title: Bacterial consortium in a nitrifying reactor replicate 2
design: The microbial population in the bioreactor was selected for an autotrophic nitrifying population by using ammonium as sole energy source and bicarbonate as carbon source. The culture was obtained from wastewater treatment plant and added to a glass continuous stirred tank reactor (CSTR, 0.14 m diameter, 0.56 m height and 5.5 L working volume). The CSTR was operated continuously under steady state for five years to study and calculate kinetic and stoichiometric parameters through respirometric techniques.  10 ml sub-samples were taken from the bioreactor with a week interval between them and total DNA was extracted with enzymatic lysis using the automated QIAcube method (Qiagen, Venlo, The Netherlands).   Total DNA was used as template for PCR amplification of 16S rRNA gene. Ribosomal libraries of V1-V6 regions were obtained using 10 bp barcoded primers 8–F (5´-AGA GTT TGA TCI TGG CTC A-3´) (Navarro-Noya et al. 2013) and 946–R (5´-CCG TCW ATT KCT TTG AGT T-3´). Each DNA sample was amplified five times, the PCR products were pooled and purified using the UltraClean™ PCR Clean-up kit (MoBio, Carlsbad, CA, USA). Equal concentrations of the purified library amplicons were sent for pyrosequencing. Pyrosequencing was done with a Roche GS–FLX Plus 454 pyrosequencer (Roche, Mannheim, Germany) by Macrogen Inc. (DNA Sequencing Service, Seoul, Korea).