Location: Bioenergy Research Unit
Title: Development and Comparison of SYBR Green Quantitative Real-time PCR Assays for Detection and Enumeration of Sulfate-reducing Bacteria in Stored Swine Manure Authors
Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 21, 2008
Publication Date: December 1, 2008
Citation: Spence, C., Whitehead, T.R., Cotta, M.A. 2008. Development and Comparison of SYBR Green Quantitative Real-time PCR Assays for Detection and Enumeration of Sulfate-reducing Bacteria in Stored Swine Manure. Journal of Applied Microbiology. 105(6):2143-2152. Interpretive Summary: Odorous compounds and emissions produced from stored swine manure create a nuisance and can influence zoning decisions about the proposed sites of new swine facilities. One such offensive odorant is hydrogen sulfide, which is produced by anaerobic sulfate-reducing bacteria (SRB). In order to reduce hydrogen sulfide emissions, it is important to determine the population of hydrogen sulfide-producing bacteria. This report describes a SYBR Green quantitative real-time polymerase chain reaction (PCR) method to detect and enumerate individual groups of SRB in stored swine manure using primer sets targeting the conserved dissimilatory sulfite reductase gene. These newly developed assays are rapid, simple, and sensitive molecular methods for the detection and enumeration of different SRB populations in swine manure. This is the first study to show population shifts of SRB in swine manure, resulting from a change in the dietary sulfur content of swine diets, or as a result of the maturity of the manure ecosystem.
Technical Abstract: A quantitative real-time polymerase chain reaction (PCR) assay for sulfate-reducing bacteria (SRB) was developed that targeted the dissimilatory sulfite reductase gene (dsrA). Degenerate primer sets were developed to detect three different groups of SRB in stored swine manure using a SYBR Green quantitative real-time PCR assay to amplify dsrA genes. These were compared to a previously reported dsrA amplifying primer set, DSR1F+ and DSR-R, for their diversity of coverage and the ability to discern SRB communities in stored swine manure. Sequenced clones isolated from swine manure were most similar to Desulfovibrio sp. and Desulfobulbus sp., and it was shown that the populations of these SRB differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus-like Group 1 SRB in manure. The newly developed assays were able to enumerate and discern different groups of SRB in swine manure. Results also suggest a richly diverse and, as yet, undescribed population of SRB in swine manure. The quantitative real-time PCR assays described here are rapid, simple, and sensitive methods for the detection and enumeration of SRB in swine manure, and provide us with improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.