Made available in DSpace on 2014-10-09T12:55:35Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:57Z (GMT). No. of bitstreams: 0 / A Prolactina (PRL) é um dos hormônios mais versáteis em termos de ação biológica. Sua ação mais conhecida está relacionada com o estímulo da lactação e regulação do crescimento e da diferenciação da glândula mamária; também apresenta importante aplicação diagnóstica. Somando os crescentes estudos sobre suas possíveis aplicações terapêuticas, fica cada vez mais notória a necessidade da obtenção desse hormônio puro, biologicamente ativo e na sua forma autêntica.O objetivo fundamental desse projeto foi a produção de hPRL em escala laboratorial a partir de bactérias (E.coli) modificadas geneticamente, utilizando um sistema de expressão baseado no promotor Lambda () PL, o mesmo utilizado com sucesso em nosso laboratório na expressão do hGH. Descrevemos nesse trabalho um processo de cultivo em biorreator, onde não foi utilizado o repressor cIts, uma proteína termo-sensível que usualmente é utilizada para inibir o funcionamento do promotor PL durante crescimento a 30ºC. O processo de cultivo apresenta basicamente três etapas: na primeira etapa o crescimento é realizado sem adição contínua de nutrientes (cultivo em batch), na segunda etapa ocorre adição contínua de nutrientes e carboidrato (cultivo em fed-batch) e na última etapa é realizada a ativação, caracterizada pelo aumento da temperatura mantendo-se a adição de nutrientes e carboidrato. Esse processo de fermentação rápido e flexível, com duração média de 20 horas, permitiu obter uma biomassa final correspondente à densidade óptica de aproximadamente 30 A600nm (unidades ópticas de absorbância em 600nm) e com uma expressão da ordem de 1g de hPRL mL-1 A600 -1, as mais altas já relatadas para secreção de prolactina no espaço periplásmico. A hPRL monomérica foi purificada e caracterizada por métodos físico-químicos e biológicos, os quais confirmaram a sua atividade biológica e imunológica, o seu correto processamento e uma massa molecular relativa (Mr) de 22.906. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Dissolved organics in wastewater samples were separated into three size fractions (0-1,000 amu, 1,000-10,000 amu, and 10,000 amu-0.22 m) using ultrafiltration (UF) membranes. The mass distribution within each fraction was adjusted by using a new permeation coefficient model to account for membrane rejection. Dissolved organic and soluble BOD (sBOD) removals in a trickling filter were studied for the different size fractions. The Logan trickling filter model was recalibrated and used to generate predicted removals by size fraction of sBOD, dissolved organic carbon (DOC), and biodegradable DOC (bDOC) for a given influent. Although there was moderate agreement between observed and predicted removals, more investigation is needed to explain shifts in material between different size fractions. Of the three parameters, bDOC may offer a better parameter for modelling trickling filter performance than sBOD.

No description available.

Bacterial biofilms consist of communities of cells encased in self-produced extracellular polymeric substance (EPS) at the interface of different phases. While most of our understanding of biofilms to date has been obtained from submerged biofilms formed at the solid-liquid (S-L) interface, the interface between air and liquid provides a niche for the formation of biofilms with unobstructed access to both liquid and gaseous phases, which is of ecological, clinical and commercial significance. But there is a paucity of information on air-liquid (A-L) interface biofilms. Cultivation and characterization of A-L interface biofilms are still limited to traditional methods created for S-L interface biofilms, mostly based on microtiter plates and tubes. In this thesis, two bioreactors have been designed, fabricated and validated to culture A-L interface and air-liquid-solid (A-L-S) interface biofilms. / The first bioreactor, named hanging drop biofilm reactor (HDBR), has been constructed on an open microfluidic platform to hold hanging droplets culturing biofilms in a suspended fashion. Hanging droplets containing microorganisms were accessed from the topside of the microfluidic platform, whilst measurement and harvesting were performed on the underside. The system was driven by evaporating flows. No additional electronic or mechanical actuation was needed. Biofilms were formed in hanging droplet biofilm reactors with bacterial suspensions. After 24 h of incubation under optimized conditions, biofilm-positive phenotype Bacillus subtilis developed A-L biofilms eliciting distinct developmental stages. Formation and dispersal of A-L interface biofilms were for the first time analytically characterized. Biofilms harvested by dripping droplets showed a spherical cap shape with porous structures. This is the first reported attempt to culture A-L interface biofilms inside a hanging droplet on an open microfluidic platform under evaporation flow. / The second bioreactor, named segmented-flow microfluidic air-liquid-solid interface biofilm reactor (SFMBR), has been implemented to grow A-L-S interface biofilms on chip. A distinct A-L-S interface was made available by introducing a gas phase into successive nutrient flows with a T-junction design, forming a segmented gas-liquid flow. Velocity fields were computed by numerical simulation, involving not only the hydrodynamic fields in the gaseous and aqueous phases, but also of the air-liquidand solid-liquid interfaces. Formation of A-L-S interface biofilms along microfluidic channels was observed and characterized by microscopy techniques non-destructively. The effect of hydrodynamics, interface topology and channel hydrophobicity on A-L-S interface biofilm formation was simulated and tested. Biofilms interacted with the shear forces introduced by the passage of A-L interfaces of an air slug with a shear stress of about 0.66 Pa and relaxed after the A-L interface passed. This device is a low-cost on-chip biofilm reactor to culture A-L-S interface biofilms and to measure in situ biological and mechanical properties of growing biofilm. / We have successfully constructed and validated two novel microfluidic strategies utilizing the interfacial phenomenon at the air-liquid interface and evaporating flow to culture and investigate the complex biology of A-L and A-L-S interfaces biofilms. / 细菌生物膜(生物膜)由生长在固、气、液相的界面上的细菌包裹于由其产生的细胞外间质聚合物内组成。固-液界面生物膜可以利用微流系统低耗材高效培养检测。但没有微流系统可研究生长在气-液界面的生物膜。为了填补这个空白，本论文设计了两种微流生物膜反应器(反应器)培养气-液和气-液-固界面生物膜。 / 一种反应器是培养气-液界面生物膜的基于蒸发产生的微流动和微循环的倒悬液滴反应器。我们创新的利用蒸发产生的微流和微循环增加微流系统中材料混合和供氧。我设计了两种倒悬液滴反应器:倒悬液滴气-液界面反应器微孔板和微流气-液界面反应器在静态和流动培养液条件下培养气-液界面生物膜。我们监控并分析了气-液界面生物膜形成过程。第一次报道了气-液界面生物膜生长阶段的划分。了解反应器的特性，如微流动和反应器形态等对应用反应器有至关重要的作用。我们使用粒子图像测这和数值模拟的方法研究了反应器特性。设计了滴落打印的方法收获并离线测量生物膜。这是第一种可以方便、可重复收获生物膜的方法。我们研究了收获后生物膜的抗药性和微结构，发观倒悬液滴反应器培养的生物膜对干燥和灭菌过程有很高的抗性。我们还首次发现了细菌由纳米管连接的气液界面细菌生物膜。 / 另一种反应器是基于微流分段气-液流的分段流微流反应器。这是首个培养气-液-固界面生物膜的微流反应器。我们创新性的应用了分段气-液流为生物膜滴定供养。我们的微流反应器模拟工业标准方法培养气-液-固界面生物膜的反应器。为了研究生物膜在反应器中的生长和流动对生物膜的影响，我们监测了生物膜的生长，用数值模拟的方法研究了分段流的流动。发现了单层菌落储水的现象。我们还首次报道了微流系统中，用粒子图像測速和数值模拟的方法研究生物膜的弹性松弛过程。 / 这里设计的基于液滴的高通量，高效率，低成本，操作简单的生物膜反应器可以加快生物膜的研究。 / Liu, Songlin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2015. / Includes bibliographical references (leaves 115-130). / Abstracts also in Chinese. / Title from PDF title page (viewed on 19, September, 2016). / Liu, Songlin. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Bacterial strain Pseudomonas sp. ADP is capable of degrading atrazine via an enzymatic pathway in six sequential steps to yield carbon dioxide and ammonia. Atrazine is a persistent herbicide that frequently contaminates soil, drinking water, and ground water throughout areas of heavy use in the United States. A biological remediation approach using Pseudomonas sp. APD is considered as an effective, cost-efficient, and environmentally conscious method of decreasing atrazine concentration in areas of high contamination. Each enzyme in the degradation pathway is encoded by a corresponding gene, AtzA-AtzF, and is located on a self-transmissible 108-kb plasmid. Due to their prevalence in nature, and their unique genetic and physical characteristics, biofilms are of great interest in the field of bioremediation. Biofilms exhibit high tolerance for harsh environmental stressors/conditions, prodigious potential for recalcitrant compound entrapment via an extracellular polymeric matrix, quorum sensing, and increased horizontal gene transfer compared to their planktonic counterparts. Despite frequent genetic and chemical analyses performed on atrazine-degrading genes on planktonic cells of strain Pseudomonas sp. APD, the genetics and degradation potential of Pseudomonas sp. ADP biofilms is relatively unexplored. Real-time quantitative PCR was used to differentiate the expression of six genes involved in the process of atrazine degradation. Relative expression experiments revealed no statistically significant difference in the expression of atrazine-degrading genes in Pseudomonas sp. ADP cells grown as biofilms relative to Pseudomonas sp. ADP cells grown as planktonic cells. In biofilms alone, the expression of genes AtzDEF was differentiated via temperature of biofilm growth in cells grown at 25, 30, and 37 degrees. Analytical techniques, including GC-MS and HPLC, were used to elucidate atrazine remediation potential of Pseudomonas sp. ADP biofilms and our previously collected genetic data. Stable decreases in atrazine degradation following a first-order kinetic model have been demonstrated for planktonic cells compared to a complex degradation pattern, including transient increases, observed for corresponding biofilm-mediated cells. This is attributed to the unique structure of the biofilm and the potential of atrazine to be entrapped in the substances of the extracellular polymeric matrix and subsequently released into the effluent. Overall, the biodegradation efficiency was higher for Pseudomonas sp. ADP biofilm-mediated cells compared to their planktonic counterparts. A novel methodology of using confocal microscopy and in situ reverse transcription was proposed for optimization to visualize the expression of atrazine-degrading genes in fixed Pseudomonas sp. ADP biofilms. The sugar composition of Pseudomonas sp. ADP was evaluated using fluorescent lectin binding analysis and was determined to exhibit a prominent level of diversity and dependent upon growth medium. The results from these experiments will play a role in application of biofilms grown in bioreactors for atrazine remediation throughout areas of persistent and high contamination throughout the US. The new step in methodology development of an in situ visual gene expression technique can be extended to bioremediation of alternate recalcitrant compounds. The results may also be aid progress in alternate biofilm-related studies in medicine & human health, metallurgy, and engineering.

Graduation date: 1994

Graduation date: 1994

This study involves the development of a biofilm reactor that supports growth of a deep biofilm on a gas permeable membrane. The reactor solution is not aerated, and oxygen is supplied through the membrane. The reactor is termed a substratum-aerated-biofilm reactor or SAB. With adequate concentrations of electron-donors and electron-acceptors, a deep biofilm grows on the membrane and is comprised of different layers of bacterial activity. The aerobic layers are near the membrane support, while the anaerobic layers are near the biofilm-liquid boundary. In the SAB, the substrate diffuses from the bulk liquid into the biofilm to react. Oxygen diffuses through the membrane into the biofilm. All products likewise are transported by molecular diffusion through the biofilm and into the bulk liquid. The reactors consisted of a reactor wall made of a plexiglass cylinder with the gas permeable membrane supported on a shallow rotating cup. The cup was designed so that the cup and the membrane function as a flat plate. The flat plate was utilized for support of the biological growth, transfer of oxygen, and mixing of the bulk liquid and the gas phase. The experiments were conducted in completely mixed, continuous-flow reactors maintained at 25°C with a hydraulic detention of 8 hours. Pure oxygen was delivered to a gas compartment under the membrane. All reactors were fed a synthetic waste buffered to pH 7.0. The background solution for the feed solution was made from distilled water combined with adequate inorganic nutrients and vitamins. The background solution was supplemented with acetate and ammonia to obtain the desired substrate compositions. Combined nitrification and heterotrophic oxidation activity resulted when the SABs where fed 5 or 10 mg/1 acetate, and 10 mg-N/1 ammonia. Combined nitrification, heterotrophic oxidation, and denitrification resulted with acetate concentration of 20, 40, and 100 mg/1, and 10 mg- N/1 ammonia. Combined heterotrophic oxidation and fermentation resulted with acetate concentration of 800 mg/1, and 10 mg-N/1 ammonia. A series of mass balances were developed to determine the fate of the nitrogen compounds and acetate. These results showed that the flux values for carbon oxidation, nitrification, denitrification, and fermentation are higher than those reported for competing technologies such as rotating biological contactors. / Graduation date: 1990

L’activitat de recerca i desenvolupament en el camp de la biotecnologia genera de forma altament dinàmica noves cèl· lules i productes d’interès en múltiples sectors. En aquest context, cal explorar en temps raonables i amb mitjans altament tecnificats i estandarditzats, el potencial productor de les diferents cèl· lules modificades, en quant al seu creixement, la capacitat producció i la qualitat dels productes obtinguts. Establir les condicions d’un procés productiu basat en cèl· lules requereix completar un nombre d’etapes molt elevat: des de la selecció del model cel· lular i la realització de treballs de biologia molecular fins a assolir un procés productiu amb unes condicions optimitzades. En aquest treball es presenta el disseny i desenvolupament d’un sistema de múltiples minibioreactors orientats a escometre les primeres fases del desenvolupament del bioprocés de forma ràpida, sistemàtica i amb informació rellevant sobre l’evolució de les principals variables de cultiu, de manera que sigui possible realitzar, entre altres experiments, la selecció de clons, caracterització del seu comportament en cultiu, optimització de medis de cultiu, i proves de toxicitat de molècules sobre els perfils dels cultius. El disseny del sistema de múltiples minibioreactors presentat reuneix els avantatges dels sistemes habituals de cultiu a petita escala, com són el treball amb volums reduïts i la capacitat de realitzar diversos cultius en paral· lel, així com els avantatges del treball amb reactors de major volum, com són l’homogeneïtat i el coneixement que les principals variables del cultiu estan en valors no limitants per a aquest. Així doncs, s’ha donat una elevada importància a que els minibioreactors disposin d’un sistema d’agitació que permeti assegurar l’homogeneïtat del seu contingut, i a disposar de sistemes de seguiment. Aquests permeten d’una banda conèixer els valors de les variables dels cultius, i d’altra banda la caracterització del comportament de les cèl· lules estudiades, obtenint dades útils per al disseny dels bioprocessos corresponents que puguin ser transferires amb èxit els processos cap a majors escales, i permetent prosseguir el camí cap a l’escala de producció sense que apareguin incidències, bàsicament pèrdues de productivitat, degudes a la manca de coneixement existent habitualment en les cultius a petita escala. Prèviament a iniciar l’etapa de desenvolupament tecnològic, s’ha realitzat un estudi preliminar per tal de tenir un major coneixement de les prestacions necessàries dels equips, tant pel que fa a la funcionalitat dels sistemes que proporcionen les condicions necessàries per al creixement dels cultius (agitació i aeració), com pel que fa a la potencialitat i limitacions dels sistemes de seguiment seleccionats. D’entrada, l’objectiu ha estat seguir el pH i l’oxigen dissolt com a variables del cultiu, la concentració cel· lular per a conèixer el creixement, i el consum d’oxigen com a indicador metabòlic, si bé el pH també és una variable que dóna informació interessant relativa al metabolisme i fisiologia dels cultius. Donats els diferents enfocaments de desenvolupament possibles en funció dels models cel· lulars a emprar, se n’ha seleccionat un d’ells, les cèl· lules animals. Així, el disseny detallat del sistema de minibioreactors s’ha orientat cap a aplicacions amb aquest model cel· lular, i aquesta elecció també s’ha tingut en compte en el capítol de desenvolupament, on s’han posat a punt els sistemes per a permetre la realització d’aquest tipus de cultius (bàsicament agitació i aeració), realitzant una caracterització hidrodinàmica dels minibioreactors desenvolupats. D’altra banda, també en aquest capítol es presenta el desenvolupament de les tècniques per a seguir la concentració cel· lular, el pH, l’oxigen dissolt i consum d’oxigen. La darrera part d’aquest treball, una vegada desenvolupades i validades les diferents tecnologies, consta de la realització de cultius emprant diferents tipus de cèl· lules, valorant la reproduïbilitat de l’equip i discutint la informació que aquest proporciona quan una línia cel· lular es cultiva a condicions diferents. Finalment, també cal remarcar que aquest treball obre les portes al desenvolupament de noves prestacions per al sistema de múltiples minibioreactors desenvolupat, com són el control del pH i de l’oxigen dissolt, i la possible utilització del sistema per al cultiu de bacteris i llevats. / The research and development in the biotechnological field generates a vast amount of new cells and products of interest for various economical sectors. In this framework, it is needed to explore, in reasonable times and with automated means, the potential of the generated, regarding its growth, productivity and quality of the obtained products. Establishing the culture conditions of a productive process based in cells required accomplishing several stages: from the selection of the cellular system and the realization of molecular biology works to the achievement of a bioprocess having optimized conditions. In this work the design and development of a multiple minibioreactor system is presented, having the aim of fastening the first stages of the bioprocess development, in a systematic way, and acquiring relevant information regarding the evolution of the main culture variables, making possible the realization of various sorts of experiments such as clone selections, the characterization of its culture performance, the culture medium optimization, and the toxicological evaluation of molecules. The design of the multiple minibioreactor system combines the benefits of the usual small scale culture systems, such as the consumption of small amounts of medium ad the capability of performing multiple parallel cultures, and the benefits of larger scale culture systems, such as the homogeneity and the knowledge of the main culture variables, ensuring these do not have limiting values. Consequently, special attention has been paid to the development of a stirring system ensuring the homogeneity of the bioreactor content and to the monitoring systems allowing the characterization of the cell growth and metabolism, which permits having sufficient data to upscale the process, minimizing the risks of such duty. Before starting the technological development stage, a preliminary study has been done with the aim of identifying and characterizing the required equipment features, regarding the auxiliary equipment supplying the culture conditions (stirring, aeration), and also regarding the potentiality and limitations of the monitoring systems. The main culture environmental variables to be monitored are pH and dissolved oxygen, whereas to monitor growth cellular concentration is to be followed. Additionally, oxygen consumption is to be used as a metabolic indicator, and pH is also a variable from which it can be obtained interesting information regarding culture physiology and metabolism. Given the different design possibilities for the required technologies depending on the cellular models to be used, one of them, precisely mammalian cells have been chosen. Thus, the detailed design and development of the minibioreactor system has been directed towards applications with such cellular model, paying special attention to the characteristic requirements of mammalian cells (basically agitation and aeration), and to the hydrodynamic characteristics of the system. On the other hand, also in the technological development chapter, techniques to monitor cell concentration, pH, dissolved oxygen and oxygen consumption have been implemented. Once the technological development has been completed and the required functionalities validated, in the last part of this work various cultures are performed with the aim of evaluating the culture reproducibility, and the information obtained by the equipment as it is used for culturing a certain cell line at various conditions. Finally, it is also interesting to underline the potential fields of development based on this work: new features of the multiple minibioreactor system, such as the pH and dissolved oxygen controls, and the potential use of the system for the culture of bacteria and yeast.

Thesis (DTech (Chemical Engineering))--Cape Peninsula University of Technology, 2009. / Includes bibliographical references. Also available online.