, 2012). Ammonium, nitrite, and nitrate were extracted from the soil with 2 M KCl and measured using a SAN++ Continuous Flow Analyzer (Skalar Analytical, The Netherlands). Total nitrogen, soil organic matter, Mn2+, and Mn4+ were measured according to standard methods (Bao, 2000). Soil pH was determined at a soil/water ratio of 1 : 2.5. All analyses were performed in triplicate on each sample. The in situ measurement of oxygen concentration was achieved by OXY Meter S/N 4164 with stainless electrode sensor (Unisense, Denmark) (Gundersen et al., 1998). Statistical analyses were performed using program spss for Windows. DNA in soil and sediment samples were extracted

from 0.25 g PI3K inhibitor samples using the Powersoil DNA isolation kits (Mobio). DNA from enriched anammox biomass was extracted according to the method described previously (Schmid et al., cancer metabolism inhibitor 2008). For the specific PCR amplification of the anammox hzsB gene,

newly designed primer pair of hzsB_396F and hzsB_742R was applied based on our new findings in anammox molecular mechanism (Kartal et al., 2011; Harhangi et al., 2012). The pmoA gene of n-damo bacteria was amplified using a nested approach (first-step primer pair A189_b-cmo682, followed by primer pair cmo182-cmo568) according to Luesken et al. (2011c). The 16S rRNA gene of n-damo was amplified using a nested approach (first-step primer pair 202F-1545R, followed by primer pair qP1F-qP2R) according to Juretschko et al. (1998) and Ettwig et al. (2009). The sequences of primers and thermal profiles were shown in Table 1. PCRs

were performed with the PerfeCTa SYBR Green FastMix (Quanta). 10 min at 95 °C, followed by 35 cycles of 60 s at 95 °C, 60 s at 59 °C and 45 sat 72 °C (PCR) 3 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 59 °C and 30 sat 72 °C (qPCR) 10 min at 95 °C, followed by30 cycles of 60 s at 95 °C, 60 s at 63 °C and 45 sat 72 °C (PCR, qP1F – qP2R) 3 min at 95 °C, followed Cyclic nucleotide phosphodiesterase by 40 cycles of 30 s at 95 °C, 30 s at 63 °C and 30 sat 72 °C (qPCR, qP1F – qP1R) PCR amplified fragments were cloned using the pGEM-T Easy cloning kit (Promega) according to the manufacturer’s instructions. Plasmid DNA was isolated with the GeneJET Plasmid Miniprep kit (Fermentas, Lithuania). Plasmids were digested with EcoRI enzyme, and the digestion products were examined for an insert with expected size by agarose (1%) gel electrophoresis. Selected clones were sequenced using primer of M13f targeting vector sequences adjacent to the multiple cloning sites. Phylogenetic analysis was performed using mega 5.0 software (Tamura et al., 2011) by neighbor-joining (NJ) with the Jukes-Cantor correction. Diversity indices, including Chaol, Shannon, and Simpson, were generated by DOTUR for each clone library (Schloss & Handelsman, 2005). Quantitative PCR was performed on a Bio-Rad iQ5 real-time PCR instrument (Bio-Rad) with a SYBR Green qPCR kit (Quanta).