The production of second generation biofuels -- cellulosic ethanol from renewable lignocellulosic biomass has the potential to lead the bioindustrial revolution necessary to the transition from a fossil fuel-based economy to a sustainable carbohydrate economy. Effective release of fermentable sugars through biomass pretreatment followed by enzymatic hydrolysis is among the most costly steps for emerging cellulosic ethanol biorefineries. In this project, two pretreatment methods (dilute acid, DA, and cellulose solvent- and organic solvent-lignocellulose fractionation, COSLIF) for corn stover were compared. It was found that glucan digestibility of the corn stover pretreated by COSLIF was much higher, along with faster hydrolysis rate, than that by DA- pretreated. This difference was more significant at a low enzyme loading. Quantitative measurements of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were established to find out the cause. The COSLIF-pretreated corn stover had a CAC nearly twice that of the DA-pretreated biomass. Further supported by qualitative scanning electron microscopy images, these results suggested that COSLIF treatment disrupted microfibrillar structures within biomass while DA treatment mainly removed hemicelluloses, resulting in a much less substrate accessibility of the latter than of the former. It also concluded that enhancing substrate accessibility was the key to an efficient bioconversion of lignocellulose. A simple method for determining the adsorbed cellulase on cellulosic materials or pretreated lignocellulose was established for better understanding of cellulase adsorption and desorption. This method involved hydrolysis of adsorbed cellulase in the presence of 10 M of NaOH at 121oC for 20 min, followed by the ninhydrin assay for the amino acids released from the hydrolyzed cellulase. The major lignocellulosic components (i.e. cellulose, hemicellulose, and lignin) did not interfere with the ninhydrin assay. A number of cellulase desorption methods were investigated, including pH adjustment, detergents, high salt solution, and polyhydric alcohols. The pH adjustment to 13.0 and the elution by 72% ethylene glycol at a neutral pH were among the most efficient approaches for desorbing the adsorbed cellulase. For the recycling of active cellulase, a modest pH adjustment to 10.0 may be a low-cost method to desorb active cellulase. More than 90% of cellulase for hydrolysis of the pretreated corn stover could be recycled by washing at pH 10.0. This study provided an in-depth understanding of biomass saccharification for the production of cellulosic ethanol for cellulose hydrolysis and cellulase adsorption and desorption. It will be of great importance for developing better lignocellulose pretreatment technologies and improving cellulose hydrolysis by engineered cellulases. / Master of Science

Biomass recalcitrance has been recognized as one of the major barriers that hided the cost-effective conversion of lignocellulosic biomass to bioethanol, therefore the current bioconversion process require an essential step known as pretreatment to increase the cellulose accessibility. This thesis provides information about changes in cellulose accessibility upon different pretreatments, along with how these pretreatments alter the chemical and physical structures of biomass, will be extremely helpful to further optimize the current pretreatment process. Multiple promising analytical techniques including Simons’ stain, NMR cryoporometry, relaxometry, mercury porosimetry was introduced and successfully applied on pretreated biomass samples to characterize the cellulose accessible surface area and biomass porosity. Different pretreatments increase cellulose accessibility through different mechanisms to different extent. Dilute acid pretreatment is more effective than steam explosion in terms of increasing accessible surface area of cellulose as reflected by Simons’ stain and NMR cryoporometry, while NMR relaxometry suggested steam explosion is more effective at pore expansion for the cell wall water pools detected by changes in NMR relaxation time. Alkaline pretreatment decreased cellulose degree of polymerization, cellulose crystallinity, lignin content and subsequently increased cellulose accessibility, with sodium hydroxide pretreatment proved to be much more effective compared lime or soaking in ammonia pretreatment. Delignification through alkaline-based pretreatment is found less effective than removal of hemicellulose using acid in terms of cellulose accessibility increase. Lignin didn’t directly dictate cellulose accessibility but rather restricted xylan accessibility which in turn controls the access of cellulase to cellulose. Pore size distribution analysis based on mercury porosimetry also indicated that the most fundamental barrier in terms of biomass porosity scale for efficient enzymatic hydrolysis is the nano-pore space formed between coated microfibrils, despite some of the porous architecture such as cell lumen and pit could be severely destroyed after pretreatment. The action of cellulase on the characteristics of cellulosic fractions obtained from pretreated biomass was also investigated. Cellulose accessibility was found to increase at the beginning of hydrolysis, and after reaching a maximum value then starting to decrease. Enzymatic hydrolysis resulted in a rapid decrease in the cellulose degree of polymerization then gradually leveled off, suggesting the existence of a synergistic action of endo- and exo-glucanases that contribute to the occurrence of a peeling off type mechanism.

No presente trabalho, avaliou-se o efeito limitante da lignina residual de bagaço de cana submetido a diferentes pré-tratamentos na hidrólise da celulose. O bagaço foi submetido a cinco pré-tratamentos: NaOH (5%), Na2SO3/NaOH (10%/5%), H2SO4 (0,75%), NaHSO3/H2SO4 (5%/0,75%) e explosão a vapor catalisada por SO2 (3%). Os pré-tratamentos resultaram em bagaços com diferentes composições químicas, sendo que os tratamentos alcalinos favoreceram a solubilização de lignina e os tratamentos ácidos favoreceram a solubilização de hemicelulose. Os bagaços tratados com Na2SO3/NaOH e por explosão a vapor resultaram em rendimentos de hidrólise de celulose superiores a 80% ao utilizar altas cargas de enzima (Celluclast), indicando a maior acessibilidade da celulose desses materiais. Isso foi confirmado pela técnica de coloração de Simons, que mostrou que a área superficial acessível da celulose desses dois bagaços foi maior que a dos demais. Boas correlações (R2>0,8) entre o rendimento de hidrólise da celulose em 72 h e a celulose superficial acessível só foram obtidos com altas cargas de enzima, evidenciando que fatores além da acessibilidade limitaram a hidrólise da celulose com quantidade menor de enzimas. As ligninas dos bagaços pré-tratados foram isoladas e a capacidade adsortiva de proteínas foi determinada. A lignina de bagaço tratado por explosão a vapor apresentou maior capacidade adsortiva, seguida da lignina de bagaço tratado com NaOH. Os pré-tratamentos com íons sulfito (Na2SO3/NaOH e NaHSO3/H2SO4) resultaram em ligninas com capacidades adsortivas inferiores quando comparado aos seus análogos sem sulfito (NaOH e H2SO4), possivelmente pela sulfonação da lignina residual, confirmada pela presença de grupos ácidos fortes nesses bagaços. A adição de BSA prévia à adição de celulases confirmou o forte efeito da adsorção improdutiva causada pela lignina nos bagaços tratados com NaOH e por explosão a vapor, materiais cujas ligninas apresentaram maiores capacidades adsortivas. A adsorção improdutiva foi menor ao utilizar o extrato enzimático Cellic CTec3, evidenciando que as enzimas desse coquetel são menos sensíveis à presença da lignina. Do extrato Celluclast, a enzima que mais adsorveu nas ligninas de bagaço tratado com NaOH e por explosão a vapor foi a ?-glicosidase, seguida da endoglucanase. As enzimas purificadas CBHI e EGII de T. longibrachiatum adsorveram menos à lignina de bagaço tratado por explosão a vapor que as mesmas enzimas de T. reesei. A ?-glicosidade de A. niger não adsorveu a essa lignina. Independentemente do pré-tratamento, a presença de fenóis solubilizados a partir da lignina, em baixas concentrações, resultou em aumento no rendimento de conversão de celulose ao utilizar baixas cargas de enzima, possivelmente pelo efeito positivo causado por esses na atividade de enzimas oxidativas, ou pela presença de lignossulfonatos. Em concentrações mais elevadas, independente da carga enzimática, os fenóis liberados foram inibitórios em todos os pré-tratamentos. A atividade de CBHI foi mais sensível à presença dos compostos fenólicos que a atividade de ?-glicosidase. A partir dos resultados, conclui-se o aumento da acessibilidade, seja pela remoção ou pela relocalização da lignina, é o fator que mais influencia a hidrólise eficiente da celulose do bagaço. A adsorção improdutiva foi dependente do pré-tratamento, e resultou em diminuição significativa dos rendimentos de hidrólise da celulose com baixas cargas de enzima. / The present work evaluated the limiting effect of the residual lignin of sugarcane bagasse submitted to different pretreatments: NaOH (5%), Na2SO3/NaOH (10%/5%), H2SO4 (0.75%), NaHSO3/H2SO4 (5%/0.75%) and SO2-catalyzed steam explosion (3%). The pretreatment resulted in bagasses with different chemical compositions, wherein the alkaline treatments resulted in a more efficient solubilization of lignin, and the acidic treatments solubilized the hemicellulose. The bagasses treated with Na2SO3/NaOH and by steam explosion resulted in cellulose hydrolysis yields above 80% using higher enzyme loadings (Celluclast), indicating the increased accessibility of the cellulose in these materials. This was confirmed by Simons\' Stain, which showed that the accessible surface area of the cellulose in these bagasses was higher than in the others. Good correlations (R2>0.80) between the 72 h hydrolysis yields and the accessible surface area of cellulose were only observed at higher enzyme loadings, indicating that other factors than accessibility, limit the hydrolysis at lower enzyme loadings. The lignins were isolated from the pretreated bagasses and the protein binding capacity was determined. The lignin from steam exploded bagasse showed the highest binding capacity, followed by the lignin extracted from NaOH-treated bagasse. The sulfite pretreatments (Na2SO3/NaOH e NaHSO3/H2SO4) resulted in lignins with lower binding capacities, when compared to their analogues without sulfite (NaOH e H2SO4), possibly because of the sulfonation of the residual lignin, confirmed by the presence of strong acid groups in these bagasses. BSA addition prior to the cellulases confirmed the strong effect of unproductive binding caused by the lignin in the NaOH-treated and steam exploded bagasses, materials with the highest binding capacity lignins. Less unproductive binding was observed using the enzyme extract Cellic CTec3, showing that the enzymes in this cocktail are less sensitive to the presence of lignin. From the extract Celluclast, the enzyme that most adsorbed to the lignins isolated from NaOH-treated and steam exploded bagasses was ?-glucosidase, followed by the endoglucases. The purified CBHI and EGII from T. longibrachiatum were less adsorbed to steam exploded bagasse lignin that the same enzymes from T. reesei. ?-glucosidase from A. niger did not bind to this lignin. Despite of the pretreatment, the presence of phenols solubilized from lignin, at lower concentrations, increased the cellulose hydrolysis yields with lower enzyme loading, possibly because of the positive effect of these compounds in the activity of oxidative enzymes, or because of the presence of lignosulfonates. At higher concentrations, despite of the enzyme loading, the phenols were inhibitory in all the pretreatments. The CBHI activity was more sensitive to the presence of phenolic compounds than the ?-glucosidase activity. From these results, it is possible to conclude that the increase in the accessibility, due to the lignin removal or relocation, influences the most the efficient hydrolysis of bagasse cellulose. The unproductive binding was pretreatment dependent and resulted in a significant decrease in the hydrolysis yields of the cellulose at lower enzyme loadings.

No presente trabalho, avaliou-se o efeito limitante da lignina residual de bagaço de cana submetido a diferentes pré-tratamentos na hidrólise da celulose. O bagaço foi submetido a cinco pré-tratamentos: NaOH (5%), Na2SO3/NaOH (10%/5%), H2SO4 (0,75%), NaHSO3/H2SO4 (5%/0,75%) e explosão a vapor catalisada por SO2 (3%). Os pré-tratamentos resultaram em bagaços com diferentes composições químicas, sendo que os tratamentos alcalinos favoreceram a solubilização de lignina e os tratamentos ácidos favoreceram a solubilização de hemicelulose. Os bagaços tratados com Na2SO3/NaOH e por explosão a vapor resultaram em rendimentos de hidrólise de celulose superiores a 80% ao utilizar altas cargas de enzima (Celluclast), indicando a maior acessibilidade da celulose desses materiais. Isso foi confirmado pela técnica de coloração de Simons, que mostrou que a área superficial acessível da celulose desses dois bagaços foi maior que a dos demais. Boas correlações (R2>0,8) entre o rendimento de hidrólise da celulose em 72 h e a celulose superficial acessível só foram obtidos com altas cargas de enzima, evidenciando que fatores além da acessibilidade limitaram a hidrólise da celulose com quantidade menor de enzimas. As ligninas dos bagaços pré-tratados foram isoladas e a capacidade adsortiva de proteínas foi determinada. A lignina de bagaço tratado por explosão a vapor apresentou maior capacidade adsortiva, seguida da lignina de bagaço tratado com NaOH. Os pré-tratamentos com íons sulfito (Na2SO3/NaOH e NaHSO3/H2SO4) resultaram em ligninas com capacidades adsortivas inferiores quando comparado aos seus análogos sem sulfito (NaOH e H2SO4), possivelmente pela sulfonação da lignina residual, confirmada pela presença de grupos ácidos fortes nesses bagaços. A adição de BSA prévia à adição de celulases confirmou o forte efeito da adsorção improdutiva causada pela lignina nos bagaços tratados com NaOH e por explosão a vapor, materiais cujas ligninas apresentaram maiores capacidades adsortivas. A adsorção improdutiva foi menor ao utilizar o extrato enzimático Cellic CTec3, evidenciando que as enzimas desse coquetel são menos sensíveis à presença da lignina. Do extrato Celluclast, a enzima que mais adsorveu nas ligninas de bagaço tratado com NaOH e por explosão a vapor foi a ?-glicosidase, seguida da endoglucanase. As enzimas purificadas CBHI e EGII de T. longibrachiatum adsorveram menos à lignina de bagaço tratado por explosão a vapor que as mesmas enzimas de T. reesei. A ?-glicosidade de A. niger não adsorveu a essa lignina. Independentemente do pré-tratamento, a presença de fenóis solubilizados a partir da lignina, em baixas concentrações, resultou em aumento no rendimento de conversão de celulose ao utilizar baixas cargas de enzima, possivelmente pelo efeito positivo causado por esses na atividade de enzimas oxidativas, ou pela presença de lignossulfonatos. Em concentrações mais elevadas, independente da carga enzimática, os fenóis liberados foram inibitórios em todos os pré-tratamentos. A atividade de CBHI foi mais sensível à presença dos compostos fenólicos que a atividade de ?-glicosidase. A partir dos resultados, conclui-se o aumento da acessibilidade, seja pela remoção ou pela relocalização da lignina, é o fator que mais influencia a hidrólise eficiente da celulose do bagaço. A adsorção improdutiva foi dependente do pré-tratamento, e resultou em diminuição significativa dos rendimentos de hidrólise da celulose com baixas cargas de enzima. / The present work evaluated the limiting effect of the residual lignin of sugarcane bagasse submitted to different pretreatments: NaOH (5%), Na2SO3/NaOH (10%/5%), H2SO4 (0.75%), NaHSO3/H2SO4 (5%/0.75%) and SO2-catalyzed steam explosion (3%). The pretreatment resulted in bagasses with different chemical compositions, wherein the alkaline treatments resulted in a more efficient solubilization of lignin, and the acidic treatments solubilized the hemicellulose. The bagasses treated with Na2SO3/NaOH and by steam explosion resulted in cellulose hydrolysis yields above 80% using higher enzyme loadings (Celluclast), indicating the increased accessibility of the cellulose in these materials. This was confirmed by Simons\' Stain, which showed that the accessible surface area of the cellulose in these bagasses was higher than in the others. Good correlations (R2>0.80) between the 72 h hydrolysis yields and the accessible surface area of cellulose were only observed at higher enzyme loadings, indicating that other factors than accessibility, limit the hydrolysis at lower enzyme loadings. The lignins were isolated from the pretreated bagasses and the protein binding capacity was determined. The lignin from steam exploded bagasse showed the highest binding capacity, followed by the lignin extracted from NaOH-treated bagasse. The sulfite pretreatments (Na2SO3/NaOH e NaHSO3/H2SO4) resulted in lignins with lower binding capacities, when compared to their analogues without sulfite (NaOH e H2SO4), possibly because of the sulfonation of the residual lignin, confirmed by the presence of strong acid groups in these bagasses. BSA addition prior to the cellulases confirmed the strong effect of unproductive binding caused by the lignin in the NaOH-treated and steam exploded bagasses, materials with the highest binding capacity lignins. Less unproductive binding was observed using the enzyme extract Cellic CTec3, showing that the enzymes in this cocktail are less sensitive to the presence of lignin. From the extract Celluclast, the enzyme that most adsorbed to the lignins isolated from NaOH-treated and steam exploded bagasses was ?-glucosidase, followed by the endoglucases. The purified CBHI and EGII from T. longibrachiatum were less adsorbed to steam exploded bagasse lignin that the same enzymes from T. reesei. ?-glucosidase from A. niger did not bind to this lignin. Despite of the pretreatment, the presence of phenols solubilized from lignin, at lower concentrations, increased the cellulose hydrolysis yields with lower enzyme loading, possibly because of the positive effect of these compounds in the activity of oxidative enzymes, or because of the presence of lignosulfonates. At higher concentrations, despite of the enzyme loading, the phenols were inhibitory in all the pretreatments. The CBHI activity was more sensitive to the presence of phenolic compounds than the ?-glucosidase activity. From these results, it is possible to conclude that the increase in the accessibility, due to the lignin removal or relocation, influences the most the efficient hydrolysis of bagasse cellulose. The unproductive binding was pretreatment dependent and resulted in a significant decrease in the hydrolysis yields of the cellulose at lower enzyme loadings.

L'objectif de la thèse est de caractériser des populations bactériennes susceptibles de dégrader la lignine des résidus de culture afin d'optimiser la biodisponibilité du compartiment holocellulosique. L'originalité du sujet réside dans la recherche de bactéries ligninolytiques en utilisant des approches combinant à la fois de l'isolement de souches, des techniques de biologie moléculaire et de la mise en œuvre de ces micro-organismes à l'échelle de réacteur pilote, dans le cadre de prétraitements des résidus de culture. Les travaux de recherche ont été réalisés pour mettre en évidence et identifier des micro-organismes potentiellement intéressants et caractériser leurs systèmes enzymatiques susceptibles d'intervenir dans la dégradation de la lignine. Suite à l'isolement de plusieurs souches d'intérêts et à la mise en place de réacteurs, des expérimentations ont été réalisées afin de suivre l'effet des prétraitements biologiques sur la dégradation de la biomasse et notamment par l'étude des transformations biochimiques, de l'accessibilité à la cellulose ainsi que de l'évolution des communautés microbiennes. Les résultats de ces travaux ont montré que la communauté endogène joue un rôle primordial dans la biodégradation de la paille de colza. Bien que l'inoculation de bactéries exogènes ait eu peu d'influence, il est possible d'améliorer l'accessibilité à cellulose par un mélange de lignine et des sels minéraux permettant d'enrichir la communauté endogène active. Cette thèse s'articule autour de plusieurs analyses multi-échelles et complémentaires permettant de développer un prétraitement biologique de résidu de culture, d'améliorer sa mise en œuvre et son utilisation pour la digestion anaérobie par voie sèche ou la production de bioéthanol. / The aim of the PhD project is to characterize bacterial populations potentially capable of degrading lignin within crop residues to optimize the bioavailability of holocellulose compartment to be valorized by anaerobic digestion. The originality of the subject lies in finding ligninolytic bacteria using approaches combining the isolation of strains and technique of molecular biology, and the application of these microorganisms on a reactor scale, as part of pretreatment of crop residues. Firstly, research work was carried out to find and identify potentially interesting micro-organisms and characterize their specific enzyme systems. Secondly, studies have been conducted on the application of micro-organisms isolated for a biological pretreatment of rape straw. Pretreatments were carried out in a series of laboratory reactors dedicated to follow the effect in real time. A method allowing the quantification of the cellulose accessibility for cellulases in substrate was established. In parallel, high-throughput sequencing analysis was performed to monitor and/or characterize the bacterial and archaeal communities in the reactors. The results obtained have shown that the main factor influencing the degradation of the straw was not the presence of ligninolytic strains which could not influence the development of endogenous community, but the supply of some nutrients included in a mixture with lignin and mineral salts which could enrich a particularly active community. The data obtained in the thesis allowed to capitalize knowledge on the biodegradation of lignocellulosic biomass. This prospective study allowed the consideration of using the biological pretreatments developed at the upstream of biogas and biofuel sectors.