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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

Crude glycerol is a major byproduct if the biodiesel industry. Biodiesel manufacturers are currently facing the challenges of appropriate disposal of this waste material. Crude glycerol is expensive to purify for use in food, cosmetic, and pharmaceutical industries and therefore, alternative methods for use of this crude glycerol are needed. A promising alternative is to use this crude glycerol as a carbon source for microalgae fermentation. In this project, we investigated the use of crude glycerol as a less expensive substrate for the fermentation of the microalgae Schizochytrium limacinum and Pythium irregulare which are prolific producers of omega-3 polyunsaturated fatty acids. Omega-3 fatty acids have many beneficially effects on treating human diseases such as cardiovascular diseases, cancers, and neurological disorders. In addition, the omega-3 fatty acids docosahexaenoic acid (DHA) has been shown to be an important factor in infant brain and eye development. The first part of this study focused on the continuous fermentation of S. limacinum, a prolific producer of DHA. The objective of this study was to examine the algal cellular physiology and maximize its DHA productivity. Two important parameters used in continuous fermentation were studied: dilution rate (D) and feed glycerol concentration (S₀). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L. The cells had a true growth yield of 0.283 g/g, a maximum specific growth rate of 0.692 day⁻¹, and a maintenance coefficient of 0.2216 day⁻¹. The second part of this study focused on morphology issues with P. irregulare, a prolific producer of eicosapentaenoic acid (EPA). P. irregulare has a filamentous morphology, which can make fermentation difficult. The mycelium can stick to the agitation blades resulting in mechanical problems. In addition, this filamentous morphology prevents adequate amounts of oxygen from reaching some cells resulting in decreased productivities. The focus of this research was to control the fermentation conditions to make the algae grow in small pellets, a morphology more suitable for fermentation. In flask culture studies, pellets were formed at an agitation speed of 110 rpm in both regular and baffled flasks. Baffled flasks resulted in pellet formation at 90 and 130 rpm as well. Fermentation studies resulted in pellet formation at agitation speeds of 150 and 300 rpm. Pellets were better able to form when a baffle was not in place. In addition, agitation speed influenced pellet size, with smaller pellets forming at the higher agitation speed. Overall, this study showed that crude glycerol can be used as a carbon source for the continuous fermentation of S. limacinum with high DHA productivity and the morphology of P. irregulare could be controlled by manipulating culture conditions, mainly agitation speed. These results show the potential for scale-up studies for both algal species. Master of Science

Omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA, C20:5, n-3) and docosahexaenoic acid (DHA, C22:6, n-3), have many medically established benefits against cardiovascular diseases, cancers, schizophrenia, and Alzheimer's. Currently, fish oil is the main source of omega-3 fatty acids, but there are many problems associated with it such as undesirable taste and odor, and heavy metal contamination. As a result, it is necessary to seek alternative production sources based on various microorganisms. In this thesis we have developed a novel microfungal culture process to produce EPA from the crude glycerol byproduct generated in biodiesel industry. This process provides both an alternative source of omega-3 fatty acids and a benefit to the biodiesel industry. Indeed, as oil prices reach historical highs, biodiesel has attracted increasing interest throughout the United States. The disposal of the crude glycerol byproduct has been a challenge faced by the biodiesel producers. Crude glycerol presents a cheap carbon source for growth of many microorganisms. In this thesis, we tested the feasibility of using crude glycerol for producing eicosapentaenoic acid (EPA, 20:5, n-3) by one algal species, Phaeodactylum tricornutum and two fungal species, Mortierella alpina and Pythium irregulare. We observed that the algal growth is inhibited in the crude glycerol while the fungi can grow very well in crude glycerol-containing medium. The fungus M. alpina produced significant amount of ARA but negligible amount of EPA. P. irregulare produced significant amount of biomass as well as a relatively high level of EPA. The maximum dry biomass for the P. irregulare culture was 2.9 g/L with an EPA productivity of 7.99 mg/L-day. Based on these results, we concluded that P. irregulare was a promising candidate for EPA production from biodiesel derived crude glycerol. Further optimization work showed that P. irregulare grown 30 g/L crude glycerol and 10g/L yeast extract results in the highest level of EPA production. A temperature of 20o C is optimal for high fungal biomass and EPA levels. Addition of vegetable oil (at 1%) enhanced the EPA production and almost doubled the amount of biomass reached. Soap inhibits growth as well as EPA production severely even in small amounts. Methanol completely inhibits growth. The final optimized growth conditions for the fungus P.irregulare were a medium with 30g/L of crude glycerol, 10 g/L of yeast extract at a pH of 6 with 1% supplementation of oil, at a temperature of 20o C for a period of 7 days.Thus we have established that the fungus P.irregulare can be used successfully to produce high mounts of EPA from crude glycerol. Master of Science