Time-dependent kinetic models for glutamic acid fermentation are presented. These can divide cell growth into three stages: the positive acceleration stage, negative acceleration stage, and equilibrium stage. By comparing data from the laboratory and facto9, good agreement between experimental values and predicted values is found.
Time-dependent kinetic models for glutamic acid fermentation are presented. These can divide cell growth into three stages: the positive acceleration stage, negative acceleration stage, and equilibrium stage. By comparing data from the laboratory and facto9, good agreement between experimental values and predicted values is found.
Time-dependent kinetic models for glutamic acid fermentation are presented. These can divide cell growth into three stages: the positive acceleration stage, negative acceleration stage, and equilibrium stage. By comparing data from the laboratory and facto9, good agreement between experimental values and predicted values is found.
glutamic acid fermentation Xue-Wu Zhang, * Tao Sun,* Zhong-Yi Sun, Xin Liu,* and De-Xiang Gu* *Food Engineering Research Center, Zhongshan University, Guangzhou, Peoples Republic of China: Educational College of Guizhou. Guiyang. Peoples Republic of China A group of time-dependent kinetic models for glutumic acid fermentatiort are presented. These can divide cell growth into three stages: the positive acceleration stage. negative acceleration stage, and equilibrium stage. The substrate consumption is also divided into three stages: the negative acceleration state, positive acceleration stage. and exhaustion stage. Product formation is also separated into three stages: the lag stage, positive acceleration stage. and negative acceleration stage. By comparing data from the laboratory and facto9, good agreement between experimental values and predicted values is found and in comparisons to reported models in the documents about glutamic acid fermentation, the present models are most satisfactov. 0 I998 ElselGer Science inc. Keywords: Glutamic acid, fermentation kinetics. time-dependent models Introduction The glutamic acid fermentation is a key process of sodium glutamate production. The kinetic models play an important role in monitoring and predicting fermentation processes. A great number of dynamic models for the glutamic acid fermentation have been reported.lm7 The common point for the reported models is that these models basically existed in the form of differential equations. These models are con- stantly difficult to solve by integral form and only express the implicit functions of time. They cannot directly reflect the effect of fermentation time on cell growth, substrate consumption, and product formation, the model-based real- time control and monitor of the process variables (i.e., the concentrations of biomass, substrate, and products) cannot be determined. Especially for the glutamic acid fermenta- tion, the results during the earlier period produce great effects on results during the later period, so a predictive model is greatly needed which can be used to estimate the state of the later stage process based upon that of the earlier stage process. Consequently, problems are found in a timely manner. This guarantees the running of the process in the desired route. On the other hand, when simulating replacement of Ay/At Address reprint requests to Dr. X.-W. Zhang, Zhongshan University, Food Engineering Research Center, Guangzhou 5 10275, Peoples Republic of China Received I May 1997; revised 21 July 1997; accepted 5 August 1997 for dyldt, a greater discrepancy between the theoretical values and the experimental values are produced; hence, an influence on the accuracy of the model is observed. Moreover, the structured model completely based upon mechanism is very complicated and not as adaptable as expected, so to search for or a new and simpler model structure which is suitable for monitoring and predicting both laboratory-scale and factory- scale fermentation processes, this becomes the urgent task of researchers in biochemical engineering. A fermentation process is a nonlinear and time-depen- dent process; thus, the kinetic models describing the process should also be nonlinear and time dependent. The objective of this study is just an attempt to solve this problem. Based upon the integral form of an existing model, a group of empirically nonlinear and time-dependent kinetic models for cell growth, substrate consumption, and product forma- tion for the glutamic acid fermentation are proposed. By these models, the detailed description of the glutamic acid fermentation process is made. This lays the theoretical foundation for the design, optimization, and scaleup of the process. Theoretical aspects The reported models for glutamic acid fermentation (1) Cell growth models dX dt = (1) Enzyme and Microbial Technology 22:205-209, 1998 0 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010 0141-0229/98/$19.00 PII SOl41-0229(97)00168-3 Papers dX SX -= dt I lrnK, + S (2) where Model (2) is just the well-known Monod equation. (2) Substrate consumption models dS 1 dX 1 dP --_= dt y,dt+y,dt (3) dS v,sx --= dt K, + S (4) (3) Product formation model dP VJX dX dt= K,,, + S - % (5) Construction of time-dependent models Eq. (I) can be integrated in the form: = 1 - (X,/X,)[ 1 - exp (p&) (6) Starting with Eq. (6) and screening repeatedly, a group of empirical, simple, and uniform equations are chosen to describe the cell growth, substrate consumption, and prod- uct formation for the glutamic acid fermentation: X = XL exp ( - b,(t - tx) _ y) (7) S = S, exp ( - b, (t - tJ) (8) P = PL exp ( - bp(t - t,J - ) (9) where X, S, and P are the cell, substrate, and product concentrations respectively. X, is the limit concentration of the cell (t = m); P, is the limit concentration of product (t = ~0); S, is the ideal concentration of substrate: t is the fermentation time; t, is the time when X = 0; t, is the time when S = St; t, is the time when P = 0. Usually, t,, ts, t, 5 0. If the inequality holds, it shows that the cell and product concentration have an initial value (though very low probably) and that the initial substrate concentration (t = 0) tends to be low. The ideal substrate concentration (S,) should be higher than this. All b and IE are constants which are responsible for the convergent rate of curves, i.e., the speed of change for X, S, and P. By Eq. (7), the process of cell growth can be divided into three stages: PAS:0 5 t 5 tx + NAS:t 2 tXrP D t,,, then $5 e, (11) Here, PAS represents the positive acceleration stage in which the cell grows with a positive acceleration; NAS represents the negative acceleration stage in which the cell grows with a negative acceleration; ES represents the equilibrium stage in which the rate of the cell growth (dX/dt) is less than or equal to a small and positive number (e,). So to speak, the cell growth arrives at the equilibrium under the ex level. The symbol D means is defined as. By Eq. (8), the process of substrate consumption can be divided into three stages (n, 2 1): NAS:O 5 t I ts + (12) PAS:t 2 tsrp NES:t I ts + Dt,,,, then S % es (13) If 0 < n, < 1, then there are two stages left: PAS (0 < t < 00) and NES. Here, NAS and PAS mean the same as above. In NAS, substrate decreases at a negative acceleration, and at a positive acceleration in PAS. NES is the nearly exhausting stage of substrate under the es level where es is a positive number. Finally, by Eq. (9), product formation can be divided into three stages: PAS:0 5 t 5 tp + NAS:t 2 tPrP LS:O 5 t f t, + ( ln GL,l,,)..Dt,,,, then P 5 where PAS and NAS are the same as above. In PAS, the product is formed at a positive acceleration, and at a negative acceleration for NAS. LS stands for the lag stage in which a little product is formed. The concentration of product is not larger than a small and positive number (Ip). In fact, the division of PAS and NAS can be understood as follows. In the beginning of the fermentation, sufficient substrate makes the cells grow at a positive acceleration and the product accumulates at a positive acceleration after lag time while the lower base number both for cell concentra- tion and product concentration make the substrate decrease only at a negative acceleration. As the fermentation goes on, the increasing cell and product concentration enlarge the needs for substrate so that substrate decreases at a positive acceleration after a period of time. On the other hand, the decreasing substrate limits the growth of cells and the formation of product so that they have to slow down gradually. The cell grows at a negative acceleration and arrives at equilibrium in time. The product accumulates at a negative acceleration during the middle or late stage of the fermentation; thus, from the theoretical analysis, the present models conform to the laws of the fermentation process and are expected to describe the process of glutamic acid fermentation well. Below are applications in production for the proposed time-dependent models. 206 Enzyme Microb. Technol., 1998, vol. 22, February 15 Time-dependent kinetic models: X. W. Zhang et al. Table 1 Parameter values for different models Item Model Laboratory data Parameters Error Factory data Parameters Error Cell Growth Models (X) Substrate Consumption Models (S) Product Formation Models (P) (I) (2) (6) (3) (4) (8) (5) (9) X,,, = 2.204 IL,,, = 0.4406 JL, = 0.20 KS = 0.70 X, = 3.7679 tx = -4 b, = 64.0077 n, = 1.4 Ye = 20.4166 Y, = 0.322 V,,, = 0.0123 K,,, = 0.3303 S, = 1.3236 t, = -4 b, = 7.45x 1O-5 n, = 2.8 V, = 0.0084 K,,, = 0.5676 a = 0.0129 PL = 1.0696 tp = -36 bp = 3191391.88 np = 3.0 0.0130 0.4395 0.009 0.0666 0.1097 0.0235 0.0412 0.0310 X = 9.256 ).I,,,, = 0.2072 )L., = 0.4146 KS = 1860.28 X, = 9.270 tx = -12 b, = 186557.009 n, = 4.5 T, = 0.4456 Y, = 0.0575 v, = 0.5545 K,,, = 7.245 S, = 125.984 t, = 0 b, = 2.29x 10m3 n, = 2 V,,, = 0.0286 K,,, = 2.278 a = 0.1380 PL = 10.7839 tp = -12 b, = 3393.02 np = 2.3 0.4949 1.0632 0.4445 2.1584 2.8595 1.9705 0.1046 0.0946 Units: X, S, P, X,, S,, s, X,,,, KS, K,,, are all in g I-; r.+,, (h-l), V,,,(h-),Y, (g g-,Y, (g 9-l) Results and discussion Two sets of data are used for simulation. One comes from a laboratory 2 and the other from a factory.8 Cell density was measured turbidometricahy at 610 nm and converted into a cell mass concentration using a calibration curve. The concentration of glucose was measured by the glucose oxidase method (Wako Pure Chemical Industries, Japan) and the concentration of glutamic acid by the calorimetric method using glutamate dehydrogenase (Boehringer Mann- heim, Mannheim, Germany). The total sugar concentration was measured by the phenol sulfuric acid method. The generalized least square method is applied for the parameter estimation in the different models. The simula- tions are evaluated by the fitting error: _ \ n I where Yi and Yi* are the original and the predicted value, respectively, and n is the number of data used. The values of parameters and the fitting errors for different models are listed in Table 1. The simulation curves with different models for cell, substrate, and product are illustrated in Figures IA-IF. It can be seen from Table I that the fitting errors for different models that fit the data from laboratory and factory can be sequenced as below: For cell growth: Model (2) > Model (1) > Model (6) For substrate consumption: Model (3) > Model (4) > Model (8) For product formation: Model (5) > Model (9) Thus, the time-dependent Models (7)-(9) presented in this paper have the smallest error in all the situations. Obvi- ously, the simulation curves with different models (Figures lA-1F) show that the time-dependent models are best. Meanwhile, the critical values appearing in the inequal- ities (lo)-( 15) can be calculated. For the laboratory: tXtp = 9.3134, t+ = 48.7557 (e, = 1% X, = O.O25g/L), tstp = 21.4537, t,, = 3 1.3473 (e, = 20% S, = 0.26g/L), tptp = 30.4596, t,p = 7.4606 (lr = 3% P, = O.Olg/L) For the factory: tXtp = 2.1881,t,, = 15.7374(ex = 1% X, = 0.09gU ts,p = 24.48/L), t,, = 14.7786,&? = 26.7780 (es = 20% S, = 17.3002. t,p = 6.0822 (It, = 3% P, = O.l4g/L) Here, S,, X,,,, and P, are the initial concentration of substrate (t = O), and the maximum concentration of cell and product, respectively. A detailed description of the glutamic acid fermentation in the laboratory or factory (corresponding to the figures in parentheses) can be made: 1. The cell grows at a positive acceleration when 9.3 134 2 (2.1881) Th ago, and- this time backward grows at a negative acceleration. The cell growth will arrive at an equilibrium under the level of ex = 1% X, = 0.025g I- (e, = 1% X, = 0.09 g l-l), i.e., after 48.7557 (15.7374) h, dX/dt < 0.025 g I- hh (0.09 g I- hh). In fact within the interval of 28 (15) - 32 (16) h, the increasing rate of cell growth AX/At is 0.033 (0.1) g 1-l hh = 1.3% X, (1 .l% X,), really near to equilibrium. The substrate decreases at a negative acceleration when 21.4537 (14.7786) h ago, and this time backward de- creases at a positive acceleration. After 31.3473 (26.7780) h the concentration of substrate will be lower Enzyme Microb. Technol., 1998, vol. 22, February 15 207 Papers 0 4 8 I.2 16 20 24 0 4 8 I2 16 20 ti E Time t(h) F Time tfh) Fi gur e 1 Simulation curves of the glutamic acid fermentation by using different models to fit the experimental data. For A-C, the data are from a laboratory. For D-F, the data from a factory.* than the level of es = 20% S, = 0.26 (24.4)g 1-l. In fact, fermentation is about 7.4606 h (6.0822 h). Within this the concentration of substrate at t = 32 h (25h) is equal to 0.25 (28) g 1-l = period of time, the concentration of product is under the 18.94% S, (22.95% S,), which is level of 1, = 3% P, = 0.01 (0.14) g 1-l. In fact, the basically near the predicted level. concentration of product at t = 8h (5 h) is equal to 0.017 3. The lag time for product formation in the glutamic acid (0.11) g 1-l = 5.6% P, (2.4% P,) which is basically 208 Enzyme Microb. Technol., 1998, vol. 22, February 15 near the predicted level. As the fermentation goes on, the product accumulates at a positive acceleration 30.4596 (17.3002) ago, and this moment later accumulates at a negative acceleration. From the above research, it can be seen that the structure of time-dependent models is adaptable both to the labora- tory and factory. Different processes simply correspond to different values for parameters. In the course of the glutamic acid fermentation, the results in the beginning of the fermentation have a great impact on those at the end of the fermentation; hence, the time-dependent model can be used to predict what happens toward the end of the fermentation based upon results from the early fermentation so that problems can be discovered in a timely manner and some measures can be taken to assure that the fermentation proceeds as expected. Apparently looking at the expression of time-dependent models (7)-(9), the interrelations among cell, substrate, and product cannot be displayed in the models; however, after mathematical calculations, the time-dependent models can be transferred into the form of a differential equation which implies interactions among cell, substrate, and product. The transferred equations can be written as (Jb/2)K~ (.fPf2)P.s pp = .fx + OP - t?i)lJ cY +fs + CtfJ - t.dl% (17) (fs/2)px (fs/2)l-b ps = .fY + (ts - tx)l-h +fP + (ts - t,)l-b (18) 1 dX where kx = x x is the specific growth rate of cell, kp = IdP 1 dS ~dt is the specific formation rate of product, ks = -- - S dt is the specific consumption rate of substrate, and fx = n, ln(X,/X), fp = np ln(P,/P), fs = n,ln(S,/S). Thus, the time-dependent models (7)-(9) presented in this paper can describe the interactions among X, S, P, k,_ ps, and kp by their derivative equations (16)-(18). Conclusions Beginning with the integral form of known equation, a series of time-dependent kinetic models for the glutamic acid fermentation have been proposed in this study. Com- pared to the reported models, the present models have the following advantages: (1) They are the explicit functions of fermentation time; hence, they can directly reflect the effect of time on cell growth, substrate consumption, and product formation. In other words, realtime monitoring and control can be realized; (2) They can make a more detailed description of the fermentation process, i.e., the positive or negative acceleration stage and the equilibrium time of the cell, nearly exhausting time of substrate and the lag time of product under the given level of critical value can be determined, which is very important in real production; (3) Time-dependent kinetic models: X. W, Zhang et al. They have a simple and unified structure of model: Y = aexp [ -b(t - c)], where n < 0 for cell growth and product formation and n > 0 for substrate. It is easy to estimate the parameters. In this study, the fitting results are best; (4) They can also be transferred into the form of a differential equation as in the reported models. The interactions among cell, substrate, product, and their specific rate can be described while this makes one of the advantages of the known differential models disappear; however. the pre- sented models need further verification and development. cl = Constant in Eq. (51 (g I-) ;&.fP 1 = i, P,. P, = s, S,, S, = f = fXCP> CstP~ fP,P = x, x,, x, = yc7 YM = fJ,xs I.9 CLP = Fitting error Functions in Eqs. ( 16)-( 18) Constant in inequality ( 15) (g I- ) Concentration. limit concentration, and maximum concentration of product (g l--l) Concentration, ideal concentration, and initial concentration of substrate (g I- ) Fermentation time (h) Constants in inequalities (IO), (l2), (14) (h) Concentration. limit concentration. and maximum concentration of cell (g I-) Constants in Eq (3) (g g- 1 Specific growth rate of cell (h- ), specific consumption rate of substrate (h-l), spe- cific formation rate of product (h-l) Constants in Eqs. (7)-(9) Constants in inequalities (1 I), (13) (g I--) Constants in Eqs. (21, (5) (g I-~) Constants in Eqs. (7)-(9) Constants in Eq. (7)~(9) (h) Constant in Eq. (4) (h-l) Maximum growth rate (h- ) b,, 6,. b, = = z;, ;m = ilx, n,, np = rv !u f,, = nl = P,, References 4. 5. 6. 7. 8. 9. Gaden. E. L. Fermentation process kinetics. Biotechnol. Bioeng. 1958, I. 413-429 Hu, Z. D. and Tao. C. Q. Study of the kinetic models of glutamic acid fermentation. Chinese J. 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