Quantifying the parametric sensitivity of ethanol production by Scheffersomyces (Pichia) stipitis: development and verification of a method based on the principles of growth on mixtures of complementary substrates.

Under aerobic conditions, Crabtree-negative yeasts grow but do not ferment, and under anaerobic conditions, they ferment but do not grow. It is therefore believed that fermentation by these yeasts is sensitive to small variations of the operating parameters, e.g. dilution rate D, mass transfer coefficient kla and oxygen solubility co*. However, this parametric sensitivity has never been quantified. Here, we present a method to quantify the parametric sensitivity of ethanol production in the Crabtree-negative yeast Scheffersomycesstipitis. The method is based on our experimental observation that S. stipitis cultures follow the principles of growth on mixtures of complementary substrates. Specifically, if a chemostat operating at fixed D, kla and co* is fed with progressively increasing glucose feed concentrations sf, the culture passes through three regimes. (1) At low sf, the culture is carbon-limited and no ethanol is produced. (2) At high sf, the culture is oxygen-limited and ethanol is produced, but unused glucose is lost with the effluent. (3) At intermediate sf, both glucose and oxygen are limiting, and ethanol is produced without loss of glucose. Ethanol must therefore be produced in this dual-limited regime. The dual-limited regime can be predicted by simple unstructured models. It is characterized by the relation Yos<Dsf/kla⋅co∗<Yos', where Yos and Yos' denote the g of glucose consumed per g of oxygen during carbon- and oxygen-limited growth. Hence, the parametric sensitivity of fermentation by Crabtree-negative yeasts can be improved by targeting the yields Yos and Yos'.