previously, absent adsorption of lignin to TpBgl1 and adsorption of lignin to TpBgl3 of 70-50%. The ELS tests showed that lignocresol and lignin present negative zeta potential (mV) between pH range 4-7. It is worth mentioning that, between pH 5-7, lignocresol presents higher values negatively when compared to lignin. The adsorption of lignocresol from Kraft lignin with Cellic Ctec 3 cocktail was efficient, cellulase was effectively immobilized by lignocresol. The performance of the enzymatic hydrolysis of immobilized cellulases in lignocresol is approximately 33% in relation to that of free cellulase in the conversion of sugars. Cellulase was active even after adsorption on lignocresol. The stability of the lignocresol cellulase complex was also tested and activity was maintained from the complex formation. Therefore, all these results show good adsorption of lignocresol to the enzymes, demonstrating that lignocresol can be used as support for enzymatic recovery, being an alternative to reduce costs in the production of 2G ethanol
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Palavras-chave
Recuperação enzimática, Lignina, Lignocresol, Enzimas, Enzymatic recycling, Lignin, Enzymes