Wat. Res. Vol. 32, No. 5, pp.

1355±1362, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(97)00366-7 0043-1354/98 $19.00 + 0.00

EFFECTS OF COPPER AND ZINC ON THE ACTIVATED

SLUDGE BACTERIA GROWTH KINETICS
ALBERTO CABRERO*, SARA FERNANDEZ, FERNANDO MIRADA and
JULIAN GARCIA
Chemical Engineering Department, Faculty of Chemistry, Complutense University of Madrid, 28040
Madrid, Spain

(First received July 1996; accepted in revised form September 1997)

AbstractÐA simple experimental set-up has been proposed to study the in¯uence of Cu(II) and Zn(II)
on the activated sludge growth kinetics by following the rate of change on biomass concentration
during batch growth experiments. Data on biomass were ®tted to a sigmoidal equation providing us
the main biokinetics parameters (Yxs, and mm). Inocula seeded to the system was obtained from a
wastewater treatment plant operating at 14 days cell residence time. A synthetic feed solution contain-
ing 2000 mg/l gelatin (corresponding to 1630 mg/l COD) served as source of carbon. Di€erent concen-
trations of Cu(II) and Zn(II) (1, 5, 10 and 20 mg/l) were introduced singly in the reactors keeping all
environmental parameters constant (pH, Ta, basic nutrients). The combined e€ects of Cu(II) and Zn(II)
were determined by mixing these metallic ions (5/5, 5/10, and 10/5 mg/l of Cu(II)/Zn(II) respectively).
Experimental data showed that Zn(II) was less toxic than Cu(II), as expressed by a slight stimulating
e€ect for 1 mg/l Zn(II). Moreover, biokinetic parameters were not adversely a€ected by the presence of
Cu(II) up to a concentration of 5 mg/l. However, a concentration of 10 mg/l and higher, caused serious
upsets in the system. Combined e€ects of copper and zinc on the activated sludge growth kinetics, indi-
cated that these two heavy metals acted neither synergistically nor antagonistically. The sigmoidal
model very well ®ts the experimental data and could be used in a simulation study. # 1998 Elsevier
Science Ltd. All rights reserved

Key wordsÐactivated sludge, growth kinetics, heavy metals, wastewater, sigmoidal equation

NOMENCLATURE Since microorganisms are key components for de-

MLSS=mixed liquor suspended solids, g/l
COD=chemical oxygen demand, mg/l
Cx(t)=population density at each moment, g/l
Cxm=maximum population density, g/l
Cx0=initial population density, g/l
S(t)=substrate concentration with time, mg/l
S0=initial substrate concentration, mg/l
tm=half time to reach this speci®c growth rate, h
rmax=maximum growth rate of biomass, g/l
h
Yxs=biomass yield coecient
mm=maximum speci®c growth rate, hÿ1.
INTRODUCTION
Even though complex chemical manufacturing
processes can be operated e€ectively, consistent suc-
cessful wastewater treatment has been slow to
develop, and in some respect has not kept up with
advances in manufacturing technology.
For a long time, operational parameters for
wastewater systems were based on empirical optim-
ization. But before improving the biodegradation
process either by better designing treatment plants
or engineering more ecient microorganisms, the
limits of microbial degradation must be identi®ed.
*Author to whom all correspondence should be addressed
[Fax: +34-1-3944243].
1355
composition of organic matter, metal toxicity
towards microorganisms has received special atten-
tion in recent years, not only because of the en-
vironmental incidence that heavy metal discharges
into a receiving water course can generate, but also
because an important reduction in eciency of bio-
logical wastewater treatment can occur. In natural
ecosystems, a variety of factors probably alter the
shape of growth curves. These factors may include
predation by protozoa, the time for induction of
the active organisms to the accumulation of toxins
produced by other microorganisms depletion of
inorganic nutrients or growth factors, the presence
of other substrates that may repress utilization of
the compound of interest, and binding of the com-
pound to colloidal matter. The impacts or inter-
actions of such potentially important factors may
make it dicult to predict the activated sludge
growth kinetics.
Although the mechanisms by which heavy metals
a€ect biological treatment processes are not well
de®ned, the general response to varying concen-
trations is well documented (McCarty, 1964;
Neufeld and Hermann, 1975; Bagby and Sherrad,
1981; Sujarittanonta and Sherrard, 1981; Chang et
al., 1986).
1356 Alberto Cabrero et al.
McDermott et al. (1963) studied extensively the
e€ect of copper on the activated sludge system. In
this way, a copper sulphate solution was added to
the system to provided 10 to 25 mg/l Cu(II) concen-
trations. In every case, reduction in removal e-
ciency of the BOD or COD was less than 4%.
Moulton and Shumate (1963) emphasized the im-
portance of acclimation and indicated that an accli-
mated activated sludge was not adversely a€ected
by 45 mg/l Cu(II). Similarly, Neufeld and Hermann
(1975), demonstrated that it was possible to achieve
a satisfactory level of e‚uent water quality by vary-
ing the sludge age in an activated sludge system
that has been previously acclimated to heavy
metals.
A study of the e€ect of organic load on the tox-
icity of copper to the activated sludge process was
developed by Salotto et al. (1964). In this study, a
relationship between organic load and copper tox-
icity in the activated sludge processes was estab-
lished. They reported that moderate variations of
organic loading did not markedly alter the copper
toxicity.
In 1965, the results of an extensive bench and
®eld study of the activated sludge process with
chromium, copper, nickel and zinc were published
by the U.S. Public Health Service. In these studies,
changes in chemical oxygen demand (COD), bio-
chemical oxygen demand (BOD), suspended solids
(SS) and turbidity were used as indications of the
e€ects of heavy metal toxicity. The report indicated
that concentrations of the above metals either singly
or in combinations of up to 10 mg/l in in¯uent
wastewater would produce at most a 5% reduction
in treatment eciency in a continuous activated
sludge plant.
Dilek et al. (1991) reported that the substrate
removal eciency was not adversely a€ected when
di€erent copper concentrations (0.5 to 10 mg/l)
were dosed to a chemostat unit treating a synthetic
wastewater. Moreover, a slight increase in the bio-
mass yield can be detected up to a concentration of
10 mg/l Cu(II).
The discussed researches studying metal toxicity
on activated sludge are related to the acclimatiz-
ation period of microorganisms to heavy metals.
However, sewage contaminated by heavy metals
come from a wide variety of industrial sources, and
their complex mixtures varying along the time.
Changes in the metal concentration make it dicult
to achieve a complete acclimation of the microor-
ganisms to the new medium.
Other important aspect that has to be considered
for an optimal operation of a waste treatment plant
receiving heavy metal solutions is the period of time
necessary to the attainment of the biokinetic par-
ameters. In this way, a quick determination of these
parameters is desirable in order to achieve an opti-
mal operation of an aerobic biological process.
However, in most of the research discussed above,
the period of time necessary to gather all data
required for the experiments varied from 1 to
4 weeks.
In this paper, a simple experimental set-up was
used to determine the cause by which heavy metals
copper and zinc either singly or in combination
a€ect microbial batch-growth curves in response to
shock dosed metal concentration in the medium.
Moreover, no more than 3 days were spent for
gathering all data required in the experiments. The
®tting of biomass production data to the sigmoidal
equation in presence of di€erent metal concen-
trations allowed the determination of the main bio-
kinetic parameters. The system used in this research
was constituted by two independent units that oper-
ated under de®ned environmental conditions (pH
and temperature) and at di€erent metal concen-
trations. Data obtained were recorded and com-
pared to a reference reactor charged with the same
synthetic wastewater but without copper and zinc.
Theoretical considerations
Several formulations have been purposed to
express the growth dynamics of a microbial popu-
lation that is only limited by a particular substrate
(Powell, 1967; Koch, 1982), but the most popular
kinetic expression used today is the Monod
equation. This relation is based on the assumption
that only the total amount of the biomass in the
culture is sucient to specify the activities of the
microorganisms. This model can be simpli®ed to
others based on various assumptions found in the
literature (Monod, 1950; Esener et al., 1983;
Robinson and Tiedje, 1983). However, Monod
equation is referred to a single type of microorgan-
ism which grows in a well-de®ned medium where all
nutrients are in excess except one (carbon source)
which acts as limiting substrate. Investigators typi-
cally ®tted biomass formation data to the integrated
form of the Monod equation describing biomass
production using a systematic procedure, which
involved numerical adjustment of the biokinetic
parameter, until a good ®t could be obtained.
However, in practice, Monod model cannot provide
an adequate description of microbial growth beha-
vior for this particular case because of the mixed
culture character of the activated sludge in which a
variety of factors probably alter the shape of mi-
crobial biomass curves. Also, a simple sigmoidal
equation 1 had to be used to accurately describe the
di€erent steps in the microbial growth (lag phase,
exponential phase and stationary phase):
Cx0 ÿ Cxm
Cx …t† ˆ ‡ Cxm …1†
1 ‡ …t=tm †…mm
2tm †
where Cx(t) is the population density at each
moment (MLSS), Cx0 is the initial population den-
sity, Cxm is the maximum population density
achieve, mm is the maximum speci®c growth rate
 Activated sludge bacteria growth kinetics
and tm is the half time necessary to reach this
speci®c growth rate.
Initial estimates of mm and tm were entered into a
nonlinear regression computer program that ®ts
data obtained to equation (1) by the Marquardt
method. The program estimated, additionally, the
standard errors of parameters. Then, true yield
coecient Yxs, may be obtaining from equation (2):
Cx …t† ÿ Cx0
Yxs …t† ˆ …2†
S0 ÿ S…t†
equation (2) can be simpli®ed to equation (3) when
the substrate concentration is nearly exhausted
(S(t)1 0):
Cxm ÿ Cx0
Ymax ˆ …3†
S0
where Ymax is the maximum true yield, Cxm is the
maximum biomass concentration and S0 is the in-
itial substrate concentration (referred to COD).
Moreover, values for maximum growth rate (rmax)
were estimated from numerical di€erentiation of the
curves to allow us extract additional information.
MATERIALS AND METHODS
Experimental batch-growth system
The schematic diagram of the batch growth system used
in the inhibition kinetics of the activated sludge process
due to the presence of copper and zinc is shown in Fig. 1.
The equipment was constituted of 10 Erlenmeyer ¯asks
acting as growth reactors. These reactors were placed into
a thermostatic bath of plexiglass material to remain oper-
Fig. 1. Schematic diagram of the batch-growth system.
1357
Table 1. Composition of wastewater solution fed to the reactors
Constituent Concentration (mg/l)
a
Gelatin from porcine skin 2000
K2HPO4 1750
KH2PO4 250
NH4Cl 250
Other micronutrients Ðb
a
COD of waste = 1630 mg/l.
b
Micronutrients were supplied using tap water as diluent.
ating temperature constant at 258C. The operating volume
for each reactor was of 2 litres.
Compressed air was used to supply oxygen to each ¯ask
through porous di€user stones. The air also served to pro-
vide good mixing of suspended solids. An adequate volu-
metric air ¯ow rate was supply in order to maintain
saturated oxygen conditions in the medium. There was
and additionally magnetic stirrer module that achieved an
e€ective mix in the reactors.
Bacterial growth medium
The same synthetic wastewater was used throughout the
experiments. Components of wastewater used in this inves-
tigation are listed in Table 1. In this solution, gelatin
served as the main carbon and energy source. Phosphate
bu€er was added as a phosphorous source, as well as to
maintain a stable pH. A solution of NH4Cl was added to
the medium to provide the source of nitrogen. All other
micro-nutrients were added in sucient quantities to make
carbon the growth-limiting substrate. Metals studied (cop-
per and zinc) were added as sulphate salts to provide a
concentration of 1, 5, 10 and 20 mg/l in the reactors. The
combinations of these metals were 5/5, 5/10 and 10/5 mg/l
of copper and zinc respectively. All inorganic salts were of
analytical reagent grade.
1358 Alberto Cabrero et al.
Analytical techniques
Microbial growth evolution was carried out by monitor-
ing the MLSS concentration along the time in a
VARIAN-CARY 1E spectrophotometer. During batch-
growth, liquid samples were withdrawn and centrifuged
immediately to separate bacteria from the liquid medium.
Depending on turbidity, samples were diluted with water
by a factor of 2±8 and then centrifuged at 4000 rpm for
20 min to precipitate the bacteria. After centrifugation, the
supernatant was removed, cell pellet was resuspended into
water and cell density was then determined by optical den-
sity at 450 nm. Optical density measurements of the acti-
vated sludge culture were converted into MLSS
concentration by means of this calibration curve:
[MLSS] = ÿ 2.118(20.941) + 548.74(24.68)
ABS, where
ABS is the absorbance value and [MLSS] is the mixed
liquor suspended solids concentration (expressed as mg
biomass/l). The coecient of correlation for this linear re-
gression was r2=0.998. Copper and zinc ion concen-
trations were determined using an atomic absorption
spectrophotometer.
Experimental procedures
Activated sludge inocula was obtained from a biological
treatment pilot plant. This plant was operated under
steady-state conditions with a MLSS concentration of
2200 mg/l. Steady-state was identi®ed by invariant total
biomass concentrations over a month. A 350 ml/d purge
was done from the aerated reactor to maintain a desired
mean cell residence time value of 14 days. This cell resi-
dence time was ®xed according to the studies performed
by Bagby and Sherrad (1981). Components of the waste-
water fed to the biological treatment pilot-plant are listed
in Table 2.
In order to achieve optimal experimental conditions in
the medium, some batch growth experiments were made at
di€erent pH, temperature, suspended solids' inocula con-
centration (MLSS) and limiting substrate concentration
(gelatine).
Once the optimal conditions in the medium were
achieved (pH = 7.5; Ta=258C; MLSS inocula = 0.025 g/l;
limiting substrate = 2 g/l), metal toxicity studies were per-
formed by measuring the total suspended solids concen-
tration along the time. During the experiment, the sludge
was suddenly shock dosed to 1, 5, 10 and 20 mg/l of the
appropriate heavy metal added in a soluble sulfate form.
Immediately after dosing of the sludge and for about
3 days thereafter, MLSS measurement was made by
sampling (4 ml) the mixed content of the di€erent ¯asks at
intervals of 2 h. The pH measurements in the reactors
were made at the beginning and at the end of the exper-
iments. It was controlled at 72 0.2 with a phosphate buf-
fer solution.
Accordingly to other studies, (Sierp and Fransemeier,
1933; Pettet, 1956) an increase in the growth medium tur-
bidity was observed after 2 days. An explanation of this
phenomenon was given by LombranÄa et al. (1993). In this
study, discrepancies between data for biomass yield and
speci®c rate of substrate removal (referred to COD) seem
to be caused by the fact that not all of the MLSS increase
Table 2. Composition of wastewater solution fed to the pilot-plant
Constituent Concentration (mg/l)
D-(+)-glucosea 500
K2HPO4 1750
KH2PO4 250
NH4Cl 250
Other micronutrients Ðb
a
COD of waste = 430 mg/l.
b
Micronutrients were supplied using tap water as diluent.
is a real and active biomass increase. They consider that a
considerable amount of soluble and suspended COD is
trapped in the ¯ocks but not digested, producing an
apparent biomass growth. The problem described was
solved by using growth solution supernatant obtained
after centrifugation of the suspended solids as a blank.
RESULTS AND DISCUSSION
Biomass production curve and biokinetic parameters
Due to the mixed culture character of the acti-
vated sludge, they present a great variance in toler-
ance towards the di€erent metal studied. In this
study, it has been found that relatively lowered con-
centrations of certain heavy metals could promote a
stimulating e€ect on the microbial population
growth, as re¯ected by an increase in biomass yield.
This phenomenon is consistent with observations
made by other authors and will be discussed later.
Conversely, for higher metal concentrations (5 mg/
l), microbial growth began to be inhibited, as
re¯ected by a decrease in biokinetic parameters
values.
The study allowed to make qualitative and semi-
quantitative assessments of the in¯uence of copper
and zinc, singly or in combination, on activated
sludge growth kinetic parameters by ®tting data
obtained to the previously established sigmoidal
equation. Figures 2±4 show comparisons between
the data and the ®tted curves (drawn as solid lines).
The estimations for the parameters of the logistic
model at di€erent metal concentrations are sum-
marized in Table 3. The adequate ®tness of the data
obtained to the logistic equation con®rms a good
choice of the model, which states that the presence
of metals a€ected both growth rate and biomass
yield.
The study of the individual and combined e€ect
of copper and zinc on the activated sludge growth
phenomena was carried out under the predeter-
mined conditions described above. All the exper-
iments were repeated three times. Included below is
a presentation of the most signi®cant results of the
experiments.
Control. The experimental set-up was divided into
two identical units, each one was constituted of ®ve
Erlenmeyer ¯asks. One of them was charged with
the same constituents that the others but void of
metals, operating at the same pH and temperature
as the others. Control reactor served to obtain base-
line data to compare the toxic e€ect produced by
the addition of di€erent amounts of heavy metals.
Microbial growth curve was characterized by a
non-exponential increase for the ®rst 20 h. There-
after, the culture in the reactor was considered ac-
climatized to the new medium. In this way 48±50 h
were necessary to reach the stationary phase, in
which the nutrients were nearly exhausted. It was
taken about three days to observe the ®rst evidence
of endogenous metabolism accordingly with that
 Activated sludge bacteria growth kinetics 1359

Fig. 2. Batch-growth experiments in presence of Zn(II). Symbols show the data and curves show model
predictions.

found by Herbert (1958). It should be noted on
Figs 2±4 that the sigmoidal equation used in this
study did not describe the last part of the growth
curve (declining phase), however, declining growth
phase was not decisive for the attainment of the
kinetic parameters. Values of kinetic parameters for
the control reactor are shown in Table 3. The
results of biomass yield, Yxs, obtained were in
agreement with those found by Bagby and Sherrad
(1981).
E€ect of zinc. The time course data at four di€er-
ent zinc concentrations ranging from 1 to 20 mg/l
are shown in Fig. 2. This ®gure showed that there
was no signi®cant change neither in the acclimation
phase nor in the exponential phase for all the metal
concentrations studied if compared with the refer-
ence reactor. Thus, in comparison to the control
reactor, a lower MLSS concentration was observed
for all the experiments except that carried out with
1 mg/l of zinc, where a slight stimulating e€ect was
detected. When the amount of zinc was increased to
5 mg/l a small drop in biomass yield occurred. For
10 mg/l Zn(II) a sharply decrease on biomass pro-
duction (15%) was observed, while higher amounts
of zinc (20 mg/l) caused similar e€ects. This seems
to indicate that 10 mg/l of zinc are more than
enough to inhibit sludge growth, however, up to
this metal concentration, a precipitation and/or

Fig. 3. Batch-growth experiments in presence of Cu(II). Symbols show the data and curves show model
predictions.
1360 Alberto Cabrero et al.

Fig. 4. Batch-growth experiments in presence of Cu(II) and Zn(II). Symbols show the data, and curves
show model predictions.

complexation process may occur. Otherwise, when
the reactor was charged with zinc concentrations
from 1 to 20 mg/l, values for mm were nearly equal
to that found for the control. For this parameter,
variations in the value of speci®c growth rate ran-
ged into experimental error.
The stimulating phenomena occurred at 1 mg/l of
zinc was also observed by several authors but for
another metals as copper, nickel and cadmium
(Bagby and Sherrad, 1981; Yetis and Gokcay, 1989;
Gokcay and Yetis, 1991; Dilek et al., 1991).
E€ect of copper. Results of biomass concentration
evolution carried out by varying metal concen-
trations from 1 to 20 mg/l are shown in Fig. 3.
From these plots it can be observed that in every
case, the maximum MLSS concentration was lower
than in the reference reactor. There was no copper
concentration that caused any stimulating e€ect as
happens with 1 mg/l of zinc. These results showed
that, comparatively, the toxic inhibitory e€ect of
copper was substantially higher than zinc.
As it may be seen in Fig. 3, at low Cu(II) concen-
tration (<20 mg/l) the period of time for the sub-
strate hydrolysis (lag phase) was similar to the
other assays. Over to 10 mg/l Cu(II), however, the
lag phase increase by a 30%. This phenomenon was
not observed for any zinc concentration assayed.
The inhibitory e€ect of copper causes a very im-
portant decrease of the maximum speci®c growth
rate, mm, specially when 20 mg/l of copper were
dosed to the ¯asks, decreasing this parameter by a
20% if compared with the reference reactor.
A series of batch-growth cultures was run with
the metal concentrations described above. In the
presence of 1 to 5 mg/l of Cu(II), the slight decrease
in the biomass yield was accompanied by a similar
decrease in the exponential phase curve slope
(Fig. 3). For all the assays, a continuous decrease in

Table 3. Mean value parameters of logistic model ®t to data on the bacteria cell growth and standard deviations
Kinetic parameters and standard deviations
Metal concentration (mg/l) Yxs (g/g) Cxm (g/l) mm (hÿ1) rmax (g/l
h)

Reference 0.448 20.024 0.916 20.048 0.1022 0.014 0.0402 0.0010

Cu (II)
1 0.378 20.004 0.770 20.008 0.0962 0.003 0.0392 0.0009
5 0.357 20.006 0.759 20.012 0.0942 0.005 0.0372 0.0007
10 0.188 20.006 0.394 20.012 0.0912 0.010 0.0222 0.0012
20 0.171 20.005 0.353 20.010 0.0782 0.005 0.0142 0.0006
Zn (II)
1 0.469 20.013 0.965 20.027 0.0982 0.008 0.0472 0.0005
5 0.412 20.007 0.852 20.015 0.0972 0.006 0.0412 0.0003
10 0.348 20.004 0.723 20.009 0.0992 0.004 0.0352 0.0004
20 0.333 20.008 0.693 20.016 0.0942 0.006 0.0322 0.0008
Cu(II)/Zn(II)
5/5 0.335 20.005 0.689 20.011 0.1002 0.006 0.0342 0.0007
5/10 0.246 20.011 0.507 20.023 0.1002 0.015 0.0262 0.0014
10/5 0.213 20.007 0.445 20.014 0.0892 0.009 0.0212 0.0010
 Activated sludge bacteria growth kinetics
the maximum microbial growth rate (rmax) and bio-
mass yield (Yxs) was observed. This means that true
yield (expressed as g biomass/g substrate) dimin-
ished from an average value of 0.448 for the refer-
ence reactor, to about 0.17 when 20 mg/l of copper
were added, which is the lowest value obtained, just
as maximum growth rates (expressed as g biomass/
h) decreased to 0.020 for 10 mg/l of copper and to
about 0.014 for 20 mg/l of copper, respectively. As
can be seen in Figs 5 and 6, sharp decreases in the
maximum growth rate and biomass yield par-
ameters were observed for the wastewater contain-
ing over to 5 mg/l Cu(II), showing that this metal
concentration is the limit for which microbial
growth begins to be substantially inhibited.
The results of this study were di€erent to the
®ndings of other authors. For example, Barth et al.
(1965) found that amounts over 10 mg/l of copper
caused signi®cant inhibitory e€ects on microorgan-
isms. Conversely, Pettet (1956) founds that inhi-
bition took place for only 1 mg/l of this metal.
Neufeld and Hermann (1975), demonstrated that
the toxicity of heavy metals depends on cell resi-
dence time. Otherwise, Directo and Moulton (1962)
and Lamb and Tollefson (1973), established a re-
lationship between the amount of organic loading
on copper toxicity. Both parameters, might have
importance to explain these di€erences, but the
comparison of the results obtained by these authors
was not allowed because of the di€erent experimen-
tal conditions they used. This also happened with
our ®ndings, however, all agree that copper can
cause negative e€ects on activated sludge.
E€ect of copper and zinc. Curves of microbial
growth evolution for three metal mixtures (5/5, 5/
10, and 10/5 mg/l of Cu(II)/ Zn(II) respectively)
were represented in Fig. 4. An extended lag was
also ensued for that metal combination, in which
coppers was the main constituent (10 mg/l of cop-
per). It was observed that MLSS concentration was
lower than in the control reactor along the entire
metal concentration range studied and began to be
Fig. 5. In¯uence of Zn, Cu and Cu/Zn metal mixtures on
biomass yield (Yxs).
1361
Fig. 6. In¯uence of Zn, Cu and Cu/Zn metal mixtures on
maximum growth rate (vmax).
very noticeably lowered for the last metal combi-
nation described above.
Estimated maximal speci®c growth rates, mm, are
presented in Table 3. It is readily read from this
table that only for the case in which the metal mix-
ture of 10/5 mg/l Cu(II)/Zn(II) is added, a substan-
tial decrease in mm is observed as compared with
the reference reactor. This ®nding suggests a higher
inhibitory e€ect of copper on activated sludge
growth in relation to zinc. The results of Yxs carried
out by changing the metal dosage are shown in
Fig. 5. At 5/5 mg/l Cu(II)/Zn(II), the biomass yield
was 25% lower as compared with the reference
reactor. Decrease in true yield was similar than the
one established when 5 mg/l Cu(II) were present.
Again, these results reassess the higher toxicity of
copper in respect to zinc. The relationship between
the maximum rate of growth and metal concen-
tration is represented in Fig. 6. The comparison
between 5/10 mg/l Cu(II)/Zn(II) and 10/5 mg/l
Cu(II)/Zn(II) which have the same total metal con-
centration, shows clearly the higher inhibitory e€ect
of copper in respect to zinc attending to growth
rate. Our ®ndings are in concordance with the
results obtained by Barth et al. (1965), McDermott
et al. (1963) and Adams (1975).
CONCLUSIONS
The presence of copper and zinc, singly or in
combination, in a sewage that need to be treated
using activated sludge process, produced di€erent
e€ects on the microbial growth kinetics. This toxic
e€ect depends on the characteristic and concen-
tration of the metal, and is re¯ected by a decrease
of the main biokinetic parameters obtained from
the model (Yxs, mm, and rmax). However, copper
and zinc in low concentrations act as micronutrients
that are essential for the bacteria as constituents of
special enzymes. This theory is supported by
Brynhildsen et al. (1988).
1362 Alberto Cabrero et al.
Kinetic parameters from sigmoidal equation (1),
which describe the microbial growth curves, have
been evaluated at several heavy metal concen-
trations. For zinc, these parameters were found to
be virtually una€ected until a threshold concen-
tration of metal in the sludge was reached
(>10 mg/l). For copper, a higher toxic e€ect
towards the microorganisms was observed. It would
probably be that this metal inhibits the substrate
hydrolysis rate as can be deduced from the increase
in the lag growth phase when 20 mg/l Cu(II) was
dosed to the system. This phenomenon was not ap-
preciated when 20 mg/l of zinc was added to the
reactor.
Introduction of combinations of copper and zinc
concentrations to wastewater, can produce serious
upsets in the biological treatment system. This
negative e€ect caused by copper and zinc mixtures
increases when the main metal-dosed in the system
was copper. Moreover, metal combinations acted
neither synergistically nor antagonistically as
deduced from data obtained.
In this study, it has been proved that the two
heavy metals studied have quanti®cable in¯uences
on the biokinetics parameters, which are so import-
ant for an optimal operation and design of an
aerobic biological process. The use of this simple
experimental set-up permitted us to obtain, in only
three days, the main kinetics parameters in order to
evaluate the degree of toxicity of heavy metals to
biological wastewater treatment processes. This in-
formation can be of great interest to operators of
an activated sludge treatment plant.
AcknowledgementsÐThis study was conducted within the
framework of the Complutense University of Madrid
(Industrial Chemistry Department) as part of a Ph. D.
Thesis. The author thanks analytical support from the
UCM Laboratory sta€. This paper was presented at the
7th Mediterranean Congress of Chemical Engineering in
Barcelona.
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