3rd AMIREG International Conference (2009): Assessing the Footprint of 237

Resource Utilization and Hazardous Waste Management, Athens, Greece

Precipitation of metals in a fixed-bed sulphate-reducing reactor under

theoretical stoichiometric lactate/sulphate ratio

P. Kousi, E. Remoundaki, A. Hatzikioseyian and M. Tsezos

Laboratory of Environmental Science and Engineering, School of Mining and Metallurgical Engi-
neering, National Technical University of Athens, Greece

ABSTRACT (Dvorak et al., 1992; Foucher et al., 2001; Hul-

An upflow fixed-bed sulphate-reducing reactor
has been set up and monitored for the treatment
of wastewater containing divalent iron
(100 mg/L), zinc (100 mg/L), copper (50 mg/L
and 100 mg/L), nickel (50 mg/L and 100 mg/L)
and sulphates (1,700 mg/L) at initial pH 3.5-4,
using lactate as electron donor. This work pre-
sents the results obtained from the runs of the
reactor at the theoretically calculated stoichio-
metric lactate/sulphate ratio. The experimental
results showed that the reduction of sulphates
was limited to about 30% due to the incomplete
oxidation of the intermediately produced ace-
tate. However, the concentration of the pro-
duced sulphides was enough for the quantitative
precipitation of copper, zinc and nickel. Resid-
ual iron was observed in the effluent due to lack
of available S2-. The order of precipitation of
metal sulphides (Cu > Zn > Ni > Fe) was also
experimentally observed from the operation of
the reactor at stoichiometric conditions. XRD
and SEM-EDS analyses performed on samples
of sludge produced during the operation of the
reactor showed that amorphous phases of metal
sulphides are predominant.
1. INTRODUCTION
Wastewater originating from mining and metal-
lurgical industries is often acidic and typically
characterized by a significant content of sul-
phates and soluble metals, such as Zn, Fe, Cu,
Ni, Pb and Cd. Soluble metal- and sulphate-
bearing wastewater treatment schemes are usu-
ally based on sulphate-reducing reactors re-
cently developed in both pilot and full scale
shoff et al., 2001; Kaksonen et al., 2003a).
In such reactor schemes, Sulphate-Reducing
Bacteria (SRB) (Postgate, 1965) obtain energy
for cell synthesis and growth by coupling the
oxidation of organic substrates or molecular hy-
drogen (H2) under anaerobic conditions to the
reduction of sulfate (SO42-) to sulfide (H2S, HS-)
(Rabus et al., 2007).
The preferred carbon sources for SRB are
low molecular-weight compounds such as or-
ganic acids (e.g. lactate, pyruvate, formate and
malate), fatty acid (e.g. acetate) and alcohols
(e.g. ethanol, propanol, methanol and butanol)
(Hao, 2003).
Lactate, a relatively common substrate, can
be incompletely oxidized to acetate (1) or com-
pletely oxidized to CO2 (2), depending on the
SRB species (Rabus et al., 2007).
2CH 3CHOHCOO − + SO 24 − →
(1)
→ 2CH 3COO − + 2HCO3− + H 2S
2CH 3CHOHCOO − + 3SO 24 − → 6HCO3− + 3H 2S (2)
Sulphates are usually present in mining and
metallurgical industrial wastewater. The simul-
taneous removal of sulphates and soluble metal
species is a main advantage of the SRB reactor
schemes. The organic substrate is the main ma-
terial to be supplied to the system.
Hydrogen sulphide reacts with divalent solu-
ble metals which are then sequestered from
wastewater as insoluble metal sulphides in the
form of various mineral phases (Herbert et al.,
1998; Kaksonen et al., 2003b):
H 2S + Me 2 + → MeS(s) + 2H + (3)
3rd AMIREG International Conference (2009): Assessing the Footprint of
Resource Utilization and Hazardous Waste Management, Athens, Greece
where Me stands for divalent metals such as Zn,
Fe, Cu, Ni, Pb, Cd, etc.
Bicarbonate ions react with protons to form
CO2 and water; thus, removing acidity from so-
lution as CO2:
HCO3− + H + → CO 2(g) + H 2 O (4)
H2S and HCO3- formed during sulphate re-
duction equilibrate into a mixture of H2S, HS-,
S2-, CO2, HCO3- and CO32-. This mixture buffers
the solution pH typically around neutral to
slightly alkaline values (Dvorak et al., 1992).
Minimum consumption of materials to be
supplied (i.e. organic substrate) and zero dis-
charge of soluble metals and organic residuals
are main targets for the optimization of the op-
erating conditions of such reactor schemes. In
the present work, the efficiency of the reactor
running in stoichiometric lactate/sulphate ratio
is experimentally examined and discussed.
2. MATERIALS AND METHODS
2.1 Sulphate-reducing fixed-bed reactor
The sulphate-reducing fixed-bed reactor, operat-
ing in upflow mode, is presented in Figure 1.
The biofilm was established on porous ceramic
pipes 1cm long (Cermec®, JBL Germany), after
transferring sufficient support material with al-
ready grown bacterial biomass from another re-
actor (Kousi et al., 2007). The mixed sulphate-
Figure 1: Sulphate-reducing fixed-bed reactor. Height:
50 cm, diameter: 10 cm, bed height: 45 cm, head space
(necessary for gas collection and removal): 5 cm.
238
reducing bacteria culture has already been char-
acterized and described in detail (Remoundaki
et al., 2008).
The reactor operated first in batch mode
(close loop) for biofilm development. The reac-
tor was fed with a variation of Postgate's me-
dium (DSMZ GmbH, Desulfovibrio medium
no.63) using lactate as major electron donor.
Following the period of the biofilm devel-
opment on the fixed bed of the reactor, the sys-
tem was operated in continuous mode, fed with
an influent after the mixing of two streams
(Fig. 1):
- nutrient solutions containing sodium lactate
at appropriate concentrations so that the lac-
tate/sulphate molar ratio in the final stream
would be the one indicated by the
stoichiometry of the redox reaction (2) as-
suming complete oxidation of lactate.
- synthetic, sulphate-rich and metal-bearing so-
lutions containing divalent iron (100 mg/L,
added as FeSO4.7H2O), zinc (100 mg/L,
added as ZnCl2), copper (50 and 100 mg/L,
added as CuCl2.2H2O), nickel (50 and
100 mg/L, added as NiSO4.6H2O) and sul-
phates (1,700 mg/L, added as Na2SO4,
MgSO4.7H2O and NiSO4.6H2O). The pH of
these solutions was adjusted to 2.5-3 by addi-
tion of HCl (Merck, analytical grade) so that
the pH of the reactor influent, after mixing of
the two streams, would be 3.5-4.
The reactor operated continuously for 5
months at ambient temperature at a constant hy-
draulic residence time (HRT) value of 9 h.
2.2 Liquid phase monitoring
During the operation of the reactor, sampling
was systematically performed at the following
points: inlet, outlet and 2 sampling ports along
the column length. The liquid phase samples
were vacuum filtered through 0.45 μm sterilized
membranes before any chemical determination.
The pH of the feed and the outlet solutions were
systematically monitored.
The sulphate concentration was determined
by turbidimetry at 450 nm after formation of
BaSO4 (Hach, DR/2000, Method 8051). TOC
was determined by colorimetry after persulphate
oxidation of carbon to carbon dioxide and col-
our change of a pH indicator (Hach, DR/2500,
Method 10129). Copper, nickel, zinc and iron
3rd AMIREG International Conference (2009): Assessing the Footprint of 239
Resource Utilization and Hazardous Waste Management, Athens, Greece

concentrations were determined by ICP (Lee- 7

man Labs, Direct Reading Echelle). All deter- 6
minations were performed in duplicate.
5
2.3 Solids characterization 4

pH
Powder XRD analysis of the bulk solid samples 3
was performed on a Bruker D8 Focus X-Ray 2
Diffractometer. The samples were step scanned pH (Fe,Zn,Ni,Cu: 100mg/L)
1
from 2° to 80° (2θ), at a step of 0.02° and step pH (Fe,Zn: 100mg/L & Ni,Cu: 50mg/L)

time of 1s. 0
The solids morphology study was performed 0 10 20 30 40 50
using a Scanning Electron Microscope (JEOL Distance from bottom [cm]
JSM 6380-LV) in low vacuum (15 kV) mode on
non-coated material. Semi-quantitative elemen- Figure 2: Typical pH profiles along the reactor column
(HRT=9h).
tal analysis of the SEM samples was performed
by Energy Dispersive X-Ray Spectrometry
(EDS system by Oxford Instruments), employ- 3.2 Metal removal and sulphides precipitation
ing Oxford INCAEnergy 250 software interface. The reactor operated with synthetic metal-
bearing solutions containing initially 100 mg/L
3. RESULTS AND DISCUSSION Fe, 100 mg/L Zn, 50 and 100 mg/L Cu as well
as 50 και 100 mg/L Ni. Figure 3 presents the
3.1 pH variation profiles of the metal ions along the reactor col-
Figure 2 presents a typical pH profile along the umn for two different sets of metal concentra-
column of the reactor. The pH, initially adjusted tions tested. From Figure 3(a), it can be seen
at 3.5-4 in the feeding solution, always reaches that the three metals are quantitatively precipi-
a value of about 6.0 as a result of the alkalinity tated. Figure 3(b) shows that iron precipitates by
generated during the SRB metabolism. Figure 2 80-90%.
also shows that the influent flow is neutralized According to the solubility order of the cor-
within the first 15 cm of the column length, in- responding sulphides (Dean, 1999), copper sul-
dicating an intense microbial activity at the bot- phide is the less soluble compound and iron sul-
tom of the reactor. The process is not inhibited phide is the more soluble among the four metals
by copper and nickel present at concentrations when they coexist in an aquatic solution
up to 100 mg/L. (Cu>Zn>Ni>Fe). This explains the results pre-
sented in Figure 3 where Cu, Zn and Ni precipi-
tate quantitatively while there is residual soluble

100 100
Zn (Fe,Zn,Ni,Cu: 100mg/L) Fe (Fe,Zn,Ni,Cu: 100m g/L)
Zn (Fe,Zn: 100m g/L & Ni,Cu: 50m g/L)
Fe concentration [mg/L]
Zn, Ni, Cu concentration

80 80 Fe (Fe,Zn:100m g/L & Ni,Cu: 50m g/L)

Ni (Fe,Zn,Ni,Cu: 100m g/L)
Ni (Fe,Zn: 100mg/L & Ni,Cu: 50mg/L)
60 Cu (Fe,Zn,Ni,Cu: 100mg/L) 60
[mg/L]

Cu (Fe,Zn: 100m g/L & Ni,Cu: 50m g/L)

40 40

20 20

0 0
0 10 20 30 40 50 0 10 20 30 40 50
Distance from bottom [cm] Distance from bottom [cm]

(a) (b)
Figure 3: Metal removal along the reactor column for two different sets of metal concentrations.
3rd AMIREG International Conference (2009): Assessing the Footprint of 240
Resource Utilization and Hazardous Waste Management, Athens, Greece

Figure 4: XRD analysis on sludge sample taken from the bottom of the reactor.
Fe. This can also be attributed to the deficiency
of available sulphides in solution. In the follow-
ing paragraphs, it will be presented that there is
limited sulphate reduction due to the choice of
stoichiometric conditions. Such behaviour has
also been reported in literature (Jong et al.,
2003; Samaranayake et al., 2002).
Moreover, software applications for the
simulation of the chemical equilibrium at the
experimental conditions (ΕSHE ≈ -250 mV,
pH ≈ 6.6, alkalinity based on the lactate/acetate
oxidation reactions: 1830 mg/L CO32-) (Gustafs-
son, 2009; Puigdomenech, 2009) confirm the
observed mechanism for the removal of the four
metals precipitating as sulphides following the
order Cu>Zn>Ni>Fe.
Solid samples analyses by XRD and SEM-
EDS confirm that amorphous phases of metal
sulphides are predominant (Remoundaki et al.,
2008). From the analyses of sludge samples
taken from the bottom of the reactor on XRD
(Fig. 4), it seems that there are phases which can
be characterised as wurtzite (ZnS) and geerite
(Cu8S5).
SEM-EDS results showed almost pure crys-
talline forms of copper sulphide (Fig. 5), almost
pure forms of zinc sulphide (Fig. 6) and several
aggregates of iron-zinc-nickel sulphides
(Fig. 7).
3.3 Sulphate reduction and organic carbon deg-
radation
Figure 8 shows the profiles for the sulphate re-
duction and the degradation of organic carbon
along the reactor column for different concen-
trations of metal ions in the feed stream. The

Figure 5: SEM-EDS analysis on sludge sample taken from the bottom of the reactor: almost pure crystalline forms of
copper sulphide.
3rd AMIREG International Conference (2009): Assessing the Footprint of 241
Resource Utilization and Hazardous Waste Management, Athens, Greece

Figure 6: SEM-EDS analysis on sludge sample taken from the bottom of the reactor: almost pure forms of zinc sulphide.

Figure 7: SEM-EDS analysis on sludge sample taken from the bottom of the reactor: aggregates of iron-nickel-zinc sul-
phides.

data depicted in Figure 8 show that the reduc- 100 mg/L, indicating that they do not pose any
tion of sulphates is limited to 30% related to a toxicity effect on the SRB-culture.
critical value for the concentration of the total
organic carbon. As lactate is incompletely oxi-
dized to acetate (1) by three of the four genera 4. CONCLUSIONS
which species were identified in the system The systematic monitoring of the sulphate-
SRB population (Castro et al., 2000; Rabus reducing reactor leads to the following conclu-
et al., 2007), this critical concentration may be sions:
attributed to the concentration of remaining ace-
tate. 1800
Complete reduction of the sulphates of the 1600
metal-bearing solution was observed for lactate
Sulphates / TOC [mg/L]

1400
excess in the influent for lactate/sulphate molar
1200
ratio equal to 1.06 while the stoichiometric lac-
tate/sulphate molar ratio is 0.64. It is apparent 1000
that for complete sulphate removal, the lac- 800
Sulphates (Fe,Zn,Ni,Cu: 100mg/L)
Sulphates (Fe,Zn: 100mg/L & Ni,Cu: 50mg/L)
tate/sulphate ratio must be higher than the 600 TOC (Fe,Zn,Ni,Cu: 100mg/L)

stoichiometric (Kousi et al., 2007; Velasco 400

TOC (Fe,Zn: 100mg/L & Ni,Cu: 50mg/L)

et al., 2008).
200
Moreover, from Figure 8 it is apparent that
the degradation of lactate to acetate and the con- 0
sequent reduction of sulphates are completed 0 10 20 30 40 50
Distance from bottom [cm]
within the first 15 cm of the reactor column.
This is not affected by the simultaneous pres- Figure 8: Sulphate reduction and organic carbon degrada-
ence of the metals at concentrations up to tion.
3rd AMIREG International Conference (2009): Assessing the Footprint of
Resource Utilization and Hazardous Waste Management, Athens, Greece
- An excess of sodium lactate above stoichio-
metry is necessary for the complete reduction
of the sulphates existing in the synthetic sul-
phate- and metal-bearing solution.
- The sulphides, generated during the reduc-
tion of sulphates by 30%, are sufficient for
the quantitative precipitation of 100 mg/L
Zn, 100 mg/L Cu και 100 mg/L Ni as well as
80-90% of the added quantity of divalent iron
(100 mg/L).
- The mechanism of the metals precipitation
was experimentally confirmed. The precipita-
tion order of the metals complies with the
solubility products order for the correspond-
ing metal sulphides and is also confirmed by
simulation of the chemical equilibrium at the
experimental conditions.
ACKNOWLEDGMENT
This work was carried out in the frame of
BioMinE (European project contract NMP1-
CT-500329-1). The authors acknowledge the fi-
nancial support given to this project by the
European Commission under the Sixth Frame-
work Programme for Research and Develop-
ment.
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