Neste trabalho investigamos a porosidade nanométrica de materiais lignocelulósicos derivados do bagaço de cana-de-açúcar que foram submetidos à compressão úmida. A produção dos materiais estudados a partir do bagaço de cana-de-açúcar utilizou processos de tratamento hidrotérmico seguido de processos de deslignificação organossolve (etanol-água) e soda (hidróxido de sódio). Os tratamentos hidrotérmicos utilizaram a fração fibra do bagaço de cana-de-açúcar, no estado bruto e moído, seguindo planejamento experimental fatorial de 2² com ponto central mais configuração estrela. O tratamento hidrotérmico ocorreu em temperaturas de 150-190°C com tempos de 20-60 minutos. Os processos de deslignificação utilizaram temperaturas de 160°C e 190°C para o processo soda e organossolve respectivamente, com tempos de 20, 40, 60, 80 e 100 minutos de tratamento. Os ensaios de compressão úmida foram realizados com cargas de 5, 10, 15, 20 e 25 toneladas e mostraram que materiais mais homogêneos e com menor granulometria, como o material moído e as polpas, são mais fáceis de comprimir. No entanto, os ensaios mostraram que os materiais comprimidos não são homogêneos, apresentando uma variação no teor de humidade do material comprimido (o centro da pastilha é mais seco que a periferia). O adensamento dos materiais também não é homogêneo, sendo o centro mais denso que a periferia das pastilhas. A perda de água durante a compressão foi de 74-85% para o material tratado hidrotermicamente, 66-85% para as polpas obtidas no processo soda e 81-94% para as polpas obtidas no processo organossolve. As análises de termoporometria mostraram que fração apreciável da porosidade nanométrica dos materiais deslignificados é colapsada com as menores pressões aplicadas (21 MPa). Incrementos de pressão (até carga de 107 MPa) promovem reduções comparativamente muito menores na porosidade nanométrica. / In this work we investigate the nanometric porosity of lignocellulosic materials derived from sugarcane bagasse and tested for wet pressing. The production of the studied materials from sugarcane bagasse employed hydrothermal treatments followed by organosolv (ethanol-water) and soda (sodium hydroxide) delignifications. For the hydrothermal treatments, we used bagasse fiber fractions in crude and milled states, following the factorial experimental design of 2² with central point plus star configuration. Hydrothermal treatment used temperatures of 150-190°C and times of 20-60 minutes. The delignification processes used temperatures of 160°C and 190°C for the soda and organosolv, respectively, with treatment times of 20, 40, 60, 80 and 100 minutes. Wet pressing was carried out with loads of 5, 10, 15, 20 and 25 tons and showed that materials with small and homogeneous particles such as the ground materials and the pulps are easier to compress and form a mass of material. However, the tests showed that the compressed materials are not homogeneous, presenting variations in the moisture content of the compressed materials (the center of the tablets were dryer than the periphery). Densification of the materials is also not uniform, the center being denser than the periphery. Water loss during compression was 74-85% for hydrothermally treated material, 66-85% for soda pulps and 81-94% for organosolv pulps. Thermoporometry analysis showed that appreciable fraction of the nanoscale porosity of the delignified materials collapse with the lowest applied pressures (21 MPa). Pressure increments (up to 107 MPa) promotes comparatively much lower reduction on nanoscale porosity.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22854号 / 農博第2437号 / 新制||農||1082(附属図書館) / 学位論文||R2||N5314(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / Production of bioethanol from lignocellulosic biomass has gained significant attention worldwide as an alternative fuel source for the transportation sector without affecting food supply. Efficient conversion of pentose sugars (L-arabinose and D-xylose) produced during hydrolysis of hemicellulose to ethanol can enhance the economic viability. In this study, a total of 390 yeasts isolated from Marula wine, the gut of dung beetles, herbal concoctions and banana residues were screened for the ability to ferment L-arabinose and D-xylose. Fourteen yeasts were able to ferment both pentose sugars and ten strains were subjected to an adaptation process in the presence of acetic acid using L-arabinose as carbon source. Four adapted strains of Meyerozyma caribbica were able to ferment L-arabinose to ethanol and arabitol in the presence of 3 g/L acetic acid at 35 °C. Meyerozyma caribbica Mu 2.2f fermented D-xylose, L-arabinose and a mixture of D-xylose and L-arabinose to produce 1.7, 3.0 and 1.9 g/L ethanol, respectively, compared to the parental strain with 1.5, 1.0 and 1.8 g/L ethanol, respectively, in the absence of acetic acid. The adapted strain of M. caribbica Mu 2.2f produced 3.6 and 0.8 g/L ethanol from L-arabinose and D-xylose, respectively in the presence of acetic acid while the parental strain failed to grow. In the bioreactor, the adapted strain of M. caribbica Mu 2.2f produced 5.7 g/L ethanol in the presence of 3 g/L acetic acid with an ethanol yield and productivity of 0.338 g/g and 0.158 g/L/h, respectively at a KLa value of 3.3 h-1. The adapted strain produced 26.7 g/L arabitol with a yield of 0.900 g/g at a KLa value of 4.9 h-1. Meyerozyma caribbica Mu 2.2f could potentially be used to produce ethanol and arabitol under stressed conditions. / National Research Foundation (NRF)

Thesis (Ph.D. (Microbiology)) -- University of Limpopo, 2021 / Biofuel production from lignocellulose material is an attractive alternative to fossil fuel. The use of lignocellulose material for biofuel production is imperative because of the numerous advantages that it offers. Biofuel is environmentally friendly and in developing countries such as South Africa, it has the potential to reduce the use of imported fuel and create jobs. Currently, several constraints are affecting the implementation of biofuel. One of the constraints is the cost-effectiveness and the efficiency of the enzymes involved in the enzymatic degradation of lignocellulose polymers to monomers, which can further be fermented to bioethanol. The potential way to reduce enzymatic degradation cost could be by supplementing the fungal enzymes with accessory enzymes such as endo-xylanase. The enzyme production cost is also dependent on the carbon source used. Lignocellulose materials that are regarded as waste must be assed for their use as enzyme inducer carbon sources and as biomass for biofuel production. This is a potential route that will reduce enzyme and biofuel production costs. Biofuel production cost can further be reduced by finding a yeast that can ferment xylose and ferment in the presence of inhibitors released during lignocellulose pretreatment. This study sought to tackle the enzymatic hydrolysis constraints and also search for xylose-fermenting yeast by exploring the gut microbiota of dung beetle. The gut of the dung beetle has recently received great attention since it is proposed to be a bioreactor for lignocellulolytic microorganisms that can be used in biofuel applications. This is because dung beetles feed on the dung of herbivorous animals and the dung is composed of 80% undigested plant material. In this study the guts of four Scarabaeidae dung beetles Kheper nigroanaeus Boheman, Heteronitis castelnaui, Pachylomerus femoralis, Anachalcos convexus and dung beetle larvae, Euoniticellus intermedius were screened for hemicellulolytic fungi and xylose-fermenting yeast. Hundred and thirty-two yeast isolates and two-hundred and twenty-two filamentous fungi were isolated and identified using ITS and D1/D2 regions. The yeast isolates were assigned to 8 genera and 18 species, Trichosporon was the most dominant genus while Candida tropicalis was the most dominant specie. Some of the yeast isolates were identified as uncultured fungi. This yeast must be characterised to be certain if they are novel species. The fungal isolates were assigned to 12 genera and 25 species, Aspergillus was the most dominant genus while Hypocrea lixii was the most dominant specie. The yeast isolated could assimilate xylose and could grow at a maximum temperature of 40 °C. Furthermore, these yeast isolates could also grow in the presence of 3 g/L acetic acid. Most of the fungal isolates had xylanolytic activity. The phylogenetic analysis revealed close genetic relatedness between isolates from the different dung beetle species and dung beetle larvae. The profile of the fungal genera was similar in the different dung beetles. Both guts and the larvae had Aspergillus, Hypocrea, Trichoderma, Talaromyces and Penicillium. The filamentous fungi that showed good xylanolytic activity were further screened for their ability to produce xylanase enzyme using thatch grass as an inductive carbon source. Thatch grass was selected in this study since it is in-house plant-based biomass. Thatch grass is abundantly available in South Africa; it is used for animal grazing but the more it grows it loses its nutritional content. Once it reaches this stage, it is no longer used and most of it is burnt. The fire from burning grass contains higher levels of nitrogen-containing chemicals that pollute the environment. Its compositional analysis (cellulose 46%, hemicellulose 27% and lignin 10%) also attributed to its selection as potential inductive carbon and attractive lignocellulose biomass for biofuel production. The higher xylanase activity of 283.43, 270 and 287.03 nkat/ml were observed from Aspergillus fumigatus L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus), respectively. This was achieved when acid pretreated thatch grass was used as an inductive carbon source. Aspergillus fumigatus L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus) showed xylanase activity of 393,22, 313,06 and 200 nkat/ml when grown on synthetic xylan. Neosartotya sp AB2XYL20 showed higher xylanase activity on thatch grass. The suitable production process for xylanase enzyme on acid pretreated thatch grass was assessed by conducting a comparative study on solid-state and submerged fermentation using L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus) as the best xylanase producer on acid pretreated thatch grass. The strain showed better xylanase activity when submerged fermentation was used. In this study, Hypocrea lixii AB2A3 was selected for further studies since it was the most dominant species and also showed good xylanase activity. Thatch grass was pretreated differently to evaluate the suitable chemical for pretreating thatch grass. Thatch grass was pretreated with dilute sulphuric acid 1.2% and maintained the pH of 5.5 by using sodium hydroxide while another batch was pretreated the same way and was washed with distilled water till pH of 5.5. The other batch was then pretreated with ammonium solution and was also washed with distilled water to maintain a pH of 5.5. The above-mentioned pretreated thatch grass was tested as an inductive carbon source as well as untreated thatch grass. The xylanase activity was determined to assess a good inductive carbon. All the thatch grass pretreated and washed with distilled water showed very low xylanase activity. The untreated thatch grass resulted in lower xylanase activity as compared to xylanase activity achieved when pretreated thatch grass was used. Parameters such as agitation speed and initial inoculum size were also assessed during xylanase production by Hypocrea lixii AB2A3 on acid pretreated thatch grass. Xylanase activity increased from 525 nkat/ml (Inoculum size 2×106 spore/ml and agitation speed 150 rpm) to 584.8 nkat/ml (Inoculum size 2×106 spore/ml and agitation speed 200 rpm). The crude xylanase from Hypocrea lixii AB2A3 was used to hydrolyse acid pretreated thatch grass. This resultant in xylose yield of 138 mg/g of substrate and glucose yield of 49 mg/g of substrate. Crude xylanase was mixed with commercial celluclast™. This enzyme mixture resulted in a xylose yield of 128 mg/g substrate and a glucose yield of 549 mg/g of substrate. The results obtained in this study show that indeed gut of the dung beetles and dung beetle larvae are a rich source of microorganisms that can play an important role in biofuel application and remediating the environment by degrading plant-based biomass regarded as waste into valuable products. It is imperative to evaluate the gut microbiota of dung beetles from different regions in South Africa for their application in the biofuel industry to reinforce its implementation. Thatch grass is a potential inductive carbon and lignocellulose biomass for biofuel production. / NRF (National Research Foundation)

Le compostage est un procédé anthropique basé sur le processus naturel de décomposition de la biomasse qui exploite l'activité enzymatique des microorganismes sous le contrôle de plusieurs facteurs environnementaux. Les résidus lignocellulosiques de par leur composition et leur faible pourcentage d'humidité sont particulièrement adaptés au compostage dans lequel ils jouent le rôle d’élément structurant. Bien que majoritairement d’origine végétale, la matière organique dirigée vers les sites de compostages est très diversifiée, tout comme les types de contaminants qu’elle peut incidemment contenir et dont l’impact sur les processus de biodégradation, et de surcroit leur rémanence dans l’environnement, reste largement à investiguer. L’objectif de cette thèse vise ainsi à faire état de l’effet de la composition de la biomasse lignocellulosique et de la présence d’un contaminant fréquent tel que le glyphosate sur le compostage. Pour ce faire, le suivi de la transformation de la matière organique végétale et de la dégradation du glyphosate, l’évolution des paramètres physicochimiques et la dynamique de recrutement des populations bactériennes ont été effectués tout au long du processus. Deux expériences menées sur le terrain visaient dans un premier temps à mesurer l’effet de l’âge d’une plante ligneuse, dans ce cas-ci le saule arbustif (Salix), et d’une période d’entreposage hivernal sur la transformation de la biomasse, et dans un deuxième temps à étudier les dynamiques de succession bactériennes impliquées dans le cycle du carbone et de l’azote lors du compostage de résidus végétaux. Les résultats obtenus ont révélé une différence dans la composition de la biomasse des tiges âgées de 2 ans et de 3 ans. Alors que les premiers contenaient plus de composés extractibles, les seconds étaient plus riches en sucres structuraux. Ces différences expliquent une hausse des températures plus forte et plus rapide dans le tas de copeaux de tiges plus jeunes. La diminution des composés extractibles, la conservation des sucres structuraux et l’augmentation de la proportion de lignine démontrent l’importance de la source de carbone soluble pour l’initiation de la décomposition du bois et la récalcitrance des éléments lignocellulosiques durant l’entreposage hivernal. La seconde expérience a mis en évidence une très grande diversité de bactéries responsables de la décomposition de la cellulose, des hémicelluloses et de la lignine durant la phase thermophile du compostage. Cette phase qui était le théâtre d’une activité intense comptait moins d’espèces, mais ces dernières étaient très abondantes, une tendance qui s’est inversée avec la maturation de la matière organique. La dynamique observée traduit une redondance fonctionnelle des communautés qui semblent évoluer selon la température, le taux d’oxygène et la nature du substrat disponible. Une troisième expérience menée en milieu contrôlé a ensuite démontré l’impact négligeable du glyphosate sur l’activité microbienne et l’évolution des paramètres physicochimiques lors du compostage. Le glyphosate était presque ou entièrement dégradé à l’issue du compostage et la présence du principal produit de dégradation, l’acide aminométhylphosphonique (AMPA) n’a d’ailleurs même pas pu être quantifiée durant l’expérience. L’impact du glyphosate sur les communautés bactériennes était également négligeable. Seules quelques bactéries étaient différentiellement abondantes entre les deux traitements, la grande majorité étant moins abondante dans le traitement contenant du glyphosate. La richesse en espèces aux différents temps d’échantillonnage était la même entre le traitement témoin et le traitement contenant du glyphosate « pur » et l’analyse de la bêta-diversité n’a relevé aucune différence significative entre les communautés présentes dans le traitement témoin et le traitement glyphosate. Cette thèse a ainsi fait valoir l’importance de la nature initiale de la matière organique sur l’activité microbienne, le recrutement et la dynamique des communautés durant le compostage, tandis que la présence du contaminant glyphosate s’est présenté comme un facteur beaucoup moins déterminant sur les processus de décomposition et l’abondance des espèces bactériennes. Ces informations devraient non seulement permettre d’optimiser le traitement de la matière organique par compostage, mais aussi de mieux évaluer les risques potentiels associés au compostage de biomasse contaminé. / Composting is an anthropic process based on the natural decay of biomass that exploits the enzymatic activity of microorganisms under the control of several environmental factors. Due to their composition and low moisture content, lignocellulosic residues are particularly suitable for composting and serve as a structuring element, which confers them an important role in the process. Although mostly of plant origin, the organic matter (OM) directed towards composting sites is highly diversified, as are the types of contaminants it can contain. The impact of these contaminants, such as glyphosate, on the biodegradation process and their persistence in the environment remain to be investigated. The objective of this thesis is thus to report on the effect of the composition of the lignocellulosic biomass and the presence of glyphosate on the evolution of the physicochemical parameters and the recruitment of bacteria during composting, while ensuring the follow-up of the transformation of the vegetable organic matter and the degradation of glyphosate during the process. Two field studies were conducted to measure the effect of stem age and winter storage on the transformation of wood chips, and to study the dynamics of bacterial succession involved in the carbon and nitrogen cycle during the composting of plant residues. The results obtained revealed a difference in the composition of 2-year-old and 3-year-old stems from shrub willow (Salix sp.), with the younger ones containing more extractable compounds and the more mature ones richer in structural sugars. These differences were reflected in a higher and faster temperature rise in the younger chip pile. A decrease in extractives, retention of structural sugars, and an increase in the proportion of lignin demonstrate the importance of the soluble carbon source for the initiation of wood decomposition and recalcitrance of lignocellulosic elements. The second experiment revealed a very high diversity of bacteria responsible for the decomposition of cellulose, hemicelluloses and lignin during the thermophilic phase of composting. This phase, during which intense activity took place, had fewer species, but they were very abundant, a trend that reversed as the organic matter matured. The observed dynamics reflect a functional redundancy of the communities, which seems to evolve according to the temperature, oxygen level and nature of the available substrate. A third experiment conducted in a controlled environment demonstrated the negligible impact of glyphosate on microbial activity and the evolution of physicochemical parameters during composting. Glyphosate was almost or completely degraded after composting, while the main product of degradation, aminoethylphosphonic acid (AMPA), was not detected. The impact of glyphosate on bacterial communities was also negligible, while species richness at different sampling times was the same when comparing the control treatment and the treatment containing "pure" glyphosate. The beta-diversity analysis found no significant difference between the communities present in the control and glyphosate treatments, while a few bacteria were differentially abundant between the two treatments, the vast majority being less abundant in the glyphosate treatment. This thesis has thus highlighted the importance of the initial nature of the organic matter on microbial activity as well as on the recruitment and dynamics of bacterial communities during composting, while the presence of glyphosate was shown to be a weak determinant of decomposition processes and species abundance. This information should help to optimize the treatment of organic matter by composting and to better assess the potential risks associated with composting contaminated biomass.

Den ökande efterfrågan på energi och den förväntade nedgången i råoljeproduktion har lett till ett enormt sökande efter nya energikällor.Cellväggen i växter består till stor del av lignocellulosa som i sin tur innehåller cellulosa och hemicellulosa. Dessa polysackarider är av stor betydelse för sökandet efter förnyelsebar energi.Cellväggen måste förbehandlas innan den kan brytas ner till enkla sockerarter. Efter nedbrytning kan monosackariderna användas till produktion av etanol eller biogas genom väl etablerade fermenteringstekniker. Syftet med denna studie var att jämföra och utvärdera några metoder som används vid degradering av lignocellulosa. Tre behandlingar har jämfört för att se vilken som ger mest avkastning i form av monosackarider. Vetehalm användes som substrat och hydrolyseras med hjälp av tre kommersiella enzymblandningar. Proverna förbehandlades före den enzymatiska reaktionen med antingen mikrovågor eller ångexplosion.Resultaten visade att en behandling med mikrovågsbestrålning eller ångexplosion kombinerad med enzymhydrolys gav högst avkastning. De slutsatser som kan dras är att en mekanisk förbehandling ökar utbytet drastiskt men är otillräcklig i sig. Ytterligare enzymatisk behandling är nödvändig att erhålla större mängder enkla sockerarter från lignocellulosa. / The increasing energy demand and the anticipated decline in crude oil production has led to an immense search for new energy sources. Plant cell walls contain lignocellulose that conserve great amounts of energy. These polysaccharides are of high importance for the search of renewable energy sources. Pretreatment of the cell wall is necessary in order to hydrolyse it to its component sugars. Once degraded to monomeric sugars it can be fermented to either ethanol or biogas through established fermentation technologies.The aim of this thesis was to compare and evaluate some of the methods used for sacchrification of lignocellulose. Three treatments where compared to determine which is highest yielding. These are enzymatic hydrolysis, microwave irradiation and steam explosion.Wheat straw was used as substrate and hydrolysed by three commercial enzyme mixtures. Samples were pretreated before the enzymatic reaction with either microwave or steam explosion. Results showed that a treatment of either microwave irradiation or steam explosion combined with enzyme hydrolysis gives the highest yield in monomeric sugars. The conclusions that can be drawn are that mechanical pretreatment increases yield drastically but is insufficient in its self. Further enzymatic treatment of wheat straw is necessary to obtain high amounts of simple sugars.

Thesis (M. Sc. (Microbiology)) -- University of Limpopo, 2016 / Smart hydrogels could facilitate immobilisation of cellulases to allow recovery and decrease enzyme cost in the biofuel industry, as they have a soluble-gel transition. The aim of the study was to design and evaluate the use of smart hydrogels for immobilisation of cellulase system that can be recovered after hydrolysis of cellulosic biomass. Cellulases from Aspergillus niger FGSC A733 produced under solid state fermentation and commercial cellulases were used in immobilisation. Various support matrices prepared were poly-N-isopropylacrylamide (p-NIPAAm), poly-N isopropylacrylamide-co-Methacrylic acid (p-NIPAAm-co-MAA) and supermacroporous poly-crosslinked-Acrylamide-co-N,N’-Methylenebisacrylamide (p-crosslinked-AA-co MBA). Cellulases were coupled onto the support matrices by covalent attachment method through reactive groups of N-acryloxysuccinimide (NAS) or Methacrylic acid N-hydroxysuccinimide (NMS). The low critical solution temperature (LCST) of formed p-NIPAAm-co-MAA copolymer was determined by the inflection point method. The shrinking and swelling kinetics and pH sensitivity of p-NIPAAm-co-MAA copolymer and conjugates were characterised using a cloud point method. Hydrolysis of CMC using cellulase-microbeads-p-NIPAAm and cellulase-crosslinked-p-NIPAAm with different percentage gel showed activity trend of 0.05>1>10>5>0.1% and 5>2>10% respectively. HPLC analysis showed that supplementation of β-glucosidase in cellulase-crosslinked-p-NIPAAm conjugates increased glucose by 12 and 14-fold at 30 and 50 °C respectively in the avicel hydrolysate in comparison with no β glucosidase supplementation. In the hydrolysis of avicel using cellulase-crosslinked p-NIPAAm-co-MAA conjugate a total of 13.6 g/L of reducing sugar was liberated after three cycles. In comparison a total of 21.4 g/L of reducing sugars were released from avicel hydrolysis using cellulase-crosslinked-p-AA-co-MBA conjugate after 3 cycles. In contrast, reducing sugars released in thatch grass hydrolysis using free enzyme were 8 times greater than in cellulase-crosslinked-p-AA-co-MBA conjugate. Cellulase crosslinked-p-NIPAAm-co-MAA conjugates were more stable than free enzyme at 50 and 60 °C after 24 hour and 120 minutes of incubation respectively, but lost activities at 65 °C after 120 minute. Therefore the activity loss in the immobilised enzymes was more due to thermal inactivation during precipitation and recovery than incomplete recovery during precipitation cycles. The results show that cellulases immobilised on smart polymers with sol-gel transition could be used in hydrolysis of cellulose due to ease of recovery. Hydrolysis kinetics was efficient for both immobilised enzyme system (cellulase-crosslinked-p-AA-co-MBA and cellulase-crosslinked-p-NIPAAm-co MAA conjugate) since were re-used in hydrolysis of avicel. Therefore the use of these smart polymers for cellulase immobilisation can contribute in cost reduction of the enzymatic hydrolysis process in the biofuel industry. / National Research Foundation (NRF) , University of Limpopo financial aid office and Flemish Interuniversity Council (VLIR-UOS) fo

In order to meet the energy needs of a growing world in a sustainable manner, new high efficiency, carbon-neutral fuels and chemical feedstocks are required. An emerging approach that shows promise for high efficiency production of renewable fuels and chemicals is the use of purified enzymes combined in one pot to catalyze complex conversions: synthetic pathway biotransformations (SyPaB). An exemplary technology in this burgeoning field is the production of hydrogen from biomass sugars. Lignocellulosic biomass, which includes agricultural residues, energy crops, and industrial waste streams, is an ideal substrate for SyPaB conversion, as it is abundant and cheap, second only to untaxed coal on a $/energy content basis. But the structure of biomass is highly recalcitrant, making high-yield biological conversion difficult to achieve. In order to increase susceptibility to enzymatic digestion, thermochemical pretreatments are applied, with the goals of removing of lignin, the simplest example being soaking in aqueous ammonia (SAA); hemicellulose removal, most often using dilute acid (DA); and increasing cellulose accessibility by cellulose solvent-based pretreatments, such as cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF). In a comparison of the lignin removal (SAA) and accessibility increase (COSLIF) approaches, we found that certain levels of lignin removal (~15%) were important, but further lignin removal was less effective at achieving digestibility gains than increasing cellulose accessibility. Pretreated biomass forms an excellent substrate for SyPaB hydrogen generation, due to low cost and high sugar content. Following experiments demonstrating the high yield conversion of sucrose to hydrogen (97%) and SyPaB generation of hydrogen at a rate commensurate with the best biological rates achieved, 157 mmol/L/h. SyPaB was combined with enzymatic hydrolysis to enable the direct conversion of cellulosic biomass, including untreated, DA, and COSLIF corn stover. In addition, an updated kinetic model of the system was used to rationally increase the maximum hydrogen production rate by 70% while minimizing total enzyme loading and without increasing substrate concentration. Together, these results demonstrate the high level of engineering control in cell-free systems, which can enable conversion of a variety of substrates to hydrogen at the highest possible yield and rates as high as any biohydrogen production method. / Ph. D.

Cette thèse se décline en six parties. Le premier volet porte sur la compréhension du procédé de rotomoulage et une description des matériaux composites à base de fibres lignocellulosiques. Le second volet comporte une revue de la littérature sur les composites produits par rotomoulage, tandis que le troisième volet se consacre à la compréhension de la modification des fibres d’érables par le polyéthylène maléisé (MAPE) en solution et son influence et de la teneur en fibre sur les propriétés morphologiques et mécaniques des composites. Le quatrième volet étudie les effets de la taille des particules et la modification en solution, ainsi que la teneur en fibre sur les propriétés morphologiques, thermiques, physiques et mécaniques des composites. Le cinquième volet se penche sur l’effet de la modification, la teneur et la taille des fibres d’érable sur la morphologie et les propriétés physiques et mécaniques des auto-hybrides. Finalement, le dernier volet étudie l’influence de la modification en solution avec du MAPE, la teneur et la taille des fibres sur la morphologie et les propriétés mécaniques des composites hybrides à base de fibres d’érable et de chanvre. Les résultats montrent que les fibres (érable et chanvre) ont été modifiées avec succès par le MAPE en solution, ce qui a amélioré la qualité de l’interface fibre-matrice des composites, conduisant à de meilleures propriétés mécaniques. En outre, les résultats ont prouvé que l’effet de la taille de particule était significatif. En effet, le module de traction augmente jusqu’à 73% lors de l’utilisation de fibres d’érable de 355-500 μm à 30% en poids. Une augmentation de 52% de la résistance au choc a été réalisée avec l’utilisation de 30% en poids de fibre d’érable (355-500 μm) comparé à ceux produits avec 125-250 μm. D’autre part la production de composites auto-hybrides a été en mesure d'améliorer les propriétés mécaniques comparées aux composites simples. Enfin, une augmentation du module de traction (63%), de la contrainte maximale (17%) et de la densité (17%) a été réalisée lors de l’utilisation d’un ratio de 75/25 de fibre d’érable/chanvre à 20% en poids total de fibre par rapport à la matrice seule. / This thesis is divided into six parts. The first part is related to the understanding of the rotomolding process and a description of composite materials based on lignocellulosic fibers. In the second part, a literature review on composites produced by rotomolding is presented. The objective of the third part is to understand the modification of maple fibers in solution by maleated polyethylene (MAPE) and its effect combined with fiber content on the morphological and mechanical properties of the composites. The fourth part studies the effects of fiber size, modification in solution as well as fiber content on the morphological, thermal, physical and mechanical properties of the composites. The fifth part investigates the effect of MAPE modification in solution, fiber content and particle size of maple fibers on the morphological and mechanical properties of selfhybrid composites. Finally, the effect of the surface treatment in solution, the content and the fiber size (maple and hemp) on hybrid composites is presented. The results showed that the fibers (maple and hemp) were successfully modified by MAPE in solution, which improved the interface quality between the matrix and fibers, leading to better mechanical properties. Moreover, the results showed that the effect of fiber size was significant as the tensile modulus increased by up to 73% with the use of 355-500 μm at 30% wt. of maple fiber compared to those produced with 125-250 μm. The production of self-hybrid composites was able to improve the mechanical properties compared to simple composites. An increase in the tensile modulus (63%), tensile strength (17%) and density (17%) was obtained by using a 75/25 ratio of maple/hemp fibers at a total fiber content of 20% wt. compared to the neat matrix.

La présente étude a eu pour objectif principalde mettre en place un procédé de prétraitement de la fibre naturelle de lin, de fabrication de nanofibres lignocellulosiques et leur modification de surface subséquente, environnementalement irréprochable sur toute la ligne. Afin de répondre à cet objectif principal, les nanofibres lignocellulosiques de lin ont été préparées en utilisant un procédé respectueux de l'environnement, soit une combinaison de prétraitement au dioxyde de carbone (CO2) dans les conditions supercritiqueset d'hydrolyse enzymatique. Le prétraitement au CO2 supercritique visait à surmonter la récalcitrance de la biomasse lignocellulosique et à donner accès aux enzymes hydrolytiques. Il a été démontré que le prétraitement au CO2 supercritique des fibres de lin a aidé à déstructurer la biomasse tout en évitant son fractionnement et à faciliter l’hydrolyse enzymatique subséquente du substrat. Un cocktail d’enzymes hydrolytiques comprenant la cellulase, la xylanase, la pectinase et la viscozyme a été utilisé et a permis d’extraire des fibres lignocellulosiques ayant des dimensions nanométriques.Ces nanofibres lignocellulosiques extraites ainsi que les résidus solides de l’hydrolyse sont de nature hydrophile en raison de l'attraction / interaction entre les groupes hydroxyles des composants fibreux et des molécules d'eau. La nature hydrophile de ces nanofibres lignocellulosiques aboutit souvent à une mauvaise compatibilité avec des matrices polymères hydrophobes. Une modification de surface s’impose donc afin de les rendre plus hydrophobes et donc compatibles avec les matrices hydrophobes. La laccase, une enzyme spécifique a été utilisée pour catalyser le greffage de composés phénoliques naturels, le gaïacol et le syringaldéhyde, rendant ainsi les nanofibres lignocellulosiques et les résidus solides de l’hydrolyse plus hydrophobes et compatibles avec lesmatrices hydrophobes. Aucun changement significatif dans la composition chimique des fibres de lin n’a été observé après le prétraitement tel que suggéré par les analyses par spectroscopie infrarouge. Ces dernières ont démontré par ailleurs le greffage de surface induit par la laccase, du guaïacol et du syringaldéhyde sur les nanofibres extraites et sur les résidus de l’hydrolyse. La technique de diffraction des rayons X a révélé que la cristallinité augmentait suite au prétraitement de la fibre avec le CO2 supercritique ainsi que suite à l’extraction des nanofibres. La microscopie électronique à balayage a révélé les dommages physiques causés à la surface des fibres suite au prétraitement alors que la microscopie électronique à transmission démontrait que les nanofibres lignocellulosiques extraites étaient en forme des filaments, avec un diamètre de 5 –10 nm et plusieurs micromètres de longueur. Enfin les fibres fonctionnalisées ont montré une meilleure stabilité thermique et un caractère hydrophobe comparativementaux fibres brutes non traitées. / The main goal of this research was to set up an environmentally friendly process for the pretreatment of natural flax fibres in view to produce lignocellulosic nanofibers and modify their surface for their use as compatible fillers in polymer composites. To achieve this main objective, lignocellulosic nanosized flax fibres were prepared using an environmentally friendly process based on a combination of supercritical carbon dioxide pretreatment and enzymatic hydrolysis conditions. Supercritical CO2 pretreatment aimed to overcome the recalcitrance of lignocellulosic biomass and to provide access to hydrolytic enzymes. It was shown that the supercritical CO2 pretreatment of raw flax fibers helped to deconstruct biomass, avoiding its fractionation and increased access to hydrolytic enzymes such as cellulase, xylanase, pectinase and viscozyme leading to extraction of lignocellulosic fibres having nanometric dimensions. These extracted lignocellulosic nanofibres as well as the solid residues of the hydrolysis are hydrophilic in nature because of the attraction / interaction between the hydroxyl groups of the fibrous components and water molecules. The hydrophilic nature of these lignocellulosic nanofibers often results in poor compatibility with hydrophobic polymeric matrices. Surface modification is therefore necessary to make them more hydrophobic and compatible with the hydrophobic matrices. Laccase mediated grafting of natural phenolic compounds, i.e. guaiacol and syringaldehyde, onto lignocellulosic fiber was achieved, thus making lignocellulosic nanofibers and hydrolysis solids residues more hydrophobic and compatible with hydrophobic matrices. No significant changes in the chemical composition of flax fibres were observed after pretreatment. This was confirmedby FTIR analysis, which also demonstrated laccase-induced grafting of guaiacol and syringaldehyde onto lignocellulosic nanofibers and solid residues hydrolysis surfaces. Moreover, X-ray diffraction revealed that crystallinity increased for supercritical CO2 pretreated fibres as well asenzymatically produced lignocellulosic nanofibers. Scanning electron microscopy revealed the physical damages in the form of holes, cracks and erosions onto the surface of supercritical CO2 pretreated flax fibres, while transmission electron microscopy evidencedthe production of filament-shaped nanosized fibrils with a diameter of 5-10 nm and several micrometers length. Finally, bio-grafted fibers showed better thermal stability and hydrophobicity if compared to untreated raw analogues.