Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-08-16T11:47:39Z No. of bitstreams: 1 texto completo.pdf: 828724 bytes, checksum: 25366a317fbcede6addce4a82965b471 (MD5) / Made available in DSpace on 2017-08-16T11:47:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 828724 bytes, checksum: 25366a317fbcede6addce4a82965b471 (MD5) Previous issue date: 2017-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bactérias oxidantes de formas reduzidas de S são utilizadas na indústria para lixiviar metais, como ouro e cobre. A geração de acidez, a partir da dissolução oxidativa do S, é o principal mecanismo pelo qual consegue-se solubilizar minerais utilizando a bactéria Acidithiobacillus thiooxidans. Neste estudo, avaliou-se a solubilização de uma rocha silicatada potássica, regionalmente conhecida como rocha Verdete (RV), por meio de um ensaio biológico utilizando A. thiooxidans . Frascos erlenmeyers contendo meio 9 K, cultura bacteriana e RV foram incubados a 30 °C em tempos variando de 0 a 49 d. Após os tempos de incubação, coletaram-se o filtrado e a fase sólida. As fases sólidas foram submetidas a Difração de Raios X (DRX) para caracterizações mineralógicas. Já no filtrado, mediram-se pH, potencial redox e teores solúveis de alguns elementos (K, Al, Fe, Si e S). Na presença de A. thiooxidans o pH inicial estimado de 4,2 foi reduzido para 0,57 aos 49 d após a incubação (dai). Sob condições ácidas, promovida pela presença bacteriana, ocorreu solubilização parcial da RV, com expressivo aumento nas concentrações em solução de elementos estruturais de minerais da rocha. As quantidades liberadas de K, Al, Fe e Si, aos 49 d, no meio de cultura contendo bactérias corresponderam a 6,6; 5,8; 14,1 e 1,7 % dos seus teores totais na RV. A análise mineralógica da RV, aos 49 dai, revelou alterações semelhantes nos espectros de Difração de Raio-X para as amostras da RV, na ausência e presença de bactéria A. thiooxidans. No entanto, o estudo confirmou que a solubilização da RV, como indicado pela lixiviação dos elementos rochosos (K, Al, Fe e Si), é devida à acidificação do meio promovida pela catálise biológica da bactéria A. thiooxidans na oxidação do S 0 , e sugere o uso potencial dessa bactéria para solubilizar rochas silicitadas ricas em K, ou em outros metais de interesse, uma vez que é uma tecnologia de baixo consumo de energia em relação aos processos convencionais. Estudos adicionais devem ser realizados para otimizar a biossolubilização de minerais de silicato de K, incluindo o uso de biorreatores mais sofisticados, processo de recuperação de elementos solúveis e associações microbianas. / Bacteria that oxidize reduced forms of S are used in industry to leach metals, such as gold and copper. The generation of acidity, from the oxidative dissolution of S, is the main mechanism by which it is possible to solubilize minerals using the bacterium Acidithiobacillus thiooxidans. In this study, the solubilization of a potassium silicate rock, regionally known as Verdete rock (VR), was evaluated by means of a biological assay using A. thiooxidans. Erlenmeyer flasks containing 9 K medium, bacterial culture and VR were incubated at 30 °C at times ranging from 0 to 49 d. After the incubation times, the filtrate and solid phase were collected. The solid phases were subjected to X-Ray Diffraction (XRD) analysis for mineralogical characterization. In the filtered, pH, redox potential and soluble contents of some elements (K, Al, Fe, Si and S) were measured. In the presence of A. thiooxidans the estimated initial pH of 4.2 was reduced to 0.57 at 49 d after incubation (dai). Under acidic conditions, with the presence of the bacterium, there was partial solubilization of the VR, evidenced by a significant increase in the concentrations of the structural elements of rock minerals. The released amounts of K, Al, Fe and Si, at 49 d, in the culture medium corresponded to 6.6, 5.8, 14.1 and 1.7 % of their total contents in VR. Mineralogical analysis showed similar changes in DRX spectra for VR samples in absence and presence of A. thiooxidans at 49 dai. In addition, the study confirmed that the solubilization of VR, as indicated by leaching of rock elements (K, Al, Fe and Si), is due to the acidification of the medium promoted by the biological catalysis of A. thiooxidans in the oxidation of S 0 , and suggests the potential use of this bacterium to solubilize silicate rocks rich in K, or other metals of interest, since it is a technology of low energy consumption compared to conventional processes. Further studies should be carried out into optimize the biosolubilization of K-silicate minerals, including the using of more sophisticated bioreactors, processes for recovering soluble elements and microbial associations.

Submitted by Laíze Fernanda Pereira null (laize_fernandapereira@yahoo.com.br) on 2016-10-03T11:51:03Z No. of bitstreams: 1 Dissertação Laize Fernanda Pereira_versão final.pdf: 1765685 bytes, checksum: c480da42091df39cbc80f15e56710bec (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-10-05T17:05:35Z (GMT) No. of bitstreams: 1 pereira_lf_me_araiq.pdf: 1765685 bytes, checksum: c480da42091df39cbc80f15e56710bec (MD5) / Made available in DSpace on 2016-10-05T17:05:35Z (GMT). No. of bitstreams: 1 pereira_lf_me_araiq.pdf: 1765685 bytes, checksum: c480da42091df39cbc80f15e56710bec (MD5) Previous issue date: 2016-09-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O carvão é a mais importante fonte de energia não renovável de origem fóssil do planeta. Um dos maiores problemas em sua utilização como fonte energética se deve à presença de enxofre na forma orgânica e inorgânica (pirita FeS2). Durante a combustão do carvão, são lançados na atmosfera vários gases, entre eles o SO2 que causam problemas ambientais como a formação de chuva ácida além da corrosão de “boilers”, oleodutos subterrâneos, instalações metálicas e maquinários de minas. Há vários processos físicos e químicos para redução do enxofre antes da combustão do carvão, no entanto, tais métodos são dispendiosos, pois necessitam de condições extremas de temperatura e pressão. Os métodos biológicos de dessulfurização deste combustível fóssil têm demonstrado serem mais eficazes para tal fim. Neste contexto o presente trabalho utilizou bactérias oxidantes de ferro e/ou enxofre, Acidithiobacillus ferrooxidans e Acidithiobacillus thiooxidans a fim de obter uma redução no conteúdo de enxofre pirítico do carvão fornecido pela Carbonífera do Cambuí. Ensaios de biolixiviação em frascos sob agitação foram conduzidos em diferentes condições com a finalidade de acompanhar a cinética de dissolução do enxofre pirítico na amostra mineral. Na fase líquida a evolução dos seguintes parâmetros foi monitorada: pH, Eh e íons ferrosos. A fase sólida foi caracterizada posteriormente através de medidas de difração de raio-X (DRX) e espectroscopia de infravermelho (FTIR). A quantificação parcial do enxofre total foi avaliada utilizando Analisador Preiser Mineco de acordo com as normas ABNT NBR 8295 e suas formas (pirítica, sulfática e orgânica) foram analisadas por normas ASTM. Os ensaios utilizando tais microrganismos obtiveram redução de 44% no teor de enxofre total e de 90% no teor de enxofre pirítico. Como resultado das análises de raio-X observou-se a redução de picos característicos da pirita e o surgimento de uma nova fase cristalina durante o tratamento biológico. Os espectros de infravermelho indicaram que os microrganismos utilizados neste estudo foram capazes de interagir apenas com a fase inorgânica do carvão. Estes ensaios de bancada forneceram parâmetros para um posterior trabalho em escala de bancada utilizando colunas de biolixiviação. / Coal is the most important non-renewable energy source of fossil origin in the world. One of the major problem in using coal as an energy source is the presence of sulfur in the organic and inorganic form (pyritic FeS2). During combustion of coal, SO2 is released in the atmosphere, causing many environmental problems such as the formation of acid rain, beside this sulfur also causes the corrosion of boilers and installations of underground metal pipelines. There are several physical and chemical processes to reduce sulfur in the coal before combustion, however, such methods are very costly, with the use of extreme temperatures and pressures. The biological method for desulfurization of the fossil fuel has proved to be more effective for such purpose. In this context, the present work evaluated the use iron and/or sulfur oxidizing bacteria, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans in order to obtain a maximum reduction in the pyritic sulfur content of the coal supplied by the Carbonífera Cambuí. For this purpose, the bioleaching tests were performed in shake flasks and conducted under different conditions in order to follow the pyritic sulfur kinetics of dissolution of the mineral sample. In the liquid phase, the following parameters such as pH, Eh and ferrous ions were monitored. The solid phase was characterized subsequently by means of measurements of X-ray diffraction (XRD) and infrared spectroscopy (FTIR). The partial quantification of total sulfur was evaluated using Preiser Mineco analyzer according to ABNT NBR 8295 standards and other forms such as pyritic, sulfatic and organic were analyzed by ASTM. The tests using such microorganisms obtained 44% reduction in total sulfur and 90% pyritic sulfur. The result of ray-X analyzes showed the reduction of characteristic peaks of pyrite and the emergence of a new crystalline phase during biological treatment. Infrared spectra indicated that the microorganisms used in this study were able to interact only with the inorganic phase coal. These bench tests provided parameters for further laboratory scale using bioleaching column.

Dans un premier temps, nous avons synthétisé et caractérisé une chondrite à enstatite reconstituée. En étudiant cet objet, nous avons pu identifier et comprendre le signal Raman des sulfures présents dans les météorites à enstatite. La réponse Raman de ces phases est obtenue via l'activation des modes infra-rouge suite à une modification de la symétrie dans le réseau cristallin. Dans un second temps, nous avons utilise cet analogue pour comprendre le processus de bio-alteration des chondrites à enstatite dans les conditions terrestres actuelles, donc en aérobiose. Afin de bien comprendre ce phénomène de bio altération sur ce matériau complexe, il a été indispensable d'étudier ce mécanisme sur chacune des phases présentes dans celui-ci. Ainsi l'analogue de chondrite à enstatite, l'enstatite, la troilite, l'alliage Fe-Si et aussi de l'olivine ont été altérés abiotiquement et bio altérés par les souches Acidithiobacillus ferrooxidans et Acidithiobacillus thiooxidans, ¨¤ pH ¡Ö 2 ¨¤ T = 20¡ãC. Cette étude a permis d'obtenir et d'interpréter à l'échelle de la microscopie électronique à balayage les principales phases et microstructures qui se développent lors de l'altération abiotique ou biologique d'une chondrite à enstatite. En parallèle, des mesures régulières de la chimie du milieu aqueux ont permis d'étudier la cinétique de lixiviation de ces expériences de bio-alteration et de la comparer à une modélisation thermodynamique et cinétique, que nous avons effectué avec le programme Jchess. Nos résultats montrent qu'en opposition avec ce qui a été observe sur les phases séparées, les cinétiques de dissolution sont très différentes lorsque les différentes phases sont associées dans l'analogue de chondrite à enstatite : la troilite se dissout bien plus lentement que dans les expériences sur phases séparées alors que l'enstatite se dissout plus vite. La dissolution plus lente de la troilite est attribuée à la présence de monosulfures très solubles dans le matériau de départ. Ces observations pourront être utilisées pour modéliser et interpréter l'évolution d'une chondrite à enstatite à la surface de la Terre et, au delà, de matériaux réduits associant métaux, sulfures et silicates. Les chondrites à enstatite constituent un substrat approprié pour les deux souches bactériennes étudiées qui, en présence de cet assemblage minéral, ont montré de l'activité biologique, en particulier la formation de biofilms, et ont accéléré les vitesses de dissolution / The aim of this study is to investigate the mineralogical and chemical processes which take place during the bioweathering of an enstatite chondrite by bacteria. Synthetic enstatite chondrites were made in laboratory in order to begin this complex study with simplified and well-defined materials. These analogs were shown to contain the major phases of enstatite chondrites: enstatite, Si-rich kamacite, troilite and unusual sulfides such as oldhamite or niningerite. First, the Raman study of this analog allowed to identify and understand the specific Raman signal of sulphides present in enstatite meteorites. The Raman signal of these phases is explained by infrared activation due to a symmetry modification in the crystal lattice. Then, in order to better understand the aqueous alteration of enstatite chondrites, each major phase and olivine were separately submitted to aqueous and aerobic alteration with or without Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans at pH ¡Ö 2 ¨¤ T = 20¡ãC. All experiments were characterized before and after alteration using scanning electron microscopy. Regular measurements og the chemistry of the aqueous medium were used to study the kinetics of leaching experiments of bio-alteration and compared to thermodynamic and kinetic modeling with JChess. Our results show that contrary to the observations on the separate phases, the dissolution kinetics are very different when the whole phases are associated in the enstatite chondrite analogue: troilite dissolves more slowly than in experiments on separate phases while enstatite dissolves faster. The slower dissolution of troilite is ascribed to the presence of oldhamite and niningerite in the starting material. These observations will be useful to understand and model the evolution of enstatite chondrites at the surface of the Earth and, beyond that, of reduced assemblages containing metals, sulphides and silicates. Enstatite chondrites are shown to be an appropriate substrate for the two bacterial strains which have shown biological activity, especially by the formation of biofilms and which have accelerated the dissolution kinetics

Doctorado en Ciencias mención Microbiología. / Acidithiobacillus thiooxidans es una bacteria que obtiene energía metabólica y poder reductor a partir de la oxidación de compuestos reducidos de azufre. En estado natural, esta bacteria se encuentra junto con otros microorganismos participantes del consorcio de biolixiviación de sulfuros metálicos formando estructuras multicelulares llamadas biopelículas sobre la superficie de sulfuros minerales. Las células están embebidas en una matriz de sustancias poliméricas extracelulares (EPS) las cuales se componen principalmente de exopolisacáridos, proteínas y lípidos. Se ha descrito que la solubilización de los minerales sulfurados metálicos está mediada por estos EPS. Sin embargo, la composición de los EPS y su rol exacto en la solubilización de sulfuros metálicos ha sido caracterizado solamente en la bacteria oxidadora de hierro y azufre Acidithiobacillus ferrooxidans, aunque las demás especies de Acidithiobacillus que son capaces de oxidar solamente compuestos reducidos del azufre son fundamentales en estos procesos de lixiviación. En base a estos antecedentes, nos propusimos caracterizar alguno de los EPS y los mecanismos moleculares involucrados en la formación de biopelículas en la especie modelo At. thiooxidans. En un trabajo previo, se identificó un gen putativo que codifica para una proteína de unión a c-di-GMP PelD, la cual está caracterizada en Pseudomonas aeruginosa formando parte de un complejo multiproteico de membrana involucrado en vii la biosíntesis del exopolisacárido Pel. Al analizar el contexto genético del gen pelD, se identificó un putativo operón con una estructura similar al operón canónico identificado en P. aeruginosa. El análisis comparativo entre los genomas disponibles de las bacterias acidófilas que están presentes en los entornos minerales ácidos reveló que este operón sólo está presente en los genomas de At. thiooxidans y At. caldus. Ante este resultado, este trabajo se enfocó en comprender cuál sería la importancia de este exopolisacárido en la formación de biopelículas de At. thiooxidans sobre la superficie de azufre elemental. La medición de los niveles de transcrito mediante qPCR mostró que los genes pelA, pelD y wcaG del putativo operón presentan un mayor de expresión en células adheridas que en células planctónicas. El análisis de azúcares de superficie mediante el uso de lectinas identificó la presencia de N-acetil-galactosamina y N-acetil-glucosamina. Tratando de dilucidar algunas de las señales que regulan la producción de Pel, se descubrió que la adición exógena de la molécula de Quorum Sensing (QS) 3-oxo-C8 AHL incrementa la transcripción de los genes pelA y pelD. Finalmente, se procedió a obtener una cepa mutante nula en el gen pelD mediante recombinación homóloga. Luego de analizar el DNA de esta cepa, se realizaron análisis de adherencia y formación de biopelículas sobre la superficie de azufre elemental mediante microscopía electrónica y de epifluorescencia. La mutación ΔpelD afectó la estructura de la biopelícula al reducir la presencia de N-acetil-galactosamina y N-acetilglucosamina en la superficie, pero adicionalmente provocó un incremento en la viii producción de estructuras filamentosas que rodean a las células. Para resolver la incógnita sobre la naturaleza de estas fibras, se realizaron análisis comparativos preliminares de la expresión de proteínas extracelulares. El análisis del sobrenadante de los cultivos y de las proteínas del EPS reveló un cambio en producción de proteínas secretadas al medio. En base a estos resultados es posible concluir que el exopolisacárido Pel producido por la bacteria biominera At. thiooxidans juega un rol estructural en la formación de biopelículas sobre la superficie de azufre elemental y que la regulación de su síntesis involucra a las vías del QS y del c-di-GMP. / Acidithiobacillus thiooxidans is a bacterium that obtains metabolic energy and reducing power from the oxidation of reduced sulfur compounds. In the natural state, this bacterium is found along with other microorganisms that are members of the metal sulfides bioleaching consortium forming multicellular structures called biofilms on the surface of mineral sulfides. The cells are embedded in a matrix of extracellular polymeric substances (EPS) which are composed mainly of exopolysaccharides, proteins and lipids. It has been described that the solubilization of the metal sulfide minerals is mediated by these EPS. However, the composition of the EPS and their exact role in the solubilization of metal sulfides has been characterized only in the iron and sulfur oxidizer bacterium Acidithiobacillus ferrooxidans, although the other Acidithiobacillus species that are only capable of oxidizing reduced sulfur compounds are important in these leaching processes. Based on this background, we set out to characterize some of the EPS and the molecular mechanisms involved in biofilm formation in the model species At. thiooxidans. In a previous work it was identified a putative gene encoding a c-di-GMP binding PelD protein, which is characterized as part of a multiprotein membrane complex involved in Pel exopolysaccharide biosynthesis in Pseudomonas aeruginosa. x When we were analyzing the genetic context of the pelD gene, a putative operon with a structure similar to the canonical operon identified in P. aeruginosa was identified. Comparative analysis between available genomes of acidophilic bacteria that are present in acidic mineral environments revealed that this operon is only present in the genomes of At. thiooxidans and At. caldus. Given this result, this work focused on understanding the importance of this exopolisaccharide in biofilm formation of At. thiooxidans on the surface of elemental sulfur. Measurement of transcript levels by qPCR showed that pelA, pelD and wcaG genes of the putative operon exhibit a greater expression in attached cells than in planktonic cells. The analysis of surface sugars by the use of lectins identified the presence of N-acetyl-galactosamine and N-acetyl-glucosamine. Trying to elucidate some of the signals that regulates the production of Pel, it was discovered that the exogenous addition of Quorum Sensing (QS) molecule of 3-oxo-C8 AHL increases the transcription of pelA and pelD genes. Finally, a pelD gene null mutant strain was obtained by homologous recombination. After analyzing the DNA of this strain, adherence and biofilm formation analysis were performed on the elemental sulfur surface by electronic and epifluorescence microscopy. The ΔpelD mutation affected the structure of the biofilm by reducing the presence of N-acetyl-galactosamine and N-acetyl glucosamine on the surface, but additionally caused an increase in the production of fibrillar structures xi surrounding the cells. To solve the unknown about the nature of these fibers, preliminary comparative analysis of extracelluar protein expression were performed. Analysis of the culture supernatant and proteins from EPS revealed a change in production of secreted proteins to the medium. Based on these results it is possible to conclude that the Pel exopolisaccharide produced by the biomining bacterium At. thiooxidans plays a structural role at biofilm formation on the surface of elemental sulfur and that the regulation of its synthesis involves QS and c-di-GMP pathways. / Proyectos regulares Fondecyt 1120295 y 1160702 y beca Conicyt 21120064 por aportar el financiamiento durante el desarrollo de la tesis.

Submitted by JOSIANE APARECIDA GASPAR null (josianegaspar@yahoo.com.br) on 2016-04-26T21:06:10Z No. of bitstreams: 1 Tese Josiane Gaspar final 26 04 2016.pdf: 2896477 bytes, checksum: bae3bd8eaa7bebba113a4f4c308cc51b (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-28T20:42:46Z (GMT) No. of bitstreams: 1 silva_eo_me_guara_par.pdf: 795191 bytes, checksum: eaa53defa424680464e90f198b5c1635 (MD5) / Made available in DSpace on 2016-04-28T20:42:46Z (GMT). No. of bitstreams: 1 silva_eo_me_guara_par.pdf: 795191 bytes, checksum: eaa53defa424680464e90f198b5c1635 (MD5) Previous issue date: 2016-04-18 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Atualmente, a indústria sucroalcooleira é tida como uma das indústrias mais importantes do cenário socioeconômico brasileiro. Entretanto, este setor gera grandes quantidades de resíduos. Dentre esses resíduos encontra-se a torta de filtro (TF), um resíduo orgânico sólido representativo. Uma alternativa para a disposição final da TF tem sido a reciclagem agrícola. Contudo, uma das principais preocupações em relação à aplicação deste composto no solo é a presença de contaminantes, como os elementos potencialmente tóxicos (EPTs). Estudos têm mostrado que a biolixiviação, um processo que se baseia na capacidade dos microrganismos transfomarem os EPTs ligados a resíduos sólidos em formas solúveis extraíves, é uma alternativa promissora para o tratamento de diversos resíduos. Assim sendo, o presente trabalho foi desenvolvido com o objetivo de avaliar a remoção de elementos potencialmente tóxicos presentes no composto de torta de filtro produzido por uma usina sucroalcooleira utilizando a biolixiviação. Para isso, foram realizados ensaios de biolixiviação, utilizando inicialmente as bactérias Acidithiobacillus ferrooxidans - LR e Acidithiobacillus thiooxidans – FG01. Iniciou-se a pesquisa com duas amostras do composto de torta de filtro de safras diferentes (2014 e 2015). As amostras foram caracterizadas quanto ao seu potencial agronômico e EPTs. Os ensaios de biolixiviação foram realizados em frascos agitados (150 rpm, 30ºC), utilizando uma bactéria oxidante de ferro e a bactéria oxidante de enxofre suplementadas com FeSO4 ou S°, respectivamente, bem como seus controles abióticos correspondentes. Foi utilizada uma concentração inicial de densidade de polpa de 2% (m/v), e posteriormente com 10% (m/v) de torta de filtro. Observou-se que amostras coletadas na safra de 2014 inibiram a oxidação do Fe2+ e a produção de ácido, parâmetros relacionados com as atividades microbianas. Sugere-se que a concentração de carbono orgânico presente nas amostras coletadas em 2014 seja um dos responsáveis pela inibição observada. Todos os ensaios subsequentes foram realizados com as amostras da safra de 2015. As solubilizações dos EPTs na TF, em ordem decrescente, utilizando At. ferrooxidans – LR como inóculo foram: 91% de Mn, de 65% de Zn, 43% de Pb, 34% de Al, 32% de Cr, 22% de Cu, e em frascos inoculados com At. thiooxidans - FG01 foram de 93% de Mn, 62% de Zn, 38% de Cr, 35% de Al, 35% de Pb, e 24% de Cu. Os resultados mostraram que as solubilizações dos EPTs nos grupos experimentais inoculados não foram significativamente diferentes exceto para os íons metálicos Cr e Pb. Os EPTs foram correlacionados negativamente com a variação do pH e positivamente com potencial redox. O modelo Logístico foi usado para acompanhar o estudo do processo de biolixiviação. Extração sequencial dos EPTs mostrou que a maioria dos elementos estavam ligadas a frações disponíveis ao ambiente. Após o tratamento microbiano a maioria dos EPTs remanescentes da TF apresentou-se ligadas às frações mais estáveis e, por conseguinte, o resíduo biolixiviado poderia ser aplicado sobre o solo. As extrações observadas do nitrogênio e fósforo após o processo estão relacionadas à condição ácida do sistema. Por fim, os ensaios de fitotoxicidade aguda mostraram que os elutriatos obtidos dos resíduos biolixiviados foram classificados como uma baixa toxicidade sobre a germinação e crescimento das radículas de Lactuca sativa (alface), enquanto que os elutriatos sem tratamento foram classificados com uma elevada toxicidade. Os resultados obtidos nesta pesquisa mostraram evidências de que a biolixiviação é eficaz para atenuar a concentração final de elementos potencialmente tóxicos na torta de filtro, evitando a sua acumulação no solo, podendo assim ser utilizada como condicionador na agricultura. / Currently, the sugar industry is considered one of the most important industries of the Brazilian socioeconomic scenario. However, this sector generates large amount of waste products, among which the filter cake (FC) is a representative solid waste. Agricultural recycling can be considered as an alternative to the final disposal of such solid waste. However, one of the main concerns regarding its application in the soil is the presence of contaminants such as trace elements. Studies have shown that bioleaching is a promising alternative for the treatment of various wastes. Bioleaching is efficient for the removal of potentially toxic elements (PTE), allowing the compost to be used as soil conditioner, without causing additional problems. The objectives of the present work were to evaluate the removal of potentially toxic elements present in the filter cake produced by a sugar and alcohol industry, through bioleaching. To accomplish this objective, we used bioleaching tests performed initially with the bacteria Acidithiobacillus ferrooxidans - LR and Acidithiobacillus thiooxidans - FG01. Initially two samples of the filter cake of different harvests in 2014 and 2015 were used. The samples were characterized for their agronomic potential and presence of potentially toxic elements. Bioleaching experiments were carried out in flasks shaked (150 rpm at 30 °C) supplemented with FeSO4 or So depending on the bacteria, as well as their corresponding abiotic controls. An initial concentration of 2% pulp density (w/v) followed by 10% (w/v) concentration of the filter cake along with T&K and 9K media were used. The results showed that samples from the 2014 crop inhibited the growth of microorganisms in comparison with the 2015 one. All subsequent tests were performed with samples from 2015 crop. The potentially toxic elements solubilization in the filter cake was, in descending order using At. ferrooxidans – LR, 91%, Mn; 65%, Zn; 43%, Pb; 34%, Al; 32%, Cr and 22% of the Cu, and with At. thiooxidans - FG01 93%, Mn; 62%, Zn; 38%, Cr; 35%, Al; 35%, Pb and 24% of the Cu. The solubilization of potentially toxic elements in the inoculated experimental groups did not show significant difference except for Cr and Pb. Potentially toxic elements were negatively correlated with pH and positively correlated with redox potential. The Logistic model was shown to accompany the study of the bioleaching process. The sequential extraction of potential toxic elements showed that most of the elements were connected to fractions considered available to organisms, and after the microbial treatment, most of the remaining potential toxic elements of the filter cake were present as more stable fractions. Therefore, we suggest that the bioleached residue can be applied to the soil. Nitrogen and phosphorus leaching were observed, and this is related to the acidic condition of the system. Finally, we tested the bioleached filter cake in lettuce through acute phytotoxicity tests that showed lower toxicity when compared with untreated filter cake. For these tests, we used elutriates obtained from the bioleached filter cake and the untreated filter cake. The Lactuca sativa germination and rootlests were evaluated. In general, we demonstrated that the bioleaching with At. ferrooxidans and At. thiooxidans have attenuated the potentially toxic elements concentrations in the filter cake, so presenting no final toxicity to lettuce. This has important environmental implications, since a residue can be treated and safely used as soil conditioner in agriculture. / FAPESP: 2012/19115-0

Les impacts environnementaux dues à l'extraction minière sont considérables. C'est l'action des microorganismes, en utilisant leur métabolisme du soufre sur les déchets miniers, qui engendre les plus grands défis. Jusqu'à présent, peu de recherches ont été effectués sur les microorganismes environnementaux pour la compréhension globale de l'action du métabolisme du soufre dans une optique de prévention et de rémédiation des impacts environnementaux de l'extraction minière. Dans cette étude, nous avons étudié une bactérie environnementale, Acidithiobacillus thiooxidans, dans le but de comprendre le métabolisme du soufre selon le milieu de culture et le niveau d'acidité du milieu. Nous avons utilisé la transcriptomique à haut débit, RNA-seq, en association avec des techniques de biogéochimie et de microscopie à électrons pour déterminer l'expression des gènes codants les enzymes du métabolisme du soufre. Nous avons trouvé que l'expression des gènes des enzymes du métabolisme du soufre chez ce microorganisme sont dépendantes du milieu, de la phase de croissance et du niveau d'acidité présent dans le milieu. De plus, les analyses biogéochimiques montrent la présence de composés de soufre réduits et d'acide sulfurique dans le milieu. Finalement, une analyse par microscopie électronique révèle que la bactérie emmagasine des réserves de soufre dans son cytoplasme. Ces résultats permettent une meilleure compréhension de son métabolisme et nous rapprochent de la possibilité de développer une technique de prédiction des réactions ayant le potentiel de causer des impacts environnementaux dus à l'extraction minière. / The environmental impact of mining extraction is important. The action of microorganisms using their sulfur metabolism to metabolise compounds in mining waste contributes to reactions that may impact water quality and the environment. Few studies have been conducted on environmental microorganisms to advance the global comprehension of their sulfur metabolism in an attempt to study their impact on the environment. In this study, we cultivate an environmental bacterium, Acidithiobacillus thiooxidans, in an attempt to understand its sulfur metabolism in different growth media and at different levels of acidity. We used high-throughput RNA sequencing in association with sulfur biogeochemistry and electron microscopy to determine the expression of the genes encoding sulfur metabolism enzymes. The expression of genes encoding sulfur metabolism enzymes was media and pH-dependent. Also, the biogeochemical analysis showed the presence of reduced sulfur intermediates and of sulfuric acid in the medium. Finally, an electron microscopic analysis revealed that the bacteria stock sulfur in the cytoplasm. These results resulted in a better comprehension of its sulfur metabolism and it opens the possibility to predict reactions in mining operations that have impact on the environment.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-02-13T14:57:58Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoPotencialBiotecnologico.pdf: 2998358 bytes, checksum: 8f3a932c518a34e03b009f67d86b48bc (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-02-15T15:18:42Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoPotencialBiotecnologico.pdf: 2998358 bytes, checksum: 8f3a932c518a34e03b009f67d86b48bc (MD5) / Made available in DSpace on 2017-02-15T15:18:42Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoPotencialBiotecnologico.pdf: 2998358 bytes, checksum: 8f3a932c518a34e03b009f67d86b48bc (MD5) Previous issue date: 2016-04-08 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A bioxidação é um dos pré-tratamentos utilizados antes da cianetação convencional para promover a solubilização de metais como cobre, ferro através da ação oxidante de micro-organismos nos sulfetos metálicos. Esta técnica consolidada é utilizada antes do processo de cianetação para reduzir o consumo de cianeto livre e tornar o ouro acessível aos processos posteriores de recuperação, elevando significativamente a extração deste metal. Desta forma, o presente trabalho consiste em estudar a melhor rota biotecnológica para o pré-tratamento de um minério aurífero e o efeito destes tratamento na avaliação do consumo de cianeto durante a cianetação . Para este fim, testes de bioxidação foram conduzidos em escala de laboratório utilizando um minério aurífero (90g/ton Au) proveniente de uma mina situada no Estado do Amapá, Brasil. As culturas bacterianas utilizadas nos testes biológicos de 20 dias foram Acidithiobacillus ferrooxidans, linhagem LR e Acidithiobacillus thiooxidans, linhagem FG01. Os produtos do processo de bioxidação foram submetidos a ensaios de cianetação para testes de avaliação de consumo de cianeto e recuperação de ouro. Os estudos experimentais realizados mostraram que após 24h de cianetação da amostra mineral sem tratamento a recuperação de ouro foi de 93% (32 mg/L) com o consumo de NaCN de 2,86 kg.t-1, enquanto a melhor extração entre as amostras bioxidadas foi a do biorreator R2 de condição oxidante com 87% (30 mg/L) de ouro, e com consumo de 2,64 kg.t-1 de NaCN. Os resultados obtidos mostram a viabilidade da técnica de bioxidação antes da cianetação para a redução no consumo de cianeto. / One of the most commonly used pre-treatments prior conventional cyanidation is the biooxidation. This technical is a biological process capable of promote solubilization of metals such as copper, iron through the oxidative action of microorganisms in metal sulfides and it is applied as a pretreatment in the cyanidation process to reduce free cyanide consumption making this a feasible and economic process. Thus, the aim of this work was to study the best biotechnology route for the pretreatment of the gold ore in order to reduce cyanide consumption during the convencional cyanidation and increasing the gold recovery. For this purpose, biological tests has been investigated at laboratory scale on a gold ore sample coming from Estado do Amapá, Brazil (90 g/ton). Bacterial cultures utilised in the biological tests of 20 days consisted of Acidithiobacillus ferrooxidans-LRe Acidithiobacillus thiooxidans-LR. The biooxidated samples were submitted to cyanidation tests for gold recovery and tests of consumption of cyanide. Experimental studies demonstrate that after 24h leaching time by direct cyanidation, the gold recovery was 93% (32 mg/L) with a cyanide consumption of 2,84 kg.t-1, while the best gold extraction between biooxidated samples was from bioreactor R2 with 87% (30 mg/L), and the cyanide consumption of 2,64 kg.t-1. Experimental results have shown the technical feasibility of the biooxidative prior to convencional leaching for for reducing the consumption of reagent cyanide.

Dans un premier temps, nous avons synthétisé et caractérisé une chondrite à enstatite reconstituée. En étudiant cet objet, nous avons pu identifier et comprendre le signal Raman des sulfures présents dans les météorites à enstatite. La réponse Raman de ces phases est obtenue via l'activation des modes infra-rouge suite à une modification de la symétrie dans le réseau cristallin. Dans un second temps, nous avons utilise cet analogue pour comprendre le processus de bio-alteration des chondrites à enstatite dans les conditions terrestres actuelles, donc en aérobiose. Afin de bien comprendre ce phénomène de bio altération sur ce matériau complexe, il a été indispensable d'étudier ce mécanisme sur chacune des phases présentes dans celui-ci. Ainsi l'analogue de chondrite à enstatite, l'enstatite, la troilite, l'alliage Fe-Si et aussi de l'olivine ont été altérés abiotiquement et bio altérés par les souches Acidithiobacillus ferrooxidans et Acidithiobacillus thiooxidans, ¨¤ pH ¡Ö 2 ¨¤ T = 20¡ãC. Cette étude a permis d'obtenir et d'interpréter à l'échelle de la microscopie électronique à balayage les principales phases et microstructures qui se développent lors de l'altération abiotique ou biologique d'une chondrite à enstatite. En parallèle, des mesures régulières de la chimie du milieu aqueux ont permis d'étudier la cinétique de lixiviation de ces expériences de bio-alteration et de la comparer à une modélisation thermodynamique et cinétique, que nous avons effectué avec le programme Jchess. Nos résultats montrent qu'en opposition avec ce qui a été observe sur les phases séparées, les cinétiques de dissolution sont très différentes lorsque les différentes phases sont associées dans l'analogue de chondrite à enstatite : la troilite se dissout bien plus lentement que dans les expériences sur phases séparées alors que l'enstatite se dissout plus vite. La dissolution plus lente de la troilite est attribuée à la présence de monosulfures très solubles dans le matériau de départ. Ces observations pourront être utilisées pour modéliser et interpréter l'évolution d'une chondrite à enstatite à la surface de la Terre et, au delà, de matériaux réduits associant métaux, sulfures et silicates. Les chondrites à enstatite constituent un substrat approprié pour les deux souches bactériennes étudiées qui, en présence de cet assemblage minéral, ont montré de l'activité biologique, en particulier la formation de biofilms, et ont accéléré les vitesses de dissolution

Acid mine drainage (AMD) is a worldwide environmental problem associated with the mining wastes, generated from active and inactive mining sites from mineral processing activities. AMD is defined as the drainage that occurs as a result of oxidation of sulphide minerals/wastes/tailings when exposed to air and water in the presence of chemolithotrophs namely the Acidithiobacillus sp. AMD is characterised by low pH and increased acidity due to elevated heavy metals and sulphate concentration. The acid production potential was carried out for sulphide minerals such as pyrite and chalcopyrite and copper tailings sample in the absence and presence of bacteria namely Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Acidity was generated in all the cases due to the oxidation of the mineral samples. The oxidation was found to occur at a higher rate in the presence of the bacteria compared to the control samples. Bioremediation experiments were conducted on acid mine water collected from the Ingaldahl Mines, Chitradurga, Karnataka, India, using organic and inorganic substrates. In the experiments with rice husk, complete removal of metal ions from the acid mine water was achieved with an attendant increase in the pH of the acid mine water from 2.3 to 5.5. About 21% of sulphate could be removed using Dsm. nigrificans from acid mine water pretreated with rice husk at pH 5.5 and this was further increased to 40% by the supplementation of organic components. The rice husk filtrate was found to serve as a good growth medium for Dsm. nigrificans. About 96 % of Fe, 75 % of Zn, 92 % of Cu and 41 % of sulphate removal was achieved from the acid mine water of pH 2.4 with a concomitant increase in the pH value by two units after interaction with the tree bark. About 56 % and 71 % of sulphate reduction could be achieved at initial pH values of 4.1 and 5.5 respectively of the acid mine water pretreated with E. tereticornis (Sm) bark, after inoculation with Dsm. nigrificans. The complete removal of Fe2+ and Fe3+, 80% of Zn, 83% of Cu and 62% of sulphate could be removed from acid mine water using fly ash as the substrate with an increase in pH of acid mine water from 2.3 to 7. About 68% of sulphate reduction at pH 6.8 could be achieved in acid mine water pretreated with fly ash in the presence of Dsm. nigrificans. With red mud as the substrate, complete removal of all the metal ions namely Fe2+, Fe3+, Zn, and Cu from acid mine water was achieved with a concomitant increase in the pH from 2.3 to 8. The sulphate reduction was increased to about 51% at pH 7.2 when the acid mine water pretreated with red mud was inoculated with Dsm. nigrificans. The adsorption experiments carried out on the acid mine water using either organic or inorganic substrates indicated that the free energy of adsorption was negative for all the chosen metal ions attesting to favorable interaction. The adsorption isotherms of the metal ions for rice husk exhibited Langmuirian behaviour, while those for the other substrates adhered to both Langmuir and Freundlich relationships. The adsorption process was found to be endothermic in nature for rice husk, fly ash and red mud. On the contrary, the adsorption onto tree bark showed exothermic behaviour. The adsorption kinetics of the metal ions onto the various substrates adhered to the first order Lagergren equation. The metal uptake processes by the organic and inorganic substrates chosen for this study involve ionic, chemical and physical forces of adsorption. The different types of functional groups present on the surface of the substrates such as carboxyl, hydroxyl and carbonyl, as revealed by FTIR spectroscopic studies, partake in metal binding. The metal ions will also be adsorbed by complexing with the negatively charged reaction sites on the substrate surfaces. Furthermore, the complex solution chemistry of the metals as a function of pH has also to be taken into consideration. The mechanism of sulphate reduction by Dsm. nigrificans in the presence of organic carbon can be illustrated as: 2CH2O + SO42- + 2H+  2CO2 + 2H2O + H2S M2 + H2S  MS  + 2H+ where, CH2O represents the organic matter and M represents the metal ion.