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As a potential source of biofuel, the green colonial microalga Botryococcus braunii produces large amounts of hydrocarbons that are accumulated in the extracellular matrix. Generally, pretreatment such as drying or heating of wet algae is needed for sufficient recoveries of hydrocarbons from B. braunii using organic solvents. In this study, the Showa strain of B. braunii was cultured in media derived from the modified Chu13 medium by supplying artificial seawater, natural seawater, or NaCl. After a certain period of culture in the media with an osmotic pressure corresponding to 1/4-seawater, hydrocarbon recovery rates exceeding 90% were obtained by simply mixing intact wet algae with n-hexane without any pretreatments and the results using the present culture conditions indicate the potential for hydrocarbon milking.HighlightsSeawater was used for efficient hydrocarbon extraction from Botryococcus braunii. The alga was cultured in media prepared with seawater or NaCl. Hydrocarbon recovery rate exceeding 90% was obtained without any pretreatment.

The green colonial microalga Botryococcus braunii (race B) has attracted considerable attention because it is known to produce many hydrocarbons. The hydrocarbons are present in the extracellular matrix that connects the cells located in the interior of a B. braunii colony; however, the surface of each colony is covered with a retaining wall and a fibrillar colony sheath. This unique colony structure is thought to be associated with hydrocarbon storage and known to affect the extractability of hydrocarbons by using an organic solvent. In this study, we investigated the relation between hydrocarbon extractability and changes in the colony surface structure after two distinct treatments: culture of the algae in brackish medium or thermal pretreatment of the algae before solvent extraction. Both the treatments improved hydrocarbon extractability when non-polar solvents such as n-hexane were used. Thermal pretreatment inhibited the formation of fibrils on the surface of algal colonies, as revealed by rapid-freezing and freeze-substitution electron microscopy. However, when the alga was cultured in brackish medium, the fibrils on the surface shortened and became less dense. Thus, the fibrillar colony sheath that mainly consisted of saccharic components prevented the entry of organic solvents into the colony interior, and thus hydrocarbon extraction.

Hydrocarbons are easily extracted by organic solvents such as n-decane from wet samples of Botryococcus braunii by thermal pretreatment at 90°C even after being cooled to room temperature. However, hydrocarbon recoveries are not as readily achieved at room temperature from samples pretreated at temperatures lower than 80°C. This suggests that there is the point of no return for pretreatment temperature that enables effective solvent extraction of hydrocarbons at room temperature from wet algal samples of B. braunii. To elucidate the mechanism of hydrocarbon recovery from B. braunii following thermal pretreatments, we investigated the thermophysical properties of the water phase separated from heated algal slurry. Differential scanning calorimetry (DSC) measurements revealed sol-gel transitions in the water phase of algal slurry after protein denaturation at 64°C in samples that was pretreated at 70 or 80°C but not in those pretreated at 90°C. Furthermore, the pretreated >70°C water-soluble polymers revealed polysaccharides composed of galactose, arabinose, and uronic acid. These results suggest that the transition from sol state to gel state of water-soluble polysaccharides in algal slurry prevented hydrocarbon recovery with organic solvents since hydrocarbons were easily recovered from sol state samples pretreated at 70 or 80°C when the extraction temperature was kept the same as the pretreatment temperature. These results reveal that the presence of water-soluble polymers with gelation ability in the water phase and removal of these polymers in sol state enable effective recovery of hydrocarbons at room temperature after thermal pretreatments.

The B race of the green microalga Botryococcus braunii produces triterpene hydrocarbons, botryococcenes and methylsqualenes that can be processed into jet fuels with high heating values. In this alga, squalene is also converted into membrane sterols after 2,3-epoxidation. In the present study, cDNA clones of two distinct squalene epoxidases (BbSQE-I and -II) were isolated. Predicted amino acid sequences encoded on these genes are 45% identical with each other. Introduction of BbSQE-I or -II into Saccharomyces cerevisie erg1 mutants resulted in the complementation of ergosterol auxotrophy. The relative expression level of SQE-II increased 3.5-fold from the early stage to the middle phase of a culture period of 42 days, while that of SQE-I was almost constant throughout the culture period. Southern blot analyses suggested that these genes are single-copied genes. This is the first report on the isolation of functional SQEs that are encoded in duplicated loci in the algal genome.

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