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Heavy metal removal in anaerobic semi-continuous stirred

tank reactors by a consortium of sulfate-reducing bacteria
Hoa T.Q. Kieu*, Elizabeth Muller, Harald Horn
Institute of Water Quality Control, Technische Universitat Munchen, Am Coulombwall, 85748 Garching, Germany

article info

abstract

Article history:

Removal of heavy metals by an enriched consortium of sulfate-reducing bacteria (SRB) was

Received 9 February 2011

evaluated through the abundance of SRB, sulfate reduction, sulfide production and heavy

Received in revised form

metal precipitation. Five parallel anaerobic semi-continuous stirred tank reactors (CSTR,

17 April 2011

V 2 L) (referred as R1eR5) were fed with synthetic wastewater containing mixtures of

Accepted 23 April 2011

Cu2, Zn2, Ni2, and Cr6 in the concentrations of 30, 60, 90, 120, and 150 mg L1 of each

Available online 11 May 2011

metal and operated with a hydraulic retention time of 20 days for 12 weeks. The loading
rates of each metal in R1eR5 were 1.5, 3, 4.5, 6, and 7.5 mg L1 d1, respectively. The results

Keywords:

showed that there was no inhibition of SRB growth and that heavy metal removal effi-

Sulfate-reducing bacteria (SRB)

ciencies of 94e100% for Cu2, Zn2, Ni2, and Cr6 were achieved in R1eR3 throughout the

Heavy metal removal

experiment and in R4 during the first 8 weeks. The toxic effect of heavy metals on the SRB

Continuous stirred tank reactor

consortium was revealed in R5, in which no SRB could survive and almost no heavy metal

(CSTR)

precipitation was detected after four weeks of operation.

Fluorescent in situ hybridization

2011 Elsevier Ltd. All rights reserved.

(FISH)

1.

Introduction

The environmental pollution caused by wastewaters containing high concentrations of dissolved heavy metals and
low pH from mining and industrial processing (e.g. metallurgical, electronic, electroplating and metal finishing industries)
negatively impacts to living organisms as well as humans. The
toxic effects of heavy metals include ion displacement and/or
substitution of essential ions from cellular sites and blocking
of functional groups of important molecules, e.g. enzymes,
polynucleotides, and essential nutrient transport systems.
This results in denaturation and inactivation of enzymes and
disruption of cell organelle membrane integrity, as well as
damage to the structure of DNA, nerves, livers and bones (Sani
et al., 2001). Unlike organic contaminants, which can be

degraded into harmless chemical species, heavy metals

cannot be degraded. However, they can be transformed from
mobile and toxic forms into their stable immobile and less
toxic forms (Uhrie et al., 1996; Beyenal and Lewandowski,
2004). Many methods have been used for treatment of heavy
metal contaminated wastewaters. Among them, the classical
physicochemical methods were widely applied (e.g. precipitation, absorption, ion exchange and complex formation).
Despite effective treatment, these methods are expensive and
generate large amounts of residual sludge (Gallegos-Garcia
et al., 2009; Tekerlekopoulou et al., 2010). Therefore, immobilization of heavy metals through microbial mediated reduction and precipitation is now of considerable interest.
Especially, metal sulfide precipitation by SRB has promise as
an attractive alternative over physico-chemical and other

Abbreviations: SRB, sulfate-reducing bacteria; CSTR, continuous stirred tank reactor; FISH, fluorescent in situ hybridization.
* Corresponding author. Tel.: 49 0 89 289 13714; fax: 49 0 89 289 13718.
E-mail addresses: h.kieu@bv.tu-muenchen.de, kieuthiquynhhoa@gmail.com (H.T.Q. Kieu).
0043-1354/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2011.04.043

3864

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methods. This method involves two stages: (i) Sulfatereducing bacteria (SRB), under anaerobic conditions, oxidize
simple organic compounds (e.g. lactate, acetate, propionate,
butyrate, etc.) by utilizing sulfate as an electron acceptor and
generating hydrogen sulfide and bicarbonate ion (Eq. (1)), and
(ii) the biologically produced hydrogen sulfide reacts with
dissolved heavy metals such as Cu, Zn, and Ni to form insoluble metal sulfide precipitates (Eq. (2) (Hulshof et al., 2006;
Neculita et al., 2007).

2CH2 O SO2
4 /H2 S 2HCO3

(1)

Me2 H2 S/MeSY 2H Me Metal

(2)

SRB are able to reduce indirectly Cr via the production of

H2S (Eq. (3)) (Chang and Kim, 2007; Neculita et al., 2007) and by
a direct enzymatic process involving hydrogenases and c3
cytochromes (Lovley and Phillips, 1994; Goulhen et al., 2006).
3HS 2Cr6 /3So 2Cr3 Y 3H

(3)

In comparison with chemical precipitation of metals as

hydroxides or oxyhydroxides heavy metal precipitation by
using biological sulfide has advantages which include low
amounts of residual sludge and more important a lower
solubility of the produced sulfides even at acidic pH, highly
reactive efficiency, and cost effectiveness (Tsukamoto et al.,
2004; Gallegos-Garcia et al., 2009). This method not only
eliminates toxic heavy metals from heavy metal contaminated wastewater, but also enables the recovery of valuable
metals as metallic sulfide (Gallegos-Garcia et al., 2009).
The metal resistance of microorganisms varies with the
species by developing a variety of specific resistance mechanisms such as metal exclusion by permeability barrier, active
transport of the metal away from the cell, intracellular
sequestration of the metal by protein binding, extracellular
sequestration, enzymatic detoxification of the metal to a less
toxic form, and reduction in metal sensitivity of cellular
targets (Bruins et al., 2000). In addition, the ability of heavy
metal resistance of organisms is also dependent on the
mobility, bioavailability, and toxicological effect of each heavy
metal.
Heavy metal treatment using anaerobic sulfate reduction is
influenced by a variety of parameters such as electron donor,
pH, temperature, sulfate concentration, and heavy metal
species. Therefore, the use of an active treatment method as
a sulfidogenic bioreactor for heavy metal removal is probably
preferable to passive treatment. The advantage of this method
is the ease of control during the treatment process resulting in
the permanent removal of heavy metals as compared to
passive process.
In recent years, heavy metal removal by sulfate reduction
has been studied in various bioreactor-types such as continuously stirred tank reactor (CSTR) (Nagpal et al., 2000;
Sahinkaya, 2009), upflow anaerobic sludge blanket reactor
(UASB) (Lenz et al., 2008), off-line sulfidogenic bioreactors
(Goncalves et al., 2007), fixed bed reactor (FBR) (Kaksonen
et al., 2003; Viggi et al., 2010), and permeable reactive
barriers (PRB) (Bartzas et al., 2006).
The aim of the present study was to investigate the heavy
metal removal efficiency of selected SRB consortium in five

parallel semi-continuous stirred tank reactors spiked with

different concentrations of heavy metal mixtures (Cu2, Ni2,
Zn2, and Cr6).

2.

Materials and methods

2.1.

Inoculum

A heavy metal tolerant SRB consortium obtained from heavy

metal contaminated sediment in Tong Xa, a settlement known
for bronze, iron casting and electroplating in Nam Dinh, Vietnam was used as an inoculum. Element composition measured
in the water phase of the sample used as the inoculum in this
study comprised of 82 mg L1 total Cr, 64 mg L1 Ni2, 76 mg L1
total Fe, 18.5 mg L1 Zn2, 38 mg L1 Cu2, 4 mg L1 Mn2; and
202 mg L1 SO42-. This culture was cultivated under anaerobic
condition using Postgates medium B (Postgate, 1984). All
procedures during preparation of the medium and cultivation
were performed according to the modified Hungates method
for anaerobes (Miller and Wolin, 1974). To enrich the bacteria
number the cultivation step was repeated three times before
inoculating the bioreactors.

2.2.

Experimental set-up

The schematic diagram of semi-continuous stirred tank

reactor is present in Fig. 1

2.2.1.

Bioreactors

The experiments were carried out in five glass anaerobic semicontinuous stirred tank reactors (CSTR, V 2 L). This reactor
type was selected because of making the concentration
uniform throughout the reactor maintains the culture in
a constant average physiological state. The reactors were kept
at 30  C in a heated water bath and mixed by magnetic stirrers
at a speed of 400 rpm. All reactors were soaked in a 3 M HNO3
solution for 72 h and rinsed with de-ionized before use to
avoid metal contamination.

2.2.2.

Feeding tanks

Synthetic wastewater (see below) was prepared aseptically

every week to avoid the contamination and then fed continuously at the top of the bioreactors by a peristaltic pump
(Ismatec SA, Zuerich, Switzerland) with a volumetric flow rate
of 100 mL d1.
To maintain the anaerobic condition, feeding tanks were
purged with filter sterilized nitrogen gas (0.22 mm). Gas
produced during the treatment process was trapped by gas
collection bags.

2.2.3.

Synthetic wastewater composition

Most heavy metal contaminated wastewater contains low

concentration of sulfate ions and relatively little dissolved
organic carbon. Thus, synthetic wastewater was used to make
the heavy metal removal process efficient by supplementing
an adequate carbon and energy source for SRB. The composition of the synthetic wastewater was prepared as follows:
KH2PO4, 0.5 g L1; NH4Cl, 1.0 g L1; Na2SO4, 3.7 g L1; Sodium
lactate, 4.42 g L1; and Trisodium citrate, 0.3 g L1. The

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Fig. 1 e Schematic diagram of semi-continuous stirred tank reactor.

synthetic wastewater was not supplemented with Fe2 and

reducing agents such as ascorbic acid, sodium thioglycolate,
Na2S to allow the valuation of the precipitation of other metals
under investigation. Trisodium citrate was added to prevent
any initial metal precipitation. The pH was adjusted to
6.0  0.2 using HCl and NaOH. For continuous operation,
synthetic wastewater was spiked with a mixture of Cu2, Ni2,
Zn2, and Cr6 in different concentrations as shown in Table 1.
Solution for each heavy metal was prepared from the dissolution of chloride salt for Cu2, Ni2, Zn2 and potassium
dichromate (K2Cr2O7) for Cr6 and then sterilized by
membrane filtration (0.22 mm).

as electron acceptor and donor, respectively, without heavy

metals. After pre-incubation (start of the experiment), five
reactors were fed continuously with synthetic wastewater
containing mixtures of Cu2, Ni2, Zn2, and Cr6 in the
influent concentrations of 30, 60, 90, 120 and 150 mg L1 each,
respectively. Reactor 1 (R1) was spiked with the loading rate of
1.5 mg L1 d1 each metal. The loading rate of each metal was
increased up to 3, 4.5, 6 and 7.5 mg L1 d1 for reactor 2 (R2), 3
(R3), 4 (R4), and 5 (R5), respectively. The reactors were operated
semi-continuously with a hydraulic retention time (HRT) of 20
days for 12 weeks, with the exception of R5 which was only
operated for 6 weeks (Table 1).

2.3.

2.4.

Experimental procedure

The bioreactors were first inoculated with 10% (v/v) of the

enriched SRB consortium containing 1  108 cells mL1 and
incubated for 9 days at batch operating conditions using
synthetic wastewater containing sulfate and sodium lactate

Analytical methods

Samples were taken weekly to measure sulfate, dissolve

sulfide, dissolved heavy metals, and pH according to standard
methods (APHA, 1998). Dissolved sulfide and pH were immediately measured after collection. For sulfate and dissolved

Table 1 e Reactor operating characteristics.

Reactor

Conc. of each heavy metala

spiked in synthetic wastewater (mg L1)

Loading rate of each heavy metala

(mg L1 d1)

Total time of operation

(weeks)

30
60
90
120
150

1.5
3.0
4.5
6.0
7.5

12
12
12
12
6

R1
R2
R3
R4
R5
a Cu2, Zn2, Ni2 and Cr6.

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heavy metal analyses the samples were filtered through

0.45 mm cellulose nitrate - membrane filters (Germany) before
measuring. Dissolved sulfide, sulfate and Cr6 were analyzed
by using Dr. Lange kit LCK-653, LCK-153, and LCK- 313,
respectively. For the Dr. Lange analyses, a spectrophotometer
Dr. 2800 was used (Hach Lange GMBH, Germany). The
concentrations of Cu2, Zn2, and Ni2 were determined by an
atomic absorption spectrophotometer (AAS) after acidifying
with concentrated nitric acid (pH < 2) to prevent metal
precipitation and adsorption to surfaces.

2.4.1.

EDS analysis

Qualitative analysis of precipitates was realized by energy

dispersive spectrometry (EDS) analysis using a Leica/Cambridge, model StereoScan 360. The precipitates were obtained
after filtering the effluent sample of R2 (3 mg L1 d1) through
0.45 mm cellulose nitrate - membrane filters (Germany). The
filter paper was dried in an oven for 2 h at 105  C and then thin
coated by Au for EDS analysis.

2.4.2. Total cell counts and fluorescent in situ hybridization

(FISH)
The abundance of SRB was estimated by total cell count using
40 , 60 diamidino-2-phenylindole (DAPI) staining and FISH
technique using 16S rRNA-target oligonucleotide probe
(SRB385) labeled at the 50 end with indocarbocyanine (Cy3)
reactive fluorescent dye. All procedures such as fixation of
samples by paraformaldehyde and EtOH, DAPI staining,
drying and washing were performed using modified standard
procedure described previously (Amann et al., 1995). Hybridized and DAPI stained cells were visually detected by using
Axioplan epifluorescence microscope (Carl Zeiss, Jena,
Germany). Samples of R1-R4 were collected at the start, after
two, six, and 11 weeks and of R5 at the start, after one and four
weeks and immediately fixed with 4% (w/v) paraformaldehyde
(PFA) and stored at 20  C until analysis.

3.

Results

The obtained results showed that Cu2, Zn2, Ni2, and Cr6
were removed effectively throughout the experiment in R1,
R2, and R3 with the heavy metal loading rate of 1.5, 3, and
4.5 mg L1 d1, respectively. The behavior of the SRB consortium in these three reactors was similar and marked by high
levels of heavy metal removal, sulfate reduction, and sulfide
production. However, the inhibition of the SRB consortium
began to be detected in R4 (6 mg L1 d1) from week 9 onwards.
A toxic effect on the SRB consortium was observed in R5
(7.5 mg L1 d1). No growth of SRB and almost no heavy metal
precipitation were detected after four weeks of the experiment. Therefore, the results of sulfate reduction, sulfide
production and heavy metal removal obtained from R2, R4,
and R5 were shown representatively in Fig. 2.

3.1.

Heavy metal removal

Heavy metal removal efficiencies of 96e100% for Cu2,

94e100% for Zn2 and Ni2, and 96e100% for Cr6 were achieved in R1 (1.5 mg L1 d1), R2 (3 mg L1 d1), and R3
(4.5 mg L1 d1) during 12 weeks of operation. The final heavy

metal concentrations for all experiments are summarized in

Table 2. Furthermore, the results for R2, R4 and R5 are shown
in Fig. 2. Although Cu2, Zn2, Ni2, and Cr6 were also
removed effectively (98e100%) in R4 (6 mg L1 d1) during the
first 8 weeks, the decrease in heavy metal removal was
observed from week 9 (91e97%). The removal of heavy metals
in R5 (7.5 mg L1 d1) was significantly lower than the 98e100%
achieved during the first week to 78e91% at the second week
and no heavy metal precipitation was detected in the effluent
of R5 after four weeks of operation.

3.2.
Effect of heavy metals on sulfate reduction and
sulfide production
Table 2 displays the final concentrations achieved in all five
reactors at the end of operation. The production of sulfide
correlates nicely with the removal of heavy metals. Sulfate was
converted by about 43e67% of initial concentration to dissolved
sulfide of 145e310 mg L1 in R1- R3 (1.5e4.5 mg L1 d1)
throughout the experiment (Fig. 2b). In R4 (6 mg L1 d1) this high
sulfide production could only be observed within the first 8
weeks of experiment. After that time point only 17e39% of initial
sulfate concentration was reduced in R4 (Fig. 2d). This led to
a gradual decrease of dissolved sulfide in R4 with a sulfide
concentration of 26 mg L1 at the end of experiment (Fig. 2d). In
R5 only 33% of the initial sulfate concentration was reduced
(7.5 mg L1 d1) in the first week. At the end of experiment R5
nearly no sulfate reducing activity has been observed (see Fig. 2f).
The results indicated that the efficiency of sulfate reduction and
sulfide production greatly decreased with the increase of heavy
metal loading rate up to 6 (R4) and 7.5 mg L1 d1 (R5).

3.3.

Sulfur balance

A sulfur balance was estimated from an average value of the

obtained results of R2 (heavy metal loading 3 mg l1 d1)
throughout 12 weeks of operation. The balance was only done
for one reactor to check the general plausibility of the measured
sulfate and sulfide concentrations. The balance was based on
elemental sulfur (S) by assuming that the total initial sulfur as
1
sulfate SO2
4 (w762 mg L ) was converted partly to total dis2

solved sulfide (S HS dissolved H2S). Therefore, the total
final sulfur existing in R2 effluent includes unconsumed sulfate
(w305 mg L1) and generated sulfide (w269 mg L1). The percent
ratio of total final sulfur to total initial sulfur was only 75%. The
difference between total initial and final sulfur might be
explained as follows: (i) The loss of w48 mg L1 (w6.2%) of sulfur
for the precipitation of Cu2, Zn2, Ni2, and Cr6 in the R2 (ii)
The loss of sulfur as volatile sulfide through air oxidation in
transferring samples and diffusion of H2S gas.

3.4.
Effect of heavy metals on sulfate-reducing bacteria
population
The hybridized positive cells detected by FISH using the specific
probe for SRB (SRB385) and total DAPI stained cells of the
enriched sulfidogenic consortium were estimated in all five
reactors. The three representative reactors R2, R4, and R5 are
shown in Fig. 3. In general the abundance of SRB decreased with
increasing heavy metal load. The ratio of SRB detected by FISH to

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2
1.8

Cu2+

Zn2+

1.6

Ni2+

Cr6+

350

2500

300

2000

250

1.4
1.2

200

1500

0.8

150

1000

0.6

100

Sulfide
Sulfate

0.4

50

0.2
0

0
0

500

5
6
7
Time (weeks)

10

11

12

0
Inf. 0

10 11 12

Time (weeks)

Cu2+
Ni2+

4.5
4

Zn2+
Cr6+

2500

300
250

3.5

2000

200
1500

150

2.5

1000

100

1.5
1

Sulfide
Sulfate

50

0.5
0

500
0

0
0

10

11

12

Inf. 0

Time (weeks)

120

Cu2+

Zn2+

Ni2+

Cr6+

10 11 12

Time (weeks)

160
140

300

2500

250

100

2000

200
1500

80

150

60

1000
100

40

Sulfide
Sulfate

50

20

500

0
Inf.

Time (weeks)

Time (weeks)

Fig. 2 e Residual concentrations of heavy metals (Cu2D, Zn2D, Ni2D, and Cr6D), sulfate, and sulfide in R2 (3 mg LL1 dL1),
R4 (6 mg LL1 dL1) and R5 (7.5 mg LL1 dL1) with time.

the total cell counts was 58% at the start. In R2 the abundance
ratio of SRB increased from 58% at the start to 83e84%. In R3
a similar behavior has been detected. The SRB achieved a fraction of 83e86% at the end of the experiment. As can already been
observed from the heavy metal concentrations (see Fig. 2c) the
abundance ratio in R4 shifted from 58% at the start to 79% after 2
weeks, 63% after 6 and only 14% after 11 weeks of operation.
Obviously R4 has been operated with transient heavy metal load
which more and more inhibits the activity of SRB.
R5 finally shows an immediate inhibition. The SRB abundance significantly decreased from 58% at the start to 30%
after one week; no positive hybridized cell was detected by
specific probe for SRB (SRB385) after four weeks of operation.
In addition, a distinct biomass loss through the significant

decrease of the total cell counts from 1.3  108 cell mL1 at the
start to 3.5  104 cell mL1 after four weeks of experiment was
observed in R5. FISH results are consistent with the obtained
results of sulfate reduction, dissolve sulfide production and
heavy metal removal, indicated that SRB played a key role in
heavy metal removal.

3.5.

Effect of heavy metals on pH value

The effects of heavy metals on the SRB consortium were also

observed by the change of pH value (Fig. 4). The pH increased
from an initial value of 6 in the influent to more than 7 at the
start of continuous operation (after 9 day pre-incubation) and
in the effluent of R1eR3 (1.5e4.5 mg L1 d1) throughout the

3868

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Table 2 e Dissolved metal concentrations in the effluent

at the end of the experiments.

7.6

Reactor

7.2
pH value

7.4

1

1

experiment as well as in the effluent of R4 (6 mg L d )

during the first seven weeks. However, the effluent pH of R4
dropped gradually to between 6.91 and 6.58 toward the end of
the experiment. The effluent pHs of R5 (7.5 mg L1 d1) were
more than 7 at only during the first two weeks and dropped
gradually to 6.88 after three weeks and to 6.55 after six weeks
of operation.

3.6.

Qualitative EDS analysis

Qualitative EDS analysis of the precipitate of R2 (3 mg L1 d1)

experiment was performed. The precipitates as CuS, ZnS, and
NiS were confirmed by the EDS spectrum with strong peaks of
copper, zinc, nickel and sulfur shown in Fig. 5.

4.

Discussion

The impact of heavy metals on microorganisms is classified as

toxic (causing death) and/or inhibitory (causing a reduction in
metabolic activity). Toxic concentration is the lowest initial
dissolved metal concentrations at which no bacterial growth
1.00E+09
1.00E+08
1.00E+07

Cells mL

-1

1.00E+06

R1
R2
R3
R4
R5

6.6
6.4
6.2
6

12

280
281
265
26.3
0

11

834
917
902
1860
2200

0.1
0.24
0.55
1.25
135

10

0.36
0.4
0.5
3.5
138

0.3
0.3
0.6
1.9
141

<0.2
0.26
0.25
1.3
135

6.8

S2

SO42

Cr6

Ni2

Zn2

Cu2

In
f.
st
ar
t

R1
R2
R3
R4
R5

Dissolved heavy metal, sulfate and sulfide

concentrations in the effluent of R1-R4 after
12 weeks and of R5 after 6 weeks (mg L1)

Time (weeks)

Fig. 4 e The changes of pH value in the effluent of R1-R5

(1.5e7.5 mg LL1 dL1) with time.

is observed. The obtained results of this study showed that

heavy metal mixtures (Cu2, Zn2, Ni2 and Cr6) were
removed effectively (94e100%) without any inhibition to the
growth of SRB throughout the experiment in R1eR3 with
heavy metal loading rate ranging from 1.5 to 4.5 mg L1 d1.
However, SRB abundance and heavy metal removal began to
decrease at heavy loading rate of 6 mg L1 d1 after 8 weeks of
experiment (91e97%). A toxic effect on the growth of SRB that
resulted in failure of hybridized positive cell detection and
heavy metal precipitation was observed at a heavy metal
loading rate of 7.5 mg L1 d1 (R5). The heavy metal loading
rate of 6 mg L1 d1 (R4) and 7.5 mg L1 d1 (R5) therefore are
considered to be the inhibitory and toxic concentrations to the
SRB consortium in the present study, respectively. The toxic
concentrations of heavy metals to single SRB species or SRB
consortia ranging from a few mg L1 to as much as one
hundred mg L1 were reported by other authors. A Cu2
concentration of 1.92 mg L1 causing death of organism was
shown by Sani et al., 2001 who used a single SRB species
(Desulfovibrio desulfuricans) and a specific medium to prevent
metal ions from abiotic precipitation. Lack of growth of SRB
consortium at 12 mg L1 of Cu2 and 20 mg L1 of Zn2 was
shown by Utgikar et al., 2001. Hao et al., 1994 reported that the
toxic concentrations of individual heavy metal for SRB

1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01

DAPI
SRB385

1.00E+00
Start R2-2 R2-6 R2-11 R4-2 R4-6 R4-11 R5-1 R5-4
Samples

Fig. 3 e Relative number of positive hybridized cells

detected by specific probe for sulfate-reducing bacteria
(SRB385) and total DAPI-stained cells in three
representative anaerobic semi-continuous bioreactors
(referred as R2, R4, and R5) with heavy metal (Cu2D, Ni2D,
Zn2D, and Cr6D) loading rate of 3, 6, and 7.5 mg LL1 dL1,
respectively. Samples of R2 and R4 were analyzed after L2,
L6, and L11 weeks and of R5 after L1 and L4 weeks of
operation. Cell counts are on a logarithmic scale.

Fig. 5 e EDS spectrum of the precipitate of R2 (3 mg LL1 dL1)

experiment.

w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 3 8 6 3 e3 8 7 0

consortium in batch test were 20 mg L1 Cd2, 20 mg L1 Cu2,

20 mg L1 Ni2, 25 mg L1 Zn2, and 60 mg L1 Cr3,
75 mg L1 Pb2, but only 10 mg L1 heavy metal mixture.
Azabou et al., 2007a who studied heavy metal inhibition and
precipitation by a mixture of SRB in batch condition showed
that more than 72 mg L1 of Zn2 caused death of organisms.
Goncalves et al., 2007 reported that more than 99% of
70 mg L1 Zn and 2 mg L1 Cd were removed from synthetic
wastewater in a continuous bench-scale upflow anaerobic
sludge blanked reactor. The results obtained from different
studies were not similar, suggesting that the toxic and inhibitory effect of heavy metals on SRB cultures are influenced by
many factors such as the chemical and physiochemical
properties of the surrounding SRB environment, the species
composition of microbial community, and configuration of
bioreactor. Moreover, the simultaneous presence of heavy
metals could induce synergistic or cumulative toxic effects.
Utgikar et al., 2004 reported that the toxic effects of binary
mixtures of Cu and Zn were significantly higher than the toxic
effect of individual heavy metal. This was also demonstrated
by the study of Hao et al., 1994 mentioned above. In the
present study, 94e100% of w10e30 mg L1 each of heavy
metal mixture (Cu2, Zn2, Ni2 and Cr6) was removed
effectively by SRB consortium weekly throughout 12-week
operational period. The obtained results might be due to the
use of an indigenous SRB consortium isolated from heavy
metal contaminated sediment, resulting in an effective heavy
metal removal. Possible explanations for why the use of
indigenous consortia may be more advantageous than the use
of single species could be: (i) indigenous consortia containing
multi-species have adapted to a heavy metal polluted environment by developing a variety of resistance mechanisms
(see introduction). They are less liable to mutate and to be
contaminated from other microorganisms, and (ii) they
contain more than one kind of organisms that facilitate the
formation of reducing conditions by oxidizing completely
carbon sources (Bruins et al., 2000). In addition, it is difficult to
maintain culture purity due to the ubiquity of microorganisms
in the environment. Therefore, using a consortium instead of
single species is an optimum option and widely applied for
heavy metal treatment bioreactors.
An increase of heavy metal loading rate of up to 6 and
7.5 mg L1 d1 in this study resulted in a decrease or even no
detection of hybridized positive cells, sulfate reduction, sulfide
production, and heavy metal precipitation. Contrary to common
belief that only soluble metallic ion can be toxic or inhibitory, the
insoluble metallic compounds, especially metal sulfides, could
affect the activity of SRB by deposition on the surface of the cells
and blocking the access to the substrate and other nutrients
(Utgikar et al., 2002). At low levels of sulfide precipitate, the
bacteria themselves may directly accelerate metal sulfide
precipitation and facilitate settling of the solids by binding the
metal in their cell walls and extracellular polymeric substances
(EPS) (Beech and Cheung, 1995). This was demonstrated by Jalali
and Baldwin, 2000 and Azabou et al., 2007b, who evaluated the
influence on copper and zinc removal, respectively. The
precipitation of copper and zinc was detected more quickly in
the presence of bacteria cells than without bacteria cells in these
both studies. Thus, association of copper and zinc with
the bacterial cells could promote the precipitation rate.

3869

Although the presence of bacterial cells may facilitate metal

precipitation, the high level of sulfide precipitate can act as
a barrier between the cells and their essential growth nutrients.
Therefore, the influent with heavy metal concentrations below
the inhibitory level to maintain a maximum rate of sulfidogenesis are required to have a successful operation for heavy
metal sulfidogenic bioreactors.
Metal sulfides have been attributed to the major precipitation of Cu, Zn, and Ni by biological activity of SRB in the present
study. This is confirmed by a simultaneous decrease of sulfate
and heavy metal concentrations in the effluent and EDS spectrum with strong peaks of copper, zinc, nickel and sulfur. This is
in agreement with Azabou et al., 2007a,b who showed that zinc
removal was possible only in the presence of sulfate. This
suggests that sulfide produced from sulfate reduction by activity
of SRB is responsible for zinc removal as ZnS. In addition to
precipitation with sulfide, heavy metals may also have been
removed through sorption to the biomass or by other precipitation mechanisms i.e., hydroxide and carbonate precipitation,
as well as generated alkalinity (Goncalves et al., 2007; Neculita
et al., 2007). However, sulfide precipitation is the dominant
mechanism, whereas other mechanisms play only a minor role
for the removal of heavy metals in anaerobic sulfidogenic
bioreactors (Machemer and Wildeman, 1992; Radhika et al.,
2006). As mentioned in the introduction, soluble Cr6 can be
reduced into much less toxic and insoluble Cr3 by reacting with
bacterially produced hydrogen sulfide or enzymatic reduction.
However, precipitation of Cr as metal sulfide is not stable in
aqueous medium in comparison with Cu, Zn, and Ni. Thus, the
reduction to Cr3 is likely to be followed by rapid deposition as
hydroxides with the concentration of soluble Cr3 in equilibrium with Cr(OH)3 is w6.3  1031 mol L1 (Dean, 1999).

5.

Conclusions

There was no inhibition of SRB growth and the effective removal

(94e100%) of heavy metal mixture (Cu2, Zn2, Ni2, and Cr6)
can be achieved in R1eR3 with heavy metal loading rates
ranging from 1.5 to 4.5 mg L1 d1 throughout 12 weeks of
experiment. The heavy metal removal efficiency (91e97%) was
reduced from week 9 onwards and the inhibition of SRB growth
was observed at the heavy metal loading rate of 6 mg L1 d1 (R4).
The toxic effect of heavy metals on SRB growth was revealed in
R5 (7.5 mg L1 d1), in which no SRB could survive and no heavy
metal precipitation was detected after four weeks of operation.
This implied that the investigated SRB consortium might have
potential application for heavy metal sulfidogenic bioreactors.
To have a successful heavy metal treatment process, the heavy
metal loading rates below the inhibitory level to SRB activity
ranging from 1.5 to 4.5 mg L1 d1 should be selected.

Acknowledgment
The authors acknowledge financial support provided by
Federal Ministry for Education and Research (BMBF/Germany),
Institute of Water Quality Control, Faculty of Civil Engineering
and Geodesy, Technische Universitat Munchen, Germany.

3870

w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 3 8 6 3 e3 8 7 0

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