Metais em áreas portuárias e sua influência na dinâmica microbiana: da caracterização do problema à busca de soluções
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Data
2020-03-12
Autores
Del Busso Zampieri, Bruna
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Universidade Estadual Paulista (Unesp)
Resumo
Atividades portuárias são consideradas fontes de contaminação por metais, que em altas concentrações são tóxicos aos microrganismos, podendo mudar o tamanho, a composição e a atividade da comunidade microbiana. Essa pressão seletiva faz com que bactérias desenvolvam mecanismos para combater o estresse causado por eles. Diversos grupos de microrganismos que desenvolvem sistemas de tolerância a metais podem fazer sua biorremediação. Bactérias redutoras de sulfato (BRS) podem, devido ao seu metabolismo, precipitar metais facilitando sua remoção. A hipótese é que em áreas portuárias exista uma alteração na comunidade microbiana onde são favorecidos microrganismos resistentes, aumentando a chance do isolamento de bactérias resistentes com potencial biotecnológico. Portanto o objetivo dessa tese foi avaliar a influência da contaminação de metais na comunidade microbiana em áreas portuárias e na seleção de cepas resistentes, e isolar cepas de BRS com potencial biotecnológico. Para isso, foram coletados sedimentos de 2 áreas portuárias (Santos e São Sebastião) e uma área pristina (Ubatuba). Foram caracterizadas variáveis ambientais do sedimento (como pH, salinidade, temperatura, oxigênio dissolvido, porcentagem de matéria orgânica, concentrações de Cr, Cu, Cd e Zn). Foi extraído DNA total do sedimento para caracterização da comunidade utilizando sequenciamento Mi-Seq. Os sedimentos serviram como inóculo para isolamento de cepas que foram submetidas a testes de resistência aos metais. Os sedimentos mais contaminados também serviram como inóculo para enriquecimento de BRS em diferentes concentrações de Cr, Cd, Cu e Zn. Esses enriquecimentos foram monitorados através da redução de sulfato, produção de sulfeto e precipitação de metais. Os enriquecimentos onde houve crescimento de BRS em concentrações altas de metais foram utilizados para isolamento de BRS. As cepas isoladas foram identificadas, caracterizadas quanto as suas exigências nutricionais, resistência aos metais, seu genoma foi sequenciado e os genes envolvidos na resistência aos metais analisados. Houve diferença significativa na composição microbiana das áreas portuárias com a área pristina. A diversidade foi menor nas áreas portuárias. Alguns grupos demonstraram ser mais sensíveis ao Cu, Zn e Cr como Flavobacteriaceae, Pseudoalteromonadaceae, Desulfobacteraceae e Saprospiraceae; ou filo como Chloroflexi, Nitrospirae, Plactomyces. Os metais apresentaram forte influência no metabolismo nos enriquecimentos de BRS, onde havia maior concentração de metal ocorreu menor porcentagem de remoção de sulfato. Cd e Cu foram os metais que mais influenciaram na atividade de BRS. Desulfovibrio foi o gênero de BRS encontrado em maior abundância. Foi isolada a cepa Desulfovibrio sp Fe33, uma nova espécie altamente resistente aos metais testados. A cepa Desulfovibrio sp Fe33, apresenta crescimento rápido e fácil em laboratório. Seu crescimento ótimo ocorre em pH neutro e à 30°C. Apresenta grande versatilidade de crescimento pois utiliza diversos tipos de aceptores e doadores de elétrons. Portanto, em áreas portuárias existe uma alteração na comunidade microbiana devido a presença de metais onde são favorecidos microrganismos resistentes e onde a diversidade microbiana pode ser comprometida. Nessas áreas foi possível o isolamento de uma cepa de BRS altamente resistente, capaz de realizar a precipitação de metais mesmo em concentrações tóxicas desse componente fazendo com que seu uso futuro para tratamento de efluentes contaminados seja eficiente.
Port activities are considered sources of metal contamination, which in high concentrations are toxic to microorganisms, and can change the size, composition and activity of the microbial community. This selective pressure forces bacteria to develop resistance mechanisms. Several microorganisms groups that develop metal tolerance systems can perform their bioremediation. One example is Sulfate-reducing bacteria (SRB). Because of their metabolism, this group can precipitate metals facilitating their removal. In this way, the hypothesis is that in port areas there is a change in the microbial community where resistant microorganisms are favored, increasing the chance of isolation of resistant bacteria with biotechnological potential. Therefore, the objective of this thesis was evaluate the influence of metal contamination in the microbial community in port areas and in the selection of resistant strains, and to isolate strains of SRB with biotechnological potential. For this, sediments were collected from two port areas (Santos and São Sebastião) and one pristine area (Ubatuba). Sediment environmental variables were characterized (such as pH, salinity, temperature, dissolved oxygen, percentage of organic matter, concentrations of Cr, Cu, Cd and Zn). Total DNA was extracted from the sediment for community characterization using Mi-Seq sequencing. The sediments was used as inoculum for strain isolation, and the metal resistance was checked. The most contaminated sediments also was used as inoculum for SRB enrichments in different concentrations of Cr, Cd, Cu and Zn. The enrichments were monitored through the measurements of sulfate reduction, sulfide production, and metals precipitation. The enrichments where there was SRB growth even at high metal concentrations were used for isolation of SRB strains. The isolated strains were identified, characterized by their nutritional requirements, resistance to metals, their genome was sequenced and the genes involved in resistance to metals analyzed. There was a difference in the microbial composition of the port areas in comparison with the pristine area. Diversity was lower in the port areas. Some groups were more sensitive to Cu, Zn and Cr such as Flavobacteriaceae, Pseudoalteromonadaceae, Desulfobacteraceae and Saprospiraceae; or phylum like Chloroflexi, Nitrospirae, and Plactomyces. The metals had a strong influence on the BRS metabolism presents is the enrichments. The higher was metal concentration in the enrichments, the lower was the percentage of sulfate removal. Cd and Cu were the metals that most influenced SRB activity. Desulfovibrio was the SRB genus found in higher abundance. The strain Desulfovibrio sp Fe33 was isolated, a new species highly resistant to the metals tested. This strain presents fast and easy growth in laboratory. Its optimal growth occurs at neutral pH and at 30° C. The strain has growth versatility because it uses several types of electron acceptors and donors. Therefore, in port areas there is a change in the microbial community due to the presence of metals where resistant microorganisms are favored and where the microbial diversity can be compromised. In these areas, it was possible to isolate a highly resistant SRB strain, capable of precipitating metals even in toxic concentrations of this component. This hability makes its future use, for the treatment of contaminated effluents efficient.
Port activities are considered sources of metal contamination, which in high concentrations are toxic to microorganisms, and can change the size, composition and activity of the microbial community. This selective pressure forces bacteria to develop resistance mechanisms. Several microorganisms groups that develop metal tolerance systems can perform their bioremediation. One example is Sulfate-reducing bacteria (SRB). Because of their metabolism, this group can precipitate metals facilitating their removal. In this way, the hypothesis is that in port areas there is a change in the microbial community where resistant microorganisms are favored, increasing the chance of isolation of resistant bacteria with biotechnological potential. Therefore, the objective of this thesis was evaluate the influence of metal contamination in the microbial community in port areas and in the selection of resistant strains, and to isolate strains of SRB with biotechnological potential. For this, sediments were collected from two port areas (Santos and São Sebastião) and one pristine area (Ubatuba). Sediment environmental variables were characterized (such as pH, salinity, temperature, dissolved oxygen, percentage of organic matter, concentrations of Cr, Cu, Cd and Zn). Total DNA was extracted from the sediment for community characterization using Mi-Seq sequencing. The sediments was used as inoculum for strain isolation, and the metal resistance was checked. The most contaminated sediments also was used as inoculum for SRB enrichments in different concentrations of Cr, Cd, Cu and Zn. The enrichments were monitored through the measurements of sulfate reduction, sulfide production, and metals precipitation. The enrichments where there was SRB growth even at high metal concentrations were used for isolation of SRB strains. The isolated strains were identified, characterized by their nutritional requirements, resistance to metals, their genome was sequenced and the genes involved in resistance to metals analyzed. There was a difference in the microbial composition of the port areas in comparison with the pristine area. Diversity was lower in the port areas. Some groups were more sensitive to Cu, Zn and Cr such as Flavobacteriaceae, Pseudoalteromonadaceae, Desulfobacteraceae and Saprospiraceae; or phylum like Chloroflexi, Nitrospirae, and Plactomyces. The metals had a strong influence on the BRS metabolism presents is the enrichments. The higher was metal concentration in the enrichments, the lower was the percentage of sulfate removal. Cd and Cu were the metals that most influenced SRB activity. Desulfovibrio was the SRB genus found in higher abundance. The strain Desulfovibrio sp Fe33 was isolated, a new species highly resistant to the metals tested. This strain presents fast and easy growth in laboratory. Its optimal growth occurs at neutral pH and at 30° C. The strain has growth versatility because it uses several types of electron acceptors and donors. Therefore, in port areas there is a change in the microbial community due to the presence of metals where resistant microorganisms are favored and where the microbial diversity can be compromised. In these areas, it was possible to isolate a highly resistant SRB strain, capable of precipitating metals even in toxic concentrations of this component. This hability makes its future use, for the treatment of contaminated effluents efficient.
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Palavras-chave
Metais, Comunidade microbiana, Diversidade, Bactérias redutoras de sulfato, Biorremediação, Metals, Microbial community, Diversity, Sulfate reducing bacteria, Bioremediation