Chapter 15

Efficient Development of a Mixed Feed Process for Pichia

pastoris
David Johannes Wurm and Oliver Spadiut

Abstract
Pichia pastoris is one of the most important host organisms for the recombinant production of proteins in
industrial biotechnology. A prominent promoter system for recombinant protein production in P. pastoris
is the promoter of alcohol oxidase (PAOX1) which is induced by methanol, but repressed by several other
carbon sources, like glucose and glycerol. Thus, typical cultivation strategies for such P. pastoris strains
describe two different phases: growth on a carbon source, like glycerol, to get a high biomass concentra-
tion, followed by the induction of recombinant protein production by methanol. However, cells barely
grow on methanol resulting in only moderate productivity in such bioprocesses. To enhance productivity,
it is common to employ mixed substrate feeding strategies. The knowledge of certain strain-specific
parameters is required to be able to set up such mixed feed fed-batch cultivations to avoid methanol
accumulation and guarantee highest productivity. Here, we present an efficient strategy comprising only
one experiment to determine the settings of such a mixed feed system based on the physiology of the
respective yeast strain.

Key words Pichia pastoris, Methanol pulse, Specific substrate uptake rate, Dynamic fed-batch strat-
egy, Strain characterization

1 Introduction

The methylotrophic yeast Pichia pastoris, also known as Komaga-

taella phaffii, is extensively used as host organism for recombinant
protein production (e.g., [1–7]). The main advantages of P. pastoris
are its fast growth, its ability to use the cheap substrate methanol as
sole carbon source, its ability to perform typical eukaryotic post-
translational modifications, and the possibility of secreting the
recombinant product [5, 6, 8–10]. Usually, recombinant protein
production in P. pastoris is either regulated by a constitutive pro-
moter, like the promoter of glyceraldehyde-3-phosphate dehydro-
genase (PGAP), or an inducible promoter, like the promoter of
alcohol oxidase (PAOX1) (e.g., [7, 8, 11, 12]).

Brigitte Gasser and Diethard Mattanovich (eds.), Recombinant Protein Production in Yeast, Methods in Molecular Biology,
vol. 1923, https://doi.org/10.1007/978-1-4939-9024-5_15, © Springer Science+Business Media, LLC, part of Springer Nature 2019

323
324 David Johannes Wurm and Oliver Spadiut

The well-known PAOX1 is induced by methanol but repressed

by several other carbon sources, like glucose and glycerol, which
causes accumulation of methanol [13]. Thus, commonly used cul-
tivation strategies for P. pastoris describe two different phases:
growth on a carbon source, like glycerol or glucose, to get a high
biomass concentration, followed by the induction of recombinant
protein production by methanol. However, cells barely grow on
methanol resulting in moderate productivity in such bioprocesses.
To enhance the productivity of such recombinant P. pastoris pro-
cesses, it is common to employ mixed substrate feeding strategies
[14, 15]. A mixed feed strategy gives different benefits, like lower
oxygen consumption and lower heat production [16], it facilitates
biomass growth due to higher biomass yields on the second sub-
strate [16] and leads to an increased cell density resulting in
increased volumetric productivity. A prominent C-source for
these approaches is glycerol which represses PAOX1 if a critical
specific uptake rate is exceeded [17, 18]. Thus, it is crucial to find
the physiological limits of the respective strain and thus the optimal
substrate feeding ratio of methanol and glycerol [13, 18, 19]. Usu-
ally, several fed-batch cultivations and time-consuming continuous
cultivations have to be performed for that purpose. Here, we
present an efficient strategy comprising only one experiment to
determine the settings of a mixed feed system based on the physi-
ology of the respective yeast strain. This dynamic strategy is sche-
matically depicted in Fig. 1.

2 Materials

Prepare all media and solutions with analytical grade reagents and
deionized water.

2.1 Medium Yeast nitrogen base (YNB) medium per L: 1.0 M potassium phos-
for Preculture phate buffer (dissolve 118.1 g KH2PO4 and 23.0 g K2HPO4 in
1000 mL distilled water, pH 6.0), 3.4 g YNB without amino acids
and ammonium sulfate, 10 g (NH4)2SO4, 400 mg biotin, and 20 g
glucose. Weigh YNB without amino acids and ammonium sulfate
(NH4)2SO4, biotin, and glucose in a beaker. Add 100 mL of 1.0 M
potassium phosphate buffer (pH 6.0), dissolve by stirring, and set
with water to 1 L. Filter-sterilize through a 0.2 μm cutoff filter into
a sterile flask (see Note 1). Store at 4
C.

2.2 Stock Solutions 1. Basal salt medium (BSM) for 1 L of Cultivation Medium:
for Batch and Fed- 26.7 mL of 85% (v/v) phosphoric acid, 1.17 g CaSO4 · 2H2O,
Batch Cultivation 18.2 g K2SO4, 14.9 g MgSO4 · 7H2O, 4.13 g KOH, 44 g
C6H12O6 · H2O, and 0.3 mL antifoam. Weigh the chemicals in
a beaker, dissolve in around 600 mL of water, and then fill up to
725 mL in a measuring cylinder. Fill the bioreactor with this
medium and autoclave.
 Efficient Development of a Mixed Feed Process for Pichia pastoris 325

Fig. 1 Dynamic strategy to determine the settings for a mixed feed strategy with glycerol and methanol for a
Pichia pastoris strain. (a) Course of specific uptake rates qs during cultivation; (b) course of concentrations
during cultivation
326 David Johannes Wurm and Oliver Spadiut

2. Glycerol stock: prepare sterile glycerol by autoclavation.

3. Methanol stock: prepare sterile methanol by sterile filtration
(see Note 2).
4. Trace metal solution (PTM1) per L: 6.0 g CuSO4 · 5H2O,
0.08 g NaI, 3.0 g MnSO4 · H2O, 0.2 g Na2MoO4 · 2H2O,
0.02 g H3BO3, 0.5 g CoCl2, 20.0 g ZnCl2, 65.0 g
FeSO4 · 7H2O, 0.2 g biotin, 5 mL H2SO4. Weigh the chemi-
cals in a beaker and fill with water to 1 L. Filter-sterilize
through a 0.2 μm cutoff filter into a sterile flask and store at
room temperature.
5. Base solution to set the pH: 2–3 M NH4OH.

2.3 Medium for Fed- For P. pastoris strains based on the PAOX1 system, glycerol is a
Batch and Mixed Feed prominent C-source for biomass formation, whereas methanol is
Cultivation used for the induction of protein expression.
1. Glycerol feed per L: 250 g glycerol, 12 mL PTM1, 0.3 mL
antifoam. Sterilize by autoclavation.
2. Methanol feed per L: 300 g methanol (use a balance), 4 mL
PTM1, 0.3 mL antifoam. Sterile-filtered through a 0.2 μm
cutoff filter into a sterile flask (see Note 2).

2.4 Methanol For methanol pulses, use pure methanol supplemented with
Solution for Adaption PTM1. For this purpose, add 12 mL PTM1 solution into 1 L
and Pulses pure methanol, filter-sterilize this solution into a sterile bottle,
and store this solution at 4
C.

2.5 Equipment For a standard fed-batch cultivation, the following equipment are
at least required:
1. Bioreactor (e.g., 5 L working volume glass bioreactor; Infors,
Switzerland).
2. pH and dO2 probe.
3. Pressurized air and oxygen supply lines.
4. Off-gas analyzer (e.g., infrared cell for CO2 and a zirconium
dioxide sensor for O2 concentration; DasGip, Germany).
5. Pumps and tubings for base and feed.
6. If available: balances (reactor balance, feed balance, base bal-
ance) - connected to the process information management
system.
7. Process information management system (PIMS; e.g., Lucul-
lus, Securecell, Switzerland).
8. Spectrophotometer, centrifuge, and dry oven for sample
preparation.
 Efficient Development of a Mixed Feed Process for Pichia pastoris 327

9. HPLC for exact determination of methanol concentrations

(e.g., Agilent Technologies, USA) equipped with a SUPEL-
COGEL C-610H ion-exchange column (Sigma-Aldrich,
USA), and a refractive index detector (Agilent Technologies,
USA).

3 Methods

3.1 Preculture Start a preculture of the P. pastoris strain of interest in 100 mL of

of Pichia pastoris YNB medium in 1 L baffled shaking flasks at 220 rpm and 28
C for
maximum 24 h (to guarantee good aeration, only 1/10 of the total
volume of the flask is filled with medium). The preculture is inocu-
lated with 1 mL of frozen glycerol stock (see Notes 1, 3, and 4) and
should yield an OD600 of about 15–20 (ca. 8–10 g/L dry cell
weight) after 20–24 h.

3.2 Batch Phase After autoclaving the BSM in the bioreactor vessel, aseptically add
the sterile glycerol to a final concentration of 40 g/L. Then adjust
the temperature and the stirring speed to the desired values, before
the pH in the bioreactor is adjusted to pH 5.0 using 25% (v/v)
ammonia solution (NH4OH) by manually pumping the solution
into the bioreactor. Aseptically transfer sterile PTM1 solution into
the bioreactor (4.5 mL/L BSM). Aseptically transfer the preculture
into a sterile inoculation flask (a vessel providing a connection to
the bioreactor, see Note 5). The inoculum should be 10% of the
final volume in the bioreactor thus leading to a starting OD600 of
1.5–2 (about 1 g/L dry cell weight). Dissolved oxygen (dO2) is
measured with a sterilizable polarographic dissolved oxygen elec-
trode. The pH is measured online with a sterilizable electrode and
maintained constant with a step controller using 2–3 M NH4OH
which also represents the N-source during cultivation. The exact
concentration of NH4OH in the base bottle is determined by
titration with 0.25 M potassium hydrogen phthalate (KHP; see
Note 6). Base consumption is determined gravimetrically by put-
ting the base bottle on a balance and recording the loss in weight
over time. Set the cultivation temperature (e.g. 30
C), and aerate
the culture with 2.0 vvm dried air (volume per volume per minute;
in 1 L cultivation volume 2.0 vvm correspond to 2 L of in-gas per
minute). Always keep the dO2 above 30% by increases the stirrer
speed and, if necessary, by increasing the ratio of oxygen in the
in-gas by addition of pure oxygen. Measure the off-gas of the
culture by using an infrared cell for CO2 and a paramagnetic cell
for O2 concentration. Temperature, pH, dO2, agitation in the
vessel, as well as CO2 and O2 in the off-gas are measured online
and logged in a process information management system.
328 David Johannes Wurm and Oliver Spadiut

3.3 Fed-Batch Phase After the complete consumption of the C-source glycerol in the
for Biomass batch (indicated by an increase of dO2 and a drop in off-gas activity
Generation and verifiable by atline HPLC) an exponential fed-batch phase on
glycerol, e.g., at 90% of qs,Gly,max, is performed (Fig. 1). Assuming a
biomass yield YX/S of about 0.5, this corresponds to a certain
specific growths rate μ (see Note 7). Therefore, the biomass con-
centration after the batch phase must be known (see Note 7). If the
biomass yield of the strain on glycerol is known (this can be ana-
lyzed from the data in the batch; see Note 7), the biomass concen-
tration can be calculated based on the amount of glycerol used in
the batch (see Notes 8 and 9). The feed rate can then be determined
by Eqs. 1 and 2 and controlled using a gravimetrically based PID
flow controller.
X 0  V 0  q s  δFeed
F0 ¼ ð1Þ
c Feed

F ¼ F 0  e ðq s Y X =S t Þ ¼ F 0  e ðμt Þ ð2Þ
F0 ¼ initial feed rate [g/h]; X0 ¼ calculated biomass concen-
tration at the start of the fed-batch [g/L]; V0 ¼ volume in the
bioreactor at the start of the fed-batch [L]; qs ¼ specific substrate
uptake rate [g/g/h]; μ ¼ specific growth rate [1/h]; δFeed ¼ density
glycerol feed [g/L]; YX/S ¼ biomass yield on glycerol [g/g];
cFeed ¼ concentration glycerol feed [g/L]; F ¼ feed rate [g/h];
e ¼ Euler constant; t ¼ time [h].
During the glycerol fed-batch, dO2 levels should be kept above
30% at all time points, which is why pure oxygen is added in case
airflow is not sufficient (see Note 10). The fed-batch on glycerol is
stopped when the biomass concentration reaches about 50–60 g/L
(Fig. 1).

3.4 Methanol After the complete consumption of the C-source glycerol, which is
Adaption indicated by an increase of dO2 and a drop in off-gas activity, a
methanol adaptation pulse of a final concentration of 0.5% (v/v) is
added using the sterile methanol solution supplemented with
PTM1 (Fig. 1).

3.5 Determination After the complete uptake of MeOH in the adaptation pulse, the
of qs,MeOH,max by cells are regarded to be adapted to MeOH and to take up MeOH at
Methanol Pulses their maximum specific methanol uptake rate (qs,MeOH,max). The
complete uptake of the MeOH can be determined in real time by a
drop in the CO2 off-gas signal, an increase in the O2 off-gas signal,
as well as an increase in the dO2 signal. Furthermore, it can be
verified by a MeOH sensor in the off-gas in online mode or by
HPLC analytics at-line. To determine the qs,MeOH,max, at least two
MeOH pulses of a final concentration of 1–2.0% (v/v) are added
(Fig. 1). To obtain the specific rates for MeOH uptake during each
pulse, a minimum of two samples has to be taken: one directly after
 Efficient Development of a Mixed Feed Process for Pichia pastoris 329

methanol addition and the other close to complete methanol deple-

tion. The samples are used to determine the concentration of
residual substrate, OD600, and the dry cell weight. Determined
values at the beginning and the end of the respective MeOH pulse
are used to calculate an average value of the specific substrate uptake
rate (qs) according to Eq. 3 (in short: pulse methanol; take a
sample; when the off-gas signal starts to drop, indicating depletion
of methanol, take another sample; measure the exact methanol
concentration in these two samples by HPLC; calculate the volu-
metric methanol uptake rate; and relate it to the total biomass
content at the latter sample point).
ΔMeOH
qs, MeOH, max ¼ BMΔt
 1000 ð3Þ
M ðMeOHÞ
qs,MeOH,max ¼ maximum specific methanol uptake rate [mmol/
g/h]; ΔMeOH ¼ amount of methanol which was taken up [g];
Δt ¼ duration of pulse [h]; BM ¼ average biomass during the pulse
[g], M(MeOH) ¼ molecular weight of methanol (32.04 [g/mol]).
The strain-specific physiological parameters which can be deter-
mined by this strategy are (1) the adaptation time of P. pastoris to
methanol, (2) the qs during the adaptation pulse, and (3) a maxi-
mum qs for methanol (qs,MeOH,max) (see Notes 11 and 12).

3.6 Mixed Feed To find the physiological limits of the respective P. pastoris strain in
a mixed feed environment as well as optimum feeding conditions
(highest productivity or qp), a MeOH feed at 90% of qs,MeOH,max is
fed (Fig. 1). During this MeOH feed another, separately controlled
glycerol feed is started. Initially, the glycerol feed is adjusted to a
rather low qs,Gly (e.g., 1.0 mmol/g/h). In regular time intervals
(e.g., every 10 h), the feed is adjusted to correspond to a higher qs,
Gly; thus, different qs,Gly steps are performed (Fig. 1). At a certain
qs,Gly, PAOX is repressed and MeOH accumulates (see Note 13). In
case MeOH accumulates, the glycerol feed can be stopped, and
accumulated MeOH is metabolized. Regular sampling during these
different steps allows the determination of physiology, productivity,
and MeOH accumulation and thus reveals the optimal mixed feed
settings for the respective P. pastoris strain (see Note 14).

3.7 Sampling During the cultivation, samples have to be taken to characterize the
strain regarding biomass growth, substrate uptake, possible metab-
olite formation, and product formation (see Note 15).
For dry cell weight determination, harvest 5 mL of culture
broth, centrifuge in 10 mL glass tubes (4500  g, 4
C, 10 min),
wash the pellet twice with 5 mL physiological salt solution (0.9%
NaCl in deionized water), and determine the dry cell weight
(DCW) after drying at 105
C to a constant weight in an oven
(approximately 2–3 days). Optical density of the culture broth
330 David Johannes Wurm and Oliver Spadiut

throughout the process is measured using a spectrophotometer at a

wavelength of 600 nm (OD600). Dry cell weight measurement and
OD600 have to be correlated to be able to use the measured OD600
values for qs adaptation in subsequent fed-batch cultivations (see
Notes 8 and 9).
For methanol determination, samples are centrifuged
(20,000  g, 15 min), and concentration of methanol is deter-
mined in cell-free samples by HPLC on a SUPELCOGEL C-610H
ion-exchange column (Sigma-Aldrich, USA) with a refractive index
detector. The mobile phase is 0.1% H3PO4 with a constant flow rate
of 0.5 mL/min, and the system is run isocratic. Calibration is done
by measuring standard points in the range of 0.1–10 g/L
methanol.

4 Notes

1. If necessary, add antibiotics specific for the selection markers

harbored by the strain (e.g., Zeocin, Kanamycin) to the pre-
culture in appropriate concentrations (e.g., 100 μg/mL
medium) to further reduce the risk of contamination.
2. Do not autoclave methanol because of evaporation.
3. When combining the sterile solutions for the preculture in a
baffled shaking flask, work in the laminar flow hood and be
careful to work sterile and avoid contaminations.
4. The glycerol cryo-cultures are prepared by mixing 1 mL of a
fresh overnight culture of the respective P. pastoris strain with
0.5 mL sterile 75% glycerol (v/v), and snap-freezing it in liquid
N2. The frozen glycerol stocks are then stored at 80
C.
5. Before inoculating the bioreactor with the appropriate amount
of preculture, the following actions should be taken:
(a) Aseptically add the C-source to the sterile BSM in the
bioreactor.
(b) Set the desired temperature (typically 30
C) and stirring
speed (e.g., 1500 rpm).
(c) Set the pH value of the BSM to pH 5.0 with NH4OH and
note the amount of base which is required to determine
the overall content in the bioreactor vessel.
(d) Add PTM1 aseptically to the cultivation broth.
(e) Calibrate the dO2 electrode according to manufacturer’s
instructions.
(f) Adjust the weight of the bioreactor balance to the weight
of the bioreactor content—the bioreactor weight is
logged in the process information management system,
and by adjusting it correctly at this stage of the bioprocess,
the final data analysis will be facilitated.
Efficient Development of a Mixed Feed Process for Pichia pastoris 331

6. For base titration, the following materials are required: base

(NH4OH), 0.25 M KHP, bromothymol blue (indicator),
burette and beaker with magnetic stirrer. Add 2 mL of base
to the beaker (dilute NH4OH 1:10) and use the burette to add
0.25 KHP. At the point of equivalence, the color of the indica-
tor will turn from blue to gray and then to green.
7. The biomass yield on glycerol can be calculated from the data
in the batch. It is known how much glycerol was used in the
batch, e.g., 60 g/L. Based on the off-gas signals, the time,
which the cells needed to consume the substrate glycerol, can
be determined, e.g., 60 h. From these values, the volumetric
uptake rate of glycerol (rs,glycerol [g/L/h]) can be calculated:

60 Lg  0 Lg g
rs ¼ ¼1
60 h Lh
In analogy, the volumetric biomass formation rate (rx) can
be calculated from the dry cell weight values before (e.g.,
1.5 g/L) and after the batch (e.g., 31.5 g/L) and the respective
time period:
31:5 Lg  1:5 Lg g
rx ¼ ¼ 0:5
60 h Lh
To calculate the biomass yield on glycerol (YX/S [g/g]),
these two volumetric rates are put in relation:
rX g
Y X =S ¼ ¼ 0:5
rS g
The specific growth rate during the fed-batch can thus be
calculated by the set specific substrate uptake rate and the yield:
μ ¼ q s  Y X =S
8. It is important to know the biomass concentration before the
fed-batch phase to be able to implement a correct feed strategy.
There are several ways to measure, calculate or estimate the
biomass concentration in a bioreactor. Some of the possibilities
are listed here:
(a) Measure online by near-infrared spectroscopy [20].
(b) Measure online by capacitance probe [21].
(c) Calculate by using a soft sensor tool based on the feed and
the off-gas signal [22, 23].
(d) Calculate based on the amount of glycerol used in the
batch and the biomass yield on the respective substrate.
(e) Estimate by correlating the measured OD600 values to the
biomass dry cell weight (see Note 9).
332 David Johannes Wurm and Oliver Spadiut

9. To be able to use the OD600 values to set the feeding rate to the
desired qs set point, it is crucial to have a good and reliable
calibration curve of the OD600 and the biomass content (dry
cell weight) in (g/L). Before starting the fed-batch bioreactor
cultivation, generate such a calibration curve by using the
biomass from batch cultivations in different dilutions. During
cultivations, use the same photometer for OD600 measure-
ments as for the calibration curve. Do not switch photometers
during the experiment.
10. During batch and fed-batch on glycerol, the dO2 level should
be >30% to avoid anaerobic metabolism and thus the produc-
tion of undesired metabolites. This can be done by increasing
the oxygen ratio in the in-gas by supplementing the airflow
with pure oxygen.
11. The strain characteristic parameters, which can be analyzed by
the batch experiment with methanol pulses, are:
(a) Δtimeadapt: time period from induction until the off-gas
(CO2) has reached its maximum.
(b) qs,adapt: specific uptake rate for methanol during the adap-
tation pulse.
(c) qs,MeOH,max: the maximum specific uptake rate for metha-
nol during consecutive pulses.
12. To get even more precise data for qs, the methanol stripping
from the bioreactor can be considered according to the
Antoine’s equation [24, 25].
13. Methanol accumulation can be easily be detected without great
time delay by online HPLC or online GC.
14. This strategy allows high flexibility. During the MeOH pulses
as well as during the mixed feed phase, different process para-
meters (e.g., temperature, dO2, pH, etc.) can be adjusted and
thus tested.
15. To quantify productivity, product-specific assays must be used.
In case such assays are not available, the total extracellular
protein content can be used to get an idea about productivity,
since P. pastoris mainly secretes the target product.

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