Comparative Methods for Quantification of Sulfate-Reducing Bacteria in Environmental and Engineered Sludge Samples

SIMPLE SUMMARY: Sulfate-reducing bacteria (SRB) are important microorganisms in natural ecosystems and are widely utilized in engineered processes for removing heavy metals and sulfate from acid rock drainage and other wastewater. Conventional methods for quantifying SRB are time consuming and display variable results, while molecular techniques require specialized equipment and analysis. We compared microscopic cell counting, culture, and quantitative or real-time PCR (qPCR) methods for the absolute enumeration of SRB populations in engineered and environmental sludge samples. qPCR analysis showed the best performance regarding specificity, precision, and accuracy. However, it did not work for all samples due to their complex physical, chemical, and microbiological characteristics. Using a qPCR method normalized to dsrA gene copies and a synthetic double-stranded DNA fragment as a calibrator could be a better solution for enumerating SRB in samples from diverse origins. ABSTRACT: This study aimed to compare microscopic counting, culture, and quantitative or real-time PCR (qPCR) to quantify sulfate-reducing bacteria in environmental and engineered sludge samples. Four sets of primers that amplified the dsrA and apsA gene encoding the two key enzymes of the sulfate-reduction pathway were initially tested. qPCR standard curves were constructed using genomic DNA from an SRB suspension and dilutions of an enriched sulfate-reducing sludge. According to specificity and reproducibility, the DSR1F/RH3-dsr-R primer set ensured a good quantification based on dsrA gene amplification; however, it exhibited inconsistencies at low and high levels of SRB concentrations in environmental and sulfate-reducing sludge samples. Ultimately, we conducted a qPCR method normalized to dsrA gene copies, using a synthetic double-stranded DNA fragment as a calibrator. This method fulfilled all validation criteria and proved to be specific, accurate, and precise. The enumeration of metabolically active SRB populations through culture methods differed from dsrA gene copies but showed a plausible positive correlation. Conversely, microscopic counting had limitations due to distinguishing densely clustered organisms, impacting precision. Hence, this study proves that a qPCR-based method optimized with dsrA gene copies as a calibrator is a sensitive molecular tool for the absolute enumeration of SRB populations in engineered and environmental sludge samples.