Use of qPCR to monitor 2,4-dinitroanisole degrading bacteria in water and soil slurry cultures.

Unlabelled: Prediction and process monitoring during natural attenuation, bioremediation, and biotreatment require effective strategies for detection and enumeration of the responsible bacteria. The use of 2,4-dinitroanisole (DNAN) as a component of insensitive munitions leads to environmental contamination of firing ranges and manufacturing waste streams. Nocardioides sp. strain JS1661 degrades DNAN under aerobic conditions via a pathway involving an unusual DNAN demethylase. We used the deeply branched sequences of DNAN degradation functional genes as a target for development of a molecular method for detection of the bacteria. A qPCR assay was designed for the junction between dnhA and dnhB, the adjacent genes encoding DNAN demethylase. The assay allowed reproducible enumeration of JS1661 during growth in liquid media and soil slurries. Results were consistent with biodegradation of DNAN, accumulation of products, and classical biomass estimates, including most probable number and OD600. The results provide a sensitive and specific molecular method for prediction of degradation potential and process evaluation during degradation of DNAN.

One-sentence Summary: A unique target sequence in functional genes enables the design of a simple and specific qPCR assay for enumeration of aerobic 2,4-dinitroanisole-degrading bacteria in soil and water.