Ziel der Arbeit war die Nutzbarmachung von Bioethanol zur Wandlung in Strom und Wärme in einer Hochtemperaturbrennstoffzelle. Dazu waren neben der Entwicklung eines langzeitstabilen, effektiven Katalysators zur Synthesegaserzeugung und dessen Testung sowie der Übertragung gewonnener Erkenntnisse auf in einem Reformer einsetzbare Konzepte die Verifizierung kommerzieller Katalysatorsysteme für die partielle Oxidation von Ethanol notwendig. Außerdem ist für die Entwicklung eines ethanolbetriebenen SOFC-Systems eine pulsations- und ablagerungsfreie Verdampfung von unvergälltem und vergälltem Ethanol – welche nicht Stand der Technik ist – erforderlich, für die ein geeignetes Verdampferkonzept entwickelt und getestet wurde. Experimentell konnte die Betreibbarkeit eines SOFC-Systems mit Ethanol an einem für den Betrieb mit LPG ausgelegten System nachgewiesen werden.

Master of Science / Department of Grain Science and Industry / Praveen V. Vadlani / In our study, five different bioenergy crops: wheat straw (Triticum aestivum), forage sorghum stover (sorghum bicolor), switchgrass (Panicum virgatum), miscanthus (Miscanthus giganteus) and sweet sorghum baggase (Sorghum bicolor) were evaluated for bio-ethanol production at 20% (w/v) initial substrate concentration under separate hydrolysis and fermentation (SHF) process. The substrates were ground to pass through 600µm mesh size and treated with 2% (w/v) NaOH at 121oC for 30 minutes. The washed and neutralized pretreated residues were subjected to saccharification using cellulase and β-glucosidase enzymes (ratio 1:1.25) at concentrations of 25 filter paper unit (fpu)/g and 31.25fpu/g, respectively, in pH 5.0 citrate buffer in an orbital incubator shaker at 150 rpm for 72 h. The hydrolysate obtained was centrifuged and supernatant was collected for fermentation. Fermentation was performed in shake flasks using Saccharomyces cerevisiae at 10% (w/v) inoculum concentration at 100 rpm for 24 h. Alkali treatment was effective in delignification of all the biomass feedstocks. The highest percent removal on raw biomass basis was attained for sorghum stover BMR-DP (81.3%, w/w) followed by miscanthus (79.9%, w/w), sorghum stover BMR-RL (69.2 %, w/w), wheat straw (68.0 %, w/w), switchgrass (66.0%, w/w), and sorghum baggase (65.4%, w/w). Glucan saccharification varied from 56.4-72.6 % (w/w) corresponding to a glucose levels of 0.45-0.34 g/g of dry substrate. Highest saccharification was observed for wheat straw while lowest was observed for miscanthus after 48 hours of hydrolysis. A maximum final ethanol concentration of 4.3% (w/v) was observed for wheat straw followed by sorghum baggase (4.2%), sorghum RL-BMR (3.6%), miscanthus (3.4%), sorghum DP-BMR (3.4%), and switchgrass (3.2%). From our studies, it is evident that high substrate concentration used for enzymatic hydrolysis was able to provide high final ethanol concentration. The lignin content and its arrangement in different biomass feedstocks may have affected saccharification and subsequent ethanol production. Bulk density and flowability are the two major key parameters that should be addressed to reduce processing cost of biomass for bioethanol production. Pelleting of biomass can increase the bulk density, thereby reducing the handling and transportation costs. In addition to above study, I analyzed the changes in chemical composition due to pelletization and pretreatment, and its effect on ethanol production by comparing unpelleted and pelleted biomass ethanol production efficiency. Wheat straw and big bluestem pelleted and unpelleted biomass were compared for their ethanol production efficiency. Pelleted and unpelleted wheat straw (Triticum aestivum) and bigblue stem (Andropogon gerardii Vitman) at a substrate concentration of 10% (w/v) were subjected to 2% NaOH treatment at 1210C for 30 min and the resulting residues were analyzed for changes in chemical composition. Saccharification of residue was done at substrate concentration of 12% (w/v) for 48 h. The sugars obtained were fermented to ethanol using Saccharomyces cerevisiae. Pelletization did not significantly affect the chemical composition of biomass in terms of glucan, xylan and lignin content. Delignification of pelleted biomass was greater than unpelleted biomass. Pelletization did not influence final ethanol production for both substrates.

Doctor of Philosophy / Department of Grain Science and Industry / Sajid Alavi / The Renewable Fuel Standard (RFS) in the Energy Independence and Security Act of 2007 has set the goal of 36 billion gallons of annual ethanol production in the U.S. by 2022, which is equivalent to 17.5% of the current gasoline consumption in the U.S. However, corn ethanol is expected to plateau at a level of 7.3% of current gasoline consumption on an energy-equivalent basis. Thus, it is essential to utilize a variety of substrates including lignocellulosic biomass from perennial energy crops such as switch grass, crop residues such as corn and sorghum stover, and agri-industrial co-products such as soybean hulls and wheat bran. Lignocellulosic substrates have a recalcitrant nature and require a pretreatment step that is critical for efficient enzymatic hydrolysis of cellulose and hemicellulose to fermentable sugars. In this study, soybean hulls were used as a model substrate for cellulosic ethanol. A novel thermo-mechanical pretreatment process using extrusion was investigated and compared with two traditional pretreatment methods, dilute acid and alkali hydrolysis, with regard to structural changes in the lignocellulosic substrate, and glucose and ethanol yields. The effect of extrusion parameters, such as barrel temperature, in-barrel moisture and screw speed, on glucose yield from soybean hulls was determined. Optimum processing conditions were screw speed of 350 rpm, maximum barrel temperature of 80C and 40% in-barrel moisture content, resulting in 95% cellulose conversion to glucose. Compared with untreated soybean hulls, the cellulose to glucose conversion of soybean hulls increased by 69.5, 128.4 and 132.2% for dilute acid, alkali and thermo-mechanical pretreatments, respectively. Glucose and other hexose sugars such as mannose and galactose were effectively fermented by Saccharomyces cerevisiae, resulting in ethanol yields of 13.04–15.44 g/L. Fermentation inhibitors glycerol, furfural, 5-(hydroxymethyl)-2-furaldehyde (HMF) and acetic acid were found in the thermo-mechanically pretreated substrate, ranging in concentrations from 0.072–0.431, 0–0.049, 0–0.023 and 0.181–0.278 g/L, respectively, which were lower than those reported from acid hydrolyzed substrates. The economic feasibility of commercial cellulosic ethanol production processes employing dilute acid hydrolysis and thermo-mechanical pretreatment were compared using a system dynamics modeling approach. It was concluded that low feedstock cost and high sugar conversion are important factors that can make cellulosic ethanol production commercially viable. Thermo-mechanical pretreatment was a more promising technology as compared to dilute acid hydrolysis because of the lower capital and operating costs, and higher sugar conversion.

Doctor of Philosophy / Department of Civil Engineering / Kyle A. Riding / The cement industry is an important component in the quest to reduce global greenhouse gas emissions because of vast amounts of cement used annually. Incorporating supplementary cementitious materials (SCMs) into concrete is one alternative to reduce cement production and thereby reduce greenhouse gas emissions. This study investigated three types of agricultural residues, namely corn stover, wheat straw, and rice straw, in addition to bioethanol byproducts as potential resources for SCM production for concrete applications. Pretreatments, commonly used in bioethanol production, were used to improve pozzolanic reactivity of corn stover ash (CSA), wheat straw ash (WSA), and rice straw ash (RSA) in cementitious systems. In the first part of this research, the impact of distilled water and dilute hydrochloric acid pretreatments on pozzolanic reactivity of WSA, RSA, and CSA were studied. Results showed that pretreatments, particularly dilute acid, improved pozzolanic properties of CSA, WSA, and RSA by removing potassium and phosphorous from the biomass prior to ashing. In addition, WSA and RSA were shown to have similar pozzolanic reactivity to that of silica fume. In the second part of this study, suitability of high lignin residue (HLR), a bioethanol byproduct, for SCM production was investigated. It was shown that burning high lignin residue produces HLR ash that is very reactive in cementitious materials and can be used as a reactive SCM in concrete. The impact of each step in the production of bioethanol on the quality of bioethanol byproduct for subsequent burning and use in concrete was also studied. Sodium hydroxide and sulfuric acid pretreatments and enzymatic hydrolysis were used. Results revealed that sodium hydroxide pretreatment of the biomass have negative impact on biomass ash properties for concrete use because sodium hydroxide pretreatment did not remove phosphorous and other crystalline phases out of the biomass. However, sulfuric acid pretreatment of biomass greatly improved ash properties. It was also shown that enzymatic hydrolysis could have beneficial impact on ash properties because, during enzymatic hydrolysis, some phosphorous was leached out of the biomass.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Complete degradation of cellulose produces mainly glucose, which can be fermented to ethanol. Therefore cellulose presents an abundant renewable energy resource for the production of an alternative, environmentally friendly, transportation fuel. Enzymatic degradation of cellulose is achieved by the synergistic action of three cellulase enzyme groups: endoglucanases, exoglucanases and -glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol. Therefore, a need has arisen to develop a recombinant microorganism with the ability to produce cellulolytic enzymes, hydrolyze cellulose and ferment the resulting sugars to ethanol in a single process step, referred to as “Consolidated Bioprocessing” (CBP). This would provide a cost-effective, economically feasible strategy for the production of bioethanol. The naturally fermentative yeast, Saccharomyces cerevisiae, is often used as host for the expression of recombinant proteins due to several characteristics, including its robustness in industrial processes, the well developed genetic tools available for manipulation and its proven safety status. A number of cellulase genes have previously been successfully expressed by recombinant S. cerevisiae strains. In this study, all three components of the cellulase system were co-expressed in S. cerevisiae to test the ability of the yeast to effectively produce the heterologous proteins, and consequently produce enough glucose for growth on an amorphous cellulosic substrate. The Trichoderma reesei endoglucanase gene egII (Cel5A) was successfully expressed by a S. cerevisiae Y294 strain. Recombinant EGII displayed activities of 19.6 nkat.ml-1 and 22.3 nkat.ml-1 towards CMC and barley -glucan, respectively. The major endoglucanase gene, egI (Cel7B) from T. reesei was subjected to random mutagenesis by propagating the egI-containing plasmid in an E. coli mismatch repair deficient strain. Screening of S. cerevisiae transformants revealed a strain, S. cerevisiae Y294[pLEM1], with improved levels of endoglucanase activity (21.8 nkat.ml-1), compared to S. cerevisiae Y294[pAZ40], expressing the wild type gene (10.3 nkat.ml-1). Through subcloning of the mutated ENO1 promoter region and the mutated egI gene fragment, it was established that the mutations located in both the promoter- and gene sequences were responsible for the improved levels of activity displayed by S. cerevisiae Y294[pLEM1]. The egII gene and the altered egI gene were co-expressed with a codon optimised T. reesei cellobiohydrolase (sCBHI) and a -glucosidase from Saccharomycopsis fibuligera. This resulted in a reduction in endoglucanase levels, possibly due to the metabolic burden placed on the yeast by co-expressing the different cellulases. The hydrolysis products produced by cellulase co-expressing strains were cellotriose, cellobiose and glucose, although the glucose yield was insufficient to enable growth on cellulose as sole carbon source. As the major hydrolysis product was cellobiose, it is likely that a bottleneck exists at its conversion to glucose, suggesting inadequate -glucosidase activity. This study has provided insight into co-expression of cellulase enzymes by the yeast S. cerevisiae. The knowledge obtained could be applied in optimizing cellulase cocktails for efficient cellulose degradation and eventual production of ethanol by recombinant yeast. It has also demonstrated the applicability of random mutagenesis for improving the activity of cellulases.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The search for a cost-effective, environmentally friendly replacement for fossil fuels resulted in bio-ethanol production receiving a lot of attention. Lignocellulose, is considered to be the most abundant renewable source on earth, and consists of cellulose, hemicellulose and lignin. Exploitation thereof as a substrate for ethanol production, can serve as solution in producing bio-ethanol as an adequate replacement for fossil fuels. Hemicelluloses, contributing up to a third of the lignocellulosic substrate, consists mainly of xylan and mannan and can be degraded by hemicellulolytic enzymes that are produced by plant cell wall degrading organisms. Galactoglucomannan is the most complex form of mannan and requires a consortium of enzymes for complete hydrolysis. These enzymes include β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and galactomannan acetylesterases. Saccharomyces cerevisiae is a well-known fermentative organism that has been used in various industrial processes and is able to produce ethanol from hexose sugars. Although this organism is unable to utilize complex lignocellulosic structures, DNA manipulation techniques and recombinant technology can be implemented to overcome this obstacle. Strains of S. cerevisiae pose other shortcomings like hyperglycosylation and therefore other non-conventional yeasts (such as Kluyveromyces lactis) are now also being considered for heterologous protein production. The mannanase gene (manI) of Aspergillus aculeatus was expressed in K. lactis GG799 and S. cerevisiae Y294. K. lactis transformants were stable for two weeks in consecutive subcultures and secreted a Man1 of 55 kDa. The recombinant Man1 displayed an optimum temperature of 70°C and a pH optimum of 5 when produced by K. lactis. Activity levels of about 160 – 180 nkat/ml was obtained after 86 hours of cultivation, which was similar to the activity observed with S. cerevisiae under the same conditions. Disruption of the ku80 gene did not contribute to the stability of the cultures and a heterogeneous culture developed for 10 days of consecutive subculturing. The mannosidase gene (man1) from A. niger and mannanase gene (manI) from A. aculeatus were constitutively expressed in S. cerevisiae Y294 and S. cerevisiae NI-C-D4. The MndA and Man1 proteins appeared as a 140 kDa and 58 kDa species on the SDS-PAGE analysis when expressed in S. cerevisiae Y294, respectively. MndA had an optimum temperature of 50°C and optimum pH 5. Man1 produced by S. cerevisiae Y294 indicated a pH optimum of 6 and temperature optimum of 70°C. The MndA displayed low levels of endomannanase activity and no β-mannosidase activity could be detected. Co-expression of man1 and mndA in either S. cerevisiae Y294 and S. cerevisiae NI-C-D4, resulted in less hydrolysis of galactoglucomannan. An increase in the size of the plasmid generally results in a decrease in the copy number, leading to a decrease in the amount of ManI protein being produced. The co-expression of ManI and MndA could also have resulted in a higher metabolic burden on the cell, hence the amount of ManI are produced. This study confirms that more research should be done on the evaluation of alternative hosts for expression of foreign proteins. Furthermore, producing enzymes cocktails for industrial application should be considered rather than co-expression of various enzymes in one host. / AFRIKAANSE OPSOMMING: ‘n Behoefte na ‘n koste-effektiewe en omgewingsvriendelike vervoer brandstof is besig om toe te neem. Lignosellulose word beskou as die volopste hernubare bron vir biobrandstof en lignosellulose bestaan uit sellulose, hemisellulose en lignien. Die gebruik daarvan vir die produksie van bio-etanol kan ’n voldoende alternatief vir fossielbrandstowwe bied. Verbruik van lignosellulose as bron vir die produksie van biobrandstof bied ’n oplossing vir die energie krises. Hemisellulose vorm ’n derde van lignosellulose substraat en bestaan uit xilaan en mannaan en word deur hemisellolitiese ensieme afgebreek wat algemeen by plantselwand-verterende organismes voorkom. Galaktoglukomannaan is die mees komplekse vorm van mannaan en benodig verskeie ensieme vir volkome hidroliese. Hierdie ensieme sluit in β-mannanase, β-mannosidase, α-galaktosidase, β-glukosidase en galaktomanaan asetielesterases. Saccharomyces cerevisiae is ‘n bekende fermenterende organisme wat gereeld in verskeie industriële prosesse gebruik word en kan etanol van heksose suikers produseer. Die organisme beskik nie oor die vermoë om komplekse polisakkarides wat in lignosellulose voorkom te hidroliseer nie maar. DNS-manipuleringstegnieke en rekombinante tegnologie maak dit egter moontlik die probellm te oorbrug. S. cerevisiae het nogtans tekortkominge soos hiperglikosilering en daarom word ander nie-konvensionele giste (soos Kluyveromyces lactis) tans ook vir die produksie van rekombinante proteine ondersoek. Die mannanase geen (manI) vanaf Aspergillus aculeatus is in K. lactis GG799 en S. cerevisiae Y294 uitgedruk. K. lactis transformante was stabiel vir twee weke in opeenvolgende subkluture en het ‘n Man1 van 55 kDa geproduseer. Die rekombinante Man1 ensiem het ‘n temperatuur optimum van 70°C en pH optimum van 5.0 getoon in K. Lactis. Aktiwiteitsvlakke van 160 – 180 nkat/ml was bereik na 86 uur klutivering, In vergelyking met S. cerevisiae was aktiwiteitsvlakke eenders oor ‘n periode Die disrupsie van die ku80 geen het geen effek op die stabiliteit van die transformante in 10 dae opeenvolgende sub-kulture getoon nie. Die mannosidase geen (mndA) vanaf Aspergillus niger en die mannanase geen (man1) van Aspergillus aculeatus is konstitutief in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk. Uitdrukking van die MndA en Man1 proteïen in S. cerevisiae Y294 het onderskeidelik ‘n 140 kDa en 58 kDa spesie getoon met SDS-PAGE analisering. Die MndA ensiem het ‘n temperatuur optimum van 50°C and pH optimum van 5.0 getoon. Man1 het ‘n pH optimum van 6.0 en ‘n temperatuur optimum van 70°C getoon. MndA het lae hidrolitiese aktiwiteit op galaktoglukomannaan, maar geen β-mannosidase aktiwiteit getoon nie. Wanneer man1 and mndA saam in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk is, het die hidroliese van galaktoglukomannan dramaties afgeneem. ‘n Toename in die grootte van ‘n plasmied veroorsaak dikwels ‘n afname in kopiegetal wat die produksie van ManI verlaag. Die ko-uitdrukking van ManI en MndA kan ook tot ’n hoër metaboliese las lei en dus die laer produksie van ManI. Resultate in hierdie studie wys daarop dat meer navorsing benodig word in die soeke na alternatiewe gashere vir uitdrukking van mannanases. Ensiem mengsels vir industriële toepassings behoort eerder gebruik te word as die ko-ekspressie van verskeie ensieme in ’n enkel gasheer.

Tirtas kukurūzų frakcijų kiekio pasiskirstymo priklausomybė nuo atstumų dydžio tarp trupintuvo valcų. Tyrimai atlikti 2013-2014 metais Aleksandro Stulginskio universitete. Tyrimo objektas – diskinių valcų trupintuvas-traiškytuvas, kurio našumas iki 15 t.h-1. Valcai – apvalūs, dantyti, sukimosi dažnis ns = 4000 min-1. Trupintuvą-traiškytuvą sudaro keturi valcai, dvi poros – pakopos. Trupintuvo diskiniams valcams sukti, reikalinga aktyvioji galia, matuota elektros energijos tinklo analizavimo prietaisu ME-MI2492 („Metrel“). Frakcijų atskyrimui buvo naudojami penki sietai: 3,5 mm; 3 mm; 2,4 mm; 2 mm; 1 mm. Iš viso gautos šešios frakcijos. Valcų atstumas kito nuo 4,5 iki 6 mm pirmos pakopos ir nuo 1,5 iki 3 mm antrosios pakopos. Tyrimais nustatytas žymus, frakcijų didesnių nei 3,5 mm, kiekio didėjimas. Grūdų drėgniui sumažėjus pastebėtas galios poreikio padidėjimas esant minimaliems atstumams tarp valcų. Apdorojus duomenis nustatytas esminis skirtumas tarp frakcijų didesnių nei 3,5 mm kiekio procentinės dalies, esant santykiniam grūdų drėgniui ωg1=37,77±0,5% ir frakcijų kiekio kai drėgnis ωg3=33,25±0,88%. / Studied corn fraction pattern on the cracker rolls gap wide. Investigations were carried out in 2013-2014 at Aleksandras Stulginskis University. Research object was a disc roller crusher, with a capacity of up to 15 t.h-1. Rollers - rounded, toothed, rotation speed n = 4000 min -1. Crusher consists of four rolls, two pairs in all. Crusher disc rolls turn required active power, measured electricity network analysis device ME- MI2492 ( Metrel ) sievs was used in five screens: 3.5 mm; 3 mm; 2.4 mm; 2 mm; 1 mm. A total of six groups. The results demonstrated significant groups larger than 3.5 mm, the increase in volume. Grain moisture decreased observed a increase in power consumption at the minimum roller distance. After processing the data a fundamental difference been set between the groups of larger than 3,5 mm the percentage of grain moisture ωg1 = 37.77 ± 0.5% and the volume fraction when humidity ωg3 = 33.25 ± 0.88 %.

The work of the present thesis focused on conversion of the cellulosic part of waste textiles into biogas and ethanol, and its challenges. In 2009, the global annual fiber consumption exceeded 70 Mt, of which around 40% consisted of cellulosic material. This huge amount of fibers is processed into apparel, home textiles, and industrial products, ending up as waste after a certain time delay. Regretfully, current management of waste textiles mainly comprises incineration and landfilling, in spite of the potential of cellulosic material being used in the production of ethanol or methane. The volume of cellulose mentioned above would be sufficient for producing around 20 billion liters of ethanol or 11.6 billion Nm3 of methane per year. Nevertheless, waste textiles are not yet accepted as a suitable substrate for biofuel production, since their processing to biofuel presents certain challenges, e.g. high crystallinity of cotton cellulose, presence of dyes, reagents and other materials, and being textiles as a mixture of natural and synthetic fibers. High crystallinity of cotton cellulose curbs high efficient conversion by enzymatic or bacterial hydrolysis, and the presence of non-cellulosic fibers may create several processing problems. The work of the present thesis centered on these challenges. Cotton linter and blue jeans waste textiles, all practically pure cellulose, were converted to ethanol by SSSF, using S. cerevisiae, with a yield of about 0.14 g ethanol/g textile, only 25% of the theoretical yield. To improve the yield, a pretreatment process was required and thus, several methods were examined. Alkaline pretreatments significantly improved the yield of hydrolysis and subsequent ethanol production, the most effective condition being treatment with a 12% NaOH-solution at 0 °C, increasing the yield to 0.48 g ethanol/g textile (85% of the theoretical yield). Waste textile streams, however, are mixtures of different fibers, and a separation of the cellulosic fibers from synthetic fibers is thus necessary. The separation was not achieved using an alkaline pretreatment, and hence another approach was investigated, viz. pretreatment with N-methyl-morpholine-N-oxide (NMMO), an industrially available and environment friendly cellulose solvent. The dissolution process was performed under different conditions in terms of solvent concentration, temperature, and duration. Pretreatment with 85% NMMO at 120 °C under atmospheric pressure for 2.5 hours, improved the ethanol yield by 150%, compared to the yield of untreated cellulose. This pretreatment proved to be of major advantage, as it provided a method for dissolving and then recovering the cellulose. Using this method as a foundation, a novel process was developed, refined and verified, by testing polyester/cellulose-blended textiles, which predominate waste textiles. The polyesters were purified as fibers after the NMMO treatments, and up to 95% of the cellulose content was regenerated. The solvent was then recovered, recycled, and reused. Furthermore, investigating the effect of this treatment on anaerobic digestion of cellulose disclosed a remarkable enhancement of the microbial solubilization; the rate in pretreated textiles was twice the rate in untreated material. The overall yield of methane was, however, not significantly affected. The process developed in the present thesis appears promising for transformation of waste textiles into a suitable raw material, to subsequently be used for biological conversion to ethanol and biogas. / <p>Thesis to be defended in public on Friday, May 27, 2011 at 13.00 at KC-salen, Kemigården 4, Göteborg, for the degree of Doctor of Philosophy.</p>

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.

Les procédés de bioraffinerie, et notamment les agrocarburants, sont aujourd'hui reconnus comme essentiels pour sortir de l'économie actuelle basée sur le pétrole. Dans le cas du bioéthanol produit à partir de biomasse lignocellulosique, l'hydrolyse enzymatique par les enzymes de Trichoderma reesei est le principal point faible du procédé et doit être améliorée. Ces travaux de thèse s'intègrent dans le cadre du projet Futurol, et ont pour objectif d'identifier de nouvelles enzymes capables d'améliorer l'activité de T. reesei sur la lignocellulose. Une analyse post-génomique réalisée sur les secrétomes de vingt souches fongiques s'est révélée particulièrement prometteuse pour l'identification d'enzymes lignocellulolytiques d'intérêt. Une approche de génomique comparative a également abouti à la sélection de deux endo-mannanases de famille GH5 et GH26 chez le champignon Podospora anserina. Ces hémicellulases ont permis d'améliorer significativement la libération de glucose par T. reesei à partir d'épicéa. Une étude fondamentale approfondie a permis de résoudre les structures cristallographiques et de mettre en évidence les relations entre les spécificités enzymatiques de chaque enzyme et leurs caractéristiques structurales. La structure tridimensionnelle de la mannanase GH26 couplée à son CBM35 présente un linker court et rigide et une organisation du site actif atypique. Les deux mannanases ont également fait l'objet d'un travail d'ingénierie aléatoire qui a abouti à des variants des deux enzymes présentant une amélioration de l'efficacité catalytique et/ou une modification de spécificité. / Biorefineries such as biofuels are nowadays considered as essential to reduce our dependence on oil products. In the production process of bioethanol from lignocellulosic biomass, enzymatic hydrolysis performed by Trichoderma reesei enzymes is the main bottleneck of the process and requires improvements.The present work is part of the Futurol project, and aims at identifying new enzymes to improve the activity of T. reesei toward lignocellulose. Post-genomic analyses on twenty fungal strains have revealed the potential of this approach to identify lignocellulolytic enzymes of interest. Comparative genomics also led to the selection of two endo-mannanases from families GH5 and GH26 from the fungus Podospora anserina. These hemicellulases significantly improved glucose release upon T. reesei hydrolysis of spruce. An in-depth fondamental study allowed the solving of cristallographic structures and revealed the relationships between enzymatic specificities and structural characteristics. The structure of GH26 catalytic module appended to CBM35 highlighted a short and rigid linker and an atypical active site organization. The two mannanases were subjected to molecular engineering. Variants displaying improved catalytic efficiency and/or modified specificity were identified for both enzymes.