A quantitative method for detecting ammonia-oxidizing bacteria in coastal aquaculture systems

Abstract

There is a need to quantify autotrophic nitrifiers in coastal aquaculture systems for evolving a bioremediation strategy. Autotrophic nitrifiers are extremely slow-growing organisms, which cannot be detected by traditional methods as they are notoriously difficult to culture. Molecular techniques based on functional genes could be deployed for the detection of nitrifiers. Ammonia monooxygenase (amoA), that catalyses the oxidation of ammonia to hydroxylamine in the rate-determining step of nitrification 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, different 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, biofilm formation of the consortium of nitrifying bacteria onto bagasse has also been quantified. The results demonstrate that the developed method is a rapid and sensitive tool for the quantitative detection of nitrifying bacteria in aquatic and related environment. This helps in making the bioremediation approach for ammonia removal by immobilization of nitrifying bacteria onto the natural substrate.