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.
1003328
Grant No.
2014-33610-22275
Project No.
MICK-2014-02631
Proposal No.
2014-02631
Multistate No.
(N/A)
Program Code
8.8
Project Start Date
Sep 1, 2014
Project End Date
Aug 31, 2016
Grant Year
2014
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
Whenever biodiesel is made, ten percent of the reaction is the by-product glycerol (also called glycerin). Glycerol has many uses, but it is not a fuel: burning glycerol in any engine will cause irreversible damage. So glycerol must be removed from biodiesel before it can be sold or used. ASTM Intermational, the trade association that sets standards for purity of many industrial products, has set the limit of glycerol in biodiesel at 0.02 percent. There is no wuick and easy way to measure the glycerol content of biodiesel. NECi has identified an enzyme that can do the job. In order to make the test practical, commercial quantities of enzyme of consistently reliable quality is needed. The goal of this project is to develop protein expression processes for making pure and stable enzyme. At the same time, we will develop reagents and simple test kits. Reagents are targeted to biodiesel process development groups and producers. Simple kits for use on site are aimed at small scale producers such as on-farm sites, and fuel testers. The goals will help producers design better fuel production processes, help regulatory personnel monitor fuel QC, and prevent engine damage from partially purified biodiesel. The overall goal is to help make biodiesel a more practical, commercially competitive fuel choice.
Animal Health Component
60%
Research Effort Categories
Basic
10%
Applied
60%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5117410100020%
5111899104050%
4041899200030%
Goals / Objectives
Glycerol is a byproduct of biodiesel production that must be removed from fuel to prevent damage to engines. The goal of this proposal is to develop commercial scale production of the enzyme Glycerol oxidase (GlyOx), which we have shown can be used to measure the glycerol content of crude and finished biodiesel. GlyOx is a large and complex glycosylated protein, making recombinant expression of active enzyme a technical challenge. Assay protocols for glycerol detection were developed in Phase I and will be optimized and validated in Phase II. Reagents and test kits designed for a variety of laboratory instrumentation will be developed. Finally, we will design simplified test kits for use onsite by nonskilled users, patterned on the Company line of test kits for agriculture. There are no quick and accurate methods for glycerol detection on the market today.
Project Methods
The goal of this proposal is to develop commercial scale production of the enzyme Glycerol oxidase (GlyOx), which we have shown can be used to measure the glycerol content of crude and finished biodiesel. GlyOx is a large and complex glycosylated protein, making recombinant expression of active enzyme a technical challenge. Assay protocols for glycerol detection were developed in Phase I and will be optimized and validated in Phase II. Reagents and test kits designed for a variety of laboratory instrumentation will be developed. Finally, we will design simplified test kits for use onsite by nonskilled users, patterned on the Company line of test kits for agriculture. There are no quick and accurate methods for glycerol detection on the market today.The protein expression aspect of the project is challenging. GlyOx hasnever been characterized fully, and there is no known amino acid sequence. Advanced methods in protein expression and purification will be applied, advancing the state of the art in this area. The product development aspects of the project involve analytical chemistry skills and methods.

Progress 09/01/14 to 08/31/16

Outputs
Target Audience:The goals of the project were express a recombinant enzyme for use in development of an assay for quantitation of glycerol in biodiesel. There were numerous targets, but the main set has remained the biofuel industry and companies who sell analytical products into this industry. Hobbyists, agricultural producers making their own fuel from waste materials, and academic aspects of alternative fuel production are additional targets. Changes/Problems:As the project progressed, we determined that a different enzyme was better suited to the goals of the project. We expressed this enzyme and developed reagents and kits with the required capabilties to detect glycerol in the difficult matrices of raw and finished biodiesel and in fermentation media. What opportunities for training and professional development has the project provided?This is an SBIR project, so these are not defined goals of this grant. However, NECi takes pride in training interns and employees in advanced techniques in protein expression, analytical chemistry, and lab practices in general. How have the results been disseminated to communities of interest?The initial public presentation was to the analytical chemistry community at The Pittsburgh Conference, March 2016 (pittcon.org). We have made presentations to three commercialization partners with customers in the biofuel community. We plan to submit articles for trade publications now that the project is finalized. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impacts: Recombinant enzyme, reagents, kits, and a device for reading test results on site were developed under this Grant. At the end of the grant period, biodiesel production was at an ebb due to reduced costs for conventional fuel. However, biodiesel remains a goal of the US military and is an important fuel internationally. We have one company which has tested and qualified the new enzyme for use in their own products for glycerol testing, and a scientific supply catalog company with plans to introduce these reagents in India in early 2017. Additionally, we developed other applications for this enzyme: many biomedical products are produced by fermentation and use glycerol as a feedstock for microbial growth. Monitoring the glycerol content of the fermentation media is critical to yield of medical product. Finally, the additional company goal of making science - and chemistry - accessible and interesting to a broader range of users has been achieved by development of a handheld device (a photometer) that pairs with Smartphones to read and record test results.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Podium presentation, NEMC2016 Complete PowerPoint presentation available at conference website: nemc.us
  • Type: Websites Status: Other Year Published: 2016 Citation: nitrate.com has complete info on USDA support.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster, PittCon 2016 Abstract available at pittcon.org


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:Target audience for this SBIR project is biodiesel producers, including small installations producing fuel for their own use. Efforts for Year 1 of the project concentrated on R&D. We maintain contact with large and small producers to keep in touch with their needs and potential applications of our technology. We also talk to the equipment manufacturers who would incorporate our reagents into their instruments so that they can increase their penetration into the biofuels area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?NECi has always employed at least one college or area high school student in order to provide real world biotech research experience to interested undergrads. For this project, we trained Kayla Warko, chemical engineering student, in analytical method devevopment. Kayla graduated in May 2015. Starting in June 2015, we hired Hayden Henderson, environmental engineering junior. He has assisted in assay development and photometer validation (for nitrate and phosphate). He will return for his senior year, following a 3-week stint in Panama with Engineers Without Borders. Sabrina Swanson is between high school and college and is working as a general lab assistant. Half of her efforts are dedicated to this project; the other half to more business-related activities. How have the results been disseminated to communities of interest?We've maintained contact and discussions with potential customers for successful glycerol detection products throughout the Year I project. We have not engaged in public outreach efforts on biofuel-related work, concentrating on nitrate and phosphate products in 2014 and 2015. We're making plans for participation at one or more biofuel events in 2015 or 2016. What do you plan to do during the next reporting period to accomplish the goals?Aside from continuing efforts as described in the proposal and Interim report, we are making progress in development of manufacturing methods for the handheld photometer. This will be based on "distributed manufacturing", that is making the case and optical bench of the devices by 3D printing (modified from the PLOS ONE paprt), with electronics assembly done in house by our computer and coder technicians. This way we can start test marketing the devices without huge upfront capital requirements.

Impacts
What was accomplished under these goals? The objective of this project is to develop new tools for biodiesel process monitoring, quality control, and quality analysis via the application of biotechnology. When biodiesel is produced from any feedstock, approximately 10% of the resulting product will be glycerol (glycerin). Glycerol must be removed from the fuel to avoid irreversible engine damage. ASTM International has set the limit for glycerol in biodiesel at 0.020%. Existing technology for detection of glycerol in biodiesel requires expensive equipment or, for small and medium producers, requires samples to be sent to specialized analytical chemistry labs. Small and large producers contacted in preparing the original proposal agreed that more analytical options are needed to help biodiesel become a viable, cost-effective alternative fuel. NECi Superior Enzymes is developing a series of tools based on the enzyme Glycerol Oxidase, produced by recombinant protein expression. The enzyme (GlyOx) itself will be sold to other companies for incorporation into online monitoring equipment; for example, Xylem, Inc. provided a Letter of Support for this project and will be the first customer. We are also developing simplified test kits that any producer can use to monitor processes and perform onsite quality control. The kits pair with NECi's proprietary handheld photometer, whose first iteration was funded by a Phase I STTR award from NSF. A product line parallel to our nitrate test products will be developed over time. NECi Superior Enzyme's nitrate test line can be viewed at our website, nitrate.com. An example of a simplified test kit can be seen in our Open Access publication on development of test kits for phosphate here: http://www.sciencedirect.com/science/article/pii/S2215016115000266 The Open Source version of our Photometer, for nitrate detection, is described here: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134989 Modification of this device for glycerol detection is one of the Objectives of this Phase II project. The glycerol assay itself was developed in Phase I, and included confirmation that the native GlyOx enzyme can indeed detect glycerol in raw biodiesel samples; we obtained biodiesel samples at varying stages of processing from a small producer in Michigan. We have therefore concentrated on the recombinant protein expression Objective in Year 1; progress is described below. We've also begun the modification of the photometer for detection of assay results.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Powerpoint available at nemc.us
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: http://www.sciencedirect.com/science/article/pii/S2215016115000266