Berlin, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Ingeniero en Biotecnología Molecular. / La chaperona bacteriana periplasmática CusF es parte del sistema de resistencia a cobre CusCFBA. CusF une cobre (I) en el periplasma bacteriano mediante una histidina y dos metioninas muy conservadas en las bacterias. En Acidithiobacillus ferrooxidans, la histidina del sitio de unión a cobre de CusFAf se encuentra sustituída por metionina. Se ha determinado que a pesar que la afinidad de la histidina por cobre es mayor que la de la metionina a pH neutro, esta última presenta una mayor afinidad por cobre a pH ácido. Siendo A. ferrooxidans una bacteria que vive en ambientes ácidos, esta sustitución podría estar relacionada con la adaptación al periplasma ácido de la bacteria. Se analizó bioinformáticamente el sitio de unión a cobre de CusF para determinar las especies en las que se encuentra esta sustitución. Se encontró que sólo el género Acidithiobacillus presenta esta sustitución. Adicionalmente se midió la unión de cobre a la CusF de A. ferrooxidans y el efecto que tiene esta sustitución. Para ello se clonó el gen cusF3Af en E. coli y se realizó una mutación sitio dirigida de su metionina 66 cambiándola por histidina. Luego de expresar y purificar ambas proteínas (silvestre y mutada) se midió in vitro su capacidad de unir cobre tanto a pH ácido como neutro. A pH 7, la CusF3 de A. ferrooxidans presentó unión a cobre con un valor experimental de 0.7 átomos de cobre por proteína mientras que la mutante M66H unió 0.3 átomos de cobre por proteína (la misma cantidad de cobre que el control). A pH ácido no fue posible medir la unión de cobre con el método utilizado. Se concluye que la sustitución de histidina por metionina en la CusF3Af es exclusiva del género Acidithiobacillus y que esta chaperona tiene capacidad de unir cobre a pH neutro. / The periplasmatic metallochaperone CusF is part of the CusCFBA copper resistance system. CusF binds Cu(I) through a conserved site consisting of 1 histidine and 2 methionines. In Acidithiobacillus ferrooxidans, this histidine present in the position 66 of CusFAf is found substitued by a methionine. It has been determined that while the affinity of copper for histidine is higher than that for methionine at neutral pH, the latter has higher affinity for the metal at low pH. Since A. ferrooxidans lives in acidic environments, it is proposed that this subsitution could be an adaptation to the acid periplasm of this bacterium. The copper binding site of CusF was analyzed bioinformatically to determine the species in which this subsitution is found. This substitution was only present in the CusF of species from the genus Acidithiobacillus. Additionally, copper binding capacity of the A. ferrooxidans CusF was measured, and the effect of this substitution was investigated. To fulfill that purpose, the cusF3Af gene was cloned into E. coli and a site directed mutation was performed to replace methionine 66 with histidine. Both wild type and mutated proteins were expressed and purified to measure in vitro their copper binding capacity at both neutral and acid pH. A copper binding capacity of 0,7 copper atoms per protein was found for the wild type CusF3Af, while the M66H mutant was able to bind 0,3 copper atoms per protein (the same amount of copper present in the negative control). It was not possible to perform this measurement under acidic conditions. It was concluded that the subsitution of histidine for methionine in CusF3Af is exclusive of the Acidithiobacillus genus, and that this copper chaperone has the capacity to bind copper at neutral pH. / Enero 2018

No description available.

No description available.

Orientador: Laura Maria Mariscal Ottoboni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T18:11:30Z (GMT). No. of bitstreams: 1 Bergamo_RogerioFaria_D.pdf: 5816487 bytes, checksum: c55c4aa07d6b8202428bd0d045c5779f (MD5) Previous issue date: 2007 / Resumo: Acidithiobacillus ferrooxidans é uma bactéria não patogênica, Gram-negativa, que obtém a energia necessária para o seu crescimento pela oxidação de compostos inorgânicos. Essa bactéria tem despertado interesse por fazer parte de um consórcio de microrganismos envolvidos na biolixiviação de metais. O processo de biolixiviação é influenciado por vários fatores, dentre eles o pH. Apesar desse fator ser importante no crescimento e sobrevivência da bactéria, existem poucas informações sobre os mecanismos moleculares acionados pela bactéria para suportar mudanças envolvendo este parâmetro. Assim sendo, nesse trabalho foram realizados experimentos de respirometria e curvas de crescimento para analisar-se o comportamento da linhagem brasileira de A. ferrooxidans LR em situações que envolviam alterações no pH ideal de cultivo. Com esses experimentos, observou-se que, na presença de pHs acima (2,5 e 3,0) e abaixo (1,5 e 1,2) de 1,8, tanto a respiração quanto o crescimento de A. ferrooxidans LR são afetados. Os pHs dos experimentos de respirometria e curvas de crescimento foram utilizados para obtenção de células de A. ferrooxidans para o isolamento do RNA que foi analisado por RAP-PCR com os primers arbitrários OPF01, OPF03, OPF04, OPF08, OPJ04, OPJ14. Como controle, foi utilizado RNA isolado de células obtidas em pH ideal de cultivo da bactéria. Os cDNAs isolados tiveram a expressão diferencial confirmada por slot blot e foram, a seguir, clonados e seqüenciados. As seqüências obtidas foram comparadas em bancos de dados. As seqüências deduzidas de aminoácidos de quatro cDNAs com expressão induzida em pHs acima ou abaixo de 1,8 apresentaram similaridade com as seguintes proteínas: proteína hipotética, proteína small heat shock, L-2-haloalkanoic acid dehalogenase e ResB. As seqüências deduzidas de aminoácidos de quatro cDNAs com expressão reprimida em pHs acima ou abaixo de 1,8 apresentaram similaridade com as proteínas fructose/tagatose biphosphate aldolase, DedA, proteina ribosomal e DNA helicase dependente de ATP. Esses resultados sugerem que alterações no pH ideal de cultivo de A. ferrooxidans resultam em alterações da expressão de genes que codificam para proteínas pertencentes a diferentes categorias funcionais / Abstract: Acidithiobacillus ferrooxidans are non pathogenic, Gram-negative bacteria that obtain energy for growth from the oxidation of inorganic compounds. These bacteria are part of a consortium of microorganisms involved in the bioleaching of metals. Among others, pH alterations can affect bioleaching. The pH is very important in the survival and growth of the bacteria, but very little is known about the molecular mechanisms used by the bacteria to bare pH alterations. This way, in this work respirometric experiments and growth curves were conducted to analyze the behavior of the A. ferrooxidans Brazilian strain LR in situations involving changes in the bacteria ideal pH. In pHs above (2.5 and 3.0) and below (1.5 and 1.2) 1.8 both oxygen consumption and growth were affected. The pHs used in the respirometric experiments and growth curves were selected to grow A. ferrooxidans cells to isolate the RNA that was used in the RAP-PCR experiments with the arbitrary primers OPF01, OPF03, OPF04, OPF08, OPJ04 and OPJ14. The differential expression of the isolated cDNAs was validated by slot blot. The cDNAs were cloned, sequenced and the obtained sequences were compared to the ones present In GenBank. The deduced amino acids sequences of four cDNAS up-regulated in pHs above and below 1.8 presented similarity with the following proteins: hypothetical protein, small heat shock, L-2-haloalkanoic acid dehalogenase and ResB. The deduced amino acids sequences of four cDNAS down-regulated in pHs above and below 1.8 presented similarity with the proteins: fructose/tagatose biphosphate aldolase, DedA, ribosomal protein and ATP-dependent DNA helicase. These results suggest that alterations in the A. ferrooxidans ideal pH change the expression of genes that encode proteins from different functional categories / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Resumo: Devido ao esgotamento progressivo das jazidas de minérios de cobre com teores elevados (>2%), a aplicação de processos convencionais para a extração de cobre desses minérios, como a pirometalurgia, torna-se inviável devido ao alto custo dos gastos de energia envolvidos. Em função desse esgotamento, bem como do acúmulo de rejeitos de baixos teores (< 0,5%), a busca de processos alternativos para atender a demanda crescente desse metal em todo mundo tornou-se uma necessidade. Uma das alternativas mais viáveis envolve o uso de microrganismos capazes de solubilizar sulfetos metálicos de cobre, em decorrência de seus processos metabólicos oxidativos. O processo que utiliza esses microrganismos na recuperação do cobre de minérios de baixo teor ou de rejeitos é conhecido como biolixiviação e tem sido aplicado em escala industrial em vários países, em especial EUA e Chile, onde estão as maiores reservas mundiais de minério de cobre. Biolixiviação ou lixiviação bacteriana é o processo pelo qual bactérias, sobretudo as do gênero Acidithiobacillus e, principalmente, a espécie Acidithiobacillus ferrooxidans, oxidam sulfetos metálicos (CuFeS2, CuS, CuS2, PbS, ZnS entre outros) como fonte de energia, levando à solubilização desses metais. O A. ferrooxidans é uma espécie mesofílica, quimiolitotrófica e acidofílica que, além de sulfetos metálicos, oxida também íons ferrosos e compostos reduzidos de enxofre. Esse processo é uma alternativa viável para a extração de cobre, pois requer poucos gastos com insumos (ácidos e agentes oxidantes), apresenta reduzido gasto energético, baixo investimento de capital, baixo custo operacional e reduzida mão de obra especializada na operação. Dentre os minerais de cobre, o mais abundante e também o mais refratário ao ataque químico ou bacteriano é a calcopirita. Assim, muitos estudos foram desenvolvidos...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: High-grade copper ores deposits (>2%) have been depleted for centuries. So, conventional processes to recover copper from low-grade ores such as pirometallurgy have become prohibitive due the elevated costs, since large amount of energy is spent in this process. As a result of this depletion, a huge amount of low-grade ores (<0.5%) as well waste materials from conventional process have been accumulated for years, waiting for an economic alternative method. In order to match up a constant increase in the copper world demand, the search for new methodologies has become an obligation. Among these alternatives, the use of microorganisms capable to copper sulfides dissolution is the most feasible one. This process, known as bioleaching or bacterial leaching, has been applied in industrial scale in several countries, such as USA and Chile, where the highest copper deposits are located. Bacterial leaching is developed by certain bacteria belong to the Acidithiobacillus genus, among others, mainly by species Acidithiobacillus ferrooxidans, which oxidize metal sulfides (CuFeS2, CuS, CuS2, PbS, ZnS, etc) as its energy source, solubilizing the corresponding metal. It is a mesophilic, chemolithotrophic and acidophilic bacterium that besides metal sulfides oxidize also ferrous iron and other reduced sulphur compounds as energy source. The viability of the process is related with the low costs operation: low capital investments, reduced needs for specialized works, low reagents and materials consumption, etc. Chalcopyrite is most abundant copper mineral and, at same time, the most refractory to chemical or bacterial attack. In this way, several studies have been devoted to understand the mechanisms involved in the mineral dissolution, as well to developed new methodologies to improve chalcopyrite dissolution. It was investigated in this study the effect of some chemical agents...(Complete abstract click electronic access below) / Orientador: Oswaldo Garcia Junior / Coorientador: Denise Bevilaqua / Banca: Assis Vicente Benedetti / Banca: Wilson Cervi da Costa / Mestre

Biogas, composed principally of methane, has limited use in energy generation due to the presence of hydrogen sulphide (H2S). Biogas cannot be burned directly in an engine as H2S present causes corrosion in the reaction chamber. There currently exist various technologies for the removal of H2S from a gas stream, but most are chemically based, expensive, and are limited in use. The purpose of this study was to determine a biogas purification technique suitable for a small scale farm application; including using a technology inexpensive, efficient, robust and easy to operate. As such, biofiltration was investigated for H2S removal from biogas. Factors considered in the design of the biofiltration system included the source and conditioning of inoculum, type of packing material, and general operating conditions including inlet gas flow rate and H2S loading rate to the biofilter. Activated sludge conditioned in A. ferrooxidans media was an effective inoculum source. This was tested for growth support compatibility with gravel packing material, to be used in the biofilter. The inoculated packing material was loaded into the biofilter initially during start-up and acclimatization. In this study, synthetic biogas (49.9%volCH4, 49.9%volCO2, 2000ppmv H2S) mixed with air (totalling 4%vol O2) was added at 5-10L/hr to a biofilter of 0.4L gravel packing inoculated with conditioned activated sludge. Baseline H2S removal studies in a non-inoculated biofilter were performed with anticipated operating conditions, including an inlet gas stream at 7.5L/h (25oC, 1atm), resulting in 31-56% H2S removal. A factorial test performed found that air content in the inlet gas stream was the significant factor affecting the removal of H2S in the non-inoculated biofilter. Operation of the biofilter with biogas was done for 61 days, including 41 days for start-up and acclimatization and 20 days of H2S loading tests. Start-up and acclimatization with biogas resulted in complete H2S removal after 2 days, with an average overall H2S removal of 98.1%±2.9 std deviation over 34 days. Loading tests performed on the system ranged 5-12.4L/h (25oC, 1atm), with a loading rate of 27.8 to 69.5gH2S/m3h of filter bed. Throughout this test the average H2S removal rate was 98.9%±2.1 std deviation over 20 days. Although complete H2S breakthrough studies were not performed, these results indicate that biofiltration is a promising technology for H2S removal from biogas in a small scale application.

Biogas, composed principally of methane, has limited use in energy generation due to the presence of hydrogen sulphide (H2S). Biogas cannot be burned directly in an engine as H2S present causes corrosion in the reaction chamber. There currently exist various technologies for the removal of H2S from a gas stream, but most are chemically based, expensive, and are limited in use. The purpose of this study was to determine a biogas purification technique suitable for a small scale farm application; including using a technology inexpensive, efficient, robust and easy to operate. As such, biofiltration was investigated for H2S removal from biogas. Factors considered in the design of the biofiltration system included the source and conditioning of inoculum, type of packing material, and general operating conditions including inlet gas flow rate and H2S loading rate to the biofilter. Activated sludge conditioned in A. ferrooxidans media was an effective inoculum source. This was tested for growth support compatibility with gravel packing material, to be used in the biofilter. The inoculated packing material was loaded into the biofilter initially during start-up and acclimatization. In this study, synthetic biogas (49.9%volCH4, 49.9%volCO2, 2000ppmv H2S) mixed with air (totalling 4%vol O2) was added at 5-10L/hr to a biofilter of 0.4L gravel packing inoculated with conditioned activated sludge. Baseline H2S removal studies in a non-inoculated biofilter were performed with anticipated operating conditions, including an inlet gas stream at 7.5L/h (25oC, 1atm), resulting in 31-56% H2S removal. A factorial test performed found that air content in the inlet gas stream was the significant factor affecting the removal of H2S in the non-inoculated biofilter. Operation of the biofilter with biogas was done for 61 days, including 41 days for start-up and acclimatization and 20 days of H2S loading tests. Start-up and acclimatization with biogas resulted in complete H2S removal after 2 days, with an average overall H2S removal of 98.1%±2.9 std deviation over 34 days. Loading tests performed on the system ranged 5-12.4L/h (25oC, 1atm), with a loading rate of 27.8 to 69.5gH2S/m3h of filter bed. Throughout this test the average H2S removal rate was 98.9%±2.1 std deviation over 20 days. Although complete H2S breakthrough studies were not performed, these results indicate that biofiltration is a promising technology for H2S removal from biogas in a small scale application.

La oxidación bacteriana del ión ferroso con Acidithiobacillus ferrooxidans en presencia de carbón activado es un proceso útil tanto para la generación de ión férrico como para la generación de inóculos de alta concentración bacteriana. El objetivo del presente trabajo es mejorar el entendimiento del mecanismo mediante el cual la presencia del carbón activado afecta los procesos de oxidación del ión ferroso. En particular, en este trabajo se evalúa por primera vez la influencia que el efecto químico del carbón activado sobre el ión férrico pueda tener en el proceso. El trabajo experimental se efectuó a 30 ºC. Experimentos preliminares en que se contactó medio basal MC, de composición igual a 0,4 g/L de (NH4)2SO4; 0,056 g/L de K2HPO4·3H2O y 0,4 g/L de MgSO4·7H2O,.a pH = 1.6 y 3 g/l de ión férrico con carbón activado, confirmaron que el ión férrico es parcialmente reducido a ión ferroso por la acción química del carbón. Estudios cinéticos conducidos con el mismo medio basal MC, pero con concentraciones de ión férrico en el rango entre 100 a 1200 mg/L, y con 0.2 g carbón en 100 ml de solución, demostraron que la cinética de reducción del ión férrico con carbón se rige por la expresión: Con : k =7,68x10-6 [(mg/L)-0,731/min] ; n=3,369 Los estudios de crecimiento bacteriano en presencia de carbón activado se efectuaron con 6,2 g/L de carbón, con una cepa pura de Acidithiobacillus ferrooxidans con concentración inicial de 2,9 x 109 [bact/L], en un medio basal MC a pH 1,6 que contenía una concentración inicial de 3 g/L de ión férrico, sin ión ferroso. El experimento se efectuó en un frasco agitado modificado que impedía el contacto directo entre los microorganismos y el carbón, de modo que el crecimiento bacteriano se pudo monitorear por conteo directo. El resultado de este experimento permitió constatar que, a pesar de no haber inicialmente ión ferroso, la población bacteriana después de 19 días creció hasta 4x1010 [bact/L]. En un experimento control efectuado en condiciones similares, pero sin agregar carbón, no se observó crecimiento durante el mismo período. Los resultados de este estudio permitieron confirmar que en el proceso de oxidación bacteriana del ión ferroso con Acidithiobacillus ferrooxidans en presencia de carbón activado, el carbón contribuye adicionalmente al crecimiento bacteriano a través de la regeneración del ión ferroso por reducción química de ión férrico con el carbón. El trabajo de simulación efectuado permitió demostrar, además, que la evolución de la población bacteriana, concentración de ión ferroso, ión férrico y Eh durante el proceso se puede describir adecuadamente en base a un modelo que describe la oxidación bacteriana del ión ferroso con una cinética tipo Monod que incluye el efecto inhibitorio del ión férrico y describe la reducción de ión férrico con la expresión cinética deducida en este trabajo. El efecto químico del carbón activado sobre el ión férrico detectado en este estudio es un aspecto que no ha sido considerado en los artículos reportados hasta el momento en este tema. El resultado de este estudio contribuirá entonces a aclarar las contradicciones entre los diferentes resultados reportados y posibilitará el estudio de la optimización del proceso sobre bases conceptuales más sólidas.

Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-02Bitstream added on 2014-06-13T19:08:53Z : No. of bitstreams: 1 silva_dr_me_araiq.pdf: 984256 bytes, checksum: bcc952300b822c0cddcc1c2d5f5eec53 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A biolixiviação (lixiviação bacteriana) é uma alternativa para a extração de metais presentes em sulfetos minerais empregando microorganismos. Dentre as principais vantagens da biohidrometalurgia em relação aos métodos convencionais podem ser destacadas a não-emissão de SO2 para a atmosfera, pequeno gasto com insumos, pois são produzidos pelos próprios microorganismos, baixa demanda energética e o tratamento de minérios contendo metais em baixo teor. Uma das espécies envolvidas na biolixiviação de sulfetos minerais mais estudadas é Acidithiobacillus ferrooxidans. Essa bactéria é gram negativa, aeróbia, acidófila, mesófila e obtém energia através da oxidação de íons Fe2+ e compostos reduzidos de enxofre. A calcopirita é a principal fonte de cobre presente na crosta terrestre. Uma das principais propriedades desse sulfeto é a sua alta refratariedade ao ataque químico e bacteriano, conseqüência da sua elevada energia de rede cristalina. Além disso, a formação de uma camada protetora, constituída basicamente por polissulfetos, sobre a sua superfície diminui a dissolução desse minério. Por isso, diversos grupos de pesquisa buscam alternativas para a resolução desses problemas visando elevar as extrações de cobre a partir desse sulfeto. Com esse objetivo, o presente trabalho estudou o efeito da adição de nanopartículas, polares e apolares, na biolixiviação da calcopirita por A. ferrooxidans LR. O único estudo relacionado à lixiviação envolvendo nanopartículas demonstrou que a adição de nanosílica elevou as taxas de dissolução da calcopirita mediante utilização de agentes oxidantes, meio reacional ácido e temperatura moderada. Para avaliar o efeito da adição de nanopartículas no crescimento bacteriano, foi monitorada a concentração de íons Fe2+ presentes em sistemas contendo A. ferrooxidans... / Bioleaching is an alternative for metal extraction from mineral sulfides using microorganisms. The main advantages of the biotechnological processes in comparison with usual methods of metal extraction are the non-emission of SO2 to atmosphere, lower inputs cost, as they are produced by the microorganisms themselves, lesser energy requirements and treatment of low-grade ores. One of the most studied microbes involved in the bioleaching of mineral sulfides is Acidithiobacillus ferrooxidans. This bacterium is Gram-negative, aerobic, acidophilic, mesophilic and oxidizes Fe2+ ions and reduced forms of sulfur to obtain energy. Chalcopyrite is the main copper source present in the world. One of its main properties is the high resistance to the chemical and bacterial attacks, result of its great crystal lattice energy. Also, the formation of a protective layer, mainly constituted by polysulfides, over its surface reduces the dissolution of that ore. For those reasons, several research groups throughout the world are investigating alternatives to solve these problems and promote the higher copper extraction from this sulfide. This work studied the effect of adding nanoparticles, polar and non-polar, in the bioleaching of chalcopyrite by A. ferrooxidans LR. The only study related to leaching involving nanoparticles, published in 2005, demonstrated that the addition of nano-sized silica raised the dissolution rates of chalcopyrite when using oxidizing agents, acidic medium and moderate temperature. To evaluate the effect of adding nanoparticles in bacteria growth, it was monitorated the Fe2+ ions concentration present in flasks that contained A. ferrooxidans LR and different concentrations of nanoparticles. Thus, it was observed that high amounts of nanoparticles (> 2.5% m/v) affected the bacteria growth. Shake flasks... (Complete abstract click electronic access below)