RESUMO
Hydrocarbon-degrading microorganisms from natural environments have been isolated and identified using culture-dependent or molecular techniques. However, there has been little research into the occurrence of microorganisms incorporated into crude oil in the initial steps of extraction and handling, which can reduce the quality of stored petroleum. In the present study, a packed-column reactor filled with autoclaved perlite soaked with crude oil was subjected to a continuous flow of sterile medium in order to determine the presence of potential hydrocarbon degraders. Microorganisms developed on the surface of the perlite within a period of 73 days. DNA was extracted from the biofilm and then PCR-amplified using 16S rRNA bacterial and archaeal primers and 18S rRNA eukaryotic primers. No amplification was obtained using archaeal primers. However, denaturing gradient gel electrophoresis (DGGE) revealed the presence of unique bands indicating bacterial and eukaryotic amplification. Excision of these bands, sequencing, and subsequent BLAST search showed that they corresponded to Bacillus sp. and Aspergillus versicolor. The fungus was later isolated from intact perlite in agar plates. A bacterial clone library was used to confirm the presence in the biofilm of a unique hydrocarbon-degrading bacterium closely related to Bacillus sp. Analysis of the petroleum components by gas chromatography showed that there n-alkanes, aromatic hydrocarbons, and carbazoles were degraded.
Assuntos
Aspergillus/metabolismo , Bacillus/metabolismo , Hidrocarbonetos/metabolismo , Petróleo/metabolismo , Aspergillus/genética , Aspergillus/isolamento & purificação , Bacillus/genética , Bacillus/isolamento & purificação , Biofilmes , Reatores Biológicos/microbiologia , Ecossistema , Eletroforese em Gel de Poliacrilamida/métodos , Hidrocarbonetos/química , Reação em Cadeia da Polimerase/métodosRESUMO
A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.
