Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas

Detalhes bibliográficos
Autor(a) principal: Corradini, Felipe de Almeida Silva
Data de Publicação: 2019
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UFSCAR
Texto Completo: https://repositorio.ufscar.br/handle/ufscar/12060
Resumo: The production of ethanol from xylose with the yeast Saccharomyces cerevisiae was initiated in the group with the SIF process (simultaneous isomerization and fermentation). This process allows total conversion of xylose, but has been shown to be vulnerable to bacterial contamination. The process of simultaneous hydrolysis, isomerization and fermentation (SHIF) of xylooligomers was then proposed as an alternative for the fermentation of pentoses by commercial yeast. In this process, a xylanase cocktail hydrolyses the substrate to xylose, which, by the action of xylose isomerase, is transformed into xylulose, which is then fermented to ethanol by S.cerevisiae. All reactions take place within the biocatalyst where enzymes and yeast are co-immobilized. It was possible to produce ethanol by this process, but the first tests showed that the substrate conversion was incomplete, requiring investigation of the possible causes of the problem. The inefficiency of the hydrolysis stage, catalyzed by endo- / exoxylanases and β-xylosidase, a non-existent step in the SIF process, was the first hypothesis verified. The need to produce a standard substrate for the study of this reaction stage was the first challenge faced, since birchwood xylan, a commercial substrate previously used, was discontinued. A substrate was then produced in the laboratory from bleached eucalyptus pulp. Xylan was extracted with 4% NaOH solution at 25 °C, precipitated with glacial acetic acid and lyophilized. An autoclave heat pretreatment (15 min, 121 °C, 1 atm) allowed to increase the solubility of the carbohydrate chains. The xylan thus obtained was hydrolyzed by xylanases at the same rate as the commercial birchwood xylan, and made it possible to begin the investigation of the hydrolysis reaction. The observed xylobiose accumulation indicated that the inefficiency of the hydrolysis was due to the low concentration of β-xylosidase in the commercial enzymatic complexes. After cloning the Bacillus subtilis β-xylosidase gene in Escherichia coli, the recombinant enzyme was produced, purified and characterized. The results showed reduced enzyme activity at the process pH and low operational stability. The immobilization and stabilization of β-xylosidase in agarose-glyoxyl support proved to be efficient, as it considerably increased the thermal stability of the enzyme at 35 °C (164x) and 50 °C (3605x), with no significant loss of activity of the derivative after 10 consecutive hydrolysis cycles. The commercial xylanolytic complex Multifect, mainly responsible for the endoxylanase action, was immobilized on chitosan-glutaraldehyde. This technique does not allow a significant increase in the stability of complex enzymes with immobilization, but since soluble xylanases already have good stability at 35 °C, no new protocols have been tested. The obtained derivatives presented recovered activities maximum of 50%. Activity assays at different temperatures did not indicate the presence of diffusive effects, possibly being the enzymatic distortion due to the formation of multiple bonds with the support a probable reason for the 50% loss of immobilization activity. The obtained derivative was capable to hydrolyze the hydrothermal liquor in smaller XOS (X<6), with XOS yields of 56.3% and xylose of 12.9%. The high concentration of xylobiose (28.8 g.L-1) accumulated indicated a need to complement the enzymatic cocktail with more β-xylosidase. Hydrolyses supplemented with β-xylosidase derivative increased yields of xylose to 34.2%, yielding 46.5 g.L-1 xylose and reducing xylobiose to 7.4 g.L-1. The failure to obtain complete conversion of xyloligomers into xylose may be due to inhibitory effects of the product or limitation of the action of endoxylanases by their specificity to the substrate. The results obtained, however, were considered satisfactory for initial studies of the SHIF process. Another possible explanation for the SHIF halt would be the inhibitory effects of calcium ions and / or the hydrolysis reaction byproducts (e.g. XOS) on the action of xylose isomerase. Ca2+ isomerization tests showed that the reduction of the isomerization reaction rate truly occurs, with competition between the Ca2+ and Mg2+ ions for the metal site inside the enzyme. The isomerization velocity was affected by X2 only at high substrate concentrations (CS> 50 g.L-1), characterizing the competitive inhibition (R² = 0.99). The isomerization rate was significantly reduced under SHIF conditions (pH 5.6 35 ° C) and was even more affected by the combined effect of the presence of Ca2+ and X2 ions, although the reaction did not completely stop. These effects should therefore not be responsible for the incomplete conversion of the substrate. New assays of the SHIF process confirmed the importance of the β-xylosidase derivative in the biocatalyst composition, with an increase in ethanol production. The addition of more yeast to the reactor increased the consumption of the pentoses and led to an increase in ethanol production to 0.221 g.g-1 and 0.153 g.L-1.h-1), indicating that the SHIF process stagnation could also be related to the reduced speed of xylulose fermentation. It was decided at this point to compare the performance of genetically modified S. cerevisiae with that of commercial yeast in the fermentation of xylooligomers obtained by hydrothermal treatment of sugarcane bagasse. The SHIF process again showed stagnation, reaching only 5 g.L-1 of ethanol. Using the recombinant yeast, the simultaneous hydrolysis and fermentation-SHF process is performed, since this microorganism does the in vivo isomerization. In this process, ethanol concentration was increased to 15 g.L-1, generated from xylose already present in the hydrothermal liquor and the partial hydrolysis of xylooligomers. However, with the recombinant yeast stagnation was also observed, with accumulation of xylose in solution. These results thus indicate that the hydrolysis process generates some condition affecting the fermentation. The pH control allowed a small increase of the conversion in the SHIF and SHF processes, but did not prevent the stagnation. After the tests, 95% of the wild yeast and 50% of the genetically modified yeast remained viable. It has been observed that the reduction in fermentation rate may be related to an observed increase in acetic acid concentration generated in the hydrolysis step and which would be affecting both commercial and recombinant yeast. Thus, although it has not achieved full elucidation of the phenomena present in this complex process, this work allowed a great increase in the knowledge of the SHIF process, providing important indications for the necessary continuity of its study, in view of the total conversion of xylooligomers.
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spelling Corradini, Felipe de Almeida SilvaGiordano, Raquel de Lima Camargohttp://lattes.cnpq.br/9695542424889786http://lattes.cnpq.br/1435227207179790b69ee152-e7db-42d8-a856-79041c7491d42019-11-21T19:26:08Z2019-11-21T19:26:08Z2019-04-24CORRADINI, Felipe de Almeida Silva. Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas. 2019. Tese (Doutorado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12060.https://repositorio.ufscar.br/handle/ufscar/12060The production of ethanol from xylose with the yeast Saccharomyces cerevisiae was initiated in the group with the SIF process (simultaneous isomerization and fermentation). This process allows total conversion of xylose, but has been shown to be vulnerable to bacterial contamination. The process of simultaneous hydrolysis, isomerization and fermentation (SHIF) of xylooligomers was then proposed as an alternative for the fermentation of pentoses by commercial yeast. In this process, a xylanase cocktail hydrolyses the substrate to xylose, which, by the action of xylose isomerase, is transformed into xylulose, which is then fermented to ethanol by S.cerevisiae. All reactions take place within the biocatalyst where enzymes and yeast are co-immobilized. It was possible to produce ethanol by this process, but the first tests showed that the substrate conversion was incomplete, requiring investigation of the possible causes of the problem. The inefficiency of the hydrolysis stage, catalyzed by endo- / exoxylanases and β-xylosidase, a non-existent step in the SIF process, was the first hypothesis verified. The need to produce a standard substrate for the study of this reaction stage was the first challenge faced, since birchwood xylan, a commercial substrate previously used, was discontinued. A substrate was then produced in the laboratory from bleached eucalyptus pulp. Xylan was extracted with 4% NaOH solution at 25 °C, precipitated with glacial acetic acid and lyophilized. An autoclave heat pretreatment (15 min, 121 °C, 1 atm) allowed to increase the solubility of the carbohydrate chains. The xylan thus obtained was hydrolyzed by xylanases at the same rate as the commercial birchwood xylan, and made it possible to begin the investigation of the hydrolysis reaction. The observed xylobiose accumulation indicated that the inefficiency of the hydrolysis was due to the low concentration of β-xylosidase in the commercial enzymatic complexes. After cloning the Bacillus subtilis β-xylosidase gene in Escherichia coli, the recombinant enzyme was produced, purified and characterized. The results showed reduced enzyme activity at the process pH and low operational stability. The immobilization and stabilization of β-xylosidase in agarose-glyoxyl support proved to be efficient, as it considerably increased the thermal stability of the enzyme at 35 °C (164x) and 50 °C (3605x), with no significant loss of activity of the derivative after 10 consecutive hydrolysis cycles. The commercial xylanolytic complex Multifect, mainly responsible for the endoxylanase action, was immobilized on chitosan-glutaraldehyde. This technique does not allow a significant increase in the stability of complex enzymes with immobilization, but since soluble xylanases already have good stability at 35 °C, no new protocols have been tested. The obtained derivatives presented recovered activities maximum of 50%. Activity assays at different temperatures did not indicate the presence of diffusive effects, possibly being the enzymatic distortion due to the formation of multiple bonds with the support a probable reason for the 50% loss of immobilization activity. The obtained derivative was capable to hydrolyze the hydrothermal liquor in smaller XOS (X<6), with XOS yields of 56.3% and xylose of 12.9%. The high concentration of xylobiose (28.8 g.L-1) accumulated indicated a need to complement the enzymatic cocktail with more β-xylosidase. Hydrolyses supplemented with β-xylosidase derivative increased yields of xylose to 34.2%, yielding 46.5 g.L-1 xylose and reducing xylobiose to 7.4 g.L-1. The failure to obtain complete conversion of xyloligomers into xylose may be due to inhibitory effects of the product or limitation of the action of endoxylanases by their specificity to the substrate. The results obtained, however, were considered satisfactory for initial studies of the SHIF process. Another possible explanation for the SHIF halt would be the inhibitory effects of calcium ions and / or the hydrolysis reaction byproducts (e.g. XOS) on the action of xylose isomerase. Ca2+ isomerization tests showed that the reduction of the isomerization reaction rate truly occurs, with competition between the Ca2+ and Mg2+ ions for the metal site inside the enzyme. The isomerization velocity was affected by X2 only at high substrate concentrations (CS> 50 g.L-1), characterizing the competitive inhibition (R² = 0.99). The isomerization rate was significantly reduced under SHIF conditions (pH 5.6 35 ° C) and was even more affected by the combined effect of the presence of Ca2+ and X2 ions, although the reaction did not completely stop. These effects should therefore not be responsible for the incomplete conversion of the substrate. New assays of the SHIF process confirmed the importance of the β-xylosidase derivative in the biocatalyst composition, with an increase in ethanol production. The addition of more yeast to the reactor increased the consumption of the pentoses and led to an increase in ethanol production to 0.221 g.g-1 and 0.153 g.L-1.h-1), indicating that the SHIF process stagnation could also be related to the reduced speed of xylulose fermentation. It was decided at this point to compare the performance of genetically modified S. cerevisiae with that of commercial yeast in the fermentation of xylooligomers obtained by hydrothermal treatment of sugarcane bagasse. The SHIF process again showed stagnation, reaching only 5 g.L-1 of ethanol. Using the recombinant yeast, the simultaneous hydrolysis and fermentation-SHF process is performed, since this microorganism does the in vivo isomerization. In this process, ethanol concentration was increased to 15 g.L-1, generated from xylose already present in the hydrothermal liquor and the partial hydrolysis of xylooligomers. However, with the recombinant yeast stagnation was also observed, with accumulation of xylose in solution. These results thus indicate that the hydrolysis process generates some condition affecting the fermentation. The pH control allowed a small increase of the conversion in the SHIF and SHF processes, but did not prevent the stagnation. After the tests, 95% of the wild yeast and 50% of the genetically modified yeast remained viable. It has been observed that the reduction in fermentation rate may be related to an observed increase in acetic acid concentration generated in the hydrolysis step and which would be affecting both commercial and recombinant yeast. Thus, although it has not achieved full elucidation of the phenomena present in this complex process, this work allowed a great increase in the knowledge of the SHIF process, providing important indications for the necessary continuity of its study, in view of the total conversion of xylooligomers.A produção de etanol a partir de xilose com a levedura Saccharomyces cerevisiae se iniciou no grupo com o processo SIF (simultâneas isomerização e fermentação). Esse processo permite total conversão da xilose, mas se mostrou vulnerável a contaminação por bactérias. Propôs-se então como alternativa para a fermentação de pentoses por levedura comercial o processo de hidrólise, isomerização e fermentação simultâneas (SHIF) de xilooligômeros. Nesse processo, um complexo de xilanases hidrolisa o substrato a xilose, a qual, pela ação de xilose isomerase, é transformada em xilulose, que é fermentada então a etanol por S.cerevisiae. Todas as reações ocorrem dentro do biocatalizador onde se encontram co-imobilizados enzimas e levedura. Foi possível a produção de etanol por esse processo, mas os primeiros ensaios mostraram que a conversão de substrato era incompleta, requerendo investigação das possíveis causas do problema. A ineficiência da etapa de hidrólise, catalisada por endo-/exoxilanases e β-xilosidase, etapa não existente no processo SIF, foi a primeira hipótese verificada. A necessidade de produzir um substrato padrão para estudo dessa etapa reacional foi o primeiro desafio enfrentado, pois a xilana de bétula, substrato comercial até então usado, foi descontinuada. Um substrato foi desenvolvido em laboratório, a partir da polpa branqueada de eucalipto. Xilana foi extraída com solução de NaOH a 4% m.v-1 a 25°C, precipitada com ácido acético glacial e liofilizada. Um pré-tratamento térmico em autoclave (15 min, 121 °C, 1 atm) permitiu aumentar a solubilidade das cadeias de carboidrato. A xilana assim obtida foi hidrolisada por xilanases na mesma velocidade que a xilana comercial de bétula, e tornou possível iniciar-se a investigação da reação de hidrólise. O acúmulo de xilobiose observado indicou que a ineficiência da hidrólise era devida à pequena concentração de β-xilosidase nos complexos enzimáticos comerciais. Após se obter Escherichia coli clonada com gene de β-xilosidase de Bacillus subtillis, a enzima recombinante foi produzida, purificada e caracterizada. Os resultados obtidos mostraram reduzida atividade da enzima no pH do processo e baixa estabilidade operacional. A imobilização e estabilização de β-xilosidase em suporte agarose-glioxil mostrou-se eficiente, pois aumentou consideravelmente a estabilidade térmica da enzima a 35 °C (164x) e 50 °C (3605x), não havendo perda significativa de atividade do derivado após 10 ciclos de hidrólise consecutivos. O complexo xilanolítico comercial Multifect, responsável principalmente pela ação endoxilanase, foi imobilizado em quitosana-glutaraldeído. Essa técnica não permite significativo aumento na estabilidade das enzimas do complexo com a imobilização, mas como as xilanases solúveis já têm boa estabilidade a 35 °C, não foram testados novos protocolos. Os derivados obtidos apresentaram atividades recuperadas máximas de 50%. Testes de atividades em diferentes temperaturas não indicaram presença de efeitos difusivos, sendo, possivelmente, a distorção da enzima devido à formação de múltiplas ligações com o suporte a provável justificativa para a perda de 50% de atividade com a imobilização. O derivado obtido permitiu hidrolisar o licor hidrotérmico em XOS menores (X<6), com rendimentos em XOS de 56,3% e de xilose de 12,9%. A elevada concentração de xilobiose (28,8 g.L-1) acumulada indicou necessidade de complementar o complexo com mais β-xilosidase. Hidrólises complementadas com derivado de β-xilosidase aumentaram os rendimentos de xilose para 34,2%, produzindo 46,5 g.L-1 de xilose e reduzindo xilobiose para 7,4 g.L-1. A não obtenção de conversão completa de xiloligômeros em xilose pode ser devida a efeitos inibitórios de produto ou limitação da ação das endoxilanases pela sua especificidade frente ao substrato. Os resultados obtidos, contudo, foram considerados satisfatórios para estudos iniciais do processo SHIF. Outra possível explicação para a parada SHIF viriam dos efeitos inibitórios de íons cálcio e/ou subprodutos da reação de hidrólise (XOS) sobre a ação de xilose isomerase-XI. Ensaios de isomerização com Ca2+ mostraram realmente ocorrer redução da velocidade da reação de isomerização, havendo competição entre os íons Ca2+ e Mg2+ pelo sítio metálico no interior da enzima. A velocidade de isomerização foi afetada por X2 apenas em elevadas concentrações de substrato (CS>50 g.L-1), caracterizando a inibição incompetitiva (R² = 0,99). A velocidade de isomerização se mostrou bastante reduzida nas condições de SHIF (pH 5,6 35 °C), sendo ainda mais afetada pelo efeito combinado das presenças de íons Ca2+ e X2, embora não cesse completamente a reação. Esses efeitos não devem, portanto, serem os responsáveis pela conversão incompleta do substrato. Novos ensaios do processo SHIF confirmaram a importância do derivado de β-xilosidase na composição do biocatalisador, observando-se aumento da produção de etanol. A adição de mais levedura ao reator elevou o consumo das pentoses e conduziu ao aumento da produção de etanol para 0,221 g.g-1 e 0,153 g.L-1.h-1), indicando que a estagnação do processo SHIF poderia estar também relacionada à reduzida velocidade de fermentação de xilulose. Decidiu-se, nesse ponto, comparar o desempenho de S. cerevisiae modificada geneticamente com o da levedura comercial na fermentação de xilooligômeros obtidos por tratamento hidrotérmico de bagaço de cana. O processo SHIF novamente mostrou estagnação, atingindo apenas 5 g.L-1 de etanol. Utilizando-se a levedura recombinante, tem-se o processo de hidrólise e fermentação simultâneas (SHF), uma vez que esse microrganismo faz a isomerização in vivo. Nesse processo houve aumento da concentração de etanol para 15 g.L-1, o qual foi gerado de xilose já presente no licor hidrotérmico e da hidrólise parcial de xilooligômeros, mas também com a levedura recombinante observou-se estagnação, com acúmulo de xilose em solução. Esses resultados indicam assim que o processo de hidrólise gera alguma condição que afeta a fermentação. O controle do pH permitiu pequeno aumento da conversão nos processos SHIF e SHF, mas não impediu a estagnação. Após os ensaios, 95% da levedura selvagem e 50% da modificada geneticamente permaneceram viáveis. Observou-se que a redução na velocidade de fermentação pode estar relacionada com um observado aumento na concentração de ácido acético gerado na etapa de hidrólise e que estaria afetando tanto a levedura comercial quanto a recombinante. Assim, embora não tenha conseguido total elucidação dos fenômenos presentes nesse complexo processo, este trabalho permitiu um grande aumento no conhecimento do processo SHIF, fornecendo importantes indicações para a necessária continuidade do seu estudo, tendo em vista a total conversão de xilooligômeros.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CNPq: 146438/2015-9CAPES: código de financiamento - 001porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Engenharia Química - PPGEQUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessSHIFSHFβ-xilosidaseXilanaseXilose IsomeraseEtanolβ-xylosidaseEthanolXylose IsomeraseXylanaseENGENHARIAS::ENGENHARIA QUIMICA::PROCESSOS INDUSTRIAIS DE ENGENHARIA QUIMICAEstudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadasStudy of ethanol production from xylose and xylooligomers using co-immobilized xylases, xylose isomerase and yeastinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis60060087b60e6c-591e-4a38-94f3-e75e2beebea0reponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALFelipe Corradini_tese2019.pdfFelipe Corradini_tese2019.pdfTese finalapplication/pdf6541760https://repositorio.ufscar.br/bitstream/ufscar/12060/2/Felipe%20Corradini_tese2019.pdfcbada4b65aff850cdc9def6a481b77b1MD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufscar.br/bitstream/ufscar/12060/8/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD58TEXTFelipe Corradini_tese2019.pdf.txtFelipe Corradini_tese2019.pdf.txtExtracted texttext/plain459374https://repositorio.ufscar.br/bitstream/ufscar/12060/9/Felipe%20Corradini_tese2019.pdf.txtb316386d95c25a0d0b1fb0cd231ca6b2MD59THUMBNAILFelipe Corradini_tese2019.pdf.jpgFelipe Corradini_tese2019.pdf.jpgIM Thumbnailimage/jpeg8141https://repositorio.ufscar.br/bitstream/ufscar/12060/10/Felipe%20Corradini_tese2019.pdf.jpg49991be49fc58c73fc2f658c0f94e428MD510ufscar/120602023-09-18 18:31:46.368oai:repositorio.ufscar.br:ufscar/12060Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:46Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false
dc.title.por.fl_str_mv Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
dc.title.alternative.eng.fl_str_mv Study of ethanol production from xylose and xylooligomers using co-immobilized xylases, xylose isomerase and yeast
title Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
spellingShingle Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
Corradini, Felipe de Almeida Silva
SHIF
SHF
β-xilosidase
Xilanase
Xilose Isomerase
Etanol
β-xylosidase
Ethanol
Xylose Isomerase
Xylanase
ENGENHARIAS::ENGENHARIA QUIMICA::PROCESSOS INDUSTRIAIS DE ENGENHARIA QUIMICA
title_short Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
title_full Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
title_fullStr Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
title_full_unstemmed Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
title_sort Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas
author Corradini, Felipe de Almeida Silva
author_facet Corradini, Felipe de Almeida Silva
author_role author
dc.contributor.authorlattes.por.fl_str_mv http://lattes.cnpq.br/1435227207179790
dc.contributor.author.fl_str_mv Corradini, Felipe de Almeida Silva
dc.contributor.advisor1.fl_str_mv Giordano, Raquel de Lima Camargo
dc.contributor.advisor1Lattes.fl_str_mv http://lattes.cnpq.br/9695542424889786
dc.contributor.authorID.fl_str_mv b69ee152-e7db-42d8-a856-79041c7491d4
contributor_str_mv Giordano, Raquel de Lima Camargo
dc.subject.por.fl_str_mv SHIF
SHF
β-xilosidase
Xilanase
Xilose Isomerase
Etanol
topic SHIF
SHF
β-xilosidase
Xilanase
Xilose Isomerase
Etanol
β-xylosidase
Ethanol
Xylose Isomerase
Xylanase
ENGENHARIAS::ENGENHARIA QUIMICA::PROCESSOS INDUSTRIAIS DE ENGENHARIA QUIMICA
dc.subject.eng.fl_str_mv β-xylosidase
Ethanol
Xylose Isomerase
Xylanase
dc.subject.cnpq.fl_str_mv ENGENHARIAS::ENGENHARIA QUIMICA::PROCESSOS INDUSTRIAIS DE ENGENHARIA QUIMICA
description The production of ethanol from xylose with the yeast Saccharomyces cerevisiae was initiated in the group with the SIF process (simultaneous isomerization and fermentation). This process allows total conversion of xylose, but has been shown to be vulnerable to bacterial contamination. The process of simultaneous hydrolysis, isomerization and fermentation (SHIF) of xylooligomers was then proposed as an alternative for the fermentation of pentoses by commercial yeast. In this process, a xylanase cocktail hydrolyses the substrate to xylose, which, by the action of xylose isomerase, is transformed into xylulose, which is then fermented to ethanol by S.cerevisiae. All reactions take place within the biocatalyst where enzymes and yeast are co-immobilized. It was possible to produce ethanol by this process, but the first tests showed that the substrate conversion was incomplete, requiring investigation of the possible causes of the problem. The inefficiency of the hydrolysis stage, catalyzed by endo- / exoxylanases and β-xylosidase, a non-existent step in the SIF process, was the first hypothesis verified. The need to produce a standard substrate for the study of this reaction stage was the first challenge faced, since birchwood xylan, a commercial substrate previously used, was discontinued. A substrate was then produced in the laboratory from bleached eucalyptus pulp. Xylan was extracted with 4% NaOH solution at 25 °C, precipitated with glacial acetic acid and lyophilized. An autoclave heat pretreatment (15 min, 121 °C, 1 atm) allowed to increase the solubility of the carbohydrate chains. The xylan thus obtained was hydrolyzed by xylanases at the same rate as the commercial birchwood xylan, and made it possible to begin the investigation of the hydrolysis reaction. The observed xylobiose accumulation indicated that the inefficiency of the hydrolysis was due to the low concentration of β-xylosidase in the commercial enzymatic complexes. After cloning the Bacillus subtilis β-xylosidase gene in Escherichia coli, the recombinant enzyme was produced, purified and characterized. The results showed reduced enzyme activity at the process pH and low operational stability. The immobilization and stabilization of β-xylosidase in agarose-glyoxyl support proved to be efficient, as it considerably increased the thermal stability of the enzyme at 35 °C (164x) and 50 °C (3605x), with no significant loss of activity of the derivative after 10 consecutive hydrolysis cycles. The commercial xylanolytic complex Multifect, mainly responsible for the endoxylanase action, was immobilized on chitosan-glutaraldehyde. This technique does not allow a significant increase in the stability of complex enzymes with immobilization, but since soluble xylanases already have good stability at 35 °C, no new protocols have been tested. The obtained derivatives presented recovered activities maximum of 50%. Activity assays at different temperatures did not indicate the presence of diffusive effects, possibly being the enzymatic distortion due to the formation of multiple bonds with the support a probable reason for the 50% loss of immobilization activity. The obtained derivative was capable to hydrolyze the hydrothermal liquor in smaller XOS (X<6), with XOS yields of 56.3% and xylose of 12.9%. The high concentration of xylobiose (28.8 g.L-1) accumulated indicated a need to complement the enzymatic cocktail with more β-xylosidase. Hydrolyses supplemented with β-xylosidase derivative increased yields of xylose to 34.2%, yielding 46.5 g.L-1 xylose and reducing xylobiose to 7.4 g.L-1. The failure to obtain complete conversion of xyloligomers into xylose may be due to inhibitory effects of the product or limitation of the action of endoxylanases by their specificity to the substrate. The results obtained, however, were considered satisfactory for initial studies of the SHIF process. Another possible explanation for the SHIF halt would be the inhibitory effects of calcium ions and / or the hydrolysis reaction byproducts (e.g. XOS) on the action of xylose isomerase. Ca2+ isomerization tests showed that the reduction of the isomerization reaction rate truly occurs, with competition between the Ca2+ and Mg2+ ions for the metal site inside the enzyme. The isomerization velocity was affected by X2 only at high substrate concentrations (CS> 50 g.L-1), characterizing the competitive inhibition (R² = 0.99). The isomerization rate was significantly reduced under SHIF conditions (pH 5.6 35 ° C) and was even more affected by the combined effect of the presence of Ca2+ and X2 ions, although the reaction did not completely stop. These effects should therefore not be responsible for the incomplete conversion of the substrate. New assays of the SHIF process confirmed the importance of the β-xylosidase derivative in the biocatalyst composition, with an increase in ethanol production. The addition of more yeast to the reactor increased the consumption of the pentoses and led to an increase in ethanol production to 0.221 g.g-1 and 0.153 g.L-1.h-1), indicating that the SHIF process stagnation could also be related to the reduced speed of xylulose fermentation. It was decided at this point to compare the performance of genetically modified S. cerevisiae with that of commercial yeast in the fermentation of xylooligomers obtained by hydrothermal treatment of sugarcane bagasse. The SHIF process again showed stagnation, reaching only 5 g.L-1 of ethanol. Using the recombinant yeast, the simultaneous hydrolysis and fermentation-SHF process is performed, since this microorganism does the in vivo isomerization. In this process, ethanol concentration was increased to 15 g.L-1, generated from xylose already present in the hydrothermal liquor and the partial hydrolysis of xylooligomers. However, with the recombinant yeast stagnation was also observed, with accumulation of xylose in solution. These results thus indicate that the hydrolysis process generates some condition affecting the fermentation. The pH control allowed a small increase of the conversion in the SHIF and SHF processes, but did not prevent the stagnation. After the tests, 95% of the wild yeast and 50% of the genetically modified yeast remained viable. It has been observed that the reduction in fermentation rate may be related to an observed increase in acetic acid concentration generated in the hydrolysis step and which would be affecting both commercial and recombinant yeast. Thus, although it has not achieved full elucidation of the phenomena present in this complex process, this work allowed a great increase in the knowledge of the SHIF process, providing important indications for the necessary continuity of its study, in view of the total conversion of xylooligomers.
publishDate 2019
dc.date.accessioned.fl_str_mv 2019-11-21T19:26:08Z
dc.date.available.fl_str_mv 2019-11-21T19:26:08Z
dc.date.issued.fl_str_mv 2019-04-24
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dc.identifier.citation.fl_str_mv CORRADINI, Felipe de Almeida Silva. Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas. 2019. Tese (Doutorado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12060.
dc.identifier.uri.fl_str_mv https://repositorio.ufscar.br/handle/ufscar/12060
identifier_str_mv CORRADINI, Felipe de Almeida Silva. Estudo da produção de etanol a partir de xilose e xilooligômeros usando xilanases, xilose isomerase e levedura co-imobilizadas. 2019. Tese (Doutorado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12060.
url https://repositorio.ufscar.br/handle/ufscar/12060
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dc.rights.driver.fl_str_mv Attribution-NonCommercial-NoDerivs 3.0 Brazil
http://creativecommons.org/licenses/by-nc-nd/3.0/br/
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rights_invalid_str_mv Attribution-NonCommercial-NoDerivs 3.0 Brazil
http://creativecommons.org/licenses/by-nc-nd/3.0/br/
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dc.publisher.none.fl_str_mv Universidade Federal de São Carlos
Câmpus São Carlos
dc.publisher.program.fl_str_mv Programa de Pós-Graduação em Engenharia Química - PPGEQ
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