Zugl.: Braunschweig, Techn. Univ., Diss., 2007

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-30T14:47:31Z No. of bitstreams: 1 DissVMV.pdf: 2967521 bytes, checksum: 9bebee517d7d20c1b36810aa5e5744df (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-04T17:34:08Z (GMT) No. of bitstreams: 1 DissVMV.pdf: 2967521 bytes, checksum: 9bebee517d7d20c1b36810aa5e5744df (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-04T17:34:16Z (GMT) No. of bitstreams: 1 DissVMV.pdf: 2967521 bytes, checksum: 9bebee517d7d20c1b36810aa5e5744df (MD5) / Made available in DSpace on 2016-10-04T17:34:24Z (GMT). No. of bitstreams: 1 DissVMV.pdf: 2967521 bytes, checksum: 9bebee517d7d20c1b36810aa5e5744df (MD5) Previous issue date: 2015-02-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / The enzymes involved in the conversion process of lignocellulosic biomass into second generation (2G) ethanol add a high cost to the process. Studies reported in the literature indicate that some metal ions, easily found in nature, improve the activity and stability of enzymes and the biomass saccharification process when added to the enzymatic cocktail. In this context, this work aimed to evaluate the strategy of addition of metal ions in the crude enzymatic extract in order to increase the enzyme activity and stability and to improve the saccharification process. For the production of (hemi)cellulase enzymatic complex step, a selected strain of Aspergillus niger was cultivated under solid-state fermentation (SSF), submerged fermentation (SmF) and sequential fermentation (FS). Enzyme production and thermostability were evaluated also considering the effects of pretreatment (using steam-explosion and liquid-hot-water) of the sugarcane bagasse used as carbon source and inducer. For endoglucanase and xylanase enzymes, cultivation under SSF favored production when using steam-exploded and liquid-hot-water pretreated bagasse (both washed). Removal of inhibitors from the pretreated biomass employed in the cultivation media was necessary, because the presence of phenolic compounds restricted fungal growth. The findings revealed that endoglucanase and β-glucosidase produced under SSF were less prone to thermal deactivation. From these results, enzyme complex produced by SSF using the hydrothermal bagasse was selected to evaluate the influence of the divalent metal ions Ca, Co, Cu, Fe, Mg, Mn, Ni, Zn in two concentrations (2 and 10 mM) on enzymatic activity and stability. Most of the ions influenced differently the enzymatic activity and stability, at both concentrations. The most favorable influence was obtained by the Mn+2 ion, increasing by 57% the endoglucanase activity and maintaining the enzyme stable for 72 hours, besides it also showed no negative effects on β-glucosidase and xylanase. The Mn+2 ion was then selected for the saccharification of sugarcane bagasse submitted to different pretreatments. The addition of Mn2+ ion (10 mM) in the saccharification process using enzymatic extracts produced "in house" proved to be very effective by increasing the release of glucose up to 120% when using the acid pretreated bagasse (Bác). The increase was 55% for the washed exploded bagasse (BEXL) and 70% for the liquid-hot-water pretreated bagasse (BHT). Therefore, it was possible to validate that the addition of metal ions is able to positively influence the activity and stability of enzymes, and also the process of enzymatic saccharification of lignocellulose biomass. / As enzimas envolvidas no processo de degradação da biomassa lignocelulósica para a produção do etanol de segunda geração (2G) agregam um alto custo ao processo. Estudos já reportados na literatura indicam que alguns íons metálicos, facilmente encontrados na natureza, ao serem adicionados ao coquetel enzimático atuam na melhoria da atividade e estabilidade das enzimas e no processo de sacarificação da biomassa. Nesse contexto, este trabalho teve como objetivo avaliar a estratégia de adição de íons metálicos ao extrato enzimático bruto com o intuito de aumentar a atividade e estabilidade enzimática e a melhoria na conversão do processo de sacarificação. Para a etapa de seleção do complexo (hemi)celulásico foi utilizada uma cepa selecionada de Aspergillus niger cultivada em fermentação em estado sólido (FES), fermentação submersa (FSm) e fermentação sequencial (FS). A produção de enzimas e a termoestabilidade foram avaliadas considerando os efeitos do pré-tratamento (explosão a vapor e hidrotérmico) do bagaço de cana-de-açúcar utilizado como fonte de carbono e indutor. A produção de endoglucanase e xilanase foi favorecida pelo cultivo em FES a partir do bagaço explodido e hidrotérmico (submetidos ao processo de lavagem). A remoção de inibidores da biomassa prétratada mostrou-se necessária, pois a presença de compostos fenólicos restringiu o crescimento fúngico em FES. Os resultados revelaram que endoglucanase e β-glicosidase produzidas sob FES foram menos propensas à desativação térmica. A partir desses resultados, foi selecionado o complexo enzimático produzido por FES utilizando o bagaço hidrotérmico lavado, para avaliação da influência dos íons metálicos bivalentes Ca, Co, Cu, Fe, Mg, Mn, Ni, Zn em duas concentrações (2 e 10 mM) na atividade e estabilidade enzimática. A maioria dos íons metálicos utilizados influenciou, de forma distinta, a atividade e estabilidade enzimática em ambas as concentrações para as enzimas estudadas. O íon Mn2+ se destacou, propiciando um aumento de 57% na atividade de endoglucanase e mantendo a enzima estável por 72 horas, além disso não apresentou efeito negativo para β-glicosidase e xilanase. Assim, o íon Mn2+ foi selecionado para o estudo da sacarificação dos bagaços de cana submetidos a diferentes pré-tratamentos. A adição do íon Mn2+ (10 mM) no processo de sacarificação utilizando extratos enzimáticos produzidos “in house” mostrou-se bastante eficaz no aumento da liberação de glicose, com ganhos percentuais de até 120% para o bagaço pré-tratado em meio ácido (Bác). Para o bagaço explodido lavado (BEXL) o aumento foi de 55 % e para o hidrotérmico (BHT) foi de 70%. Desse modo, foi possível validar que a adição de íons metálicos é capaz de influenciar positivamente tanto a atividade e estabilidade, como o processo de sacarificação enzimática da biomassa lignocelulósica.

Orientador: Rubens Monti / Coorientador: Marco Filice / Banca: Daniela Parreira Marques / Banca: Jonas Contieiro / Banca: Ariela Veloso de Paula / Banca: Juliana Cristina Bassan / Doutor

Orientador: Luciana Francisco Fleuri / Resumo: As inulinases catalisam a hidrólise da inulina, produzindo inulo-oligossacarídeos, frutose e glicose, por isso, possuem várias aplicações industriais. A fermentação submersa é mais usada na produção industrial de inulinase, contudo a fermentação em estado sólido tem demonstrado bons resultados quanto à obtenção de enzimas a baixo custo. O yacon (Smallanthus sonchifolius) é um tubérculo com alta concentração de antioxidantes e frutanos e por isso tem atraído o interesse na área de alimentos. A fermentação em estado sólido utilizando o yacon como substrato possui potencial para a produção de enzimas e também para utilização na alimentação animal. Objetivou-se com este trabalho avaliar a produção de inulinases utilizando 16 espécies fúngicas filamentosas cultivadas em fermentação em estado sólido utilizando resíduos agroindustriais como substrato, bem como identificar a melhor combinação substrato/micro-organismo para utilização do preparado enzimático sólido como prebiótico em rações de frango de corte. A atividade da inulinase foi muito superior com a utilização da farinha de yacon como substrato em fermentação em estado sólido em relação ao farelo de trigo, farelo de soja e bagaço de cana-de-açúcar, independente do fungo utilizado. As inulinases provenientes da farinha de yacon demonstraram grande amplitude de pH e temperatura, independente do micro-organismo fermentante. O preparado enzimático sólido de farinha de yacon com Aspergillus niger 40018 apresentou alta atividade d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Inulinases catalyze the inulin hydrolysis, yielding inulo-oligosaccharides, fructose and glucose, therefore, they have many industrial applications. The submerged fermentation is more used at inulinase industrial production, however the solid state fermentation (SSF) has showed good results regarding to low cost enzymes. The yacon (Smallanthus sonchifolius) is a tuber root with high content of antioxidants and fructans and because of this it has attracted interest of food area. The solid state fermentation using the yacon root as substrate has potential to produce enzymes and to use in animal feeding. The aim of this work was to evaluate the inulinase production of 16 filamentous fungal species growing in solid state fermentation (SSF) on agroindustrial wastes as substrate, as well as to identify the best combination substrate/microorganism of the solid enzymatic preparation (SEP) to use as prebiotic in broilers diets. The inulinase activity was higher with yacon flour than wheat bran, soybean bran and sugarcane bagasse, independently of fungus. The inulinases from yacon flour showed large pH and temperature amplitude, independently of fungus. The SEP of yacon flour and Aspergillus niger 40018 showed high lipase and antioxidant activity and high fructooligosaccharides and it was selected to further trials with broilers. There was no alteration on performance, caecum pH and lipid profile with the SEP. The SEP promoted a stronger reduction of Salmonella Enteritidis number afte... (Complete abstract click electronic access below) / Doutor

Demand for lithium (Li) and cobalt (Co) is on the rise, due in part to their increased use in rechargeable Li-ion batteries (RLIB). Current recycling processes that utilize chemical leaching efficiently recover in Li and Co from the cathode material in spent batteries; however, these processes are costly and emit hazardous waste into the environment. Therefore, a more sustainable process for recycling Li and Co is needed, and bioleaching may provide a solution. Fungal bioleaching has been shown in previous studies to effectively mobilize metals (Pb, Al, Mn, Cu, and Zn) from mine tailings, electronic scrap, and spent batteries with organic acids. However, little is known regarding fungal tolerance to Li and Co, and if the concentrations of organic acids excreted by fungi can effectively leach Li and Co from the cathode material. In order to address these questions, experiments were first conducted to test the Li and Co leaching efficiency with organic acids at concentrations similar to what has been previously reported in fungal cultures. The remaining experiments were performed with three fungal species: Aspergillus niger, Penicillium chrysogenum, and Penicillium simplicissimum. First, fungal biomass production, pH and organic acid excretion were examined when the fungi were grown in Czapek dox broth (CDB) or Sabouraud dextrose broth (SDB). Second, fungal biomass production and pH were examined when the fungi were grown in the presence of Li or Co. This determines tolerance of the fungi to the metals, and if fungal processes were inhibited by the metals. Third, bioleaching was performed with cathode material from RLIB in batch cultures to test the ability of organic acids excreted by A. niger to mobilize Li and Co. Three bioleaching strategies, one-step, two-step, and spent-medium leaching techniques were used to mobilize Li and Co from the cathode in RLIB. Low concentrations of organic acids similar to what is excreted by fungi have not been tested to leach Li and Co from the cathode in RLIB. Results from chemical leaching with low concentrations of organic acids in this study indicate that organic acid leaching efficiency can be increased by utilizing higher concentrations (above 50 mM) of citric or oxalic acid to mobilize Li or Co from the cathode in RLIB. Furthermore, 100 mM of citric acid or 100 mM of oxalic acid mobilized more Co or Li than mixtures of organic acids. Notably the addition of hydrogen peroxide to mixed concentrations of organic acids significantly improved mobilization of Li and Co under abiotic conditions. Different growth media may alter biomass production and potentially organic acid excretion by the three fungal species. Analysis of biomass production by A. niger and P. simplicissimum showed that differences in media composition between CDB and SDB did not affect collected biomass for each species. However, CDB cultures with P. chrysogenum had significantly less biomass than SDB cultures after 10 days of growth. Differences in growth by P. chrysogenum between CDB and SDB may be attributed to preferred nutrients and/or low pH present in SDB cultures. Biomass production by the three fungi increased up to day 10 in CDB or SDB. This result indicated that nutrients in CDB or SDB were not limiting toward fungal growth. Cultures with A. niger had the highest concentrations of organic acids (50 mM of oxalic acid), followed by cultures with P. simplicissimum (30 mM oxalic acid), and P. chrysogenum (less than 5 mM oxalic acid). Organic acids excreted by all three fungal species were detected in cultures in CDB, while only A. niger and P. chrysogenum excreted organic acids in SDB cultures. Metals such a Li or Co present in the cathode of RLIB may be toxic to fungal processes when exposed to high metal concentrations. Metal tolerance experiments indicate that biomass production by the three fungi was significantly inhibited by 100 mg/L Co compared to controls, which contained no metal. Li at a concentration of 1000 mg/L inhibited biomass production by A. niger and P. simplicissimum. However, biomass production by P. chrysogenum was not significantly inhibited by 1000 mg/L Li. I found that P. simplicissimum was the most susceptible to toxic effects of Li and Co among the three fungi. In A. niger cultures amended with 100 mg/L Li or Co, pH at day 5 was similar to control cultures of A. niger without metals (pH 3.0 – 3.4), whereas pH was significantly higher in cultures with 1000 mg/L of Li or Co (pH 7.1 – 7.3). Cultures of A. niger were exposed to the cathode material from RLIB to test the leaching efficiency of excreted organic acids after mobilizing Li and Co. In bioleaching experiments with A. niger, organic acids excreted in the presence of cathode material from RLIB were quantified at concentrations under 50 mM. At the end of bioleaching experiments with A. niger, 40 mM tartaric acid was detected and was the highest produced organic acid in bioleaching cultures. However, with conditions set in this study, organic acids excreted by A. niger mobilized only ̴7% of Co and 20% of Li when using spent medium with cathode material from RLIB. According to findings in chemical leaching experiments, concentrations of organic acids higher than 50 mM will be required in fungal cultures to increase mobilization of Li or Co from the cathode material in RLIB. Modifying growth media to include higher concentrations of sucrose will potentially increase organic acid excretion as demonstrated in previous publications. Future studies should focus on how to maximize organic acid excretion by fungi when exposed to metals found in the cathode of RLIB.

This diploma thesis is focused on studying of the effect of the dielectric barrier discharge (DBD) on eucaryotic microorganisms. Plasma sterilization is considered to be an alternative method to conventional sterilization processes. Contrary to standard decontamination methods it doesn´t stress exposed material by heat, pressure and chemicals. Plasma acts on eucaryotic and procaryotic systems by means of synergy of three inactivation mechanisms. They are various reactive species, UV radiation and heat. The Aspergillus niger has been chosen as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. Plasma was generated in dielectric barrier discharge (DBD) at atmospheric pressure. Nitrogen and argon were used as working gases, paper and PET foil were used as carrying media. The influence of various working conditions on the sterilization effect was studied. Namely it was the influence of plasma exposition time, plasma power density, the type of operating gas and type of supporting medium. The effect of UV radiation in combination with temperature, temperature and direct plasma were studied separately. According to our results the efficiency of DBD increases with plasma power density, resp. plasma exposition time. When comparing sterilization efficiency of nitrogen and argon operating at the same conditions, the higher sterilization effect was observed in argon. The influence of the carrying medium on sterilization effectiveness was proved. It was caused by the different structure of surface. It was found out, that in our experimental setup the active species are probably the main inactivation mechanism. The influence of temperature on inactivation of microorganisms was negligible. The combination of UV radiation and temperature reached the decontamination level about 2 orders. The discharge parameters were studied by means of optical emmision spectroscopy. Scanning electron microscopy enabled to evaluate possible damage of exposed materials through DBD.

The study of production of hydrolytic enzymes dealt with the production of cellulase and polygalacturonase by two microbial strains - Aspergillus niger and Aureobasidium pullulans. The enzymes were produced in solid-state fermentation system. The wheat straw and sugar beet pulp were used as a substrate. The substrates were moistened by water, mineral solution or by medium with glucose. The effect of mineral solution and glucose on production of these enzymes were monitored during cultivation. The highest production of polygalacturonase was achieved by Aspergillus niger during cultivation on sugar beet pulp moistened by mineral solution. The highest production of cellulase was achieved by Aspergillus niger during cultivation on wheat straw moistened by medium with glucose.

Study of the employment of waste paper to microbial production of cellulase was carried out using Aspergillus niger cultivation on carbon sources that have been waste office paper and cardboard, humidified by no – carbon medium or distiled water. Cultivation took place in the SSF way in Erlenmeyer flasks and columns. Concentration of extracellular proteins, cellulase and protease activity for selected samples were monitored. It was found that the most advantageous method of cultivation in terms of cellulase activity production is the cultivation in the column washing by no – carbon medium in three day intervals.

Publicación a texto completo no autorizada por el autor / Investiga la bioadsorcion del plomo (II) usando como material bioadsorbente las biomasas fúngicas Aspergillus niger y Aspergillus fumigatus aislados del pueblo joven Puerto Nuevo-callao. El material bioadsorbente se obtuvo de las muestras tomadas de la zona contaminada del pueblo joven Puerto Nuevo-Callao. El material tratado fue secado en una estufa a la temperatura de 80 °C por 2 horas. Se determinó la bioadsorcion de plomo (II) en solución por las biomasas celular de dos hongos por el método instrumental de espectroscopia de absorción atómica. Los experimentos sobre el efecto del pH en el proceso de bioadsorción de Pb (II) por las biomasas fúngicas demostraron que el pH óptimo es 5.0; así como el experimento del efecto de la temperatura optima por las biomasas fúngicas demostraron una temperatura optima de 25°C para la biomasa fúngicas; la concentración del plomo (II) que presenta la mejor bioadsorcion es a 1000ppm. Del estudio de la cinética del proceso de bioadsorción, se determinó que la biomasa de Aspergillus niger 98% fue más eficiente en la remoción del plomo (II) que el Aspergillus fumigatus 96%. El equilibrio se alcanzó a las 100 minutos del inicio del proceso de bioadsorción logrando un porcentaje de remoción de Plomo (II) para Aspergillus niger 98% y para Aspergillus fumigatus 96%. Se concluye que las biomasas fúngicas remueven eficientemente plomo (II) en solución y pueden utilizarse para descontaminar nichos acuáticos contaminados con este metal. / Tesis

El presente trabajo investigo la actividad antifúngica in vitro de doce extractos etanolicos correspondientes a diez plantas medicinales peruanas; Annona cherimolia Mill. (hojas), Annona muricata L. (corteza y hojas), Bidens pilosa L. (partes aéreas), Hypericum laricifolium L. (partes aéreas), Juglans neotropica Diels (corteza), Piper spp. (hojas), Plantago major L. (hojas), Psidium guajava L. (hojas), Schinus molle L. (corteza y hojas) y Spartium junceum L. (planta entera). Las especies fueron recolectadas en el departamento de Amazonas, excepto Schinus molle L. (Apurímac) y Annona muricata L. (Lima). La actividad antifúngica se evaluo mediante los métodos de difusión en agar y dilución en agar para la determinación de la concentración mínima inhibitoria (CMI). Los microorganismos de prueba utilizados fueron las levaduras Candida albicans ATCC 10231 y Candida albicans cepa clínica, así como, el hongo filamentoso Aspergillus niger ATCC 16404; las cepas fueron proporcionadas por la Cátedra de Microbiología de la Facultad de Farmacia y Bioquímica de la Universidad Nacional Mayor de San Marcos. De doce extractos investigados, seis presentaron actividad antifúngica consistente con un diámetro de halos de inhibición ≥18mm (Prueba de Difusión en agar) frente a Candida albicans ATCC 10231. Ningún extracto mostró actividad consistente frente a la cepa clínica de Candida albicans y Aspergillus niger ATCC 16404. La CMI de los extractos que presentaron actividad consistente frente a Candida albicans ATCC 10231, fue de 250 µg/mL para Hypericum laricifolium L., Juglans neotropica Diels, Psidium guajava L. y Schinus molle L. (un extracto de corteza y uno de hojas) y de 500µg/mL para Piper spp. No se determino la CMI de los extractos (Juglans neotropica Diels y Psidium guajava L.) que presentaron halos frente al Aspergillus niger ATCC 16404 por considerarlos sin actividad significante (<18mm.). Los antifúngicos Nistatina y Fluconazol fueron incluidos en el estudio como controles positivos. -- Palabras clave: Actividad antifúngica, plantas medicinales, plantas del Perú, Amazonas, concentración mínima inhibitoria, Candida albicans, Aspergillus niger. / -- The present work investigated the antifungal activities in vitro of twelve ethanolic extracts corresponding to ten peruvian medicinal plants; Annona cherimolia Mill. (leaves), Annona muricata L. (bark and leaves), Bidens pilosa L. (aerial parts), Hypericum laricifolium L. (aerial parts), Juglans neotropica Diels (bark), Piper spp. (leaves), Plantago major L. (leaves), Psidium guajava L. (leaves), Schinus molle L. (bark and leaves) and Spartium junceum L. (whole plant). The plants were collected in the department of Amazonas, except Schinus molle L (Apurímac) and Annona muricata L. (Lima). The antifungal activities were determinated by the methods of agar diffusion and agar dilution for the determination of the Minimal Inhibitory Concentration (MIC). The used microorganisms of test were the yeasts Candida albicans ATCC 10231 and Candida albicans clinical isolate, as well as, the filamentous fungus Aspergillus niger ATCC 16404; the microorganism were provided by the Chair of Microbiology of Faculty of Pharmacy and Biochemistry of the Universidad Nacional Mayor de San Marcos. Of twelve investigated extracts, six presented significant antifungal activity with a diameter of inhibition haloes ³18mm (Agar Diffusion) against Candida albicans ATCC 10231. No extract showed significant activity against to Candida albicans clinical isolate and Aspergillus niger ATCC 16404. The MIC of the extracts that presented significant activity against Candida albicans ATCC 10231, were of 250µg/mL for Hypericum laricifolium L., Juglans neotropica Diels, Psidium guajava L. and Schinus molle L.(bark and leaf extracts) and of 500µg/mL for Piper spp. We do not determine the MIC of the extracts (Juglans neotropica Diels and Psidium guajava L.) that presented haloes against Aspergillus niger ATCC 16404 for considering them without significant activity (<18mm.). The antifungal agents Nistatin and Fluconazole were included in the study as positive controls. -- Key words: Antifungal activity, medicinal plants, Peruvian plants, Amazonas, minimal inhibitory concentration, Candida albicans, Aspergillus niger. / Tesis