Laboratory experiments were carried out to evaluate the effectiveness of antibacterial substances and antibiotics against Thiobacillus ferrooxidans, the organisms responsible for bacterial mediated acidic mine drainage. Twenty two antibiotics (obtained from Lilly and Co.) and two antibacterial substances were added to: bacterial culture ATCC 19859 grown in 9K medium. Appropriate controls were maintained. Inhibition of iron oxidizing bacteria was recorded in terms of changes in Eh of the medium treated with the compound. Seven antibiotics (A38533A:, A38533B, 197506, 13780, 171541, chloramphenicol and cephalexin) and the two antibacterial substances [N-serve(nitrapyrin) and Dicyandiamide] effectively inhibited the oxidation of Fe²⁻ ions in the medium. The kinetics of Fe²⁻ oxidation with the addition of antibiotics and the antibacterial substances was studied. N-serve [2-chloro-6-(trichloromethyl) pyridine], used as a nitrification inhibitor in agriculture, was highly effective at concentrations greater than 0.1 ml/l. Iron oxidation levels were reduced to levels close to that in uninoculated controls (abiotic oxidation). The use of N-serve to inhibit acid mine drainage (AMD) causing bacteria seems to be both economical and environmentally safe. / Master of Science

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

Les piles à combustible microbiennes (PCMs) sont une technologie convertissant l’énergie chimique stockée dans la matière organique en énergie électrique à l’anode à l’aide de bactéries dites électroactives. A l'inverse des bioanodes, l’intérêt porté aux cathodes biologiques dans les PCMs est récent et ces dernières sont encore peu étudiées. Les objectifs de ces recherches ont donc été d’identifier et de décrire des bactéries ainsi que les mécanismes permettant de catalyser une réduction cathodique notamment celle de l’oxygène. La première partie des travaux portant sur la réalisation d’un criblage pour la formation de biocathodes a révélé l’inefficacité du métabolisme hétérotrophe pour la conception de biocathodes performantes. De ce fait, la suite des travaux a porté sur la bactérie chimiolithoautotrophe acidophile Acidithiobacillus ferrooxidans. Deux mécanismes de transfert d’électrons dépendant du fer ont été mis en évidence générant un courant maximal de -3,8 A.m-2. Nous avons également abordé les mécanismes de transfert d’électrons en milieu neutre chez une bactérie hétérotrophe, Pseudomonas brassicacearum NFM421 bien que peu performante en terme de production de courant. Cette dernière permet une catalyse indirecte de la réduction de l’oxygène à la cathode à pH neutre via un métabolite secondaire le 2,4-diacéthylphloroglucinol associé au fer, mettant encore une fois en lumière l’importance du fer dans le transfert d’électrons. Ce travail a également porté sur la potentielle application des biopiles dans des environnements anthropisés en vue de l’extraction de métaux et plus particulièrement de terre rares qui s’avèrent être une voie de recherche prometteuse. / Microbial fuel cells (MFCs) are devices that convert chemical energy contained in organic matter into electrical energy using electroactive bacteria that act at the anode. Currently, MFCs performances are limited by the use of abiotic cathode. The interest in biological cathode has recently started and less is known about bacterial diversity and mechanism that catalyze cathodic reduction. The aims of the research work are therefor to identify and describe potential bacteria and mechanism involved in such catalysis. The first part of the work is the realization of a screening that did not show conclusive results and might indicate that heterotrophic metabolism was not an efficient choice. Next, Acidithiobacillus ferrooxidans had been used and shown two extracellular electron transfer mechanism depending on iron. A maximum current intensity of -3,8 A.m-2 had been reached. To be close to operational condition of MFC, Pseudomonas brassicacearum NFM 421 has also be use and shown capacity to indirectly catalyze the oxygen reduction at a neutral pH using the 2,4-diacethylphloroglucinol, a secondary metabolite, associated to iron. However, current reach remained weak. Considering difficulty to build efficiant biocathode at a neutral pH, the end of this work had been focused on a new application of the MFC: metal and rare earth extraction from soil and contaminated site that appeared to be a great research opportunity to follow.

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 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.

<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>

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 Acidithiobacillus ferrooxidans) 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. 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. 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 &gt; 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. 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.

Techn. University, Diss., 2001--Berlin.

El presente trabajo de título tiene por objetivo el estudio de la biolixiviación de pirita por un cultivo puro de Acidithiobacillus ferrooxidans y por un consorcio de cepas nativas provenientes de la solución lixiviante de la pilas de biolixiviación de Minera Escondida (PLS). El estudio se efectuó con una muestra de pirita purificada y de fina granulometría proveniente de relaves de Minera Escondida, la cual fue lixiviada en reactores de frascos agitados. El porcentaje de disolución del mineral se determinó a partir del monitoreo del hierro disuelto desde el mineral, cuantificado como la suma del hierro en solución más el hierro precipitado como jarositas. Los resultados de conversión del mineral en el tiempo se analizaron según el modelo del núcleo sin reaccionar. Se comparó también la eficiencia de la adherencia a pirita y la capacidad de oxidar azufre de ambos tipos de microorganismos. Los resultados indicaron que la cinética de biolixiviación de pirita con At. ferrooxidans en soluciones con 2,4g/l de férrico se comporta de acuerdo a un control por difusión. El tiempo para alcanzar disolución total del sulfuro, τ, en estas condiciones es de 833 días. Al aumentar la concentración de ion férrico a 5g/l la cinética de disolución de la pirita con este microorganismo pasa a ser controlada por reacción química y el valor de τ se reduce a 141 días. Por otra parte la cinética de biolixiviación de pirita con las cepas nativas en soluciones con 1,8g/l de férrico se comporta de acuerdo a un control por reacción química y el valor de τ en estas condiciones es de 122 días. Los experimentos de adherencia mostraron que el 100% de las cepas nativas se adhiere a pirita mientras que la adherencia máxima de At. ferrooxidans alcanza a solo un 30% de los microorganismos presentes. Por otra parte la capacidad oxidativa de azufre a sulfato en las cepas nativas se registró de forma inmediata en los experimento de oxidación de azufre, mientras que para At. ferrooxidans la actividad se observó posterior a la semana de monitoreo. Los resultados indican que la velocidad de biolixiviación de la pirita con los microorganismos nativos es mucho mayor que con la cepa pura de At. ferrooxidans. Este resultado parece estar determinado por la mayor capacidad de adherencia y de oxidación de azufre de los microorganismos nativos, lo que posibilita una eficiente disolución de subproductos pasivantes desde la superficie de la pirita. Sin embargo, esta capa también puede ser removida en presencia de At. ferrooxidans si se aumenta la concentración de ion férrico a 5g/l. Finalmente, los resultados indicaron que la adherencia bacteriana en el caso de las cepas nativas favorece la disolución de la pirita, alcanzando una recuperación y velocidad de lixiviación más alta que en el caso sin adherencia.

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