Glycerol Detection Enzyme - Case Study

BACKGROUND

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.

APPLICATION

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.

PROGRESS

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.