Detection of SRP Activity by Quantification of mRNA for Dissimilatory (bi) Sulfite Reductase Gene (dsrA) by Reverse Transcription Quantitative PCR

Abstract

Detection of SRP Activity by Quantification of mRNA for the Dissimilatory (bi) Sulfite Reductase Gene (dsrA) by Reverse Transcription Quantitative PCR

Molecular biological methods have been used for some years to identify and quantify active microorganisms present in a commercial oil reservoir where biogenic sulfide production is routinely controlled by nitrate injection. In order to gain a more complete understanding of the effects of nitrate injection on the activity of sulfate reducing prokaryotes (SRP, (which encompasses sulfate reducing Bacteria (SRB) and sulfate reducing Archaea (SRA), the mRNA for dsrA present in produced water samples was quantified by reverse transcription quantitative PCR (RT-qPCR); mRNA for dsrA should only be produced by SRP actively reducing sulfate. The aims of this study were: to help further our understanding on the mode of action of nitrate on SRP activity e.g. competitive inhibition by nitrate utilising Bacteris (NUB), nitrite toxicity, change in reduction-oxidation potential or a metabolism switch from sulfate to nitrate reduction, and; to provide a rapid monitoring tool for SRP activity.

Since messenger RNA is known to be unstable and is rapidly processed within cells, the first task was to design a laboratory experiment to demonstrate that mRNA for dsrA could be detected and quantified in produced water samples. Produced water samples were spiked with a SRP culture grown from the produced water sample and the mRNA for dsrA was successfully detected and quantified.

For the field study, fresh produced water samples were obtained from two wells where direct seawater and nitrate breakthrough has occurred. DAPI, FISH & RT-qPCR analyses were performed directly on the water samples. This paper describes the use of RT-qPCR and detection of mRNA for dsrA as a tool for monitoring SRP activity in biogenic sulfide-producing reservoirs. In order to gain an understanding of the SRB sulfidogenesis activity, a combination of quantifying SRB numbers by FISH, and quantification of the levels of dsrA mRNA, in order to calculate the number of dsrA transcripts per cell is required. The technique has the potential to ascertain the effects of nitrate injection on SRP populations, for instance, in the case of Desulfovibrio; do species of this bacterium preferentially reduce nitrate rather than sulfate? The technique may also be used to determine the recovery of SRP activity following nitrate or biocide dosing.