Environmentally Benign Automated Nitrate Analysis

Nitrate is one of the most widely determined anions in all spheres of agriculture. Economical and environmentally responsible crop production now requires that farmers know the concentration of nitrate in soil-and increasingly, in the sap of developing plants-to determine the amount and timing of fertilizer application to their fields. Nitrate is classified as a primary contaminant under the Clean Water and Safe Drinking Water Acts, and it has been identified as a major water quality problem in many agricultural regions where runoff and drainage from croplands contaminate surface water and groundwater. The demand for nitrate analysis in these regions to support monitoring and remediation activities is therefore likely to increase substantially. Nutrient management programs now under development in many parts of the country to regulate the discharge of wastes from confined animal feeding operations (CAFOs) will further increase the demand for nitrate analysis. This burgeoning demand for agriculturally related nitrate analysis provides the economic incentives to develop faster, cheaper, more environmentally benign reagents and analytical systems for laboratory and on-site nitrate analysis despite the ready availability of time honored and effective nitrate determination methods. Automated methods for nitrate determination, U.S. EPA method 353.2 for example, are widely used in the United States and throughout the world. In such methods, nitrate is reduced to nitrite by small, packed-bed, cadmium reactors. Nitrite is subsequently reacted with Griess reagents-sulfanilamide and N-(1-Naphthyl)ethylenediamine-to form a stable and highly colored product that is ideal for determination by photometry. Cadmium required by these methods, however, is a highly toxic heavy metal. Its use exposes analysts to a potent health hazard, and generates a cadmium contaminated waste stream, which must be collected for proper disposal. Enzymatic nitrate reduction is an attractive alternative to the cadmium-based chemistry. Enzymatic reactions are rapid, extremely selective, and nontoxic. They are safer for the user and for the environment, and can improve lab economy by reducing costs of hazardous waste disposal. NECi has developed reliable nitrate analysis tools based on its proprietary NADH:nitrate reductase (NaR) enzyme, which is isolated from corn seedlings. Our company is also developing new and potentially less expensive NaR reagent tools using genetic engineering and protein expression technologies, under funding from the SBIR program of NIH (Phase II award #R44 GM56598). The goal of this project is to develop manufacturable and cost-competitive reagent systems using NADH:Nitrate Reductase (NaR) as a direct replacement for the cadmium now widely used for nitrate determinations in standard CF (air-segmented continuous flow) and FI (flow injection) methods. Reagent packages using enzyme in solution will be compared to immobilized enzyme reactors. Technical feasibility was demonstrated in Phase I.