Water Research Vol. 9, pp. 637 to 642. Pergamon Press 1975. Printed in Great Britain.

KINETICS O F M U L T I C O M P O N E N T SUBSTRATE
R E M O V A L BY ACTIVATED SLUDGE

P. GRAU,M. DOH,~NYOSand J. CHUDOBA

Prague Institute of Chemical Technology, Department of Water
and Environmental Technology, 166 28 Prague, Czechoslovakia.

(Received 10 October 1974)

Abstract--A kinetic model for multicomponent substrate removal by activated sludge has been presented.
The model is based on the linear removal concept which is a special case of the broader Monod equation.
Both the simultaneous and sequential removals have been considered in the kinetic model. It maintains
the "order of reaction" analogical to chemical reaction kinetics, which in this case simulates random
and gradual diminuation of individual components with time, and is not limited to the integers only.
The model has been verified experimentally and it has been found that the variation coefficients of the
substrate removal kinetics constants were 3-5 times lower than those of the chemical reaction kinetics
constants, calculated from the same data.

NOMENCLATURE which are directly transportable into the cell; (b) multi-
a coefficient component substrates, which are represented by a mix-
b coefficient ture of several single substrates; and (c) complex sub-
C complex substrate strates, which have to be changed externally prior to
K complex constant the transportation into the cell.
k~ IVtonod's constant
k' constant
k constant
M initial number of components
m number of components remaining SINGLE COMPONENT SUBSTRATE REMOVAL
P reaction products KINETICS
p slope
S substrate concentration Single substrates are transported into a cell accord-
AS substrate removed ing to the M o n o d equation which can be transformed
t time to a differential equation describing the removal rate
V relative reaction rate of a substrate:
X biomass concentration
Y biomass yield dS _ / i X S
~t,fl constants (1)
maximum growth rate of microorganisms dt Y k s + S"
Subscripts:
i the i-th component Its integration gives two different formulae depend-
j denotes specific constants per unit of biomass ing upon whether the biomass activity X, usually in-
o variable at zero time troduced as biomass concentration, is constant during
max maximum value the time period considered or if it increases substan-
s denotes substrate removal kinetics constants
r removable substrate tially. It has been shown [1] that if the biomass con-
1,2. . . . . n order of differential equation. centration is used for X, the substrate removal activity
and rate remain constant if the initial specific substrate
concentration So/X o is smaller than 2. Under such con-
INTRODUCTION ditions equation (1) integrates to:

The mechanism of substrate utilization by a bacterial

cell can be generally described as a sequence of three
complex processes: contact of a cell with the molecule
o f a substrate; transport of the molecule into the cell;
and intermediate metabolism of the substrate.
Large or sterically incompatible molecules which
cannot be readily transported into a cell have to be
first broken down or transformed externally to trans-
portable fractions by exoenzymes or wall bound
enzymes.
O n the basis of the described general mechanism it
is practical to classify various types of substrates into
three main groups: (a) single component substrates.
637
ksln(So/S) + So - S = ~X t. (2)
Y
For low values of ks in relation to S equation (2)
turns to the zero order kinetics because the logarithmic
expression multiplied by the low value of k~ is negli-
gible. If the specific initial substrate concentration is
higher than about 2, the biomass activity increases
during the period of the substrate utilization and equa-
tion (1) integrates to that proposed by Gates and Mar-
lar [2]. It can be expected that their equation would be
applicable also at lower specific initial substrate con-
centrations, but only to populations from very high
638 P. GRAIl, M. DOH~.NYOSand J. CHUI)OBA
loaded processes with low sludge age. Recent findings
of Speece, Engelbrecht and Aukamp [3] confirm this.
It should be also noted at this moment that the values
of [~ and k~ represent in mixed cultures mean values
and that any shift in the mixed population composi-
tion would alter the values of these constants [4].
MULTICOMPONENT SUBSTRATE REMOVAL
Substrates in multicomponent mixtures are removed
KINETICS
from the solution and transported into the cells simul-
taneously, as expected by Wuhrmann [5] and verified
by Tischler and Eckenfelder [6] and others, or sequen-
tially. Sequential growth and removal has been de-
scribed for homogeneous cultures as diauxie [7] and
found also in heterogenous cultures [8, 9].
Both sequential and simultaneous removal of each
individual substrate from a mixture is characterized by
a broken line in total substrate concentration versus
time plots. Each breakpoint indicates depletion of one
component from the mixture.
Complex substrates kinetics
Many constituents of sewage and industrial wastes
behave as complex substrates (polysaccharides, pro-
teins, fats etc.). Their removal kinetics can be described
as a sequence of several reactions in a simple case:
C ~ ~Si k,,,' , P
where k is the rate constant of external break-down;
ko.~ are individual rate constants of different products
of break-down which are transported into the cells.
Depending on relative magnitude of k and ko,~ each
of the two processes can control the overall removal
rate. F r o m the experiments carried out by the authors
and not described herein it seems that if the first reac-
tion is hydrolysis, as it is for starch used in our exper-
iments, the overall rate is controlled by the transport
of the degradation products.
COMPARISON OF SUBSTRATE REMOVAL
CHEMICAL REACI'ION KINETICS
Chemical kinetics
A general differential equation of simple chemical
reaction kinetics, if the concentrations of reacting com-
ponents are expressed in a cumulative form, is:
dS
..... k,, S".
dt
This type of kinetic equation has been applied for years
also for microbial processes. In most cases better
agreement was achieved if the rate constant was
expressed as specific constant for unit of biomass.
Then:
k, = k,~X. (5)
This modification, however, does not eliminate the
major discrepancies between the substrate removal
data and the chemical reaction kinetics equations.
V1
S 0,6-
S-* o.4
/ v2 ¸ .
I I I I
~
I
/ ~¸ /
I
t,h v / 9 ¢1 h-~
Fig. I. Graphical model of multicomponent substratc
removal,
Multicomponent substrate removal kinetic~s
Figure 1 shows a generalized model based on our ex-
perimental data as well as the data presented by other
authors. The model was constructed to fulfill the con-
dition that the rate of total substrate removal would
decrease with time according to the first order kinetics.
For simplicity we are considering only simultaneous
removal, because the final conclusions for sequential
removal are similar. The left part of Fig. 1 shows con-
centration versus time and the right side the rate vs
time presentation. It can be seen that at any time the
(3) total rate of substrate removal and the total substrate
concentration are sums of all single substrate removal
rates and all substrate concentrations, respectively. As
soon as any single substrate is exhatisted, the rate
drops instantaneously, what can be seen on the left side
of the figure as a breakpoint on the line, on the right
side as a step. This shows why the kinetic data for mul-
tiple substrate mixtures can be interpreted under some
conditions as M o n o d kinetics, under other conditions
as chemical kinetics. If only the initial concentration of
the total substrate is changed experimentally and the
measurements are carried out during such a short time
period that all single substrates are still present in the
mixture, the total substrate removal kinetics can be de-
AND
scribed as M o n o d kinetics by [~ and k, (equation 1). On
the contrary, if the course of substrate remowfl is mea-
sured for such a long time period that some of" the ori-
ginally present individual substrates would disappear
from the mixture, the rate of removal would decrease,
because the composition of the mixture is reflected in
smaller values of the mean values of fi and k~. Then, of
course, some type of chemical reaction kinetics can fit
(4)
the data, because it is based on the assumption that the
rate decreases in some way with the decrease in con-
centration of the total substrate. The weak point in ap-
plication of the chemical kinetics, however, is that it
presumes also an increase in remowd rate with any in-
crease in the total substrate concentration. But if we in-
crease the initial concentration of a multiple substrate
and maintain all the single substrates present and in
the same proportion, no other increase than that corre-
sponding to M o n o d equation can be observed. Practi-
cally, in the zero order region there would be no rate
 Kinetics of multicomponent substrate 639

increase at all in spite of a substantial increase in the
rate predicted by chemical reaction kinetics.
We feel that the facts resulting from the kinetic
model have not been c o m m o n l y understood a n d b o t h
types of measurements can be found in the literature
as competing concepts, accordingly. In fact, however,
b o t h approaches are different a n d limited alternatives
of one general concept of multiple substrate removal
kinetics only.
MULTIPLE SUBSTRATE REMOVAL KINETICS MODEL
It has been shown that the instantaneous removal
rate of multiple substrate is related to the n u m b e r of
remaining single substrates. To consider the initial
rate, which is constant for a specific culture a n d a s p e -
cific mixture of the individual substrates, we can
express the rate as a function of remaining n u m b e r of
c o m p o n e n t s to the original one. Then:
where: V is the relative rate of removal; M is n u m b e r
of c o m p o n e n t s originally present; a n d m is n u m b e r of
c o m p o n e n t s remaining.
In general, we do not know neither the original
n u m b e r of c o m p o n e n t s nor the rule describing their
removal from the mixture. Assuming great n u m b e r of
c o m p o n e n t s we can substitute the n u m b e r of com-
p o n e n t s by the concentration. The function F would be
represented in the concentration region of zero order
kinetics by the m a x i m u m removal rate at t = O:
-- ~ max - - -~ -- ~jX.
The latter equation has been c o m p a r e d to the em-
pirical formula proposed earlier by Tu~ek, C h u d o b a
a n d M a d ~ r a [10]. Both equations have been verified
experimentally.
DESCRIPTION OF EXPERIMENTS
Cultivation of stock activated sludge
The activated sludge used for kinetic tests was cultivated
in two semicontinuous units on a once-a-day feeding sched-
ule. The units were aerated for 23 h and after that left to
settle for 1 h. The sludge age of 10 days (peptone system) and
5 days (peptone-starch system) was maintained by with-
drawing an appropriate volume of mixed liquor daily before
feeding. At the start, both units were seeded with activated
sludge from the Prague sewage treatment plant. After an in-
terval greater than three times the sludge age the kinetic
tests were carried out using only the harvested daily sludge.
Cultivation media and substrates
In the first semicontinuous unit, peptone was used as a
substrate (1200 mg 1-1 day- l) to simulate the first order
kinetics. A mixture of peptone (500mgl 1 day 1) and
starch (500 mg 1- 1 day- 1) was used in the second unit to
simulate the second order kinetics. All components were
dissolved in tap water to which a necessary amount of phos-
phates was added.
Kinetic tests
Excess activated sludge from each unit was decanted and
washed twice using tap water adjusted to 20°C at which
temperature also the tests were performed. Then the washed
sludge was mixed with peptone or peptone-starch solutions
balanced with phosphates and the mixture was made up to
1 or 21. with tap water. Immediately after the thorough mix-
ing the first sample was withdrawn for determination of in-
itial values of soluble COD and MLVSS. Then mixed liquor
samples were periodically withdrawn from the testing unit,
filtered through a paper filter and analysed for soluble
COD. The kinetic tests were carried out with various initial
(7) concentrations of substrates and MLVSS. The substrate
concentration was measured and expressed in COD units.

It must also express in some way the decrease of re-

moval rate caused by a reduced n u m b e r of c o m p o n e n t s
RESULTS AND DISCUSSION
a n d thus the decrease in total substrate concentration
with time. To reflect this we are using a n exponent The results of the experiments are summarized in
similar to the order of chemical reactions. Because this Tables 1 a n d 2. Figures 2 a n d 3 are presented as
exponent has to fit any r a n d o m line of gradual com- examples of the typical courses of C O D removal. The
p o n e n t s diminuation, it is not limited to the integers. data from Fig. 3 are shown in Fig. 4 as a second order
The kinetic equation would be eventually:
Table l. Summary of the results of the experiments with

-dt
dS
=
k,ts)X
(s).
~ "
(8)
EXp. So
peptone

Xo SO kl(sj klj x 103

No. %'
mg 1-I mg 1"I mg mg "I h-I mg -I 1 h -I
Integrating equation (8) for n = 1 a n d n = 2 yields
the most c o m m o n l y existing cases of multiple substrate 1 285 1926 0.148 0.352 1.236
removal kinetics. First order: 2 570 1953 0.292 0.478 0.840
3 670 2592 0.258 0.354 0.529

S exp . (9) 4 705 1382 0.510 0.371 0.528

So So 5 800 747 1.071 0.363 0.455

6 860 1980 0.434 0.456 0.530
Second order:
Mean : 0.396 0.686
S 1
- (10) Stan~rd deviation: * 0.056 _~ 0 . ~
So l + k2(s)X
- - o t" Variation coefficient: 14.1 43.9
So
640 P. GRAU, M. DOH,~NYOSand J. CHUI)OBA

Table 2. Summary of the results of the experiments with a peptone starch mixture.
Exp. No. 1 12: 4(~48th day of cultivation Exp. No. 13 17: 81st~83rd day of
cultivation

Exp. So Xo So k2Cs) k2j x 103

.... &
~o. xo
mg 1-I ~g 1-I mg m~-I h -I mg -I m~ -I 12 h "l

1 327 428 0,764 0.984 0.776 7.26

2 286 815 0.351 0.561 0.626 7.65
3 226 1586 0.142 0.309 0.461 9.03
4 600 801 0.749 0.779 0.961 2.67
5 524 1584 0.331 0.316 1.047 3.81
6 462 3276 0.141 0.210 0.671 3.14
7 884 860 1.028 1.173 0.876 i~12
8 804 1746 0.460 0.506 0.910 1.41
9 720 3654 0.197 0.208 0.948 1.83
I0 1180 805 1.466 1.710 0.858 0.62
II II00 1649 0.667 0.660 1.011 0.84
12 900 3469 0.259 0.345 0.752 0.93
Mean: 0.825 3.359
Standard deviation: _* 0.174 _~ 2.976
Variation coefficient: 21.1 88.6

13 258 1460 0.177 0.248 0.713 10.70

14 198 2760 0.073 0.114 0.629 16.04
15 527 1310 0.402 0.497 0.812 2.92
16 437 2810 0.156 0.190 0.819 4.29
17 774 1270 0.610 0.641 0.951 1.59
Mean: 0.785 7.108
Standard deviation: 2 0.121 ~ 6.095
Variation coefficient: 15.4 85.7

plot. It can be seen in both cases that the experimental The second order equation was linearized in the
data fit sufficiently the proper orders. form:
The first order equation was linearized in the form: t S,,
- a + bt (12)
S So - S
- lOg~o = p t (11)
where

The rate constants were then calculated as follows: t

chemical kinetics: k: i Xo So

kli = p/Xo ; for chemical kinetics; and

substrate kinetics: s,,

k2~ Xo
So
k 1(~) = P X~o" for substrate kinetics.

4~

Xo, mg (I

6O0 300 k • 428

$00
l~ 0 815
_ WOO
.%_
r2oo
l=

100

I I I i
I00 I I i I 1 2 3 4
! 2 3 4 5 t,h
t,h

Fig. 2. Linearization of the first order kinetic equation Fig. 3. Courses of COD removal from a peptone starch
(peptone). mixture.
 Kinetics of multicomponent substrate 641

1.8
Q ¢
I.It DAY OF CULTIVATION /

-y
/
5 I.l
t.At. 1.1
SiS
I.@
ul

0,~

O.4
0 ! 2 3 4 5
I,k O.2
Fig. 4 Linearization of the second order kinetic equation. I I I I * I I I

(The data from Fig. 3.) 04 OJ 1.2 1.6

!,
Xo
The rate constants are then:
Fig. 5. Dependence of the coefficient a on the initial specific
1 1 substrate concentration So/Xo. (Correlation coefficient 0'8.)
k2j - a Xo So
and
Our experiments have proved again that a relation
1so
k2(s) a Xo exists between a and the So/Xo ratio. This relation is
shown in Fig. 5 and can be described by the equation:
The coefficient b in equation (12) is close to one and
generally reflects the impossibility of attaining a zero So
value of COD. a = 1.1 xoo + 0.04. (15)
It clearly follows from the data summarized in
Tables 1 and 2 that the proposed equation of substrate The constant 13 in equations (14) and (15) has a
kinetics fits the experimental data much better than the special meaning. It represents a limit of the coefficient
equation of chemical kinetics. It was also found that a for Xo---,oo. In continuously operated activated sludge
the rate constant of chemical kinetics sharply de- process higher concentrations of MLSS can be
creased with the increasing values of the initial con- attained, under given conditions, by increasing the
centration of the multicomponent substrate. sludge age only. By this means it is impossible to in-
The substrate removal kinetics equation of second crease the substrate removal rate, which is reciprocal
order is in a good agreement with the kinetic equation to the coefficient a, unlimitedly. With increasing sludge
proposed by Tuc+ek et al. [10] for municipal sewage: age, namely, a proportion of viable and active micro-
organisms in a weight unit of sludge decreases [11].
dS,
- K S2 (13)
dt
SUMMARY
where:
A kinetic model for multicomponent substrate
S, = ~ - AS removal by activated sludge has been presented. The
model is based on the linear removal concept which
is removable BOD 5 remaining; is a special case of the broader Monod equation. Both
the simultaneous and sequential removals have been
k,b 2 considered in the kinetic model. It maintains the
K-
Soa "order of reaction" analogical to chemical reaction
kinetics, which in this case simulates random and grad-
is a complex constant; and
ual diminuation of individual components with time,
so and is not limited to the integers only. The model has
a = ~+ t~. (14) been verified experimentally and it has been found that
the variation coefficients of the substrate removal kine-
Neglecting the constant fl, the value of which is very
tics constants were 3-5 times lower than those of the
low, and substituting for a leads to the rate constant
chemical reaction kinetics constants, calculated from
of substrate kinetics: the same data.
k' b 2 Xo
K = So a So/X--o k2(,, S-~ REFERENCES

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where Linear Substrate Removal in the Activated Sludge
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~t Prague.
642 P. GRAtJ, M. Dor~/~yvos and J. CHUI)OBA
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