Abstract
Quantitative PCR (qPCR) methods (fluorogenic 5′-nuclease assay and SYBR Green I assay) were developed to detect and quantify bacteria and some critical sub-populations related to gas and oil pipeline corrosion. 16S rRNA genes were targeted for the detection and quantification of total bacteria using fluorogenic 5′-nuclease assays. The functional genes, dissimilatory sulfite reductase gene (dsrAB), nitrite reductase gene (nirS), and methyl-coenzyme M reductase gene (mcrA), were targeted for sulfate reducing bacteria (SRB), denitrifying bacteria, and methanognes, respectively. The methods were evaluated in the growth cultures of pipeline samples for accuracy and sensitivity, and then applied directly to pipeline samples. The spiking tests indicated the qPCR methods can accurately quantify all 4 target microorganisms with an accuracy of ±10% of their true value (95% confidence level). The assay sensitivities were 2.7 x 103, 20, 13, and 100 gene copies per reaction for bacteria, SRB, denitrifying bacteria, and methanogens, respectively. Several methodologies aimed to increase the detection sensitivity for bacteria by reducing or eliminating the excessive background DNA present in the reaction did not show the significant improvement. The quantification results indicated that methanogens were more abundant in most of pipeline samples than denitrifying bacteria, and SRB the least abundant bacteria tested in this study. The results also showed the dramatic changes of microbial community composition after inoculating the pipeline samples to the growth media. The study indicated that functional gene-based quantitative PCR is a fast, accurate, and sensitive method, and can be successfully applied to elucidate the distribution, abundance, and functional interactions of microbial communities in various environments, including corrosion-related microbial communities in gas and oil pipelines.
