Analytical determinations
Cultures were centrifuged for 5 minutes at 6000 rpm, the supernatants
were filtered through 0.22 µm nylon membranes (MSI, USA) and stored at
-20 ºC for analytical determinations.
Organic acids were determined by high-performance liquid chromatography
(HPLC) using an LC-20AT Prominance equipment (Shimadzu Corp., Kyoto,
Japan) with an HPX-87-H Aminex column (Cat no. 125- 170 0140; Bio Rad
Laboratories Inc., Hercules, CA) at 50ºC. The mobile phased consisted of
5mM sulfuric acid at a flow rate 0f 0,6 ml/min. The metabolites were
quantified with a SPD-20AV UV detector (Shimadzu 171 Corp.).
Ethanol and butanol were measured by Manual Head Space GC-FID using an
Agilent 7820A GC–FID with manual head space injection. The separation
was conducted on a HP-INNO- WAX capillary column (30 m, 0.25 µm film
thickness and 0.25 mm ID). The supernatants were diluted ½ in
K2CO3 1g/ml, and 5 µl of isobutanol 5g/L
was added as an internal standard. The samples were heated at 60°C for
1h and 1ml of the gas phase was injected manually in a GC-FID. The GC
oven was initially heated at 35 °C for 2 min, then to 45 °C at 30 °C/min
and held for 1 min, finally, was heated to 120ºC at 50ºC/ml and
maintained for 1 min. The injector and FID temperatures were set at 150
and 300°C, respectively. Nitrogen was used as the carrier gas at a
column flow of 2 ml/min with a 5:1 split ratio.