Thesis (Ph. D.)--University of Washington, 1994. / Vita. Includes bibliographical references (leaves [123]-131).

Techn. Hochsch., Diss.--Aachen.

Various methods for the production of bioethanol using different feedstocks have been researched on. In most work on bioethanol synthesis from sugar cane, tops and leaves have been regarded as waste and generally removed and thrown away. In this work, lignocellulosic sugarcane leaves and tops were not discarded but instead used as biomass to evaluate their hydrolyzate content. The leaves and tops were hydrolysed using different methods, namely concentrated acid, dilute acid pre-treatment with subsequent enzyme hydrolysis and compared with a combination of oxidative alkali pretreatment and enzyme hydrolysis. Subsequent fermentation of the hydrolyzates into bioethanol was done using the yeast saccharomyces cerevisae. Acid hydrolysis has the problem of producing inhibitors, which have to be removed and this was done using overliming with calcium hydroxide and compared to sodium hydroxide neutralization. Oxidative alkali pre-treatment with enzyme hydrolysis gave the highest yields of fermentable sugars of 38% (g/g) using 7% (v/v) peroxide pre-treated biomass than 36% (g/g) for 5% (v/v) with the least inhibitors. Concentrated and dilute acid hydrolysis each gave yields of25% (g/g) and 22% (g/g) yields respectively although for acid a neutralization step was necessary and resulted in dilution. Alkaline neutralization of acid hydrolyzates using sodium hydroxide resulted in less dilution and loss of fermentable sugars as compared to overliming. Higher yields of bioethanol, 13.7 (g/l) were obtained from enzyme hydrolyzates than 6.9 (g/l) bioethanol from dilute acid hydrolyzates. There was more bioethanol yield 13.7 (g/l) after 72h of fermentation with the yeast than 7.0 (g/l) bioethanol after 24h. However, the longer fermentation period diminishes the value of the increase in yield by lowering the efficiency of the process.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21157号 / 農博第2283号 / 新制||農||1059(附属図書館) / 学位論文||H30||N5131(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 吉村 剛, 教授 髙野 俊幸, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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The potential of enhancing lipid accumulation in municipal sewage activated sludge via fermentation of lignocellulose biomass hydrolyzate was investigated. The overall objective was to increase the levels of feedstock lipids in the activated sludge biomass and increase its biodiesel yield via in situ or ex situ transesterification; and improve its cost competitiveness as an abundant feedstock source for biofuels production. To reduce production costs and maintain environmental sustainability, influent wastewater and waste lignocellulose biomass hydrolyzate were used as cultivation media and substrate, respectively. However, lignocellulose hydrolyzates also contain degradation by-products such as furfural and acetic acid that are known to exert inhibitory effects on microorganisms; hence their effects on the fermentative performance of activated sludge were investigated and fermentation strategies were proposed and evaluated to counteract the microbial toxicity of these compounds. The utilization rate and efficiency of xylose by activated sludge microorganisms for lipid production was also evaluated as pentose sugars such as xylose usually constitute a major percentage of lignocellulose hydrolyzates. Furthermore, variations in the population profile of activated sludge microbiota were determined via 16S rRNA sequence analysis to determine the effect of sugar fermentation at different initial conditions. Results show that activated sludge lipid contents and biodiesel yield could be enhanced by fermentation of sugars at a high initial C:N ratio (70:1). Furfural was found to be highly inhibitory to microbial growth and lipid accumulation while high initial acetic acid concentrations enhanced biomass production but not lipid formation. Xylose was also utilized more efficiently than glucose by the activated sludge microorganisms for biomass and lipid production albeit at relatively slower rates; hence sugar mixtures derived from lignocellulose could be utilized for the process. Semicontinuous and continuous fermentation modes were proposed and evaluated as strategies to reduce inhibitory effect of furfural and acetic acid and improve lipid productivity, but the lack of nutrient supplementation prevented the cultures from sustaining microbial growth and lipid production, leading to cell death and washout. Finally, the reduction in the diversity of the activated sludge microbiota could point to specific microbial strains that are mainly responsible for lipid accumulation.

Ce travail est consacré au développement de biocomposites de caoutchouc naturel (NR) performants pour produire des composites ayant des propriétés similaires aux formulations conventionnelles à base de noir de carbone (CB). Le projet est divisé en deux parties principales selon les types de charges: les charges lignocellulosiques de taille macro et la nanocellulose. Dans un premier temps, le remplacement du CB par de la lignine et de la cellulose (avec et sans modification) est étudié. Les résultats montrent que la lignine et la cellulose ont leurs propres avantages et limites, mais le remplacement partiel du CB par les deux macro-biocharges peut fournir de meilleures propriétés mécaniques et dynamiques par rapport au CB seul. Néanmoins, des propriétés améliorées sont également obtenues après une modification de surface de la cellulose par de l'anhydride maléique greffé sur du polyisoprène (MAPI). Cependant, il n'est pas possible de remplacer complètement le CB par ces charges en raison des fortes interactions entre les charges. Ensuite, l'effet de la nanocellulose sur le renforcement du NR est étudié. Ce travail comprend également un système de renforcement hybride à base de nanocellulose et de nanotubes de carbone (CNT) qui montre la formation d'un réseau conducteur 3D solide à l'intérieur de la matrice de caoutchouc. La présence de ce réseau conduit à d'excellentes propriétés (propriétés mécaniques dynamiques et conductivité thermique) qui peuvent être facilement contrôlées en ajustant la teneur en charges. Enfin, un nouveau système hybride contenant de la lignine et de la nanocellulose a été développé pour renforcer le NR. Dans ce cas, une concentration élevée (40 parties pour cent de caoutchouc, phr) de lignine est utilisée comme biocharge non-renforçante pour réduire les coûts et augmenter la durabilité, tandis que la nanocellulose est ajoutée pour renforcer ces biocomposites NR. On constate que l'ajout de 7,5 phr de nanocellulose aux composés lignine/NR (contenant 40 phr de lignine) augmente la teneur en caoutchouc lié (37%), la résistance à la traction (36%) et le module à 100 % de déformation (101%), tout en diminuant le temps de durcissement (14%) et le facteur de perte (55% à 10% de déformation). Malgré sa biodégradabilité et sa durabilité, le bionanocomposite nanocellulose/lignine/NR présente des propriétés mécaniques similaires et même de meilleures propriétés mécaniques dynamiques (53% à 10% de déformation) que les composites NR conventionnels renforcés avec du CB seul. / This work is devoted to the development of high-performance natural rubber (NR) biocomposites to produce composites having similar properties as conventional formulations based on carbon black (CB). The project is divided into two main parts depending on the types of fillers: macro-sized lignocellulosic fillers and nanocellulose. Firstly, the effect of replacing CB by lignin and cellulose (with and without modification) is studied. The results show that both lignin and cellulose have their own advantages and limitations, but partial replacement of CB with both macro-biofillers can provide better mechanical and dynamic mechanical properties compared to CB alone. Nevertheless, improved properties are also obtained after surface modification of the cellulose with maleic anhydride grafted to polyisoprene (MAPI). However, it is not possible to completely replace CB with these fillers due to strong filler-filler interactions. Then, the effect of nanocellulose on NR reinforcement is studied. This work also includes a hybrid reinforcing system based on nanocellulose and carbon nanotube (CNT) which is showing the formation of a strong 3D conductive network inside the rubber matrix. The presence of this network leads to excellent properties (mechanical and dynamic properties, and thermal conductivity), which can be easily controlled by tuning the fillers content ratio. Finally, a novel hybrid system containing lignin and nanocellulose is developed to reinforce NR. In this case, a high concentration (40 parts per hundred rubber, phr) of lignin is used as a non-reinforcing biofiller to reduce the costs and increase the sustainability, while nanocellulose is added to reinforce these NR biocomposites. It was found that adding 7.5 phr of nanocellulose to the lignin/NR compounds (containing 40 phr lignin) increased the bound rubber content (37%), tensile strength (36%) and modulus at 100% strain (101%), while decreasing the curing time (14%) and loss factor (55% at 10% strain). Despite its biodegradability and sustainability, the nanocellulose/lignin/NR bionanocomposite exhibits similar mechanical properties and even better dynamic mechanical properties (53% at 10% strain) than conventional NR composites reinforced with CB alone.

For the first time, the bonding patterns of specific carbon atoms in woody plant lignin have been identified in situ. This was accomplished by administering and incorporating into the lignin fraction of Leucaena leucocephala, a tropical hardwood, ferulic acid enriched with ¹³C at either the 1-, 2-, or 3-C atom of the side chain. The plants were grown hydroponically over extended periods of time (28 days) under aseptic conditions in media containing the ferulic acid precursor, and then the tissues were examined by solid-state ¹³C NMR spectroscopy. Consequently, resonances due to the bonding patterns of the specific carbon atoms were determined. These resonances differ substantially from similarly labelled synthetic dehydrogenatively polymerized (DHP) lignin in both spectral profile and relative peak intensities. Subsequent studies using phenylalanine as precursor showed that it was better translocated into the aerial portions of the plant, and that its uptake did not result in distortion of lignification in those tissues, both in amount and monomeric composition. Consequently, the difference spectra obtained by ¹³C NMR analyses of phenylalanine-treated plants confirmed and extended the results obtained with ferulic acid. Evidence for the conversion of both precursors to the monolignols was shown by the difference spectra of [1-¹³C]-precursor-fed tissues, where the dominant resonance at 61-63 ppm is consistent with substructures containing the hydroxymethyl functionality. The spectrum obtained with roots administered [1-¹³C] ferulic acid showed the presence of a minor resonance (170-174 ppm) attributable to carboxylic acids/esters. By allowing the plant to undergo further metabolism by growing in hydroponic media without the precursor, these signals disappeared from the resulting spectrum. The first direct evidence for the dominance of the β-O-4’ linkage of lignin in situ was shown by the appearance of the resonance at 83 ppm corresponding to this substructure in both [2-¹³C] ferulic acid-treated roots and [2-¹³C] phenylalanine-treated roots and stems. Evidence for the occurrence of α-O-carbohydrate or α-O-aryl linkage in intact plant tissues was obtained in the spectra of tissues administered [3-¹³C] ferulic acid and [3-¹³C] phenylalanine. The effect of horseradish peroxidase/H₂O₂ in organic medium (dioxane/aqueous acetate buffer, pH 5, 95:5) on dehydrogenatively polymerized (DHP) lignin was reinvestigated. We found no evidence for vigorous depolymerization of DHP lignin under these conditions, contrary to claims made by Dordick, Marletta and Klibanov (1986, Proc. Natl. Acad. Sci. USA 83:6255-6257). Furthermore, we did not detect ferulic acid as a degradation product following treatment of DHP lignin with HRP/H₂O₂. Both coniferyl alcohol and DHP lignin were used in incubation experiments to determine effects of lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and H₂O₂ on these substrates. Gel filtration chromatography showed that polymeric materials of high molecular weights were the result of these treatments. Incubation of [1-¹³C], [2-¹³C] and [3-¹³C] coniferyl alcohol with lignin peroxidase/H₂O₂ resulted in products similar to-DHP lignins prepared by horseradish peroxidase/H₂O₂ with respect to occurrence of identical resonances in corresponding solution-state ¹³C NMR spectra. Consequently, the role of polymerization of low molecular weight phenolics as a mechanism for detoxification was ascribed to these fungal peroxidases. / Ph. D.

The polyhydroxy character of lignocellulosics and their natural abundance make them good candidates for the manufacture of polyurethanes. The cure characteristics of hydroxypropyl-cellulose and hydroxypropyl lignin (HPC and HPL, respectively) with polymeric methylene diphenyl diisocyanate (MDI) was studied via dynamic mechanical thermal analysis (DMTA). HPC/MDI and HPL/MDI resins flow at 30°C and proceed to cure at 50°C. The latter has excellent thermal stability over the former. Crosslinking of HPL and HPC with MDI follow an nth order kinetics, with an order of reaction of 2 and an apparent activation energy in the range of 12.9 kcal/mol - 14.7 kcal/mol. The rate of cure with time is higher in HPL-based polymers than HPC-based ones at the initial stage of cure; the difference vanishes at later stages. This demonstrates that the hydroxyl groups in HPC are less accessible to the NCO groups, and that cure rate might be dependent on diffusion limitations at later stages. Degree of cure, under all cure schedules, follows a parallel trend, and has to do with the fact that the hydroxyl groups of HPC are less accessible to isocyanate. Both HPL and HPC react with MDI at a reduced rate in comparison to a synthetic polyol: caprolactone triol. Time-glass transition temperature superposition was used to calculate times to vitrification of the HPL-based polymers, and is presented in a TTT cure diagram. This bio-based polymer displays the s-shaped vitrification pattern characteristics of thermosets. A similar approach did not work with HPC-based polymers. HPC- and HPL-based polymers did not display damping transitions, in isothermal cure, typical of gelation and vitrification. As the isocyanate to hydroxyl ratio (NCO:OH) increased, the glass transition temperature of the polymers increased, and the transition amplitude and width decreased and increased, respectively. In practical terms, this study illustrates that it is advantageous to use a) to use high isocyanate to hydroxyl ratios in order to produce polyurethanes which retain desirable damping behavior over a wider range of temperature. b) to use HPC/MDI resins in those situations where retention of stiffness at temperatures below 230° is required. c) to use HPL where rapid cure is desired. The study also reveals that the relative reactivity of water, HPL and HPC with isocyanate takes the form water > HPL > HPC. / Master of Science

Cellulose to lignin ratio is one of the widely used indicators of degree of landfill stabilization. This ratio shows the amount of carbohydrate or cellulose consumed by anaerobes compared to relatively inert lignin. However, the method of lignin measurement contains an intrinsic error. Plastics are contained in the landfill samples and these are characterized as lignaceous materials due to their acid-insolubility. Lignin is typically measured as the organic residue that is acid insoluble but is combustible upon ignition. Additionally lignin may degrade under anaerobic, high temperature conditions associated with wet conditions in sediments and bioreactor landfills. In this study, it has been found that the typical measure of lignin, a gravimetric measure, also includes plastics, leading to erroneous measures of both lignin and the cellulose/lignin (C/L) ratio. Typically, 100% of the plastic will be measured as lignin. Since plastic amounts to approximately 10% of landfill contents, lignin measurements will be 10% greater than actual amounts. Laboratory reactors were set up with known amounts of paper and plastic. The degradation of the cellulose and lignin in paper was measured and compared to plastics, which was collected by hand and weighed. Ratios of cellulose to plastics and lignin to plastics were obtained. It was found, based on the cellulose to plastic ratio and lignin to plastic ration that lignin degrades under anaerobic conditions although at a much slower rate than cellulose. These findings indicate that the cellulose to lignin ratio cannot be used as the sole indicator of stabilization in the landfills. The inclusion of the biochemical methane potential test data along with C/L is thought to provide a better indication of landfill stabilization. / Master of Science