The potential of high digestible grain sorghum (HDGS) with a modified starch protein endosperm matrix to replace corn in ethanol production was investigated using dry grind simultaneous saccharification and fermentation (SSF). Preliminary experiments showed that HDGS yielded higher amounts of glucose and ethanol than normal digestible grain sorghum (NDGS) and corn particularly in the first 48 hrs of fermentation. It was hypothesized that fast conversion of starch to glucose and ethanol during hydrolysis and fermentation are results of improved protein digestibility of HDGS. The invagination of protein structures in HDGS produced a flourier endosperm texture, softer kernels and lower starch content than the normal digestible protein (ND) lines. Highly digestible protein (HD) lines have better pasting properties (significantly lower pasting temperature, faster rate of gelatinization and higher peak viscosity) than ND lines based on the RVA profile. Increasing protein digestibility of the HDGS improved starch digestibility (increased rate of glucose conversion and total glucose yield during saccharification), which is supported by highly significant correlation of turbidity with rate of glucose conversion and efficiency of enzymatic conversion. The efficiency of ethanol conversion is significantly correlated with starch digestibility, pasting properties, and protein digestibility. Results also showed that HD sorghum lines had significantly faster rate of conversion and shorter reaction time needed to achieve completion than ND sorghum lines and corn. Increasing the dry solid concentration from 22% to 30% (w/v) increased the ethanol yield from 8% v/v to 13%v/v. This will allow considerable saving of water, reduced distillation cost and increased ethanol production for a given plant capacity and labor cost. Fineness of grind influences the amount of sugar formed due to variation in surface area of the flour. The hypothesis that finer particles has faster and higher glucose yield, defined as g of glucose converted per g of theoretical glucose, is supported by highly significant correlation of mass fraction of 3 to 60 mu m size range and mass median diameter (MMD) of 60 to 1000 mu m size range with glucose conversion efficiency and glucose conversion rate during saccharification and fermentation.

Made available in DSpace on 2017-05-12T14:36:23Z (GMT). No. of bitstreams: 1 DISSERTACAO AMANDA ALVES MESTRADO EM CIENCIAS FARMACEUTICAS _UNIOESTE 2015.pdf: 10598736 bytes, checksum: 51f0f3eb83858fee62392b7892930766 (MD5) Previous issue date: 2015-12-07 / Lignocellulosic biomass are the raw material most abundant and promising as a natural and renewable resource. These plant materials are complex carbohydrate polymer composed mainly of cellulose, hemicellulose and lignin, which are linked by covalent bonds and can be transformed into value-added products, such as biofuels. The degradation of lignocellulosic material is made mainly from enzymes produced by microorganisms such as filamentous fungi, yeast and bacteria. Ethanol production from agricultural residues, based on the enzymatic hydrolysis, it takes basically four stages: production of enzymes, pretreatment, enzymatic hydrolysis and fermentation. Pretreatment is a work that will break the lignin cellulose complex, reducing the degree of crystallinity of the cellulose and increase the porosity of the material, by increasing the surface area of the biomass. However, pre-treatment products can generate inhibitors which include phenolic and other aromatic, aliphatic acids, aldehydes, furans, inorganic ions. The fermentation and simultaneous saccharification is an important approach for producing cellulosic or ethanol of second generation, where the enzymatic hydrolysis of cellulose and fermentation are simultaneously carried out in the same reactor, in order to obtain ethanol at a high rate and decrease formation of inhibitor compounds. Enzymatic hydrolysis requires, first, that the lignocellulosic biomass is pretreated to increase access to enzymatic attack, so that later the cellulose is broken down by cellulase action. Xylanases include the group of enzymes responsible for the hydrolysis of xylan, the major constituent of hemicellulose. The key enzymes involved in this process are β-1,4-endoxylanase and β-D-xylosidase. Endoxylanase cleave glycosidic linkages of the main chain of xylan releasing xylo-oligosaccharides, which are used by β-xylosidase to produce monomers of xylose. The alfaproteobacteria Caulobacter crescentus is non pathogenic, Gram negative, mainly found in aquatic environments and on many types of soils. This bacterium has about seven genes directly associated with xylan degradation and five of them encoding β-xylosidases. To date, there are only three studies on the β-xylosidase II from C. crescentus. The first characterization of this enzyme showed that it is capable of hydrolyzing substrates such as xylobiose, xylotriose and xilopentose whose optimum pH is 6 and optimum temperature is 55°C, although it is stable at 50°C, which shows a thermotolerance, indicating strong enough to be used in different biotechnological applications. The stability and reusability of enzymes are of fundamental importance, since they reflect significantly on the cost of the final product, and one way to achieve this is with the immobilization of enzymes, consisting of confinement thereof in a matrix or support, which can be inert polymers or inorganic materials, so that its catalytic activity is retained and the enzyme can be used repeatedly and continuously. In the present report, it was found that the β-xylosidase II (CcXynB2) of Caulobacter crescentus increased by 62% of its activity in 5 mM KCl probably as a consequence of a positive role of K+ ions. CCxynB2 was measured against various compounds described as inhibitors of hydrolysis and fermentation of lignocellulosic biomass and showed 61% more tolerant incubation with ethanol (200 mM) at 37 °C for 48 h in the absence of alcohol. The specific activities of CcXynB2 were evaluated in the presence of 10mM phenol or galacturonic acid, 100 mM hydroxymethylfurfural or ferulic acid, 1 mM acetic acid, 200 mM arabinose, glucose or xylose and it was found that were equal (100%) or much higher than the values obtained in the total absence of these compounds after 48 h. When the inhibitors were used in combination, the CcXynB2 retained 67% of its initial activity after testing at 37°C during 48 h. The enzymatic hydrolysis of hemicellulose from corncob was conducted with CcXynB2 alone or in synergism with xylanase and commercial β-glycosidase, which were more efficient in performed the saccharification of hemicellulose from 37-50 °C. The immobilized CcXynB2 in mobile phase resin led to a protective effect of specific activity, which was proportionally parallel to decreased temperatures (60 to -20°C). The data presented here indicate that CcXynB2 is promising and has potential to work in simultaneous saccharification and fermentation processes for cellulosic ethanol production. To our knowledge, is the first time that similar results are reported in the literature to bacterial β-xylosidases. Thus, this work contribute positively by providing essential information to improve the use of β-xylosidase II of Caulobacter crescentus. / Biomassas lignocelulósicas constituem a matéria-prima mais abundante e promissora como recurso natural e renovável. Esses materiais vegetais são polímeros de carboidratos complexos compostos basicamente por celulose, hemicelulose e lignina, que estão unidos entre si por ligações covalentes e podem ser convertidos em produtos de valor agregado, como os biocombustíveis. A degradação dos materiais lignocelulósicos é feita a partir de enzimas produzidas principalmente por micro-organismos como fungos filamentosos, leveduras e bactérias. Para obter etanol a partir de resíduos agroindustriais, baseando-se na hidrólise enzimática, são necessárias, basicamente, quatro etapas: produção de enzimas, pré-tratamento, hidrólise enzimática e fermentação. O pré-tratamento é o processo que irá dissociar o complexo lignina-celulose, reduzir o grau de cristalinidade da celulose e aumentar a porosidade dos materiais, através do aumento da área superficial da biomassa. No entanto, o pré-tratamento pode gerar produtos inibidores, que incluem compostos fenólicos e outros aromáticos, ácidos alifáticos, aldeídos, furanos, íons inorgânicos. A fermentação e sacarificação simultânea é uma estratégia importante para a produção de etanol celulósico ou de segunda geração, onde a hidrólise enzimática da celulose e a fermentação são desenvolvidas simultaneamente no mesmo reator, com o intuito de obter etanol em altas taxas e diminuir a formação de compostos inibidores. A hidrólise enzimática necessita, primeiramente, que a biomassa lignocelulósica seja pré-tratada para aumentar o acesso ao ataque enzimático, para que posteriormente a celulose seja quebrada pela ação de celulases. As xilanases compreendem o grupo de enzimas responsáveis pela hidrólise do xilano, principal constituinte da hemicelulose. As principais enzimas envolvidas nesse processo são β-1,4-endoxilanase e a β-D-xilosidase. Endoxilanases clivam as ligações glicosídicas da cadeia principal do xilano liberando xilo-oligossacarídeos, que são utilizados pelas β-xilosidases para liberar xilose. A alfaproteobactéria Caulobacter crescentus é não patogênica, Gram negativa, encontrada principalmente em ambientes aquáticos e em muitos tipos de solos. Essa bactéria apresenta cerca de sete genes envolvidos diretamente na degradação do xilano, sendo que cinco deles codificam para β-xilosidases. Até o momento, existem apenas três trabalhos sobre a β-xilosidase II de C. crescentus. A primeira caracterização da enzima mostrou que esta é capaz de hidrolisar substratos como xilobiose, xilotriose e xilopentose, cujo pH ótimo é 6 e temperatura ótima é 55ºC, embora seja mais estável em 50ºC, o que demonstra uma modesta termotolerância, indicando ser suficientemente resistente para diferentes aplicações biotecnológicas. A estabilidade e a possibilidade de reutilização de enzimas são de fundamental importância, pois refletem significativamente no custo do produto final, e uma forma de conseguir isso é com a imobilização de enzimas, que consiste no confinamento da mesma em uma matriz ou suporte, que podem ser polímeros inertes ou materiais inorgânicos, de modo que sua atividade catalítica fique retida e a enzima possa ser usada repetidamente e continuamente. No presente trabalho, verificou-se que a β-xilosidase II (CcXynB2) de Caulobacter crescentus aumentou 62% da sua atividade em 5 mM de KCl provavelmente em consequência de um papel positivo dos íons K+. CcXynB2 foi avaliada frente a diferentes compostos descritos como inibidores do processo de hidrólise e fermentação da biomassa lignocelulósica e mostrou-se 61% mais tolerante a incubação com etanol (200 mM) a atividades específicas da CcXynB2 foram avaliadas na presença de 10 mM fenol ou ácido galacturônico, 100 mM de hidroximetilfurfural ou ácido ferúlico, 1 mM de ácido acético, 200 mM de arabinose, glicose ou xilose, e verificou-se que foram iguais (100%) ou muito superiores aos valores obtidos na ausência total destes compostos após 48 h. Quando os inibidores foram usados em associação, a CcXynB2 reteve 67% da sua atividade inicial após 48 h de ensaio a 37ºC. A hidrólise enzimática da hemicelulose de sabugo de milho foi conduzida com CcXynB2 isoladamente ou em sinergismo com xilanase e β-glicosidase comerciais, as quais foram mais eficientes em sacarificar a hemicelulose entre 37-50ºC. A imobilização da CcXynB2 em resina de fase móvel levou a um efeito protetor da atividade específica, que ocorreu de forma paralela à diminuição de temperatura (60 a -20ºC). Os dados apresentados aqui indicam que a CcXynB2 é promissora e possui potencial para atuar em processos de sacarificação e fermentação simultânea para produção de etanol celulósico. Segundo nosso conhecimento, é a primeira vez que resultados similares são relatados na literatura para β-xilosidases bacterianas. Dessa forma, este trabalho pode contribuir positivamente, fornecendo informações fundamentais para aprimorar o uso da β-xilosidase II de Caulobacter crescentus

Made available in DSpace on 2017-07-10T13:59:26Z (GMT). No. of bitstreams: 1 DISSERTACAO AMANDA ALVES MESTRADO EM CIENCIAS FARMACEUTICAS _UNIOESTE 2015.pdf: 10598736 bytes, checksum: 51f0f3eb83858fee62392b7892930766 (MD5) Previous issue date: 2015-12-07 / SIM(não especificado) / Lignocellulosic biomass are the raw material most abundant and promising as a natural and renewable resource. These plant materials are complex carbohydrate polymer composed mainly of cellulose, hemicellulose and lignin, which are linked by covalent bonds and can be transformed into value-added products, such as biofuels. The degradation of lignocellulosic material is made mainly from enzymes produced by microorganisms such as filamentous fungi, yeast and bacteria. Ethanol production from agricultural residues, based on the enzymatic hydrolysis, it takes basically four stages: production of enzymes, pretreatment, enzymatic hydrolysis and fermentation. Pretreatment is a work that will break the lignin cellulose complex, reducing the degree of crystallinity of the cellulose and increase the porosity of the material, by increasing the surface area of the biomass. However, pre-treatment products can generate inhibitors which include phenolic and other aromatic, aliphatic acids, aldehydes, furans, inorganic ions. The fermentation and simultaneous saccharification is an important approach for producing cellulosic or ethanol of second generation, where the enzymatic hydrolysis of cellulose and fermentation are simultaneously carried out in the same reactor, in order to obtain ethanol at a high rate and decrease formation of inhibitor compounds. Enzymatic hydrolysis requires, first, that the lignocellulosic biomass is pretreated to increase access to enzymatic attack, so that later the cellulose is broken down by cellulase action. Xylanases include the group of enzymes responsible for the hydrolysis of xylan, the major constituent of hemicellulose. The key enzymes involved in this process are β-1,4-endoxylanase and β-D-xylosidase. Endoxylanase cleave glycosidic linkages of the main chain of xylan releasing xylo-oligosaccharides, which are used by β-xylosidase to produce monomers of xylose. The alfaproteobacteria Caulobacter crescentus is non pathogenic, Gram negative, mainly found in aquatic environments and on many types of soils. This bacterium has about seven genes directly associated with xylan degradation and five of them encoding β-xylosidases. To date, there are only three studies on the β-xylosidase II from C. crescentus. The first characterization of this enzyme showed that it is capable of hydrolyzing substrates such as xylobiose, xylotriose and xilopentose whose optimum pH is 6 and optimum temperature is 55°C, although it is stable at 50°C, which shows a thermotolerance, indicating strong enough to be used in different biotechnological applications. The stability and reusability of enzymes are of fundamental importance, since they reflect significantly on the cost of the final product, and one way to achieve this is with the immobilization of enzymes, consisting of confinement thereof in a matrix or support, which can be inert polymers or inorganic materials, so that its catalytic activity is retained and the enzyme can be used repeatedly and continuously. In the present report, it was found that the β-xylosidase II (CcXynB2) of Caulobacter crescentus increased by 62% of its activity in 5 mM KCl probably as a consequence of a positive role of K+ ions. CCxynB2 was measured against various compounds described as inhibitors of hydrolysis and fermentation of lignocellulosic biomass and showed 61% more tolerant incubation with ethanol (200 mM) at 37 °C for 48 h in the absence of alcohol. The specific activities of CcXynB2 were evaluated in the presence of 10mM phenol or galacturonic acid, 100 mM hydroxymethylfurfural or ferulic acid, 1 mM acetic acid, 200 mM arabinose, glucose or xylose and it was found that were equal (100%) or much higher than the values obtained in the total absence of these compounds after 48 h. When the inhibitors were used in combination, the CcXynB2 retained 67% of its initial activity after testing at 37°C during 48 h. The enzymatic hydrolysis of hemicellulose from corncob was conducted with CcXynB2 alone or in synergism with xylanase and commercial β-glycosidase, which were more efficient in performed the saccharification of hemicellulose from 37-50 °C. The immobilized CcXynB2 in mobile phase resin led to a protective effect of specific activity, which was proportionally parallel to decreased temperatures (60 to -20°C). The data presented here indicate that CcXynB2 is promising and has potential to work in simultaneous saccharification and fermentation processes for cellulosic ethanol production. To our knowledge, is the first time that similar results are reported in the literature to bacterial β-xylosidases. Thus, this work contribute positively by providing essential information to improve the use of β-xylosidase II of Caulobacter crescentus. / Biomassas lignocelulósicas constituem a matéria-prima mais abundante e promissora como recurso natural e renovável. Esses materiais vegetais são polímeros de carboidratos complexos compostos basicamente por celulose, hemicelulose e lignina, que estão unidos entre si por ligações covalentes e podem ser convertidos em produtos de valor agregado, como os biocombustíveis. A degradação dos materiais lignocelulósicos é feita a partir de enzimas produzidas principalmente por micro-organismos como fungos filamentosos, leveduras e bactérias. Para obter etanol a partir de resíduos agroindustriais, baseando-se na hidrólise enzimática, são necessárias, basicamente, quatro etapas: produção de enzimas, pré-tratamento, hidrólise enzimática e fermentação. O pré-tratamento é o processo que irá dissociar o complexo lignina-celulose, reduzir o grau de cristalinidade da celulose e aumentar a porosidade dos materiais, através do aumento da área superficial da biomassa. No entanto, o pré-tratamento pode gerar produtos inibidores, que incluem compostos fenólicos e outros aromáticos, ácidos alifáticos, aldeídos, furanos, íons inorgânicos. A fermentação e sacarificação simultânea é uma estratégia importante para a produção de etanol celulósico ou de segunda geração, onde a hidrólise enzimática da celulose e a fermentação são desenvolvidas simultaneamente no mesmo reator, com o intuito de obter etanol em altas taxas e diminuir a formação de compostos inibidores. A hidrólise enzimática necessita, primeiramente, que a biomassa lignocelulósica seja pré-tratada para aumentar o acesso ao ataque enzimático, para que posteriormente a celulose seja quebrada pela ação de celulases. As xilanases compreendem o grupo de enzimas responsáveis pela hidrólise do xilano, principal constituinte da hemicelulose. As principais enzimas envolvidas nesse processo são β-1,4-endoxilanase e a β-D-xilosidase. Endoxilanases clivam as ligações glicosídicas da cadeia principal do xilano liberando xilo-oligossacarídeos, que são utilizados pelas β-xilosidases para liberar xilose. A alfaproteobactéria Caulobacter crescentus é não patogênica, Gram negativa, encontrada principalmente em ambientes aquáticos e em muitos tipos de solos. Essa bactéria apresenta cerca de sete genes envolvidos diretamente na degradação do xilano, sendo que cinco deles codificam para β-xilosidases. Até o momento, existem apenas três trabalhos sobre a β-xilosidase II de C. crescentus. A primeira caracterização da enzima mostrou que esta é capaz de hidrolisar substratos como xilobiose, xilotriose e xilopentose, cujo pH ótimo é 6 e temperatura ótima é 55ºC, embora seja mais estável em 50ºC, o que demonstra uma modesta termotolerância, indicando ser suficientemente resistente para diferentes aplicações biotecnológicas. A estabilidade e a possibilidade de reutilização de enzimas são de fundamental importância, pois refletem significativamente no custo do produto final, e uma forma de conseguir isso é com a imobilização de enzimas, que consiste no confinamento da mesma em uma matriz ou suporte, que podem ser polímeros inertes ou materiais inorgânicos, de modo que sua atividade catalítica fique retida e a enzima possa ser usada repetidamente e continuamente. No presente trabalho, verificou-se que a β-xilosidase II (CcXynB2) de Caulobacter crescentus aumentou 62% da sua atividade em 5 mM de KCl provavelmente em consequência de um papel positivo dos íons K+. CcXynB2 foi avaliada frente a diferentes compostos descritos como inibidores do processo de hidrólise e fermentação da biomassa lignocelulósica e mostrou-se 61% mais tolerante a incubação com etanol (200 mM) a atividades específicas da CcXynB2 foram avaliadas na presença de 10 mM fenol ou ácido galacturônico, 100 mM de hidroximetilfurfural ou ácido ferúlico, 1 mM de ácido acético, 200 mM de arabinose, glicose ou xilose, e verificou-se que foram iguais (100%) ou muito superiores aos valores obtidos na ausência total destes compostos após 48 h. Quando os inibidores foram usados em associação, a CcXynB2 reteve 67% da sua atividade inicial após 48 h de ensaio a 37ºC. A hidrólise enzimática da hemicelulose de sabugo de milho foi conduzida com CcXynB2 isoladamente ou em sinergismo com xilanase e β-glicosidase comerciais, as quais foram mais eficientes em sacarificar a hemicelulose entre 37-50ºC. A imobilização da CcXynB2 em resina de fase móvel levou a um efeito protetor da atividade específica, que ocorreu de forma paralela à diminuição de temperatura (60 a -20ºC). Os dados apresentados aqui indicam que a CcXynB2 é promissora e possui potencial para atuar em processos de sacarificação e fermentação simultânea para produção de etanol celulósico. Segundo nosso conhecimento, é a primeira vez que resultados similares são relatados na literatura para β-xilosidases bacterianas. Dessa forma, este trabalho pode contribuir positivamente, fornecendo informações fundamentais para aprimorar o uso da β-xilosidase II de Caulobacter crescentus

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Second-generation bioethanol is an alternative transportation fuel currently being investigated whereby cellulose, specifically lignocellulosic (woody) portions, of any plant mass can be converted to ethanol. To date, the technology had only been successfully implemented with demonstration scale facilities. Despite intensive research efforts at laboratory scale, no-one is certain what the secondary effects of scale-up to large systems are. The objective of this project was to develop threedimensional numerical models of a laboratory scale fermenter which could predict the effects of particulate mixing and reaction kinetics for future scale-up investigations. A numerical model of the reaction kinetics for simultaneous saccharification and fermentation of Avicel (microcrystalline cellulose) particles to ethanol is presented. The novelty of this model is the separation of the two primary cellulase enzyme-kinetics, which generated the capability to predict the heterogeneous behaviour of the enzyme-substrate interactions. This model improves the understanding of these systems while maintaining sufficient simplicity for implementation alongside a commercial computational fluid dynamics environment. Effects of the various fermentation medium constituents and the influence of each on the dynamic viscosity of the medium were also investigated. Results indicated that particle volume fraction had the dominant effect on the apparent dynamic viscosity resulting in further research of the particle properties. Due to the irregular shapes of Avicel particles, tests were conducted to determine drag and settling behaviour, which led to the development and modification of models to account for these phenomena. This investigation is unique as it allows a more accurate calculation of particle transportation through a three-dimensional environment including the effects of natural packing density. At lower particle volume fraction the concentration of ethanol and glycerol had the greatest effect on the apparent dynamic viscosity and was calculated from models obtained from literature. Validation of the physics and the incorporation thereof in the simulations resulted in the modification of various generic models which either improved numerical stability or accuracy, or both. Contributions included a modified form of the pressure force model, which proved significantly more stable and accurate than previous models proposed in literature. The models developed for capturing the effects of particles on the apparent dynamic viscosity proved effective for this specific substrate. Results from cross-coupling the reaction models with computational fluid dynamic simulations provide a novel approach to capturing the secondary effect of substrate conversion and particle distribution on the performance of the fermentation vessels. This is the first time where that biological reactions were successfully combined with particle dynamics and fluid flow fields to investigate the secondary effects which occur in fermenters. This work served as a foundation for future research and development within the bioethanol field with significant potential for expansion into other biochemical disciplines. / AFRIKAANSE OPSOMMING: Tweede-generasie bioetanol is ’n alternatiewe vervoerbrandstof wat tans ondersoek word waar sellulose, spesifiek lignosellulosiese (houtagtige) gedeeltes, van enige plantmassa na etanol omgesit kan word. Tot op hede was die tegnologie slegs suksesvol geïmplimenteer in demonstrasieskaal fasiliteite. Ten spyte van intensiewe navorsingpogings op laboratoriumskaal, is niemand seker wat die sekondêre effekte van die opskaal tot groot stelsels sal wees nie. Die doelwit van die projek was om drie-dimensionele modelle te ontwikkel van ’n laboratoriumskaal fermentor wat die effekte van partikulêre vermenging en reaksiekinetika kan voorspel vir toekomstige opskaal navorsing. ’n Numeriese model van die reaksiekinetika vir gelyktydige versuikering en fermentasie van Avicel (mikrokristallyne sellulose) partikels tot etanol word aangebied. Die oorspronklikheid van die model is geleë in die skeiding van die twee primêre sellulase ensiemkinetika, wat lei tot die vermoë om die heterogene gedrag van die ensiem-substraat interaksies te voorspel. Hierdie model verbeter die kennis van die stelsels, terwyl voldoende eenvoud behoue bly vir implementering parallel aan kommersiële berekeningsvloeidinamika sagteware. Effekte van die verskillende bestanddele van die fermentasiemedium en die invloed van elk op die dinamiese viskositeit van die medium is ook ondersoek. Resultate dui aan dat partikel volume fraksie die dominante invloed op die skynbare dinamiese viskositeit het, wat gelei het tot verdere ondersoek van die partikel eienskappe. As gevolg van die onreëlmatige vorms van Avicel partikels, is toetse gedoen om die sleur-en uitsakkingsgedrag te bepaal, wat gelei het tot die ontwikkeling en aanpassing van modelle om hierdie verskynsels in ag te neem. Hierdie ondersoek is uniek, want dit laat meer akkurate berekening van partikelvervoer deur ’n drie-dimensionele omgewing toe, insluitend die effekte van natuurlike verpakkingsdigtheid. By laer partikel volume fraksie het die konsentrasie van etanol en gliserol die grootste effek op die skynbare dinamiese viskositeit gehad en was bereken vanaf modelle in die literatuur. Bevestiging van die fisika en die insluiting daarvan in die simulasies het gelei tot die aanpasing van verskillende generiese modelle wat óf numeriese stabiliteit óf akkuraatheid óf beide verbeter. Bydraes gemaak sluit ’n aangepaste vorm van die drukkragmodel in, wat heelwat meer stabiel en akkuraat was as die vorige modelle voorgestel in die literatuur. Die modelle wat ontwikkel is om die effek van partikels op die skynbare viskositeit vas te vang, was effektief bewys vir hierdie spesifieke substraat. Resultate van die kruiskoppeling van inligting vanaf die reaksiemodelle met berekeningsvloeidinamika simulasies lewer ’n nuwe benadering tot die bepaling van die sekondêre effek van substraatomskakeling en partikeldistribusie op die uitvoering van die fermentasie toestel. Hierdie is die eerste poging om biologiese reaksies met partikel dinamika en vloeivelde te kombineer om die sekondêre effekte wat in fermenter plaasvind, te ondersoek. Hierdie werk dien as ’n grondslag vir toekomstige navorsing en ontwikkeling binne die bioetanolveld, met beduidende potensiaal vir uitbreiding na ander biochemiese dissiplines.

Diploma thesis deals with use of Zymomonas mobilis for the production of bioethanol from waste paper. There were used three kinds of substrate (cardboard, drawing and office paper) to optimize of bioethanol production. Individual papers were subjected to the same pre treatment, namely a milling, a combination of microwave irradiation and NaOH, a combination of microwave irradiation and H2SO4 and combination microwave irradiation, H2SO4 and NaOH. The substrates were decomposed by enzymatic hydrolysis after pre treatment to evaluate the best pre-treatment. Simultaneous saccharification and fermentation was carried out for each substrate (with two of the best pre-treatment). The samples were taken during the hydrolysis and the simultaneous saccharification and fermentation, and were determined by HPLC. Growth curves of Zymomonas mobilis were constructed, as the most appropriate for SSF was chosen temperature of 40 ° C in which the exponential phase took place at the time of 6 15 hours. During hydrolysis was monitored glucose concentration in the solution. The maximum concentration of glucose was in the cardboard (microwaves + H2SO4 + NaOH) 16.46 gdm-3, a drawing (microwaves + H2SO4 + NaOH) 31.78 gdm-3, and office paper (microwaves + H2SO4) 25.04 gdm-3. The concentration of ethanol for SSF was highest in the same cases as in the hydrolysis. The cardboard was the maximum concentration of bio ethanol 9.5 gdm-3, for the drawing 16.1 gdm-3 and for the office paper 12.13 gdm-3.

This diploma thesis deals with the optimization of the production of ethanol from waste paper using yeast. There were used 4 kinds of paper as a substrate - office paper, non-recycled workbook, recycled workbook and newspaper. All papers were pretreated with the following procedures: grinding, microwaves + NaOH, microwave + H2SO4 and microwave + H2SO4 + NaOH. The glucose concentration was determined in enzymatic hydrolysis by HPLC. Saccharomyces cerevisiae were chosen for ethanol production. The production of ethanol was carried out with all the pretreated papers in simultaneous saccharification and fermentation. During hydrolysis, the pretreated papers have reached the highest results in the combination with microwave + H2SO4 + NaOH. Non-recycled workbook was the only exception, where the highest concentration of glucose has been obtained by the pretreatment of microwaves + H2SO4. Following results have been acquired: office paper 24,69 gdm-3, non-recycled workbook 22,47 gdm-3, recycled workbook 16,94 gdm-3 and newspapers 15,36 gdm-3. SSF was carried out again with all the papers and their pretreatments. The highest concentration of ethanol has been achieved in microwave pretreatment + H2SO4 + NaOH. The highest overall concentration has been gained from the office paper, amounted to 16,98 gdm-3. The maximum concentration of ethanol for non-recycled workbook has been 15,25 gdm-3, for recycled workbook 12,2 gdm-3 and for newspapers 12,59 gdm-3.

O presente estudo teve como objetivo avaliar a conversão dos açúcares do bagaço de cana em etanol, com foco na integração entre o pré-tratamento com H2SO4 diluído, sacarificação com celulases comerciais e fermentação com Scheffersomyces stipitis DSM- 3651, buscando-se uma configuração simplificada. Previamente aos ensaios de integração, o bagaço foi moído para a redução de sua heterogeneidade granulométrica. Após a moagem, o bagaço foi submetido a fracionamento, por fluxo de ar, separando frações de fibras e finos. Estas frações foram submetidas a analise composicional e observou-se que a fração de finos possui elevados teores de cinzas e extrativos, além de baixo teor de lignina em relação à fração de fibras, entretanto a estratégia de fracionamento do bagaço não foi seletiva. Os experimentos de definição da condição de pré-tratamento mais adequada para a avaliação entre integração entre sacarificação e fermentação foram conduzidos em reator Parr (2 gal) com 2Kg de mistura reacional, incluindo bagaço (10%), água e ácido. Três condições operacionais de pré-tratamento foram avaliadas: 140°C/1%H2SO4/20 min, 150°C/2% H2SO4/30 min e 160°C/3% H2SO4/40 min. Após o pré-tratamento, o slurry foi separado em sólido pré-tratado e hidrolisado hemicelulósico, por filtração. Em seguida o bagaço pré-tratado foi lavado exaustivamente com água destilada e submetido à hidrólise enzimática com celulases (10 FPU/g bagaço) por 72h. Paralelamente, o hidrolisado foi submetido à fermentação com S. stipitis (3 g/L) por 120h. As composições das frações geradas após o pré-tratamento (bagaço pré-tratado, hidrolisado e água de lavagem), quanto aos teores de açúcares e sólidos (solúveis e insolúveis) foram determinadas. As três condições de pré-tratamento levaram a remoção completa de hemicelulose do bagaço, e o aumento da severidade do pré-tratamento acarretou em aumento da eficiência de sacarificação dos sólidos pré-tratados, porém em menor recuperação de glicose após hidrólise enzimática. Por outro lado, o aumento da severidade do pré-tratamento prejudicou a recuperação de açúcares no hidrolisado hemicelulósico, elevando concentrações de ácido acético, furfural e HMF, resultando em inibição completa de fermentação daqueles obtidos nas condições de 150°C e 160°C. A conversão do sólido prétratado (lavado ou não-lavado) + hidrolisado (destoxificado ou não), e do slurry completo (destoxificado ou não) ambos obtidos após pré-tratamento à 140°C, foi avaliada em duas configurações, respectivamente: \"Fermentação do hidrolisado hemicelulósico (FHH), em separado da sacarificação do sólido pré-tratado e da fermentação do hidrolisado celulósico, separadas (SHF)\" e \"Sacarificação do sólido pré-tratado, em separado da co-fermentação dos hidrolisados celulósico e hemicelulósico, integradas (ISHCF)\". Na primeira, observouse que a destoxificação do hidrolisado com lacase (1U/mL) aumentou a produção de etanol, e possibilitou a redução do tempo de conversão em 48h. A sacarificação de sólidos não-lavados foi prejudicada pela presença de compostos solúveis e o condicionamento com lacase não influenciou a eficiência de sacarificação. A eficiência máxima de conversão em configuração de SHF+FHH foi de 28,4%, devido a não lavagem do sólido pré-tratado (13,7%) e destoxificação do HH (14,7%). Em ISHCF, observou-se que a eficiência de conversão de slurry integral destoxificado com lacase é mais alta (38,9%) do que em SHF+FHH, devido a não separação sólido-líquido e maior disponibilidade de açúcares. / This study aimed to evaluate the conversion of sugarcane bagasse into ethanol, focusing on integration between pretreatment with dilute H2SO4, saccharification with commercial cellulases and fermentation with Scheffersomyces stipitis DSM-3651, in a simplified design process. Prior to integration assays, the bagasse was milled to reduce its granulometric heterogeneity. After milling, the fractions of bagasse (fiber and pith) were separated by air flow. These fractions had their chemical composition determined and it was observed that pith fraction has higher content of ash, soluble extractives and low lignin content than fiber fraction; however the separation method was not selective. The experiments to define the most suitable pretreatment condition for evaluating the integration between saccharification and fermentation were conducted in a Parr reactor (2 gal) with 2 kg of reaction mixture, including bagasse (10%), water and acid. Three operational conditions of pretreatment were evaluated: 140°C/1% H2SO4/20min, 150°C/ 2% H2SO4/30 min and 160°C/3% H2SO4/40min. After pretreatment, the slurry was separated in pretreated solid and hemicellulosic hydrolyzate, by filtration. The pretreated solid was thoroughly washed with distilled water and submitted to enzymatic hydrolysis with cellulases (10 FPU / g residue) for 72h. In parallel, the hemicelullosic hydrolyzate was submitted to fermentation with S. stipitis (3 g / L) for 120h. The compositions of the fractions generated after pretreatment (pretreated bagasse, hemicellulosic hydrolyzate and washing water), regarding sugars and solids (soluble and insoluble) contents were determined. All pretreatment conditions led to complete removal of hemicellulose from the bagasse, and the increase of pretreatment severity improved the saccharification yield of pretreated solid, but, leaded to low recovery of glucose after enzymatic hydrolysis. On the other hand, the increase of pretreatement severity impaired the recovery of sugars in hemicellulosic hydrolysate besides increasing the concentrations of acetic acid, furfural and HMF, resulting in complete inhibition of fermentation of those obtained under the pretreatement conditions of 150°C and 160°C. The conversion of pretreated solid (washed and non-washed) + hemicellulosic hydrolyzate (with or without detoxification) and of the whole slurry, both obtained at 140°C was evaluated in two process designs, respectively: \"Fermentation of hemicellulosic hydrolyzate (FHH), separated from hydrolysis of pretreated solid and fermentation of cellulosic hydrolyzate, separately (SHF)\" and \"Integrated hydrolysis of pretreated solid separated from co-fermentation of cellulosic and hemicellulosic hydrolysates (ISHCF)\". In the first, it was observed that the detoxification of the hydrolyzate with laccase (1U / ml) improved the ethanol production, particularly for conversion time. The saccharification of non-washed solid was hampered by the presence of soluble compounds and its conditioning treatment with laccase did not influence the saccharification yield. The maximum conversion efficiency in SHF + FHH configuration was 28.4% due to not washing the pretreated solid (13.7%) and detoxification of hemicellulosic hydrolysate (14.7%). In the second process design, it was observed that the conversion efficiency of whole slurry, with laccase detoxification, was higher (38.9%) than in the separate configuration, with a lower conversion time.

O presente estudo teve como objetivo avaliar a conversão dos açúcares do bagaço de cana em etanol, com foco na integração entre o pré-tratamento com H2SO4 diluído, sacarificação com celulases comerciais e fermentação com Scheffersomyces stipitis DSM- 3651, buscando-se uma configuração simplificada. Previamente aos ensaios de integração, o bagaço foi moído para a redução de sua heterogeneidade granulométrica. Após a moagem, o bagaço foi submetido a fracionamento, por fluxo de ar, separando frações de fibras e finos. Estas frações foram submetidas a analise composicional e observou-se que a fração de finos possui elevados teores de cinzas e extrativos, além de baixo teor de lignina em relação à fração de fibras, entretanto a estratégia de fracionamento do bagaço não foi seletiva. Os experimentos de definição da condição de pré-tratamento mais adequada para a avaliação entre integração entre sacarificação e fermentação foram conduzidos em reator Parr (2 gal) com 2Kg de mistura reacional, incluindo bagaço (10%), água e ácido. Três condições operacionais de pré-tratamento foram avaliadas: 140°C/1%H2SO4/20 min, 150°C/2% H2SO4/30 min e 160°C/3% H2SO4/40 min. Após o pré-tratamento, o slurry foi separado em sólido pré-tratado e hidrolisado hemicelulósico, por filtração. Em seguida o bagaço pré-tratado foi lavado exaustivamente com água destilada e submetido à hidrólise enzimática com celulases (10 FPU/g bagaço) por 72h. Paralelamente, o hidrolisado foi submetido à fermentação com S. stipitis (3 g/L) por 120h. As composições das frações geradas após o pré-tratamento (bagaço pré-tratado, hidrolisado e água de lavagem), quanto aos teores de açúcares e sólidos (solúveis e insolúveis) foram determinadas. As três condições de pré-tratamento levaram a remoção completa de hemicelulose do bagaço, e o aumento da severidade do pré-tratamento acarretou em aumento da eficiência de sacarificação dos sólidos pré-tratados, porém em menor recuperação de glicose após hidrólise enzimática. Por outro lado, o aumento da severidade do pré-tratamento prejudicou a recuperação de açúcares no hidrolisado hemicelulósico, elevando concentrações de ácido acético, furfural e HMF, resultando em inibição completa de fermentação daqueles obtidos nas condições de 150°C e 160°C. A conversão do sólido prétratado (lavado ou não-lavado) + hidrolisado (destoxificado ou não), e do slurry completo (destoxificado ou não) ambos obtidos após pré-tratamento à 140°C, foi avaliada em duas configurações, respectivamente: \"Fermentação do hidrolisado hemicelulósico (FHH), em separado da sacarificação do sólido pré-tratado e da fermentação do hidrolisado celulósico, separadas (SHF)\" e \"Sacarificação do sólido pré-tratado, em separado da co-fermentação dos hidrolisados celulósico e hemicelulósico, integradas (ISHCF)\". Na primeira, observouse que a destoxificação do hidrolisado com lacase (1U/mL) aumentou a produção de etanol, e possibilitou a redução do tempo de conversão em 48h. A sacarificação de sólidos não-lavados foi prejudicada pela presença de compostos solúveis e o condicionamento com lacase não influenciou a eficiência de sacarificação. A eficiência máxima de conversão em configuração de SHF+FHH foi de 28,4%, devido a não lavagem do sólido pré-tratado (13,7%) e destoxificação do HH (14,7%). Em ISHCF, observou-se que a eficiência de conversão de slurry integral destoxificado com lacase é mais alta (38,9%) do que em SHF+FHH, devido a não separação sólido-líquido e maior disponibilidade de açúcares. / This study aimed to evaluate the conversion of sugarcane bagasse into ethanol, focusing on integration between pretreatment with dilute H2SO4, saccharification with commercial cellulases and fermentation with Scheffersomyces stipitis DSM-3651, in a simplified design process. Prior to integration assays, the bagasse was milled to reduce its granulometric heterogeneity. After milling, the fractions of bagasse (fiber and pith) were separated by air flow. These fractions had their chemical composition determined and it was observed that pith fraction has higher content of ash, soluble extractives and low lignin content than fiber fraction; however the separation method was not selective. The experiments to define the most suitable pretreatment condition for evaluating the integration between saccharification and fermentation were conducted in a Parr reactor (2 gal) with 2 kg of reaction mixture, including bagasse (10%), water and acid. Three operational conditions of pretreatment were evaluated: 140°C/1% H2SO4/20min, 150°C/ 2% H2SO4/30 min and 160°C/3% H2SO4/40min. After pretreatment, the slurry was separated in pretreated solid and hemicellulosic hydrolyzate, by filtration. The pretreated solid was thoroughly washed with distilled water and submitted to enzymatic hydrolysis with cellulases (10 FPU / g residue) for 72h. In parallel, the hemicelullosic hydrolyzate was submitted to fermentation with S. stipitis (3 g / L) for 120h. The compositions of the fractions generated after pretreatment (pretreated bagasse, hemicellulosic hydrolyzate and washing water), regarding sugars and solids (soluble and insoluble) contents were determined. All pretreatment conditions led to complete removal of hemicellulose from the bagasse, and the increase of pretreatment severity improved the saccharification yield of pretreated solid, but, leaded to low recovery of glucose after enzymatic hydrolysis. On the other hand, the increase of pretreatement severity impaired the recovery of sugars in hemicellulosic hydrolysate besides increasing the concentrations of acetic acid, furfural and HMF, resulting in complete inhibition of fermentation of those obtained under the pretreatement conditions of 150°C and 160°C. The conversion of pretreated solid (washed and non-washed) + hemicellulosic hydrolyzate (with or without detoxification) and of the whole slurry, both obtained at 140°C was evaluated in two process designs, respectively: \"Fermentation of hemicellulosic hydrolyzate (FHH), separated from hydrolysis of pretreated solid and fermentation of cellulosic hydrolyzate, separately (SHF)\" and \"Integrated hydrolysis of pretreated solid separated from co-fermentation of cellulosic and hemicellulosic hydrolysates (ISHCF)\". In the first, it was observed that the detoxification of the hydrolyzate with laccase (1U / ml) improved the ethanol production, particularly for conversion time. The saccharification of non-washed solid was hampered by the presence of soluble compounds and its conditioning treatment with laccase did not influence the saccharification yield. The maximum conversion efficiency in SHF + FHH configuration was 28.4% due to not washing the pretreated solid (13.7%) and detoxification of hemicellulosic hydrolysate (14.7%). In the second process design, it was observed that the conversion efficiency of whole slurry, with laccase detoxification, was higher (38.9%) than in the separate configuration, with a lower conversion time.

The diploma thesis is focused on production possibilities of bioethanol from waste paper by yeast Kluyveromyces marxianus. Waste cardboard was used as a potential substrate for bioethanol production. Several methods for cardboard preparation were introduced and compared as well as methods of fermentation. Simultaneous sacharification and fermentation and separate hydrolysis and fermentation of preprepared cardboard paper were performed in different pH buffer (4,8-7). Simultaneous sacharification and fermentation was held at a temperature of 45°C. Hydrolysis in separate hydrolysis and fermentation was performed at 50°C and fermentation at 25°C. Procedures outputs were obtained by sampling in specific time intervals and samples were analyzed by HPLC for presence and concentration glucose and ethanol. The results of the analysis have shown that the highest concentration of glucose produced by enzymatic hydrolysis was achieved by using microwaves, 2% H2SO4 and 2% NaOH pretreated paperboard at pH 4,8. The highest yield of ethanol was obtained by separate hydrolysis and fermentation of pulp pretreated by microwaves, 2% H2SO4 and 2% NaOH in pH 5,4 buffer. The method SHF proved to be more effective for the production of ethanol than SSF.

>Magister Scientiae - MSc / Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method. We first utilized the CRISPR systems for targeting the T.r.eg2 gene to single locus chromosomal sites for single copy integration. Subsequently, we then targeted the same gene to repeated sequences in the genome, namely the delta sites, for multi-copy integration. The procedure was repeated with a different gene, T.e.cbh1, integrated into the same sites to ascertain reporter gene specific effects. High integration efficiency was achieved, since all the strains successfully integrated the genes. However, we discovered significant differences in enzyme activities between the two genes when targeted to different loci, as well as varying copy numbers as determined by qPCR. The T.e.cbh1 gene was highly expressed by yeast transformants targeted at the repeated delta sequences used for multi-copy integration, reaching maximum levels of 248 mU/gDCW. The T.r.eg2 gene was highly expressed in yeast transformants targeted to the single locus site on chromosome 12, reaching a maximum of 160U/gDCW, though it was shown that off-target integration likely occurred. We then used the information from these observations to construct a CBP yeast strain containing three cellulase genes: T.r.eg2, T.e.cbh1, and S.f.BGL1. Significant differences in enzyme activities were observed between the three genes, and it was shown that the S.f.BGL1 gene was poorly expressed by the CBP yeast strain, whereas the T.r.eg2 gene was highly expressed. Notably, due to the fact that marker containing plasmids could be cured from these strains, many additional genetic changes can still be made. Overall, our two CRISPR-Cas9 systems were efficient at engineering strains that produce recombinant proteins and can be used in future studies for a variety of applications, including metabolic engineering in S. cerevisiae