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A novel method for the measurement of oxygen mass transfer rates in small-scale vessels
Biochemical Engineering Journal 25 (2005) 63–68
A novel method for the measurement of oxygen mass transfer rates in small-scale vessels Kenny Ortiz-Ochoa a , Steven D. Doig a , John M. Ward b , Frank Baganz a,∗ a
b
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Received 26 October 2004; received in revised form 31 March 2005; accepted 10 April 2005
Abstract A novel method for the measurement of the volumetric oxygen transfer coefficient, kL a, using the catechol-2,3-dioxygenase (XylE) bio-oxidation of catechol yielding 2-hydroxymuconic semialdehyde (2-HS), has been developed for small-scale systems. This method was kinetically characterized and validated by comparison to other established techniques. The bio-oxidation rate was found to be zero order over a catechol concentration from 2 to 7 mM l−1 . When the enzyme concentration was in excess and the bio-oxidation rate was mass transfer limited, indicated by a percent DOT of zero, the oxygen transfer rate was determined from the linear increase in product concentration. The method was validated in a 2 l stirred tank vessel equipped with a DOT probe and connected to a gas mass spectrometer. The novel method yielded similar kL a values when compared to the dynamic gassing out method under the same conditions. The applicability of this novel method for small-scale devices was demonstrated by measuring kL a values of up to 150 h−1 in shaken microplates with a working volume of only 200 l. © 2005 Elsevier B.V. All rights reserved. Keywords: Enzyme kinetics; Bio-oxidation; Mass transfer; Small-scale
1. Introduction In aerobic cell cultivation oxygen transfer is an important parameter [1]. The volumetric oxygen transfer coefficient, kL a, relates the concentration driving force to the oxygen transfer rate and therefore measuring kL a is key to the design of an aerobic cell cultivation process. The kL a depends on many factors including the fluid properties (density, viscosity and diffusivity), the size of gas–liquid interfacial area and the system geometry and operating conditions (e.g. stirrer speed in a stirred tank) [2]. There are many methods to determine kL a [3] and these are briefly described (see also Table 1). Dynamic methods measure the rate at which oxygen-depleted liquid can be reoxygenated and use a probe to measure the rate of increase of oxygen concentration over time. The main problems with ∗
Corresponding author. Tel.: +44 20 7679 2968; fax: +44 20 7290 0723. E-mail address: [email protected] (F. Baganz).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.04.003
this method are the need for an intrusive probe and the effect of the probe response time on the measurement [4]. Another approach, useful during active cell respiration, is to measure the oxygen concentration of the inlet and outlet gas streams and from a simple mass balance the oxygen uptake rate can be determined. Assuming that the liquid oxygen concentration can be measured, then the kL a can be determined. This method is often termed the oxygen balance method. A third method is to measure the reaction rate of an oxygen consuming process (e.g. cellular respiration or chemical oxidation). If the reaction rate is oxygen limited, i.e. the liquid oxygen concentration is zero, then the kL a can be determined if the reaction stoichiometry is known. Common examples of this type of method include measuring the oxygen limited growth rate of a strictly aerobic microorganism [5] and measuring the rate of sodium sulphite oxidation [6]. The use of small-scale and high throughput cell cultivation devices has recently become prominent in bioprocess
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K. Ortiz-Ochoa et al. / Biochemical Engineering Journal 25 (2005) 63–68
Table 1 Comparison of different methods for kL a evaluation Method
Scale applied
kL a range (h−1 )
Assay time
Assumptions/limitations
Dynamic gassing out
>100 ml only
0 to >1000
Several minutes
Oxygen balance
>100 ml only
0 to >1000
Several minutes
Sodium sulphite oxidation
Lab scale only
0 to >1000
Many hours
Linear growth of strict aerobe
Any scale
20 to 300
Many hours
Bio-oxidation of catechol Enzymatic oxidation of glucose
<100 ml Small scale
<300 <200
5–10 min <1 min
Invasive probes are necessary; probe response time must be considered; gassing time can be significant at larger scales Invasive probes are necessary; only possible during active oxygen consumption; requires large gas flows and gas analysis Alterations to concentration driving force, diffusion coefficient and coalescence properties; complex kinetics and boundary layer reduction Assumptions about growth kinetics are required; experimentally laborious Availability of oxidative enzyme; limited to small scales Sample handling and fast reaction; expensive reagents and complex reaction kinetics; limited to small scales
development so that large numbers of cell lines can be quantitatively evaluated [8,15]. In this way cultivation processes can be developed quicker whilst using less material. Until recently shake flasks were the most commonly used cultivation vessel, but now microtiter plates and microfluidic chips are being considered as they provide greater experimental throughput and the potential for automation. The measurement of oxygen transfer rates in these devices is clearly important for their further development as quantitative tools in bioprocess application. However, due to the small scale and non-conventional geometry, the use of well-established measurement techniques is either not possible or not ideal. For example, the use of conventional oxygen probes is unrealistic due to their large size. Although novel fluorescent probes have been developed (http://www.presens.de) their application with shaken vessels is complicated since the agitation must be paused for the duration of each measurement [9]. Likewise, the oxygen balance method cannot be used due to the very small volumes of gas flow and the fact that most small-scale vessels are open and no direct sweep gas is supplied. The sulphite oxidation method has been used to measure kL a values in shaken microwells [6], but the kinetics of the reaction are complex, the analysis time is long (many hours) and the liquid physical properties (diffusivity and saturation concentration) are significantly altered [10]. Attempts have been made to address these concerns by using a dilute enzymatic oxidation of glucose coupled to a secondary reaction yielding a colourful product that can be easily measured in a spectrophotometer [11]. Although, this method overcomes the disadvantages of the sulphite oxidation, difficulties still arise from the low volumetric oxygen uptake capacity (2 mM l−1 ) and that the reaction is quick with samples needed to be taken every 15 s. The implication of these two problems is that the environment and handling time between taking a sample and measuring it are critical and thus highly prone to error. The main aim of this research was therefore to develop a novel method for determination of kL a, which could be used for small-scale systems.
1.1. Proposed method The proposed method consists of an oxygen consuming reaction, operating under mass transfer limited conditions. The primary requirements of the method are that it should be rapid, controllable, kinetically robust and simple to perform. Ideally measurements should take in the order of 5–10 min, thus making them practically manageable. The system should be dilute to avoid significant alterations to the fluid properties. Specifically the method is based on the bio-oxidation of catechol forming 2-hydroxymuconic semialdehyde (2-HS), catalyzed by the enzyme catechol-2,3-dioxygenase (XylE) [12] (see Fig. 1). XylE forms part of the toluene oxidation pathway expressed from the Tol plasmid in Pseudomonas putida [13]. A Xhol restriction fragment, containing the XylE gene, was inserted into the multicopy plasmid pUC8 to yield pQR109 and gene expression was controlled by the lac promoter in Escherichia coli JM107. The enzyme uses 1 mol of molecular oxygen to convert 1 mol of catechol into 1 mol of 2-hydroxymuconic semialdehyde, which is a bright yellow compound that can be quantified at 425 nm. We suggest that if the enzyme concentration is in excess and the bio-oxidation rate is zero order, then the rate at which the product is formed, d(2-HS)/dt, will equal the oxygen transfer rate (OTR) and since, OTR = kL a(C* − C), and C = 0, then the kL a can be determined by measuring the linear rate of formation of the brightly coloured 2-HS (t is time, C* is the saturation concentration of oxygen, and C is the actual concentration of oxygen in the liquid).
Fig. 1. Bio-oxidation of catechol by the enzyme catechol-2,3-dioxygenase (XylE).
K. Ortiz-Ochoa et al. / Biochemical Engineering Journal 25 (2005) 63–68
2. Materials and methods 2.1. Microorganism and media E. coli JM107 pQR109 was constructed by JMW at University College London, UK, using standard methods of cloning and expression such as agarose gel electrophoresis, DNA purification, restriction digestion, ligation, transformation and cell cultivation [14]. The growth media consisted of: 10 g l−1 d-glucose, 10 g l−1 tryptone, 10 g l−1 yeast extract, and 50 mg l−1 ampicillin. All components were obtained from Sigma–Aldrich (Gillinghan, Dorset, United Kingdom). For induction of XylE, 1 mM l−1 isopropyl -d-1 thiogalactopyranoside (IPTG) was added to the media. The ampicillin and IPTG were filter sterilized by 0.2 m pore filter.
65
chol was then added as a 10% (v/v) of a 70 mM l−1 aqueous solution. The bio-oxidation was monitored by taking samples every minute and recording the optical density at 425 nm (Ultraspec 400, Pharmacia Biotech). Samples were diluted 100 times with deoxygenated water (nitrogen purged) and the optical density was converted into concentration using an external calibration curve, with an extinction coefficient of 15,750 l mol−1 cm−1 . The large dilution factor not only allowed for more accurate measurement of the product concentration, but also quenched the bio-oxidative reaction. The time taken to remove a sample and dilute it was typically less than 10 s and after this point the reaction ceased and could be measured.
3. Results and discussion 2.2. Cell cultivation and XylE preparation 3.1. Reaction kinetics One hundred millilitres of media in a 500 ml Erlenmeyer flask was inoculated with a frozen stock (−80 ◦ C, 50%, v/v glycerol) and grown overnight in an orbital shaker (K¨uhner, Birsfelden, Switzerland) at 30 ◦ C and 280 rpm. This culture was then used to inoculate either another 100 ml shake flask or a 1.5 l stirred tank fermenter (LH 210 series, Bioprocess Engineering Services, Charing, Kent, UK). IPTG was added prior to inoculation. For the larger scale an airflow rate of 1 VVM was used, and the pH was controlled at 7 by the metered addition of 4 M KOH. The impeller speed was set at 1200 rpm and the oxygen concentration was measured by a Clark type oxygen electrode (Ingold Messtechnik, Urdorf, Switzerland). The biomass concentration was determined by monitoring optical density at 600 nm (Ultraspec 400, Pharmacia Biotech, Cambridge, UK) and converted to a dry cell weight concentration using a calibration curve. Typically the fermentation was completed once the OD (600 nm) had reached 16–18 resulting in a dry cell weight of 6–7 g l−1 . In shake flask cultivation the final dry cell weight achieved was lower at between 3.5 and 4 g l−1 . In either scale the cells were subsequently harvested using a Beckman J2M1 centrifuge, fitted with a JA10 rotor (High Wycombe, Buckinghamshire, United Kingdom) at 10,000 rpm for 10 min. The biomass was then resuspended in one third (except where stated) of the initial volume in 50 mM phosphate buffer (pH 7) and disrupted either in a sonicator, soniprep 150 MSE (Beckenham, Kent, United Kingdom) or in a homogenizer Lab 60 APV Manton Gaulin (Crawley, W. Sussex, United Kingdom) depending of the volume. Sonication was carried out at 4 × 10 s at an amplitude of 6 m with 10 s cooling periods. Large-scale homogenization was carried out in five passes at 500 bar. Subsequently, cell debris was separated by centrifugation at similar conditions and the supernatant, containing XylE, was collected. 2.3. Catechol bio-oxidation The enzyme preparation was placed in the reaction vessel and once the temperature had equilibrated at 30 ◦ C, the cate-
Studies of the bio-oxidation kinetics were performed in 4 ml plastic cuvettes where 2.7 ml of the oxygen saturated enzyme preparation (oxygen concentration ≈0.2 mM l−1 [1]) was mixed with 0.3 ml of catechol solution. The reaction was monitored on-line. In this way the maximum specific activity of the enzyme preparation was determined. These experiments were executed without concentrating the enzyme during resuspension and were routinely performed with each new batch of enzyme preparation in order to ensure that sufficient activity was available. Reducing the volume of the resuspension buffer prior to disruption and thus increasing the concentration factor resolved cases of low specific activity. Fig. 2 shows the reaction rate attained at different catechol concentrations in this system. The highest bio-oxidation rate achieved was 0.63 mM l−1 min−1 at a catechol concentration of 2 mM l−1 and therefore the maximum specific activity was
Fig. 2. Effect of catechol concentration on the bio-oxidation activity of the enzyme catechol-2,3-dioxygenase (XylE) at 30 ◦ C and pH 7. Inset: product formation profile during a batch bio-oxidation in a 4 ml cuvette. (- - -) 15 mM l−1 catechol, (—) 2 mM l−1 catechol.
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180 U g DCW−1 obtained from 3.5 g DCW l−1 . Fig. 2 (inset) shows the product formation profile from which this value was obtained and for comparison also shows the rate when the catechol concentration was 15 mM l−1 . At catechol concentrations between 2 and 7 mM l−1 the bio-oxidation rate ranged from 0.58 to 0.63 mM l−1 and since the percentage variation was less than 10% the bio-oxidation rate was considered to be zero order with respect to catechol in this concentration range. At higher concentrations some inhibition was observed. The oxygen uptake capacity of the system in the zero order range was 5 mM l−1 . Since we considered a minimum analysis time of 5 min to be practically manageable, and assuming that the stoichiometry shown in Fig. 1 is valid, the maximum accurately measurable oxygen transfer rate is 60 mM l−1 h−1 . If the dissolved oxygen concentration is assumed to be zero, and the driving force is 0.2 mM l−1 , the maximum kL a that can be accurately measured using this novel method is about 300 h−1 . 3.2. Characterization of a typical catechol bio-oxidation The oxidation stoichiometry was experimentally determined and the assumption of zero DOT during the biotransformation was verified in a 1 l scale reaction in which product formation, dissolved oxygen concentration and the oxygen uptake rate were measured, see Fig. 3. Upon addition of catechol to the system, 2-HS was produced immediately and the rate of formation was constant at 22.4 mM l−1 h−1 . The kL a was calculated from this product formation rate as 112 h−1 . Also, the dissolved oxygen concentration decreased to zero due to its rapid uptake by the enzymatic oxidation. Concomitant with this was a decrease in the oxygen content of the air exiting the reactor and thus the oxygen uptake rate (OUR) increased sharply to a constant value of about 20 mM l−1 h−1 shown in Fig. 3. There was a time delay of about 3–4 min in
Fig. 3. A typical catechol bio-oxidation profile by the enzyme catechol-2,3dioxygenase (XylE) in a 2 l stirred tank using a 1 l working volume, 1vvm aeration rate, 30 ◦ C, pH 7 and impeller speed of 900 rpm. (- - -) DOT%, (—) OUR, () [2-HS] considered for kL a determined, () [2-HS] not considered for kL a determination.
these OUR data and this was due to distant location of the mass spectrometer in relation to the bioreactor. In this experiment, 7.5 mM l−1 of catechol was initially added to the bioreactor and the final concentration of 2-HS was 7.12 mM l−1 and thus a 95% conversion was achieved. These experiments verify the reaction stoichiometry shown in Fig. 1, i.e. that 1 mol of catechol is converted to 1 mol of 2-HS using 1 mol of molecular oxygen. The assumption of mass transfer limiting conditions (i.e. a DOT of zero) was also demonstrated since this is vital for kL a determination from OTR values in small-scale vessels without DOT probes. 3.3. Method validation The aim of this section was to compare this novel biooxidation technique to the established dynamic gassing out method. This was considered important as the chemical sulphite oxidation method, also applicable in small-scale systems without probes, often leads to incorrect kL a values [10]. Fig. 4 compares the results from both methods at different stirrer speeds in the 2 l bioreactor. The dynamic method resulted in slightly lower kL a values than those determined from the bio-oxidation method, but the difference was less than 15% in all cases. This difference was considered acceptable and therefore mass transfer coefficients obtained using the novel method are valid. 3.4. Application in microplates Fig. 5 compares kL a values determined in shaken 96well microplates measured using a variety of techniques. The methods compared with the catechol bio-oxidation were: (a) the sulphite oxidation technique (data shared from [6]); (b)
Fig. 4. Parity plot comparing the kL a values determined using the enzymatic method and dynamic method over a range of impeller speeds (, 700; , 900; , 1000; and ♦, 1100 rpm) in a 2 l stirrer tank. The dashed lines represent ±15% either side of parity.
K. Ortiz-Ochoa et al. / Biochemical Engineering Journal 25 (2005) 63–68
Fig. 5. Comparison of kL a values determined using the bio-oxidation against other methods in a 96-well shaking microplate (liquid volume = 0.2 ml). () kL a values determined using the sulphite oxidation method from [6]; () kLa values determined using the linear growth rate of Bacillus from [7]; (—) parity line, the dashed lines represent ±25% either side of parity.
the oxygen limited linear growth of a strict aerobe, Bacillus subtilis [7]. Results from the bio-oxidation of catechol are in good agreement with data from the linear growth rate method. However, results from the sulphite oxidation were slightly higher at low shaking speeds and slightly lower at high shaking speeds. It is established that the sulphite oxidation method tends to over estimate the kL a [3]. However, it provided under-estimates when the shaking frequencies were at their highest and where the gas–liquid surface area was deformed most. We suggest that this is due to the significance of surface tension in these small-scale systems and therefore at higher shaking frequencies the deformation in the surface area, increasing the kL a, will be higher when using biological process liquids due to the presence of surface-active agents. Therefore the bio-oxidation method presented in this paper provides a closer approximation to the conditions in a ‘real’ fermentation broth than the sulphite oxidation method and therefore we suggest is a more appropriate method of measuring oxygen mass transfer rates.
4. Conclusions The proposed novel bio-oxidation method, based on the oxidation of catechol, was kinetically characterized and shown to be zero order over the catechol concentrations 2–7 mM l−1 and if the enzyme was in excess, the DOT during the bio-oxidation was 0%. The simple reaction stoichiometry was experimentally verified and we estimate that kL a values of up to 300 h−1 can be accurately measured. Table 1 compares this novel method with other established techniques. Advantages and disadvantages exist for all the methods, but in general either the dynamic gassing out or
67
the oxygen balance methods are most suitable for lab and pilot scale systems. In these cases the use of conventional oxygen probes is manageable and problems with probe response time can be accounted for. In small-scale devices kL a determination from the linear growth rate of a strict aerobe is valid and offers the advantage that the mass transfer rate is being determined in a representative bio-medium. However, the technique is very time consuming and therefore other mass transfer limited reaction rate strategies are preferred. Endpoint measurement of the sulphite oxidation is complicated by the adverse effect on the physical properties of the liquid and is mostly unfavoured. The bio-oxidation of catechol is both rapid and unencumbered by these problems and since no kinetic assumptions are required, it provides a true evaluation of the kL a. We expect that the wider availability of the enzyme catechol-2,3-dioxygenase should result in the novel method presented here to be commonly used during kL a evaluation in small-scale devices.
Acknowledgements The authors would like to thank the UK Joint Infrastructure Fund (JIF), the Science Research Investment Fund (SRIF) and the Gatsby Charitable Foundation for funds to establish the UCL Centre for Micro Biochemical Engineering. Financial support for the research work presented in this paper is acknowledged from the UK Engineering and Physical Sciences Research Council (EPSRC). Prof. Dr. Jochen B¨uchs of the Biochemical Engineering Department at Aachen University (Germany) is gratefully acknowledged for sharing his oxygen transfer data used in comparing to our developed method.
References [1] J.E. Bailey, D.F. Ollis, Biochemical Engineering Fundamentals, second ed., McGraw Hill International Editions, 1986. [2] H.W. Blanch, D.S. Clark, Biochemical Engineering, Marcel Dekker Inc., 1996. [3] P.M. Duran, Bioprocess Engineering Principles, xth ed., Academic Press, 2003. [4] I.J. Dunn, A.J. Einsele, Oxygen transfer coefficients by the dynamic method, J. Appl. Chem. Biotechnol. 25 (1975) 707–720. [5] W.A. Duetz, L. Ruedi, R. Hermann, K. O’Connor, J. Buchs, B. Witholt, Methods for intense aeration, growth, storage and replication of bacterial strains in microtiter plates, Appl. Environ. Microbiol. 66 (2000) 2641–2646. [6] R. Hermann, M. Lehmann, J. Buchs, Characterization of gas–liquid mass transfer phenomena in microtiter plates, Biotechnol. Bioeng. 81 (2002) 178–186. [7] G.T. John, I. Klimant, C. Wittmann, E. Heinzle, Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation, Biotechnol. Bioeng. 81 (2003) 829– 836. [8] S.D. Doig, A. Diep, F. Baganz, Characterisation of a novel miniaturised bubble column bioreactor for high throughput cell cultivation, Biochem. Eng. J. 23 (2005) 97–105.
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[9] K. Van’t Riet, Review of measuring methods and results in nonviscous gas–liquid mass transfer in stirred vessels, Ind. Eng. Chem. Process Des. Dev. 18 (1979) 357–364. [10] W.A. Duetz, B. Witholt, Oxygen transfer by orbital shaking of square vessels and deepwell microtiter plates of various dimensions, Biochem. Eng. J. 17 (2004) 181–185. [11] J.M. Sala-Trepat, W.C. Evans, The meta cleavage of catechol by Azotobacter species. 4-Oxalocrotonate pathway, Eur. J. Biochem. 20 (1971) 400–413. [12] S. Inouye, A. Nakazawa, T. Nakazawa, Molecular cloning of TOL genes xylB and xylE in Escherichia coli, J. Bacteriol. 145 (1981) 1137–1143.
[13] J. Sambrook, E.F. Fritsch, T. Maniatis, in: C. Nolan (Ed.), Molecular Cloning, A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, USA, 1989. [14] S.D. Doig, F. Baganz, G.J. Lye, High throughput screening and process optimsation, in: C. Ratledge, B. Kristiansen (Eds.), Basic Biotechnology, third ed., Cambridge University Press, Cambridge, UK, 2004. [15] G.J. Lye, P. Ayazi-Shamlou, F. Baganz, P.A. Dalby, J.M. Woodley, Accelerated design of bioconversion processes using automated microscale processing techniques, Trends Biotechnol. 21 (2003) 29– 73.
A novel method for the measurement of oxygen mass transfer rates in small-scale vessels Kenny Ortiz-Ochoa a , Steven D. Doig a , John M. Ward b , Frank Baganz a,∗ a
b
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Received 26 October 2004; received in revised form 31 March 2005; accepted 10 April 2005
Abstract A novel method for the measurement of the volumetric oxygen transfer coefficient, kL a, using the catechol-2,3-dioxygenase (XylE) bio-oxidation of catechol yielding 2-hydroxymuconic semialdehyde (2-HS), has been developed for small-scale systems. This method was kinetically characterized and validated by comparison to other established techniques. The bio-oxidation rate was found to be zero order over a catechol concentration from 2 to 7 mM l−1 . When the enzyme concentration was in excess and the bio-oxidation rate was mass transfer limited, indicated by a percent DOT of zero, the oxygen transfer rate was determined from the linear increase in product concentration. The method was validated in a 2 l stirred tank vessel equipped with a DOT probe and connected to a gas mass spectrometer. The novel method yielded similar kL a values when compared to the dynamic gassing out method under the same conditions. The applicability of this novel method for small-scale devices was demonstrated by measuring kL a values of up to 150 h−1 in shaken microplates with a working volume of only 200 l. © 2005 Elsevier B.V. All rights reserved. Keywords: Enzyme kinetics; Bio-oxidation; Mass transfer; Small-scale
1. Introduction In aerobic cell cultivation oxygen transfer is an important parameter [1]. The volumetric oxygen transfer coefficient, kL a, relates the concentration driving force to the oxygen transfer rate and therefore measuring kL a is key to the design of an aerobic cell cultivation process. The kL a depends on many factors including the fluid properties (density, viscosity and diffusivity), the size of gas–liquid interfacial area and the system geometry and operating conditions (e.g. stirrer speed in a stirred tank) [2]. There are many methods to determine kL a [3] and these are briefly described (see also Table 1). Dynamic methods measure the rate at which oxygen-depleted liquid can be reoxygenated and use a probe to measure the rate of increase of oxygen concentration over time. The main problems with ∗
Corresponding author. Tel.: +44 20 7679 2968; fax: +44 20 7290 0723. E-mail address: [email protected] (F. Baganz).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.04.003
this method are the need for an intrusive probe and the effect of the probe response time on the measurement [4]. Another approach, useful during active cell respiration, is to measure the oxygen concentration of the inlet and outlet gas streams and from a simple mass balance the oxygen uptake rate can be determined. Assuming that the liquid oxygen concentration can be measured, then the kL a can be determined. This method is often termed the oxygen balance method. A third method is to measure the reaction rate of an oxygen consuming process (e.g. cellular respiration or chemical oxidation). If the reaction rate is oxygen limited, i.e. the liquid oxygen concentration is zero, then the kL a can be determined if the reaction stoichiometry is known. Common examples of this type of method include measuring the oxygen limited growth rate of a strictly aerobic microorganism [5] and measuring the rate of sodium sulphite oxidation [6]. The use of small-scale and high throughput cell cultivation devices has recently become prominent in bioprocess
64
K. Ortiz-Ochoa et al. / Biochemical Engineering Journal 25 (2005) 63–68
Table 1 Comparison of different methods for kL a evaluation Method
Scale applied
kL a range (h−1 )
Assay time
Assumptions/limitations
Dynamic gassing out
>100 ml only
0 to >1000
Several minutes
Oxygen balance
>100 ml only
0 to >1000
Several minutes
Sodium sulphite oxidation
Lab scale only
0 to >1000
Many hours
Linear growth of strict aerobe
Any scale
20 to 300
Many hours
Bio-oxidation of catechol Enzymatic oxidation of glucose
<100 ml Small scale
<300 <200
5–10 min <1 min
Invasive probes are necessary; probe response time must be considered; gassing time can be significant at larger scales Invasive probes are necessary; only possible during active oxygen consumption; requires large gas flows and gas analysis Alterations to concentration driving force, diffusion coefficient and coalescence properties; complex kinetics and boundary layer reduction Assumptions about growth kinetics are required; experimentally laborious Availability of oxidative enzyme; limited to small scales Sample handling and fast reaction; expensive reagents and complex reaction kinetics; limited to small scales
development so that large numbers of cell lines can be quantitatively evaluated [8,15]. In this way cultivation processes can be developed quicker whilst using less material. Until recently shake flasks were the most commonly used cultivation vessel, but now microtiter plates and microfluidic chips are being considered as they provide greater experimental throughput and the potential for automation. The measurement of oxygen transfer rates in these devices is clearly important for their further development as quantitative tools in bioprocess application. However, due to the small scale and non-conventional geometry, the use of well-established measurement techniques is either not possible or not ideal. For example, the use of conventional oxygen probes is unrealistic due to their large size. Although novel fluorescent probes have been developed (http://www.presens.de) their application with shaken vessels is complicated since the agitation must be paused for the duration of each measurement [9]. Likewise, the oxygen balance method cannot be used due to the very small volumes of gas flow and the fact that most small-scale vessels are open and no direct sweep gas is supplied. The sulphite oxidation method has been used to measure kL a values in shaken microwells [6], but the kinetics of the reaction are complex, the analysis time is long (many hours) and the liquid physical properties (diffusivity and saturation concentration) are significantly altered [10]. Attempts have been made to address these concerns by using a dilute enzymatic oxidation of glucose coupled to a secondary reaction yielding a colourful product that can be easily measured in a spectrophotometer [11]. Although, this method overcomes the disadvantages of the sulphite oxidation, difficulties still arise from the low volumetric oxygen uptake capacity (2 mM l−1 ) and that the reaction is quick with samples needed to be taken every 15 s. The implication of these two problems is that the environment and handling time between taking a sample and measuring it are critical and thus highly prone to error. The main aim of this research was therefore to develop a novel method for determination of kL a, which could be used for small-scale systems.
1.1. Proposed method The proposed method consists of an oxygen consuming reaction, operating under mass transfer limited conditions. The primary requirements of the method are that it should be rapid, controllable, kinetically robust and simple to perform. Ideally measurements should take in the order of 5–10 min, thus making them practically manageable. The system should be dilute to avoid significant alterations to the fluid properties. Specifically the method is based on the bio-oxidation of catechol forming 2-hydroxymuconic semialdehyde (2-HS), catalyzed by the enzyme catechol-2,3-dioxygenase (XylE) [12] (see Fig. 1). XylE forms part of the toluene oxidation pathway expressed from the Tol plasmid in Pseudomonas putida [13]. A Xhol restriction fragment, containing the XylE gene, was inserted into the multicopy plasmid pUC8 to yield pQR109 and gene expression was controlled by the lac promoter in Escherichia coli JM107. The enzyme uses 1 mol of molecular oxygen to convert 1 mol of catechol into 1 mol of 2-hydroxymuconic semialdehyde, which is a bright yellow compound that can be quantified at 425 nm. We suggest that if the enzyme concentration is in excess and the bio-oxidation rate is zero order, then the rate at which the product is formed, d(2-HS)/dt, will equal the oxygen transfer rate (OTR) and since, OTR = kL a(C* − C), and C = 0, then the kL a can be determined by measuring the linear rate of formation of the brightly coloured 2-HS (t is time, C* is the saturation concentration of oxygen, and C is the actual concentration of oxygen in the liquid).
Fig. 1. Bio-oxidation of catechol by the enzyme catechol-2,3-dioxygenase (XylE).
K. Ortiz-Ochoa et al. / Biochemical Engineering Journal 25 (2005) 63–68
2. Materials and methods 2.1. Microorganism and media E. coli JM107 pQR109 was constructed by JMW at University College London, UK, using standard methods of cloning and expression such as agarose gel electrophoresis, DNA purification, restriction digestion, ligation, transformation and cell cultivation [14]. The growth media consisted of: 10 g l−1 d-glucose, 10 g l−1 tryptone, 10 g l−1 yeast extract, and 50 mg l−1 ampicillin. All components were obtained from Sigma–Aldrich (Gillinghan, Dorset, United Kingdom). For induction of XylE, 1 mM l−1 isopropyl -d-1 thiogalactopyranoside (IPTG) was added to the media. The ampicillin and IPTG were filter sterilized by 0.2 m pore filter.
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chol was then added as a 10% (v/v) of a 70 mM l−1 aqueous solution. The bio-oxidation was monitored by taking samples every minute and recording the optical density at 425 nm (Ultraspec 400, Pharmacia Biotech). Samples were diluted 100 times with deoxygenated water (nitrogen purged) and the optical density was converted into concentration using an external calibration curve, with an extinction coefficient of 15,750 l mol−1 cm−1 . The large dilution factor not only allowed for more accurate measurement of the product concentration, but also quenched the bio-oxidative reaction. The time taken to remove a sample and dilute it was typically less than 10 s and after this point the reaction ceased and could be measured.
3. Results and discussion 2.2. Cell cultivation and XylE preparation 3.1. Reaction kinetics One hundred millilitres of media in a 500 ml Erlenmeyer flask was inoculated with a frozen stock (−80 ◦ C, 50%, v/v glycerol) and grown overnight in an orbital shaker (K¨uhner, Birsfelden, Switzerland) at 30 ◦ C and 280 rpm. This culture was then used to inoculate either another 100 ml shake flask or a 1.5 l stirred tank fermenter (LH 210 series, Bioprocess Engineering Services, Charing, Kent, UK). IPTG was added prior to inoculation. For the larger scale an airflow rate of 1 VVM was used, and the pH was controlled at 7 by the metered addition of 4 M KOH. The impeller speed was set at 1200 rpm and the oxygen concentration was measured by a Clark type oxygen electrode (Ingold Messtechnik, Urdorf, Switzerland). The biomass concentration was determined by monitoring optical density at 600 nm (Ultraspec 400, Pharmacia Biotech, Cambridge, UK) and converted to a dry cell weight concentration using a calibration curve. Typically the fermentation was completed once the OD (600 nm) had reached 16–18 resulting in a dry cell weight of 6–7 g l−1 . In shake flask cultivation the final dry cell weight achieved was lower at between 3.5 and 4 g l−1 . In either scale the cells were subsequently harvested using a Beckman J2M1 centrifuge, fitted with a JA10 rotor (High Wycombe, Buckinghamshire, United Kingdom) at 10,000 rpm for 10 min. The biomass was then resuspended in one third (except where stated) of the initial volume in 50 mM phosphate buffer (pH 7) and disrupted either in a sonicator, soniprep 150 MSE (Beckenham, Kent, United Kingdom) or in a homogenizer Lab 60 APV Manton Gaulin (Crawley, W. Sussex, United Kingdom) depending of the volume. Sonication was carried out at 4 × 10 s at an amplitude of 6 m with 10 s cooling periods. Large-scale homogenization was carried out in five passes at 500 bar. Subsequently, cell debris was separated by centrifugation at similar conditions and the supernatant, containing XylE, was collected. 2.3. Catechol bio-oxidation The enzyme preparation was placed in the reaction vessel and once the temperature had equilibrated at 30 ◦ C, the cate-
Studies of the bio-oxidation kinetics were performed in 4 ml plastic cuvettes where 2.7 ml of the oxygen saturated enzyme preparation (oxygen concentration ≈0.2 mM l−1 [1]) was mixed with 0.3 ml of catechol solution. The reaction was monitored on-line. In this way the maximum specific activity of the enzyme preparation was determined. These experiments were executed without concentrating the enzyme during resuspension and were routinely performed with each new batch of enzyme preparation in order to ensure that sufficient activity was available. Reducing the volume of the resuspension buffer prior to disruption and thus increasing the concentration factor resolved cases of low specific activity. Fig. 2 shows the reaction rate attained at different catechol concentrations in this system. The highest bio-oxidation rate achieved was 0.63 mM l−1 min−1 at a catechol concentration of 2 mM l−1 and therefore the maximum specific activity was
Fig. 2. Effect of catechol concentration on the bio-oxidation activity of the enzyme catechol-2,3-dioxygenase (XylE) at 30 ◦ C and pH 7. Inset: product formation profile during a batch bio-oxidation in a 4 ml cuvette. (- - -) 15 mM l−1 catechol, (—) 2 mM l−1 catechol.
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180 U g DCW−1 obtained from 3.5 g DCW l−1 . Fig. 2 (inset) shows the product formation profile from which this value was obtained and for comparison also shows the rate when the catechol concentration was 15 mM l−1 . At catechol concentrations between 2 and 7 mM l−1 the bio-oxidation rate ranged from 0.58 to 0.63 mM l−1 and since the percentage variation was less than 10% the bio-oxidation rate was considered to be zero order with respect to catechol in this concentration range. At higher concentrations some inhibition was observed. The oxygen uptake capacity of the system in the zero order range was 5 mM l−1 . Since we considered a minimum analysis time of 5 min to be practically manageable, and assuming that the stoichiometry shown in Fig. 1 is valid, the maximum accurately measurable oxygen transfer rate is 60 mM l−1 h−1 . If the dissolved oxygen concentration is assumed to be zero, and the driving force is 0.2 mM l−1 , the maximum kL a that can be accurately measured using this novel method is about 300 h−1 . 3.2. Characterization of a typical catechol bio-oxidation The oxidation stoichiometry was experimentally determined and the assumption of zero DOT during the biotransformation was verified in a 1 l scale reaction in which product formation, dissolved oxygen concentration and the oxygen uptake rate were measured, see Fig. 3. Upon addition of catechol to the system, 2-HS was produced immediately and the rate of formation was constant at 22.4 mM l−1 h−1 . The kL a was calculated from this product formation rate as 112 h−1 . Also, the dissolved oxygen concentration decreased to zero due to its rapid uptake by the enzymatic oxidation. Concomitant with this was a decrease in the oxygen content of the air exiting the reactor and thus the oxygen uptake rate (OUR) increased sharply to a constant value of about 20 mM l−1 h−1 shown in Fig. 3. There was a time delay of about 3–4 min in
Fig. 3. A typical catechol bio-oxidation profile by the enzyme catechol-2,3dioxygenase (XylE) in a 2 l stirred tank using a 1 l working volume, 1vvm aeration rate, 30 ◦ C, pH 7 and impeller speed of 900 rpm. (- - -) DOT%, (—) OUR, () [2-HS] considered for kL a determined, () [2-HS] not considered for kL a determination.
these OUR data and this was due to distant location of the mass spectrometer in relation to the bioreactor. In this experiment, 7.5 mM l−1 of catechol was initially added to the bioreactor and the final concentration of 2-HS was 7.12 mM l−1 and thus a 95% conversion was achieved. These experiments verify the reaction stoichiometry shown in Fig. 1, i.e. that 1 mol of catechol is converted to 1 mol of 2-HS using 1 mol of molecular oxygen. The assumption of mass transfer limiting conditions (i.e. a DOT of zero) was also demonstrated since this is vital for kL a determination from OTR values in small-scale vessels without DOT probes. 3.3. Method validation The aim of this section was to compare this novel biooxidation technique to the established dynamic gassing out method. This was considered important as the chemical sulphite oxidation method, also applicable in small-scale systems without probes, often leads to incorrect kL a values [10]. Fig. 4 compares the results from both methods at different stirrer speeds in the 2 l bioreactor. The dynamic method resulted in slightly lower kL a values than those determined from the bio-oxidation method, but the difference was less than 15% in all cases. This difference was considered acceptable and therefore mass transfer coefficients obtained using the novel method are valid. 3.4. Application in microplates Fig. 5 compares kL a values determined in shaken 96well microplates measured using a variety of techniques. The methods compared with the catechol bio-oxidation were: (a) the sulphite oxidation technique (data shared from [6]); (b)
Fig. 4. Parity plot comparing the kL a values determined using the enzymatic method and dynamic method over a range of impeller speeds (, 700; , 900; , 1000; and ♦, 1100 rpm) in a 2 l stirrer tank. The dashed lines represent ±15% either side of parity.
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Fig. 5. Comparison of kL a values determined using the bio-oxidation against other methods in a 96-well shaking microplate (liquid volume = 0.2 ml). () kL a values determined using the sulphite oxidation method from [6]; () kLa values determined using the linear growth rate of Bacillus from [7]; (—) parity line, the dashed lines represent ±25% either side of parity.
the oxygen limited linear growth of a strict aerobe, Bacillus subtilis [7]. Results from the bio-oxidation of catechol are in good agreement with data from the linear growth rate method. However, results from the sulphite oxidation were slightly higher at low shaking speeds and slightly lower at high shaking speeds. It is established that the sulphite oxidation method tends to over estimate the kL a [3]. However, it provided under-estimates when the shaking frequencies were at their highest and where the gas–liquid surface area was deformed most. We suggest that this is due to the significance of surface tension in these small-scale systems and therefore at higher shaking frequencies the deformation in the surface area, increasing the kL a, will be higher when using biological process liquids due to the presence of surface-active agents. Therefore the bio-oxidation method presented in this paper provides a closer approximation to the conditions in a ‘real’ fermentation broth than the sulphite oxidation method and therefore we suggest is a more appropriate method of measuring oxygen mass transfer rates.
4. Conclusions The proposed novel bio-oxidation method, based on the oxidation of catechol, was kinetically characterized and shown to be zero order over the catechol concentrations 2–7 mM l−1 and if the enzyme was in excess, the DOT during the bio-oxidation was 0%. The simple reaction stoichiometry was experimentally verified and we estimate that kL a values of up to 300 h−1 can be accurately measured. Table 1 compares this novel method with other established techniques. Advantages and disadvantages exist for all the methods, but in general either the dynamic gassing out or
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the oxygen balance methods are most suitable for lab and pilot scale systems. In these cases the use of conventional oxygen probes is manageable and problems with probe response time can be accounted for. In small-scale devices kL a determination from the linear growth rate of a strict aerobe is valid and offers the advantage that the mass transfer rate is being determined in a representative bio-medium. However, the technique is very time consuming and therefore other mass transfer limited reaction rate strategies are preferred. Endpoint measurement of the sulphite oxidation is complicated by the adverse effect on the physical properties of the liquid and is mostly unfavoured. The bio-oxidation of catechol is both rapid and unencumbered by these problems and since no kinetic assumptions are required, it provides a true evaluation of the kL a. We expect that the wider availability of the enzyme catechol-2,3-dioxygenase should result in the novel method presented here to be commonly used during kL a evaluation in small-scale devices.
Acknowledgements The authors would like to thank the UK Joint Infrastructure Fund (JIF), the Science Research Investment Fund (SRIF) and the Gatsby Charitable Foundation for funds to establish the UCL Centre for Micro Biochemical Engineering. Financial support for the research work presented in this paper is acknowledged from the UK Engineering and Physical Sciences Research Council (EPSRC). Prof. Dr. Jochen B¨uchs of the Biochemical Engineering Department at Aachen University (Germany) is gratefully acknowledged for sharing his oxygen transfer data used in comparing to our developed method.
References [1] J.E. Bailey, D.F. Ollis, Biochemical Engineering Fundamentals, second ed., McGraw Hill International Editions, 1986. [2] H.W. Blanch, D.S. Clark, Biochemical Engineering, Marcel Dekker Inc., 1996. [3] P.M. Duran, Bioprocess Engineering Principles, xth ed., Academic Press, 2003. [4] I.J. Dunn, A.J. Einsele, Oxygen transfer coefficients by the dynamic method, J. Appl. Chem. Biotechnol. 25 (1975) 707–720. [5] W.A. Duetz, L. Ruedi, R. Hermann, K. O’Connor, J. Buchs, B. Witholt, Methods for intense aeration, growth, storage and replication of bacterial strains in microtiter plates, Appl. Environ. Microbiol. 66 (2000) 2641–2646. [6] R. Hermann, M. Lehmann, J. Buchs, Characterization of gas–liquid mass transfer phenomena in microtiter plates, Biotechnol. Bioeng. 81 (2002) 178–186. [7] G.T. John, I. Klimant, C. Wittmann, E. Heinzle, Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation, Biotechnol. Bioeng. 81 (2003) 829– 836. [8] S.D. Doig, A. Diep, F. Baganz, Characterisation of a novel miniaturised bubble column bioreactor for high throughput cell cultivation, Biochem. Eng. J. 23 (2005) 97–105.
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[9] K. Van’t Riet, Review of measuring methods and results in nonviscous gas–liquid mass transfer in stirred vessels, Ind. Eng. Chem. Process Des. Dev. 18 (1979) 357–364. [10] W.A. Duetz, B. Witholt, Oxygen transfer by orbital shaking of square vessels and deepwell microtiter plates of various dimensions, Biochem. Eng. J. 17 (2004) 181–185. [11] J.M. Sala-Trepat, W.C. Evans, The meta cleavage of catechol by Azotobacter species. 4-Oxalocrotonate pathway, Eur. J. Biochem. 20 (1971) 400–413. [12] S. Inouye, A. Nakazawa, T. Nakazawa, Molecular cloning of TOL genes xylB and xylE in Escherichia coli, J. Bacteriol. 145 (1981) 1137–1143.
[13] J. Sambrook, E.F. Fritsch, T. Maniatis, in: C. Nolan (Ed.), Molecular Cloning, A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, USA, 1989. [14] S.D. Doig, F. Baganz, G.J. Lye, High throughput screening and process optimsation, in: C. Ratledge, B. Kristiansen (Eds.), Basic Biotechnology, third ed., Cambridge University Press, Cambridge, UK, 2004. [15] G.J. Lye, P. Ayazi-Shamlou, F. Baganz, P.A. Dalby, J.M. Woodley, Accelerated design of bioconversion processes using automated microscale processing techniques, Trends Biotechnol. 21 (2003) 29– 73.