Dissertations / Theses on the topic 'Solid-state bioreactor'

Solid state fermentation (SSF) refers to the microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment to which the selected microorganisms, especially fungi, are naturally adapted. The current status of SSF research globally was discussed in terms of articles publication. This was followed by discussion of the advantages of SSF and the reason for interest in SSF as a notable bioprocessing technology to be investigated and compared to submerged fermentation (SmF) for the production of various added-value products. SSF also proved to be a potential technology to treat solid waste produced from food and agricultural industry and to provide environmental benefits with solid waste treatment. A summary was made of the attempts at using modern SSF technology for future biorefineries for the production of chemicals. Many works were carried out in the Satake Centre for Grain Process Engineering (SCGPE), University of Manchester, to prove the strategy of using SSF for the production of hydrolysate rich in nutrients for sequel microbial fermentation with or without adding any commercial nutrients. The research findings presented in this thesis are based on a series of SSF experiments carried out on systems varying in complexity from simple petri dishes to our own design of bioreactor systems. They were conducted to assess a solution for biomass estimation, enzymes production, and successful mass and heat transfer. A proper technique for inoculum transfer prior to the start of the fermentation process was developed. In SSF, estimation of biomass presents difficulties as generally the fungal mycelium penetrates deep and remains attached with the solid substrate particles. Although many promising methods are available, the evaluation of microbial growth in SSF may sometimes become laborious, impractical and inaccurate. Essentially, this remains another critical issue for monitoring growth. In these studies, measurement of colour changes during SSF are presented as one of the potential techniques that can be used to describe growth, complementary to monitoring metabolic activity measurement, such as CER, OUR and heat evolution, which is directly related to growth. For the growth of Aspergillus awamori and Aspergillus oryzae on wheat bran, soybean hulls and rapeseed meal, it was confirmed that colour production was directly proportional to fungal growth. This colourimetric technique was also proved to be a feasible approach for fungal biomass estimation in SmF. This new approach is an important complementation to the existing techniques especially for basic studies. The key finding is that the colourimetric technique demonstrated and provided information of higher quality than that obtained by visual observation or spores counting. The effect of aeration arrangements on moisture content, oxygen (O2), mass and heat transfer during SSF was investigated. A. awamori and A. oryzae were cultivated on wheat bran in newly designed four tray solid state bioreactor (SSB) systems. The new tray SSB systems were: (1) single circular tray SSB, (2) multi-stacked circular tray SSB, (3) Single rectangular tray SSB and (4) multi-square tray SSB. The purpose was to study the effect, on heat and water transfer, of operating variables, fermentation on the perforated base tray and internal moist air circulation under natural and forced aeration. Temperature, O2 and carbon dioxide were measured continuously on-line. Enzyme activity, moisture content and biomass were also measured. The results suggest that the air arrangements examined have a remarkable effect on the quantity of biomass produced using measurement of spores and enzymes production. The strategy presented in these studies allowed quantitative evaluation of the effect of forced internal moist air circulation on the removal of metabolic heat. With the proposed strategy, it was possible to maintain the bed temperatures at the optimum level for growth. However, the effect on moisture content was very different for the two fungi. It was found that the ability of A. oryzae to retain moisture was much higher than that of A. awamori. This is possibly due to the higher levels of chitin in A. oryzae. Greater spores and enzymes (glucoamylase, xylanase and cellulase) production was observed for A. awamori in multi-stacked circular tray and multi-square tray SSB systems compared to the conventional petri dishes and the other two systems. A. oryzae was excellent in producing protease in the same bioreactors. A direct technique of establishing a correlation between fungal growth and CER, OUR, heat evolved was proven successful in this work. The information obtained from CER and OUR led to the estimation of respiratory quotient (RQ). RQ describes the state of the fungal population in the tray SSB and gives an indication of fungal metabolic behaviour. RQ values < 1 were obtained from 38 experiments using four tray SSB systems for the two fungi. A kinetic model based on CO2 evolution instead of biomass concentration was examined in order to simplify the required experiments for kinetic model development. A Gompertz model was used to fit the integrated CO2 data and predict the quantity of CO2 evolution in all experiments. A correlation was found between the heat evolution and CER. The performances of tray SSB systems can be improved by constructing them as multi-trays. The multi-tray systems improved the mass transfer considerably compared with single tray systems. In addition, the multi-tray systems allowed precise measurement of the gradients of CO2, enzymes, spores and fungal biomass. In addition, the air arrangements using moistened air were successful in maintaining moisture content, adequate O2 supply and control of temperature, and hence, increased the productivity of both fungi. Overall A. awamori and A. oryzae have their own ability and performance to degrade and utilise the complex compositions contained in the solid substrate and fermentation conditions may lead to possible comparisons. In addition, multi-stacked circular tray and multi-square tray SSB systems demonstrated an excellent system for further investigations of mass transfer and possibly for large scale operation, though considerable optimisation work remains to be done, for example the height/diameter ratio and total number of trays should be optimised.

Doctor of Philosophy
Department of Grain Science and Industry
Praveen V. Vadlani
The purpose of this study was to investigate micro- and macro-scale aspects of solid state fermentation (SSF) for production of cellulolytic enzymes using fungal cultures. Included in the objectives were investigation of effect of physicochemical characteristics of substrate on enzymes production at micro-scale, and design, fabrication and analysis of solid-state bioreactor at macro-scale. In the initial studies response surface optimization of SSF of soybeans hulls using mixed culture of Trichoderma reesei and Aspergillus oryzae was carried out to standardize the process. Optimum temperature, moisture and pH of 30ºC, 70% and 5 were determined following optimization. Using optimized parameters laboratory scale-up in static tray fermenter was performed that resulted in production of complete and balanced cellulolytic enzyme system. The balanced enzyme system had required 1:1 ratio of filter paper and beta-glucosidase units. This complete and balanced enzyme system was shown to be effective in the hydrolysis of wheat straw to sugars. Mild pretreatments– steam, acid and alkali were performed to vary physicochemical characteristics of soybean hulls – bed porosity, crystallinity and volumetric specific surface. Mild nature of pretreatments minimized the compositional changes of substrate. It was explicitly shown that more porous and crystalline steam pretreated soybean hulls significantly improved cellulolytic enzymes production in T. reesei culture, with no effect on xylanase. In A. oryzae and mixed culture this improvement, though, was not seen. Further studies using standard crystalline substrates and substrates with varying bed porosity confirmed that effect of physicochemical characteristics was selective with respect to fungal species and cellulolytic activity. A novel deep bed bioreactor was designed and fabricated to address scale-up issues. Bioreactor’s unique design of outer wire mesh frame with internal air distribution and a near saturation environment within cabinet resulted in enhanced heat transfer with minimum moisture loss. Enzyme production was faster and leveled within 48 h of operation compared to 96 h required in static tray. A two phase heat and mass transfer model was written that accurately predicted the experimental temperature profile. Simulations also showed that bioreactor operation was more sensitive to changes in cabinet temperature and mass flow rate of distributor air than air temperature.

Thesis submitted in fulfilment of the requirements for the degree of Doctor Technologiae: Chemical Engineering, Faculty of Engineering at the Cape Peninsula University of Technology - Cape Town, South Africa
Polycyclic aromatic hydrocarbons (PAHs) are one of the most common and recalcitrant environmental contaminants – known for their potential toxicity, mutagenicity, and carcinogenicity to humans. Biosurfactant application can enhance the biodegradation of PAHs. The main object of this work was to explore the novelty of biosurfactant produced by the isolated strains of Bacillus sp and Pseudomonas aeruginosa grown exclusively on Beta vulgaris, and the modification of the zeolites nanoparticles by the biosurfactant, for enhanced biodegradation of PAHs in soil. Novel biosurfactant-producing strains were isolated from hydrocarbon-contaminated environments, while several agrowaste were screened as primary carbon sources for the expression of biosurfactants, which were quantified using various standardized methods......

Wastewater, recycled irrigation water, and agricultural runoff can contain high levels of pathogenic bacteria, which pose a threat to human and ecosystem health. The use of a bioreactor containing mycelial mats of filamentous fungi is a novel treatment technology that incorporates physical, biological, and biochemical processes to remove bacterial pathogens from influent water. Although a relatively new concept, fungal bioreactors have demonstrated the ability to dramatically reduce fecal coliform bacteria in water, but no studies have attempted to explicitly identify the bacterial pathogen removal mechanisms exhibited by the fungi. This study evaluated several different species of fungi for use in fungal bioreactor systems and aimed to identify the modes of action responsible for the removal of bacterial pathogens. The species evaluated were Daedaleopsis confragosa, Pleurotus eryngii, and Piptoporus betulinus. Experimental results showed that all species of fungi assessed were capable of removing E. coli in a synthetic water solution. Significant concentrations of hydrogen peroxide, an antiseptic, were produced by all species of fungi evaluated. The fungal bioreactors containing P. eryngii produced the highest concentrations of hydrogen peroxide, generating a maximum concentration of 30.5 mg/l or 0.896 mM. This maximum value exceeds reported minimum concentrations required to demonstrate bacteriostatic and bactericidal effects when continually applied, providing evidence that a major bacterial removal mode of action is the production of antimicrobial compounds. In addition to its promising application to improve water quality, fungal bioreactors are a low cost and passive treatment technology. The development a hyper-functional system could be a have a substantial impact on the use of recycled irrigation water and on the water/wastewater treatment industry, for both municipal and agricultural wastewater.
Master of Science

Stale bread contributes to the biggest volume of food waste in Sweden. Current method on recovering bread waste is by producing biogas or bioethanol. Despite advantages in the energy sector, the bread which still has relatively high quality could be recovered into new products with higher value, such as food for human consumption. Development of a product, termed ‘fungal cake’ by solid state fermentation on bread waste using Neurospora intermedia in small scale petri dishes have previously been successfully conducted. This study aims to scale up the production of fungal cake into bench scale production. Two systems using different bioreactors were used in this study. The first system operated in batch mode using a tray bioreactor, in which the effect of particle size, mixing, and bread loading were evaluated. The fermentation was conducted during 5 days. Bread crumb with a larger particle size of 2 mm resulted in similar outcomes as bread crumb with a smaller particle size of 0.5 mm in terms of CO2 evolution rate, cumulative CO2 production, starch, and protein content of the final product. However, larger particle size resulted in a more homogeneous growth of the fungus throughout the product, which is preferred. The presence of daily mixing had no significant effect compared to static condition for all measured variables. Thus, mixing could be introduced to promote product homogeneity. Likewise, bread loading had no significant effect on the measured variables, which implies that a higher productivity can be achieved using a higher bread loading. The second system operated in continuous mode using a newly developed continuous tubular bioreactor with product recycle. Two experiments, in which the residence time (48h and 24h) and recycle ratio (10/65 and 20/55) were conducted. Both experiments yielded product with stable starch and protein content, indicated by a stable CO2 evolution rate over time. The performance using continuous tubular bioreactor was compared to batch fermentation in tray bioreactor using the same ratio of inoculum and both system yielded product with the same starch and protein content. Successful operation in continuous bioreactor certainly improved the productivity of fungal cake production.

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Ao contrário dos bioprocessos submersos, que são amplamente utilizados e estudados, bioprocessos em estado sólido (BES) ainda são carentes de estudos de modelagem e simulação, o que aponta para o grande potencial de otimização. A dificuldade no aperfeiçoamento de BES está associada a problemas com a dissipação do calor gerado pelas atividades metabólicas do microrganismo durante o crescimento. Esta dificuldade na transferência de calor dentro do biorreator pode levar a zonas de altas temperaturas, que afetam adversamente a produtividade. A modelagem matemática é uma ferramenta essencial para otimizar bioprocessos. Através de modelos matemáticos é possível otimizar as variáveis operacionais para controle do bioprocesso e também analisar o design do biorreator. A otimização geométrica, de acordo com a Teoria Constructal, visa melhorar o desempenho do biorreator através, por exemplo, de minimizar a temperatura no interior do leito a níveis ótimos para o cultivo. O presente trabalho apresenta projetos de biorreatores para BES, todos com geometria otimizada, obtidos a partir de experimentação numérica, através de um software de computational fluid dynamics (CFD). O modelo matemático utilizado era preditivo e significativo ao nível de confiança de 95%. A otimização geométrica foi apresentada em função das condições operacionais do cultivo. Para o biorreator de coluna e leito fixo com paredes isoladas, foram apresentadas as geometrias ótimas em função da velocidade, da vazão e da temperatura do ar de admissão. Para uma temperatura do ar de admissão de 29,5 ºC, as configurações ótimas ((D/L)opt) variaram entre 1,0 e 2,4 para uma faixa de velocidade de admissão do ar entre 0,003 e 0,006 m s-1 . Relacionando com vazão, as razões mostraram-se ótimas entre 2,2 ≤ (D/L) ≤ 2,6 quando operando sob 3,3 a 3,5 10-5 m3 s-1 . Outro biorreator estudado foi o biorreator modular, composto de módulos elementares com geometria otimizada, sendo adaptável a diferentes escalas de produção e de fácil montagem. As configurações ótimas dos módulos de geometria retangular e seção quadrada foram apresentadas para diferentes volumes de módulos, em função da temperatura e da velocidade do ar de admissão. Foi observado que o volume máximo do módulo sem resfriamento externo é 5 L, para uma velocidade do ar de admissão acima de 0,0045 m s-1 e temperatura inferior ou igual 29,0 ºC O último biorreator proposto foi o biorreator hollow, semelhante a um biorreator de coluna e leito fixo, porém com um duto oco inserido nele. O duto interno tem inúmeros furos perpendiculares às suas paredes, mas sua saída é isolada, permitindo que o ar penetre no meio poroso. A geometria otimizada do biorreator hollow foi apresentada em função da fração de volume do duto interno, da razão entre os diâmetros de entrada e saída do duto interno, da vazão e da temperatura do ar de admissão. Em comparação com o biorreator de coluna convencional de mesmas dimensões e sob mesmas condições operacionais, o biorreator hollow apresentou temperatura máxima mais baixa, demonstrando que o projeto é eficiente para resfriar o meio poroso. Concluiu-se, enfim, no presente trabalho, que a geometria é um parâmetro importante e a sua otimização pode beneficiar o desempenho do biorreator.
Unlike the submerged bioprocesses, that was wildly used and studied, solid state bioprocess (SSB) are still poorly studied with respect to modeling and simulation, what indicates a big potential for optimization. The difficulty in the BES improvement is associated to problems with the dissipation of heat generated by metabolic activities of microorganisms during growth. This difficulty in transferring heat from the bioreactor could lead to areas with high temperature, which usually affect the productivity adversely. The mathematical modeling is an essential tool for optimizing bioprocesses. Using mathematical models it is possible to optimize operational variables to control the bioprocess and also explore the design of the bioreactor. The geometric optimization, according Constructal Theory, aims to improve the performance of the bioreactor through, for example, minimizing the temperature inside the bed to optimum levels for the bioprocess. The present work presents designs of bioreactors to SSB, all with optimized geometry, obtained from numerical experiments, by computational fluid dynamics (CFD) software. The mathematical model used has been predictive and significant at .95 level of confidence. The geometric optimization was presented as function of operational conditions of the cultivation. For the column fixed bed bioreactor with isolated wall, the optimal configurations are shown as function of flow, velocity and temperature of inlet air. For a inlet air temperature of 29.5 ºC, the optimal configurations ((D/L)opt) varied between 1.0 e 2.4 to a range of inlet velocity between 0.003 e 0.006 m s-1 . Relating with the volumetric flow, the optimal ratios presented between 2.2 ≤ (D/L) ≤ 2.6 when operating under 3.3 a 3.5 10-5 m3 s-1 . Other studied bioreactor was the modular bioreactor, consisting of elementary modules with optimized geometry, being adaptable to different scales of production and easy assembly. The optimal configurations of the modules with rectangular geometry and square section were shown depending on the volume of modules and the temperature and velocity of inlet air. It was observed that the maximum volume of the module without external cooling was 5 L, for a inlet velocity upper 0.0045 m s-1 and temperature smaller or equal to 29.0 ºC. The last proposed bioreactor was the hollow bioreactor, similar to a column fixed bed bioreactor, but with an empty duct inserted on it. The internal duct has innumerable holes perpendicular to its wall (the inlet port), but its end is insulated, allowing the air penetrates into the porous medium. The optimized geometry of hollow bioreactor was presented in function of the volume fraction of internal duct, the ratio between the diameters of inlet and outlet of the internal duct, the flow rate and temperature of the inlet air. Comparing with the conventional column bioreactor with the same configuration and same operational conditions, the hollow bioreactor showed a lower maximum temperature. This demonstrates that the project is efficient at cooling the porous medium. Finally, it was concluded that the geometry is an important parameter and its optimization can benefit the performance of the bioreactor.

Aspergillus niger souche G131 est un champignon qui produit en quantité des métabolites secondaires appartenant à la famille des naphtho-gamma-pyrones (NγPs). Ces NγPs sont des pigments qui présentent des intérêts industriels de par leurs importants potentiels antiradicalaires. L’objectif de ce doctorat est la production à l’échelle pilote et en continu de NγPs à travers la culture du champignon sur milieu solide. Le choix de la fermentation en milieu solide (FMS) comme processus de culture repose sur des aspects d’ordre qualitatif et quantitatif de production, ainsi que sur des raisons économiques et éthiques, relatives à la protection de l’environnement avec notamment la possibilité de valoriser des coproduits agricoles comme milieu de culture pour le champignon. Dans un premier temps, ce travail s’intéresse à la caractérisation de la composition et des potentialités associées aux molécules produites par la souche. Ces potentialités incluent les activités anti-radicalaires et les mesures de cytotoxicité. La thèse porte également sur la caractérisation de la physiologie de croissance de la souche en FMS et sur l’optimisation des conditions de culture par la méthodologie des plans d’expériences pour l’augmentation de la production de NγPs. Une stratégie originale d’optimisation adaptée aux contraintes posées par la FMS est d’ailleurs proposée. Finalement, un transfert d’échelle de production est réalisé au moyen d’un bioréacteur prototype innovant permettant la production à l’échelle pilote de milieu fermenté en continu. Dans son dernier chapitre, ce travail s’intéresse donc à la mise au point des paramètres opératifs qui entourent la production continue de NγPs par FMS
Aspergillus niger strain G131 is a non-ochratoxigenic filamentous fungus producing high quantities of secondary metabolites known as naphtha-gamma-pyrones (NγPs). NγPs are pigments of industrial interest in reason of their high antioxidant properties. The aim of this dissertation is the continuous, pilote-scaled production of these NγPs through the cultivation of the fungus on solid medium. The choice of solid state fermentation (SSF) as cultivation method is not only driven by quantitative and qualitative considerations, but also by economical and ethical concerns related to environmental protection. SSF allows, in fact, a direct valorization of agricultural byproducts as the solid medium for the fungal growth. First, this work deals with the characterization of the composition and potentialities associated with the molecules produced by the strain, which include antioxidant and cytotoxic activities. Second, the dissertation focuses on the characterization of the fungal growth’s physiology on solid medium and on the optimization of the culture conditions using experimental methodology in order to increase NγPs production. For this purpose, an original optimization strategy is proposed to overcome specific constraints connected to SSF. Finally, a scale transfer of the production is advanced by means of an innovative prototype bioreactor continuously producing fermented material. The final chapter of this work addresses the development of parameters regarding the continuous NγPs production using SSF

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Financiadora de Estudos e Projetos
The main challenge on the conversion of lignocellulosic biomass into liquid fuels is the economic viability of this process. Thus, the commercialization of lignocellulosic ethanol is hindered mainly by the high costs of the enzyme preparations currently available cellulases - enzymes used in the saccharification step. Some strategies that can be adopted to reduce the enzymes costs include selecting microorganisms, use of cheaper raw materials and more efficient fermentation strategies such as the solid state fermentation (SSF) and efficient techniques for saccharification and fermentation. The aim this work was evaluate the use of the whole fermentation medium containing lignocellulosic biomass, fungal mycelium and enzymes in the hydrolysis of sugarcane bagasse pretreated by steam explosion for cellulosic ethanol production. In this context, a selection of filamentous fungi highly producing cellulases and hemicellulases, optimization operating conditions, such as humidity and temperature, were carried out for in house enzyme production using an instrumented bioreactor. Then, the efficiency of the whole fermentation medium and enzyme extract in enzymatic hydrolysis of lignocellulosic biomass for cellulosic ethanol production was evaluated. Among the 40 fungal strains evaluated, two strains of A. oryzae (P6B2 and P27C3A) stood out. In addition, one strain of A. niger 3T5B8 and another of T. reesei RUT C30 were also evaluated in this study. The influence of the substrate initial moisture content and temperature on efficiency of cellulase and xylanase production by strains of A. oryzae, A. niger and T. reesei grown in SSF under conditions of forced aeration and static were evaluated. The initial moisture content of the substrate did not affect the production of cellulases and xylanases by strain of A. oryzae P27C3A, however higher moisture was better for enzyme production by strains of A. oryzae P6B2 and A. niger and lower moisture were better for the production of cellulases and xylanases by T. reesei in both cultive systems. Temperature 28°C was best for xylanase production by all the fungal strains, while higher temperatures was better cellulases production in both culture systems. The use of whole fermented medium of A. niger or T. reesei obtained in the bioreactor were better in the hydrolysis sugarcane bagasse pretreated by steam explosion (BPSE) than the enzymatic extract with a final conversion of 41.3 and 24.9% of theoretical, respectively. The combination of whole fermentation medium of strains of A. oryzae (P6B2 or P27C3A) obtained in flasks and ½ commercial enzyme hydrolysis also were efficient on BPSE hydrolysis (26.1 and 42.4% of theoretical, respectively). Nevertheless, the combination of whole fermented medium of A. oryzae P6B2 and enzymatic extract of A. niger obtained in flasks promoted a conversion of 65% and an ethanol yield of 84% of the theoretical value. As overall conclusion it was found that the use of whole fermented medium produced by fungi cultivated under solid state fermentation (SSF) in the BPSE hydrolysis resulted in similar or higher yields compared to the hydrolysis using the enzyme extract, giving clear indication that the extraction/filtration step of the enzyme can be eliminated. The use of the enzyme complex of A. oryzae P6B2 in combination with the enzymes of A. niger resulted in a BPSE hydrolysis more efficient when compared with other combinations, showing the importance of selecting microorganisms for high enzymes production. Moreover, the use of a single reactor system for performing enzyme production steps by SSF, saccharification and alcoholic fermentation may be performed, avoiding the need for steps separation.
A discussão dominante sobre a transformação da biomassa lignocelulósica a combustível líquido é a sua viabilidade econômica. Assim, a comercialização do etanol a partir de biomassa lignocelulósica é dificultada principalmente pelos custos proibitivos das preparações de celulases enzimas usadas na sacarificação. Algumas estratégias que podem ser adotadas para a redução do custo das enzimas utilizadas na degradação da biomassa incluem a seleção de micro-organismos altamente produtores de celulases e hemicelulases, utilização de matéria-prima mais barata e estratégias de fermentação a um custo efetivo - como a fermentação em estado sólido (FES) e técnicas mais eficientes de sacarificação e fermentação alcoólica. O objetivo deste trabalho foi avaliar a utilização do meio fermentado integral (MFI), contendo biomassa lignocelulósica, micélio fúngico e enzimas na hidrólise do bagaço de cana pré-tratado por explosão a vapor para produção de etanol celulósico. Neste contexto, realizou-se a seleção de fungos filamentosos isolados do solo de madeira em decomposição da Região Amazônica produtores de celulases e hemicelulases, otimizou-se as condições operacionais, como umidade e temperatura para a produção de enzimas in house utilizando biorreator de coluna instrumentado e por fim, avaliou-se a eficiência do MFI e (EE) na hidrólise enzimática da biomassa lignocelulósica para produção de etanol celulósico. Entre os 40 fungos caracterizados quanto à produção de enzimas envolvidas na degradação da lignocelulose, duas linhagens de A. oryzae (P6B2 e P27C3A) se destacaram em relação às demais. Além das linhagens de A. oryzae outras duas linhagens de fungos, uma de A. niger 3T5B8 e outra de T. reesei RUT C30 foram avaliadas neste trabalho, a fim de verificar a eficiência das linhagens isoladas do solo da Floresta Amazônica. A umidade inicial do substrato não influenciou na produção de celulases e xilanases pela linhagem de A. oryzae P27C3A, no entanto umidades elevadas foram melhores para a produção de enzimas pelas linhagens de A. oryzae P6B2 e A. niger e umidades baixas foram melhores para a produção de celulases e xilanases por T. reesei em ambos os sistemas de cultivo, forçado e estático. Com relação à temperatura de fermentação, 28ºC foi melhor para a produção de xilanases por todas as linhagens fúngicas e temperaturas mais elevadas favoreceram a produção de celulases pelos fungos. A utilização do MFI de A. niger ou T. reesei obtido em biorreator de coluna instrumentado foram melhores na hidrólise do bagaço de cana pré-tratado por explosão a vapor (BEX) do que o EE, com uma conversão final de 41,3 e 24,9% do valor teórico, respectivamente. A combinação de MFI das linhagens de A. oryzae (P6B2 ou P27C3A) obtida em Erlenmeyer e ½ de enzima comercial também favoreceram a hidrólise do BEX (26,1 e 42,4% do valor teórico, respectivamente). No entanto, a combinação de MFI de A. oryzae P6B2 e EE de A. niger obtido em Erlenmeyer promoveram uma conversão final de 65% e um rendimento de etanol de 84% do valor teórico. Vale salientar que foi utilizado na fermentação alcoólica o meio hidrolisado na íntegra, contendo açúcares, enzimas, biomassa lignocelulósica e micélio fúngico. Como conclusões gerais, constatou-se que a utilização de MFI produzido pelos fungos por FES na hidrólise do BEX resultou em rendimentos semelhantes ou mais elevados quando comparado com a hidrólise do BEX utilizando EE, dando a clara indicação de que o passo de extração/filtração das enzimas pode ser eliminado; a utilização do complexo enzimático de A. oryzae P6B2 em combinação com o complexo enzimático de A. niger resultou em uma hidrólise mais eficiente do BEX quando comparado com outras combinações, mostrando a importância da seleção de micro-organismos produtores de enzimas envolvidas na degradação da lignocelulose, para que a produção de etanol celulósico possa se tornar economicamente viável; e por fim, a utilização de um único sistema de reator para a realização das etapas de produção de enzimas por FES, sacarificação e fermentação alcoólica pode ser realizada, evitando-se a necessidade de etapas de filtração.

Suite aux désordres écologiques engendrés par l’utilisation des pesticides chimiques, le marché des biopesticides est en plein essor, même s’il ne représente encore que 2 % du marché des produits phytosanitaires. Le contrôle biologique des ravageurs des cultures représente une solution alternative fiable pour lutter contre les nuisances. Parmi les champignons filamenteux (Beauveria bassiana, Trichoderma harzianum) il existe des souches homologuées pour leurs utilisations dans la lutte biologique. Parmi celles-ci, on trouve les souches de Trichoderma peuvent produire la 6 Pentyl-alpha-Pyrone (6-PP), métabolite secondaire antifongique volatile à forte odeur de noix de coco. Ces molécules bioactives, ainsi que les enzymes et les conidies de Trichoderma présentent un potentiel antifongique intéressant susceptible d’être utilisées pour lutter contre des ravageurs et donc de limiter l'apport de pesticides chimiques. Pour l'ensemble de ces propriétés nous avons étudié la croissance et le métabolisme de 23 souches de Trichoderma utilisées comme agents de contrôle biologique contre d'autres champignons phytopathogènes (Botrytis cinerea). Pour la culture de ces souches nous avons utilisé la Fermentation en Milieu Solide (FMS) sur différents mélanges de coproduits agro-industriels (bagasse de canne à sucre ou sarments de vigne, son de blé ou purée de pommes de terre, moringa ou jatropha, chitine ou huile d’olive grignons d'olive ou grignon d’argan). L’objectif recherché est l'obtention d'un produit fermenté contenant un cocktail d'enzymes lytiques, une concentration élevée en 6-PP et une concentration très élevée de conidies viables et virulentes
Biopesticides are placed as a viable alternative to control pests and as a possible substitute for the traditional chemical that causing severe damages to human health, to ecology by non-target organisms eliminated and the creation of pests resistant to pesticides. Trichoderma is a filamentous fungus considered as biocontrol agent (BCA). During their development, Trichoderma produce biomass, lytic enzymes, conidia and secondary metabolites like 6-pentyl-alpha-pyrone (6-PP), a fungicidal compound. The aim of this thesis is the production of these 6-PP, lytic enzymes (cellulases, lipases, amylases) and conidia through the cultivation of the fungus on solid medium. The choice of solid state fermentation (SSF) as cultivation method is not only driven by quantitative and qualitative considerations, but also by economical aspect. In fact, SSF allows the valorization of agroindustrial wastes having an impact on the worldwide ecology. First, this work deals the screening and identification based on internal transcribed spacer (ITS) sequence analysis of Trichoderma strains derived from different natural sources on solid media for 6-PP and conidia production. The second part of this work focuses on the optimization of culture condition using experimental methodology in order to increase enzymes (cellulases, lipases, amylases) conidia and 6-PP production. For this purpose, an original optimization strategy is proposed to overcome specific valorization of wastes generated by agroindustrial. Finally, a scale transfer of the production is advanced by means of an innovative prototype single used bioreactor producing fermented material

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Sugarcane bagasse is an abundant lignocellulosic residue in traditional regions of sugar and ethanol production in Brazil. It is not only a potential substrate for second generation ethanol production but also have structural features to be classified as good inducer for cellulases production by microorganisms. However, the high cost of cellulases industrial production is the major bottleneck in the hydrolysis of this raw material for subsequent fermentation, which makes unfeasible in large scale the ethanol production using this process. In this context, the development of more efficient and less expensive fermentation processes for industrial cellulases production, as well as better alternatives of enzymatic hydrolysis of lignocellulosic material is crucial to achieve economic feasibility in this process. For this purpose, this work aims to develop a cellulase production process using Trichoderma reesei, as well as assessing the use of produced enzymatic extract in sugarcane bagasse hydrolysis, in order to evaluate the ultrasound effects in the hydrolysis process. The optimized process of cellulases production consisted in five days of grow in pre-inoculum Petri dishes, followed by two days of grow in optimized liquid medium and four days of solid state fermentation, using sugarcane bagasse supplemented with 1% of soybean bran and 15% (v/w) of corn steep liquor as substrate, moisture of 65%, 28±1°C and 0.5 mL of inoculum per gram of substrate. This experimental condition in bench scale (5 g) resulted in a production of 1.4 FPU/g of cellulases, and the production was approximately three-fold high in a fixed-bed bioreactor with forced aeration for 70 g of substrate capacity. For ultrasound assisted enzymatic hydrolysis using an ultrasound bath, the condition that achieved higher efficiencies were 43.4±2°C and 18.5% (v/v) of enzyme concentration, resulting in a maximal hydrolysis efficiency of 229 grams of reducing sugar per kilogram of used substrate, achieving an average increase of 12% in efficiency in those experiments where the hydrolysis was assisted by ultrasound compared with those without sonication. Regarding the saccharification using the ultrasonic probe, results using the indirect sonication during process were, on average, 158% higher than those using the direct sonication. Thus, it can be concluded that indirect sonication is more suitable to be used as an auxiliary in the hydrolysis, since the direct sonication can cause denaturation of the enzyme, reducing the process efficiency.
O bagaço de cana-de-açúcar (BC) é um resíduo lignocelulósico abundante em regiões sucroalcooleiras no Brasil e é um potencial substrato para produção de etanol de segunda geração, além de possuir características estruturais que o classificam como bom indutor para produção de celulases por microrganismos. O alto custo da produção industrial de celulases, no entanto, é um grande empecilho na hidrólise desse tipo de material para posterior fermentação, o que inviabiliza a utilização desse processo na produção de etanol em larga escala. Nesse contexto, o desenvolvimento de processos de fermentação mais eficientes e de menor custo para a produção de celulases em escala industrial, bem como alternativas mais eficazes de hidrólise enzimática desse material são necessários a fim de viabilizar economicamente o processo. Para essa finalidade, esse trabalho tem como proposta o desenvolvimento de um processo para produção de celulases utilizando uma cepa do fungo filamentoso Trichoderma reesei, bem como a utilização do extrato enzimático produzido na hidrólise enzimática de bagaço a fim de avaliar os efeitos do ultrassom no processo. O processo otimizado de produção das celulases consistiu em cinco dias de crescimento do pré-inóculo em placas de Petri, seguido de dois dias de crescimento em meio líquido otimizado, e quatro dias de FES de BC suplementado com 1% de farelo de soja (FS) e 15% de água de maceração de milho (AMM), 65% de umidade, 28±1°C e densidade de 0,5 mililitros de inóculo por grama de substrato. Essa condição experimental em escala de bancada (5 g) resultou em uma produção de 1,4 FPU/g, valor esse que aumentou aproximadamente três vezes com o aumento de escala de produção em um biorreator de leito fixo com aeração forçada com capacidade para 70 g de substrato. Para hidrólise enzimática assistida por banho de ultrassom, a condição que atingiu melhores eficiências foi de 43,4±2°C e 18,5% (v/v) de concentração de enzima, atingindo um máximo de 229 gramas de açúcares redutores por quilograma de substrato utilizado, e foi observado um aumento médio de 12% na eficiência de hidrólise naqueles experimentos em que a hidrólise foi assistida por ultrassom. Já nas sacarificações utilizando a sonda ultrassônica, os resultados utilizando sonicação indireta durante a sacarificação foram, em média, 158% maiores que aqueles utilizando sonicação direta. Dessa forma, conclui-se que a utilização de sonicação indireta é mais indicada como auxiliar nas hidrólises, uma vez que a sonicação direta pode causar desnaturação da enzima e diminuir a eficiência do processo.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Esta dissertação apresenta um estudo sobre a ampliação de escala de biorreatores de leito empacotado para produção de celulases e hemicelulases por fermentação em estado sólido (FES) empregando o fungo termofílico Myceliophthora thermophila I-1D3b e substrato composto por bagaço de cana de açúcar e farelo de trigo. As etapas de desenvolvimento do projeto consistiram em ensaios em biorreator de diâmetro interno e altura iguais a 7,62 e 10 centímetros, de modo que foram realizados ensaios variando tamanho das partículas de bagaço de cana, densidade bulk do leito, taxas de aeração e modo de distribuição da vazão de ar. Com base nos resultados obtidos, observou-se que partículas de bagaço de tamanho reduzido fornecem bons rendimentos de atividades enzimáticas quando submetidas a altas taxas de aeração em leitos de elevada densidade. Partículas maiores forneceram melhores resultados para leitos de �bulk= 0,395 g/cm3 , bem como a utilização de vazão de ar determinada com base no número de Damköhler modificado igual a 1. Resultados obtidos em ensaios em biorreatores de 0,8 metros, com e sem distribuição de ar, mostraram que inserir ar ao longo do leito é uma estratégia que permite aeração eficiente ao longo do sistema fermentativo, sem que ocorra secagem excessiva do substrato. Atividades relativas de CMCase e xilanase atingiram cerca de 1,2 e 0,7, respectivamente. As etapas finais consistiram em fermentações realizadas em biorreator de diâmetro interno e altura iguais a 20 centímetros. Nessas condições, foram testadas duas formas de distribuição de ar ao longo do leito de quatro módulos fermentativos: utilizando um tubo interno perfurado no módulo central inserindo metade da vazão total, e a outra metade inserida pelo inferior do leito e utilizando um tubo interno central, com quatro orifícios por módulo para espalhamento da vazão. Em termos de produção de celulase, o tubo no módulo central se mostrou a melhor alternativa para o aumento de escala, chegando a apresentar 0,8 de atividades enzimáticas relativas de CMCase, e em termos de atividades relativas de xilanase, a melhor opção foi o tubo interno longitudinal, apresentando valores de 0,55. Diante do exposto, o número de Damköhler modificado é interessante do ponto de vista de aumento de escala, no entanto é preciso ainda entender o mecanismo de atividade respiratória dos microrganismos para que os conceitos possam ser aplicados em sinergia, podendo-se assim esperar bom desempenho do processo em escala industrial.
This work presents a study of scale–up packed bed bioreactors for the production of cellulases and hemicellulases by solid-state fermentation (SSF) using the thermophilic fungus Myceliophthora thermophila I-1D3b and substrate composed by sugar cane bagasse and wheat bran. The development phases of the project consist in tests using bioreactors composed by internal diameter and height equal to 7.62 and 10 centimeters, respectively, so that the assays vary the size of the sugar cane bagasse particles, the bulk density, the airflow rate and how’s distributed along the bed. Based on the results obtained, it was defined that the particles of small size provide good results of enzymatic activity yields when submitted to high aeration rates in high bulk density beds. When testing normal size sugarcane bagasse particles, the best results were obtained for the beds with �bulk= 0,395 g/cm3 as well as airflow rate based on the modified Damköhler number equal to 1. Results obtained in tests in 0,8 meter height bioreactors, with and without airflow distribution, show that inserting air in different bed heights is a strategy to ensure efficient aeration throughout the fermentation system without excessive drying of the substrate. Activities related to CMCase and xylanase reached about 1.2 and 0.7, respectively. The final stages consisted in perform fermentation assays in a bioreactor of internal diameter and height equal to 20 centimeters. Under these conditions, two forms of long-term distribution were tested: using an internal ring to insert half of the total flow, together with the other half inserted in the lower part of the bed and using a central internal pipe with 4 holes per module for airflow distribution. In terms of cellulase production, the internal ring showed a better alternative for scaling-up, reaching a valeu of 0.8 CMCase relative enzymatic activities, and with respect to xylanase relative enzymatic activities, the best option was the central pipe, presenting values of 0,55. On the above, the modified Damköhler number is interesting when it refers to scale-up, although it’s necessary to understand the mechanism of respiratory activity of the microorganisms so that the concepts can be applied in synergy, and it can be expected good process performance in industrial scale.
CNPq: 132957/2016-7

Este trabalho teve por objetivo verificar a influência da agitação no cultivo em meio sólido, FES, quanto a crescimento microbiano, homogeneização do meio e remoção de calor. As correlações obtidas contribuem na definição de critérios para ampliação de escala da FES. O modelo adotado foi o cultivo do fungo Monascus sp. em arroz. Os experimentos foram conduzidos em reator tubular horizontal de 40 l, com agitação interna intermitente, camisa de resfriamento e vazão de ar de 2 l.min-1.Kgms-1. Os cultivos foram realizados com base num planejamento fatorial rotacional: 12 a 60 rotações das pás em 24 horas; 2 a 12 horas de intervalo entre os eventos de agitação. A intensidade do crescimento celular foi considerada com base no consumo de O2, produção de CO2 , concentrações de proteína e ergosterol. O consumo de O2 apresenta correlação de 81% com os padrões de agitação sendo que tanto o número de rotações quanto o intervalo entre os eventos de agitação influenciam negativamente o crescimento celular assim estimado. Por outro lado, a máxima velocidade de consumo de oxigênio, OUR, obtida por volta de 24 horas, em cultivos com menores intervalos entre os eventos de agitação, indica efeito positivo da agitação sobre a velocidade do crescimento de fungos em superfície, enquanto não ocorre compactação do meio de cultivo. Conclui-se, portanto, que a natureza do substrato empregado, arroz, cuja reologia é sensivelmente alterada pela agitação, contribuiu de modo deletério à respiração celular e que a adoção de reatores com agitação na FES, requer substrato com baixo teor de amido e elevado teor de fibras. As medidas de ergosterol apresentaram correlação de 85% com os padrões de agitação mostrando que o intervalo entre os eventos de agitação é o fator com maior impacto nesta resposta e os ensaios com maiores intervalos entre os eventos de agitação e maior número de voltas apresentaram concentrações aproximadamente dez vezes maiores de ergosterol em relação aos outros ensaios. Os coeficientes de variação de umidade em cinco pontos do reator representam a homogeneidade, pois relacionam-se com os padrões de agitação com correlação de 95%.
This investigation aimed to verify the influence of mixing microbial growth, medium homogenization and heat removal within a solid state fermentation (SSF) bioreactor. The correlations obtained will help to establish the scale-up criteria. The model system involved the cultivation of the fungi Monascus sp. on rice. The assays were performed in a 40 l bioreactor under internal intermittent mixing with a cooling jacket and an air flux of 2 l.min-1 kgdm-1. The cultivations followed a rotational factorial plan: 12 to 60 paddle revolutions in 24 hours; with an interval of 2 to 12 hours between mixing events. Cellular growth rate was estimated by O2 consumption, CO2 production, and protein and ergosterol concentrations. The O2 consumption showed an 81% correlation with the revolutions pattern, and both the number of revolutions and interval between mixing events, influenced cell growth negatively. The maximal oxygen consumption rate (OUR) was reached after about 24 hours in cultivations submitted to shorter intervals between mixing events which indicates a positive effect of shaking on the fungal growth rate on the particle surface, as long as no medium compaction occurs. Thus it was concluded that the kind of used substrate (rice), whose reology was perceptively modified by the mixing process, acted harmfully on microbial respiration. If mixing is to be used in SSF bioreactors, the substrate used should have a low starch content and a high fiber content Ergosterol content showed an 85% positive correlation with the revolution pattern, indicating that the interval between mixing events is the most important factor. Assays performed with longer intervals between mixing events and greater numbers of turns achieved about 10 times higher ergosterol concentration than the others. The coefficient of variation of the moisture at five sites of the reactor represents the homogeneity, since they are related to the revolution patterns by 95%.

Orientador: Ranulfo Monte Alegre
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Este trabalho se propôs a estudar a produção da iturina A por Bacillus subtilis em fermentação semi-sólida em biorreatores de leito empacotado. O trabalho foi desenvolvido em quatro partes. Em uma primeira parte foi feito um screening com cepas silvestres e seus mutantes obtidos a partir da exposição de luz UV e acridina laranja. A cepa Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi a que apresentou maior atividade antagônica contra os fungos Aspergillus fumigatus Fresenius NRRL 164, Aspergillus fumigatus Fresenius NRRL 166 e Aspergillus flavus var. oryzae NRRL 484. O extrato metanólico obtido da fermentação semi-sólida do Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi analisado através da espectrometria de massas encontrando-se lipopeptídeos com massa molecular entre m/z 1021,43 e m/z 1087,48, mas sem a presença da iturina A. Em uma segunda etapa a cepa Bacillus Iso 1 foi isolada a partir das raízes de soja, e ante a dificuldade de identificar a iturina A através da cromatografia liquida de alta eficiência (HPLC), foi desenvolvida a metodologia de purificação da iturina A utilizando a cromatografia em coluna de vidro preenchida com sílica gel 60. A iturina A foi eluída com três sistemas de solventes compostos por 20 mL de clorofórmio-metanol-água (65:25:4,v/v/v), fração 1, seguido de 20 mL de clorofórmio-metanol-água (30:50:10, v/v/v), fração 2, e a fração final composta por 10 mL de clorofórmio-metanol-água (20:60:15, v/v/v). As frações obtidas foram analisadas através da HPLC e da espectrometria de massa, identificando 5 isômeros da iturina A (C13-C16). Na terceira etapa, foi feito um delineamento composto central rotacional (DCCR) para avaliar o efeito da casca de arroz como suporte inerte e da vazão volumétrica de ar na produção de iturina A; como substratos foram utilizados o farelo de soja desengordurado e o farelo de trigo. Nenhuma variável do DCCR foi estatisticamente significativa, mas operacionalmente foram importantes, devido à redução da oxigenação do Bacillus Iso 1 pela baixa vazão de ar e menor concentração de casca de arroz, favorecendo a produção de iturina; nestas condições obteve-se 6,88 g/kg de substrato seco de iturina A.Esta é a maior quantidade de iturina A produzida em biorreatores de leito empacotado (coluna) com aeração forçada até hoje. Na quarta etapa, a partir dos resultados obtidos no DCCR foram estudados os parâmetros do processo: queda de pressão, consumo de oxigênio e perfis de temperatura, visando entender o comportamento da fermentação a 0,4 L/min e 0,8 L/min. A máxima produção de iturina obtida foi 5,58 g/kg de substrato seco com a vazão de 0,4 L/min. O incremento na queda de pressão é ocasionado não unicamente pelo incremento da vazão volumétrica, mas também pela produção do biopolímero ?-PGA o qual ocupa os espaços livres entre as partículas, dificultando o fluxo normal de ar através do leito, reduzindo o consumo de oxigênio. A baixa oxigenação favoreceu a alta produção da iturina A e gerou baixo calor metabólico (5,75 W/kg-dry substrato·min). Os resultados obtidos podem ser úteis na elaboração de estratégias para ampliação de escala do processo em fermentadores aerados de leito empacotado
Abstract: This work covers a study of the production of iturin A by Bacillus by solid-state fermentation in packed bed bioreactors. The study was conducted in four parts. At first a screening was conducted with wild strains and their mutants obtained from exposure to UV light and mutagenic agent acridine orange. The strain Bacillus subtilis subsp subtilis NRRL NRS 1270 showed the highest antagonistic activity against Aspergillus fumigatus NRRL 164, Aspergillus fumigatus NRRL 166 and Aspergillus flavus var . oryzae NRRL 484. A methanolic extract obtained by solid state fermentation of Bacillus subtilis subsp subtilis NRRL NRS 1270 was analyzed with mass spectrometry showing lipopeptides with molecular mass between m/z 1021.43 and m/z 1087.48, but without the presence of iturin A. In the second stage, the strain Bacillus Iso 1 was isolated from soybean roots. Given the difficulty of identifying iturin A by high performance liquid chromatography (HPLC), a iturin A purification methodology was developed using glass column chromatography packed with activated Silica gel 60 and alumina. This methodology involved three solvent systems for elution of the iturin A from the column. A first fraction consisted of 20 ml of chloroform-methanol-water (65:25:4 , v/v/v) and was followed by 20 ml of chloroform - methanol- water (30:50 : 10, v/v/v), that was then followed by a final fraction consisting of 10 ml of chloroform-methanol-water (20:60:15, v/v/v). The fractions obtained of fermentation were analyzed by both HPLC and mass spectrometry, identifying five iturin A isomers (C13-C16). In the third stage of the study, an experimental design was constructed in the form of a central composite rotational design (CCRD) to evaluate the effect of rice husk as an inert support and air flow rates to the iturin A production, using defatted soybean meal and wheat bran as substrate. Although none of the studied variables showed statistical significance, the operational importance of reduction of oxygenation of the Bacillus Iso 1 fermentation due to the low concentration of rice husk and air flow rate was observed to favor the production of iturin; in these conditions high productivity was obtained reaching 6.88 g/kg-dry substrate of iturin A. Concluding from available literature, this is the highest concentration of iturin A ever produced in packed bed bioreactor (column) with forced aeration to date. In the fourth stage, in order to understand the behavior of the fermentation under aeration conditions between 0.4 L/min and 0.8 L/min, the following process parameters were studied, based on the results obtained from the CCRD: pressure drop, oxygen consumption and temperature profiles. The maximum production of iturin obtained was 5.58 g/kg-dry substrate with the air flow rate at 0.4 L/. The increase of the pressure gradients is caused not only by increasing the volumetric air flow rate but also by the production of biopolymer ?-PGA by Bacillus iso 1, which occupies the free interparticle space, hindering or preventing the normal flow of air through the bed and thus leading to reduced oxygen consumption. The low oxygenation favored the high iturin A production and resulted in low metabolic heat generation (5.75 W/kg-dry substrate.min). The results of this work are expected to be conducive for designing strategies to scale up the process in aerated packed bed bioreactors
Doutorado
Engenharia de Alimentos
Doutor em Engenharia de Alimentos

Orientador: João Cláudio Thoméo
Resumo: Este trabalho tem como objetivo produzir esporos do fungo Metarhizium anisopliae IBCB 425 em biorreator de leito empacotado visando o aumento da escala, propondo ainda alternativas para a extração dos esporos do meio de cultivo. Também se propõe compreender a preferência do fungo pelo arroz como substrato, procurando-se alternativas para redução dos custos de produção relativos a este substrato. Para isso, realizou-se o cultivo e o reuso do substrato em embalagens plásticas, onde se observou que o cultivo em arroz tipo 1 ainda é o melhor substrato para o fungo, quando comparado com os utilizados neste trabalho sendo, quirera de arroz e farelo de arroz, porém para o aumento de escala e reuso do substrato, a melhor alternativa é a mistura de arroz com bagaço de cana-de-açúcar na proporção de 9:1. Também se realizou ensaios em biorreatores de leito empacotado de 7.62 cm e 20 cm de diâmetro, utilizando a mistura de arroz e bagaço como substrato, e observou-se que, para ambas as configurações, o bagaço evitou a compactação do leito, não se observando temperaturas elevadas que interferissem no desenvolvimento do microrganismo. Com as análises de degradação do amido e da atividade das enzimas amilase total, alfa – amilase e protease, foi possível concluir que o fungo se desenvolve de maneira distinta a cada uso dos grãos e que, o maior consumo de amido ocorre no primeiro cultivo (R1), reduzindo-o em cerca de 30%. Análises de microscopia óptica foram realizadas nos grãos cultivados, ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This work aimed to produce spores of the entomopathogenic fungus Metarhizium anisopliae IBCB 425 in packed-bed bioreactors targeting the scale-up, and also to propose alternatives to the extraction of the spores from the cultivation medium. The understanding of the preference of the fungus for the long rice as substrate was also verified in order to reduce the production costs associated to the rice. Therefore, the rice was cultivated in plastic packages and it was noticed that rice is still the best substrate for the fungal growth, even though for the scale-up and reutilization of the substrate it was necessary to mix it with sugarcane bagasse at a proportion 9:1 (rice:bagasse). Experiments in packed-beds of 7.62 and 20 cm internal diameter have been carried out using the mixture rice:bagasse and for both sizes no compaction was noted and no excessive temperature took place. From starch analysis and from the activities of total amylase, alpha-amylase and protease it was noticed that the microbe develops distinctly in each consecutive cultivation of rice, and that after the first cultivation (R1), the starch content was reduced by 30%. Optical microscopy of the cultivated grains revealed that during R1 the fungal hyphaes penetrated the grains, but during the second (r2) and third (R3) cultivations the fungal growth is restricted to the grain surface. It was observed a substrate mass reduction in the packed-bed experiments of 24%, 20% and 17% after R1, R2 and R3, respectively.... (Complete abstract click electronic access below)
Doutor

Os processos de fermentação em estado sólido (FES) existem há muitos séculos nas civilizações orientais, onde têm sido amplamente utilizados na produção de gêneros alimentícios. No ocidente, a indústria tem trabalhado preferencialmente com os processos de Fermentação Submersa (FS) porque, devido ao meio ser aquoso, existem facilidades para se controlar esse tipo de processo. No entanto, novas demandas (tais como o tratamento de resíduos sólidos) não são inteiramente contempladas pela FS. Por outro lado, os processos de FES podem ser descritos como o crescimento de microorganismos em substratos sólidos na ausência de água livre, podendo suprir essas demandas. Entretanto, também devido a essa característica, a maior dificuldade encontrada é o controle das variáveis internas do biorreator (como, por exemplo, a remoção do calor produzido pela atividade biológica). As pesquisas nesse campo mostram que essa remoção é mais fácil através das trocas pelo ar, por causa das dificuldades de condução térmica em meio sólido. Portanto, torna-se necessário o desenvolvimento de sistemas de controle da aeração que permitam a avaliação dos processos em escala de bancada, diminuindo assim o número de incertezas na modelagem e simulação do processo. Com melhores modelos do processo em escala de bancada, torna-se mais fácil o controle da temperatura no leito de um biorreator de maior escala. Esse trabalho tem por objetivo aplicar uma técnica de controle robusto que seja capaz de garantir os índices de desempenho do sistema em toda a faixa operacional do fluxo e da temperatura ar do biorreator. A planta do sistema foi modelada em nove diferentes condições de temperatura e aeração através de modelos de primeira ordem sem atraso. Esses índices são: tempo de acomodação inferior a 12000 segundo e sobressinal inferior a 10%. O controlador utilizado foi do tipo Proporcional Integrativo (PI). Esse controlador foi sintonizado utilizando a metodologia LMI (do inglês Linear Matrix Inequalities) ou Desigualdades Matriciais Lineares, através das restrições elaboradas no algoritmo iterativo V-K. Os resultados da implementação mostram que as restrições utilizadas no algoritmo são capazes de sintonizar o controlador, mesmo não se conhecendo todas as dinâmicas do sistema de aeração.
The solid-state fermentation (SSF) processes have existed for centuries in Eastern civilizations and have been widely used in the production of foodstuffs. In Western, the industry has worked preferably with the submerged fermentation (SF) processes, because it occurs in aqueous medium and it facilitates the bioreactor control. However, new demands, such as solid waste management, are not fully covered by FS. On the other hand, the processes of FES can be described as the growth of microorganisms on solid substrates in the absence of free water, which can meet this demand. But because of this characteristic, the greater difficulty is the bioreactors internal variables control and the major one the removal of the heat produced by biological activity. Researches in this field show that removal is easier through air exchange, because of the difficulties of thermal conduction in a solid medium. Therefore, it becomes necessary to develop an aeration control system that allows processes evaluation in bench scale, thereby reducing the number of uncertainties in modeling and simulation process. Thus, facilitating the temperature control of a larger-scale bioreactors bed. The aim of this work is to apply a robust control technique that guarantees the systems performance indexes throughout the air flow and temperature operational range. The plant was modeled on a first-order system without delay, at nine different conditions of temperature and aeration. These indixes are: settling time less than 12000 seconds and overshoot less than 10%. The controller used was a Proportional Integrative (PI) type. This controller was tuned using the LMI methodology (Linear Matrix Inequalities) through the V-K iterative algorithm restrictions. The implementation results show that the restrictions used in the algorithm are able to tune the controller, even not knowing all the dynamics of the aeration system.

碩士
東海大學
化學工程與材料工程學系
107
Hericium erinaceus has been historically as edible and medicinal mushroom. It contains a variety of physiologically active ingredients in which erinacines and hericenones have been demonstrated to posses the function of protection and repairs of nerve cells. In this study, different solid-state fermentation bioreactor that explore the mycelium growth and the metabolites formatiom of Hericium erinaceus. 　　In the first part of this research is using different additives as an inducer to enhance the production of erinacines A, which included: tomato juice and carrot juice, the results show that although tomato juice and carrot juice have no positive effect on the concentration of erinacines A, tomato juice and carrot juice can slow down the degradation of erinacines A. In the respect of antioxidant capacity, when the carrot juice is added at the concentration of 100% was proved to achieve the highest level of reducing power (0.28 mg of Vitamin C/g D.W.), which was 1.13 times more than control. Secondly, different air contact surface area on the mycelium growth and the metabolites formatiom of Hericium erinaceus, the results show that the concentration of erinacines A of container which has laregest air contact surface area was 1.06 times more than smallest container and the protein of container which has laregest air contact surface area was 2.3 times more than basal media. The third part is the design of the new multi-layer solid state fermentation bioreactor and explore the different ventilation volumes for the mycelium growth and the metabolites formatiom of Hericium erinaceus, the results show that uneven growth of the mycelium, but on the whole, the multi-layer solid state fermentation bioreactor can effectively shorten the culture time and was reduced from 30 days to21 days, and the concentration of erinacines A which growth with forced ventilation was 1.13 times more than natural ventilation. Aiming at the problem of uneven growth, the improved multi-layer solid state fermentation bioreactor have achieved the goal of uniform growth for each layer. In this study, the new type of forced ventilation multi-layer solid state fermentation bioreactor can enhance the production of solid state fermentation and facilitate future scale up.

The objective of this thesis is to evaluate the engineering properties of Municipal Solid Waste (MSW) that are necessary in the design of landfills. The engineering properties of MSW such as compressibility, shear strength, stiffness and hydraulic conductivity are crucial in design and construction of landfills. The variation of the engineering properties with time, age and degradation are of paramount importance in the field of landfill engineering. There is a need to address the role of the engineering properties in landfill engineering as it is not apparent how the engineering characteristics vary with time. The thesis presents the results of study of the engineering properties of MSW comprehensively and develops experimental data for design of MSW landfills. The work includes the study of the index properties and the engineering properties of MSW such as compressibility, shear strength, shear modulus and damping ratio and a detailed experimental study of the bioreactor landfill. The components of settlements, variation of shear strength with respect to unit weight and particle size are determined experimentally and analyzed. The dynamic properties such as shear modulus and material damping ratio and its variation with parameters such as unit weight, load, amplitude, degradation and moisture content are studied and analyzed. The normalized shear modulus reduction curve which is used in the seismic analysis of the landfills is developed for MSW based on the experimental results and previous studies. A pilot-scale bioreactor was setup in the laboratory for long term monitoring of the settlement, temperature variation and gas production simultaneously. The parameters of interest viz, pH, BOD, COD, conductivity, alkalinity, methane and carbon-di-oxide were determined. The generated data can be effectively used in the engineered design of landfills. For a better understanding, the present thesis is divided into the following eight chapter Chapter 1 provides a general introduction to the thesis with respect to the importance of engineering properties of MSW and presents the organization of the thesis. Chapter 2 presents a detailed review of literature pertaining to the basic, index and the engineering properties of MSW namely compressibility, shear strength, shear modulus and damping ratio, bioreactor landfill and also the scope of the study. Chapter 3 includes the materials and methods followed in the thesis. Chapter 4 presents the evaluation of compressibility characteristics of MSW including the components of settlement and the settlement model parameters. Chapter 5 presents the determination of the shear strength properties of MSW using direct shear tests and triaxial tests. The variation of the strength with respect to unit weight and the particle size is examined. The results are examined in terms of strength ratio and stiffness ratio and the implications are discussed. Chapter 6 presents the study of the dynamic characters of MSW. The variation of the shear modulus and damping ratio with respect to unit weight, confining pressure, loading frequency, decomposition and moisture content are analyzed. Normalized shear modulus reduction and damping curves are proposed for seismic analysis. Chapter 7 presents the study of the conventional and the bioreactor landfill in a small scale laboratory setup. A large scale experimental setup is fabricated to study the characteristics of a bioreactor landfill and includes the long term monitoring and analysis of temperature, gas, settlement and leachate characteristics periodically. The results of the comprehensive study are presented in this chapter. Chapter 8 summarizes the important conclusions from the various experimental studies reported in this dissertation. Conclusions and the scope of future work are presented. A detailed list of references and the list of publications from the thesis are presented at the end. Appendix A presents the life cycle analysis and life cycle cost analysis of MSW land disposal options. The land disposal options such as open dumps, engineered landfills and bioreactor landfills are analyzed in this study.

Polymer hydrolysis is the first (and rate limiting) step for biomethanation of heterogeneous biomass feedstock’s. Satisfactory hydrolysis has been difficult to achieve, understand and predict adequately, to run anaerobic bioreactors with such feedstock’s efficiently. The fraction of hot water soluble extracts (crude proteins and extractables, Fcpe), the nature and material of intercellular binding and the extent and complexity of lignin present have been considered as key parameters for hydrolysis and has been analyzed for a variety of biomass degradation data available at the Centre for Sustainable Technologies, Indian Institute of Science. Feedstocks were grouped into those bound with high levels of pectic/protein materials or lignin-bound types. The data on the initial (10-15d) as well as the overall rates of hydrolysis (0-50d) has been analyzed. The extent of hydrolysis achieved for pectin bound substrates were high (≥65%) and that of lignin bound substrate was low (≤30% VS, Acacia). The initial hydrolysis rates were strongly correlated to the content of extractables (=0.117Fcpe). Subsequently, the hydrolysis rates rise to reach maxima and then begin to fall. Most fresh feedstock had somewhat similar rates of the increase in hydrolysis rates but the time to reach maximum and its value varied among feed stocks. Many lignin bound feed stocks did not have such a pattern. With regards to the overall hydrolysis rate constant, it was found that these clustered into two groups that represented pectin bound (0.154/d) and lignin bound (0.045/d) types. Therefore from this study it was concluded that anaerobic decomposition of heterogeneous biomass could be predicted using two rate parameters and one intrinsic property of the biomass feedstock, namely, a. the initial rate of hydrolysis (based on the extent of extractables =0.117 Fcpe) b.the maximum rate achieved and the time when it is reached (an intrinsic property based on feed stock and but not determined in this study) c. the overall hydrolysis rate (choosing between 0.154 /d or 0.045 /d depending upon the nature of inter-cellular binding material, pectin or lignin, respectively). This research provides new insights into the prediction of hydrolysis rate a key limiting step for heterogeneous biomass biomethanation (hydrolysis) based on the level of extractables, the type of cellular cementing material and the maxima that can be achieved.