Nucleic acid extraction and in vitro transcription. Two sets of mixed liquor samples (14 ml each sample) were centrifuged at 3,200 g for 5 min and stored at 80°C for RNA and DNA extractions. RNA was extracted using a modified low-pH hot-phenol extraction procedure (38). DNA was extracted using a PowerSoil DNA isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA) according to the manufacturer’s instructions. Nucleic acids concentrations were measured
spectrophotometrically.
Since pure cultures of Nitrospira were not available, in vitro-transcribed 16S rRNA was used as reference rRNA in membrane hybridizations as previously described (30).