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Sample Preparation Instruction for Petroleum Microbiome Sequencing

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Petroleum Reservoir and Its Microorganisms

An unusual subsurface biosphere, where indigenous microbes have lived and developed for millions of years, is the petroleum reservoir. However, through the implementation of novel electron acceptors, donors, and exogenous microbes, continuous water injection has changed the situation.

Due to their conjunction of remarkably significant amounts of hydrocarbons, comparatively low inputs of oxygen, and a long history of segregation from surface components, petroleum reservoirs are uncommon areas. The reservoirs are inhabited by groups of microorganisms with diverse phylogenetic affiliations. These microorganisms with different metabolic capacities and their encounters with the intra-community can have a massive impact on the resources of petroleum and the process of extraction. On the down side, in Alberta oil fields in Canada, the hydrocarbon-degrading bacterial (HDB) degraded the consistency of crude oil by changing light to heavy crude oil, which is more difficult to manipulate; corrosion and increased refining expenses are caused by the sulfate-reducing bacterial (SRB) in the Halfdan oil field. On the bright side, for bioremediation, bioconversion, and improving oil recovery, the microbes in petroleum reservoirs are beneficial. These microbial impacts encourage the oil industry to spend a great deal in investigating the microbial communities of petroleum.

Petroleum-Contaminated Ecosystems

Soil contaminated with petroleum is among the soil ecosystems most researched for the restoration of microorganisms and the exploration of enzymes. Due to its high content of organic hydrocarbons, it is abundant in indigenous microorganisms with the potential for hydrocarbon degradation. In order to discover the application of soil microorganisms for bioremediation, a significant amount of research has been done. In order to dissolve a vast array of oil hydrocarbons, a variety of bacteria from numerous genera have been segregated and assayed. In order to degrade specific organic contaminants, several metabolic pathways have also been formed and many novel enzymes have been found to be involved in degrading activities.

The metagenomic approach, combined with amplicon sequencing, has recently published an option for a thorough examination of the microbial community and metabolic potential. In order to study various types of microbiome, large-scale metagenomic sequencing has been widely used. Due to its complex features formed by many key variables, the high-throughput metagenomic study of petroleum microbiome also received numerous study interest.

Sampling and Sample Preparation for Sequencing

Specimens are taken from each wellhead (oil-water mixture) of the production wells. Before compilation, the fluids in the production line are expelled for a few minutes to remove any kind of toxicity. Specimens are taken directly from the injection system pipes of the injection wells before the water is infused into the wells. In order to prevent contamination and oxygen interference, the samples that filled sterilized plastic bottles are immediately secured with screw caps. For more assessment, the bottles are then transferred to the laboratory room as soon as possible.

Samples are removed into aqueous and oil phases prior to DNA extraction by centrifugation at 4 °C for 15 min at 10000g in a high-speed centrifuge. In the aqueous and oil phases, genomic DNA is extracted and combined for analysis. The final DNA concentration and purification are dictated, and 1 percent agarose gel electrophoresis verified the DNA quality. Qualified DNA samples can then be used for conventional sequencing protocols.


  1. Wang X, Li X, Yu L, et al. Characterizing the microbiome in petroleum reservoir flooded by different water sources. Science of The Total Environment. 2019.
  2. Bao YJ, Xu Z, Li Y, et al. Song H. High-throughput metagenomic analysis of petroleum-contaminated soil microbiome reveals the versatility in xenobiotic aromatics metabolism. Journal of environmental sciences. 2017.
  3. Vigneron A, Alsop EB, Lomans BP, et al. Succession in the petroleum reservoir microbiome through an oil field production lifecycle. The ISME journal. 2017, 11(9).
* For Research Use Only. Not for use in diagnostic procedures.

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