Water Research 36 (2002) 3341–3349

Effect of chromium(VI) on bacterial kinetics of heterotrophic

biomass of activated sludge
Athanasios S. Stasinakisa,*, Daniel Mamaisb, Nikolaos S. Thomaidisa,
Themistokles D. Lekkasa
a
Department of Environmental Studies, University of the Aegean, Theofrastou and Alkaiou Street, Mytilene 81 100, Greece
b
Department of Water Resources, Faculty of Civil Engineering, National Technical University of Athens, 5 Iroon Polytechniou Street,
Zografou, Athens 15773, Greece
Received 13 July 2001; accepted 20 December 2001

Abstract

The effect of hexavalent chromium, Cr(VI), on the maximum specific growth rate, mm and biomass yield, YH ; of
heterotrophic biomass was studied in batch tests conducted under high (=10) and low (=1.5) substrate-to-biomass
ratios ðS0 =X0 Þ: The effects of sludge age and biomass acclimatization to Cr(VI) on the bacterial kinetics were also
studied. The mm values were determined by measuring oxygen uptake rate (OUR) and volatile suspended solids (VSS)
increase. Cr(VI) concentrations equal or greater than 10 mg l
1 inhibited the growth of unacclimatized activated sludge
and caused a significant decrease in mm and YH values. The acclimatization of biomass and the selection of a high
operating sludge age reduced the inhibitory effect of Cr(VI). At a sludge age of 20 days, Cr(VI) concentrations of
o10 mg l
1 stimulated bacterial growth as evidenced by an increase in both the mm and YH values. Determining mm
values by OUR and VSS methods, revealed that the presence of Cr(VI) in unacclimatized biomass caused an inhibitory
effect mostly on substrate oxidation, while in acclimatized biomass, anabolic pathways were inhibited more. r 2002
Elsevier Science Ltd. All rights reserved.

Keywords: Toxicity; Cr(VI); Activated sludge; Bacterial kinetics; Batch methods; S0 =X0 ratio

1. Introduction plating, leather tanning and chemical manufacturing.

Each of the above oxidation states has very different
The increasing trend towards combining industrial biological and toxicological properties. Cr(III) accumu-
and municipal wastes for treatment in sewage plants lates in the cell membrane, while Cr(VI) is transported
increases the possibility of contamination of the influent into the cells, where it is reduced to three valent form
by metal ions. Since microorganisms are key compo- and react with intracellular material [1].
nents for the decomposition of organic matter, the effect There are numerous methods for evaluating metal
of metal toxicity on microorganisms has received special toxicity in activated sludge systems. The most widely
attention in recent years. used are enzymatic and nitrification inhibition [2],
Chromium is usually encountered in the environment inhibition of respiratory activity [3], influence on
in the oxidation states of (III) and (VI). It is released by activation process efficiency [4,5], influence on activated
effluent discharge from steelworks, chromium electro- sludge microfauna [6] and influence on activated sludge
bacterial kinetics [7]. Various techniques have been
*Corresponding author. Tel.: +30-251-36225; fax: +30-251- developed to determine the kinetic constants of the
36226. biomass growth. However, in the literature, the values of
E-mail address: astas@env.aegean.gr (A.S. Stasinakis). kinetic parameters vary considerably for growth and

0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 3 - 1 3 5 4 ( 0 2 ) 0 0 0 1 8 - 0
3342 A.S. Stasinakis et al. / Water Research 36 (2002) 3341–3349

Nomenclature yc sludge age (day)

m specific growth rate (day
1)
COD chemical oxygen demand (mg l
1)
MLVSS mixed liquor volatile suspended solids (mg l
1) Subscripts
OUR oxygen uptake rate (mg O2 l
1 day
1) m maximum
S substrate concentration (mg COD l
1) OUR OUR-based estimation of mm in experiments
S0 initial substrate concentration (mg COD l
1) with high S0 =X0
SBR sequencing batch reactor our OUR-based estimation of mm in experiments
VSS volatile suspended solids (mg l
1) with low S0 =X0
X biomass concentration (mg VSS l
1) VSS volatile suspended solids estimation of mm
X0 initial bacterial biomass (mg COD l
1) H heterotrophic
Y yield coefficient (mg VSS (mg COD oxi-
dized)
1)

substrate removal determined in batch reactors. This observed a continuous decrement of mm in the presence
discrepancy may be attributed to culture history of the of 2–11 mg l
1 Cr(VI).
microorganisms [8], the parameter identifiability [9] and These controversial results on the effect of Cr(VI)
the nature of kinetic assay [10]. Under high substrate-to- could be attributed to the fact that the impact of heavy
biomass ratios ðS0 =X0 Þ; fast grown bacteria appeared to metals and other toxic compounds on activated sludge is
gain a competitive advantage, resulting in higher mm influenced by several factors such as the sludge age ðyc Þ
values than those obtained under low S0 =X0 [10,11], an [21–23], the acclimatization of biomass to the toxic
observation consistent with ‘‘r’’ strategist microorga- compound [18,24], the concentration of suspended solids
nisms [12]. Moreover, it was proposed that the oxygen [5], the species of microorganisms presented in activated
uptake rate (OUR)-based estimate of mm ðmmðOURÞ Þ is a sludge [25] and the exposure time of biomass to the toxic
measure of substrate oxidation and under certain compound [22,3].
circumstances is differentiated from mm determined by These controversial results lead us to undertake the
measuring exponential bacterial growth ðmmðVSSÞ Þ current research. Thus, the purpose of this study was to
[13,14,11]. investigate the toxic effects of Cr(VI) on the bacterial
The data presented in the literature describing the kinetics of heterotrophic biomass (maximum specific
Cr(VI) toxicity effects on activated sludge process and growth rate, mm ; and biomass yield, YH ) and to estimate
specifically on substrate removal, respiration activity the effect of acclimatization of biomass and sludge age
and bacterial growth, are often controversial. Early on observed toxicity. For this reason, experiments were
works by Barth et al. [15] and Moore et al. [16] on conducted with Cr(VI) acclimatized and unacclimatized
substrate removal supported that aerobic biological biomass, grown at various sludge ages that ranged from
treatment processes could tolerate, without significant 2.5 to 20 days. Based on our previous study [11], values
loss in treatment efficiency, Cr(VI) concentrations in the of mm were obtained from oxygen uptake (OUR) and
range of 10–50 mg l
1. Moreover, Moore et al. [16] biomass growth (volatile suspended solids (VSS)) in
showed that at concentration of 5 mg l
1 Cr(VI), the unit batch tests conducted at high S0 =X0 (=10) and low
performed better than the control reactor. However, S0 =X0 (=1.5) ratios in order to evaluate the sensitivity
Lamb and Tollefson [5] reported that shock loading of a of different methods to assess Cr(VI) toxicity and to
completely mixed activated sludge system with 5 mg l
1 estimate the effect of Cr(VI) on substrate oxidation and
CrO2
4 reduced organic substrate removal by 50%. bacterial growth.
Literature data on biomass respiration activity are also
contradictory. Vankova et al. [3] reported that the 1-h
EC50 value was in the range of 40–90 mg l
1, whereas 2. Materials and methods
Madoni et al. [17] reported that 1 h exposure of activated
sludge to 100 mg l
1 Cr(VI) (as initial concentration) 2.1. Activated sludge cultivation
reduced the OUR only by 21.5%. Gokcay and Yetis [18]
and Yetis et al. [19] showed that an acclimatized sludge Four parallel 10 l sequencing batch reactors (SBRs)
was stimulated in the presence of Cr(VI) and observed were used to simulate activated sludge process and to
an approximately two times increase in mm value and provide biomass for evaluating bacterial kinetics.
stimulatory effects on biomass yield in the presence of Activated sludge from a municipal wastewater treatment
25 mg l
1 Cr(VI). On the contrary, Mazierski [20] plant (Plomari, Lesvos) was used to seed the reactors.
 A.S. Stasinakis et al. / Water Research 36 (2002) 3341–3349 3343

Table 1 was measured on the filtrate through a 0.45 mm

Composition of wastewater solution fed to the SBRs membrane filter (Millipore). Mixed liquor volatile
Constituents Concentration (mg l
1) suspended solids (MLVSS) were determined by filtering
the sample through a 0.45 mm glass fiber filter (Whatman
CH3COOH 470 GF/C). The initial Cr concentration was determined by
NH4Cl 250 graphite furnace atomic absorption spectrometry (Per-
K2HPO4 1750 kin Elmer 5100ZL, with autosampler AS 72).
KH2PO4 250
Other micronutrients Fa
a
2.4. Determination of maximum specific growth rate
Micronutrients were supplied using tap water as diluent.
Maximum specific growth rate, mm, was determined
Synthetic wastewater was used as feed, supplemented for different substrate-to-biomass ratios, S0 =X0 ;
with a phosphate buffer to maintain a constant pH of (S0 =X0 ¼ 10 and S0 =X0 ¼ 1:5). Microbial growth was
7.070.2 (Table 1). Initially, the four reactors operated carried out by monitoring the OUR and the MLVSS
for 2 weeks on synthetic wastewater devoid of chro- concentration [27,28,13,14,11].
mium. Following that, different quantities of hexavalent
chromium, added as potassium dichromate (K2Cr2O7,
2.5. Determination of heterotrophic yield coefficient, YH
Merck), were added to the four systems to achieve a
Cr(VI) concentration of 0 mg l
1 (Reactor A), 1 mg l
1
The values of heterotrophic yield coefficient, YH ; were
(Reactor B), 10 mg l
1 (Reactor C) and 25 mg l
1
obtained from Eq. (1) [7,27]:
(Reactor D), respectively. The temperature of the
activated sludge units was maintained at 20711C. Xm
X0
YH ¼ ; ð1Þ
The SBRs were operated in a 24-h fill and draw cycle, S0
Sm
each cycle consisting of four stages: Fill (10 min), React
(22.5 h with aeration), Settle (1 h) and Decant (20 min). where Xm and X0 are the maximum and initial biomass
A minimum period of 3 yc was allowed to achieve concentration (mg l
1 VSS), S0 is the initial substrate
biomass acclimatization to each Cr(VI) concentration. concentration and Sm is the substrate concentration at
time when biomass concentration is maximum (mg
2.2. Batch experiments for the determination of kinetic COD l–1).
constants

To estimate the effect of Cr(VI) on the measured 3. Results and discussion

kinetics of unacclimatized biomass, activated sludge
samples from the Reactor A (0 mg l
1 Cr(VI)) were 3.1. Effect of Cr(VI) on YH
introduced in four parallel batch experiments. Each
batch experiment was conducted using the same YH values obtained from experiments with unaccli-
synthetic wastewater, supplied with the required Cr(VI) matized and acclimatized biomass are presented in Table
quantity to achieve concentrations of 0, 1, 10 and 2. YH values obtained from reactors that received no
25 mg l
1, respectively. To determine the kinetic para- Cr(VI) ranged from 0.31 to 0.34 mg VSS mg COD
1 and
meters of acclimatized biomass, activated sludge sam- did not depend on sludge age. Similar dependence was
ples from the three SBRs that received Cr(VI) were reported by Sollfrank and Gujer [29] and Kappeler and
introduced in batch reactors supplied with synthetic Gujer [28]. On the contrary, YH values obtained at
wastewater containing the amount of Cr(VI) that various Cr(VI) concentrations appeared to depend on
biomass was acclimated to. sludge age. A stimulating effect on unacclimatized
To inhibit nitrification in batch reactors, thiourea microbial population, reflected by an increase in
(Merck) was added to a final concentration of 10 mg l
1 biomass yield (Table 2), was observed at a Cr(VI)
[14]. All batch experiments were run in duplicate and concentration of 1 mg l
1 and a sludge age of 20 days.
conducted at a constant pH equal to 7.070.2, a The above stimulating effect was not observed at other
temperature of 20711C, a DO concentration of sludge ages. At yc values of 10 and 5 days and Cr(VI)
4 mg l
1. The experiments were repeated for sludge ages concentration of 1 mg l
1, YH was approximately equal
ðyc Þ of 2.5, 5, 10 and 20 days. to the values obtained in the control reactor. At lower yc
(2.5 days), Cr(VI) concentration of 1 mg l
1 resulted in a
2.3. Analytical methods significant YH decrease. Increase of Cr(VI) concentra-
tion to 10 and 25 mg l
1 led to a gradually decrement of
All analyses were done according to standard YH values, with the maximum inhibition observed at
methods [26]. Soluble chemical oxygen demand (COD) lower sludge ages.
3344 A.S. Stasinakis et al. / Water Research 36 (2002) 3341–3349

Similar effects of hexavalent chromium on YH were 100

observed for acclimatized biomass. The only difference 80
was that stimulating effects on sludge aged 20 days were 60

Inhibition (%)
observed not only for Cr(VI) concentration of 1 mg l
1
40
but also for 10 mg l
1. Further increase of Cr(VI)
concentration decreased the YH value below the value 20

obtained in the control reactor. 0

The increase of biomass yield obtained at low heavy -20 0 5 10 15 20 25 30
metals concentrations and the inhibitory effects at -40
higher concentrations are reported in the literature for Cr(VI) (ppm)
other heavy metals, such as Zn [7] and Cd and Ni [30]. θc=20 days θc=5 days θc=2.5 days
The higher values of YH could be attributed to the
enrichment of selected resistant microorganisms in the Fig. 1. Cr(VI) toxicity in acclimatized biomass expressed as
process and/or the stimulatory effects of low concentra- proportional reduction of mmðourÞ (% inhibition) at various
tions of heavy metals on microbial activity. sludge ages and at S0 =X0 ¼ 1:5:

3.2. Effect of Cr(VI) on mm

Cr(VI) concentration constantly decreases within the
The effects of Cr(VI) addition on the maximum first 10 h of contact time [3].
specific growth rate of unacclimatized and acclimatized The effects of Cr(VI) presence on microbial growth
biomass, evaluated by conducting batch assays at low were better quantified in batch experiments conducted
and high substrate-to-biomass ratios (S0 =X0 ¼ 1:5 and using acclimatized biomass, where exposure time of
S0 =X0 ¼ 10), are described in the following paragraphs. biomass to Cr(VI) is equal to the sludge age. As shown
in Table 3 and Fig. 1, the presence of chromium
3.2.1. Batch tests at initial S0 =X0 ¼ 1:5 influenced mmðourÞ values and toxicity in acclimatized
Table 3 summarizes the obtained data for S0 =X0 ¼ biomass appeared to be a function of sludge age. Cr(VI)
1:5 and various operating sludge ages (2.5, 5 and 20 toxicity, determined as the ratio of the maximum specific
days). All the experiments conducted with unacclima- growth rate in the presence of Cr(VI) to the rate in the
tized biomass in the presence of Cr(VI) exhibit minor absence of Cr(VI) (% inhibition), decreased with
effects on the mmðourÞ values for concentrations up to increasing sludge age. Particularly, for sludge age of
25 mg l
1. Similar observations in experiments at low 2.5 days, Cr(VI) concentration equal to 1 mg l
1
S0 =X0 ratio have been recorded in a previous study for inhibited the growth of heterotrophic microorganisms
Ni toxicity [11] and are probably due to the low by more than 60%. On the other hand, for sludge age of
exposure time of microorganisms to the toxic com- 20 days it was found that concentrations of Cr(VI) equal
pound. Experiments at low S0 =X0 ratio, lasted not more to 1 and 10 mg l
1 had stimulating effects on the
than 2 h, whereas it has been reported that the EC50 microbial population. This stimulating effect is probably
due to the fact that at low Cr(VI) concentrations, cells
are respiring under physiological stress [13].
Table 2
Values of yield coefficient, YH ; that were obtained in various
sludge ages, for unacclimatized and acclimatized biomass
3.2.2. Batch tests at initial S0 =X0 ¼ 10
Table 4 summarizes the maximum growth rates, mm ,
Cr(VI) YH (mg VSS/mg COD) determined by both OUR and VSS measurements at
(mg l
1) S0 =X0 ¼ 10 and various operating sludge ages. Cr(VI)
yc ¼ 20 yc ¼ 10 yc ¼ 5 yc ¼ 2:5 concentration equal to 1 mg l
1 stimulated mm ; deter-
days days days days
mined by both VSS and OUR measurements of
Unacclimatized biomass acclimatized and unacclimatized activated sludge cul-
0 0.34 0.32 0.33 0.31 tures grown at a long sludge age of 20 days (Figs. 2 and
1 0.41 0.33 0.33 0.25 3, respectively). At a Cr(VI) concentration equal to
10 0.32 0.27 0.24 0.22 10 mg l
1 the effect of Cr(VI) depended on the acclima-
25 0.23 0.24 0.21 0.19 tization of biomass; Cr(VI) presence resulted in a
stimulatory effect on acclimatized biomass and an
Acclimatized biomass
inhibitory effect on unacclimatized biomass. Several
1 0.37 0.29 0.31 0.25
10 0.41 0.26 0.29 0.19
researchers have reported the stimulation effect that
25 0.24 0.19 0.22 0.13 heavy metals exert on microbial growth. Gokcay and
Yetis [18] observed that Cr(VI) concentrations as high as
 A.S. Stasinakis et al. / Water Research 36 (2002) 3341–3349 3345

Table 3 25 mg l
1 increased significantly mm values. In this study

Values of maximum specific growth rate, mmðourÞ , that were however, increasing chromium concentration to
obtained in batch tests with S0 =X0 ¼ 1:5 25 mg l
1 reduced remarkably mm values. It should be
Cr(VI) (mg l
1) mmðourÞ (d
1) mentioned that at this concentration it was not possible
to determine mmðOURÞ values because OUR did not
yc ¼ 20 days yc ¼ 5 days yc ¼ 2:5 days increase during the test, suggesting that aerobic growth
Unacclimatized biomass was completely inhibited.
0 1.28 2.09 2.44 The above stimulating effects of Cr(VI) were not
1 1.30 2.10 2.33 observed at lower sludge ages, where Cr(VI) concentra-
10 1.25 1.79 2.33 tion as low as 1 mg l
1 inhibited mm values. In fact at a
25 1.27 1.64 2.07 sludge age of 2.5 days, SBRs failed at Cr(VI) concentra-
tions of 10 mg l
1 (Reactor C) and 25 mg l
1 (Reactor
Acclimatized biomass D), indicating zero growth rate.
1 1.60 1.73 0.68
Based on the data presented above, obtained from
10 1.34 1.05 0.20
25 0.37 0.76 0.10
both acclimatized and unacclimatized biomass experi-
ments, it can be concluded that at high sludge ages
chromium toxicity is significantly reduced (Figs. 2 and

Table 4
Values of maximum specific growth rate, mmðOURÞ and mmðVSSÞ that were obtained in batch tests with S0 =X0 =10

Cr(VI) (mg l
1) mmðOURÞ (day
1) mmðVSSÞ (day
1)

yc ¼ 20 days yc ¼ 10 days yc ¼ 5 days yc ¼ 2:5 days yc ¼ 20 days yc ¼ 10 days yc ¼ 5 days yc ¼ 2:5 days

Unacclimatized biomass
0 1.06 2.05 2.84 3.46 0.96 1.73 1.83 1.80
1 1.77 1.79 2.46 3.10 1.57 1.71 1.63 1.25
10 0.46 1.17 0.53 1.70 1.12 1.04 0.59 0.78
25 0a 0a 0a 0a 0.69 0.36 0.22 0.16

Acclimatized biomass
1 1.95 1.44 3.04 2.95 1.09 0.93 1.76 1.29
10 1.82 0.76 1.92 0b 1.17 0.61 0.86 0b
25 0.4 0.72 0.95 0b 0.33 0.46 0.48 0b
a
Activated sludge growth was completely inhibited as indicated by a zero increase in OUR values.
b
SBRs failed and neither mmðOURÞ nor mmðVSSÞ could be determined.

100
80
Inhibition (%) of µm(VSS)

60
40
20
0
-20 0 5 10 15 20 25 30
-40
-60
-80
Cr(VI) (ppm)

θ c=20 days θ c=10 days θ c=5 days θ c=2.5 days

Fig. 2. Cr(VI) toxicity in unacclimatized biomass expressed as proportional reduction of mmðVSSÞ (% inhibition) at various sludge ages
and at S0 =X0 ¼ 10:
3346 A.S. Stasinakis et al. / Water Research 36 (2002) 3341–3349

100
80

Inhibition (%) of µm(OUR)

60
40
20
0
-20 0 5 10 15 20 25 30

-40
-60
-80
-100
Cr(VI) (ppm)

θc=20 days θc=10 days θ c=5 days θc=2.5 days

Fig. 3. Cr(VI) toxicity in acclimatized biomass expressed as proportional reduction of mmðOURÞ (% inhibition) at various sludge ages
and at S0 =X0 ¼ 10:

2 100

1.8
1.6 non-acclimated biomass 80

1.4 acclimated biomass

Inhibition (%)
µm(VSS) (1/d)

60
1.2
1
40 θc=10 days, µm(OUR)
0.8
θc=10 days, µm(VSS)
0.6 θc=5 days, µm(OUR)
20
0.4 θc=5 days, µm(VSS)
0.2
0
0 0 5 10 15 20 25 30
0 5 10 15 20 25 30 Cr(VI) (ppm)
Cr(VI) (ppm)
Fig. 5. Comparison of inhibition on values of mmðOURÞ 2mmðVSSÞ
Fig. 4. Effect of acclimatization on mmðVSSÞ values for sludge in the presence of Cr(VI) for unacclimatized biomass (sludge
age of 5 days (batch tests with So =Xo ¼ 10). age of 5 and 10 days) determined by batch tests conducted at
S0 =X0 ¼ 10:

3). The above effect of sludge age has been reported by In this study, comparison of chromium toxicity
several researchers [31,32,22] and can be attributed to determined via experiments with acclimatized and
the higher amount of extracellular polymers excreted by unacclimatized biomass showed that the acclimatized
activated sludge at long yc that tend to adsorb inhibitors microorganisms are less sensitive in the presence of
[21,24] rendering them unavailable to bacteria. chromium than unacclimatized ones (Fig. 4). This
According to the literature the effect of biomass phenomenon is probably due to the fact that during
acclimatization to hexavalent chromium has been acclimatization, resistant microorganisms are selected
mostly studied by determining the kinetic parameters and/or microorganisms are adapted metabolically to the
in experiments with acclimatized biomass and not in heavy metal concentration. According to the hypothesis
parallel experiments, where acclimatized and unaccli- of metabolic adaptation, the metal ion-damaged en-
matized biomass is used simultaneously [18,19]. Only zymes are replaced by new enzyme synthesis or new
Gokcay and Dilek [33] conducted parallel experiments shunt pathways are created to substitute the inoperative
at Cr(VI) concentrations of 1 and 50 mg l
1 and reported ones [34,18]. The above effect of acclimatization was not
that the effect of Cr(VI) was two times higher in cultures observed at a sludge age of 10 days, where Cr(VI)
seeded with unacclimatized biomass. toxicity appeared to be approximately the same for
 A.S. Stasinakis et al. / Water Research 36 (2002) 3341–3349 3347

100
80
60
Inhibition (%) 40
20
0
-20 0 5 10 15 20 25 30
-40
-60
-80
-100
Cr(VI) (ppm)

θc=20 days, µm(OUR) θc=5 days, µm(OUR)

θc=20 days, µm(VSS) θc=5 days, µm(VSS)

Fig. 6. Comparison of inhibition on values of mmðOURÞ 2mmðVSSÞ in the presence of Cr(VI) for acclimatized biomass (sludge age of 5 and
20 days) determined by batch tests conducted at S0 =X0 ¼ 10:

100
80
60
40
Inhibition (%)

20
0
-20 0 5 10 15 20 25 30
-40
-60
-80
-100
Cr(VI) (ppm)

θ c=20 days, µm(our) θ c=5 days, µm(our) θ c=2.5 days, µm(our)

θ c=20 days, µm(OUR) θ c=5 days, µm(OUR) θ c=2.5 days, µm(OUR)

Fig. 7. Comparison of inhibition on values of mmðOURÞ 2mmðourÞ in the presence of Cr(VI) for acclimatized biomass (sludge age of 2.5, 5
and 20 days) determined by batch tests conducted at S0 =X0 ¼ 10 and S0 =X0 ¼ 1:5:

acclimatized and unacclimatized bacteria. This behavior acclimatized biomass. According to Stasinakis et al.
of acclimatized biomass was attributed to insufficient [11] mmðOURÞ provides information on oxygen consump-
biomass acclimatization period (15 days). tion and substrate oxidation by bacteria, while mmðVSSÞ is
Cr(VI) toxicity evaluated by the OUR ðmmðOURÞ Þ and related to microbial growth. Pollard et al. [13,14]
VSS ðmmðVSSÞ Þ methods is illustrated in Figs. 5 and 6. The underlined the importance of developing novel methods
results revealed that in the case of unacclimatized to describe bacterial growth that can differentiate from
biomass, the OUR method appears to exhibit a higher the bacterial metabolic activities determined by the
negative effect on growth rates than the VSS method, OUR method. Therefore the results of our study
while the opposite was observed in the case of indicate that the presence of Cr(VI) in unacclimatized
3348 A.S. Stasinakis et al. / Water Research 36 (2002) 3341–3349

biomass inhibits more catabolic activities, whereas in Batch experiments conducted at low S0 =X0 appeared
acclimatized biomass Cr(VI) is causing a greater to be a sensitive method for evaluating Cr(VI) toxicity
decrease in bacterial growth. only in cases that acclimatized biomass is used. The
insensitivity of this method for evaluating inhibition in
3.2.3. Comparison of mm determined at low and high unacclimatized biomass is probably due to the short
S0 =X0 ratios contact time between biomass and the toxic compound.
Fig. 7 illustrates that for acclimatized biomass mmðourÞ Determining mm values by OUR, at high and low
values determined at high S0 =X0 ratios ðS0 =X0 ¼ 10Þ are substrate-to-biomass ratios, showed that fast growing
influenced less by Cr(VI) than mmðOURÞ values deter- bacteria appeared to be less sensitive to chromium
mined at low S0 =X0 ratios ðS0 =X0 ¼ 1:5Þ: High S0 =X0 toxicity than the original bacterial culture. Further
ratios tend to support cell multiplication and favor the investigation is needed to evaluate the effect of substrate
bacterial species growing faster. Therefore, kinetic oxidation on Cr(VI) reduction to the less toxic Cr(III),
constants determined at high S0 =X0 reflect the char- which may also affect the evaluation of Cr(VI) toxicity.
acteristics of the culture developed at the end of the Batch experiments conducted with unacclimatized
batch assay and less the original culture. On the other and acclimatized biomass illustrated that the presence
hand, at low S0 =X0 ratio no significant cell replication of Cr(VI) in unacclimatized biomass inhibits more
occurs as storage phenomena prevail and therefore the catabolic activities, whereas in acclimatized biomass
obtained kinetic constants are more representative of the Cr(VI) causes a greater decrease in bacterial growth.
original culture [10,35,11]. Thus the results indicate that
fast growing microorganisms seem to be less sensitive to
hexavalent chromium than the initial activated sludge Acknowledgements
biocenosis.
However, two more alternative explanations should A.S. Stasinakis would like to thank the Greek
be considered. High S0 =X0 ratio could promote the Scholarship Foundation for financial support of this
higher production of biomass at the end of the work. The authors would also like to thank Mrs. Anna
experiment, thus extracellular polymers could be present Vassalou and Mrs. Eustathia Nakou for their valuable
at higher concentrations, leading to a lower bioavail- help during the experiments.
ability of inhibitors. Moreover, it should also be taken
into account that Cr(VI) reduction to the less toxic
Cr(III) may occur during oxidation of organic matter.
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