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Culture of Escherichia coli under dissolvedoxygen gradients simulated in a two‐
compartment scale‐down system: Metabolic
response and production of recombinant
protein
Article in Biotechnology and Bioengineering · March 2005
Impact Factor: 4.13 · DOI: 10.1002/bit.20383 · Source: PubMed
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Culture of Escherichia coli Under DissolvedOxygen Gradients Simulated in aTwo-Compartment Scale-Down System:Metabolic Response and Production ofRecombinant Protein
Edgar A. Sandoval-Basurto,1 Guillermo Gosset,2 Francisco Bolı́ var,2
Octavio T. Ramı́ rez1
1Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico (UNAM),
Apdo. Post. 510-3, Cuernavaca, Mor. 62250, México;telephone: (52) (777) 329-1617; fax: (52) (777) 313-8811;e-mail: tonatiuh@ ibt.unam.mx2Department of Cellular Engineering and Biocatalysis, Institute of Biotechnology, National Autonomous University of Mexico (UNAM),
Apdo. Post. 510-3, Cuernavaca, Mor. 62250, México
Received 29 June 2004; accepted 5 October 2004
Published online 17 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20383
Abstract: A significant problem of large-scale cultures, butscarcely studied for recombinant E. coli , is the presence ofgradients in dissolved oxygen tension (DOT). In this study,the effect of DOT gradients on the metabolic responseof E. coli and production of recombinant pre-proinsulin,accumulated as inclusion bodies, was determined. DOTgradients were simulated in a two-compartment scale-down system consisting of two interconnected stirred-tank bioreactors, one maintained at anoxic conditions andthe other at a DOT of at least 6%. Cells were continuouslycirculated between both vessels to simulate circulation
times (tc) of 20, 50, 90, and 180 sec. A complete kineticand stoichiometric characterization was performed in thescale-down system as well as in control cultures main-tained at constant DOT in the range of 0– 20%. Theperformance of E. coli cultured under oscillating DOT wassignificantly affected, even at a tc of 20 sec correspondingto transient exposures of only 13.3 sec to anaerobicconditions. Specific growth rate decreased linearly with tcto a maximum reduction of 30% at the highest tc tested.The negative effect of DOT gradients was even morepronounced for the overall biomass yield on glucose andthe maximum concentration and yield of pre-proinsulin.In these cases, the losses were 9%, 27%, and 20%,respectively, at tc of 20 sec and 65%, 94%, and 87%, re-spectively, at tc of 180 sec. Acetic, lactic, formic, and
succinic acids accumulated during oscillatory DOT cul-tures, indicating that deviation of carbon flow to anaero-bic metabolism was responsible for the observed losses.The results of this study indicate that even very short ex-posures to anaerobic conditions, typical of large-scale op-erations, can substantially reduce recombinant proteinproductivity. The information presented here is useful for
establishing improved rational scale-up strategies andunderstanding the behavior of recombinant E. coli exposedto DOT gradients. B 2004 Wiley Periodicals, Inc.
Keywords: scale-down; dissolved oxygen gradients; cir-culation time; recombinant protein; Escherichia coli ;anaerobic-aerobic metabolism; inclusion body; mixed-acidfermentation
INTRODUCTION
Large-scale fermentation and cell culture processes are often
characterized by the presence of gradients in relevant
parameters, such as pH, dissolved oxygen tension (DOT),
dissolved carbon dioxide concentration, glucose concen-
tration, and other substrates commonly fed to the cultures
(Amanullah et al., 2004; Palomares and Ramı́rez, 2000a,b).
DOT gradients can be particularly detrimental to E. coli, as
exposure to transient anaerobic conditions, of even a few
seconds (13 sec), are sufficient to induce genes of anaerobic
pathways (Schweder et al., 1999) and divert essential carbon
skeletons to undesirable by-products (Xu et al., 1999b). A
heterogeneous environment is caused by a deficient mixing
that results from limitations inherent to large-scale bio-
reactor design and operation. It can also result from adrawback, recognized a long time ago, of the classical
approach for scaling-up fermentations by the geometrical
similarity concept. Namely, only one fundamental variable
can be maintained constant, whereas the rest will vary as the
process is translated to the larger scale (Palomares and
Ramı́rez, 2000a). Performance differences between large-
and small-scale fermentations have been well documented
and are usually attributed to the heterogeneous conditions
that prevail at the large scale compared to the well-mixed
B 2004 Wiley Periodicals, Inc.
Correspondence to: O.T. Ramı́rez
Contract grant sponsors: CONACYT-México; DGAPA-UNAM
Contract grant numbers: NC-230; IN118904
https://www.researchgate.net/publication/12840671_Monitoring_of_genes_that_respond_to_process-related_stress_in_large-scale_bioprocesses?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12908036_Glucose_overflow_metabolism_and_mixed_acid_fermentation_in_aerobic_large_scale_fed_batch_processes_with_E_coli?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12840671_Monitoring_of_genes_that_respond_to_process-related_stress_in_large-scale_bioprocesses?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12908036_Glucose_overflow_metabolism_and_mixed_acid_fermentation_in_aerobic_large_scale_fed_batch_processes_with_E_coli?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==
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situation of laboratory vessels. In general, processes at the
larger scale show inferior performances, such as reduced
yields and productivity, and increased cell lysis and by-
product formation (Bylund et al., 1998, 2000; Geraats, 1994;
Takebe et al., 1994). For instance, Onyeaka et al. (2003)
reported a 35% loss in biomass yield of an E. coli
fermentation in 20 m3 compared to the equivalent 5 L
laboratory-scale case. Likewise, Bylund et al. (1998)
observed a 15–20% lower final biomass concentration and
biomass yield on glucose when scaling a recombinant E. coli
fermentation from 3 L to 9 m3.
The scale-down methodology, proposed 20 years ago by
Oosterhuis (1984), is a rational approach for determining
the effects of environmental heterogeneities present in
large-scale fermentors (Amanullah et al., 2004; Palomares
and Ramı́rez, 2000a). Information derived from scale-down
studies can be used to improve scale-up procedures and to
anticipate or identify problems at the production level. The
effect of gradients in DOT, glucose, pH, and molasses has
been determined by oscillating the variable of interest in
scale-down systems (Amanullah et al., 2001; Cortés et al.,
2005; George et al., 1993; Larsson and Enfors, 1988;
Oosterhuis et al., 1985; Osman et al., 2002; Serrato et al.,2004; Takebe et al., 1994; Träger et al., 1992; Trujillo-
Roldán et al., 2001; Yegneswaran and Gray, 1991). Such
studies have been performed on a wide variety of micro-
organisms, including Gluconobacter oxydans, Saccharomy-
ces cerevisiae, Aspergillus niger, Streptomyces clavuligerus,
S. hygroscopicus, Bacillus subtilis, Azotobacter vinelandii,
Kluyveromyces marxianus, Penicillium chrysogenum, hy-
bridomas and mouse myeloma cells, among others. Re-
combinant E. coli is one of the most relevant hosts for
heterologous protein expression (Palomares et al., 2004),
and thus its fermentations at large scale have become
increasingly important. Nonetheless, there is a notorious
lack of scale-down studies using recombinant E. coli, as
most employ wildtype strains (Enfors et al., 2001; Hewitt
et al., 2000; Neubauer et al., 1995; Onyeaka et al., 2003;
Schweder et al., 1999; Xu et al., 1999b). Substantial differ-
ences can exist between the behavior of wildtype and
recombinant E. coli, and even between the same recombi-
nant strain if the gene coding for the product of interest is
induced or not (Lin and Neubauer, 2000). Accordingly,
reports of wildtype E. coli under environmental gradients
can be of limited value for understanding the behavior of
recombinant strains.
To our knowledge, the only scale-down studies employ-
ing recombinant E. coli are those of De León et al. (1995),Namdev et al. (1993), Lin and Neubauer (2000), and
Bylund et al. (1999, 2000). Among them, only De León et al.
(1995) directly controlled DOT in an oscillatory mode. In
such a report, a one-compartment system was used to
continuously measure and control DOT at a relatively long
oscillation period of 400 sec under various amplitudes and
oscillation axes. Namdev et al. (1993) used a Monte Carlo
method to set a fluctuating on/off supply of air in a one-
compartment scale-down system, but without an actual
control of DOT. Thus, no oscillating DOT profiles were
obtained. In general, the characteristic slow response time
of one-compartment systems prevents an accurate feedback
control of DOT at very short oscillation periods. DOT
fluctuations have been generated indirectly by changes
in oxygen uptake rate induced by oscillating glucose con-
centration in one- (Lin and Neubauer, 2000) or two-
compartment (Bylund et al., 2000) systems. Such studies
are of great relevance when trying to mimic the feed zone
of a fed-batch fermentation. However, in the former case
the relatively slow DOT oscillation occurred in a range well
above the critical concentration, and thus was inconse-
quential for cell physiology and metabolism, whereas in the
latter no information on DOT profiles was presented. In any
case, simultaneous variations in both DOT and glucose
concentration complicates data interpretation. Furthermore,
oxygen limitation following glucose overflow, rather than
glucose overflow itself, has been found to be the critical
parameter in studies with fluctuating glucose concentration
(Bylund et al., 2000). Together, the existing information
indicates that scale-down studies of recombinant E. coli
with independent control of DOT under rapid oscillations
around the critical concentration are necessary.In this work, a recombinant E. coli producing inclusion
bodies of a precursor hybrid protein of human insulin was
subjected to rapid DOT oscillations in a two-compartment
scale-down system composed of two interconnected stirred-
tank reactors. The effect of circulation time on production
of biomass, recombinant protein, and organic acid by-
products, as well as other relevant kinetic and stoichio-
metric parameters, was determined. The results obtained
are useful for predicting the behavior of the biological
model studied when cultured in batch mode under DOT
gradients typically found in large-scale fermentors.
MATERIALS AND METHODS
Bacterial Strain and Plasmids
A trp+ derivative of E. coli strain W3110, transformed with
the pTEXP-MMRPI plasmid, was used in this study. The
pTEXP-MMRPI plasmid carries a tetracycline resistance
gene and a gene coding for the fusion protein TrpLE-hybrid
human proinsulin under control of the trp operon promoter.
Details and construction of the plasmid can be found
elsewhere (Olmos et al., 1994).
Culture Medium and Inoculum Preparation
The working cell bank was maintained in 2-mL cryo-
conservation tubes at –70jC. Each tube contained 1 mL of
Luria liquid medium with glycerol (25%). Inoculum was
prepared by transferring the total content of a tube to a
shake flask containing culture medium at a volume equal to
10% of the total volume used in the bioreactor cultures. The
total working volumes of cultures at constant and oscil-
latory DOT were 500 and 1,100 mL, respectively. Shake
454 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 89, NO. 4, FEBRUARY 20, 2005
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-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/8266548_Heterogeneous_conditions_in_disolved_oxygen_affect_N-glycosylation_but_not_productivity_of_a_monoclonal_antibody_in_hybridoma_cultures?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12165400_Physiological_Responses_to_Mixing_in_Large_Scale_Bioreactors?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/11264099_The_response_of_GS-NS0_Myeloma_cells_to_single_and_multiple_pH_perturbations?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/15432773_Production_in_Escherichia_coli_of_a_rat_chimeric_proinsulin_polypeptide_carrying_human_A_and_B_chains_and_its_preparative_chromatography?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/5252049_Experimental_simulation_of_dissolved_oxygen_fluctuations_in_large_fermentors_Effect_onStreptomyces_clavuligerus?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12501297_Influence_of_controlled_glucose_oscillations_on_a_fed-batch_process_of_recombinant_Escherichia_coli?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12501297_Influence_of_controlled_glucose_oscillations_on_a_fed-batch_process_of_recombinant_Escherichia_coli?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==
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flasks were incubated at 37jC and 300 rpm, and their
contents were transferred to the bioreactors when an optical
density of 3.5 was reached. The culture medium for inocu-
lum propagation contained (per liter of deionized water):
K 2HPO4, 3 g ; N a2HPO4, 3 g;yeast extract, 5 g;(NH4)2HPO4,
1.7 g; glucose, 2 g; MgSO4 7 H2O, 2 g; tetracycline, 30 mg.
The initial pH was adjusted to 7.4 with NaOH. A similar
medium was used for bioreactor cultures, except for the
following differences: glucose, 10 g; (NH4)2HPO4, 17 g; and
2 ml of trace element solution containing (per liter of deion-
ized water) CaCl2 2 H2O,1g;CuCl22H2O,1g;CoCl26H2O,
2 g; FeCl36H2O, 27 g; ZnCl24H2O, 2 g; H3BO3, 0.5 g;
Na2MoO42H2O, 2 g; HCl concentrated, 100 ml.
Two-Compartment Scale-Down Bioreactor System
A two-compartment system was designed to simulate
dissolved oxygen tension (DOT) gradients typically present
in large-scale fermenters (Fig. 1). The system consisted of
two interconnected 1.5 L baffled stirred-tank bioreactors
(Virtis, Gardiner NY), each equipped with two six-blade
Rushton turbines and maintained at 37jC. Culture medium
was circulated between the two bioreactors through a lowgas permeability tubing (Norprene, 12.7 mm internal
diameter) and using a peristaltic pump equipped with a
double head (Masterflex, Cole-Parmer, Vernon Hill, IL).
The medium inlets and outlets were placed at a height of
3 cm with respect to the bottom of the vessels and separated
180j from each other. The first bioreactor (BAnaerobic) was
maintained under anaerobic conditions by sparging pure
nitrogen through two stainless steel porous diffusers at a
flow rate of 1 to 1.5 vvm, using two six-blade Rushton
turbines. Aerobic conditions were maintained in the second
bioreactor (BAerobic) by sparging an oxygen/nitrogen mix-
ture through a five-orifice diffuser at a constant total gas
flow rate of 1 vvm and using one six-blade Rushton tur-
bine. The operation volumes of the aerobic and anaerobic
vessels were set to 350 and 700 mL, respectively. This
resulted in an aerobic to anaerobic volume ratio of 0.5. The
volume in the circulation loop was f50 mL. Circulation
times (total culture volume divided by medium circulation
flow rate) in the range of 7–180 sec were tested.
DOT was measured in both bioreactors using steril-
izable polarographic probes (Ingold, Columbus, OH). In
BAerobic the DOT was controlled at a constant value by a
proportional-integral-derivative (PID) algorithm that au-
tomatically varied the flow rate of the individual gases
through two mass flow controllers (Brooks Instruments,
Halfield, PA). PID constants were determined from pro-
cess reaction curves and following the Ziegler-Nichols cri-
teria (De León et al., 2003). The DOT setpoint for BAerobicin all cultures was 10% with respect to air saturation. Agi-
tation rate was varied between 600–750 rpm in BAnaerobic,
whereas it remained constant at 500 rpm in BAerobic. A
detailed description of the computerized data acquisition
and control system for maintaining constant DOT in each
vessel can be found elsewhere (Trujillo-Roldan et al., 2001).
The pH was measured (pHoenix sterilizable glass elec-
trode) only in BAerobic and maintained at 7.2 F 0.2 by
automatic addition of a 2 N NaOH solution with a peristal-tic pump connected to an on/off pH controller (Ingold).
No pH differences were found between the two bioreactors
as determined in samples taken simultaneously from both
vessels. A 0.1% w/v silicon solution was used for foam
control in both vessels.
Control Cultures at Constant DissolvedOxygen Tension
Control cultures at constant DOT (0, 0.2, 1, 5, 10, and 20%
with respect to air saturation) were performed using only
the aerobic bioreactor section of the system described
above. Cultures were performed without medium recircu-lation by sealing the inlet and outlet of the circulation loop.
Oxygen and nitrogen mixtures were used to control DOT as
low as 5%, whereas air and nitrogen were used to control
DOT below 5%. The same DOT and pH control system as
described above was used. A constant agitation rate was
used for each culture but was different (in the range of
300– 500 rpm) for the various DOT tested. In addition,
possible effects due to cells passing through the circulation
loop and pump heads were tested. For this, cultures were
performed at 20% DOT with medium circulated at a flow
rate of 6 L/min through the same loop of the scale-down
system but excluding the anaerobic bioreactor.
Analytical Methods
Biomass concentration was determined from optical den-
sity readings at 600 nm (OD600) (Beckman Instruments,
Palo Alto, CA, DU 610 spectrophotometer) converted to
dry cell weight using a standard curve. One OD600 unit
corresponded to 0.32 g/L of dry cell weight. Glucose
concentration was determined off-line with a YSI 2700
biochemical analyzer (Yellow Spring Instruments, Youngs-Figure 1. Schematic of the two-compartment scale-down fermenta-
tion system.
SANDOVAL-BASURTO ET AL.: RECOMBINANT PROTEIN PRODUCTION BY E. COLI 455
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town, OH). Total protein concentration was determined by
the Bradford method (BioRad kit, Hercules CA) using
bovine serum albumin as standard. The concentration of
recombinant protein (TrpLE-proinsulin) was determined
in sonicated cell pellets by SDS-polyacrylamide (15%)
gel electrophoresis (PAGE) under denaturing conditions
(PAGE BioRad kit). Gels were scanned and area and in-
tensity of bands were quantified by densitometric analysis
(NIH Image 1.61/Fat software) using bovine carbonic an-
hydrase (Amersham Pharmacia Biotech, Piscataway, NJ)
as standard. Acetate, lactate, succinate, formate, and eth-
anol were determined from culture supernatants by HPLC
on an AMINEX HPX87H column (300 7.8 mm; BioRad)
at 50jC. A 5 mM H2SO4 solution at a constant flow of
0.5 ml/min was used as the mobile phase and analytes were
detected at 210 nm in a photodiode array detector as well
as by refractive index.
RESULTS
Control Cultures at Constant Dissolved Oxygen
Tension (DOT)To characterize the metabolic behavior of the recombinant
E. coli used in this study, a series of control cultures were
performed at constant DOT in the range of 0– 20%.
Duplicate cultures were performed at each condition tested.
Typical kinetics of growth, consumption of glucose, and
production of protein and organic acids at 10% DOT are
shown in Figure 2. Similar trends were obtained for the
cultures maintained at 0.2, 1, 5, and 20% DOT, although
rates of the various parameters measured, their maximum
concentrations, and times to reach them were different in
each case. No growth, substrate consumption, or product
production was observed for the cultures at 0% DOT. As
seen in Figure 2B, biomass concentration increased ex-
ponentially until exhaustion of glucose after 7 h of cul-
ture. The gene coding for the hybrid recombinant protein
TrpLE-proinsulin was under the trp operon promoter; thus,
it was induced upon tryptophan depletion. Production of
TrpLE-proinsulin initiated after 4 h and reached a maxi-
mum of 0.31 g/L (Fig. 2C). After this, a small decrease
in both total and recombinant protein concentrations was
observed. Accumulation of acetic, lactic, succinic, and for-
mic acids was observed from the beginning of the culture.
The concentrations of lactic, succinic, and formic acids de-
creased after 6 h, when glucose concentration was below
2 g/L, whereas acetic acid concentration decreased until 1 hafter glucose depletion (Fig. 2D).
Relevant kinetic and stoichiometric parameters of
cultures maintained at constant DOT are summarized in
Figures 3– 5. The effect of constant DOT on specific
growth rate (A) during exponential phase and overall
biomass yield on glucose (Yx/s) of E. coli batch cultures is
Figure 2. Typical kinetics of recombinant E. coli at constant 10% DOT.
A: pH and DOT. B: Biomass and glucose concentration. C: Total and
recombinant protein concentration. D: Concentration of organic acids.
Figure 3. Effect of constant DOT on kinetic and stoichiometric
parameters of batch cultures of recombinant protein produced by E. coli
maintained at constant DOT. A: Specific growth rate. B: Overall biomass
yield on glucose. Data points represent average of two independent cultures
and error bars the difference between cultures. In some cases data symbols
are larger than error bars. Line in A represents Monod’s model fit to data
points. Line in B drawn only to show trend.
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shown in Figure 3. Data points correspond to the average of
two independent cultures. A maximum specific growth
rate (Am) of 0.39 h1 and a Monod saturation constant for
oxygen (K O2) of 1% were obtained by fitting the experimen-
tal values of A to Monod kinetics through linear regression
of Lineweaver-Burk plots. Similar to A, Yx/s also followed
a saturation-type behavior as DOT increased, reaching a
maximum value of 0.4 g/g (Fig. 3B). In comparison, Da
Silva and Bailey (1986) reported for E. coli maximum
theoretical yields for secreted and intracellular recombinant
proteins of 0.42 and 0.64 g/g, respectively.
The effect of DOT on specific glucose uptake rate, (qs),
and specific production rate of organic acids (qO.A.) during
exponential growth phase, and maximum concentration of
organic acids is shown in Figure 4. For DOT above 5%, qsremained relatively constant around 1 g/g-h; however, it
increased to a maximum of 1.6 g/g-h as DOT decreased to
0.2%. Such a value agrees with the maximum reported by
Lin et al. (2001) of 1.86 g/g-h for strain W3110. The
maximum specific production rate of acetic (qaa), lactic
(qla), and succinic (qsa) acids occurred at 1% DOT, whereas
the maximum specific production rate of formic acid (qfa)
occurred at 0.2% DOT. The maximum values for qaa, qfa,
qla, and qsa were 0.52, 0.51, 0.48, and 0.11 g/g-h, respec-
tively. As DOT increased above 1%, the specific produc-
tion rates of organic acids decreased; however, it remained
at detectable levels even at 20% DOT. For qfa, qla, and qsasuch a decrease was substantial (i.e., only between 0.5–4%
of the maximum rates remained at nonlimiting DOT
conditions). In comparison, a substantial amount of ace-
tic acid was still produced at 20% DOT (33% of maxi-
mum qaa). The maximum concentration of organic acids
followed a similar trend as that of their specific production
rates with respect to DOT (Fig. 4C). The maximum
concentration of acetic, lactic, formic, and succinic acids
was 3.6, 3.34, 2.91, and 0.83 g/L, respectively. Similar
to A and Yx/s, the recombinant protein concentration and
recombinant protein yield on biomass increased with
DOT in a saturation-type behavior (Fig. 5). The maximum
recombinant protein concentration and maximum recom-
binant protein yield on biomass were f0.32 g/L and
0.09 g/g, respectively, at 20% DOT. No growth, substrate
consumption, product or metabolite production was ob-served at 0% DOT.
Cultures Under Oscillating DOT Conditions
To simulate DOT gradients that can occur in large-scale
fermentors, E. coli cultures were continuously circulated
between two interconnected stirred-tank reactors, one
Figure 4. Effect of constant DOT on kinetic and stoichiometric
parameters of batch cultures of recombinant protein produced by E. coli.
A: Specific glucose uptake rate. B: specific organic acid production rate.
C: Maximum organic acid concentration. Data points represent average of
two independent cultures and error bars the difference between cultures. In
some cases data symbols are larger than error bars. Line in B drawn only to
show trend.
Figure 5. Effect of constant DOT on: (A) maximum recombinant protein
concentration; (B) recombinant protein yield on biomass. Data points
represent average of two independent cultures and error bars the difference
between cultures. In some cases data symbols are larger than error bars.
Lines are drawn only to show trend.
SANDOVAL-BASURTO ET AL.: RECOMBINANT PROTEIN PRODUCTION BY E. COLI 457
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maintained at anaerobic conditions and the other at aerobic.
The DOT control set points for the anaerobic (BAnaerobic)
and aerobic (BAerobic) bioreactors were 0 and 10%,
respectively. Controlling DOT at such setpoints for each
vessel can be a challenge for very short tc (
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Such results are in agreement with Onyeaka et al. (2003),
who found no hydrodynamic damage to wildtype E. coli
at power inputs of 1–30 kW/m3 associated with agitation
or aeration.
Relevant kinetic and stoichiometric parameters of cul-
tures maintained at oscillating DOT are summarized in
Figures 7–9. Data points at tc of 0 sec represent values
obtained from control cultures maintained at constant 10%
DOT (Figs. 2 –5). The performance of E. coli was neg-
atively affected when cultured under oscillating DOT,
even at the lowest tc tested. As shown in Figure 7A, A
decreased linearly with increasing tc, from 0.37 h1 at
constant DOT to 0.27 h-1 at the highest tc tested (180 sec).
Such a decrease corresponded to a maximum reduction in A
of about 30% with respect to the constant DOT cultures. In
comparison, Amanullah et al. (1992) reported for B. subtilis
a 55% decrease in A as tc increased from 0 to 300 sec for an
aerobic to anaerobic volume ratio of 0.43 and a decrease of
as much as 70% when the volume ratio was 0.25. The Yx/salso decreased with increasing tc, but the effect was more
pronounced than for A as f65% of the yield was lost even
at tc of 90 sec (Fig. 7B). As tc further increased to 180 sec,
Yx/s leveled off between 0.12–0.19 g/g.
Specific glucose uptake rate increased from 1.1 g/g-h to
a maximum of 2.7 g/g-h as tc increased from 0 to 90 sec
(Fig. 8A). In comparison, Xu et al. (1999b) reported for a
wildtype E. coli qs values in the range of 2.7– 4.4 and
0.33–0.69 g/g-h for the oxygen-limited and oxygen-
sufficient zones, respectively, of a scale-down system.
For tc above 90 sec specific glucose uptake rate decreased.
The specific production rate of organic acids followed a
trend similar to that observed for specific glucose uptake
rate (Fig. 8B). Acetic acid had the highest specific pro-
duction rate at 1.05 g/g-h, followed by lactic, formic, and
succinic acids with 0.98, 0.48, and 0.33 g/g-h, respectively.
The acetic acid concentration reached a maximum of
3.9 g/L at tc of 50 sec and then remained relatively constant
as tc increased (Fig. 8C). In turn, the maximum concen-
tration of lactic, formic, and succinic acids was 2.36, 1.52,
and 0.77 g/L, respectively, and occurred at or above a tc of
90 sec. The effect of circulation time on maximum recom-
binant protein concentration and maximum yield of re-
combinant protein on dry cell weight is shown in Figure 9.As seen, simulated DOT gradients had a drastic negative
effect on both parameters. Maximum recombinant protein
concentration and recombinant protein yield decreased
from 0.3 to 0.02 g/L and from 0.08 to 0.01 g/g, re-
spectively, as tc increased from 0–180 sec. Such reductions
represent a loss of about 94 and 87% for each parameter
Figure 8. Effect of circulation time on kinetic and stoichiometric pa-
rameters of batch cultures of recombinant E. coli maintained at oscillating
DOT in the two-compartment scale-down system. A: Specific glucose
uptake rate. B: Specific organic acid production rate. C: Maximum organic
acid concentration. Data points represent average of two independent
cultures and error bars the difference between cultures. In some cases data
symbols are larger than error bars. Data points at tc of 0 sec represent values
obtained from control cultures maintained at constant 10% DOT.
Figure 9. Effect of circulation time on: (A) maximum recombinant
protein concentration; (B) recombinant protein yield on biomass. Batch
cultures of recombinant E. coli maintained at oscillating DOT in the two-
compartment scale-down system. Data points represent average of two
independent cultures and error bars the difference between cultures. In
some cases data symbols are larger than error bars. Lines are drawn only to
show trend. Data points at tc of 0 sec represent values obtained from
control cultures maintained at constant 10% DOT.
SANDOVAL-BASURTO ET AL.: RECOMBINANT PROTEIN PRODUCTION BY E. COLI 459
https://www.researchgate.net/publication/9041036_Further_Studies_Related_to_the_Scale-up_of_High_Cell_Density_Escherichia_coli_Fed-Batch_Fermentations_The_Additional_Effect_of_a_Changing_Microenvironment_When_Using_Aqueous_Ammonia_to_Control_pH?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/9041036_Further_Studies_Related_to_the_Scale-up_of_High_Cell_Density_Escherichia_coli_Fed-Batch_Fermentations_The_Additional_Effect_of_a_Changing_Microenvironment_When_Using_Aqueous_Ammonia_to_Control_pH?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==
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at the largest tc tested. Furthermore, even at a tc of 20 sec
the reduction was significant, as a loss of 27% and 20%
was observed for maximum recombinant protein concen-
tration and yield, respectively, compared to the constant
DOT cultures.
DISCUSSION
It is remarkable that in spite of the paramount importance
that oxygen has on microbial physiology and metabo-
lism (Konz et al., 1998), reports on detailed kinetic and
stoichiometric characterization of the effect of DOT on
production of recombinant proteins by E. coli are not
abundant. Furthermore, no generalization can be made
based on existing information, as the various proteins and
host/vector systems reported behave very differently (De
León et al., 2003). Accordingly, the comprehensive char-
acterization performed here under controlled DOT con-
ditions, summarized in Figures 3– 5, provide relevant
information to the field. The K O2 of 1% obtained here is
in good agreement with the scarce information available.
For instance, K O2 of 0.54% and 0.48% have been reported
by De León et al. (2003) and Li et al. (1992), respectively,for recombinant E. coli under different medium and culture
conditions. In contrast, Chen et al. (1985) reported a K O2 of
0.16%, although carbon dioxide was sparged in such a
study, permitting better growth at limiting DOT due to
possible replenishment of TCA cycle intermediates by
heterotrophic CO2 fixation. The DOT range for aerobic/
anaerobic transitions can also be inferred from other ev-
idence. For instance, half-maximal synthesis of fermen-
tation products (acetate, formate, ethanol, lactate, and
succinate) and expression of key enzymes in the fermenta-
tive pathway (alcohol dehydrogenase and pyruvate formate-
lyase) occur in a DOT range of 0.2–5.0 mbar (or 0.1–2.4%
w.r.t. air sat.) (Becker et al., 1996, 1997). Moreover, at a
DOT as low as 6% most kinetic and stoichiometric pa-
rameters shown in Figures 3–5 had reached at least 80% of
the values attained at nonlimiting DOT conditions (20%
DOT). Together, the information and data described above
indicate that oscillating the DOT between 0% to at least 6%,
as was done in this study (Fig. 6), is enough to switch
between fermentative and aerobic pathways.
Several important differences distinguish the scale-down
study conducted here from previous reports employing
E. coli. Namely, an actual independent feedback DOT
control was established in a two-compartment system at
very short tc and with continuously measured DOT pro-files. Accordingly, the magnitude and duration of DOT
gradients were known and could be manipulated to mimic
potential conditions that can occur under real operation
of large-scale cultures (Amanullah et al., 2004; Palomares
and Ramı́rez, 2000a,b). In comparison, previous reports
employing one-compartment scale-down simulators are
limited by their inherently slow dynamic response. In such
systems only relatively long DOT oscillation periods can
be obtained (De León et al., 1995) or no oscillations are
possible at all (Namdev et al., 1993). Faster DOT oscil-
lations, relevant to large-scale operations, can be generated
in two-compartment systems consisting of interconnected
stirred-tank (STR) and plug flow (PFR) reactors (Bylund
et al., 1999, 2000). Such systems are important for simu-
lating fed-batch operations and determining the impact of
pH control, as they can accurately mimic the zone of nu-
trients and/or acid and base addition. Nonetheless, in these
cases, no DOT profiles have been reported and interpre-
tation of the physiological response is more complex, as
DOT oscillations are forced indirectly through changes in
oxygen uptake rate induced by glucose spikes. DOT
oscillations have also been induced by glucose oscilla-
tions in a one-compartment system (Lin and Neubauer,
2000), but a combination of the drawbacks mentioned pre-
viously limits such an approach. In comparison, the sys-
tem used here is most applicable to simulate large-scale
batch operations.
A suitable scale-down system must simulate the relative
volumes of both the well-mixed highly oxygenated zones
and the poorly aerated areas, present at the large scale.
Well- to poorly aerated volume ratios in the range of 0.25–
0.5 have been considered to replicate actual large-scaleconditions (Oosterhuis, 1984; Oosterhuis et al., 1985;
Amanullah et al., 1992, 2004). Accordingly, in this study
an aerobic-to-anaerobic volume ratio of 0.5 was selected to
simulate a batch operation. Scale-down systems based on a
combination of STR-PFR have been successfully employed
for simulating local heterogeneities caused by point ad-
dition of high substrate concentration in fed-batch cultures
(Bylund et al., 1999, 2000; George et al., 1993; Hewitt
et al., 2000; Neubauer et al., 1995; Onyeaka et al., 2003;
Schweder et al., 1999; Xu et al., 1999b). STR-PFR systems
are also useful for assessing the effect of acid/base addition
during pH control. Thus, such systems have been designed
with very small volume fractions (typically 5–9% of total
volume) for the combined high-glucose and low-oxygen
PFR section and very large residence times (up to 9 min) in
the high-oxygen STR compartment. Accordingly, results of
such reports, although very relevant for fed-batch studies,
should be interpreted with caution when investigating DOT
gradients not caused by local surges of glucose concen-
tration. In addition to the relative compartment volumes,
both mean and distribution of circulation times present at
the large scale must be reproduced in the scale-down system.
The use of a two interconnected STR system satisfied the
latter condition as distribution of circulation times has been
shown to closely follow that of single large-scale vessels(Amanullah et al., 2004). Furthermore, a range of mean tc(between 20 to 180 sec), commonly reported for large-scale
fermenters (Amanullah et al., 2004; Palomares and Ramı́rez,
2000a,b), was examined in this study.
Relatively few reports have investigated DOT gradients
under a range of different tc. Most of them have found a
detrimental effect of oxygen oscillations; for instance, in
maximum concentration of B. subtilis (Amanullah et al.,
2004) and Saccharomyces cerevisiae (Sweere et al., 1988),
460 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 89, NO. 4, FEBRUARY 20, 2005
https://www.researchgate.net/publication/13661036_Effects_of_Oxygen_on_Recombinant_Protein_Expression?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/21787685_Effect_of_the_levels_of_dissolved_oxygen_on_the_expression_of_recombinant_proteins_in_four_recombinant_Escherichia_coli_strains?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/14446547_O2_as_the_regulatory_signal_for_FNR-dependent_gene_regulation_in_Escherichia_coli_J_Bacteriol_1784515-4521?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/13924751_Regulatory_O2_tensions_for_synthesis_of_fermentation_products_in_Escherichia_coli_and_relation_to_aerobic_respiration?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/222913271_Effect_of_dissolved_oxygen_tension_on_the_production_of_recombinant_penicillin_acylase_in_Escherichia_coli?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/5242484_Effect_of_oxygen_fluctuations_on_recombinant_E_coli_fermentation?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/225677417_Scale_Down_of_Recombinant_Protein_Production_-_A_Comparative_Study_of_Scaling_Performance?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/225677417_Scale_Down_of_Recombinant_Protein_Production_-_A_Comparative_Study_of_Scaling_Performance?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12458092_Influence_of_scale-up_on_the_quality_of_recombinant_human_growth_hormone_Biotechnol_Bioeng?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12501297_Influence_of_controlled_glucose_oscillations_on_a_fed-batch_process_of_recombinant_Escherichia_coli?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12501297_Influence_of_controlled_glucose_oscillations_on_a_fed-batch_process_of_recombinant_Escherichia_coli?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/41674157_Scale-up_of_bioreactors_A_scale-down_approach?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/225677417_Scale_Down_of_Recombinant_Protein_Production_-_A_Comparative_Study_of_Scaling_Performance?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/12458092_Influence_of_scale-up_on_the_quality_of_recombinant_human_growth_hormone_Biotechnol_Bioeng?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/227208866_A_scale-down_two-compartment_reactor_with_controlled_substrate_oscillations_Metabolic_response_of_Saccharomyces_cerevisiae_Bioproc_Eng_9249-257?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/227973345_Studies_Related_to_the_Scale-Up_of_High-Cell-Density_E_coli_Fed-Batch_Fermentations_Using_Multiparameter_Flow_Cytometry_Effect_of_a_Changing_Microenvironment_with_Respect_to_Glucose_and_Dissolved_Oxyg?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e48e58480e0&enrichSource=Y292ZXJQYWdlOzgxMjEzNDA7QVM6OTc0MDU5NDkxODYwNTFAMTQwMDIzNDc5NjIzOA==https://www.researchgate.net/publication/227973345_Studies_Related_to_the_Scale-Up_of_High-Cell-Density_E_coli_Fed-Batch_Fermentations_Using_Multiparameter_Flow_Cytometry_Effect_of_a_Changing_Microenvironment_with_Respect_to_Glucose_and_Dissolved_Oxyg?el=1_x_8&enrichId=rgreq-de9f0086-2bf5-4fb0-8f0d-6e4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8/16/2019 Cultures of E. Coli Under Dissolved Oxygen Gradients Simulated in A
10/12
in maximum concentration and growth of hybridomas and
N-glycosylation of monoclonal antibody (Serrato et al.,
2004), in maximum mean molecular weight of alginate
by Azotobacter vinelandii (Trujillo et al., 2001), and in
product productivity by Gluconobacter oxydans (Ooster-
huis et al., 1985; Buse et al., 1992). Yet a positive influ-
ence of oscillatory DOT has also been reported, such as
increased glucose oxidase induction and transport in
Aspergillus niger (Träger et al., 1992) and increased h-
galactosidase activity by Kluyveromyces marxianus (Cortés
et al., 2005) compared to control cultures at constant DOT.
In this study, DOT oscillations had a detrimental effect on
A, maximum concentration of biomass and recombinant
protein, yield of biomass on glucose, and yield of
recombinant protein on biomass (Figs. 7–9). This was a
direct consequence of carbon flow deviation to mixed-acid
fermentation as inferred from the increased specific glucose
uptake rate and specific production rate of organic acids as
tc increased. Nonetheless, in both constant and oscillating
DOT cultures a substantial amount of acetic acid originated
from overflow metabolism rather than mixed-acid fermen-
tation as a high concentration of this acid (f2.2 g/L) ac-
cumulated even at 20% constant DOT.Overflow metabolism results when high glucose con-
centration causes glucose uptake to exceed a critical rate
corresponding to maximum respiration rate or oxidative
capacity of the Krebs cycle (Xu et al., 1999a). In turn,
mixed-acid fermentation occurs as a response to DOT
limitation and is transcriptionally regulated by the system
ArcA/ArcB and FNR (Becker et al., 1996; Unden and
Bongaerts, 1997). As seen in Figure 8, mixed-acid fer-
mentation was turned on even at the lowest tc of 20 sec,
corresponding to transient exposures of only 13.3 sec to
anaerobic conditions. Onset of fermentative metabolism as
fast as 24 and 54 sec after oxygen limitation has been
reported previously by Bylund et al. (2000) and Xu et al.
(1999b), respectively. A fast induction of the fermentative
pathways is also in agreement with Schweder et al. (1999),
who observed increased mRNA levels of pyruvate formate-
lyase, fumarate reductase, and acetate kinase after only
13 sec of exposure to high-glucose and low-oxygen con-
centrations in an STR-PFR scale-down system. In such a
system, the residence time in the well-oxygenated STR
section was about 10 min. This allowed sufficient time for
mRNA of key fermentative enzymes to decay, as their
average half-life is about 2 min (Schweder et al., 1999). In
contrast, the residence times studied here for the aerobic
compartment ranged from 7–60 sec, much less than themean half-life of mRNA. Short residence times in the
aerobic compartment can also prevent substantial reassim-
ilation of organic acids generated within the anaerobic
compartment (Xu et al., 1999b; Neubauer et al., 1995). Such
differences can explain the higher accumulation of organic
acids and specific glucose uptake rates obtained here
compared to STR-PFR scale-down systems that rely on
addition of glucose at high concentration (Xu et al., 1999b;
Bylund et al., 1999; Neubauer et al., 1995). It also under-
lines the pertinence of the system employed here for
studying the independent effect of DOT gradients in con-
trast to scale-down studies intended for simulating high
substrate addition in fed-batch cultures through simulta-
neous variation in glucose and oxygen concentrations.
Acetic acid accumulated to the highest concentration,
followed by lactic, formic, and succinic acids. In contrast,
formic acid accumulated to the highest level followed by
acetate and lactate in a PFR-STR scale-down system (Xu
et al., 1999b). Such differences are a result of the different
production and reassimilation rates of organic acids in each
scale-down system. For instance, whereas higher acetic
acid was produced in our configuration by overflow
metabolism due to the overall higher glucose concentration,
acetate and lactate were reassimilated much faster than
formate in the relatively large oxygen-sufficient zone of the
PFR-STR system used by Xu et al. (1999b). Such differ-
ences should be taken into account when designing a repre-
sentative scale-down simulator. No ethanol was detected in
any culture, either at constant or oscillating DOT, which
agrees with the results of Xu et al. (1999b). It is interesting
to note that even at 20% DOT formic, lactic, and acetic
acids, although low, remained at detectable levels. In anycase, the range of DOT where organic acids accumulated
to the highest values are comparable with those for half-
maximal synthesis of fermentation products and expression
of fermentative pathway enzymes reported by Becker et al.
(1996, 1997).
Escherichia coli is a facultative microorganism; how-
ever, no growth or product formation was observed at 0%
DOT. De León et al. (2003) found a similar behavior for a
recombinant E. coli JM101 strain and attributed it to a lack
of nitrate and selenium necessary for growth under an-
aerobic conditions. Formate dehydrogenase H (FDHH) is
a selenium- and molybdenum-containing enzyme that
together with hydrogenase 3 forms part of the formate –
hydrogen lyase complex (FHL) (Sawers, 1994). Under
anaerobic conditions the FHL complex catalyzes the
conversion of formate to H2 and CO2, contributing to the
maintenance of pH homeostasis. If a culture medium lacks
selenium, no FDHH activity will be present under anaerobic
conditions, and therefore growth will be impaired. On the
other hand, nitrate is needed as electron acceptor during
anaerobic respiration (Becker et al., 1997; Unden and
Bongaerts, 1997).
All stoichiometric and kinetic parameters of oscillatory
cultures fell within those of cultures kept at constant DOT
between 0–5%. Accordingly, in oscillatory cultures E. colicells