It is widely known that fossil fuels are limited; consequently, the generation of new sources of energy in a clean and environmentally friendly manner is a research priority. Bioethanol appears to be one potential solution, especially second-generation production from renewable biomass. In order to use lignocellulosic feedstock to produce bioethanol, its polysaccharide components, cellulose and hemicellulose, must be hydrolysed into soluble sugars, which can then be converted into ethanol by fermentative microorganisms such as Geobacillus thermoglucosidasius TM242 used by the company ReBio Technologies Ltd. To date, the cost of commercial enzymes used during the hydrolysis process remains a major economic consideration in the production of second-generation bioethanol as an alternative fuel. The research project presented in this thesis aims to improve this rate-limiting step of microbial bioethanol production through an investigation of the different enzymes associated with hemicellulose hydrolysis. Firstly, the TM242 genome sequence revealed a number of genes encoding glycoside-hydrolases. Six of these genes were cloned and expressed in E. coli and the recombinant enzymes characterised; three of them, two β-xylosidases and an α arabinofuranosidase, are relevant to xylan hydrolysis, and were found to be highly active and thermostable. Crystallisation of one of the β-xylosidases permitted the determination of a high-resolution (1.7 Å) structure of the apo-enzyme along with a lower resolution (2.6 Å) structure of the enzyme-substrate complex, resulting in the first reported structure of a GH52 family member (Espina et al., 2014). Secondly, as the TM242 microorganism lacks xylanase enzymes, four genes encoding xylanases from closely-related Geobacillus strains were cloned and expressed in E. coli, with one of them being also successfully cloned and expressed in G. thermoglucosidasius TM242. This heterologous xylanase was secreted in active form representing an enhanced biomass utilisation by TM242. In conclusion, it is felt that the findings presented here have the potential to make a valuable contribution towards second-generation bioethanol production.

A maceração do milho é uma das etapas mais importantes para seu processamento através da moagem por via úmida. Consiste em uma série de processos que envolvem fermentação e hidrólise química de forma a permitir a separação adequada de seus componentes. Trata-se de uma etapa demorada que requer alto investimento em tanques e utilidades. No presente trabalho buscou-se avaliar o uso de enzimas para auxiliar na redução do tempo de maceração de milho dentado, em escala laboratorial, produzido na região do triângulo mineiro. Para tal, os grãos foram submetidos a diferentes tratamentos com as enzimas xilanase e protease. A ação enzimática foi comparada a padrões de maceração convencionais. Os resultados obtidos mostraram que o uso de enzima permite diminuir o tempo de maceração em torno de 18 horas assim como reduzir a quantidade de SO2 adicionada ao processo de maceração. / Corn steeping is one of the most important stage in the corn wet milling process. It consists of several processes involving fermentation and chemical hidrolysis to enable separation of different fractions of the grain. This process is time and capital investment intense. In the present work it was studied the use of enzymes to reduce the steeping time for dent corn, produced in the southeast of Brazil, in laboratory scale. The evaluated enzymes were xilanase and protease. The enzyme performance was compared with the regular steeping process. The final result has shown that the use of enzymes enabled to reduce steeping time in 18 hours as weel as the amount of added SO2 in the steeping process.

Orientador: Sâmia Maria Tauk Tornisielo / Banca: Jonas Contiero / Banca: Luis Henrique Souza Guimarães / Resumo: Neste estudo, foram isoladas 67 linhagens de fungos filamentosos de solo de área de caatinga, sendo as coletas efetuadas em períodos seco e chuvoso, com o objetivo de se conhecer a biodiversidade deste bioma não explorado e avaliar o potencial destes fungos na produção de xilanase. Algumas linhagens não foram identificadas por inexistência de metodologias específicas e outras foram identificadas através de métodos microscópicos e bioquímicos. Foi efetuada uma triagem dos fungos potencialmente produtores desta enzima em meio de Vogel contendo xilano 1% como única fonte de carbono e avaliou-se através do Método Turbidimétrico Automatizado (Bioscreen-C), a melhor fonte de carbono para crescimento dos fungos selecionados. Os resultados obtidos nos cultivos em MFS, foram inexpressivos quando comparados aos obtidos em MFT. Os melhores produtores de xilanase em MFT foram cultivados em meio líquido de Vogel e MFT com adição individual de outras fontes de carbono a 1% (carbometilcelulose- CMC, xilano e a melhor fonte de carbono para crescimento, determinada pelo Bioscreen-C) em temperaturas apropriadas, pH 5, por 5 dias, inóculo de 1 x 107 esporos/mL. O CMC causou repressão catabólica na síntese de xilanase por estes fungos tendo a adição do xilano apresentado o mesmo efeito, com exceção de Trichoderma viride, que teve sua atividade aumentada para 143,0 U/mL e Aspergillus niger 11 que teve sua atividade aumentada de 33,4 U/mL para 57,1 U/mL / Abstract: In this study, 67 strains of filamentous fungi were isolated from caatinga area, the collections were performed during the dry and rainy period, aiming to know the biodiversity of this not explored bioma and to evaluate the potential of these fungi to produce xylanase. Some of the strains were not identified due to the lack of specific methodologies and others were identified through microscopic and biochemical methods. A selection of the fungi which were considered potentially producers of xylanase was carried out in Voguel medium containing 1% of xylan as an only carbon source, and through a Automatized Turbidimetric Method (Bioscreen - C) the best carbon source for the fungi growth was evaluated. The results obtained in SBM were not expressive when compared to the ones obtained in WBM. The best xylanase producers in WBM were cultivated in Voguel liquid medium and WBM adding individually other carbon sources at 1% (carboxymethylcellulose -CMC; xylan; and the best growth carbon source determined by Bioscreen-C) at appropriated temperatures, pH 5,0 ; for 5 days, and a spore concentration of 1 x107 spores/mL. The CMC addition caused catabolic repression in xylanase production by these fungi, xylan addition showed the same effect, but Trichoderma viride 13 and Aspergillus niger 11 which have their activities increased from 39,21 U/mL to 143,0 U/mL and from 33,4 U/mL to 57,1 U/mL, respectively / Mestre

Orientador: Francisco Maugeri Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T15:34:14Z (GMT). No. of bitstreams: 1 Motta_FelipeBastos_M.pdf: 972824 bytes, checksum: a462e688ab46d2def7974c05146d274b (MD5) Previous issue date: 2008 / Resumo: A endo-1,4-ß-xilanase (E.C. 3.2.1.8), comumente chamada de xilanase, possui grande aplicação em diferentes tipos de indústrias tais como a de ração animal, alimentos, têxteis, de papel, de produção de etanol a partir de biomassa, entre outras. Isso se deve ao fato de que esta enzima atua na hidrólise da xilana, o segundo mais abundante polissacarídeo encontrado na natureza, que está presente ligado a hemicelulose da parece celular de plantas. O objetivo deste trabalho foi selecionar, através do método de screening em placas de Petri, e avaliar a atividade enzimática de leveduras silvestres isoladas de diversas regiões do Brasil (Mata Atlântica, Floresta Amazônica, Cerrado e Pantanal) quanto à produção de xilanase. Para o screening em placas, utilizou-se um meio de cultura no qual a única fonte de carbono era a xilana para induzir a produção da enzima. De um total de 349 microrganismos analisados, foram obtidas 84 cepas produtoras, porém somente 37 possuíam um índice de relação enzimática (diâmetro do halo descolorido / diâmetro da colônia) maior que 2,5. Em função deste resultado prosseguiu-se com a seleção dos melhores microrganismos para a produção da enzima em meio líquido. Após observações preliminares apenas 9 cepas obtiveram um crescimento satisfatório em meio líquido a 30ºC e 150 rpm. Estes foram avaliados em dois meios de culturas diferentes em relação à atividade enzimática do caldo enzimático à 50ºC por 5 min em shaker. Dentre estes, os microrganismos AAD5 e AY10 se destacaram por apresentarem a atividade enzimática em torno de 2 µmol/mL.min . Posteriormente, foi realizada a caracterização das enzimas produzidas, sendo que a proveniente do microrganismo AAD5 possui condições ótimas na faixa de 57,5 ¿ 67,5 ºC e pH entre 4,7 ¿ 5,5, além de meia vida de 21,33 horas a 52ºC e pH 5,3, com vmax de 1,77 µmol/mL.min e Km de 0,44 g/L. Já a enzima produzida pela cepa AY10 possui condições ótimas na faixa de pH entre 4,8 ¿ 4,1 à temperatura de 80°C, além de meia vida de 11,21 horas a 72ºC e pH 5,3, com vmax de 5,47 µmol/mL.min e Km de 1,37 g/L. Estes resultados demonstram o potencial de aplicação de leveduras na produção de xilanase, porém, para as cepas estudadas, há a necessidade de purificação das enzimas produzidas para que estas sejam aplicadas industrialmente / Abstract: Endo-1,4-ß-xylanase (E.C. 3.2.1.8), commonly called xylanase, has great application in different types of industries such as feed, food, textiles, paper, ethanol from biomass, among others. These enzymes act in hydrolysis of the xylan, one of the most abundant polysaccharides found in the nature, present in cell plants linked to the hemicelullose. The main goal of this work was to select, through a screening method using Petri dishes, and to evaluate the enzymatic activity of isolated wild yeasts from several brazilian regions (Atlantic Forest, Amazonian Forest, Cerrado and Pantanal) about the production of xylanase. For the screening in Petri dishes, the culture medium used has xylan as the only carbon source to induce the production of the enzyme. From 349 evaluated microorganisms, 84 could produce xylanase, although only 37 showed an enzymatic ration (diameter of the undye halo/diameter of the colony) higher than 2.5. And, after initial assays in submerged method, 9 strains showed a satisfactory growth at 30ºC and 150 rpm. Among them, the microorganisms AAD5 and AY10 presented a higher potential, with the enzymatic activity above 2 µmol/mL.min. The characterization of the produced enzymes was carried out and the one produced by the strain AAD5 has, as optimal conditions, temperature between 57.5 ¿ 67.5 ºC and pH 4.7 ¿ 5.5. Also, it was shown 21.33 hours of half-life at 52ºC and pH 5.3, with vmax of 1.77 µmol/mL.min and Km of 0.44 g/L. The enzyme produced by the strain AY10 has, as optimal conditions, pH between 4.8 ¿ 4.1 and temperature around 80ºC. The half-life of the enzyme was 11.21 hours at 72ºC and pH 5.3, with vmax of 5.47 µmol/mL.min and Km of 1.37 g/L. These results demonstrate the potential application of the yeasts for xylanase production, although, for those enzymes, further works are necessary to establish the actual industrial and technical potentialities / Mestrado / Mestre em Engenharia de Alimentos

Os ingredientes de origem vegetal que compõem as dietas de frangos de corte possuem em suas composições uma fração indigestível, os polissacarídeos não amiláceos (PNAs). Estas macromoléculas poliméricas de açúcares simples (monossacarídeos) são resistentes à hidrólise no trato gastrointestinal dos animais monogástricos, caracterizando-se como fatores antinutricionais. O objetivo deste trabalho foi avaliar o desempenho zootécnico e rendimento de carcaça de frangos de corte criados de 1 a 42 dias de idade, alimentados com dietas à base de milho ou trigo, com ou sem inclusão de xilanase e valorização da matriz energética. No experimento foram utilizados 1.152 frangos de corte machos, da linhagem Cobb 500. Os animais foram distribuídos em blocos casualizados, com os tratamentos em esquema fatorial 2 x 2 x 2 (duas rações : uma com milho e outra com trigo, 2 níveis de xilanase: 0 e 100 g/ton e 2 níveis de energia: de acordo com as recomendações para cada fase e reduzido em 60 kcal/kg em relação às recomendações), totalizando oito tratamentos com 12 repetições de 12 aves por unidade experimental. As dietas experimentais, com milho ou trigo, foram formuladas para atender as exigências nutricionais das aves segundo Rostagno et al. (2011). Os dados de rendimento de carcaça e desempenho de crescimento foram analisados pelo software SAS 9.3, com análise de variância, e as médias comparadas pelo teste de Tukey (P 0,05). Os resultados indicaram melhorias nos parâmetros de desempenho com a adição de enzima em dietas com base de trigo, e melhor conversão alimentar aos 42 dias de idade com o uso da enzima nas dietas com nível de energia padrão / Ingredients of vegetal sources, which compose the diets of broilers, have in its composition an indigestible fraction known as the non-starch polysaccharides (NSP). These polimeric macromolecules of simple sugars (monosaccharides) are resistant to hydrolisis in the gastrointestinal tract of monogastric animals, and are characterized as anti-nutritional factors. The objective of this work was to evaluate the performance and the carcass yield of broilers raised from 1 to 42 days of age, fed with diets based in corn or wheat, with or without inclusion of xylanase and valorization of the energetic matrix. In the experiment it were used 1.152 male broilers, from the Cobb 500 lineage. The animals were distributed in randomized blocks, with treatments in a factorial 2 x 2 x 2 (two feeds: one with corn and the other with wheat, 2 levels of xylanase: 0 and 100 g/ton, and 2 levels of energy: according to recommendations for each phase, and reduced in 60 kcal/kg in relation to the recommendations), completing eight treatments with twelve replications, and twelve birds in each experimental unit. The experimental diets, with corn or wheat, were formulated to supply the nutritional requirements of the birds according to Rostagno et al. (2011). Carcass yield and growth performance data were analyzed by SAS 9.3 software, with analysis of variance, and the means compared by the Tukey test (P 0.05). Results presented improvements in parameters of performance with inclusion of the enzyme in diets based in wheat, and best feed conversion rate for the period of 1 to 42 days of age with the use of enzyme on standard level energy of diet

Bagasse is the fibrous derivative of sugar cane, that is grown on a commercial scale in many tropical and sub-tropical countries, where ideal climatic conditions are experienced. The seasonality of sugar cane presents storage problems for bagasse, since this lignocellulosic material is susceptible to degradation by a diverse range of microorganisms, mainly fungi. The decay that is brought about contributes largely to the losses of fibre in a bagasse pile. The surrounding microclimate, and conditions within the pile, needs to be carefully monitored in order to understand the factors that support the fungal populations and biochemical activity. The microclimate at the surface and inside the bagasse pile at a paper mill in Stanger (South Africa) was carefully monitored over a one-year storage period. Significant changes were noted in temperature, pH and moisture content, between the surface and the inside of the pile, as the pile aged. The data were compared to established parameters for bagasse preservation, and it was found that the temperature was lower than expected, thus promoting fungal growth. The pH was much higher (promoting bacteria and actinomycetes) and the moisture content was too low to produce anaerobic conditions. The environmental conditions in the bagasse pile at Stanger, therefore, promoted the proliferation of microbes, and consequently decay. Fungi that were present in the pile, were enumerated in order to investigate the diversity and fungal succession. There was a wider variety of species and higher numbers of fungi at the surface than inside the bagasse pile and the Shannon and Berger-Parker diversity confirmed these observations. Sorensons measure also showed that the types of fungal communities at the surface and inside the pile only started becoming similar toward the latter part of storage. When compared to models for abundance of species, conditions on the surface of the pile allowed maximum niche occupation at the beginning of storage, followed by the establishment of a mature community. The inside of the pile displayed minimal niche pre-emption followed by a state where most fungal species shared the domain. This study indicated that, as the storage time increased, the microbial communities became better established. Bagasse is rich in holocellulose, the basic raw material used for paper-making. Since there were many species of holocellulolytic fungi found growing on the surface and the inside of the bagasse pile, the activity of cellulases and xylanases were determined. These enzymes were found to be active at the surface and inside the pile. However, higher activities of both enzymes were noted inside the bagasse pile than on the surface. The higher levels of activity inside the pile, despite lower fungal numbers, suggested that fungal counts were not a clear indication of biomass or biochemical activity. It appeared that the environment on the inside of the bagasse pile promoted the establishment of specific fungal populations that bring about a high degree of degradation to fibre inside the bagasse pile. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

Pour réduire notre empreinte carbone sur l’environnement, il est urgent de développer des procédés industriels utilisant une source de carbone renouvelable comme la biomasse lignocellulosique. La paroi cellulaire végétale est un enchevêtrement complexe de cellulose, d’hémicelluloses et de lignines. Elle résiste aux attaques biologiques et chimiques mais limite le développement d’une bioéconomie responsable. Dans la Nature, des enzymes multimodulaires produites par certains microorganismes peuvent la déconstruire. Toutefois, ces enzymes sont majoritairement étudiées sur des substrats artificiels ou purifiés. Dans cette thèse, nous proposons de les étudier sur des substrats broyés puis des coupes de paille de blé brutes. Les enzymes multimodulaires sont préparées à façon en utilisant la propriété d’association covalente des protéines Jo et In. Nous avons ainsi associé la xylanase NpXyn11A de N. patriciarum avec deux modules non catalytiques : le CBM3a de C. thermocellum ou le CBM2b1 C. fimi ciblant la cellulose ou les xylanes respectivement. Les propriétés biochimiques de ces protéines chimériques ont été comparées aux modules sauvages. L’activité enzymatique des protéines chimériques a ensuite été étudiée sur des substrats solubles, jusqu’à des substrats insolubles comme le son et la paille de blé, notamment par immunocytochimie. Ce travail a mis en évidence l’importance de la relation enzymes/substrats pour une caractérisation in muro d’activité enzymatique et une meilleure compréhension de la déconstruction de la biomasse végétale. / In order to reduce our carbon footprint on the environment, it is more than urgent to develop new industrial process using a renewable carbon source such as lignocellulosic biomass. Plant cell walls consist of a complex network of cellulose, hemicelluloses and lignins that cross-link with each other mainly via non-covalent bonds. It is thus hardly surprising that plant biomass is rather recalcitrant to chemical or biological degradation. In the present era marked by the desire to build a green bioeconomy, this recalcitrance remains a key point. In Nature, the plant-based organic carbon contained within plant cell walls is mainly recycled by the action of cellulolytic microorganisms, producing multimodular enzymes. However, these enzymes are mainly characterized on artificial or purified substrates. In this thesis, we proposed to study multimodular enzymes on raw substrates such as wheat straw sections. The studied multimodular enzymes were associated thanks to the use of two small proteins Jo and In. Thus, we associated the xylanase NpXyn11A from N. patriciarum with two non-catalytic modules: CBM3a from C. thermocellum or CBM2b1 from C. fimi targeting cellulose or xylans respectively. Biochemical properties of these chimeric proteins and wild-type modules have been compared. The enzymatic activity of chimeric proteins has been studied on soluble substrates and compared to the activity on insoluble substrates, mainly by immunocytochemistry. This work highlighted the importance of the relationship enzymes/substrates and its key role to better understand the biomass deconstruction in muro.

Xylanase was purified from the crude culture of Aspergillus oryzae DSM1863 by sephadex G200 and DEAE – cellulose ion exchange chromatography. The molecular mass of the purified xylanase determined by SDS–PAGE was 21 kDa with a specific activity of 6768 U/mg towards 1% (w/v) of birch wood xylan. The optimum temperature was observed at 60°C. The enzyme was thermostable in the temperature range of 37-50°C with a high residual activity of 62-74% (650.6- 775.9 U/mg protein). / Enzyme xylanase được tinh sạch từ dịch lên men của chủng Aspergillus oryzae DSM1863 sau khi qua cột sắc ký lọc gel sephadex G200 và sắc ký trao đổi ion DEAE – cellulose. Khối lượng phân tử của enzyme xylanase tinh sạch được xác định bằng điên di đồ SDS- PAGE. Xylanase tinh sạch có kích thước là 21 kDa với hoạt tính đặc hiệu đạt 6768 U/mg sau khi được xác định với nồng độ cơ chất là 1% birch wood xylan. Nhiệt độ tối ưu để enzyme hoạt động mạnh nhất là 60°C. Enzyme xylanase khá bền nhiệt. Hoạt tính của enzyme vẫn còn duy trì 62-74% (hoạt tính đặc hiệu đạt 650.6-775.9 U/mg protein) sau khi 8 giờ ủ ở 37-50°C.

Le veau nouveau-né possède un rumen peu développé et non fonctionnel. Au cours des premiers mois, les fonctions digestives s’établissent, avec l’implantation du microbiote composé majoritairement de bactéries, archées et protozoaires. Les objectifs de ce travail étaient doubles : i) caractériser et comprendre la séquence d’implantation taxonomique des microorganismes du rumen chez le veau par des techniques de biologie moléculaire et de dénombrement, ainsi que la séquence de mise en place des paramètres fermentaires (AGV et ammoniac) et des activités principales enzymatiques chez le veau en périodes pré- et post-sevrage, ii) étudier l’effet de l’addition de levures vivantes sur la mise en place de cet écosystème ruminal en périodes pré- et post-sevrage. D’une part, nos travaux ont permis de confirmer qu’à la naissance, le rumen chez le veau, est dépourvu de micro-organismes, d’AGV, d’activité xylanasique et amylasique, avec un pH proche de la neutralité et un Eh fortement positif. La colonisation du rumen se fait dès la naissance, pendant les 15 premiers jours de la vie de l’animal par un microbiote complexe prédominé par les bactéries (phyla Proteobacteria et Bacteroidetes) et comprenant aussi des archées (majoritairement Methanobrevibacter). En même temps, le Eh devient fortement négatif. Ces communautés entraînent la production de produits fermentaires grâce à leurs activités enzymatiques. Entre 15 jours et le sevrage, avec l’ingestion d’aliments solides, la composition du microbiote du rumen évolue pour se rapprocher de celle de ruminants adultes, sans atteindre pour autant la maturité en termes de densités et abondances relatives. A cette période, le phylum Bacteroidetes est majoritaire, avec le genre Prevotella. Après le sevrage de légers changements apparaissent sur certains paramètres fermentaires comme les AGV sans doute en raison d’une évolution du microbiote qui devient moins diversifié et plus adapté à la dégradation d’aliments solides. L’apparition, à partir de 90 jours, des protozoaires ciliés dans le rumen semble conditionnée par la présence d’animaux adultes à proximité. A 4 mois d’âge, l’écosystème ruminal tend à devenir proche de celui observé chez les animaux adultes en matière de paramètres fermentaires, activités enzymatiques et composition taxonomique du microbiote. D’autre part, nos travaux ont permis de conclure que, avant sevrage, une supplémentation en levures vivantes (Saccharomyces cerevisiae) diminue l’ingestion de concentrés, conduit à une apparition plus précoce de la communauté des protozoaires et une plus grande densité d’archées, mais a peu d'effets sur la densité et la diversité de la communauté bactérienne, à l'exception de variations d’abondances de quelques taxa mineurs. L’apport de levures entraîne une diminution de la protéolyse, une augmentation de la proportion d'acétate ruminal et une diminution de la proportion de propionate. Au cours de la période post-sevrage, les veaux supplémentés en levures consomment plus de foin et la densité en archées est plus importante alors qu’une réduction de la diversité et de la densité de la communauté bactérienne est observée, mais accompagnée d’une augmentation de l’abondance relative des bactéries dégradant l’amidon, les pectines, les protéines et majoritairement les parois cellulaires en fonction des substrats présents dans le rumen. Ces changements sont probablement à relier aux augmentations de l'activité xylanasique et de la proportion d'acétate. L’ensemble des résultats acquis dans ce travail de thèse a permis d’apporter un certain nombre de connaissances et une meilleure compréhension de la mise en place de l’écosystème ruminal chez le veau de race laitière en périodes pré- et post-sevrage, ainsi que quelques pistes pour orienter ou améliorer cette implantation pour une meilleure maîtrise de l’élevage des veaux d’élevage. / The newborn calf has a little and non-fonctional rumen. During the first months of life, digestive functions establish, in relationship with the colonization by a microbiota mainly composed of bacteria, archaea and protozoa. This study had two objectves: i) characterize and understand the sequence of establishment of ruminal microbiota in calves by molecular biology and counting techniques abd describe the appearance of fermentation parameters (VFA and ammonia) and enzyme activities during the pre- and post-weaning periods, ii) define the effect of yeast supplementation on the establishment of the ruminal ecosystem in pre-and post-weaning periods. On the one hand, our work confirmed that at birth, calf rumen is devoid of micro-organisms, AGV, xylanase and amylase activities, with a pH close to neutrality and a strongly positive Eh. From 2 to 15 days of age, the rumen is colonized by a complex microbiota dominated by bacteria (Proteobacteria and Bacteroidetes phyla) and also containing archaea (with mainly the Methanobrevibacter genus), and Eh becomes strongly negative. These communities result in the production of fermentation products due to their enzymatic activities. Between 15 days and weaning, with the ingestion of solid food, the composition of rumen microbiota changes to become closer to that of adult ruminants without reaching maturity in term of densities and relative abundances. At this time, the phylum Bacteroidetes is predominant with the Prevotella genus. After weaning, slight differences occurs on some fermentative parameters such as VFA, probably related to a change in the microbiota that becomes less diversified and more adapted to the degradation of solid food. From 90 days, the establishment of ciliated protozoa in the calf rumen seems conditioned by the proximity with adult animals. From 4 months of age, considering fermentation, enzymatic and taxonomic composition of the microbiota, the ruminal ecosystem tends to be similar to that observed in adult animals. On the other hand, our work showed that during the pre-weaning period, the supplementation with live yeast (Saccharomyces cerevisiae) results in a lower concentrate intake, an earlier establishment of ciliated protozoa and a higher archaeal community density, but poorly affects the density and diversity of the bacterial community, with the exception of changes in abundances of some minors taxa. Yeast supplementation reduces proteolysis, increases the proportion of acetate and decreases that of propionate. During the post-weaning period, yeast supplemented calves consumed more hay, had a higher archaeal density, but a lower diversity and density of the bacterial community with an increased relative abundance of fiber, protein, starch, pectine degrading bacteria compared to control according to the substrates present in the rumen. These changes are probably related to increases of the xylanolytic activity and the proportion of acetate. Taken together, results obtained in his thesis have improved knowledge and understanding of the establishment of the ruminal ecosystem in the dairy calf in pre- and post-weaning periods, and carried some possibilities to orientate or improve the ruminal establishment for better control of calves rearing.

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.