Source: NECi: The Nitrate Elimination Company, Inc submitted to
GLYCEROL DETERMINATION IN BIODIESEL FUELS USING GLYCEROL OXIDASE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0233776
Grant No.
2013-33610-20904
Project No.
MICW-2013-00092
Proposal No.
2013-00092
Multistate No.
(N/A)
Program Code
8.8
Project Start Date
Jul 15, 2013
Project End Date
Mar 14, 2014
Grant Year
2013
Project Director
Campbell, W. H.
Recipient Organization
NECi: The Nitrate Elimination Company, Inc
334 Hecla Street
Lake Linden,MI 49945
Performing Department
(N/A)
Non Technical Summary
Glycerol, also called glycerin(e), is a byproduct or contaminant of biofuel production. Glycerol must be removed from biofuels before they can be burned in order to avoid damage to combustion engines. The standard for biodiesel is less than 0.02% by weight (ASTM D6751; ASTM International sets voluntary consensus standards for quality in many industry sectors). Standard methods for glycerol determination require skilled operators, complex sample preparation, and expensive lab equipment. Biofuel producers need facile and inexpensive methods for glycerol detection, especially in biodiesel. NECi has identified an unusual fungal enzyme, glycerol oxidase, and demonstrated that it is capable of glycerol determination in raw biodiesel at varying production stages. The Phase I project focuses on assay development and improved production/purification of the native enzyme. Prototype test kits and reagents for high-throughput labs based on the purified native enzyme will be developed in Phase I. Recombinant Glycerol Oxidase enzyme is the key target product of the Phase I/II project. Test kits based on the new enzyme will in incorporated into NECis test kit product line and marketed directly to biofuels producers. Enzyme reagent packages will be developed for autoanalyzer equipment in collaboration with the manufacturers.
Animal Health Component
50%
Research Effort Categories
Basic
20%
Applied
50%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5117410100050%
5117410104050%
Goals / Objectives
Glycerol, also called glycerin(e), is a byproduct or contaminant of biofuel production. Glycerol must be removed from biofuels before they can burned in order to avoid damage to combustion engines. The standard for biodiesel is less than 0.02% by weight (ASTM D6751; ASTM International sets voluntary consensus standards for quality in many industry sectors). Standard methods for glycerol determination require skilled operators, complex sample preparation, and expensive lab equipment; e.g. ASTM D6584-09, glycerol by gas chromatography. Several of NECis current customers are engaged in some aspect of biofuel production, from catalyst development to process monitoring to systems for growth of marine algae. These customers need facile and inexpensive methods for glycerol detection, especially in biodiesel. NECi has identified an unusual fungal enzyme, glycerol oxidase, and demonstrated that it is capable of glycerol determination in raw biodiesel at varying production stages. The Phase I project will focus on assay development and improved production/purification of native enzyme. Recombinant Glycerol oxidase enzyme is the key target product of the Phase I/II project. Test kits based on the new enzyme will in incorporated into NECis test kit product line and marketed directly to biofuels producers. Enzyme reagent packages will be developed for autoanalyzer equipment in collaboration with the manufacturers.
Project Methods
The project is an application of advanced protein purification methods for production of an enzyme, Glycerol Oxidase, to be used as a tool for analytical chemistry. Glycerol Oxidase (GlyOx) is a complex protein that has not been fully studied in the technical literature. GlyOx is rare, and difficult to purify in stable form. Advanced techniques in biochemistry and protein production and characterization are the initial methods of interest in the initial stages of the project. As material is produced, development of analytical methods using the purified protein will come into play. The GlyOx-based glycerol assay will be tested in-house on biofuel process samples obtained from biofuel processors and other potential customers. Biochemistry and analytical chemistry methods are keys to the Phase I project. Molecular biology and other biotechnology techniques (e.g. cloning and expression of GlyOx) will be used in Phase II.

Progress 07/15/13 to 03/14/14

Outputs
Target Audience: This is a Phase I project, so there was little opportunity for outreach over the period. We continued our relationship with YSI/Xylem, Inc, who will be an initial customer for the Glycerol Oxidase reagent itself. We've kept in touch wuth biodiesel producers who have expressed interest in glycerol test kits. We presented the glycerol test kits as an upcoming new product at PittCon 2014 (The Pittsburgh Conference, Chicago, IL 2 - 6 March). PittCon is the trade show for analytical chemistry; we shared a booth with our contract packaging company. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? NECi and our molecular biology technician, MSc J L Ladd, was trained in new techniques in protein identification from DNA gel analyses. We also trained two undergraduate students from Michigan Technological University in bioanalytical techniques and protein expression and purification. Michael Briseno is pursuing a Biomedical Engineering degree with emphasis on optics. He will return in Phase II as we develop a proprietary photometer to sell with our test kits. Kayla Warsko is a Chemical Engineering student with interest and experience in biofuel production. NECi was able to fund additional hours for these students due to a 1:1 matching grant program. Through the Michigan Corporate Relations Network (MCRN), and with funds provided by the Michigan Economic Development Corporation, the Office of Innovation and Industry Engagement at Michigan Technological University has access to a program targeted at helping small- to mid-sized Michigan companies grow. Through the Small Company Internship Award (SCIA) program, qualified companies can access student talent to help move a project, product or service forward. Through the Michigan Corporate Relations Network (MCRN), and with funds provided by the Michigan Economic Development Corporation, the Office of Innovation and Industry Engagement at Michigan Technological University has access to a program targeted at helping small- to mid-sized Michigan companies grow. Through the Small Company Internship Award (SCIA) program, qualified companies can access student talent to help move a project, product or service forward. How have the results been disseminated to communities of interest? We have kept in touch with the commercialization partners who continue to survive the tumultuous biofuels market. We engaged with new potential customers in the course of preparing the Phase II proposal. And we talked about enzyme-based glycerol testing to the analytical chemistry community from our booth at PittCon 2014, Chicago, IL 2 - 6 March. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Phase I project went well and close to its planned schedule. No major obstacles have been encountered to date.

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