Submitted by VIVIANE CRISTINA PADILHA LOPES null (vivianelops@gmail.com) on 2016-12-05T17:47:42Z No. of bitstreams: 1 tese vivi BIBLIOTECA.pdf: 2903583 bytes, checksum: d8537b21926462f650e1ff21b8ca9c26 (MD5) / Rejected by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido não contém o certificado de aprovação. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2016-12-07T12:15:05Z (GMT) / Submitted by VIVIANE CRISTINA PADILHA LOPES null (vivianelops@gmail.com) on 2016-12-12T19:24:22Z No. of bitstreams: 1 tese vivi repositório.pdf: 3145051 bytes, checksum: 8c5d16d927b1c2cbac9b9320e9e9b7ce (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-13T12:41:26Z (GMT) No. of bitstreams: 1 lopes_vcp_dr_rcla.pdf: 3145051 bytes, checksum: 8c5d16d927b1c2cbac9b9320e9e9b7ce (MD5) / Made available in DSpace on 2016-12-13T12:41:26Z (GMT). No. of bitstreams: 1 lopes_vcp_dr_rcla.pdf: 3145051 bytes, checksum: 8c5d16d927b1c2cbac9b9320e9e9b7ce (MD5) Previous issue date: 2016-11-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A degradação biológica da lignina pode ser realizada por fungos de podridão branca, parda e mole, os quais produzem enzimas oxidativas especialmente lacases, manganês peroxidase (MnP) e lignina peroxidase (LiP). Neste trabalho, foi investigada a interação entre diferentes fungos filamentosos para formar consórcios capazes de remover a lignina de resíduos agroindustriais. Após triagens enzimáticas realizadas para LiP, MnP, Lacase e glioxal oxidase (GLOX), 8 isolados apresentaram potencial para aplicação de degradação lignocelulósica e 5 fungos foram selecionados como os melhores produtores das enzimas estudadas. As capacidades de interação e inibição de crescimento dos isolados selecionados foram estudadas para posterior montagem dos consórcios. Desta maneira, 6 diferentes consórcios foram estruturados e submetidos a testes de degradação de lignina utilizando três diferentes resíduos derivados da agroindústria: sabugo de milho, casca de arroz e bagaço de cana. Dados de seletividade (celulose/lignina) foram também utilizados como parâmetro de avaliação dos consórcios. Os consórcios C5 (Tinctoporellus sp. e Marasmiellus sp. e Aspergillus sect. flavi) e C6 (Tinctoporellus sp. e Marasmiellus sp. Fusarium sp.) apresentaram resultados mais coerentes de degradação de lignina, seletividade e atividades enzimáticas no bagaço de cana após 7 dias a 28ºC. A casca de arroz apresentou menor degradabilidade devido a uma maior resistência ao ataque microbiano. O sabugo de milho foi melhor degradado por consórcios que apresentaram habilidade em reduzir a lignina e os polissacarídeos. Sendo assim, os consórcios C5 e C6 foram submetidos aos experimentos de degradação do bagaço de cana por 15 dias. Os resultados revelaram que o consórcio C5 foi mais eficiente no processo de deslignificação e apresentou melhores resultados de seletividade. Para aumentar o rendimento da deslignificação, 3 experimentos de otimização do processo foram realizados utilizando o planejamento experimental do tipo Plackett-Burman (PB). Aumentos sequenciais da deslignificação foram observados a cada experimento, onde as duas melhores condições de cultivo encontradas (PB3) apresentavam: i) inóculo fúngico (7), 8 mM de CuSO4, 0,4 g/L de KH2PO4 e 2,5 g de bagaço de cana (ensaio 10) e ii) inóculo fúngico (7), 20 g/L de glicose, 2 g/L de extrato de levedura, 8 mM de CuSO4 e 2,5 g de bagaço de cana (ensaio 5). As condições de cultivo dos ensaios 10 e 5 permitiram uma porcentagem de deslignificação correspondente a 57,65% e 49,73%, respectivamente. Os melhores resultados de seletividade (celulose/lignina e xilana/lignina) foram, de maneira geral, condizentes com os melhores resultados de deselignificação. Experimentos adicionais foram conduzidos visando avaliação da influência do sulfato de cobre no crescimento dos fungos dos consórcios e nos fungos de maneira isolada, e avaliação da deslignificação em cultivos contendo extrato de malte e bagaço de cana. Os resultados revelaram que a presença do sulfato de cobre não interferiu na eficiência dos consórcios frente à deslignificação. Na presença do sulfato de cobre a porcentagem de seletividade da lignina do bagaço de cana pelo consórcio C5 (53,18%) foi muito próxima à obtida no PB3. Neste teste, houve produção de LiP e MnP, enquanto a lacase foi detectada na ausência do sulfato de cobre. Os resultados da deslignificação dos experimentos em meio malte e bagaço de cana foram inferiores aos resultados com o meio otimizado, destacando a importância da aplicação do planejamento experimental para a otimização de processos. Os resultados do presente trabalho evidenciam o potencial do consórcio C5 para a deslignificação do bagaço de cana em 7 dias de cultivo. / Biological lignin degradation can be performed by white rot, brown, and soft fungi, which are able to produce oxidative enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP) and Laccase. In this work, the interaction between different filamentous fungi (isolated from different environments) was investigated aiming to the structuration of consortia able to remove the lignin from agro-industrial wastes. After the enzymatic screening accomplished to LiP, MnP, laccase, and glyoxal oxidase (GLOX), 8 isolates presented potential application to lignocellulose degradation and 5 were selected as the best producers of the studied enzymes. The interaction and inhibition growth ability of the selected isolates was investigated for the subsequent consortia assembling. In this way, 6 different consortia were pooled and subjected to lignin degradation tests using 3 different waste materials derived from agro-industry: corn cobs, rice husks, and sugarcane bagasse. Data related to selectivity (cellulose/lignin) were also used for the consortia´s evaluation. The consortia C5 (Tinctoporellus sp., Marasmiellus sp., and Aspergillus sect. Flavi) and C6 (Tinctoporellus sp., Marasmiellus sp., and Fusarium sp.) presented more coherent results of lignin degradation, selectivity, and enzymatic activities in sugarcane bagasse after 7 days incubation at 28°C. The rice husk showed lower degradability due to the higher resistance to the microbial attack. The corncob was better degraded by the consortia that showed ability to reduce the amount of lignin and polysaccharides (cellulose and xylan). In this sense, the consortia C5 and C6 were subjected to the experiments related to sugarcane bagasse degradation during 15 days. Results showed that the consortium C5 was more efficient during the delignification process and showed better results of selectivity. In order to increase the delignification efficiency, three experiments related to the process optimization were conducted using Plackett-Burman (PB) experimental design. Sequential improvements in delignification were observed to each experiment and the two better culture conditions found comprised: i) inoculum disks (7), 8 mM CuSO4, 0.4 g/L of KH2PO4, and 2.5 g of sugarcane bagasse (assay 10), and ii) inoculum (7), 20 g/L glucose, 2 g/L yeast extract, 8 mM CuSO4, and 2.5 g of sugarcane bagasse (assay 5). The culture conditions of assay 10 and 5 allowed a delignification corresponding to 57.65% and 49.73%, respectively. In general, the better results of selectivity (cellulose /lignin and xilan/lignin) were consistent with the better results of delignification. Additional experiments were conducted in order to evaluate the effect of copper sulfate on the growth of the consortia fungal strains and the each single fungus, and to evaluate the delignification in cultures containing malt extract and sugarcane bagasse. Results reveal that the presence of copper sulphate did not interfere in the consortia delignification efficiency. In the presence of copper sulphate the percentage of sugarcane bagasse delignification by the consortium C5 (53.18%) was almost the same that one obtained in the PB3. In general, in the presence of copper sulphate LiP and MnP were produced, while laccase was detected in the absence of this compound. Results related to the delignification in medium containing malt extract and sugarcane bagasse were lower than that ones with the optimized medium, highlighting the relevance of the experimental design to the process optimization. Results from the present study show the potential of the consortium C5 for the delignification of sugarcane bagasse in 7 days of cultivation.

Submitted by MARÍLIA GABRIELA DE SANTANA COSTA null (mariliagabrielasc@hotmail.com) on 2017-06-01T19:21:17Z No. of bitstreams: 1 TESE_MARILIA_GABRIELA_SANTANA_COSTA.pdf: 4170522 bytes, checksum: 8ff9e42b797cdb58ebfce7e345bfe167 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-06-02T13:34:14Z (GMT) No. of bitstreams: 1 costa_mgs_dr_jabo.pdf: 4170522 bytes, checksum: 8ff9e42b797cdb58ebfce7e345bfe167 (MD5) / Made available in DSpace on 2017-06-02T13:34:14Z (GMT). No. of bitstreams: 1 costa_mgs_dr_jabo.pdf: 4170522 bytes, checksum: 8ff9e42b797cdb58ebfce7e345bfe167 (MD5) Previous issue date: 2017-04-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A indústria de papel e celulose brasileira ocupa uma posição de destaque no mercado mundial. O Eucalyptus grandis é a espécie mais plantada pelo setor florestal e a principal fonte de matéria-prima para a indústria de papel e celulose. Contudo, seu crescimento em regiões de baixa temperatura é limitado. Contrariamente, a espécie Eucalyptus globulus apresenta melhor crescimento em baixas temperaturas, tornando-se cada vez mais interessante para a indústria nacional de papel e celulose. Tendo em vista que a temperatura é um modulador chave no metabolismo das plantas, uma análise global do proteoma foi realizada em plântulas cultivadas em temperaturas controladas, visando a identificação de proteínas diferencialmente reguladas entre as espécies e entre as diferentes condições de cultivo, detecção de vias metabólicas estimuladas pela temperatura e detecção de alterações no metabolismo antioxidante e de lignificação. A estratégia adotada permitiu a identificação de 3.111 proteínas caulinares, representando aproximadamente 9% do proteoma predito para a espécie modelo E. grandis. O perfil de expressão gênica, em termos de número de proteínas identificadas, corroborou com o padrão de densidade gênica para os cromossomos de Eucalyptus. Foram identificadas proteínas envolvidas em diversas vias metabólicas, com destaque para as vias relacionadas com o metabolismo energético, de aminoácidos e nucleotídeos, de lipídeos e carboidratos. Com relação às proteínas diferencialmente reguladas, 16 proteínas apresentaram alteração em sua abundância somente em E. grandis, 38 somente em E. globulus e 12 apresentaram alterações em ambas as espécies. Além disso, um total de 103 proteínas antioxidantes foram detectadas nos caules de ambas as espécies. O perfil de regulação frente ao estímulo térmico, revelou que as alterações nas proteínas antioxidantes são mais proeminentes quando as mudas de Eucalyptus foram expostas a altas temperaturas. Com relação à biossíntese de lignina, a nossa abordagem proteômica resultou na identificação de 27 das principais enzimas envolvidas neste metabolismo, corroborando com as predições de genes e o “toolbox” de lignificação proposto. A análise quantitativa revelou diferenças na abundância de isoformas específicas dependendo do estímulo térmico em que as plantas foram expostas; contudo, o tratamento a alta temperatura foi aquele que induziu mais diferenças entre os proteomas relacionados com a lignificação de E. globulus e de E. grandis. A partir dos resultados obtidos, conclui-se que o estímulo térmico promove alterações importantes no proteoma caulinar jovem de Eucalyptus, evidenciando aumento na atividade antioxidante em plantas expostas ao calor e expressão diferencial mais proeminente em plantas submetida ao frio. / The Brazilian pulp and paper industry occupies a prominent position in the world market. The Eucalyptus grandis is the most used species in the forest sector and the main source of raw material for Brazilian industry. However, its growth in low temperature regions is limited. In contrast, the Eucalyptus globulus species shows a better growth at low temperatures, for whom interest has increased within the national pulp and paper industry. Since temperature is a key modulator in plant metabolism, a global proteome analysis was performed on seedlings grown at controlled temperatures, aimed at the identification of differentially regulated proteins between the species and between the different culture conditions, detection of metabolic pathways stimulated by temperature and detection of alterations in antioxidant metabolism and lignification. The strategy used here allowed the identification of 3,111 stem proteins, representing approximately 9% of the predicted proteome of the E. grandis model species. The gene expression profile, in terms of the number of proteins identified, corroborated with the gene density pattern for the Eucalyptus chromosomes. Proteins involved in several metabolic pathways have been identified, especially the pathways related to energy metabolism, and metabolism of amino acids, nucleotides, lipids and carbohydrates. Regarding the differentially regulated proteins, 16 proteins showed alterations in their abundance only in E. grandis, 38 only in E. globulus and 12 presented alterations in both species. In addition, a total of 103 antioxidant proteins were detected in the stems of both species. The regulation profile upon thermal stimulus revealed that changes in antioxidant proteins are more prominent when Eucalyptus seedlings were exposed to high temperatures. Regarding the biosynthesis of lignin, our proteomic approach resulted in the identification of 27 of the main enzymes involved in this metabolism, corroborating with the gene predictions and the proposed lignin toolbox. Quantitative analysis revealed differences in the abundance of specific isoforms depending on the thermal stimulus to which the plants were exposed; however, the high temperature treatment was the one that induced more differences between the proteomes related to the lignification of E. globulus and E. grandis. The results demonstrate that thermal stimuli cause important alterations in the Eucalyptus stem proteome, evidenced by the increase in the antioxidant activity in plants exposed to heat and the most prominent differential expression in plants submitted to cold environments.

A busca por novas tecnologias que visam à produção de biocombustíveis renováveis, especialmente bioetanol e outros biomateriais, tem se intensificado nos últimos anos. Há um interesse mundial crescente na limitação dos impactos ambientais e mudanças climáticas através da substituição de produtos petroquímicos por análogos ambientalmente corretos, a fim de alcançar uma economia mais sustentável. Além disso, as plataformas biorrefinarias lignocelulósicas necessárias para a produção de bioetanol representam uma oportunidade de estimular novos mercados para o setor agrícola e aumentar os empregos locais, contribuindo para o desenvolvimento das economias emergentes. No entanto, a maioria dos processos de conversão são baseados no conhecimento empírico, exigindo estudos mais aprofundados sobre os fatores envolvidos na hidrólise enzimática da celulose, tais como características biomassas, a otimização da etapa de pré-tratamento, bem como das atividades das enzimas e seus mecanismos de ação. Assim, com o objetivo de contribuir para a viabilização e implantação das tecnologias de produção do etanol lignocelulósico, na primeira parte deste trabalho de doutorado, foi realizada a purificação da β-glicosidase do fungo Aspergillus Níger (NaBgl1), principal enzima do coquetel comercial Novozymes 188, e sua caracterização bioquímica e biofísica. As análises de espalhamento de raios-x a baixo ângulo revelaram uma organização multidomínios desta enzima, com uma estrutura molecular de girino semelhante ao encontrado para as celulases. A sua estrutura é composta por um domínio catalítico N-terminal e um domínio fibronectina de tipo III (FnIII) na região C-terminal, conectados entre si por um longo linker com uma inserção de 100 resíduos de aminoácidos numa conformação estendida. Apesar desta estrutura molecular incomum, os ensaios de eletroforese capilar revelaram um perfil processividade característico de β-glucosidases, e os ensaios enzimáticos confirmaram, também, a ausência de atividade em substratos poliméricos. Nos ensaios adosrção com diferentes compostos poliméricos, a enzima β-glicosidase mostrou uma capacidade de adsorção elevada em lignina. Os mecanismos de ligação FnIII-lignina foram elucidados por simulações de dinâmica molecular, que confirmaram apresença de vários sítios de ligação à lignina no domínio FnIII da enzima. Como segunda parte da presente tese, diferentes biomassas, como bagaço de cana, resíduos de casca de eucalipto e gramíneas (Panicum maximum, Pennisetum purpureum e Brachiaria brizantha) foram submetidas a vários métodos de pré-tratamento (ácido diluído, alcalino, sulfito e água quente) em diferentes condições de tratamento e avaliadas quanto ao seu potencial para a produção de bioetanol. As biomassas in natura e pré-tratadas foram caracterizadas quanto à sua composição química por métodos cromatográficos, ressonância magnética nuclear e espectroscopia de infravermelho por transformada de Fourier; o índice de cristalinidade das amostras foi determinado por método químico e difração de raios-x; as análises morfológicas foram realizadas por microscopia eletrônica de varredura; e os resultados da caracterização foram correlacionados com os perfis de sacarificação enzimática encontrados para cada uma delas. / The search for new technologies aimed at the production of renewable biofuels, specially bioethanol, and other biomaterials has intensified in recent years. There is an increasing world-wide interest in the limitation of environmental impact and climate change by replacing petrochemical products with environment-friendly analogues in order to move towards a sustainable economy. In turn, the lignocellulosic biorefining platforms required for ethanol production present an opportunity to stimulate new markets for the agriculture sector and increase domestic employment, contributing to the development of emerging economies. However, most of conversion processes are based on empirical knowledge, demanding thorough studies about the factors involved on enzymatic hydrolysis of cellulose, such as biomasses characteristics, optimization of pretreatment steps and enzymes activities and molecular action mechanisms. Aiming to contribute for the viability and establishment of lignocellulosic ethanol technologies, on the first part of the present thesis, we performed the purification of main Aspergillus niger β-glucosidase (AnBgl1) from the commercial cocktail Novozymes 188 and its biochemical and biophysical characterization. The small angle x-ray scattering analysis revealed a multidomain organization, with a tadpole-like molecular shape similar to that found for cellulases. Its structure is composed by a N-terminal catalytic domain and a fibronectin type III-like (FnIII) C-terminal domain, connected by a long linker with a 100 aminoacids residues insertion in a extended conformation. In spite of this uncommon molecular structure, capilar zone electrophoresis assays revealed a processivity profile characteristic of β-glucosidases and the enzymatic assays confirmed no-activity on polymeric substrates. On the pull-dowm assays with different polymeric compounds, the β-glucosidase showed a high adsorption ability to lignin. The FnIII-lignin binding mechanisms were elucidated by molecular dynamics simulations, confirming the multiple binding sites to lignin in the enzyme FnIII domain. As a second part of the present thesis, different biomasses such as sugarcane bagasse, eucalyptus bark residues and grasses (Panicum maximum, Pennisetum purpureum and Brachiaria brizantha) were submitted to several pretreatment methods (diluted acid, alkaline, sulfite and hot water) at various conditions and evaluated about their potential to bioethanol production. The raw and pretreated biomasses were characterized about their chemical composition by chromatographic methods, nuclear magnetic ressonance and Fourier transformed infrared spectroscopy; the crystallinity index was determined by chemical method and x-ray diffraction; morphological features were analysed by scanning electron microscopy; and the characterization results were correlated to their enzymatic saccharification profiles.

Para tornar a biomassa disponível para aplicações em biocombustíveis, produtos químicos e materiais, algumas propriedades indesejáveis, como alto teor de umidade e baixa densidade, devem ser corrigidos. Uma solução para estes problemas seria a densificação da biomassa. O processo de densificação da biomassa e seus resíduos, como a briquetagem e a peletização, são muito conhecidos e utilizados, entretanto, não existe um consenso sobre os mecanismos de ligação que ocorre entre as partículas da biomassa densificada e a influência que as variáveis da matéria-prima e do processo de densificação exerce sobre eles. Compreender os mecanismos de ligação entre as partículas da biomassa densificada é fundamental para determinar os parâmetros da matéria-prima e do processo de densificação, assim como os ensaios que devem ser realizados para medir a qualidade do produto densificado. Este trabalho teve como objetivo principal analisar o efeito das variáveis do material e do processo de densificação da serragem de madeira nas propriedades do produto densificado e a sua influência sobre os mecanismos de ligação entre as partículas do material. O material utilizado neste trabalho foi a serragem de Eucalyptus sp. Foram analisados o efeito do teor de umidade do material e a temperatura do processo de densificação na densidade e resistência mecânica do produto densificado, como também a influência da composição química da serragem na transição vítrea do material. A serragem de eucalipto foi caracterizada por análise termogravimétrica (TGA), caracterização química e espectroscopia de infravermelho por transformada de Fourier (FTIR). A caracterização de serragem densificada foi realizada por densidade aparente, expansão volumétrica, ensaio mecânico de compressão, microscopia eletrônica de varredura (MEV), microscopia ótica (MO), espectrometria de massa de íons secundários por tempo de voo (ToF-SIMS) e análise dinâmico-mecânica (DMA). Os resultados mostraram que no processo de densificação sem aquecimento, o teor de umidade ideal da serragem de eucalipto foi o de aproximadamente 10%, favorecendo as forças intermoleculares entre as partículas do material. Na densificação da serragem de eucalipto em diferentes temperaturas, a lignina agiu como um aglutinante natural, formando pontes sólidas entre as partículas do material em temperaturas superiores à 140°C. A transição vítrea encontrada para a lignina da serragem de eucalipto foi da ordem de 135°C. Em conclusão, as variáveis do material e do processo de densificação influenciaram nos mecanismos de ligação, afetando as propriedades da serragem de madeira densificada. Os principais mecanismos de ligação encontrados na serragem de madeira densificada foram interações intermoleculares e pontes sólidas. / Making biomass available for investments in biofuels, chemicals and materials, some undesirable properties as high moisture content and low density must be corrected. One solution to these problems would be densification of biomass. The densification process of biomass and waste materials, such as briquetting and pelletizing, are well known and used, however, there is no consensus about the connection mechanism occurs between the particles of the densified biomass and the influence of the variables of raw material and densification process has on them. Understanding the connection mechanism between the particles of the densified biomass is critical in determining the parameters of the raw material and the densification process, as well as the tests that should be performed to measure the quality of densified product. This study aimed to analyze the effect of varying the material and the densification process of sawdust in the densified product features and their influence on the binding mechanism between the particles of the material. The material in this work was the sawdust Eucalyptus sp. It was analyzed the effect of the material moisture content and temperature of the densification process in the density and mechanical strength of the densified product, but also the influence of the chemical composition of sawdust on the glass transition of the material. The eucalyptus sawdust was characterized by thermogravimetric analysis (TGA), chemical analysis and Fourier transform infrared spectroscopic (FTIR). The characterization of densified sawdust was performed by bulk density, volumetric expansion, compression mechanical testing, scanning electron microscopy (SEM), optical microscopy (OM), mass spectrometry of secondary ions time of flight (ToF-SIMS) and dynamical mechanical analysis (DMA). The results showed that without the heat densification process, the ideal moisture content eucalyptus sawdust was about 10%, favoring the intermolecular forces between particles of the material. In eucalyptus sawdust densification at different temperatures, lignin acted as a natural binder to form solid bridges between particles of the material at temperatures higher than 140°C. The glass transition temperature of the lignin found to eucalyptus sawdust was of the order of 135°C. In conclusion, the variables of material and densification process influenced the binding mechanism affecting the properties of the densified wood sawdust. The main binding mechanism found in densified wood sawdust were intermolecular interactions and solid bridges.

C'est dans un contexte marqué par une demande croissante en énergie primaire, une diminution des ressources et dans un souci de protection de l'environnement que le biocarburant de 2ème génération est développé. Cependant, ce biocarburant est non viable économiquement. L’optimisation, le contrôle et la connaissance des cinétiques régissant les procédés de fabrication de ce bioéthanol sont donc des éléments capitaux. Dans cette étude, le potentiel de la calorimétrie isotherme pour surveiller les réactions d'hydrolyse et de fermentation est testé.Les résultats montrent que cette méthode est efficace. En effet, celle-ci a permis de mettre en évidence l'importance du ratio enzyme/substrat pour maximiser le rendement et de déterminer un meilleur cocktail composé de cellulases + cellobiose déshydrogénase (CDH) qui permet la production d'une certaine quantité d'acide gluconique, qui pourrait améliorer l'attractivité de ce biocarburant. Ces mêmes essais ont également permis de déterminer la chaleur de l'hydrolyse de la paille de blé, qui est 32,18 ± 3,18 J.g-1 (gramme de sucres produits).Les mesures obtenues ont été utilisées pour déterminer les constantes cinétiques des cellulases + CDH sur la paille de blé et les résultats montrent que ce cocktail enzymatique est plus rapide à 45 °C dans la gamme de températures testée (40-55°C) avec une vitesse de 7,36 ± 0,62 mmol/L.min.Par ailleurs, les essais avec un calorimètre à échelle laboratoire ont montré que même si celui-ci ne mesure pas avec précision les chaleurs engendrées par les réactions d'hydrolyse et de fermentation, celui-ci donne de bonnes indications sur le déroulement et l'avancement de ces réactions. / Second generation biofuel is developed in a context marked by an increasing demand for primary energy, a decrease in resources and in environmental protection concernsHowever, this biofuel is not economically viable. Optimization, control and knowledge of the kinetics governing this bioethanol production processes are crucial elements.In this study the potential of isothermal calorimetry to monitor hydrolysis and fermentation reactions is tested.The results show that the isothermal calorimetry is an effective method. Indeed this method allowed determining that the substrate/enzyme ratio is an important parameter of the hydrolysis yield.Furthermore it has determined a better enzyme cocktail consisting of Cellulases + Cellobiose Dehydrogenase (CDH) which allows the production of a certain amount of gluconic acid, which could improve the attractiveness of these second-generation biofuels. These same tests also determined the hydrolysis heat of wheat straw which is 32.18 ± 3.18 J.g-1 (gram reducing sugars product).The measurements obtained were used to determine kinetic constants cellulases + CDH on wheat straw and the results show that this enzyme cocktail is faster at 45 ° C in the range of temperatures tested (40 - 55°C) with a speed of 7.36 ± 0.62 mmol/L.min.In addition, testing with a laboratory-scale calorimeter showed that even if this tool does not accurately measure the heat generated by the hydrolysis reaction and fermentation, it gives a good indication of the development and advancement of these reactions.

L'Analyse de Cycle de Vie (ACV) a été explicitement employée, dans le cadre législatif en Europe et aux Etats-Unis afin de quantifier les bénéfices de filières biocarburants vis-à-vis des objectifs de réduction d'émissions des gaz à effet de serre (GES) et d'indépendance énergétique. Nous investiguons, au cours de cette thèse, la concordance du développement des biocarburants avancés (éthanol lignocellulosique, Biomass to Liquids - BTL, EMHV/HVO à partir de microalgues) avec ces objectifs par le biais de deux travaux indépendants. Le premier est une méta-analyse (synthèse de littérature) des études ACV appliquées aux filières de biocarburants avancés. Dans cette partie de la thèse, en utilisant des méthodes économétriques, nous identifions et quantifions les facteurs qui impactent le plus les résultats d'émissions de GES au long du cycle de vie des produits étudiés et calculons l'intervalle de confiance pour ces résultats. Le deuxième travail est une ACV conséquentielle et prospective illustrée par une étude de cas concernant l'introduction du BTL en France. Nous avons adapté un modèle prospectif de type TIMES pour la réalisation d'évaluations environnementales. De cette façon, nous prenons en compte des mécanismes économiques à l'origine d'impacts sur d'autres systèmes de la production de biocarburants. Ainsi, nous contribuons aux développements méthodologiques autour de l'ACV conséquentielle et prospective avec des discussions sur la définition de l'unité fonctionnelle, la définition des frontières du système, l'inclusion d'aspects dynamiques dans la caractérisation d'impacts, l'utilisation de scénarios (différents contextes politiques et économiques) et la réalisation des analyses de sensibilité sur les frontières du système pour mettre en évidence les limites du modèle utilisé. / Life Cycle Assessment (LCA) has been used in legislative texts in Europe and in the United States to quantify the benefits of biofuel production in terms of greenhouse gas (GHG) emissions reductions and energy security. In this thesis, we propose two independent approaches to investigate the compliance of advanced biofuels (cellulosic ethanol, Biomass to liquids - BTL, microalgae FAME/HVO) production with these objectives. The first one is a meta- analysis (literature synthesis) of LCA studies concerning advanced biofuels. Using econometric methods, we are able to identify and quantify the main factors impacting GHG emission LCA results. Also, we estimate a confidence interval for these results for each type of advanced biofuel in question. The second part of this work is a consequential and prospective LCA illustrated by a case study about the introduction of BTL technology in France. A long-term TIMES-type energy model was adapted for environmental evaluations in order to capture impacts occurring in affected systems through economic mechanisms. We contribute, with this work, for a proper systematization of consequential and prospective LCA. We discuss the functional unit and system boundaries definitions, the use of dynamic impact characterization factors, the use of scenarios (different political and economical contexts) and the exploration of the system's boundaries to bring attention to the limits of the model employed.

Dans un contexte du développement durable, la bioproduction de synthons (molécules plateformes, ou building blocks) de façon biosourcée à partir de biomasse végétale, constitue une voie de remplacement des actuelles molécules prétrosourcées. Ce travail de thèse concerne spécifiquement l’utilisation de la biomasse lignocellulosique, renouvelable et abondante, pour la production de deux acides organiques d’intérêt : l’acide itaconique et l’acide fumarique. Ces molécules ont été choisies notamment car elles peuvent générer des polymères aux propriétés intéressantes. Les travaux expérimentaux ont consisté à utiliser le son de blé et les rafles de maïs, déchets agricoles, comme substrats pour la fermentation de quatre souches de champignons filamenteux du genre Aspergillus. Des criblages des meilleures conditions fermentaires montrent que les rafles de maïs permettent d’atteindre des rendements plus élevés, surtout en fermentation en milieu solide. Parmi d’autres résultats marquants, nous avons montré pour la première fois la capacité d’Aspergillus oryzae à produire l’acide itaconique. L’ensemble de nos résultats montrent que l’utilisation de la biomasse lignocellulosique est une alternative prometteuse pour la production de ces deux synthons d’intérêt industriel. / In the context of sustainable development, the bioproduction of building blocks (chemical platforms) from biomass is way to substitute the current fossil-based chemical molecules. This thesis is focused on the use of lignocellulosic biomass, renewable and abundant, towards the production of two organic acids (potential building blocks): itaconic acid and fumaric acid. These molecules have been chosen especially because they can generated polymers with interesting properties. The experimental work consisted in using wheat bran and corn cobs, agricultural wastes, as substrates for fermentation by four strains of filamentous fungi from Aspergillus genus. Screenings of the best fermentation conditions show that enzymatically pretreated corn cobs, especially in solid state fermentation achieve higher yields, especially in solid state fermentation. Among other notable results, we have shown for the first time the ability of Aspergillus oryzae to produce itaconic acid. Overall, our results show that the use of lignocellulosic biomass is a promising alternative for the production of these two building blocks of industrial interest.

Over the last several decades, there have been a tremendous developments and greatinnovations in photocatalysis process along with the development of efficient nanosized catalysts for simple approach and economic viability. In this study, magnetic core@doubleshell nanomaterials were investigated and synthesized in lab with three-step innovative approach where Fe3O4 nanoparticles (NPs) were produced first to act as cores without using any surfactants. The magnetite/silica core–shell structure was then prepared by hydrolysis of tetraethoxysilane (TEOS) in the presence of core particles under alkaline conditions. And the outermost shell, the α-Fe2O3/TiO2 nanoparticles, were grown over magnetic core of Fe3O4@SiO2 using coprecipitation and calcination method. Furthermore, the Fe3O4@SiO2@α-Fe2O3/TiO2 NPs were then loaded on the reduced graphene oxide (r-GO) using hydrothermal method and are also mixed by kneading with the layered double hydroxides (LDH) of Mg2+ and Al3+. These nanoparticles were characterized with scanning electron microscope (SEM), transmission electron microscope (TEM), dynamic light scattering (DLS), and energy dispersive X-ray spectroscopy (EDS). Different model compounds like microcrystalline cellulose (90 μm), D-xylose, and sodium lignosulfonate representing cellulose, hemicellulose, and lignin, respectively, were converted to valuable chemicals with different NPs under visible light for different time periods. For example, valeric acid (VA) and vanillylmandelic acid (VMA) were produced when cellulose was used for the conversion with core-double shell NPS which were quantified using high performance liquid chromatography (HPLC). Similar approach was adopted for the conversion of brewers’ spent grain (BSG), a lignocellulosic biomass, without oxygen under visible light, which yielded ethanol as the main product along with other sugars and acids of very low concentrations. The magnetic property of the nanomaterials made it easy for recycle and reuse. From a sustainability point of view, this study will fill a large need in the biomass photocatalysis field by developing core-shell multi-functional photocatalysts for direct transformation of lignocellulose into valuable chemicals under low temperatures, atmospheric pressure, and visible light from the sun.

Les levures oléagineuses ascomycètes font partie des productrices de lipides les plus connus de notre époque. Elles peuvent produire des lipides, des molécules chimiques dérivées et des acides organiques à partir de sucres simples ou complexes. Nous avons choisi les levures du genre Blastobotrys afin de définir un nouvel organisme modèle pour la production d'acides gras et de lipides, car ces levures sont capables de synthétiser et de stocker naturellement 15 à 25% de lipides dans leur biomasse sèche à partir de glucose et xylose, soit plus que Yarrowia. lipolytica dans les mêmes conditions. La plupart des études d'ingénierie métabolique connues ont utilisé les levures du genre Blastobotrys dans une logique de production de molécules différentes des lipides. Nous avons caractérisé les traits oléagineux de deux souches appartenant à deux espèces du genre Blastobotrys, en utilisant comme substrats du glucose, xylose, glycérol, fructose, cellobiose, saccharose, galactose avec un rapport C/N de 60. La plus forte production de lipides vient du cellobiose (35%) et du glucose (32%).Ensuite, afin de mieux comprendre le métabolisme des lipides des levures du genre Blastobotrys, nous avons exploré l'effet de la température sur leur physiologie, production de lipides et le profil lipidique en utilisant un milieu YNB contenant 30 g/L de glucose. Nous n'avons pas trouvé de différence marquée de transition de formes entre les formes hyphes et les levures en milieu YNB sous l’effet de quatre températures (28°C, 37°C, 42°C, 45°C), mais la production des lipides est favorisée à 28°C et le C18:1 est l'acide gras le plus abondant dans le profil lipidique. Nous avons transformé avec succès l’espèce B. raffinosifermentans grâce au système Xplor2. Nous avons pu augmenter l'accumulation de lipides en sur-exprimant deux diacylglycérol acyltransférase endogènes, DGA1 et DGA2. Le niveau d’expression élevé de DGA1 dans nos mutants n’est pas corrélé à une production élevée de lipides alors que celui de DGA2 l’est. Notre meilleure souche, dérivée de la souche parentale G1212, a produit 26,5% de lipides à partir de 30 g/L de glucose en culture en flasque. Ce travail représente l’une des premières ingénieries métaboliques de souches de Blastobotrys pour la production de lipides. Ce sont donc des levures oléagineuses comme Y. lipolytica avec un potentiel biotechnologique avéré. / Ascomycetous oleaginous yeasts are among the highest known producers of lipids of our era that may supply lipids compounds, derived chemicals and organic acids from simple or complex carbon sources. We chose oleaginous yeasts species of Blastobotrys genus for defining a new model organism for fatty acid production and lipids, because these oleaginous yeasts natively produce higher lipids rate than Yarrowia lipolytica in the same conditions and can metabolize glucose and xylose. Most of the metabolic engineering studies on these yeast species focused on other molecules compounds than lipids. We characterized the oleaginous traits of two strains belonging to two different species of genus Blastobotrys, using glucose, xylose, glycerol, fructose, cellobiose, sucrose, galactose, starch and oleic acid as substrates with a C/N ratio of 60. We found the higher lipid production (35%) on cellobiose and glucose (32%).Next, in order to further understand the lipid metabolism in Blastobotrys, we explored the effect of temperature on cell physiology, lipid production and lipid profile using YNB medium with 30 g/L glucose. No markedly transition were found from the hyphae to budding form or reversely on YNB medium under four temperatures (28°C, 37°C, 42°C, 45°C). The lipids production is favored at 28°C and C18:1 is the most abundant fatty acid in the lipid profile. We successfully transformed the yeast species B. raffinosifermentans using the Xplor2 system. We increased lipid accumulation by over-expressing two native diacylglycerol acyltransferase genes, DGA1 and DGA2. Our best strain, derived from the parental strain G1212, produced 26.5 g/L lipid from 30g/L glucose in shake-flask experiments. This strain also produced citric acid like Y. lipolytica. We didn’t find significant overall elevated expression in lipid synthesis pathway for DGA1 gene when lipid production was favored on contrary to DGA2 gene. This work represents one of the first metabolic engineering of B. adeninivorans for lipid production.

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