Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices

ABSTRACT Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.

Bacterial diversity characterization in petroleum samples from Brazilian reservoirs / Caracterização da diversidade bacteriana em amostras de petróleo provenientes de reservatórios brasileiros

This study aimed at evaluating potential differences among the bacterial communities from formation water and oil samples originated from biodegraded and non-biodegraded Brazilian petroleum reservoirs by using a PCR-DGGE based approach. Environmental DNA was isolated and used in PCR reactions with bacterial primers, followed by separation of 16S rDNA fragments in the DGGE. PCR products were also cloned and sequenced, aiming at the taxonomic affiliation of the community members. The fingerprints obtained allowed the direct comparison among the bacterial communities from oil samples presenting distinct degrees of biodegradation, as well as between the communities of formation water and oil sample from the non-biodegraded reservoir. Very similar DGGE band profiles were observed for all samples, and the diversity of the predominant bacterial phylotypes was shown to be low. Cloning and sequencing results revealed major differences between formation water and oil samples from the non-biodegraded reservoir. Bacillus sp. and Halanaerobium sp. were shown to be the predominant components of the bacterial community from the formation water sample, whereas the oil sample also included Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. and Acidithiobacillus ferrooxidans. The PCR-DGGE technique, combined with cloning and sequencing of PCR products, revealed the presence of taxonomic groups not found previously in these samples when using cultivation-based methods and 16S rRNA gene library assembly, confirming the need of a polyphasic study in order to improve the knowledge of the extent of microbial diversity in such extreme environments.

Este estudo teve como objetivo comparar as comunidades bacterianas de amostras de água de formação e de óleo de reservatórios de petróleo brasileiros com diferentes graus de biodegradação usando a técnica de PCR-DGGE. O DNA ambiental foi isolado e empregado em reações de PCR com primers bacterianos, com subseqüente separação dos fragmentos de DNAr 16S em DGGE. Os produtos de PCR foram também clonados e seqüenciados, visando à afiliação taxonômica dos membros da comunidade. Os fingerprints obtidos permitiram a comparação direta entre as comunidades bacterianas das amostras de óleo com diferentes graus de biodegradação, assim como entre as comunidades da água de formação e do óleo do reservatório não biodegradado. Perfis de DGGE muito similares foram observados para todas as amostras, e a diversidade dos filotipos bacterianos predominantes mostrou-se baixa. Os resultados de clonagem e seqüenciamento revelaram diferenças mais significativas entre as amostras de água de formação e de óleo do reservatório não biodegradado. Bacillus sp. e Halanaerobium sp. mostraram-se os componentes predominantes da comunidade bacteriana da presente na amostra de água de formação, ao passo que a amostra de óleo incluiu também Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. e Acidithiobacillus ferrooxidans. A técnica de PCR-DGGE, combinada com clonagem e seqüenciamento dos produtos de PCR, revelou a presença de grupos taxonômicos não encontrados anteriormente nestas amostras quando métodos baseados em cultivo e na construção de bibliotecas de genes RNAr 16S foram utilizados, evidenciando a necessidade de um estudo polifásico a fim de contribuir para o conhecimento da diversidade microbiana em ambientes extremos como reservatórios de petróleo.