Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR

Abstract

Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results, the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities.