Fermentation studies
The precultures were inoculated into double strength SC-URA media with the appropriate carbon sources to a final concentration of 1×106 cells ml-1. Ampicillin (100 μg ml-1) and streptomycin (15 μg ml-1) were added to inhibit bacterial contamination. Agitation and incubation were performed on a magnetic multi-stirrer at 30°C, with regular sampling through a syringe needle pierced through the rubber stopper. Fermentation with high substrate loading was performed similarly, but the double strength SC-URA media containing 200 g l-1 raw corn starch and 5 g l-1 glucose, was inoculated with a 50 g l-1 inoculum (wet weight). The wet cell weight was determined by weighing a cell pellet obtained from centrifugation of the pre-culture at 3 000 × g for 5 minutes.
Ethanol, glycerol, acetic acid, maltose and glucose concentrations were quantified with HPLC, using a Surveyor Plus liquid chromatograph (Thermo Scientific) consisting of a liquid chromatography pump, autosampler and Refractive Index Detector. The compounds were separated on a Rezex RHM Monosaccharide 7.8 × 300 mm column (00H0132-K0, Phenomenex) at 60°C with 5 mM H2SO4 as mobile phase at a flow rate of 0.6 ml min-1. The theoretical CO2 yields were calculated based on the ethanol concentrations.