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Extended Monod Kinetics for Substrate, Product, and Cell Inhibition

Keehyun Han* and Octave Levenspiel

Department of Chemical Engineering, Oregon State University, Corvallis, Oregon 9733 1

Accepted for publication August 6, 1987

A generalized form of Monod kinetics is proposed to ac- count for all kinds of product, cell, and substrate inhibi- tion. This model assumes that there exists a critical inhibitor concentration above which cells cannot grow, and that the constants of the Monod equation are func- tions of this limiting inhibitor concentration. Methods for evaluating the constants of this rate form are presented. Finally the proposed kinetic form is compared with the available data in the literature, which unfortunately is very sparse. In all cases, this equation form fitted the data very well.

INTRODUCTION

Although microbial growth is a very complex phenome- non, the overall growth can often be regarded as a single chemical reaction with a simple rate expression. Many equations are used for this purpose. Among these, the sim- plest and most popular is the one proposed by Monod who assumed that a single essential substrate is the growth limiting factor. I Monod kinetics can be expressed as

Substrate (A) -

Cells (C)

more Cells

(C) + Product (R)

r,

=

k

cell mass produced

where r,

is the growth rate of cells; k is the reaction rate

constant; CA is the substrate concentration; C, is the

cell

concentration; and C, is the Monod constant. Unfortunately, there are often inhibitory effects in the real world. In this article, we propose a generalization of the Monod expression which takes into account inhibition effects caused by high concentration of either substrate, or cell, or product, or other inhibitory substance. This pro- posed expression is

where C, is the inhibitor concentration, C: is the critical inhibitor concentration above which reaction stops, and n and m are constants.

* Present address: Biochemical Engineering Program, School of Engi- neering, University of California, Irvine, CA 92717.

For substrate inhibition:

for product inhibition:

for cell inhibition:

In the extreme where C, -G C ,: the above eqs. (2)-(5) all reduce to the simple Monod expression of eq. (1).

HOW TO EVALUATE THE CONSTANTS OF THE GENERALIZED MONOD EQUATION FOR PRODUCT OR CELL INHIBITION

As with Monod kinetics, the constants in eq. (2) with

C, =

C ,

or C, =

C ,

can be evaluated from a Lineweaver-

Burk plot of C,/r, vs l/CA.Thus inverting eq. (2) gives

cc -

--

r,

C,(1

- c,/c:)m1

-+

k(l - C,/Cf)"

CA

1

k(l - C,/CF)"

This expression can account for and represent the six com- mon patterns of inhibition illustrated by the Lineweaver- Burk plot of Figure 1:

  • (a) represents noncompetitive inhibition, where n > 0 and m = 0;

  • (b) represents competitive inhibition, where n = 0 and m < 0;

  • (c) represents generalized uncompetitive inhibition, where n>m>O;

  • (d) represents generalized uncompetitive inhibition, where m>n>0;

Biotechnology and Bioengineering, Vol. 32, Pp. 430-437 (1988)

Calculate C,,, from eq. (17), and plot as in Figure 6. The slope and intercept on this graph gives C, and m.

Substrate Inhibition: Example 1

Uemura and co-workers6 studied the microbial attack on

n -pentane by a pentane-consuming bacterium (JTB-27 1). The results were read from Figure 5 of Edwards' paper,7 and are shown in Table 11. A plot of these data points in Figure 7 clearly shows substrate inhibition since the rate of reaction slows at high substrate concentration. When C,

2

  • 65 mmHg, assume that C, S CMobs, in which case eq. (3)

is well approximated by eq. (15). It will be shown later that this is a reasonable assumption. Following the procedure outlined above, we first make the plot of Figure 8, from which we find C,* = 700 mmHg, k = 0.117 h-', and n = 1.82. CMobsvalues are then calcu- lated from eq. (17) and listed in Table 111. Finally, the plot of Figure 9 gives C, = 30 mmHg and m = 66.8. At C, =

  • 65 mmHg, we find CMobs= 0.045 mmHg, hence the as-

sumption that C, S CMobsand m > 0 is well satisfied. The final result is then

0: data

from Uemura et aI6

eq. 18, best fit

of

the

general Monod equation

0'

0

1

1

200

1

400

1

600

partial pressure of n-pentane (mm Hg)

Figure 7. Data of ref. 6 clearly shows that high concentration of n-pentane, the substrate, inhibits the reaction; see Table 11.

(18)

This expression fits the reported data well, as shown in Figure 7.

-2.8

 

-3.0

-0.4

-0.2

0

 

Figure 8. This graph gives k, n, and C,* for tion; data of ref. 6.

n-pentane substrate inhibi-

 

( ::)

I--

0

 

In

   

Table III.

C,,

value extracted from ref. 6.

Figure 6.

For substrate inhibition m and C, are evaluated at low C,

 

Table 11.

Experimental result of ref. 6 taken from Figure 5 of ref. 7.

5.000

 
  • 0.0227 20.44

 

6.875

  • 0.0378 14.03

10.938

  • 0.0564 11.11

15.313

  • 0.0792 6.42

19.688

0.091 1

4.32

 

5.000

6.875

0.0227

0.0378

25.625

  • 0.0992 2.61

10.938

0.0564

15.313

0.0792

19.688

0.0911

Substrate Inhibition: Example 2

 

25.625

0.0992

30.625

0.0992

Kortan* reported substrate inhibition by n -butanol to

65.000

1oo.OOo

135.625

0.0991

0.0817

0.0786

Arthrobacter AK 19. The experimental results of Table IV

are from Figure 2 from the article by Wayman and Tseng.'

As in the previous example C,*, k, and n values are ob-

199.375

0.0633

268.7 50

0.0489

tained from Figure 10. In this example, we assume that eq. (3) is well approximated by eq. (15) when C, 3

HAN AND LEVENSPIEL: EXTENDED MONOD KINETICS

433

product inhibition, as shown in Table VI. However the ki- netic form proposed here should be useful for all forms of inhibition. In addition if there are no inhibitory effects (n = 0 and m = 0), the generalized equation reduces to simple Monod kinetics. Finally, the key characteristic of the proposed model is its assumption that there exits a critical inhibitor concentra- tion above which reaction ceases. This is what actually happens in many, if not most situations. Many of the prod- uct inhibition models incorporate a critical inhibitor con- centration. On the contrary, for substrate inhibition only Wayman and Tseng’s model includes this feature. Conse- quently, those models which do not have a critical inhibitor

concentration predict that inhibitor concentration should be infinite to stop the reaction.

Substrate Inhibition

Edwards’ compared the goodness of fit of five kinetic models for substrate inhibition to the reported data of Uemura et aL6 Table VII lists these models, Table VIII gives the best-fit value of the constants of these models, and Figure 13 displays the results as dotted or dashed lines. Also included in this figure is the curve from the model developed here, or eq. (18). This figure shows that one should be able to more clearly discriminate between models at high substrate concentration.

Table VI.

Kinetic models for inhibition; equations adapted from p. 256 and p. 259 of ref. 10.

 

Equation

Reference

For substrate inhibition:

 

rC

-k

--

 

1

 

Andrews and Noack

CC

+ cM/cA

+ CAIK/

 

Webb

Yano and co-workers

Aiba and co-workers

‘c = k [exp(-CA/Kl) - exp(-CA/C~)]

 

Teissier-type

CC

 

(u is ionic strength)

Webb

 

Wayman and Tseng

‘c =k-

CA

- K1(CA- CJ,

when CA> Ci

CC

cM

+ CA

For product inhibition:

 

’c=k-

CA

(1 -

KCR)

Dagley and Hinshelwood

CC

CM + CA

 

rC - k

--

CC

-

   

Holzberg and co-workers

K(CR - KI)

%=k

CC

(

I--

-

Ghose and Tyagi

 

‘c=k-

CA

exp( -KCR )

Aiba and co-workers

CC

CM + cA

 

-rc

=k--

CA

KI

Jerusalimsky and Neronova

Cc

CM

+ CA Kl + CR

 

‘c = k(1 -3T5L

 

Bazua and Wilke

cc

CM + CA

 

Levenspiel

HAN AND LEVENSPIEL: EXTENDED MONOD KINETICS

435

0.10

0.08

0.06

0.04

present

model predicts

this limiting point

0.02

0

0

200

400

600

700

Figure 13.

partial pressure of

n-pentane (mm Hg)

Fit of various proposed substrate inhibition equations of

Table VII to the data of ref. 6. Note that data at high pentane concentra-

tions would sharply discriminate between models.

NOMENCLATURE

CA

substrate concentration (substrate/volume)

CC

cell concentration (cell/volume)

c,

chf

inhibitor concentration (inhibitor/volume) Monod constant

Chf,

observed value of C,

Clf

product concentration (product/volume)

C*

critical concentration above which reaction cannot pro-

iandj

ceed; C: for inhibitor, C: for cell, Ci for substrate, C,* for product positive integers

k

reaction rate constant (time-')

k*s

observed value of k

K, K,,

and Ks

constants in Table VI

and Table VII

nandm

constants

rC

rate of cell

formation (cell mass/volume . time)

This work was carried out under NSF Grant no.: CBT-851.8737.

References

  • 1. J. Monod, Ann. Rev. Microbiol., 3, 371 (1949).

  • 2. C. N. Hinshelwood, The Chemical Kinetics of the Bacterial Cell (Clarendon, Oxford, 1952).

  • 3. C. D. Bazua and C. R. Wilke, Biotechnol. Bioeng. Symp., 7, 105 (1977).

  • 4. 0. Levenspiel, Biotechnol. Bioeng., 22, 1671 (1980).

  • 5. S. Aiba, M. Shoda, and M. Nagatani, Biotechnol. Bioeng., 10, 845 (1968).

  • 6. N. Uemura, J. Takahashi, and K. Ueda, J. Ferment. Technol., 47, 220 (1969).

  • 7. V. H. Edwards, Biotechnol. Bioeng., 12, 679 (1970).

  • 8. K. Kortan, M. S. thesis, University of Toronto, Ontario, Canada,
    1972.

  • 9. M. Wayman and M. C. Tseng, Biotechnol. Bioeng., 18, 383 (1976).

10. A. Moser, in Biotechnology, Volume 2, H. Brauer, Ed. (Verlag Chemie, Weinheim, 1985).

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