Combination Analysis of Reverse Transcription (RT) -PCR and DGGE to Monitor Expression of Ammonia Monooxygenase Gene in Domestic Wastewater Treatment Process

Abstract

Expression of ammonia monooxygenase (amoA) gene in domestic wastewater treatment process was monitored by combination analysis of reverse transcription (RT) -PCR and denaturing gradient gel electrophoresis (DGGE) . Sludge samples were collected from a bench-scale reactor treating synthetic domestic wastewater. After total RNA was extracted, RT-PCR was performed. DGGE analysis was used to separate the amplified products according to their sequence and thereby to visualize individual community members. Ammonia oxidizers that were demonstrated by the presence of amoA mRNA were regarded as metabolically active ones. The DGGE profiles of amoA mRNA RT-PCR products obtained at the initial stage of the operation showed decrease of the diversity of amoA mRNA. When influent load was changed with reduction of hydraulic retention time (HRT) or increase of concentration of wastewater, new DGGE bands newly appeared on only the short HRT condition. The amoA sequences of DGGE bands disappeared in the beginning of the operation affiliated to the Nitrosomonas eutropha lineage. On the other hand, all the amoA sequences of new DGGE bands affiliated to the genus Nitrosomonas, and formed phylogenetic lineage for which no cultured representative exists.