Towards the commercialization of microalgal production: the role of environmental factors in production of triterpenoids from Botryococcus braunii

Abstract

Production of hydrocarbons and biofuels from microalgae is an emerging field that shows great potential for carbon neutral production. The green alga Botryococcus braunii is widely recognized as a potential carbon-neutral source of hydrocarbons and biofuels. However a number of issues hamper its commercial development including, low growth rates and biomass production, low hydrocarbon yield, problems associated with scaling up of production, harvesting of biomass, and recovery of hydrocarbons and/or biofuel. This study addressed these issues using a series of laboratory-scale and large-scale experiments. Examination of the effects of three growth-media on biomass and hydrocarbon production in two B. braunii Race-B strains, Kossou-4 and Overjuyo-3, previously reported to produce high yields of hydrocarbons including triterpenoids showed that growth of B. braunii in Blue-green-11 medium (BG11) resulted in significantly higher biomass (dry-weight) compared to Jaworski’s medium (JM) and Bold-base-3-nitrogen medium (BBM-3N) after 15 days for both strains. Oil production was also significantly higher in BG11 whether measured as oil weight or absorbance. Examination of the effects of nutrients (nitrogen, iron) and environmental conditions (temperature, light intensity, photoperiod) on biomass and hydrocarbon production in Kossou-4 and Overjuyo-3 grown in BG11 showed highest biomass and oil production was obtained at nitrogen concentration 750mg l-1, iron concentration 6mg l-1, 25 oC, and 135μmol m-2 s-1 with photoperiod of 16h light/8h dark. Culturing the strains in modified BG11 containing optimized nutrients resulted in up to ~10.6 fold increase in biomass from 0.16g l-1 and 0.31g l-1 in normal BG11 to 1.74g l-1 and 2.17g l-1 in optimized BG11 and growth conditions for Kossou-4 and Overjuyo-3 respectively. This was accompanied by ~8-10 fold increase in oil production compared to that in normal BG11, with final oil yields of 264.2mg l-1 (Kossou-4) and 220.6mg l-1 (Overjuyo-3). Growth of Overjuyo-3 and Kossou-4 in 500L open tanks with 24h light (54μmol photons m-2s-1) at 25°C in modified BG11 for 60 days were then carried out. Maximum growth was reached after 40 days with Overjuyo-3 producing more biomass (3.05g l-1) than Kossou-4 (2.55g l-1). However, Kossou-4 produced more hydrocarbons (29% of dry weight) compared to Overjuyo-3 (20% of dry weight). The final stage investigated the feasibility of harvesting large-scale B. braunii using co-culture with a fungus to induce flocculation. Co-culture with the fungus Aspergillus fumigatus resulted in a flocculation efficiency of over 90% through co-pelletisation with both Overjuyo-3 and Kossou-4. Investigation of the ratio of fungus to algal broth found that 1:40 was optimal, resulting in large-scale flocculation efficiencies of 97% for Kossou-4 and 98% for Overjuyo-3 after 12h following inoculation. To determine the effects of co-culture with A. fumigatus potential energy yields, pyrolysis was conducted. Proximate analysis showed the proportions of bio-oil, bio-char and bio-gas were very similar for the pure Kossou-4 biomass and the co-cultured pellets resulting from harvest using A. fumigatus. Similar results were found for Overjuyo-3 confirming that the co-culture harvest method did not compromise the end product. This research demonstrates for the first time that large-scale production using bio-flocculation for harvesting of high value triterpenoids for commercial application is feasible using these B.braunii Race-B strains