Biomass determination
For the chemostat cultures, biomass growth was monitored by optical
density (OD) measurement at a wavelength of 660 nm with a Libra S11
spectrophotometer (Biochrom, Cambridge, United Kingdom). For the
fed-batch cultures, biomass growth was monitored by optical density (OD)
measurement at a wavelength of 600 nm with a Thermo Genesys
spectrophotometer (Thermo Fisher Scientific, MA, USA).
For the chemostat cultures, dry weight was determined by filtering 10 ml
culture broth over a pre-weighed nitrocellulose filter with a pore size
0.45 μm, washing the filter with demineralized water and drying the
filter for 20 min at 360 W in a microwave oven before weighing again
(Postma et al., 1989). Duplicate measurements varied less than 3.5%
throughout the cultivation. For the fed-batch cultures, dry weight was
determined by centrifuging 2 x 5 mL of culture broth at 6,000 × g for 15
min. The pellet was washed once by resuspending in deionized water and
centrifuged again at 6,000 × g for 15 min. After washing, the pellet was
dried for 24 hours at 105°C and weighed.
Gas
analysis
For the chemostat cultures, the offgas was cooled (2 °C) in a condenser
and dried, prior to the analysis of O2 and
CO2 concentrations using an NGA 2000 analyzer. For the
fed-batch cultures, the offgas was analysed with a Thermo Fischer Prima
BT Mass Spectrometer (Thermo Fisher Scientific, MA, USA).
Substrate and metabolite
analysis
During the runs, rapidly temperature-quenched samples were taken from
the fermenters in order to instantly stop metabolism and obtain
representative measurements of extracellular metabolites such as formic
acid. Immediately after sampling the samples were cooled in syringes
filled with precooled steel beads for fast cooling of the sample
(Mashego et al. , 2003). The cells were immediately removed by
filtration and samples were stored frozen.
Extracellular concentrations of glucose and formic acid in culture
filtrates were analysed by high performance liquid chromatography (HPLC)
on an Agilent 1260 HPLC, equipped with a Bio-Rad HPX 87H column,
operated at 60°C with 5 mM H2SO4 as
mobile phase at a flow rate of 0.600 mL min-1.
Detection was performed by means of an Agilent refractive index detector
and an Agilent 1260 VWD detector at 210 nm.