Science of the Total Environment 412-413 (2011) 380–385

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Science of the Total Environment

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Short Communication

Toxicity of copper(II) ions to microorganisms in biological wastewater

treatment systems
Valeria Ochoa-Herrera a, b,⁎, Glendy León a, Qais Banihani a, Jim A. Field a, Reyes Sierra-Alvarez a
a
Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ 85721-0011, USA
b
Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito, Diego de Robles y Vía Interoceánica S/N, Cumbayá, Ecuador

a r t i c l e i n f o a b s t r a c t

Article history: Copper is an essential element, however, this heavy metal is an inhibitor of microbial activity at relatively low
Received 18 October 2010 concentrations. The objective of this study was to evaluate the inhibitory effect of copper(II) towards various
Received in revised form 16 September 2011 microbial trophic groups responsible for the removal of organic constituents and nutrients in wastewater
Accepted 19 September 2011
treatment processes. The results of the batch bioassays indicated that copper(II) caused severe inhibition
Available online 26 October 2011
of key microbial populations in wastewater treatment systems. Denitrifying bacteria were found to be very
Keywords:
sensitive to the presence of copper(II). The concentrations of copper(II) causing 50% inhibition (IC50) on
Copper the metabolic activity of denitrifiers was 0.95 mg L−1. Copper was also inhibitory to fermentative bacteria,
Microbial toxicity aerobic glucose-degrading heterotrophs, and nitrifying bacteria (IC50 values= 3.5, 4.6 and 26.5 mg L−1, respec-
Nitrification tively). Nonetheless, denitrifying and nitrifying bacteria showed considerable recovery of their metabolic activity
Denitrification after only several days of exposure to high copper levels (up to 25 and 100 mg Cu(II) L−1 for denitrification and
Fermentation nitrification, respectively). The recovery could be due to attenuation of soluble copper or to microbial adaptation.
Activated sludge treatment © 2011 Elsevier B.V. All rights reserved.

1. Introduction Copper is an essential element for all living organisms (Burgess et

The potential impact of heavy metals on the environment, public
health and economy has been a major concern over the last decades.
Heavy metals such as copper, zinc, nickel and cadmium are commonly
present in untreated wastewaters coming from the mining, smelting,
semiconductor, metallurgical, electroplating and metal-finishing indus-
try. The concentrations of copper in liquid discharges vary significantly
depending on the industrial activities. For instance, copper levels as
high as 1550 mg L −1 were reported in mining effluents from the copper
mine “Cerovo” in Serbia (Stankovic et al., 2009). The concentration of
copper in semiconductor effluents is also high, often ranging from 5 to
100 mg L −1 (Sierra-Alvarez et al., 2007). However, copper levels in mu-
nicipal wastewaters are expected to be considerable lower because in-
dustrial effluents will be diluted by wastewaters from residential
sources. In addition, adsorption and precipitation reactions can lead to
removal of copper in biological wastewater treatment systems. A
study conducted by Crane et al. (2010) reported considerably removal
of copper in different types of secondary biological wastewater treat-
ment processes. In bench assays spiked with up to 40 μg Cu L −1, copper
removals of 99, 84 and 47% were obtained for activated sludge, trickling
filters and membrane bioreactors, respectively.
⁎ Corresponding author at: Colegio de Ciencias Biológicas y Ambientales, Universidad
San Francisco de Quito, Diego de Robles y Vía Interoceánica S/N, Cumbayá, Ecuador.
Tel.: + 593 2 2971700; fax: + 593 2 2890070.
E-mail address: vochoa@usfq.edu.ec (V. Ochoa-Herrera).
al., 1999; Cervantes and Gutierrez Corona, 1994); however, high con-
centrations of this heavy metal inhibit cell metabolism. Copper and
copper-containing compounds are widely used as bactericides and
fungicides. The antimicrobial action of copper is believed to result
from the ability of copper ions to chelate sulfhydryl groups, thereby
interfering with the cell proteins or enzymes (Yeager, 1991). Several
studies have been conducted to determine the microbial inhibition
potential of copper during wastewater treatment. The inhibitory effect
of copper to aerobic heterotrophic bacteria and to microorganisms in-
volved in sulfate-reduction and other anaerobic degradation processes,
including acetate- and hydrogen-utilizing methanogens, has been studied
extensively (Ahring and Westermann, 1985; Jalali and Baldwin, 2000;
Karri et al., 2006; Lawrence and McCwq, 1965; Mori et al., 2000;
Mosey and Hughes, 1975; Utgikar et al., 2001). In contrast, information
on the toxicity of this metal to other microbial populations that play key
roles in wastewater treatment, such as nitrifying and denitrifying mi-
croorganisms and glucose fermenting bacteria, is limited.
The concentrations of copper(II) reported to inhibit microbial ac-
tivity vary widely, even for microorganisms in the same trophic
group. As an example, 50% inhibitory levels of copper(II) ranging
from only 0.84 mg L −1 to as much as 200 mg L −1 have been observed
in studies with sulfate reducing bacteria (Jalali and Baldwin, 2000;
Karri et al., 2006; Sani et al., 2001; Utgikar et al., 2001). In the same
manner, a significant variation in the inhibitory levels of copper to
methanogenic microorganisms in anaerobic sludge is found in the
literature, with values ranging several orders of magnitude from
2.2 mg L −1 to as much as 400 mg L −1 (Ahring and Westermann,

0048-9697/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2011.09.072
 V. Ochoa-Herrera et al. / Science of the Total Environment 412-413 (2011) 380–385
1985; Karri et al., 2006; Lawrence and McCwq, 1965; Mori et al., 2000;
Mosey and Hughes, 1975). The significant variation observed in the in-
hibitory levels reported for copper are most likely due to differences in
the experimental conditions utilized in the various assays (e.g., compo-
sition of the medium, pH values, temperature, concentration of copper
ligands such as sulfide, etc) which are known to impact copper specia-
tion and bioavailability.
The objective of this study is to evaluate the toxicity effect of copper
(II) towards various microbial populations involved in the removal of
organic matter and nitrogen nutrient during the biological treatment
of wastewaters. The toxicity bioassays utilized were designed to mini-
mize experimental variation (i.e., pH value, trace element solution, tem-
perature, shaking conditions were the same in all bioassays) in order to
facilitate comparison of the inhibitory levels determined for the various
microbial populations.
2. Materials and methods
2.1. Chemicals
Potassium nitrate and sodium acetate (N99.0% purity) were
obtained from Spectrum Chemicals and Laboratory Products (Gardena,
CA, USA). Copper(II) chloride dehydrate (99.0% ACS grade), sodium ni-
trite (N99.5%) and sulfuric acid (95–98% ACS grade) were purchased
from Sigma Chemicals Co. (St. Louis, MO, USA). Ammonium bicarbonate
was obtained from MP Biomedicals (Solon, OH, USA). Phenol (99.0%
ACS grade) was obtained from EMD chemicals (Gibbstown, NJ, USA).
D-glucose anhydrous was purchased from Mallinckrodt Chemical
(Paris, KY, USA). N2/CO2 gas (80/20, v/v) were delivered from US Air
(Phoenix, AZ, USA). All chemicals were used as received.
2.2. Sludge sources
Two types of anaerobic methanogenic granular sludge (Eerbeek
and Aviko sludges) were evaluated in this study. Eerbeek sludge
was obtained from an industrial anaerobic sludge blanket (UASB) re-
actor treating recycle paper effluent (Industriewater, Eerbeek, The
Netherlands), and the Aviko sludge from a UASB reactor treating po-
tato processing wastewater (Aviko, Steenderen, The Netherlands).
Both inocula were washed and sieved to remove fine particles and
they were stored under nitrogen gas at 4 °C. The content of volatile
suspended solids (VSS) in the Eerbeek and Aviko sludges was 12.9
and 11.5%, respectively.
The inoculum utilized in the nitrification assays, Randolph Park
sludge I, was obtained from the nitrification stage of a full-scale sewage
treatment facility (Randolph Park Wastewater Reclamation Facility,
Tucson, AZ, USA). The VSS content in the sludge was 4.8%. Glucose-
degrading enrichment cultures were obtained under anaerobic and
aerobic conditions using Aviko granular sludge and Randolph Park
sludge II, respectively. The enrichment cultures were developed by suc-
cessive transfer of the culture supernatant to fresh glucose containing
basal medium at a rate of 5% (v/v) upon glucose depletion. The aerobic
sewage sludge, Randolph Park sludge II (0.13% VSS), was obtained from
the aeration tank of the Randolph Park Wastewater Reclamation Facili-
ty. All sludge samples were stored under nitrogen gas at 4 °C.
2.3. Culture media
The composition of the basal mineral medium supplied in the ni-
trification bioassays (BM-1) was (in mg L −1): NaHCO3 (2000), NaH2-
PO4·H2O (1200) and Na2HPO4 (715). The basal mineral medium
employed in the denitrification bioassays (BM-2) contained (in
mg L −1): K2HPO4 (250); (NH4)HCO3 (417) and NaHCO3 (1500). The
basal mineral medium utilized in both the aerobic heterotrophic bio-
assays and fermentation toxicity bioassays (BM-3) contained (in
mg L −1): NH4Cl (280); NaHCO3 (2000); K2HPO4 (250); KH2PO4
381
(2050); CaSO4·2 H2O (10), MgSO4·7 H2O (100) and yeast extract (50).
All media were supplemented with yeast extract (10 mg L−1) and with
1 mL L−1 of trace element solution containing (in mg L−1): H3BO3 (50),
FeCl2·4 H2O (2000), ZnCl2 (50), MnCl2·4 H2O (50), (NH4)6Mo7O24·4
H2O (50), AlCl3·6 H2O (90), CoCl2·6 H2O (2000), NiCl2·6 H2O (50),
CuCl2·2 H2O (30), NaSeO3·5 H2O (100), EDTA (1000), resazurin (200)
and 36% HCl (1 mL L−1). All media were adjusted to pH 7.2 with HCl or
NaOH, as required.
2.4. Batch microbial toxicity assays
The experimental conditions utilized in the various toxicity bioas-
says are summarized in Table 1. Batch bioassays were conducted
using glass serum flasks (165 mL) supplemented with 50 mL of medium,
unless otherwise indicated. The desired amount of copper(II) was
added to flasks using concentrated stock solutions of copper chloride
(CuCl2·2 H2O). The stock solutions were prepared in diluted HCl
(10 mM) to ensure complete solubilization of copper. Flasks lacking
copper were also incubated and served as uninhibited controls. In an-
aerobic and anoxic bioassays, flasks were sealed with butyl rubbers
stoppers and aluminum crimp seals and, subsequently, the headspace
was flushed with N2:CO2 gas (80:20, v/v) to assure anaerobic condi-
tions. The headspace of the aerobic heterotrophic and nitrification bio-
assays was atmospheric air, and it was replenished daily to prevent
oxygen limitations. In all bioassays, the final pH of the medium after ad-
dition of copper stock solution was approximately 7.2 due to the high
buffering capacity of the basal mineral medium.
The maximum specific nitrifying (mg NH4+-consumed g −1
VSS d −1), denitrifying (mg NO3ˉ g −1 VSS d −1) and glucose degrading
(mg glucose-degraded g −1 VSS d −1) activities were calculated from
the slope of the ammonium concentration, nitrate concentration
and glucose consumption; respectively, and biomass concentration
versus time (d), as the mean value of triplicate or duplicate assays.
In each case, the maximum specific activity at a given copper concen-
tration was determined during the time period when the copper-free
control displayed the maximum specific activity. The inhibition ob-
served was calculated as shown below.
 
Inhbitionð%Þ ¼ 100− 100∗
Maximum Specific Activity of the Tested Concentration
Maximum Specific Activity of the Control
The initial concentrations of copper(II) causing 20, 50 and 80% re-
duction in activity compared to an uninhibited control were referred
to as IC20, IC50 and IC80, respectively. These values were calculated by
interpolating in the graph plotting the inhibition observed (%) as a
function of the inhibitor concentration. Unless otherwise indicated,
reported inhibitory concentrations are average values of triplicate as-
says and corresponding standard deviations.
2.5. Analytical methods
Copper(II) in liquid samples was analyzed by inductively coupled
plasma-mass spectrometry (ICP-MS) using an Agilent 7500a. The ana-
lytical system was operated at a Rf power of 1500 W, a plasma gas
flow of 15 L min−1 and a carrier gas flow of 1.2 L min−1. Samples for
copper determination were membrane filtered (0.45 μm) immediately
after sampling and diluted with dilute nitric acid.
Sugars were determined colorimetrically as described elsewhere
(Dubois et al., 1956). Briefly, 0.5 mL of centrifuged sample was trans-
ferred into a test tube and then 0.5 mL of 5% (v/v) phenol and 2.5 mL
of concentrated sulfuric acid was added. The sample was vortexed
and allowed to rest for 7 min. Subsequently, the tube was heated at
45 °C for 20 min. The solution was allowed to cool down and then
the concentration of glucose in the bioassays was determined by
measuring the color intensity of the sample at 490 nm. Calibration
curves containing glucose concentrations ranging from 2.9 to
382 V. Ochoa-Herrera et al. / Science of the Total Environment 412-413 (2011) 380–385

Table 1
Summary of experimental conditions utilized in the various inhibition batch bioassays.

Inhibition bioassaya Substrate Substrate conc. Inoculum conc. Copper(II) conc. Basal mediumb Headspace contentc Monitoring
(g L−1) (g VSS L−1) (g Cu(II) L−1)

Nitrification NH4+ 0.05 0.50 0–100 BM-1 Air Ammonia

Nitrate
Denitrification Acetate 0.62 1.00 0–25 BM-2 N2/CO2 (80/20, v/v) Nitrate
Nitrate 1.01
Aerobic glucose degradation Glucose 1.00 1.50 0–25 BM-3 Air Glucose
Glucose fermentation Glucose 1.00–3.00 1.50 0–25 BM-3 N2/CO2 (80/20, v/v) Glucose
a
All bioassays were incubated in a climate-controlled chamber at 30 ± 2 °C in an orbital shaker (110 rpm).
b
The composition of the basal medium is detailed in Materials and methods section.
c
The headspace pressure in all the bioassays using N2/CO2 or air atmosphere was 102.3 kPa.

14.3 mg L −1 were previously constructed. Blank samples were ana- 3.1. Glucose fermenting bacteria
lyzed to correct for background noise.
Nitrate and nitrite were analyzed by suppressed conductivity ion Copper(II) was inhibitory to glucose-fermenting microorganisms
chromatography using a Dionex 3000 system (Sunnyvale, CA, USA) in anaerobic sludge at very low concentrations (Table 2). A considerable
fitted with a Dionex IonPac AS18 analytical column (4 × 250 mm) decrease in the rate of glucose utilization by the anaerobic cultures with
and an AG18 guard column (4 × 40 mm). The column was maintained increasing copper concentrations in the range of 0–10 mg L −1 was ob-
at 35 °C. The eluent used was 10 mM KOH at a flow rate of served (Fig. 3A). The concentrations of copper(II) causing 50% inhibition
1.0 mL min −1. The injection volume was 25 μL. Before measurement,
all samples were either, centrifuged (10,000 rpm) for 10 min or
passed through a membrane filter (0.45 μm). Ammonium was deter- A
mined using an Orion Thermo combination ion-selective electrode. 120
The pH was determined immediately after sampling with an Orion
model 310 PerpHecT pH-meter with a PerpHecT ROSS glass combina-
100
tion electrode. Volatile suspended solids and other analytical parame-
Activity (% of Control)

ters were determined according to Standard Methods for Examination

of Water and Wastewater (APHA, 1998). 80

3. Results and discussion 60

Fig. 1 shows an illustrative example of the time course of nitrate

40
formation from ammonia for the nitrifying activity assay in presence
of 0, 1, 2.5, 5, 10, 25, 35, 50, 80 and 100 mg copper(II) L −1. The nitri-
fying activities in treatments containing copper(II) were normalized 20
based on the activity of the uninhibited control lacking copper.
Fig. 2A shows the normalized activity of nitrifying microorganism as 0
a function of the initial copper(II) concentration. The nitrifying activities 0 20 40 60 80 100
were calculated during the maximum nitrate formation activity of the Copper (II) (mg/L)
control, approximately 90 to 180 h. The same procedure was utilized
to calculate the microbial activities of the different microorganisms B
evaluated in this study. 120

100
250
Activity (%of Control)

80
200

60
Nitrate (mg/L)

150
40

100
20

50 0
0 5 10 15 20 25
Copper (II) (mg/L)
0
0 30 60 90 120 150 180 Fig. 2. Inhibitory effect of copper(II) on the specific nitrifying activity of a mixed micro-
Time (h) bial culture obtained from the nitrification stage of a full-scale municipal wastewater
treatment plant as measured by nitrate formation (A); and on the denitrifying specific
Fig. 1. Time course of nitrate formation from ammonia by a mixed microbial culture activity of an industrial anaerobic granular sludge as determined by nitrate depletion
obtained from the nitrification stage of a full-scale municipal wastewater treatment (B). The specific nitrifying and denitrifying activities were normalized with respect to
plant in the presence of increasing copper(II) concentrations in (mg L−1): (●) 0, (○) the activity of an uninhibited control. Error bars (shown if larger than the symbols)
1.0, (−) 2.5, (◊) 5.0, (▲) 10.0, (□) 25.0, ( ) 35.0, (■) 50.0, (Δ) 80.0, and (—) 100.0. represent standard deviations of duplicate assays.
 V. Ochoa-Herrera et al. / Science of the Total Environment 412-413 (2011) 380–385 383

Table 2
Inhibitory effect of copper(II) on the key microbial populations in biological wastewater treatment systems. IC20, IC50 and IC80 are the concentrations of copper(II) causing 20, 50
and 80% decrease in the activity of the target microorganisms, respectively.

Substrate Redox Toxicity Inoculuma IC20 IC50 IC80

conditions
(mg/L)

Nitrification
Ammonium Aerobic Activity (NH4+ consumption) Randolph Park I sludge 16.0 26.5 34.0
Activity (nitrate formation)

Denitrification
Nitrate Anoxic Activity (nitrate reduction) Eerbeek granular sludge 0.40 0.95 2.5

Heterotrophic aerobes
Glucose Aerobic Growth Randolph Park II (enrichment culture) 3.2 4.6 6.9

Glucose fermentative bacteria

Glucose Anaerobic Growth Aviko (enrichment culture) 0.60 1.40 2.90
Glucose Anaerobic Activity Aviko granular sludge 1.20 3.50 6.80
a
All inocula used consisted of dispersed biomass unless indicated.

of glucose utilization by an anaerobic enrichment culture and by anaer-
obic granular sludge (Aviko) were 1.4 and 3.5 mg L −1, respectively.
These results indicate that the growth of fermentative microorganisms
A magnitude (6.5 mg L−1) of those determined in this study. Two studies
100
Activity (% of Control)
was somewhat more affected by the presence of copper(II) compared to
their metabolic activity. The IC50 values of copper towards fermentative
bacteria reported in the literature vary substantially. Lin and Shei
(2008) evaluated the effect of copper on the fermentative hydrogen
production using sucrose and found an IC50 value in the same order of
evaluating the impact of copper on H2 production from sucrose (Li and
Fang, 2007) and one from dairy wastewater (Yu and Fang, 2001) report
IC50 values one order of magnitude higher (65 and 30 mg L −1, respec-

80 tively). Zheng and Yu (2004) found a considerably higher IC50 value

(350 mg L −1) in assays evaluating the effect of copper on the conver-
sion of a dairy wastewaster to H2. Fermentative bacteria appear be
60
more susceptible to the inhibitory effects of copper compared to other
microorganisms involved in the degradation of organic matter in anaer-
40 obic environments such as methanogens and sulfate-reducing bacteria.
IC50 values reported for soluble copper in studies with both microbial
communities are often one or more orders of magnitude higher com-
20
pared to those determined here for fermentative bacteria (Ahring and
Westermann, 1985; Jalali and Baldwin, 2000; Karri et al., 2006; Law-
0 rence and McCwq, 1965; Mori et al., 2000; Mosey and Hughes, 1975;
0 5 10 15 20 25 30 Utgikar et al., 2001; Utgikar et al., 2003).
Copper (II) (mg/L) During the glucose fermentation toxicity assays, soluble copper(II)
levels were observed to decline rapidly. Fig. 4 shows the concentra-
B tions of soluble copper(II) determined in the various treatments at
120 the beginning and end of the experiment. As an example, only 0.60
and 4.4 mg Cu(II) L −1 were detected in the treatments initially
100 amended with 2 and 50 mg Cu(II) L −1, respectively. The mechanisms
responsible for the removal of copper are probably due to precipita-
Activity (% of Control)

tion and adsorption to sludge (Artola et al., 1997; Karri et al., 2006). Bi-
80
carbonate, phosphate and hydroxyl groups are available in the mineral
medium; therefore, chemical precipitation of copper can occur due to
60 the low solubility products of CuCO3, Cu(OH)2, and Cu3(PO4)2 which
are 2.5× 10−10, 2.2× 10−20, and 1.4 × 10 −37, respectively (Stumm and
40 Morgan, 1981). Copper can also form highly insoluble precipitates
with sulfides (solubility product of CuS = 8 × 10 −37, Stumm and
20
Morgan, 1981), a process that is responsible for (partial) copper detox-
ification in anaerobic bioreactors. However, formation of CuS is not
expected because sulfate and sulfide salts were excluded from the
0
0 5 10 15 20 25 30 basal mineral medium in all bioassays to prevent precipitation of copper
Copper (II) (mg/L) with (biogenic) sulfide. Soluble copper concentrations can also be re-
duced by adsorption of copper onto the biomass (Hu et al., 2004;
Fig. 3. Inhibitory effect of copper(II) on glucose fermentation by different mixed anaerobic White et al., 1995), and several studies have shown that the inhibitory
cultures (A), and on the specific glucose-degrading activity of an enrichment culture effect of copper decreased with increasing biomass (expressed as vola-
obtained from aerobic wastewater treatment sludge (B). Legends for panel A: (■) anaero- tile solid concentration) to copper ratios (Braam and Kkapwijk, 1981;
bic granular sludge, and (●) an anaerobic glucose-degrading enrichment culture. The
glucose-degrading specific activities were normalized with respect to the control in
Pamukoglu and Kargi, 2007). Adsorption and precipitation reactions
which no contaminant was added. Error bars (shown if larger than the symbols) represent can lead to considerable removal of copper in biological wastewater
standard deviations of triplicate assays. treatment systems and aquatic environments. A review paper reported
384 V. Ochoa-Herrera et al. / Science of the Total Environment 412-413 (2011) 380–385
60
50
Copper (II) (mg/L)
40
30
20
10
0
Bioassay Treatment
Fig. 4. Concentration of soluble copper in the various treatments of the bioassays con-
ducted to evaluate the inhibitory effect of copper(II) on glucose fermentation. Concen-
trations supplied (empty blocks); concentrations determined at the end of the
bioassays (filled blocks).
33 to 98% copper removal during activated sludge treatment (Lester,
1983).
3.2. Nitrification and denitrification
Copper(II) was found to inhibit nitrifying microorganisms in aero-
bic sewage sludge. Shaken batch bioassays showed a significant de-
crease in the rate of ammonia consumption and nitrate formation
with increasing copper(II) levels (Fig. 2A), with near complete inhibi-
tion of nitrification occurring at added copper concentrations of
50 mg L −1 and higher. The IC20, IC50 and IC80, values determined are
summarized in Table 2. Interestingly, nitrifying bacteria acclimated
rapidly to copper(II), and the metabolic activity of treatments that
were initially inhibited by copper increased substantially after 140 h
of exposure (Fig. 1). Assays with copper concentrations ranging
from 50 to 100 mg L −1 were completely inhibited during the initial
120 to 140 h, but their activity increased sharply thereafter reaching
levels close to those in the uninhibited control. This recovery could
be attributed to a physiological adaptation of the predominant nitrifying
strains to copper(II) or a shift in the microbial population to another
strain that was more tolerant to copper(II). Gradual decrease in the con-
centration of copper present in the medium might be another reason
contributing to the microbial adaptation to toxicity. Literature studies
have demonstrated that the toxicity of metals is related to the free-ion
concentration in solution rather than to the total metal concentration
(Campbell, 1995). Similarly to other toxicity bioassays a considerable
decrease in the concentration of soluble copper(II) was determined. An-
alyses of soluble copper(II) performed for all the nitrification treatments
at the end of the experiment confirmed that the Cu concentrations were
greatly reduced to values of only 0.25 to 0.51 mg L−1.
A noteworthy observation is that low concentrations of copper(II)
(b10 mg L −1) were found to have a moderate stimulatory effect on
the microbial oxidation of ammonia to nitrate. Stimulation of nitrifi-
cation has been reported earlier in the literature. Barber and Stuckey
(2000) observed that nitrification efficiency was improved greatly
(68% increase) upon addition of copper (32 mg L −1) to the influent
of a bioreactor. However, continued supplementation of copper virtual-
ly stopped nitrification after four weeks. Nitrification resumed soon
after copper was removed from the wastewater. Copper is required
for nitrification, since a number of enzymes involved in electron trans-
fer chains as well as the primary enzyme, ammonia mono-oxygenase,
contain copper (Jones, 1982; Painter, 1970).
Copper was found to be a potential inhibitor on the activity of
denitrifying microorganisms (Fig. 2B). The IC20, IC50 and IC80 values
determined were 0.40, 0.95 and 2.50 mg Cu(II) L −1, respectively.
However, similarly to the nitrification bioassays, recovery of the meta-
bolic activity was evident after 49 to 60 h of incubation for all the treat-
ments containing less than 25 mg Cu(II) L −1 probably due to a shift in
the structure of microbial population resulting in the dominance of
denitrifying species which were less sensitive to copper. Again, gradual
precipitation of copper(II) could be another possible explanation for the
observed behavior.
Few literature studies have considered the inhibitory effect of cop-
per towards denitrifying microorganisms. The only study found
reported no inhibition of denitrifying biomass in a suspended-
growth biological system by copper concentrations of up to
26 mg L −1 (Kamath et al., 1991). Copper is a required trace element
for denitrification. Several enzymes involved in denitrification, e.g.,
some nitrite reductases and nitrous oxide reductases, are copper-
dependent enzymes (Stouthamer, 1991; Vanniel et al., 1992). Based
on these results, it can be concluded that denitrifiers will be more sus-
ceptible to the inhibitory effect of copper(II) as compared to nitrifiers.
However, in both cases, a significant recovery of the microbial popu-
lation will be expected during the biological processes of nitrogen re-
moval from wastewater.
3.3. Aerobic heterotrophs
Copper(II) was found to inhibit glucose degradation by aerobic
heterotrophic microorganisms in activated sludge at low concentra-
tions. The specific rate of glucose utilization (expressed as percent
of the rate calculated for the uninhibited control) determined in the
various assays as a function of the copper(II) concentration is
shown in Fig. 3B. The IC20, IC50 and IC80 values determined were 3.2,
4.6 and 6.9 mg L −1. Analysis of soluble copper at the beginning and
end of the experiment revealed a considerable decrease in the con-
centration of the toxicant with time (results not shown). As an exam-
ple, only 4.6 and 5.3 mg Cu(II) L −1 were detected in the treatment
initially amended with 10 and 25 mg Cu(II) L −1, respectively. There
are numerous literature reports investigating the toxic effects of Cu(II)
ions against aerobic heterotrophs in activated sludge. Inhibition of respi-
ration and microbial growth has been reported at total copper concen-
trations ranging from 10 to 50 mg L−1 (Almanza et al., 1996; Braam
and Kkapwijk, 1981; Cabrero et al., 1998; Kumarc, 2009; Neumegen et
al., 2005). Some of these concentrations are higher compared to those
determined in our study.
Taken together the results of this study demonstrate that soluble
copper can potentially be a severe inhibitor of various microbial
populations involved in the removal of organic matter (i.e., fermenta-
tive bacteria, aerobic heterotrophs) and nitrogen nutrient removal
(i.e., nitrifying and denitrifying bacteria) during biological wastewa-
ter treatment. However, the inhibitory effect of Cu(II) is limited by
precipitation reactions with anions common in aerobic and/or anaero-
bic treatment systems such as carbonates, sulfides, and hydroxides.
Our results also indicated a considerable ability of nitrifying and denitri-
fying bacteria to adapt to the inhibitory effects of copper ions.
4. Conclusions
The results of this study confirm that copper(II) is inhibitory to-
wards a wide variety of microbial populations, including fermentative
bacteria, aerobic heterotrophs, as well as denitrifying and nitrifying
bacteria, at concentrations commonly found in industrial effluents.
However, the inhibitory impact of copper on denitrifying and nitrifying
bacteria decreased considerably with exposure time, probably due to
attenuation of soluble copper or microbial adaptation. Such recovery
was not evident in assays with glucose fermenters. In order to prevent
microbial inhibition of the highly susceptible fermenters by copper,
measures to reduce the concentration of this ion such as precipitation
and wastewater dilution might be required during the biological treat-
ment of copper-containing streams.
 V. Ochoa-Herrera et al. / Science of the Total Environment 412-413 (2011) 380–385
Acknowledgments
This study was conducted with financial support from the Semi-
conductor Research Corporation/Sematech Engineering Research
Center for Environmentally Benign Semiconductor Manufacturing.
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