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).