Production of Eicosapentaenoic acid from biodiesel derived crude glycerol using fungal culture

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.