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Friedrich Käß, Stefan Junne, Peter Neubauer, Wolfgang Wiechert, Marco

Oldiges

Process inhomogeneity leads to rapid side

product turnover in cultivation of

Corynebacterium glutamicum

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Suggested Citation

Käß, Friedrich ; Junne, Stefan ; Neubauer, Peter ; Wiechert, Wolfgang ; Oldiges, Marco : Process

inhomogeneity leads to rapid side product turnover in cultivation of Corynebacterium glutamicum. - In:

Microbial Cell Factories. - ISSN 1475-2859 (online). - 13 (2014), art. 6. - doi:10.1186/1475-2859-13-6.

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Process inhomogeneity leads to rapid side

product turnover in cultivation of

Corynebacterium glutamicum

Käß et al.

Käß et al. Microbial Cell Factories 2014, 13:6

http://www.microbialcellfactories.com/content/13/1/6

RESEARCH Open Access

Process inhomogeneity leads to rapid side

product turnover in cultivation of

Corynebacterium glutamicum

Friedrich Käß

, Stefan Junne

, Peter Neubauer

, Wolfgang Wiechert

and Marco Oldiges

Abstract

Background: Corynebacterium glutamicum has large scale industrial applications in the production of amino acids

and the potential to serve as a platform organism for new products. This means the demand for industrial process

development is likely to increase. However, large scale cultivation conditions differ from laboratory bioreactors,

mostly due to the formation of concentration gradients at the industrial scale. This leads to an oscillating supply of

oxygen and nutrients for microorganisms with uncertain impact on metabolism. Scale-down bioreactors can be

applied to study robustness and physiological reactions to oscillating conditions at a laboratory scale.

Results: In this study, C. glutamicum ATCC13032 was cultivated by glucose limited fed-batch cultivation in a

two-compartment bioreactor consisting of an aerobic stirred tank and a connected non-aerated plug flow reactor

with optional feeding. Continuous flow through both compartments generated oscillating profiles with estimated

residence times of 45 and 87 seconds in the non-aerated plug flow compartment. Oscillation of oxygen supply

conditions at substrate excess and oscillation of both substrate and dissolved oxygen concentration were compared

to homogeneous reference cultivations. The dynamic metabolic response of cells within the anaerobic plug flow

compartment was monitored throughout the processes, detecting high turnover of substrate into metabolic side

products and acidification within oxygen depleted zones. It was shown that anaerobic secretion of lactate into the

extracellular culture broth, with subsequent reabsorption in the aerobic glucose-limited environment, leads to

mixed-substrate growth in fed-batch processes. Apart from this, the oscillations had only a minor impact on growth

and intracellular metabolite characteristics.

Conclusions: Carbon metabolism of C. glutamicum changes at oscillating oxygen supply conditions, leading to a

futile cycle over extracellular side products and back into oxidative pathways. This phenomenon facilitates a

dynamic and flexible shift of oxygen uptake at inhomogeneous process conditions. There is no loss of process

characteristics at oscillation times in the minute range, which emphasizes the robustness of C. glutamicum in

comparison to other industrial microorganisms. Therefore, the metabolic phenotype of C. glutamicum seems to be

particularly well-suited for cultivation at inhomogeneous process conditions for large-scale fed-batch application,

which is in good accordance with the respective industrial experiences.

Keywords: Scale-down, Oxygen supply limitation, Two-compartment reactor, STR-PFR, Oxygen uptake

redistribution, Metabolic robustness

* Correspondence: [email protected]

Institute of Bio- and Geosciences, IBG-1: Biotechnology, Systems

Biotechnology, Forschungszentrum Jülich GmbH, Jülich D-52425, Germany

Full list of author information is available at the end of the article

Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly cited.

Käß et al. Microbial Cell Factories 2014, 13:6

http://www.microbialcellfactories.com/content/13/1/6

Background

Corynebacterium glutamicum is an important organism

for industrial biotechnology. Application in large scale

amino acid production is state of the art in the food and

feed industries, with several established amino acid prod-

ucts [1]. Currently, millions of tons of amino acids for

food and feed application (e.g. continuously improving

L-lysine strains [2], etc.) are produced using C. glutamicum

every year. Also, the increasing demand for bio-based

fine-chemicals is fuelling the search for new and efficient

platform organisms, with C. glutamicum as one promising

candidate (e.g. succinate production [3], L-valine [4-6],

1,2-propanediol [7], L-alanine [8], and other organic acids

[9,10]). Typically, industrial products of C. glutamicum

are bulk chemicals produced in mostly aerobic processes

using reactors of up to 500 m

. Due to the high metabolic

activity of microorganisms, cultivation is performed in

fed-batch mode in stirred tank reactors. The limited car-

bon source feeding reduces oxygen demand and heat gen-

eration. This is beneficial in large scale reactors, which are

often restricted in these aspects due to technical and com-

mercial limitations.

A direct consequence of large scale cultivation is in-

creased gradient formation of nutrients and environmen-

tal process parameters (e.g. pO

, pH, pCO

, substrate

concentration), which represents a major challenge in in-

dustrial biotechnology [11]. Gradients are caused by insuf-

ficient mixing, combined with the high metabolic activity

of microbial cells. One example is oxygen supply distribu-

tion: the solubility of oxygen is very low at typical biopro-

cess conditions, and therefore oxygen is rapidly depleted

in zones of low aeration. An individual microorganism

within a large-scale culture is exposed to gradients and os-

cillating changes of its environment in terms of substrate

availability and dissolved oxygen. Depending on pa-

rameters such as mixing or homogeneity time, i.e. time

required for a defined depletion of gradients [12], oscilla-

tions can vary in their duration and statistical distribution

for individual cells in a microbial culture.

In contrast to large scale bioreactors, gradients and inho-

mogeneities are rarely observed under lab-scale conditions.

Most procedures for strain selection during screening and

early process development are carried out with shaken or

stirred tank bioreactors in a volume range of milliliters to

liters, which is typically associated with mixing times in

the range of seconds [13,14]. Depending on the final pro-

duction scale, industrial homogeneity times of up to sev-

eral minutes can be expected for typical bioprocesses [12],

which results in substantial gradients and an oscillating

exposure of the microbial cells to these gradients. This

constitutes a pitfall for scale-up of microbial processes,

since strain choice and process engineering are derived

from well-mixed laboratory experiments. As a result, se-

lected strains can show decreasing performance during

sequential scale-up, i.e. increasing homogeneity time and

oscillation, resulting in time- and cost-intensive additional

strain or process iteration. Procedures for evaluating meta-

bolic robustness against oscillations can therefore facilitate

the selection of robust strains during laboratory develop-

ment, and can improve transferability of processes from

laboratory to production scale [15,16].

Scale-down simulators are a promising tool for investi-

gating the metabolic impact of industrial production con-

ditions. They are gaining increasing attention, as has been

shown in recent perspective publications [17,18]. They

are mostly used to generate oscillating supply condi-

tions within laboratory bioprocesses. Technical setups

vary from pulse-profile addition of substrate (e.g. de-

scribed in Neubauer et al. [19,20]) to deliberate mixing

time enhancement (e.g. Schilling et al. [21]) and compart-

mented reactors, which allow the investigation of several

environmental inhomogeneities in parallel. Scale-down

designs can be adjusted to represent mixing properties of

technical scale reactors through modeling and simulation,

as was shown for two-compartment reactors by Delvigne

et al. [22], which facilitates the prediction of large-scale

performance.

The significance of scale-down approaches for biotech-

nology is mirrored by the large number of successfully

characterized metabolic properties in industrial microor-

ganisms (e.g. overview in Neubauer et al. [17] and Takors

[18]). Results clearly indicate that almost all aspects of mi-

crobial metabolism, growth and production properties are

affected by bioreactor inhomogeneity. With increased un-

derstanding of metabolic effects in response to oscilla-

tions, scale-down simulation can help to identify better

production strains and optimize industrial operating con-

ditions in the future.

Surprisingly, despite its industrial importance, few publi-

cations have focused on the scale-down characteristics of

C. glutamicum [21,23,24]. The only literature source that

investigates reactor inhomogeneity is Schilling et al. [21],

who cultivated an auxotrophic L-lysine producer in a modi-

fied stirred tank setup. This study focused on increased

mixing times without further gradient characterization,

thus making it rather difficult to relate the described effects,

i.e. decrease of growth and enzyme activities, to the specific

source of the disturbance. Multi-compartment reactors

have not been applied so far.

We have therefore chosen to use a well characterized

two-compartment reactor [25] as a promising experi-

mental alternative. The study aims to assess the process

engineering and metabolic consequences of oxygen and

substrate supply oscillation for Corynebacterium gluta-

micum. Combining bioprocess engineering and distinct

industrial bioreactor-like conditions with a systems biol-

ogy perspective and modern bioanalytics, the effects on

growth, metabolic activity, and net carbon utilization

Käß et al. Microbial Cell Factories 2014, 13:6 Page 2 of 10

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can be characterized. The controlled application of de-

fined reactor inhomogeneity reveals metabolic and physio-

logical effects within the separated reactor compartments,

i.e. aerobic stirred tank reactor (STR) vs. non-aerated plug

flow reactor (PFR), as well as the macroscopic effect for

entire cultures of C. glutamicum.

Results

Oxygen uptake at inhomogeneous supply conditions

In order to study oscillation of oxygen supply during

substrate excess, batch growth was analyzed in a two-

compartment bioreactor (TCR, setup see Figure 1,

process data see Figure 2). During the batch phase sub-

strate was present in excess. Initial batch glucose con-

centration was 22 g/L which was completely consumed

at the end of batch phase (time t = 0, Figure 2) when

fed-batch phase was started. Residence times within the

non-aerated plug flow compartment were set to 45 s

(cultivation TCR1) and 87 s (cultivation TCR2) in differ-

ent experiments, respectively. For each residence time

we have performed one experimental run as a set of ex-

periments with gradually increasing process inhomogen-

eity. The aim was to characterize basic principles of C.

glutamicum in an oscillatory scale-down bioreactor en-

vironment, which can best be demonstrated with such

an approach.

Anaerobic conditions appeared in the plug flow part

of the reactor due to the high glucose concentration and

the resulting high metabolic activity of C. glutamicum,

which could be verified by absence of dissolved oxygen

signals at all plug flow sensor ports throughout the two-

compartment cultivation (Figure 1). In contrast, the aer-

obic stirred tank compartment was kept at high dissolved

oxygen levels (DO > 30%). Reference cultivations were

performed in an aerobic stirred tank without the plug flow

element. After batch glucose had been used up, feed was

added at the entrance of the plug flow compartment. This

led to conditions of glucose excess and simultaneous oxy-

gen limitation within the plug flow compartment, whereas

the overall cultivation remained substrate-limited, and

gives a lab-scale representation of the typical stress condi-

tions in inhomogeneous fed-batch processes (Figure 2). At

batch phase exponential growth and subsequent switch to

substrate limited fed-batch, similar growth activity was ob-

served at homogeneous and oscillating process conditions.

From the very similar growth curve data a good reprodu-

cibility of the experiments can be deduced. Net respiration

activity changed only slightly at batch conditions, as will

be discussed below. This is a clear indication that cells

were not affected by the substrate and oxygen oscillation,

considering the balanced data of both compartments,

i.e. the whole inhomogeneous process.

However, there is a zonal inhomogeneity of oxygen up-

take between the two-compartment cultivations, which

was monitored by determining the specific oxygen uptake

rate (q

) within the individual reactor compartments,

and the whole process setup (Figure 3). Two different phe-

nomena can be distinguished: (i) for batch conditions, i.e.

100

012345

τ[s]

port position

TCR2

Figure 1 Two-compartment scale-down reactor setup. (A) = two-compartment scale-down reactor for analysis of oxygen supply and substrate

oscillations as described in Junne et al. [25] and position of sampling/sensor ports (P-[0–5]), F = feed line entry, color gradient represents accumulation

of fermentative by-products over anaerobic residence time (from blue to red); (B) = mean residence time τat individual port positions for experimental

condition TCR2.

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substrate excess, the net uptake rate of the oscillating cul-

tures is lowered by approximately 13% in comparison to

the homogeneous reference cultivation, which is also the

volume fraction of the PFR in relation to the total two-

compartment volume. A maximum oxygen uptake rate

) of 4 mmol g

-1

is reached in the fully aerobic

referencecultivation,aswellasintheaerobicSTR

compartments of the two-compartment reactor. In con-

trast, oxygen supply is limited in the plug flow compart-

ment, i.e. 0.5 to 1 mmol g

-1

depending on anaerobic

residence time. (ii) For substrate-limited feed conditions,

the net oxygen uptake is identical for the two-compartment

process and the homogeneous reference culture. Inter-

estingly, the q

was higher in the STR compartment of

oscillation

batch feed

-8 -6 -4 -2 0 2 4 6

OTR; feed [mmol·L-1·h-1]

CDW [g·L-1]

t [h]

CDW_REF CDW_TCR1 CDW_TCR2 CDW_mean

OTR_REF OTR_TCR1 OTR_TCR2 feed

Figure 2 Process overview for two-compartment scale-down cultivations of C. glutamicum ATCC13032. First phase: oscillating oxygen

supply at substrate excess (batch, from t=−4 h until t= 0 h); second phase: oscillating oxygen/substrate supply (feed, after t= 0 h); scale-down

cultivations with residence time τ= 45 s (TCR1) and τ= 87 s (TCR2); reference experiment without oscillation in aerobic stirred tank (REF); cell dry

weight (CDW, left axis, samples taken from stirred tank compartment), glucose feed rate (feed, right axis), oxygen transfer rate (OTR, right axis,

same unit as feed); initial batch phase from t = −8 until t = 0 with 22 g/L glucose, fed-batch phase started at t = 0; dissolved oxygen (DO) levels

were always > 30% for aerobic stirred tank compartment and absence of DO signals at all sensors in the plug flow compartment showed

anaerobic conditions.

total STR only PFR only total STR only PFR only

qO2 [mmol·g-1·h-1]

REF TCR1 TCR2

batch feed

Figure 3 Biomass specific oxygen uptake rate q

in batch and feed phase. Balance-based mean uptake rate (= net uptake rate) for stirred

tank compartment (STR, highlighted in green), plug flow compartment (PFR, highlighted in red) and full reactor volume (total) of C. glutamicum

scale-down cultivation for reference experiment (REF, first bars in compartment groups), scale-down cultivation with τ= 45 s (TCR1, second bars),

scale-down cultivation with τ= 87 s (TCR2, third bars), error bars indicate standard deviation over time (see Methods).

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the TCR system, compared to the reference cultivation.

It seems that oxygen uptake is redistributed within the

TCR, from anaerobic to aerobic zones. The q

in the

PFR compartment is limiting, but is compensated for by

increased uptake in the aerobic zone.

Thus, a metabolic difference in the effect of inhomogen-

eous oxygen supply can be demonstrated, depending on

the process mode: under substrate excess batch conditions

the oxygen uptake in the STR compartment reaches the

maximum oxygen uptake capacity of C. glutamicum,and

therefore is not further increased to compensate for miss-

ing oxygen uptake in the non-aerated PFR compartment.

On the other hand, under substrate limited fed-batch

conditions the oxygen uptake is increased to compen-

sate for the missing oxygen uptake in the non-aerated

PFR compartment.

Metabolic impact of anaerobic residence time and

glucose perturbations

Secondary metabolic effects were identified, which re-

sulted from the perturbations in the glucose and oxygen

availability in the TCR. They were monitored in the PFR

compartment at the different sampling positions.

Substrate uptake could be identified by decreasing glucose

concentrations between plug flow ports, with a biomass-

specific uptake rate of q

GLC

= 0.90 (± 0.17) g g

-1

,i.e.

5.0 (± 1.0) mmol g

-1

during the feed phase. This value

is higher than the typical aerobic substrate uptake capacity

of C. glutamicum (own data) and reported microaerobic

uptake rates [26] (both ca. 3mmolg

-1

). Interestingly,

in the PFR there was also a significant pH difference be-

tween the different ports, as is shown for the longer resi-

dence time (τ= 87 s) in Figure 4A. During both the batch

and the feed phase, the pH decreased along the PFR com-

partment. This shows that C. glutamicum expresses fast

acidification of culture broth in response to glucose excess

and oxygen depletion. However, base addition did not in-

crease between reference and oscillating process, indicat-

ing reversibility of this acidification effect, i.e. formation of

acids in the PFR and subsequent consumption of these

acids under glucose limitation and aerobic conditions in

the STR part of the TCR system.

Along with the pH drop, analysis of typical anaerobic

side products of C. glutamicum revealed increasing extra-

cellular lactate concentration as a major by-product over

the anaerobic residence time in the PFR compartment

(Figure 4B). During the feed phase, concentrations kept

increasing up to a maximum of approximately 2.7 mM at

the last (upper) port of the PFR compartment. However,

extracellular lactate could not be detected within the aer-

obic stirred tank compartment. Therefore, we conclude

that the lactate which accumulated under oxygen-limited

conditions in the PFR part was rapidly assimilated at re-

stored oxygen supply in the STR compartment.

In both experimental setups with residence times in

the PFR part of 45 s (TCR1) and 87 s (TCR2), the

oscillation-induced accumulation of lactate increased

over the time of cultivation. The specific rate of lactate

production (q

LAC

) increased with process time, reaching

the highest values at long anaerobic residence times

(TCR2, Figure 5A). The carbon fraction of glucose which

was converted into extracellular lactate during the pas-

sage through the PFR reached considerably high values

of up to 80% (Figure 5B), pointing to a double substrate

process with lactate as a second major substrate at in-

homogeneous process conditions. Since the lactate con-

sumption rate in STR always equals the lactate formation

rate in PFR, no net accumulation of lactate is observed in

the overall TCR setup.

Impact of oscillation on intracellular metabolite pools

The influence of oscillations on intracellular metabolites

was analyzed after cold-methanol quenching of cell

suspensions with an LC-MS/MS procedure [27], using

C-labeled internal standards for absolute quantification.

For this purpose, samples from the STR (P-0) and in the

last (upper) port of the PFR in a two-compartment reactor

6.6

6.7

6.8

6.9

7.0

7.1

7.2

-3 -2 -1 0 1 2 3 4 5 6

pH [-]

t [h]

P-0

P-1

P-2

P-3

P-5

P-0

P-1

P-2

P-3

P-4

P-5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

-3 -2 -1 0123456port

Lactate [mM]

t [h]

Figure 4 pH (A) and lactate (B) profiles along anaerobic plug

flow compartment. Two-compartment scale-down cultivation of

C. glutamicum for batch and feed phase; oscillation of oxygen supply

(t< 0 h, batch) and oscillation of oxygen and substrate supply

(t> 0 h, fed-batch) with τ= 87 s (TCR2), pH (A) and extracellular lactate

concentration (B) indicated for individual port positions (P-[0–5]) as

assigned in Figure 1.

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cultivation with long residence time in the PFR (TCR2) were

compared for the energy related adenosine phosphates

(ATP, ADP, AMP) and redox related cofactors (NAD(H),

NADP(H), Figure 6). Interestingly, the differences between

batch and fed-batch conditions were pronounced, indicating

that the extracellular glucose level is a decisive factor. For

batch conditions, the pool sizes and the calculated energy

charge were at least 2-3 times higher than in fed-batch con-

ditions. In contrast, the metabolite concentrations before

and after anaerobic oscillation were very similar. The ener-

getic state of the cells seemed to be more related to glucose

concentration than to the presence of oxygen. Also, no sig-

nificant changes were observed for redox-related cofactors,

with very similar values seen under all investigated condi-

tions. Therefore, no pattern or trend of oscillation correlat-

ing to either glucose or oxygen presence could be identified.

Discussion

This study illustrates the metabolic robustness of C. gluta-

micum against substrate and oxygen oscillations, which it

is claimed appear within the feed zone of large-scale in-

dustrial bioreactors. It also provides insight into metabolic

principles within inhomogeneous processes in general.

The lack of change in growth and metabolic activity shows

C. glutamicum possesses a high robustness against an os-

cillating oxygen supply, in conditions of both substrate ex-

cess and substrate limitation. Oscillations with anaerobic

residence times τof 45 or 87 s did not result in a decrease

of the final biomass yield or net oxygen uptake at fed-

batch conditions. Since sensitivity against oscillation has

been documented in similar approaches for several other

industrial organisms (e.g. E. coli [28], S. cerevisiae [29] B.

subtilis [25], see also: Lara et al. [11]), this may underline

the special suitability of C. glutamicum for large scale in-

dustrial applications, and even may explain the good prac-

tical experiences during the long history of its use in bulk

0.1

0.2

0.3

0.4

0.5

0.6

200

400

600

800

1000

P-0 P-5 P-0 P-5

energy charge [-]

cintra [µM]

AMP ADP ATP energy charge

batch feed

0.1

1.0

10.0

100.0

1000.0

P-0 P-5 P-0 P-5

cintra [µM]

NAD NADH NADP NADPH

Figure 6 Intracellular concentration of metabolites under aerobic

conditions (P-0) and after anaerobic residence time (P-5) in

two-compartment cultivation TCR2. Quantified by metabolic

quenching/LC-MS/MS [27] in batch and fed-batch phase of C. glutamicum

cultivation with oscillation of τ=87s(TCR2);(A) = adenosine phosphates

(left axis) and resulting energy charge (right axis, calculation and

definition of energy charge see Methods); (B) = redox cofactor

equivalents, batch = substrate excess and oscillation of oxygen

supply, feed = substrate limitation and oscillation of both substrate and

oxygen supply; AMP = adenosine monophosphate, ADP = adenosine

diphosphate, ATP = adenosine triphosphate, NAD = nicotinamide

adenine dinucleotide (oxidized), NADH = reduced, NADP = nicotinamide

adenine dinucleotide phosphate (oxidized), NADPH = reduced.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0123456

qLAC[mmol·g-1·h-1]

t [h]

TCR2

TCR1

REF

20%

40%

60%

80%

100%

123456

C-mol fraction

lactate/glucose [-]

t [h]

TCR2

Figure 5 Reversible lactate turnover at oscillating oxygen

supply in two-compartment cultivation of C. glutamicum.

(A) = lactate turnover rates at reference cultivation of C. glutamicum

without oscillation (REF), scale-down cultivation with short oscillations

(τ= 45 s, TCR1) and scale-down cultivation with long oscillations

(τ= 87 s, TCR2); lactate turnover rate q

LAC

represents the production

rate within non-aerated plug flow compartment and equals the lactate

uptake rate (r

LAC,STR

) within aerobic stirred tank compartment

LAC,PFR

LAC,STR

)withq

LAC

· flow through PFR;(B) =carbon

fraction of feed glucose being converted into extracellular lactate

[mol

C_in_lactate

/mol

C_in_glucose

] within anaerobic plug flow compartment

for experimental condition TCR2.

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chemical synthesis. Connected to this robustness, this

study identified several unique metabolic properties under

inhomogeneous oxygen/glucose supply.

One important difference to homogeneous cultivation

is the shift of oxygen uptake from zones of limited oxygen

supply to aerobic zones. This mechanism is not functional

under conditions of substrate excess, where the net oxy-

gen uptake has already reached the maximum capacity of

=4mmolg

-1

. Under glucose limitation, however,

the demand for oxygen uptake is lower, allowing a com-

pensation of process inhomogeneity to occur. Under

these conditions, a net oxygen uptake similar to that in

homogeneous conditions is preserved, due to the in-

creased uptake of oxygen within the aerobic zones of

the STR. Even at longer residence times in the anaerobic

part of the PFR, the relatively small volume fraction in

the two-compartment system facilitates an efficient

compensation by the larger aerobic bulk volume. This

mechanism provides a basis for metabolic robustness

against partial oxygen supply limitation, and illustrates

the superiority of the fed-batch mode under inhomo-

geneousprocessconditions.

The basis for oxygen uptake compensation was identi-

fied in the rapid metabolic switch from aerobic substrate

utilization to fermentative pathways. The necessity for

this is evident, because substrate uptake is sustained

during anaerobic oscillation. The rapid redirection of

substrate carbon flow was observed directly by the in-

crease in extracellular lactate concentrations over PFR

residence time. The anaerobic metabolism in homoge-

neous cultures, which has already been described exten-

sively (e.g. publications by Inui et al. [30] and Yamamoto

et al. [31]), was shown to be rapidly activated during the

anaerobic oscillations: C. glutamicum switches from aer-

obic respiration to fermentative pathways with lactate as

a predominant side product among other organic acids

(e.g. acetate, succinate). A pH decrease is a secondary

effect of side product accumulation; this could also be

observed in this study. The results therefore demon-

strate the robustness and flexibility of the aerobic/an-

aerobic carbon metabolism. This is substantiated by the

physiological reaction at fully anaerobic conditions, at

which C. glutamicum undergoes growth arrest and sub-

strate uptake decrease [26], which is not observed under

oscillating conditions. It can be concluded that the physio-

logical changes at anaerobic conditions follow a slow re-

sponse, and are not triggered by short-term depletion in

inhomogeneous environments.

In comparison to studies of e.g. E. coli [28,32], some of

which made similar observations about side product

turnover [33], the extent of carbon flow redirection in

the different zones of the TCR is surprisingly high. Even

at moderate biomass, the majority of glucose is rapidly

transformed into lactate during the passage through the

oxygen-limited PFR. Later, in the STR part of the TCR,

lactate is metabolized; however, this kind of futile transport

cycle does not affect the biomass growth. Theoretically,

while lactate secretion is thermodynamically favorable, the

reassimilation step should lead to metabolic energy loss

(aspects on transport described in Stansen et al. [34]). The

formation of lactate from pyruvate by lactate dehydrogen-

ase is a reversible reaction, i.e. the NADH used for lactate

formation is regenerated in the reaction back to pyruvate.

From this point of view the intermediary formation of lac-

tate does not consume or generate energy.

This is different for the transport of lactic acid over the

cell membrane. Currently, it is not fully understood in lit-

erature how lactic acid is excreted or taken up in C. gluta-

micum. The excretion of lactic acid could be managed

without use of energy, simply due to the concentration

gradient under conditions of lactic acid formation. How-

ever, the following uptake (i.e. re-assimilation) of lactic

acid against the concentration gradient would require en-

ergy to overcome the thermodynamically unfavorable con-

centration gradient. Assuming that one energy equivalent

in form of ATP would be required for the uptake mechan-

ism this would require one ATP equivalent for each mol-

ecule lactic acid formed during the cultivation.

Since there is no negative impact on process perform-

ance observed in this case, the required additional energy

for lactate utilization seems to be negligible in the context

of the overall energy metabolism. The information that

there is, in fact, a high turnover of substrate into side

product at oscillating oxygen supply conditions is import-

ant for the characterization of inhomogeneous processes:

instead of growing on primary substrate alone, the degree

of inhomogeneity defines the composition of primary sub-

strate and secondary, organic acid carbon sources for

growth in the bulk culture.

From the metabolic perspective, the rapid switch of sub-

strate utilization during oscillating oxygen supply means

that NADH reoxidation shifts from aerobic respiratory

phosphorylation to detoxification through action of the

enzyme lactate dehydrogenase. This should affect cellular

energy levels, which are represented in the adenosine

phosphates, because substrate level phosphorylation gener-

ates only minor amounts of ATP compared to the aerobic

pathways. In the intracellular concentrations, however, the

changes during anaerobic residence time do not seem to be

of a considerable magnitude, and seem instead to be related

to the level of substrate availability in the entire process.

Most strikingly, the metabolism maintains its NAD(H)

and NADP(H) levels throughout the oscillation phase, as

the reduced side product lactate is transported out of the

cells. This avoids a disturbance of the metabolic network

and seems to provide a fast and flexible intermediary op-

tion for NADH reoxidation. This rapid action effectively

avoids any negative effects of NADH accumulation in the

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cytoplasm, such as redox imbalance or decreased glyco-

lytic substrate consumption due to reduced glyceraldehyde

3-phosphate dehydrogenase activity at an unfavorable

NAD/NADH ratio. Also, the cellular energy charge (EC)

is maintained at a constant level, which is in the range of

previously reported studies for batch growth [5], and

drops along with absolute pool sizes of adenosine phos-

phates at substrate limitation. This phenomenon is a typ-

ical effect of the lower substrate-to-biomass ratio, as was

previously observed in other cultivations (not shown).

Notably, the energy charge remains at a similar magnitude

over anaerobic residence time in the feed phase, even

though the cells experience substrate limitation before ex-

posure to the oxygen supply limitation/substrate excess

step change. There is, however, a generally lower pool size

of adenosine phosphates observed for the substrate limited

process phase, which might also influence the speed and

efficiency of energy-generating reactions. In any case, the

seemingly similar pool size of important metabolites dur-

ing anaerobic oscillation is an indication of metabolic ro-

bustness for C. glutamicum.

The main advantage of the applied scale-down method,

i.e. assessment of metabolic robustness in a scale-down

bioreactor with two compartments, is that changes in

microenvironment, e.g. pH or carbon flux redirection, can

be monitored along the plug flow reactor while also

checking for performance parameters of the whole pro-

cess setup, e.g. productivity or growth. Scale-down simula-

tors with plug flow compartments can form the link

between understanding microbial responses to oscillating

conditions and simulation of industrial performance. Since

anaerobic residence times of 87 s are in the range of real-

istic mixing or homogeneity times for industrial reactors

[12], this study provides an example for the assessment of

microbial robustness against application-oriented process

inhomogeneity. As described by Delvigne et al. [22],

two-compartment reactors can be adjusted to mimic

realistic mixing conditions of industrial reactors. With

this study showing one example, a two-compartment

system can also be applied for microbial robustness

assessment by comparing different degrees of oscilla-

tion. In upcoming studies, this strategy can be pursued

further by increasing residence times within anaerobic

zones and thereby studying the metabolic robustness of

C. glutamicum against more challenging inhomogenei-

ties. Knowing the threshold of an organism for adapta-

tion to oscillating conditions provides valuable insight

for all process transfers, especially at large-scale indus-

trial applications, and could give some estimation of the

scalability of the particular biological system.

Conclusions

The results of this study indicate that Corynebacterium

glutamicum is robust against oscillating oxygen supply

limitation of fed-batch environments with anaerobic

residence times in the lower minute range. The reason

for oxygen starvation is usually high metabolic activity,

which is triggered by local substrate excess and high

cell densities in large scale bioreactors. The presented

scale-down approach for this phenomenon can identify

metabolic properties of process inhomogeneity. The mi-

crobial response to oscillation involves a fast adaptation

to the conditions in the different reactor compartments,

with a high rate of lactate formation in the high glucose/

low oxygen zone, and a resulting mixed-substrate uptake

(joint use of glucose and lactate) in the bulk culture. The

robustness of the fed-batch is mainly caused by compen-

sation of process inhomogeneity in a rapid, reversible

switch to fermentative anaerobic metabolism, which sur-

prisingly leaves no negative impact on metabolic proper-

ties. Therefore, the native phenotype of C. glutamicum is

well-adjusted to oscillation at typical fed-batch process

conditions, which makes it particularly well-suited for

large-scale application. This is unique among the indus-

trial organisms which have previously been subjected to

similar scale-down analysis. Further research should focus

on the underlying physiological properties which facilitate

this extraordinary robustness, in the hope of exploiting

them for future bioprocess development in metabolic and

process engineering.

Methods

Reactor setup and operation

The chosen two-compartment bioreactor setup (scale-down

reactor) consists of a stirred tank bioreactor (Biostat E,

Sartorius SA, Goettingen, Germany) and a connected plug

flow compartment built from commercial elements, as

has been previously described by Junne et al. [25]. It fea-

tures sampling and sensor ports for pH and dissolved oxy-

gen, both within the stirred tank and at five distinct

positions within the plug flow compartment (Figure 1).

The volume proportion of the two compartments is 82%

for the aerobic stirred tank (8.2 L working volume) and

18% for the anaerobic plug flow compartment (1.8 L).

Static mixer elements are installed along the plug flow

compartment. Mean residence times τat the individual

ports are indicated for the experiment TCR2 with mean

residence time τ= 87 s (Figure 1). Mean residence times

were determined by extrusion experiments, as described

in Levenspiel [35], and are slightly higher than hydro-

dynamic residence times (τhyd ¼VPFR

:) due to backmixing

effects. Plug flow characteristics were maintained at all ex-

perimental conditions, as demonstrated by the determin-

ation of Bodenstein numbers above 10 for the plug flow

compartment at experimental flow conditions [29]. Due to

the plug flow behavior and optimized geometry of the plug

flow setup, a hypothetical impact of stagnant zones on the

reactor performance can be neglected. Circulation was set

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to a constant flow using a peristaltic pump at flow rates of

2.64 L · min

-1

(τ

(P-5)

= 45 s) and 1.32 L · min

-1

(τ

(p-5)

=87s).

For reference cultivation without oscillation, the plug flow

compartment was omitted, resulting in a full volume of

10 L in stirred tank aerobic process with top feeding. The

feed line was equipped with a backpressure valve and intro-

duced behind the pump (reference: top feeding). Circula-

tion through the plug flow compartment was initiated 4 h

before feed start, resulting in oscillation of oxygen supply

conditions at remaining batch glucose within the non-

aerated plug flow compartment, before entering the feed

phase with oscillation of oxygen and substrate supply.

pH and dissolved oxygen sensors within the reactor

setup were calibrated as described in Junne et al. [25].

Oxygen transfer rates (OTR) were calculated according

to Junne et al. [36] from off-gas measurements per-

formed with paramagnetic oxygen analyzer and spectro-

scopic infrared CO

sensor (Binos, Fisher-Rosemount,

Wessling, Germany). Biomass specific oxygen uptake

rates were calculated by dividing OTR by biomass con-

centration, with batch phase t=[−2, -0.5] and feed

phase t = [0.5, 2] (biomass fitted exponentially from

growth curve in batch phase, linear in feed phase). Total

oxygen uptake for the plug flow compartment is calculated

assuming maximum solubility c

O2,max

= 225 μmol · L

-1

CgXII minimal medium, as can be estimated according to

media component salt effects [37,38], and assuming complete

consumption from initial c

init

=DO

STR

[%] · c

O2,max

Strain, media, and culture conditions

Cultivation of C. glutamicum ATCC13032 in the two com-

partment scale-down reactor was performed in CgXII

minimal medium [39] containing 22 g L

-1

of initial batch

glucose · H

O. Constant feed was applied after the end of

the batch phase (identified by increase in DO-signal) with

double concentrated CgXII, 440 g L

-1

glucose · H

Oata

flow rate of 60 mL · h

-1

. pH was maintained at pH = 7 by

addition of 25% (v/v) NH

OH solution. Antifoam AF204

(Sigma, Missouri, U.S.A.) was added to the medium before

inoculation in 0.5‰(v/v). Temperature was maintained at

30°C. Aeration rate was set to 0.3 vvm. In order to

maintain aerobic conditions within the stirred tank,

stirrer speed was regulated for DO > 30%. Reactors

were inoculated with OD

600

= 0.005 for an initial batch

phase of 15 h before start of feed phase at approxi-

mately OD

600

= 30, t = 0 h.

Sampling

Sampling of cell free culture supernatant from ports of the

stirred tank reactor and the ports along the plug flow re-

actor was facilitated using the self-locking Monovette®

port system (Sarstedt AG, Nümbrecht, Germany) with a

needle adapter remaining in every port septum throughout

cultivation. Samples were taken with syringes equipped

with 25 mm, 0.8 μm pore size CA-syringe filters (Carl

Roth, Karlsruhe, Germany) connected to Monovette

adapters. Immediate cell separation was necessary to avoid

further anaerobic substrateconversionaftersampling

(e.g. lactate).

Analytics

Cell dry weight was determined as the mean of threefold

determination from cells washed in 0.9% (w/w) NaCl

solution, after > 24 h of drying at 80°C. Supernatants

were assayed for organic acid side products (pyruvate,

succinate, malate, lactate, acetate, fumarate and citrate)

with an Agilent 2100 Infinity HPLC system in 0.1 mol · L

-1

at flow rate 0.5 mL min

-1

, 34 min/sample isocratic

separation with column and precolumn of organic acid resin

(300 × 8 mm, CS Chromatographie Service, Langerwehe,

Germany). For the investigation of intracellular metabo-

lites, the method as described in Paczia et al. [27] was

applied, which is based on isotope dilution mass spec-

trometry with

C-labeled internal standards from C. glu-

tamicum ATCC13032 cell extracts, reaching quantitative

determination after LC-ESI-MS/MS analysis. Metabolic

quenching for immediate inactivation of enzymatic action

was performed in a cold methanol solution, using pre-

cooled syringes with 60% (V/V) methanol in 1:4 dilution

(2 mL culture suspension + 6 mL pre-cooled quenching

solution, resulting temperature approximately −20°C)

and Monovette® port adapters, centrifugation (−20°C),

and subsequent chloroform extraction from biomass

(50% chloroform, 25% TE buffer, 25% methanol [V/V]).

Extraction was performed in 2 mL of extraction volume

with 4 h of incubation time (agitation on shaker, -20°C),

centrifugation (-20°C), and aqueous phase separation.

Measurement was performed as specified in Paczia

et al. [27]. Intracellular concentrations were calculated

accounting for leakage of metabolites into quenching

supernatant (measured separately), and excluding me-

tabolite leakage into culture supernatant (measured

separately, leakage into culture supernatant was negli-

gible for all presented metabolites). Cellular energy

charge was calculated according to Atkinson et al. [23]:

ergy charge ECðÞ¼

ATP½þ

2ADP½

ATP½ADP½AMP½

;(squarebracketsindi-

cating intracellular concentrations).

Competing interests

The authors have declared that no competing interest exists.

Authors’contributions

FK prepared the manuscript. FK and SJ designed and performed the

experiments, developed and validated the experimental methods. SJ and PN

designed and validated the two-compartment reactor concept. FK and SJ

conducted the experiments. MO and WW initiated the project. MO is the

principal investigator, and supported with design and manuscript preparation.

All authors read and approved the final manuscript.

Käß et al. Microbial Cell Factories 2014, 13:6 Page 9 of 10

http://www.microbialcellfactories.com/content/13/1/6

Acknowledgements

The authors thank the Bundesministerium für Bildung und Forschung (BMBF)

for funding in the cluster project “Corynebacterium: Improving flexibility and

fitness for industrial production”(grant no. 0315589A), and the fruitful

cooperation with industrial project partner Evonik Industries. The study was

partially supported by a project from the German Research Foundation (DFG

project no. 1360/2-1). We also thank Hamilton (Bonaduz, Switzerland) for the

donation of the pH and DO sensors for the PFR module. The authors thank

Florian Glauche, Arjun Prasad, Petra Geilenkirchen and Dr. Nicole Paczia for

assistance with conducting fermentation experiments and analytical

procedures.

Author details

Institute of Bio- and Geosciences, IBG-1: Biotechnology, Systems

Biotechnology, Forschungszentrum Jülich GmbH, Jülich D-52425, Germany.

Chair of Bioprocess Engineering, Department of Biotechnology, Technische

Universität Berlin, Ackerstrasse 71-76, Berlin D-13355, Germany.

Received: 5 July 2013 Accepted: 9 December 2013

Published: 10 January 2014

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doi:10.1186/1475-2859-13-6

Cite this article as: Käß et al.:Process inhomogeneity leads to rapid side

product turnover in cultivation of Corynebacterium glutamicum.Microbial

Cell Factories 2014 13:6.

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