Dans le contexte de la bioraffinerie végétale pour la production de molécules et d'énergie, des prétraitements sont nécessaires pour augmenter la réactivité de la biomasse ligno-cellulosique. Cette thèse s'insère dans une thématique dont l'objectif général est d'établir les bases d'une raffinerie du végétal par des procédés par voie sèche. Cette étude a pour objectif de développer et comprendre le fractionnement mécanique poussé de la paille de blé jusqu'à des tailles sub-millimétriques et d'évaluer les effets sur des procédés de bioconversions énergétiques. La paille de blé présente une grande hétérogénéité à plusieurs niveaux d'échelle (du cm au µm). Un diagramme de broyage multi-étapes à l'échelle pilote (>1kg) a permis d'obtenir une large gamme de tailles de particules, par 3 modes de sollicitation distincts: i) broyages à grille sélective produisant des tailles du grossier (800 µm) au fin (50 µm), ii) broyage à jet d'air (ultrafin, ~20 µm) et iii) broyage à boulets (ultrafin, ~10 µm). Une méthodologie d'analyse morphologique des particules a été développée par analyse d'images de microscopie optique. La paille soumise aux mécanismes complexes de rupture lors de broyages produit une forte variabilité des formes et compositions des particules. L'analyse multiple de co-inertie a permis d'évaluer de façon globale les morphologies des particules. Globalement, le broyage diminue la taille et les facteurs de forme des particules, avec quelques exceptions dues aux configurations matérielles. La dégradabilité enzymatique (saccharification) des poudres produites a été améliorée par la réduction de la taille des particules. Jusqu'à ~100 µm la solubilisation des polysaccharides augmente puis se stabilise à 36 % des polysaccharides totaux et 40 % de la cellulose. Seuls les échantillons issus du broyage à boulets dépassent cette limite et atteignent 46 % d'hydrolyse des polysaccharides totaux et 72 % de la cellulose. Ceci est lié à une augmentation de l'efficacité enzymatique due à la diminution de la cristallinité de la cellulose (de 22 à 13%). Ces résultats du broyage à boulets sont comparables à ceux de l'explosion à la vapeur, avec une meilleure préservation des hémicelluloses. Cette amélioration d'efficacité enzymatique s'est traduite par des dégradations anaérobies (biogaz) accélérées et légèrement augmentées (cas du broyeur à boulets). La décomposition aérobie dans le sol a été améliorée par le broyage grossier, mais les broyages plus fins n'ont pas entrainé de gain. Les caractéristiques de la paille broyée peuvent varier selon l'intensité et le mode de broyage. Bien que tous les broyages permettent la réduction de la taille, le broyage à grilles et le broyage à jet d'air n'engendrent pas de changements dans la structure fine des polymères pariétaux. Seul le broyage à boulets a engendré des changements de la structure interne des particules notamment en réduisant la cristallinité de la cellulose et en solubilisant partiellement les hémicelluloses. Ces résultats permettent de mettre en évidence que la fragmentation mécanique poussée par voie sèche est une alternative possible aux prétraitements utilisés en raffinerie végétale. / In a context of plant biorefinery for the production of molecules and energy, pretreatments are necessary to increase the reactivity of the lignocellulosic biomass. This thesis is part of a general project aiming to establish the bases for a dry plant refinery. This study aimed to develop and understand advanced mechanical fractionation of wheat straw down to sub-millimeter sizes and to assess its effects on bioconversion processes for bioenergy. Wheat straw exhibited a high heterogeneity at several scale levels (from cm to μm). A multistep diagram of dry grinding at pilot-scale (> 1 kg) produced a wide range of particle sizes by three distinct mode of action: i) sieve-based grinding producing particle sizes from coarse (800 μm) to fine (50 μm), ii) air-jet milling (ultra-fine, ~ 20 μm) and iii) ball milling (ultra-fine, ~ 10 "m). A morphological analysis of particles was developed by image analysis from light microscopy. Subjecting wheat straw to the complex breaking mechanisms during grinding produced particles highly variable in shapes and compositions. A multiple co-inertia analysis allowed the evaluation of the overall particle morphologies. Generally, grinding reduced the size and shape descriptors of particles, with some exceptions due to equipment configurations. The enzymatic degradability (saccharification) of produced powders was improved by reducing their particle size. Until ~ 100 μm the polysaccharides solubilisation was increased and then stabilised at 36% total polysaccharides and 40% cellulose. Only samples from ball milling overcome this limit and attained hydrolysis yields of 46% total polysaccharides and 72% cellulose. This is due to an increase in enzymatic efficiency by the reduction of cellulose crystallinity (from 22 to 13%). These results of ball milling are comparable to those of steam explosion process, with a better preservation of hemicelluloses. This improved enzymatic efficiency resulted in faster and slightly more extensive (ball milling case) anaerobic degradations (biogas). Aerobic decomposition in the soil was improved by coarse grinding, but finer grinding did not result in a further increase. The characteristics of ground straw varied depending on grinding intensity and mode. Although all grindings could reduce the size, sieve-based grinding and air-jet milling did not allow changes in the fine structure of cell wall polymers. Only ball milling led to changethe internal structure of particles especially reducing cellulose crystallinity and partially solubilising hemicelluloses. These results demonstrate that advanced mechanical fragmentation by dry processes is a possible alternative for pretreatments in a plant refinery

Lecythophora (Coniochaeta) hoffmannii, a soil- and lignocellulose-inhabiting sordariomycete (Ascomycota) that can also live as a facultative tree pathogen causing soft rot, belongs to the family Coniochaetaceae. The strain CBS 245.38 sequenced here was assembled into 869 contigs, has a size of 30.8 Mb, and comprises 10,596 predicted protein-coding genes.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Cellulose and hemicellulose represents the two most abundant groups of renewable polysaccharides known to man. Apart from their presence in plant material, they also contribute to a significant portion of inexpensive readily available material, such as wastes and bypro ducts from forestry / agricultural origin. The chemical composition of plant material varies, but the biomass content consists of approximately 75% carbohydrate polymers (cellulose and hemicellulose) and 25% lignin. The enzymes required for the degradation of cellulose and hemicellulose are collectively called cellulases and hemicellulases. These enzymes have a broad spectrum of industrial applications including the production of fuel ethanol through fermentations, reducing the amount of chlorine required for bleaching in the pulp and paper industry, increasing dough volume in the baking industry, improving digestion and nutritional value of animal feed, increasing clarification and enhancing the filterability of wine, beer and fruit juice, etc. Therefore, a large potential market exists for cellulases and hemicellulases provided their production is economical and the product, authentic. Aspergilli occur in a wide variety of habitats including soil, stored food and feed products and decaying vegetation. The advantages for using A. niger as host for heterologous enzyme production include good protein secretion, industrial fermentation technology dating as far back as 1919, being a non-pathogenic fungus with GRAS status, no special substrate or cultivation requirements, FDA approval of numerous enzymes (homologous and heterologous) produced, etc. In this study an Aspergillus expression vector was constructed using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdp) of A. niger and the glucoamylase terminator (glaAT) of Aspergillus awamori. The cDNA copies of the eg! and xyn2 genes of Trichoderma reesei, cbhl-4 of Phanerochaete chrysosporium, man! of Aspergillus aculeatus and xyn3 of Aspergillus kawachii were introduced into the expression vector, respectively. All the plasmids were co-transformed with plasmid p3SR2 to A. niger and transformants selected for stable plasmid integration into the genome of the host. The recombinant enzymes EgI, Xyn2, Cbhl-4, Man! and XynC were successfully expressed and secreted at activity levels of 2300, 8000, 500, 6000 and 900 nkatlml, respectively. The enzymes were produced as functional entities and were subsequently characterized. The EgI, Xyn2 and ManI were evaluated as feed additives for the possible use in the animal feed industry. Improved biomass gain was observed with in vivo studies on poultry. With the possible mass production of heterologous enzymes in mind, a simple medium had to be devised for their inexpensive production. Molasses medium (available from the South African sugar industry) was therefore evaluated and the cultivation conditions optimized for it's possible use as cultivation substrate for A. niger. The evaluation was done on the grounds of EgI and Xyn2 activity produced which was monitored over time. This study highlighted the possible use of A. niger for the heterologous production of enzymes, the use of industrial substrate for cultivation and paved the way for the high level expression of industrially important genes at low cost and a positive environmental impact. / AFRIKAANSE OPSOMMING: Sellulose en hemisellulose verteenwoordig die twee vollopste herwinbare polisakkariede bekend. Behalwe vir hul teenwoordigheid in plantmateriaal, dra hulle ook by tot 'n beduidende fraksie van goedkoop, maklik bekombare materiaal soos afval- en byprodukte van bosbou I landbou oorsprong. Soos te verwagte, varieër die chemiese samestelling van die plantmateriaal, maar die biomassa-inhoud bestaan uit naastenby 25% lignien en 75% koolhidraatpolimere (sellulose and hemicellulose). Die ensieme benodig vir die afbraak van sellulose en hemisellulose staan gesamentlik as sellulases en hemisellulases bekend. Hierdie ensieme het 'n breë spektrum van industriële toepassings insluitende die produksie van brandstofalkohol d.m.v. fermentasies, vermindering in die hoeveelheid chloor benodig vir die bleikproses in die pulp-en-papier industrie, toename in deegvolume in die bakkersindustrie, verbetering van verteerbaarheid en verhoging van voedingswaarde van dierevoer, toename in verheldering en verbeterde filtreerbaarheid van wyn, bier en vrugtesap, ens. Dus bestaan daar 'n groot potensiële mark vir sellulases en hemisellulases, mits hul produksie ekonomies en die produk outentiek is. Aspergilli kom in 'n wye verskeidenheid van omgewings voor, insluitende grond, gestoorde voedsel- en voerprodukte asook ontbindende plante materiaal. Die voordele vir die gebruik van A. niger as gasheer vir heteroloë ensiemproduksie sluit in 'n goeie proteïen produseerder, industriële fermentasietegnologie dateer sover terug as 1919, 'n nie-patogeniese fungus met GRAS-status, benodig geen spesiale substrate of kwekingskondisies nie, FDA goedkeuring vir 'n groot aantal ensieme (homoloog sowel as heteroloog) wat reeds geproduseer word, ens. In hierdie studie is 'n Aspergillus uitdrukkingsvektor gekonstrueer deur van die konstitutiewe gliseraldehied-3-fosfaat dehidrogenase promoter (gpdp) van A. niger en die glukoamilase termineerder (glaAT) van Aspergillus awamori gebruik te maak. Die cDNA kopiee van die die eg! en xyn2 van Trichoderma reesei, cbhl-4 van Phanerochaete chrysosporium, man! van Aspergillus aculeatus en die xynC van Aspergillus kawachii was onderskeidelik na die uitdrukkingsplasmied oorgedra. Alle plasmiede is gesamentlik met die p3 SR2 plasmied na A. niger getransformeer en vir stabiele integrasie in die gasheergenoom geselekteer. Die rekombinante ensieme Egl, Xyn2, Cbhl-4, Manl en Xyn3 is suksesvol uitgedruk en teen aktiviteitsvlakke van 2300, 8000, 500, 6000 en 900 nkat/ml, onderskeidelik uitgeskei. Die ensieme is as funksionele entiteite geproduseer en vervolgens gekaraktiriseer. Die Egl, Xyn2 en Manl is as voertoevoegings vir die moontlike gebruik in die dierevoerindustrie geëvalueer. Verbeterde biomassa toename is in die in vivo studie op pluimvee waargeneem. Met die moontlikheid van grootskaalse heteroloë ensiemproduksie in gedagte, moes 'n eenvoudige substraat vir hul goedkoop produksie gevind word. Molasse medium (verkrygbaar vanaf die Suid Afrikaanse suiker industrie) was derhalwe geëvalueer en die kwekingskondisies geoptimiseer vir die moontlike gebruik as kwekingssubstraat vir A. niger. Vir die evaluasie is die Egl en Xyn2 aktiwiteite onder verskillende toestande geproduseer en oor tyd gemonitor. Hierdie studie beklemtoon die moontlike gebruik van A. niger vir heteroloë produksie van ensieme, die gebruik van industriële substrate as kwekingsmedium en baan die weg vir ekonomiese, hoëvlakuitdrukking van industrieelbelangrike ensieme met 'n positiewe implikasie op die omgewing.

Submitted in fulfillment of the academic requirements for the degree of Master of Applied Sciences (Chemistry), Durban University of Technology, Durban, South Africa, 2016. / Globally there is a drive for the use of renewable materials for the production of biofuels or high-end value chemicals. The current production of chemicals from crude oil refining is unsustainable and leads to global warming effects. Biomass is the most attractive renewable energy source for biofuel or fine chemical production. Sugarcane bagasse is a by-product of the sugar milling industry and is abundantly available. In this study lignin was sequentially extracted using ionic liquids. The ionic liquids (ILs) 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) and triethylammonium hydrogen sulfate ([HNEt3][HSO4]) were used to fractionate the sugarcane bagasse. The pre-treatment of sugarcane bagasse was carried out at different temperatures ranging from 90 - 150 0C and reaction times ranging from 1 - 24 h in a convection oven at a 10 % biomass loading. Both ILs were able to dissolve the raw bagasse samples at 120 0C with [Emim][OAc] giving a lignin maxima of 28.8 % and a low pulp yield of 57 % after 12 h; [HNEt3][HSO4] gave a lignin recovery of 17.2 % and low pulp yield of 58.5 % after 6 h. Regenerated lignin was obtained by adding ethanol/ water to the mixture followed by vacuum filtration. The regenerated pulp materials were characterized by Scanning Electron Microscope (SEM) to study the morphology; Fourier Transform Infrared Spectroscopy (FTIR) to study the characteristic bands and thermal analysis to study the thermal stability. / M

A dissertation submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Engineering. October 2017 / Increasing concerns over environmental and geo-political issues on resources’ sustainability have driven the industries to shift their efforts to produce chemicals from renewable biomass. Amongst the lignocellulosic biomass, corncob contains cellulose, hemicellulose and lignin that are built in a compact structure which makes it difficult to access. Pre-treatment is then applied to make the content to be accessible to enzymatic hydrolysis which breaks down the polysaccharides to monomers. The sugar monomers can be converted to a wide range of bioproducts such as biofuels and bio-chemicals. The objective of the study was to determine, evaluate and optimize the best solvent system to pre-treat corn cob. In addition, the study evaluated the effect of pre-treatment parameters on the yield of cellulose and hemicellulose and attempt to develop a kinetic model to explain the dissolution. Lithium perchlorate, zinc chloride, phosphoric acid, sulphuric acid and sodium hydroxide were used during the pre-treatment, which was carried out at 70-80 ° C for 6 hours. Characterization of pre-treated samples showed a significant change in structure after pretreatment indicating disruption in cell wall of the lignocellulosic material. FTIR revealed a reduction in phenolic group; indicating that the lignin content has been reduced. The XRD patterns show that crystallinity was considerably reduced; this was shown by an increase in calculated crystallinity index (CrI) after LiClO4, ZnCl2, H3PO4 and NaOH pre-treatment. The CrI of raw corncob (CrI= 32.7%) increased to 46.2 %, 42.3 %, 55.6 % and 53.4 % of LiClO4, ZnCl2, H3PO4 and NaOH, respectively. The crystallinity index increased for pre-treated material, indicating that the amorphous cellulose is dissolved in the liquor, as well as lignin and hemicellulose removal This study has shown that LiClO4.2H2O pretreatment agent is an efficient solvent system to pretreat corncob which consecutively increase the accessibility of cellulose and hemicellulose from the solid fractions. The accessibility was confirmed by an ease hydrolysis of cellulose & hemicellulose to glucose & xylose respectively. An increase of nearly four times compared to the untreated corncob. The effect of reaction operating parameters i.e. Reaction time, temperature and solvent concentration was carried out and then optimized by response surface methodology (RSM) using Minitab 16. The target was to maximize the yield of cellulose and hemicellulose. It was discovered that the increase in temperature and reaction time increase the accessibility of cellulose and hemicellulose until an equilibrium is reached at 3 & half hours and 176 °c. The pretreatment solvent concentration was discovered to have an effect on the accessibility but not as much as temperature and time. The best pretreatment conditions to obtain high polysaccharides conversions to monomers were at 176°c for 3.5 hours using LiClO4.2H2O for 10 g of corncob. The results obtained from RSM were used to evaluate the temperatures profile, kinetic model for the corncob pretreatment as a function of temperature. The kinetics of pretreatment were studied by the amount of glucose, xylose and the lignin removed from the pretreated solids. The kinetic model of lignin removal and sugars accessibility was identified as a first-order reaction corresponding to the bulk phase for pretreatment time up to 24 hours. The rate constant results show that the kinetic rate increased with temperature. The activation energy for glucose, xylose and lignin were calculated to be 15.0 kJ/mol, 14.2 kJ/mol and 36.54 kJ/mol, respectively. / MT 2018

Lo Sze Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 100-113). / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.iv / List of Figures and Tables --- p.vii / Abbreviations --- p.xii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Edible mushrooms --- p.1 / Chapter 1.1.1 --- Pleurotus sajor-caju --- p.1 / Chapter 1.1.2 --- Flammulina velutipes --- p.2 / Chapter 1.2 --- Lignocellulose and phenolic monomers --- p.4 / Chapter 1.2.1 --- Sources of phenolic monomers --- p.4 / Chapter 1.2.2 --- Toxicity of phenolic monomers --- p.10 / Chapter 1.3 --- Fungal laccases --- p.13 / Chapter 1.3.1 --- Occurrence --- p.13 / Chapter 1.3.2 --- Laccase reaction --- p.14 / Chapter 1.3.3 --- Physiological functions --- p.18 / Morphogenesis --- p.18 / Pathogenicity --- p.19 / Lignin degradation --- p.20 / Chapter 1.4 --- Purpose of study --- p.22 / Chapter 2. --- Materials and Methods --- p.24 / Chapter 2.1 --- General --- p.24 / Chapter 2.1.1 --- Organisms --- p.24 / Chapter 2.1.2 --- Culture medium --- p.24 / Chapter 2.1.3 --- Addition of phenolic compounds --- p.24 / Chapter 2.2 --- Effect of phenolic monomers on the growth of mushroom mycelium on agar plates --- p.25 / Chapter 2.3 --- Effect of phenolic monomers on the production of fungal biomass in liquid culture --- p.25 / Chapter 2.4 --- Effect of phenolic monomers on the extracellular laccase produced by P. sajor-caju and F. velutipes --- p.26 / Chapter 2.5 --- Assay of laccase activity --- p.26 / Chapter 2.6 --- Electrophoresis patterns of laccase proteins --- p.27 / Chapter 2.6.1 --- Non-denaturing polyacrylamide gel electrophoresis --- p.27 / Chapter 2.6.2 --- Localization of laccase activity --- p.27 / Chapter 2.7 --- Purification of extracellular laccases from P. sajor-caju --- p.28 / Chapter 2.7.1 --- Inoculum preparation --- p.28 / Chapter 2.7.2 --- Culture conditions --- p.28 / Chapter 2.7.3 --- Concentration of culture supernatant --- p.29 / Chapter 2.7.4 --- Ammonium sulphate fractionation --- p.29 / Chapter 2.7.5 --- Anion exchange chromatography --- p.29 / Chapter 2.7.6 --- Preparative polyacrylamide gel electrophoresis --- p.30 / Chapter 2.7.7 --- Protein detection and quantification --- p.30 / Chapter 2.8 --- Characterization of Laccase Protein --- p.31 / Chapter 2.8.1 --- "Effect of pH, temperature and substrate concentration" --- p.31 / Chapter 2.8.2 --- Effect of inhibitors --- p.32 / Chapter 2.8.3 --- Determination of isoelectric point --- p.32 / Chapter 2.8.4 --- Determination of molecular weight --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Effect of phenolic monomers on the growth of P. sajor-caju and F. velutipes --- p.34 / Chapter 3.1.1 --- P. sajor-caju --- p.34 / Chapter 3.1.2 --- F. velutipes --- p.38 / Chapter 3.2 --- Effect of phenolic monomers on laccase production by P. sajor-caju and F. velutipes --- p.41 / Chapter 3.2.1 --- P. sajor-caju --- p.45 / Chapter 3.2.2 --- F. velutipes --- p.49 / Chapter 3.3 --- Electrophoretic patterns of extracellular laccase --- p.53 / Chapter 3.3.1 --- P. sajor-caju --- p.53 / Chapter 3.3.2 --- F. velutipes --- p.56 / Chapter 3.4 --- Purification of laccase protein from P. sajor-caju --- p.58 / Chapter 3.4.1 --- Separation of laccase proteins --- p.58 / Chapter 3.4.2 --- Purification of laccase IV --- p.59 / Chapter 3.5 --- Characterization of laccase IV from P. sajor-caju --- p.64 / Chapter 3.5.1 --- Optimum temperature and thermostability --- p.64 / Chapter 3.5.2 --- Optimum pH and pH stability --- p.67 / Chapter 3.5.3 --- Substrate concentration --- p.70 / Chapter 3.5.4 --- Effect of inhibitors --- p.74 / Chapter 3.5.5 --- Isoelectric point --- p.74 / Chapter 3.5.6 --- Molecular weight --- p.74 / Chapter 4. --- Discussion --- p.78 / Chapter 4.1 --- Phenolic monomers and the growth of P. sajor-caju and F. velutipes --- p.78 / Chapter 4.2 --- Phenolic monomers and laccase production by P. sajor- caju and F. velutipes --- p.81 / Chapter 4.3 --- Electrophoretic patterns of laccase proteins --- p.83 / Chapter 4.4 --- Physiological functions of laccase --- p.85 / Chapter 4.5 --- Purification of selected laccase protein from P. sajor-caju --- p.88 / Chapter 4.6 --- Properties of laccase IV from P. sajor-caju --- p.89 / Chapter 4.6.1 --- Optimum temperature and thermostability --- p.89 / Chapter 4.6.2 --- Optimum pH and pH stability --- p.90 / Chapter 4.6.3 --- Effect of inhibitors --- p.92 / Chapter 4.6.4 --- Km --- p.93 / Chapter 4.6.5 --- Isoelectric point --- p.94 / Chapter 4.6.6 --- Molecular weight --- p.94 / Chapter 4.7 --- Future works --- p.96 / Chapter 5. --- Conclusion --- p.98 / Chapter 6. --- References --- p.100

Currently, the production of palm oil leads to the destruction of rainforest. A more sustainable source of lipids could be obtained using abundant lignocellulosic waste (e.g. wheat straw) as a source of carbon in the form of polysaccharides. Some species of oleaginous yeast, grown on sugars, can be made to accumulate between 20-80% of their biomass as oil and so offer a promising alternative to terrestrial crops. In this thesis, the yeast Metschnikowia pulcherrima was selected for its resilience to contamination. Although not previously classified as oleaginous, a combination of low temperature and restricted nutrient availability prevented sporulation and consequently triggered levels of oil production in M. pulcherrima cultures of up to 47%. The potential of this yeast to produce lipids inexpensively on waste resources was investigated. This yeast was grown under non-sterile conditions at pilot scale with minimal temperature control. The possibility of growing M. pulcherrima on lignocellulose was studied on models and showed that it was tolerant to a range of sugars and inhibitors commonly found in hydrolysed lignocellulose. The yeast produced 6.04 g L-1 lipid when cultivated on a mixture of hexoses and pentoses. This was corroborated by demonstrating that the yeast could be cultured on oligomers and sugars produced by hydrolysing wheat straw. Evidence of cellulase production was observed, and this was utilised in a process involving mildly pretreated wheat straw, using a range of pretreatment processes and culture conditions to yield a maximum of 1.12 g L-1 lipid. The usefulness of the products of this cultivation in forming oil in water emulsions was evaluated, and some evidence of surface-active effects was found. Overall, M. pulcherrima was found to have phenotypes that would be highly beneficial in reducing the capital and running costs of a putative lipid production process.

Modern society depends on fossil fuels to fulfill the increasing energy demand, which could cause further damages in the environment. Therefore, it is mandatory to develop technologies for the production of efficient alternative renewable energy sources, like biofuels. Agricultural waste streams, such as wheat straw, produced in larger amount can be utilized for these purposes, and can be used as raw materials for both ethanol and biogas production. However, the compact structure of lignocelluloses present in these materials makes their biological degradation difficult.In order to enhance the biogas production, wheat straw milled to 2mm size was pretreated with N-methylmorpholine-N-oxide (NMMO) at 90°C for 7-30 hours in this study. 90°C was applied as treatment temperature for the treatment to be able to investigate if the district heating system could be utilized in the treatment process in the future. The effects of the different pretreatment conditions were then evaluated by running batch anaerobic digestion assays for 45 days at 55°C.The best result was achieved after 7h treatment, leading to 0.47 Nm3/kg VS of straw methane production, corresponding to an increase by 47% compared to methane production obtained for the untreated straw.Furthermore, it was possible to recover up to 91.33% of the straw after the NMMO pretreatment. There were no significant changes in the chemical composition of the straw caused by the treatment, however structural analyses, like FTIR for the determination of crystallinity and and Simon Staining for the determination of porosity of the material showed that the crytallinity decreased, while the porosity increased as results of the treatment.Soaking in water for 72h prior to NMMO treatment was also applied to see whether it can improve the diffusion of NMMO into the material, but without success, due to the dilution of the treatment chemical caused by the soaking. The findings made during the experimental stage indicated that it is possible to run the NMMO pretreatment at an industrial temperature, further evaluation for the economical feasibility of this treatment method is however needed in the future. / Program: MSc in Resource Recovery - Industrial Biotechnology

L’extrusion biocatalytique, ou bioextrusion, est une technique d’extrusion réactive utilisant des enzymes comme catalyseurs. Cette technique est considérée en temps qu’étape intermédiaire, subséquente au prétraitement physico-chimique et précédente à l’hydrolyse enzymatique enréacteur fermé. L’utilisation de l’extrusion permet un procédé continu, facilement modulable et adaptable à des conditions de hautes consistances, de nombreuses biomasses et facilement transférable à l’échelle industrielle. Néanmoins, les données bibliographiques font ressortir la complexité des entrants et leurs interactions lors de la bioextrusion de biomasses lignocellulosiques. Les conclusions des bioextrusions de biomasses amidonnées soulignent l’importance de l’étude de l’influence de la concentration en substrat et en enzymes. Les résultats obtenus à partir de la bioextrusion des biomasses lignocellulosiques valident l’existence d’une activité enzymatique en extrudeuse malgré la contrainte thermomécanique et le temps de séjour limité. Lors de cette étape, l’hydrolyse de la fraction cellulosique est favorisée pour des milieux concentrés en substrat et en enzymes. Des modifications significatives des fractions cellulosiques cristallines et amorphes en surface, des réductions des tailles de particules, une dégradation visuelle des structures de la biomasse et l’augmentation de la sensibilité à la décomposition thermique, sont aussi observées sur la fraction solide. L’hydrolyse enzymatique des bioextrudats est prolongée en réacteur fermé. La bioextrusion permet des améliorations significatives des taux et vitesses de conversion des sucres sur le long terme, jusqu’à 48 h. Les gains observés sont relativement constants pour la paille de blé et augmentent avec le temps pour les écorces de bouleau et les résidus de maïs. Post-extrusion, la concentration en substrat influence négativement la conversion des sucres. Cependant, les plus-values de conversion du glucose lié à la bioextrusion de paille de blé sont principalement observables pour des concentrations en substrat et en enzymes élevées. À partir de 4 h, des baisses significatives de la conversion du xylose sont observées après bioextrusion. Les déstructurations de la fraction solide, déjà observées au cours la bioextrusion, se poursuivent en réacteur fermé. Les meilleurs résultats hydrolytiques aux niveaux des hautes charges en enzymes et en substrat sont associables aux bonnes conditions de mélanges caractéristiques des éléments bilobes. L’ensemble enzymatique est probablement réparti de façon plus homogène (mélange distributif) pour cibler plus de sites disponibles. De plus, le mélangé dispersif limite la proximité entre enzymes de même type et les gênes associées. Le procédé d’extrusion permet une agitation efficace, un bon transfert de masse et probablement un meilleur contact entre enzymes et substrat. Les moins bons résultats de conversion du xylose sont probablement à relier à des phénomènes d’adsorption non-spécifique, ou encore de désactivation des hémicellulases, provoqués par l’intensité des contraintes thermomécaniques et les résidus ligneux. Les bons résultats de déstructuration après bioextrusionsont associables à une action synergétique des contraintes mécanique et biochimique. Les analyses d’autofluorescence montrent l’évolution de la fraction ligneuse dans le processus de déconstruction de la fraction solide. Une production progressive de particules très fines,visiblement associée à la fraction ligneuse, est observée. Des complexes lignine-carbohydratessont aussi détectés dans la fraction liquide. Etant peu, voire pas hydrolysable par voie enzymatique, ces fractions hétéropolymériques sont un frein à la déconstruction. Si la déstructuration des lignines est probablement majoritairement liée au prétraitement alcalin, le procédé de bioextrusion provoque une diminution de la teneur en hétéropolymères de plus hautes masses moléculaires.

<p>To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. &alpha / -L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones.</p>