Inhibition kinetics of iron oxidation by Leptospirillum ferriphilum in the presence of

ferric, nickel and zinc ions

P. Nurmi
a,

,1
, B. zkaya
a,2
, A.H. Kaksonen
a
, O.H. Tuovinen
a,b
, J.A. Puhakka
a
a
Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101 Tampere, Finland
b
Department of Microbiology, Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA
a b s t r a c t a r t i c l e i n f o
Article history:
Received 7 January 2009
Received in revised form 7 February 2009
Accepted 9 February 2009
Available online 28 February 2009
Keywords:
Iron oxidation
Bioleaching zinc and nickel concentrates
Kinetics
Metal ion inhibition
Leptospirillum ferriphilum
Kinetic modelling
The inhibition kinetics of iron oxidation was studied in a Leptospirillum ferriphilum-dominated, uidized
bed bioreactor culture, which was challenged with various concentrations of Fe
3+
, Zn
2+
and Ni
2+
. Iron(II)
was oxidized in the presence of binary combinations of 40 g/L Fe
3+
+10 g/L Ni
2+
, 30 g/L Fe
3+
+40 g/L Zn
2+
,
60 g/L Zn
2+
+10 g/L Ni
2+
and 60 g/L Ni
2+
+10 g/L Zn
2+
. The data showed that L. ferriphilumwas insensitive to
the test metals, suggesting that the bacterium has application in ferric ion regeneration processes in circuits
involving tank bioleaching of zinc and nickel sulde concentrates. A combined method for determining the type
andcoefcients of inhibitionwas developedandvalidatedwithdata frombatchFe
2+
oxidationexperiments at pH
1.10.2. Iron(III) competitively inhibited Fe
2+
oxidation, whereas Zn
2+
and Ni
2+
conformed to non-competitive
inhibitionmodel withinhibitioncoefcients (K
ii
) of 49.1and62.7g/L, respectively. However, Mg
2+
andNa
+
hada
compounding effect on Zn
2+
toxicity. The combined competitive and non-competitive inhibition model was
applied for data on iron oxidation in the presence of Fe
3+
+Zn
2+
, Fe
3+
+Ni
2+
and Zn
2+
+Ni
2+
and good
agreement was generally found between the model predictions and experimental data. The combined model can
be used to predict the kinetics of bacterial iron oxidation in ferric ion regeneration for zinc and nickel concentrate
tank leaching.
2009 Elsevier B.V. All rights reserved.
1. Introduction
Microbiological leaching of sulde ores in acid solutions involves
soluble iron as a central electron shuttle (Rawlings, 2004). Ferric iron
acts as an electron sink and may be regenerated as a separate step in
some bioleaching operations. Many candidate organisms have been
proposed for ferric ion regeneration steps as the number of known
iron-oxidizing prokaryotes has increased in recent years (Hallberg and
Johnson, 2001; Hallberg and Johnson, 2003; Dopson et al., 2004;
Kupka et al., 2007). Leptospirillum ferriphilum meets with the
requirements for a mesophilic iron oxidation process. This bacterium
can oxidize iron over a broad pH range including values below pH 1
and becomes selectively enriched at high Fe
3+
concentrations
(Rawlings et al., 1999; Kinnunen and Puhakka, 2004; Plumb et al.,
2008).
There are numerous studies inthe literature onthe toxicity of metals
to acidophilic iron oxidizers. Grishin and Tuovinen (1988) used a xed
lm bioreactor to oxidize Fe
2+
by Acidithiobacillus ferrooxidans and
found substrate inhibition at high dilution rates. Kinnunen and Puhakka
(2005) studied the effects of pH, Fe
2+
, Fe
3+
and Cu
2+
concentrations on
Fe
2+
oxidation by an enrichment culture dominated by Leptospirillum
ferriphilum, which was tolerant to relatively high metal concentrations.
A. ferrooxidans was also determined to tolerate high levels of the heavy
metals tested (Cabrera et al., 2005a) whilst Kupka and Kupskov
(1999) studied the effects of Ni
2+
and Cu
2+
on the kinetics of Fe
2+
oxidation by A. ferrooxidans. Furthermore, Leduc et al. (1997), De et al.
(1997) and Sampson and Phillips (2001) have reported the resistance to
heavy metals in different strains of A. ferrooxidans and Leptospirillum
ferrooxidans. Recently, Watkin et al. (2009) demonstrated the metal
tolerances of isolates froma spent copper sulde heap, closely related to
Acidithiobacillus caldus, Acidimicrobium ferrooxidans and Sulfobacillus
thermosuldooxidans, and foundadaptationto Cu, Zn, Ni and Co. Dopson
et al. (2003) summarized the characteristics of metal resistance in
acidophilic iron-oxidizers.
Given the importance of iron oxidation by acidophilic bacteria in
bioleaching processes, the oxidation kinetics has been addressed in
several studies. A number of kinetic models for the bacterial Fe
2+
oxidation have been proposed and they can be broadly classied as
either empirical or based on MichaelisMenten/Monod rate expres-
sions (Lacey and Lawson, 1970; Kelly and Jones, 1978; Boon et al., 1999;
Ojumu et al., 2006). Generally, experimental data on Fe
2+
oxidation
kinetics with acidophilic microorganisms and the corresponding
growth rates have been adjusted to the Monod equation (Ojumu
Hydrometallurgy 97 (2009) 137145
Corresponding author. Tel.: +358 50 351 8211.
E-mail address: pauliina.nurmi@inbox.com (P. Nurmi).
1
Present address: MTTAgrifood ResearchFinland, Biotechnologyand Food Research, FI-
31600 Jokioinen, Finland.
2
Present address: Department of Environmental Engineering, Faculty of Civil
Engineering, Davutpasa Campus, Yildiz Technical University, TR-34342, Istanbul, Turkey.
0304-386X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.hydromet.2009.02.003
Contents lists available at ScienceDirect
Hydrometallurgy
j our nal homepage: www. el sevi er. com/ l ocat e/ hydr omet
et al., 2006), which is most frequently used to describe microbial
growth rate:
dX
dt
= Y
dS
dt
_ _
bX 1
dS
dt
=
q
m
XS
K
s
+ S
2
where dX/dt =bacterial growth rate, dS/dt =oxidation rate, S=sub-
strate concentration (g/L Fe
2+
), X=biomass concentration (g/L VS),
Y=yield coefcient (g VS/g Fe
2+
), q
m
=maximum specic substrate
oxidation rate (g Fe
2+
/gVSh), and K
s
=the half saturation concen-
tration (g/L Fe
2+
).
The Monod-based data analysis is applicable only to initial rates
and does not consider inhibitory effects. In the presence of toxicants,
both rates in Eqs. (1) and (2) can become slower. A common type of
inhibition is the so-called self-inhibition (Haldane kinetics), in which
the rate of enzyme-catalyzed degradation of the substrate is slowed
down because of high concentration of the substrate. Considering
substrate inhibition, Eq. (2) can be written as
dS
dt
=
q
m
XS
K
s
+ S + S
2
= K
i
3
whereK
i
(g/L) = the inhibitionconstant of the self-inhibitory substrate.
When additional inhibition occurs with the presence of a competi-
tive inhibitor at a concentrationof I, inhibitor binds tothe catalytic site of
the enzyme, which excludes substrate binding in proportion to the
degree to which the inhibitor is bound (Rittman and McCarty, 2001). In
the presence of a competitive inhibitor and considering self-inhibition,
Eq. (3) converts to following form:
dS
dt
=
q
m
SX
K
s
1 + I = K
iic
+ S + S
2
= K
i
4
where K
iic
(g/L)=the competitive inhibition constant, and I (g/L)=
the inhibitor concentration. A low value of K
iic
indicates strong
inhibition.
Another type of inhibition is non-competitive inhibition, in which
the inhibitor binds to the enzyme at a site other than the active site,
altering the enzyme conformation (Rittman and McCarty, 2001). In
the case of non-competitive inhibition, Eq. (3) converts to the
following form:
dS
dt
=
q
m
SX
K
s
+ S + S
2
= K
i
_ _
1 + I = K
iinc

5
where K
iinc
(g/L)=the non-competitive inhibition constant.
Third type of inhibition is un-competitive, in which the inhibitor
binds only to the complex formed between the enzyme and the
substrate (Rittman and McCarty, 2001). In the case of un-competitive
inhibition, Eq. (3) converts to the following form;
dS
dt
=
q
m
SX
K
s
+ S + S
2
= K
i
_ _
1 + I = K
iiuc

6
where K
iiuc
(g/L)=the uncompetitive inhibition constant, and I (g/L)=
the inhibitor concentration.
In the present study, a kinetic expression for Fe
2+
oxidation was
developed that takes into account the inhibitory effects caused by high
concentrations of Fe
3+
anddissolvedheavymetals. As reviewedbyOjumu
et al. (2006) there is a discrepancy in the literature about the competitive
versus non-competitive inhibitiontypes todescribe the inhibitingeffect of
Fe
3+
on acidophilic Fe
2+
oxidizers. In our previous study (zkaya et al.,
2007), Fe
2+
oxidation by a Leptospirillum ferriphilum dominated culture
was competitively inhibitedby Fe
3+
, andoxidationkinetics was described
as Haldane inhibition. The inhibitory effect of other metals on the
biological Fe
2+
oxidation was not, however, considered in that study.
Acidophilic Fe
2+
oxidizers are exposed to highconcentrations of Fe
3+
and
other metals inbioleachingprocesses, yet thecombinedeffects of multiple
metals have not been systematically examined. The previous kinetic
models of Fe
2+
oxidation and inhibition are applicable to only single
inhibitors, for example, aheavymetal. This workaimedat determiningthe
potential of ferric ionregenerationunder conditions applicabletoleaching
of nickel andzinc sulde concentrates, whichare among the future targets
of application (Morin, 2007; Viera et al., 2007). Further, it was
hypothesized that a model can be developed that can describe the
kinetics of ironoxidationinthepresence of multiple, potentiallyinhibitory
metals. Thus the study aimed also at developing and examining a
combined kinetic model of iron oxidation by bacteria based on the
Haldane (self-inhibition) and competitive (Fe
3+
) and non-competitive
(Zn
2+
, Ni
2+
) inhibition.
2. Materials and methods
2.1. Culture
The acidophilic iron-oxidizing culture was obtained from a
uidized bed bioreactor (FBR), which had been maintained for several
years with a mineral salts solution that contained (per L) 7 g Fe
2+
(added as ferrous sulfate), 0.35 g (NH
4
)
2
HPO
4
, 0.05 g K
2
CO
3
, and
0.05 g MgSO
4
at pH 0.9. Due to the high recycle ratio of the FBR, iron
was present as Fe
3+
during long-term operation. The microbial
community in the bioreactor was monitored by phase contrast
microscopy and by denaturing gradient gel electrophoresis (DGGE)
of PCR-amplied partial 16S rRNA genes. DGGE analysis was
performed with DNA extracted from the bioreactor carrier matrix
(activated carbon) at different time intervals. The enrichment culture
was dominated by L. ferriphilum (zkaya et al., 2007).
At the beginning of the experiments, the amount of biomass (as
volatile solids, VS) in the inoculum, originating from the FBR, was
measured using the detachment procedure described by Kinnunen
and Puhakka (2004). The VS concentration of the detached bio-
mass was determined according to the Standard Methods (1992).
In batch experiments, the concentration of initial biomass was kept
high (12520 mg/L VS) to accelerate the Fe
2+
oxidation rate. The
initial Fe
2+
concentration was 4 g/L Fe
2+
(added as ferrous sulfate) in
all cases. Considering the quantity of Fe
2+
oxidized and using a net
yield coefcient of 2.4 mg VS/g Fe
2+
(Smith et al., 1988), the biomass
yield was estimated as 10 mg/L VS. Therefore, the biomass growth
in our batch assays was insignicant due to high initial biomass
concentration.
2.2. Experimental
Batch experiments for Fe
2+
oxidation were conducted in 250 mL
conical asks containing 200 mL of mineral salts solution and 3 mL of
carrier material from the bioreactor. Flasks were incubated at 27 C
and at 160 rpmon a rotary shaker. To study the effect of Ni
2+
and Zn
2+
on iron oxidation, the L. ferriphilum bioreactor culture received
different concentrations (060 g/L) of metals. To determine the
simultaneous effects of Fe
3+
and Ni
2+
or Zn
2+
on Fe
2+
oxidation, the
culture was supplemented with different levels of Fe
3+
(added as Fe
2
(SO
4
)
3
) at xed zinc or nickel concentrations (10 g/L, added as
ZnSO
4
7H
2
O and NiSO
4
6H
2
O). Furthermore, the culture was sub-
jected to multiple inhibitory metals concentrations of different
levels of Zn
2+
(060 g/L) and xed Ni
2+
(10 g/L), different levels of
Ni
2+
(060 g/L) and xed Zn
2+
(10 g/L) and different levels of Fe
3+
(030 g/L) and Zn
2+
(040 g/L). These high levels reect concentra-
tions that may be attained in tank bioleaching applications with zinc
and nickel sulde concentrates. Additionally, the effect of Mg
2+
and
Na
+
ions on Zn
2+
toxicity was studied by simultaneously exposing the
culture to Zn
2+
(10 g/L) and Mg
2+
(added as MgSO
4
7H
2
0) or Na
+
(added as Na
2
SO
4
). The initial pH of the mineral salts solution was 1.0,
therefore ferric iron did not precipitate in these experiments.
138 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145
Fig. 1. Substrate (Fe
2+
) utilizationproles inthepresence of A: Zn
2+
; B: Ni
2+
; C: 10g/L Ni
2+
+varyingZn
2+
; D: 10g/L Zn
2+
+varyingNi
2+
; E: 10g/L Zn
2+
+varyingFe
3+
; F: 20g/L Zn
2+
+
varying Fe
3+
; G: 30 g/L Zn
2+
+varying Fe
3+
; H: 40 g/L Zn
2+
+varying Fe
3+
; I: 10 g/L Ni
2+
+varying Fe
3+
(Variable key: :0, :5, :10, x:20, :30, :40, +:50, :60 g/L).
139 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145
Samples were ltered through membrane lters (0.45 m pore
size). Fe
2+
concentrations were measured immediately after sampling
using the colorimetric ortho-phenanthroline method, according to
modied 3500-Fe (Standard Methods, 1992) to accommodate a 10 L
sample size. For pH measurements, the pH meter was calibrated with
citrate buffer solutions (pH 4 and 2) using two point calibration.
Redox potential was measured using an Ag/AgCl electrode.
2.3. Kinetic modeling
The baseline Fe
2+
oxidation studies in the absence of other metals
were described in our previous study (zkaya et al., 2007). The kinetic
parameters of Fe
2+
oxidation were determined by using the Haldane
model to describe the specic Fe
2+
oxidationrate. The inhibitionof Fe
2+
oxidation by Fe
3+
was described by combining the competitive
inhibition and Haldane models.
The inhibition models described above are applicable only to a single
inhibitor. In the present study, the following additional combined
competitive (Fe
3+
) and non-competitive (Zn
2+
and Ni
2+
) model was
used;

dS
dt
=
q
m
SX
K
s
1 +
I
Fe
3+
K
iiFe
3+
_ _
+ S + S
2
= K
i
_ _
1 +
I
1
K
ii1
+ 1 +
I
2
K
ii2
+ N + 1 +
I
n
K
iin
_ _ 7
where I
Fe
3+
=Fe
3+
i.e. competitive inhibitor concentration, and I
n
=the
concentration of the non-competitive inhibitors, e.g., I
1
=Zn
2+
or
I
2
=Ni
2+
concentration. This model can be used for solutions containing
high substrate concentrations (Fe
2+
) and competitive (Fe
3+
) and non-
competitive inhibitors (both Ni
2+
and Zn
2+
) at the same time. Thus it is
Fig. 2. Specic substrate utilization rates (g Fe
2+
/g VS
.
h) in the presence of A: 10 g/L Zn
2+
and varying amount of Fe
3+
or Ni
2+
, B: 10 g/L Ni
2+
and varying amount of Fe
3+
or Zn
2+
,
and C: 1040 g/L Zn
2+
and varying amount of Fe
3+
.
140 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145
applicable to the bioleaching conditions for multi-metal ores. The
competitive andnon-competitive kinetic expressions were developed to
a combined model by Jin and Bhattacharya (1996). In the present study,
the model was modied to describe the combined effects of two non-
competitive inhibitors. Conceptually, the combined model can be
extended to additional inhibitors by derivation and reiterating the
combined denominator approach in Eq. (7).
2.4. A combined method for determining inhibition type and kinetic
parameters
In the present study, single (Zn
2+
and Ni
2+
) and combined (Fe
3+

Zn
2+
, Fe
3+
Ni
2+
and Zn
2+
Ni
2+
) effects were determined on Fe
2+
oxidation using the L. ferriphilum dominated culture. Kinetic para-
meters and inhibition types were determined by nonlinear tting;
whilst the performance of the basic inhibition models and the
combined inhibition model were tested by solving the Fe
2+
oxidation
rate from the differential equations.
In determining the inhibition types, a common technique is to
compare the values of K
s
and q
m
determined in the presence and
absence of inhibitors. According to this technique, K
s
should change in
competitive inhibition, but not q
m
. Incontrast, K
s
valueremains constant,
but q
m
should decrease in non-competitive inhibition. However, to
examine the type of Zn
2+
and Ni
2+
inhibitions, the following two steps
were followed: (1) Competitive (Eq. (4)), non-competitive (Eq. (5)) and
un-competitive ((Eq. (6)) inhibition constants of K
iic
, K
iinc
and K
iiuc
were
determined by nonlinear curve ttings of specic substrate utilization
rates (g Fe
2+
/g VSh) corresponding to different levels of inhibitor
concentrations (Zn
2+
, Ni
2+
). To evaluate the goodness of the ts, the
sumof squares due to error (SSE), R
2
, residual degrees of freedom(DFE),
adjusted R
2
, and root mean square errors (RMSE) associated with the
output model result were calculated. (2) The differential equations of the
competitive and non-competitive inhibition models were solved and the
results compared to the experimental results, and the most appropriate
inhibition model was chosen by comparing the values of R
2
and other
goodness of t parameters (adjusted-R
2
, root mean squared error
(RMSE), variance and standard deviation). Similar kinetic studies have
been presented that examine inhibition types by solving the integrated
form of substrate utilization (Jin and Bhattacharya, 1996; Molchanov et
al., 2007). Moreover, measured and predicted Fe
2+
concentrations
including 122 data points from eight batch assays with Zn
2+
or Ni
2+
as
the only inhibitor were subjected to regression analysis to conrm inhi-
bitiontypes. ThePOLYMATH6.1packageprogramwas usedfor regression
analysis. For nonlinear equation solving in step 1, the MATLAB

7 (R14)
packageprogramwas used, andtheinhibitionmodels werettedtobatch
kinetic data by nonlinear regression with a TrustRegion Reective
Newton algorithm. The model results and coefcients were calculated
with 95% condence interval. The best tting model for the data set was
dened. To evaluate the goodness of the ts, the sum of squares due to
error (SSE), R
2
, residual degrees of freedom (DFE), adjusted R
2
, and root
meansquare errors (RMSE) associatedwiththe output model result were
calculated. Inhibition type performance was tested based on Fe
2+
oxidation rates (Eqs. (4) and (5)) by POLYMATH 6.1, which used the
MATLAB

7 and the RungeKuttaFehlberg (RFK) numerical integration

routine (step 2). The program integrates the system of differential
equations using the RFK algorithm. All differential equations describing
the inhibition were solved; and POLYMATH 6.1 was used to test the
performance of the equations with observed kinetic constants.
3. Results and discussion
In the previous study by zkaya et al. (2007), the kinetic
parameters of Fe
2+
oxidation by L. ferriphilum dominated culture
were determined in the range of 0.120 g/L Fe
2+
, and the effect of
ferric iron (060 g/L Fe
3+
) on Fe
2+
oxidation was examined at pH 0.8.
The specic Fe
2+
oxidation rates increased up to about 4 g/L Fe
2+
and
then started to decrease above this threshold concentration. The
L. ferriphilum culture oxidized Fe
2+
at pH 0.8 even in the presence of
60g/L Fe
3+
but soslowly that it was not consideredfor kinetic modeling
in the work. For the present study, the initial Fe
2+
concentration was
selectedas 4g/L Fe
2+
abovewhichsubstrate inhibitionoccurs. Substrate
inhibition was not considered any further in the present study.
3.1. Effect of metals on iron oxidation
The ferrous iron oxidation in the test bottles in the presence of
different levels of the metals is presented in Fig. 1. The specic iron
oxidation rates are summarized in Fig. 2 showing that the L. ferriphilum
culture tolerated highconcentrations of the metals tested. Concerning the
tolerance to individual metals, the culture was able to oxidize ferrous iron
evenat thepresenceof thehighest concentrations of Zn
2+
andNi
2+
tested
(Fig. 1A,B). The initial 4 g/L Fe
2+
was completely oxidized within 70 days
(at the rate of 0.50 g Fe
2+
/g VSh) in the presence of 60 g/L Zn
2+
and
within45days (at therateof 0.71gFe
2+
/gVSh) inthepresenceof 50g/L
Ni
2+
. It canbeconcludedthat theculturetolerates higher amounts of Ni
2+
than Zn
2+
. Cabrera et al. (2005a) observed that 30 g/L Ni
2+
, 15 g/L Ni
2+
,
30 g/L Zn
2+
and40 g/L Zn
2+
were the highest toleratedconcentrations of
Zn
2+
for a pure culture of A.ferrooxidans and a mixed culture of A.
ferrooxidans and Acidiphilium spp., respectively. The maximum tolerated
concentration of Ni
2+
by A. ferrooxidans has been reported to vary from
10 g/L Ni
2+
to upto 60g/L withsome strains (Cabrera et al., 2005a; Leduc
et al., 1997; Dopson et al., 2003). Also the combined effects of the metals
weretested(Fig. 1CI). Combiningvarious concentrationof Zn
2+
to10g/L
of Ni
2+
hadonlya relativelysmall effect comparedtotheindividual effects
of Zn
2+
. For the combination of various concentrations of Ni
2+
and 10 g/L
of Zn
2+
the combining effect was slightly larger and the iron oxidation
rates werethus smaller. Therate of ironoxidationinthepresenceof 10g/L
Ni
2+
+60g/L Zn
2+
was 0.47gFe
2+
/gVSh, and0.49gFe
2+
/gVShinthe
presence of 10 g/L Zn
2+
+60 g/L Ni
2+
. Iron was oxidized even in the
presence of the highest concentrations tested.
From the experiments combining the effects of Zn
2+
or Ni
2+
and
ferric iron (Fig. 1EI) it can be seen that ferric ion is more inhibitory to
ironoxidationby the culture thanZn
2+
or Ni
2+
. Inthe presence of 10g/L
Zn
2+
the culture tolerated additionally 40 g/L of Fe
3+
(oxidation rate
0.32 g Fe
2+
/g VSh, Fig. 1E). Inthe presence of 20g/L Zn
2+
and40 g/L of
Fe
3+
, iron(II) was still oxidized at a rate of 0.07 g Fe
2+
/g VSh (Fig. 1F).
When the Zn
2+
was increased to 30 g/L the iron oxidation rate
decreased to a negligible 0.03 g Fe
2+
/g VSh in the presence of 40 g/L
Fe
3+
(Fig. 1G).
With 40 g/L Zn
2+
(Fig. 1H) the maximum concentration of Fe
3+
tested was 30 g/L since it was known from previous experiments that
the culture cannot tolerate higher concentrations. For the combined
effect of Ni
2+
and Fe
3+
the maximum concentrations tolerated were
10 g/L Ni
2+
+40 g Fe
3+
(oxidation rate 0.21 g Fe
2+
/g VSh, Fig. 1I).
Very little is available in the literature on combined effects of
several metals on iron oxidation kinetics by acidophilic leptospirilli or
acidithiobacilli. Das et al. (1997) and Li and Ke (2001a) tested binary
and ternary combinations of potentially toxic metals on iron oxidation
by A. ferrooxidans. While the results showed synergistic toxicity and
acclimation for improved leaching, no kinetic analysis of inhibition
was undertaken.
3.2. Types of inhibition and kinetic constants
In our past observations (zkaya et al., 2007), predictions of Fe
2+
oxidation in the presence of Fe
3+
by competitive inhibition model
have given better ts than the other models. When the kinetic data
with different initial metal concentrations at a constant initial
substrate concentration (4 g Fe
2+
/L) were applied to (Eqs. (4)(6)),
the competitive inhibition model gave better t for Fe
3+
toxicity,
yielding the kinetic constants of q
m
=2.4 g/g VSh, K
s
=0.413 g/L,
K
i
=8.65 g/L and K
ii
=0.83 g/L. The Zn
2+
toxicity conformed, however,
141 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145
to the non-competitive inhibition model with the kinetic constants of
q
m
=2.04 g/g VSh, K
s
=0.410 g/L, K
i
=8.65 g/L and K
ii
=49.1 g/L. This
was consistent with the q
m
, K
s
, K
i
values at different substrate
concentrations described by the Haldane substrate inhibition model in
our previous study (zkaya et al., 2007). The Ni
2+
toxicity was non-
competitive with the kinetic constants of q
m
=2.01 g/g VSh,
K
s
=0.410 g/L, K
i
=8.65 g/L and K
ii
=62.7 g/L. A low value of K
ii
indicates strong inhibition.
The Fe
2+
oxidation rates (dS/dt) were tested by the observed kinetic
constants for competitive and non-competitive inhibition models in the
presence of Zn
2+
and Ni
2+
(Fig. 3). The prediction of competitive
inhibition model did not t well the Zn
2+
and Ni
2+
toxicity kinetic data,
whereas the non-competitive inhibition model gave good ts. The
competitive inhibition model runs yielded R
2
values of 0.89 and 0.95
(adj-R
2
values of 0.88 and 0.95) inthe presence of 50 g/L Zn
2+
and 50 g/
L Ni
2+
(Fig. 3A), and the corresponding non-competitive model runs
were characterized by R
2
values of 0.94 and 0.99 (adj-R
2
values of 0.92
and 0.99), respectively. In addition, the root mean squared errors of the
ts by the non-competitive inhibition (0.105, 0.043) were smaller than
those obtained by tting the competitive model (0.147, 0.079). The
variance of the model curves and closely-related standard deviation, the
square root of the variance, were also evaluated, and the variance of the
non-competitive modeling curves were considerably smaller than that
of the competitive curves (Fig. 3). Fig. 3C shows regression analysis
between measured and predicted data with the competitive and non-
competitive inhibition models of Fe
2+
concentrations in all the
experiments conducted with Zn
2+
or Ni
2+
as the only inhibitor.
Predicted non-competitive data are very close to the measured data
(giving a linear line, y=x, with high correlation). Therefore, it can be
concluded that Zn
2+
and Ni
2+
non-competitively inhibited Fe
2+
oxidation by L. ferriphilum dominated culture. The un-competitive
inhibition model was also tested, but the predictions of the un-
competitive inhibition model did not t well the Zn
2+
and Ni
2+
toxicity
kinetic data (data not shown). The ts were not as good as they were for
the competitive inhibition model presented in this study.
3.3. Effect of Zn
2+
and Ni
2+
on Fe
2+
oxidation
The competitive and non-competitive inhibition models were
evaluatedfor different Zn
2+
concentrations (060 g/L) andinitial Fe
2+
concentrationof 4 g/L. The best t K
ii
value (R
2
=0.97) was 49.1 g/L for
Zn
2+
with the non-competitive inhibition model (Fig. 4A). The K
ii
value of Ni
2+
was 62.7 g/L (R
2
=0.97). These non-competitive
inhibition constants showed that the L. ferriphilum culture tolerated
high Zn
2+
and Ni
2+
concentrations and that Zn
2+
was more toxic than
Ni
2+
. The values of K
ii
obtained by Cabrera et al. (2005b) by applying
the non-competitive inhibition models were 132 and 125 g/L Zn
2+
for
A. ferrooxidans in pure and in a mixed culture with Acidiphilium spp.,
respectively. The corresponding K
ii
values for Ni
2+
were 89.2 and
98.6 g/L Ni
2+
(Cabrera et al., 2005b). For reference, Kupka and
Kupskov (1999) reported a much lower K
ii
value of 13.7 g/L Ni
2+
.
Iron(II) oxidation kinetics (dS/dt) was predicted with the help of the
observed kinetic constants (q
m
, K
s
, K
i
and K
ii
) and the differential
equation (Eq. (5)) was numerically solved. The Fe
2+
oxidation prole
with respect to the incubation time in the presence of different
Fig. 3. Competitive (dash line) and non-competitive (solid line) inhibition model
predictions for substrate utilization in the presence of A: 50 g /L Zn
2+
; B: 50 g /L Ni
2+
.
In Fig. 3C non-competitive () and competitive () data are marked for 122
observations. The solid line shows: y (predicted)=x (measured).
Fig. 4. A: Specic Fe
2+
oxidation rates (g Fe
2+
/g VSh) versus 060 g/L Fe
3+
(); Zn
2+
(); and Ni
2+
(), B and C: Measured and predicted Fe
2+
concentrations in the
presence of Zn
2+
and Ni
2+
(Key: :10, :20, :30, :50, :60 g/L). Lines represent non-
competitive inhibition model predictions.
142 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145
concentration of Zn
2+
(060 g/L) and Ni
2+
(060 g/L) are shown in
Fig. 4B and C. Goodness of tting parameters in Fig. 4B: R
2
=0.97, RMSE:
0.0458, SSE: 0.0147; andFig. 4C: R
2
=0.97, RMSE: 0.038, SSE: 0.0102. The
stoichiometric elemental formula for microbial cell is approximately
C
5
H
9
O
2.5
N (Jones and Kelly, 1983; Rittmann and McCarty, 2001). In the
present study, the maximum specic iron oxidation rate (q
m
) can be
converted to 5.27 mol Fe
2+
/mol cellh considering the formula given
above. As a point of reference, Boon (1996) determined a q
max
of 8.8 mol
Fe
2+
/mol cellhfor continuous culture of A. ferrooxidans and Breedet al.
(1999) reported 8.65 mol Fe
2+
/mol cellh for L. ferrooxidans.
3.4. Simultaneous effects of Fe
3+
and heavy metals (Zn
2+
or Ni
2+
) on
Fe
2+
oxidation
A combined competitive and non-competitive inhibition model
(Eq. (7)) was used to examine the simultaneous effects of Fe
3+
, Zn
2+
,
and Ni
2+
on Fe
2+
oxidation. The t of the combined model with the
experimental data from the combined Fe
3+
and Zn
2+
and Ni
2+
experiments is shown in Fig. 5AD. The highest Fe
3+
concentration
data displayed some deviation especially in the case of Ni
2+
.
Fig. 6shows theFe
2+
oxidationproles withrespect totimeat different
initial levels of Fe
3+
(1030 g/L Fe
3+
) and Zn
2+
(1040 g/L Zn
2+
).
Simultaneous maximum tolerable concentrations of Fe
3+
and Zn
2+
were 30 g/L Fe
3+
and 40 g/L Zn
2+
and the tted curves of the developed
model matched the experimental data. Ferrous ionwas slowly oxidized at
4060 g/L Fe
3+
concentrations but these data did not t the com-
bined model and are therefore excluded from this paper. Thus the
models cannot be used to describe situations that involve concentra-
tions N40 g/L Fe
3+
.
Figs. 7 and 8 show the ts in the presence of only two non-
competitive inhibitors (Zn
2+
and Ni
2+
). The culture oxidized Fe
2+
in
the presence of two non-competitive inhibitors at concentrations of
60 g/L Zn
2+
+10 g/L Ni
2+
and 60 g/L Ni
2+
+10 g/L Zn
2+
.
The proposed model is a useful tool to predict the Fe
2+
oxidation
rate in the presence of Fe
3+
and other metals. Such predictions will
help to determine the solids retention time and the volume of
bioreactors for Fe
2+
oxidation in bioleaching applications, in which
acidophilic iron oxidizers are in contact with high Fe
3+
and metal
concentrations. At present, the designer of a bioleaching operation has
to depend on very limited qualitative information on inhibition. The
Fig. 5. Specic substrate utilization rates (g Fe
2+
/g VS
.
h) in the presence of 030 g/L Fe
3+
and A: 10 g/L Zn
2+
or B: 10 g/L Ni
2+
. Experimental data (); combined competitive and
non-competitive inhibition model predictions for Fe
2+
concentration proles in the
presence of Fe
3+
(g/L Fe
3+
:10, *:20, +:30) and C: 10 g/L Zn
2+
, or D: 10 g/L Ni
2+
.
Fig. 6. Combined kinetics model prediction performances for Fe
2+
concentration
proles in the presence of high concentrations of Zn
2+
and Fe
3+
(g/L Fe
3+
; :10, :20,
:30) A: 10 g/L Zn
2+
; B: 20 g/L Zn
2+
; C: 30 g/L Zn
2+
and D: 40 g/L Zn
2+
.
143 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145
Fe
2+
oxidation rates of the L. ferriphilum culture can be calculated
using the following equation with observed kinetic constants in the
presence of Fe
3+
, Zn
2+
and Ni
2+
.

dS
dt
=
2:15 F 0:25 Fe
2 +
_ _
X
0:413 1 +
Fe
3 +

0:828
_ _
+ Fe
2 +
_
+ Fe
2 +
_
2
= 8:65
_ _
1 +
Zn
2 +

49:1
+ 1 +
Ni
2 +

62:6
_ _
8
This equation was derived from the parameters of the competitive
and non-competitive kinetic models for single toxicants.
3.5. Effect of Mg
2+
and Na
+
ions on Zn toxicity
Metal toxicity may be the result of metal ions reacting with binding
sites onthecell surface. Thesemaybeactivebindingsites or transport sites
associatedwithmetal uptakesystems. Furthermore, Ca
2+
, Mg
2+
, Na
+
and
K
+
cancompetewithmetal ions for binding sites at the cell surface (Plette
et al., 1996). The results showed that high levels of Mg
2+
and Na
+
adverselyaffectedthespecic rates of Fe
2+
oxidation. BothMg
2+
andNa
+
at 10g/Lhadacompoundingeffect onZn
2+
toxicity(Fig. 9AandB). Dueto
their toxicity at these levels, partial alleviation of Zn
2+
toxicity through
competition by Mg
2+
or Na
+
for binding sites could not be demonstrated
in this study. Li and Ke (2001a,b) also reported on the toxicity of Mg
2+
to iron oxidation, with complete inhibition observed at 20 g/L Mg
2+
.
These data suggested that the accumulation of Mg
2+
and Na
+
in leach
solution circuits may have a negative impact on the biological regenera-
tion of iron.
4. Conclusions
The study was standardized to relatively high biomass concentration
to avoid the effect of selection pressure on culture composition. The pH
was kept below the threshold for precipitation of ferric iron and the
other test metals. This way the bioavailability of metals was not affected
by formation of poorly soluble complexes. A combined method for
determining inhibition types of toxic metals (Fe
3+
, Zn
2+
, Ni
2+
) was
developed and applied to data analysis in this study. Iron(II) oxidation
was non-competitively inhibited by Zn
2+
and Ni
2+
ions with the
inhibition constants (K
ii
) of 49.1 and 62.7 g/L, respectively but it
was competitively inhibited by Fe
3+
. The order of increasing toxicity
was Fe
3+
NZn
2+
NNi
2+
. Iron(II) oxidation was relatively insensitive to
Fe
3+
, Zn
2+
and Ni
2+
. Iron(II) oxidation proceeded in the presence of
binary combinations of 30 g/L Fe
3+
+40 g/L Zn
2+
, 40 g/L Fe
3+
+10 g/L
Ni
2+
, 60 g/L Zn
2+
+10 g/L Ni
2+
and 60 g/L Ni
2+
+10 g/L Zn
2+
. Thus it
is possible to use high concentrations of ferric sulfate in bio-
hydrometallurgical applications including tank leaching systems
where the concentrations of dissolved metals can approach levels that
were tested for Zn
2+
and Ni
2+
in this study.
The results of this study demonstrate that L. ferriphilum is tolerant
to high levels of metal ions in ferrous sulfate medium. The low pH
value of 1.0 prevents metal precipitation and thus the metal additions
represented the bio-available levels. Precipitation of Fe
3+
in the form
of Fe(III) hydroxysulfates occurs at pHN1.5.
Kinetic evaluation of Fe
2+
oxidation in the presence of multiple
metals is one of the keys to reactor design because the kinetics must
be considered when determining the dimensions and residence times.
Lag phases were not considered in the kinetic models and therefore
the initial iron oxidation data deviated from the predicted lines in
some experiments. The models also failed to describe iron oxidation at
strongly inhibitory concentrations of metals (N40 g/L) because the
type of inhibition deviated from the three kinetic models used in this
study. This nding suggests that the models cannot predict mixed
inhibitory effects affecting substrate oxidation and growth at very
high toxicant concentrations. Despite these limitations, the examples
in this study demonstrate that the developed model is applicable in
bioleaching processes as long as the kinetic constants are determined
for process-specic metals and organisms.
Fig. 7. Kinetics model performances for substrate (Fe
2+
) utilization proles in the
presence of 10 g/L Ni
2+
and increasing Zn
2+
(g/L Zn
2+
; :0, :10, :30, :50, :60).
Fig. 8. Kinetics model performances for substrate (Fe
2+
) utilization proles in the
presence of 10 g/L Zn
2+
and increasing Ni
2+
(g/L Ni
2+
; :0, :10, :30, :50, :60).
Fig. 9. Iron oxidation rates as inuenced by 10 g/L Zn
2+
, Mg
2+
or Na
+
. A: Fe
2+
concentration proles (:control (without inhibitor), :Zn
2+
, +:Mg
2+
, x:Na
+
, :Zn
2+
+
Na
+
, :Zn
2+
+Mg
2+
. B: Specic Fe
2+
oxidation rates (g Fe
2+
/g VSh) in the assay with
10 g/L metal ions; a: control (without inhibitor), b: Zn
2+
, c: Mg
2+
, d: Na
+
, e: Zn
2+
+Na
+
,
f: Zn
2+
+Mg
2+
.
144 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145
Acknowledgements
This work was carried out in the frame of BioMinE (European
project contract NMP1-CT-500329-1). The authors acknowledge the
nancial support given to this project by the European Commission
under the Sixth Framework Programme for Research and Develop-
ment. Partial salary and research support were provided to B. zkaya
by the Scientic and Technological Research Support Council of Turkey
(TUBITAK) and to O.H. Tuovinen by the Finnish Funding Agency for
Technology and Innovation (Finland Distinguished Professor Program,
402/06).
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Glossary
Term denition
I: Inhibitor concentration (g/L)
K
i
: Inhibitor constant for substrate inhibition (g/L Fe
2+
)
K
ii
: Inhibition constant (g/L)
K
S
: Half saturation constant (g/l Fe
2+
)
q
m
: Maximum specic substrate oxidation rate (g Fe
2+
/g VSh)
S: Substrate concentration (g/L Fe
2+
)
VS: Volatile solids
X: Biomass concentration (g VS/L)
Y: Yield coefcient (g VS/g Fe
2+
)
145 P. Nurmi et al. / Hydrometallurgy 97 (2009) 137145