Community Analysis of Ammonia Oxidizing Bacteria through Molecular Genetics in Activated Sludge of Effluent Treatment Plant

Abstract

We investigated the communities of Ammonia-Oxidizing Bacteria (AOB) in activated sludge using Polymerase Chain Reaction (PCR) followed by Terminal Restriction Fragment Length Polymorphism (T-RFLP), cloning, and sequencing of the alpha-subunit of the ammonia monooxygenase gene (amoA). In this study the techniques of specific amplification of ammonia oxidiser 16S rDNA fragments by PCR, separation of mixed PCR samples by Denaturing Gradient Gel Electrophoresis (DGGE), and band identification by specific hybridization with oligonucleotide probes were combined to allow for the comparison of the community composition of multiple samples over space and time. DGGE bands of interest were also excised for DNA isolation, reamplification, sequence determination and phylogenetic analysis. We compared monthly samples by the emergent macrophyte Glyceria maxima to determine the seasonal effects that the plant roots and the oxygen availability might have on the β-subgroup ammoniaoxidiser populations present. Similarly, five soil or sediment samples, varying in oxygen availability, from different locations were compared. Although the presence of two previously defined Nitrosospira sequence clusters could be differentially detected in the samples examined, there was no evidence for a particular group which was specific to periodically anoxic environments.