Thesis (M.Sc. Biological Science)--University of Waikato, 2008. / Title from PDF cover (viewed September 18, 2008) Includes bibliographical references (p. 84-96)

Thesis (Ph. D.)--Rijksuniversiteit te Groningen, 1983. / Forword and summary in Dutch. Errata sheet inserted. Includes bibliographical references.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

O hidrogênio (H2) produzido a partir de microrganismos fotossintetizantes (microalgas e cianobactérias) é considerado um vetor energético sustentável do ponto de vista ambiental. Quando comparado a outras formas de produção são encontradas limitações técnicas e econômicas principalmente pela geração de pequenos volumes de gás e pela inexistência de um ciclo de vida completo que assegure a produção contínua de H2 por esses organismos. Nesse sentido, o presente projeto teve por propósito avaliar a capacidade de produção de H2, sob condições mixotróficas, em três estirpes de cianobactérias: Anabaena sp. UTEX 1448, Anabaena sp. PCC7120 (selvagem) e Anabaena sp. PCC 7120 &Delta;hypF (mutante). O primeiro capítulo tratou da otimização de biomassa da estirpe UTEX 1448, utilizando meio de cultura BG-11 (Rippka, 1979), em condições controladas de pH (10,2), temperatura (32 ºC), radiação (30 &micro;mol.m-2.s-1), com cinco diferentes substratos orgânicos (ácido lático, glicerol, glicose, frutose e sacarose), em três concentrações de carbono (0,20; 0,52 e 0,84 gC.L-1). No segundo capítulo, investigou-se a produção de H2, pelas enzimas hidrogenase bidirecional, de assimilação e nitrogenase, com medições in vivo, a partir do uso do eletrodo de H2 (Hansatech, Ltd) e análises de clorofila a, proteínas (Western Immunoblotting) e géis de poliacrilamida desnaturantes. Os ensaios de H2 foram realizados para as três estirpes, em triplicata, com réplicas biológicas nos dias 0h, 24h, 48h, 72h e 7 dias, após passagem das culturas de condições não fixadoras de nitrogênio para condições fixadoras de H2, considerando a condição otimizada encontrada no Capítulo 1. Utilizou-se BG-11 para aumento da densidade celular em níveis que viabilizassem a realização dos ensaios e BG-110 (sem nitrato) para estímulo à diferenciação celular em heterocistos, estrutura importante por conter a enzima nitrogenase, diretamente relacionada à geração de H2. A fonte de carbono orgânico frutose a 0,84 gC.L-1 foi a condição otimizada encontrada, com produtividade de biomassa de 190 ± 18 mg.L-1.dia-1 (Anova, Tukey, p < 0,05). A estirpe mutante não cresceu nas condições otimizadas do cultivo e consequentemente não foi possível quantificar a geração de H2. Em fase clara, aos 7 dias, a maior produtividade de H2 foi de 0,50 ± 0,38 &micro;molH2.mg clorofila a-1.h-1 para a cepa PCC 7120 (selvagem) e na fase escura obteve-se produtividade média de H2 de 0,147 ± 0,00 &micro;molH2.mg clorofila a-1.h-1, ao dia 0 (0h) para a estirpe UTEX 1448. / Hydrogen (H2) produced from photosynthetic microorganisms (microalgae and cyanobacteria) is considered an environmentally sustainable energy vector. When compared to other production ways, some technical and economic limitations are found mainly because of the small amount of gas generated and also due the lack of a complete life cycle that assures the constant generation of hydrogen by these organisms. Regarding to this, the following study intended to evaluate the capacity of hydrogen production under mixotrophic conditions in three cyanobacteria strains: Anabaena sp. UTEX 1448, Anabaena sp. PCC7120 (wild type) and Anabaena sp. PCC 7120 &Delta;hypF (mutant). The first chapter refers to the optimization of the biomass of the UTEX1448 strain, using BG-11 as a mean of culture (Rippka, 1979) under controlled conditions of pH (10,2), radiation (30 &micro;mol.m-2.s-1), temperature (32ºC), with no photoperiod, five different organic substrates (lactic acid, glycerol, glucose, fructose and sucrose) in three different carbon concentrations (0.20, 0.52 and 0.84 gC.L-1). The second chapter investigated the hydrogen production by the bidirectional hydrogenases enzymes, by uptake and nitrogenase, with in vivo measurements using the hydrogen electrode (Hansatech, Ltd), chlorophyll a and proteins analyses (Western Immunoblotting and SDS-Polyacrylamide gels). The H2 assays were performed for the three strains, in triplicate, with biological replicates on days 0h, 24h, 48h, 72h and 7 days, considering the optimized condition found in Chapter 1. BG-11 medium was used to increase cell density at levels that would be viable to perform the tests and BG-110 (without nitrate) to stimulate cell differentiation in heterocysts, an important structure that contains the nitrogenase enzyme, directly related to H2 generation. The source of organic carbon fructose at 0.84 gC.L-1 was the optimized condition found, with biomass productivity of 190 ± 18 mg.L-1.day-1 (ANOVA, Tukey, p < 0.05). The mutant strain did not grow under optimized culture conditions and consequently it was not possible to quantify H2 generation. In the light phase, at 7 days, the highest yield of hydrogen was 0.50 ± 0.38 &micro;molH2.mg chlorophyll a-1.h-1 for the strain PCC 7120 (wild) and in the dark phase yielded average productivity of hydrogen from 0.147 ± 0 &micro;molH2.mg chlorophyll a-1.h-1, at day 0 (0h) for strain UTEX 1448.

O hidrogênio (H2) produzido a partir de microrganismos fotossintetizantes (microalgas e cianobactérias) é considerado um vetor energético sustentável do ponto de vista ambiental. Quando comparado a outras formas de produção são encontradas limitações técnicas e econômicas principalmente pela geração de pequenos volumes de gás e pela inexistência de um ciclo de vida completo que assegure a produção contínua de H2 por esses organismos. Nesse sentido, o presente projeto teve por propósito avaliar a capacidade de produção de H2, sob condições mixotróficas, em três estirpes de cianobactérias: Anabaena sp. UTEX 1448, Anabaena sp. PCC7120 (selvagem) e Anabaena sp. PCC 7120 &Delta;hypF (mutante). O primeiro capítulo tratou da otimização de biomassa da estirpe UTEX 1448, utilizando meio de cultura BG-11 (Rippka, 1979), em condições controladas de pH (10,2), temperatura (32 ºC), radiação (30 &micro;mol.m-2.s-1), com cinco diferentes substratos orgânicos (ácido lático, glicerol, glicose, frutose e sacarose), em três concentrações de carbono (0,20; 0,52 e 0,84 gC.L-1). No segundo capítulo, investigou-se a produção de H2, pelas enzimas hidrogenase bidirecional, de assimilação e nitrogenase, com medições in vivo, a partir do uso do eletrodo de H2 (Hansatech, Ltd) e análises de clorofila a, proteínas (Western Immunoblotting) e géis de poliacrilamida desnaturantes. Os ensaios de H2 foram realizados para as três estirpes, em triplicata, com réplicas biológicas nos dias 0h, 24h, 48h, 72h e 7 dias, após passagem das culturas de condições não fixadoras de nitrogênio para condições fixadoras de H2, considerando a condição otimizada encontrada no Capítulo 1. Utilizou-se BG-11 para aumento da densidade celular em níveis que viabilizassem a realização dos ensaios e BG-110 (sem nitrato) para estímulo à diferenciação celular em heterocistos, estrutura importante por conter a enzima nitrogenase, diretamente relacionada à geração de H2. A fonte de carbono orgânico frutose a 0,84 gC.L-1 foi a condição otimizada encontrada, com produtividade de biomassa de 190 ± 18 mg.L-1.dia-1 (Anova, Tukey, p < 0,05). A estirpe mutante não cresceu nas condições otimizadas do cultivo e consequentemente não foi possível quantificar a geração de H2. Em fase clara, aos 7 dias, a maior produtividade de H2 foi de 0,50 ± 0,38 &micro;molH2.mg clorofila a-1.h-1 para a cepa PCC 7120 (selvagem) e na fase escura obteve-se produtividade média de H2 de 0,147 ± 0,00 &micro;molH2.mg clorofila a-1.h-1, ao dia 0 (0h) para a estirpe UTEX 1448. / Hydrogen (H2) produced from photosynthetic microorganisms (microalgae and cyanobacteria) is considered an environmentally sustainable energy vector. When compared to other production ways, some technical and economic limitations are found mainly because of the small amount of gas generated and also due the lack of a complete life cycle that assures the constant generation of hydrogen by these organisms. Regarding to this, the following study intended to evaluate the capacity of hydrogen production under mixotrophic conditions in three cyanobacteria strains: Anabaena sp. UTEX 1448, Anabaena sp. PCC7120 (wild type) and Anabaena sp. PCC 7120 &Delta;hypF (mutant). The first chapter refers to the optimization of the biomass of the UTEX1448 strain, using BG-11 as a mean of culture (Rippka, 1979) under controlled conditions of pH (10,2), radiation (30 &micro;mol.m-2.s-1), temperature (32ºC), with no photoperiod, five different organic substrates (lactic acid, glycerol, glucose, fructose and sucrose) in three different carbon concentrations (0.20, 0.52 and 0.84 gC.L-1). The second chapter investigated the hydrogen production by the bidirectional hydrogenases enzymes, by uptake and nitrogenase, with in vivo measurements using the hydrogen electrode (Hansatech, Ltd), chlorophyll a and proteins analyses (Western Immunoblotting and SDS-Polyacrylamide gels). The H2 assays were performed for the three strains, in triplicate, with biological replicates on days 0h, 24h, 48h, 72h and 7 days, considering the optimized condition found in Chapter 1. BG-11 medium was used to increase cell density at levels that would be viable to perform the tests and BG-110 (without nitrate) to stimulate cell differentiation in heterocysts, an important structure that contains the nitrogenase enzyme, directly related to H2 generation. The source of organic carbon fructose at 0.84 gC.L-1 was the optimized condition found, with biomass productivity of 190 ± 18 mg.L-1.day-1 (ANOVA, Tukey, p < 0.05). The mutant strain did not grow under optimized culture conditions and consequently it was not possible to quantify H2 generation. In the light phase, at 7 days, the highest yield of hydrogen was 0.50 ± 0.38 &micro;molH2.mg chlorophyll a-1.h-1 for the strain PCC 7120 (wild) and in the dark phase yielded average productivity of hydrogen from 0.147 ± 0 &micro;molH2.mg chlorophyll a-1.h-1, at day 0 (0h) for strain UTEX 1448.

The majority of power generated worldwide is from combustion of fossil fuels. The sustainability and environmental impacts of this non renewable process are severe. Alternative fuels and power generation systems are needed, however, to cope with increasing energy demands. Ammonia shows promise for use in power generation, however it is costly to produce and very few methods of using it as a fuel are developed. To address the need for alternative methods of ammonia synthesis, this research designed and tested a bioelectrochemical device that generates NH3 through electrode induced enzyme catalysis. The ammonia generating device consists of an electrode modified with a polymer that contains whole cell Anabaena variabilis, a photosynthetic cyanobacterium. A. variabilis contains nitrogenase and nitrate/nitrite reductase, catalysts for the production of ammonia. In this system, the electrode supplies driving force and generates a reductive microenvironment near cells to facilitate enzymatic production of NH3 at ambient temperatures and pressures. Farm animal wastes contain significant amounts of NO2- and NO3-, which can leech into groundwater sources and contaminate them. The system described here recycles NO2- and NO3- to NH4sup+ by the nitrate/nitrite reductase enzyme. Unlike nitrogen fixation by the nitrogenase enzyme whose substrate is atmospheric N2, the substrates for nitrate/nitrite reductase are NO2- and NO3-. The ammonia produced by this system shows great potential as a crop fertilizer. While the substrates and enzymatic basis for ammonia production by nitrogenase and nitrate/nitrite reductase are very different, there is utility in the comparison of commercially produced ammonia by the Haber Bosch synthesis and by the bioelectrocatalytic device described here. In one day, the Haber Bosch process produces 1800 tons of NH3 at an energetic cost of $500/ton. Per ton of ammonia, the Haber Bosch process consumes 28 GJ of energy. The bioelectrocatalytic device produces 1 ton of NH3 for $10/ton, consuming only 0.04 GJ energy, which can be obtained by sunlight via installation of a photovoltaic device. Thus, the system presented here demonstrates ammonia production with significant impact to the economy. NH3 production by the bioelectrocatalytic is dependent upon A. var. cell density and electrode polarization. The faradaic current response from cyclic voltammetry is linearly related to cell density and ammonia production. Without electrode polarization, immobilized A. var. do not produce ammonia above the basal level of 2.8 ± 0.4 ΜM. Ten minutes after cycled potential is applied across the electrode, average ammonia output increases to 22 ± 8 ΜM depending on the mediator and substrate chemicals present. Ammonia is produced by this system at 25 °℃ and 1 atm. The electrochemical basis for enhanced NH3 by immobilized cyanobacteria is complex with multiple levels of feedback.

Ammonia is an important compound to many industries around the world. Most of the fertilizers used by crop growers have ammonia as an essential ingredient. It can also be useful as a fuel source, offering greater energy density per unit than hydrogen and greater safety. Currently, the predominant method for producing ammonia on an industrial scale is by the Haber-Bosch process. This process uses steam evolution of methane to provide H2 gas, which is then combined with N2 gas over an iron catalyst to form NH3. This process requires large amounts of energy as well as high temperatures and pressures. Here, an alternative method for ammonia production is explored. With Anabaena Variabilis, a photosynthetic cyanobacteria, on a carbon electrode, ammonia can be generated at ambient temperatures and pressures at little energy cost, a few tenths of a volt. A bioelectrocatalytic device has been constructed by immobilizing whole cell a. variabilis in a Nafion film modified with a trimethyl octadecyl ammonium bromide (TMODA) salt at an electrode surface [3]. The polymer modified electrode provides the driving force and reductive microenvironment to facilitate production of NH3 by nitrogenase and nitrate/nitrite reductase enzymes present in a. variabilis. Ammonia production by cyanobacteria were increased from basal levels of 2.8 ± 0.4 µM produced over a two week period, to 22 ± 8 µM produced in 20 minutes under mild voltage perturbation, roughly 104% increase in rate. Control of ammonia producing structures (nitrogenase in heterocystic cells or nitrate/nitrite reductase in vegetative cells) can be accomplished by growing the algae with and without fixed sources of nitrogen in the growth media. With the addition of various nitrogen-containing gases to the electrolyte solution during cyclic voltammetry, there is evidence that biofilms containing a mixture of cell types increases ammonia production above controls when the nitrogen is present as NO2-, NO, or N2O. Chronoamperometric perturbation studies show increased ammonia production at near +600 mV and -300 mV vs SCE. In cyclic voltammetric studies, nitrate/nitrite reductase in vegetative-only biofilms responds favorably to positive voltage ranges, while isolated heterocyst biofilms containing nitrogenase can be effectively targeted with the application of a negative voltage profile. References: [1] Johna Leddy and Timothy M. Pashkewitz, Ammonia Production Using Bioelectrocatalytic Devices, US Patent Application 20140011252 [2] Timothy M. Paschkewitz, Ammonia Production at Ambient Temperature and Pressure: An Electrochemical and Biological Approach, Ph.D., University of Iowa, 2012.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2000. / Bibliography: p. 158-173.

Univ., Diss.--Regensburg, 1992.

Made available in DSpace on 2016-06-02T19:32:03Z (GMT). No. of bitstreams: 1 DissRBC.pdf: 967066 bytes, checksum: 70991b6b98884e4372060fd788d5c4d4 (MD5) Previous issue date: 2004-05-13 / Financiadora de Estudos e Projetos / Human specie can alter deeply and very fast the enviroment in which it is in. Because of the industrial development, the water, air and soil contamination have become cause of concern, chiefly in big cities with great populations. Among the contaminants figure the heavy metals, which levels at aquatic and terrestrial ecossistems are growin up every year. These elements are able to bioaccumulate in the organisms and biomagnified on the food webs. The bioavailability of metals can be influencied by several factors like the complexation with dissolved organic matter, e.g. algal exudates, that generally decreases the toxicity of this elements. The scope of this work was evaluate the potencial use of exopolysccharides of Anabaena spiroides (Cyanophyceae) as food source of Ceriodaphnia cornuta (Cladocera, Daphnidae), and to establish the influence of this organic matter on copper toxicity and uptake to this cladoceran. Initially, it was estabilished the C. cornuta length-weigth relation. After this, it was investigated ingestion of exopolysccharide and its influence in life history parameters of C. cornuta. Results showed that A. spiroides exopolysccharide is able to sustain a population of this zooplanktonic specie. Individuals fed with this compound exhibited rate of population growth very significant to this specie (r = 0,263). The copper acute toxicity and uptake by C. cornuta assay revealed that addition of 30mg L-1 of A. spiroides exopolysccharide increased about 4 times copper EC/50 (calculated by Trimmed Spearman-Karber method) to C. Cornuta (from 8,11x10-8M ±9,80x10-9M without exopolysccharide to 3,25x10-7M ±5,30x10-8M with addition of exopolysccharide). Copper concentration in the organisms after 24 hours exposure to several metal concentration was determined by DPASV using a polarograph and showed little variation among concentrations and treatments with and without exopolysccharide. It suggests that organisms of this study were able to regulate copper body contents. / A espécie humana altera profundamente e com grande rapidez o ambiente no qual se insere. Com o desenvolvimento industrial, a contaminação da água, do ar e do solo tornou-se preocupante, sobretudo nas grandes cidades densamente povoadas. Dentre os contaminantes, estão os metais pesados, cujos níveis nos ecossistemas aquáticos e terrestres vêm aumentando a cada ano. Esses elementos podem ser bioacumulados nos organismos e biomagnificados nas cadeias tróficas. A biodisponibilidade de metais pode ser influenciada por vários fatores, entre eles, a formação de complexos com a matéria orgânica dissolvida, como exudatos algais, que geralmente diminui a toxicidade desses elementos. O escopo deste trabalho foi avaliar o uso potencial de exopolissacarídeos de Anabaena spiroides (Cyanophyceae) como fonte alimentar de Ceriodaphnia cornuta (Cladocera, Daphnidae), bem como determinar a influência dessa matéria orgânica na toxicidade e captura do cobre por esse cladócero. Inicialmente, foi confeccionada uma regressão peso seco (µg) comprimento (mm) para Ceriodaphnia cornuta. Em seguida, foi investigada a ingestão do exopolissacarídeo por C. cornuta e a influência desse tipo de alimento em parâmetros bionômicos dessa espécie zooplanctônica. Os resultados demonstraram que o exopolissacarídeo A. spiroides é capaz de sustentar uma população de C. cornuta. Os animais alimentados com esse composto apresentaram taxa de crescimento populacional (r) de 0,263, bastante significativa para a espécie. O experimento de toxicidade e captura de cobre por C. cornuta revelou que a adição de 30mg L-1 de exopolissacarídeo de A. spiroides aumentou em aproximadamente 4 vezes a EC/50 (calculada pelo método Trimmed Spearman-Karber ) do cobre para C. Cornuta (de 8,11x10-8M ±9,80x10- 9M - sem exopolissacarídeo - para 3,25x10-7M ±5,30x10-8M com exopolissacarídeo). As concentrações de cobre nos organismos após 24 horas de exposição a diferentes concentrações do metal no meio experimental foram determinadas em polarógrafo através da técnica de DPASV e demonstraram pouca variação entre concentrações e entre os tratamentos com e sem exopolissacarídeos, o que sugere que os organismos testados regulem o conteúdo de cobre no corpo.