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Η λιγνοκυτταρίνη είναι, περίπου, η μισή από τη συνολική ποσότητα ύλης που παράγεται από τη φωτοσύνθεση. Αποτελείται από τρία είδη πολυμερών: την κυτταρίνη, την ημικυτταρίνη και τη λιγνίνη. Η κυτταρίνη είναι το απλούστερο από τα συστατικά που βρίσκονται σε λιγνοκυτταρινικά υλικά. Είναι η πιο διαδεδομένη οργανική ένωση στη φύση. Μεγάλα ποσοστά κυτταρίνης συναντάμε στα διάφορα είδη χαρτιού. Έχει βρεθεί πως το 80% των αποβλήτων ενός σπιτιού σχετίζεται με την κατανάλωση χαρτιού. Τίθεται, λοιπόν, άλλο ένα ερώτημα πως μπορεί να γίνει δυνατή η αξιοποίηση αυτής της οργανικής ύλης. Αρκεί να διασπαστεί η κυτταρίνη σε γλυκόζη. Η παραγωγή γλυκόζης είναι το πιο ακριβό βήμα κατά την παραγωγή αιθανόλης από βιομάζα. Συνεπώς, κρίνεται απαραίτητη η μείωση του κόστους της παραγωγής της γλυκόζης για την βιομηχανική εκμετάλλευση της μεθόδου. Κατά τη διάρκεια της πειραματικής διαδικασίας διερευνήθηκε η βέλτιστη απόδοση 2 συγκεκριμένων βιομηχανικών ενζύμων για την παραγωγή γλυκόζης από 5 διαφορετικά είδη χαρτιού: Α4, ανακυκλωμένο, χαρτοπετσέτα, χαρτί κουζίνας και χαρτόκουτο. Τα ένζυμα αυτά είναι μια κυτταρινάση (Celluclast 1.5 L FG) και μια κελλοβιάση (Novozym 188), τα οποία πρέπει να χρησιμοποιηθούν μαζί γιατί δρουν συνεργειακά, το πρώτο αποικοδομώντας τη μεγαλομοριακή κυτταρίνη σε μικρού μεγέθους ολιγοσακχαρίτες και κυρίως κελλοβιόζη, και το δεύτερο υδρολύοντας την κελλοβιόζη σε γλυκόζη. Τα είδη χαρτιού επιλέχθηκαν λόγω της ευρείας χρήσης τους, αλλά και της αδυναμίας πολλαπλής ανακύκλωσής τους ή ακόμα απλής (χαρτοπετσέτα, χαρτί κουζίνας) ανακύκλωσής τους. Το πρόβλημα υπό εξέταση σε αυτά τα πειράματα ήταν διττό: η διερεύνηση, και κατά πόσο είναι απαραίτητη, ειδικής επεξεργασίας των χαρτιών πριν την εφαρμογή τους προς παραγωγή γλυκόζης και η χρήση διασυνδεδεμένων ενζύμων για τη μείωση του κόστους εφαρμογής τους Αρχικά μελετήθηκε η απόδοση αυτών των ενζύμων, σε ελεύθερη μορφή (μη διασυνδεδεμένα), στην παραγωγή γλυκόζης από απόβλητα χαρτιού. Παρόλα αυτά, συνέχισαν να υπάρχουν 2 βασικά μειονεκτήματα: α. η ενζυμική δραστικότητα χάνονταν με γρήγορο ρυθμό, με αποτέλεσμα να περιορίζεται η παραγωγή της γλυκόζης, β. αρκετά μεγάλο οικονομικό κόστος των ενζύμων. Γι’ αυτό στη συνέχεια μελετήθηκε η απόδοση των ενζύμων αφού αυτά είχαν μερικώς ακινητοποιηθεί μέσω διασύνδεσης, το Celluclast με EDAC και το Novozym με γλουταραλδεϋδη, όπως είχε διαπιστωθεί από προηγούμενες μελέτες ότι είναι ο καλύτερος συνδυασμός, ο οποίος μάλιστα επιτρέπει σε υψηλό ποσοστό τη διατήρηση της ενζυμικής δραστικότητας. Κάτω από αυτές τις συνθήκες επιτεύχθηκε σταθεροποίηση των ενζύμων και μείωση της χρονικής διάρκειας της κατεργασίας σε δύο ημέρες από πέντε που απαιτούνταν με τη χρήση μη διασυνδεδεμένων ενζύμων. / The ligno is about half the total quantity of matter produced by photosynthesis. It consists of three types of polymers: cellulose, the hemicellulose and lignin. Cellulose is the simplest of ingredients are ligno-cellulosic materials. It is the most common organic compound in nature. Large percentages of cellulose found in different types of paper. It found that 80% of waste a house associated with the consumption of paper. The question, then, another question that may be possible to use this organic matter. Just to break the cellulose into glucose. The production of glucose is the most expensive step in the production of ethanol from biomass. Therefore, it is necessary to reduce the cost of production of glucose for the industrial exploitation of the method. During the experimental procedure to investigate the optimal performance of these 2 enzymes for industrial production of glucose from 5 different types of paper: A4, recycled, napkin, kitchen paper and cardboard box. The enzyme is a cellulase (Celluclast 1.5 L FG) and a kelloviasi (Novozym 188), which must be used together because they act synergistic, the first deconstructing the macromolecular cellulose into small oligosaccharides and especially cellobiose, and the second hydrolyzing the cellobiose glucose. The paper items were selected because of their widespread use, and the inability of multiple recycling or even simple (napkin, kitchen paper) recycling. The problem under consideration in these experiments was twofold: to investigate, and whether it is needed, special processing of papers prior to their use for glucose production and use of enzyme grid to reduce the cost of their implementation First, the performance of these enzymes in free form (not connected), the glucose production from waste paper. Nevertheless, they continued to have 2 main drawbacks: a. The enzyme activity lost at a rapid pace, thereby limiting the production of glucose, b. sizable economic cost of enzymes. So then we studied the efficiency of enzymes after they had been partially paralyzed by liaison with the Celluclast EDAC and Novozym with glutaraldehyde, as found by previous studies that the best combination, which allows even high conservation enzyme activity. Under these conditions achieved stabilization of enzymes and reducing the duration of treatment in two days from five required the use of non-connected enzymes.

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

Cellulolytic microorganisms are considered to be key players in biorefinery, especially for the utilization of plant biomass. These organisms have been isolated from various environments. The Red Sea is one of the seas with high biodiversity and a unique environment, characterized by high water temperature and high salinity . However, there is little information regarding cellulases in Red Sea environments. The aim of the present study is to evaluate the Red Sea as a gene resource for microbial cellulase. I first surveyed microbial cellulases in the Red Sea using a method called metagenomes, and then investigated their abundance and diversity. My survey revealed that the Red Sea biome has a substantial abundance and a wide range of cellulase enzymes with substantial abundance, when compared with those in other environments. Next, I tried to isolate cellulase-active microorganisms from the Red Sea and I successfully obtained seven strains of four different taxonomic groups. These strains showed a similarity of 99% identity to Aspergillus ustus, 99% to Staphylococcus pasteuri, 99% to Bacillus aerius and 99% to Bacillus subtilis. The enzyme assay I conducted, revealed that these strains actually secreted active cellulases. These results suggest that the Red Sea environment can be, indeed, an excellent gene resource of microbial cellulases.

Non-ionic surfactants are known to enhance the biochemical conversion of lignocellulosic (LC) biomass to bioethanol. Their mechanisms of action, however, are incompletely understood. This research was conducted to elucidate the effects of the non-ionic surfactant Tween 80 on the enzymatic hydrolysis of cellulose and LC substrates. Model cellulose substrates were prepared from cellulose nanocrystals (CNCs) obtained by sulfuric acid hydrolysis of wood pulp. Two methods were developed for the removal of the sulfate groups on the CNCs, resulting from the use of sulfuric acid in their preparation. The effect of sulfate groups, which may be introduced into LC biomass during pretreatment with sulfuric acid, on the enzymatic hydrolysis of cellulose was studied with model cellulose substrates prepared from CNCs with different sulfate group densities. Adsorption of cellulases onto sulfated substrates increased with increasing sulfate group density but their rate of hydrolysis decreased. The decrease indicated an inhibitory effect of sulfate groups on the enzymatic hydrolysis of cellulose, possibly due to non-productive binding of the cellulases onto the substrates through electrostatic interactions instead of their cellulose binding domains. The effect of Tween 80 on the adsorption of cellulases onto lignin, often present as residual lignin in pretreated biomass, was studied with model lignin substrates, prepared from kraft lignin, organosolv lignin, and milled wood lignin. Cellulases appeared to adsorb onto the lignin substrates via both hydrophobic and polar interactions. Tween 80 molecules on the lignin substrates seemed to hinder cellulase adsorption via hydrophobic interactions and reduced the adsorption rate. Finally, the effects of lignin and Tween 80 on the enzymatic hydrolysis of cellulose and LC substrates were studied. Lignin hindered both the adsorption of cellulases onto the substrates and the enzymatic hydrolysis of the substrates. Tween 80 was found to form surfactant–protein complexes with cellulases in solution without compromising cellulase activity. Either substrate-adsorbed or in solution, Tween 80 had no effect on the hydrolysis of cellulose by cellulases. Substrate-adsorbed Tween 80 increased the apparent enzymatic hydrolysis rates of LC substrates but the ability of Tween 80 to increase their apparent hydrolysis rate depended strongly on their structural properties and the chemical properties of the lignin. Hence, Tween 80 may be able to mitigate the inhibitory effect of lignin on the enzymatic hydrolysis of pretreated biomass. / Ph. D.

En réponse aux préoccupations environnementales, les procédés industriels comme la production de bioéthanol de deuxième génération sont apparus. Basés sur la conversion enzymatique de la cellulose, ces processus font face à un problème majeur, la réticence de la biomasse lignocellulosique à l'hydrolyse. Afin de résoudre ce problème et celui lié aux coûts d'utilisation de cocktails de cellulases, les recherches se sont axées sur diverses méthodes permettant d'augmenter l'hydrolyse de la cellulose. Les champignons filamenteux sont connus pour être des dégradeurs naturels du bois et, par conséquent, sont utilisés dans de nombreuses applications biotechnologiques. Récemment, quelques études ont révélé l'importance d'enzymes fongiques telles que la CDH et les GH61 dans la dégradation oxydative de la lignocellulose. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose. L'utilisation de ces enzymes oxydatives offre de réelles voies d'amélioration de la production de bioéthanol et de compréhension de la dégradation in vivo des lignocelluloses par les champignons. / In response to environmental concerns, industrial processes such as second generation bioethanol production have emerged. Based on enzymatic cellulose conversion, these processes are confronted with a major problem, the recalcitrance of lignocellulosic biomass. To solve the problem caused by substrate recalcitrance and high cost of cellulase cocktails, research has focused on various methods to enhance cellulose hydrolysis. Fungi are known to be natural degraders of wood and consequently are used in derived biotechnological applications. Recently, several studies have revealed the importance of fungal enzymes such as GH61 and CDH in the oxidative degradation of lignocellulose. During the work done on this thesis, we demonstrated implication of these oxidative enzymes in lignocellulose deconstruction to enhance hydrolysis performed by more classical cellulases. Utilization of oxidative enzymes offers a suitable way for bioethanol processing enhancement and comprehension of the in vivo lignocellulosic degradation by fungi.

Made available in DSpace on 2016-06-02T19:02:45Z (GMT). No. of bitstreams: 1 6663.pdf: 3464569 bytes, checksum: 56765698cbf2bedabdd5b1e555a404ae (MD5) Previous issue date: 2015-03-20 / Financiadora de Estudos e Projetos / Currently the great challenge for the production of second generation ethanol is to reduce thecost of the enzymes. It is possible to reduce part of this cost by carrying out an optimization ofthe process fermentation using sources of cellulase-induction that allow further growth of fungalbiomass, and increased secretion of proteins. The use of a mutant strain with overexpression ofhemicelulolytics activators could increase expression of the enzymes of interest and therebycontribute to cost reduction. This work aimed to study the production of enzymes involved inthe degradation of biomass by the newly isolated strain of Trichoderma harzianum P49P11focusing on the improvement of the submerged fermentation processes and on the use molecularbiology tools to improve the fungal strain. Regarding the fermentation process, the effects ofdifferent inducing sources were evaluated in flasks and bioreactor using statistical experimentaldesign tools and strategies to enhance biomass in the pre-culture step. For fungal strainimprovement, it was used molecular biology tools for the overexpression of two activators ofcellulases (xyr1 and lae1). A proteomic analysis of the T. harzianum enzymatic extract obtainedusing sugarcane bagasse pretreated by steam explosion followed by delignification (BED) wasperformed. The results showed that the best source for inducing cellulase was BED + sucrose(3: 1), reaching values of 1.21 FPU/mL 80.0U/mL of xylanase and 17.30 U/mL of β-glucosidase. The proteomic analyis identificated 24 different glycoside hydrolases and fourCBM proteins, within 12 different CAZy families. From this study, the enzymatic cocktailproduced "on site" could be supplemented using two accessory enzymes, pectinase and α-Larabinofuranosidase,leading to an increase of 100% of the hydrolysis yield. Regarding thestudy of carbon sources in the pre-culture step, it was possible to increase cellulases productionin 2 times using glycerol as the initial carbon source, followed by inducing carbon source(BED). The xyr1 and lae1 overexpression influencied positively the FPase, CMCase, xylanaseand β-glucosidase production, representing a new approach to increase production of theseenzymes. / Atualmente o grande desafio para a produção de etanol de segunda geração consiste emdiminuir o custo das enzimas degradantes da fibra lignocelulósica. Uma alternativa quepode levar a redução de parte desse custo é a otimização do processo fermentativoutilizando fontes indutoras das enzimas (hemi)celulolíticas que permitam um maiorcrescimento da biomassa fúngica e maior secreção de proteínas. A utilização de umalinhagem mutante com a superexpressão dos ativadores dessas enzimas também podecontribuir nesse sentido. Portanto, o objetivo deste trabalho foi estudar a produção deenzimas envolvidas na degradação da biomassa pelo fungo recém-isolado Trichodermaharzianum P49P11 com foco no melhoramento dos processos fermentativos submersose no uso de ferramentas de biologia molecular para o melhoramento da linhagemfúngica. Em relação ao processo fermentativo foi avaliado o efeito de diferentes fontesindutoras em frascos agitados e em biorreator utilizando ferramentas estatísticas deplanejamento de experimentos e estratégias de aumento de biomassa na fase de préinóculo.Em relação ao melhoramento da linhagem fungica foram utilizadas ferramentasde biologia molecular para a superexpressão de dois ativadores de celulases (xyr1 elae1). Para um melhor entendimento das proteínas produzidas em cultivo submerso foirealizado o proteôma do extrato enzimático secretado pelo fungo selvagem em bagaçode cana pré-tratado. Os resultados mostraram que a melhor fonte de carbono indutora decelulases foi o bagaço de cana explodido e deslignificado (BED) + sacarose naproporção 3:1, alcançando valores de 1,21 FPU/mL, 80.0U/mL de xilanse e 17,30 U/mL de β-glicosidase. O proteôma do extrato enzimático de T. harzianum resultou naidentificação de 24 hidrolases glicosídicas diferentes, 4 proteínas CBM dentro de 12diferentes famílias do CAZy. A partir deste estudo pode-se suplementar o coquetelenzimático on site com duas enzimas acessórias, pectinase e α-L-arabinofuranosidaseque aumentaram o rendimento de hidrólise em mais de 100%. Em relação ao estudo dasfontes de carbono na fase de pré-inóculo foi possível aumentar a produção de celulasesem 130% utilizando o glicerol como fonte de carbono inicial, seguida de fonte de18carbono indutora (BED). A aplicação de técnicas de biologia molecular para a mutaçãodo T. harzianum visando a superexpressão de xyr1 e lae teve influência positiva naprodução das enzimas FPase, CMCase, xilanase e β-glicosidase, representando umanova abordagem para aumentar a produção dessas enzimas.

En réponse aux attentes actuelles des consommateurs pour des produits de haute qualité nutritionnelle et environnementale, et face aux impératifs industriels conduisant à minimiser les risques et l’impact environnemental lors de l’extraction à l'hexane de l’huile de la graine de colza, l’étude de l’extraction aqueuse avec assistance enzymatique de cette huile a été reprise avec 2 objectifs principaux : déterminer les enzymes et mélanges d'enzymes adaptés à la meilleure déstructuration du tissu adipeux végétal, et d'autre part étudier les propriétés et la déstabilisation de l'émulsion formée. De l'optimisation de ces 2 séquences du process dépendent les rendements finaux en huile de l'extraction et les propriétés du tourteau, qui constituent les clés économiques de l'émergence de cette nouvelle technologie. Pour cela, après caractérisation physicochimique des constituants de la graine, protéases et polysaccharides hydrolases ont été testées seules ou en combinaison afin d’optimiser le rendement en huile libre et en huile contenue dans l’émulsion engendrée lors de l’extraction et obtenue par centrifugation. Après caractérisation de l’émulsion (rhéologie, diffusion statique de la lumière, pH, conductivité), des tests de déstabilisation physicochimiques ou thermo-mécaniques ont été mis en œuvre afin de séparer les constituants de l’émulsion formée, et obtenir ainsi la libération de l'huile. Les tests réalisés ont permis de retenir trois procédés de déstabilisation: l’addition de talcs, l’inversion de phase par addition d’huile exogène en présence de NaCl dans la phase aqueuse, et les cycles de congélation/décongélation. Afin d’apporter les premiers éléments de l’optimisation de ce dernier procédé, une étude par planification expérimentale a été mise en œuvre / Consumer's concerns about the quality and environmental impact of the products as well as the industrial requirements regarding the risk assessment and the environmental and health repercussions of the solvent extraction of rapeseed oil using hexane led us to work on the optimisation of the aqueous enzymatic extraction of this oil. The study has been carried out to determine the best combination of enzymes able to achieve the disruption of the vegetal adipose tissue, and to characterize the emulsion obtained after centrifugation. The final objective was to maximize the yields of the oil extraction and to obtain adequate nutritional properties of the cake. After the physicochemical characterization of the rapeseed raw material, several proteases and polysaccharide hydrolases have been tested individually and in combination in order to optimize the removal of free oil and the emulsion oil yield occurring during the aqueous extraction process. The physicochemical properties of the emulsion have been determined: rheological properties, pH, conductivity, spectroscopy by Short Angles Light Scattering). Thereafter some physicochemical and thermo-mechanical treatments have been carried out to destabilize the oil-in-water emulsion obtained after the centrifugation, which contained a large part of the total oil of the reaction mixture. Three destabilization processes appeared particularly interesting to increase the free oil removal from the emulsion: talc addition before centrifugation, phase inversion by addition of exogenous oil in presence of NaCl in the aqueous phase, and freezing/thawing cycles. Finally, an optimisation trial of the freezing/thawing process using a Doehlert experimental design has been done as an example

>Magister Scientiae - MSc / Bioethanol is currently the most widely used biofuel and can be used as a direct replacement for current fossil fuel based transportation fuels. Consolidated bioprocessing (CBP), in which bioethanol is produced in a single reactor by a single microorganism, is a cost-effective way of producing bioethanol in a second generation process using lignocellulosic biomass as feedstock. The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and is able to produce heterologous cellulases, which are required for CBP. However, S. cerevisiae produces low titres of cellulases and one suspected reason for this is the stress caused by the heterologous proteins that induce the unfolded protein response (UPR). The UPR is a stress response pathway that will either lead to increased folding capacity within the ER or to degradation of these proteins and possibly apoptosis of the cell. It is thus beneficial to be able to determine when and to what extent the UPR is active during CBP organism development. Current methods of measuring the UPR include RNA and reverse transcriptase qPCR (r.t.qPCR) measurements, which can be cumbersome and expensive. The purpose of this study was to develop a vector based biosensor that will detect and quantify UPR activation. The vector consisted of either the T.r.xyn2 or eGFP reporter genes under the control of the S. cerevisiae HAC1p or KAR2p promoters. HAC1 and KAR2 are important regulators of UPR as their activation allows the UPR to achieve its function. The eGFP reporter under the transcriptional control of KAR2p was shown to be the superior combination due to the improved dynamic range when the UPR was induced in transformed S. cerevisiae strains by the stress inducer, tunicamycin. This UPR biosensor also proved to be more sensitive when measuring UPR induction in cellulase producing strains, depicting significant differences, compared to previous r.t.qPCR based tests which were unable to detect these differences. We thus developed a UPR biosensor that has greater sensitivity for changes in UPR induction compared to RNA based methods and the first KAR2p based UPR biosensor plasmid that allowed for more accurate detection and measurement of the UPR in cellulase secreting S. cerevisiae strains. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them ideal to use in developing CBP yeasts.