Minerals Engineering 20 (2007) 173–178

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Zinc precipitation by heavy-metal tolerant sulfate-reducing bacteria

enriched on phosphogypsum as a sulfate source
Samia Azabou, Tahar Mechichi *, Sami Sayadi
Laboratoire des Bio-procédés, Centre de Biotechnologie de Sfax, BP ‘‘K’’, 3038 Sfax, Tunisia

Received 28 April 2006; accepted 17 August 2006

Available online 2 October 2006

Abstract

In the present study, heavy-metal tolerance and precipitation by a mixed culture of sulfate-reducing bacteria (SRB) were evaluated.
These bacteria have been enriched during a previous study from a sewage sludge using phosphogypsum as sulfate source. Taking into
account that both sulfate and zinc are naturally occurring in phosphogypsum, zinc tolerance of SRB was tested in synthetic media con-
taining 20 mM sulfate and zinc chloride at concentrations ranging from 0 to 200 mg L1. Zinc tolerance was determined by bacterial
growth susceptibility and zinc removal monitoring. Bacterial growth and sulfate reduction were possible between 10 and 150 mg L1
of initial zinc concentration. Zinc concentrations more than 150 mg L1 were lethal to SRB. Zinc was removed effectively by SRB to
less than 5% from medium containing 150 mg L1 initial zinc concentrations or less. Energy-dispersive X-ray analysis showed that pre-
cipitation of zinc occurred in the form of sulfide. The results presented in this paper have shown that this mixed culture might be of use
for bioremediation of sulfate and heavy-metals containing wastewaters.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: SRB; Zinc tolerance; Zinc precipitation; Phosphogypsum

1. Introduction with their hydroxides. However, chemically produced H2S

Heavy-metals are present in industrial wastewaters dis-
charged from industries such as battery, paint, chemical
manufacturing and also from mining industry in acid-mine
drainage, posing significant disposal problems that require
urgent solution to avoid serious environmental contamina-
tion. Conventional technologies for removing metals from
contaminated wastewaters include precipitation, ion
exchange and absorption. The most effective precipitant is
sulfide (H2S) because it precipitates complex metals almost
regardless of wastewater characteristics and is far less pH-
dependent than hydroxide (Tunay and Kabdasli, 1994).
Moreover, many toxic metals such as lead, cadmium and
zinc have solubility products with their sulfide which are
several orders of magnitude lower than solubility products
*
Corresponding author. Tel.: +216 74 440 816; fax: +216 74 440 818.
E-mail address: mechichi.tahar@cbs.rnrt.tn (T. Mechichi).
is relatively expensive to handle and to use safely. The pre-
cipitation of metals with biologically produced H2S by SRB
has been proposed as an alternative process (Foucher et al.,
2001). Moreover, biological processes are more effective at
removing metals at lower concentrations. Indeed, there is
an increasing interest in the potential biotechnological
applications of bacterial sulfate reduction as an alternative
method for sulfate and heavy-metal removal from environ-
mental contamination, particularly from mining activities
(Garcia et al., 2001; Chang et al., 2000; Dvorork et al.,
1992). SRB are anaerobes characterized by their ability to
perform dissimilatory sulfate reduction with the simulta-
neous oxidation of the organic substrates (Postgate,
1984). They include representatives of the genera Desulf-
ovibrio, Desulfomicrobium, Desulfobacter, Desulfosarcina,
Desulfotomaculum, Thermodesulfobacterium, Archaeoglo-
bus, etc. (Odom and Singleton, 1993). Sulfate reduction
leads to the production of sulfide, which can readily react
with metals and form insoluble metal sulfide precipitates

0892-6875/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mineng.2006.08.008
174 S. Azabou et al. / Minerals Engineering 20 (2007) 173–178
(Eqs. (1) and (2)) since the solubility of most toxic metal sul-
fides is generally very low (Gadd and White, 1993).
þ  SRB
4H2 þ SO2
4 þ H ! HS þ 4H2 O
Me2þ þ HS ! MeSð#Þ þ Hþ ðMe2þ the metal cationÞ
Thus, SRB play an important role in both natural (White
et al., 1997) and engineered (Jong and Parry, 2003;
Tuppurainen et al., 2002) bioremediation of toxic wastes.
Nevertheless, selection of the most efficient and the most
heavy-metal resistant SRB requires their long exposure to
toxic wastes containing heavy-metal ions. Phosphogypsum
is produced during the process of phosphoric acid produc-
tion when Ca3(PO4)2 ore (apatite) is reacted with H2SO4.
Phosphogypsum is composed mainly of CaSO4 (calcium
sulfate) and contains impurities such as Al, P, F, Si, Fe,
Mg as well as many trace elements, including the rare earth
elements and naturally occurring radioactive materials
(Taha and Seals, 1991; Rutherford et al., 1994). This by-
product was shown, during a previous study, to be an
attractive substrate for the enrichment of SRB by using it
as a sulfate source (Azabou et al., 2005). The mixed culture
of SRB, exposed to phosphogypsum for six months, was
used during the present work in order to evaluate the abil-
ity of SRB for both heavy-metal tolerance and removal.
For this purpose, a heavy-metal-contaminated model solu-
tion was prepared containing sodium sulfate as a sulfate
source and different zinc chloride concentrations as the
heavy-metal contaminant.
2. Material and methods
2.1. Origin of the SRB mixed culture
The mixed culture of SRB used in this study was from
an enrichment culture which was previously developed on
phosphogypsum as a sulfate source (Azabou et al., 2005).
The mixed culture of SRB was enriched from a sludge
obtained from a wastewater treatment plant (Mahres,
Tunisia). The enrichment culture was developed as follow:
Serum bottles were prepared containing 50 mL basal med-
ium and supplemented with 10 g L1 phosphogypsum and
20 mM of organic acids mixture. The cultures were seeded
with 10% sludge and incubated at 30 °C. Throughout the
incubation period, microscopic examination of the cultures
and sulfide production were also routinely performed. Cul-
ture which presented high sulfide production concomitant
with high microbial density was used for the inoculation
of fresh medium containing 10 g L1 phosphogypsum.
After 10 days of incubation (corresponding to steady pro-
duction rate of sulfide), a fresh medium was inoculated
from this culture and the procedure was repeated three
more times. Ten days were chosen since this was the time
required for the SRB to reach stationary growth as
observed by the leveling of the sulfate concentration and
the steady production rate of sulfide.
2.2. Medium culture preparation
The culture medium was prepared oxygen free according
ð1Þ to Hungate (1969), but modified for use with syringes
(Macy et al., 1972; Miller and Wollin, 1974). The basal
medium contained (per liter of deionized water): 0.3 g
ð2Þ
KH2PO4, 0.3 g K2HPO4, 1 g NH4Cl, 0.1 g CaCl2 Æ 2H2O,
0.2 g MgCl2 Æ 6H2O, 0.1 g KCl, 1 g yeast extract (Difco
Laboratories), 1.5 mL trace element solution (Widdel and
Pfennig, 1981), and 1 mg resazurin. The pH was adjusted
to 6.8 with 10 M KOH solution, the medium boiled under
a stream of O2-free N2 gas and cooled to room tempera-
ture. 50 mL of aliquots were dispensed into serum bottles
under N2 and subsequently sterilized by autoclaving at
120 °C for 20 min. Prior to use, 1 mL 5% (w/v) NaHCO3
was injected from sterile stock solutions into the pre-steril-
ized basal medium.
2.3. Experimental cultures
Experimental cultures were carried out over 10 days to
investigate the zinc removal kinetics by the consortium of
SRB in the presence of different initial zinc concentrations.
Zinc was chosen since it is present at high amount in phos-
phogypsum (50–100 mg kg1). Moreover, zinc is a good
and safe representative of metals (Krishnan et al., 1993).
Indeed, zinc gives stable precipitates with low solubility
when reacting with sulfide and is less toxic to handle in
comparison with other heavy-metals such as cadmium or
lead. Experimental cultures were grown with 20 mM
sodium lactate as carbon source and 20 mM sodium sulfate
in 120 mL crimp-top vials with butyl rubber stoppers. A
mixed culture of SRB, previously enriched on phosphogyp-
sum as a sulfate source (Azabou et al., 2005), was used to
inoculate 50 mL medium culture. All cultivations were car-
ried out under identical conditions except that appropriate
volumes of ZnCl2 were added to the culture medium from
anaerobic stock solutions to yield the required concentra-
tion: 10, 50, 100, 150 or 200 mg L1. ZnCl2 anaerobic stock
solutions were prepared from analytical grade chemicals in
ultrapure water.
Na2S was omitted from both control culture (culture
without adding zinc) and zinc-containing cultures to avoid
premature precipitation of the added zinc.
2.4. Analytical methods
Sulfate was measured according to the turbidimetric
method (APHA, AWWA, APCF, 1995) with slight modifi-
cation. Dissolved sulfide was measured according to
Cord-Ruwisch (1985). Bacterial growth was monitored by
measuring optical densities at 580 nm using a Shimadzu
UV-110-01 spectrophotometer. Zinc concentrations were
measured using an atomic absorption analyzer, a Perkin–
Elmer 1101 B, with a hollow cathode lamp. The lamp
current was 8 mA. Absorption wave length was 213.9 nm.
Eighty percent air/20% (v/v) acetylene was used as
 S. Azabou et al. / Minerals Engineering 20 (2007) 173–178 175

oxidizing fuel flame at a flow rate of 5 L min1. Qualitative
and quantitative analysis of precipitates were realized by
EDS (energy-dispersive spectroscopy), using an instrument
consisting of a scanning electron microscope BS300 and
X-ray microanalyzer EDAX 9100/60 (Philips). Precipitates
were obtained by filtering the culture through Whatman
filter paper number 42 and washed with ultrapure water.
The filter paper with precipitate was dried in an oven for
2 h at 60 °C. Small sections of the filter paper were thin
coated by carbon and then used for EDS analysis (Jalali
and Balwin, 2000).
growth was observed. We can also notice that the higher
the initial concentration of ZnCl2, the higher was the inhi-
bition of the growth.
The amounts of sulfate reduced during the 10 days batch
experiments with different initial zinc concentrations are
shown in Fig. 3. It can be seen that the rate of sulfate
reduction was less than that for culture without zinc. The
sulfate reduction rate decreases with increasing initial con-
centrations of zinc. Moreover changes in optical density at
580 nm showed that SRB growth was inhibited by increas-
ing initial zinc concentrations. It should be noted that no

2.5. Precipitation of zinc in the presence and in the absence

50

Residual zinc concentration (mgL )

-1
of bacterial cells

A new set of experiments were carried out to evaluate 40

the influence on zinc removal of the bacteria cells them-
selves. SRB were cultivated, in one vial that did not contain 30
zinc. After 10 days of bacterial growth, the culture solution
was divided in two equal volume vials. The bacteria cells 20
were removed by centrifugation from one vial and the
SRB cells were retained in the other vial. ZnCl2 10
(50 mg L1) were added to each vial and zinc concentra-
tions were monitored with time. 0
With sulfate Without sulfate
3. Results Fig. 1. Effect of sulfate on residual zinc concentration. Initial zinc
concentration = 50 mg L1.
A culture of SRB has been previously enriched on phos-
phogypsum (10 g L1) as the sole sulfate source. A high
density of bacteria of different morphological types was 0.25
noted in this culture (Azabou et al., 2005). During the pres-
0.2
ent work, this enrichment culture of SRB, previously
exposed to phosphogypsum for six months, was tested
OD 580 nm

0.15
for both tolerance and removal of heavy-metal in response
to increasing zinc concentrations. Blank assays were per- 0.1
formed in order to determine the role of SRB among the
microorganisms forming in the mixed culture for the 0.05
removal of zinc ions. Two vials each containing 50 mg L1
ZnCl2 were studied for zinc removal in the presence and in 0
0 10 25 50 100 150 200
the absence of sulfate. Results showed that zinc removal -1
Initial zinc concentration (mgL )
was possible only in the presence of sulfate which suggests
Fig. 2. Optical density in the supernatant after 10 days growth of SRB
that sulfides produced from sulfate reduction by means of versus initial zinc concentrations.
SRB are responsible for zinc removal (Fig. 1). After inocu-
lating the vials with SRB enriched on phosphogypsum as a
2500
sulfate source, some interesting observations were noted. A
Residual sulfate (mgL )
-1

very fine, grey, suspended precipitate appeared, the 2000

intensity of which increased with zinc concentration. The
precipitate in the cultures with initial zinc concentrations 1500
of 10–50 mg L1 settled to the bottom of the vials during
1000
the first incubation day. The precipitate in the vials with
the higher initial zinc concentrations started to settle after 500
three days of incubation.
The effect of initial zinc concentrations on the growth of 0
0 10 25 50 100 150
SRB was investigated in batch experiments in the presence
of increasing concentrations of ZnCl2. Fig. 2 shows that Initial zinc concentration (mgL-1)
after 10 days of incubation at 30 °C, growth was possible Fig. 3. Residual sulfate concentration in the supernatant after 10 days
from 10 to 150 mg L1 of ZnCl2 while at 200 mg L1 no growth of SRB versus initial zinc concentrations.
176 S. Azabou et al. / Minerals Engineering 20 (2007) 173–178

sulfate reduction was observed at 200 mg L1 of zinc. This
confirms the results obtained for bacterial growth at this
concentration.
Sulfide production versus initial zinc concentration is
illustrated in Fig. 4. The sulfide formation rate was par-
tially inhibited with increasing initial zinc concentrations.
The amount of sulfide produced, in the absence of zinc,
was generally in good agreement with sulfate consumed.
The S2 2
produced =Sconsumed ratio is equal to 0.75 in control con-
taining zinc. However for batch experiments containing
10.6 mg L1. However, at the end of the experiment, the
final concentrations reached in both of the vials with and
without SRB cells were very similar.
Qualitative EDS analyses of the precipitate were per-
formed at the end of the run. The mineral content of the
precipitate was confirmed as zinc sulfide (ZnS) by the
EDS spectrum shown in Fig. 7. Table 1 illustrates that both
60

Zinc concentration (mgL-1)

zinc metal this ratio ranged from 0.5 to 0.3. 50
Fig. 5 shows the removal efficiency of zinc by the means
of the mixed SRB versus the initial zinc concentration. Zinc 40
removal to more than 95% was achieved after 10 days for 30
all the cultures grown in media containing initial zinc con-
centrations of 150 mg L1 or less. On the other hand, for 20
an initial zinc concentration of 200 mg L1, no zinc 10
removal was observed and therefore all the initial metal
concentration remained in the culture. 0
0 1 2 3 4 5
Zinc removal was studied in the presence and the Time (d)
absence of SRB cells. Zinc concentrations versus time are
plotted in Fig. 6. Zinc was removed faster from the vial Fig. 6. Comparison of zinc removal kinetics with (j) and without (h)
SRB cells. Zinc concentration (mg/L) versus time (days).
that contains SRB cells than that without SRB cells. After
one day, the zinc concentration in the vial with SRB cells
dropped from 50 to 1.5 mg L1, whereas in the vial without
SRB cells, the zinc concentration had only dropped to

18
Sulfide concentration (mM)

15
12

6
3

0
0 10 25 50 100 150 200
-1
Initial zinc concentration (mgL )

Fig. 4. Sulfide concentration in the supernatant after 10 days growth of

SRB versus initial zinc concentrations.

Fig. 7. The EDS qualitative analysis of solids originating at precipitation

120 of soluble zinc by the biologically produced H2S. The horizontal axis
represents X-ray energy (0–15 KeV) and the vertical axis shows X-ray
100 fluorescence intensity.
% zinc removal

80

60 Table 1
The EDS quantitative spectra of ZnS precipitated by the action of the
40 SRB on soluble Zn
Element Composition (%)
20
C 3.51
0 O 20.16
10 25 50 100 150 200 Zn 31.63
-1
Initial zinc concentration (mgL ) P 4.03
S 38.9
Fig. 5. Removal of zinc in the supernatant after 10 days growth of SRB
Ca 1.78
versus initial zinc concentrations.
 S. Azabou et al. / Minerals Engineering 20 (2007) 173–178
zinc and sulfur were found to be present at almost equal
proportions (31.63% and 38.90% respectively), in the
precipitates.
4. Discussion
The metabolic properties of SRB enable them to play an
important role in the bioremediation of harmful pollutants.
A number of studies have been carried out regarding
heavy-metal resistance in SRB (Hard et al., 1999; White
and Gadd, 2000). Selection of the most efficient and the
most heavy-metal resistant SRB requires their long expo-
sure to toxic wastes containing heavy-metal ions. Phospho-
gypsum, a residue of the phosphoric acid production from
apatite, seems to be an alternative possibility for enrich-
ment of heavy-metal resistant SRB. Apart from the
amount of phosphogypsum produced each year (thousands
of tons), phosphogypsum presents an environmental prob-
lem because of its acidic nature and the presence of several
heavy-metals and trace elements such as cadmium, nickel
and zinc. In a previous study, we have demonstrated that
phosphogypsum is a good source of sulfate for SRB culti-
vation except when the culture medium was supplemented
with an external carbon source (Azabou et al., 2005). Dur-
ing this study, a mixed culture of SRB showed growth in
the presence of zinc at initial zinc concentrations ranging
from 10 to 150 mg L1. An initial zinc concentration of
200 mg L1 was shown to be lethal for SRB. SRB growth,
monitored by optical density at 580 nm and measurement
of the sulfate reduction rate, was inhibited by increasing
initial zinc concentrations. The rate of sulfide formation
was found to decrease as the initial zinc concentration
was increased. The S2 2
produced =Sreduced ratio in the presence
of zinc ranged from 0.5 to 0.3 where this ratio was in good
agreement with the control assay (0.75). The low values of
the S2 2
produced =Sconsumed could be explained by the fact that
some of the biogenic sulfide was used in precipitating zinc
ions to form insoluble zinc sulfide (ZnS) and can not be
measured as soluble sulfide.
The ability of this mixed culture of SRB to grow with
phosphogypsum as sulfate source suggests that these
microorganisms should tolerate high concentrations of
heavy-metals. The reported toxic concentrations of
heavy-metals to SRB range from a few ppm (mg L1) to
as much as 100 ppm (Utgikar et al., 2002). Hao et al.
(1994) studied the toxic concentrations of zinc among other
heavy-metals for a mixed culture of SRB. They have
reported that zinc concentrations between 25 and 40 ppm
cause death of microorganisms. Taking into account the
results obtained, we can assume that this mixed culture cul-
tivated with phosphogypsum as a sulfate source, and which
can remove effectively zinc to less than 5 mg L1 from sul-
fate solutions containing up to 150 mg L1 initial zinc con-
centrations, consists of zinc-tolerant SRB.
In the present study, the mechanism for zinc removal was
apparently precipitation with sulfide. This was confirmed by
a simultaneous reduction in the concentration of S and Zn
177
and by EDS analysis of the precipitate. On the basis of the
EDS analysis the most abundant compound in the precipi-
tate from the batch experiments at the end of the run was
zinc sulfide, ZnS. In addition to precipitation with sulfide,
zinc might also have been removed through sorption on
the biomass. Indeed, the presence of SRB cells was shown
to facilitate metal precipitation. This result was in agree-
ment with other author’s observations who demonstrate
that in addition to providing sulfide for metal sulfur forma-
tion, SRB have an additional, important role in facilitating
metal precipitation. It has been suggested that the bacteria
themselves may directly accelerate metal sulfide precipita-
tion and facilitate settling of the solids by binding the metal
in their cell walls (Jalali and Balwin, 2000) and extracellular
polymeric substances (EPS) (Beech and Cheung, 1995).
These EPS comprise a mixture of polysaccharides, muco-
polysaccharides and proteins which varies in composition
between species and culture conditions (Zinkevich et al.,
1996) and can take up soluble metals.
5. Conclusion
In view of the results obtained in the present work, it can
be concluded that the mixed culture of SRB was able to
grow and thus to reduce sulfate when the medium culture
contains initial zinc concentrations ranging between 10
and 150 mg L1. Zinc was removed effectively by SRB to
less than 5% from medium containing 150 mg L1 initial
zinc concentrations or less. The mechanism of zinc
removal, observed in the present study, is a precipitation
with sulfide as it was confirmed by the simultaneous reduc-
tion in sulfate and removal of zinc and by EDS analysis of
the precipitate.
Acknowledgements
This work was supported by a grant from ‘‘Contracts
Programmes MRSTCD’’ Tunisia. We thank Pr. Zouheir
Fakhfakh from the Faculty of Science of Sfax for technical
assistance EDS analysis.
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