Occurrence of nitrifiers and diversity of ammonia-oxidizing bacteria in developing drinking water biofilms

We studied the population dynamics of nitrifying bacteria during the development of biofilms up to 233 or 280 days on polyvinylchloride pipes connected to two full-scale drinking water distribution networks supplying processed and chloraminated surface water. The numbers of nitrifiers in biofilms were enumerated at intervals of 10–64 days by the most probable number (MPN) method at waterworks and at several study sites in distribution network areas. The numbers of nitrifiers increased towards the distal sites. The highest detected MPN counts of ammonia-oxidizing bacteria (AOB) for study areas 1 and 7 were 500 MPN cm⁻² and 1.0×10⁶ MPN cm⁻², and those of nitrite-oxidizing bacteria (NOB) 96 MPN cm⁻² and 2.2×10³ MPN cm⁻², respectively. The diversity of AOB was determined by PCR amplifying, cloning and sequencing the partial ammonia monooxygenase (amoA) gene of selected biofilm samples presenting different biofilm ages. The PCR primers used, A189 and A682, also amplified a fragment of particulate methane monooxygenase (pmoA) gene of methane-oxidizing bacteria. The majority of biofilm clones (24 out of 30 studied) contained Nitrosomonas amoA-like sequences. There were only two pmoA-like sequences of Type I methanotrophs, and four sequences positioned in amoA/pmoA sequence groups of uncultured bacteria. From both study area very similar or even completely identical Nitrosomonas amoA-like sequences were obtained despite of high difference in AOB numbers. The results show that the conditions in newly formed biofilms in drinking water distribution systems favor the growth of Nitrosomonas-type AOB.