Source: THE NITRATE ELIMINATION COMPANY, INC. submitted to
ENZYME-BASED TEST KITS FOR PHOSPHATE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0226062
Grant No.
2011-33610-30778
Project No.
MICW-2011-02354
Proposal No.
2011-02354
Multistate No.
(N/A)
Program Code
8.4
Project Start Date
Sep 1, 2011
Project End Date
Aug 31, 2014
Grant Year
2011
Project Director
Campbell, W. H.
Recipient Organization
THE NITRATE ELIMINATION COMPANY, INC.
334 HECLA STREET
LAKE LINDEN,MI 49945
Performing Department
(N/A)
Non Technical Summary
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.
Animal Health Component
30%
Research Effort Categories
Basic
20%
Applied
30%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110200020%
1020210200020%
1027410100020%
1027410104020%
1027410200020%
Goals / 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.
Project Methods
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.

Progress 09/01/11 to 08/31/14

Outputs
Target Audience: We participated in or attended a number of agricultural nutrient management and analytical chemistry events during the course of this grant. Highlight events include a demonstration/workshop at the Lake Linden High School, for their science club, Feb 2014. This event was organized by MI Technoloogical University. ER Campbell gave a talk on science entrepreneurship at MI Tech for their entrepreneur program in Fall 2013. ERC participated in an event at the American Chemical Society headquarter, in DC, on how to make chemistry less intimidating to consumers. We partnered with our contract packaging company on a booth at PittCon 2014, Chicago, IL, in early March. We showed our nitrate test products in addition to the new reagents for Phosphate and the prototype photometer. We continued our policy of shipping sample kits and reagents to potential customers at no charge. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? NECi always hires one or two college interns for grant projects. The student who worked during Year I of this project is now a biotech production factility designer at an engineering firm. The Year II student, a biomedical engineer, will use his experience to help understand how labs use optics in their work. How have the results been disseminated to communities of interest? Info on the new products developed under this grant will be available on our website. Current activities are targeted at labs - a new line of phosphate reagents for lab use. The results of the phosphate assay are in the UV, not visible to the eye, so a photometer will always be required even for the test kits for agriculture. (Results of the nitrate assay are bright pink, and color density can be estimated with semi-quantitative accuracy without equipment). One marketing partner will assemble a group of key decision makers in the upper Midwest when we have the 3rd generation smartphone-linked photometers ready for beta-testing in early 2015. We'll have a booth at PittCon 2015. ER Campbell will talk about the new phosphate assay at the Environmental Measurement Symposium/NEMC/EPA conference in DC, 3 - 7 Aug 2014. Trade publications and workshops are planned for 2015, starting in time for the Spring 2015 planting season. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goals were either achieved or means to continue toward achievement acquired. All marketing intelligence confirmed that users need to see a device that provides info in digital form that can be entered into other ag software packages. They also want the test reading device - the photometer - to be compelling to hold and use, so we are partnered with a professor at MI Technological University to develop 3D printed models. An NSF STTR, starting 1 July 2014, will get us to this point. The initial device will work with our nitrate and phosphate test kits (dual wavelength). Software that can integrate into ag packages is nearing completion. We'll continue adding functionality to the software so that advice on how to use the data provided by the test kits is right there on the smartphone that displays kit results.

Publications