Biotechnol. Bog. 1992, 8, 580-582
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Bioreactor Scale-up for the Oxygen-Sensitive Culture Bacillus subtilis The Influence of Stirrer Shaft Geometry John R. Bourne, Eddy P. Zurita,?and Elmar Heinzle' Technisch-chemisches Laboratorium, ETH Ztirich, Switzerland
The relative amounts of the metabolites acetoin and 2,&butanediol, formed in a Bacillus subtilis culture, depend strongly on the dissolved oxygen concentration (DO). Hence any factor determining DO can influence the product ratio Ac/Bu. Experiments in a 0.045 m3 stirred bioreactor, fitted with three Rushton turbines, have shown different product ratios when the diameter of the drive shaft was changed and all other conditions were kept constant. This unexpected result was attributed to changes in the power consumption and the pumping and gas handling capacities of the turbines. B. subtilis was cultivated in three geometrically similar vessels (0.045,0.45,and 4.5 m3) using a constant superficial gas velocity (0.0075 m s-9. The product ratio and the volumetric mass transfer coefficient were essentially independent of scale when the gassed power input per unit volume was constant. This scale-up result reflects the importance of adequate oxygenation of this culture, which was violated when the shaft diameter was not scaled with the vessel diameter. Geometrical similarity was an important factor in the scale-up of this fermentation.
Introduction Shibai et al. (1973) reported the production of 2,3butanediol (Bu) in a culture of Bacillus subtilis, which was oxidized to acetoin (Ac) after the glucose had been exhausted. The product concentration was found to depend upon the dissolved oxygen (DO) concentration in the medium. The relevant metabolic pathways for the production of acetoin (CH3CHOHCOCH3)and butanediol (CH3CHOHCHOHCH3) during the culture of B. subtilis are given by Moes et al. (1986). Moes et al. (1985) conducted controlled DO fermentations and found that Bu was the principal product when DO < 70 ppb, whereas Ac was predominant when DO > 100ppb. Griot et al. (1986) confirmed and extended these findings. Thus, at DO levels corresponding to 1-2 % of air saturation there is a dramatic change in the product distribution Ac/Bu. Factors influencing the DO in this culture can profoundly influence the product ratio, e.g., stirrer speed, air flow rate and occurrence of dead zones (Griot et al., 19861, bioreactor size (Griot et al., 1988b), impeller design, and mediumviscosity (Griotetal., 1988a). Conversely,product distribution is a measure of oxygenation, which as a working hypothesis will be expressed by Ac/Bu = f(kLa) (1) The liquid-film volumetric mass transfer coefficient, kLa, dependsupon the superficialgas velocity,us,and the gassed . a given ferpower input per unit volume, P g / V ~For mentation and a specified bioreactor geometry, kLa a ( P g / V L ) " u ~ (2) Equation 2 predicts an unchanged kLa value when scaling up with a constant specific power input and a constant superficial gas velocity, while eq 1 predicts a constant product ratio under these conditions.
* Corresponding author. + Current address: Centro de Quimica Farmaceutica,Cubanacan,
Havana, Cuba. 87567938/92/3008-0580$03.00/0
At constant superficial gas velocity, us, eq 2 reduces to kLa = y(Pg/VL)a (3) Fermentations have been carried out in 0.045, 0.45,and 4.5m3vessels, each fitted with three Rushton turbines, at a constant superficial gas velocity of us = 0.0075 m s-l (Griot et al., 1988b). The Ac/Bu ratio for the two larger tanks fell in the range 1-19 and was almost the same when Pg/VL was constant, but the results for the smallest tank were not consistent with those from the 0.45 and 4.5 m3 tanks. This deviation was attributed to a lack of geometrical similarity, whereby the drive shafts of the 0.045 and 0.45 m3 bioreactors had almost the same diameters. (Allother dimensions were properly scaled.) The space between the shaft and the turbine blades was consequently greatly reduced in the 0.045m3 tank, thus reducing the pumping and gas handling capacities of the impeller. This was reflected in a fall in the ungassed power number from 14.6 for the 0.45 m3 tank to 10.9 for the 0.045 m3 vessel (Griot et al., 198813). It was concluded that the Ac/Bu ratio during the culture of B. subtilis is so sensitive to oxygen supply that it responds to the reduced performance of the three turbines in the 0.045 m3 bioreactor. The present contribution checks this conclusion directly. By reducing the shaft diameter in the 0.045 m3 vessel, three geometrically similar tanks could be compared to decide whether the AcIBu ratio is independent of scale when PglVL and us are held constant.
Materials and Methods Figure 1 shows the types of stirrers used at the three scales. The most important reactor dimensions and operating conditions were reported by Griot et al. (1988b). The only difference between the previously and currently employed 0.045 m3 bioreactors was the impeller shaft diameter (ds). The new vessel is geometrically similar to the 0.45 and 4.5 m3 tanks. The shaft diameters of the larger reactors were 0.029 and 0.063 m. ds of the old wide shaft in the 0.045 m3 reactor was 0.028 m, and ds of the corresponding new narrow shaft was 0.015 m.
0 1992 American Chemical Society and American Institute of Chemical Engineers
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1 0 0 2 0 0 3 0 0 4 0 0 "
.,
n [min-11
Figure 2. Product ratio as function of stirrer speed (rpm) at three scales: 4.5 m3;0,0.45 m3;A, 0.045 m3 (narrow shaft); A, 0.045 m3 (wide shaft). uB= 0.0075 m s-l in all runs.
Figure 1. Shaft and stirrer geometries of the 0.045m3bioreactor with dimensions in millimeters. Left, old shaft, right, new shaft.
The power number of the three Rushton turbines in the new 0.045 m3vessel was measured (De Boni, 1988),giving 14.8f 0.2 for ungassed conditions. This is consistent with the value of 14.6 measured for the 0.45 m3 tank. B. su btilis fermentations have been fully described earlier (Griot et al., l986,1988a, 1988b),including the GC analysis of Ac and Bu (Griot et al., 1988~) and gas analysis by mass spectrometry (Heinzleet al., 1990). The same B. subtilis strain (AJ 1992)with 1% inoculum of spores and the same complex medium was used, and rapid growth under nonsterile conditions was again obtained. Temperature was controlled within better than 1K, and NaOH (5N) was added to hold the pH at 6.5. An Ingold electrode measured DO in a zone of turbulent flow. A Brooks mass flowmeter measured the air flow, while a strain gage determined the torque in the drive shaft. Liquid samples were taken periodically during the fermentation, and the biomass and glucose concentrations were determined photometrically and enzymatically, respectively. After centrifugation and filtration, dissolved Ac and Bu were analyzed by capillary GC within 10 min.
Experimental Results As found earlier (Griotet al., 1988a),fermentations were highly reproducible, and rapid and reversible interconversionsbetween Ac and Bu were observeddependingupon DO. Figure 2 shows as a function of stirrer speed n (rpm) the product ratio measured 0.5 h before sporulation. The results from the 4.5, 0.45, and old 0.045 m3 bioreactors were taken from Griot et al. (1988b), while those for the new 0.045 m3bioreactor, having the smaller shaft (Figure l),were obtained in the current work. Figure 3 representsthe product distribution as a function of the gassed power per unit volume. Figures 2 and 3 show an effect of shaft diameter on product distribution, which means that an apparently unimportant change in geometry was significant for this culture. The highest measured Ac/Bu ratio was 17.5, which refers to the new 0.045 m3bioreactor. The capillary GC method has limited resolution for low Bu concentrations, and it is possible that the true product ratio was lower (Griot et al., 1988~). When the superficial gas velocity and gassed power per unit volume were both constant, Figure 3 shows that the product distribution was almost independent of scale. This
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1
1
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I
2
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I
.
3
I
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P,/V L [kW m -31
Figure 3. Product ratio as function of measured gassed power uptake per unit of culture volume. Symbols and superficial gas velocity as in Figure 2.
is consistent with the known sensitivity of the B. subtilis culture to oxygenation and the constancy of the mass transfer coefficient kLa under these fermentation conditions. Volumetric mass transfer coefficients (kLa) were determined during the fermentations at the same time samples were taken to analyze for Ac and Bu. Figure 4 shows these results as a function of the specific gassed power consumption when the superficial gas velocity was constant. The kLa values at the three different scalesagree within experimental accuracy for geometrically similar stirrer shafts. Equation 2 was well satisfied, and kLa primarily depended on the gassed specific power input to the culture, when geometrical similarity was maintained. Using the product distributions already plotted in Figures 2 and 3, Figure 5 was prepared to test the hypothesis that the product distribution of this oxygensensitive culture depends upon kLa. Large symbols indicate directly measured kLa values, whereas small symbols connected with lines indicate kLa values derived from empiricalcorrelations. These were established using the Hughmark correlation (Joshi et al., 1982) for the estimation of P g / V ~ .Parameter values of eq 3 were as follows: 4.5 m3, a! = 0.481, y = 1.23 X 0.45 m3, a! = 0.736, y = 2.28 X 0.045 m3 (wide shaft), a! = 0.87, y = 1.09 X For the 0.045 m3 tank with the narrow shaft, there were not enough kLa values available to establish a correlation. Furthermore, the Pg/VL values measured during fermentations were larger than those predicted by the correlations. Therefore, the measured Pg/VLwith a! = 0.9 and y = 5 X low5was used. As is also evident in the earlier two figures, product distributions
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became geometrically similar to the 0.45 and 4.5 m3vessels. It was then found that, when the superficial air velocity was held constant, the product ratio at the three scales depended upon the mass transfer coefficient, kLa, or upon the specific gassed power input. This scale-up result is accounted for by the high oxygen sensitivity of this culture.
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Notation
0 . 0 0 - 1 1 0 1
2
3
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PgN L [kW m-31
Figure 4. Mass transfer coefficient as a function of measured gassed power uptake per unit culture volume. Symbols and superficial gas velocity as in Figure 2. 20
acetoin concentration, kg m-3 butanediol concentration, kg m-3 dissolved oxygen concentration, ppb or mg m-3 volumetric oxygen mass transfer coefficient, s-1 gassed power input, kW liquid volume, m3 superficial gas velocity, m s-l coefficients in eq 3
Acknowledgment The stay of E.Z. at ETH Ziirich was supported by UNIDO.
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Literature Cited 10
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0.02
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0.08
0.10
0.12
kLa [h-11
Figure 5. Product ratio as a function of mass transfer coefficient. Symbols and superficial gas velocity as in Figure 2. Large symbole: measured kLa. Small symbols and lines: kLa estimated from correlations (also see text).
scatter somewhat. The use of correlationsbrings the curves closer together except at high kLa values, where the 0.45 m3t a n k results deviate considerably from the others. This is at least partly caused by analytical difficulties when the butanediol concentration is low, which is most pronounced at high Ac/Bu ratios. Figure 5 nevertheless shows that kLa has an important effect on the product ratio. I n comparing Figures 4 and 5 the kLa value offers a slightly better correlating parameter than the gassed power input.
Conclusions The high sensitivity to oxygenation of the product distribution Ac/Bu, arising during the culture of B. subtilis, showed itself here in two ways. Changing from a 28- to a 15-mm drive shaft (upon which three Rushton turbines were mounted) in a 0.045 m3 bioreactor, b u t keeping all other conditions constant, was found to change Ac/Bu. Using t h e smaller drive shaft, t h e 0.045 m3 bioreactor
De Boni, S. Diplomarbeit, Laboratorium fcir Technische Chemie, ETH, Zcirich, Switzerland, 1988. Griot, M.; Moes,J.; Heinzle, E.; Dunn, I. J.; Bourne, J. R. A microbial culture for the measurement of macro- and micromixing phenomena in biological reactors. Proceedings of Conferenceon Bioreactor Fluid Dynamics; BHRA: Cranfield, England, 1986;pp 203-216. Griot, M.; Galindo, E.; Heinzle, E.; Dunn, I. J.; Bourne, J. R. Investigation of bioreactor mixing and mass transfer using an oxygen-sensitivemicrobial culture. Proceedings of Conference on Mixing; BHRA Cranfield, England, 1988a;pp 435-442. Griot, M.; Saner, U.; Heinzle, E.; Dunn, I. J.; Bourne, J. R. Fermenter scale-up using an oxygen-sensitiveculture. Chem. Eng. Sci. 1988b,43,1903-1908. Griot, M.; Dettwiler, B.; Heinzle, E.; Mayer, F.,Dunn, I. J. Simple and rapid gas-chromatographicanalysisof volatile metabolites in fermentation broths. Anal. Chim. Acta 19880,213,ll-22. Heinzle, E.; Oeggerli, A.; Dettwiler B. On-line fermentation gas-analysis: error analysis and application of mass spectroscopy. Anal Chim. Acta 1990,238,101-115. Joshi, J. B.; Pandit, A. B.; Sharma, M. M. Mechanically agitated gas-liquid reactors. Chem. Eng. Sci. 1982,34,813-844. Moes, J.; Griot, M.; Keller, J.; Heinzle, E.; Dunn, I. J.; Bourne, J. R. A microbial culture with oxygen-sensitive product distribution as a potential tool for characterizing bioreactor oxygen transport. Biotechnol. Bioeng. 1985,27,482-489. Moes, J.; Griot, M.; Heinzle, E.; Dunn, I. J.; Bourne, J. R. A microbial culture as an oxygen sensor for reactor mixing effects. Ann. N.Y. Acad. Sci. 1986,469,118-130. Shibai, H.; Ishizaki, A.; Hirose, A. Conversion of microbial products in relation to oxygen supply in inosine fermentation. Agric. Biol. Chem. 1973,37, 2083. Accepted August 24, 1992. R e g i s t r y No. CHsCHOHCOCH3, 513-86-0;CH3CHOHCHOHCH3, 513-85-9; 02,7782-44-7.
