When biodiesel is made - from any feedstock - approximately 10% of the product will be glycerol (also called glycerin). Glycerol has many uses, but it destroys engines because it's a sugar alcohol, and therefore must be removed from biodiesel before it can be used or sold as fuel. ASTM International has set a limit of 0.02% glycerol in biofuels (ASTM Standard D6751). ASTM's standard analytical chemistry method for glycerol detection in biodiesel is based on gas chromatography (GC). This is not a barrier for large producers with well-equipped labs and trained technicians, but nonetheless has limitations that biofuel producers must work around, such as being excessively complicated for production personnel and inaccuracy in certain important circumstances. This is assuming that producers are equipped with a GC and trained personnel to operate it, which most producers are not. Challenges of current testing solutions have been identified in the biodiesel production market:

Many samples are shipped far away to a lab for glycerol and other analysis, with a wait time of one week.

The time in delay in QA is a barrier to process development; it is difficult to make timely decisions for method improvement if real-time data isn't available.

A quick and accurate means to monitor glycerol content on-site could benefit producers of any size, according to the editor of an online biofuels newsletter. "A 3% increase in efficiency would translate into significant improvements in margins."

QA Manager for a biodiesel producer in Colorado states that their current and rather sophisticated testing system gives an accurate reading when free glycerin is over tolerance. He sees cost and efficiency benefits if they accurately determine glycerol levels before spending time and energy returning their product through the distillation process again.

This enzyme is unique; there is nothing comparable on the market anywhere. Anything that improves processing, QA, or QC will help the growing biodiesel industry flourish. Test kits for onsite use will help producers of any size improve process timings and conditions. Reagents for higher end analytical instruments will make this possible for small and medium scale biofuel producers to perform some of their own testing on-site, saving time and testing costs. This enzyme will result in faster, better, and cheaper results - especially for on-site production spot checks.

This enzyme-based glycerol analysis method will benefit biodiesel producers of all sizes, from individual farm-based operations to large producers with their own QA/QC laboratories. We envision that this method will also be employed at commercial laboratories performing multiple analysis per day for contract analysis.

Producing an enzyme-based chemistry method for the biodiesel industry is in line with NECi Superior Enzymes' priorities and values. We believe in benefiting rural communities that thrive on agriculture, and we plan to do so by helping them become more self-sufficient and sustainable. Biofuels are produced using agricultural waste, used cooking oil, and animal fats which can be converted into sustainable fuel that will be used for local transportation. This limits fossil fuel consumption by providing a fuel that minimizes bio-waste, all while keeping production within the communities. For example, a small biofuel producer we've interviewed for this project takes waste from local restaurants and processes the used cooking oil to produce biodiesel which fuels the local K-12 school buses. This is a self-sustaining cycle which we hope to benefit by making biodiesel production more efficient and accessible with our enzyme-based glycerol test method.

During a USDA Phase I grant, our researchers determined that the native form of this enzyme can quantify glycerol in biodiesel samples during all steps of production. Since native enzymes aren't stable and reproducible, our scientists successfully developed and produced a recombinant form of this enzyme. Recombinant enzymes are stable and are reproducible for lot-to-lot consistency.

Our research team has identified an ideal clone of the enzyme, manufactured in Pichia pastoris, after many growth trials. This process involves examining stability, growth and purification efficiency, and specific enzyme properties. The enzyme has been successfully purified and demonstrates the superior qualities of NECi's commercially available enzymes. Assay development, stability studies, production improvements, and market research are currently underway. The photometer development team will then optimize our handheld device for compatibility with the enzyme-based glycerol test kits.



This project was prompted by feedback from the Michigan Department of Agriculture & Rural Development (MDARD). Ethanol has been added as an octane enhancer in some Michigan gasoline for some time, it increases the octane almost 3 Anti Knock Index points. If gasoline contains ethanol, there is a limit of 10% of total volume for conventional fuels. [1] Around 13 billion gallons of fuel ethanol were added to motor gasoline produced in the United States in 2014.[2] Most gasoline in the U.S. contains up to 10% ethanol by volume (E10), also known as the "blend wall", which all gasoline vehicles can use. Gasoline containing 15% ethanol can only be used by light-duty vehicles with a model year 2001 or newer, and only flex-fuel vehicles can use gasoline with greater than 15% ethanol.

Congress has mandated increasing volumes of ethanol to be added to U.S. fuel to improve U.S. energy independence and also to support a move away from fossil fuels. In addition to these initiatives, more ethanol production from bio-stock in the U.S. means an increase in jobs for U.S. citizens and lower transportation costs since it's produced and used within the country and limits the need for overseas shipping. Ethanol is considered to be greener than gasoline, because corn and other plants absorb carbon dioxide from the atmosphere as they grow. The fuel still releases CO2 when it burns, but the net increase is lower.

There are cons, however, to using ethanol and ethanol blends as a domestic fuel source. Ethanol has a lower energy content than gasoline and therefore delivers less power when burned. This converts to higher fuel consumption and less miles per gallon in a conventional vehicle. Therefore, it is becoming increasingly important to monitor ethanol levels in fuel blends for Quality Assurance and Quality Control measures.

Another market to note when mentioning QA/QC ethanol testing is the alcoholic beverage industry. Alcoholic beverages are made by converting sugars to alcohol through a process called fermentation. Extracts from grapes, barley, and more are fermented, which converts the naturally occurring glucose to ethanol. Monitoring ethanol content is essential during the fermentation process and concentration can affect taste, microbial activity, solubility of other constituents, and is also important for federal and state labeling statutes.

Most ethanol test kits for on-site analysis of gasoline use densitometry measurement, which are rudimentary disposable test tubes that measure ethanol by the difference in density between ethanol and water. This method requires careful calibration and an eyeball's guess using units printed or marked on the side of the tubes. Considering these circumstances brings a reliability and accuracy concern into question. With an increasing interest and importance in determining ethanol levels in conventional fuels and biofuels, more reliable and accurate test methods need to be employed on-site and in analytical laboratories.

Current quantification methods for ethanol in the alcoholic beverage industry include gavimetric analysis, ebulliometry, HPLC, FTIR, gas chromatography, and IR. Many of these methods described require a large investment in special equipment or rely on methods which are outdated, inaccurate, or not reliable. Many times, distilleries, breweries, and wineries do not have their own analytical laboratories, and therefore must send their samples to an external laboratory and wait for results, which can be very costly. Offering a colorimetric, on-site, easy to use, and cost-effective test kit to these beverage producers will allow them to make more timely decisions and save by doing these testing procedures internally.

An optimal clone of the enzyme has been chosen and successfully produced in our lab. We've completed numerous assays with ethanol standards, and have shown the enzyme to be effective in accurately quantitatively measuring ethanol concentration. We're testing the enzyme's compatibility with real-world samples, including various alcoholic beverage samples and plan to begin testing fuel samples soon.

What else is on the docket? We're putting our enzymes through the wringer, which includes checking for potential interferences, optimizing test kit ranges, enzyme stability for storage and use, and working temperatures. We're also gathering input from the fuel and alcoholic beverage industries and researching other potential uses for this enzyme. Our photometer team will soon be adding in a function to the software which will enable users to spot-check ethanol with a digital readout on mobile devices, and the ability to track and store data, then export it for further analysis. Have suggestions, questions, or comments regarding this project? Please contact us!



In 1998, Dr. Bill Campbell and his team published a peer-reviewed journal article in Analytical Chemistry on the construction and characterization of Nitrate Reductase-Based Amperometric Electrode and Nitrate Assay of Fertilizers and Drinking Water. This publication set the stage for the nitrate biosensor project. In this publication, we found that nitrate reductase was indeed capable of being employed in an electrode based system and assays conducted with the electrode compared well with colorimetric and potentiometric assays of the same samples.

In 2012, Nicolas Plumere, Jorg Henig, and Dr. Campbell published another peer-reviewed article in Analytical Chemistry on an Enzyme-Catalyzed O2 Removal System for Electrochemical Analysis under Ambient Air. This technology eliminates the problem of oxygen interference with an electrochemical Nitrate Reductase catalyzed Nitrate Biosensor. Dr. Bill Campbell and his wife, Ellen R. Campbell submitted a Phase 1 USDA Proposal for funding research to develop the Nitrate Biosensor which would employ the oxygen removal system.

During our funded project from NSF, we collaborated with Dr. Joshua Pearce and his team at Michigan Technological University to develop an open-source, handheld, spectrophotometer. This device improves the utility of NECi's on-site Nitrate Test Kits, and sends data directly to mobile devices via Bluetooth interface where it can be geographically tracked, stored, and exported as a CSV file for further analysis. When beta-testing of these devices was completed during a pilot project with a major agricultural company, feedback was that the enzyme-based test kits simply weren't fast or easy enough, and they wanted results within seconds. Our solution to this was to develop a Nitrate Biosensor that would provide users with data within minutes on-site using enzyme based analytical chemistry technology. The concept for the biosensors was inspired by the design of glucose meter for diabetes point-of-care management, using interchangeable "test strips". Ideally, the Nitrate Biosensor will be a two component system with a disposable point-of-use nitrate sensor and nitrate meter, which will be interfaced via Bluetooth transmission to a smartphone or tablet PC. We plan to adapt this technology to our phosphate measurement enzymes as well.

Nitrate supplies nitrogen for plant growth and crop productivity and is a limiting factor for obtaining maximum yield in many crops. Phosphate is also a key factor for plant growth and crop productivity since phosphorus is central to many biochemical processes and components. Both nitrogen and phosphorus are supplied to plants in a variety of forms through fertilizers, mainly applied to soils in fields. However, not all of the nitrogen and phosphorus ends up as a usable form in crop plant tissue which will in turn support growth and productivity. Excess nutrients leach into nearby groundwater, leading to potential adverse affects to environmental and human health.

In order to properly manage nutrients, better and more frequent analysis of soil and water need to be implemented. Two goals are achievable with nutrient status information: optimization of crop productivity for maximum yield with minimum fertilizer application, and minimizing nutrient loss to nearby groundwater from the field to avoid nutrient pollution.

In 2012, our team designed and constructed a potentiostat for use with commercial screen-printed 3-electrode sensors in a USDA SBIR funded project. The schematics of the printed circuit board from this project will be used as a guide for designing the new potenitiostat as the central component of the Nitrate Meter. NECi has patented the oxygen removal system to circumvent oxygen interfering with the sensor's operation.

We are currently seeking funding and interest in this project in order to continue research.