In situ identification of ammonia-oxidizing bacteria

A 16S rRNA-targeted oligonucleotide probe (NEU) specific for some representatives of the lithoautotrophic ammonia-oxidizing bacteria was developed based on comparative sequence analysis. Whole cell hybridization of target cells in combination with digital microscopy was used to determine the optimal hybridization stringency. In situ hybridization of several activated sludge samples and a trickling filter biofilm with probe NEU allowed the detection of dense cell clusters exclusively in those samples originating from sewage treatment plants with stable nitrification. Scanning confocal laser microscopy revealed that these aggregates were built up by up to 3,000 cells. Specific arrangements of intracytoplasmic membranes detected in these clusters in activated sludge ultrathin sections by transmission electromicroscopy independently confirmed the presence of ammonia-oxidizing bacteria. In activated sludge samples of the animal waste processing treatment plant Kraftisfied up to 20% of total bacteria could be identified in situ as ammonia-oxidizers. The specific activity (k(o)) per in situ detected ammonia-oxidizing cell was calculated to be in the range of 0.00022 ± 0.000045 pmol/cell/hr. Allylthiourea and sodium chlorate were used for specific inhibition of autotrophic ammonia- and nitrite-oxidiziers in samples of the plant Kraftisried. No heterotrophic nitrification was detected. Inhibition of ammonia-oxidizing bacteria over a period of five hours did not result in a measurable decrease of the cellular rRNA content.