Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Made available in DSpace on 2014-11-10T11:09:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-06-13Bitstream added on 2014-11-10T11:57:42Z : No. of bitstreams: 1 000790107_20150511.pdf: 338150 bytes, checksum: a68b2a9eb314d27a58d6c9cc6e3d1e9c (MD5) Bitstreams deleted on 2015-05-11T13:13:25Z: 000790107_20150511.pdf,Bitstream added on 2015-05-11T13:13:55Z : No. of bitstreams: 1 000790107.pdf: 1190313 bytes, checksum: 3985054a66230646f5c4ccfafc0565a2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Devido à crescente demanda por combustíveis, pesquisas vêm sendo desenvolvidas com o intuito de se obterem novos tipos de biocombustíveis provenientes de fontes renováveis. O etanol celulósico tem sido cogitado como complemento da produção de etanol, entretanto, a viabilização desse processo requer o desenvolvimento de tecnologias de sacarificação dos materiais lignocelulósicos, por via bioquímica, como a enzimática, ou por métodos abióticos. Os fungos filamentosos são um grupo de microrganismos secretores de enzimas que atuam na degradação de biomassa. As enzimas do complexo celulolítico e hemicelulolítico clivam as ligações que formam os polímeros de polissacarídeos liberando hexoses e pentoses fermentescíveis a etanol, porém, enzimas acessórias como a celobiose desidrogenase, são importantes na eficiência do processo. O presente projeto visou o isolamento de cepas fúngicas termofílicas com habilidade de secretar aquelas enzimas, assim como, a obtenção de preparados enzimáticos com estabilidade às variações de temperatura e pH que ocorrem durante as etapas da hidrólise. Foram isoladas 32 linhagens de fungos capazes de crescer em temperaturas entre 45 e 60ºC, tendo sido selecionadas cinco para as etapas seguintes do trabalho (Thermomyces lanuginosus S1, T. lanuginosus S3, Rasamsonia emersonii S10, T. lanuginosus FZI e R. emersonii BC) em função de apresentarem crescimento a 60ºC. Entre essas linhagens, as maiores atividades de β-glicosidase (52 U/g) e endoglucanase (579 U/g) foram obtidas em meio de cultivo da cepa R. emersonii S10 em 240h para ambas as enzimas. A melhor atividade de FPase foi apresentada pelo fungo T. lanuginosus S1 em 168h de cultivo, tendo produzido 262,8 U/g da enzima. O fungo R. emersonii S10 foi o único que apresentou a produção detectável das enzimas avicelase (9,2 U/g) e β-xilosidase (910,7 U/g). Esse isolado também produziu as maiores atividades de xilanase com ... / Owing the growing demand for fuels, research is being done with the aim of obtaining new types of biofuels from renewable resources. Cellulosic ethanol, obtained from residual biomass from agricultural and agro-industrial processes has been considered as a complement of ethanol production by decreasing the need for expansion of sugarcane areas. However, the viability of the process requires the development of technologies saccharification of lignocellulosic materials by biochemical route, such as enzymatic or abiotic methods. The filamentous fungi are a group of microorganisms that produce (secretory) of enzymes involved in the degradation of biomass. The enzymes cleave hemicellulolytic and cellulolytic complex connections that form polymers of polysaccharides present in the biomass releasing fermentable hexoses and pentoses to ethanol, however, accessory enzymes such as cellobiose dehydrogenase are important in the process efficiency. This design aimed at the isolation of thermophilic fungal strains with the ability to secrete those enzymes, as well as the enzyme preparations to obtain stability to pH and temperature variations that occur during the hydrolysis step. 32 fungal strains capable of growing at temperatures between 45 and 60 °C were isolated, five were selected to continue the following work steps (Thermomyces lanuginosus S1, T. lanuginosus S3, Rasamsonia emersonii S10, R. emersonii BC and T. lanuginosus FZI) as a function of experiencing growth at 60 °C. Among these strains, the highest activities of β-glucosidase (52 U/g) and endoglucanase (579 U/g) were obtained from the culture medium of strain R. emersonii S10 in 240h for both enzymes. The best activity FPase (cellulase that hydrolyzes filter paper) was presented by the fungus T. lanuginosus S1 in 168h of cultivation, having produced 262.8 U/g enzyme. The R. emersonii S10 was the only fungus that showed detectable avicelase production of enzymes (9.2 U/g) and ...

Orientador: Eleni Gomes / Banca: Adalberto Pessoa Júnior / Banca: Gustavo O. Bonilla / Resumo: Devido à crescente demanda por combustíveis, pesquisas vêm sendo desenvolvidas com o intuito de se obterem novos tipos de biocombustíveis provenientes de fontes renováveis. O etanol celulósico tem sido cogitado como complemento da produção de etanol, entretanto, a viabilização desse processo requer o desenvolvimento de tecnologias de sacarificação dos materiais lignocelulósicos, por via bioquímica, como a enzimática, ou por métodos abióticos. Os fungos filamentosos são um grupo de microrganismos secretores de enzimas que atuam na degradação de biomassa. As enzimas do complexo celulolítico e hemicelulolítico clivam as ligações que formam os polímeros de polissacarídeos liberando hexoses e pentoses fermentescíveis a etanol, porém, enzimas acessórias como a celobiose desidrogenase, são importantes na eficiência do processo. O presente projeto visou o isolamento de cepas fúngicas termofílicas com habilidade de secretar aquelas enzimas, assim como, a obtenção de preparados enzimáticos com estabilidade às variações de temperatura e pH que ocorrem durante as etapas da hidrólise. Foram isoladas 32 linhagens de fungos capazes de crescer em temperaturas entre 45 e 60ºC, tendo sido selecionadas cinco para as etapas seguintes do trabalho (Thermomyces lanuginosus S1, T. lanuginosus S3, Rasamsonia emersonii S10, T. lanuginosus FZI e R. emersonii BC) em função de apresentarem crescimento a 60ºC. Entre essas linhagens, as maiores atividades de β-glicosidase (52 U/g) e endoglucanase (579 U/g) foram obtidas em meio de cultivo da cepa R. emersonii S10 em 240h para ambas as enzimas. A melhor atividade de FPase foi apresentada pelo fungo T. lanuginosus S1 em 168h de cultivo, tendo produzido 262,8 U/g da enzima. O fungo R. emersonii S10 foi o único que apresentou a produção detectável das enzimas avicelase (9,2 U/g) e β-xilosidase (910,7 U/g). Esse isolado também produziu as maiores atividades de xilanase com ... / Abstract: Owing the growing demand for fuels, research is being done with the aim of obtaining new types of biofuels from renewable resources. Cellulosic ethanol, obtained from residual biomass from agricultural and agro-industrial processes has been considered as a complement of ethanol production by decreasing the need for expansion of sugarcane areas. However, the viability of the process requires the development of technologies saccharification of lignocellulosic materials by biochemical route, such as enzymatic or abiotic methods. The filamentous fungi are a group of microorganisms that produce (secretory) of enzymes involved in the degradation of biomass. The enzymes cleave hemicellulolytic and cellulolytic complex connections that form polymers of polysaccharides present in the biomass releasing fermentable hexoses and pentoses to ethanol, however, accessory enzymes such as cellobiose dehydrogenase are important in the process efficiency. This design aimed at the isolation of thermophilic fungal strains with the ability to secrete those enzymes, as well as the enzyme preparations to obtain stability to pH and temperature variations that occur during the hydrolysis step. 32 fungal strains capable of growing at temperatures between 45 and 60 °C were isolated, five were selected to continue the following work steps (Thermomyces lanuginosus S1, T. lanuginosus S3, Rasamsonia emersonii S10, R. emersonii BC and T. lanuginosus FZI) as a function of experiencing growth at 60 °C. Among these strains, the highest activities of β-glucosidase (52 U/g) and endoglucanase (579 U/g) were obtained from the culture medium of strain R. emersonii S10 in 240h for both enzymes. The best activity FPase (cellulase that hydrolyzes filter paper) was presented by the fungus T. lanuginosus S1 in 168h of cultivation, having produced 262.8 U/g enzyme. The R. emersonii S10 was the only fungus that showed detectable avicelase production of enzymes (9.2 U/g) and ... / Mestre

Este projeto de pesquisa visou à investigação do desempenho do reator UASB (10 L de volume) no tratamento da vinhaça quando submetido a aumento progressivo da carga orgânica em condições termofílicas. A carga orgânica afluente média foi de 2,79 gDQO/L.dia (desvio de 1,5 gDQO/L.dia) e média efluente bruto de 1,24 gDQO/L.dia (desvio de 0,93 gDQO/L.dia). A operação atingiu o limite de carregamento de 6,5 gDQO/L.dia, limitado pela produção excessiva de ácidos voláteis totais que atingiram concentrações da ordem de 1.200 mgHac/L, tóxicas para biomassa metanogênica. A adaptação do lodo mesofílico às condições termofílicas ocorreu no período de 55 dias, que pode ser considerado um período curto. A operação com ácidos orgânicos para enriquecimento do lodo não se mostrou eficaz, com acúmulo desses ácidos para a COV de 10 gDQO/L.dia. A operação com etanol mostrou-se eficaz para a recuperação do lodo, com COV de 8,0 gDQO/L.dia, obtendo-se eficiência máxima de remoção de DQO de 80%. As eficiências máximas na operação foram durante a adaptação do lodo, com COV de 1,20 gDQO/L.dia (92,0%) e durante a operação do sistema, com COV de 3,50 gDQO/L.dia (83,0%). O TDH médio desenvolvido neste trabalho foi de 1,34 dias. Este dado levantou questionamentos a respeito de um TDH ótimo utilizado para este tipo de tratamento, devido a grande variação de TDHs encontrados na literatura em trabalhos correlatos (entre 1,0 e 6,5 dias). / This research project had the objective to investigate the performance of the UASB reactor (10 L of volume) in the vinasse treatment submitted to progressive organic loadings under thermophilic conditions. The organic matter upload average was 2,79 gDQO/L.day (standard deviation of 1,5 gDQO/L.day) and the full organic matter average was 1,24 gDQO/L.dia (standard deviation of 0,93 gDQO/L.day). The operation reached the organic limit loading in 6,5 gCOD/L.day, limited by the excessive production of total volatile acids that reached concentrations beyond 1.200 mgHac/L, toxic to metanogenic biomass. Adaptation of mesophilic sludge to thermophilic conditions occurred in a period of 55 days, what can be considered a short period. The operation with organic acids for sludge enrichment did not seem to be efficient, accumulating these acids in a 10 gCOD/L.day. The operation with ethanol seemed to be efficient for sludge recuperation, of 8,0 gCOD/L.day, reaching maximum COD reduction efficiencies of 80%. The maximum operation efficiency was during sludge adaptation with 1,20 gCOD/L.day (92,0%) and during operation system, with 3,50 gCOD/L.day (83%). The HDT average developed in this research was 1,34 days. These data bring questions about the HDT optimum for this kind of anaerobic treatment, in correlation with significant variation of HDT used in another studies in the literature (between 1,0 and 6,5 days of HDT).

>Magister Scientiae - MSc / The negative environmental impact of fossil fuels and growing concerns about petroleum supplies has driven the search for alternative, renewable transportation fuels. An 'ideal' fuel replacement would be a biofuel produced from lignocellulosic biomass. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides into simple sugars and the subsequent conversion of these sugars into useable fuels. One of the most common fates of lignin in nature is to be metabolized by lignin peroxidases (LiPs), predominantly of microbial origin. This study aims to isolate and characterise microorganism(s) involved in the degradation of lignocellulose. Thermophilic bacteria were isolated from straw-based compost and screened for lignin peroxidase activity. One isolate, CP11, showed significant lignin peroxidase activity and based on 16S rRNA gene sequence analysis, the isolate was found to be most closely related to Bacillus thermoamylovorans. Morphological, physiological and biochemical characterisation was conducted to determine whether the isolate was a novel species. Morphologically, CP11 was characterised as an endospore-forming, Gram positive rod. In addition, the isolate was found to be a facultative anaerobe, catalase positive and capable of utilising a range of carbon sources including glucose, sucrose and arabinose. Isolate CP11 was moderately thermotolerant and grew between 37°C and 55°C, with an optimum growth temperature of 45°C. Based on its phenotypic characteristics CP11 could be clearly distinguished from its closest phylogenetic neighbours. Preliminary characterisation of the lignin peroxidase was conducted using crude enzyme extract and Azure B dye as the substrate. Activity was detected in the supernatant only and a growth curve was constructed to determine the growth phase of lignin peroxidase production. In order to identify the gene encoding the lignin peroxidase a small insert library was constructed and screened for ligninase activity using Azure B as the substrate. / National Research Foundation

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2000 / M

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

This master thesis deals with conventional methods of bacterial cell analysis, polyhydroxyalkanoates, Raman spectroscopy and electron microscopy in the theoretical part. The production of polyhydroxybutyrate by selected thermophilic bacteria and their analysis by gas chromatography, cryogenic scanning electron microscopy and Raman spectroscopy is described in the experimental part. The chosen sample was analyzed by a transmission electron microscope. Comparing the results from previous mentioned methods it was found that the bacteria Schlegelella thermodepolymerans accumulated the highest amount of PHB. The lowest amount of PHB was obtained by bacteria Rubrobacter xylanophilus. The assumption that the PHB granules formed so-called needle-like plastic deformations during freeze-fracturing was affirmed by cryo-SEM photos analysis. Moreover, it was found that the bacterial cell characterization deduced from microscopic observation of samples corresponded to the description in the literature. TEM provided better resolution photos and in consequence the cells and PHB are more visible. The thesis is also focused on chemical fingerprint analysis of cells by Raman spectroscopy. Several biomolecules were identified by measured Raman spectra for the particular samples.

The plant cell wall consists of several layers: cellulose, hemicellulose, lignin and pectin. These biopolymers are degraded by many microorganisms. Extracellular enzymes are used for biodegradation by microorganisms. This thesis was focused on studying the impact of cultivation conditions on the production of extracellular enzymes at carboxymethyl cellulase and pectin when a mixed thermophilic culture containing Bacillus and Thermus microorganisms is used. The cultivation was carried out in flasks on a shaking machine with a shaking speed of 99 min-1 at a temperature of 60°C. The monitoring covered cellulolytic and polygalacturonase activities, protein concentration by the Biuret method, concentration of reducing substances by the Somogyi and Nelson methods, and the temperature optimum.

This thesis is focused on study of biodegradability of modified polyurethane elastomeric films in synthetic medium with minerals and vitamins on tempered shaker by mixed thermophilic aerobic bacterial culture Bacillus and Thermus genera. In most cases addition of all used fillers (carboxymethyl cellulose, hydroxyethyl cellulose, acetylated cellulose, acetylated starch and glutein) led to increased biodegradability of elastomeric films with modifying agent in comparison with elastomeric films without modifying agent (referential). The growth of cultures was strongly increased in presence of elastomeric films modified by 10 % acetylated cellulose and 10 % carboxymethyl cellulose. Elastomeric film biodegradation mechanism included probably two stages: abiotic destruction of elastomeric films and consequent utilization of degradation products by bacterial culture.