Caracteriza??o de cons?rcio e isolados bacterianos associados a biorremedia??o do mangan?s.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.O mangan?s ? um contaminante comum em ?guas residu?rias e drenagens produzidas por v?rias atividades mineradoras. A remo??o deste metal ? notoriamente dif?cil, devido ? elevada estabilidade do ?on Mn(II) em solu??es aquosas. Uma alternativa para remo??o do ?on Mn(II) destes efluentes ? a utiliza??o de micro-organismos. Assim, a hip?tese desse trabalho foi que esp?cies bacterianas presentes em ?gua residu?rias com elevada concentra??o de Mn(II) s?o capazes promoverem a eleva??o do pH do meio de cultura e, por sua vez, catalisar a oxida??o do ?on Mn(II) pelo oxig?nio. Al?m disso, tamb?m foi objetivo determinar a participa??o ou n?o de enzimas pertencentes ? fam?lia multicobre oxidase (MCO), como observado em alguns estudos. Dessa forma, n?s investigamos o potencial de oxida??o indireta do Mn(II) por um cons?rcio e por isolados bacterianos obtidos a partir de duas amostras de ?gua de minas denominadas: (i) CL e (ii) BIA. O cons?rcio bacteriano, enriquecido a partir da ?gua CL, utilizando-se meio K, foi capaz de remover 99,7% do Mn(II) do meio de cultura. A an?lise filogen?tica dos isolados obtidos a partir do cons?rcio CL demonstrou a predomin?ncia de membros dos g?neros Stenotrophomonas, Bacillus e Lysinibacillus. Foram observadas remo??es de Mn(II) entre 58,5% e 82,7%, em sete dias de experimento para os isolados selecionados. Al?m disso, dois isolados bacterianos foram obtidos durante o isolamento de fungos a partir da amostra de ?gua CL. Ambos foram identificados como Serratia marcescens atrav?s de abordagens moleculares e bioqu?micas. A remo??o do Mn(II) obtida por esses isolados foi 55% em sete dias de experimento, a partir de uma solu??o contendo 45 mg L-1 do ?on Mn(II). Posteriormente, quatro cepas foram isoladas da amostra de ?gua BIA e identificados como Klebsiella oxytoca. Dentre eles, o isolado SA8 foi testado quanto ? sua capacidade para remover o ?on Mn(II) a partir do meio de cultura, obtendo-se 82,7% de remo??o. A oxida??o catal?tica de Mn(II) mediada por enzimas MOC e o envolvimento de prote?nas extracelulares n?o foram detectados para todos os isolados estudados nesta tese. Experimentos realizados em condi??es aer?bicas demonstraram que a oxida??o do Mn(II) n?o foi relacionada a presen?a de bact?rias, uma vez que o reagente azul Leucoberbelin I revelou a presen?a de mangan?s oxidado em ambas as condi??es bi?ticas e abi?ticas, desde que o pH estivesse acima de 8,0. No entanto, em todas os experimentos bi?ticos, foi detectado um aumento significativo no pH do meio de cultura, o que n?o ocorreu nos frascos de controle, sugerindo um mecanismo indireto. An?lises das c?lulas microbianas por microscopia eletr?nica n?o indicaram a presen?a de mangan?s no interior das c?lulas, mas demonstraram dep?sitos de mangan?s em estruturas em torno dos isolados 13P, CL11 e CL35. Em resumo, esta tese identificou novas esp?cies (Stenotrophomonas, Lysinibacillus, Serratia e Klebsiella) com a capacidade de oxidar o ?on Mn(II) atrav?s de um mecanismo sem a participa??o de MCO em que tanto o metabolismo quanto o crescimento bacteriano resultaram no aumento do pH da solu??o, catalisando a remo??o do ?on Mn(II) por um mecanismo qu?mico.Manganese is a contaminant in wastewaters and drainages produced in several mining operations. The removal of this metal is notoriously difficult because of the high stability of the Mn(II) ion in aqueous solutions. An alternative for Mn(II) removal from effluents is the use of microorganisms. Thus, the hypothesis of this thesis was that bacterial species present in wastewaters with high Mn(II) concentrations are able to increase the pH of the culture medium and, in turn catalyze Mn(II) oxidation by oxygen. Another objective was to determine whether the bacterial manganese oxidation mechanism involved the participation of enzymes belonging to the multicopper oxidase family (MCO), as was observed in some studies. We investigated the potential for indirect Mn(II) oxidation by a consortium and bacterial isolates obtained from two mine water samples named: (i) CL and (ii) BIA. The bacterial consortium enriched from the CL water, using K medium was able to remove 99.7% of the Mn(II) from a solution containing 45 mg.L-1. A phylogenetic analysis of the isolates from the consortium CL demonstrated the predominance of members of the Stenotrophomonas, Bacillus and Lysinibacillus genera. Mn(II) removal between 58.5% and 82.7% were observed in a seven-day-long experiment with selected isolates. In addition, two bacterial isolates were found during fungal isolation from the water sample CL. Both were identified as Serratia marcescens by a phylogenetic analysis and biochemical tests. Both isolates promoted 55% of Mn(II) removal in a seven-day-long experiment, from a solution containing 45 mg.L-1 of Mn(II). Subsequently, four strains were isolated from the water sample BIA and identified as Klebsiella oxytoca. Among them, the isolated SA8 was tested for its ability to remove the Mn(II) ion from the culture medium and the outcome was 82.7% removal. The catalytic oxidation of Mn(II) mediated by MCO and the involvement of extracellular proteins were not detected for all isolates studied in the current thesis. In the aerobic conditions of the experiments, Mn(II) oxidation was not bacterially related because the Leucoberbelin blue I reagent revealed oxidized manganese in both abiotic and biotic conditions provided the pH was above 8.0. However, in all of the biotic experiments, a significant increase in the pH of the culture medium was detected, which did not occur in the control flasks, hence suggesting an indirect mechanism. The observation of microbial cells in electron microscopy did not indicate the presence of manganese inside the cells but showed manganese deposits in the structures around isolates 13P, CL11 and CL35. Summarizing, this thesis identified new species (Stenotrophomonas, Lysinibacillus, Serratia and Klebsiella) with the ability to oxidize the Mn(II) ion by a non-MCO-driven mechanism in which either bacterial growth or cell metabolism resulted in the increase of solution pH, which catalyzed Mn(II) removal by a chemical mechanism