Hydrometallurgy 48 Ž1998.

171–186

The oxidation kinetics of zinc sulphide with

Thiobacillus ferrooxidans
M. Boon ) , M. Snijder, G.S. Hansford, J.J. Heijnen
KluyÕer Laboratory of Biotechnology, Department of Biochemical Engineering, Technical UniÕersity Delft,
Julianalaan 67, 2628 BC Delft, The Netherlands
Received 10 July 1997; revised 4 November 1997; accepted 9 November 1997

Abstract

The kinetics and rate limiting sub-processes in the bacterial oxidation of zinc sulphide with
Thiobacillus ferrooxidans were examined. The oxidation rate of synthetic ZnS in the presence and
absence of bacteria at equal ferrous and ferric iron concentration and pH, was measured in parallel
batch experiments. Bacteria oxidize Fe 2q produced in the chemical oxidation of ZnS with Fe 3q.
In the fermenter without bacteria hydrogen peroxide was added to regenerate ferric iron and
maintain the Fe 2q concentration equal to that in the fermenter with bacteria. No significant
differences in the oxidation rates of zinc sulphide were found between the slurry with and without
bacteria at equal Fe 2q and Fe 3q concentrations. Consequently, an indirect mechanism Ži.e.
chemical oxidation of ZnS with Fe 3q to Zn2q, S 0 and Fe 2q, and bacterial oxidation of S 0 to
SO42y and Fe 2q to Fe 3q . determines the oxidation rate of ZnS in the bacterial oxidation process.
From the experiments it was shown that T. ferrooxidans prefers S 0 as a substrate and the
regeneration of Fe 3q can even become terminated. From the experimental data several sugges-
tions for improved process design and experimental research are given. q 1998 Elsevier Science
B.V.

1. Introduction

For optimization of bioleaching processes of metal sulphides it needs to be known

which sub-processes are involved and how the process conditions determine the rate of
sub-processes. Examples of sub-processes in the bio-oxidation of sulphide minerals are
chemical and bacterial oxidation processes, electrochemical interactions, gas–liquid
mass transfer of oxygen and carbon dioxide, liquid–solid mass transfer, the formation of

)
Corresponding author. E-mail: m.boon@stm.tudelft.nl.

0304-386Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

PII S 0 3 0 4 - 3 8 6 X Ž 9 7 . 0 0 0 8 1 - 9
172 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186

precipitates, etc. In order to determine optimal process conditions the mechanism of the
bio-oxidation reaction needs to be known. This paper discusses the bacterial and
chemical oxidation reactions of metal sulphides. In the literature the discussion on
whether a direct mechanism is involved is still lively w1x. In the direct mechanism it is
assumed that attached bacteria directly oxidize the mineral surface to Zn2q and SO42y
for example. According to the indirect mechanism solid metal sulphide is chemically
oxidized with ferric iron to ferrous iron and reduced sulphur species Že.g. S 0 or S 2 O 32y .
which are oxidized further by bacteria. Arguments in favour of a direct mechanism have
been reviewed and discussed w1x. It was argued that the presented experimental observa-
tions do not unambiguously show the occurrence of a direct mechanism. An argument
against the direct mechanism was based on the comparison of sterile chemical and
bacterial oxidation rates of sulphide minerals reported in the literature w2x. It was shown
that reported bacterial and sterile chemical zinc sulphide oxidation rates were similar
when compared at equal process conditions. However, the data were obtained by many
different research groups who worked at different conditions with different minerals,
size fractions, temperatures, etc. and several data transformations were needed to make
these kinetic data mutually comparable. Moreover, most data sets presented in the
literature contained very limited information concerning the kinetics due to the use of
off-line measurement methods only. Therefore, in the research presented in this paper
kinetic experiments were carried out to measure the bacterial and Žsterile. chemical
oxidation rate of zinc sulphide at equal conditions ŽpH, ferric and ferrous iron concentra-
tion and temperature.. Verbaan and Huberts w3x applied a redox-stat method to examine
the role of the direct mechanism in bacterial oxidation of synthetic nickel sulphide,
Ni 3 S 2 . In this study an adapted method was used for examining the role of the direct
mechanism in the bacterial oxidation of synthetic ZnS. In a redox-stat experiment zinc
sulphide was oxidized in two parallel batches in which the ferric to ferrous iron ratios
were kept equal by means of continuous redox potential measurement and off-line
ferrous iron analyses. The one batch was inoculated with Thiobacillus ferrooxidans, the
other was kept without bacteria. In both slurries ferrous iron was produced in the
chemical reaction of ferric iron with zinc sulphide. Bacteria will oxidize ferrous iron,
whereas in the reactor without bacteria ferrous iron was oxidized by the addition of
hydrogen peroxide and, thus, kept equal to the Fe 2q concentration in the fermenter with
T. ferrooxidans. The production of zinc ions in the two batches was compared. If
additionally to the oxidation of chemically produced ferrous iron and elemental sulphur,
T. ferrooxidans also oxidizes zinc sulphide in a direct mechanism, more zinc sulphide is
expected to be oxidized than in a reactor without bacteria. This experimental method w3x
was extended with the application of on-line off-gas analyses of oxygen and carbon
dioxide to the fermenter with bacteria. This allows the accurate determination of the
biomass concentration and the oxygen and carbon dioxide consumption rate in the
fermenter with bacteria w4x.
Besides examining whether the direct mechanism is a relevant sub-process, the
kinetics of the bacterial oxidation of zinc sulphide were examined. In the case of an
indirect mechanism at least three sub-processes are involved: Ži. the chemical oxidation
of zinc sulphide with ferric iron, Žii. the bacterial oxidation of ferrous iron and Žiii. the
bacterial oxidation of elemental sulphur. It is expected that the kinetics of the chemical
 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186 173

oxidation of zinc sulphide are independent of the kinetics of the bacterial oxidation of
the produced substrate. On the other hand, the oxidation kinetics of ferrous iron by T.
ferrooxidans w5x will probably change when elemental sulphur is available.

2. Theory

2.1. Chemical oxidation kinetics of ZnS with ferric iron

In an indirect mechanism zinc sulphide is chemically oxidized with ferric iron and it
is assumed that the kinetics are equal to those in the absence of bacteria. Kinetic models
for the chemical oxidation of zinc sulphide reported in the literature have been reviewed
w2x. Essentially three different kinetic models were proposed:
Ži. the electrochemical mechanism in which the chemical oxidation rate of ZnS, r ZnS
Žmolrlrs., is proportional to the square root of the ferric iron concentration and to the
mineral surface area. In our calculations the mineral specific ZnS oxidation rate, n ZnS
Žmolrmolrs., is estimated from:
0.5
n ZnS s kX1 P w Fe 3q x Ž 1.
Žii. Verbaan and Crundwell w6x proposed the electrochemical charge-transfer model
with shrinking particle model in which the observed inhibition by ferrous iron is
accounted for. In this model the oxidation rate of ZnS is related to the ferric to ferrous
iron ratio:
2r3
X
yEact w ZnSx
yr ZnS s k P exp ž RT / ž
P A0
w ZnS 0 x / P exp Ž K P E h . Ž 2.

with A 0 s initial BET surface of the mineral and E h s redox potential of the Fe 2qrFe 3q
couple which are related according to the Nernst equation:
RT w Fe 3q x
E h s E h0 P ln Ž 3.
nF w Fe 2q x
Accordingly, rewriting Eq. Ž2. by elimination of E h yields an equation in which the
chemical oxidation rate of ZnS is proportional to the ratio between the ferric and ferrous
iron concentration. This would agree with the kinetics recently found in chemical pyrite
oxidation w7x.
Žiii. Crundwell w8x corrected this model because it was shown that inhibition by
ferrous iron is only an apparent effect caused by the decrease of active ferric ions at
increasing ferrous iron concentration. In the corrected model the mineral specific
oxidation rate is related to the concentration of active ferric ions. However, the corrected
model was too complex for our use. Therefore, only the electrochemical mechanism ŽEq.
Ž1.. and the Verbaan–Crundwell model ŽEqs. Ž2. and Ž3.. are used in our work because
the two equations will, at the phenomenological level, show a different effect of
wFe 3q xrwFe 2q x. Firstly, Eqs. Ž1. and Ž2. are compared in their description of a simulated
batch experiment of the chemical oxidation of zinc sulphide. The specific ZnS oxidation
174 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186

Fig. 1. Simulations of the specific ZnS oxidation rate, n ZnS ŽnuZnS., in a sterile chemical batch experiment
with initial concentration of 0.21 M ZnS Žwith BET surface is 7.2 m2 rg ™ A 0 s144 m2 rl., 0.16 M Fe 3q
and 0.03 Fe 2q at 308C. Ž- - -. Electrochemical mechanism ŽEq. Ž1.. with k 1 s6.1)10y6 My0.5Psy1 .
Ž . Verbaan–Crundwell model ŽEq. Ž2.. with kX s 40 molrm2 rs, Eact s 79 kJrmol, E h0 s 0.45 V
and K s17.3.

rate, n ZnS , is plotted as a function of the ferric to ferrous iron concentration ratio ŽFig.
1A. and against time ŽFig. 2B.. From these graphs it is concluded that Žfor the chosen
kinetic constants. the difference of the two models is only significant at ferric to ferrous
iron concentration ratios larger than 4. These circumstances only occur in the initial
phase Ž- 5 h. of this chemical batch experiment. Consequently, determining whether the
chemical oxidation rate depends on either the ferric iron concentration Želectrochemical
mechanism., or the ferric to ferrous iron concentration ratio ŽVerbaan–Crundwell
model., is only possible when measuring in a range of the ferric to ferrous iron
 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186 175

concentration ratio between 100 and 10. In this range the electrochemical mechanism
predicts a constant mineral specific rate, whereas the Verbaan–Crundwell model pre-
dicts a steep decrease of the rate. In bacterial oxidation of zinc sulphide bacteria are able
to maintain relatively high ferric to ferrous iron concentration ratios. This explains why
it is important to determine whether the chemical oxidation rate is determined by either
the ferric iron concentration or the ferric to ferrous iron ratio. Therefore, both the
electrochemical and the Verbaan–Crundwell models are considered here to describe the
measured oxidation rate of zinc sulphide.

2.2. Bacterial oxidation kinetics of the mixed substrate, S 0 and Fe 2 q

The ferrous and ferric iron concentrations are determining parameters of the chemical
oxidation rate of zinc sulphide. Therefore, in bacterial oxidation of zinc sulphide, the
bacterial oxidation kinetics of ferrous iron need to be known. The simplified kinetic
equation for ferrous iron oxidation Žin the absence of elemental sulphur. by T.
ferrooxidans is w5x:
q Fe 2q ,max
q Fe 2qs Ž 4.
1 q Ž K srK i . P Ž w Fe 3q x r w Fe 2q x .
In this equation q Fe 2q ,max is the maximum biomass specific ferrous iron oxidation rate,
indicating the specific oxygen consumption rate at an excess of ferrous iron and no
inhibition effects from ferric iron. K srK i is the affinity coefficient for the ferric to
ferrous iron concentration ratio. In continuous cultures it was measured w5x that
q Fe 2q ,max s 9 mol Fe 2qrC-molrh and K srK i s 0.05. ŽThus q Fe 2q is already close to
q Fe 2q ,max at wFe 2q xrwFe 3q x ) 0.5..
Only limited research has been carried out to study the behaviour of T. ferrooxidans
on the mixed substrate, Fe 2q and S 0 . Espejo et al. w9x measured bacterial oxidation rates
on ferrous iron and on the mixed substrate in colorimetric experiments. They found that
the ferrous iron oxidation rate is significantly less in the presence than in the absence of
elemental sulphur. Wiertz w10x came to the same results in respiration experiments. Also
from measurement of the yield per ferrous iron or sulphur electron it is clear that the
affinity of T. ferrooxidans for the two substrates is different. Arkesteyn w11x and Hazeu
et al. w12–14x determined the yield of T. ferrooxidans on both ferrous iron and reduced
sulphur compounds and concluded that the yield of bacteria per mole electron trans-
ferred to oxygen through the electron transport chain ŽC-molrmol electron. is signifi-
cantly less for ferrous iron than for sulphur electrons w1,12–14x. Apparently, elemental
sulphur electrons enter the electron transport at a higher energy level than ferrous iron
electrons, which explains the higher yield per mol of sulphur electrons w15x. More details
on the mechanisms in bacterial oxidation of reduced sulphur compounds are given
elsewhere w16–18x. Consequently, if elemental sulphur is present in bioleaching pro-
cesses, for energetic reasons, the bacteria will prefer this substrate to ferrous iron. Thus,
it must be expected that the kinetics of the oxidation of the mixed substrate by T.
ferrooxidans cannot be constructed from the kinetics of two independent sub-processes
Ži.e. the bacterial oxidation of Ži. ferrous iron and Žii. elemental sulphur.. In this work
the bacterial oxidation kinetics of the mixed substrate ŽFe 2q and S 0 . are only measured
176 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186

in the bacterial oxidation experiments with zinc sulphide; these kinetics will be
qualitatively described.

2.3. Elemental and charge balances

Dutrizac and MacDonald w19x in their review of leaching with ferric iron, claim that
from thermodynamic data one would expect the production of sulphate if a metal
sulphide is immersed in an acidified ferric ion solution. However, according to them it is
known that in acidic ferric solution the dissolution reaction often produces elemental
sulphur and very little sulphate. From measurements of the stoichiometry this was
confirmed for ZnS w20x. Therefore, in the indirect mechanism zinc sulphide is incom-
pletely oxidized by ferric iron to ferrous iron and elemental sulphur:
ZnS q 2Fe 3q™ Zn2qq S 0 q 2Fe 2q Ž 5.
T. ferrooxidans is able to simultaneously oxidize ferrous iron and elemental sulphur.
Eqs. Ž6. and Ž7. give simplified Žcatabolic. stoichiometric equations in which the growth
of bacteria is neglected.
Fe 2qq 0.25O 2 q Hq™ Fe 3qq 0.5H 2 O Ž 6.
S 0 q 1.5O 2 q H 2 O ™ SO42y q 2Hq Ž 7.
When the stoichiometric equations of the bacterial ferrous iron and elemental sulphur
oxidation are used, the degree of reduction balance w21x is achieved which relates the
simultaneous bacterial ferrous iron and elemental sulphur oxidation to the oxygen and
carbon dioxide consumption w2x. In deriving this equation a biomass composition of
CH 1.8 O 0.5 N0.2 was assumed w21x:
bio bio
2qy 6 r 0 s y4r
yr Fe S O 2 y 4.2 rCO 2 Ž 8.
The zinc ion production rate is equal to the zinc sulphide oxidation rate:
r Zn 2qs yr ZnS Ž 9.
When all chemically produced ferrous iron and elemental sulphur are consumed by the
bacteria, the zinc sulphide oxidation is also expressed in terms of the degree of reduction
balance:
y8r ZnS s y4O 2 y 4.2 rCO 2 Ž 10 .
Accordingly, if no accumulation of ferrous iron or elemental sulphur occurs Ži.e. reaction
rate limited regime. the zinc sulphide oxidation rate is directly related to the oxygen and
carbon dioxide consumption rates. Under those constraints the bacterial oxidation rates
of ferrous iron and elemental sulphur are also stoichiometrically related:
bio bio
yr Fe 2qs y2 r 0 Ž 11 .
S

In the biomass limited regime, the zinc sulphide, ferrous iron and elemental sulphur
oxidation rates can only be calculated from the changes in concentrations over the time
interval between two off-line analyses. In the measurements only ferrous iron and zinc
 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186 177

ion concentrations were analyzed. The integrated degree of reduction balance is used to
determine the concentration of elemental sulphur:
t2 t2
y6DS 0 Ž bio . y DFe 2q Ž bio . s y4ÝrO 2 y 4.2ÝrCO 2 Ž 12 .
t1 t1

The amount of ferrous iron that is chemically produced is determined from the measured
concentration of zinc ions:
DFe 2q Ž chem . s 2 Ž w Zn2q x t 2 y w Zn2q x t 1 . Ž 13 .
and for chemically produced elemental sulphur there holds:
DS 0 Ž chem . s Ž w Zn2q x t 2 y w Zn2q x t 1 . Ž 14 .
The bacterially oxidized ferrous iron within a time interval is calculated from:
yDFe 2q Ž bio . s 2 Ž w Zn2q x t 2 y w Zn2q x t 1 . y Ž w Fe 2q x t 2 y w Fe 2q x t1 . Ž 15 .
The concentration of elemental sulphur is equal to:
w S 0 x s D w S 0 x Ž chem. y D w S 0 x Ž bio. Ž 16 .
In redox-stat experiments with bacterial and chemical oxidation of zinc sulphide the
redox potential in the reactor without bacteria is kept equal to that in a parallel reactor
with bacteria. The chemically produced ferrous iron in the reactor without bacteria is
then regenerated with hydrogen peroxide:
2Fe 2qq H 2 O 2 q 2Hq™ 2Fe 3qq 2H 2 O Ž 17 .
In this reaction protons are consumed. Therefore, the addition of sulphuric acid is
required to keep pH in the reactor without bacteria equal to that in the parallel reactor
with bacteria.
In acid ferric iron solutions part of the added hydrogen peroxide decomposes:
H 2 O 2 ™ H 2 O q 0.5O 2 Ž 18 .
Therefore, it is not possible to calculate the accumulation of zinc ions from a mass
balance which accounts for the amount of hydrogen peroxide.

2.4. Biomass specific rates

The biomass specific ferrous iron and elemental sulphur oxidation rates within a time
interval Ž t 2 y t 1 . are calculated from:
yD w Fe 2q x Ž bio .
q Fe 2qs Ž 19 .
Ž t 2 y t1 . P C x
and
yD w S 0 x Ž bio .
qS 0 s Ž 20 .
Ž t 2 y t1 . P C x
178 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186

2.5. Mineral specific rates

The zinc sulphide specific rate of compound i, n i , is defined as:

< ri <
ni s Ž 21 .
w ZnSx
and if no accumulation of intermediate products occurs the mineral specific zinc
sulphide, oxygen and carbon dioxide consumption rates are related according to the
degree of reduction balance for zinc sulphide ŽEq. Ž10.: 8 n ZnS s 4n O 2 q 4.2 n CO 2 ..
Otherwise the specific zinc sulphide oxidation rate within that time interval is calculated
from:
D w Zn2q x
n ZnS s Ž 22 .
Ž t 2 y t1 . P w ZnSx

3. Materials and methods

3.1. Redox-stat

Redox-stat experiments were carried out in stirred and aerated baffled fermenters that
were kept at 308C. The reactor without bacteria was gassed with nitrogen and from TOC
measurements it was shown that no bacterial growth occurred during the experiments.
The reactor with bacteria was aerated with air that was enriched with carbon dioxide in
order to prevent exhaustion of CO 2 in the gas phase w22x. The aeration rate was
manually controlled at a flow rate between 6 and 15 lrlrh, Ž0.1 or 0.25 vvm. to acquire
sufficient aeration w22x, and the carbon dioxide addition was manually controlled to
obtain a carbon dioxide concentration in the off-gas between 100 and 500 cm3rm 3. The
oxygen and carbon dioxide in the off-gas and aeration air of the fermenter with bacteria
were continuously measured. It is assumed that the ferric and ferrous iron concentrations
Žor the concentration of ferric iron and the redox potential. and the pH are the relevant
process conditions for the comparison of the chemical and bacterial oxidation rates of
ZnS, which therefore need to be maintained equal in the two fermenters. The measured
redox potentials ŽMetrohm 691 E h meter. in the parallel reactors with and without
bacteria were used to control the hydrogen peroxide addition. Because deviations
between the redox potentials occurred at equal ferrous iron concentration in the parallel
batches, the set-point of the Eh meter in the sterile reactor was set equal to that in the
bacterial fermenter after each wFe 2q x analysis. It was found that the ferrous iron
concentrations in the parallel reactors agreed during the course of the experiments. It
was not examined whether the deviations in the redox potential were due to real changes
in the redox potential of the slurries, or to shifts and fouling of the electrodes. A
Metrohm 614 Impulsomat and 665 Dosimat was used to add hydrogen peroxide Ž30%..
A Metrohm pH stat was used to maintain pH 1.8. Technical details on the equipment
and analytical procedures are presented elsewhere w4x.
 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186 179

3.2. Inoculum

T. ferrooxidans was adapted to synthetic zinc sulphide for about three months in an
aerated batch culture Ž0.21 M total iron, wZnSx 0 0.205 M, 308C, pH 1.8., in which the
bacteria were at a low growth rate Ži.e. substrate limited regime.. As an inoculum the
pellet of 1 l centrifuged slurry was used, while, in order to obtain equal solids
concentrations, a pellet of 1 l centrifuged slurry was sterilized in an autoclave and added
to the reactor without bacteria. The pellets were resuspended in 2 l ferric iron medium
Ž0.16 M Fe 3q and 0.03 M Fe 2q ., and ZnS was added Žfinal concentration 0.205 M
ZnS.. Six redox-stat experiments ŽI–VI. were carried out within a time period of 270 h.
When terminating an experiment pellets were obtained from the two slurries and added
to the subsequent experiment. However, during the course of the experiments I to IV,
the bacteria lost their ability to oxidize ferrous iron. Therefore, after experiment IV the
bacteria were grown for 24 h in a batch culture on ferrous iron only Žwithout elemental
sulphur., after which the iron oxidizing capacity was regained Žexperiments V and VI..

3.3. Analytical procedures

Samples were taken every 30 min. The zinc ion concentration ŽAAS., total iron and
ferrous iron concentrations Žortho-phenanthroline method, which is adapted for samples
containing zinc ions w4x. are determined in filtered supernatant. The total organic carbon
ŽTOC. was determined in the slurry. Off-gas analyses were used to determine the
oxygen and carbon dioxide consumption rates and also the concentration of elemental
sulphur Žfrom degree of reduction balance, Eqs. Ž12. – Ž16.. and biomass Žfrom carbon
balance. in the fermenter with bacteria.

3.4. Media and chemicals

In order to avoid electro-chemical reactions in the solid, synthetic ZnS was used
instead of mineral sphalerite, ZnS ) 98%, Fluka Chemika, BET surface area of 7.2
m2rg Žat 0.205 M ™ A 0 s 144 m2rl.. In order to remove organic carbon the zinc
sulphide needed to be washed with medium solution before use. The iron-free medium
contained ŽNH 4 . 2 SO4 , 2 grl; KCl, 0.2 grl; K 2 HPO4 , 1.0 grl and MgSO4 P 7H 2 O. 0.4
grl. A solution of 2 mlrl trace elements w23x was added. Further details are reported
elsewhere w4x.

4. Results and discussion

It was found that the elemental sulphur and zinc ion concentrations, as well as the
oxygen and carbon dioxide consumption rates agreed in the six duplicate bacterial batch
experiments. On the other hand, the iron-oxidizing capacity of the bacteria is not
reproducible. In several of the six redox-stat experiments the observed bacterial ferrous
iron oxidation rate was very low and even disappeared Žin experiments III and IV...
Then the ratio between the zinc ions and ferrous iron concentration in the fermenter with
180 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186

Fig. 2. Typical plot of wZn2q x in parallel reactors of redox-stat experiments. Ž'. wZn2q x in fermenter without
bacteria. Žq. wZn2q x in fermenter with bacteria.

bacteria was 1:2 during the course of the experiment and no addition of hydrogen
peroxide was needed in the reactor without bacteria.
The zinc ion concentrations in the parallel reactors with and without bacteria were
very close during the course of each redox-stat experiment. Fig. 2 shows a typical plot.
Consequently, the amount of zinc ions produced by a direct bacterial oxidation
mechanism of zinc sulphide is negligible as compared with the chemically produced
zinc ions. These results give strong evidence in favour of the indirect mechanism for
ZnS, which is in accordance with the result of Verbaan and Huberts for synthetic nickel
sulphide w3x. While in the absence of bacterial or chemical ferrous iron oxidation the
zinc ions and ferrous iron concentration was 1:2, the chemical oxidation of ZnS with
Fe 3q is incomplete. Therefore, the bacterial oxidation of ZnS consists of the chemical
oxidation of ZnS with Fe 3q and the oxidation of Fe 2q and S 0 by T. ferrooxidans.
When an indirect mechanism is assumed two regimes are distinguished in a batch
culture of T. ferrooxidans on zinc sulphide: Ži. the biomass limited regime in which the
chemical production rate of Fe 2q and S 0 is much larger than the microbial capacity to
oxidize Fe 2q and S 0 , causing that ferrous iron andror elemental sulphur accumulate, Žii.
the regime that is limited by the chemical reaction rate in which the microbial capacity
to oxidize Fe 2q and S 0 is much larger than the chemical production rate of Fe 2q and
S 0 ; in this regime the concentrations of ferrous iron and elemental sulphur decrease and
will approach a pseudo-steady state.
Experiment III Žabsence of bacterial ferrous iron oxidation. and experiment V Žlargest
bacterial ferrous iron oxidation. are used in this discussion; more details of the results
are presented elsewhere w24x. Fig. 3 shows that the ferrous iron concentration in
experiment III continuously increases, whereas it decreases in experiment V. Also the
elemental sulphur concentration Žfrom degree of reduction balance. is shown in Fig. 3
and it can be seen that after about 15 h S 0 becomes a limiting substrate in both
 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186 181

Fig. 3. ŽB. wS 0 x, Ž'. wFe 2q x in bacterial batch culture of redox-stat experiment III Žbacterial Fe 2q oxidation
is absent. with wZnSx0 s 210 mM, wFe 2q x 0 s8 mM, wFe 3q x 0 s180 mM, C x,0 s 5.7)10y3 C-molrl and V
Žhighest Fe 2q oxidation capacity. with wZnSx 0 s 210 mM, wFe 2q x 0 s8 mM, wFe 3q x 0 s180 mM, C x,0 s
8.1)10y3 C-molrl. Note different scaling of wFe 2q x axes in III and V.

experiments. This explains why the oxygen and carbon dioxide consumption rate, after a
steep increase in the biomass limited regime, dramatically dropped after about 15 h in all
six experiments ŽFig. 4.. This result is consistent with the higher energy content of
sulphur electrons compared with Fe 2q.
In Fig. 5 the chemical oxidation rate of zinc sulphide and the ferric to ferrous iron
ratios in experiments III and V are shown. Despite the different ranges of wFe 3q xrwFe 2q x
Že.g. in III and V. the zinc sulphide oxidation was almost constant Žabout 0.0025 mol
ZnSrLrh. in all experiments. This would suggest that the Verbaan–Crundwell model is
incorrect and the electrochemical mechanism ŽEq. Ž1.. applies to the chemical oxidation
kinetics of ZnS. However, experiments with mineral sphalerite are needed to verify this
182 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186

Fig. 4. Typical plot of Ž . rO and ŽP P P. rCO in bacterial batch of redox-stat experiments.

2 2

result. Synthetic ZnS slurry used in these experiments was viscous and mass transfer
limitation at the reaction surface might have affected the chemical oxidation rate. It is
assumed that despite this effect the conclusion with respect to the indirect mechanism
still holds.
Because the bacterial ferrous iron oxidation was largely affected by elemental
sulphur, little can be concluded about the bacterial ferrous iron oxidation kinetics in
these experiments. Fig. 6 shows q Fe 2q Žexperiment V with highest bacterial Fe 2q
oxidation. calculated with Eq. Ž4.. The value of q Fe 2q ,max s 1 molrC-molrh, is a factor
of 9 lower than the value of q Fe 2q ,max for T. ferrooxidans on Fe 2q in the absence of
elemental sulphur. The strong decrease of q Fe 2q ,max in the presence of elemental sulphur
agrees with the findings of other authors w9,10x. It is unlikely that the loss of the ferrous
iron oxidizing capacity is caused by Zn2q inhibition. The ferrous iron oxidation kinetics
by T. ferrooxidans in the presence of zinc ions was independently measured and at
much higher zinc ion concentrations q Fe 2q ,max only decreased by a factor of 2 w5x.
These results show that independently measured ferrous iron oxidation kinetics of T.
ferrooxidans is of limited value to describe the ferrous iron oxidation kinetics in the
presence of elemental sulphur. Further research is needed to examine the ferrous iron
oxidation kinetics of T. ferrooxidans in the presence of elemental sulphur in pseudo-
steady state Ži.e. both ferrous iron and elemental sulphur are limited substrate..
It is desirable to examine the bio-oxidation kinetics of ZnS in the pseudo-steady state.
An improved experimental method to measure the chemical oxidation kinetics of ZnS
can be suggested from the present experiments. Because an indirect mechanism
determines the bacterial ZnS oxidation, it is possible to measure the chemical oxidation
kinetics of ZnS with Fe 3q in the presence of bacteria. Previously, an experimental
method for measuring the chemical oxidation kinetics of FeS 2 at high steady state values
of wFe 3q xrwFe 2q x has been developed w7,25x. This method is also applicable to measure
the chemical ZnS oxidation kinetics at high wFe 3q xrwFe 2q x. In this method L. ferrooxi-
 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186 183

Fig. 5. ŽB. wFe 3q xrwFe 2q x and Ž'. r ZnS , in experiment III Žbacterial ferrous iron oxidation is absent. and V
Žhighest Fe 2q oxidizing capacity., see Fig. 3.

dans instead of T. ferrooxidans is used to oxidize ferrous iron in batch experiments with
ZnS, and on-line off-gas analysis of O 2 and CO 2 is used to measure the bacterial
oxygen and carbon dioxide consumption rate in the slurry. Because L. ferrooxidans is
unable to oxidize reduced sulphur compounds ŽS 0 ., the bacterial ferrous iron oxidation
kinetics are not affected by elemental sulphur in the slurry. The bacterial O 2 and CO 2
consumption rates are directly coupled to the bacterial Fe 2q consumption rate. The
proposed method has several advantages over the measurement of the chemical ZnS
oxidation kinetics using the addition of hydrogen peroxide to regenerate ferric iron and
obtain high wFe 3q xrwFe 2q x: Ž1. in the case of an overshoot, H 2 O 2 might also chemically
react with ZnS. Ž2. A much higher ferric to ferrous iron ratios can be achieved. Ž3. In
the substrate limited regime a pseudo-steady state can be achieved in which
184 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186

Fig. 6. ŽB. Biomass specific ferrous iron consumption rate, q Fe 2q in the bacterial batch fermenter of
redox-stat experiment V. Ž . q Fe 2q calculated with q Fe 2q ,max s1.0 molrC-molrh, K s r K i s 0.05.

wFe 3q xrwFe 2q x remains constant over a longer period of time. In this pseudo-steady state
the ratio of wFe 3q xrwFe 2q x is varied by the staged addition of ZnS. Ž4. The most
important advantage is that at a pseudo-steady state the chemical oxidation rate of zinc
sulphide, r ZnS , is accurately and continuously determined from the on-line measured rO 2
and rCO 2 and using the degree of reduction balance for ZnS ŽEq. Ž10...

5. Conclusions

From the redox-stat experiments it was shown that an indirect mechanism determines
bio-oxidation of ZnS with T. ferrooxidans, where ZnS chemically reacts with Fe 3q, and
Fe 2q and S 0 are bacterially oxidized. Because the ferrous iron oxidation capacity of T.
ferrooxidans largely decreases in the presence of elemental sulphur it is important to
know the chemical oxidation kinetics of ZnS at high ferric to ferrous iron ratios.
However, kinetic models in the literature disagree about the effect of high wFe 3q xrwFe 2q x
ratios. Because synthetic ZnS formed a viscous slurry, mass transfer problems at the
reaction surface probably occurred. Therefore, the effect of high ferric to ferrous iron
ratios on the chemical oxidation rate of ZnS could not be determined from the
experimental data and mineral sphalerite should be used in future kinetic experiments.
Furthermore, it was suggested to use batch experiments with L. ferrooxidans to measure
the chemical oxidation kinetics of sphalerite at high ferric to ferrous iron concentration
ratios. The use of on-line O 2 and CO 2 measurement in the off-gas together with the use
of elemental and charge balances ŽEqs. Ž8. – Ž11.., is very valuable in the measurement
of the bio-oxidation kinetics of ZnS: in the transient state the concentration of elemental
sulphur is known, and in a pseudo-steady state Ži.e. substrate limited regime with
constant low concentrations of Fe 2q and S 0 ., the chemical zinc oxidation rate, r ZnS , and
bio bio
bacterial oxidation rates, r Fe 2q and r
S , are continuously known.
 M. Boon et al.r Hydrometallurgy 48 (1998) 171–186 185

The previously determined kinetic model for ferrous iron oxidation with T. ferrooxi-
dans was not applicable to describe the ferrous iron oxidation kinetics in the bacterial
batch experiments with ZnS because the Fe 2q kinetics of T. ferrooxidans were largely
affected in the presence of elemental sulphur. Another important finding was that T.
ferrooxidans can even lose its ferrous iron oxidizing capacity when elemental sulphur is
available. It is suggested that for industrial bio-oxidation applications the use of mixed
cultures of T. ferrooxidans and L. ferrooxidans is more appropriate. Then, L. ferrooxi-
dans will only oxidize ferrous iron, while T. ferrooxidans will only oxidize elemental
sulphur. Consequently, the chemical oxidation of ZnS with Fe 3q, the oxidation of
elemental sulphur by T. ferrooxidans and the oxidation of ferrous iron by L. ferrooxi-
dans, are three independent sub-processes, and from using the proposed experimental
and theoretical methods in this work the kinetics of these three sub-processes can be
accurately determined.

6. List of symbols

A0 Initial BET surface of the mineral Žm2BETrl.

Cx Biomass concentration ŽC-molrl.
Eh Redox potential ŽV.
wFe 2q x Ferrous iron concentration Žmolrl.
wFe 3q x Ferric iron concentration Žmolrl.
wFe 2q xŽ t 1 . Ferrous iron concentration at t 1 Žmol Fe 2qrl.
m Specific growth rate Žhy1 .
mmax Maximum specific growth rate Žhy1 .
ni Mineral specific rate of compound i Žmol irmol ZnSrh.
qi Bacterial specific consumption rate of compound i Žmol irC-molrh.
q Fe 2q ,max Maximum specific Fe 2q consumption rate Žmol Fe 2qrC-molrs.
rCO 2 Carbon dioxide consumption rate Žmol CO 2rlrs.
rO 2 Oxygen consumption rate Žmol O 2rlrs.
bio
r Fe 2q Bacterial ferrous iron oxidation rate Žmol Fe 2qrlrs.
bio
rS Bacterial oxidation rate of sulphur Žmol S 0rlrs.
rx Bacterial growth rate ŽC-molrlrs.
r Zn 2q Zinc ion production rate Žmolrlrs.
r ZnS Zinc sulphide oxidation rate Žmolrlrs.
YZnS, x Yield of biomass on ZnS ŽC-molrmol ZnS.
wZnSx Concentration zinc sulphide Žmol ZnSrl.

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