Optimization of biomolecular techniques for detection of nitrite-oxidizing bacteria

Abstract: Nitrification is a natural occurring, oxidative process which is essential for plants´ ability to take up nitrogen in the form of nitrate. The oxidation is divided into two steps. First ammonia is oxidized to nitrite by ammonia-oxidizing bacteria (AOB) or archaea (AOA) and then the nitrite is further oxidized to nitrate by nitrite-oxidizing bacteria (NOB). The enzyme used by NOB for the oxidation is nitrite oxidoreductase (nxr). One of few bacteria that catalyze this reaction is Nitrobacter sp. The purpose of this study has been to optimize the detection of Nitrobacter in samples of activated sludge from municipal wastewater treatment plants (WWTPs) in Eskilstuna and Västerås (Sweden). This was done by PCR, cloning and sequencing. Primers used were nor F/nor R that are specific for the functional gene encoding nxr. This optimization has been compared to a different PCR-system where nor F/nor R were exchanged for another primerpair consisting of a 16S rDNA-primer (NIT3), which was specific for Nitrobacter and a universal 16S-primer (U2, Rit388). In addition to this, a semi quantitative analyze has also been conducted. The result of the study was two PCR-programmes, one optimized for each set of sludge samples. The quantitative analysis showed that the concentration Nitrobacter in the sludge samples was approximately the same as a pure culture, which was used as a positive control and contained ~104 CFU/ml. Cloning and sequencing revealed the presence of 3 different Nitrobacter. Surprisingly half of the clones from one of the Västerås samples, taken in December, were most likely Methylibium petroleiphilum. The matter of fact that we were able to detect this bacterium with primers specifically designed for Nitrobacter made this discovery even more interesting. With NIT3/U2 Methylocella sp. was also detected in samples from Västerås, which confirmed the presence of methylotrophic bacteria in the Västerås samples.