Beruflich Dokumente
Kultur Dokumente
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London,
Torrington Place, London, WC1E 7JE, UK
b Bioxplore, 50 Moxon Street, Barnet, Hertfordshire, EN5 5TS, UK
Received 2 August 2007; accepted 3 September 2007
Abstract
The establishment of a high productivity microbial fermentation process requires the experimental investigation of many interacting variables.
In order to speed up this procedure a novel miniature stirred bioreactor system is described which enables parallel operation of 416 independently
controlled fermentations. Each miniature bioreactor is of standard geometry (100 mL maximum working volume) and is fitted with a magnetically
driven six-blade miniature turbine impeller (di = 20 mm, di /dT = 1/3) operating in the range 1002000 rpm. Aeration is achieved via a sintered sparger
at flow rates in the range of 02 vvm. Continuous on-line monitoring of each bioreactor is possible using miniature pH, dissolved oxygen and
temperature probes, while PC-based software enables independent bioreactor control and real-time visualisation of parameters monitored on-line. In
addition, a new optical density probe is described that enables on-line estimation of biomass growth kinetics without the need for repeated sampling
of individual bioreactors. Initial characterisation of the bioreactor involved quantification of the volumetric oxygen mass transfer coefficient as
a function of agitation and aeration rates. The maximum kL a value obtained was 0.11 s1 . The reproducibility of E. coli TOP10 pQR239 and
B. subtilis ATCC6633 fermentations was shown in four parallel fermentations of each organism. For E. coli (1000 rpm, 1 vvm) the maximum
specific growth rate, max , was 0.68 0.01 h1 and the final biomass concentration obtained, Xfinal , was 3.8 0.05 g L1 . Similarly for B. subtilis
(1500 rpm, 1 vmm) max was 0.45 0.01 h1 and Xfinal was 9.0 0.06 g L1 . Biomass growth kinetics increased with increases in agitation and
aeration rates and the oxygen enrichment for control of DOT levels enabled max and Xfinal as high as 0.93 h1 and 8.1 g L1 respectively to be
achieved. Preliminary, scale-up studies with E. coli in the miniature bioreactor (100 mL working volume) and a laboratory scale 2 L bioreactor
(1.5 L working volume) were performed at matched kL a values. Very similar growth kinetics were observed at both scales giving max values of
0.94 and 0.97 h1 , and Xfinal values of 5.3 and 5.5 g L1 respectively. The miniature bioreactor system described here thus provides a useful tool
for the parallel evaluation and optimisation of microbial fermentation processes.
2007 Elsevier B.V. All rights reserved.
Keywords: Miniature bioreactor; Parallel operation; Fermentation; On-line monitoring
1. Introduction
The design and optimisation of industrial fermentation
processes requires the experimental investigation of many interacting biological and physical variables. Advances in metabolic
engineering and protein evolution techniques now enable the
rapid creation of large libraries of recombinant microorganisms
[1]. These are normally evaluated in parallel microwell cultures
and a small number of promising strains identified based on simple initial screens for product yield or activity [2]. For the chosen
1369-703X/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2007.09.001
strains product synthesis can be further enhanced by study of culture medium composition, nutrient feeding regimes and physical
variables such as temperature, pH and dissolved oxygen levels.
Here large numbers of parallel shake flask or stirred-bioreactor
experiments must be performed because of the number of experimental variables requiring investigation per strain. Finally, once
a production strain is identified, further experimental investigation over a range of scales is necessary to establish the operating
boundaries and robustness of the process for validation purposes
[2]. At this stage virtually all processes would be performed in
stirred bioreactors because of the dominance of this design in
the chemical and biopharmaceutical sectors.
Small scale bioreactor systems, that enable parallel and automated operation of several fermentations simultaneously, have
Nomenclature
CL
the potential to increase the rate at which the necessary experiments are performed thus reducing fermentation development
times and costs [3]. In recent years various designs of parallel miniature bioreactor systems have been reported including
stirred tank bioreactors, bubble columns and shake flasks [47].
The key design features of many of these will be discussed in
detail later (Section 3.5). While each bioreactor design aims to
satisfy all the requirements for rapid fermentation process development there is normally a trade-off between throughput and the
information content of the data obtained from each experiment
[2]. This is most clear when comparing related developments
in microwell fermentations [610] with the small scale stirred
bioreactor designs considered here. Irrespective of the approach
taken for high throughput fermentation process development
however, it will be important that advances in throughput are
matched by the creation of microscale downstream processing operations. In this regard the generation of quantitative
process data from a number of microwell-based downstream
unit operations has recently been reported [3,11,12] as has the
automated operation of linked process sequences [13,14]. The
automated linkage of upstream and downstream operations at
such scales now offers the potential for the integrated optimisation of the entire process from fermentation through to purified
product.
165
166
167
168
kL a = K
S
(2)
V
where Pg /V is the impeller gassed power per unit volume, S is
the superficial gas velocity, K is a constant and and are exponents in the range of 0.4 < < 1 and 0 < < 0.7, respectively [18].
For kL a measurements in ionic solutions, the reported values of
the constants K, and were 2.0 103 , 0.7 and 0.2, respectively. Although the vant Riet correlation was determined for
much larger vessels than used here, fitted with standard Rushton
turbine impellers, the stronger dependency of kL a on Pg /V (and
hence stirrer speed) than on uS shown in Fig. 4 initially suggests
that results for the miniature bioreactor are consistent with the
established theory. At present the Power number of the miniature
bioreactor is not known and so absolute values of Pg /V cannot
be estimated.
3.3. Parallel fermentations and in-situ monitoring
3.3.1. Reproducibility of parallel E. coli fermentations
A key requirement of any parallel bioreactor system is that
cultivations in separate bioreactors should be highly repro-
169
Fig. 5. Parallel batch fermentation kinetics of E. coli TOP10 pQR239 grown under identical conditions: () off-line biomass concentration; (-) DOT. Experiments
performed at 1000 rpm and 1 vvm as described in Section 2.4. B1B4 refer to bioreactors one to four, respectively.
170
Table 1
Reproducibility of batch E. coli TOP10 pQR239 fermentation kinetics from four parallel fermentations
Parameter
(h1 )
max
Xfinal (g L1 )
tDOT = 0 (min)
OURmax (mmol L1 h1 )
B1
B2
B3
B4
BAV
0.68
3.7
290
18.1
0.66
3.8
300
18.4
0.68
3.8
300
18.4
0.69
3.8
243
18.4
0.68
3.8
283
18.3
0.01
0.05
27.2
0.15
Kinetic parameters derived from the fermentation profiles shown in Fig. 5. B1B4 refer to bioreactors one to four respectively, while BAV indicates the mean values
of the kinetic parameters (error indicated represents one standard deviation).
The time at which the measured DOT reached zero was also comparable in the majority of cases. Overall these results indicate
excellent reproducibility of the four-pot system.
At the point where the measured DOT first reaches zero, it
can be assumed that the maximum oxygen uptake rate (OURmax )
and the maximum oxygen transfer rate (OTRmax ) are equal. The
OURmax can thus be estimated from:
OURmax =
X
+ m O2 X
YX/O2
(3)
where is the specific growth rate at the time point when the
DOT first reaches zero, X is the corresponding biomass concentration, YX/O2 is the yield of biomass on oxygen, which was
taken to be 1.92 g g1 [20] and m is the oxygen required for cell
maintenance which was taken to be 0.003 mol O2 g1 h1 [20].
(4)
where C* is the saturated dissolved oxygen concentration, estimated to be 6.9 mg L1 [21] and CL is the actual concentration of
dissolved oxygen in the fermentation broth. At the point where
the measured DOT first reaches zero, CL is also zero and so Eq.
(4) reduces to:
OTRmax = kL aC
(5)
Fig. 6. Parallel batch fermentation kinetics of B. subtilis ATCC6633 grown under identical conditions: () off-line biomass concentration; (-) DOT. Experiments
performed at 1500 rpm and 1 vvm as described in Section 2.4. B1B4 refer to bioreactors one to four, respectively.
171
(6)
max
Xfinal (g L1 )
tDOT = 0 (min)
kL a (s1 )
B1
B2
B3
B4
BAV
0.46
9.0
529
0.061
0.45
9.0
572
0.062
0.45
9.0
550
0.061
0.44
9.1
608
0.059
0.45
9.0
565
0.061
0.01
0.06
33.7
0.001
Fig. 7. On-line measurement of optical density in E. coli TOP10 pQR239 fermentations. (A) Comparison of biomass growth kinetics from an individual
fermentation: () off-line optical density; () on-line optical density. (B) Parity plot of on-line and off-line biomass concentration data from nine identical
fermentations. Experiments performed at 1000 rpm and 1 vvm as described in
Section 2.4.
172
Fig. 8. Influence of agitation and aeration rates and oxygen enrichment on batch fermentation kinetics of E. coli TOP10 pQR239: () off-line biomass concentration;
(-) DOT. Experimental conditions: (A) 2000 rpm, 1 vvm; (B) 1000 rpm, 2 vvm; (C) 2000 rpm, 1 vvm and DOT controlled at 30%; (D) 2000 rpm, 1 vvm and DOT
controlled at 50%. Fermentations performed as described in Section 2.4.
173
Table 3
Variation of bioreactor oxygen mass transfer coefficient and E. coli TOP10 pQR239 fermentation kinetics as a function of agitation and aeration conditions including
the use of oxygen enrichment to control DOT levels
Parameter
Gas blending
kL a (s1 )
max (h1 )
Xfinal (g L1 )
OURmax (mmol L1 h1 )
1500 rpm
1 vmm
2000 rpm
1 vmm (A)
1000 rpm
1.5 vmm
1000 rpm
2 vmm (B)
0.04
0.68
3.8
18.4
0.06
0.83
5.1
35.7
0.08
0.94
5.3
46.8
0.06
0.75
5.6
26.3
0.06
0.79
5.6
27.5
DOT = 30%
0.86
7.6
ND
DOT = 50%
0.93
8.1
ND
(A)(D) correspond to the fermentation profiles shown in Fig. 8. Oxygen mass transfer coefficient and values derived from Fig. 3. N/D = not determined.
Table 4
Comparison of E. coli TOP10 pQR239 fermentations carried out in miniature
and conventional laboratory bioreactors using constant kL a as a basis for scale-up
(based on aeration with atmospheric air)
Parameter
Miniature bioreactor
Laboratory bioreactor
100
2000
1.00
0.08
2.0
0.94
5.3
1500
1000
0.67
0.08
6.7
0.97
5.5
174
175
[10] G.T. John, I. Klimant, C. Wittmann, E. Heinzle, Integrated optical sensing of dissolved oxygen in microtitre plates: a novel tool for microbial
cultivation, Biotechnol. Bioeng. 81 (2003) 829836.
[11] K. Rege, M. Pepsin, B. Falcon, L. Steele, M. Heng, High-throughput process development for recombinant protein purification, Biotechnol. Bioeng.
93 (2006) 618630.
[12] N.B. Jackson, J.M. Liddell, G.J. Lye, An automated microscale technique
for the quantitative and parallel analysis of microfiltration operations, J.
Membrane Sci. 276 (2006) 3141.
[13] C. Ferreira-Torres, M. Micheletti, G.J. Lye, Microscale process evaluation
of recombinant biocatalyst libraries: application to Baeyer-Villiger monooxygenase catalysed lactone synthesis, Bioprocess Biosystems Eng. 28
(2005) 8393.
[14] M. Micheletti, T. Barrett, S.D. Doig, F. Baganz, M.S. Levy, J.M.
Woodley, G.J. Lye, Fluid mixing in shaken bioreactors: implications for scale-up predictions from microlitre scale microbial
and mammalian cell cultures, Chem. Eng. Sci. 61 (2006) 2939
2949.
[15] S.D. Doig, L.M. OSullivan, S. Patel, J.M. Ward, J.M. Woodley, Large
scale production of cyclohexanone monooxygenase from Escherichia coli
TOP10 pQR239, Enzyme Microb. Technol. 28 (2001) 265274.
[16] S.D. Doig, A. Diep, F. Baganz, Characterisation of a novel miniature bubble
column bioreactor for high throughput cell cultivation, Biochem. Eng. J.
23 (2004) 97105.
[17] M.E. Van Loo, P.E. Lengowski, Automated workstations for parallel synthesis, Org. Process Res. Dev. 6 (2002) 833840.
[18] K. vant Riet, Review of measuring methods and results in non-viscous
gas liquid mass transfer in stirred vessels, Ind. Eng. Chem. Process Design
Dev. 18 (1979) 357364.
[19] I.J. Dunn, A.J. Einsele, Oxygen transfer coefficients by the dynamic
method, J. Appl. Chem. Biotechnol. 25 (1975) 707720.
[20] B. Atkinson, F. Mavituna, Biochemical Engineering and Biotechnology
Handbook, second ed., Stockton press, 1999.
[21] Bailey, Ollis, Biochemical Engineering Fundamentals, McGraw-Hill International, Singapore, 1986.
[22] A. Morao, C.I. Maia, M.M.R. Fonseca, J.M.T. Vasconcelos, S.S. Alves,
Effect of antifoam addition on gas-liquid mass transfer in stirred fermenters,
Bioprocess Eng. 20 (1999) 165172.
[23] R. Puskeiler, K. Kaufmann, D. Weuster-Botz, Development, parallelisation, and automation of a gas inducing millilitre-scale bioreactor for
high-throughput bioprocess design (HTBD), Biotechnol. Bioeng. 89 (2005)
512523.
[24] R. Puskeiler, A. Kusterer, G.T. John, D. Weuster-Botz, Miniature bioreactors for automated high-throughput bioprocess design
(HTBD): Reproducibility of parallel fed-batch cultivations with
Escherichia coli, Biotechnol. Appl. Biochem. 42 (2005) 227
235.
[25] J.I. Betts, S.D. Doig, F. Baganz, Charaterisation and application of a miniature 10 mL stirred-tank bioreactor, showing scale-down equivalence with
a conventional 7 L reactor, Biotechnol. Prog. 22 (2006) 681688.
[26] S.R. Lamping, H. Zhang, B. Allen, P. Ayazi-Shamlou, Design of a prototype
miniature bioreactor for high throughput automated bioprocessing, Chem.
Eng. Sci. 58 (2003) 747758.
[27] Y. Kostov, P. Harms, L. Randers-Eichhorn, G. Rao, Low-cost microbioreactor for high-throughput bioprocessing, Biotechnol. Bioeng. 72 (2001)
346352.
[28] P. Harms, Y. Kostov, J.A. French, M. Soliman, M. Anjanappa, A. Ram, G.
Rao, Design and performance of a 24-station high throughput microbioreactor, Biotechnol. Bioeng 93 (2006) 613.
[29] S. Dilsen, W. Paul, D. Herforth, A. Sandgathe, J. Altenbach-Rehm, R.
Freudl, C. Wandrey, D. Weuster-Botz, Evaluation of parallel operated
small-scale bubble columns for microbial process development using
Staphylococcus carnosus, J. Biotechnol. 88 (2001) 7784.
[30] D. Weuster-Botz, J. Altenbach-Rehm, A. Hawrylenko, Processengineering characterisation of small-scale bubble columns for microbial
process development, Bioprocess Biosystems Eng. 24 (2001) 311.
[31] S.D. Doig, K. Ortiz-Ochoa, J.M. Ward, F. Baganz, Characterization of oxygen transfer in miniature and lab-scale bubble
176
column bioreactors and comparison of microbial growth performance based on constant kL a, Biotechnol. Prog. 21 (2005) 1175
1182.
[32] T. Anderlei, J. Buchs, Device for sterile online measurement of the oxygen
transfer rate in shaking flasks, Biochem. Eng. J. 7 (2001) 157162.