>Magister Scientiae - MSc / Biofuels are currently recognised as the most viable source of energy to replace depleting fossil fuel reserves, with bioethanol the most popular alternative alcohol fuel. Producing bioethanol from agricultural waste residues is a feasible socio-economic industrial process. Lignocellulose, from which plant material is composed, is highly recalcitrant to enzymatic degradation and therefore requires a suite of enzymes for complete hydrolysis of the biomass. Metagenomes, particularly from extreme environments, represent an unlimited resource for the discovery of novel biocatalysts for inclusion in industrial processes. Here we report on the cloning and functional characterisation of a novel thermophilic cellulase identified by the functional screening of a Malawian, hotspring sediment metagenomic library. The gene encoding the cellulase, celMHS, composed of 2,705 nucleotides and encoded a polypeptide of 905 amino acids with a predicted molecular mass of about 98 kDa. The in silico translated protein, CelMHS, contained a putative transmembrane domain, a family 4 carbohydrate binding motive (CBM 4), a truncated glycoside hydrolase family 42 (GH42) domain and a N-terminal region that does not have sequence similarity to any previously described domains. Functional characterisation of the recombinant CelMHS demonstrated that the protein displayed an optimal pH of 6.0 and temperature of 100°C. CelMHS had high specific activity toward substrates comprising of β-1,4 linked glucose subunits such as carboxymethyl cellulose, β-D-glucan from barley and lichenan, however, some activity was also observed against avicel, a crystalline cellulose substrate. HPLC analysis of the hydrolysis products produced by CelMHS indicates that this particular enzyme prefers longer chain oligosaccharides. This is, to the best of our knowledge, the first investigation describing the cloning and characterization of a carbohydrate hydrolysing enzyme comprised of the unique sequence architecture: a partial GH42 catalytic domain, a CBM 4 and a unique N-domain sequence. Key words: cellulose, cellulases, lignocellulosic biomass, bioethanol, saccharification, hydrolysis, metagenomic library, thermophilic

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13347号 / 論農博第2890号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5254(農学部図書室) / (主査)教授 杉山 淳司, 教授 髙部 圭司, 教授 渡邊 隆司 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Parmi les ressources d'origines agricole et forestière utilisables aujourd'hui en tant que biomasse à destination énergétique, le miscanthus apparait comme l'une des espèces de graminées les plus prometteuses pour la production de bioéthanol de seconde génération grâce à son haut potentiel en biomasse. Ce procédé dit "2G" convertit la cellulose contenue dans ces biomasses lignocellulosiques en bioéthanol et ce via un procédé intégrant prétraitement physico-chimique, hydrolyse enzymatique et fermentation. Le principal objectif de ce projet de thèse visait à étudier l'impact de l'hétérogénéité tissulaire et structurale du miscanthus sur sa saccharification et s'est décliné en différents volets liés à l'étude de l'efficacité des prétraitements et à l'analyse des performances de différents cocktails enzymatiques de Trichoderma reesei. L'hydrolyse enzymatique est essentiellement limitée par la structure et la porosité des complexes pariétaux qui réduisent l'accessibilité de la cellulose aux cellulases. En plus des constituants hémicelluloses et lignines qui recouvrent la cellulose, les parois cellulaires du miscanthus sont riches en acides hydroxycinnamiques (pCA et FA) qui jouent un rôle important dans la cohésion du réseau pariétal complexe. L'application de prétraitements acide et alcalin sur le miscanthus a ainsi révélé une différence de réactivité en fonction des types cellulaires. Les parois secondaires du sclérenchyme sont plus facilement dégradées par les cellulases fongiques après prétraitement acide. L'étude de la distribution des composés phénoliques au niveau cellulaire par micro spectrophotométrie UV a rapporté une nette diminution de l'absorbance UV dans tous les tissus après chaque prétraitement. Ceci n'expliquant pas totalement les différences de réactivité observées, d'autres facteurs physicochimiques seraient donc impliqués. Une approche visant à évaluer la progression des cellulases au sein des parois par immunocytochimie a également été initiée mais elle s'est heurtée à des problématiques techniques liées à la nature des tissus et aux anticorps employés. Les performances en terme de conversion de la cellulose ont été évaluées avec des cocktails enzymatiques de T. reesei comprenant des activités (hemi-)cellulolytiques variables. Une meilleure efficacité du prétraitement par explosion à la vapeur a ainsi pu être montrée par réduction de la quantité d'enzymes mises en œuvre. Comme c'est le cas pour d'autres graminées, ces travaux ont permis de confirmer le rôle crucial de l'enzyme β-glucosidase, permettant de limiter l'inhibition par le cellobiose et améliorant la cinétique initiale de saccharification. L'amélioration du rendement d'hydrolyse par l'utilisation d'un sécrétome comprenant une bonne activité hémicellulolytique a pu être ensuite démontrée. L'utilisation de cocktails enzymatiques reconstitués à partir d'enzymes pures a enfin permis de définir un mélange "optimal" composé des quatre principales cellulases de T. reesei (CBH1, CBH2, EG1 et EG2) associées à une hémicellulase (XYN1). / Among agricultural and forest resources, the grass specie miscanthus has emerged as one of the most promising feedstock candidates for 2G-biofuel production due to its high biomass yield. The biofuels 2G-production process is based on cellulose conversion into bioethanol via physicochemical pretreatment, enzymatic hydrolysis and fermentation. The main objective of this Ph.D. project was to evaluate the effect of tissue and structure heterogeneity of miscanthus on its saccharification by evaluating pretreatment efficiency and analyzing the performance of different Trichoderma reesei cellulolytic cocktails.Enzymatic hydrolysis is mainly hindered by cell wall structure and porosity which limit cellulose accessibility to cellulase. In addition to hemicelluloses and lignin polymers, miscanthus cell walls, contain high amounts of hydroxycinnamic acids (pCA and FA) that play a significant role in cross-linking polymers into cohesive network. Applying acid and alkali pretreatments on miscanthus revealed a distinctive reactivity depending on cell types. Secondary cell walls of sclerenchyma appeared more digested by fungal cellulases after acid pretreatment. Addressing phenolics distribution (lignin and hydroxycinnamic acids) at cell level by UV micro spectrophotometry highlighted a significant decrease in UV absorbance after both pretreatments irrespective to cell type indicating that other physicochemical and structural features are involved in distinct cell wall reactivity. We have also attempted to evaluate cellulase progression into miscanthus cell walls by immunocytochemistry but we have had many technical problems due to the nature of miscanthus tissues and used antibodies. Cellulose conversion ability was then evaluated using enzymatic cocktails of T. reesei which vary in their (hemi-)cellulolytic activities. Higher efficiency of the steam explosion pretreatment was demonstrated by reducing enzymes loading. As reported previously on other grasses, β-glucosidase plays a crucial role by limiting the inhibiting effect of cellobiose and improving the initial saccharification step. We furthermore showed that the use of hemicellulases-improved cocktails allowed significant increase in saccharification yields. We finally identified an optimal reconstituted enzyme mixture composed of four major cellulases of T. reesei (CBH1, CBH2, EG1 and EG2) and the hemicellulase XYN-1.

Com o intuito de elucidar os efeitos das propriedades físicas e morfológicas na efetividade da sacarificação enzimática das fibras de bagaço de cana-de-açúcar, este trabalho propõe o uso de diferentes métodos de processamento físico-químicos e -mecânicos para a modificação da estrutura da parede celular. Os tratamentos físico-mecânicos, através de fenômenos de fibrilação e delaminação, promovem a abertura estrutural das fibras e aumentam a acessibilidade às enzimas hidrolíticas, porém sem modificar a composição química do material. Para uma compreensão abrangente da ação dos métodos físico-mecânicos propostos nas características estruturais, as fibras de bagaço foram previamente tratadas por métodos físico-químicos - hidrotérmico e organossolve - de modo a obter cinco materiais de diferentes composições químicas. O estudo dos tratamentos físico-mecânicos foi realizado empregando-se equipamentos de diferentes configurações, cujos modos de ação e consequente impacto nas fibras diferem entre si, sendo estes dois tipos de refino - refinador de discos Bauer e moinho Jokro, e um tipo de moagem - moinho criogênico. A variável do refino em moinho Jokro foi o tempo de tratamento, enquanto as variáveis do processamento em refinador de discos foi o tempo de refino e a distância entre os discos. Já as condições da moagem criogênica foram definidas de modo a obter amostras homogêneas do ponto de vista macroscópico. As modificações provocadas na estrutura das fibras foram determinadas a partir das análises das áreas superficiais externa (dimensões das fibras) e interna (porosidade da parede celular), além da organização cristalina das fibrilas celulósicas. Primeiramente, estudou-se de modo detalhado os efeitos dos métodos mecânicos nas propriedades estruturais das amostras de bagaço, interpretando-se como os efeitos primários do refino evoluem de acordo com a severidade e o tipo tratamento empregado a partir das caracterizações dos efeitos secundários. Posteriormente, confrontaram-se os resultados obtidos nas análises físicas e morfológicas com o rendimento de açúcares obtido na hidrólise enzimática. Os resultados permitiram constatar que, enquanto o moinho Jokro promoveu um grande aumento no rendimento de glicose obtido por culminar, simultaneamente, no aumento da estrutura capilar pela intensa fibrilação interna, e da área superficial externa tanto pela formação de elementos finos quanto pela redução das dimensões das fibras por corte, o refinador de discos Bauer levou a uma melhoria menos pronunciada na hidrolisabilidade por resultar no aumento da porosidade, porém sem expressivos corte e fibrilação externa das fibras. Diferentemente, a moagem criogênica promoveu apenas a drástica e heterogênea redução das dimensões das fibras, enquanto não permitiu mudanças significativas na hidrolisabilidade das amostras. Por fim, os valores dos parâmetros estruturais determinados foram analisados pelo método estatístico de componentes principais (PCA) visando quantificar por qual fator cada uma destas características influencia na extensão da hidrólise da celulose do bagaço. A PCA permitiu visualizar que os fatores relacionados à superfície interna da parede celular, como a área de poros acessíveis e a dimensão lateral do cristalito de celulose, são os principais aspectos que regem o rendimento de sacarificação da biomassa lignocelulósica. Os resultados deste estudo permitiram assim a proposta de um modelo de predição do comportamento de hidrólise das amostras de bagaço. / In order to elucidate the effects of the physical and morphological properties on the effectiveness of enzymatic saccharification of sugarcane bagasse fibers, this work proposes different methods of physico-chemical and -mechanical processing to modify the cell wall structure. Through fibrillation and delamination phenomena, physico-mechanical treatments promote the structural opening of the fibers and increase the accessibility to hydrolytic enzymes, but without modifications on the chemical composition of the processed material. For a thorough comprehension about the action of the proposed physico-mechanical methods on the structural characteristics, the bagasse fibers were previously treated by physico-chemical methods - hydrothermal and organosolv - to obtain five materials with different chemical composition. The study of the physico-mechanical treatments was performed by equipment with different configuration, which operating modes and consequent impact on the fibers differ from each other. The equipment were two types of refiner - Bauer discs refiner and Jokro mill - and one type of mill - cryogenic mill. The refining variable considered for the Jokro mill was the refining time, while the processing variables for the disc refiner were the refining time and the discs gap. Concerning the cryogenic mill, the operation conditions were defined to achieve macroscopic homogeneous samples. Modifications on the fibers structure were assessed by analysis of the external and internal surfaces (fibers dimension and the cell wall porosity, respectively), as well as the crystalline organization of the cellulosic fibrils. Firstly, it was performed a thorough study concerning the effects of the mechanical methods in the structural properties of the bagasse samples. In this study, it was interpreted how the primary effects of refining evolve according to severity and type of treatment from the characterization of the secondary effects. Then, the results acquired in the physical and morphological analysis were confronted with the glucose yield obtained in the enzymatic hydrolysis. The results showed that the Jokro mill promoted a great increase in the glucose yield by culminating, simultaneously, in the increase of the capillary structure by the intense internal fibrillation, and of the external surface area both by the formation of fines as by the reduction of the dimensions of fibers by cutting. In turns, the Bauer discs refiner leaded to a lower improvement of the bagasse pulps hydrolysability, which was a consequence of the increased porosity, but without expressive cut and external fibrillation of the fibers. In a different way, the cryogenic milling promoted just a drastic and heterogeneous reduction of the fibers dimensions, without any significant change in the hydrolysability of the samples. Finally, the determined values of the structural parameters were analyzed by the statistical method of the principal component analysis (PCA), aiming to quantify by which factor each one of these characteristics influences in the extent of hydrolysis of bagasse cellulose. The PCA showed that the factors related to the internal surface of the cell wall, such as the accessible pore area and the lateral dimension of the cellulose crystallite, are the main aspects that govern the saccharification yield of the lignocellulosic biomass. The results of this study allowed the proposal of an empiric prediction model of the hydrolysis behavior of the bagasse samples.

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.

Com o intuito de elucidar os efeitos das propriedades físicas e morfológicas na efetividade da sacarificação enzimática das fibras de bagaço de cana-de-açúcar, este trabalho propõe o uso de diferentes métodos de processamento físico-químicos e -mecânicos para a modificação da estrutura da parede celular. Os tratamentos físico-mecânicos, através de fenômenos de fibrilação e delaminação, promovem a abertura estrutural das fibras e aumentam a acessibilidade às enzimas hidrolíticas, porém sem modificar a composição química do material. Para uma compreensão abrangente da ação dos métodos físico-mecânicos propostos nas características estruturais, as fibras de bagaço foram previamente tratadas por métodos físico-químicos - hidrotérmico e organossolve - de modo a obter cinco materiais de diferentes composições químicas. O estudo dos tratamentos físico-mecânicos foi realizado empregando-se equipamentos de diferentes configurações, cujos modos de ação e consequente impacto nas fibras diferem entre si, sendo estes dois tipos de refino - refinador de discos Bauer e moinho Jokro, e um tipo de moagem - moinho criogênico. A variável do refino em moinho Jokro foi o tempo de tratamento, enquanto as variáveis do processamento em refinador de discos foi o tempo de refino e a distância entre os discos. Já as condições da moagem criogênica foram definidas de modo a obter amostras homogêneas do ponto de vista macroscópico. As modificações provocadas na estrutura das fibras foram determinadas a partir das análises das áreas superficiais externa (dimensões das fibras) e interna (porosidade da parede celular), além da organização cristalina das fibrilas celulósicas. Primeiramente, estudou-se de modo detalhado os efeitos dos métodos mecânicos nas propriedades estruturais das amostras de bagaço, interpretando-se como os efeitos primários do refino evoluem de acordo com a severidade e o tipo tratamento empregado a partir das caracterizações dos efeitos secundários. Posteriormente, confrontaram-se os resultados obtidos nas análises físicas e morfológicas com o rendimento de açúcares obtido na hidrólise enzimática. Os resultados permitiram constatar que, enquanto o moinho Jokro promoveu um grande aumento no rendimento de glicose obtido por culminar, simultaneamente, no aumento da estrutura capilar pela intensa fibrilação interna, e da área superficial externa tanto pela formação de elementos finos quanto pela redução das dimensões das fibras por corte, o refinador de discos Bauer levou a uma melhoria menos pronunciada na hidrolisabilidade por resultar no aumento da porosidade, porém sem expressivos corte e fibrilação externa das fibras. Diferentemente, a moagem criogênica promoveu apenas a drástica e heterogênea redução das dimensões das fibras, enquanto não permitiu mudanças significativas na hidrolisabilidade das amostras. Por fim, os valores dos parâmetros estruturais determinados foram analisados pelo método estatístico de componentes principais (PCA) visando quantificar por qual fator cada uma destas características influencia na extensão da hidrólise da celulose do bagaço. A PCA permitiu visualizar que os fatores relacionados à superfície interna da parede celular, como a área de poros acessíveis e a dimensão lateral do cristalito de celulose, são os principais aspectos que regem o rendimento de sacarificação da biomassa lignocelulósica. Os resultados deste estudo permitiram assim a proposta de um modelo de predição do comportamento de hidrólise das amostras de bagaço. / In order to elucidate the effects of the physical and morphological properties on the effectiveness of enzymatic saccharification of sugarcane bagasse fibers, this work proposes different methods of physico-chemical and -mechanical processing to modify the cell wall structure. Through fibrillation and delamination phenomena, physico-mechanical treatments promote the structural opening of the fibers and increase the accessibility to hydrolytic enzymes, but without modifications on the chemical composition of the processed material. For a thorough comprehension about the action of the proposed physico-mechanical methods on the structural characteristics, the bagasse fibers were previously treated by physico-chemical methods - hydrothermal and organosolv - to obtain five materials with different chemical composition. The study of the physico-mechanical treatments was performed by equipment with different configuration, which operating modes and consequent impact on the fibers differ from each other. The equipment were two types of refiner - Bauer discs refiner and Jokro mill - and one type of mill - cryogenic mill. The refining variable considered for the Jokro mill was the refining time, while the processing variables for the disc refiner were the refining time and the discs gap. Concerning the cryogenic mill, the operation conditions were defined to achieve macroscopic homogeneous samples. Modifications on the fibers structure were assessed by analysis of the external and internal surfaces (fibers dimension and the cell wall porosity, respectively), as well as the crystalline organization of the cellulosic fibrils. Firstly, it was performed a thorough study concerning the effects of the mechanical methods in the structural properties of the bagasse samples. In this study, it was interpreted how the primary effects of refining evolve according to severity and type of treatment from the characterization of the secondary effects. Then, the results acquired in the physical and morphological analysis were confronted with the glucose yield obtained in the enzymatic hydrolysis. The results showed that the Jokro mill promoted a great increase in the glucose yield by culminating, simultaneously, in the increase of the capillary structure by the intense internal fibrillation, and of the external surface area both by the formation of fines as by the reduction of the dimensions of fibers by cutting. In turns, the Bauer discs refiner leaded to a lower improvement of the bagasse pulps hydrolysability, which was a consequence of the increased porosity, but without expressive cut and external fibrillation of the fibers. In a different way, the cryogenic milling promoted just a drastic and heterogeneous reduction of the fibers dimensions, without any significant change in the hydrolysability of the samples. Finally, the determined values of the structural parameters were analyzed by the statistical method of the principal component analysis (PCA), aiming to quantify by which factor each one of these characteristics influences in the extent of hydrolysis of bagasse cellulose. The PCA showed that the factors related to the internal surface of the cell wall, such as the accessible pore area and the lateral dimension of the cellulose crystallite, are the main aspects that govern the saccharification yield of the lignocellulosic biomass. The results of this study allowed the proposal of an empiric prediction model of the hydrolysis behavior of the bagasse samples.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / In nature, saccharification is done by a variety of microorganisms, secreting a variety of cellulase in addition to other proteins. Co-culturing enables the production of more efficient enzyme preparations that would mimic the natural decomposition of lignocelluloses. During the decay of banana (Musa paradisiaca) pseudostem, a potential feedstock for second-generation biofuels, there may be a number of microorganisms producing cellulolytic enzymes, and other factors, which in combination might decompose the lignocelluloses more efficiently. The aim of the study was to establish a microbial co-culture for the production of highly active cellulase preparations. Banana pseudostems (BPS) and microbial samples from decaying banana pseudostems were collected in the Mopani District Allesbeste Nursery, Limpopo Province, South Africa. Fungi and bacteria were isolated using CMC agar plates. The best cellulase producing fungi and bacteria were tested for cellulase activity in monocultures and in various combinations (fungi-fungi, fungi-bacteria, bacteria-bacteria, fungi-live bacterial cells and fungi-dead bacterial cells) in submerged fermentation, using Avicel™ as a carbon source. Solid-state fermentation was also performed using banana pseudostem as a carbon source. Zymography was done in studying the variety of cellulase in the secretions from co-cultures/ mixed cultures. Identification of the bacterial and fungal isolates from decomposing banana pseudostems was also done using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) or DNA sequencing. A mixed culture of fungi in combination with dead bacterial cells was the best combination to produce higher levels of endoglucosidase and β-glucosidase activities in both submerged fermentation and solid-state fermentation. During SmF, endoglucosidase was (0.229 after 144 h) and β-glucosidase (4.519 after 96 h) activities and SSF, endoglucosidase (12.793 after 48 h) and β-glucosidase (37.45 after 144 h). Endoglucosidase zymography showed that monocultures and co-cultures produced four active bands for endoglucanase, except for the monoculture Trichoderma longibrachiatum 1B that produced a faint or unclear band. The current study demonstrated that three fungal strains namely, T longibrachiatum 1B, Aspergillus fumigatus 5A, and Aspergillus flavus 2A and one bacterial strain Enterobacter asburiae 1 are capable of producing a variety of endoglucanases. It seems that a combination of fungi with dead cells could significantly improve endoglucosidase and v β-glucosidase activities. The use of A. fumigatus in mixed cultures is highly recommended in order to produce high levels of β-glucosidases, no matter the combination used. / Foodbev Seta

Glycerol thermal processing (GTP) is studied as a novel biomass pretreatment method in this research with the purposes to facilitate biopolymer fractionation and biomass saccharification. This approach is performed by treating sweet gum particles on polymer processing equipment at high temperatures and short times in the presence of anhydrous glycerol. Nine severity conditions are studied to assess the impact of time and temperature during the processing on biopolymer structure and conversion. The GTP pretreatment results in the disruption of cell wall networks by increasing the removal of side-chain sugars and lignin-carbohydrate linkages based on severity conditions. After pretreatment, 41% of the lignin and 68% of the xylan is recovered in a dry powdered form by subsequent extractions without additional catalysts, leaving a relatively pure cellulose fraction, 84% glucan, as found in chemical pulps. Lignin structural analysis indicated GTP processing resulted in extensive degradation of B-aryl ether bonds through the C-y elimination, followed by abundant phenolic hydroxyl liberation. At the same time, condensation occurred in the GTP lignin, providing relatively high molecular weight, near to that of the enzymatic mild acidolysis lignin. Better thermal stability was observed for this GTP lignin. In addition to lignin, xylan was successfully isolated as another polymer stream after GTP pretreatment. The recovered water insoluble xylan (WIX) was predominant alkali soluble fraction with a maximum purity of 84% and comparable molecular weight to xylan isolated from non-pretreated fibers. Additionally, the narrow molecular weight distribution of recovered WIX, was arisen from the pre-extraction of low molecular weight water-soluble xylan. Additionally, a 20-fold increase of the ultimate enzymatic saccharification for GTP pretreated biomass was observed even with significant amounts of lignin and xylan remaining on the non-extracted fiber. The shear and heat processing caused a disintegrated cell wall structure with formation of biomass debris and release of cellulose fibrils, enhancing surface area and most likely porosity. These structural changes were responsible for the improved biomass digestibility. Additionally, no significant inhibitory compounds for saccharification are produced during GTP processing, even at high temperatures. While lignin extraction did not promote improvement in hydrolysis rates, further xylan extraction greatly increases the initial enzymatic hydrolysis rate and final level of saccharification. The serial of studies fully demonstrate glycerol thermal processing as a novel pretreatment method to enhance biomass saccharification for biofuel production, as well as facilitate biopolymer fractionation. Moreover, the study shows the impact of thermally introduced structural changes to wood biopolymers when heated in anhydrous environments in the presence of hydrogen bonding solvent. / Ph. D.

At present, the world's transportation sector is being principally supplied by fossil fuels. However, energy consumption in this sector is drastically increasing and there are concerns with supply, cost, and environmental issues with the continuing use of fossil fuels. Utilizing non-petroleum ethanol in the transportation sector reduces the dependence on oil, and allows for cleaner burning of gasoline. Lignocellulose materials are structurally composed of five types of polymeric sugars, glucan, galactan, mannan, arabinan, and xylan. NREL has developed a quantitative saccharification (QS) method for determining carbohydrate composition. We proposed a new protocol based on the NREL 2006 Laboratory Analytical Procedure "Determination of Structural Carbohydrates and Lignin in Biomass" (Sluiter et al. 2006a) with a slight modification, in which xylose concentration was determined after the secondary hydrolysis by using 1% sulfuric acid rather than 4% sulfuric acid. We found that the current NREL protocol led to a statistically significant overestimation of acid-labile xylan content ranging from 4 to 8 percent. Lignocellulosic biomass is naturally recalcitrant to enzymatic hydrolysis, and must be pretreated before it can be effectively used for bioethanol production. One such pretreatment is a fractionation process that separates lignin and hemicellulose from the cellulose and converts crystalline cellulose microfibrils to amorphous cellulose. Here we evaluated the feasibility of lignocellulose fractionation applicable to the hurds of industrial hemp. Hurds are the remaining material of the stalk after all leaves, seeds, and fiber have been stripped from the plant. After optimizing acid concentration, reaction time and temperature, the pretreated cellulosic samples were hydrolyzed to more than 96% after 24 hours of hydrolysis (enzyme loading conditions of 15 FPU/g glucan Spezyme CP and 60 IU/g glucan Novozyme 188) at the optimal pretreatment condition (> 84% H₃PO₄, > 50 °C and > 1 hour). The overall glucose and xylose yields were 89% (94% pretreatment; 96% digestibility) and 61%, respectively. All data suggest the technical feasibility of building a biorefinery based on the hurds of industrial hemp as a feedstock and a new lignocellulose fractionation technology for producing cellulosic ethanol. The choice of feedstock and processing technology gives high sugar yields, low processing costs, low cost feedstock, and low capital investment. / Master of Science

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