No description available.

Vascular grafts are used to repair, replace, or bypass diseased arteries, and there is a growing need for tissue-engineered blood vessels (TEBVs) as replacement grafts. Three-dimensional, self-assembled smooth muscle cell (SMC) rings can be fabricated and fused to create SMC tissue tubes with a structure similar to native vessels; however, this approach is limited by the underdeveloped mechanical integrity of the tissue. Thus, the goal of this research is to design, manufacture, and validate a cyclic circumferential stretch bioreactor to mechanically stimulate SMC tissue rings, with the goal of developing rings that can withstand the physiological forces of the in vivo environment. The bioreactor consists of a closed cam-syringe-tubing system that forces fluid into the tubing with each rotation of the cam, thereby distending and relaxing the tubing. Various sized cams were implemented to modify the distension of the tubing (5%, 7.5%, 10%, and 15% stretch magnitudes). Tissue rings are placed on the tubing, which is housed in a custom culture chamber. The tubing was validated using DVT® imaging technology to distend approximately 5, 7.5, 10, and 15% under static conditions. High density mapping was used to analyze the dynamic distension of the tubing and tissue rings. During bioreactor operation, the tubing distends 1-2% less than expected for the fabricated cams (5, 7.5, 10, 15%), and the tissue ring distends 31-56% less than the tubing on which it is located. To assess the effects of cyclic distension, 7-day-old SMC rings were cultured dynamically for 7 days and exposed to 0%, 5%, 7.5%, 10%, or 15% cyclic stretch (1 Hz, 100% duty cycle). Histology and immunohistochemistry indicate that both stretched and non-stretched rings synthesized collagen and glycosaminoglycans, but the contractile proteins á-smooth muscle actin and calponin were not synthesized. A decrease in cell density was observed as the magnitude of stretch increased, and the 5-15% stretched samples demonstrated more cellular alignment than the 0% stretch control samples. Mechanical testing analysis concluded that the stretched rings exhibited a reduction in ultimate tensile strength, maximum tangent modulus, maximum strain, and maximum load compared to unstretched control samples. It is anticipated that future work, including modifications of the culture medium and mechanical stimulation parameters (eg. reduced duty cycle, reduced frequency), has the potential to achieve the expected outcome of this research - a strong, aligned, contractile vascular smooth muscle cell tissue ring through dynamic culture using a cyclic circumferential stretch bioreactor.

Em caso de envenenamento ofídico, o tratamento no Brasil hoje é realizado pela administração de soros geralmente produzidos por equinos, que apresentam eficácia limitada: são úteis para os efeitos sistêmicos, mas não inibem efetivamente a evolução dos danos locais, podem causar reações adversas e apresentam alto custo de produção. De acordo com a Organização Mundial da Saúde (OMS), trata-se de uma doença negligenciada pelas autoridades científicas mundiais. O presente projeto, em parceria com o Instituto de Pesquisas em Patologias Tropicais da Fundação Oswaldo Cruz - Rondônia, propõe a produção por Escherichia coli de fragmentos de anticorpos de cadeia pesada de camelídeos, denominados VHH, contra as toxinas do veneno de Bothrops jararacussu, utilizando biorreator. Neste trabalho há interesse em produzir VHH, através da otimização do crescimento desta E. coli. A cinética do crescimento bacteriano foi realizada em shaker orbital sob diferentes condições, variando tamanho do frasco, rotação do shaker, composição do meio de cultura e concentração de substrato; e em biorreatores, alternando meios de cultura e modo de operação do reator (descontínuo e descontínuo alimentado), alterando a vazão de alimentação (linear e exponencial) O processo cinético é fortemente limitado pela formação de acetato, por condições auxotróficas da célula e pela transferência de oxigênio. Nos ensaios em frascos agitados, uma melhor condição de crescimento foi obtida utilizando frascos de 1 L, sob rotação a 270 rpm e 5,0 g/L de glicose. Nos ensaios em reator, quando operados em batelada obtiveram-se cerca 5,5 g/L de células finais, contra 9,3 g/L de células em batelada alimentada com vazão constante. Um maior crescimento foi ainda obtido em um reator de 2 L em regime de batelada alimentada exponencialmente. O biorreator varia a agitação do meio e mantém um nível pré-definido de oxigênio dissolvido, evitando a limitação de oxigênio e controlando a oferta de glicose para o crescimento celular. Neste processo, atingimos 25,6 g/L de células e 0,35 g/L de proteína total após purificação, utilizando meio M9 suplementado. / Nowadays in Brazil, the treatment for snakebite poisoning is carried out by the administration of horse sera, which have limited effectiveness: they are useful for systemic effects, but do not effectively inhibit local damage, cause adverse reactions, and present high production costs. According to the WHO, this disease is neglected by the world`s scientific authorities. This project, in partnership with the Institute for Research in Tropical Diseases of Oswaldo Cruz Foundation - Rondonia, proposes the production of heavy chain antibody fragments from camelids, called VHH, using Escherichia coli, to be used against the toxins from the Bothrops jararacussu poison, using a bioreactor. This work is interested in producing VHH through the use of E. coli. The kinetics of bacterial growth were performed in orbital shaker under different conditions, varying vial size, shaker rotation, composition of the culture medium and substrate concentration; and bioreactors, alternating culture media and operation mode of reactor (batch and fed-batch), changing feeding rate (linear and exponential). The kinetic process is statically bound to acetate formation, auxotrophic conditions of the cell and oxygen transfer. In assays in shaken flasks, an output of 270 rpm and 5.0 g/L of glucose. In reactor runs, when operated in a batch 5.5 g/L of final cells were obtained, against 9.3 g/L of final cells in fed-batch with constant flowrate. A larger value was obtained in an exponentially fed-batch reactor of 2 L. The bioreactor varies the agitation of oxygen and controls glucose addition for cell growth. In this process, 25.6 g/L cells and 0.35 g/L total protein after purification were reached, using supplemented M9 medium.

O etanol é o combustível que mais se destaca como alternativa à gasolina no setor do transporte. No entanto, a melhoria da eficiência da fermentação, aprimorando o processo de conversão de biomassa em etanol combustível e reduzindo o custo de produção são desafios ainda pertinentes. Neste cenário, as fermentações com alta concentração de açúcar ou VHG (very high gravity) estão sendo cada vez mais estudadas. A adição de compostos como fonte de nitrogênio pode gerar benefícios para a produção de etanol, entretanto, a maioria dos estudos com suplementação de fontes nitrogenadas é realizada em sistema descontínuo simples e em escala laboratorial, embora os processos fermentativos industriais sejam conduzidos em sistema descontínuo alimentado. Neste contexto, este trabalho visou avaliar o perfil fermentativo da linhagem CAT-1 em mosto contendo alta concentração de açúcar fermentescível e suplementado com as fontes nitrogenadas peptona e sulfato de amônio, nas concentrações 5 e 10 g/L, em sistema de descontínuo simples e descontínuo alimentado com reciclo de células, utilizando diferentes escalas produtivas. Nos experimentos em sistema descontínuo simples, os tratamentos controle e peptona (5 g/L) apresentaram uma melhor produção de etanol, maior rendimento e produtividade em comparação com os demais tratamentos. Nos experimentos em sistema descontínuo alimentado não houve diferença significativa entre os tratamentos controle e peptona 5 e 10g/L, porém, o tratamento com sulfato de amônio (10 g/L) apresentou parâmetros fermentativos de produção de etanol e produtividade superiores ao tratamento controle, o oposto do descontínuo simples. Os resultados mostraram que quando as células de levedura foram submetidas a concentrações crescentes de açúcar, como é o caso do sistema descontínuo alimentado, ocorre uma adaptação das mesmas, proporcionando melhores resultados, com uma redução no tempo de fermentação para 16h. Além disso, a alta concentração de etanol no meio fermentativo causa um efeito prejudicial às células, já que nos tratamentos onde houve teores mais elevados de etanol ocorreu menor viabilidade e maior quantidade de açúcar residual. / Ethanol is the fuel that stands out as an alternative to gasoline in the transportation sector. However, improving the efficiency of fermentation, improving the process of converting biomass to fuel ethanol and reducing the cost of production are still relevant challenges. In this scenario, fermentations with high sugar concentration or very high gravity (VHG) are being studied more and more. The addition of compounds as a source of nitrogen can generate benefits for ethanol production, however, most of the studies with nitrogen supplementation are carried out in a simple discontinuous system and in laboratory scale, although the industrial fermentation processes are conducted in a fed batch system . In this context, the objective of this work was to evaluate the fermentation profile of the CAT-1 strain in must containing high fermentable sugar concentration and supplemented with nitrogen peptone and ammonium sulfate at 5 and 10 g / L in a simple discontinuous system and fed batch system, fed with recycle of cells, using different productive scales. In the simple discontinuous system experiments, the control and peptone treatments (5 g / L) presented better ethanol production, higher yield and productivity in comparison to the other treatments. However, the treatment with ammonium sulfate (10 g / L) showed fermentative parameters of ethanol production and productivity higher than the control treatment, the opposite of simple discontinuity. The results showed that when the yeast cells were submitted to increasing concentrations of sugar, as is the case of the fed batch system, an adaptation occurs, providing better results, with a reduction in the fermentation time to 16h. In addition, the high concentration of ethanol in the fermentation medium causes a detrimental effect on the cells, since in the treatments with higher levels of ethanol; less viability and more residual sugar were observed.

Os polissacarídeos possuem diversas aplicações industriais devido a sua ampla variedade de propriedades físico-químicas. Além desses empregos tradicionais, pesquisas mais recentes estão impulsionando o uso de polissacarídeos para novas aplicações, principalmente na área de terapia farmacêutica. Com o objetivo de ampliar a produção industrial de polissacarídeos microbianos, pesquisas têm se concentrado nos exopolissacarídeos (EPS), que apresentam produtividade elevada e processos de extração e purificação mais simples quando comparados aos polímeros tradicionais. A lasiodiplodana, uma ?-(1->6)-D-glucana, é um EPS produzido por Lasiodiplodia theobromae, um fungo filamentoso característico de regiões tropicais e patógeno de mais de 500 espécies vegetais. Na produção de EPS observa-se que, como resultado do crescimento e da produção do biopolímero, o meio de cultivo torna-se mais viscoso, tornando difícil a manutenção da homogeneidade no biorreator e, consequentemente, prejudicando a transferência de massa e oxigênio no meio. Nos cultivos em que ocorrem mudanças na reologia do meio e nos quais são utilizados fungos filamentosos, sensíveis ao cisalhamento, o uso de biorreatores convencionais, como o Stirred Tank Reactor (STR) e o Airlift, não é adequado. Nesse contexto, no presente estudo tem-se como objetivo estudar a cinética de crescimento, consumo de substrato e produção de EPS pelo fungo filamentoso Lasiodiplodia theobromae, a partir de glicose e de glicerol, no Biorreator Agitado e Aerado de Baixo Cisalhamento (BAABC) e compará-lo com o STR. No estudo inicial, realizado em frascos agitados, a maior produção de EPS (6,49 ± 0,03 g/L) foi alcançada pelo ensaio G, que continha a maior concentração glicose testada (50 g/L) e a menor concentração de extrato de levedura (3 g/L). Quando se utilizou glicerol como fonte de carbono, a maior produção de EPS (3,39 ± 0,06 g/L) foi observada no Ensaio O, que continha 30 g/L de glicerol e a maior concentração de fonte de nitrogênio testada (12 g/L de extrato de levedura). Nos ensaios em biorreatores, quando utilizou-se glicose como fonte de carbono observou-se que o BAABC com controle de temperatura proporcionou uma produção de 3,17 ± 0,16 g/L de EPS, concentração inferior à obtida em frascos, porém bastante superior à obtida no biorreator STR (0,70 ± 0,12 g/L). Para os meios contendo glicerol, o biorreator STR proporcionou uma produção de EPS de 3,02 ± 0,19 g/L, enquanto no BAABC a concentração de EPS obtida foi muito menor (1,45 ± 0,25 g/L). Analisando-se, porém, a concentração máxima de biomassa obtida (28,86 ± 1,46 g/L) bem como os parâmetros cinéticos relacionados (YX/S e QX), nota-se que a produção de biomassa foi muito superior e mais eficiente no BAABC. Apesar das diferenças morfológicas visualmente observadas e confirmadas por meio de microscopia óptica nos EPS obtidos a partir de glicose e de glicerol, as análises de Difração de raios-X e Espectroscopia de absorção na região do infravermelho com Transformada de Fourier permitiram evidenciar a similaridade estrutural entre ambos os EPS. / Polysaccharides have several industrial applications because of their wide variety of physicochemical properties. In addition to these traditional applications, recent research is driving the use of polysaccharides towards new applications, especially in the field of pharmaceutical therapy. With the aim of increasing the industrial production of microbial polysaccharides, research has focused on exopolysaccharides (EPS), which have high productivity and simpler extraction and purification processes when compared to traditional polymers. Lasiodiplodana, a ?-(1->6)-D-glucan, is an EPS produced by Lasiodiplodia theobromae, a filamentous fungus characteristic of tropical areas and pathogenic of more than 500 plant species. In EPS production, as a result of the growth and biopolymer production, the culture medium becomes more viscous, making it difficult to maintain homogeneity inside the bioreactor and, consequently, harming the transfer of mass and oxygen in the medium. In cultures where changes in media rheology occur and in which shear-sensitive filamentous fungi are used, the use of conventional bioreactors, such as Stirred Tank Reactor (STR) and Airlift, is not appropriate. In this context, the objective of the present study was to study the growth kinetics, substrate consumption and EPS production by the Lasiodiplodia theobromae filamentous fungus, from glucose and glycerol, in the Low-Shear Aerated-Agitated Bioreactor (LSAAB) and compare it with the STR. In the initial study, conducted in shaken flasks, the highest EPS production (6.49 ± 0.03 g/L) was achieved by the G test, which contained the highest glucose concentration tested (50 g/L) and the lowest concentration of yeast extract (3 g/L). When glycerol was used as the carbon source, the highest EPS yield (3.39 ± 0.06 g/L) was observed in Test O, which contained 30 g/L glycerol and the highest concentration of nitrogen source tested (12 g/L of yeast extract). In the bioreactor trials, when glucose was used as the carbon source, it was observed that the LSAAB with temperature control provided a production of 3.17 ± 0.16 g/L of EPS, a concentration lower than that obtained in flasks, but rather higher than that obtained in the STR bioreactor (0.70 ± 0.12 g/L). For the glycerol-containing media, the STR bioreactor produced 3.02 ± 0.19 g/L of EPS, while in LSAAB the EPS concentration obtained was much lower (1.45 ± 0.25 g/L). However, analyzing the maximum concentration of biomass obtained (28.86 ± 1.46 g/L) as well as the related kinetic parameters (YX/S and QX), biomass production was much higher and more efficient in LSAAB. In spite of the visually observed morphological differences confirmed by optical microscopy in EPS obtained from glucose and glycerol, X-ray diffraction and absorption spectroscopy analyzes in the infrared region with Fourier transform showed the structural similarity between both EPS.

The recent surge in biodiesel production has resulted in a huge surplus of crude glycerol, a by-product of the process to the level of 10% by weight. This is turn has caused the price of glycerol to fall dramatically, and there are now few economically viable channels for using this resource: waste glycerol is usually combusted. Therefore, much interest has arisen in the possibility of making use of glycerol with biotechnology, as this would not only be a more efficient use of resources but also make biodiesel itself more commercially viable. The purple bacterium Rhodopseudomonas palustris is able to metabolize glycerol through photofermentation and thereby produce hydrogen, a commercially useful commodity. R. palustris is of particular interest for this purpose as, in contrast to many other species which have been investigated with a view to fermenting glycerol, it is highly tolerant of crude glycerol. The feedstock requires little purification or dilution to be made suitable for cultivation of R. palustris. Furthermore, the hydrogen gas produced by R. palustris when grown on glycerol is of high purity, and the organism's great metabolic diversity suggests it may be a useful strain for remediation of other waste materials. However, much groundwork is needed to establish R. palustris as a viable chassis organism for biotechnological purposes. This work sets out to establish optimal conditions for cultivating R. palustris in the laboratory, including the design of a suitable batch photobioreactor system. It also determines optimal conditions for electroporation of R. palustris for the purpose of knocking out endogenous genes or introducing heterologous genes. Furthermore, the introduction of heterologous genes is attempted in order to demonstrate the possibility of producing other high-value compounds with R. palustris, and several deletion strains with potential benefits for hydrogen production are created. Finally, several existing deletion strains are investigated to establish their suitability as chassis strains for further genetic manipulation.

O Streptococcus pneumoniae, também conhecido como pneumococo, é um importante agente etiológico causador de pneumonia, bacteremia, meningite e otite aguda do ouvido médio, acometendo especialmente crianças e idosos. A cápsula polissacarídica (PS) é o fator de virulência mais importante e localiza-se na superfície do microrganismo e tem ação anti-fagocítica in vivo. O pneumococo expressa pelo menos 91 cápsulas diferentes, que são química e sorologicamente distintas. Usualmente, a cápsula é o antígeno das vacinas anti-pneumocócicas, tanto como polissacarídeo livre ou conjugado a proteínas. O sorotipo 6B acomete principalmente crianças e é o segundo mais prevalente no Brasil, e a otimização das condições de cultivo buscando maior produtividade em PS é o alvo desta tese. Simultaneamente, também foi investigada a influência do inóculo e da glicose residual no instante do pulso ou do início da alimentação em cultivos descontínuos alimentados. Nos cultivos descontínuos alimentados foi observado que quanto menor a densidade óptica da cultura do inóculo, maior a concentração de PS obtida. A concentração da glicose residual no momento do pulso provavelmente influenciou a produção de PS: a produção foi maior quando o pulso foi dado ainda em altas concentrações de substrato. Por isso, obteve-se uma produção de PS ~390 mg/L em cultivo descontínuo alimentado com pulso, quando DO 600 nm da cultura do inóculo ~1,6 e o pulso de glicose e acetato de amônio ~15 g/L. Observou-se também que, mesmo com DO do inóculo ~1,65, a produção de PS foi somente 248 mg/L, devido à aplicação do pulso quando a concentração de glicose residual era ~4,5 g/L. A menor produção de PS foi obtida quando a DO da cultura do inóculo foi ~2,6 e a concentração de glicose residual ~3,4 g/L, gerando somente 194 mg/L de PS. Assim, cultivos descontínuos alimentados com pulso renderam melhores resultados que os cultivos descontínuos alimentados. A alimentação constante rendeu maior quantidade de PS quando comparada à alimentação exponencial. E tanto os ensaios descontínuos com ou sem pulso e os descontínuos alimentados foram melhores do que os cultivos contínuos, principalmente se considerarmos a facilidade de operação. O efeito da concentração residual da glicose no instante da alimentação na produção de PS é provavelmente influenciado pela presença de outros componentes do meio, devido aos requerimentos nutricionais do pneumococo. O estado fisiológico do inóculo determinou uma importante correlação entre a produção de PS nos cultivos descontínuos alimentados do sorotipo 6B: cultura do inóculo no meio da fase exponencial rendeu maior produção de PS. Esta correlação poderia ser conseqüência do perfil de crescimento do microrganismo in vitro e da ação de enzimas líticas após a fase exponencial. Foi observado, portanto, um efeito sinérgico entre a DO da cultura do inóculo, a concentração de glicose residual no momento de alimentação e produção de PS nos cultivos descontínuos alimentados. / Streptococcus pneumoniae is a major pathogen commonly responsible for pneumonia, bacteraemia, meningitis and otitis media, especially among young children and older adults. The most prominent pneumococcal virulence factor is the capsular polysaccharide (PS), which coats the surface of the bacterium and acts as an antiphagocytic factor in vivo. S. pneumoniae express at least 91 distinct capsules which are chemically and serologically distinct. The capsule is currently used as antigen in pneumococcal vaccines, either as free polysaccharide or conjugated to proteins. Pneumococcal serotype 6B is the second most prevalent in Brazil and the optimization of cultivation conditions is part of this study. Simultaneously we investigated the influence of the growth phase of the inoculum and the residual glucose concentration at the instant of the pulse or the start of feeding on PS production in batch and fed-batch cultivation. Streptococcus pneumoniae serotype 6B strain ST 433/03 was used. Bench scale experiments were carried out using a variant of the Hoeprich medium, containing glucose, acid-hydrolyzed casein, dialyzed yeast extract, L-glutamine and asparagine as nitrogen sources, choline as a growth factor and salts. The experiments were conducted in bioreactors monitored by the LabView 7.1program. It was observed in fed-batch cultivation, that the lower was the OD of the starter culture, the higher was the PS concentration obtained. The residual glucose concentration at the moment of the pulse also influenced the PS production: the PS production was higher when the pulse was given at higher residual glucose concentration. Hence, in fed-batch culture the highest PS production (387 mg/L) was obtained using an OD=1.6 of the starter culture and giving the pulse when the residual glucose was ~15 g/L. Using a similar inoculum (OD=1.65), the PS concentration reached 248 mg/L after giving the pulse when the residual glucose was 4.5 g/L. The lowest PS production was obtained when a culture with an OD=2.6 was inoculated into the reactor and the pulse was given when the residual glucose was 3.4 g/L (PS=194 mg/L). The effect of the residual glucose concentration at the instant of the start of feeding on PS production was probably influenced by the presence of other components in the concentrated feeding medium, which could better fit the nutritional requirements of the microorganism. The physiological state of the inoculum showed an important correlation to the PS production in batch and fed-batch cultivation of S. pneumoniae serotype 6B: mid-log phase inocula yielded high PS production. This correlation is consequence of the in vitro growth profile, the action of lytic enzymes after the log phase. In fed-batch cultivation, it was also observed a synergic effect of the inoculum OD and the residual glucose concentration in the moment of the pulse on PS production.

No description available.

Anaerobic membrane bioreactor (AnMBR) serves as a more sustainable form of wastewater treatment. However, biofouling is particularly detrimental to the performanceof AnMBRs. This dissertation focuses on understanding more about the biofouling in nMBR, and to devise strategies to control or make use of these biofoulant layers. First, we aim to investigate the microbial community structure of sludge and biofilm from 13 different AnMBRs. Our findings indicate 20 sludge core genera and 12 biofilm core genera (occurrence ≥ 90% samples) could potentially account for the AnMBR performance. Sloan neutral model analysis indicates the anaerobic microbial consortium between sludge and biofilm is largely affected by stochastic dispersal and migration processes (i.e., neutral assembly), suggesting that the majority of these core genera are not selectively enriched for biofilm formation. Therefore, the second part of this dissertation aims to minimize the growth of the overall bacterial cells attached on the membranes. For this, membranes embedded with zinc oxide (ZnO) and copper oxide (CuO) nanoparticles were examined for their antifouling efficacies. Our findings indicate both CuO and ZnO nanoparticles embedded membranes could delay biofouling formation without significantly triggering the overall expression/abundance of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in biofilm. Furthermore, CuO and ZnO nanoparticles could inhibit the expression of quorum sensing associated genes, resulting in lower quorum sensing signal molecules production. Despite the positive results demonstrated from this study as well as those from others, we recognize that no control strategies are likely to achieve total prevention of anaerobic biofouling. Therefore, the last part of this dissertation focuses on exploring the effects of different foulant layers on antibiotic-resistant bacteria (ARB) and ARGs removal. Our findings suggest both ARB and ARGs could be absorbed by membrane foulant. Transmembrane pressures and the foulant layer synergistically affected ARB removal, but the foulant layer is the main factor that contributed to ARG removal through adsorption. Overall, the collective findings could bring new insights to the anaerobic membrane biofouling phenomenon, and offer pragmatic approaches to minimize biofouling without compromising the post-AnMBR effluent quality.

Biodegradation is the breakdown of a compound by a biological organism. Over the past few decades, the biodegradation of compounds such as phenol has been researched extensively. Phenol research has shown that certain organisms are capable of utilising it as an energy source, and a variety of methods are available for its removal. Unfortunately, there is lack of research on phenol degradation at low concentrations. The majority of research performed on phenol degradation has used concentrations above 500 mg, while phenol is highly toxic at levels below 25 mg. The aim of this research was to pursue the problem of phenol degradation at below 100 mg and develop a system able to degrade phenol at such levels. The system consisted of a bioreactor developed to run in continuous mode, using Ps. putida immobilised in calcium alginate. A standard method was modified to quantitatively analyze effluent phenol levels, and a medium designed to increase the longevity of calcium alginate beads in continuous culture. A continuous flow bioreactor was also designed using an overflow weir for use with immobilised cells. Based on the results obtained, immobilisation offers increased stability and increased protection for cells under extreme conditions and is able to use higher dilution rates than cells under continuous culture / Master of Science (Hons)