Nessa pesquisa avaliou-se a produção fermentativa de hidrogênio e ácidos orgânicos a partir do bagaço de cana-de-açúcar (BCA) usado como substrato em reatores em batelada. Três condições de pré-tratamento (hidrotérmico, autoclave e hidrotérmico mais autoclave) do BCA e condição in natura foram avaliadas a fim de favorecer a produção de hidrogênio. Verificou-se produção molar de hidrogênio de 3,79 mmol/L, 3,47 mmol/L, 1,67 mmol/L e 1,01 mmol/L para BCA autoclavado, BCA in natura, BCA pré-tratado em sistema hidrotérmico e BCA pré-tratado em sistema hidrotérmico seguido de autoclave, respectivamente. A partir desses valores, optou-se por usar o BCA autoclavado como substrato para otimização da produção de hidrogênio e ácidos orgânicos a partir de metodologias de delineamento do composto central e superfície de resposta. Foram monitorados 10 reatores em batelada (R1 a R10), em triplicatas, com diferentes concentrações de substrato (0,8 a 9,2 g/L) e pH (de 4,6 a 7,4). A maior produção de hidrogênio (24,1 mmol/L) e 6,4 g/L de ácidos orgânicos foram obtidos em R4 (8,0 g BCA/L e pH 7,0). Os açúcares glicose, arabinose, xilose, manose e galactose foram observados ao longo do tempo de operação em todos os reatores, sendo arabinose observado em maior concentração nas condições dos reatores R3 (8,0 g BCA/L e pH 5,0) e R8 (5,0 g BCA/L e pH 7,4), respectivamente, 1.415,3 e 1.372,5 mg/L. A produção de hidrogênio foi concomitante à formação de ácidos orgânicos, principalmente butírico (de 14,6 a 33,8% em R1 e R6, respectivamente) e succínico (de 19,5 a 26,4% em R3 e R9, respectivamente). Os dois fatores analisados, concentração de substrato e pH, exerceram efeitos significativos na produção de hidrogênio, ácido butírico e succínico. A partir dos resultados obtidos com o planejamento fatorial, foi possível verificar que o valor máximo de produção de hidrogênio estimado pelo modelo foi de 23,10 mmol/L, para 7,0 g BCA/L e pH 7,2. O valor obtido no experimento de otimização (Rotm) foi de 19,84 mmol/L, com grau de precisão do modelo de 85,9% para produção de hidrogênio a partir de BCA autoclavado. Sequenciamento massivo via plataforma Illumina (Miseq) foi realizado para a identificação de bactérias do reator do ponto central, (R9, 5,0 g BCA/L e pH 6,0), do reator otimizado (Rotm, 7,0 g BCA/L e pH 7,2), de amostras do BCA autoclavado e inóculo. No inóculo foram identificadas principalmente bactérias semelhantes a Clostridium bifermentans (62,69% de abundância relativa), Bacillus coagulans (31,67%) e Enterobacter aerogenes (2,72%). No BCA foram identificadas bactérias semelhantes a C. bifermentans (31,91%), C. cellobioparum (32,29%), C. cellulolyticum (5,69%), C. sartagoforme (14,63%) e Paenibacillus spp. (11,67%). Estas bactérias não foram favorecidas sob as condições impostas em R9 (5,0 g BCA/L e pH 6,0) e Rotm (7,0 g BCA/L e pH 7,2), uma vez que a abundância relativa das bactérias nas amostras dos reatores foram completamente diferentes. Em R9, bactérias semelhantes a Lactobacillus paracasei e Escherichia hermannii foram as principais identificas com 37,50 e 34,32% de abundância relativa, respectivamente. Em Rotm, as principais bactérias identificadas foram semelhantes a Bacteroides sp. e Enterobacter aerogenes, com 37,35 e 27,72% de abundância relativa, respectivamente. Assim, as populações bacterianas, bem como a produção de metabólitos, foram alteradas em função das condições impostas; ou seja, concentração de BCA, pH em reatores em batelada com BCA autoclavado como substrato. / This study evaluated the hydrogen and organic acids fermentative productions from sugarcane bagasse (SCB) as substrate in batch reactors. Three pre-treatment conditions (hydrothermal, autoclave and hydrothermal plus autoclave) of BCA and the in natura condition were evaluated in order to favor the hydrogen production. Hydrogen molar productions of 3.79 mmol/L, 3.47 mmol/L, 1.67 mmol/L and 1.01 mmol/L was found for SCB pretreated in autoclave, BCA in natura, SCB pretreated in hydrothermal system and SCB pretreated in hydrothermal system followed by autoclaving, respectively. From these values, it was decided to use autoclaved BCA as a substrate for optimization of hydrogen and organic acids productions from the design methodologies of the central compound and response surface. Ten batch reactors (R1 to R10) were monitored in triplicates with different substrate concentrations (0.8 to 9.2 g/L) and pH (4.6 to 7.4). The highest production of hydrogen (24.06 mmol/L) and 6.42 g/L of organic acids were obtained in R4 (8.0 g BCA/L and pH 7.0). Glucose, arabinose, xylose, mannose and galactose were produced and consumed throughout the operating time of all reactors, and arabinose was observed at higher concentration, 1,415.26 and 1,372.45 mg/L in R3 (8.0 g BCA/L and pH 5.0) and R8 (5.0 g BCA/L and pH 7.4), respectively. The production of hydrogen was concomitant to the formation of organic acids, mainly butyric (from 14.6 to 33.8% in R1 and R6, respectively) and succinic (from 19.5 to 26.4% in R3 and R9, respectively). The two factors analyzed, substrate concentration and pH, had significant effects on the production of hydrogen, butyric acid and succinic acid. From the results obtained with the factorial design, it was possible to verify that the maximum value of hydrogen production estimated by the model was 23.10 mmol/L, to 7.0 g BCA L and pH 7.2. The value obtained in the optimization experiment (Rotm) was 19.84 mmol/L, with an accuracy of 85.9% for hydrogen production from autoclaved BCA. Sequencing by the Illumina platform (Miseq) was performed for the identification of bacteria from the central point reactor (R9, 5.0 g BCA/L and pH 6.0), optimized reactor (Rotm, 7.0 g BCA/L and pH 7.2), autoclaved BCA and inoculum samples. In the inoculum were identified mainly bacteria similar to Clostridium bifermentans (62,69% of relative abundance), Bacillus coagulans (31,67%) and Enterobacter aerogenes (2,72%). Bacteria similar to C. bifermentans (31.91%), C. cellobioparum (32.29%), C. cellulolyticum (5.69%), C. sartagoforme (14.63%) and Paenibacillus spp. (11.67%). These bacteria were not favored under the conditions imposed on R9 (5.0 g BCA/L and pH 6.0) and Rotm (7.0 g BCA/L and pH 7.2), since the relative abundance of the bacteria in the reactor samples were completely different. In R9, bacteria similar to Lactobacillus paracasei and Escherichia hermannii were the main identified with 37.50 and 34.32% of relative abundance, respectively. In Rotm, the main bacteria identified were similar to Bacteroides sp. and Enterobacter aerogenes, with 37.35 and 27.72% relative abundance, respectively. Thus, bacterial populations, as well as the production of metabolites, were altered as a function of the imposed conditions; ie, BCA concentration, pH in batch reactors with autoclaved BCA as substrate.

Dans le contexte de la bioéconomie, la découverte et la caractérisation des enzymes capables de dégrader la paroi végétale est particulièrement intéressante pour l’utilisation de la biomasse lignocellulosique dans l’industrie. A cet égard, la métagénomique fonctionnelle est un outil puissantpour découvrir de nouvelles enzymes à partir d’écosystèmes microbiens variés, comme l’illustrent les travaux sur le tube digestif du termite Pseudacanthotermes militaris. Cette étude a fourni une mine d’informations et identifié un hypothétique locus d’utilisation du xylane (XUS), codant pour cinq glycosides hydrolases (GH) et une carbohydrate esterase (CE) de Bacteroidales.Le XUS du métagénome de Pseudacanthotermes militaris contient une xylanase de la famille GH10 qui possède une organisation modulaire complexe dans laquelle la séquence du domaine GH10 est interrompue par une insertion de deux carbohydrate binding modules (CBM). Des travaux préliminaires ont montré que cette enzyme modulaire, désignée Pm25, est active sur xylane. Par conséquent, un des objectifs de cette étude a été la caractérisation détaillée des propriétés biochimiques et catalytiques de Pm25. Le rôle des CBM a également été examiné en quantifiant les interactions protéines-sucres et permettant ainsi une meilleure compréhension du rôle spécifique de ces modules, les résultats obtenus permettent de cerner l’impact de la modularité de Pm25 sur ses propriétés fonctionnelles.Dans une deuxième partie de l’étude, nous avons entrepris d’étudier la fonction de Pm25 dans le contexte du cluster XUS. Pour ce faire, nous avons étudié les enzymes adjacentes à Pm25 sur le locus,une autre GH10, une GH11, une GH115 et une GH43. La comparaison des paramètres cinétiques et une étude détaillée des produits d’hydrolyse ont été analysés par spectrométrie de masse et ont révélé que la GH10 et la GH11 étaient les enzymes clefs de la dépolymérisation en étant 20 fois plus efficaces que Pm25. En parallèle, nous avons développé un protocole pour l’utilisation de la micro-thermophorèse (MST) pour quantifier les interactions CBM-sucres, une approche intéressante qui nécessite peut d’échantillon et de ligand contrairement à d’autres méthodes biophysiques. Dans l’ensemble, cette étude a révélé le rôle important de Pm25 et ses homologues dans les locus d’utilisation des xylanes chez les Bacteroidetes et a permis d’identifié le sens de cette architecture particulière. / In the context of bioeconomy, the discovery and study of plant-cell wall degrading enzymes is particularly relevant for the use of lignocellulosic biomass for industrial purposes. In this respect, functional metagenomics has proven to be a powerful tool to discover new enzymes from a variety of microbial ecosystems, as exemplified by work performed on the gut of the termite Pseudacanthotermes militaris. This study provided a wealth of information and identified an interesting hypothetical xylan utilization system, encoding five glycoside hydrolases (GH) and one carbohydrate esterase (CE) annotated from bacteroidales. The Pseudacanthotermes militaris-derived putative XUS cluster contains a GH10 xylanase that displays a quite complex modular arrangement wherein the GH10 catalytic module contains two insertional carbohydrate binding modules (CBM). During the preliminary work, this modular enzyme, designated Pm25, was shown to be active on xylan, thus in the present research we set out to more thoroughly characterize its biochemical and catalytic properties.The role of the CBM was also investigated, quantifying protein-carbohydrate interactions and thus providing better insight into the specific role of the modules. Taken together, the results obtained provide insight into how Pm25 modularity translates into functional properties. In second part of our study, we set out investigate the function of Pm25 in the context of the XUS cluster. To achieve this we studied a xylan utilization system, which is constituted by another GH10, GH11, GH115 and GH43. The comparison of kinetic parameters and a detailed end product analysis by mass spectrometry showed that GH10 and GH11 outweigh over 20 fold Pm25 catalytic efficiency. In parallel, we developed the use of MicroScale Thermophoresis (MST) to quantify CBM-carbohydrates interactions, an interesting approach requiring smaller concentration of proteinsand ligands compared to other biophysical methods. Overall this study highlighted the important role of Pm25 homologs in the xylan utilization system in Bacteroidetes, and pinpointed the meaning of its unusual architecture.

La méthanisation est un processus biologique de transformation des matières organiques libérant principalement du méthane et du dioxyde de carbone. Cette technologie connaît un essor important pour la production de biométhane, source d’énergie renouvelable. Elle présente cependant des rendements de dégradation faibles lorsque de la biomasse lignocellulosique est utilisée comme matière première. Pour optimiser son rendement, la paille de blé a été sélectionnée comme biomasse représentative et l’impact sur la méthanisation de chacune des fractions (extractibles, hémicelluloses, cellulose et lignine) a été évalué. Une biomasse de synthèse a été construiteà partir des constituants pures de la paille de blé afin d’évaluer l’impact des interactions lignine-holocellulose. Le potentiel de méthanisation de différentes molécules phénoliques,provenant de la dégradation de la lignine, a été déterminé. Elles inhibent la méthanisation à l’exception de trois d’entres elles qui présentent un rendement de méthanisation élevé : les acides vanillique, l’acide férulique et le syringaldéhyde. Différents prétraitements physique (le chauffage par échangeur thermique ou par irradiation aux micro-ondes ainsi que la sonication et le raffinage papetier) et chimique (la soude, l’ammoniaque et l’ozone) ont ensuite été sélectionnés, et leurs impacts sur lacomposition de la paille et sa méthanisation ont été mesurés. Les meilleurs rendements de méthanisation ont été obtenus suite à l’exposition aux micro-ondes en présence de soude. / Methanization or anaerobic digestion is a biological process to transform organicmatter into a gas mixture composed by a majority of methane and carbon dioxide. Thistechnology is developing rapidly for the production of biomethane as renewable energysource. However this biotechnological route has low performances when lignocellulosicbiomass is used as raw material.Wheat straw has been chosen as typical biomass and the role of each lignocellulosicfraction (extractives, cellulose, hemicelluloses and lignin) has been determined on theperformance of anaerobic digestion. A synthetic biomass has been built with different pureconstituents of the wheat straw to assess the impact of holocellulose-lignin interactions onmethanization. Then methane potential of various lignin degradation products (phenolicmolecules) has been studied. Majority of them have been shown an inhibitory effect butthree of them have been converted to methane: ferulic and vanillic acids andsyringaldehyde.Various physical pretreatments (heating, microwave irradiation, sonication andrefining) and chemical pretreatments (sodium hydroxide, ammonia and ozone) have beenselected to prepare the biomass to anaerobic digestion and their impacts on wheat strawcomposition have been evaluated. The best methanization yield has been obtained afterpretreatments by sodium hydroxide heating by microwave irradiation.

Nesta tese, a ressonância magnética nuclear do estado sólido (SSNMR) foi utilizada para estudar a composição química e estrutura dos componentes da parede celular de plantas. Visando contribuir no desenvolvimento de estratégias de despolimerização da biomassa, SSNMR foi inicialmente utilizada para estudar efeitos dos pré-tratamentos químicos e físicos, e da ação de enzimas sobre algumas biomassas. Os resultados mostraram que, em baixas concentrações, tratamentos ácidos são altamente efetivos na remoção das frações de hemicelulose, com pouco efeito nas frações de lignina e celulose. Já tratamentos alcalinos promovem eficiente deslignificação da biomassa, sendo que a mínima concentração da solução alcalina necessária para obter a máxima deslignificação depende do tipo de biomassa e da temperatura do tratamento. Os estudos por SSNMR foram correlacionados com estudos por outras técnicas, contribuindo para um entendimento mais profundo sobre o efeito dos pré-tratamentos e da hidrolise enzimática em diferentes biomassas. Outra parte da tese aborda a determinação da cristalinidade de celulose nativa (não extraída) de biomassa de bagaço de cana-de-açúcar. Utilizando a técnica de polarização cruzada em múltiplas etapas (Multi-CP) e um procedimento de subtração espectral, foi possível isolar os sinais de RMN da celulose nativa e a partir daí avaliar o índice de cristalinidade (CI). Esse método foi utilizado para avaliar o CI da celulose nativa de bagaço de cana-de-açúcar submetido à pré-tratamentos com H2SO4 e NaOH e os resultados não mostraram variações significativas do CI da celulose nas concentrações utilizadas, apesar do aumento da eficiência da hidrólise. Assim, ao contrário de muitos trabalhos encontrados na literatura, não parece que a cristalinidade da celulose seja um fator primordial no aumento de eficiência da hidrólise enzimática. Na parte final da tese, as interações intermoleculares entre os dois principais polissacarídeos da biomassa: celulose e xilano foram investigadas utilizando uma variedade de técnicas avançadas de RMN bidimensional. Neste trabalho, a arquitetura molecular de hastes de plantas de Arabidopsis Thaliana, sem nunca serem seca foi estudada. Utilizando a técnica refocused J-INADEQUATE (Increadible Natural Abundance Double Quantum Transfer Experiment via J coupling) observamos dois conjuntos de deslocamentos químicos distintos para o xilano, sendo um deles coincidente com aquele observado em solução. Em seguida, utilizamos experimentos SSNMR com o intuito de investigar se algum desses domínios de xilano estaria vinculado com a celulose. Experimentos CP-PDSD (Proton Driven Spin Diffusion detected via 13C through Cross-Polarization) demonstram a existência de proximidade espacial entre o novo domínio do xilano e o domínio da celulose. A comparação de resultados entre as amostras de padrão e o seu mutante deficiente em celulose (irx3) indicaram que o xilano com novo deslocamento químico é fortemente dependente da presença de celulose. A análise da mobilidade molecular pela técnica Dipolar Chemical Shift Correlation (DIPSHIFT), mostrou que as moléculas do novo domínio do xilano são altamente rígidas - uma característica partilhada com a celulose. Combinados, esses dados fornecem evidências de uma arquitetura molecular específica entre os dois polissacarídeos majoritários da parede celular. / Solid-state nuclear magnetic resonance (SSNMR) was used to study the chemical composition and structure of plant cell wall components. Aiming the development of depolymerization strategies, SSNMR was initially used to study the effects of chemical and physical pre-treatments, as well as the enzymatic action on the structure and composition of biomasses. The results showed that, at low concentrations, pre-treatments with acids are highly effective for removal of hemicellulose without significant effect on lignin and cellulose. In turn, the alkaline pre-treatment promotes efficient delignification of the biomass. The minimum concentration of the alkaline solution required to achieve the maximum delignification depends on the type of biomass and treatment temperature. SSNMR studies were correlated with studies using other techniques, contributing to an in-depth understanding of the effect of pre-treatments and enzymatic hydrolysis in different biomasses. Another part of the thesis discusses is the determination of native cellulose crystallinity (not extracted) of sugarcane bagasse biomasses. Using the cross-polarization technique in multiple blocks (Multi-CP) and a spectral subtraction approach, it was possible to isolate the NMR signals of the native cellulose and to evaluate the crystallinity index (CI). This method was used to accessof the CI of cellulose in sugarcane bagasse samples pre-treated with H2SO4 and NaOH. The results did not show significant variations of the cellulose CI, at the concentration used here, despite the increase in the hydrolysis efficiency. Thus, in contrast to some studies in the literature, it does not appear that the crystallinity of cellulose is a primary limiting factor concerning the enzymatic hydrolysis efficiency in biomasses. In the final part of this thesis, the intermolecular interactions between the two main polysaccharides of the plant cell wall, cellulose and xylan, were investigated using advanced two-dimensional NMR techniques. The molecular architecture of 13C labelled never-dried Arabidopsis Thaliana stems was studied. Using refocused J-INADEQUATE (Increadible Natural Abundance Double Quantum Transfer Experiment via J coupling) we observed two distinct chemical shifts in xylan, one of which coincides with that observed in solution. Next, we used SSNMR experiments toinvestigate the interaction between the novel xylan and cellulose domains. CP-PDSD (Proton Driven Spin Diffusion detected via 13C through Cross-Polarization) experiments demonstrated spatial proximity between the new xylan and cellulose domains. The same approach was used to study cellulose deficient (irx3) mutants and the comparison between the results indicate that the new xylan domain is cellulose-dependent. Dipolar Chemical Shift Correlation (DIPSHIFT) experiments were performed to analyse the molecular mobility of these polysaccharides showing that the novel xylan is highly rigid - a characteristic which is shared with cellulose. Combined, these data provide evidence for a specific molecular architecture between the two most common polysaccharides in plant cell walls.

Biofuels produced from lignocellulosic biomass via the fermentation platform are sustainable energy alternatives to fossil fuels. Process Systems Engineering (PSE) uses computer-based tools and methods to design, simulate and optimize processes. Application of PSE tools to the design of economic biorefinery processes requires the development of simulation approaches that can be integrated with existing, mature PSE tools used to optimize traditional refineries, such as Aspen Plus. Current unit operation models lack the ability to describe unsteady state fermentation processes, link unsteady state fermentation with in situ separations, and optimize these processes for competing factors (e.g., yield and productivity). This work applies a novel architecture of commercial PSE tools, Aspen Plus and MATLAB, to develop techniques to simulate time-dependent fermentation without and with in situ separations for process design, analyses and optimization of the operating conditions. Traditional batch fermentation simulations with in situ separations decouple these interdependent steps in a separate “steady state” reactor followed by an equilibrium separation of the final fermentation broth. A typical mechanistic system of ordinary differential equations (ODEs) describing a batch fermentation does not fit the standard built-in power law reaction kinetics model in Aspen Plus. To circumvent this challenge, a novel platform that links the batch reactor to a FORTRAN user kinetics subroutine (incorporates the ODEs) combined with component substitution (to simulate non-databank components) is utilized to simulate an unsteady state batch and in situ gas stripping process. The resulting model system predicts the product profile to be sensitive to the gas flow rate unlike previous “steady state” simulations. This demonstrates the importance of linking a time-dependent fermentation model to the fermentation environment for the design and analyses of fermentation processes. A novel platform linking the genetic algorithm multi-objective and single-objective optimizations in MATLAB to the unsteady state batch fermentation simulation in Aspen Plus through a component object module communication platform is utilized to optimize the operating conditions of a typical batch fermentation process. Two major contributions are: prior concentration of sugars from a typical lignocellulosic hydrolysate may be needed and with a higher initial sugar concentration, the fermentation process must be integrated with an in situ separation process to optimize the performance of fermentation processes. With this framework, fermentation experimentalists can use the full suite of PSE tools and methods to integrate biorefineries and refineries and as a decision-support tool to guide the design, analyses and optimization of fermentation-based biorefineries.

Increasing demand for energy and transportation fuel has motivated researchers all around the world to explore alternatives for a long-term sustainable source of energy. Biomass is one such renewable resource that can be converted into various marketable products by the process of biorefining. Currently, research is taking strides in developing conversion techniques for producing biofuels from multiple bio-based feedstocks. However, the greatest concern with emerging processes is the long-term viability as a sustainable source of energy. Hence, a framework is required that can incorporate novel and existing processes to validate their economic, environmental and social potential in satisfying present energy demands, without compromising the ability of future generations to meet their own energy needs. This research focuses on developing a framework that can incorporate fundamental research to determine its long-term viability, simultaneously providing critical techno-economic and decision support information to various stakeholders. This contribution links various simulation and optimization models to create a decision support tool, to estimate the viability of biorefining options in any given region. Multiple disciplines from the Process Systems Engineering and Supply Chain Management are integrated to develop the comprehensive framework. Process simulation models for thermochemical and biochemical processes are developed and optimized using Aspen Engineering Suite. Finally, for validation, the framework is analyzed by combining the outcomes of the process simulation with the supply chain models. The developed techno-economic model takes into account detailed variable costs and capital investments for various conversion processes. Subsequently, case studies are performed to demonstrate the applicability of the decision support tool for the Jackson Purchase region of Western Kentucky. The multidisciplinary framework is a unique contribution in the field of Process Systems Engineering as it demonstrates simulation of process optimization models and illustrates its iterative linking with the supply chain optimization models to estimate the economics of biorefinery from multi-stakeholder perspective. This informative tool not only assists in comparing modes of operation but also forecasts the effect of future scenarios, such as, utilization of marginal land for planting dedicated energy crops and incorporation of emerging enzymatic processes. The resulting framework is novel and informative in assisting investors, policy makers and other stakeholders for evaluating the impacts of biorefining. The results obtained supports the generalizability of this tool to be applied in any given region and guide stakeholders in making financial and strategic decisions.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-03-15T17:17:40Z No. of bitstreams: 1 REBECA DE ALMEIDA SILVA - DISSERTAÇÃO PPGEQ 2014..pdf: 2095528 bytes, checksum: 2f418390669f1b14f0649337a5231873 (MD5) / Made available in DSpace on 2018-03-15T17:17:40Z (GMT). No. of bitstreams: 1 REBECA DE ALMEIDA SILVA - DISSERTAÇÃO PPGEQ 2014..pdf: 2095528 bytes, checksum: 2f418390669f1b14f0649337a5231873 (MD5) Previous issue date: 2014-09-11 / Com a atual busca mundial por fontes renováveis, o uso de resíduos de biomassa lignocelulósica apresenta uma perspectiva bastante promissora para a produção de etanol. O processo deriva da fermentação de açúcares de origem hemicelulósica e celulósica, frações de materiais lignocelulósicos, através de um pré-tratamento adequado e da hidrólise enzimática da celulose. O resíduo da acerola constitui de 40 % do volume processado, sendo 24,7 % de celulose, 19,27 % de hemicelulose e 28,37 % de lignina. O objetivo deste trabalho foi estudar a cinética da hidrólise enzimática do bagaço de acerola in natura e do bagaço pré-tratado e avaliar o efeito desse prétratamento sobre a hidrólise. Inicialmente foi feita a caracterização lignocelulósica do bagaço in natura e do bagaço pré-tratado e a caracterização microestrutural por meio das análises de DRX e MEV. Ao bagaço de acerola foi aplicado o pré-tratamento ácido seguido de básico. Em seguida foi realizada a hidrólise enzimática do bagaço in natura e pré-tratado. Foram usadas as enzimas comerciais Celluclast 1.5L da Novozyme e betaglicosidase da Proenzyme. Como ferramenta para avaliação das variáveis que influenciam no processo usou-se um planejamento fatorial 22 com 3 pontos centrais, onde as variáveis analisadas foram carga enzimática e relação de massa seca de bagaço por volume do meio reacional. A caracterização lignocelulósica mostrou que o bagaço de acerola é um substrato viável para obtenção de açúcares fermentescíveis e sua subseqüente conversão em etanol. O pré-tratamento ácido seguido de básico mostrou-se bastante eficiente em concentrar a celulose, pela remoção de parte da hemicelulose e lignina, provocando um aumento da celulose de 25 % para 50 %. Evidenciou-se a cristalinidade do bagaço de acerola, comprovada por Difração de Raios X e a modificação na morfologia do bagaço, verificada por Microscopia Eletrônica de Varredura. Por meio da cinética de hidrólise enzimática do bagaço de acerola in natura e pré-tratado foram obtidos rendimentos de 100 % na conversão da celulose em glicose. A melhor produção de glicose foi de 22,3 g/L alcançada em 36 horas de hidrólise para o bagaço de acerola pré-tratado, onde ocorreu nas maiores condições de carga enzimática e relação massa seca de bagaço por volume reacional. / With the current worldwide search for renewable sources, the use of lignocellulosic biomass waste has a very promising perspective for ethanol production. The process derives from the fermentation of sugar from hemicellulose and cellulose origin, fractions of lignocellulosic materials, by a suitable pre-treatment and enzymatic hydrolysis of cellulose. The waste of acerola is constituted by 40 % of the processed volume, and 24,7 % of cellulose, 19,27 % of hemicellulose and 28,37 % of lignin. The aim of this work was to study the kinetics of enzymatic hydrolysis of acerola’s waste in natura and waste pretreated, and also to evaluate the effect of this pretreatment on the hydrolysis. Initially, a lignocellulosic characterization was made with the waste in natura and waste pretreated, and microstructural characterization by means of XRD and SEM analysis. To the waste of acerola, an acid pretreatment was applied, followed by alkaline pretreatment. Then, the enzymatic hydrolysis of waste in natura and pretreated was performed. Commercial enzymes Celluclast 1.5L (Novozyme) and beta-glucosidase (Proenzyme) were used. As a tool to evaluate the variables that influence the process, a factorial design 22 was used with three central points, and the analyzed variables were enzymatic load and ratio of dry weight of waste per volume of reaction. This lignocellulosic characterization showed that the waste of acerola is a viable substrate for obtaining fermentable sugars and its subsequent conversion to ethanol. The acid pretreatment followed by the alkaline showed to be very effective in concentrating the cellulose, by the removal of part of the hemicellulose and lignin, causing an increase of the cellulose from 25 % to 50 %. The crystallinity of waste of acerola was evidenced by X-ray Diffraction and the modification in the morphology of waste, verified by Scanning Electron Microscopy. Through the kinetics of enzymatic hydrolysis of waste of acerola in natura and pretreated, yields of 100 % were obtained, in the conversion of cellulose to glucose. The best glucose production was 22,3 g/L, reached in 36 hours of hydrolysis of waste of acerola pretreated, which occurred in the best conditions of enzyme load and dry weight of waste per reaction volume ratio.

L'assainissement a de tout temps été une préoccupation majeure pour des questions d'hygiène et de santé publique. Une installation d'assainissement non collectif (ANC) assure la collecte, le transport, le traitement et l'évacuation des eaux usées domestiques. En France, l’ANC représente 20 % des installations de traitement des eaux usées domestiques. Cela concerne une population de 12 millions d'habitants, soit environ cinq millions d'installations en zones rurales. Parmi les technologies utilisées dans l’ANC, les systèmes de filtration biologique utilisant un milieu filtrant sont souvent mis en oeuvre. Le traitement des eaux usées (élimination des matières en suspension et de la matière organique) est effectué par les actions conjointes de filtration, et de biodégradation par des bactéries se développant au sein du milieu filtrant. Historiquement, des matériaux comme le sable et la tourbe sont les plus utilisés, et plus récemment des milieux à base de copeaux de coco venus remplacer la tourbe dont l’extraction est interdite, sont en développement croissant. Cependant, l’empreinte carbone produite par l'importation depuis l'étranger de matériaux tels que ce dernier est très importante. Dès lors l’utilisation de sousproduits agro-industriels locaux pourrait la diminuer et rendre l’utilisation de ces nouveaux milieux filtrants plus compatible avec des critères environnementaux. Le premier objectif de cette thèse est de déterminer le degré de fiabilité en conditions réelles d’un milieu filtrant innovant d’origine végétale ayant fait ses preuves en conditions de laboratoire. L’étude est basée sur la mise en parallèle de résultats obtenus en laboratoire et sur le terrain. Au laboratoire, une approche hydraulique a permis de caractériser le fonctionnement du lit filtrant. Sur le terrain, l’évaluation d’un filtre compact qui fonctionne de façon continue depuis plus de quatre ans et de cinq autres installations a été réalisée. Pour l’ensemble des installations, le milieu filtrant testé permet d’obtenir une qualité des eaux rejetées en accord avec la règlementation en vigueur. Les pourcentages moyens de réduction estimés de la DCO, de la DBO5 et des MES sont respectivement de plus de 79 %, 98 % et 88 %, confirmant ainsi la forte dégradation de la matière carbonée et de la pollution particulaire. Le second objectif de cette thèse consiste à établir un jeu de paramètres physicochimiques permettant de guider le choix de nouveaux matériaux potentiellement utilisables en ANC. Pour ce faire, la caractérisation des paramètres physicochimiques clés de nouveaux matériaux et des paramètres hydrauliques des lits filtrants correspondants a été effectuée. Une analyse statistique a permis d’étudier les corrélations entre ces paramètres et les performances épuratoires des matériaux. Au final, une méthodologie de sélection a été proposée. / Sanitation has always been a major concern for hygiene and public health issues. An on-site sewage facility (OSSF) ensures the collection, transportation, treatment and disposal of domestic wastewater. In France, the OSSF represents 20% of domestic wastewater treatment facilities. This represents a population of 12 million or about five million rural settlements. Among the technologies used in the OSSF, biological filtration systems using a filter media are often implemented. The treatment of wastewater (removal of suspended solids and organic matter) is carried out by the joint actions of filtration, and biodegradation by bacteria developing within the filter media. Historically, materials such as sand and peat are the most used. More recently coconut-based media to replace peat, the extraction of which is prohibited, are in increasing development. However, the carbon footprint of foreign imports of materials such as coconut is very important. Therefore, the use of local agro-industrial by-products could reduce the carbon footprint and make the use of these new filter media more compatible with environmental criteria. The first objective of this PhD thesis is to determine the degree of reliability under real conditions of an innovative filtering medium of lignocellulosic origin that has been proven successful in laboratory conditions. The study is based on the comparison of results obtained in the laboratory and in the field. In the laboratory, a hydraulic approach allowed the characterisation of the filter bed operation. In the field, the evaluation of a compact filter that has been running continuously for more than four years and five other installations has been completed. For all installations, the filter media tested allowed a quality of the discharged water in accordance with the current regulations. The estimated average reduction percentages of COD, BOD5 and TSS, are respectively over 79%, 98% and 88%, thus confirming the important degradation of the organic matter and particulate pollution. The second objective of this PhD thesis is to establish a set of physicochemical parameters to guide the choice of new materials potentially usable in OSSF. To do this, the characterisation of the key physicochemical parameters of new materials and the hydraulic parameters of the corresponding filter beds were carried out. Statistical analysis made it possible to study the correlations between these parameters and the purification performances of the materials. In the end, a selection methodology has been proposed.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Atualmente, tem-se estudado diversos tipos de pré- tratamentos para reduzir a recalcitrância da biomassa lignocelulósica com intuito de aumentar sua digestibilidade química/enzimática, para que esta possa ser utilizada na produção de etanol e/ou outros bioprodutos de valor agregado. Neste estudo avaliou-se o efeito do pré-tratamento de explosão a vapor catalisada por ácido cítrico e hidróxido de sódio, e do pré-tratamento de deslignificação alcalina nas propriedades químicas e estruturais do bagaço de cana-de-açúcar (BCA), bem como sobre o processo de hidrólise enzimática. O pré-tratamento de explosão a vapor foi realizado em reator de 1,4L sob temperatura de 180 °C com tempo de retenção de 5 min. A etapa de deslignificação com NaOH (2% m:m) foi realizada a 120 °C, sob refluxo de 4h. A caracterização química e estrutural da biomassa in natura e pré-tratada foi realizada por FTIR, MEV, DRX. A hidrólise enzimática foi realizada com volume final de 20 mL constituído de 3% de BCA (massa seca), tampão de citrato de sódio 50 mM (pH=5,0) e 10 FPU do complexo enzimático Cellic® CTec 3. Os frascos foram mantidos sob agitação de 150 rpm a 50ºC por 72 h. Retirou-se alíquotas de 1,5 mL após 0, 12, 24, 36, 48 e 72h para determinação de açúcares redutores totais (ART) pelo método do ácido 3,5-dinitrosalicílico (DNS). O bagaço de cana-de-açúcar apresentou 24,22% de lignina, 27,61% de hemiceluloses e 42,77% de celulose, no entanto após o pré-tratamento de explosão a vapor catalisado com ácido cítrico, obteve-se uma biomassa com menor quantidade de hemiceluloses (16,16%) e com formação de fissuras na parede celular da fibra. O bagaço pré-tratado por explosão a vapor com NaOH apresentou completa desestruturação da fibra, remoção de 65% da lignina e preservação da fração hemicelulósica. Depois de submeter à biomassa lignocelulósica sem tratamento e pré-tratada por explosão a vapor ao processo de deslignificação alcalina observou-se a completa desestruturação da matriz lignocelulósica e a solubilização de 85-90% da lignina em todas as amostras. O índice de cristalinidade da biomassa após os prétratamentos teve uma aumento quando comparado ao material in natura, podendo este ser associado à remoção de componentes amorfos, como a lignina e hemiceluloses, e também da fração amorfa da celulose. Frente ao percentual mássico de biomassa utilizada no processo de hidrólise enzimática valor teórico correspondente a 100% de sacarificação equivale, aproximadamente, a 33,0 g.L-1. Após a hidrólise enzimática (72h) da biomassa in natura e pré-tratada por explosão a vapor obteve-se uma maior concentração de ART e um maior percentual de sacarificação para o bagaço de cana obtido a partir da explosão a vapor com NaOH (23,05 g.L-1, 69,15% de sacarificação). Já na hidrólise da biomassa in natura após processo de deslignificação alcalina a concentração de ART aumentou em 18,33 g.L-1, enquanto que para o bagaço pré-tratado por explosão a vapor com água, ácido cítrico e NaOH seguido da deslignificação alcalina o aumento de ART, correspondeu a 19,67 g.L-1, 19,93 g.L-1 e 6,87 g.L-1. Diante da produção de ART após deslignificação notou-se que o percentual de sacarificação para a biomassa sem tratamento elevou-se de 11,88% para 69,15%, enquanto que para o bagaço após explosão a vapor e deslignificação este percentual ficou entre 82,05% - 89,79%. Por fim, cabe ressaltar que no BCA previamente pré-tratado por explosão a vapor com NaOH, a remoção de lignina após o segundo pré-tratamento teve um acréscimo de apenas 20% e a concentração de ART de 6,87 g.L-1. Dessa forma, acredita-se que ao aumentar a concentração da solução de NaOH para realizar a explosão a vapor poderia-se não necessitar da realização da etapa de deslignificação. / Nowadays, there is various types of studied pre-treatments to reduce the lignocellulosic biomass recalcitrance in order to increase its chemical / enzymatic digestibility, so that it can be used in ethanol production and / or other bioproducts value. This study evaluated the effect of steam explosion pretreatment catalyzed by citric acid and sodium hydroxide, and alkaline delignification pretreatment over chemical and structural properties of sugarcane bagasse, as well as the enzymatic hydrolysis process. Steam explosion pretreatment was conducted in a 1.4 L reactor at temperature of 180 °C and 5 min hold time. The delignification step with NaOH (2% m:m) was performed at 120 °C under reflux for 4 hours. Chemical and structural characterization of raw and pretreated biomass was performed by FTIR, SEM and XRD. Enzymatic hydrolysis was performed with final volume of 20 mL consisting 3% SCB on dryweight basis, sodium citrate buffer 50 mM (pH = 5.0) and 10 FPU of Cellic® Ctec 3 enzyme complex. Flasks were kept under agitation of 150 rpm at 50 °C during 72 h. It was analyzed 1.5 mL aliquots after 0, 12, 24, 36, 48 and 72 hours to determinate total reducing sugars (TRS) by acid 3,5-dinitrosalicílic method (DNS). Sugarcane bagasse showed 24.22% lignin, 27.61% hemicelluloses and 42.77% of cellulose, however after steam explosion pretreatment catalyzed by citric acid, it was obtained a biomass with lesser hemicelluloses amount (16.16%) and cracks formation on fiber cell wall were observed. The bagasse pretreated by steam explosion by NaOH showed complete destructuring of fiber and lignin removal was 65%, while hemicellulosic fraction was preserved. After submitting untreated and pretreated lignocellulosic biomass by steam explosion to alkaline delignification process, complete destructuring and solubilization of the lignocellulosic matrix was observed with 85-90% lignin removal in all samples. The crystallinity index of biomass after pretreatments increased when compared to raw material and this could be associated to amorphous components removal, such as lignin and hemicelluloses, and also the amorphous cellulose fraction. The theoretical value corresponding to 100% of saccharification corresponds to approximately 33.0 g·L-1, compared to the mass percentage of biomass used in the enzymatic hydrolysis process. After 72 h enzymatic hydrolysis of raw and pretreated biomass by steam explosion, the highest concentration of total reducing sugars (TRS) and the highest percentage of saccharification were obtained for sugarcane bagasse (23.05 g·L-1, 69,15% saccharification). In in nature biomass hydrolysis, after alkaline delignification process, ART concentration increased 18.33 g·L-1, while pretreated biomass by steam explosion with water, citric acid and NaOH followed by alkaline delignification increase TRS to 19.67 g·L-1, 19.93 g·L-1 and 6.87 g·L-1. After delignification, it was noted that saccharification percentage for untreated biomass increased from 11.88% to 69.15%, while for bagasse after steam explosion and delignification this percentage was between 82.05% - 89.79%. Lastly, it should be noted that in biomass previously pretreated by NaOH steam explosion, lignin solubilization after the second pretreatment had an increase of only 20% and TRS concentration of 6.87 g·L-1. Thus, it is believed that increasing NaOH solution concentration to perform steam explosion could not need to implement delignification step. / Dissertação (Mestrado)

Submitted by Rosângela Cristina Alves (rosangela.alves@etec.sp.gov.br) on 2018-09-26T02:51:48Z No. of bitstreams: 1 Dissertacao-FINAL Rosangela C. Alves - repositorio.pdf: 1876874 bytes, checksum: 2f37062ec0152ad7c5ccde681207d93b (MD5) / Approved for entry into archive by Ana Paula Santulo Custódio de Medeiros null (asantulo@rc.unesp.br) on 2018-09-26T11:31:00Z (GMT) No. of bitstreams: 1 alves_rc_me_rcla.pdf: 1836538 bytes, checksum: 3798050a36146a32df2e62013e4e7c2a (MD5) / Made available in DSpace on 2018-09-26T11:31:00Z (GMT). No. of bitstreams: 1 alves_rc_me_rcla.pdf: 1836538 bytes, checksum: 3798050a36146a32df2e62013e4e7c2a (MD5) Previous issue date: 2018-07-27 / A grande capacidade da agricultura sucroalcooleira, aliada às suas dimensões continentais, faz do Brasil o maior produtor de cana-de-açúcar do mundo. O bagaço desta matéria-prima é um material lignocelulósico, remanescente da moagem dos seus colmos, é composto principalmente por celulose, hemicelulose e lignina, numa estrutura vegetal organizada que deve ser desestruturada mediante pré-tratamentos para disponibilizar a fração polissacarídica (celulose e hemicelulose). Objetivou-se com a presente pesquisa determinar a influência da granulometria do bagaço de cana-de-açúcar na extração/solubilização de hemicelulose na forma de polissacarídeo (macromolécula) e monossacarídeo (xilose). A extração da hemicelulose macromolecular foi realizada em condições otimizadas para bagaço de cana-de-açúcar, 6 % H2O2 m/v a 25 °C durante 4 h, utilizando bagaço selecionado em peneiras de 16, 30, 40, 50 mesh e base (material que passou pela peneira de 50 mesh). Para extração de hemicelulose na forma monomérica foi utilizado um pré-tratamento com ácido sulfúrico diluído 20 % (m/m), sendo que as amostras foram autoclavadas a 121 ºC por 1 h; a xilose no filtrado foi quantificado por HPLC. Foi realizada também a condutividade, determinação do teor de cinzas totais, solubilidade da hemicelulose. Os resíduos sólidos dos pré-tratamentos foram submetidos a hidrólise enzimática (12 FPU/g – Cellic Ctec) para determinação do rendimento em glicose. O bagaço in natura apresentou conteúdo de glucana de 42,25 %, arabinana de 2,62 %, xilana de 25,39 %, ácido acético de 3,38 % e lignina de 13,83 %. A xilana solubilizada em meio alcalino apresentou variação de 36,18 % (bagaço retido na peneira de 16 mesh) a 71,43 % (bagaço retido na base, inferior a 50 mesh), quanto menor o tamanho da partícula, maior a quantidade de xilana solubilizada. Houve uma tendência nos valores obtidos de glucana, conforme diminuiu o tamanho de partícula, aumentou o teor desse polissacarídeo no bagaço pré-tratado em meio ácido. Os valores variaram de 44,28 % (bagaço retido em peneira de 16 mesh) a 66,70 % (bagaço retido na peneira de 50 mesh). Para os materiais pré-tratados em meio alcalino retidos em peneiras de 40, 50 mesh e base ficaram acima do valor encontrado para o bagaço in natura. Considerando como uma característica importante da hemicelulose, todas as amostras apresentaram solubilidade acima de 96 %. A hidrólise enzimática do material pré-tratado em meio alcalino resultou em maior rendimento em glicose (65,37 % de glicose) para todas as frações/granulometrias estudadas, em comparação com meio ácido (32,12 % de glicose), mostrando tendência em aumento com diminuição da granulometria. Os resultados evidenciaram que a granulometria do bagaço de cana-de-açúcar tem influência na extração/solubilização de hemicelulose na forma de polissacarídeo (polimérica) e monossacarídeo (xilose). Conclui-se que, um melhor aproveitamento em conjunto da hemicelulose e celulose ocorreu com as condições experimentais de estudo do pré-tratamento alcalino. / The great capacity of sugar-alcohol agriculture, combined with its continental dimensions, makes Brazil the largest producer of sugar cane in the world. The bagasse of this raw material is a lignocellulosic material, reminiscent of the milling of its stems, is composed mainly of cellulose, hemicellulose and lignin, in an organized vegetable structure that must be destructured by means of pre-treatments to provide the polysaccharide fraction (cellulose and hemicellulose). The objective of this research was to determine the influence of sugarcane bagasse granulometry on the extraction/solubilization of hemicellulose in the form of polysaccharide (macromolecule) and monosaccharide (xylose). Macromolecular hemicellulose extraction was performed under optimized conditions for sugarcane bagasse, 6 % H2O2 m / v at 25 °C for 4 h, using bagasse selected in 16, 30, 40, 50 mesh sieves and base (material which passed through the 50 mesh screen). For the extraction of hemicellulose in the monomeric form, a pretreatment with sulfuric acid diluted 20 % (m/m) was used, and the samples were autoclaved at 121 ºC for 1 h; the xylose in the filtrate was quantified by HPLC. Conductivity, determination of total ash content and solubility of hemicellulose were also performed. The solid residues of the pre-treatments were submitted to enzymatic hydrolysis (12 FPU/g - Cellic Ctec) to determine the glucose yield. The bagasse in natura had glucan content of 42.25 %, arabinana of 2.62 %, xylan of 25.39 %, acetic acid of 3.38 % and lignin of 13.83 %. The solubilized xylan in alkaline medium showed a variation of 36.18 % (bagasse retained in the 16 mesh sieve) at 71.43 % (bagasse retained at the base, less than 50 mesh), the smaller the particle size, the greater the amount of solubilized xylan. There was a tendency in the values obtained of glucana, as the particle size decreased, the content of this polysaccharide increased in the acid treated bagasse. The values varied from 44.28 % (bagasse retained in 16 mesh sieve) to 66.70 % (bagasse retained in the 50 mesh sieve). For materials pretreated in alkaline medium retained in 40, 50 mesh and base sieves were above the value found for the in natura bagasse. Considering as an important characteristic of hemicellulose, all samples had solubility above 96 %. The enzymatic hydrolysis of the pretreated material in alkaline medium resulted in a higher glucose yield (65.37 % of glucose) for all fractions/granulometry studied, compared to acid medium (32.12 % glucose), showing a trend in increase with decreasing granulometry. The results showed that the sugarcane bagasse granulometry influences the extraction/solubilization of hemicellulose in the form of polysaccharide (polymeric) and monosaccharide (xylose). It was concluded that a better use of hemicellulose and cellulose occurred with experimental conditions of the alkaline pretreatment.