Camacho, D. J.; Moseley, J. L.; Merchant, S. S.

Auxenochlorella sp. (strain UTEX 250-A) is a fast-growing, oleaginous alga that is an emerging reference organism for basic research and broad biotechnological applications. Advancing UTEX 250-A as a biotechnological workhorse requires a deeper understanding of its nutritional demands, particularly under different trophic conditions. However, little is known about the specific acclimations that allow UTEX 250-A to thrive when essential nutrients are scarce. Here, we describe the formulation of a defined growth medium that supports the cultivation of UTEX 250-A with controlled trace levels of the essential micronutrients iron, copper, and zinc. Special attention was given to ensure that the medium was compatible with inductively coupled plasma mass spectrometry (ICP-MS) analysis, enabling the detection and minimization of metal contamination in the nanomolar range. The medium was designed to provide sufficient nutrients to support the mixotrophic growth of UTEX 250-A from an initial density of 105 cells/mL to a stationary density of 2.4x108 cells/mL, with additional nutrients supplied to accommodate metabolic and trophic transitions during stationary phase when photosynthesis is restored due to carbon limitation. This replete medium provides a foundation for robust nutrient limitation studies in Auxenochlorella sp. (UTEX 250-A).