Fermentation parameters determination
Fermentation parameters containing the concentrations of residual glucose, ethanol, glycerol, succinate, biomass, cell viability, and oxidation reduction potential (ORP) were investigated to assess the CVEF system.
Triplicate samples were withdrawn and centrifuged at 10,000 rpm at ambient temperature for 5 min. The cell pellet was collected for biomass determination, and the supernatant was frozen at −20 °C for HPLC analysis. An HPLC system with RI-detector (Waters 410, Waters, USA) and an Aminex HPX 87-H column (300 ×7.8 mm, Bio-Rad Laboratories, Hercules, CA, USA) was used to analyze glucose, ethanol, glycerol, and succinate in the effluent according to Wang et al.(2013). Biomass was measured as dry cell weight (DCW), wash the cell pellet obtained previously twice with deionized water, overnight at 80°C to constant weight, and weigh the DCW. Cell viability was evaluated via methylene blue stain described by Bai et al. (2004). Living cells were calculated from biomass×viability. ORP value was monitored by an ORP electrode.