Дисертації з теми "System of anaerobicaerobic bioreactors"

The thesis involves the development and implementation of a new and robust control system based on permeability trends but at the same time capable of reducing aeration proportionally to permeate flux. Permeability was made a key parameter for directly comparing temporary changes in membrane performance. Transmembrane pressure and flux were gathered every 10 seconds and permeability values were automatically calculated; different mathematical algorithms were applied for the signal filtering of on-line data. Short term and long term permeability trends were compared once a day, and a control action was applied proportionally to the short term/long term permeability ratio without exceeding the aeration flow recommended by the membrane suppliers.
El treball presentat a la tesi inclou el desenvolupament i la implementacio d’un nou sistema de control robust basat en les tendencies de la permeabilitat i, al mateix temps, capac de reduir l’aeracio de forma proporcional al flux de permeat. S’ha seleccionat la permeabilitat com el parametre clau per comparar directament els canvis temporals en el funcionament de les membranes. La pressio transmembrana i el flux es mesuren cada 10 segons i llavors la permeabilitat es calcula automaticament. El senyal de les dades recollides en linia es filtra adequadament mitjancant diversos algoritmes matematics. L’algoritme de control compara diariament una tendencia a curt termini de la permeabilitat amb una tendencia a llarg termini de la permeabilitat, i s’aplica una accio de control proporcional al quocient de les dues tendencies, sense excedir mai el cabal d’aeracio recomanat pels fabricants de membranes.

Composting is an increasingly popular method of municipal and residential waste management. Uniform composting is necessary to obtain a consistent product and to ensure the destruction of pathogens. It requires that a homogeneous temperature be maintained throughout the compost. To accomplish this, a compost vessel with a heat redistribution system (HRS) was designed, constructed and tested. This system was composed of a heat exchanger, plastic tubing and a copper coil filled with water. The system moved heat from the warmer center of the compost bed to the cooler areas at the outside and bottom of the bed without external inputs of energy. Once composting began, the temperature of the water inside the heat exchanger increased and buoyancy effects caused the water to flow through the copper tubing, distributing the core heat throughout the compost. Heat was also redistributed by conduction along the copper tubing. The HRS can be used in applications requiring assurance of uniform composting conditions and a high-quality product. Previously obtained test data suggested that the HRS system accomplished its goal, but high amounts of heat loss occurred through the four-inch exhaust vent. An air heat exchanger (AES) was added to reduce heat loss from the top aeration vent. A total of twelve experiments were run: four with the HRS, four with the AES and four controls. The vessels were fitted with thermocouples at 33, 54 and 84 cm from the bottom. The HRS vessels demonstrated higher temperatures during the first 10 days of the experiment (p<0.001).
Le compostage est une méthode de plus en plus populaire pour la gestion municipale et résidentielle des déchets. Le compostage uniforme est nécessaire pour obtenir un produit homogène de haute qualité et assurer la destruction des agents pathogènes. Il est donc essentiel de maintenir une température uniforme dans tout le compost. Pour mieux y parvenir, un récipient de compost équipé d'un système de redistribution de la chaleur (HRS) a été conçu, construit et vérifié. Ce système est composé d'un échangeur de chaleur, un tube en plastique, et une bobine de cuivre rempli d'eau. La digestion bactérienne des matières organiques cause une augmentation de la température de l'eau à l'intérieur du HRS et provoque un effet de flottabilité qui enchaîne un déplacement d'eau à l'intérieur du tube de cuivre, distribuant la chaleur du centre le plus chaud du compost vers les zones plus froides et ce, sans apport d'énergie externe. La chaleur est également redistribuée par conduction le long du tube de cuivre. Les résultats obtenus suggèrent que le HRS atteint son objectif, mais des pertes de chaleur ont été découvertes à la sortie d'air de 4 ̎. Un échangeur de chaleur à air (AES) a été ajouté pour réduire la perte de chaleur. Un total de douze expériences ont été effectuées : quatre avec le HRS, quatre avec l'AES et quatre contrôles. Les vaisseaux été équipés de thermocouples placés à 33, 54 et 84 cm du sol. Les vaisseaux équipés du HRS ont démontré des températures plus élevées au cours des 10 premiers jours de l'expérience (p < 0,001).

The work presented in this PhD thesis includes various partial studies aimed at developing a decision support system for membrane bioreactor integrated control. The decision support systems (DSS) have as a main goal to facilitate the operation of complex processes due to the multiple variables that are processed. For this reason, the research used has focused on aspects related to nutrient removal, and on the development of indicators or sensors capable of facilitating, automating and controlling the filtration process in an integrated way with the biological processes that taking place. Work has also been done on the design, development, implementation and validation of tools based on the knowledge made available by the automatic control and the supervision of the MBRs
El treball presentat en aquesta tesi doctoral inclou diversos estudis parcials amb l’objectiu final de desenvolupar un sistema d’ajuda a la decisió pel control integrat dels bioreactors de membrana. Els sistemes d’ajuda a la decisió, SAD o DSS de l’anglès, decision support system, tenen com a objectiu facilitar l’operació de processos complexes degut a multitud de variables de procés. Per aquest motiu, la recerca realitzada s’ha centrat en aspectes relacionats amb l’eliminació de nutrients, i en el desenvolupament d’indicadors o sensors pel procés de filtració capaços d’integrar-se amb els processos biològics que hi tenen lloc. També s’ha treballat en el disseny, desenvolupament, implementació i validació d’eines basades en el coneixement que facilitin el control automàtic i la supervisió dels MBR mitjançant un DSS.

The combination of two membrane technologies coupled together in series has become a standard technology when it comes to producing reclaimed water of high quality for potable reclamation or industrial applications. This combination of two membrane processes is referred to as integrated membrane systems (IMS). Despite the widespread experience gained utilizing such a process technology around the world, there are a number of aspects of the process technology which require further investigation including the fate of compounds of emerging concern (CEC), the control of N-Nitrosodimethylamine (NDMA) formation, the use of energy associated with the process and the total cost of producing the reclaimed water, and monitoring membrane integrity in RO treatment processes. The objective of this work was to further the knowledge in one aspect related to each of these four challenges and then bring each of these areas together in the discussion to understand whether proposing a decision support system for the online monitoring and operation of integrated systems would allow improvements to the current state-of-the-art.
La combinació de dos tecnologies de membrana acoblades en sèrie ha esdevingut un tecnologia consolidada degut a la capacitat de produir aigua d’elevada qualitat i potencialment reutilitzable per aplicacions industrials com fins i tot per ser potabilitzada. Tot i l’elevada experiència adquirida en aquests processos combinats, encara hi ha aspectes del procés que calen una investigació més profunda que inclogui el coneixement sobre l’eliminació dels compostos emergents, el control de la formació de N-Nitrosodimetilamines (NDMA), l’ús de l’energia associada amb el procés incloent el cost total de produir l’aigua reutilitzable, i el seguiment de la integritat de la membrana en el tractament amb osmosi inversa (OI). L’objectiu d’aquest treball recau en avançar en el coneixement dels aspectes relacionats amb cada un dels quatre reptes esmentats, per aconseguir discutir de forma conjunta la millor forma d’integrar aquest nou coneixement adquirit proposant un sistema d’ajuda a la decisió pel control i seguiment de l’operació de sistemes integrats de membrana (SIM).

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2006.
Wick, Tim, Committee Member ; Neitzel, Paul, Committee Member ; Fyhrie, David, Committee Member ; Garcia, Andres, Committee Member ; Guldberg, Robert, Committee Chair. Vita.

Les domaines d'application de la levure sont vastes, allant de la nourriture, de la brasserie à l'énergie verte. La levure Saccharomyces cerevisiae est l'espèce dominante à l’échelle mondiale. En outre, S. cerevisiae est également un organisme modèle important dans la recherche sur la biologie cellulaire moderne et est l'un des micro-organismes eucaryotes les plus étudiés.Ce travail se concentre sur le comportement des cultures de levures exposées à la pression induite par le CO₂ et l'O₂. La pression maximale étudiée est de 9 bar (A) car la pression la plus élevée pouvant être atteinte dans les bioréacteurs à l'échelle industrielle est de 8 bar (A). Afin d'exposer la culture de levure à des conditions de pression, de nouveaux bioréacteurs ont été construits et caractérisés. Deux expériences sont conçues : des expériences pour étudier la croissance des levures et des métabolites sous pression, ainsi que les expériences de biologie moléculaire pour mieux comprendre le comportement des cellules de levures sous différentes pressions d'O₂.La première expérience a permis de mieux comprendre l'influence des pressions de CO₂ et O₂ sur le comportement de culture de S. cerevisiae. En ce qui concerne l’impact du CO₂, l'étude a montré que la culture de levure a des comportements constants sous différentes pressions. Alors qu'en termes de pression O₂, sous 2 à 5 bars (A) de pression atmosphérique, les cellules de levure présentent des taux de croissance plus élevés par rapport à la pression atmosphérique. De plus, le glutathion moléculaire antioxydant a maintenu un équilibre redox. En dessous de 6 à 9 bars (A), la croissance cellulaire est inhibée et 9 bars (A) entraînent une accumulation excessive de glutathion oxydé.D'un autre côté, l'expérience moléculaire a permis de mieux comprendre le comportement de la culture sous les pressions d'O₂. L'étude de plusieurs gènes induits par le stress oxydatif a mis en évidence les effets cellulaires du stress oxydatif et la réponse des mécanismes moléculaires dans les cellules de levure. Il a été démontré que plusieurs gènes induits par le stress oxydatif étaient régulés à la hausse : le gène du facteur de transcription Msn2/4 et Yap 1, les gènes du métabolisme du glutathion GSH2 et GLR, ainsi qu'un gène de synthèse de la superoxyde dismutase SOD2
Yeast fields of application are extensive, ranging from food, brewing to green energy. The yeast Saccharomyces cerevisiae is the worldwide dominating species. In addition, S. cerevisiae is also an important model organism in modern cell biology research and is one of the most thoroughly studied eukaryotic microorganisms.This work focuses on the behaviour of yeast culture exposing to pressure induced by CO2 and O2. The pressure is set up to 9 bar (A) due to the highest pressure can be reached in industrial scale bioreactors is 8 bar (A). In order to expose yeast culture to pressure conditions, new bioreactors were built and characterised. Two experiments are designed: an experiment to investigate the yeast growth and the metabolites under pressure, as well as the molecular biology experiments to better understand yeast cells behaviour under various O₂ pressure.The first experiment has offered a better understanding of the influence of CO₂ and O₂ pressures on S. cerevisiae culture behaviour. Regarding the impact of CO₂, the study has shown that the yeast culture has consistent behaviours under different pressures. While, in terms of O₂ pressure, under 2 to 5 bar (A) air pressure, yeast cells show higher growth rates compared with atmospheric pressure. Furthermore, the antioxidant molecular glutathione kept a redox balance. Under 6 to 9 bar (A), the cells growth is inhibited and 9 bar (A) leads to the excessive oxidised glutathione accumulation.On the other hand, the molecular experiment has derived further insights on the culture behaviour under O2 pressures. The investigation of several oxidative stress induced genes has highlighted the cellular effects of oxidative stress induced by oxygen pressure and molecular mechanisms of oxidative stress response in yeast cell. It was shown that several oxidative stress induced genes were upregulated: transcription factor gene Msn2/4 and Yap 1, glutathione metabolism genes GSH2 and GLR, as well as a superoxide dismutase synthesis gene SOD2

Articular cartilage has a limited ability to heal due to its avascular, aneural, and alymphatic nature. Currently, there is a need for alternative therapies for diseases that affect articular cartilage such as osteoarthritis. Recently, it has been shown that tissue constructs, which resemble cartilage in structure and function, can be cultured in vitro in a cell-polymer bioreactor system. Bioreactors provide a three dimensional environment that promotes cell proliferation and matrix production. The primary objective of this study is to accurately simulate fluid mechanics using the lattice Boltzmann method for application to a cell-polymer bioreactor system. Lattice Boltzmann (LB) is a flexible computation technique that will allow for the simulation of a moving construct under various bioreactor conditions. The method predicts macroscopic hydrodynamics by considering virtual particle interactions. Derived from the Lattice Gas Automata, lattice Boltzmann allows for mass transfer, complex geometries, and particle dynamics. A primary goal is to characterize the accuracy of the LB implementation and eventually the shear stresses felt by a tissue construct in this dynamic environment. This information is important since recent studies show that chondrocytic function may depend on the mechanical stimuli produced by fluid flow. Hence, shear stress may affect the final mechanical properties of tissue constructs. In this study, numerical simulations are done first in 2D and then extended to 3D to test the LB implementation. Simulations of the rotating wall vessel (RWV) bioreactor are then undertaken. The results are benchmarked against computations done with a commercial CFD package, FLUENT, and compared with analytic solutions and experimental data.

Orientadores: Lucimara Gaziola de La Torre, Reinaldo Gaspar Bastos
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: A microfluídica é uma ciência que opera em pequenos volumes de fluídos dentro de canais em dimensões de micrômetros (10-6 m). Estes sistemas permitem controlar moléculas no espaço e no tempo, gerando resultados rápidos e confiáveis num sistema precisamente controlado e capaz de mimetizar ambientes celulares. Os dispositivos microfluídicos apresentam uma diversidade de geometrias aplicáveis para diversas áreas de pesquisas, sendo que a capacidade de formar gradientes permite avaliar as condições e o desempenho celular microbiano. Assim, este trabalho teve como objetivo desenvolver dispositivos microfluídicos capazes de formar gradiente de concentração difusivo e investigar sua aplicabilidade em bioprocessos. Diante disso, foram propostos três modelos de dispositivos usando materiais biocompatíveis: (i) dispositivo em base de vidro, denominado de Vidro-vidro; (ii) em base de vidro e poli dimetilsiloxano (PDMS), chamado de Vidro-PDMS e (iii) vidro e PDMS modificado quimicamente para tornar a superfície hidrofílica, Vidro-mPDMS. Os três dispositivos foram avaliados quanto à capacidade de formação de gradiente de concentração difusivo, os quais apresentaram um perfil linear. Além disso, validou-se o estudo do comportamento de Saccharomyces cerevisiae ATCC 7754 num gradiente de concentração de glicose de 0 a 40 g/L de glicose, sendo usado o dispositivo vidro-vidro. Foi observado que houve crescimento de células ao longo das câmaras microfluídicas, e isso possibilitou na determinação de parâmetros cinéticos, os quais não apresentaram diferença estatisticamente significativa com o cultivo em batelada convencional. As condições da microfluídica possibilitaram também a determinação da cinética de Monod, usando menores intervalos de gradiente. Portanto, este dispositivo microfluídico mostrou-se uma ferramenta com potencial para investigar comportamento celular frente à diferença de concentração e contribuirá para a otimização de bioprocessos através da determinação de parâmetros cinéticos
Abstract: Microfluidic is a science that operates in small amounts of fluids inside channels in dimensions of micrometers (10-6 m). These systems allow the precise control of molecules in space and time, generating fast and reliable results and it can also be used to mimics environment cellular . Microfluidic devices can be produced in diversity of geometries, it can be applied in several scientific areas and especially the formation of concentration gradients can be used to evaluate conditions and performance of microbial cell. Therefore, this work had the objective to develop microfluidic devices that are able to generate diffusive concentration gradients and investigate their applicability in bioprocesses. In this context, we propose three models of microfluidics devices using biocompatible materials: (i) Glass-based device, named glass-glass; (ii) glass and poli dimetilsiloxane (PDMS) based device, Glass-PDMS and (iii) glass and chemically modified PDMS (hydrophilic surface), Glass-mPDMS. The three devices were evaluated by their capacity of generating difusive concentration gradient, demonstrating linear concentration profile. Furthermore, the behavior of Saccharomyces cerevisiae ATCC 7754 inside of glucose concentration gradient ranging from 0 to 40 g/L were validated, using the glass-glass device . It was observed that cell growth along the microfluidic chambers, having determined the kinetic parameters, which was considered statistically similar to conventional batch cultivation. Conditions of microfluidics also allowed determination of the Monod kinetic, using smaller intervals gradient Therefore, the use of concentration gradient in microfluidic device is a potential tool for investigate of microbial cell behavior against the concentration difference and it can contribute to the optimization of bioprocesses through the determination of kinetic parameters
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestra em Engenharia Química

Ischemic heart disease is a major cause of human death worldwide owing to the heart minimal ability to repair following injury. Other than heart transplantation, there are currently no effective or long-lasting therapies for end-stage heart failure. Therefore, it is crucial to develop not only alternative therapies that potentiate heart regeneration or repair, but also new tools to study human cardiac physiology and pathophysiology in vitro. In this context, cardiac tissue engineering arises a promising strategy, as it is aimed at generating cardiac tissue analogues that would act as in vitro models of human cardiac tissue or as surrogates for heart repair. Thus, having 3D human cardiac tissue constructs resembling human myocardium could revolutionize drug discovery and toxicity testing, cardiac disease modelling and regenerative medicine. An strategy to obtain reliable cardiac tissue constructs is to mimic the native cardiac environment. The classical approach is based on seeding cardiomyocytes in biocompatible 3D scaffolds, and then culturing the construct in a biomimetic signaling system, usually a bioreactor. Although major advances have been made, the generation of thick and mature tissue constructs from human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) is still a challenge. Therefore, the hypothesis of our study is that the combination of hiPSC-CM with 3D scaffolds and appropriate regulatory signals may lead to the generation of mature human cardiac tissue constructs resembling human myocardium, both functionally and structurally. To address this, we have characterized a collagen-based 3D scaffold and established an efficient method for cell seeding into the scaffold. We have also developed a parallelized perfusion bioreactor system, which ensures an effective mass transport between cells and culture medium and allows culturing multiple replicas of tissue constructs. In addition, we have designed and fabricated a perfusion chamber including electrodes to electrically stimulate cells during culture, as well as to monitor tissue function. With this advanced 3D culture system, we have been able to generate thick 3D human cardiac constructs with tissue-like functionality. Our results indicate that perfusion of culture medium combined with electrical stimulation and collagen-based scaffold improve the structural and functional maturation of hiPSC-CM. In general terms, electrical stimulation has improved the structural organization, alignment and coupling of cardiomyocytes in our cardiac tissue constructs. Moreover, electrical stimulation has promoted the formation of synchronous contractile constructs at the macroscale with improved electrophysiological functions. Through the development of a new electrophysiological recording system, we report for the first time to our knowledge a technique that provides information about the electrical activity of intact cardiac tissue constructs in real time. Specifically, the combination of action potentials generated by hiPSC-CM composing cardiac constructs produces ECG-like signals, which could be monitored online. Finally, we have demonstrated the ability of stimulated human cardiac tissue constructs to detect drug-induced cardiotoxicity, as typical features of arrhythmias (e.g. prolongation of RR intervals and regular blockades) could be observed upon treatment with sotalol. Taken together, results indicate that macroscopic human cardiac tissue constructs with tissue-like functionality can be obtained through the use of our advanced 3D culture system. We have studied the effects of electrical stimulation on cardiomyocytes at multiple levels: molecular (presence, distribution and expression of cardiac proteins), ultrastructural (sarcomere width and presence of specialized cellular junctions), cellular (morphology and alignment), and functional (amplitude, directionality and strain of contractions, and electrophysiological recordings). Findings validate our in vitro approach as a valuable system to obtain 3D cardiac patches with an improved maturity and functionality. Importantly, the online monitoring system developed in this study can provide essential electrophysiological information of intact cardiac tissue constructs, which opens up myriad possibilities in the field of cardiovascular research.
La cardiopatia isquèmica és una de les principals causes de mort a nivell mundial. Exceptuant el trasplantament de cor, les teràpies actuals són insuficients per restablir la funció cardíaca. Per tant, cal desenvolupar teràpies alternatives que fomentin la regeneració i/o reparació del cor, així com també noves eines per estudiar la fisiologia i fisiopatologia cardíaca in vitro. Una de les estratègies més prometedores és l’enginyeria tissular cardíaca, ja que té com a finalitat generar constructes de teixit cardíac que mimetitzin el teixit real. Aquests constructes podrien utilitzar-se com a models in vitro del miocardi humà i també com a empelts per reparar el cor malmès. Per obtenir constructes de teixit cardíac humà cal reproduir l’entorn cardíac real. Una de les estratègies més habituals consisteix en sembrar cardiomiòcits en una estructura 3D (bastida), i després cultivar el constructe en un sistema de senyalització biomimètic, normalment un bioreactor. Tanmateix, generar constructes grans i semblants al miocardi humà adult a partir de cardiomiòcits humans derivats de cèl·lules mare de pluripotència induïda (hiPSC-CM) segueix sent un repte. Així doncs, la hipòtesi d’estudi és que combinant hiPSC-CM amb una bastida 3D i estímuls biofísics adequats, es podrien generar constructes de teixit cardíac semblants al miocardi humà tant a nivell estructural com funcional. Per abordar la hipòtesi, en aquest treball s’ha caracteritzat una bastida 3D constituïda principalment per col·lagen i s’ha definit un mètode eficient per sembrar cardiomiòcits dins l’estructura. A més a més, s’ha desenvolupat un bioreactor de perfusió de sistema en paral·lel que assegura un transport de massa efectiu entre les cèl·lules i el medi de cultiu. També s’ha dissenyat una càmera de perfusió que inclou elèctrodes per estimular elèctricament les cèl·lules durant el cultiu, així com també per monitorar la funció del teixit artificial. Amb aquest avançat sistema de cultiu, s’han generat constructes de teixit cardíac humà 3D amb una funcionalitat semblant a la del teixit real. A més a més, el sistema ha permès monitorar l’electrofisiologia del teixit artificial en temps real, així com també demostrar el paper crucial de l’estimulació elèctrica per obtenir constructes amb una funcionalitat òptima.

Bioprocesses often require considerable expertise to insure proper operation and performance. However, they are also rich in both data and factual knowledge. Supervision and control can be improved by utilizing information from all available sources. Knowledge about dynamics may be incomplete, particularly during upsets or equipment failure. Additional information derived from past data trends, expert operators and the literature can be very useful in process analysis, fault detection, process salvaging and optimization. In this work an expert system which utilizes on-line real-time data as well as archived data trends and operator expertise to analyze a process is developed. A modular format (the hierarchical modular structure) is designed to organize the large body of information--typical of the knowledge likely to be encountered in a bioprocess. The highly modular format allows for knowledge to be classified as general, process configuration specific or system specific. The expert system is challenged with process faults which are not easily noticed without a qualitative understanding of process dynamics. The supervisor is able to detect the unusual situations by first generating a process assessment based on a synthesis of all available on-line measurements in conjunction with known qualitative information. Once a global process picture is established, the expert system can decide upon appropriate strategies for recovery or backup control. Process analysis, recovery and optimization were experimentally demonstrated using Clostridium acetobutylicum fermentation as a sample system. Successful expert system process analysis was able to detect sensor failures and media feed flow failures. In addition, effective backup pH control and process recovery strategies were suggested and implemented in real-time by the expert system supervisor. The expert system ability to aid on-line optimization by shortening the time needed to analyze process transients may result in faster attainment of the region of optimal performance.

碩士
國立臺灣大學
應用力學研究所
106
Disease-on-a-chip is one of the leading research fields in personalized medicine for future health care. Among the major chronical diseases, cancer is the second leading cause of death next to cardiovascular disease. It is due to its high heterogeneity, and identification of an effective treatment sets a big challenge to cure cancer. Hence, the method to develop tumor-on-a-chip has drawn much attention in this field. By using a tumor-on-a-chip system for drug screening or genetic screening, we can design personalized medical treatment for different patients according to the screening results. In this study, two kinds of microfluidic systems to generate two different culture environments through generating different flow patterns are designed: (1) Nutrition-deprived condition, which offers a slow nutrient support through diffusive flow, (2) Convection-dominated condition, which offers a rapid nutrient support through interstitial convective flow to achieve a rapid nutrient supply. In this thesis, different initial cell numbers and nutrition supply to investigate the growth of a microtumor are studied and compared. A long microfluidic channel is designed to create a diffusion dominant microtumor chamber, and the level of nutrition deprivation is controlled by two methods. One is to control the initial loading concentration of cells, and the other is to control the perfusion rate of the nutrition supply diffuses from two side channels. Our experimental finding suggests that a lower initial cell concentration can developed into continuous microtumors across the 1 mm by 2 mm microchamber with a uniform growth rate. The growth of microtumors can be regulated by the level of nutrition deprivation. It suggests that nutrition deprivation could play an important role on the initial development of a microtumor. Applying this model system, it could potentially be optimized for the tumor growth among different kinds of tumor cells. Thus, we can provide a personalized system to develop patient specific microtumors to study the heterogeneity of tumors between patients and to identify an effective treatment. In the second study we attempt to connect microvasculature into microtumor tissue to mimic nutrition and oxygen supply of tumor. Once the tumor is formed, cancer cells can directly uptake surrounding nutritions or exclude the metabolic waste by the diffusion between the cells; however, once the tumor grows beyond the diffusion limit of 100 μm, the diffusion between the cells is insufficient, resulting in hypoxic conditions. Thus, tumors can tirger angiogenic process to create an environment for tumor growth. Otherwise, the tumor is anoxia (<2%) due to limited oxygen induction, and acidification due to reprogramming of metabolic procss, it eventually creates apoptosis and necrosis. We further design two kinds of microfluidic systems to generate two different microenvironments through different stages of tissue development. Both are nutritionally deprived and provide slow nutritional support through the diffusion transport. We successfully develop a large-scale vascular network and microtumor. It was shown that the angiogenic process is induced by the adjacent fibroblast chamber. In the second study, we successfully developed a large-scale microtumor next to developed vasculature. This platform can provide complete physiological and engineering conditions, and it is believed that vascular biomimetic cancer can be realized on this bioreactor. All microfluidic platforms are verified with finite element analysis. The microfluidic and driving pressures of different chamber height are analyzed. The flow velocity of the microfluid in the microchannel, the pressure gradient of the chamber and the velocity distribution in the chamber also are discussed. Finally, time-dependent concentration analysis is performed. The results show that we successfully developed microfluidic systems that can be applied to microtissues. These newly developed systems break through the limitations of the mm size of microtumor and blood vessels in lab-on-a-chip systems. The 3-D tissue not only improves human compatibility, and in the future, patient-specific tumor models or different cancer cell types also can be applied for personalized health before clinical applications.

As doenças ósseas são um problema que afeta grande parte da população mundial e têm tendência a aumentar no futuro. Apesar de o tecido ósseo ter capacidade de regeneração, quando uma fratura ultrapassa um ponto crítico o osso não possui capacidade de autorreparar esse defeito. Atualmente estes defeitos são tratados a nível médico através da utilização de implantes cerâmicos ou metálicos que originam respostas imunológicas por parte do hospedeiro e como consequência são rejeitados ao fim de algum tempo pós implantação. Além deste facto, a não bioatividade destes biomateriais restringe a reparação total do tecido e a recuperação das suas propriedades biológicas e funcionais. Terapias como a injeção in situ de células encapsuladas em biomateriais bioativos e biodegradáveis para aplicação na regeneração óssea têm surgido nos últimos anos como uma abordagem alternativa e vantajosa. Neste contexto, os sistemas capsulares apresentam-se como os mais vantajosos pois não só protegem as células administradas, mas também permitem a troca de nutrientes/metabolitos de uma forma eficaz. Este facto garante a viabilidade do sistema ao longo de maiores períodos de tempo, contribuindo assim para uma melhor regeneração do tecido lesado. No entanto, a implantação de microcápsulas contendo células tem-se revelado bastante desafiante devido à sua fraca interação com os tecidos circundantes, sendo comum o seu deslocamento do local inicial da implantação. Como forma de ultrapassar estas limitações, este trabalho teve como objetivo desenvolver uma cápsula magneticamente responsiva como sistema de entrega de células aderidas a micropartículas. A inclusão de reposta a estímulos magnéticos tem como objetivo permitir a fixação das cápsulas in situ no local de implantação através da utilização de um campo magnético externo. Para tal foram inicialmente produzidas nanopartículas magnéticas de óxido de ferro (≈42.69 nm), que foram ressuspendidas numa solução de Policaprolactona que por emulsão óleo-em-água deram origem a micropartículas magneticamente responsivas (μPCL[MNPs]) cujo tamanho médio foi de 40.5±13.2 μm antes de serem peneiradas. A gama de tamanhos a ser utilizada foi de 40-63μm para potenciar a adesão celular daí a necessidade de peneirar as partículas (55.3±9.00 μm). As micropartículas μPCL[MNPs] foram depois sujeitas a um revestimento de colagénio I para promover a adesão de células pre-osteoblásticas (MC3T3-E1) após a encapsulação. A encapsulação foi feita utilizando uma mistura de alginato, μPCL[MNPs] e células, que por gelificação ionotrópica originou a formação de estruturas esféricas como demonstrado pelas imagens de microscopia ótica. As microesferas foram posteriormente sujeitas a um processo de revestimento por deposição sequencial de polielectrólitos utilizando a técnica camada-a-camada (LBL). Esta abordagem permitiu a obtenção de cápsulas com um núcleo liquefeito e com integridade devido ao seu revestimento LBL. De seguida foi avaliada a viabilidade celular e a atividade metabólica das células encapsuladas. Resumindo, as cápsulas produzidas mantiveram a viabilidade celular e atividade metabólica ao longo de 7. Estudos ex vivo demonstraram que a utilização de um campo magnético externo permite a fixação das cápsulas no local onde foram colocadas mesmo quando sujeitas a lavagem com um fluído. Os resultados obtidos evidenciam que é possível fixar as cápsulas in situ após serem implantadas e abrem a oportunidade de utilizar estes sistemas em aplicações biomédicas no futuro próximo
Bone related disorders are a problem which affects most of the world population with a tendency to increase. Although the bone tissue has regenerative capacity, when a fracture exceeds a critical point the bone does not have the capacity to self-repair this defect. Currently these defects are treated at the medical level using ceramic or metallic implants which give rise to immune responses by the host and as a consequence are rejected after some time after implantation. Furthermore, the non-bioactivity of these biomaterials restricts the total repair of the tissue and the recovery of its biological and functional properties. Therapies such as the in situ injection of cells encapsulated in bioactive and biodegradable biomaterials have emerged in recent years as an alternative and advantageous approach for bone regeneration. In this context, capsular systems are the most advantageous because they not only protect the cells administered, but also allow the exchange of nutrients / metabolites in an effective way. This ensures the viability of the system over longer periods of time, thus contributing to a better regeneration of the damaged tissue. However, the implantation of microcapsules containing cells has proved to be quite challenging because of its poor interaction with surrounding tissues, and its displacement from the initial site of implantation is common. As a way of overcoming these limitations, this work aimed to develop a magnetically responsive capsule as a delivery system for cells adhered to microparticles. The inclusion of a response to magnetic stimuli aims to allow the capsules to be fixed in situ at the implantation site through an external magnetic field. For this purpose, iron oxide magnetic nanoparticles (≈42.69 nm) were initially produced, which were resuspended in a solution of polycaprolactone which, by oil-in-water emulsion, gave rise to magnetically responsive microparticles (μPCL [MNPs]) with an average size of 40.5 ± 13.2 μm. The range of sizes to be used was 40-63μm to enhance cell adhesion, hence the need to sieve the particles (55.3 ± 9.00 μm). Microparticles μPCL [MNPs] were then subjected to a coating of collagen I to promote adhesion of pre-osteoblastic cells (MC3T3-E1) after encapsulation. The encapsulation was done using a mixture of alginate, μPCL [MNPs] and cells, which by ionotropic gelation gave rise to the formation of spherical structures as demonstrated by optical microscopy images. The microspheres were then subjected to a coating process by sequential deposition of polyelectrolytes using the layer-by-layer (LBL) technique. This approach allowed to obtain capsules with a liquefied nucleus and with integrity due to their coating LBL. Cell viability and metabolic activity of the encapsulated cells were then evaluated. In summary, the capsules produced maintained cell viability and metabolic activity over 7 years. Ex vivo studies demonstrated that the use of an external magnetic field allows the capsules to be fixed where they were placed even when subjected fluid wash. The results show that it is possible to fix the capsules in situ after being implanted and open the opportunity to use these systems in biomedical applications in the near future
Mestrado em Biotecnologia

碩士
國立屏東科技大學
動物疫苗科技研究所
97
The industrial enzymes and therapeutic recombinant proteins are often produced by E. coli expression systems, the physiology and metabolic mechanism of E. coli were mainly investigated, and utilized in the biological technology. However, the traditional production vessels of bacterium recombinant protein antigens are flask systems, the culture space limited, the medium share to be able gradually to consume unable to supplement the oxygen. Certainly, they do not suit the long-term culture and the large-scale production process. Streptococcus suis, S. suis was based on capsule polysaccharide, 35 serotypes of the S. suis have been identified, causing arthritis, meningitis, septicemia and sudden death, now it is also an zoonotic agent for humans. Recently research indicated that a new surface protein Sao of S. suis reacted with 30 serotypes of convalescent-phase sera from pigs clinically infected by S. suis. Our rSao (recombinant surface antigen one) was recognized by challenged swine serum. The antibody titers of immunized inactivated S. suis plus rSao mice and swine were significantly higher than control (p&lt;0.01). The antibody titers of immunized swine with rSao plus subunit inactivated vaccine were also be enhanced. After challenge non-vaccinated as control swine showed symptoms of fever, anorexia, depression, and arthritis. The anatomical lesions were reduced 60% in rSao group than control after challenge with serotype 1; and reduced 43% after challenge with serotype 2. These data indicated that rSao can induce cross-protection with different serotypes of S. suis. The immunofluorescences analysis CD4+/CD8+ cells showed that immunized rSao swine were significantly higher than control (p&lt;0.01) and toward the Th1 pathway. In this study, we used 10-liter stirred tank bioreactor to culture E. coli with designed medium and increase the production efficiency by batch culture. To explore the relationship between carbon and oxygen in the fermentation process and E. coli growth condition, leading to increase the yield of recombinant proteins, to develop the production technique platform for the low cost vaccine antigens.

碩士
國立中央大學
環境工程研究所在職專班
105
The purpose of this paper study was to investigate the effluent of membrane bioreactors for treatment industrial park sewerage system. Long-term experimental results show that the main function of membrane bioreactors is to remove the suspended solids in the wastewater and by the way remove the insoluble chemical oxygen demand, but the average removal rate of dissolved chemical oxygen demand is less than 50% and the removal rate of heavy metals in sewage also showed unstable results. To enhance the industrial park sewerage system membrane bioreactors for chemical oxygen demand, heavy metal removal rate, the first implementation of membrane bioreactors and the traditional biological treatment system similar process of chemical oxygen demand and dissolved chemical oxygen removal efficiency detection and comparison, and to identify the two systems of different treatment unit chemical coagulation-flocculation dosing reaction unit with chemical oxygen demand and heavy metal removal rate function, and by the simulated Jar tests to verify the hypothesis, Collecting the whole operation test and experimental result evaluation to seek improvement policy. It is hoped that by membrane bioreactors to deal with industrial park sewerage system wastewater, the first need for the industrial park manufacturers pre-treatment processes and into the membrane bioreactors raw wastewater quality control to reduce the formation of membrane fouling substances and high concentrations of raw wastewater into the plant is absolutely the key factor of membrane bioreactors smooth operation. And no treatment system can be completely effective for any pollutions to remove from the raw wastewater, the results show that membrane bioreactors for treatment industrial park sewerage system at least with chemical coagulation-flocculation dosing reaction unit, and investigate the other into the plant wastewater characteristics need to configure other wastewater treatment unit with the membrane bioreactors can make the full advantage of processing function.

This study revealed that ammonium ion exchange of natural zeolite could be a feasible method of nitrogen removal and recovery from permeate from anaerobic membrane bioreactors (AnMBRs). NaCl concentrations optimized for chemical regeneration in batch experiments did not match those in continuous column tests. Instead, the mass ratio of Na+ to Zeolite-NH4+-N was significant for improving regeneration efficiency in column experiments; this mass ratio was 750 g Na+/g Zeolite-NH4+-N required for regeneration efficiency over 90% in 2 hours at pH 9. ???To decrease the NaCl dose in regeneration of exhausted zeolite, a high pH regeneration method was developed using an NaCl concentration of 10 g/L at pH 12 (the mass of Na+ to Zeolite-NH4+-N of 4.2 ) which achieved a regeneration efficiency about 85%. The recovery of ammonium nitrogen from the exhausted zeolite was assessed with air stripping followed by ammonia collection in an acid scrubber. The effects of shaking and air stripping were investigated in batch tests and the results showed the superiority of air stripping over shaking. Liquid circulation and air flow rates were varied for optimization of ammonia recovery in a continuous zeolite-packed column combined with a regeneration chamber and a stripping column. The liquid circulation rate had no significant effect on either the regeneration efficiency or the ammonia transfer efficiency from ammonium nitrogen to ammonia gas, while the ammonia transfer efficiency significantly increased with the air flow rate.??? Furthermore, the effect of pH on ammonia recovery was tested. Both the regeneration efficiency and the ammonia transfer efficiency were significantly improved with increasing pH. When the pH was increased from 9.5 to 12, the regeneration efficiency increased from 9.2% to 84% and the ammonia transfer efficiency increased from 54% to 92%. The nitrogen recovery process that combines zeolite ammonium exchange and air stripping can decrease chemical costs for regeneration of exhausted zeolite and efficiently collect ammonium nitrogen to be reused as fertilizers. Hence, the integrated nitrogen process can resolve the challenge of nitrogen removal in anaerobic membrane bioreactors treating organic wastewater in sustainable manners.

Many industries use, or produce, metal-containing solutions which must be treated for reuse or discharge to sewer. One such treatment is biological and both living and dead materials have been investigated for the abstraction of metal ions from solution. Studies on systems containing only a single biosorbent are well documented, and mostly involve optimisation of biosorption capacities and metal uptake rates through modification of Biological Support Particle (BSP) size and surface characteristics. Literature on dual biosorbent studies is sparse. The commercial application of biosorption technology in wastewater treatment remains largely unexplored and unexploited. The primary objective here was to assess the potential of forced-upflow packed-bed bioreactors, containing dual biological sorbents, for treating a synthetic wastewater containing copper, zinc and cadmium, at both laboratory- and pilotscale. Pine bark was selected as BSP since it is an abundant, relatively cheap, agricultural waste product in South Africa, and is known to sorb metal ions. Initial experiments aimed to optimise biofilm development on the pine bark surfaces, since microbial biomass is also known to sequester metal ions. Systems comprising either one, or both, these biosorbents were compared for their efficiency in metal removal. The effects of type, size, and state of decomposition, of the pine bark, the addition of supplementary nutrients (Voermolas) and the mixing conditions, on the metal biosorption capacity and reaction kinetics of the systems were also studied. All experiments were conducted at an initial metal concentration of 100mg.ℓ⁻¹with both composted and uncomposted pine bark as BSP. The former supported microbial colonisation and resisted biofilm sloughing, but degraded rapidly causing engineering difficulties. Uncomposted pine bark showed the same ability, but was also physically more robust. Organic compounds leached from the pine bark did not hinder microbial colonisation of the BSP; rather they served as additional nutrients. Literature studies suggest that these compounds would not significantly compromise the COD or increase the toxicity of the final effluent. Biofilms developed without supplementary nutrients, but Cd²⁺ and Zn²⁺ were sorbed more effectively in bioreactors containing Voermolas (39% and 38% Cd²⁺ removal, 36% and 32% Zn²⁺ removal, in 0.2% and 0.1% Voermolas solutions respectively) than in unsupplemented systems (25% Cd²⁺ removal and 20% Zn²⁺ removal). Conversely, Cu²⁺ was removed most efficiently in the absence of supplementary nutrients. Based on biosorption of the target metal ions, 0.1% (v/v) Voermolas was the most effective concentration of supplementary nutrients. Raw, un-colonised pine bark nuggets (16-24mm), and plastic bioballs (commercially available, bespoke BSP), were compared in laboratory-scale bioreactors by measuring the decrease in residual metal ion concentrations over time, and changes in the solution pH. These experiments showed that the two BSPs did not differ significantly in their performance as a support matrix, or as a metal sorbent (30.6% and 32.6% of metal ion remained in solution when using bioballs and pine bark respectively). However, the presence of a biofilm on both these BSPs, improved the overall performance of the bioreactors significantly (for the bioball BSP, residual metal ion levels decreased from 30.6%, in the absence of a biofilm, to 11.0% with a biofilm present. Similarly, for the pine bark BSP, residual metal ion levels decreased from 32.6%, in the absence of a biofilm, to 7.3% with a biofilm present). A cost comparison of the two BSPs showed that raw pine bark nuggets were available at less than 0.1% of the cost of the bioballs. At pilot-scale, modelled kinetic data compared poorly with experimentally determined results, but minimum residual metal concentrations for Cu (1.7mg.ℓ⁻¹) and Zn (4.2 mg.ℓ⁻¹) were below South African (eThekwini Municipality) regulatory limits for discharge to sewer (5mg,ℓ⁻¹ for both), and sea outfall (3mg.ℓ⁻¹ Cu and 20mg.ℓ⁻¹ Zn). However, for Cd the final residual metal concentration (5.6mg.ℓ⁻¹) was above the regulatory discharge threshold for any receiving system. Although some of the effluents from the system investigated could not be legally released into the municipal sewer system without further remediation, the study showed that a system combining living and dead biomass in a single reactor is capable of significantly reducing dissolved metal concentrations in synthetic wastewaters without temperature or pH adjustment. Furthermore, such a system can operate at pilot-scale, where a pine bark matrix represents a significant cost saving over conventional plastic BSPs.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.