Cancer study and drug selection research attract more and more researchers, which need a significant laboratory technique, named cell injection. Hundreds of cells are loaded on devices and injected to investigate the behavior of the cells. Traditionally, cell injection is performed manually, which leads to human fatigue, is time-consuming and has a low success rate. Therefore, a system which can replicate the actions of what technicians do, such as to aspirate cells, transfer cells, immobilize cells, and release cells automatically, is needed. This system must be accurate, reliable, and efficient and operate without human intervention. A cell-transfer-cover and a cell-holder have been fabricated and a cell injection system has been set up to investigate the performance of the newly created device. Simulations and experiments have proven that this system would carry out the entire process of cell injection with the result of enhancing the speed of this important activity.

Depuis la découverte des cheminées hydrothermales, de multiples travaux ont été menés afin d’en étudier la diversité microbienne. Les inventaires moléculaires réalisés ont ainsi mis en évidence une grande diversité d’espèces qui contraste avec la faible proportion (1 %) d’espèces isolées par approche culturale. Une nouvelle approche d’immobilisation cellulaire par inclusion dans une matrice de polymères (gellane et xanthane) a ainsi été développée pour permettre l’étude de ces communautés thermophiles anaérobies marines. Le système, basé sur la formation d’une émulsion entre une solution de polymères inoculée et de l’huile, permet le piégeage de cellules dans des billes de gel de 1 à 2 mm de diamètre. Les conditions optimales d’immobilisation ont été obtenues pour une émulsion réalisée à 80 °C sous agitation (150 tr/min) à partir d’une solution de gellane (2,5 %) et de xanthane (0,25 %) avec 12 g/L de NaCl et 4 g/L de citrate de sodium, bullée à l’azote et réduit au Na2S avant inoculation. Les billes ont montré une bonne résistance mécanique après 5 semaines d’incubation à des pH compris entre 5,4 et 8, des températures allant jusqu’à 90 °C et des concentrations en NaCl et soufre allant jusqu’à respectivement 80 et 5 g/L. Des cultures en batch de Thermosipho sp. AT1272 et Thermococcus kodakarensis KOD1 immobilisées ont permis d’obtenir des concentrations allant jusqu’à 107 cellules/g de billes et 108 cellules/mL de fraction liquide. Une culture en continu réalisée en bioréacteur gas-lift pendant 41 jours à partir d’une communauté synthétique immobilisée composée de 8 souches (hyper)thermophiles a démontré la capacité de l’immobilisation cellulaire à protéger les cellules face à un stress oxique et à les maintenir (3 des 8 souches) dans le bioréacteur jusqu’à ce que les conditions de culture soient propices à leur croissance. La réactivité de la communauté immobilisée face aux changements environnementaux (température) a également été démontrée. Enfin, la culture en continu réalisée pendant 64 jours d’un échantillon immobilisé de diffuseur du site Rainbow a permis la croissance de plusieurs espèces bactériennes et archéennes (Oceanithermus sp., Thermococcus sp.) dont une partie n’a été détectée que dans les billes (Sulfurimonas sp., Nitratifractor sp., Vibrio sp.) par clonage-séquençage. L’ensemble de ces résultats ont permis de valider l’utilisation d’un protocole d’immobilisation par inclusion dans une matrice de polymères pour l’étude des communautés hydrothermales, de leur diversité et de leur dynamique. / Since the discovery of hydrothermal vents, multiple studies have been conducted in order to study microbial diversity. Molecular inventories realized have thus demonstrated a great diversity of species that contrasts with the low proportion (1%) of species isolated by culture approach. A new cell immobilization approach by inclusion in a polymer matrix (gellan and xanthan) has been developed for the study of these thermophilic anaerobic marine communities. The system, based on the formation of an emulsion between an inoculated polymer solution and oil, allows the entrapment of cells in gel beads with a diameter between 1 and 2 mm. The optimal immobilization conditions were obtained for emulsion performed at 80 °C with stirring (150 rpm) with a polymer solution composed of gellan (2.5%) and xanthan (0.25%) with 12 g/L of NaCl and 4 g/L of sodium citrate, bubbled with nitrogen and reduced with Na2S before inoculation. The beads showed a good mechanical stability after a 5-week incubation at pH between 5.4 and 8, temperatures up to 90 °C and NaCl and sulfur concentrations up to respectively 80 and 5 g/L. Batch cultures of immobilized Thermosipho sp. AT1272 and Thermococcus kodakarensis KOD1 yielded concentrations up to 107 cells/g of beads and 108 cells/mL of liquid fraction. A continuous culture performed in a gas-lift bioreactor for 41 days of an immobilized synthetic community composed of 8 (hyper)thermophilic strains demonstrated the capacity of cell immobilization to protect cells from oxique stress and to maintain them (3 of 8 strains) in the bioreactor until having suitable culture conditions for their growth. The reactivity of the immobilized community to environmental change (temperature) was also demonstrated. Finally, the continuous culture performed for 64 days of an immobilized diffuser sample from Rainbow site allowed the growth of several bacterial and archaeal species (Oceanithermus sp., Thermococcus sp.), part of which was detected only in the beads (Sulfurimonas sp., Nitratifractor sp., Vibrio sp.) by cloning-sequencing. All these results have validated the use of an immobilization protocol by inclusion in a polymer matrix for the study of hydrothermal communities, of their diversity and their dynamics.

Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T19:09:12Z : No. of bitstreams: 1 covizzi_lg_me_sjrp.pdf: 1482206 bytes, checksum: 2f1c1f77dc261f160aba2bc3a1d1ffea (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O uso de células microbianas imobilizadas para aumentar a produção de metabólitos fúngicos em processos fermentativos tem mostrado altos rendimentos. Nesse trabalho foi avaliado pela primeira vez, a imobilização de células do Botryosphaeria rhodina, um fungo ligninolitico produtor constitutivo de lacases. Três suportes foram avaliados: Fibra Acrílica Fina (FAF); Espuma de Poliuretano Expandido (EPE); Espuma de Poliuretano Fibroso (EPF). O EPF foi o melhor suporte por ter mostrado uma imobilização mais homogenia das células. Um planejamento fatorial foi desenvolvido para otimizar a produção de lacases por células livres, na presença de álcool veratrílico (AV). A análise da superfície de resposta mostrou que 18mM como a melhor concentração de AV para a produção de lacases, usando-se 3 mL de homogeinato de células como inóculo (DOλ400nm 0.4-0.6) para 25 mL de meio de cultura em frascos de 125mL, a 180 rmp, durante 126 horas a 28ºC. O perfil de crescimento do fungo, associado a produção de lacase foram comparados na presença e na ausência de AV, usando-se células livres e células imobilizadas do B. rhodina. A imobilização aumentou aproximadamente 3 vezes a produção de lacases e manteve estável o nível de produção durante 6 reciclos. A imobilização de células do B. rhodina mostrou-se útil uma vez que economizou 72horas para atingir a maior produção de lacase, quando comparada com células livres e também aumentou a tolerância do fungo a concentrações mais altas de AV (500mM) / The use of microbial immobilized cells to increase the production of fungal metabolites in fermentation processes has showed higher yields. This work evaluated by the first time, the immobilization of Botryosphaeria rhodina cells, a ligninolytic fungus that produces laccase. Three carriers were evaluated: acrylic fine fiber (FAF), expanded polyurethane foam (EPE), and fiber polyurethane foam (EPF). The EPF was the best carrier because showed a homogeneous immobilization cells. A factorial design was developed in order to optimize the laccase production by free cells in the presence of the laccase inducer veratryl alcohol (VA). The analysis by response surface answer showed 18 mM as the best VA concentration to produce laccase using 3 mL of a cell homogenate (ODλ400nm 0.4-0.6) as inoculum, to 25 mL of culture medium in shaked flasks (125 mL) at 180 rpm, during 126 hours at 28 °C. A growth profile for laccase and fungal biomass production were compared with and without VA using free and immobilized cells of B. rhodina. The cell immobilization increased approximately 3 folds the laccase production and maintained it stable during 6 consecutive recycles. The cell immobilization of B. rhodina showed to be useful once saved 72 hours to achieve the higher laccase production when compared to the one with free cells, and also increased the fungal cell tolerance at higher VA concentrations (500 mM)

Orientador: Roberto da Silva / Banca: Crispin Humberto Garcia Cruz / Banca: Aneli de Melo Barbosa / Resumo: O uso de células microbianas imobilizadas para aumentar a produção de metabólitos fúngicos em processos fermentativos tem mostrado altos rendimentos. Nesse trabalho foi avaliado pela primeira vez, a imobilização de células do Botryosphaeria rhodina, um fungo ligninolitico produtor constitutivo de lacases. Três suportes foram avaliados: Fibra Acrílica Fina (FAF); Espuma de Poliuretano Expandido (EPE); Espuma de Poliuretano Fibroso (EPF). O EPF foi o melhor suporte por ter mostrado uma imobilização mais homogenia das células. Um planejamento fatorial foi desenvolvido para otimizar a produção de lacases por células livres, na presença de álcool veratrílico (AV). A análise da superfície de resposta mostrou que 18mM como a melhor concentração de AV para a produção de lacases, usando-se 3 mL de homogeinato de células como inóculo (DOλ400nm 0.4-0.6) para 25 mL de meio de cultura em frascos de 125mL, a 180 rmp, durante 126 horas a 28ºC. O perfil de crescimento do fungo, associado a produção de lacase foram comparados na presença e na ausência de AV, usando-se células livres e células imobilizadas do B. rhodina. A imobilização aumentou aproximadamente 3 vezes a produção de lacases e manteve estável o nível de produção durante 6 reciclos. A imobilização de células do B. rhodina mostrou-se útil uma vez que economizou 72horas para atingir a maior produção de lacase, quando comparada com células livres e também aumentou a tolerância do fungo a concentrações mais altas de AV (500mM) / Abstract: The use of microbial immobilized cells to increase the production of fungal metabolites in fermentation processes has showed higher yields. This work evaluated by the first time, the immobilization of Botryosphaeria rhodina cells, a ligninolytic fungus that produces laccase. Three carriers were evaluated: acrylic fine fiber (FAF), expanded polyurethane foam (EPE), and fiber polyurethane foam (EPF). The EPF was the best carrier because showed a homogeneous immobilization cells. A factorial design was developed in order to optimize the laccase production by free cells in the presence of the laccase inducer veratryl alcohol (VA). The analysis by response surface answer showed 18 mM as the best VA concentration to produce laccase using 3 mL of a cell homogenate (ODλ400nm 0.4-0.6) as inoculum, to 25 mL of culture medium in shaked flasks (125 mL) at 180 rpm, during 126 hours at 28 °C. A growth profile for laccase and fungal biomass production were compared with and without VA using free and immobilized cells of B. rhodina. The cell immobilization increased approximately 3 folds the laccase production and maintained it stable during 6 consecutive recycles. The cell immobilization of B. rhodina showed to be useful once saved 72 hours to achieve the higher laccase production when compared to the one with free cells, and also increased the fungal cell tolerance at higher VA concentrations (500 mM) / Mestre

Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE / Polycyclic aromatic hydrocarbons (PAHs) are a class of fused-ring aromatic compounds that are ubiquitous environmental pollutants due to their toxic properties. The importance of preventing PAHs contamination has been recognized by the Unites States Environmental Protection Agency which has proposed 16 PAHs as priority pollutants, including fluoranthene. Biodegradation is considered as one of the major removal ways of these compounds. In this context, the search for new sources of natural microorganisms is extremely important. One of the most promising source is Peat, which consists in a organic soil formed through a complex process of decomposition and humification of plant residues by microbial oxidation. In addition to the huge biological and genetic pool which can be explored directly from this natural resource, biotechnologic applications using immobilized microbial cells is also an aspect that has gained importance due to various advantages. Thus, this study aims to investigate the biodegradation of in vitro fluoranteno using microorganisms isolated from Santo Amaro das Brotas peat-Sergipe, as well as, develop immobilized chitosan beads for the same purpose. Using this PAH as sole carbon source, it was possible to isolated 8 bacteria and 3 fungi identified as belonging to Bacillus sp., Serratia sp., Penicillium sp. and Fusarium sp. All strains were tested for their ability to degrade 100 mg L-1 of fluoranthene during different incubation periods: 5 and 10 days for bacteria, and 14 and 28 days for fungi. It was found that the duration of the incubation period was proportional to the degree of biodegradation. Analysis of metabolites enabled identification of three compounds common to all the microorganisms: 2,3-dimethyl-9H-fluoren-9-one, carbazole, and bis (octyl) benzene-1,2-dicarboxylate. In general, it was possible to observe the formation of aliphatic metabolites, probably showing a particular feature of these microorganisms from tropical peat. Regarding to microbial immobilization,the Serratia sp. AC-11 strain was selected for trapping in chitosan beads that were then used to biodegrade fluoranthene (at 100 mg L-1). The beads produced were uniform in size, with an average diameter of 3 mm, and were characterized by SEM and FTIR. The immobilized bacteria were able to degrade 76% of fluoranthene in 5 days and 84% in 10 days, at a degradation rate that was almost 50% higher than achieved using free-living cells. Furthermore, the beads with immobilized bacteria had the advantage of being reusable, with satisfactory biodegradation obtained during continuous cycles of use. The numbers of viable cells in the chitosan beads revealed the capacity of the strain to grow and multiply during the biodegradation process. The findings revealed that the peat environment could provide a useful source of PAH-degrading microorganisms.. The new bioproduct produced represents a low cost, efficient, eco-friendly, and practical solution for use in the bioremediation of areas contaminated by fluoranthene. / Os Hidrocarbonetos Policíclicos Aromáticos (HPAs) são uma classe de compostos poluentes formados por dois ou mais anéis aromáticos fundidos, que possuem risco significativo de contaminação da biota por apresentar propriedades tóxicas. Devido a isso, a Agência de Proteção Ambiental Americana selecionou 16 HPAs prioritários para o monitoramento ambiental, dentre eles o Fluoranteno. Uma das formas de se remediar estes compostos do ambiente é pela biodegradação. Nesse contexto, a procura por novas fontes naturais de micro-organismos é de suma importância. Uma das possíveis fontes promissoras é a turfa, a qual consiste em um solo orgânico formado através de oxidação microbiológica de restos de plantas. Além do enorme pool biológico e genético que pode ser explorado diretamente desse recurso natural, aplicações biotecnológicas com o uso de células microbianas imobilizadas também são uma vertente que tem ganhado importância. Diante disso, este estudo propõe investigar a biodegradação in vitro do fluoranteno utilizando micro-organismos isolados da turfa de Santo Amaro das Brotas - Sergipe, assim como desenvolver um bioproduto centrado na produção de esferas de quitosana imobilizadas com bactéria para o mesmo fim. Utilizando este HPA como única fonte de carbono, foi possível isolar 8 bactérias e 3 fungos, os quais foram identificados como pertencentes aos gêneros Bacillus sp., Serratia sp., Penicillium sp. e Fusarium sp. Todos eles foram testados quanto à sua capacidade em degradar o fluoranteno (100 mg L-1) durante diferentes períodos de incubação: 5 e 10 dias para as bactérias; 14 e 28 dias para fungos. Foi verificado que a duração do período de incubação foi proporcional ao grau de biodegradação. As melhores taxas de biodegradação entre as bactérias e os fungos foram alcançadas, respectivamente, pelo isolado Bacillus sp. AC-25 em 10 dias (49%) e pelo Penicillium sp. AC-1 em 28 dias (64%). A análise dos metabólitos identificou a presença de compostos com diferentes grupos funcionais, incluindo três metabólitos comuns a todos os micro-organismos: 2,3-dimetil-9H-fluoreno-9-ona, carbazol, e bis (octil) benzeno-1,2-dicarboxilato. Em geral, foi possível observar a formação de metabólitos alifáticos, provavelmente, mostrando uma particularidade no metabolismo destes micro-organismos, o qual está relacionado ao ambiente da turfa.Com relação à imobilização em esferas de quitosana, foi observado que o isolado Serratia sp. AC-11 foi o que melhor se adaptou as condições necessárias. Estas esferas imobilizadas produzidas foram caracterizadas por MEV e FT-IR. Em apenas 1 dia, elas foram capazes de degradar 56% do fluorantreno, 76% em 5 dias e 84% em 10 dias, apresentando uma taxa de degradação de quase 50% a mais quando comparada a célula livre. Além disso, estas esferas apresentaram a vantagem de reutilização durante ciclos contínuos com biodegradação satisfatória do HPA. Diante dos resultados obtidos, observa-se que a turfa foi uma fonte útil de micro-organismos capazes de degradar o fluoranteno e possibilitou a geração de um novo bioproduto caracterizado principalmente por ser eco-amigável, possuir baixo custo associado, eficiência de degradação e possível aplicação industrial destinado a biorremediação de áreas contaminadas por HPAs.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O aproveitamento integral do pedÃnculo de caju, uma matÃria prima vastamente encontrada no Cearà e pouco aproveitada, aliado a produÃÃo de etanol, produto de valor energÃtico sÃo o foco desse projeto. O suco de caju integral, rico em frutose, glicose, sais e vitaminas, mostrou-se bastante adequado para o uso da levedura Saccharomyces cerevisiae no processo fermentativo alcoÃlico. A busca por melhorias na obtenÃÃo de etanol se deu atravÃs do estudo do uso de leveduras imobilizadas em suporte de bagaÃo de caju (SBC). A imobilizaÃÃo celular vem sendo estudada por inÃmeros pesquisadores e tem se mostrado uma tÃcnica importante devido aos benefÃcios e vantagens possibilitados como o aumento na produtividade. O bagaÃo de caju surge como uma matÃria prima barata e de fÃcil obtenÃÃo para a aplicaÃÃo da tÃcnica de imobilizaÃÃo. Para comparaÃÃo foram estudados os resultados com cÃlulas livres e imobilizadas em alginato de cÃlcio, um conhecido agente de imobilizaÃÃo de cÃlulas por encapsulamento e cÃlulas imobilizadas por adsorÃÃo em bagaÃo de caju. Utilizando cÃlulas imobilizadas em bagaÃo foram realizadas fermentaÃÃes preliminares, consecutivas e de estocagem. Foram verificados os seguintes parÃmetros cinÃticos: produtividade (Qp, g.L-1h-1), eficiÃncia (Ef, %), conversÃo substrato/biomassa (Yx/s, g.g-1) e conversÃo substrato/produto (Yp/s, g.g-1). O suporte de bagaÃo de caju atingiu vantagens com relaÃÃo a adesÃo e altas densidades celulares, gerando elevadas produtividades (≈ 5 g.L-1h-1). Com relaÃÃo a eficiÃncia e a taxa de conversÃo substrato/produto, cÃlulas imobilizadas atingiram valores similares aos obtidos com cÃlulas livres no suco de caju integral (em torno de 90% e 0,47 respectivamente). Outra vantagem foi a durabilidade e possibilidade de reutilizaÃÃo do suporte tornando o processo mais econÃmico, rÃpido e eficiente. O SBC mostrou-se apto a reutilizaÃÃo por no mÃnimo 10 bateladas consecutivas e se manteve estÃvel, em geladeira, por pelo menos 6 meses / The use of cashew apple peduncle, a vast available material found in Cearà that is usually wasted, combined with production of ethanol, is the focus of this project. Cashew apple juice, rich in fructose, glucose, salts and vitamins, is an appropriate raw material for alcoholic fermentation by the yeast Saccharomyces cerevisiae. The search for improvements in ethanol production in this work was achieved through the study of the use of yeast immobilized in cashew apple bagasse support, (SBC). Cell immobilization has been studied by many researchers and has been an important technique because of the possible benefits and advantages as the increase in productivity. Cashew apple bagasse is a cheap and easily accessible raw material to the application of the immobilization technique. As comparison, results with free, immobilized cells in calcium alginate, a known agent for immobilization by encapsulation, and immobilized cells by adsorption on SBC were studied. Preliminary fermentation assays using immobilized cells on SBC were performed, as well as consecutive and storage fermentations. The following kinetic parameters were checked: productivity (Qp, gL-1h-1), efficiency (Ef,%), substrate / biomass yield (Yx / s, g.g-1) and substrate / product yield (Yp / s, g.g - 1). SBC presented advantages, such as high cell densities and adhesion, generating high yields (≈ 5 g.L-1h-1). With respect to efficiency and rate of substrate / product yield, immobilized cells reached values similar to those obtained with free cells in the integral cashew apple juice (about 90% and 0.47 respectively). Another advantage is the durability and possibility of reuse of SBC making the process more economical, fast and efficient. SBC was reused for at least 10 consecutive batches and remained stable, at refrigerator, for 6 months

Thesis (PhD (Chemical Engineering))--Cape Peninsula University of Technology, 2019 / The South African wine industry is constantly facing several challenges which affect the quality of wine, the local/global demand and consequently the revenue generated. These challenges include the ongoing drought, bush fires, climate change and several liquor amendment bills aimed at reducing alcohol consumption and alcohol outlets in South Africa. It is therefore critical for the wine industry to expand and find alternative ways in which sub-standard or surplus wine grapes can be used to prevent income losses and increase employment opportunities. Traditional Balsamic Vinegar (TBV) is a geographically and legislative protected product produced only in a small region in Italy. However, the methodology can be used to produce similar vinegars in other regions. Balsamic-styled vinegar (BSV), as defined in this thesis, is a vinegar produced by partially following the methods of TBV while applying process augmentation techniques. Balsamic-styled vinegar is proposed to be a suitable product of sub-standard quality or surplus wine grapes in South Africa. However, the production of BSV necessitates the use of cooked (high sugar) grape must which is a less favourable environment to the microorganisms used during fermentation. Factors that negatively affect the survival of the microorganisms include low water activity due to the cooking, high osmotic pressure and high acidity. To counteract these effects, methods to improve the survival of the non-Saccharomyces yeasts and acetic acid bacteria used are essential. The primary aim of this study was to investigate several BSV process augmentation techniques such as, aeration, agitation, cell immobilization, immobilized cell reusability and oxygen mass transfer kinetics in order to improve the performance of the microbial consortium used during BSV production. The work for this study was divided into four (4) phases. For all the phases a microbial consortium consisting of non-Saccharomyces yeasts (n=5) and acetic acid bacteria (n=5) was used. Inoculation of the yeast and bacteria occurred simultaneously. The 1st phase of the study entailed evaluating the effect of cells immobilized by gel entrapment in Ca-alginate beads alongside with free-floating cells (FFC) during the production of BSV. Two Ca-alginate bead sizes were tested i.e. small (4.5 mm) and large (8.5 mm) beads to evaluate the effects of surface area or bead size on the overall acetification rates. Ca-alginate beads and FFC fermentations were also evaluated under static and agitated (135 rpm) conditions. The 2nd phase of the study involved studying the cell adsorption technique for cell immobilization which was carried-out using corncobs (CC) and oak wood chips (OWC), while comparing to FFC fermentations. At this phase of the study, other vinegar bioreactor parameters such as agitation and aeration were studied in contrast to static fermentations. One agitation setting (135 rpm) and two aeration settings were tested i.e. high (0.3 vvm min−1) and low (0.15 vvm min−1) aeration conditions. Furthermore, to assess the variations in cell adsorption capabilities among individual yeast and AAB cells, the quantification of cells adsorbed on CC and OWC prior- and post-fermentation was conducted using the dry cell weight method. The 3rd phase of the study entailed evaluating the reusability abilities of all the matrices (small Ca-alginate beads, CC and OWC) for successive fermentations. The immobilized cells were evaluated for reusability on two cycles of fermentation under static conditions. Furthermore, the matrices used for cell immobilization were further analysed for structure integrity by scanning electron microscopy (SEM) before and after the 1st cycle of fermentations. The 3rd phase of the study also involved the sensorial (aroma and taste) evaluations of the BSV’s obtained from the 1st cycle of fermentation in order to understand the sensorial effects of the Ca-alginate beads, CC and OWC on the final BSV. The 4th phase of the study investigated oxygen mass transfer kinetics during non-aerated and aerated BSV fermentation. The dynamic method was used to generate several dissolved oxygen profiles at different stages of the fermentation. Consequently, the data obtained from the dynamic method was used to compute several oxygen mass transfer parameters, these include oxygen uptake rate ( 𝑟𝑟𝑂𝑂2 ), the stoichiometric coefficient of oxygen consumption vs acid yield (𝑌𝑌𝑂𝑂/𝐴𝐴), the oxygen transfer rate (𝑁𝑁𝑂𝑂2 ), and the volumetric mass transfer coefficients (𝐾𝐾𝐿𝐿𝑎𝑎). During all the phases of the study samples were extracted on weekly intervals to evaluate pH, sugar, salinity, alcohol and total acidity using several analytical instruments. The 4th phase of the study involved additional analytical tools, i.e. an oxygen µsensor to evaluate dissolved oxygen and the ‘Speedy breedy’ to measure the respiratory activity of the microbial consortium used during fermentation. The data obtained from the 1st phase of the study demonstrated that smaller Ca-alginate beads resulted in higher (4.0 g L-1 day−1) acetification rates compared to larger (3.0 g L-1 day−1) beads, while freely suspended cells resulted in the lowest (0.6 g L-1 day−1) acetification rates. The results showed that the surface area of the beads had a substantial impact on the acetification rates when gel entrapped cells were used for BSV fermentation. The 2nd phase results showed high acetification rates (2.7 g L-1 day−1) for cells immobilized on CC in contrast to cells immobilized on OWC and FFC, which resulted in similar and lower acetification rates. Agitated fermentations were unsuccessful for all the treatments (CC, OWC and FFC) studied. Agitation was therefore assumed to have promoted cell shear stress causing insufficient acetification during fermentations. Low aerated fermentations resulted in better acetification rates between 1.45–1.56 g L-1 day−1 for CC, OWC and FFC. At a higher aeration setting, only free-floating cells were able to complete fermentations with an acetification rate of 1.2 g L-1 day−1. Furthermore, the adsorption competence data showed successful adsorption on CC and OWC for both yeasts and AAB with variations in adsorption efficiencies, whereby OWC displayed a lower cell adsorption capability compared to CC. On the other hand, OWC were less efficient adsorbents due to their smooth surface, while the rough surface and porosity of CC led to improved adsorption and, therefore, enhanced acetification rates. The 3rd phase results showed a substantial decline in acetification rates on the 2nd cycle of fermentations when cells immobilized on CC and OWC were reused. While cells entrapped in Ca-alginate beads were able to complete the 2nd cycle of fermentations at reduced acetification rates compared to the 1st cycle of fermentations. The sensory results showed positive ratings for BSV’s produced using cells immobilized in Ca-alginate beads and CC. However, BSV’s produced using OWC treatments were neither ‘liked nor disliked’ by the judges. The SEM imaging results further showed a substantial loss of structural integrity for Ca-alginate beads after the 1st cycle fermentations, with minor changes in structural integrity of CC being observed after the 1st cycle fermentations. OWC displayed the same morphological structure before and after the 1st cycle fermentations which was attributed to their robustness. Although Ca-alginate beads showed a loss in structural integrity, it was still assumed that Ca-alginate beads provided better protection against the harsh environmental conditions in contrast to CC and OWC adsorbents due to the acetification rates obtained on both cycles. The 4th phase data obtained from the computations showed that non-aerated fermentations had a higher 𝑌𝑌𝑂𝑂/𝐴𝐴, 𝑟𝑟𝑂𝑂2 , 𝑁𝑁𝑂𝑂2 and a higher 𝐾𝐾𝐿𝐿𝑎𝑎 . It was clear that aerated fermentations had a lower aeration capacity due to an inappropriate aeration system design and an inappropriate fermentor. Consequently, aeration led to several detrimental biochemical changes in the fermentation medium thus affecting 𝐾𝐾𝐿𝐿𝑎𝑎 and several oxygen mass transfer parameters which serve as a driving force. Overall, it was concluded that the best method for BSV production is the use of cells entrapped in small alginate beads or cells adsorbed on CC under static and non-aerated fermentations. This conclusion was based on several factors such as cell affinity/cell protection, acetification rates, fermentation period and sensorial contributions. However, cells entrapped in Ca-alginate beads had the highest acetification rates. The oxygen mass transfer computations demonstrated a high 𝐾𝐾𝐿𝐿𝑎𝑎 when Ca-alginate beads were used under static-non-aerated conditions compared to fermentations treated with CC. Therefore, a fermentor with a high aeration capacity needs to be designed to best suit the two BSV production systems (Ca-alginate beads and CC). It is also crucial to develop methods which can increase the robustness of Ca-alginate beads in order to improve cell retention and reduce the loss of structural integrity for subsequent cycles of fermentation. Studies to define parameters used for upscaling the BSV production process for large scale productions are also crucial.

Submitted by Bruna Rodrigues (bruna92rodrigues@yahoo.com.br) on 2017-10-16T11:32:32Z No. of bitstreams: 1 TeseTSSM.pdf: 23662587 bytes, checksum: 4ef26b2b65e46560d905cc700258cd0d (MD5) / Approved for entry into archive by Ronildo Prado (producaointelectual.bco@ufscar.br) on 2017-10-31T16:29:33Z (GMT) No. of bitstreams: 1 TeseTSSM.pdf: 23662587 bytes, checksum: 4ef26b2b65e46560d905cc700258cd0d (MD5) / Approved for entry into archive by Ronildo Prado (producaointelectual.bco@ufscar.br) on 2017-10-31T16:29:42Z (GMT) No. of bitstreams: 1 TeseTSSM.pdf: 23662587 bytes, checksum: 4ef26b2b65e46560d905cc700258cd0d (MD5) / Made available in DSpace on 2017-10-31T16:41:50Z (GMT). No. of bitstreams: 1 TeseTSSM.pdf: 23662587 bytes, checksum: 4ef26b2b65e46560d905cc700258cd0d (MD5) Previous issue date: 2017-03-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / In ethanol production process from hemicellulosic fraction, the use of xylooligomers (XOS) as substrate reduce the contamination risk, favoring its application at industrial scale. Thus, a biocatalyst, containing xylanases, xylose isomerase (XI) and yeast co-immobilized in calcium alginate gel, was developed and XOS simultaneous hydrolysis, isomerization and fermentation (SHIF) process was studied. Firstly, xylanases from Multifect CX XL A03139 (XAS-5), a commercial enzyme preparation, and the recombinant xylanase from Bacillus subtilis (XynA) were selected to compose biocatalyst beads. XAS-5 presented better conversion (78.7%) and higher xylose production in the hydrolysis of beechwood xylan, while XynA showed exclusive endoxylanase activity. The immobilization and stabilization of XynA were performed in chitosan-glutaraldehyde, chitosan-glyoxyl and agarose-glyoxyl. Although the enzyme was efficiently immobilized on all supports, the agarose-glyoxyl-XynA derivative was notable for exhibiting remarkable stabilization under tested conditions (8600 times). Studies of SHIF process were carried out with birchwood xylan, leading to ethanol production (0.160 g/g and 0.092 g/L.h) and xylose accumulation, which indicated XI activity decrease. Further experiments were then performed to to identify possible inhibitors of XI (pH, Ca2+, Mg2+ and xylooligosaccharides). Ca2+ was identified as an inhibitor, while Mg2+ acts as an activator of the enzyme, and both actions are potentiated at acidic pHs. XI is also inhibited by XOS, with a decrease of 31.6% in XI activity in the presence of 7.0 g/L of xylobiose. For this reason, it was decided to evaluate SIF process with a recombinant yeast, capable of expressing XI. In batch runs, GSE16-T18 (T18) yeast encapsulated in alginate gel was capable to ferment xylose efficiently, consuming 40 g/L of xylose in 4 h and producing 14.4 g/L of ethanol, with yield of 0.422 g/g and productivity of 3.61 g/L.h. Calcium alginate gel encapsulation also contributed to protect yeast from the action of inhibitors, such as acetic acid. The encapsulated T18 was able to perform 10 consecutive cycles in repeated batch (yeast extract-peptone medium with 40 g/L of xylose), keeping the same productivity and high yields. It also fermented efficiently sugarcane bagasse hydrolysate, containing 60 g/L of fermentable sugars and high grade of inhibitors. The modified yeast to be more tolerant to acetic acid, GSE16-T18 HAA1, was also studied, exhibiting superior performance in comparison to T18 for hydrolysate fermentations. Continuous experiments were conducted in a fixed bed reactor using the T18-HAA1 yeast immobilized, with different xylose concentrations (40, 60, 80 and 120 g/L) in the feed medium. The reactor was operated up to 15 days, without bacterial contamination, with yield of 0.45 g/g, productivity of 4.8 g/L.h and selectivity of 31 gethanol/gxylitol (60 g/L of xylose in the feed). For the concentrations higher than 60 g/L, the conversion decreased after 4 days of continuous operation, indicating loss of cell viability due to hazardous effect of ethanol when present at 30 g/L or more, as well as limitation of oxygen and nutrients in the system. / No processo de produção de etanol a partir da fração hemicelulósica, a utilização de xilooligômeros como substrato reduz o risco de contaminação, favorecendo o emprego da tecnologia em escala industrial. Para isso, um biocatalisador contendo xilanases, xilose isomerase (XI) e levedura co-imobilizadas em gel de alginato de cálcio foi desenvolvido e o processo de hidrólise, isomerização e fermentação simultâneos (SHIF) de xilooligômeros foi estudado. Primeiramente, as xilanases presentes no produto Multifect CX XL A03139 (XAS- 5) e a xilanase recombinante de Bacillus subtilis (XynA) foram selecionadas para compor os beads do biocatalisador. XAS-5 apresentou melhor conversão (78,7%) e maior produção de xilose na hidrólise da xilana de faia, enquanto XynA apresentou exclusiva atividade de endoxilanase. Realizou-se a imobilização e estabilização da XynA em quitosanaglutaraldeído, quitosana-glioxil e agarose-glioxil. Apesar da enzima ser eficientemente imobilizada nos três suportes, o derivado agarose-glioxil-XynA se destacou por apresentar uma estabilização notável nas condições testadas (8600 vezes). Estudos do processo SHIF foram realizados com xilana de bétula, observando-se produção de etanol (0,160 g/g e 0,092 g/L.h) e acúmulo de xilose, indicando redução da atividade da XI. Realizou-se então, um estudo para identificar possíveis inibidores da XI (pH, Ca2+, Mg2+ e XOS), constatando-se que Ca2+ é um inibidor enquanto Mg2+ é um ativador da enzima, sendo suas ações potencializadas em pHs ácidos. Comprovou-se também que XI é inibida por XOS, observando-se queda da atividade de XI (31,6%) na presença de 7,0 g/L de xilobiose. Desta forma, tornou-se interessante avaliar o processo SIF com uma levedura recombinante, capaz de expressar XI. Em ensaios em batelada, a levedura GSE16-T18 (T18), encapsulada em gel de alginato, mostrou-se eficiente na fermentação de xilose, consumindo 40 g/L de xilose em 4 h e produzindo 14,4 g/L de etanol, com rendimento de 0,422 g/g e produtividade de 3,61 g/L.h. O encapsulamento em gel de alginato de cálcio também protegeu a levedura da ação de inibidores, como o ácido acético. A T18 encapsulada foi capaz de realizar 10 ciclos consecutivos em bateladas repetidas (meio contendo extrato de levedura, peptona e 40 g/L de substrato), mantendo mesma produtividade e elevado rendimento, além de fermentar eficientemente hidrolisado hemicelulósico de bagaço de cana, contendo 60 g/L de açúcares fermentescíveis e alto teor de inibidores. A levedura GSE16-T18 HAA1, modificada geneticamente para ser mais tolerante ao ácido acético, foi também estudada, com resultados superiores a T18 nas fermentações de hidrolisado. Fermentações em modo contínuo foram realizadas em reator de leito fixo utilizando a levedura T18-HAA1 imobilizada, com diferentes concentrações de xilose na alimentação (40, 60, 80 e 120 g/L). O reator foi operado por até 15 dias, sem ocorrência de contaminação por bactérias, com rendimento 0,45 g/g, produtividade em etanol de 4,8 g/L.h e seletividade de 31 getanol/gxilitol (60 g/L de xilose na alimentação). Para as concentrações superiores a 60 g/L, a conversão diminuiu após 4 dias de operação contínua, indicando perda de viabilidade celular devido à ação do etanol quando presente em concentrações acima de 30 g/L e da limitação de oxigênio e nutrientes no sistema.

Im Bereich der Umwelttechnik bieten sich vielversprechende Einsatzmöglichkeiten für Sol-Gel-immobilisierte Mikroorganismen sowohl für die Entfernung als auch für die Detektion von Schadstoffen an. Für einen effizienten Einsatz sind zum einen eine hohe Langzeitaktivität und -vitalität der eingebetteten Zellen als auch eine gute Lagerbeständigkeit wichtig. Neben einer hohen Makroporosität, die sowohl für einen guten Stoffaustausch mit der Umgebung sorgt sowie den Zellen Raum für Zellteilung bietet, ist vor allem auch die Aufrechterhaltung der Feuchtigkeit in der Immobilisierungsmatrix während der Immobilisierung, der Lagerung und des Einsatzes wichtig. Besonders vorteilhaft hat sich hier eine Immobilisierung in dünnen SiO2-Schichten auf Blähtonbruchstücken erwiesen, da dieses Trägermaterial neben einer hohen Makroporosität auch ein hohes Wasserspeichervermögen besitzt. Immobilisiert in dünnen SiO2-Schichten auf Blähton konnten diverse Schadstoff-abbauende Mikroorganismen mehrere Monate auch außerhalb eines flüssigen Mediums unter feuchten Bedingungen gelagert werden, ohne dass ihre Abbauleistung erheblich sank. Ein weiteres Verfahren um Immobilisierungsmaterialien mit überdurchschnittlich hoher Makroporosität bei gleichzeitig hoher Stabilität zu erzeugen, stellt das Freeze-Gelation Verfahren dar. Über einen Gefriertrocknungsschritt können hier zudem die zu immobilisierenden Zellen in eine trocken lagerfähige Form überführt werden, wodurch Handhabung sowie Lagerung und Transport vereinfacht werden. Außerdem kann durch Wahl der Einfrierbedingungen und Zugabe von Füllstoffen zu dem SiO2-Sol entscheidend die Porenstruktur der Immobilisierungsmatrix beeinflusst und den Anforderungen entsprechend eingestellt werden. Allerdings zeigte sich, dass diese Methode nicht für alle Mikroorganismen gleichermaßen geeignet ist. Trotz diverser kryoprotektiver Maßnahmen konnte keine ausreichende Überlebensrate für sensible Stämme wie Aquincola tertiaricarbonis L108 erzielt werden. Neben der Erhöhung der Makroporosität der SiO2-Matrix wurde versucht das Sol-Gel-Verfahren mit einem flexiblen organischen Polymer (Na-Alginat) zu kombinieren, um eine Immobilisierungsmatrix mit einem weichen Kern zu erzeugen, der Zellteilung zulässt. Als zusätzlicher Vorteil des entwickelten Alginat/SiO2-Hybridmaterials erwies sich, dass dieses auch auf einfache Weise mittels Drucktechniken in definierten Spots abgelegt werden kann und so die Zellen in Arrays abgelegt werden können. Das Potential dieser Methodik für die Entwicklung von Ganzzellsensoren wurde am Beispiel der Grünalge Chlorella vulgaris als Modell-Sensorzelle demonstriert und für die Detektion von Atrazin als Modellsubstanz eingesetzt.

Im Bereich der Umwelttechnik bieten sich vielversprechende Einsatzmöglichkeiten für Sol-Gel-immobilisierte Mikroorganismen sowohl für die Entfernung als auch für die Detektion von Schadstoffen an. Für einen effizienten Einsatz sind zum einen eine hohe Langzeitaktivität und -vitalität der eingebetteten Zellen als auch eine gute Lagerbeständigkeit wichtig. Neben einer hohen Makroporosität, die sowohl für einen guten Stoffaustausch mit der Umgebung sorgt sowie den Zellen Raum für Zellteilung bietet, ist vor allem auch die Aufrechterhaltung der Feuchtigkeit in der Immobilisierungsmatrix während der Immobilisierung, der Lagerung und des Einsatzes wichtig. Besonders vorteilhaft hat sich hier eine Immobilisierung in dünnen SiO2-Schichten auf Blähtonbruchstücken erwiesen, da dieses Trägermaterial neben einer hohen Makroporosität auch ein hohes Wasserspeichervermögen besitzt. Immobilisiert in dünnen SiO2-Schichten auf Blähton konnten diverse Schadstoff-abbauende Mikroorganismen mehrere Monate auch außerhalb eines flüssigen Mediums unter feuchten Bedingungen gelagert werden, ohne dass ihre Abbauleistung erheblich sank. Ein weiteres Verfahren um Immobilisierungsmaterialien mit überdurchschnittlich hoher Makroporosität bei gleichzeitig hoher Stabilität zu erzeugen, stellt das Freeze-Gelation Verfahren dar. Über einen Gefriertrocknungsschritt können hier zudem die zu immobilisierenden Zellen in eine trocken lagerfähige Form überführt werden, wodurch Handhabung sowie Lagerung und Transport vereinfacht werden. Außerdem kann durch Wahl der Einfrierbedingungen und Zugabe von Füllstoffen zu dem SiO2-Sol entscheidend die Porenstruktur der Immobilisierungsmatrix beeinflusst und den Anforderungen entsprechend eingestellt werden. Allerdings zeigte sich, dass diese Methode nicht für alle Mikroorganismen gleichermaßen geeignet ist. Trotz diverser kryoprotektiver Maßnahmen konnte keine ausreichende Überlebensrate für sensible Stämme wie Aquincola tertiaricarbonis L108 erzielt werden. Neben der Erhöhung der Makroporosität der SiO2-Matrix wurde versucht das Sol-Gel-Verfahren mit einem flexiblen organischen Polymer (Na-Alginat) zu kombinieren, um eine Immobilisierungsmatrix mit einem weichen Kern zu erzeugen, der Zellteilung zulässt. Als zusätzlicher Vorteil des entwickelten Alginat/SiO2-Hybridmaterials erwies sich, dass dieses auch auf einfache Weise mittels Drucktechniken in definierten Spots abgelegt werden kann und so die Zellen in Arrays abgelegt werden können. Das Potential dieser Methodik für die Entwicklung von Ganzzellsensoren wurde am Beispiel der Grünalge Chlorella vulgaris als Modell-Sensorzelle demonstriert und für die Detektion von Atrazin als Modellsubstanz eingesetzt.:Inhalt Abstract 1 Danksagung/Vorwort 2 Inhalt 4 Abkürzungsverzeichnis 6 Abbildungsverzeichnis 8 Tabellenverzeichnis 11 1 Einleitung und Motivation 12 1.1 Zielstellung der Arbeit 14 2 Grundlagen 16 2.1 Sol-Gel-Verfahren 16 2.1.1 Geschichte des Sol-Gel-Verfahrens 16 2.1.2 Chemische Grundlagen des Sol-Gel-Verfahrens 18 2.1.3 Prekursoren (Vorläufermaterialien) 19 2.1.3.1 Alkoxysilane (Siliciumalkoxide) 19 2.1.3.2 Alkalisilikate 23 2.1.4 Modifizierungsmöglichkeiten der SiO2-Matrix 24 2.1.4.1 Chemische Modifizierung 24 2.1.4.2 Physikalische Modifizierung 25 2.1.4.3 Organisch-anorganische Hybridmaterialien 26 2.1.4.4 Alginat/SiO2-Hybridmaterialien 26 2.2 Sol-Gel-Immobilisierung von Mikroorganismen 28 2.2.1 Sol-Gel-Beschichtung von Trägermaterialien 30 2.2.2 Sol-Gel-Immobilisierung in (Hydro-)Gelkörpern 33 2.2.2.1 Sonderform: Freeze-Gelation-Formkörper 35 2.2.3 Sol-Gel-Immobilisierung mittels Drucktechniken 37 3 Material und Methoden 38 3.1 Mikroorganismen 38 3.2 Freeze-Gelation-Verfahren 39 3.2.1 Zellimmobilisierung 39 3.2.2 Kryoprotektektive Maßnahmen 40 3.2.3 Charakterisierung der Freeze-Gelation-Formkörper 40 3.2.4 Aktivitätsuntersuchung – Schadstoffabbau 41 3.3 Beschichtung von Trägermaterialien 42 3.3.1 Synthese des SiO2-Sols 42 3.3.2 Vorbehandlung der Trägermaterialien 42 3.3.3 Zellimmobilisierung 42 3.3.4 Charakterisierung der Trägermaterialien 43 3.3.5 Aktivitätsuntersuchung – Schadstoffabbau 44 3.4 Alginat/SiO2-Hybridgele 44 3.4.1 Synthese amino-funktionalisierter SiO2-Sole 44 3.4.1.1 Charakterisierung der amino-funktionalisierten SiO2-Sole 45 3.4.2 Vernetzung von Na-Alginat mit amino-funktionalisiertem SiO2-So (Erzeugung von Alginat/SiO2-Hydrogelen) 45 3.4.2.1 Vorbehandlung der Trägermaterialien 45 3.4.2.2 Charakterisierung der Alginat/SiO2-Hydrogele 46 3.4.3 Zellimmobilisierung 47 3.4.3.1 Charakterisierung der immobilisierten Zellen 48 3.4.4 Aktivitätsuntersuchung – Schadstoffdetektion 48 3.4.4.1 PAM-Fluorometer: Atrazin 48 4 Ergebnisse und Diskussion 50 4.1 Lösungsstrategien 50 4.2 Freeze-Gelation-Formkörper 54 4.3 Sol-Gel-Beschichtung von Trägermaterialien 65 4.4 Alginat/SiO2-Hybridmaterialien 74 4.5 Zusammenfassung der Ergebnisse 93 5 Schlussfolgerung und Ausblick 99 6 Literaturverzeichnis 102 7 Verzeichnis eigener Publikationen 109 ANHANG 111