Nutrient management is a touchy subject: there has to be a balance between regulatory concerns and adequate crop nutrition. Phosphate is an essential nutrient for plant productivity but also a major pollutant when applied in excess. On-the-spot testing for phosphate as orthophosphate (available, soluble, or reactive P) is an appropriate solution to give producers and their technical service providers (Extension agents, crop consultants, and farm managers) better control of the use of phosphate fertilizer in the field. NECi, The Nitrate Elimination Company, Inc., has developed a series of enzyme-based, environmentally benign, and reliable nitrate test kits with the assistance of USDA SBIR funding. NECi Ag-NTKs (Nitrate Test Kits for Agriculture) and lab reagents are being adopted by producers, consultants, and agribusiness. We now propose to develop a similar line of test kits for detection of Phosphate. Compliance with nutrient management regulations will be less burdensome when practical, affordable tools are readily available. The photometer (color reader) being perfected in Phase II is designed to work with both nitrate and phosphate test kits. The photometer will provide data on the N and P status of soils, runoff, feed, and crops. This information can be used for making timely, knowledge-based nutrient application decisions with an eye to maximizing profit and yield while staying in compliance. NECi's develops products that enable and encourage testing as an aid to conservation of rural quality of life. In a different context, NECi is deeply committed to Green Chemistry. Many of the methods routinely used by analytical chemists in the lab or out in the field require the use of hazardous or toxic reagents. Simplified methods designed for non-skilled operators tend to lack the accuracy, sensitivity, and selectivity required in order for the information gathered to be relevant and useful. Enzyme-based methods can satisfy both sets of users as the variability and instability characteristics of enzymes are solved though the use of recombinant protein expression and state of the art stabilization techniques. OBJECTIVES: Nutrient management is a touchy subject: there has to be a balance between regulatory concerns and adequate crop nutrition. Phosphate is an essential nutrient for plant productivity but also a major pollutant when applied in excess. On-the-spot testing for phosphate as orthophosphate (available, soluble, or reactive P) is an appropriate solution to give producers and their technical service providers (Extension agents, crop consultants, and farm managers) better control of the use of phosphate fertilizer in the field. NECi, The Nitrate Elimination Company, Inc., has developed a series of enzyme-based, environmentally benign, and reliable nitrate test kits with the assistance of USDA SBIR funding. NECi Ag-NTKs (Nitrate Test Kits for Agriculture) and lab reagents are being adopted by producers, consultants, and agribusiness. We now propose to develop a similar line of test kits for detection of Phosphate. Compliance with nutrient management regulations will be less burdensome when practical, affordable tools are readily available. Field-useable test kits for available P complement NECi's kits for available N. The photometer (color reader) being perfected in Phase II is designed to work with both test kits. The photometer will provide data on the N and P status of soils, runoff, feed, and crops. This information can be used for making timely, knowledge-based nutrient application decisions with an eye to maximizing profit and yield while staying in compliance. Reagentless sensors are the long term goal. NECi's develops products that enable and encourage testing as aids in conservation of rural quality of life. In a different context, NECi is deeply committed to Green Chemistry. Many of the methods routinely used by analytical chemists in the lab or out in the field require the use of hazardous or toxic reagents. Simplified methods designed for non-skilled operators tend to lack the accuracy, sensitivity, and selectivity required in order for the information gathered to be relevant and useful. Enzyme-based methods can satisfy both sets of users as the variability and instability characteristics of enzymes are solved though the use of recombinant protein expression and state of the art stabilization techniques. NECi is working to develop and validate enzyme-based analytical chemistry through methods approval agencies such as the US EPA, the US Geological Survey, and ASTM. APPROACH: The goal of this Phase II Project is to develop a simple, reliable field test for available phosphate. When combined with a dual-wavelength hand-held portable photometer and the previously developed and implemented NECi Nitrate Test Kit, the new NECi Phosphate Test Kit will provide the end-user with easy-to-use and accurate methods for both nitrate and phosphate. NECi will develop manufacturing systems for recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1) to deploy in the NECi Field Phosphate Test Kit (PTK), which was shown to be feasible in Phase I. First, we will optimize production of recombinant Ecoli-PNP-1 cell line from the Phase I Project, in Escherichia coli utilizing a fermenter system. Next we optimize the cell disruption/enzyme extraction method and perfect the one-step enzyme purification. The optimum storage conditions for stability of PNP will be determined and long-term stability analyzed. The reagents for the field PTK will be optimized for maximum ease of use. The project will develop a handheld, portable NECi Dual Wavelength Photometer for NECi Field Nitrate and Phosphate Test Kits. In cooperation with Dr. David Coates, we will develop a dual wavelength LED handheld portable photometer for qualifying results of either NECi Nitrate Test Kit (NTK) or NECi Phosphate Test Kit (PTK) with the same instrument. Algorithms will be developed for conversion of raw data to concentrations of nitrate and phosphate in Ag samples. The analyte units reported will be familiar to Ag users. Data will be transferable via a USB port to laptop PCs or other devices such as "Smart Phones". Test kits and photometers will be evaluated in the field to demonstrate that the NECi Phosphate Test Kit (PTK) when employed with the NECi Dual Wavelength Photometer will produce accurate and precise results with real world Ag samples. Field testing of the PTK and photometer will be carried out in cooperation with technical service providers and other collaborators.
