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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.

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0141-0229/98/$19.00

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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,,

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Enzyme Microb. Technol., 1998, vol. 22, February 15 209

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