10 ml mixed liquor were centrifuged (1000 g for 5 min); the supernatant was decanted and the pellet was resuspended in 10 ml sterile TriseEDTA buffer (10.0 mM TriseHCl, 1.0 mM EDTA, pH 8.0). All samples were immediately frozen after resuspension and stored at 80 C until DNA extraction. DNA was extracted from the samples with a QIAamp DNA Mini Kit (Qiagen, Valencia, CA). To minimize variations in DNA extraction, templates used for polymerase chain reactor (PCR) amplification were prepared by mixing the DNA that was extracted in triplicate for each sample.