On the Occurrence of Anoxic Microniches, Denitrification, and Sulfate Reduction in Aerated Activated Sludge

A combination of different methods was applied to investigate the occurrence of anaerobic processes in aerated activated sludge. Microsensor measurements (O₂, NO₂⁻, NO₃⁻, and H₂S) were performed on single sludge flocs to detect anoxic niches, nitrate reduction, or sulfate reduction on a microscale. Incubations of activated sludge with ¹⁵NO₃⁻ and ³⁵SO₄²⁻ were used to determine denitrification and sulfate reduction rates on a batch scale. In four of six investigated sludges, no anoxic zones developed during aeration, and consequently denitrification rates were very low. However, in two sludges anoxia in flocs coincided with significant denitrification rates. Sulfate reduction could not be detected in any sludge in either the microsensor or the batch investigation, not even under short-term anoxic conditions. In contrast, the presence of sulfate-reducing bacteria was shown by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes and by PCR-based detection of genes coding for the dissimilatory sulfite reductase. A possible explanation for the absence of anoxia even in most of the larger flocs might be that oxygen transport is not only diffusional but enhanced by advection, i.e., facilitated by flow through pores and channels. This possibility is suggested by the irregularity of some oxygen profiles and by confocal laser scanning microscopy of the three-dimensional floc structures, which showed that flocs from the two sludges in which anoxic zones were found were apparently denser than flocs from the other sludges.