a study of oxygen transfer in shake flasks using a non-invasive oxygen sensor

A Study of Oxygen Transfer in ShakeFlasks Using a Non-InvasiveOxygen Sensor

Atul Gupta, Govind Rao

Department of Chemical and Biochemical Engineering, University ofMaryland Baltimore County, 101 ECS Building; 1000 Hilltop Circle,Baltimore, Maryland 21250; telephone: 410-455-3415; fax: 410-455-6500;e-mail: [email protected] 26 February 2003; accepted 17 April 2003

Published online 21 August 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10740

Abstract: We describe a study of oxygen transfer inshake flasks using a non-invasive optical sensor. Thisstudy investigates the effect of different plugs, presenceof baffles, and the type of media on the dissolved oxygenprofiles during Escherichia coli fermentation. We mea-sured the volumetric mass transfer coefficient (kLa) un-der various conditions and also the resistances of thevarious plugs. Finally, we compared shake flask kLa withthat from a stirred tank fermentor. By matching kLa’s wewere able to obtain similar growth and recombinant pro-tein product formation kinetics in both a fermentor and ashake flask. These results provide a quantitative com-parison of fermentations in a shake flask vs. a bench-scale fermentor and should be valuable in guiding scale-up efforts. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84:

351–358, 2003.Keywords: shake flasks; oxygen transfer; stirred tank fer-mentor; oxygen sensor

INTRODUCTION

Shake flasks are used in the biotechnology industry for avariety of tasks including the screening of wild-type strains,strain development, elucidation of metabolic pathways, me-dia optimization, investigations of basic process conditions,and evaluation of fundamental growth kinetics. These stud-ies are performed in shake flasks because of the sheer num-ber of experiments involved (Kennedy et al., 1994). Experi-mental investigations in these vessels are often the first stepin fundamental studies as well as in developing a large-scalefermentation process.

Aeration in shake flasks is achieved by simple gas liquidcontact aided by shaking the vessels in reciprocating orrotary shaking machines. Shake flasks are traditionallysealed with different types of plugs to prevent contamina-tion. These plugs however are not good closures with regardto air permeability (McDaniel and Bailey, 1969; McDanielet al., 1965; Tunac, 1989). It has been shown that a cottonplug in the shake flask can limit the mass transfer signifi-

cantly such that the oxygen in the headspace may fall to aslow as 6% and carbon dioxide may accumulate up to as highas 15% (Schultz, 1964). Oxygen transfer in shake flaskspartly depends on the flow of air through the plug and so thelength of the neck of the flask or the type of closure greatlyaffects the oxygen transfer to the surface of the liquid. Manyworkers have demonstrated that the use of baffled flasksresult in increase in oxygen transfer (Gaden, 1962; Mc-Daniel et al., 1965; Smith and Johnson, 1954). Baffles in-crease the agitation in the liquid as well as the availablesurface area for oxygen transfer at the air–liquid interface.Oxygen availability is very critical during microbial growthin submerged fermentations because of the poor solubilityof oxygen in water. Changes in oxygen availability maylead to drastic effects on fermentation kinetics (Clark et al.,1995; Delgado et al., 1989; Hopkins et al., 1987). However,neither the plugs nor the geometry of flasks have been stan-dardized for use in bioprocess development. This can beattributed to the lack of bioprocess monitoring in these ves-sels which has led to a poor understanding of physical pa-rameters that characterize the culture growth conditions(Buchs, 2001; Henzler and Wuppertal, 1991) in shakeflasks.

To assess if a given culture vessel would be able to sup-ply oxygen at a nonlimiting rate, it is essential to have agood estimate of the oxygen transfer capacity of the vessel.This can be measured in terms of the oxygen mass transfercoefficient (kLa). The kLa often serves to compare the effi-ciency of bioreactors and their mixing devices, as well asbeing an important scale-up factor in the bioprocess indus-try. It is the measure to quantify the effects of operatingvariables on the provision of oxygen. Hence, it becomesextremely important to measure the oxygen mass transfercoefficient in a bioreactor. So far, shake flasks have notbeen used to perform the scale-up of fermentation processesdue to the lack of knowledge of the conditions under whichshake-flask fermentations are performed. However, with theuse of modern optical sensors and bioprocess monitoringdevices, the role of shake flasks in bioprocess developmentmay be further improved.

The transfer of oxygen from outside of the flask to the

Correspondence to: Govind RaoContract grant sponsor: National Science Foundation; DuPont; Fluo-

rometrix; Genentech; Merck; PfizerContract grant number: BES0091705

© 2003 Wiley Periodicals, Inc.

inside takes place through diffusion through the plug. Theheadspace air diffuses into the liquid in the flask. The oxy-gen balance for the headspace in the flask can thus be writ-ten as follows:

Increase in gas phase oxygen in the flask � (rate ofdiffusion through cotton plug) − (rate of transfer across gasliquid interface)

Increase in gas phase oxygen in the flask =

k�CG − CH� − kLa�CH

M− CL� � VL (1)

where k � plug transfer coefficient; kLa � gas–liquid in-terface transfer coefficient; CH � concentration of oxygenin headspace; CG � concentration of oxygen outside flask;CL � concentration of oxygen in liquid; VL � volume ofliquid in flask; M � equilibrium constant.

When the steady state is achieved the total flux across theplug would be same as the flux across the gas liquid inter-face. Rearranging the above equation thus:

� = � 1

Mk+

1

kLaVL�−1�CG

M− CL� (2)

At equilibrium across the cotton plug MCG can be replaced

with CL* (saturation concentration of oxygen in liquid). Theresulting equation can be compared with the OTR equationgiven as:

OTR = kLa�C*L − CL� (3)

and hence the equivalent mass transfer coefficient for ashake flask can be defined as:

�kLa�eq =1

VL� 1

Mk+

1

VLkLa�−1

(4)

The above expression for mass transfer coefficient isequivalent to the sum of resistances at the sterile plug and atthe gas–liquid interface. In previous studies, others havetried to measure the two transfer coefficients separately(Maier and Buchs, 2001; Mrotzek et al., 2001) using thesulfite oxidation method (Corman et al., 1957; Smith andJohnson, 1954; Tunac, 1989) or gassing-out method with aClark-type electrode (Hirose et al., 1966; Schultz, 1964) butthe combined mass transfer coefficient has not been re-ported. The previously used methods have their limitations(Schell et al., 2001). The sulfite oxidation method requiressampling at regular intervals thus limiting its utility. Toavoid regular sampling some workers (Hermann et al.,2001) have used an optical online system for a sulfite-oxidation method of oxygen transfer measurement. Clarkelectrodes used in other studies for OTR measurements inshake flasks are not appropriate for a variety of reasons(Tribe et al., 1995). Non-invasive optical sensors are a betteralternative to measure oxygen concentration in small bio-reactors because they circumvent a variety of problems as-sociated with the conventional probes.

In this study, we have retrofitted the shake-flask systemwith a luminescence-based sensor for online measurement

of oxygen (Tolosa et al., 2002). This is a non-invasive sen-sor that measures the dissolved oxygen concentration in ashake flask with minimal perturbation of the actual condi-tions of a growing culture.

MATERIALS AND METHODS

Optical Oxygen Sensor

The optical sensor system (Fluorometrix, Stow, MA) usedfor oxygen measurement consists of two parts: a coaster anda patch (Tolosa et al., 2002). The coaster contains LEDs forexcitation and photo detector for light detection. The patchis paper-thin, autoclavable, and contains an oxygen-sensitive luminescent dye, [1,2-bis(diphenyl phosphino)eth-ane Pt[S2C2(Ch2-CH2-N-2-pyrimidine](BPh4), immobilizedin a silicone matrix. The patch is mounted on the interiorsurface of the flask (Fig. 1). The flask sits on top of thecoaster, which is connected to a computer. The computerruns a Lab View (National Instruments Inc., Austin, TX)program to acquire the data online. This sensor is based onthe dynamic quenching of excited-state of the luminescentmetal-ligand complex by molecular oxygen. Dynamicquenching usually follows the Stern-Volmer equation:

I0

I=

�0

�= 1 + ksv�0�Q� (5)

where I � emission intensity, � � decay lifetime, ksv �stern volmer constant, [Q] � quencher concentration, sub-script 0’s indicate the absence of quencher (oxygen). Theconcentration of the quencher (O2 in this case) is deter-mined by measuring the change in the lifetime of the lumi-nophore (Bambot et al., 1994; Lakowicz, 1999).

The luminescent oxygen sensor was calibrated with waterin the flask and the temperature controlled at 37°C. Thesensor was mounted in the shake flask, which was spargedwith a mixture of nitrogen and oxygen. The mixture ratio

Figure 1. The above schematic is a close view of the flask and thecoaster. The flask is equipped with a paper-thin luminescent patch mountedon the interior of the flask. Underneath the flask is the coaster with light-emitting diode (LED) with an excitation filter, which emits the excitationlight, and detector with an emission filter, which receives the emission lightfrom the patch.

352 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 84, NO. 3, NOVEMBER 5, 2003

was controlled with the help of precision gas mass flowmeters (Emerson Electric Co., Brooks Instrument division,Hatfield, PA).

Response Time Correction for kLa Measurement

The gassing-out method of kLa measurement (describedlater) is associated with a limitation. This method necessi-tates the use of membrane-type electrodes, the responsetime of which may be inadequate to reflect the true changein the rate of oxygenation over a short period of time (Tribeet al., 1995). The probe response-time is the time needed torecord 63% of a stepwise change and this should be muchsmaller than the mass transfer response time of the system(1/kLa) (Stanbury and Whitaker, 1989).

Assuming the diffusion of oxygen through the membraneof the probe as a first-order response (Brown, 2001), thediffusion process can be defined as:

dCp

dt=

1

�p�CL − Cp� (6)

where Cp � the concentration of oxygen at the probe tip, CL

� concentration of oxygen in bulk fluid and �p is the timeconstant of the probe.

The time constant (63% response) of the fluorescencesensor was calculated to be 30 s and that of the Clark elec-trode used in this study to be 3 s at 37°C.

Substituting CL from Eq. (3) in Eq. (6) and solving forCP, we get:

Cp�t� = C*L�1 +kLa

kp − kLae−kpt −

kp

kp − kLae−kLat� (7)

where, kLa � response time corrected mass transfer coef-ficient and kp � mass transfer coefficient of the probe (in-verse of the probe time constant).

All the values of transfer coefficient reported in this textwere corrected for the delay due to the response time of theoxygen-measuring device.

Mass Transfer Coefficient Measurements

In this study we used the method of gassing out for themeasurement of mass transfer coefficient due to the sim-plicity of the method. The culture vessel was first filled toworking volume with a reference medium. The medium wasthen purged of oxygen by bubbling nitrogen through it untilthe medium was oxygen-free. Finally, the system wassparged with air and the resulting oxygen concentration wasmeasured (Stanbury and Whitaker, 1989). We used thismethod of measurement of oxygen transfer coefficient inshake flasks to measure the two transfer coefficients of thetwo resistances in a shake flask separately.

Experiment 1

The gas–liquid mass transfer coefficient in a shake flaskwas measured by the method described by Suijdam et al.

(1978). In this method the oxygen in the shake flask wasfirst displaced by nitrogen. After this the gas phase abovethe liquid was replaced by air, the shaker was turned on andthe oxygen tension in the liquid in shake flask was regis-tered with time.

Experiment 2

The plug transfer coefficient was measured by placing theoxygen sensor in the headspace of the flask. The flask wasfilled to working volume with water. Oxygen in the head-space was displaced by nitrogen. When the headspace oxy-gen concentration reached zero, the shaker was switched onto allow the gas to diffuse in and the oxygen concentrationwas recorded in the gas phase.

The optical sensor system (Fluorometrix, Stow, MA) wasplaced under the 250 mL conical flask (Bellco Glass Inc.,Vineland, NJ) mounted on the Lab-line orbit environ shaker(Lab-line Instruments Inc., Melrose Park, IL) with a 1.6-cmshaking diameter. The flask equipped with the oxygen sen-sitive patch (Fluorometrix, Stow, MA) was filled with 100mL distilled water. The flask was closed with an appropriateplug (cotton plug, sponge plug, and milk filter) and a thinplastic tube [3 mm O.D. (outer diameter) and 1.5 mm I.D.(inner diameter)] was passed through the plug to spargenitrogen through it. The baffled flask used in this study hadfour indentations, each 6 cm long and 1.5 cm deep. Thevarious plugs used were cotton plug (manually made with 1g of absorbent cotton), sponge plug (S/P diSPo plugs foropenings 28- to 35-mm outer diameter; Baxter HealthcareCorporation, Deerfield, IL) and milk filters (16.5 cm disksnon-gauze; Ken AG, Ashland, OH).

For the mass transfer coefficient measurement in the fer-mentor, the 1.5 L New Brunswick Scientific Bioflo III (Edi-son, NJ) was used. The fermentor was filled with 1 L dis-tilled water and equipped with a Clark type electrode(Model 21800-022, Control Company, Friendswood, TX) tomeasure oxygen. The reactor was set at a particular agitationspeed, aeration rate, and 37°C. House air and nitrogen wasused for kLa measurement using the static gassing outmethod.

Fermentation

Escherichia coli strain JM105 was transformed with thepBAD-GFP construct. (Lu et al., 2002). The seed culturewas prepared by growing 1% inoculum overnight in 10 mLbuffered Luria Bertani (LB) in a 50 mL culture tube at 250rpm and 37°C. A 4% inoculum was used from the seedculture to start a shake-flask fermentation in LB (10 g/Lbacto tryptone, 5 g/L bacto yeast extract, 10 g/L NaCl, pH7.2, 4 g/L K2HPO4, 0.5 g/L KH2PO4) or minimal media(Glucose: 5 g/L−1, NH4Cl: 1 g/L−1, K2HPO4: 1 g/L−1,MgCl2: 200 mg/L−1, FeSO4: 10 mg/L−1, CaCl2: 10 mg/L−1)at 250 rpm and 37°C in a 250 mL baffled/unbaffled shakeflask with 100 mL media volume. This amount of mediawas used in 250 mL flasks for these experiments to avoid

GUPTA AND RAO: OXYGEN TRANSFER IN SHAKE FLASKS USING A NON-INVASIVE OXYGEN SENSOR 353

the exposure of the patch to air caused by vortex formationduring agitation.

Stirred-tank fermentations were performed in the 1.5-LNew Brunswick Scientific Bioflo III (Edison, New Jersey)fermentors. Dissolved oxygen probe (Ingold) was calibratedto read 0% with nitrogen gas and 100% with air. The fer-mentor was filled with 1 L LB media and autoclaved at121°C and 15 psi for 30 min.

The seed culture was begun using 1% inoculum in 50 mlLB media in a 250 mL flask incubated overnight at 35°Cand 250 rpm. The fermentor was inoculated with 4% seedculture of E. coli pBAD-GFP construct. Aeration in thefermentor was set at 1 volume of gas/volume of culture/minute (VVM) and temperature at 37°C for all the fermen-tations. Each fermentation was performed at a constantspeed of the impeller throughout the fermentation time.

After 5 doublings (about 3 h), when the culture reachedexponential phase, GFP was induced with 0.2% arabinosesolution. The optical density of the culture was measured at600 nm with Milton Roy Spectronic 401 spectrophotometer.The fluorescence intensity was measured offline using Var-ian Cary Eclipse fluorescence spectrophotometer (VarianInc., Australia).

RESULTS AND DISCUSSION

The phase response of the fluorescence sensor was recordedand plotted against percentage DO (Fig. 2). The hyperbolicnature of the calibration curve of the oxygen sensor leads tohigher sensitivity at low DO levels. This property of thesensor is advantageous for DO measurements in fermenta-tion and cell culture work (Tolosa et al., 2002). However,

the sensor has low sensitivity for DO levels above 60%.Thus, only the data below 60% DO were used for the masstransfer coefficient measurements.

Using the oxygen sensor the mass transfer coefficient wasmeasured for the individual resistances in a shake flask(Table I and Table II). A comparison between the oxygentransfer through the plug and the gas–liquid interface wasmade by calculating the two resistances separately. Thetransfer resistance of the shake-flask closure can be ex-pressed by (1/k); and the resistance of the gas–liquid inter-face by (M/VL � kLa). From Table I it follows that for thesponge plug the transfer resistance is: 3.7 � 103 h/m3 andfrom Table II the transfer resistance of the gas–liquid inter-face in an unbaffled flask is: 1.33 � 104 h/m3 (M � 41, VL

� 10−4 m3). Clearly, the resistance provided by the gas–liquid interface was much higher than that of the plug. Theindividual plug resistances when compared with each othershowed a large difference among them. In Table II, thetransfer coefficient of the gas–liquid interface changed two-fold with the introduction of baffles.

The corrected equivalent mass transfer coefficient values(Table III) under different conditions in the shake flask wereobtained by substituting individual transfer coefficients inEq. (4). The equivalent mass transfer coefficient is expectedto be a better estimate of the transfer capacity of a shakeflask as it accounts for both the gas–liquid interface resis-tance and the plug resistance.

The effect of baffles and different sterile closures in ashake flask was tested on the cell growth during E. colifermentation. An experiment performed with three un-baffled flasks covered with different plugs (cotton, sponge,and milk filter) and a baffled flask covered with milk filtershowed that the difference in the oxygen transfer rate in ashake flask under varying conditions led to different oxygenprofiles during fermentation (Fig. 3a). All the unbaffledflasks reached zero-dissolved-oxygen level in the brothwhile the baffled flask maintained dissolved oxygen levelabove the critical value at all times during the fermentation.(Critical value of dissolved oxygen concentration is a char-

Table I. Plug mass transfer coefficient obtained with Experiment 2 in anunbaffled shake flask with 100 mL water at 37°C and 250 rpm.

Closure k (m3/h)

Sponge plug 2.7 E −04 ± 1.4 E −04Cotton plug 9.1 E −04 ± 3.3 E −04Milk filter 5.7 E −03 ± 8.6 E −04

Table II. Gas–liquid mass transfer coefficient obtained with Experiment1 in an open shake flask with 100 mL water at 37°C and 250 rpm.

Type of flask kLa (h−1)

Baffled 59.2 ± 7.4Unbaffled 30.8 ± 6.7

Table III. Equivalent mass transfer coefficient calculated from Equation(4) using the two transfer coefficients separately from Tables I and II.

Closure Flask kLa (h−1)

Sponge plug Unbaffled 24 ± 3Cotton plug 28.4 ± 6Milk filter 30.4 ± 6Sponge plug Baffled 40.3 ± 5Cotton plug 53.9 ± 8Milk filter 57.6 ± 7

Figure 2. The calibration curve for the optical sensor shows a hyperbolicrelation with the dissolved oxygen measurement from the Clark electrode.The sensitivity of the sensor is high for low DO values.


acteristic of each microorganism and for E. coli the value isaround 4% air saturation) If the dissolved oxygen concen-tration falls below the critical level then the cells may bemetabolically disturbed.

The equivalent overall mass transfer coefficient of theunbaffled flasks with different plugs in Table III rangedfrom 24 h−1 to 30.4 h−1 while the mass transfer coefficientof the baffled flask with milk filter cap was close to 57.6

Table IV. Duration of oxygen limitation in the shake flask during E. colifermentation calculated from Figure 3a. Oxygen limitation in the flask isvery sensitive to the change of plug and the introduction of baffles.

Closure Flask Duration (h)

Sponge plug Unbaffled 10.5Cotton plug 9Milk filter 7.2

Milk filter Baffled 0

Table V. Volumetric mass transfer coefficient in a 1.5-L stirred-tankfermentor with 1 L distilled water and 37°C at an aeration rate of 1 vvm.

Impeller speed (rpm) kLa (h−1)

25 9.8 ± 2.250 10.4 ± 1.8

100 14.2 ± 1.9200 19.9 ± 3.0300 25.7 ± 1.8350 33.9 ± 2.2

Figure 3. (a) DO during E. coli fermentation in four flasks with 100 mL LB media performed in parallel at 37°C and 250 rpm. The DO in the unbaffledflasks falls below critical value while the baffled flask does not get oxygen-limited. (b) Growth curve of E. coli fermentation in four flasks with 100 mLLB media carried out in parallel at 37°C and 250 rpm. Growth rate is higher for the flask with higher oxygen transfer rate.


h−1. This difference in the oxygen transfer capacity of theshake flasks under different conditions resulted in the dif-ferent dissolved oxygen profiles during fermentation (Fig.3a). The dissolved oxygen in the unbaffled flask withsponge cap remains below critical level for around 10.5 hwhile for the same flask with milk filter cap, this duration is7.2 h (Table IV). This shows that the change of plug of ashake flask affects the mass transfer coefficient, which, inturn, changes the duration of oxygen limitation during cellgrowth in them.

The effect of different oxygen transfer rates were alsoapparent on the biomass growth in the LB media. In Figure3b the baffled shake flask with milk filter cap, which had thehighest oxygen transfer rate, resulted in the fastest biomassgrowth rate during E. coli fermentation. Shake flasks withlower oxygen transfer rate had slower growth rates in theirexponential phase. The impact of oxygen transfer was not

limited only to the biomass growth rate but it also affectedthe extent of total biomass formation. Maximum biomassaccumulated in the flask with the highest oxygen transferwhile the biomass formation was lowest in the flask with thelowest oxygen transfer (Fig. 3b).

Interestingly, the DO profile in E. coli fermentation inminimal media in a similar experiment as above was ob-served to be oscillating in all the four flasks (Fig. 4). Thesevariations in the oxygen consumption rate may be due tometabolic oscillations (Anderson et al., 2001). A compari-son between Figures 3a and 4 shows that the DO was belowcritical value for a longer duration in LB media than inminimal media. This is probably due to lower oxygen de-mand in the minimal media caused by carbon source limi-tation. However, in both the figures, duration of oxygenlimitation was minimal in the baffled flask as compared tothe other flasks. Thus, the online monitoring of DO gives a

Figure 4. DO during E. coli growth in four flasks with 100 mL minimal media run at 37°C and 250 rpm. The flasks with high equivalent mass transfercoefficient, i.e., with milk filter cap, do not get oxygen-limited. The DO keeps oscillating in all the four flasks during E. coli growth.

Figure 5. The effect of mass transfer coefficient in a shake flask and a fermentor on the duration that oxygen remains limiting during culture growth ofE. coli.


good indication of the effect of change of media compo-nents on cell metabolism.

Table V lists the corrected volumetric mass transfer co-efficient in a bench scale fermentor with 1 L distilled wateroperated at different impeller speed. The volumetric masstransfer coefficient in the stirred-tank fermentor was mea-sured using the gassing-out method, to compare shake-flaskfermentations with those performed in a stirred-tank fer-mentor. The mass transfer coefficient in the fermentor in-creased almost linearly with the speed of the impeller at aconstant aeration rate.

Figure 5 illustrates the effect of the mass transfer coeffi-cient in the reactor vessel on the duration for which oxygenremains below critical value during E. coli fermentation.Both the stirred-tank fermentor and the shake flask showeda decrease in the duration of oxygen limitation with increasein the mass transfer coefficient of the bioreactor. In thebench-scale fermentor operated at 100 rpm, the volumetricmass transfer coefficient was 14 h−1 and the oxygen remainslimited for around 25 h for E. coli under these conditions.With an increase of agitation speed to 300 rpm, the masstransfer coefficient increased to 25.7 h−1 and the duration ofoxygen limitation dropped down to around 10 h. Similarlyfor the unbaffled shake flask, with a sponge plug the masstransfer coefficient was 24 h−1 and the oxygen remainedlimiting for around 10.9 h for E. coli fermentation. With achange of plug to the milk filter under the same conditions,the mass transfer coefficient increased to 30.4 h−1 and theduration of oxygen limitation dropped down to around 6.5h. This shows that the dissolved oxygen profile in a growingculture is very sensitive to the mass transfer coefficient ofthe bioreactor.

To compare the shake-flask fermentation with a stirred-tank fermentor the two vessels were run in parallel under thesame fermentation conditions of kLa temperature, inoculumpercentage, and media composition. The plug of the shakeflask was chosen (cotton plug) such that the equivalent masstransfer coefficient was approximately equal to the kLa ofthe bench-scale fermentor operated at 300 rpm agitation and1 vvm aeration (≈ 26 h−1). Both the shake flask and thefermentor were inoculated at the same time with an over-night grown seed culture in a common shake flask. After 3h of inoculation, the GFP was induced with 2% arabinose inboth the fermentors. The fermentation in the two vesselswas compared by observing the cell growth, the dissolvedoxygen profile, and the GFP formation. Figure 6a shows thedissolved oxygen concentration profile in the shake flaskand the fermentor. The DO profile had a similar pattern forfermentation in both the bioreactors. The period of oxygendepletion and the recovery of the dissolved oxygen to 100%were similar and reproducible in the two bioreactors. Thecurves of the data comparing the biomass and GFP forma-tion in the two vessels show good agreement (Fig. 6b, c).Hence, we can deduce that the equivalent mass transfercoefficient measured using the optical oxygen sensor, canbe used as the scale-up criteria of fermentation from theshake flask to the stirred-tank fermentor. This method of

process scale-up is comparatively simpler and easier thanthe power consumption model suggested in other studies(Buchs et al., 2000a; 2000b).

Shake flasks are well established and have proven to be avery useful and valuable tool for initial culture experimentsand screening purposes. However, the lack of fundamentalknowledge of the physical background and the controlingparameters in shake flasks is still a problem. This is mainlydue to the lack of custom made bioprocess-monitoring de-vices for shake flasks. Shake flasks will quite certainly con-tinue to be the most applied bioreactors for mass screeningin the future and there is an acute need for the improvementof shake flasks by providing on-line bioprocess monitoringin them. With the use of the optical oxygen sensor used inthis study for on-line measurement of dissolved oxygen un-

Figure 6. Time profiles of fermentation variables in shake flask in com-parison with a standard 1 L bioreactor at same culture conditions during E.coli growth. (A) Dissolved oxygen profiles. (B) Optical density profiles.(C) GFP production profiles.


der sterile conditions, much information about the cultureand the fermentation conditions can be gained. In this study,the sensor was used to perform the mass transfer coefficientmeasurements in the shake flask and also to understand theprocess of oxygen transfer into the flask. The sensor wasused to compare the DO profiles in E. coli fermentationunder different conditions and to quantify the effect ofphysical conditions of operation of the shake flask on thecellular growth in them. Also, with an understanding of theequivalent mass transfer coefficient in a shake flask and theeffect of different conditions on kLa, E. coli fermentation ina shake flask was scaled-up to a bench-scale fermentor un-der identical conditions namely kLa, media composition,inoculum percentage, and temperature. Thus, it can be de-duced that the new device for online oxygen monitoring inshake flask will play an increasing and promising role inreactor characterization and bioprocess development. Thedevice will support both the screening of media or strainsand be an important part of a scale-up procedure in researchin the bioprocess industry.

We thank Dr. Yordan Kostov, Dr. Leah Tolosa and Peter Harmsfor their helpful suggestions.

NOMENCLATURE

kG Gas side mass transfer coefficient (hr−1)kL Liquid side mass transfer coefficient (hr−1)M Equilibrium constantCL Saturation oxygen concentration in liquid (g O2/m3)Cp Oxygen concentration at probe tip (g O2/m3)DO2 Diffusivity constant of oxygen through the plug� Flux of oxygen (g O2/hr)�p Time constant of probe (s)kLa Volumetric mass transfer coefficient (hr−1)(kLa)eq Equivalent mass transfer coefficient (hr−1)� Decay lifetime (ns)kq Bimolecular quenching constantOTR Oxygen transfer rateDO Dissolved oxygenI Emission intensity

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a study of oxygen transfer in shake flasks using a non-invasive oxygen sensor

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