COMMUNICATIONS TO THE EDITOR Carbon Dioxide Hold-Up as a Source of Error in Batch-Culture Calculations INTRODUCTION Construction of material balances is of prime importance in the study of bioener- getics. The consistency of expérimental data should always be checked via the known regularities of fermentation processes and elemental balances. This subject has recently received much attention in the literature.’~* However, extensive data have so far only appeared for continuous-culture systems operating at steady state In batch cultures of Klebsiella pneumoniae studied in this laboratory, systematic losses in carbon balances were observed. This discrepancy was then traced to the presence of carbon dioxide in the culture broth at concentrations much higher than anticipated. A similar observation has recently been reported for bakers’ yeast cultures by Barford and Hall.* ‘An attempt was therefore made to quantify the amount of missing carbon dioxide ‘and to evaluate the influence of this discrepancy on the calculation of the respiratory {quotient (RQ). Therefore the amount of carbon dioxide remaining in the broth was determined during the exponential growth phase and the measured overall RQ was corrected. Measured and corrected RQ values were also compared with the theo- retical RQ which can be calculated from bioenergetic considerations if the experi- ‘mental yield on substrate, degree of reduction, and carbon content of biomass and ‘substrate, are known. The corrected RQ was found to be close to the theoretically calculated RQ whereas the measured RQ deviated significantly. This phenomenon has therefore important theoretical and practical implications for the interpretation and use of metabolic data, particularly for computer-controlled batch or any other Fermentation system operating at unsteady state, where the control parameter is ‘obtained from the instantaneous gas exchange data." MATERIALS AND METHODS ‘The organism, Klebsiella pneumoniae NCTC 418, was cultured in a synthetic medium as described by Evans et al.8 Glycerol was used as substrate and assayed enzymatically (Boehringer UV method No. 148270). Dry weights were determined by the method of de Vries and Stouthamer.* Biomass was collected on a 0.2 wm pore diam filter (Sartorius), washed with distilled water and dried to constant weight at 378°K., Medium was sterilized by membrane filtration through a 0.2 am membrane filter and inoculated by an actively growing inoculum to eliminate any lag phase and the possibility of unbalanced growth. The elemental composition of the biomass (Table 1) was determined by a computer-coupled element analyzer (Perkin-Elmer 240). Experiments were carried out in a 11 x 10-* m* working volume fermentor maintained at 308°K, pH was controlled at 6.80 0.05. The air flow rate to the fermentor was controlled by a thermal mass flowmeter (Brooks 5811) at about 0.77 kg dry airhr. ‘Special attention was paid for the accurate determination of gas flows and con- Biotechnology and Bioengineering, Vol. XXII, Pp. 1979-1983 (1980) © 1980 John Wiley & Sons, Inc. (0006-35972/80/0022-1979$01.001980 BIOTECHNOLOGY AND BIOENGINEERING VOL. XXII (1980) TABLE I Elemental Composition* and Formula of Ki. pneumoniae Grown on Glycerol at its Maximum Growth Rate cH NO Formula “In % dry weight; ash-free basis. centrations. All flows were corrected for humidity and volumetric changes. Gas- hase oxygen and carbon dioxide concentrations were determined by a twin-channel paramagnetic oxygen analyzer (Servomex OA 184) and an infrared carbon dioxide analyzer (Beckman 864), respectively. Complete aerobic growth was ensured by maintaining the dissolved oxygen tension well above the limiting range. No products other than biomass, carbon dioxide, and water could be detected at levels that can be significant. 10 Cy Dry Weight (xg Jn) 10 on 001 — + Time mind 0001 5 720 Bo 360 Zao Fig. 1. Biomass vs. fermentation time.COMMUNICATIONS TO THE EDITOR 1981 CARBON DIOXIDE DETERMINATION For the system used carbon dioxide can be assumed to be the only source of inorganic carbon. Therefore for the culture broth at any instant TC= TIC + TOC a where TC is the total carbon (kg/m), TIC is the total inorganic carbon, and TOC is, the total organic carbon. Of these TC and TOC were measured by a Dohrmann DC- 50 carbon analyzer. Carbon dioxide not accounted for, by the gas-phase analysi i.e., carbon dioxide remaining in the broth, can then be calculated by the following expression: CO, (broth) = 44/12 (TC - TOC) (keim’) ¢ ‘TC and TOC determinations were carried out immediately after sampling. All sam- ples were cooled during sampling down to about 278°K by an on-line heat exchanger T T T T rob 0, in Broth (kg 73) ost —— Time (min) ° L L L L ° 120 240 360 480 Fig. 2. Carbon dioxide retained in the broth during exponential growth.1982 BIOTECHNOLOGY AND BIOENGINEERING VOL. XXII (1980) TABLE IL Overall Carbon Balance and RQ Calculations for the Exponential Growth of Kl. pneumoniae Amount Per m? of culture: (ke) Substrate carbon used up 4.05 Carbon recovered in biomass 2.66 Carbon recovered in gas phase 1.06 Carbon recovered in broth 027 Yield on substrate (ash-free basis) 0.50 “Without With correction correction Difference Crecovery (%) 92. 9 7 RQ 0.57 on 18% RQ calculated independently from yield 0.69 (on substrate by ea. (3) manufactured in our workshop. The typical residence time in the heat exchanger was about 5 sec. RESULTS AND DISCUSSION Experimental results are shown in Figures | and 2. The maximum growth rate of this organism is quite high (1.07 = 0.02 (Wh)] therefore it was not possible to obiain more extensive data due to the sampling, processing, and analysis times Tequired. From Figure 2 it can be seen that the amount of unaccounted carbon dioxide increases during the exponential growth phase. Few mechanisms that can maintain this carbon dioxide in the broth can be thouht of. These include entrapment by cell, pure physical absorption by supernatant, or participation in the carbon dioxide carbonate buffer system. At this sage, however, the relative importance of these mechanisms is not clear. Therefore only a macroanalysis is reported here. In Table II the overall carbon balance forthe exponential growth phase is presented. These calculations indicate that carbon dioxide corresponding to 7% of the total substrate carbon input remained in the broth at the end of exponential phase. This results in a large error in the measured value of RQ. Assuming complete aerobic growth with no products other than biomass, carbon dioxide, and water, RQ can. also be calculated from the experimentally determined yield on substrate by the mass-energy balance method first developed by Minkevich and Eroshin.* Using their notation and rearranging, RQ is given by 1-¥ oso) RO = Sat Fics oa) 0.51, 0, = 039, 7, = 4.67, 95 = 4.09, and ¥, = 0.50 the above expression gives an RQ of 0.69 which is in close agreement with the RQ values corrected for the carbon dioxide retained in the culture, and hence unaccounted for in the gas phase (Table 11). 8COMMUNICATIONS TO THE EDITOR 1983 ‘The measured RQ, however, deviates significantly from both the corrected and independently calculated RQ values. These results indicate that unless carbon bal- ances fit tightly or broth-phase CO; measurements are made, measured RQ values ccan be in great error. This can lead to serious problems in industries where batch, fed-batch, or any other fermentation process essentially not at steady state is mon- itored and controlled by on-line measurement of the RQ. Nomenclature RQ respiratory quotient (dimensionless) TC total carbon in broth (kg/m*) TOC total onganic carbon in broth (kg/m®) TIC total inorganic carbon in broth (kg/m) Y, substrate yield (dimensionless) ‘o> carbon weight fraction in biomass (dimensionless) a ‘carbon weight fraction in substrate (dimensionless) % reductance degree of biomass (equiv available electrons/g atom car- bon) % reductance degree of substrate (equiv available electrons/g atom car- on) ‘Thanks are due to Mr. C. Ras for performing the TCITOC analyses. References. 1. C. L. Cooney, H. Y. Wang, and D. I. C. Wang, Biotechnol. Bioeng. 19, 55 97). 2. J. A. Roels and N. W. F. Kossen, “On the modeling of microbial metabolism,” in Progress in Industrial Microbiology, M. 5. Bull, Ed. (Elsevier, Amsterdam, 1978). 3. L. E. Erickson, I. G. Minkevich, and V. K. Eroshin, Biotechnol. Bioeng., 20, 1595 (1978). 4. J. A. Roels, ““The application of macroscopic principles in microbial energetics,” in press, 1980. 5. J. P. Barford and R. J. Hall, Biotechnol. Bioeng., 21, 609 (1979). 6. H. Y. Wang, C. L. Cooney, and D. I. C. Wang, Biotechnol. Bioeng... 19, 69 asm. 7. S. Aiba, S. Nagia, and Y. Nishizawa, Biotechnol. Bioeng., 18, 1001 (1976) 8. C.G.T. Evans, D. Herbert, and D. W. Tempest, in Methods in Microbiology, J. R. Norris and W. W. Robbins, Eds. (Academic, London, 1970), Vol. 2, p. 313. 9. W. de Vries and A. H. Stouthamer, J. Bacteriol., 96, 472 (1968) A.A. ESENER IN. W. F. Kossen JA, ROELS Biotechnology Group, Department of Chemical Engineering Delft University of Technology Jaffalaan 9, P.O. Box 5029 2600 GA Delft, The Netherlands Accepted for Publication February 21, 1980