Chemostat cultivation
Aerobic, glucose-limited chemostat cultivations were performed in 2 l
laboratory bioreactors (Applikon, Delft, The Netherlands) with a working
volume of 1 l. Cultures were stirred at 800 rpm and sparged with 500 ml
air/min and the dissolved O2 concentration was monitored
via an O2 electrode, remaining above 30% of saturation
at atmospheric conditions throughout the cultivation. The pH of the
culture was maintained at 5.0 via automated addition of 2 M KOH and the
temperature was kept constant at 30°C. SM medium used in bioreactors
contained 5.0 g/L glucose and was supplemented with 0.2 g/L Pluronic PE
6100 antifoam (BASF, Ludwigshafen, Germany) for the batch phase and 0.4
g/L antifoam for the chemostat phase.
For the initial batch phase, the cultures were inoculated with an
overnight preculture to an initial optical density of approximately
0.04. After glucose depletion, indicated by a rapid drop in the
CO2% in the exhaust gas, the medium pump was switched
on to obtain a constant flow rate of 100 ml/h resulting in a dilution
rate of 0.1 h-1. The formic acid concentration in the
feed medium was set by aseptically adding formic acid to the 20 l feed
medium vessel prior to the chemostat phase. The working volume was kept
constant at 1 l using an effluent pump controlled by an electric level
sensor. Chemostat cultures were assumed to be in steady-state if after
at least 5 volume changes, the concentration of biomass in the reactor,
as well as the CO2 concentration in the exhaust gas
remained constant (<3% variation) for at least two additional
volume changes.
Fed-batch
cultivation
Aerobic, glucose / formic acid-limited fed-batch cultivations were
performed in 10 l laboratory bioreactors (L. Eschweiler and Co., Kiel,
Germany) and in 300 l pilot bioreactors (Bio Engineering AG, Wald,
Switzerland). The laboratory bioreactors were inoculated from shake
flasks. The pilot bioreactors were inoculated from 70 l inoculum
reactors (Applikon, Delft, The Netherlands), that were in turn
inoculated from shake flasks.
For the shake flasks, 0.5 mL cell stock culture was added to 400 mL of
preculture medium (Supplementary Table S1). Precultures were incubated
for 26h in flat bottom flasks with baffles, at 30 °C with a rotational
speed of 150 rpm and a throw of 2.5 cm. The pH of the media was not
adjusted before inoculation.
The four laboratory bioreactors (from here onwards denoted as LF1
through LF4) contained 3.6 kg of batch medium (Supplementary Table S4)
and 400 g of preculture. The start weight was 4 kg, while the estimated
end weight was 8.7 kg. No formic acid was dosed in the batch medium, as
high initial concentrations of formic acid were expected to be
detrimental to the cells. The media were adjusted to pH 5.0 with
NH3 before inoculation.
The process started with a batch phase until carbon depletion. The other
operating conditions are given in Supplementary Table S5. The solutions
for pH correction and foam remediation were 25 wt% NH3,
98 wt% H2SO4 and Basildon 86-013. The
carbon feed solutions of the four laboratory scale fermentations are
given in Table 1. The pH of the feed solutions was not adjusted with
alkaline titrant.
Table 1: Composition of the carbon
feeds of the laboratory (LF) and pilot (PF) fermenters