Fed-batch growth of Rhizopus oryzae: Eliminating ethanol formation by controlling glucose addition

Abstract

Rhizopus oryzae is a prominent strain for producing fumarate, where biomass growth precedes fumarate production. The natural biofilm growth of R. oryzae as fungal mat was investigated using different glucose addition strategies in a novel fed-batch fermenter. Batch growth was compared through three fed-batch runs, each with a different glucose addition strategy. The fed-batch runs involved a constant glucose feed (CGF) of 0.075 g h−1 and controlled glucose feeds in order to keep the respiration quotient (RQ) at either 1.3 mol CO2 mol−1 O2 (RQ1.3) or 1.1 mol CO2 mol−1 O2 (RQ1.1). Ethanol overflow via the established Crabtree mechanism was completely negated for the CGF and RQ1.1 runs, while the batch and RQ1.3 runs exhibited significant ethanol formation. Biomass yield on glucose was found to be 0.476 g g−1 (RQ1.1), 0.194 g g−1 (RQ1.3), 0.125 g g−1 (CGF) and 0.144 g g−1 (batch). The results indicate a three-fold improvement in biomass yield when comparing the batch run with the RQ1.1 run. In addition, the RQ1.1 run resulted in zero detectable byproducts, unlike the batch scenario where pyruvate and fumarate were associated with ethanol formation. Clear evidence is provided that glucose overflow can be fully eliminated during R. oryzae growth, significantly affecting the biomass yield on glucose.