Chemical Engineering and Processing 46 (2007) 935940

Optimization of ferrous biooxidation rate in a packed

bed bioreactor using Taguchi approach
S.M. Mousavi
a,b,
, S. Yaghmaei
a
, A. Jafari
c
, M. Vossoughi
a
, Z. Ghobadi
d
a
Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
b
Department of Chemical Engineering, Lappeenranta University of Technology, Lappeenranta, Finland
c
Department of Energy and Environmental Engineering, Lappeenranta University of Technology, Lappeenranta, Finland
d
Biochemical and Bioenvironmental Research Centre (BBRC), Sharif University of Technology, Tehran, Iran
Received 21 May 2007; received in revised form 26 June 2007; accepted 27 June 2007
Available online 3 July 2007
Abstract
The biological oxidation of ferrous ion by iron-oxidizing bacteria is potentially a useful industrial process for removal of H
2
S from industrial
gases, desulphurization of coal, removal of sulfur dioxide from ue gas, treatment of acid mine drainage and regeneration of an oxidant agent
in hydrometallurgical leaching operations. The main purpose of this study was to nd optimum values of the process parameters on the ferrous
biooxidation rate by immobilization of a native Sulfobacillus species on the surface of low density polyethylene (LDPE) particles in a packed-bed
bioreactor using Taguchi method. Five control factors, including temperature, initial pH of feed solution, dilution rate, initial concentration of Fe
3+
and aeration rate in four levels are considered in Taguchi technique. L
16
orthogonal array has been used to determine the signal to noise (S/N) ratio.
Analysis of variance (ANOVA) was used to determine the optimum conditions and most signicant process parameters affecting the reaction rate.
Analysis of the experiments using Taguchi method indicated that pH of feed solution has the most contribution in the biooxidation rate of ferrous
ion. The biological reaction rate was obtained 8.4 g L
1
h
1
by setting the control factors according to the Taguchi approach. Finally, based on the
primary results, a verication test was also performed to check the optimum condition.
2007 Elsevier B.V. All rights reserved.
Keywords: Optimization; Ferrous biooxidation rate; Sulfobacillus species; Packed bed bioreactor; Taguchi approach
1. Introduction
Hydrogen sulde and sulfur dioxide are two well-known
environmental contaminants originate from industrial oper-
ations such as coke production, viscose rayon production,
wastewater treatment, wood pulp production using sulfate
process, oil rening process, tanning of leather and during
combustion of fossil fuels containing sulfur. These are the
main causes of global environmental problems such as air
pollution and acid rain. Acid-mine drainage also is a major
environmental problem in terrains affected by untreated acid
waters.
A number of physico-chemical processes such as dry gas
redox process, liquid redox processes and liquid adsorption
process are usually employed for desulphurization of gases

Corresponding author. Tel.: +98 21 66165494; fax: +98 21 66005417.

E-mail address: mousavi@mehr.sharif.edu (S.M. Mousavi).
containing hydrogen sulde; however, they have high capital
costs, demand large energy inputs and result in the generation
of secondary hazardous wastes [1]. Several physical means of
controlling the formation of acid-mine drainage were devel-
oped but they were not very successful. Therefore, efforts were
directed towards biochemical processes, which are character-
ized by small capital costs and low energy requirements for the
contaminants removal.
The use of microorganisms capable of oxidizing H
2
S and
producing elementary sulfur or sulfate from a complete and/or
incomplete metabolism has been considered as a potential alter-
native for the large-scale treatment of this gas [24]. In the
bioprocess of H
2
S removal an aqueous Fe
2
(SO
4
)
3
solution is
used as an absorbent. H
2
S is absorbed and oxidized to elemen-
tal sulfur. At the same time, Fe
3+
is reduced to Fe
2+
according
to
H
2
S + Fe
2
(SO
4
)
3
S
0
+2FeSO
4
+H
2
SO
4
. (1)
0255-2701/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.cep.2007.06.010
936 S.M. Mousavi et al. / Chemical Engineering and Processing 46 (2007) 935940
Elemental sulfur is removed fromthe solution by a separator,
and the reactant Fe
3+
is regenerated from Fe
2+
by biological
oxidation in an aerated bioreactor according to the following
reaction
2FeSO
4
+H
2
SO
4
+0.5O
2
bacteria
Fe
2
(SO
4
)
3
+H
2
O. (2)
Biological removal of sulfur dioxide from ue gas has also
been reported in the literature [5,6]. This process is based on the
wet scrubbing of the gas stream with a ferric sulfate solution
SO
2
+Fe
2
(SO
4
)
3
+2H
2
O
bacteria
2FeSO
4
+2H
2
SO
4
. (3)
The resultant ferrous sulfate solution is deoxidized to the fer-
ric state, using iron oxidizing bacteria. The ferric sulfate solution
produced is then recycled to the wet scrubbing tower to repeat
the cycle.
The process of microbiological desulphurization has been
applied for the quality improvement of coals used as a fuel or a
raw material in the chemical industry. In nature the pyritic sul-
fur oxidation of coal is a process that happens quite slowly. This
process can be accelerated in the presence of certain microor-
ganisms. Biological desulphurization has attractions because
it operates at close to ambient temperatures and involves no
associated loss of coal carbon [7].
Acid waters from active and abandoned coal mines are high
in acidity and dissolved solids and often characterized by low
pH values. The oxidation of pyritic iron sulde to SO
4
2
and
Fe
2+
is responsible for the high acidity. Under acidic conditions,
pyritic oxidation proceeds by the reactions (2) and (4):
FeS
2
+
7
2
O
2
+H
2
O FeSO
4
+H
2
SO
4
(4)
The major inorganic oxidation reaction follows Eq. (4). The
reaction indicated in Eq. (2) is much faster than the reaction (4).
Mostly, in these biological processes the iron-sulfur oxidiz-
ing bacteria such as A. ferrooxidans and A. thiooxidans are used
[812]. Recently, with the heightened awareness of environmen-
tal problems attendant with the use of high sulfur coal and H
2
S
containing gas, there has been a renewed interest in the use of
microorganisms in the processes of coal and gas desulphuriza-
tion [13,14].
In recent years, some studies [1517] have been focused on
improving the rate of biooxidation of Fe
2+
. Many types of reac-
tors operating under both batch and continuous regimes have
been studied in order to obtain better results particularly using
A. ferrooxidans and less attention has been paid to the other
species. On the other hand, there is no scientic literature about
the application of Taguchi technique to maximize ferrous iron
biooxidation rate in the bioreactors.
The Taguchi method was developed by Genichi Taguchi
between 1950 and 1960 to improve the implementation of total
quality control in Japan [18]. The goal of this method is to nd
out the optimal and robust product or process characteristic that
has a minimized sensitivity to noises. Taguchi design can deter-
mine the effect of factors on characteristic properties and the
optimal conditions of factors. This method is a simple and sys-
tematic approach to optimize design for performance, quality
and cost [1921]. In the Taguchi approach orthogonal arrays and
analysis of variance (ANOVA) are used as the tools of analysis.
ANOVA can estimate the effect of a factor on the characteristic
properties and experiment can be performed with the minimum
replication using the orthogonal arrays. Conventional statisti-
cal experimental design can determine the optimal condition on
the basis of the measured values of the characteristic properties
while Taguchi method can determine the experimental condi-
tion having the least variability as the optimal condition. The
variability is expressed by signal to noise (S/N) ratio. The terms
signal and noise represent the desirable and undesirable val-
ues for the output characteristic, respectively. Taguchi method
uses the S/N ratio to measure the quality characteristic deviat-
ing from the desired value. The experimental condition having
the maximum S/N ratio is considered as the optimal condition
as the variability characteristics is inversely proportional to the
S/N ratio [22].
This paper describes a case study investigating the parame-
ters that inuence biooxidation rate of ferrous iron using a native
Sulfobacillus species, in a packed-bed bioreactor. Concentration
of Fe
3+
in efuent of bioreactor and rate of biological ferrous
oxidation are the key factors for evaluating the performance of
bioreactor. Factors such as temperature, initial pH, dilution rate,
initial Fe
3+
concentration and rate of aeration affect the bioox-
idation rate of ferrous ion. The main objective was using the
Taguchi approach to nd a combination of effective parameters
to achieve high ferrous biological oxidation rate.
2. Materials and methods
2.1. Microorganism and medium
The microorganism used in this study was originally iso-
lated from the sphalerite concentrate of Kooshk lead and zinc
mine (Yazd-Iran). The bacterium was determined as Sulfobacil-
lus species, which may be distinguished by its morphology and
ability to growautotrophically on reduced sulfur. This rod shape
species is Gram-positive with ironsulfur oxidizing and sporu-
lating characteristics [23]. The composition of the medium for
growth and maintenance of cells, was as follows: FeSO
4
7H
2
O:
44.2 g, (NH
4
)
2
SO
4
: 3 g, MgSO
4
7H
2
O: 0.5 g, K
2
HPO
4
: 0.5g,
KCl: 0.1 g, Ca(NO
3
)
2
: 0.01 g and yeast extract: 0.2 g in 1020 mL
solution [24]. To culture the bacteria, 200 mL of the medium
was transferred into a 500 mL Erlenmeyer ask and was incu-
bated with Sulfobacillus culture, 10%(v/v), on a rotary shaker at
180 rpmand 60

C. The initial pHwas set to 1.5 with 1NH

2
SO
4
solution.
2.2. Apparatus and experimental procedure
2.2.1. Bioreactor
The biological oxidation was studied in a bioreactor which
was based on a glass column with an inlet for air and an out-
let for the efuent at the bottom. The main part of bioreactor
was biocatalyst bed with 7 and 45 cm in diameter and length,
respectively. Total operating volume of bioreactor was about 2 L.
The temperature of bioreactor was controlled using an external
jacket. The reactor was aerated at different aeration rates and
S.M. Mousavi et al. / Chemical Engineering and Processing 46 (2007) 935940 937
the ow rates for fresh media were regulated with a peristaltic
pump during the experiments from the top. To provide a uni-
form temperature inside the bioreactor and also to increase the
residence time of the reactant in the biocatalyst bed, part of the
liquid collected in the collection container was recirculated to
the top of the bioreactor using a peristaltic pump at a ow rate
of 1.2 Lh
1
.
2.2.2. Biolm formation on supports
Sulfobacillus cells were immobilized on 3 mmLDPEsupport
particles with a density of approximately 930 kg m
3
. Batch cul-
ture for the immobilization of cells was performed in 1000 mL
Erlenmeyer ask containing 400 mL mineral medium and 600
biomass support particles. The mediumwas inoculated with cell
suspension, 10% (v/v), and incubated on a rotary shaker for
72 h at 150 rpm and 60

C. Before complete consumption of

ferrous iron had occurred, the spent medium was replaced by
fresh medium followed by three consecutive runs without any
inoculation.
2.2.3. Operation of the bioreactor
After immobilization of cells in batch culture has been
achieved to a constant level, support particles were placed into
the bioreactor. The bioreactor inuent solution contained fer-
rous sulfate, which was converted to ferric sulfate by the bacteria
present on the surface of particles. The bacteria were inoculated
to the column while it was operated as a batch reactor. Once
>95% Fe
2+
oxidation was established, the reactor column was
changed to a continuous mode of operation. Steady-state con-
ditions were used at each ow rate for estimating the rate of
ferrous iron oxidation. After a change in the ow rate, steady-
state conditions were achieved when no further change occurred
in the iron oxidation rate. The time required to achieve steady-
state conditions at each ow rate varied depending on the ow
rate. Experiments were performed in four different levels of each
control factor. It should be mentioned that the concentration of
ferrous iron in the bioreactor inuent solution was adjusted to
12 g L
1
for all of experiments.
2.3. Analysis
Determination of ferric iron concentration in bacterial solu-
tions was done using a spectrophotometer (Varian Techtron
UVvis spectrophotometer, model 635) for the colorimetric
measurement of red-colored ferricsulfosalicylate complex.
Ammonia is then added, causing the 5-sulfosalicylic acid to form
a yellow complex with all the iron ions, which gives the con-
centration of total iron in the solution [25]. Difference between
concentrations of total iron and ferric iron led to obtain ferrous
concentration in the solution. The observation of free bacte-
ria in the solution was done by visual count, using a Thoma
chamber (0.1 mm depth and 0.0025 mm
2
area) with an optical
microscope. The pH of the cultural suspensions was monitored
at room temperature with a pH meter calibrated with a low pH
buffer.
Table 1
Factors and their levels for the experiments
Factor Level 1 Level 2 Level 3 Level 4
(A) Temperature (

C) 50 55 60 65
(B) Initial pH 1 1.5 2 2.5
(C) Dilution rate (h
1
) 0.1 0.2 0.3 0.4
(D) Initial Fe
3+
concentration (g L
1
) 1 3 5 7
(E) Aeration rate (mLmin
1
) 100 150 200 250
2.4. Orthogonal array and experimental parameters
For the Taguchi design and subsequent analysis, the software
named as Qualitek-4 (version 4.82.0) was used. The appropri-
ate orthogonal array for the experiment was determined by the
software. A well designed experiment can reduce substantially
the number of experiments required. The Taguchi technique
applies fractional factorial experimental designs, called orthog-
onal arrays, to reduce the number of experiments and meanwhile
obtaining statistically meaningful results.
The most important stage in the design of an experiment lies
in the selection of control factors, therefore as many factors as
possible should be included and no signicant variables must
be identied at the earliest opportunity. Taguchi method cre-
ates an orthogonal array to accommodate these requirements.
The selection of a suitable orthogonal array depends on the
number of control factors and their levels. By inspecting prac-
tical observation, ve selected control factors and their levels
applied in this study are listed in Table 1. These control factors
include temperature, initial pH, dilution rate, initial Fe
3+
con-
centration, and aeration rate. All control factors have four levels.
With the selection of L
16
orthogonal array, using ve mentioned
parameters and their levels, shown in Table 2, the number of
experiments required can be drastically reduced to 16. It means
that 16 experiments with different combinations of the factors
should be conducted in order to study the main effects and inter-
actions, which in the classical combination method using full
factorial experimentation would require 4
5
=1024 number of
experiments to capture the inuencing parameters. However,
in general, Taguchi design is preferred because it reduces the
number of experiments signicantly.
3. Results and discussion
3.1. Analysis of variance
The main objective of ANOVA is to extract from the results
howmuch variations each factor causes relative to the total vari-
ation observed in the result. According to the ANOVA results
in Table 3, the initial pH has the largest variance and the initial
Fe
3+
concentration indicated the second place. Therefore, it can
be concluded that the most inuential factor was in the order of
the pH. On the other hand, the degree of freedom(DOF) for each
factor was 3 and total DOF was 15, so the DOF for error term
was 0, and nally the variance for the error term (Ve), obtained
by calculating error sumof squares and dividing by error degrees
of freedom, could not be calculated. Henceforth, it was impossi-
938 S.M. Mousavi et al. / Chemical Engineering and Processing 46 (2007) 935940
Table 2
L
16
orthogonal array (Levels of ve different factors and obtained results)
Experiment number A B C D E Obtained results [Fe
2+
biooxidation rate (g L
1
h
1
)]
Run S/N ratio (db)
1 2 3
1 1 1 1 1 1 5.8 5.5 5.5 14.955
2 1 2 2 2 2 6.3 6.3 6.5 16.075
3 1 3 3 3 3 7.2 7 6.9 16.939
4 1 4 4 4 4 5 5.2 5.2 14.203
5 2 1 2 3 4 5.5 5.7 5.3 14.795
6 2 2 1 4 3 5.3 5 5.5 14.41
7 2 3 4 1 2 7.8 7.8 7.7 17.804
8 2 4 3 2 1 6.6 6.8 6.4 16.382
9 3 1 3 4 2 4.9 5 5.2 14.029
10 3 2 4 3 1 6.4 6.4 6.1 15.98
11 3 3 1 2 4 7.4 7.2 6.9 17.095
12 3 4 2 1 3 5.8 5.6 6.1 15.302
13 4 1 4 2 3 5 5.1 4.9 13.975
14 4 2 3 1 4 5.6 5.5 5.9 15.055
15 4 3 2 4 1 5.6 5.5 5.6 14.91
16 4 4 1 3 2 5.1 5 5.2 14.148
Average S/N ratio (db) 15.379
Table 3
ANOVA analysis of S/N ratio
Factor Degrees of freedom (DOF) Sum of squares (S) Variance (V) F-ratio (F) Pure sum (S

) Percent, P (%)
(A) Temperature (

C) 3 4.122 1.374 4.122 19.092

(B) Initial pH 3 10.928 3.642 10.928 50.616
(C) Dilution rate (h
1
) 3 0.5 0.166 0.5 2.315
(D) Initial Fe
3+
concentration (g L
1
) 3 5.608 1.869 5.608 25.976
(E) Aeration rate (mLmin
1
) 3 0.43 0.143 0.43 1.996
Other/error 0
Total 15 21.59 100
ble to calculate the F-ratio, dened as the variance of each factor
dividing by Ve. In order to eliminate the zero DOF fromthe error
term, a pooled ANOVA was applied. The process of ignoring a
factor once it was deemed insignicant was called pooling. The
values of F-ratio were calculated after pooling of aeration rate
can be found in Table 4. The percentage contribution of each
factor to the bioreactor performance, which was calculated by
the ratio of the variance for each factor to the total variance, was
shown in Table 3. The percentage contribution of the initial pH,
was the greatest, 50.616, with those of initial Fe
3+
concentration
and temperature being 25.976 and 19.092%, respectively.
3.2. Level average response analysis
The level average response analysis can be based upon the
S/N data. The analysis is done by averaging the S/N data at each
level of each factor and plotting the values in a graphical form.
The level average responses fromthe plots based on the S/Ndata
help in optimizing the objective function under study. The peak
points in these plots correspond to the optimum condition. The
response table of S/N ratios for control factors is displayed in
Table 5 and the level average response plots for various quality
characteristics based upon the S/N ratios are shown in Fig. 1.
Table 4
Pooled ANOVA analysis of S/N ratio
Factor Degrees of freedom (DOF) Sum of squares (S) Variance (V) F-ratio (F) Pure sum (S

) Percent P (%)
(A) Temperature (

C) 3 4.122 1.374 9.564 3.691 17.096

(B) Initial pH 3 10.928 3.642 25.357 10.497 48.622
(C) Dilution rate (h
1
) 3 0.5 0.166 1.16 0.069 0.319
(D) Initial Fe
3+
concentration (g L
1
) 3 5.608 1.869 13.013 5.177 23.981
(E) Aeration rate (mLmin
1
) 3 0.43 Pooled
Other/error 3 0.43 0.143 9.982
Total 15 21.59 100
S.M. Mousavi et al. / Chemical Engineering and Processing 46 (2007) 935940 939
Table 5
Average effect response for signal-to-noise ratios
Factor
(A) Temperature (

C) (B) Initial pH (C) Dilution rate (h

1
) (D) Initial Fe
3+
concentration (g L
1
)
(E) Aeration rate
(mLmin
1
)
Level 1 15.543 14.439 15.152 15.779 15.557
Level 2 15.848 15.38 15.271 15.882 15.514
Level 3 15.601 16.687 15.601 15.465 15.157
Level 4 14.522 15.009 15.49 14.388 15.287
Maximumminimum 1.326 2.248 0.449 1.494 0.4
Rank 3 1 4 2 5
Fig. 1. Level average response graphs by S/N ratio: (a) temperature, (b) initial pH, (c) dilution rate, (d) initial ferric concentration, and (e) aeration rate.
The ranks of the ve factors for a maximum biooxidation are B
(initial pH), D (initial Fe
3+
concentration), A (temperature), C
(dilution rate), and E (aeration rate).
3.3. Conrmation experiment
The conrmation experiment is the nal step in verifying
the conclusions drawn based on Taguchis parameter design
approach. The conrmation experiment is a crucial step and
is highly recommended by Taguchi to verify the experimental
conclusions. In fact running conrmation experiment is neces-
sary to show the optimum conditions and comparing the result
with the expected performance. If the new design does not meet
the specied requirement, the process must be reiterated using
new systems until the criteria are met.
The conrmation experiment is performed by conducting a
test with specic combination of the optimum levels. In this
study three conrmation experiments were carried out at the
optimum levels of the biooxidation parameters. The nal step
is to predict and verify the improvement of the performance
characteristic. The conrmation test indicated that the Fe
2+
biooxidation rate using new design experiments is 8.4 and the
95% condence intervals for S/N ratio are 17.881 0.639. The
mean calculated from three S/N ratios for three conrmation
experiments is equal to 18.48, which is located within the con-
dence intervals. The experimental results conrmed the validity
940 S.M. Mousavi et al. / Chemical Engineering and Processing 46 (2007) 935940
Table 6
Optimum conditions and performance
Factor Level
description
Level Contribution
(A) Temperature (

C) 55 2 0.469
(B) Initial pH 2 3 1.308
(C) Dilution rate (h
1
) 0.3 3 0.222
(D) Initial Fe
3+
concentration (g L
1
) 3 2 0.503
(E) Aeration rate (mLmin
1
) 100 1 0.178
of the applied technique for optimizing the ferrous biooxida-
tion parameters. So it is possible to increase biooxidation rate
signicantly using the proposed statistical technique.
4. Conclusion
In the present attempt optimization of ferrous iron oxidation
rate using an indigenous Sulfobacillus species in a packed-
bed reactor was investigated. Following the Taguchi method
of experimental design the effects of various factors inuenc-
ing the performance characteristics, were analyzed. Analysis of
S/N ratio has been successfully applied for nding out the rela-
tive contribution and the optimum factor level combination for
the maximum Fe
2+
biooxidation rate. According to the percent
contribution of each factor, indicated in the ANOVA table, it
could be inferred that initial pH of feed solution is the most pre-
dominant factor. Importance of the factors on the biooxidation
of Fe
2+
was ranked in Table 6. The critical process parameters,
according to their relative signicance, are initial pH of feed
solution, initial Fe
3+
concentration, temperature, dilution rate
and aeration rate, respectively. The maximum biological oxida-
tion rate was obtained by setting temperature 55

C, initial pH
2, dilution rate 0.3 h
1
, initial Fe
3+
concentration 3 g L
1
and
aeration rate 100 mLmin
1
. It was resulted that the biooxida-
tion rate of ferrous iron was increased by 7.7% at the optimum
conditions, which they determined by Taguchi optimization
method.
Acknowledgements
The authors would like to acknowledge Mehrdad Hesampour
for his useful help and discussion. They also thank Jamshid
Kash and Gharibali Farzi for their technical assistance at
BBRC.
References
[1] R.A. Pandey, S. Malhotra, Desulphurization of gaseous fuels with recovery
of elemental sulphur: an overview, Crit. Rev. Environ. Sci. Technol. 29 (3)
(1999) 229268.
[2] S. Ebrahimi, R. Kleerebezem, M.C.M. van Loosdrecht, J.J. Heijnen, Kinet-
ics of the reactive absorption of hydrogen sulde into aqueous ferric sulfate
solutions, Chem. Eng. Sci. 58 (2003) 417427.
[3] C. Pagella, P. Silvestri, D.M. De Faveri, Hydrogen sulphide removal with
a biochemical process: the biological step, Chem. Biochem. Eng. Q. 10
(1996) 165174.
[4] M.E.A.G. Oprime, O. Garcia Jr., A.A. Cardoso, Oxidation of H
2
S in acid
solution by Thiobacillus ferrooxidans and Thiobacillus thiooxidans, Pro-
cess Biochem. 37 (2001) 111114.
[5] T. Imaizumi, Some industrial applications of inorganic microbial oxidation
in Japan, Biotechnol. Bioeng. Symp., NewYork, Wiley 16 (1986) 363371.
[6] J. Gasiorek, Microbial removal of sulfur dioxide from a gas stream, Fuel
Process. Technol. 40 (1994) 129138.
[7] F. Rubiera, A. Moran, O. Martinez, E. Fuente, J. Pis, Inuence of biological
desulphurisation on coal combustion performance, Fuel Process. Technol.
52 (1997) 165173.
[8] S.N. Grouder, F.N. Genchev, F. Gaidatyjiew, Observations onthe microora
in an industrial copper dump leaching operations, in: L. Murr, A. Thorma,
J. Brierley (Eds.), Metallurgical Applications of Bacterial Leaching and
Related Microbiological Phenomena, Academic Press, New York, 1978,
pp. 253274.
[9] D. Chandra, P. Roy, A.K. Mishra, J.N. Cakrabarati, N.K. Prasad, S.G.
Chaudhuri, Removal of sulphur from coal by Thiobacillus ferrooxidans
and by mixed acidophilic bacteria present in coal, Fuel 59 (1980) 249252.
[10] S.M. Mousavi, S. Yaghmaei, F. Salimi, A. Jafari, Inuence of process vari-
ables on biooxidation of ferrous sulfate by an indigenous Acidithiobacillus
ferrooxidans. Part I: Flaskexperiments, Fuel 85(1718) (2006) 25552560.
[11] S.M. Mousavi, S. Yaghmaei, M. Vossoughi, A. Jafari, R. Roostaazad, Zinc
extraction from Iranian low-grade complex zinclead ore by two native
microorganisms: Acidithiobacillus ferrooxidans and Sulfobacillus, Int. J.
Miner. Process. 80 (2006) 238243.
[12] S.M. Mousavi, S. Yaghmaei, A. Jafari, Inuence of process variables on
biooxidation of ferrous sulfate by an indigenous Acidithiobacillus ferroox-
idans. Part II: Bioreactor experiments, Fuel 86 (78) (2007) 993999.
[13] C. Pagella, P. Perego, M. Zilli, Biotechnological H
2
S gas treatment with
Thiobacillus ferrooxidans, Chem. Eng. Technol. 19 (1996) 7988.
[14] A.B. Jensen, C. Webb, Treatment of H
2
S-containing gases: a review of
microbiological alternatives, Enzyme Microbial. Technol. 17 (1995) 210.
[15] M. Nemati, S.T.L. Harrison, G.S. Hansford, C. Webb, Biological oxidation
of ferrous sulphate by Thiobacillus ferrooxidans: a review on the kinetic
aspects, Biochem. Eng. J. 1 (1998) 171191.
[16] M.M. Mesa, J.A. Andrades, M. Ma cas, D. Cantero, Biological oxidation of
ferrous iron: study of bioreactor efciency, J. Chem. Technol. Biotechnol.
79 (2004) 163170.
[17] Z. Long, Y. Huang, Z. Cai, W. Cong, F. Ouyang, Immobilization of
Acidithiobacillus ferrooxidans by a PVAboric acid method for ferrous
sulphate oxidation, Process Biochem. 39 (2004) 21292133.
[18] G. Taguchi, Y. Yokoyama, Y. Wu, Taguchi Methods/Designof Experiments,
American Supplier Institute (ASI) Press, Tokyo, 1993.
[19] S.H. Park, Robust Design and Analysis for Quality Engineering, Chapman
& Hall, London, 1996.
[20] D.R. Cox, N. Reid, The Theory of the Design of Experiments, Chapman
& Hall/CRC, 2000.
[21] C. Douglas, Montgomery, Design and Analysis of Experiments, Wiley,
2001.
[22] R. Roy, A Primer on the Taguchi Method, Van Nostrand Reinhold, New
York, 1990.
[23] P.R. Norris, D.A. Clark, J.P. Owen, S. Waterhouse, Characteristics of Sul-
fobacillus acidophilus sp. nov. and other moderately thermophilic mineral
sulphide-oxidizing bacteria, Microbiology 141 (1996) 775783.
[24] R.M. Atlas, Handbook of Microbiological Media, second ed., CRC Press,
Boca Raton, 1997.
[25] D.G. Karamanev, L.N. Nikolov, V. Mamatarkova, Rapid simultaneous
quantitative determination of ferric and ferrous ions in drainage waters
and similar solutions, Miner. Eng. 15 (2002) 341346.