On-line control of glucose concentration using an automatic glucose analyzer

[J. Ferment. Technol., Vol. 65, No. 3, 325-331. 1987]

On-Line Control of Glucose Concentration Using An Automatic Glucose Analyzer SATORU MIZUTANI,SHINJI hJIMA, MAKOTO MORIKAWA, KAZUYUKI SHIMIZU, MASAKAZU MATSUBARA, YASUMASAOGAWA*, ROKURO IZUMI*, KUNIO MATSUMOTO*, a n d TAKESHI KOBAYASHI Department of Chemical Engineering, Faculty of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464, Japan; *Toyo Jozo Co., Ltd., Ohito-cho, Shizuoka 410-23, Japan

The fundamental characteristics of an automatic glucose analyzer which consists of sampling, sensor, and operation units were examined. The glucose sensor is a dual cathode type which has an immobilized glucose oxidase membrane coupled with an oxygen sensor. Using this glucose sensor combined with art automatic sampling device, on-off control of the glucose concentration in fed-batch cultures of Saccharorayces cerevisiae was carried out. When the glucose concentration to he controlled w a s s e t at 0.3 and 10 g/l, the concentration was well maintained within the range of 0.08-0.54 gll in the former, and within 9.2-11.1 g/1 in the latter. In the former experiment, 1.67 g/l of ethanol was produced at the end of cultivation (OD~0=34). On the other hand, 12.9 g/l of ethanol was accumulated at the end of cultivation (ODsv0=43) in the latter experiment. Fed-batch cultures of Micrococcu3 ruteus were also carried out. The glucose concentration was set at 2.5 gll. The microorganism grew up to ODet0=264 and the glucose concentration was maintained within 2.0 and 3.1 g/l.

T h e c a r b o n source is one o f the most i m p o r t a n t m e d i u m c o m p o n e n t s in fermentations. H o w e v e r , there a r e few a p p r o p r i a t e sensors a v a i l a b l e for the d e t e c t i o n o f c a r b o n sources, a n d this has l i m i t e d the c o n t r o l strategy a n d the o p t i m a l use o f c a r b o n sources. T h e o n l y successful case is t h a t for a volatile c a r b o n source such as m e t h a n o l . W e h a v e d e v e l o p e d a m e t h a n o l sensor1) w i t h a Silicone t u b i n g - F I D a n a l y z e r a n d showed t h a t the m e t h a n o l c o n c e n t r a t i o n c o u l d be c o n t r o l l e d a t a c o n s t a n t level for c u l t i v a t i o n o f a m e t h a n o l utilizing b a c t e r i u m . L a t e r , T e f l o n r e p l a c e d the Silicone a n d the T e f l o n t u b i n g sensor is n o w w i d e l y used for the c o n t r o l o f volatile c a r b o n sources. H o w e v e r , few sensors h a v e b e e n r e p o r t e d for the d i r e c t m e a s u r e m e n t o f a non-volatile c a r b o n source, such as glucose, w h i c h is often used for the c u l t i v a t i o n of m i c r o o r g a n isms. T h u s , glucose has b e e n fed into m e d i u m b y e m p i r i c a l m e t h o d s such as a s s u m i n g c o n s t a n t b i o m a s s y i e l d o r b y using a drastic increase in dissolved o x y g e n con-

c e n t r a t i o n as a n i n d i c a t o r o f the s u g a r starvation. I n b a k e r ' s y e a s t cultivation, it is well k n o w n t h a t a h i g h c o n c e n t r a t i o n o f the s u g a r causes p r o d u c t i o n o f e t h a n o l a n d therefore reduces the biomass y i e l d d u e to the so-called C r a b t r e e effect. O n the o t h e r h a n d , if the sugar s u p p l y is insufficient, the p r o d u c t i v i t y of the biomass a t t a i n a b l e is limited. Therefore, A i b a et al3) a n d W a n g et al. 3) a n a l y z e d the e x h a u s t gas on line, a n d c o n t r o l l e d the sugar-feed r a t e to m a i n t a i n the r e s p i r a t o r y q u o t i e n t ( R O ) in the r a n g e o f 1.0 to 1.2. N a n b a et al.*) m o n i t o r e d the e t h a n o l c o n c e n t r a t i o n a n d r e g u l a t e d the feed r a t e of the s u g a r b y a feedforward a n d f e e d b a c k control scheme. D a i r a k u et al. 5~ c o n t r o l l e d the e t h a n o l c o n c e n t r a t i o n in the c u l t u r e b r o t h o f b a k e r ' s y e a s t with a n on-off c o n t r o l l e r a n d a P I D controller b y using a T e f l o n t u b i n g sensor. T a k a m a t s u et al. 6~ d e v e l o p e d a p r a c t i c a l a n d useful c o m p u t e r c o n t r o l scheme so t h a t the y e a s t cell conc e n t r a t i o n or the specific g r o w t h r a t e will

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1.0; MgSO,'7H20, 0.38; KC1, 0.22; sodium citrate, 2.5; yeast extract, 0.5; 1 ml of vitamin solution (biotin, 0.04 g; vitamin Ba, 0.08 g; vitamin Be, 2.0 g; calcium pantothenate, 1.0g and inositol, 20g/l); 1 ml of mineral solution (CuSO4-5H,O, 0.05 g; ZnSO4"7H,O, 0.8 g and Fe(SO4)2(NH4)2.6H,O, 0.3 g/l) at pH 6.0 adjusted with an aqueous solution of HC1 (1 N). The culture medium for M . ruteus consisted of (per liter) 10 g meat extract, 10 g peptone, 5 g yeast extract, 1 g NaCI and 2.5 g glucose. The pH was controlled at 7.0. Cultivation Microorganisms stocked on agar slants were inoculated into two shake flasks (100 ml) containing the culture medium and cultivated at 30°C for 15 h on a reciprocal shaker. This seed culture (200 ml) was harvested, suspended in 100 ml of the fresh medium and transferred into 900 ml of the culture medium in a miaifermentor equipped with a fourbladed disc-turbine impeller and three baffles (working volume: 1 l for S. cerevisiae and 2 1 for M . ruteus, Iwashiya Bio-Science Co., Type MB). Temperature was automatically controlled at 30°C and pH was maintained at 6.0 (S. cerevisiae) or 7.0 (M. ruteus) by the addition'of 14% ammonia water. The fermentor was aerated at 1 l/min and agitated at 500 rpm for S.

follow a desired profile which is specified in advance. M a n y researchersV-9) have also attempted to control the glucose concentration indirectly during baker's yeast cultivation. Recently, m a n y biosensors using microorganisms or enzymes have been developed, 1°) one of which is the glucose sensor. The sensor is equipped with an automatic sampling device and a microcomputer which enables the measurement of glucose concentration on line. In this study, the fundamental characteristics of a newly developed glucose sensor were examined, and its applicability to on line control was investigated. M a t e r i a l s and M e t h o d s M i c r o o r g a n i s m s and culture m e d i u m The microorganisms used in this study were Saccharomyces cerevisiae and Micrococcus ruteus. The culture medium for S. cerevisiae was the same as that reported by Aiba et al. 2) The semisynthetic medium contained (g/l): glucose, 0.3 or 10; (NH2)2CO, 2.15; NaHPO4.2H,O, ,'- . . . . . . . . .

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cerevisia*; 1.5 l/rain and 500 rpm for M. ruteus. Culture broth (ca. 5 ml) was sampled every hour and was centrifuged to obtain the snpernatant for the glucose and ethanol assays. O n - l i n e glucose analysis A schematic diagram of the automatic glucose analyzer is shown in Fig. 1. Culture broth was filtered continuously using a ceramic filter (12 holes, pore size: 0.Spm, Toshiba Ceramics Co.) or a membrane filter (0.2/~m). The filtrate was withdrawn with a sample-syringe-drive unit (SS), a part of which (15 #1) was sampled using a slide valve and injected by SS into the measuring cell which had been filled up with the phosphate buffer (100 mM, pH 7.0) by the buffer feed/drain drive unit (BD). The glucose sensor, which consisted of an immobilized glucose oxidase membrane and an oxygen sensor, was installed in the cell. Output voltage of the sensor was differentiated twice in order to obtain a quick response, and its value was compared with those of the standard glucose solutions. After measurement, the sample was drained out into a waste bottle by the BD. An outline of the operation procedure is presented as a flow chart in Fig. 2. The sampling and glucose analysis procedures were fully automated. The operation was controlled by an external personal computer interfaced with PIO-16/16 (98). The personal computer (PC9801, NEC Co.) communicated with the operation unit through RS-232C. The measurement interval and the concentration of the standard glucose solution could be selected freely depending on the level of the glucose concentration for measurement and the control strategy. The shortest interval of measurement was about I min. Three injection volumes (5, 15 and 25 pl) of the sample could be selected (15 pl was used in this study). The cell volume could also be selected as 1.5 or 3 ml (the former was used). By altering these volumes, the range of measurements could be

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Control of glucose concentration As described above, glucose concentration was measured on line at intervals of 1 min for S. cerevisiae and 7 min for M. ruteus. A glucose solution (100 g/l for S. cerevisia* and 200 g/l for M. ruteus) was fed into the fermentor with a peristalic pump using an on-off control scheme. The overall organization of the control system is shown

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in Fig, 3. The glucose Concentration in the fermentor was monitored by the glucose analyzer as stated above and was controlled by adding concentrated glucose by the on-off activation of a peristalic pump. The interface board required to activate the pump was WRY/RRY-16 (98). Data acquisition was carried out using the PC9801. The pH of the culture broth was monitored using a pH electrode and it was controlled automatically by adding 14% w/v of ammonia water by the on-off activation of a peristalic pump. Analytical method The concentration of the cells was determined by measuring the optical density at 570nm (ODsv0) for S. cerevisiae and at 610nm (ODsl0) for M. ruteus in the culture broth diluted with the 0.9% NaCI solution using a Shimadzu Spectronic 20 photometer. The off-line glucose concentration was measured by Glucostat reagent (Worthington Biochemicals Co.). Ethanol concentration was measured with a gas chromatograph (Model GC-7AG, Shimadzu Co.).

Results and D i s c u s s i o n Basic characteristics o f the a u t o m a t i c g l u c o s e analyzer T h e r e l a t i o n s h i p between glucose c o n c e n t r a t i o n a n d the o u t p u t v o l t a g e o f the a n a l y z e r was e x a m i n e d for various glucose concentrations. M e a s u r e m e n t s for each c o n c e n t r a t i o n were r e p e a t e d 10 times a n d the m e a n v a l u e was used as a n o u t p u t voltage. As shown in Fig. 4 A a n d 4B, the o u t p u t v o l t a g e was l i n e a r u p to

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10 g/l. Above 10 g/l, the relationship was not linear. Reproducibility of the measurement was examined at glucose concentrations of 0.5, 5 and 20 g/l. T h e measurements were repeated 100 times at each concentration. T a b l e 1 shows the m e a n output voltage and the standard deviation at each glucose concentration. It was found that the reproducibility was excellent at these glucose concentrations. T h e magnitude of output depends upon the measuring cell volume and the sample volume. Therefore, the range of linearity can be extended to higher glucose c o n c e n t r a t i o n s (30-40 g/l) b y a d j u s t i n g the s a m p l e a n d cell volumes ( d a t a not shown). F i g u r e 5 shows the results c o n c e r n i n g the stability o f the glucose a n a l y z e r over a long period. T h e a n a l y z e r was stabilized for 7 - 8 h a n d t h e n the glucose c o n c e n t r a tion was m e a s u r e d a t intervals o f 20 rain w i t h o u t c a l i b r a t i o n using a s t a n d a r d solution. T h e o u t p u t v o l t a g e i n c r e a s e d initially for 2-3 h and then decreased gradually afterwards. W h e n glucose c o n c e n t r a t i o n s were 1 a n d 10 g/l, the d r o p s in o u t p u t voltage

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per hour were 1.0 and 3.4 m V / h , respectively. During practical use, this a m o u n t of outputdrop does not affect the control quality. For long term measurements, calibration using a standard solution every one hour enhanced the accuracy. After considering the above results, standard glucose solutions at two different concentrations were used for the initial calibration and the glucose concentration was expressed as a linear function with respect to the output voltage. Since metabolite and m e d i u m components m a y affect the output voltage of the electrode during fermentation, calibrations using a single standard solution were carried out every hour during the fermentation. Fed-batch culture with on-off control of glucose concentration Fed-batch culture of S. cerevisiae with on-off control of the glucose concentration using the automarie glucose analyzer was carried out. T h e glucose concentration to be controlled was set at 10 or 0.3g/l. Figures 6 a n d 7 present the results when the concentration was kept at 10 g/l. T h e microorganism grew exponentially to OD5~0=43 and the specific growth rate was 0.41 h-1 (Fig. 6). This value was close to the m a x i m u m specific growth rate reported by O k a d a et al.~) As l

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shown in Fig. 7, the glucose concentration was kept at almost 10g// throughout the cultivation and the fluctuation was within 10%. T h e concentrations measured on-line coincided well with those obtained from offline measurement by Glucostat. Ethanol was produced exponentially according to the Crabtree effect a n d the final ethanol concentration was 12.9g/1 (Fig. 6). T h e specific production rate of ethanol was calculated to be 0.59 g ethanol/(g dry cell.h). Figures 8 and 9 show the results when the glucose concentration was set at 0 . 3 g / l . T h e microorganism grew exponentially to

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ODs~0=34 and the specific growth rate was 0.35 h -1 (Fig. 8). As shown in Fig. 9, the glucose concentration was k e p t at almost 0.3 g/l and the concentration was within the range of 0.08-0.54g//. The glucose concentration determined by the on-line system was essentially the same as that obtained by the off-line measurement. In this case, the final e t h a n o l concentration was 1.67 g/l, which was one-eighth of the value accumulated in the fermentation shown in Fig. 6 . The specific production rate of ethanol was 0 . 0 7 g ethanol/(g dry cell.h), almost one-eighth of that obtained for the high glucose concentration fermentation. Though Aiba et al.~) and Wu etal. a) obtained a specific growth rate of 0 . 2 h - I at a low glucose-concentration level, the specific growth rate obtained in this study under similar conditions was twice as large as their value. Figure 10 shows the results of the fed-batch culture of M. ruteus w i t h on-off control of glucose concentration~ The glucose concentration to be controlled was set at 2.5 g/l. The microorganism grew t o ODs10=264 and the specific growth rate in the exponential growth phase was 0 . 2 4 h -1. As shown in Fig. 10, the glucose concentration was kept at almost 2.5 g/l throughout cultivation and the range of variation was about 20%. T h e relatively long measurement intervals (7min) m a y have caused the variation. As indicated in this study, the glucose

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concentration in culture broth could be easily kept at a constant level with an on-off control scheme using an automatic glucose analyzer. However, the on-offcontrol system has some disadvantages. First, control at lower glucose concentrations or at a higher cell concentrations m a y be difficult. Second, the sampling interval which determines the range of variation in the glucose concentration must be short to obtain good control. T o overcome these problems, we are now attempting to keep the glucose concentration at a very low level (around 0.15 g/l) by both p r o g r a m m e d - P I control and optimal control combined with moving identification. Preliminary results suggest that the glucose concentration m a y be kept at a constant value, even at a high cell concentration. Until now, almost all fermentations have been performed at high sugar concentrations because no sensor for the on-line measurement of sugars has been developed. T h e characteristics of fermentation and the microorganisms themselves at low sugar concentrations have not yet been studied except for baker's yeast. Now it is possible to monitor the glucose concentration on line, enabling one to control the glucose concentration either at a constant level or at desired profiles. References

1) Yano, T., Kobayashi, T., Shimizu, S.: J. Ferment. Technol., 56, 421 (1978). 2) Aiba, S., Nagai, S., Nishizawa, Y.: Biotechnol.

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On-Line Control with Glucose Analyzer

Bioeng., 18, 1001 (1976). 3) Wang, H . Y , Cooney, C.L., Wang, D . I . C . : Biotechnol. Bioeng., 21, 975 (1979). 4) Nanba, A., Hirota, F., Nagai, S.: J. Ferment. Technol., 59, 383 (1981). 5) Dairaku, K., Izumoto, E., Morikawa, H., Shioya, S., Takamatsu, T.: J. Ferment. Technol., 61, 189 (1983). 6) Takamatsu, T., Sbioya, S., Okada, Y., Kanda, M.: Biotecfinol. Bioeng., 27, 1675 (1985).

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7) Okada, W., Fukuda, H., Morikawa, H.: J. Ferment. Technol., 59, 103 (1981). 8) Wu, W-T., Chen, K-C., Chiou, H-W.: Biotechnol. Bioeng., 27, 756 (1985). 9) Williams, D., Yousefpour, P., Wellington, E. M.H.: Biotechnol. Bioeng., 28, 631 (1986). 10) Karube, I.: Bioreactor (in Japanese) (Fukui, S.), chapter 3, Koudansha, Tokyo (1985). (Received December 8, 1986)