Orientador: Telma Teixeira Franco / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T00:32:21Z (GMT). No. of bitstreams: 1 Anschau_Andreia_D.pdf: 2032545 bytes, checksum: 8d4ec316020e160cada0e585617e1659 (MD5) Previous issue date: 2014 / Resumo: Neste trabalho foram desenvolvidos estudos visando o estabelecimento de um processo de produção de lipídeos microbianos a partir de fontes renováveis, particularmente xilose, carboidrato derivado do processo de hidrólise de bagaço de cana-de-açúcar. Foi utilizada a levedura oleaginosa Lipomyces starkeyi DSM 70296, previamente selecionada no Laboratório de Engenharia Bioquímica, Biorefino e Produtos de Origem Renovável (LEBBPOR). A partir dos resultados preliminares em frascos agitados, partiu-se para estudos de batelada alimentada em biorreator (1,3 a 3L). Foram estudadas diferentes estratégias de alimentação, sendo que em batelada alimentada repetida, foram encontradas as maiores concentrações de células (85,4 g/L) e de lipídeos (41,8 g/L). Posteriormente foram estudados modos de operação em processos contínuos em meio sintético e meio contento o hidrolisado hemicelulósico (H-H). As maiores produtividades de células (0,443 g/g) e de lipídeos (0,236 g/g) foram encontradas em cultivo contínuo a 0,03h-1. Na vazão específica de alimentação de 0,06 h-1 foram obtidas as maiores produtividades de células (0,600 g/L.h) e de lipídeos (0.288 g/L.h). Análises de cromatografia em fase gasosa dos diferentes cultivos feitos revelaram que os principais constituintes deste complexo são os ácidos graxos de cadeia longa, como o ácido palmítico (C16:0), ácido esteárico (C18:0), ácido oleico (C18:1) e ácido linoleico. Foi estimado o número de cetano em torno de 61, muito próximo do biodiesel de palma. Também foram feitos estudos de balanço de massa e de energia em cultivo batelada alimentada utilizando somente xilose como fonte de carbono. O valor de calor de combustão (Qc) de 25,7 kJ/g obtido após 142 h de cultivo representa aproximadamente 56% do conteúdo energético do óleo diesel (45,4 kJ/g), indicando o potencial da L. starkeyi para biodiesel. Cultivos contínuos subsequentes foram feitos para a compreensão do processo de acúmulo de lipídeos, utilizando a ferramenta estatística de reconciliação de dados para melhorar os dados experimentais obtidos em quimiostato, reduzindo os erros experimentais para posterior cálculo de análise de fluxos metabólicos (MFA). Nesse sentido, os lipídeos produzidos por L. starkeyi apresentam relevante importância do ponto de vista acadêmico e industrial, podendo ser utilizados como matéria-prima para biodiesel e indústria oleoquímica / Abstract: Studies attempting the establishment of a microbial lipid production process from renewable resources, mainly xylose, were developed. This pentose, obtained from sugar cane bagasse hydrolysis. The oleaginous yeast Lipomyces starkeyi DSM 70296, previously selected at the Laboratory of Biochemical Engineering, Biorefining and Products from Renewable Sources (LEBBPOR), was used throughout this thesis. After preliminary studies in shake flasks, we started fed-batch studies in fermentor (1.3 to 3L). Among the strategies studied, the highest cell mass and lipid concentrations reached up to 85.4 and 41.8 g/L, respectively, when repeated fed?batch strategy was applied. Subsequently, continuous processes were studied in synthetic medium and media containing hemicellulosic hydrolysate (H-H). The highest overall cell mass (0.443 g/g) and lipid yields (0.236 g/g) were achieved at dilution rate of 0.03 h-1. At dilution rate of 0.06 h-1, were obtained the highest productivities of cell mass (0.600 g/L.h) and lipids (0.288 g/L.h). Gas chromatography of esterified lipids revealed that the major constituents of this complex are long-chain fatty acids, such as palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) with an estimated cetane (around 61) very close to the palm biodiesel. Also have been studies of mass and energy balances from fed-batch cultivation using xylose as sole carbon source. The combustion heat (Qc) value 25.7 (kJ/g) obtained after 142 h of fed-batch cultivation, represents approximately 56% of the energy content of diesel oil (45.4 kJ/g), indicating the potential of L. starkeyi for biodiesel. Continuous cultures were made subsequently to understanding the process of lipid accumulation using a statistical tool for data reconciliation was used to improve the experimental data obtained in chemostat culture reducing the experimental errors for subsequent calculation of metabolic flux analysis (MFA). In this sense, lipids produced by L. starkeyi have relevant importance of academic and industrial point of view, as feedstock for biodiesel and oleochemical industry applications / Doutorado / Processos em Tecnologia Química / Doutora em Engenharia Quimica

Orientadores: Gustavo Henrique Goldman, Juliana Velasco de Castro Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T09:59:21Z (GMT). No. of bitstreams: 1 Utsunomia_Camila_M.pdf: 2245062 bytes, checksum: 1803b81f54da154433e6d40c34e2b884 (MD5) Previous issue date: 2014 / Resumo: A produção de etanol a partir de resíduos agrícolas, denominada Etanol de Segunda Geração ou Etanol Lignocelulósico, surge como uma alternativa para aumentar a produção de etanol sem necessariamente expandir as áreas destinadas ao cultivo da matéria-prima. A conversão da biomassa em etanol não é economicamente favorável a não ser que a hemicelulose seja utilizada juntamente com a celulose. Entretanto, a levedura Saccharomyces cerevisiae é incapaz de fermentar pentoses, açúcares liberados pela hidrólise da hemicelulose, principalmente xilose. Uma alternativa para que S. cerevisiae produza etanol utilizando pentoses seria modificá-la geneticamente através do uso de genes provenientes de micro-organismos que naturalmente realizam esta conversão. Com o intuito de encontrar novas leveduras assimiladoras de xilose, prospectamos o trato intestinal de larvas de seis insetos parasitas da cana-de-açúcar e dornas de fermentação alcoólica, identificando 49 isolados, dentre eles as leveduras Rhodotorula mucilaginosa UC11 e Ogataea polymorpha FT212L. Neste trabalho, foi feito o isolamento e a caracterização dos genes xyl1 e xyl2, que codificam respectivamente à xilose redutase (XR) e à xilitol desidrogenase (XDH) dessas duas leveduras, assim como a clonagem e a superexpressão de tais genes na cepa industrial de S. cerevisiae Pedra-2 (PE-2). Rhodotorula mucilaginosa UC11 e O. polymorpha FT212L foram avaliadas quanto a habilidade de crescerem em xilose, a expressão dos genes xyl1 e xyl2 e a medição das atividades das enzimas XR e XDH. E pela primeira vez XR e XDH de ambas as leveduras foram expressas heterologamente em S. cerevisiae PE-2 gerando as cepas PE-2 Rm, PE-2 Op 1 e PE-2 Op 2, que foram testadas quanto a capacidade de utilizar xilose para crescimento. Adicionalmente, neste trabalho também foi construída pela primeira vez uma cepa S. cerevisiae PE-2 contendo os genes de Scheffersomyces stipitis, uma vez que esta é uma das melhores leveduras conhecidas capazes de utilizar xilose. Para isso a cepa PE-2 foi modificada geneticamente com o plasmídio pRH 274, contendo os genes superexpressos de xyl1 e xyl2 de S. stipitis e o gene xks1 de S. cerevisiae, que codifica à xilulose quinase (XK), gerando a cepa denominada PE-2 274. Esta levedura recombinante foi caracterizada através de ensaios de curva de crescimento, medição das atividades de XR e XDH e capacidade de co-fermentar xilose e glicose a etanol em biorreator. A cepa PE-2 274 mostrou ser uma cepa de grande potencial para a produção de etanol a partir de meio contendo xilose e glicose, podendo ser a base para a geração de uma cepa competitiva em ambiente industrial visando à produção do etanol de segunda geração / Abstract: The ethanol production from agricultural residues, called Second Generation Ethanol or Lignocellulosic Ethanol, arises as an alternative to increase the ethanol production without necessarily expanding the areas for raw material cultivation. The biomass conversion to ethanol is not economically feasible unless hemicellulose is used in addition to cellulose. However, the yeast Saccharomyces cerevisiae is unable to ferment pentoses, released sugars from hemicellulose hydrolysis, mainly xylose. An alternative to S. cerevisiae produces ethanol from pentose is the genetic engineering with genes from microorganisms which naturally perform this conversion. In order to find new yeasts capable to use xylose, we prospected the gut of sugarcane parasitic insects larvae and alcoholic fermentation tanks, identifying 49 isolates, among them the yeasts Rhodotorula mucilaginosa UC11 e Ogataea polymorpha FT212L. In this work were made the isolation and characterization of xyl1 and xyl2 genes, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, from these two yeasts, and the cloning and overexpression of these genes in the industrial S. cerevisiae strain Pedra-2 (PE-2). Rhodotorula mucilaginosa UC11 and O. polymorpha FT212L were evaluated over the ability to grow on xylose, xyl1 and xyl2 expression analysis and XR and XDH activity. And for the first time XR and XDH from both yeasts were heterologous expressed in S. cerevisiae PE-2 generating PE-2 Rm, PE-2 Op 1 and PE-2 Op 2 strains, which were tested by the capability to grow on xylose. Additionally, in this work was constructed also for the first time a S. cerevisiae PE-2 strain with genes from Scheffersomyces stipitis, since this is one of the best known yeasts capable of utilizing xylose. For this, PE-2 was engineered with pRH 274 plasmid, containing xyl1 and xyl2 from S. stipitis and xks1 from S. cerevisiae, which encodes xylulokinase (XK), generating the PE-2 274 strain. This recombinant strain was characterized through xylose growing assays, XR and XDH activity and xylose/glucose co-fermentation to ethanol in bioreactor. PE-2 274 showed a high potential to produce ethanol from xylose and glucose, and could be the basis for the generation of industrially competitive yeast aiming the second generation ethanol production / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

Orientadores: Telma Teixeira Franco, Ana Carolina Deckmann / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T22:35:19Z (GMT). No. of bitstreams: 1 Coradini_AlessandroLuisVenega_M.pdf: 2904811 bytes, checksum: 79be0b1291d380e8c86253645ed455d8 (MD5) Previous issue date: 2014 / Resumo: A crise energética mundial aliada aos problemas ambientais tem despertado grande preocupação mundial, aumentando assim a busca por combustíveis mais "limpos" que possam substituir os já existentes. Dentre estes substitutos podemos destacar os chamados biocombustíveis, como o etanol e o biodiesel. No caso da produção de biodiesel, diversas fontes de lipídios são consideradas, incluindo óleos vegetais, gorduras animais e óleos reciclados. O uso de microrganismos oleaginosos foi apresentado como uma fonte alternativa de óleos e gorduras de baixo custo (Meng et al., 2009). O óleo produzido por microrganismos pode ter como substrato qualquer fonte de carbono, incluindo bagaços e resíduos agrícolas em geral, evitando tanto a competição por áreas de plantio como agregando valor a dejetos de outros setores agrícolas. A esta abordagem dá-se o nome de "biocombustíveis de segunda geração". Neste contexto, as leveduras surgem como fortes candidatas à produção de biodiesel, uma vez que diversas ferramentas de cultivo e manipulação já foram implantadas especialmente para o uso de leveduras em processos industriais e biotecnológicos. Entretanto, nem todas as espécies de leveduras são adequadas para a produção de óleo, uma vez que a capacidade de acumular grandes quantidades de lipídeos varia significativamente entre as espécies. As leveduras do gênero Saccharomyces, por exemplo, apresentam de 8-15 de lipídeos totais em relação à massa seca. Outras, como as espécies Rhodotorula graminis, Rhodotorula gracilis e Lipomyces starkeyi, apresentam cerca de 30%, 60% e 65% de lipídeos totais, respectivamente. Entretanto, o emprego de microrganismos selvagens (i.e., não domesticados) limita o controle dos parâmetros que afetam o processo produtivo, uma vez que as bioconversões de interesse dependem de ajustes metabólicos desconhecidos, e a falta de conhecimento genético destes microrganismos dificulta a aplicação de técnicas de manipulação genética. Assim, o presente estudo teve como objetivo "domesticar" a linhagem DSM70826 da levedura Lipomyces starkeyi tornando-a de fácil manipulação genética e tornando-a uma plataforma para posterior introdução de características que possam melhorar o seu desempenho quanto ao acúmulo de lipídeos para produção de biodiesel. Uma vez que foi realizado o sequenciamento do genoma desta levedura durante trabalho anterior conduzido em nosso laboratório, também objetivamos utilizar este banco de dados para identificar e estudar alguns genes considerados relevantes aos processos fermentativos de xilose e também ao acúmulo de lipídeos, auxiliando a elucidar as principais rotas metabólicas envolvidas nestes processos / Abstract: The global energy crisis combined with environmental problems has aroused great worldwide concern, increasing the search for "cleaner" fuels that can replace existing ones. Among these substitutes the so-called biofuels, such as ethanol and biodiesel can be highlighted. In the case of biodiesel, different fat sources are considered, including vegetable oils, animal fats and recycled oils. The use of oleaginous microorganisms was presented as an alternative source of low cost oils and fat (Meng et al., 2009). The oil produced by microorganisms can use any substrate as carbon source, including bagasse and agricultural waste in general, avoiding the competition for growing areas and adding value to waste of other agricultural sectors. This approach is called "second generation biofuels". In this context, yeasts appear as strong candidates for biodiesel production, since many manipulation and cultivation tools have been implemented especially for the use of yeast in industrial and biotechnological processes. However, not all species of yeasts are suitable for the production of oil, since the ability to accumulate large amounts of lipids varies significantly between species. Yeasts of the genus Saccharomyces, for example, show 8-15% of total lipids in the dry mass. Others, such as the species Rhodotorula graminis, Rhodotorula gracilis and Lipomyces starkeyi, have about 30 %, 60% and 65% of total lipids, respectively. However, the use of wild microorganisms (i.e., non-domesticated) limits the control parameters that affect the production process, since bioconversions of interest depend of unknown metabolic adjustments, and lack of genetic knowledge of these microorganisms hinders the application of genetic manipulation techniques. Therefore, the present study aimed to "domesticate" the yeast strain Lipomyces starkeyi DSM70826 making it easy genetic manipulation and making it a platform for subsequent introduction of features that can improve your performance on lipid accumulation for production of biodiesel. Since the genome sequencing of this yeast was performed during previous work conducted in our laboratory, we also aim to use this database to identify and study some genes considered relevant to xylose fermentation processes and also to lipid accumulation, helping to elucidate the main metabolic pathways involved in these processes / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

Abstract Lignocellulosic biomass, such as wood or agricultural residues, is a resource widely available for use in chemical production. In a lignocellulosic feedstock biorefinery, the major parts of biomass, cellulose, hemicellulose and lignin, are converted to valuable chemicals, materials and energy. Furfural production is one option for the use of the pentose sugars available in hemicellulose, and the process could be integrated with the pulp or cellulosic ethanol industry. In the past, furfural production catalysed by organic acids has been in industrial use, but no detailed studies about the kinetics exist. However, the use of organic acid would prevent the waste problems linked to the mineral acids widely used in the furfural industry. In this thesis, furfural formation in formic acid media was studied. The major part of this work concerns the kinetics of xylose dehydration into furfural and further furfural degradation. Based on the results of this thesis and a literature review, adequate prediction of furfural yield in the conditions used can be achieved using a simple kinetic model, including three reactions: 1) Xylose dehydration into furfural, 2) Furfural degradation, and 3) Xylose degradation to products other than furfural. Moreover, it was shown that the overall order of the furfural degradation reaction, usually modelled as a first order reaction, changes with acidity (H+-concentration). Suggestions for a possible reaction mechanism have been made based on the results. In the last part of this thesis, furfural formation in the presence of kraft lignin (Indulin AT) was considered. Sulphuric acid was used as a baseline for formic acid. It was shown that the lignin has an acid-neutralising capacity, but the higher pH did not explain all the changes in the xylose conversion and the furfural yield. Thus, it is highly likely the lignin inhibits the formation of furfural. Altogether, the effects were smaller in formic acid than in sulphuric acid. This thesis confirms the fact that formic acid is an effective catalyst for furfural production. The focus of the thesis was on the reaction kinetics, and the results can be used in conceptual process design. Moreover, the results emphasise the importance of including acidity explicitly in the kinetic model and monitoring acidity changes when real process streams are used. / Tiivistelmä Lignoselluloosaa, kuten puita tai maanviljelyn jäännösmateriaaleja, on laajasti saatavilla kemiallisen tuotannon raaka-aineeksi. Biojalostamossa lignoselluloosan pääjakeet, selluloosa, hemiselluloosa ja ligniini, muutetaan arvokkaiksi kemikaaleiksi, materiaaleiksi ja energiaksi. Furfuraalin tuotanto on yksi vaihtoehto hemiselluloosan sisältämien pentoosien hyödyntämiseksi. Furfuraaliprosessi voidaan yhdistää sellun tai bioetanolin tuotantoon, ja orgaanisia happoja käyttämällä voitaisiin välttää mineraalihappoihin liittyvät jäteongelmat furfuraalin tuotannossa. Tämän väitöskirjan aiheena on muurahaishappokatalysoitu furfuraalin muodostuminen ksyloosista. Pääpaino on reaktiokinetiikassa, ja työssä on kehitetty kineettinen malli ksyloosin dehydraatiolle furfuraaliksi ja sitä seuraaville furfuraalin sivureaktioille. Tehdyn tutkimuksen ja kirjallisuusselvityksen perusteella yksinkertainen kolmen reaktion malli antaa riittävän tarkan ennustuksen furfuraalisaannoista käytetyissä olosuhteissa. Reaktiot ovat 1) ksyloosin dehydraatio sekä 2) furfuraalin ja 3) ksyloosin reaktiot sivutuotteiksi. Lisäksi huomattiin, että reaktion, jossa syntyy furfuraalin häviämistuotteita, reaktioaste on riippuvainen happamuudesta (H+-konsentraatio). Työssä onkin ehdotettu mahdollisia reaktiomekanismeja furfuraalin sivureaktioille. Työn viimeisessä osassa tutkittiin ligniinin vaikutusta furfuraalin muodos¬tu¬miseen. Vertailukohtana muurahaishapolle käytettiin rikkihappoa. Tutkimuksessa selvisi, että käytetty ligniini, Indulin AT, huononsi furfuraalisaantoa. Suurin osa vaikutuksesta johtui ligniinin neutralointikapasiteetista, jolloin reaktioliuoksen happamuus laski, mutta mahdollisia sivureaktioita ei voitu sulkea pois. Kaiken kaikkiaan vaikutukset olivat pienempiä muurahaishapolla kuin rikkihapolla. Tämä väitöskirjatutkimus osoitti, että muurahaishappo katalysoi furfuraalin tuotantoa tehokkaasti. Tutkimuksessa muodostettiin reaktiokineettinen malli, jota voidaan käyttää käsitteellisessä prosessisuunnittelussa. Tulosten perusteella on tärkeää huomioida reaktioliuoksen happamuus kinetiikassa ja tarkkailla happamuuden muutoksia käytettäessä prosessisivuvirtoja.

In the forest products industry the opportunity exists to extract currently under-utilised compounds from the process or waste streams and thereby derive more value from the wood entering the process. A big portion of the hemicellulose content of wood does not form part of the final product. Extracting the hemicelluloses from the waste streams or other locations in the process would allow them to be used more effectively. The predominant hardwood hemicellulose, xylan, is polymeric xylose. Xylose is an important platform sugar in bioconversion strategies and can be converted to fuels and other valuable chemicals. The xylan polymer can be hydrolysed to its xylose monomers by a number of conversion strategies; the most widely known being chemical and enzymatic digestion. Chemical conversion is usually done using acid at elevated temperatures, but high yields are often offset by degradation of the product. On the other hand, enzymatic hydrolysis can be better regulated to prevent unwanted degradation of the monomeric sugar products. Enzymatic hydrolysis has been pronounced the environmentally friendly choice of technology, although it is hampered by low conversions and high cost of enzymes. To date commercial enzymes for biomass conversion are not readily available most of which are still in development. In understanding how to best utilise a xylan, recovered from the pulping process, the potential to convert hardwood xylan to xylose with enzymes currently available on the market was studied. A hardwood xylan extracted from fully bleached Eucalyptus pulp with a chelating agent, Nitren, was used as substrate to evaluate the ability of some commercial enzymes to degrade the extracted xylan to xylose monomers. The enzymes used in this study were not dedicated biomass conversion enzymes, but rather chosen for their xylan degrading potential, i.e. xylanase content. By means of hydrolysis profiles on commercial Birchwood and Oat Spelts xylan as substrates and enzyme characterisation, Multifect xylanase was identified as most promising enzyme for xylan conversion. Multifect contained high levels of xylanase and xylosidase activity in the enzyme preparation. Commercial Birchwood xylan and the extracted Eucalyptus xylan were found to be chemically similar, both composed predominantly of xylose. The hydrolysis profiles obtained on Birchwood xylan could therefore serve as a benchmark against which the hy-drolysis of Eucaluptus xylan could be compared. Full conversion of the Eucalyptus xylan with Multifect could not be achieved, although Multifect completely degraded the Birchwood xylan. The maximum xylose yield that could be obtained on Eucalyptus xylan was 80 % and it was concluded that the remaining 20% was unhydrolysable by the enzyme, most likely due to the limitations in the employed extraction method. It was however noted that up to the point of 80 % conversion higher hydrolysis rates were observed on Eucalyptus xylan than Birchwood xylan with equal charges of Multifect. The differences in hydrolysis rates may have indicated that the Eucalyptus xylan is more accessible to enzyme attack than the Birchwood xylan, likely as a result of the extraction methods used to prepare the xylans. A simple economic evaluation illustrated the weight of various costs in process profitability. The most economic operation of a continuous steady state reactor is at a low enzyme charge, 17 IU/ℓ, and a long retention period, five days, due to the high cost of the enzyme compared to other factors. For a reduced retention time, an investigation into enzyme immobilisation and the use of a packed-bed type reactor is recommended. / Dissertation (MEng)--University of Pretoria, 2009. / Chemical Engineering / unrestricted

Un nouveau dendron phénol trialcyne a été développé par analogie avec le dendron phénol triallyle déjà connu. En nous appuyant sur les cœurs dendritiques polyiodés du laboratoire, nous avons obtenu une famille de dendrimères polyalcynes comportant 27, 81 et 243 branches. Des ferrocènes ont ensuite été greffés par chimie « click » à la périphérie de ces dendrimères. La série obtenue permet la reconnaissance électrochimique d’oxo-anions d’intérêt biologique, comme l’ATP, et de cations métalliques. Grâce aux propriétés d’adsorption des grand dendrimères, nous avons obtenu des électrodes de platine modifiées, robustes et recyclables, permettant de réaliser cette reconnaissance. Une série de glycodendrimères comportant 27, 81 et 243 xylopyranosides terminaux a également été synthétisée. Les méthodes de caractérisation de nanoobjets ont été investiguées, et en particulier, les techniques permettant d’obtenir la taille des molécules en solution comme la RMN DOSY et la diffusion dynamique de la lumière (DLS). Enfin, une série de dendrimères robustes comportant 4 à 6 branches alcynes très longues a été développée. Le greffage périphérique par chimie « click » de beta-cyclodextrines méthylées aléatoirement est également rapporté. / A new easily accessible trialkyne phenol dendron has been developed mimicking the already known triallyl phenol dendron. A family of polyalkynyl containing dendrimers with 27, 81 and 243 terminal branches was obtained starting from the classical polyiodo dendritic cores of the laboratory. Ferrocenes were then grafted using “click” chemistry. The dendrimers obtained allowed electrochemical sensing of both biologically interesting oxo-anions like ATP and metallic cations. Robust and recyclable modified platinum electrodes were obtained thank to the adsorption properties of large dendrimers. These electrodes are able to recognize the same ions as the dendrimer in solution. A novel series of glycodendrimers with 27, 81 and 243 modified xylopyranosides termini was synthesized too. The characterization methods for such nanoobjects were investigated, and particularly technics that allow to obtain the size of the molecules like dynamic light scattering (DLS) and DOSY NMR. Finally, a family of robust polyalkynyl containing dendrimers with 4 to 6 enlarged branches was developed. The functionalization with randomly methylated beta-cyclodextrins using “click” chemistry is also reported.

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)

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18332号 / 農博第2057号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4839(農学部図書室) / 31190 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 渡邊 隆司, 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

No description available.

No description available.