Assessing Bacterial Activity under SMF

To assess the activity of bacteria during exposure to SMF, the real-time PCR was done. The AOB activity was assessed based on the amoA gene copy number, and overall bacterial community was assesses based on the 16S ribosomal DNA (rDNA) gene copy number. The activated sludge was sampled and fixed according to Cydzik-Kwiatkowska and Wnuk (2011 (link)). Samples were taken during the initial 8 h of the cycle, i.e., until the moment, in which the content of pollutants in the cycle stabilized. Samples were stored at −20 °C until RNA was extracted. RNA was isolated with Total RNA kit (A&A Biotechnology), according to the manufacturer’s protocol. The concentration of RNA was measured using NanoDrop Lite (Thermo Scientific). Identical amounts of RNA from each sample was a template to create first-strand complementary DNA (cDNA) using RevertAid™ H Minus First Stand cDNA Synthesis Kit (Fermentas). Real-time amplification of 16S rDNA and amoA genes were done in 7500 Real-Time PCR System (Applied Biosystems). Real-time PCR was performed according to Cydzik-Kwiatkowska and Wojnowska-Baryła (2011 (link)). The template for the reaction was cDNA. Standard curves for the assessment of 16S rDNA and amoA gene copies in the samples were linear within a range of 10⁵–10¹⁰ for 16S rDNA (r² = 0.9999) and 10²–10⁷ for amoA (r² = 0.9845).