Several novel methods are described to advance the study of Botryococcus braunii race B as a potential production platform for algae biofuels. A feed-forward culturing strategy to obtain and maintain high-density cultures of B. braunii race B is described. By predicting the nutrient requirements of a culture based on a nitorgen-based mass balance, B. braunii cultures were grown to densities greater than 10 grams dry weight per liter (gDW/L) in shake flasks. This strategy is combined with the use of low-density stock cultures as “back-up” inoculum for high-density cultures which are maintained using a stoichiometrically balance medium, that was developed for the high density growth and maintenance of pH balance in freshwater algal species. In this work the preferential uptake of ammonium and associated drop in pH is confirmed to occur as with previous studies of Chlorella and Chlamydomonas. Additionally, the recalcitrance of contaminating bacterial species within the algal culture is discussed along with a method for generation of an axenic culture. A novel dual-flask co-culturing strategy is then presented and shown to enable the long-term aseptic maintenance of axenic B. braunii race B cultures using elevated CO2 in a closed system. Ongoing and future work involving the newly obtained axenic B. braunii race B lines is briefly discussed.
