Quantification of the Denitrification Gene nosZ in a Full Scale Wastewater Treatment Plant Using qPCR

Managing the nitrogen cycle is one of the Engineering Grand Challenges identified by the National Academy of Engineering. The most common process for nitrogen removal from wastewater is biological nitrification coupled to denitrification. The goal of this research is to quantify the denitrifying bacteria present over time in a wastewater treatment plant. This can be accomplished using quantitative real-time polymerase chain reaction (qPCR) targeting a specific gene that encodes for denitrification enzymes. The nosZ gene encodes for the enzyme nitrous oxide reductase, which catalyzes the reduction of nitrous oxide to nitrogen gas.Samples of water and activated sludge were taken from the North Cary wastewater treatment plant over an entire cycle of operation. Dissolved oxygen and pH were measured. The nitrate and nitrite concentrations in the water samples were measured. DNA was extracted, and qPCR targeting the nosZ gene was performed to quantify microorganisms capable of denitrification and determine how this changes over the operational cycle. Next, RNA was extracted and reverse transcription polymerase chain reaction (RT-PCR) followed by qPCR targeting the nosZ gene was performed to quantify microorganisms actively denitrifying and to determine how this changes over the operational cycle. This information can give engineers and wastewater treatment plant operators a better understanding of how denitrification genes are turned on and off during cycling of aeration in the basins, which may lead to better operational control of denitrification. In addition, it will allow us to determine the effect of the absence or presence of oxygen on the activity of the organisms; in other words, we will be able to determine exactly when lower oxygen levels switch on specific denitrification genes at a full scale N-removal plant.