Crude glycerol is the major byproduct of the biodiesel industry. In general, for every 100 pounds of biodiesel produced, approximately 10 pounds of crude glycerol are created. Because this glycerol is expensive to purify for use in food, pharmaceutical, or cosmetics industries, biodiesel producers must seek alternative methods for its disposal.

Using crude glycerol as a carbon source for fermentation is a promising alternative use for this waste material. In this project, we propose to use crude glycerol in the fermentation of the microalga Schizochytrium limacinum, which is a prolific producer of docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid with proven beneficial effects on treating human diseases such as cardiovascular diseases, cancers, and Alzheimer's.

The first part of this study provided "proof-of-concept" that Schizochytrium limacinum was capable of utilizing crude glycerol while producing DHA. The results show that pretreated crude glycerol was a viable carbon source for algal growth and DHA production, with 75 g/L to 100 g/L of glycerol being the optimum concentration range. In addition to glycerol concentration, temperature, trace metal levels, ammonium acetate concentration, and NH4Cl concentration also had significant effects (P < 0.1) on algal DHA production. Optimizing these factors using response surface methodology led to a DHA yield of 4.91 g/L and a DHA productivity of 0.82 g/L-day.

The second part of this study investigated the effects of impurities within the glycerol on DHA production and algal biomass composition, with a goal of ensuring that the algal biomass produced from crude glycerol can be safely utilized as an ingredient in omega-3 fortified foods or animal feed additives. Crude glycerol samples obtained from different biodiesel producers were tested. All the samples contained methanol, soaps, and various elements including calcium, phosphorus, potassium, silicon, sodium, and zinc. Both methanol and soaps in the glycerol negatively influence algal DHA production. The two impurities can be removed from the culture medium by evaporation through autoclaving (for methanol) or by precipitation through pH adjustment (for soap). The results showed that the crude glycerol-derived algal biomass contained 45-50% lipid, 14-20% protein, and 25% carbohydrate, with 8-13% ash content. Palmitic acid (C16:0) and DHA (C22:6) were the two major fatty acids in the algal lipid. ICP analysis showed that that boron, calcium, copper, iron, magnesium, phosphorus, potassium, silicon, sodium, and sulfur were present in the algal biomass, but no heavy metals (such as mercury) were detected. The crude glycerol-derived algal biomass had a high level of DHA and a nutritional profile similar to glucose-derived commercial algal biomass, suggesting a great potential for using crude-glycerol derived algae in omega-3 fortified food or feed.

Overall, this study shows that crude glycerol can be used as a safe and effective substrate for algal culture to produce high levels of omega-3 fatty acids. With the rapid expansion of the biodiesel industry, there is an urgent need to develop new markets for large quantities of crude glycerol. This research provides an exciting opportunity to utilize a large quantity of this low grade glycerol.