(306d) Continuous Ethanol Fermentation with Microfiltration Cell Recycle Applied to a Concentrated, Solids-Free Softwood Hydrolysate

Continuous stirred-tank fermentation with cell recycle (CSTF-CR) offers several advantages over batch fermentation when applied to second generation ethanol production.  In addition to improving volumetric productivity and reducing process downtime, high cell densities quickly metabolize inhibitors such as furfural and HMF into less toxic intermediates, significantly reducing their steady-state concentrations.  This is especially important in the fermentation of high gravity hydrolysates in which inhibitors are concentrated with the sugars.  In the present work, batch fermentations and design of experiments methodology were used to develop a kinetic model that accounts for the inhibitory effects of acetic acid, furfural, HMF and ethanol on S. cerevisiae D₅A.  The kinetic model was then used to predict CSTF-CR performance, and high cell retention (85-95%) continuous fermentations were used to validate the model for feeds with and without inhibitors. CSTF-CR runs with semi-defined media (100 g/l glucose + nutrients) resulted in ethanol productivities of 30 g/l/h for an uninhibited feed and >15 g/l/h for an inhibited feed containing acetic acid (4 g/l), furfural (2 g/l) and HMF (2 g/l).  A productivity of 16.8 g/l/h was obtained when operating with a solids-free pine wood hydrolysate (with glucose supplemented to 100 g/l and nutrients added) at cell densities of ~60 gDW/l. Longer runs (> 120 hours) at higher cell densities were used to improve the model and study effects specific to high cell density, continuous fermentation including reduced cell viability, reduced cell yields, and adaptation of the fermenting organism.  Operating conditions such as feed rate, cell retention percentage and air sparging (to maintain microaerophilic conditions) were investigated with the overall goal of improving cell viability and CSTF-CR performance.