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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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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.



6/12

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.



7/12

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 (


8/12

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.


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==


9/12

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),


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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

cultures of e. coli under dissolved oxygen gradients simulated in a

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