Chow Ka-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 52-55). / Abstracts in English and Chinese. / Chapter 1 --- Introduction / Chapter 1.1 --- Chemical sensors --- p.1 / Chapter 1.2 --- Quartz crystal microbalance --- p.4 / Chapter 1.3 --- Concept of affinity mass sensor --- p.8 / Chapter 1.4 --- Film immobilization technologies --- p.9 / Chapter 1.5 --- Research outlines --- p.13 / Chapter 2 --- Experimental / Chapter 2.1 --- Sensor fabrication --- p.14 / Chapter 2.2 --- Flow-through cell --- p.16 / Chapter 2.3 --- Analysis procedures --- p.19 / Chapter 2.4 --- Response curve --- p.19 / Chapter 2.5 --- Experimental setup --- p.21 / Chapter 3 --- Detection of ascorbic acid by affinity mass sensor based on 3-aminophenylboronic acid / Chapter 3.1 --- Conventional analytical methods --- p.23 / Chapter 3.2 --- Research method - affinity mass sensor based on APBA --- p.24 / Chapter 3.3 --- To locate the binding site in ascorbic acid --- p.25 / Chapter 3.3.1 --- Steric energy calculated by molecular modeling --- p.26 / Chapter 3.4 --- Optimization of experimental variables --- p.29 / Chapter 3.4.1 --- Effect of pH --- p.29 / Chapter 3.4.2 --- Effect of sample volume --- p.30 / Chapter 3.4.3 --- Effect of flow velocity --- p.30 / Chapter 3.5 --- Calibration and Reproducibility --- p.32 / Chapter 3.6 --- Kinetic analysis --- p.33 / Chapter 3.7 --- Stability of sensor --- p.37 / Chapter 3.8 --- Interference studies --- p.37 / Chapter 3.9 --- Determination of ascorbic acid in real samples --- p.39 / Chapter 3.9.1 --- Results and Discussion --- p.39 / Chapter 3.10 --- Comparison with conventional ascorbic acid sensors --- p.42 / Chapter 3.11 --- Summary --- p.42 / Chapter 4 --- Boronic acid derivatives for the detection of sugars / Chapter 4.1 --- Scope of this work --- p.43 / Chapter 4.2 --- Results and Discussion --- p.44 / Chapter 4.3 --- Summary --- p.49 / Conclusion --- p.50 / References --- p.52 / List for tables --- p.56 / List for figures --- p.57 / Appendix I --- p.59 / Appendix II --- p.61

L'étude et le développement des procédés de fabrication de levures immobilisées en vue de la réalisation de fermentations de vins a débuté au milieu des années 80. Malgré les bénéfices potentiels que cette technologie pouvait apporter pour le secteur œnologique, peu de procédés d'immobilisation ont réussi à dépasser l'échelle laboratoire ou pilote et ceux qui sont arrivés à l'échelle industrielle n'ont pas eu le succès désiré pour des questions d'ordre technique ou économique. Le premier objectif de ce travail concerne la mise au point du procédé industriel en insistant sur les aspects les plus sensibles, et qui comme tels ont exigé des études complémentaires. Le deuxième objectif de ce travail vise à caractériser du point de vue cinétique et lorsque possible sensoriel, les fermentations avec les levures immobilisées pour la production de vins effervescents et pour la désacidification biologique de moûts. Le troisième et dernier objectif de ce travail consiste à évaluer l'utilisation de levures immobilisées pour la réalisation de la fermentation alcoolique en continu de moût. Pour cela on a fait appel à des fermenteurs continus à lit fixe et à lit fluidisé. / The study and development of yeast immobilization processes for wine fermentations started in the mid 80’s. Even though this technology could be of great benefit for the oenological sector very few process left the laboratory or pilot scale and those which arrived to industrial scale didn’t have the ambitioned success due to technical or economical constraints. The first goal of this work was to develop an industrial process for yeast immobilisation with emphasis on the most sensitive aspects which required further studies. The second objective of this work was to characterise the fermentation kinetics of immobilised yeasts cells during the production sparkling wines and during the deacidification of grape must. Whenever possible the wines produced were also characterised from a sensorial point of view. The third and last goal was to evaluate the use of immobilised yeast cells for continuous fermentation of grape must. For that we have used continuous fixed bed and fluidized bed fermenters.

Este trabalho de doutorado apresenta os resultados da avaliação de duas configurações diferentes de reatores com biomassa imobilizada em matrizes cúbicas de poliuretano, denominados reator misto radial de leito fixo (RMRLF), utilizado para o tratamento de esgoto sanitário peneirado e reator aeróbio-anaeróbio horizontal de leito fixo (RAAHLF) para o pós-tratamento de efluente de reator de manta de lodo tipo upflow anaerobic sludge blanket (UASB) tratando esgoto sanitário. O estudo avalia duas propostas de tratamento para remoção biológica de matéria orgânica e nitrogênio por meio do acompanhamento do desempenho dos reatores sob diferentes condições operacionais, quais sejam: variação na carga orgânica aplicada, diferentes dosagens de solução alcalina e variação na taxa de aplicação de ar comprimido para o fornecimento de oxigênio dissolvido aos reatores na etapa de nitrificação. Foram desenvolvidos ensaios para obtenção de dados sobre o comportamento hidrodinâmico no RMRLF, dados sobre transferência e consumo de oxigênio dissolvido e viabilidade da utilização de biogás como doador de elétrons para a desnitrificação autótrofa. Os principais parâmetros de desempenho foram avaliados através de análises físico-químicas das amostras e a observação dos microrganismos envolvidos no processo. O RMRLF operou durante todo o período dos experimentos com a temperatura do líquido no seu interior variando entre o valor mínimo de 14 'GRAUS' Celsius até máximo de 30 'GRAUS' Celsius. Os valores da demanda química de oxigênio (DQO) no efluente do reator mantiveram média inferior a 50 mg/L. A remoção de nitrogênio como nitrato aumentou em 75% após o início do fornecimento do 'H IND.2'S' por meio da injeção de biogás. O RAAHLF operou com a temperatura do líquido no seu interior variando entre mínimo de 14 'GRAUS' Celsius e máximo de 26 'GRAUS' Celsius. Os valores de DQO no efluente, em média, foram inferiores a 66 mg/L e a remoção de nitrato foi incrementada em 87% após o fornecimento do 'H IND.2'S' pela injeção de biogás na câmara anóxica do reator. Os resultados demonstram o potencial destes reatores como alternativa para o tratamento e pós-tratamento de esgoto sanitário com resultados promissores de remoção de matéria orgânica e nitrogênio. / This work presents results of the evaluation of two different immobilized biomass reactor configurations, referred as radial flow anaerobic/aerobic immobilized biomass reactor (RAAIB) utilized for the treatment of the sanitary screened wastewater and horizontal aerobic/anaerobic immobilized biomass reactor (HAAIB) for the post-treatment of an upflow anaerobic sludge blanket reactor (UASB) treating sanitary wastewater. This study evaluates the two proposals for the biological organic matter and nitrogen removal by the evaluation of the reactors performance under several operational conditions, such as organic matter load variation, alkalinity solution feeding, variation on the compressed air supply for the nitrification step and biogas supply for the denitrification process. Assays were developed in order to obtain data on the hydrodynamic behavior in the RAAIB reactor, oxygen transfer and consumption and the viability of use biogas as electron donor for the autotrofic denitrification. The main performance parameters were measured thru physical-chemical analysis and microscopic observations. The RAAIB reactor was operated during all the experiment period with its liquid temperature varying from 14 'DEGREES' Celsius to 30 'DEGREES' Celsius. The chemical oxygen demand (COD) values in the effluent presented a mean value below 50 mg/L. The nitrogen as nitrate removal efficiency incresead up to 75% after supplying 'H IND.2'S' thru biogas injection. The HAAIB reactor operated with temperatures varying from 14 'DEGREES' Celsius to 26 'DEGREES' Celsius. The COD values in the effluent presented a concentration value lower than 66 mg/L and the nitrate removal increased 87% after supplying 'H IND.2'S' thru the biogas injection in the anoxic reactor. The collected data proved the viability of the use of these reactors as an alternative for the treatment and post-treatment of sanitary wastewater due to the promising results obtained in the assays performed in the reactors.

Made available in DSpace on 2017-07-10T19:24:23Z (GMT). No. of bitstreams: 1 Carla Limberger _Lopes (Tese revisada) 2016.pdf: 2793834 bytes, checksum: bd8e57da9e9986df4f212107c57ca249 (MD5) Previous issue date: 2016-02-17 / The aim of this study was to evaluate a combined anaerobic-aerobic upflow fixed-bed reactor with recirculation of the liquid phase for the removal of nitrogen and organic matter from poultry slaughterhouse wastewater. The reactor was made of an acrylic tube of internal diameter of 93 mm and the length of 1000 mm with a useful volume of 5.6 L being 3.5 L corresponding to the anaerobic compartments and 2.1 L to the aerobic one. The bed for immobilization of the biomass was formed by expanded clay and polyurethane foam. For discussing the results, this study was divided into three articles. In the first article, was evaluated the reactor performance with respect to the elimination of nitrogen (≈65 mg.NT.L-1) and organic matter (≈600 mg.DQO.L-1) due to the recirculation rate (R = 0.5, 1 and 2) and the hydraulic retention time (HRT) of 11 h (6.8 h in anaerobic condition and 4.2 h in aerobic condition) over the time. The best operating condition was obtained at the recirculating rate of 2. In this condition, the total nitrogen removal was 65% with the effluent concentration of 6 mg.NH4+.L-1 e 12 mg.NO3-.L-1. For all condition, the organic matter removal was greater than to 95% with the effluent concentration of approximately 20 mg.COD.L-1. Thus, the increasing of the recirculation rate influenced positively in the reactor performance. In the second article, the hydraulic detention time was evaluated (HDT) at 14 h, 11 h and 8 h with the recirculation rate (R) of 0.5 (Step I), R = 1 (Step II) and R 2 = (Step III). The affluent average concentrations were 65 mg.NT.L-1, 580 mg.COD.L-1, 77 mg L-1 of total alkalinity and pH of 6.4. The samples were collected inlet and output of each compartment along the reactor height. In the Step I, the nitrification efficiencies were 76%, 70% and 41% respectively for 14 h, 8 h and 11 h, showing the effect of HRT. In all steps, the alkalinity has been regarded as the limiting factor of the process and its deficit was 10 to 30%. It was attributed to this factor the low efficiency of total nitrogen removal of about 45%. Throughout the experiment the removal efficiency of organic matter in terms of chemical oxygen demand (COD) was over 90% and made to fit the first order kinetic model for degradation of the substrate. In the third article, was evaluated the hydrodynamic behavior of this system, through stimulus response tests using eosin Y dye as tracer. It has been evaluated the hydraulic retention time of 8 h with three recirculation rates, of .0.5, 1 and 2 times. Under these conditions, the removal of organic matter was greater than 90% and nitrogen conversion was favored by applying lower loads of (0.18 Kg.N.m-3.d-1). It was found that the hydrodynamic evaluation showed the continuous-stirred reactors behavior with 2 to 2.5 reactors in series (N-CSTR). / O objetivo desse trabalho foi avaliar um reator combinado anaeróbio-aeróbio de leito fixo e fluxo ascendente com recirculação da fase líquida, para a remoção de nitrogênio e matéria orgânica proveniente de água residuária de abatedouro de aves. O reator foi confeccionado em um tubo de acrílico de diâmetro interno de 93 mm e comprimento de 1000 mm, com volume útil de 5,6 L, sendo 3,5 L correspondentes aos compartimentos anaeróbios e 2,1 L correspondentes ao compartimento aeróbio. O leito para a imobilização da biomassa foi formado por argila expandida e espuma de poliuretano. A apresentação desse trabalho foi dividida em três artigos. No primeiro artigo, avaliou-se o desempenho do reator em relação à remoção de nitrogênio (≈65 mg.N.L-1) e de matéria orgânica (≈600 mg.DQO.L-1) em função da taxa de recirculação (R=0,5; 1 e 2) e do tempo de detenção hidráulica (TDH) de 11 horas (6,8 horas na condição anaeróbia e 4,2 horas na condição aeróbia), ao longo do tempo. A melhor condição operacional foi obtida com taxa de recirculação de 2. Nessa condição, a eficiência de remoção de nitrogênio total foi de 65% com concentrações efluentes de 6 mg.NH4+.L-1 e 12 mg.NO3-.L-1. Para todas as condições testadas, a eficiência de remoção de matéria orgânica apresentou-se superior a 95%, com concentração efluente de aproximadamente 20 mg.DQO.L-1. Assim, o aumento da taxa de recirculação influenciou positivamente no desempenho do reator. No segundo artigo, avaliaram-se os tempos de detenção hidráulica (TDH) de 14 horas, 11 horas e 8 horas com taxa de recirculação (R) de 0,5 (Ensaio I), R=1 (Ensaio II) e R=2 (Ensaio III). As concentrações médias afluentes foram 65 mg.NT.L-1, 580 mg.DQO.L-1, 77 mg.L-1 de alcalinidade total e pH de 6,4. As amostras foram coletadas na entrada e saída de cada compartimento, ao longo da altura do reator. Na Etapa I, e as eficiências de nitrificação foram de 76%, 70% e 41%, respectivamente para 14 horas, 11 horas e 8 horas, evidenciando o efeito do TDH. Em todas as etapas, a alcalinidade foi considerada o fator limitante do processo e o seu déficit variou de 10% a 30%. Atribuiu-se a esse fator a baixa eficiência na eliminação de nitrogênio total de aproximadamente 45%. Durante todo o experimento, a eficiência de remoção de matéria orgânica em termos de demanda química de oxigênio (DQO) foi superior a 90% e apresentou ajuste ao modelo cinético de primeira ordem para a degradação do substrato. No terceiro artigo, avaliou-se o comportamento hidrodinâmico desse sistema, a partir de ensaios de estímulo resposta utilizando o traçador Eosina Y. Avaliou-se o tempo de detenção hidráulica de 8 horas com as três taxas de recirculação, de 0,5, 1 e 2 vezes. Nessas condições, a remoção de matéria orgânica foi superior a 90% e a conversão de nitrogênio foi beneficiada com a aplicação de cargas menores (0,18 Kg.N.m-3.d-1). Na avaliação hidrodinâmica verificou-se que o reator apresentou comportamento de reator de mistura completa com 2 a 2,5 reatores em série (N-CSTR).

O presente trabalho teve por objetivo avaliar condições de imobilização da levedura Scheffersomyces stipitis NRRL Y-7124 pelo método do aprisionamento em gel de alginato de cálcio visando à produção de bioetanol em biorreator STR tipo cesta à partir de hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Primeiramente, realizou-se as etapas de obtenção, destoxificação e caracterização do hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Realizou-se em seguida um screening objetivando a seleção de um meio de cultivo adequado para a produção de etanol por esta levedura. O meio escolhido foi aquele onde se suplementou o hidrolisado com extrato de levedura (3,0 g/L), peptona (5,0 g/L), (NH4)2SO4 (2,0 g/L) e CaCl2 (0,1 g/L), onde verificou-se um fator de conversão de xilose à etanol (Yp/s) de 0,33 g/g. As condições de imobilização da levedura foram então avaliadas por planejamento fatorial 23 completo onde os fatores concentração de alginato de sódio, concentração do cloreto de cálcio e tempo de cura foram investigados. Após a análise estatística, as condições 2% de alginato de sódio, 0,1M de cloreto de cálcio e tempo de cura de 12 horas foram fixadas para as etapas seguintes. Nestas condições, avaliou-se então a influência da concentração de células à serem imobilizadas e agitação durante a fermentação a partir de um planejamento fatorial 22 completo, definindo-se assim 10 g/L de células e 100 rpm como condições ideais. Após a determinação das condições de imobilização do processo, verificou-se a estabilidade das células imobilizadas em repetidos ciclos fermentativos, para isso cinco bateladas repetidas em frascos Erlenmeyer foram realizadas. Observou-se que apesar da levedura assimilar xilose e produzir etanol em todos os ensaios, uma diminuição na eficiência da fermentação foi verificada, diminuindo em 24% da terceira para a quarta batelada, indicando assim que a levedura imobilizada era viável para o sistema de batelada repetida em até 3 ciclos nas condições estudadas. Iniciou-se então ensaios fermentativos em biorreator STR tipo cesta, realizando-se ensaios em meio sintético e em hidrolisado hemicelulósico. Observou-se reprodutibilidade nos ensaios utilizando os diferentes meios, com um valor de Yp/s de 0,21g/g e uma produtividade volumétrica de 0,15 g/L.h em ambos os ensaios. Fermentações em sistema de bateladas repetidas foram realizadas neste biorreator STR tipo cesta. Realizou-se cinco ciclos consecutivos, ao final dos quais observou-se comportamento semelhante às bateladas repetidas realizadas em frascos Erlenmeyer, na qual a partir de três ciclos a capacidade fermentativa da levedura S. stipitis diminuiu, apresentando uma produtividade volumétrica em torno de 0,16 g/L.h nas três primeiras bateladas. O gel de alginato de cálcio apresentou considerável estabilidade em sistema de bateladas repetidas indicando a possibilidade de sua utilização nesse processo. Embora os resultados obtidos neste trabalho sejam inferiores aos observados com células livres por outros autores, os mesmos demonstraram o potencial do emprego do gel de alginato de cálcio e da levedura Scheffersomyces stipitis imobilizada para a produção de bioetanol a partir de bagaço de cana-de-açúcar e contribuíram para os conhecimentos sobre a fermentação de hidrolisado hemicelulósico à etanol. / This study aimed to evaluate immobilization conditions for the yeast Scheffersomyces stipitis NRRL Y-7124 entrapped in calcium alginate gel in basket type of STR bioreactor for ethanol production from sugarcane bagasse hemicellulosic hydrolysate. For this purpose, first the steps to obtain the hydrolysate by dilute acid pretreatment, detoxification and characterization of hydrolysate was performed. Then, a screening aiming the selection of a suitable culture medium suitable for ethanol production by this yeast was carried out. The medium which showed maximum ethanol production (Yp/s, 0.33 g/g) was selected to continue the further studies. It was composed by the hydrolyzate supplemented with yeast extract (3.0 g/L), peptone (5.0 g/L), (NH4)2SO4 (2.0 g/L), CaCl2 (0.1g/L). The immobilization conditions of the yeast were then evaluated through a 23 factorial design where the three process variables i.e. concentration of sodium alginate, concentration of calcium chloride and reaction time were investigated. After statistical analysis, the optimum set of conditions (2% of sodium alginate, 0.1 M of calcium chloride and a reaction time of 12 hrs) were set to perform the following steps of this study. Subsequently, the influence of the cell concentration for immobilization and agitation during fermentation were studied considering a factorial design 22. This study revealed that 10 g/L of cells and 100 rpm were the optimum conditions for ethanol production via immobilized systems. After determination of the conditions for immobilization procedure, the stability of the immobilized cells were evaluated by repeated fermentation cycles, for that five repeated batches were performed in Erlenmeyer flasks. It was observed that despite the yeast assimilates xylose and produces ethanol in all assays, a decrease in the efficiency of the fermentation was verified from the third batch, revealing the 65% efficiency in the second batch and 39% in the fourth batch. This behavior indicates that the immobilized yeast is viable for repeated batch system only up to 3 cycles under the employed conditions. Fermentation tests in basket type STR bioreactor were carried out using synthetic medium and hemicellulosic hydrolysate as carbon source. Reproducibility was observed in assays using the different medium with ethanol yield (Yp/s) of 0.21 g/g and a volumetric productivity of 0.15 g/L.h in both assays. Fermentation assay in repeated batch system were carried out in STR basket type bioreactor. Five consecutive fermentation cycles were performed which eventually showed the similar behavior with the repeated batches conducted in Erlenmeyer flasks. The fermentative efficiency of the yeast S. stipitis was considerably good up to three cycles with a volumetric productivity of 0.16 g/L.h followed by a concomitant down fall. The calcium alginate gel showed a considerable stability in the experiments, indicating the viability of its application in repeated batch system. Although the results of this work are inferior to that observed by other authors using free cells, the calcium alginate gel potential is evident and the yeast Scheffersomyces stipitis showed to be capable to produce ethanol in immobilized form, contributing with knowledge for second generation ethanol production from sugarcane bagasse hemicellulosic hydrolysate adopting biochemical platform.

Se(VI) and Se(IV), as the two major species of selenium in water, are toxic to aquatic lives and may cause adverse health effects to humans at high levels. Biological reduction of Se(VI) is a two-stage process first from Se(VI) to Se(IV) and then from Se(IV) to Se(0) with potential accumulation of the more toxic Se(IV) due to the slower rate of the second stage. Selenium reduction was first evaluated with batch cultures of Shigella fergusonii strain TB42616 (TB) and Pantoea vagans strain EWB32213-2 (EWB) isolated in our laboratory from sludge and coal slurry sediment samples, respectively. In order to facilitate Se(VI) reduction and reduce Se(IV) accumulation, the Se(VI)-reducing strain TB was co-cultured with a Se(IV)-reducing strain EWB. Although Se(VI) reduction rate was not affected, Se(IV) reduction was significantly enhanced with low Se(IV) accumulation in the defined co-culture. Effects of culture composition as well as nitrate and arsenate on Se(VI) reduction were also investigated. A co-culture composition of 10:1 (EWB:TB) ratio was observed to achieve the best total selenium reduction. In addition, nitrate at 50 mg/L was observed to inhibit Se(IV) reduction but not Se(VI) reduction, while arsenate at 200 mg/L exhibited slight inhibition on both Se(VI) and Se(IV) reduction. Biokinetic parameters were optimized with a Monod-type kinetic model using batch pure culture data through the Robust Global Optimization Algorithm embedded in a computer package. Se(VI) reduction by the defined co-culture was then simulated and verified over a range of culture compositions and initial Se(VI) concentrations, respectively. An inter-species inhibition term was incorporated into the model to illustrate the competition for Se(IV) during Se(VI) reduction in the co-culture. The model showed a significant increase of Se(IV) accumulation with higher initial Se(VI) concentration. However, Se(IV) accumulation can be reduced with increasing population ratio of EWB to TB in the defined co-culture. The relatively high correlation coefficients suggested that the model was robust and applicable in simulating Se(VI) reduction by the defined co-culture. Since activated alumina was reported to be more effective for Se(IV) adsorption than Se(VI), the effect of biological activities on selenium removal was investigated using continuous-flow reactors packed with alum-impregnated activated alumina (AIAA) and cultured with a Se(VI)-reducing strain TB under various influent Se(VI) concentrations and hydraulic retention times (HRTs). A selenium removal efficiency of 92% was achieved in a bioreactor with initial biomass of 2.2×106 cells/g-AIAA after a 70-day operation period. Little improvement was observed by lowering the influent Se(VI) concentration from 50 to 10 mg/L while the removal efficiency was significantly enhanced by either extending the hydraulic retention time from 3.2 to 5.0 days or increasing the attached biomass during the startup. An increase in mass ratios of Se(VI) reduction by immobilized cells to adsorption by AIAA was also observed with increasing cell mass during the operation. Se(VI) reduction using continuous-flow reactors packed with strain TB immobilized Ca2+-alginate beads was investigated under various hydraulic retention times (HRT) and influent Se(VI) concentrations. A high removal efficiency up to 98.7% was achieved under an HRT of 5 days and an influent Se(VI) concentration of 400 mg/L. The results showed that the overall selenium removal was positively correlated to the bed height of the reactor and the HRT but not related to the influent Se(VI) concentration. The steady state was analyzed using a mathematical model based on Monod-type equations with four biokinetic parameters optimized including the half-velocity constants and maximum specific reduction rates. The relatively high correlation coefficients indicate that the model is robust and valid to simulate Se(VI) reduction in the gel-beads-packed continuous-flow system.

Immobilization is an effective method to increase ethanol production, as proven by previous research. Results almost exclusively demonstrate an increase in ethanol production by and decrease in reproduction rate of immobilized Saccharomyces cerevisiae cells. Recently, research has been conducted to determine the cause of this change. The extreme variance in results due to lack of technology makes it difficult to determine the cellular changes induced by immobilization. With the advent of new technology, specifically gene expression analysis, the RNA content of cells can be easily and rapidly analyzed. S. cerevisiae cells were immobilized in 3% (w/v) calcium alginate beads and grown inside of a packed bed reactor for comparison to planktonic cells growing in batch and chemostat cultures. Temperature inside of the reactor was maintained at 33 C with a pH of 5.5. Cell concentration inside of the beads was monitored periodically in order to create growth curves. Bud scar numbers of immobilized cells were also counted and compared to suspended cells. Scanning electron microscopy images of the alginate beads were taken to determine cell growth inside of the beads. Affymetrix Yeast 2.0 gene chips were used, and the data retrieved was analyzed with GeneSpring software using the Bioconductor packages. Results indicated changes in expression of 3,559 genes with significant difference among treatments by a factor of 2-fold or greater. One-way ANOVA of the filtered data yielded 380 highly significantly different genes between immobilized and suspended cells. Many of the genes pertaining to glycolysis exhibited increased expression levels. Several genes necessary for reproduction were expressed at lower levels in the immobilized cells than in their planktonic counterparts. Many different gene ontologies are discussed, and the expressed genes are mapped onto biochemical pathways.

Hydrogen sulphide (H2S) is a serious problem for many industries, including oil production and processing, pulp and paper, and wastewater treatment. In addition, H2S is usually present in natural gas and biogas. It is necessary to control the generation and release of H2S into the environment because H2S is corrosive, toxic, and has an unpleasant odour. In addition, the removal of H2S from natural gas and biogas is essential for preventing the emission of SO2 upon combustion of these gases. Physicochemical processes have been developed for the removal of H2S. These processes employ techniques such as chemical or physical absorption, thermal and catalytic conversion, and liquid phase oxidation. In comparison, biological processes for the removal of sulphide typically operate at ambient temperature and pressure, with the feasibility for the treatment of smaller streams, and the absence of expensive catalysts. The objective of the present work was to study the biooxidation of sulphide under denitrifying conditions in batch system and a continuous immobilized cell bioreactor using a mixed microbial culture enriched from the produced water of a Canadian oil reservoir. <p>In the batch experiments conducted at various initial sulphide concentrations, an increase in the sulphide oxidation and nitrate reduction rates was observed as the initial sulphide concentration was increased in the range 1.7 to 5.5 mM. An extended lag phase of approximately 10 days was observed when sulphide concentrations around or higher than 14 mM were used. This, when considered with the fact that the microbial culture was not able to oxidize sulphide at an initial concentration of 20 mM, indicates the inhibitory effects of sulphide at high concentrations.<p>The effect of the initial sulphide to nitrate concentrations ratio (ranging from 0.3 to 4.0) was also studied. As the initial sulphide to nitrate ratio decreased, the sulphide oxidation rates increased. The increasing trend was observed for initial nitrate concentrations in the range of 1.3 to 7.3 mM, corresponding to ratios of 4.08 to 0.83. The increase in nitrate reduction rates was more pronounced than that of the sulphide oxidation rates. However at nitrate concentrations higher than 7.3 mM (ratios lower than 0.83) the nitrate reduction rate remained constant. The percentage of sulphide that was oxidized to sulphate increased from 2.4% to 100% as the initial sulphide to nitrate ratio decreased from 4.08 to 0.42. This indicated that at ratios lower than 0.42, nitrate would be in excess and at ratios exceeding 4.08, nitrate would be limiting. In the continuous bioreactor systems, at sulphide loading rates ranging from 0.26 to 30.30 mM/h, sulphide conversion remained in the range of 97.6% to 99.7%. A linear increase in the volumetric oxidation rate of sulphide was observed as the sulphide loading rate was increased with the maximum rate being 30.30 mM/h (98.5% conversion). Application of immobilized cells led to a significant increase in oxidation rate of sulphide when compared with the rates obtained in a bioreactor with freely suspended cells. At nitrate loading rates ranging from 0.19 to 24.44 mM/h, the nitrate conversion ranged from 97.2% to 100% and a linear increase in volumetric reduction rate was observed as the nitrate loading rate was increased, with the maximum rate being 24.44 mM/h (99.7% conversion). <p>A second bioreactor experiment was conducted to investigate the effects of sulphide to nitrate concentrations ratio on the performance of the system. Sulphide conversion was complete at sulphide to nitrate ratios of 1.1 and 1.3, but decreased to 90.5% at the ratio of 3.1 and 65.0% at the ratio of 5.0, indicating nitrate was limiting for sulphide to nitrate ratios of 3.1 and 5.0. The increase in the sulphide to nitrate ratio (and the resulting limitation of nitrate) caused a decrease in the volumetric reaction rate of sulphide.<p>Nitrate conversion was complete at sulphide to nitrate ratios of 1.3, 3.1, and 5.0; however, at a ratio of 1.1, the conversion of nitrate dropped to 59.6%, indicating that nitrate was in excess, and sulphide was limiting. The volumetric reaction rate of nitrate decreased as the sulphide to nitrate ratio increased for ratios of 1.3, 3.1, and 5.0; this was due to the decrease in the nitrate loading rate. For sulphide to nitrate ratios of 1.1 and 1.3, 7.2% and 19.6% of the sulphide was converted to sulphate, respectively. At ratios of 3.1 and 5.0, no sulphate was generated. For ratios between 1.3 and 5.0, an increase in the ratio caused a decrease in the generation of sulphate.

Many industrial activities discharge sulphate- and metal-containing wastewaters, including the manufacture of pulp and paper, mining and mineral processing, and petrochemical industries. Acid mine drainage (AMD) is an example of such sulphate- and metal-containing waste streams. Formation of AMD is generally the result of uncontrolled oxidation of the sulphide minerals present in the terrain in which the drainage flows with concomitant leaching of the metals. Acid mine drainage (AMD) and other sulphate- and metal-containing waste streams are amenable to active biological treatment. Anaerobic reduction of sulphate, reaction of produced sulphide with metal ions present in the waste stream, and biooxidation of excess sulphide are three main sub-processes involved in the active biotreatment of AMD. Anaerobic reduction of sulphate can be achieved in continuous stirred tank bioreactors with freely suspended cells or in immobilized cell bioreactors. The application of freely suspended cells in a continuous system dictates a high residence time to prevent cell wash-out, unless a biomass recycle stream is used. In an immobilized cell system biomass residence time becomes uncoupled from the hydraulic residence time, thus operation of bioreactor at shorter residence times becomes possible. In the present work, kinetics of anaerobic sulphate reduction was studied in continuous immobilized cell packed-bed bioreactors. Effects of carrier matrix, concentration of sulphate in the feed and sulphate volumetric loading rate on the performance of the bioreactor were investigated. The bioreactor performance, in terms of sulphate reduction rate, was dependent on the nature of the carrier matrix, specifically the total surface area which was provided by the matrix for the establishment of biofilm. Among the three tested carrier matrices, sand displayed the superior performance and the maximum volumetric reduction rate of 1.7 g/L-h was achieved at the shortest residence time of 0.5 h. This volumetric reduction rate was 40 and 8 folds faster than the volumetric reduction rates obtained with glass beads (0.04 g/L-h; residence time: 28.6 h) and foam BSP (0.2 g/L-h; residence time: 5.3 h), respectively. Further kinetic studies with sand as a carrier matrix indicated that the extent of volumetric reduction rate was dependent on the feed sulphate concentration and volumetric loading rate. At a constant feed sulphate concentration, increases in volumetric loading rate caused the volumetric reduction rate to pass through a maximum, while increases in feed sulphate concentrations from 1.0 g/L to 5.0 g/L led to lower volumetric reduction rates. The maximum volumetric reduction rates achieved in the bioreactors fed with initial sulphate concentration of 1.0, 2.5 and 5.0 g/L were 1.71, 0.82 and 0.68 g/L-h, respectively. The coupling of lactate utilization to sulphate reduction was observed in all experimental runs and the rates calculated based on the experimental data were in close agreement with calculated theoretical rates, using the stoichiometry of the reactions involved. The maximum volumetric reduction rates achieved in the immobilized cell bioreactors were significantly faster than those reported for freely suspended cells employed in the stirred tank bioreactors.

This master thesis considers an investigation of protective formulations (ointment, cream) containing enzyme inhibitors. Model experiments have been made on the enzyme trypsin. It is well accepted that feces and urine are an important causing factor for skin irritation (dermatitis) while using diaper. A protective formulation is a physical barrier that separates the harmful substances from the skin. It can also be an active barrier containing active substances, which can be active both towards the skin, and the substances from feces and urine. By preventing contact from these substances the skin will not be harmed, at least for a period of time. A number of different inhibitors were tested towards trypsin and they all showed good inhibition, two of the inhibitors were selected to be immobilized with the help of NHS-­activated Sepharose. Immobilization of these two inhibitors leads to a lesser extent of the risk of developing allergy and also that the possible toxic effect can be minimized.