An?lise das comunidades microbianas presentes em dep?sitos de minerais sulfetados com ocorr?ncia de drenagens ?cidas de minas e em sistemas de biolixivia??o.

Esse trabalho teve como objetivo identificar a diversidade microbiana de amostras s?lidas e l?quidas coletadas em uma mina de pirita abandonada nas proximidades de Ouro Preto, MG, Brasil. Para este projeto, foram utilizadas duas abordagens experimentais diferentes: uma primeira, mais convencional, baseada no cultivo de microrganismos associado a t?cnicas de identifica??o molecular FISH (Hibrida??o fluorescente?in situ?) e DGGE (Eletroforese em Gel de Gradiente Desnaturante) e a segunda abordagem, na qual as t?cnicas mencionadas anteriormente foram empregadas independentemente do cultivo pr?vio dos microrganismos visando preservar a biodiversidade natural das amostras. Os dados obtidos para essas amostras naturais foram comparados com aqueles obtidos na an?lise de sistemas de biolixivia??o de minerais de zinco e n?quel. O cultivo e isolamento de microrganismos ocorreu em meio para microrganismos heter?trofos e microrganismos aut?trofos quimiolitotr?ficos. Para a an?lise por FISH utilizaram-se sondas espec?ficas para hibrida??o na regi?o 16S do RNA ribossomal das esp?cies Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, e Leptospirillum ferrooxidans. O DNA extra?do de todas as amostras foi amplificado usando primers universais para o rDNA 16S atrav?s da Rea??o em Cadeia da Polimerase (PCR) e analisado por DGGE utilizando-se diferentes gradientes desnaturantes. As bandas observadas foram recortadas, clonadas e enviadas para o sequenciamento com finalidade de possibilitar posterior identifica??o dos microrganismos. A presen?a de Acidithiobacillus ferrooxidans e Acidithiobacillus thiooxidans nas colunas de lixivia??o foi confirmada e foram obtidos fortes ind?cios de sua presen?a tamb?m no reator de lixivia??o amostrado. Com rela??o ?s amostras de DAM, a ocorr?ncia de Acidithiobacillus thiooxidans nas amostras coletadas foi confirmada por ambas as abordagens estudadas, entretanto, a presen?a de Acidithiobacillus ferrooxidans n?o foi observada. As bandas n?o identificadas foram clonadas e enviadas para seq?enciamento para completa caracteriza??o. A presen?a de um microrganismo heter?trofo, pouco comum nesses ambientes foi observada. Trata-se de bact?ria pertencente ao grupo Burkholderia, cuja atividade na solubiliza??o de fosfato contido em min?rio de ferro foi recentemente relatada.The aim of this work was to determine the microbial diversity of liquid and solid acid mine drainage (AMD) samples collected at an abandoned pyrite mine in Ouro Preto, MG, Brazil. For this purpose two different experimental approaches were used. The first one was based on conventional and molecular analysis techniques such as Fluorescent in situ hybridization analysis (FISH) carried out using specific 16S rRNA probes and Denaturing Gradient Gel Electrophoresis (DGGE), both were used in association with culture dependent methods. In a second approach, those molecular methodologies were used without any previous cultivation. The results obtained from AMD samples were compared to a nickel sulfide column bioleaching pregnant solution which was used as reference. Isolation and cultivation experiments were carried out using culture media specific for heterothrophic and autothrofic chemiolitothrophic microrganisms. For FISH analysis Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferrooxidans specific 16S RNA ribosomal region hybridization probes were used. The extracted DNA was amplified by Protein Chain Reaction (PCR) using universal primers for bacterial 16S rRNA genes and analyzed by DGGE using different denaturating gradients. Observed DNA bands were excised, cloned and sequenced in order to identify the microorganisms. For bioleaching column systems the presence of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans was observed and there is was also a strong evidence for the same microbial profile in the thank bioleaching system studied. For all AMD samples Acidithiobacillus thiooxidans was confirmed despite the experimental approach used, however the presence of Acidithiobacillus ferrooxidans was not observed. The non-identified DNA bands were cloned and sequenced for complete characterization. One of the obtained sequences was identified as being very similar of a DNA sequence from one heterothrophic bacterium from the Burkholderia group. It is interesting to note that, those bacteria were recently described as being capable to promote phosphate solubilization from iron ore