2.6. Fermentation with Simulated Contamination

Mock fermentations were conducted with YPD containing 60 g/L glucose (YPD60) or clarified sugarcane juice. Clarification of sugarcane juice was completed via centrifugation to remove solid contents and filtration with a 0.4 nm bottle-top filter. Clarified sugarcane juice was diluted with purified water to ~6% total sugars content, and supplemented with 0.6 g/L yeast extract. The addition of yeast extract served to replace dead yeast that is typically present in industrial fermentations in Brazil [33,34,35].

S. cerevisiae cells were cultured in synthetic complete glucose broth anaerobically at 30 °C for 24 h, to stabilize the expression vectors. Meanwhile, L. fermentum ATCC 9338 and L. plantarum ATCC 14917 were cultured in MRS broth anaerobically at 30 °C for 24 h. S. cerevisiae and L. fermentum ATCC 9338 or L. plantarum ATCC 14917 were co-inoculated from their separate cultures at an OD_600nm 0.5 and 0.05, respectively for YPD60 and OD_600nm 5.0 and 0.5, respectively for sugarcane juice. All mock fermentations were performed in a total of 20 mL, and incubated anaerobically at 30 °C, with agitation at 100 rpm for 36 h. At designated intervals, the OD at 600 nm was measured, and 1 mL samples were collected for high-performance liquid chromatography (HPLC) analysis, as previously described [36].