Renewable energy in the form of bio-diesel, bio-methane, bio-hydrogen, and hydrocarbons from microalgal biomass is gaining attention for research in recent years. The use of microalgal biomass as a source of proteins, photosynthetic pigments, antioxidants, peptides, fatty acids and polysaccharides have led to many applications in biofuels, food and feed industry and for its use in the pharmaceutical and cosmetic industries.<br />

The green colony forming microalga Botryococcus braunii is well known for its ability to produce large amount of hydrocarbons (~75% of dry biomass) and exo-polysaccharides and is divided into three different races (A, B and L), based on the types of hydrocarbon they produce. The production of metabolically expensive compounds such as hydrocarbons and polysaccharides is one of the main reasons for its slow growth rate and is largely depend on the physiological state of the cells. Apart from slow growth rate, variations across different races and strains in terms of growth behavior, formation of product and biomass yield are some of the main challenges for the commercial applications of this microalga. This leads to the identification and characterization of novel strains of B. braunii which can perform better despite of challenges associated with it.<br />

Therefore, the current study focuses on the systematic metabolic characterization and comparison of B.braunii race A and race B strains in order to address some of the challenges associated with its slow growth rate and product formation. Furthermore, the result obtained in the current work shows the impact of different physiological state of the cell on product formation and on overall metabolome profile. Additionally, the strains of race A and race B were compared with respect to their growth behavior, metabolome profile and product formation abilities.<br />

The study revealed that the investigated B. braunii strains differed greatly in terms of biomass accumulation and hydrocarbon/EPS formation. Race A and race B strains have distinct growth and hydrocarbon/EPS formation phases depending on their physiological state, based on the total chlorophyll content. Comparison between race A and B strains revealed extended linear growth phase for race B strain AC 761. Moreover, the hydrocarbon production in race A strain CCAP 807/2 and race B strain AC 761 differed during the duration of cultivation. For race A strain (CCAP 807/2) the hydrocarbon biosynthesis was promoted during late linear and early stationary phase, whereas race B produces hydrocarbons continuously from the beginning of the cultivation and the hydrocarbon productivity is maximum during the linear phase.<br />

Besides, the primary metabolome analysis of race A and B strains also showed significant differences in the abundance of metabolites related to carbohydrate metabolism especially sucrose. Moreover, race B showed higher intracellular lipid content than race A strains.<br />

Strikingly, even though the race A strains CCALA 778 and CCAP 807/2 showed similar growth behavior, they significantly differed in their ability to produce hydrocarbons. CCALA 778 was majorly carbohydrate producer with presence of only detectable amount of hydrocarbons whereas CCAP 807/2 was able to produce both hydrocarbons and carbohydrates. Furthermore, the gradual increase in the hydrocarbon formation was proportional to the decrease in oleic acid. Notably, intracellular lipid content in CCAP 807/2 was two-fold more than CCALA 778. The very long chain fatty acid (VLCFAs) which are intermediates for the hydrocarbon biosynthesis in race A were detected in both race A and race B strains. In addition, the primary metabolome analysis showed sucrose as the most abundant metabolite in both the strains of race A.<br />

Altogether, this work presents for the first time the distinct and shared characteristics of growth behavior, metabolome and product formation for the B. braunii race A strains CCLA778 and CCAP 807/2 with race B strain AC761. In addition, the results in this study also show the potential link and impact of different physiological state of the cell on product formation and on the overall metabolome profile.