Nitrite (NO2-) substrate under certain conditions can cause failure of N-removal processes relying on anaerobic ammonium oxidizing (anammox) bacteria. Detoxification of NO2- can potentially be achieved by using exogenous nitrate (NO3-). In this work, continuous experiments in bioreactors with anammox bacteria closely related to “Candidatus Brocadia caroliniensis” were conducted to evaluate the effectiveness of short NO3- additions to reverse NO2- toxicity. The results show that a timely NO3- addition immediately after a NO2- stress event completely reversed the NO2- inhibition. This reversal occurs without NO3- being metabolized as evidence by lack of any 30N2 formation from 15N-NO3-. The maximum recovery rate was observed with 5 mM NO3- added for 3 days; however, slower but significant recovery was also observed with 5 mM NO3- for 1 day or 2 mM NO3- for 3 days. Without NO3- addition, long-term NO2- inhibition of anammox biomass resulted in irreversible damage of the cells. These results suggest that a short duration dose of NO3- to an anammox bioreactor can rapidly restore the activity of NO2--stressed anammox cells. On the basis of the results, a hypothesis about the detoxification mechanism related to narK genes in anammox bacteria is proposed and discussed.

Bactérias aclimatadas a cobre foram isoladas a partir de amostras de solo e água coletadas na região da Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. foi escolhida, pois, apresentou a maior capacidade sortiva de Cu2+, Q = 40 mg/g. O cultivo em regime de batelada, meio mineral, com glicerol como fonte de carbono, resultou fator de conversão de glicerol a células, YX/S, de 0,49 g/g e velocidade específica máxima de crescimento, mmax, de 0,11 h-1. Alta concentração celular, 90 g/L, foi alcançada em cultivos em regime de batelada alimentada. Promoveu-se sorção de cobre pelas células, por meio de adição contínua ou em pulsos, de solução de CuSO4. A maior sorção específica de cobre, Q, de 30 (mg de Cu2+/g de células), foi verificada na adição por pulsos. Fotos de MET da bactéria na ausência e presença de Cu2+ mostram acúmulo de cobre na membrana e internamente, caracterizando biossorção e bioacumulação. / Bacteria acclimated to copper were isolated from soil and water samples collected in Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. was chosen as it had the highest sorptive capacity for Cu2+, Q = 40 mg/g. The fed-batch culture in mineral medium with glycerol as the carbon source resulted in a glycerol-to-cell conversion factor, YX/S of 0.49 g/g and maximum specific growth rate, mmax of 0.11 h-1. High cell concentration, 90 g/L, was achieved in cultures in fed-batch regimen. Cooper sorption by cells was promoted, by continuous or pulse addition of CuSO4 solution. The highest specific copper sorption, Q, 30 (mg Cu+2/g of cells) was seen with the addition by pulses. TEM photos of the bacteria in the absence and presence of Cu+2 show copper accumulation in the membrane and internally, featuring biosorption and bioaccumulation.

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.

Macromolecular components, including protein and polysaccharides, are viewed as one type of major foulants in the complex feed membrane filtration systems such as membrane bioreactor (MBR). In this thesis, the mechanisms of macromolecular fouling including protein and polysaccharide in the complex feed solution are explored by using Bovine serum albumin (BSA) and alginate as model solution. During the filtration of BSA and washed yeast with 0.22 ????m PVDF membrane, it was found that the critical flux of mixture solution was controlled by washed yeast concentration while the existence of BSA significantly changed the cake reversibility of much larger particles. The fouling mechanisms of alginate, as a model polysaccharide solution, were investigated both in dead end and crossflow membrane filtration. In the dead end experiments, it was found that the cake model appears to fit the entire range of the ultrafiltration data while the consecutive standard pore blocking model and cake model are more applicable to microfiltration membranes. The alginate was featured with high specific cake resistance and low compressibility despite some variations between different membranes. The specific cake resistance ( c ) is similar to c of BSA and actual extracellular polymer substance (EPS) in MBR systems reported in the literature, and higher than that of many colloidal particles. In a system contained alginate-particles mixture, it was found that the existence of alginate dramatically increased the cake specific resistance and decreased the cake compressibility. The fouling mechanism of alginate was also studied using long term cross flow filtration under subcritical flux. A two-stage TMP profile similar to that typically observed in MBR was obtained, confirming the important role of EPS during membrane fouling in MBR. In addition to adsorption, trace deposition of alginate also contributed to the initial slow TMP increase during the subcritical filtration. TMP increase during the long-term filtration was found not only due to the increase of the amount of deposition, but also the increase of specific cake resistance. A combined standard pore blocking and cake filtration model, using a critical pore size for the transition time determination, was developed and fit the experimental results well.

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.

This thesis presents the study of biodegradation factors of a candidate naphthenic acid compound, the trans isomer of 4-methyl-1-cyclohexane carboxylic acid (trans-4MCHCA). Low molecular weight components of naphthenic acids such as trans-4MCHCA are known to be toxic in aquatic environments and there is a need to better understand the factors controlling the kinetics of their biodegradation. In this study, a relatively low molecular weight naphthenic acid compound and a microbial culture developed in our laboratory (primarily Alcaligenes paradoxus and Pseudomonas aeruginosa) were used to study the biodegradation of this candidate naphthenic acid. The purpose of the research was to evaluate the kinetic parameters and model the biodegradation of this compound in three bioreactor systems: batch reactors, a continuously stirred tank reactor and immobilized cell reactors. <p>In batch reactors, the maximum specific growth rate (0.52±0.04 d-1) of the consortium at 23oC and neutral pH was not highly variable over various initial substrate concentrations (50 to 750 mg/L). Batch experiments indicated that biodegradation can be achieved at low temperatures; however, the biodegradation rate at 4oC was only 22% of that at room temperature (23oC). Biodegradation at various pH values indicated a maximum specific growth rate of 1.69±0.40 d-1 and yield (0.41±0.06 mg/mg) at a pH of 10. <p>Study of the candidate substrate using a continuously stirred tank reactor and the microbial culture developed in the batch experimentations revealed that the kinetics of the candidate naphthenic acid are best described by the Monod expression with a maximum specific growth rate of 1.74±0.004 d-1 and a half saturation constant of 363±17 mg/L. The continuously stirred tank reactor achieved a maximum reaction rate of 230 mg/(L∙d) at a residence time of 1.6 d-1 (39 h).<p>Two high porosity immobilized cell reactors operating continuously over three months were found to consume trans-4MCHCA at a rate almost two orders of magnitude higher than a continuously stirred tank reactor. The immobilized cell systems attained a maximum reaction rate of 22,000 mg/(L∙d) at a residence time of 16 minutes. High porosity immobilized cell reactors were shown to effectively remove a single naphthenic acid substrate in continuously fed operation to dilution rates of 90 d-1. A plug flow model best represented the degradation in the immobilized cell systems and was demonstrated to be a useful tool for studying the effects of parameter variation and prediction of reactor performance. This work highlights the potential of augmented bioremediation systems for the degradation of naphthenic acids.

El projecte MELISSA (Micro Ecological Life Support System Alternative) estudia el desenvolupament d'un sistema de suport de vida biològic per les missions espacials de llarga durada. Per assolir aquest fi aquest projecte proposa, com a primer model, la utilització de quatre compartiments microbiològics i un compartiment de plantes superiors.Una de les vessants més importants d'aquest projecte és l'estudi de la connexió dels seus compartiments. L'objectiu d'aquest sistema és treballar de forma contínua en períodes llargs d'operació. Cal doncs estudiar l'evolució d'aquesta connexió tant en condicions òptimes de funcionament com davant de possibles pertorbacions.En aquest treball s'ha estudiat inicialment la connexió amb un medi sintètic dels compartiments II, III i IVa del bucle MELISSA a escala de laboratori. Aquesta connexió s'ha dut a terme satisfactòriament sense que s'evidenciïn efectes tòxics o nocius en cap dels compartiments al treballar en diferents condicions d'operació. S'ha procedit també a la connexió dels compartiments III i IVa a escala pilot, comprovant el seu bon funcionament i corroborant el sistema de control del compartiment IVa quan aquest treballa en connexió.També s'han estudiat dues de les pertorbacions possibles del sistema: l'entrada d'àcid acètic en el tercer compartiment i l'entrada de nitrits en el quart compartiment. Cap de les dues pertorbacions ha tingut efectes negatius en el funcionament dels compartiments on s'han dut a terme. Finalment s'ha procedit a la connexió completa en fase líquida i a escala de laboratori dels quatre compartiments microbiològics del bucle global del sistema MELISSA. Aquests estudis s'han dut a terme amb els diferents bioreactors dels quals es disposa a la planta pilot del projecte MELISSA de la UAB, lloc on s'efectua la integració i demostració dels diferents elements del bucle i del seu conjunt.Per altra banda s'ha fet un estudi del dimensionat dels diferents compartiments del projecte MELISSA i del grau de tancament assolit en el sistema, en diferents escenaris d'operació. Aquests inclouen tant la seva operació en el laboratori, utilitzant animals d'experimentació, com la seva aplicació al manteniment de la vida humana. / The MELISSA project (Microbiological Ecological Life Support System Alternative) of the European Space Agency (ESA) is a tool for the development of a biological life support system to be used during Manned Space Missions. In order to achieve this purpose the project proposes the connection between five compartments, four of which contain microbial organisms and one higher plants. To assure the satisfactory operation of the system, it is important to study the connection between these bioreactors not only at optimal conditions but also taking into account possible deviations in the behaviour of any of them.The connection between compartments II, III and IVa at laboratory scale and using an artificial medium has been studied at different operational conditions in this work. Using these different test conditions, the connection showed an excellent functioning during more than 5 residence times. Non relevant toxic effects have been observed.The connection between compartments III and IVa at pilot plant scale, as well as the feasibility of the control system of compartment IVa while it is working in connection to compartment III, have been also successfully tested. Two different deviations from the optimal operational conditions in the connection of compartments II, III and IVa have been studied: the income of volatile fatty acids in compartment III and the income of nitrite in compartment IVa. None of these two deviations produced any significant modification on the performance of these bioreactors. Finally, the connection of the whole MELISSA microbial loop at liquid stage has been performed at laboratory scale without presenting any major problem. In parallel, the compartments size and the achieved closure degree have been evaluated for different operational scenarios of the MELISSA loop. These scenarios include its use as a laboratory tool using experimental animals and its use as a life support system for a human crew.

This thesis presents the study of biodegradation factors of a candidate naphthenic acid compound, the trans isomer of 4-methyl-1-cyclohexane carboxylic acid (trans-4MCHCA). Low molecular weight components of naphthenic acids such as trans-4MCHCA are known to be toxic in aquatic environments and there is a need to better understand the factors controlling the kinetics of their biodegradation. In this study, a relatively low molecular weight naphthenic acid compound and a microbial culture developed in our laboratory (primarily Alcaligenes paradoxus and Pseudomonas aeruginosa) were used to study the biodegradation of this candidate naphthenic acid. The purpose of the research was to evaluate the kinetic parameters and model the biodegradation of this compound in three bioreactor systems: batch reactors, a continuously stirred tank reactor and immobilized cell reactors. <p>In batch reactors, the maximum specific growth rate (0.52±0.04 d-1) of the consortium at 23oC and neutral pH was not highly variable over various initial substrate concentrations (50 to 750 mg/L). Batch experiments indicated that biodegradation can be achieved at low temperatures; however, the biodegradation rate at 4oC was only 22% of that at room temperature (23oC). Biodegradation at various pH values indicated a maximum specific growth rate of 1.69±0.40 d-1 and yield (0.41±0.06 mg/mg) at a pH of 10. <p>Study of the candidate substrate using a continuously stirred tank reactor and the microbial culture developed in the batch experimentations revealed that the kinetics of the candidate naphthenic acid are best described by the Monod expression with a maximum specific growth rate of 1.74±0.004 d-1 and a half saturation constant of 363±17 mg/L. The continuously stirred tank reactor achieved a maximum reaction rate of 230 mg/(L∙d) at a residence time of 1.6 d-1 (39 h).<p>Two high porosity immobilized cell reactors operating continuously over three months were found to consume trans-4MCHCA at a rate almost two orders of magnitude higher than a continuously stirred tank reactor. The immobilized cell systems attained a maximum reaction rate of 22,000 mg/(L∙d) at a residence time of 16 minutes. High porosity immobilized cell reactors were shown to effectively remove a single naphthenic acid substrate in continuously fed operation to dilution rates of 90 d-1. A plug flow model best represented the degradation in the immobilized cell systems and was demonstrated to be a useful tool for studying the effects of parameter variation and prediction of reactor performance. This work highlights the potential of augmented bioremediation systems for the degradation of naphthenic acids.

Thesis (M.S.)--University of Hawaii at Manoa, 2004. / Includes bibliographical references (leaves 48-51).

Thesis (Ph. D.)--Mississippi State University. Department of Civil Engineering. / Title from title screen. Includes bibliographical references.