A quantative method for detecting ammonia oxidizing bacteria in coastal aquaculture systems.

Abstract

There is a need to quantify autotrophic nitri¢ers in coastal aquaculture systems for evolving a bioreme diation strategy. Autotrophic nitri¢ers are extremely slow-growing organisms, which cannot be detected by traditional methods as they are notoriously di⁄- cult to culture. Molecular techniques based on func tional genes could be deployed for the detection of nitri¢ers. Ammonia monooxygenase (amoA), that cata lyses the oxidation of ammonia to hydroxylamine in the rate-determining step of nitri¢cation is largely unique to ammonia-oxidizing bacteria (AOB). In the present study, a quantitative real-time polymerase chain reaction assay targeting amoA was developed to estimate AOB population size in coastal soil, ammonia-removing bioaugmentors and the solid matrix. To achieve this objective, di¡erent set of primers and a dual labelled probe have been designed for SYBR Green and TaqMan real-time assays. The abundance of AOB ranged from 104 to 106 order of magnitude in the samples. In the present study, bio¢lm formation of the consortium of nitrifying bac teria onto bagasse has also been quanti¢ed. The re sults demonstrate that the developed method is a rapid and sensitive tool for the quantitative detection of nitrifying bacteria in aquatic and related environ ment. This helps in making the bioremediation ap proach for ammonia removal by immobilization of nitrifying bacteria onto the natural substrate