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

Orientador: Laura Maria Mariscal Ottoboni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T21:56:47Z (GMT). No. of bitstreams: 1 Ribeiro_DanielaAlves_D.pdf: 7458236 bytes, checksum: b0e036998f9e767306b90cf0ead49a47 (MD5) Previous issue date: 2011 / Resumo: Acidithiobacillus ferrooxidans e uma bacteria quimiolitotrófica, Gram-negativa e acidofílica que exerce um importante papel no processo de biolixiviação. O processo de biolixiviação e afetado por intempéries como variações na temperatura e na composição de nutrientes e, entender os mecanismos que A. ferrooxidans dispõe para sobreviver em ambientes extremos e de suma importância para melhorar o seu emprego no processo de biolixiviação. Neste trabalho foi estudada a resposta de A. ferrooxidans ao aumento da temperatura e a privação de fosfato. Para tanto, foram utilizadas técnicas baseadas em microscopia, espectroscopia no infravermelho, ferramentas proteômicas, PCR quantitativo e bioinformática. Pela microscopia eletrônica de varredura, verificou-se o alongamento das células de A. ferrooxidans, e a espectroscopia no infravermelho (FTIR) mostrou alterações em números de ondas entre 850 e 1275 cm-1, referentes a carboidratos, fosfolipídios e fosfoproteínas. A abordagem proteômica utilizada, uma combinação de eletroforese de duas dimensões e espectrometria de massas, identificou 44 proteínas diferencialmente expressas e pertencentes a 11 categorias funcionais. Dentre as proteínas com expressão aumentada foram identificadas algumas de resposta ao choque térmico. A técnica de PCR quantitativo em tempo real foi utilizada para avaliar o padrão de Expressão de genes do choque térmico e de outros genes que codificam proteínas pertencentes a diversas categorias funcionais em A. ferrooxidans. Para tanto, A. ferrooxidans foi cultivada a 40?C ate 50% de oxidação dos íons ferrosos (aproximadamente metade fase logarítmica) e também foi submetida a um choque térmico em diferentes tempos e na mesma temperatura. Os resultados obtidos mostraram que o padrão de expressão da maioria dos genes analisados foi afetado apos o choque térmico. Contudo, o crescimento a 40?C pouco afetou a expressão gênica, com exceção de alguns genes relacionados com o transporte de ferro, os quais foram consideravelmente reprimidos. Analises in silico para predição de sítios de ligação do fator sigma 32 da RNA polimerase nos genes afetados pelo aumento da temperatura indicaram que 15 do total de genes analisados possivelmente estão sob a regulação deste fator. Adicionalmente, analises da filogenia das sequências de aminoácidos de três "small heat shock proteins" (AFE_1437, AFE_1009 e AFE_2172) revelou que elas são, possivelmente, proteínas nao-paralogas. Estudos estruturais baseados em modelagem molecular por homologia indicaram que as proteínas AFE_1437, AFE_1009 e AFE_2172 possuem um domínio ?-cristalino com características estruturais semelhantes. Contudo, a proteína codificada por AFE_2172 apresenta uma região C-terminal bastante curta. Os resultados obtidos com este trabalho mostram que A. ferrooxidans possui um conjunto de respostas ao estresse muito bem elaborado, enfatizando seu potencial para uso biotecnológico / Abstract: Acidithiobacillus ferrooxidans is a chemolithoautotrophic, Gram negative, acidophilic bacterium which plays an important role in metal bioleaching. During bioleaching, cells are subjected to changes in growth temperature and nutrient starvation, thus understanding the mechanisms used for their survival in such harsh environments could help to improve Acidithiobacillus's efficacy on such processes. This study presents the effects of heat and phosphate starvation on A. ferrooxidans physiology, analysed using microscopy, infrared spectroscopy, proteomic tools, quantitative PCR and bioinformatics. Scanning electron microscopy results showed that under the tested stress conditions A. ferrooxidans cells became elongated, and the Fourier transform infrared spectroscopy (FT-IR) analysis showed alterations in wavenumbers between 850 and 1275 cm-1, which are related to carbohydrates, phospholipids and phosphoproteins. Proteomic analyses, such as 2-DE and tandem mass spectrometry, identified 44 differentially expressed protein spots, the identified proteins belonging to 11 different functional categories. The up-regulated proteins were mainly from the protein fate category. Real time quantitative PCR was employed to analyze changes in the expression patterns of heat shock genes, as well as many other genes encoding proteins related to several functional categories in A. ferrooxidans. Cells were submitted to long-term growth and to heat shock, both at 40°C. The results evidenced that heat shock affected the expression levels of most genes while long-term growth at 40°C caused minimal changes in gene expression patterns - with exception of some iron transport related genes, which were strongly down-regulated. Further bioinformatic analysis indicated a putative transcriptional regulation, by the ?32 factor, for 15 of the 34 heat-affected genes. In addition, phylogenetic analysis of some small heat shock proteins (AFE_1437, AFE_1009 e AFE_2172) showed that sHSPs from A. ferrooxidans are possible non paralogous proteins. Homology molecular modeling structure studies indicated that the proteins encoded by AFE_1437, AFE_1009, and AFE_2172 have conserved ?-crystallin domains. However, the model for AFE_2172 showed a very short C-terminus. These results evidence that A. ferrooxidans has an efficient range of stress-responses, which explains its ability for biotechnological purposes / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Les bactéries acidophiles du genre Acidithiobacillus joue un rôle important dans les activités industrielles de récupération des métaux au sein des sites miniers. Dans cette thèse, la séquence du génome de la bactérie psychro-tolerante Acidithiobacillus ferrivorans CF27 a été re-séquencée. L’analyse comparative du génome de CF27 et des autres bactéries du genre Acidithiobacillus a permis de montrer: (i) une synthénie conservée entre 2 clusters de tRNAs trouvés dans les génomes de At. ferrivorans CF27 et At. ferrooxidans ATCC 23270, et qui ont contribué à la redondance génique des tRNAs chez ces 2 organismes. Notre analyse in silico à grande échelle de ces clusters de tRNAs au sein des génomes procaryotes a montré que les clusters de tRNAs sont présents dans très peu de phyla bactériens; (ii) la présence d’une importante proportion de gènes spécifiques chez CF27 et SS3, ce qui indique la très grande variabilité du contenu génique dans les génomes d’Acidithiobacillus et ainsi la nature unique de chaque groupe d’espèces. L’expression de ces gènes spécifiques a été confirmée chez CF27 cultivés en présence de Fer et soufre; et (iii) une composition taxonomique chimérique des génomes de la classe des Acidithiobacillia, confirmant ainsi que ce groupe appartient à une classe taxonomique particulière. Ces résultats apporte de nouvelles connaissances sur l’adaptation de CF27 à son environnement, ainsi que la nature chimérique des génomes de la classe taxonomique Acidithiobacillia. J’ai participé au projet ‘Thioredoxine réductase (TR)’ dont l’objectif est de définir la fonction biochimique, la structure moléculaire, ainsi que l’histoire évolutive de TRi, une réductase atypique. / The acidophilic Acidithiobacillus bacteria play an important role in industrial biomining operations for metal recovery. In this thesis, the genome sequence of a psychrotolerant Acidithiobacillus ferrivorans CF27 were first refined. The comparative genome analysis between CF27 and the closely related Acidithiobacillus genomes revealed: (i) a syntenic conservation of two tRNA array units which are only present in At. ferrivorans CF27 and At. ferrooxidans ATCC 23270 genomes and mainly contribute to the tRNA gene redundancy in both organisms. Moreover, our large-scale genome survey of the tRNA array units in prokaryotic organisms showed that tRNA arrays appear in few phyla; (ii) a high proportion of species-specific genes in CF27 and SS3 strains indicated the high variability of gene content in Acidithiobacillus genomes and therefore the unique nature of each group of species. Given that mRNA expression of some CF27 specific genes were confirmed in Fe(II)-grown cells and sulfur attached cells in CF27, these results highlighted the functional importance of specific genes for CF27 lifestyle; and (iii) the mosaic taxonomic composition of members of the Acidithiobacillia class, and thus confirmed that this group belongs to a particular taxonomic class, distinct to other proteobacterial groups. Taken together, our results provide insights into At. ferrivorans lifestyle as well as the chimeric genome nature of the Acidithiobacillus organisms. In addition, I also participated to the ‘Thioredoxin reductase’ project which aims to define the biochemical function, molecular structure and evolution of TRi, an atypical thioredoxin reductase.

Les protéines à cuivre jouent un rôle crucial dans de nombreux processus biologiques essentiels à la vie tels que la respiration. De nombreuses études ont été menées afin de décrypter le lien entre la structure de leur centre actif, les propriétés électroniques qui en découlent et la fonction de ces protéines.Les travaux réalisés au sein du laboratoire sur l’étude de la chaîne respiratoire d’un organisme acidophile, A. ferrooxidans, ont permis de mettre en évidence une protéine à cuivre (AcoP), appartement à la vaste famille des cuprédoxines, indispensable au fonctionnement de cette voie. Une approche pluridisciplinaire mêlant des méthodes de spectroscopies, d’électrochimie, de cristallographie aux rayons X combinée à des expériences de mutagénèse dirigée, a permis de dévoiler la présence d’un centre cuivre atypique associé à des propriétés électroniques et d’oxydoréduction rarement retrouvées au sein de cette vaste famille. Le rôle d’une telle protéine au sein de la chaîne respiratoire d’A. ferroxidans a par la suite fait l’objet de notre attention. AcoP interagit avec le cytochrome c et l’enzyme terminale de la chaîne respiratoire, la cytochrome c oxydase. L’étude du complexe cytochrome c – AcoPcytochrome c oxydase nous a permis de proposer un rôle d’AcoP dans le recrutement du cytochrome c au sein de ce complexe, ainsi que dans le transfert d’électron entre ces deux partenaires. Ces travaux de recherche démontrent que l’étude de la biodiversité permet non seulement la découverte de nouveaux systèmes permettant la vie dans des environnements extrêmes, mais également la découverte de nouvelles protéines aux propriétés remarquables. / Copper proteins play key roles in many biological processes, such as in respiratory chains. Although many studies have been carried out to decipher the relationship between their active site structure, electronic properties and function, these features are still not fully understood. Previous studies on the respiratory chain of an acidophilic organism, Acidithiobacillus ferrooxidans, have revealed the presence of a new copper-binding protein: AcoP. This cupredoxin is critical for the correct functioning of this respiratory pathway. Using site-directed mutagenesis and a wide-range of biophysical approaches, electrochemistry and X-ray crystallography, we can show that an unconventional copper-active site in AcoP might underlie its rare electronic and redox features. The function of such a protein in the respiratory chain of A. ferrooxidans has subsequently raised our curiosity. It was shown that AcoP interacts with cytochrome c and the cytochrome c oxidase. We showed that AcoP could act as a linker between the cytochrome c and the cytochrome c oxidase, by recruiting the former, and could also participate in the electron transfer between these two partners. This work shows how exploring biodiversity leads to the discovery of new systems that allow life in extreme environments, as well as of new proteins with remarkable features.

Acidophilic bacteria are able to tolerate acidic environment and also contribute to the lowering of environmental pH value, implying potential applications in metal-leaching technology extracting metals from tailings and electronic wastes. In this study, we conducted a sampling campaign in abandoned Tin mine in Ha Thuong, Thai Nguyen province, to isolate acidophilic bacteria and to study physiological characteristics of the isolated bacteria. As a result, two acidophilic bacteria were successfully isolated and identification by 16S rDNA gene sequences showed that the two bacteria are similar to Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans (98% and 94 % of similarity, respectively). Both strains are tolerant of pH in the range of 3 and have the ability to grow optimally at temperatures of 30°C. / Vi khuẩn ưa axit có ý nghĩa ứng dụng trong công nghệ tách rút kim loại từ quặng đuôi và ngay cả từ rác thải linh kiện điện tử. Trong nghiên cứu này, chúng tôi đã tiến hành thu mẫu ở mỏ thiếc bỏ hoang ở Hà Thượng, Thái Nguyên nhằm phân lập được nhóm vi khuẩn ưa axit và qua đó nghiên cứu một số đặc điểm sinh lý của vi khuẩn này. Kết quả chúng tôi đã phân lập được hai chủng vi khuẩn ưa axit. Định dạng bằng nhận diện trình tự gen 16S rADN cho thấy hai vi khuẩn này có độ tương đồng là 98% với vi khuẩn Acidithiobacillus ferrooxidans và 94% Acidithiobacillus thiooxidans. Cả hai chủng vi khuẩn đều có tính chịu pH trong khoảng 3 và có khả năng sinh trưởng tối ưu ở nhiệt độ 30oC.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / Biomining organisms are generally found in metal-rich, inorganic environments such as iron and sulfur containing ores; where they play a vital role in mineralization and decomposition of minerals. They are typically obligatory acidophilic, mesophilic or thermophilic, autotrophic, usually aerobic, iron-or sulfur oxidizing chemolithotrophic bacteria. The most prominent biomining organisms used in bioleaching of metal sulfides are Acidithiobacillus ferrooxidans, At. thiooxidans, At. caldus, Sulfobacillus spp. and Leptospirillum spp. Biomining enables us to utilize low grade ores that would not have been utilized by conventional methods of mining. Research has focused on the backbone features of plasmids isolated from bacteria of biomining environments. The aim of this study is to sequence and analyze an 18 kb region of the 66 kb plasmid pTcM1 isolated from At. caldus MNG, focusing on accessory genes carried by this plasmid. Fifteen putative genes / open reading frames were identified with functions relating to metabolism and transport systems. The genes are located in two divergently located operons. The first operon carries features related to general metabolism activities and consists of a transcriptional regulator (ORF 2), a succinate / fumarate dehydrogenase-like subunit (ORF 3), two ferredoxin genes (ORF 4 and ORF 7), a putative HEAT-like repeat (ORF 6) which is interrupted by an insertion sequence (ORF 5) and a GOGAT-like subunit (ORF 8). The second operon contains an ABC-type nitrate / sulfonate bicarbonate-like gene (ORF 9), a binding protein-dependent inner membrane component-like gene, another ABC sulfonate / nitrate-like gene (ORF 12i and 12ii) which is interrupted by an insertion sequence (ORF 13) and two hypothetical proteins with unknown functions (ORF 14 and ORF 15). Southern hybridization analysis have shown that most of the genes from the two operons are found in other At caldus strains #6, “f”, C-SH12 and BC13 from different geographical locations. Expression of the GOGAT-like subunit and the succinate / fumarate-like subunit was demonstrated in At. caldus MNG showing that these genes are functional and actively transcribed. The transcriptional regulator (ORF 2) has been shown to repress the downstream genes of putative operon 1. The persistence of these genes on plasmids together with the fact that they are being expressed, represents a potential metabolic burden, which begs the question why they have been maintained on the plasmid from geographically separated strains (and perhaps also growing under very different nutrient availability conditions) and therefore what possible role they may play.

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.

O presente estudo investigou a participação do H2S04, como agente corrosivo no carste do Grupo Una, na bacia de Irecê e na bacia do rio Salitre, tendo como base o monitoramento hidroquímico e a determinação de fácies hidroquímicas. A fácies escoamento superficial alogênico é caracterizada por baixos valores de pH (\'X BARRA\'=4,79 ; CV=3,15%) e dos principais cátions e ânions, com destaque para S\'O IND. 4 POT. 2-\' (x=0,57 mg/L ; CV10,32%); A fácies fluxo freático profundo carbonático representa a maior concentração de HC\'O IND. 3 POT. -\' de todo conjunto amostrado, com média igual a 359,08 mg/L. Na média as águas desta fácies encontram-se saturadas para calcita (S\'l IND. c\' = 0,02) e insaturadas para gipsita (S\'l IND. g\' = -1,51). A concentração de S\'O IND. 4 POT. 2-\' também é alta (\'X BARRA\'=204,50 mg/L ; CV 135,00%), destacando-se um ponto de amostragem que exibe elevado valor médio para \'SO IND. 4 POT. 2-\' (\'X BARRA\'= 746,07 ; CV = 13,79%); Na fácies percolação vadosa a concentração de S\'O IND. 4 POT. 2-\' é expressiva, com média igual a 305,09mg/L; Nos reservatórios subterrâneos destaca-se a alta concentração de HC\'O IND. 3\'(média - 305,10 mg/L), Índice de Saturação para calcita muito próximo do equilíbrio (S\'l IND. c\'=-0,02) e concentração média para S\'O IND. 4 POT. 2-\' (\'X BARRA\'=17,79 mg/L; CV = 52,03%); Nas nascentes cársticas observou-se alto teor de HC\'O IND. 3 POT. -\' (\'X BARRA\'=246,96 mg/L ; CV = 11,0%), S\'l IND. c\'=0,00 (0,28 a -0,37), em equilíbrio de saturação para o mineral calcita e concentração de S\'O IND. 4 POT. 2-\' (\'X BARRA\'= 19,30mg/L ; CV = 31,47%). As águas meteóricas são caracterizadas por baixas concentrações dos principais cátions e ânions, com médias inferiores a 0,5mg/L. Cálculos estequiométricos foram aplicados aos dados hidroquímicos e conclui-se que no balanço molar as águas não seguem as razões clássicas para dissolução exclusiva por ácido carbônico, mas sim, adequam-se ao sistema \'H IND. 2\'S\'O IND. 4\' - CaMgC\'O IND. 3\'. Minerais secundários de cavernas foram analisados e detectou-se ocorrências expressivas de gipsita, sob a forma de crostas, estalactites e filamentos, que, vinculadas ao ambiente de formação, indicam a participação de soluções ricas em sulfato em seus processos. O evento principal de deposição de gipsita foi associado ao rebaixamento do NA após fase de ampliação paragenética dos condutos. Análises isotópicas de \'\'delta\' POT. 13\'CHC\'O IND. 3 POT. -\' em amostras de águas subterrâneas indicam valores enriquecidos (-10,4%o PDB-V) em relação ao esperado para a reação de dissolução da rocha carbonática por ácido carbônico (-17,1%o), condição relacionada à influência de \'ANTPOT. 13 C\' oriundo da rocha por ação do ácido sulfúrico. O \'delta\' POT. 34\'S de sulfato em água subterrânea (\'X BARRA\'=17,7%o CDT) é levemente empobrecido em relação à pirita (\'X BARRA\'=21,3%o), com valores na faixa de amplitude prevista para o processo de oxidação, condição que atesta a participação de ácido sulfúrico na dissolução da rocha carbonática. Análises microbiológicas na água subterrânea do aqüífero cárstico indicaram a presença de bactérias do gênero Acidithiobacillus e bactérias redutoras de sulfato (BRS), ambas presentes no ciclo do enxofre e responsáveis pela produção de ácido sulfúrico. Neste sentido, as variáveis estudadas possibilitam a confirmação da participação do ácido sulfúrico na dinâmica do sistema cárstico do Grupo Una no Estado da Bahia. / The main goal of this study is to determine the effectiveness of sulfuric acid as a corrosive agent in the karst system of the Una Group (lrecê geological basin and Salitre river basin), based on hydrochemical monitoring and the respective hydrochemical facies. The allogenic surface runoff is characterized by low ion concentrations and pH (ph=4,79 ; CV=3,15%). Sulfate is also low for this facies (\'X BARRA\'=0,57 mg/L ; CV 10,32%). With respect to the HC\'O IND. 3 POT. -\' content, the deep phreatic flux in the carbonate aquifer has the highest concentration compared to the other facies, with a mean of 359,08 mg/L. This deep phreatic flux is saturated with respect to calcite and undersaturated with respect to gypsum, showing average concentrations of 204,5 mg/L (CV 135,00%) for S\'O IND. 4 POT. 2-\', including one sampling site with average concentration of 746,07 mg/L (CV = 13,79%). Expressive sulfate concentration was also detected in the vadose seepage, with a mean of 305,09 mg/L. Underground reservoirs have high HC\'O IND. 3 POT. -\' content (\'X BARRA\'=305,10 mg/L) together with calcite saturation index close to equilibrium (S\'l IND. c\'=-0,02) and lower sulfate concentration (mean of 17,79 mg/L; CV = 52.03%). The karst springs revealed similar chemistry to the underground reservoirs, with high HC\'O IND. 3 POT. -\' content (\'X BARRA\'=246,96 mg/L ; CV = 11,0%), saturated with respect to calcite (mean of S\'l IND. c\' = 0,00; varying between 0,28 and -0,37) and mean sulfate concentration of 19,30mg/L (CV = 31.47%). The meteoric recharge has a very low solute load, with means of all ions lower than 0.5 mg/L. Stoichiometric balance calculations applied to the hydrochemical results showed that the molar balances of the various water facies do not follow the classical ratios of carbonate dissolution only due to carbonic acid, but on the contrary, they follow better the chemical system of \'H IND. 2\'S\'O IND. 4\' - CaMgC\'O IND. 3\'. The study of the expressive secondary gypsum deposits in caves, represented by crusts, stalactites and filaments, showed that their origin is related to the deep phreatic water and that the major deposition event of these deposits occurred during the water table lowering after the high water table period related to the paragenetic conduit enlargement. Isotopic composition of dissolved inorganic carbon in groundwater samples showed values of \'\'delta\' POT. 13\'CHC\'O IND. 3 POT. -\' around -10,4%o PDB-V, which are relatively enriched with respect to the predicted \'\'delta\' POT. 13\'CHC\'O IND. 3 POT. -\' of -17,1%o, if the system would be affected only by carbonic acid. This enrichment is attributed to the \'ANTPOT. 13 C\' originated by the reaction between carbonate rock and sulfuric acid. The \'delta\' POT. 34\'S of sulfate in water (\'X BARRA\'=17,7%o CDT) is slightly depleted than that of pyrite disseminated in carbonate rock (\'X BARRA\'=21,3%o), but still in the range predicted for the fractionation due to pyrite oxidation, indicating that carbonate rock is dissolved by sulfuric acid. Microbiological analysis of groundwater in the karstic aquifer revealed the presence of bacteria of the genera Acidithiobacillus and sulfate reducing bacteria (SRB). Both are typical of the sulfur cycle and produce sulfuric acid. Finally, it can be stated, based on the above described results, that sulfuric acid is an important agent in the dynamics of the studied karst.

<p>Tailings are a fine-grained waste product produced during the metal recovery process. Tailings consist mostly of different silicates but also sulfides (e.g. pyrite), since 100 % metal recovery is not possible. Freshly processed tailings are deposited in large impoundments. If the mine tailings in the impoundments are exposed to water and oxygen, the sulfides will oxidize and release acidity and metals such as Fe, Cu, Zn, and Pb. The sulfide mineral oxidation reactions are catalyzed by sulfur and iron oxidizing bacteria (principally <i>Acidithiobacillus ferrooxidans</i>) that oxidize ferrous iron to ferric iron, which then oxidizes pyrite. When the leachate produced by this process discharges from the impoundment, it is called acid mine drainage, which may lead to the pollution of adjacent streams and lakes.</p><p>The intention with this thesis is to investigate and quantify mineral weathering processes and element release rates occurring in water-saturated and soil-covered sulfidic mine tailings. The study was performed in different batch and column experiments in room temperature and in the laboratory. The batch experiments were conducted for ca. three months and investigated: a) microbial and abiotic sulfide oxidation in freshly processed tailings under oxic conditions at pH 2-3 and pH 8, b) microbial oxidation of pure pyrite grains at pH 2-3 under different oxygen concentrations ranging from anoxic to oxic conditions. The column experiments, consisting of unoxidized tailings in water-saturated columns, were conducted for up to three years. In these experiments, an oxygen-saturated solution was continually pumped into the column inlet, and investigated: a) differences in oxidation rates between tailings of two different grain sizes, b) factors affecting element discharge rates, acid neutralization, and sulfide oxidation, c) the effect of ions released in a soil cover on release rates in the tailings.</p><p>Sulfide oxidation processes within the batch experiments were limited by surface kinetics. The microbial oxidation of pure pyrite at atmospheric conditions produced the most rapid rate, while the microbial oxidation of pure pyrite at anoxic conditions was slower by 1.8 orders of magnitude. Microbial and abiotic oxidation of pyrite in freshly-processed tailings resulted in pyrite oxidation rates that were intermediate between these two extremes. The results from the microbial experiments with pure pyrite indicated a positive correlation between the concentration of dissolved oxygen, ferric iron and bacterial cells (at a total cell concentration > 10⁶ cells/mL and a dissolved oxygen concentration ≥ 13.2 µM), which implies an interdependence of these factors. The results from these batch experiments support the indirect mechanism for microbial oxidation by the ferric oxidation pathway. Pyrite oxidation rates estimated from the batch experiments may be comparable with oxidation rates in the unsaturated zone and at the groundwater table in a tailings impoundment.</p><p>Acid neutralization reactions in the column experiments resulted in the release of base cations to the column leachate. Calcite was the most important neutralizing mineral despite that it was only present in minor amounts in the tailings. It was confirmed that acidity forced the calcite dissolution. Element release rates in the column experiments were controlled by the availability of dissolved oxygen, which was a function of the water flow rate into the column. These column experiments also showed that the results are comparable with results from field studies, justifying the use of column experiments to study processes within tailings impoundments.</p>