Abstract
A new enumeration method for nitrifying bacteria was developed using the 2-(p-indophenyl) -3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) dehydrogenase assay with specific inhibitors for ammonia-and nitrite oxidizing bacteria. This technique was fistly applied to artificial mixed cultures of Nitrosomonas curopaea, Nitrobacter winogradskyi and Pseudomonas fluorescens and then to environmental mixed culture samples to evaluate the validity and sensitivity of this method. Detection efficiency of nitrifying bacteria by this method was more than 1 order of magnitude and 1-2 orders of magnitude higher than that of the most probable number (MPN) method for the pure culture samples and environmental mixed culture samples, respectively. Since the INT dehydrogenase assay counts only metabolically active bacteria, the numbers of NH4- and NO2-oxidizing bacteria determined by this method were directly proportional to ammonia and nitrite oxidation rates. Furthermore, this INT dehydrogenase method was applied to biofilm samples for in situ identification of nitrifying bacteria. Fractions of nitrifying bacteria in the biofilm were more than 1-3 orders of magnitude higher than those determined by the MPN method, whereas the fractions were comparable with those determined by the fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. Therefore, it could be summarized that this newly developed INT dehydrogenase method was more rapid, sensitive and reliable over the conventional MPN method for environmental samples and could be applied for in situ identification of nitrifying bacteria in biofilms.
