Quantification of Denitrification Genes by qPCR

nirS genes were quantified by real-time PCR using the primers nirS-1F and nirS-3R (Braker et al., 1998 (link)). All 20 μl reactions were run in an iCycler (BioRad) with ca. 5–10 ng DNA, SYBR Green I super mix (BioRad), 0.5 μM of each primer, 0.008% bovine serum albumin using the following conditions for 40 cycles: 95^∘C for 15 s, 54^∘C for 20 s, 72^∘C for 30 s. To monitor product specificity, we conducted melt curve analysis (95^∘C for 1 min, 54^∘C for 1 min, and then 0.5^∘C increase every 10 s, with fluorescence read continuously) after each run. Gene abundances were estimated by comparison to known concentrations of a plasmid containing a cloned nirS gene. Concentrations of the plasmid ranged from 1 pg to 1 fg. Bacterial 16S rRNA genes and amoA genes were quantified as previously described (Moin et al., 2009 (link); Bernhard et al., 2012 (link); Peng et al., 2013 (link)). PCR efficiencies were 94.3 ± 0.14% (nirS), 93.4 ± 4.7% (AOB), 99.6 ± 10.0% (AOA), and 89.2% (bacterial 16S rRNA).