<p>  </p> <p>The potential of lignocellulose for producing fermentable sugars as feedstock to manufacture fuels, chemicals, and materials for decarbonization remains untapped due to costly logistics and conversion processes. Pelleting technology offers a solution by addressing logistical issues and impacting downstream conversion, though it comes with its own costs. An overview of recent advances in pelleting technologies and their impact on bioconversion highlights the importance of understanding variables and product attributes. The interplay between pelleting and pretreatment processes, considering various feedstocks, is crucial for future design. Practical considerations such as energy consumption, costs, and environmental impacts must not be overlooked, along with exploration of cutting-edge technologies and strategies in this field. This work further presents a comprehensive investigation into the recalcitrance of pelleted corn stover to enzymatic digestion prior to any pretreatment.</p> <p>The potential of lignocellulose for producing fermentable sugars as feedstock to manufacture fuels, chemicals, and materials for decarbonization remains untapped due to costly logistics and conversion processes. Pelleting technology offers a solution by addressing logistical issues and impacting downstream conversion, though it comes with its own costs. An overview of recent advances in pelleting technologies and their impact on bioconversion highlights the importance of understanding variables and product attributes. The interplay between pelleting and pretreatment processes, considering various feedstocks, is crucial for future design. Practical considerations such as energy consumption, costs, and environmental impacts must not be overlooked, along with exploration of cutting-edge technologies and strategies in this field. </p> <p>This dissertation further presents a comprehensive investigation into the recalcitrance of pelleted corn stover to enzymatic digestion prior to any pretreatment. One aspect focuses on the role of high moisture pelleting in enhancing the enzymatic digestibility of corn stover before pretreatment, along with the relevant substrate characteristics. The pelleting process increases the digestibility of unpretreated corn stover, resulting in a glucan conversion increase from 8.2% to 15.5% at a 5% solid loading using 1 FPU Cellic® CTec2 per gram of solids. Under the same enzymatic hydrolysis conditions, the conversion of glucan remains higher for pelleted corn stover compared to its non-pelleted counterpart, even though both samples underwent identical milling processes and passed through the same screen to minimize particle impact. Compositional analysis reveals that loose and pelleted corn stover have similar non-dissolvable compositions, albeit with differences in their extractives. Using microcrystalline cellulose (Avicel) as a substrate, the presence of corn stover extractives results in a lower sugar yield compared to using citrate buffer instead, particularly for extractives from pelleted corn stover. This indicates a more negative impact of pelleted corn stover extractives on the activity of employed enzyme, CTec2. However, pelleted corn stover still shows increased overall glucan conversion compared to loose corn stover, suggesting improved digestibility of non-dissolvable components after milling and washing. The improvement in digestibility of pelleted material can be attributed to factors such as reduced particle size, enhanced substrate accessibility, and hydrolysis of cross-linking structures induced by the pelleting process. These findings offer valuable insights for the development of processing strategies aimed at sustainable and efficient utilization of lignocellulose.</p> <p>Furthermore, this dissertation delves into the profound impact of extractives on enzymatic hydrolysis, prompting a thorough examination of the composition and characteristics of extractives derived from pelleted corn stover, as well as their effects on enzymatic conversion. In contrast to previous reports, it is discovered that water extractable materials actually enhance the enzymatic hydrolysis of extractive-free stover, while the enzyme activities diminish when using microcrystalline cellulose as a substrate. This divergent behavior of extractives is attributed to the presence of lignin, which may interact with inhibitory compounds like phenolics, thereby mitigating the detrimental effects of soluble inhibitors or insoluble lignin, or both. These findings significantly advance our fundamental understanding of the intrinsic behavior of extractives and contribute to the optimization of schemes for efficient and cost-competitive enzymatic conversion of lignocellulose. </p>

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The present thesis investigates the application of pressurized mixtures of imidazolium-based ionic liquids with traditional organic solvents for the dissolution and extraction of lignocellulosic biomass, with bamboo as a specific example of renewable biomass. The approach has been unconventional in that the focus has been on solvent mixtures in which the ionic liquid is the minor component. The objective has been to combine the solvating power of the ionic liquid with a traditional solvent such as ethanol to modulate the outcomes of solubility and extractions by tuning the parameters of fluid composition, temperature, and pressure. Working with mixtures of ionic liquids in traditional solvents as process solvents lowers the viscosity of the medium and thus reduces the transport limitations that are often encountered when working with pure ionic liquids. Among other potential advantages are the reductions in overall process cost that are associated with ionic liquids, potentially easier recovery of post-extraction products, and the recycling of the ionic liquids. This thesis has also addressed another important question regarding the thermal stability of the ionic liquids as a processing medium at elevated temperatures and pressures over time, which may negatively impact their recovery and reuse, and may lead to environmentally unacceptable consequences. The dissolution experiments were carried out in a specially designed high-pressure view-cell equipped with sapphire windows for visual or optical observations. Evaluations were made employing standard characterization tools such as Thermogravimetric Analysis (TGA), Fourier Transform Infrared Spectroscopy (FTIR), UV-Vis Spectroscopy, and Scanning Electron Microscopy (SEM). Thermal stability studies were carried out using a combination of a view-cell and fiber optic UV-Vis capability at high pressures (up to 350 bar) and temperatures (up to 150 ℃). The dissolution of bamboo was first explored using mixtures of 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) with ethanol at temperatures from 100 to 150 ℃ and pressures from 35 to 350 bar over 4 or 24 h extraction times. The fluid mixtures employed were in the range of 1 - 40 wt % ionic liquid, which is in contrast to relevant dissolution experiments reported in the literature which either use pure ionic liquids or have the ionic liquids as the majority component. The effects of changing the temperature, pressure, and solvent composition on the removal of different components of the bamboo were assessed. Temperature played the most significant role in the amount of material extracted from the bamboo, with higher temperatures resulting in the removal of more lignin than cellulose and greater conversion of crystalline cellulose to the less recalcitrant amorphous form of cellulose. The concentration of ionic liquid in solution was also important, with higher concentrations resulting in more dissolved biomass. Finally, increasing the pressure resulted in higher amounts of dissolved biomass. The next series of studies focused on rigorously assessing the stability of 1-alkyl-3-methylimidazolium acetate and chloride ionic liquids with alkyl chain lengths from 2 to 10 under both isothermal and non-isothermal conditions via thermogravimetric analysis. Isothermal degradation experiments were conducted at temperatures ranging from 100 to 225 ℃ over time periods ranging from two hours to three weeks. Non-isothermal degradation experiments were conducted at heating rates of 5, 10, 15, and 20 ℃/min from room temperature to 650 ℃. The activation energies and pre-exponential factors were assessed with isoconversional integral methods; the activation energies () ranged from 115 to 157 kJ/mol, and the pre-exponential factors (()) ranged from 24-38. The degradation reactions could be described as 1st order, as they often are in the literature, but were best fit by the 3-dimensional reaction model. Ionic liquids with longer alkyl chains on their imidazolium rings decomposed more quickly and at lower temperatures. The thermal stability of the most promising ionic liquids ([EMIM]Ac, [BMIM]Ac, [EMIM]Cl, and [BMIM]Cl) were then assessed more closely at the possible biomass processing conditions that were being considered. The primary interest was determining the effects of various cosolvents on the thermal stability of these ionic liquids at the process temperatures and pressures, from 100 to 150 ℃ and 35 to 350 bar. These evaluations were carried out in the same high-pressure view cell in which the extraction experiments were conducted. To assess the degradation of the ionic liquids, time-evolved UV spectra of the mixtures were generated. It was found that more protic solvents such as water attenuated the degradation of the ionic liquids, whereas aprotic solvents such as DMF significantly exacerbated their degradation. Among the ionic liquids evaluated, it was found that [BMIM]Cl had the greatest stability in ethanol at 150 ℃. The bamboo extraction experiments were then continued with mixtures of [BMIM]Cl in ethanol. The results showed that higher temperatures are necessary to extract lignin and cellulose, with [BMIM]Cl's thermal stability at these temperatures giving it the advantage over [EMIM]Ac. In this system as well it was shown that higher concentrations of ionic liquid facilitated the extraction of more biomass. However, biomass constituents that dissolve into mixtures with lower concentrations of ionic liquid readily precipitate back out of solution when the mixture is returned to room conditions. Along with the results of the studies with [EMIM]Ac, the experiments conducted with [BMIM]Cl show that an increase in pressure results in greater amounts of dissolved biomass holding other conditions constant. The thesis, in summary, presents for the first time (a) the use of ionic liquids as a minor component in organic solvents as a potential biomass processing media, (b) the thermal stability of ionic liquids in a cosolvents at high pressures and temperatures, and (c) experimental results showing that pressure can enhance the amount that can be extracted from biomass with mixtures of ionic liquids in a cosolvent like ethanol. / Doctor of Philosophy / The purpose of the work detailed in the present thesis is to better understand the effects of mixtures of ionic liquids and traditional solvents on woody biomass. Ionic liquids are organic salts with melting points below 100 ℃, and they possess unique physical and chemical properties that can facilitate the dissolution or extraction of otherwise recalcitrant materials. There is a rapidly growing need for greener and more sustainable methods of processing woody biomass, which consist of primarily cellulose, lignin, and hemicelluloses. Industrial use of these liquids as processing solvents for woody biomass is limited by their relatively high viscosity, cost, and the difficulty of separating dissolved materials back out of solution. One method used to address these limitations is to mix the ionic liquids with other solvents, such as ethanol. The studies detailed in this thesis also seek to understand the effects of temperature and pressure on both the dissolution of woody biomass and on the degradation of the ionic liquids. The studies employ both traditional characterization equipment and a custom-designed view-cell which allowed for observation and characterization at high temperatures and pressures. The first part of the study investigated the dissolution of bamboo with mixtures of the ionic liquid 1-ethyl-3-methylimidazolium acetate, [EMIM]Ac, and ethanol. The effects of changing the temperature, pressure, and solvent composition on the removal of different components of the bamboo were assessed. It was found that temperature played the most significant role in the amount of material extracted, with higher temperatures resulting in the removal of more lignin than cellulose. The concentration of ionic liquid in solution was also important, with higher concentrations resulting in more dissolved biomass. Finally, increasing the pressure resulted in higher amounts of dissolved biomass. The next parts of the study focused on the degradation of the ionic liquids at elevated temperatures. The type of ionic liquids used in this study do not boil or evaporate at high temperatures, but instead break down into constituents that are themselves volatile. The thermal degradation of the ionic liquid used in the initial biomass dissolution experiments was investigated along with a series of similar ionic liquids. Their degradation behavior was assessed both by measuring their mass over time at a single constant temperature, and by heating them at a constant rate until they fully degraded. This behavior was mathematically modeled. The thermal stability of the most promising ionic liquids were then investigated in mixtures with other solvents in the high-pressure experimental cell under the same temperature and pressure conditions used in the biomass dissolution experiments. The ionic liquid found to have the best stability in ethanol in those experiments was 1-butyl-3-methylimidazolium chloride, [BMIM]Cl. Further dissolution experiments were carried out with mixtures of this ionic liquid in ethanol. These experiments took the insights gained from the previous investigations to further clarify the effects of temperature, concentration, and pressure on the dissolution of bamboo in mixtures of ionic liquid and ethanol. It was again shown that higher temperatures are necessary to extract lignin and cellulose. It was also shown that higher concentrations of ionic liquid facilitate the extraction of more biomass. However, it was also shown that biomass dissolved into mixtures with lower concentrations of ionic liquid readily precipitates back out of solution when the mixture is returned to room conditions. Pressure was again shown to have a favorable effect on the amount of material extracted.

The effects of climate change have made the need to develop sustainable production practices for biofuels and other chemicals imminent. The development of the green economy has also led to many industries voluntarily improving the sustainability of the products they produce. The microbial production of fatty acid-derived chemicals allows for the opportunity to reduce petroleum-based chemicals in the marketplace. However, for microbial produced chemicals to be industrially competitive, significant work is needed to improve the production capacity of industrial strains. There are a number of bottlenecks and challenges related to the production of various fatty acid derivatives that need to be addressed. One of these key challenges relates to the source of the fermentation feedstock. While sources such as corn or sugar cane are currently common, these feedstocks compete with food supply and require nutrient-rich soils. The use of lignocellulosic feedstocks is preferred to combat this issue, however these feedstocks present their own unique challenges. Pretreatment is required to release fermentable sugars, and this process also results in various fermentation inhibitors released into the solution. A better understanding of how engineered strains utilize these fermentable sugars as well as improving resistance to the inhibitors will help to improve the chemical production capacity of these chemical products. This work will focus on describing key bottlenecks related to fatty acid-derived products, while also evaluating proposed solutions to these bottlenecks. / Master of Science / Currently, many common household products and plastics are developed using petroleum-based components. From plastic bottles to common cosmetics, these contain ingredients that are derived from petroleum. In order to combat our reliance on petroleum for these every day products, it is essential to develop alternate sources for these materials. A potential source involve using plant material and by-products to produce these same compounds that we are able to produce from petroleum. While there has been significant research to produce useful products such as bioethanol from corn, this is not an ideal crop. Corn requires more water and space than other crops such as grasses. In addition, these grasses can grow in soil that food crops are unable to grow in, so we don’t utilize valuable land to develop common household products. However, these grasses are much more difficult to treat and process in order to form these basic chemical ingredients. In order to use grass-based crops, it is possible to engineer organisms such as yeast to process the raw material into valuable chemical precursor. This work aims to genetically engineer yeast in order to produce some of these chemical precursors from a grass-like feedstock. In addition, this work also analyzes how physical characteristics of yeast affect the final product formation. Finally, a model was developed to show how yeast ferments corn-like and grass-like feedstocks differently.

Tesis por compendio / [ES] La presente Tesis Doctoral se ha centrado en el aislamiento y caracterización de materiales celulósicos y extractos activos, procedentes de las cascarillas de arroz y café, y su incorporación a películas de almidón y mezclas compatibilizadas de almidón-PLA, para mejorar sus propiedades funcionales como materiales para el envasado de alimentos. Las fibras de celulosa (CF) se obtuvieron mediante tratamiento alcalino y de blanqueo, con un rendimiento de 41 y 53 g fibras/100 g cascarilla, respectivamente para cascarilla de arroz y café. Los nanocristales de celulosa (CNC) se aislaron de las fibras mediante hidrólisis ácida, con un rendimiento del 5% respecto a las fibras y con alta cristalinidad (90-92%), resistencia térmica y relación de aspecto (L/d: 20-40). Los compuestos activos se obtuvieron mediante extracción hidrotérmica (180 ºC; 9,5 bares), con un rendimiento de 17-18 g/ 100 g de cascarilla. Dichos extractos exhibieron capacidad antioxidante (EC50: 5,37-5,29 mg sólidos extraídos/ mg DPPH) y antimicrobiana (cuantificada en términos de concentración mínima inhibitoria: MIC) frente a L. innocua (MIC: 48-52 mg polvo/mL) y E. coli (MIC: 50-66 mg polvo/mL). Los materiales celulósicos procedentes de cascarilla de arroz y café se incorporaron a películas de almidón termoplástico (TPS), obtenidas mediante mezclado en fundido y moldeo por compresión. El módulo elástico aumentó un 186 y 121% cuando se incorporó a la matriz un 1% (p/p) de CNC de cascarilla de arroz y café, respectivamente. Del mismo modo, las CF se añadieron a las películas de TPS al 1, 5 y 10 pt%. Ambas CF aumentaron la rigidez y redujeron la extensibilidad de los films, aunque las CF de cascarilla de café mantuvieron mejor la ductilidad al 1 y 5% (p/p). La permeabilidad al vapor de agua de las películas de TPS no se redujo en los materiales compuestos, aunque la permeabilidad al oxígeno se redujo en aproximadamente un 17%. Al incorporar extractos activos a los films de almidón, mejoraron sus propiedades de tracción; el módulo elástico aumentó un 350%, a la vez que se hicieron menos extensibles. Las fibras de celulosa de ambos residuos fueron más efectivas como agentes de refuerzo en los films con extractos sólidos que en los de almidón solo. Se estudiaron también mezclas de almidón-PLA utilizando como compatibilizador policaprolactona funcionalizada con anhídrido maléico y/o glicidil metacrilato (PCLMG o PCLG). Se analizó el efecto de la proporción de PLA en la mezcla (20 y 40% respecto al almidón), y la de ambos compatibilizadores (2,5 y 5%), en las propiedades de los films. Los análisis de la microestructura, el comportamiento térmico y las propiedades funcionales (mecánicas, ópticas y de barrera) de los films, demostraron que sustituir el 20% del almidón por PLA e incorporar el 5% de PCLG podría ser una buena estrategia para obtener materiales adecuados para envasado de alimentos. Además, se estudió el efecto de la adición de rellenos celulósicos (CF y CNC) y del extracto antioxidante de cascarilla de café en la mezcla de almidón-PLA compatibilizada seleccionada. Las propiedades antioxidantes de los films se probaron a través de su eficacia para preservar al aceite de girasol de la oxidación. Se observaron diferencias significativas en las propiedades funcionales de los films cuando los CNC se incorporaron mediante dos métodos diferentes. El efecto de refuerzo de los materiales celulósicos en mezclas de S-PLA fue menos notable que en las películas de almidón, probablemente debido a la superposición del efecto de refuerzo de PLA. El extracto antioxidante no mejoró el comportamiento mecánico en la mezcla, pero le confirió capacidad antioxidante, adecuada para aplicaciones en el envasado de alimentos. / [CA] La present Tesi Doctoral s'ha centrat en l'aïllament i caracteritzaciò de materials cel.lulòsics i extractes actius, procedents de pellorfa d'arròs i café, i la seua incorporació a pel·lícules de midó i mescles compatibilitzades de midò-PLA, per a millorar les seues propietats funcionals com materials per al envasat d'aliments. Les fibres de cel.lulosa (CF) s'obtingueren mitjançant tractament alcalí i de blanqueig, amb un rendiment de 41 i 53 g fibres/100g pellorfa, respectivament per a pellorfa d'arròs i cafè. Els nanocristalls de cel·lulosa (CNC) es van aïllar de les fibres de cel·lulosa per mig d'hidròlosi àcida, amb un rendiment del 5% respecte a les fibres; en tots dos casos, amb alta cristal·línitat (90-92%), resistència tèrmica i relaciò d'aspecte (L/d: 20-40). Els composts actius s'obtingueren mitjançant l'extracció hidrotèrmica (180 ºC; 9,5 bars), amb un rendiment del 17-18 g/100 g de pellorfa. Aquests composts exhibiren capacitat antioxidant (EC50: 5,37-5,29 mg extracte solit/ mg DPPH) i antimicrobiana, (quantificada en termes de concentració mínima inhibitòria: CMC) enfront a L. innocua (MIC: 48-52 mg pols/mL) i E. coli (MIC: 50-66 mg pols/ mL). Els materials cel·lulòsics procedents de pellorfa d'arròs i cafè es van incorporar a pel·lícules de midó termoplàstic (TPS), obtingudes mitjançant mesclat en fos i modelatge per compressió. El mòdul elàstic va augmentar un 186 i 121% quan es va incorporar a la matriu un 1 pt% CNC de pellorfa d'arròs i café, respectivament. De la mateixa manera, les CF es van afegir a les pel·lícules de TPS al 1, 5 i 10 pt%. Ambdues CF va augmentar la rigidesa de les pel·lícules i es va reduir la seua capacitat d'estirament. No obstant, les CF de pellorfa de cafè mantingueren millor la ductilitat al 1 i 5%. La permeabilitat al vapor d'aigua de les pel·lícules de TPS no es va reduir en els materials compostos, encara que la permeabilitat a l'oxigen es va reduir en aproximadament un 17%. A l'incorporar extractes actius a les pel·lícules de midó, milloraren les propietats de tracció de les pel·lícules ; el mòdul elàstic va augmentar un 350%, mentre que les pel·lícules es feren menys extensibles. Les CF dels dos residus foren més efectives com agents de reforç en pel·lícules que contenien extractes actius, que en pel·lícules de midó pur. També es van estudiar mescles de midò-PLA utilitzant com a compatibilitzador policaprolactona funcionalitzada amb anhídrid maleic i/o glicidil metacrilat (PCLMG o PCLG). Es va analitzar l'efecte de la proporció de PLA en la mescla (20 i 40% respecte al midó), i de la tots dues compatibilitzadors (2,5 i 5%), en les propietats de les pel·lícules. Els anàlisis de la microestructura, el comportament tèrmic i les propietats funcionals (mecàniques, óptiques i de barrera) de les pel·lícules, demostraren que substituir el 20% del midó per PLA i incorporar el 5% de PCLG podria ser una bona estratègia per a obtindré pel·lícules adequades per a l'envasat d'aliments. A demés, es va estudiar l'efecte de l'addició de reforçaments cel·lulòsics (CF i CNC) i extracte antioxidant de pellorfa de cafè, en mescles de midó-PLA compatibilitzades. Les propietats antioxidants de les pel·lícules s'analitzaren a través de la seua eficàcia per a preservar de l'oxidació l'oli de gira-sol. S'observaren diferències significatives en les propietats funcionals de les pel·lícules quan els CNC s'incorporaren mitjançant dos mètodes diferents. L'efecte de reforç dels materials cel·lulòsics en mescles de S-PLA va ser menys notable que en les pel·lícules de midó, provablement degut a la superposició de l'efecte de reforç del PLA. L'extracte antioxidant no va millorar el comportament mecànic en les mescles, però li va conferir la capacitat antioxidant adequada per a aplicacions a l'envasat d'aliments. / [EN] This Doctoral Thesis has focused on the isolation and characterisation of cellulosic materials and active extracts from coffee and rice husks, and their incorporation into starch films and starch-PLA compatibilised blend films in order to improve their functional properties as food packaging materials. Cellulose fibres were obtained through alkali and bleaching treatment with a final yield of 41 and 53 g fibres/100 g husk, respectively for rice and coffee husks. Cellulose nanocrystals were isolated from the bleached fibres by acid hydrolysis, with a yield of 5% with respect to bleached fibres, in both cases, with high crystallinity (90-92%), thermal resistance and aspect ratio (L/d: 20-40). The active compounds were obtained by hydrothermal extraction (180 ºC, 9.5 bar) with yields of 17 -18 g/100 g husks. They exhibited antioxidant properties (EC50: 5.37-5.29 mg extract solids/mg DPPH) and antibacterial activity against L. innocua (MIC: 48-52 mg powder/mL) and E. coli (MIC: 50-66 mg powder/mL), which were quantified in terms of the minimal inhibitory concentration. Cellulosic material from rice and coffee husks were incorporated into thermoplastic starch films (TPS) by melt blending and compression moulding. The elastic modulus increased by 186 and 121% when 1 wt% of cellulose nanocrystals (CNC) from rice and coffee husks, respectively, was incorporated into the matrix. Likewise, cellulose fibres (CF) were incorporated into TPS films at 1, 5 and 10 wt%. Both CF increased the film stiffness while reducing its stretchability. However, CF from coffee husk better maintained the film ductility at 1 and 5 wt%. The water vapour permeability of TPS films was not reduced in composites, although oxygen permeability was lowered by about 17%. When active extracts were incorporated into starch films, they improved the tensile properties; the elastic modulus increased by about 350%, while films became less stretchable. The cellulosic fibres from both residues were more effective as reinforcing agents in films containing extract solids than in net starch films. Starch-PLA blend films were also studied using grafted polycaprolactone with maleic anhydride and/or glycidyl methacrylate (PCLMG or PCLG) as compatibilisers. The effect of both the PLA ratio in the blend (20 and 40% with respect to starch) and the amount of both compatibilisers (2.5 and 5%) on the film properties was analysed. The analyses of microstructure, thermal behaviour and functional properties (mechanical, optical and barrier) of the films led to the conclusion that substituting 20% of the starch by PLA, and incorporating 5% of PCLG would be a good strategy to obtain films suitable for food packaging. The effect of the addition of cellulosic fillers (CF and CNC) and antioxidant aqueous extract from coffee husk to compatibilised starch-PLA blends was also studied. The antioxidant properties of the films were tested through their efficacy at preserving sunflower oil from oxidation. Significant differences were observed in the functional properties of the films when CNC was incorporated by two different methods. The reinforcing effect of cellulosic materials in S-PLA blends was less noticeable than in starch films, probably due to the overlapping of the PLA reinforcing effect. The antioxidant extract did not improve the mechanical performance in the blends, but conferred antioxidant capacity suitable for food packaging applications. / Collazo Bigliardi, S. (2019). Lignocellulosic fractions from rice and coffee husks to improve functionality of biodegradable films based on starch and poly-lactic acid [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/123055 / Compendio

A biomassa lignocelulósica é uma fonte abundante de açúcares simples passíveis de serem fermentados em uma variedade de bioprodutos de maior valor agregado, além do etanol de segunda geração. Tal diversidade é relevante ao desenvolvimento e aprimoramento do conceito de biorrefinarias e da bioeconomia, em um viés mais amplo. Todavia, a elevada recalcitrância dos lignocelulósicos dificulta a sua sacarificação enzimática, resultando em bioprocessos mais onerosos. Por isso, coquetéis com diferentes enzimas ativas em carboidrato (CAZymes) são desenvolvidos, em busca de uma maior eficiência e melhor relação custo/benefício, para processos em larga escala. Dentre as CAZymes estudadas, encontram-se as mono-oxigenases líticas de polissacarídeo (LPMOs), tendo em vista a sua atestada capacidade de otimizar a hidrólise da lignocelulose, quando em sinergismo com diversas hidrolases. Levando isto em conta, selecionou-se, ao atual estudo, o ascomiceto termofílico Thermothelomyces thermophila (anteriormente denominado Myceliophthora thermophila), pois este tem se mostrado capaz de expressar e secretar ampla gama de LPMOs ativas em diferentes substratos. Objetivando-se estudar duas LPMOs derivadas deste organismo, as mesmas foram expressas, heterologamente, por Aspergillus nidulans linhagem A773, utilizando-se o vetor de expressão pEXPYR construído para viabilizar a secreção de altas concentrações de proteínas recombinantes. As proteínas heterólogas aqui analisadas foram denominadas TtLPMO1A9 e TtLPMO2A9. Embora ambas tenham sido capazes de gerar peróxido de hidrogênio na presença de oxigênio molecular e de um doador de elétrons, apenas TtLPMO2A9 apresentou atividade contra substratos celulósicos e bagaço de cana pré-tratado hidrotermicamente, atuando, em associação com hidrolases homemade e o preparo enzimático comercial Celluclast 1.5L, a degradação de tais materiais. / The lignocellulosic biomass is an abundant source of simple sugars that can be fermented to various value-added bio-based products. This diversity is seen as relevant to the improvement of biorefinery and bioeconomy concept. Nevertheless, the significant recalcitrance of lignocellulose imposes dificulties to its enzymatic saccharification, resulting in onerous bioprocessing. This scenario stimulates studies based on the development of efficient and cost-effective customizable carbohydrate-active enzyme (CAZymes) cocktails for large-scale processes. Among the available CAZymes, there are the lytic polysaccharide monooxygenases (LPMOs), a set of oxidative proteins capable of optimizing the lignocellulose hydrolysis, when acting in synergism with various hydrolases. Based on this fact, in the current study, the thermophilic ascomycete Thermothelomyces thermophila (previously known as Myceliophthora thermophila) was adopted, because of its ability of expressing and secreting large amounts of LPMOs. Thus, two LPMOs derived from this fungus was heterologously produced by an expression system composed by Aspergillus nidulans strain A773 and the vector pEXPYR: an expression vector built to increase the secretion of recombinant proteins. The heterologous proteins herein analysed were termed as TtLPMO1A9 and TtLPMO2A9. Although both enzymes were able to produce hydrogen peroxide in the presence of molecular oxygen and an electron donor, only the second one was active in reactions with cellulosic substrates and hydrothermally pre-treated sugar cane bagasse. When tailor-made hydrolases and the commercial enzymatic mixture Celluclast 1.5L were supplemented with TtLPMO2A9, it was noticed na improvement of the deconstruction of the aforementioned substrates.

A busca por novas tecnologias que visam à produção de biocombustíveis renováveis, especialmente bioetanol e outros biomateriais, tem se intensificado nos últimos anos. Há um interesse mundial crescente na limitação dos impactos ambientais e mudanças climáticas através da substituição de produtos petroquímicos por análogos ambientalmente corretos, a fim de alcançar uma economia mais sustentável. Além disso, as plataformas biorrefinarias lignocelulósicas necessárias para a produção de bioetanol representam uma oportunidade de estimular novos mercados para o setor agrícola e aumentar os empregos locais, contribuindo para o desenvolvimento das economias emergentes. No entanto, a maioria dos processos de conversão são baseados no conhecimento empírico, exigindo estudos mais aprofundados sobre os fatores envolvidos na hidrólise enzimática da celulose, tais como características biomassas, a otimização da etapa de pré-tratamento, bem como das atividades das enzimas e seus mecanismos de ação. Assim, com o objetivo de contribuir para a viabilização e implantação das tecnologias de produção do etanol lignocelulósico, na primeira parte deste trabalho de doutorado, foi realizada a purificação da &beta;-glicosidase do fungo Aspergillus Níger (NaBgl1), principal enzima do coquetel comercial Novozymes 188, e sua caracterização bioquímica e biofísica. As análises de espalhamento de raios-x a baixo ângulo revelaram uma organização multidomínios desta enzima, com uma estrutura molecular de girino semelhante ao encontrado para as celulases. A sua estrutura é composta por um domínio catalítico N-terminal e um domínio fibronectina de tipo III (FnIII) na região C-terminal, conectados entre si por um longo linker com uma inserção de 100 resíduos de aminoácidos numa conformação estendida. Apesar desta estrutura molecular incomum, os ensaios de eletroforese capilar revelaram um perfil processividade característico de &beta;-glucosidases, e os ensaios enzimáticos confirmaram, também, a ausência de atividade em substratos poliméricos. Nos ensaios adosrção com diferentes compostos poliméricos, a enzima &beta;-glicosidase mostrou uma capacidade de adsorção elevada em lignina. Os mecanismos de ligação FnIII-lignina foram elucidados por simulações de dinâmica molecular, que confirmaram apresença de vários sítios de ligação à lignina no domínio FnIII da enzima. Como segunda parte da presente tese, diferentes biomassas, como bagaço de cana, resíduos de casca de eucalipto e gramíneas (Panicum maximum, Pennisetum purpureum e Brachiaria brizantha) foram submetidas a vários métodos de pré-tratamento (ácido diluído, alcalino, sulfito e água quente) em diferentes condições de tratamento e avaliadas quanto ao seu potencial para a produção de bioetanol. As biomassas in natura e pré-tratadas foram caracterizadas quanto à sua composição química por métodos cromatográficos, ressonância magnética nuclear e espectroscopia de infravermelho por transformada de Fourier; o índice de cristalinidade das amostras foi determinado por método químico e difração de raios-x; as análises morfológicas foram realizadas por microscopia eletrônica de varredura; e os resultados da caracterização foram correlacionados com os perfis de sacarificação enzimática encontrados para cada uma delas. / The search for new technologies aimed at the production of renewable biofuels, specially bioethanol, and other biomaterials has intensified in recent years. There is an increasing world-wide interest in the limitation of environmental impact and climate change by replacing petrochemical products with environment-friendly analogues in order to move towards a sustainable economy. In turn, the lignocellulosic biorefining platforms required for ethanol production present an opportunity to stimulate new markets for the agriculture sector and increase domestic employment, contributing to the development of emerging economies. However, most of conversion processes are based on empirical knowledge, demanding thorough studies about the factors involved on enzymatic hydrolysis of cellulose, such as biomasses characteristics, optimization of pretreatment steps and enzymes activities and molecular action mechanisms. Aiming to contribute for the viability and establishment of lignocellulosic ethanol technologies, on the first part of the present thesis, we performed the purification of main Aspergillus niger &beta;-glucosidase (AnBgl1) from the commercial cocktail Novozymes 188 and its biochemical and biophysical characterization. The small angle x-ray scattering analysis revealed a multidomain organization, with a tadpole-like molecular shape similar to that found for cellulases. Its structure is composed by a N-terminal catalytic domain and a fibronectin type III-like (FnIII) C-terminal domain, connected by a long linker with a 100 aminoacids residues insertion in a extended conformation. In spite of this uncommon molecular structure, capilar zone electrophoresis assays revealed a processivity profile characteristic of &beta;-glucosidases and the enzymatic assays confirmed no-activity on polymeric substrates. On the pull-dowm assays with different polymeric compounds, the &beta;-glucosidase showed a high adsorption ability to lignin. The FnIII-lignin binding mechanisms were elucidated by molecular dynamics simulations, confirming the multiple binding sites to lignin in the enzyme FnIII domain. As a second part of the present thesis, different biomasses such as sugarcane bagasse, eucalyptus bark residues and grasses (Panicum maximum, Pennisetum purpureum and Brachiaria brizantha) were submitted to several pretreatment methods (diluted acid, alkaline, sulfite and hot water) at various conditions and evaluated about their potential to bioethanol production. The raw and pretreated biomasses were characterized about their chemical composition by chromatographic methods, nuclear magnetic ressonance and Fourier transformed infrared spectroscopy; the crystallinity index was determined by chemical method and x-ray diffraction; morphological features were analysed by scanning electron microscopy; and the characterization results were correlated to their enzymatic saccharification profiles.