2.2. Off-line analysis
Samples were regularly taken from the bioreactor and put into pre weighed and pre dried micro centrifuge tubes. The samples without the supernatant were let in a drying oven at 103 °C for 24 h. Then they were cooled down for 30 min before weighing. The supernatant of the samples after the centrifugation were examined by HPLC (ProStar, Variant, Walnut Creek, CA, USA) to determine the concentration of ethanol. The supernatant was firstly filtrated with pore size filter, 0.45 μm, polypropylene membrane (VWR, Darmstadt, Germany). Subsequently, it was injected 20 μL into a Rezex ROA-organic acid H+ (8 %) column (Phenomenex, Aschaffenburg, Germany) and operated at 70 °C with 5 mM H2SO4 as an eluent at 0.6 mL min−1 flow rate. The concentration of ethanol was calculated by software GalaxieTM Chromatography (Varian, Walnut Creek, CA, USA).