Generalization of Monod Kinetics
for Analysis of Growth Data with
Substrate Inhibition
J. H. T. Luong
Biotechnology Research Institute, National Research Council of Canada,
Montreal, Quebec, Canada, H4C 2K3
Accepted for publication February 7, 1986
The inhibitory effect of butanol on yeast growth has been
studied for the strain Candida utilis ATCC 8205 growing
aerobically on butanol under batch conditions. A math-
ematical expression was then proposed to fit the kinetic
pattern of butanol inhibition on the specific growth rate:
P=*[l-;]"
The maximum allowable butanol concentration above
which cells do not grow was predicted to be 9.16 g/L.The
proposed model appears to accurately represent the ex-
perimental data obtained in this study and the literature
data developed for a variety of batch culture systems at
widely ranging substrate concentrations.
INTRODUCTION
Microbial growth can be inhibited in several different
ways. The presence of inhibitors such as phenol, chlor-
ine, benzenoid, antibiotics, etc., in the growing me-
dium inhibits growth and may distort the metabolism
of microorganisms. Such interesting behavior plays a
very important role in chemotherapy, and it has led to
an enormous amount of research in theoretical and
applied biology. The other inhibitory mechanisms are
recognized as substrate and product inhibitions where
the substrate and/or product concentrations are al-
lowed to increase to levels such that the growth de-
creases and ultimately ceases.
Several mathematical models have been developed
for quantifying the inhibitory effect of substrate on
growth rate, and they are generally adaptations of
equations for substrate inhibition of enzymatic reac-
tions.
Haldane Kinetics
Andrews' proposed that the effect of substrate con-
centration on specific growth rate could be governed
by the equation
Biotechnology and Bioengineering, Vol. XXIX, Pp. 242-248 (1987)
0 1987 John Wiley & Sons, Inc.
PmS
'= (Ks + S)(1 + S/Ki)
Equation (1) was used by Haldane' in 1930 to de-
scribe enzyme inhibition by the formation of an inac-
tive complex of the enzyme with two substrate mol-
ecules. Boon and Laudelout3 later found that equation
(1) was applicable for fitting their experimental results
with Nitrobacter. The Haldane equation has then been
concluded by several researchers to provide an ade-
quate fit to a plot of p vs. S for growth at high substrate
levels. As Ki approaches infinity, equation (1) ap-
proaches the familiar rectangular hyperbola of the
Monod relationship.
Edwards4 in 1970 pointed out that many other in-
hibitory models could be borrowed from enzyme ki-
netics to fit kinetic data taken from the literature:
FmS
cL=
Ks + S + (S2/Ki)(l + S / K ) (3)
E d ~ a r d showever,
,~ also found that equation (1) was
no less consistently suitable than equation (2) or (3)
since very large values were obtained for the fourth
parameter ( K ) in equations (2) and (3).
The analysis of growth curves according to equation
(1) has some complications when considering plots of
p vs. S because this model implies that the cells are
capable of growing indefinitely. This is not what is
observed in reality since there is a definite substrate
concentration limit above which growth will cease
(complete inhibition).
Linear Kinetics
Hinshelwood' studied the reduction of the growth
rate of Bacterium Iactis perogenes by alcohols and
proposed the following relationship:
CCC 0006-3592/87/020242-07$04.00

p = p, - aP (4)
where P is product concentration and a is a constant.
Equation (4)has also been employed by several in-
vestigators for assessing product inhibition in the al-
coholic fermentati~n.~.'
Based on equation (4), Tseng and W a y ~ n a npro- ~,~
posed the following relations for correlating the growth
data of Candida utilis, C . lipolytica, Arthrobacter AK
19, and Pseudomonas methanica:
PmS
i(S - S*) when S > S* (6)
p=K,+s-
Here S* is a threshold substrate concentration below
which the organism grows apparently without inhibi-
tion. The discontinuity is a major drawback of this
model. Literature data also indicate that the relation-
ship between p and S is not always a linear
Exponential Kinetics
Aiba et al.IOproposed the following equation for cor-
relating product inhibition data from the alcoholic fer-
mentation:
pms e-PIKi
p==
(7)
Edwards4 suggested that relations proposed to de-
scribe product inhibition may be borrowed to correlate
substrate inhibition:
Like equation (I), equation (8) fails to predict the
maximum substrate concentration at which growth will
be completely inhibited. It is worth noting that when
SIK, << 1, equation (8) becomes equivalent to equa-
tion ( I ) , and both these equations approach equation
(9) by a Taylor series analysis:
(9)
Teissier Kinetics
The assumption of diffusion-controlled substrate
supply leads to equation (lo), which was originally
derived by Teissier":
p = p,(l - e-s/Ks)
(10)
Combining this mechanism with a protective diffu-
sional limitation of high and inhibitory concentrations,
Edwards4proposed the following model for correlating
the growth data:
= pm (e-SlK, - e-SlKs) (1 1)
Again, equation (11) fails to predict the total inhi-
bition concentration S, the level at which growth can-
not occur, since p only approaches zero when S ap-
proaches infinity.
In this study a kinetic model has been proposed to
quantify the effect of substrate inhibition on specific
growth rates. The model takes into account the be-
havior that substrates will act as inhibitors at higher
concentrations and behave as activators at lower lev-
els. The kinetic data developed for the growth of C.
utilis on n-butanol and the literature data have been
used to assess the applicability of the proposed model.
PROPOSED KINETIC MODEL
Levenspie112 recently proposed a generalized non-
linear equation to account for the influence of ethanol
production on the rate of alcoholic fermentation:
p = E.L,s (1 - -E)n
Ks + S
If common mechanisms are assumed, relations pro-
posed to describe product inhibition may suggest ad-
ditional empirical relationships to correlate substrate
inhibition:
where S, is the maximum substrate concentration above
which growth is completely inhibited.
As illustrated in Figure 1, at low substrate concen-
trations the specific growth rate increases with increas-
ing substrate concentration in accordance with the
Monod equation. Equation (13) will have a maximum
value when dplds = 0, and the value p* can be cal-
culated as the value of p corresponding to a substrate
concentration S*:
The value of p then decreases as substrate concen-
tration is further increased and the magnitude of con-
stant n will indicate the type of relation between p
and S .
Linear. When n approaches unity, equation (13) be-
comes equivalent to equation (9) and has a maximum
value corresponding to the following substrate con-
centration:
S* = Ks [(l + SmlKs)1/2- 13 (15)
Nonlinear (concavity upward). A rapid initial drop
in the growth rate followed by a slow decrease to zero
occurs when n > 1.
LUONG: GENERALIZATION OF MONOD KINETICS 243

0
..I2 t 1, ;2 j3 ;4 rs :6
sIS,
Figure 1. p/p, plotted against S/S, as a function of n.
Nonlinear (concavity downward). A slow initial de-
crease in the growth rate followed by a rapid decrease
to zero occurs when n < 1 .
It is obvious that the proposed kinetic model is of
the generalized Monod type and accounts for both sub-
strate availability and substrate inhibition.
MATERIALS AND METHODS
Microorganism
Candida utilis ATCC 8205 was used in this study.
Culture Medium
The medium used had the following c o m p o ~ i t i o n ' ~ that the K , value obtained for microbial growth on
(for 1 L medium): 5 g (NH4)Z SO4, 0.5 g MgS04-7H20, carbon sources generally ranges from 1 to 50 mg/LI4.
3.5 g Na2HP04, 3 g KH2P04, 0.4 g yeast extract, 10
mL 0.4% Fe EDTA solution (ferric salt of ethylene-di-
aminetetraacetic acid). n-Butanol, reagent grade, was
filter sterilized and aseptically added to the fermentor
before inoculation. The same medium was used for the
inoculum preparation.
Fermentor
Batch experiments were carried out in a 2-L fer-
mentor with a 1-L working volume. During fermen-
tation the pH and the liquid temperature were auto-
matically controlled at 4.5 and 30°C respectively- Mild
9 S; is the highest experimentally observed initial substrate concen-
agitation was provided to maintain a homogeneous cul-
244 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 29, FEBRUARY 1987
ture. The dissolved oxygen concentration was main-
tained close to the saturation level by sparging air through
the bioreactor at 1 L/min (IVVM). The exit air stream
was passed through a reflux cooler in which butanol
vapor was condensed and returned to the fermentor,
thus minimizing the evaporation of butanol during the
course of the fermentation.
Measurement of Cell Growth
Growth was followed with a Klett-Summerson pho-
toelectric colorimeter at 420 nm. A relationship be-
tween optical density and dry weight was established
a priori, and it was assumed that butanol did not alter
the optical properties of the cells in suspension appre-
ciably.
I
,
Assay of n-Butanol
Butanol was determined by using a gas-liquid chro-
matography model AGC 311 (Carle Instruments, CA).
A nickel column, 183 cm long and 0.318 cm outside
diameter, packed with 80/100 mesh Chromosorb 101
(Johns-Manville, CO) was used.
RESULTS AND DISCUSSION
Values of exponential specific growth rate p were
determined for each initial butanol concentration So by
plotting In X vs. time (figure not shown). These data
were then fitted to equations (l), (8), ( 1 l), and (13) by
nonlinear least-squares regression techniques for es-
timation of the biokinetic constants. It should be noted
that the least-squares method is sensitive to the guessed
values of the biokinetic constants, and it is quite pos-
sible to obtain numerical values with no physical mean-
ing. Such a problem was partly overcome by allowing
the computer routine to search for a minimum least
squares only with a bounded domain of allowable val-
ues for the constants (Table I). An upper limit for the
constant K , value was set to be 1 g/L. It is worth noting
However, recent studies on the growth C. boidinii, a
Table I. Bounded domain of allowable values for the biokinetic
constants.a
Equations ( I ) , (S), and (11) K, 2 K,
PZ Pm 5 3PL:
Equation (13)
K, s ig / ~
s, 2 s;
a pt is the highest experimentally observed specific growth rate.
tration at which growth still occurs.
Table 11. Parameter values, substrate inhibition models for the growth of C. utilis ATCC 8205 on 1-butanol.

Proposed model, eq. (13) 0.694 0.554 0.130 - 9.16 1.03 0.89 I
Equation (1)= 1.2% 0.570 0.515 2 - 1.01 -
Equation (8)b 1.224 0.575 0.694 3.96 - 1.35 -
Equation (1 1 0.701 0.555 0.290 5.07 - 0.88 -

a S* = (K,K,)In
S* = 4 K , {[I + 4 (K,IK,)]'n - I}

methanol-utilizing yeast, have shown a K , value as high
as 0.64 g/L15.
The values for the model parameters p,, K,, K i , S,,
and n have been estimated and summarized in Table
11. The comparison of the proposed inhibition model
and the literature model with the experimental data
was illustrated in Figure 2. As shown in this figure, at
butanol concentrations of up to 5.75 g/L, both equa-
tions (1) and (8) represented the experimental data rea-
sonably well. However, at higher concentrations equa-
tion (1) was no Ionger applicable for correlating the
growth data vs. initial substrate concentrations. Figure
2 also indicates that equation (1 1) was inferior to equa-
tions ( 1 ) and (8) at low substrate concentrations (<8
g/L) but reflected the trend in the data slightly better
at higher concentrations.
The kinetic data obtained in this study were ex-
tremely well represented by the proposed model to
predict the inhibitory effect of substrate concentration
on the specific growth rate. The model also predicted
that the specific growth rate p increased with increas-
ing butanol concentrations from 0 to 1.03 g/L in ac-
cordance with the Monod equation. However, butanol
concentrations above 1.03 g/L were inhibitory since
the value of p decreased with a further increase in the
initial butanol concentration. The parameter p has a
maximum value of 0.554 h- corresponding to 1.03 g/L
initial butanol concentration. In general, such values
agreed quite well with those predicted by equations
(l), (8), and (1 1) (Table 11).
The best-fitted value for S, the maximum allowable
butanol concentration above which cells do not grow,
was estimated to be 9.16 g/L. It is worth noting that
butanol is somewhat toxic to microorganisms, and the
maximum tolerance level is about 10 g/L8.
Comparison of the goodness of fit of these equations

0 1 I
\?\
0 1 2 3 4 5 6 7 8 9
s (9iL)
Figure 2. Inhibitory effect of butanol or yeast growth: (0)Experimental data (S, p) [dL,
h-'I, (0.8,0.55), (1.6,0.54), (3,0.468), (4,0.402), (5.7,0.285), (8.2,0.106); ( - - - - - - - - - - ) eq.
(8); (0) eq. (1); (.) eq. (11); (-) proposed model.

LUONG: GENERALIZATION OF MONOD KINETICS 245

Table 111. Goodness of fit of the inhibitory kinetic models.” ent from equation (8) at both the 0.05 and 0.01 prob-
ability levels.
x (pi - pi)* df 02 = C(pi - bi)*/df
The experimental values of p vs. S reported in the
Proposed model 0.0000025 2 0.00000125 literature have also been used to evaluate the appli-
Equation ( 1 ) 0.01632 3 0.00544 cability of the proposed model. As shown in Figure 3,
Equation (8) 0.004391 3 0.00146 equation (13) represented the literature very well. From
Equation (1 1) 0.01446 3 0.00482
Tseng and Wayman’s data,8 the proposed model pre-
a Abbreviations: df = degree of freedom = number of data - dicted that the maximum ethyl acetate concentration
number of fitting parameters; p i = observed specific growth rate; above which C. lipolytica ATCC 8661 does not grow
pi = predicted specific growth rate. was 50 g/L (Table IV). The value predicted by Tseng
and Waymans by means of equation (6) was 42.1 g/L.
A plot between p vs. S derived from the growth data
to the experimental data was made using the F-test for of C. utilis on sodium acetate4 definitely indicated that
equality of variances in accordance with the methods the curve obtained did not conform to the straight-line
employed by Edwards4 (Table 111). The 02 value ob- relationship postulated by Tseng and Wayman.8 Again,
tained from the proposed model was lowest while that equation (13) was found to represent this set of ex-
of equation (1) was highest. Equation (1 1) offered little perimental data very well. The values of S , and n were
improvement in the fit to the data compared to equation predicted to be 39.22 g/L and 0.446, respectively.
(l), and equation (8) was the second best. It was, there- KortanI6 studied the growth of Arfhrobacter AK 19
fore, decided to perform the F-test for equality of var- on n-butanol and observed that the initial growth rate
iances between equation (8) and the proposed model increased and then decreased with an increasing con-
[eq. ( 1 3 1 . centration of n-butanol. The author also attempted to
The value of F was calculated as fit the experimental data to three of Edward’s functions
equations (1)-(3) and obtained a fair agreement. Based
F =
a2 = 1168
- (16) on this kinetic data, the maximum butanoi concentra-
0% tion above which cells do not grow was predicted to
where dLis the larger variance and 0% is the smaller be 10 g/L. The applicability of the proposed model for
variance. representing the experimental data of Kortan is illus-
F tables tabulated for 0.05 and 0.01 probability levels trated in Figure 2 and Table IV where the d value was
with a df(numerator) of 3 and df(denominator) of 2 very small (0.00048), indicating a very good fit.
indicate values of 19.2 and 99.2, respectively. Since Asthana” studied substrate inhibition of P . meth-
the calculated F value (1168) was very much larger m i c a by methanol and observed that equation (I) was
than either one of the tabulated values, it may be con- only applicable for fitting the experimental data for
cluded that the proposed model is significantly differ- concentrations of methanol up to 30 mL/L. As illus-

-
1

U
P
.4.

I
0

Figure 3. Inhibitory effect of substrates on growth rates (data reported in the literature):
(0) C. lipolytica on ethyl acetate*; (0)C. utilis on sodium acetate4; (0)P.methanica on
methanol1’; ( +) Arthrobacter AK-19 on n-butanolB6;(-) proposed model.

246 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 29, FEBRUARY 1987

Table IV. Model parameters for the literature data.

C . utilis on sodium acetate" 0.41 0.8% 39.22 0.446 0.00226

Arthrobacter on n-butanolb 0.335 0.113 10 1.653 0.000048
C.lipolytica on ethyl acetate' 0.335 0.7% 50 1.41 O.oooO5487
Pseudomonas methanica on methanold 0.206 0.478 x 42.43 0.835 O.oooO107
Methylomonas mucosa on methanol' 0.913 6.4 20.74 0.776 0.000425

From ref. 4.
From ref. 16.
From ref. 8.
From ref. 17.
From ref. 18.

trated in Figure 3, equation (13) was found to represent
the data of Asthana quite well. The proposed model
also predicts that the maximum methanol concentra-
tion above which cells do not grow was 42.43 g/L. It
is interesting to observe that the value of K , was very
small (0.478 x lo-' g/L). Such an observation thus
indicated that the relationship between p and S at
methanol concentrations higher than 1.58 g/L could
simply be represented by the equation
where the values of Sm and n were determined to be
42.43 g/L and 0.835, respectively.
Tam and FinnI8 studied the growth of Methylomonas
mucosa NRRL B-56% on methanol and observed that
at methanol concentrations less than 1% v/v the spe-
cific growth rate data fits a Monod model for substrate-
limited growth. The maximum specific growth rate was
determined to be 0.725 h-l, i.e., about 3 times higher
than the average value for most of the methanol-uti-
lizing bacteria reported in the literature. l9 The other
kinetic constant, K , in the Monod model, was found
to be 6.4 g/L methanol. This value is two orders of
magnitude higher than the value of 0.12 g/L reported
for Hansenula polymorpha, a thermophilic methanol-
utilizing yeast whose growth kinetics also fit the Monod
The study on the growth of C. boidinii, an-
other methanol-utilizing yeast, shows a K , value as
high as 0.64 g/L.Is However, no literature values of K ,
for methanol-assimilating bacteria are available for
comparison. Tam and Finn18also observed that meth-
anol concentrations above 1% v/v were inhibitory and
that the Monod kinetics was no longer applicable for
representing the growth rate data.
The applicability of equation (3) for fitting the ex-
perimental data of Tam and Finn'* is illustrated in Fig-
ure 4, where an upper limit for the constant K , value
was set to be 6.4 g/L. The proposed model predicts
that the maximum tolerable methanol concentration
was 20.74 g/L and that the small value of d obtained
(0.00042) indicates a good fit. In general, methanol is
a toxic substrate for bacteria; even for methanol-

0 DATA

- E O l 131

0.2

0.1

0 1

0 5 10 I5 20
s C9/LI
Figure 4. Inhibitory effect of methanol on growth rates of Methylomonas mucosa.'8

LUONG: GENERALIZATION OF MONOD KINETICS 247

utilizing organisms a concentration below 1% v/v may
inhibit the growth of many strains.'*
Undoubtedly, the proposed kinetic model appears
to be useful for representing the kinetics of substrate
inhibition. The model is of the generalized Monod type
and accounts for both substrate stimulation at low con-
centrations and substrate inhibition at high concentra-
tions. The model has the capability to predict values
of S,, the maximum substrate concentration above
which growth is completely inhibited.
References
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