Biology Reference
In-Depth Information
1 sampling port in the bottom.
Off-gas cooling at 3
C and off-gas analysis.
Air is supplied at 1 vvm. In order to remove CO
2
, the air is sparged through
a 2-M KOH solution.
Cultivations conditions: 1000 rpm stirrer speed, 30
C temperature and pH
5 controlled with addition of 2 M HCl and 2 M NaOH, no overpressure.
Inoculum
Inoculate 5 mL normal glucose minimal mediumwith yeast and grow over
night at 30
C.
Saccharomyces cerevisiae
CEN.PK,
S. cerevisiae
FY4 and
Pichia pastoris
have all been shown to be suitable (
K¨mmel
et al.
, 2010;
Carnicer
et al.
, 2011
).
Add 35 mL of U-
13
C glucose medium to a 500 mL non-baffled shake flask
and preheat 15 min in the incubator at 30
C.
Inoculate the shake flask with the overnight pre-culture. Ideally, the
pre-culture should be in exponential growth phase and has an OD
600
of
1.0-1.5.
Fermentation
Inoculate 965 mL U-
13
C glucose medium in the bioreactor with the 35 mL
of the exponentially growing shake flask pre-culture to obtain a starting
OD
600
of about 0.05.
After about 17 h, the late ethanol-growth phase is reached (verify by
growth rate and shift in the respiratory quotient, i.e. the ratio of O
2
consumption to CO
2
production).
Sample 200 mL as described below.
Add 2.5 g U-
13
C glucose and continue cultivation for 5 min.
Sample the rest of the culture in lots of 200 mL as described below.
Sampling and quenching procedure
Prepare 5 L quenching solution: 60:40 methanol/water with 10 mM
ammonium acetate. Adjust pH to pH 7.5 with 25% ammonium hydroxide.
Distribute the quenching solution in 800 mL portions over six 1-L bottles
and cool to
40
C using a cryostat.
Assemble a quenching device: half fill an ice bucket or a large beaker with
denatured ethanol (methylated spirits) and add about 1.5 kg dry ice. Place
the bucket on a magnetic stirrer and then place a 2-L measuring cylinder
with a large magnetic stirrer bar in the bucket. Make sure the level of the
ethanol in the ice bucket reaches up to the 800 mL level of the cylinder.
Switch on the stirrer and pre-cool for 15 min.
40
C) in the
cylinder. Sample 200 mL of broth directly into the quenching solution
while stirring continuously.
Transfer the quenched suspension into two 1-L centrifuge buckets and
centrifuge at 4000
Just before sampling, add 800 mL quenching solution (at
20
C until supernatant is clear.
g
and
