Exploited Application of Denaturing Gradient Gel Electrophoresis in Analysis of Ammonia Oxidizing Bacterial Community Structure

Abstract

The aim of the present study was to investigate the diversity of the ammonia-oxidizing bacterial population by identifying autochthonous bacteria from allochthonous ammonia-oxidizing bacteria from the WWTP effluents. Measurements of potential nitrifying activity, competitive PCR, and denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments specific to ammonia-oxidizing bacteria (AOB) were used to explore the succession and shifts of the ammonia-oxidizing community. The CTO primers used in this study were shown not to be completely specific to AOB of the s subclass of Proteobacteria. We further demonstrated that when DGGE patterns are interpreted, all the different bands must be sequenced, as one major DGGE band proved to be affiliated with a group of non-AOB in the s subclass of Proteobacteria. The majority of AOB (75 to 90%) present in the Amla Khadi river downstream of the effluent output belong to lineage 6a, represented by Nitrosomonas oligotropha- and Nitrosomonas ureae-like bacteria. This dominant lineage was represented by three bands on the DGGE gel. The major lineage-6a AOB species, introduced by the WWTP effluents, survived and might have grown in the receiving medium far downstream, in the estuary; it represented about 40% of the whole AOB population. The other two species belonging to lineage 6a seem to be autochthonous bacteria. One of them developed a few kilometers downstream of the WWTP effluent input in an ammonia-enriched environment, and the other appeared in the freshwater part of the estuary and was apparently more adapted to estuarine conditions. The rest of the AOB population was represented in equal proportions by Nitrosospira- and Nitrosococcus mobilis-like species.