Enhancement of Biomass and Hydrocarbon Productivity in Botryococcus braunii Kützing by Random Mutagenesis

Abstract

Because of oil price fluctuations in last decade and climate changes resulting in many researchers and national government worldwide promote the use the alternative energy instead of fossil fuels. Microalgae such as Botryococcus braunii regarded as one of the potential source for alternative energy. This microalga produces high amounts of hydrocarbons and lipids which can be used as a raw material and convert to bioenergy. However, many studies reported that the growth of B. braunii rather low as compared with other microalgae. Thus, the main objective of the thesis was to enhance biomass, lipids and hydrocarbons productivity on B. braunii using random mutagenesis approach. Furthermore, microalgal productivity improvement was performed in cultivation process after random mutagenized as well as lipid extraction step. Botryococcus sp. was isolated from various water bodies and identified by its morphological and molecular characteristics. They were confirmed as B. braunii and obtained 5 strains of B. braunii included B. braunii AARL G035, B. braunii AARL G036, B. braunii AARL G037, B. braunii AARL G038 and B. braunii AARL G039. Incubation of microalga under different media found that, CA tend to be high on biomass production and B. braunii AARL G035, B. braunii AARL G036, B. braunii AARL G037 and B. braunii AARL G039 provided maximum biomass. Therefore, these 4 strains were selected for further experiment. When selection of desired isolates after being treated wild type with ultraviolet C radiation and ethyl methane sulfonate, it was found that almost of selected mutagens induced isolates gave biomass, lipids and hydrocarbons higher than theirs wild type. B. braunii AARL G037 isolate U18N14 showed the maximum of hydrocarbon content and hydrocarbon productivity which were 78.40±0.96% and 55.53±1.11 mg.L-1.day-1, respectively. The accumulation of lipids reached the maximum at 49.16±0.91% from B. braunii AARL G037 isolate E0.6I7. The investigation of biomass and lipid productivity the results found that B. braunii AARL G036 isolate E1.0H15 exhibited the greatest both of values at the level of 103.22±1.70 mg.L-1.day-1 and 35.46±0.59 mg.L-1.day-1, respectively. Hence B. braunii AARL G036 isolate E1.0H15 was chosen for the next experiment. The finding ammonium sources for replace ammonium nitrate in CA medium was investigated before the study of two-stage cultivation. The results showed that ammonium sulfate was suitable for B. braunii AARL G036 isolate E1.0H15. Two-stage cultivation by produced biomass in first 20 days and then applied various stresses conditions for 4 days. At the end of cultivation, microalgal biomass under stress conditions were decreased, while it showed higher in the accumulation of lipids and hydrocarbons. Under the light intensity of 160 μmol photom.m-2.s-1 , microalga provided the maximum of hydrocarbon productivity at 98.11±0.83 mg.L-1.day-1. At the light intensity of 200 μmol photom.m-2.s-1, microalga seem to be produce highest lipid productivity of 58.56±1.89 mg.L-1.day-1 . Meanwhile, the control condition produced hydrocarbon productivity and lipid productivity at the values of 81.81±2.75 mg.L-1.day-1 and 47.90±2.18 ±1.89 mg.L-1.day-1 , respectively. Fatty acid was found diversely depended on stress conditions. The comparative study of lipid extraction on biomass of B. braunii, in case of reduction sample size and solvent contact time. The size of sample less than 45 μm with 24 h extraction time gave the great condition for lipid extraction. The combination various non-polar and polar solvent with different ratio, 2:1 (v/v) dichloromethane: methanol was proper for extraction. Among several pretreatment methods in this study, 1% HCl provided the maximum amount of lipids and fatty acid methyl ester. Furthermore, heating with commercial microwave with 1% HCl lysis, microalgal lipid and fatty acid methyl ester were improved in quantitatively. So far, it can be concluded that, biomass, lipid and hydrocarbon productivity of B. braunii can be enhanced by our techniques using random mutagenesis, two-stage cultivation and lipid extraction.