Nitrification in sequencing biofilm batch reactors: Lessons from molecularapproaches

The nitrifying microbial diversity and population structure of a sequencing biofilm batch reactor receiving sewage with high ammonia and salt concentr ations (SBBR 1) was analyzed by the full-cycle rRNA approach. The diversity of ammonia-oxidizers in this reactor was additionally investigated using c omparative sequence analysis of a gene fragment of the ammonia monooxygenas e (amoA), which represents a key enzyme of all ammonia-oxidizers. Despite t he "extreme" conditions in the reactor, a surprisingly high diversity of am monia- and nitrite-oxidizers was observed to occur within the biofilm. In a ddition, molecular evidence for the existence of novel ammonia-oxidizers is presented. Quantification of ammonia- and nitrite-oxidizers in the biofilm by Fluorescent In situ Hybridization (FISH) and digital image analysis rev ealed that ammonia-oxidizers occurred in higher cell numbers and occupied a considerably larger share of the total biovolume than nitrite-oxidizing ba cteria. In addition, ammonia oxidation rates per cell were calculated for d ifferent WWTPs following the quantification of ammonia-oxidizers by competi tive PCR of an amoA gene fragment. The morphology of nitrite-oxidizing, unc ulturable Nitrospira-like bacteria was studied using FISH, confocal laser s canning microscopy (CLSM) and three-dimensional visualization. Thereby, a c omplex network of microchannels and cavities was detected within microcolon ies of Nitrospira-like bacteria. Microautoradiography combined with FISH was applied to investigate the abil ity of these organisms to use CO2 as carbon source and to take up other org anic substrates under varying conditions. Implications of the obtained resu lts for fundamental understanding of the microbial ecology of nitrifiers as well as for future improvement of nutrient removal in wastewater treatment plants (WWTPs) are discussed.