Documente Academic
Documente Profesional
Documente Cultură
Abstract
The present study explores a method for scaling-up a fermentation system. The method consists of two parts. The first part investigates
the effects of the environmental state variables on cultivation. If a wide range of the state variables has no significant effect on yield or
productivity, scaling-up is not an issue in the fermentation system. If only a small range of the state variables has no substantial impact on
cultivation, then pulse or periodical change of the state variables would be employed to investigate the effects of the change on cultivation.
To illustrate the proposed method of scale-up, Bacillus thuringiensis was cultivated for thuringiensin production with the pH value as the
environmental state variable. Different pH values at 7.0 and 8.4 had a significant effect on both cell growth and thuringiensin production.
Variation of pH value for a short period of time did not have a substantial effect on either cell growth or thuringiensin production.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Scale-up; Fermentation system; Response surface; Bacillus thuringiensis
1369-703X/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 6 9 - 7 0 3 X ( 0 2 ) 0 0 0 1 6 - 5
124 Y.-L. Hsu, W.-T. Wu / Biochemical Engineering Journal 11 (2002) 123130
The cultivation of Bacillus thuringiensis for thuringiensin oxygen electrode (Ingold, Switzerland) placed in the
production with pH as the environmental state variable was fermentor.
demonstrated. The other environmental variables that are
important in scale-up, such as dissolved oxygen (DO) con- 2.4. The algorithm
centration and shear stress due to agitation [14], are not in-
vestigated in this study. Thuringiensin, produced by some In a large-scale fermentor, both transport phenomena and
serotypes of B. thuringiensis, is a heat-stable biopesticide cell kinetics have significant effects on the outcome of the
that has a broader spectrum of effectivity against many in- cultivation. The proposed algorithm is based on a series of
sects as compared to -endotoxin [15,16]. The production special operations in a small-scale fermentor to obtain the
of thuringiensin occurs in a mixed-growth-associated sys- information for scale-up. The operations include the deter-
tem that provides an appropriate example in demonstrating mination of the effect of the environmental variables and the
the proposed method of scale-up. variation of the variables. The flow chart is shown in Fig. 1.
The method of analyzing thuringiensin was modified 2.4.2. Variation of the environmental variables
from that of Campbell et al. [17] and Levinson et al. [18]. Incomplete mixing in a large-scale fermentor may cause
High performance liquid chromatography (HPLC) was em- a gradient of the environmental variables in the fermentor,
ployed for determining the concentration of thuringiensin. especially during the period of feeding for a fed-batch cul-
Glucose concentration was measured using a glucostat ture or adding alkali/acid for pH control. This incomplete
reagent (Glucostat, BGH). Detection was carried out with mixing is simulated by using pulse or periodical change of
a spectrophotometer at a wavelength of 505 nm. The num- the environmental state variables in a well-mixed small-scale
ber of cells was counted directly using a microscope with fermentor.
a counting chamber (ERMA, Japan). The concentration A fermentation process with growth-associated, non-
of DO in the broth was monitored by a polarographic growth-associated or mixed-growth-associated product
Y.-L. Hsu, W.-T. Wu / Biochemical Engineering Journal 11 (2002) 123130 125
Fig. 1. The flow chart of the proposed method of scaling-up a fermentation system.
requires different cultivation modes. For the fermentation more positions for the inputs, is required to increase homo-
with a growth-associated product, cultivation is carried out geneity in the large-scale fermentor.
by extending the exponential growth phase. On the other
hand, for the case with a non-growth-associated product, the
cultivation strategy is to prolong the period of the stationary 3. Results
phase. Therefore, simulation of the incomplete mixing in a
large-scale fermentor using pulse or periodical change in a Cultivation of B. thuringiensis for thuringiensin produc-
small-scale fermentor is employed in both the exponential tion was carried out using the pH value as the environmental
growth and the stationary phases. variable to elucidate the methods of scale-up.
If variation of the environmental variables has no signif-
icant effect on the cultivation, scaling-up the fermentation 3.1. Effect of pH on the cultivation
system is not a problem. In contrast, if the variation has a
substantial effect on the cultivation, scaling-up is a problem Fig. 2 shows the effect of pH on specific growth rate,
and careful consideration of the fermentor, such as utilizing , under the condition of the aeration of 1.5 vvm and the
126 Y.-L. Hsu, W.-T. Wu / Biochemical Engineering Journal 11 (2002) 123130
Fig. 2. The pH effect on the specific growth rate in the cultivation of B. thuringiensis with the aeration of 1.5 vvm and the agitation speed of 500 rpm.
The range marked in the plot indicates the acceptable robustness when scaling-up the fermentation system.
agitation speed of 500 rpm. The optimum pH value was sin concentration at pH 8.4 were much lower than those at
found to be around 7.0. The time courses of batch cultures of pH 7.0. In a large-scale fermentor, if the fluctuation of pH
B. thuringiensis at pH 7.0 and 8.4 are shown in Figs. 3 and value could be controlled within 7.0 0.3 (as the range
4, respectively. Obviously, the cell number and thuringien- marked in Fig. 2) throughout the cultivation, the growth of
Fig. 3. The time course of the cultivation with the pH control at 7.0 throughout the cultivation.
Y.-L. Hsu, W.-T. Wu / Biochemical Engineering Journal 11 (2002) 123130 127
Fig. 4. The time course of the cultivation with the pH control at 8.4 throughout the cultivation.
microorganisms would be almost the same as the case with due to the intermittent action of pH control. Variation of the
pH control at 7.0 in a small-scale fermentor. If the pH value fermentation system from pH 7.0 will lead to a decrease in
in the large-scale fermentor could not be well controlled the biomass and product concentration.
within the required range, the transient response of pH vari-
ation would need to be investigated. 3.2.1. Variation in the exponential growth phase
Fig. 5 shows the time course of pH change during the
3.2. Variation of pH value period between 5 and 10 h of cultivation. The period was
30 min with 2 min at pH 8.4. The final concentration of
Since thuringiensin is a mixed-growth-associated product thuringiensin was about 92% of that at pH 7.0. As demon-
in the cultivation of B. thuringiensis, variation of pH value strated in Fig. 5, periodical variation with a short duration
in the exponential growth and stationary phases was carried time (e.g. less than 2 min) has a minimal effect on thuringien-
out. All cultures were maintained at pH 7.0 as a standard cul- sin production.
tivating condition for the B. thuringiensis before subsequent
pH manipulation. The addition of acid and alkali were em- 3.2.2. Variation in the stationary phase
ployed for pH control. A certain amount of metabolic acids Fig. 6 shows the time course of pH change during the
were found to be excreted into the broth during the exponen- stationary phase (from 10.5 to 15.5 h of cultivation). There
tial growth phase. These acids were utilized by the microor- was no difference in the thuringiensin production through-
ganisms as a carbon source after the glucose was depleted out the stationary phase regardless of pH variation. An
during the stationary phase [26]. Thus, pH varied between experiment without pH control during the stationary phase
7.0 and 8.4 during the exponential growth phase, and be- was also carried out for comparison. As shown in Fig. 7,
tween 5.8 and 7.0 during the stationary phase to mimic the the final concentrations of biomass and thuringiensin were
additions of NaOH and H2 SO4 solutions during the expo- similar to those in Fig. 3.
nential growth phase and the stationary phase, respectively. In a large-scale fermentor, variation in pH value is possi-
Good mixing is essential for a large-scale fermentor in order ble due to the fact that the reactor does not provide a com-
to prevent the cells from being subjected to pH perturbation pletely well-mixed environment. The experimental results
128 Y.-L. Hsu, W.-T. Wu / Biochemical Engineering Journal 11 (2002) 123130
Fig. 5. The time course of the cultivation with the periodical variation of pH value during the exponential growth phase (from 5 to 10 h). The period
was 30 min with 2 min at pH 8.4.
Fig. 6. The time course of the cultivation with the periodical variation of pH value during the stationary phase (from 10.5 to 15.5 h of cultivation). The
period was the same as that in Fig. 5.
Y.-L. Hsu, W.-T. Wu / Biochemical Engineering Journal 11 (2002) 123130 129
Fig. 7. The time course of the cultivation without pH control during the stationary phase.
demonstrate that a short period of pH variation has little in- on either cell growth or product formation. Therefore, if a
fluence on thuringiensin production as well as biomass con- large-scale fermentor had a short mixing time to guarantee
centration, especially during the stationary phase. that the variation of pH value remained within the limit, the
environmental state variable, pH, would not cause a problem
in a large-scale fermentor.
4. Conclusions
References
The proposed method of scale-up is based on the evalu-
ation of: (i) the effect of the environmental state variables [1] M. Rizzi, U. Theobald, M. Baltes, M. Reuss, Measurement and
on the cultivation and (ii) the variation of the environmental modeling of the dynamic glucose response of Saccharomyces in the
state variables. If the fermentation system, which is tested time window of mixing times, in: A.W. Nienow (Ed.), Proceedings
of the Third International Conference on Bioreactor and Bioprocess
in a small-scale fermentor, is insensitive to a wide range of
Fluid Dynamics, Mechanical Engineering Publications, London, UK,
the environmental state variables, scale-up is not a problem. 1993, pp. 401412.
If the fermentation system is sensitive to the environmen- [2] S. George, G. Larsson, K. Olsson, S.-O. Enfors, Comparison of
tal state variables but insensitive to a short period variation the Bakers yeast process performance in laboratory and production
of the state variables, scale-up is still not a problem. If the scale, Bioprocess Eng. 18 (1998) 135142.
[3] F. Bylund, A. Castan, R. Mikkola, A. Veide, G. Larsson, Influence
fermentation system is sensitive to both the environmental
of scale-up on the quality of recombinant human growth hormone,
variables and the variation of the state variables, scale-up Biotechnol. Bioeng. 69 (2) (2000) 119128.
is a problem. Design of a large-scale fermentor should be [4] F.L. Shuler, F. Kargi, Bioprocess Engineering: Basic Concepts,
done carefully. Utilizing more positions for the inputs to in- Prentice-Hall, Englewood Cliffs, NJ, 1992 (Chapter 10).
crease homogeneity in the fermentor could be employed. In [5] M. Charles, Fermentation scale-up: problems and possibilities, Trends
Biotechnol. 3 (1985) 124139.
the illustrative example of the cultivation of B. thuringien-
[6] N.M.G. Oosterhuis, N.W.F. Kossen, A.P.C. Olivier, E.S. Schenk,
sis for thuringiensin production, different pH values had a Scale-down and optimization studies of the gluconic acid
substantial effect on cultivation. However, variation of pH fermentation by Gluconobacter oxydans, Biotechnol. Bioeng. 27
value for a short period of time did not have significant effect (1985) 711720.
130 Y.-L. Hsu, W.-T. Wu / Biochemical Engineering Journal 11 (2002) 123130
[7] S. George, G. Larsson, S.-O. Enfors, A scale-down two-compartment [17] D.P. Campbell, D.E. Dieball, J.M. Brackett, Rapid HPLC assay for
reactor with controlled substrate oscillations: metabolic response of the -exotoxin of Bacillus thuringiensis, J. Agric. Food Chem. 35
Saccharomyces cerevisiae, Bioprocess Eng. 9 (1993) 249257. (1987) 156158.
[8] F. Bylund, F. Guillard, S.-O. Enfors, C. Tragardh, G. Larsson, Scale [18] B. Levinson, K.J. Kasyan, S.S. Chiu, Identification of -exotoxin
down of recombinant protein production: a comparative study of production, plasmid encoding -exotoxin and a new exotoxin in
scaling performance, Bioprocess Eng. 20 (1999) 377389. Bacillus thuringiensis by HPLC, J. Bacteriol. 172 (1990) 31723179.
[9] A. Humphrey, Shake flask to fermentor: what have we learned? [19] G.E.P. Box, K.B. Wilson, Experimental attainment of optimum
Biotechnol. Prog. 14 (1998) 37. conditions, J. Roy. Stat. Soc. 13 (1951) 145.
[10] W.M. Hiller, C.R. Wilke, H.W. Blanch, Transient responses of [20] R.H. Myers, D.C. Montgomery, Response Surface Methodology:
hybridoma cells to nutrient additions in continuous culture. I. Glucose Process and Product Optimization Using Designed Experiments,
pulse and step changes, Biotechnol. Bioeng. 33 (1989) 477486. Wiley, New York, 1995.
[11] C. Abel, U. Hbner, K. Schgerl, Transient behaviour of Bakers [21] D. Weuster-Botz, C. Wandrey, Medium optimization by genetic
yeast during enforced periodical variation of dissolved oxygen algorithm for continuous production of formate dehydrogenase,
concentration, J. Biotechnol. 32 (1994) 4557. Process Biochem. 30 (1995) 563571.
[12] C. Abel, F. Linz, T. Scheper, K. Schgerl, Transient behaviour of [22] Z. Malik, K. Rashid, Comparison of optimization by response surface
Bakers yeast during enforced variations of dissolved oxygen and methodology with neurofuzzy methods, IEEE Trans. Magn. 36 (1)
glucose concentrations, J. Biotechnol. 33 (1994) 183193. (2000) 241257.
[13] P. Neubauer, L. Hggstrm, S.-O. Enfors, Influence of substrate [23] D. Waitier, H.C. Dubourguier, I. Leguerinel, J.P. Hornez, Response
oscillations on acetate formation and growth yield in Escherichia surface models to describe the effects of temperature, pH, and ethanol
coli glucose limited fed-batch cultivations, Biotechnol. Bioeng. 47 concentration on growth kinetics and end products of a Pectinatus
(1995) 139146. sp., Appl. Environ. Microbiol. 62 (1996) 12331237.
[14] W.-T. Wu, Y.-L. Hsu, Y.-F. Ko, L.-L. Yao, Effect of shear stress [24] S.-L. Lee, W.-C. Chen, Optimization of medium composition for the
on cultivation of Bacillus thuringiensis for thuringiensin production, production of glucosyltransferase by Aspergillus niger with response
Appl. Microbiol. Biotechnol. 58 (2002) 175177. surface methodology, Enzyme Microb. Technol. 21 (1997) 436
[15] M.B. Mohd-Salleh, L.C. Lewis, Feeding deterrent response of corn 440.
insects to -exotoxin of Bacillus thuringiensis, J. Invertebr. Pathol. [25] G. Dey, A. Mitra, R. Banerjee, B.R. Maiti, Enhanced production of
39 (1982) 323328. amylase by optimization of nutritional constituents using response
[16] G.E. Cantwell, E. Dougherty, W.W. Cantelo, Activity of the surface methodology, Biochem. Eng. J. 7 (3) (2001) 227231.
-exotoxin of Bacillus thuringiensis var. thuringiensis against the [26] D.-Y. Hsiun, Design of an airlift reactor with net draft tubes
Colorado potato beetle and the Ames test, Environ. Entomol. 12 (5) and its application in fermentation of Bacillus thuringiensis, Ph.D.
(1983) 14241427. Dissertation, National Tsing Hua University, 1995.