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. / Master of Science

Crude glycerol is the major byproduct of the biodiesel industry. There is an abundance of this byproduct and purifying it for use in industries such as food, pharmaceutical, or cosmetic is prohibitively expensive. Developing an alternative use for crude glycerol is needed. Utilizing it as a carbon source in the fermentation of algae is one potential method for using this under-utilized byproduct. In this research, crude glycerol is used in the mixotrophic production of the alga, Phaeodactylum tricornutum, which is an eicosapentaenoic acid (EPA) producing diatom. Mixotrophic growth is when cells perform autotrophic and heterotrophic modes of growth concurrently. EPA is an omega-3 polyunsaturated fatty acid that has been demonstrated to have a multitude of beneficial health effects, including maintaining human cardiovascular health, treating cancer and human depression diseases, and an anti-obesity effect. In this study, the potential of using crude glycerol in batch mode mixotrophic culture of P. tricornutum was investigated. Once the mixotrophic culture was established, parameters involved in increasing the biomass and EPA production were optimized. These included nitrogen source, level of supplemental carbon dioxide, and concentration of crude glycerol. Using nitrate, 0.08 M crude glycerol, and 3% (vol/vol) carbon dioxide led to the highest biomass productivity of 0.446 g L?? day?? and the highest EPA productivity of 16.9 mg L?? day?? in batch mode culture. The continuous culture of the mixotrophic culture was then performed following the batch culture optimization. The effects of dilution rate were observed in continuous culture with the parameters of nitrate as the nitrogen source, 0.08 M crude glycerol, and 3% (vol/vol) carbon dioxide held constant. The highest biomass productivity of 0.612 g L?? day?? was obtained at D = 0.24 day??. The highest EPA productivity of 16.5 mg L?? day?? was achieved at both D = 0.15 day?? and D = 0.24 day??. The maximum specific growth rate was estimated from the washing out dilution rate and was determined to be around 0.677 day??. Overall, it was found that crude glycerol increases the biomass and EPA productivity of Phaeodactylum tricornutum. Continuous culture with the use of crude glycerol can further increase these measurements. The potential for scaling up studies is demonstrated by these results and can help lead to a market for this abundant, little-used byproduct of the biodiesel industry. / Master of Science