Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Investigation of Acidithiobacillus ferrooxidans in pure and

mixed-species culture for bioleaching of Theisen sludge
from former copper smelting
€ mann1 and S. Schopf1
C. Klink1, S. Eisen1, B. Daus2, J. Heim3, M. Schlo
1 Institute of Biosciences, TU Bergakademie Freiberg, Freiberg, Germany
2 Department Analytical Chemistry, UFZ – Helmholtz Center for Environmental Research, Leipzig, Germany
3 Helmholtz Institute Freiberg for Resource Technology, Freiberg, Germany

Keywords Abstract
acidophilic bacteria, bioleaching of valuable
trace elements, stirred-tank bioreactors, Aims: The aim of this study was to investigate the potential of bioleaching for
Theisen sludge, toxic organic compounds. the treatment of an environmentally hazardous waste, a blast-furnace flue dust
designated Theisen sludge.
Correspondence Methods and Results: Bioleaching of Theisen sludge was investigated at acidic
Simone Schopf, Institute of Biosciences, TU
conditions with Acidithiobacillus ferrooxidans in pure and mixed-species culture
Bergakademie Freiberg, Leipziger Strasse 29,
with Acidiphilium. In shaking-flask experiments, bioleaching parameters (pH,
09599 Freiberg, Germany.
E-mail: [email protected] redox potential, zinc extraction from ZnS, ferrous- and ferric-iron
concentration) were controlled regularly. The analysis of the dissolved metals
2015/2553: received 26 May 2015, revised showed that 70% zinc and 45% copper were extracted. Investigations regarding
18 February 2016 and accepted 11 March the arsenic and antimony species were performed. When iron ions were
2016 lacking, animonate (Sb(V)) and total arsenic concentration were highest in
solution. The bioleaching approach was scaled up in stirred-tank bioreactors
doi:10.1111/jam.13142
resulting in higher leaching efficiency of valuable trace elements.
Concentrations of dissolved antimony were approx. 23 times, and of cobalt,
germanium, and rhenium three times higher in comparison to shaking-flask
experiments, when considering the difference in solid load of Theisen sludge.
Conclusions: The extraction of base and trace metals from Theisen sludge,
despite of its high content of heavy metals and organic compounds, was
feasible with iron-oxidizing acidophilic bacteria. In stirred-tank bioreactors, the
mixed-species culture performed better.
Significance and Impact of the Study: To the best of our knowledge, this
study is the first providing an appropriate biological technology for the
treatment of Theisen sludge to win valuable elements.

pogenic waste product, contains rhenium, cobalt, anti-

Introduction
mony, and germanium as trace elements (Lorenz et al.
Elements such as rhenium, cobalt, antimony and germa- 1992). This sludge was a by-product of the blast-furnace
nium are of strategic importance and of interest even in (BF) washing plant of the former copper-smelting indus-
small amounts due to their application in high-tech try in Saxony-Anhalt, Germany (Wege 2001). It is a mix-
industries. Rhenium and cobalt are, for example, needed ture of very fine-grained bituminous particles containing
in various (high-temperature) alloys and germanium in sulphide grains with a content of 19% zinc and 14% lead,
infra-red optics. With an increasing demand for high- as well as organic compounds. With a concentration of
tech products, the application of suitable extraction pro- approx. 450 lg g-1 most of the organics are polycyclic
cesses for recovery of valuable metals is becoming aromatic hydrocarbons, but biphenyls and dibenzofurans
increasingly important. Theisen sludge, an anthropho- are also present. Theisen sludge is not hazardous with

1520 Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology
C. Klink et al. Bioleaching of fine-grained waste

respect to polychlorinated dibenzo-dioxins and -furans ria, for which Acidithiobacillus ferrooxidans is a well-
(Weiß et al. 1997). However, the high contents of heavy known representative, is the regeneration of Fe(III) as
metals (such as zinc, lead and copper) in Theisen sludge metal-sulphide oxidizing agent.
caused environmental risks, especially to surface- and The mineral phases in Theisen sludge are mostly sul-
groundwater (Schreck 1998). Approximately 220 000 tons phides containing 19
5% wurtzite, 17
5% sphalerite (both
of Theisen sludge have been deposited in open basins ZnS), and 7% galena (PbS) (Weiß et al. 1997). Therefore,
and slag heaps from 1978 until 1990. Before 1987, Thei- Theisen sludge is supposed to be amenable to bioleaching.
sen sludge has been utilized as raw material feedstock for However, it is documented that iron-oxidizing microbes
a lead smelter (Schreck 1998). After closing the lead- are sensitive to small organic compounds, such as acetic
smelting facility, many proposals to treat Theisen sludge and propionic acid, especially during incubation on solid
have been made, but most of them were obviously eco- media (Johnson 1995). Moreover, phenolic compounds
nomically not feasible. Acidic pressure leaching and oxi- seem to have an inhibitory effect on autotrophic bioleach-
dation treatment have been evaluated as technically ing microbes (Watling et al. 2009). Due to the high content
possible (Gock and S€ otemann 1994; Morency et al. of organic compounds in the blast-furnace sludge the
1998). Nevertheless, an appropriate technology for the microbes could be impeded in performing metal oxidation.
treatment of Theisen sludge is still lacking. Considering The aim of the study was to establish bioleaching of
Theisen sludge as secondary resource, action towards the the organic- and heavy metal-containing Theisen sludge
development of a treatment is needed. at acidic conditions for extraction of valuable elements
Biological processes are often recommended as being (rhenium, cobalt, antimony and germanium).
more cost-effective and resource-efficient than chemical
of physical processes. Bioleaching is considered to be a
Materials and methods
possible method to treat Theisen sludge, as the micro-
organisms obtain their energy during the leaching process
Tyndallization of Theisen sludge
and act as naturally occurring catalysts. Furthermore,
bioleaching is especially suited for sources with low target For the reduction of germs, Theisen sludge was heated to
metal contents. Therefore, bioleaching may accomplish 80°C in dry air for 4 h (modified after Brickett et al.
the twin objectives of resource recycling and pollution 1995). Afterwards, the sludge was moistened with sterile
reduction (Lee and Pandey 2012). water and incubated overnight at room temperature to
Oxidative bioleaching is defined as the mobilization of support germination of spores. This moistening- and
metals from insoluble sulphide ores in the presence heating-procedure was repeated on three consecutive
of oxygen under acidic conditions due to the application days.
of microbes. The mechanism of bioleaching is referred as
‘indirect’, because the oxidation of the metal sulphides is
Chemical composition of Theisen sludge
caused by ferric iron (Fe(III)), which acts as a strong
mineral-oxidizing agent. According to the stability of The total element concentrations of the original material
minerals at acidic conditions, two mechanisms have been were investigated by energy-dispersive X-ray fluorescence
proposed: The ‘polysulphide mechanism’ for acid-soluble spectrometry (EDXRF) with an X-LAB 2000 (SPECTRO
minerals, for example, sphalerite (ZnS) and the ‘thiosul- A.I., Kleve, Germany). The results are given in Table 1.
phate mechanism’ for acid-nonsoluble minerals, for The original material was diluted with SiO2 powder
example, pyrite (FeS2). Sulphur species which occur as (Honeywell Riedel-de-Haen AG, Seelze, Germany) and
intermediates are important in both pathways. They can homogenized to ensure fitting within the calibration
either be oxidized by Fe(III), produced by iron-oxidizing range. Theisen sludge material was mixed with stearin
bacteria, or directly by sulphur-oxidizing microbes (for wax (Hoechst wax for XRF-analysis) as binder in a ratio
reviews on the mechanisms of bioleaching see Rohwerder 80 : 20 (w/w). Afterwards, the mixture was pressed into
et al. 2003; Vera et al. 2013). pellets of 32 mm in diameter.
Due to the redox potential of the ferric/ferrous couple
(E0 (Fe3+/Fe2+) = +0
77 V at pH 2), iron is mainly pre-
Source, enrichment and identification of acidophilic
sent as ferrous iron (Fe(II)) in acidic waters (Stumm and
bioleaching bacteria
Morgan 1981). Fe(II) is chemically stable in acidic oxy-
gen-rich environments at pH values <3
5. Under the A mixed-species culture (Nochten2) from an acid-mine
given conditions, microbial ferrous iron oxidation is water treatment plant located at the open-cast lignite
the main oxidation process (Klein et al. 2014). Thus, the mining pit Nochten in Eastern Germany was enriched in
main role of the acidophilic ferrous iron-oxidizing bacte- a modified 9K medium (Silverman and Lundgren 1959)

Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology 1521
Bioleaching of fine-grained waste C. Klink et al.

Table 1 Chemical composition of Theisen sludge determined via The sequence of the 16S rRNA gene was deposited at
EDXRF NCBI database (accession number KU569322).
Elements [mg kg 1] in unleached tyndallized Theisen sludge

Zn 250 400 Bioleaching in shaking flasks

S 121 600
Biotic approaches were inoculated with Nochten2 and
Pb 97 850
Fe 15 000
A. ferrooxidans Ram6F and cultivated in 9K25 medium
Al 11 000 with a solid load of 1% (w/v) tyndallized Theisen sludge.
Cu 12 100 Abiotic controls were sterile and contained 1% (w/v) tyn-
Sb 2100 dallized Theisen sludge in either 9K25 medium (abiotic
As 9100 tyndallized with ferrous iron (AT+Fe(II)) or modified 9K
Mn 640 medium without ferrous iron, designated as 0K medium
Mo 350
(referred to as AT-Fe(II); abiotic tyndallized without fer-
Cd 290
Co 130
rous iron). Nontyndallized Theisen sludge was tested in
Ge 20 sterile 9K25 medium (nontyndallized with ferrous iron,
Ag 430 NT+Fe(II)) in order to explore the effectiveness of tyn-
Re 90 dallization. Biotic experiments in small scale were per-
formed in quadruplicates and abiotic controls in
duplicates with a culture volume of 100 ml, respectively.
containing 25 mmol l 1 (final concentration) of sterile- All suspensions were incubated at 30°C and
filtered ferrous sulphate solution of pH 1
8. Conse- 100 rpm min 1 in a shaker (Multitron Standard, Infors
quently, the medium is referred to as 9K25. A pure cul- HT, Bottmingen/Basel, Switzerland).
ture of A. ferrooxidans Ram6F (kindly provided by A. Liquid samples (3 ml) were taken regularly for mea-
Schippers, Bundesanstalt f€ ur Geowissenschaften und Roh- surement of pH and redox potential with respective elec-
stoffe, Hanover) was cultivated in 9K25, too. Growth of trodes (WTW GmbH, Weilheim, Germany). Afterwards,
bacterial cells was observed via light microscopy (Nikon liquid samples were sterile filtered and stored at 4°C for
Eclipse, Nikon Metrology GmbH, Alzenau, Germany). determination of zinc, ferrous-iron and ferric-iron con-
After the growth occurred, the cultures were transferred centrations via ion exchange chromatography. Volume
into fresh 9K25 medium containing tyndallized Theisen depletion due to sampling was balanced by adding fresh,
sludge for adaption. The composition of the mixed-spe- sterile medium until the initial weight of the flasks was
cies community before and after bioleaching was investi- reached again. After finishing bioleaching, the metal-con-
gated. For that, DNA was extracted using the UltraClean taining supernatants were analysed as described below.
Microbial DNA Isolation Kit (MO BIO Laboratories,
Inc., Carlsbad, CA). Terminal restriction-fragment length
polymorphism (T-RFLP) was performed as described Bioleaching in stirred-tank bioreactors
elsewhere (Tischler et al. 2014). PCR amplification of the Precultures of Nochten2 and A. ferrooxidans Ram6F
bacterial 16S rRNA genes was done with 0
25 lmol l 1 adapted from shaking-flask experiments were cultivated in
(final concentrations respectively) of primers 27F (Lane the OxiTop® system for determination of their activity by
1991) and 1387R (Marchesi et al. 1998) using a PCR- oxygen consumption, as described by Giebner et al.
master mix from Thermo Fisher Scientific. The mix was (2015). Cells in the exponential phase were used as inocula
supplemented with 5% (v/v) dimethylsulfoxide and for bioreactors. Bioleaching in 2 l stirred-tank bioreactors
0
2 lg ll 1 bovine serum albumin. The PCR ran with (Labfors 5, Infors HT, Bottmingen/Base, Switzerland) was
the following program: 2 min initial denaturation, 30 performed with a working volume of 900 ml of 9K25 med-
cycles of 30 s at 94°C, 30 s at 55°C, 90 s at 72°C and a ium, a solid load of 4% (w/v) tyndallized Theisen sludge, a
final incubation at 72°C for 5 min. The PCR products stirring speed of 300 rpm min 1 and an air flow of
were purified with an UltraClean PCR Purification Kit 0
25 nl min 1. The temperature was kept constant at
(MO BIO Laboratories, Inc., Carlsbad, CA). Sanger 30°C. The pH and the redox potential were constantly
sequencing of the 16S rRNA gene was done by Eurofins measured with the respective electrodes (Mettler Toledo,
Genomics (Ebersberg) and the sequence was taxonomi- Gieben, Germany) and recorded with the IRIS 6 bioprocess
cally classified by a BLAST search. Alignment against refer- control software (Infors HT, Bottmingen/Basel, Switzer-
ence sequences was made with the SILVA Incremental land). Every 2–3 days, samples were taken for external
Aligner (SINA) (Pruesse et al. 2012). The phylogenetic cross-check of the pH and the redox potential, and for
tree was calculated with MEGA 6
0 (Tamura et al. 2013). determination of zinc, ferrous iron, and ferric-iron concen-

1522 Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology
C. Klink et al. Bioleaching of fine-grained waste

trations via ion exchange chromatography. The concentra- Abiotic controls contained tyndallized Theisen sludge in
tions of (further) dissolved metals and metalloids were sterile medium with (abiotic tyndallized with ferrous
determined as described below after the bioreactor experi- iron; AT+Fe(II)) or without ferrous sulphate (abiotic tyn-
ments had been finished. dallized without ferrous iron; AT-Fe(II)). To investigate
the effectivity of sludge tyndallization, a control series in
9K25 with nontyndallized Theisen sludge was performed
Measurements of dissolved metals and speciation of
(nontyndallized with ferrous iron; NT+Fe(II)).
antimony and arsenic
For bioleaching a pure culture of A. ferrooxidans
For the quantitative determination of zinc, ferrous-iron, Ram6F and an enrichment culture designated as Nocht-
and ferric-iron concentrations in solution, ion exchange en2 were applied, because these cultures had grown well
chromatography was performed (ICS-5000, 4 mm system, in the presence of Theisen sludge in preliminary tests. To
Thermo Fisher Scientific GmbH, Dreieich, Germany). Sam- elucidate possible community changes, the composition
ples were diluted with acidic ultrapure water (pH 2 with of the enrichment culture was investigated before and
H2SO4). Transition metals were separated with an IonPacâ after the experiments. As indicated by T-RFLP (Table S1;
CS5A column (Thermo Fisher Scientific GmbH, Dreieich, Figs. S1–S3, S6–S7), Acidithiobacillus was dominant while
Germany) with Met Pac PDCA (7 mmol l 1 pyridine-2,6- Acidiphilium occurred in small numbers in the enrich-
dicarboxylic acid, 66 mmol l 1 potassium hydroxide, ment cultures.
5
6 mmol l 1 potassium sulphate and 74 mmol l 1 formic The pH in the initial shaking-flask experiments was
acid) as eluent. For separation of ions the flow rate of the not adjusted after addition of Theisen sludge to the med-
eluent was 1 ml min 1. For the separation of iron as transi- ium. The starting pH values of the biotic (i.e. inoculated)
tion metal, pyridine-2,6-dicarboxylic acid in the mobile experiments (referred to as Nochten2 and A. ferrooxidans
phase was used as complexing agent. Detection was at Ram6F) and the abiotic ones in 9K25 medium (AT+Fe
530 nm after postcolumn derivatization with 0
5 mmol l 1 (II)) were in the range of 3
5–3
7. The pH values of the
4,2-pyridylazoresorcinol dissolved in MetPac PAR Post- nontyndallized controls (NT+Fe(II)) were approx. 4
2.
column Reagent Diluent (1 mol l 1 dimethy- The pH values of the abiotic controls in 0K medium were
1
laminoethanol, 0
5 mol l ammonium hydroxide, the highest (approx. pH 5 in AT-Fe(II)) (Fig. 1a). In all
0
3 mol l 1 sodium bicarbonate). Postcolumn derivatiza- biotic approaches the pH values finally dropped to 2
5.
tion was necessary for qualitative and quantitative determi- The pH values of the abiotic controls in 9K25 medium
nation of the metal complex at 530 nm with a UV/VIS and the nontyndallized controls decreased with a delay of
detector (Cardellicchio et al. 1997). 5–7 days respectively.
The total concentrations of dissolved elements (Zn, S, In all biotic samples, either inoculated with Nochten2
Pb, Fe, Cu, Sb, As, Mn, Mo) were analysed with ICP-AES or A. ferrooxidans Ram6F, the redox potential increased
(CIROS, Spectro A.I., Kleve, Germany) or, in the case of from 400 to approx. 700 mV within 11 days (data not
low concentrations (Co, Ge, Re, Ag), with ICP-MS (Agi- shown). Zinc extraction was highest in the biotic samples
lent 7700, Agilent Technologies GmbH, Waldbronn, with a recovery of 65% dissolved zinc within this period
Germany), both having pneumatic nebulization. based on the total amount of zinc in the utilized sludge
Analyses of antimony species was as described by Kolbe (Fig. 1b). Zinc was extracted in the tyndallized and non-
et al. (2012). The antimony species in solution after tyndallized controls containing iron, while in the abiotic
bioleaching were preserved by adding EDTA to a final con- sterile control without ferrous iron (AT-Fe(II)) almost no
centration of 10 mmol l 1 (Daus and Wennrich 2014). zinc was recovered. Fe(II) was rapidly and completely
The arsenic species were determined following the proce- oxidized and the concentration dropped to near zero
dure by Daus et al. (2008) with a shortened retention time (Fig. 1c). Simultaneously, the concentration of dissolved
of 7 min due to the limited number of expected species in Fe(III) increased (Fig. 1d). In the biotic samples the con-
the samples. The arsenic species in the leaching solutions centration of Fe(III) steadily declined after reaching its
were preserved by adding 10 mmol l 1 H3PO4 immedi- maximum, indicating the precipitation of ferric-iron min-
ately after sampling (Daus et al. 2006). erals under the given conditions.
Zinc extraction in the AT+Fe(II) control was a result
of iron oxidation, as under the given starting conditions
Results
(pH > 3
5), ferrous iron is not completely stable and
chemically oxidized to ferric iron (Singer and Stumm
Bioleaching in shaking flasks
1970). Consequently, the concentration of Fe(II)
Before starting the bioleaching tests, Theisen sludge was decreased with simultaneously increasing Fe(III) concen-
tyndallized to reduce the number of microorganisms. tration as shown in Figs. 1c, d. Ferric iron, which is a

Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology 1523
Bioleaching of fine-grained waste C. Klink et al.

(a) 6 (b) 80
5·5 70
5 60

Zinc recovery [%]

4·5 50
4 40
pH

3·5 30
3 20
2·5 10
2 0
0 5 10 15 20 25 0 5 10 15 20 25
Time [days] Time [days]
(c) 1250 (d)
350

300
1000
250

cFe(III) [mg l–1]

cFe(II) [mg l–1]

750 200

150
500
100
250 50

0
0 0 5 10 15 20 25
0 5 10 15 20 25
Time [days]
Time [days]

Figure 1 (a) Development of the pH in shaking flask experiments with a solid load of 1% Theisen sludge. Inoculated approaches: Acidithiobacil-
lus ferrooxidans Ram6F (♢- -) and Nochten2 (♦—). Abiotic controls: tyndallized Theisen sludge in 9K25 (○- -); tyndallized Theisen sludge in 0K
medium (●—). Unsterile control: non-tyndallized Theisen sludge in 9K25 (▲—). (b) Zinc extraction in shaking flasks with a solid load of 1% Thei-
sen sludge. Inoculated approaches: A. ferrooxidans Ram6F (♢- -) and Nochten2 (♦—). Abiotic controls: tyndallized Theisen sludge in 9K25 (○- -);
tyndallized Theisen sludge in 0K medium (●—). Unsterile control: non-tyndallized Theisen sludge in 9K25 (▲—). (c) Development of the Fe(II)-con-
centration in shaking flasks with a solid load of 1% Theisen sludge. Inoculated approaches: A. ferrooxidans Ram6F (♢- -) and Nochten2 (♦—).
Abiotic controls: tyndallized Theisen sludge in 9K25 (○- -); tyndallized Theisen sludge in 0K medium (●—). Unsterile control: non-tyndallized Thei-
sen sludge in 9K25 (▲—). (d) Development of the Fe(III)-concentration in shaking flasks with a solid load of 1% Theisen sludge. Inoculated
approaches: A. ferrooxidans Ram6F (♢- -) and Nochten2 (♦—). Abiotic controls: tyndallized Theisen sludge in 9K25 (○- -); tyndallized Theisen
sludge in 0K medium (●—). Unsterile control: non-tyndallized Theisen sludge in 9K25 (▲—).

strong oxidizing agent, leads to the abiotic dissolution of gene showed 99% sequence identity to A. ferrooxidans
zinc in this study. However, the controls containing non- ATCC23270T. By construction of a phylogenetic tree
tyndallized Theisen sludge showed similar tendencies in (Fig. 2) the new isolate, designated Hel18, could be clas-
terms of pH, zinc extraction and ferric to ferrous-iron sified as a member of A. ferrooxidans.
ratio Figs. 1a–d). Here, zinc extraction was a result of After 21 days of bioleaching, the metal-laden leaching
growth of indigenous microorganisms from nontyndal- solutions were analysed. Solutions from biotic samples
lized Theisen sludge. Only in the NT+Fe(II) control series contained significantly higher amounts of the base metals
growth of microorganisms could be observed by light zinc and copper, and slightly elevated concentrations of
microscopy. Growth did not occur in any of the other rhenium and cobalt in comparison to the tyndallized
controls. To analyse the composition of the indigenous control without ferrous sulphate (AT-Fe(II) in Table 2).
culture, several consecutive transfers into fresh 9K25 med- Based on the total amount in the utilized Theisen sludge,
ium were performed after bioleaching. The isolate 70% of zinc, 45% of copper, 40% of cobalt, and 20% of
obtained could be assigned to the genus Acidithiobacillus rhenium were extracted. It is noteworthy that manganese
by T-RFLP. Taxonomic classification of the 16S rRNA was also leached. The zinc extraction rate was calculated

1524 Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology
C. Klink et al. Bioleaching of fine-grained waste

Acidithiobacillus ferrooxidans strain GD-1 (FJ194540.1)

Acidithiobacillus ferrooxidans strain L01 (KJ648626.1)

Acidithiobacillus ferrooxidans type strain ATCC 23270 (NR_114599.1)

94
Acidithiobacillus ferrooxidans strain QXS-1 (DQ168465.1) A. ferrooxidans

Acidithiobacillus ferrooxidans Hel18 (KU569322)

Acidithiobacillus ferrooxidans strain A1 (FN686783.1)

87 Acidithiobacillus ferrooxidans strain CUMT-1 (JX266060.1)

Acidithiobacillus ferrooxidans strain B20 (FM242697.1)

Acidithiobacillus ferrooxidans strain CC1 (AJ457804.2)

Acidithiobacillus ferrooxidans strain CB5 (AJ457804.2)

Acidithiobacillus ferrooxidans strain BRGM1 (AJ457806.2) A. ferridurans

99
Acidithiobacillus ferrooxidans strain SP5-1 (HM070044.1)

Acidithiobacillus ferrooxidans (AJ278721.1)

Acidithiobacillus ferridurans strain ATCC 33020 (NR_117036)

88 Acidithiobacillus ferrivorans strain Peru6 (EU839490.2)

Acidithiobacillus ferrivorans strain PQ510 (LN650697.1)

Acidithiobacillus ferrivorans SS3 (NR_074660.1)

A. ferrivorans
Acidithiobacillus ferrivorans strain ACH (KJ679874.1)
100
Acidithiobacillus ferrivorans strain CF27 (EU839489.2)

Acidithiobacillus ferrivorans strain Py-F1 (KC208495.1)

Acidithiobacillus thiooxidans type strain ATCC19377 (Y11596.1)

Acidithiobacillus caldus type strain ATCC 51756 (NZ_CP005986.1)

0·005

Figure 2 Maximum Likelihood phylogenetic tree. Derived from 16S rRNA gene sequences the relationship of Acidithiobacillus ferrooxidans Hel18
to other groups of Acidithiobacillus species is shown. The classification into groups was according to literature (Amouric et al. 2011; Hedrich and
Johnson 2013). Gene bank accession numbers are given in parantheses, bootstrap values at the respective nodes. The evolutionary distances
were based on the Hasegawa-Kishino-Yano model. A. caldus type strain ATTC 51756 was used as outgroup.

Table 2 Analyses of the metal-containing solution after bioleaching of Theisen sludge in shaking flasks with a solid load of 1% (mean  stan-
dard deviation of 4 replicates)

Element Acidithiobacillus ferrooxidans Ram6F Nochten2 AT+Fe(II) AT-Fe(II) NT+Fe(II)

Concentrations [mg l 1] according to ICP-AES

Zn 1710  61 1640  70 1285  5 141  2 1565  35
S 2170  80 1950  90 1760  10 1095  5 1945  7
Pb 2
9  0
05 2
9  0
04 2
7  0
01 0
5  0
03 2
9  0
04
Fe 121  30 226  21 73
4  9
0 <0
03 81  9
Cu 53
0  4
2 53
5  6
4 28
4  0
5 <0
05 51  5
Sb 0
6  0
07 0
6  0
12 1
4  0
08 2
5  0
01 0
5  0
2
As 1
1  0
12 1
2  0
44 2
1  0
36 11
0  0
3 0
4  0
07
Mn 3
5  0
1 3
50  0
02 3
0  0
02 0
8  0
01 3
3  0
06
Mo <0
06 <0
06 <0
06 0
12  0
1 <0
06
Concentrations [lg l 1] according to ICP-MS
Co 520  19 523  36 388  4 164  1 455  35
Ge 264  9 260  21 325  4 196  1 215  21
Re 209  12 206  16 135  1 75  1 213  13
Ag 0
1  0
12 1
0  0
21 0
7  0
04 <0
3 0
8  0
06

Inoculated approaches: A. ferrooxidans Ram6F and Nochten2.

Abiotic controls: tyndallized Theisen sludge in 9K25 (AT+Fe(II)); tyndallized Theisen sludge in 0K medium (AT-Fe(II)).
Unsterile controls: nontyndallized Theisen sludge in 9K25 (NT+Fe(II)).

Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology 1525
Bioleaching of fine-grained waste C. Klink et al.

from the slope of the linear range of the respective curve tained 9K25 medium and tyndallized Theisen sludge
(Fig. 1b) and was 5
6 mg l 1 h 1 for A. ferrooxidans (AT+Fe(II)). The starting pH values after addition of
Ram6F and 6
6 mg l 1 h 1 for Nochten2. Theisen sludge were 4
5 (Fig. 4a). After a relatively long
In all leached solutions, antimonate (Sb(V)) was the lag-phase in comparison to shaking flasks, dissolution of
dominant antimony species (Fig. 3). Concentrations of ZnS was especially high in the biotic bioreactor with
antimonite (Sb(III)) were less than 5 lg l 1 in all sam- Nochten2, as shown by increasing dissolved zinc concen-
ples and therefore are not shown in the diagram. In the tration (Fig. 4b) and redox potential (Fig. 4c), and by a
control without ferrous sulphate the Sb(V) concentration decreasing concentration of dissolved Fe(II) (data not
was highest with more than 2 mg l 1. (AT-Fe(II)). Under shown). ICP measurements showed that the dissolution
the specified conditions, the total concentration of dis- of zinc, copper, antimony, germanium, and rhenium was
solved arsenic was more than 12 mg l 1, because iron considerably higher in the inoculated bioreactors
was lacking to remove arsenic by sorption to iron- (Table 3). This was especially observed for bioleaching
(hydroxides) which precipitate from the solution. How- with Nochten2. Here, the calculated zinc extraction rate
ever, the sorption onto precipitated iron hydroxides was in the linear range was with 7
7 mg l 1 h 1 higher than
not completely efficient for arsenic, because dissolved in shaking flasks. Concentrations of dissolved antimony
arsenite (As(III)) and, to a lesser extent, arsenate (As(V)) were approx. 23 times, of cobalt, germanium, and
could be detected in all other ferrous sulphate-laden solu- rhenium three times higher than in shaking flasks experi-
tions. ments, when considering the difference in solid load of
Theisen sludge. As in the shaking-flask experiments, the
Nochten2 preculture was a consortium of Acidithiobacil-
Scale up in bioreactors
lus and, to a minor extent, Acidiphilium. After finishing
Bioreactor systems are more homogeneous in comparison the bioleaching experiment, only Acidithiobacillus could
to shaking flasks due to an enhanced gas transfer by con- be detected by T-RFLP (Table S1; Figs. S4, S5, S8, S9).
stant aeration and stirring. For scale up, the solid load
was increased to 4%. Two stirred-tank bioreactors
Discussion
(STBRs) were inoculated either with Nochten2 or A. fer-
rooxidans Ram6F adapted from small scale experiments. The study presented here demonstrates that bioleaching
A third bioreactor served as control reactor and con- is a suitable method for recycling of valuable metals from
Theisen sludge. Data on bioleaching of wastes are already
AT – Fe(II)

12 000
available, which are reviewed, for example, by Lee and
Pandey (2012). However, studies on bioleaching of blast
furnance sludges (BF) are scarce. In the study of Banerjee
10 000 (2007) metal extraction from a BF sludge and flue dust
from iron- and steel-industry was investigated under lab-
oratory conditions, showing that A. ferrooxidans was
8000 more effective than fungal strains. To the authors’ best
knowledge none of the studies published on BF sludge so
c [µg l–1]

far dealt with bioleaching of rhenium, cobalt, antimony

6000 and germanium in the presence of high amounts of
AT – Fe(II)
A. ferrooxidans Ram6F

A. ferrooxidans Ram6F
A. ferrooxidans Ram6F

organic compounds (approx. 10% total carbon content).

AT – Fe(II)

However, some constraints have yet to be considered,

AT + Fe(II)

4000
which may be summarized by poor extracting yields for
AT + Fe(II)
Nochten 2

AT + Fe(II)

target metals in combination with the long total reaction

NT + Fe(II)
Nochten 2
NT + Fe(II)

Nochten 2

NT + Fe (II)

times. In comparison, Baba et al. (2011) showed 92% of

2000
zinc extraction from sphalerite within 5 days, when leach-
ing under acidic conditions. In the study presented here,
0 the cultures terminated bioleaching of Theisen sludge
As(III) As(V) Sb(V) after extracting a maximum of 70% zinc in shaking
flasks. In bioreactor experiments, the pure culture of
Figure 3 Antimony- and arsenic speciation. Inoculated experiments:
Acidithiobacillus ferrooxidans Ram6F and Nochten2. Abiotic controls: A. ferrooxidans Ram6F did not grow well, although it had
tyndallized Theisen sludge in 9K25 (AT + Fe(II)); tyndallized Theisen been isolated from a mining affected site where elevated
sludge in 0K medium (AT Fe(II)). Unsterile control: non-tyndallized concentrations of heavy metals may be expected. Culture
Theisen sludge in 9K25 (NT+Fe(II)). Nochten2 showed a relatively long lag-phase although

1526 Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology
C. Klink et al. Bioleaching of fine-grained waste

(a) 4·75 (b) 3500

4·5
3000
4·25

Dissolved zinc [mg l–1]

4 2500

3·75
2000
pH

3·5
3·25 1500

3
1000
2·75
500
2·5 0 5 10 15 20 25
0 5 10 15 20 25
Time [days]
Time [days]
(c) 700

650

600
Redox potential [mV]

550

500

450

400

350

300
0 5 10 15 20 25
Time [days]

Figure 4 (a) Development of the pH in STBRs with a solid load of 4%. Inoculated reactors: Acidithiobacillus ferrooxidans Ram6F (♢- -) and Nocht-
en2 (♦—). Control reactor: tyndallized Theisen sludge in 9K25 (○- -). (b) Development of the concentration of dissolved zinc in STBRs with a solid
load of 4%. Inoculated reactors: A. ferrooxidans Ram6F (♢- -) and Nochten2 (♦—). Control reactor: tyndallized Theisen sludge in 9K25 (○- -). (c)
Development of the redox potential in STBRs with a solid load of 4%. Inoculated reactors: A. ferrooxidans Ram6F (♢- -) and Nochten2 (♦—).
Control reactor: tyndallized Theisen sludge in 9K25 (○- -).

that this culture had been adapted to Theisen sludge by respectively). This might explain the fact that after
subculturing, as recommended by Haghshenas et al. bioleaching with the mixed-species culture Nochen2 only
(2009) for adaption of A. ferrooxidans to high zinc con- Acidithiobacillus could be detected. To explore an inhibi-
centrations. With regard to heavy metal toxicity, maxi- tory effect of organic compounds like phenol, as
mum concentrations, in which growth or enzyme activity described in literature (Watling et al. 2009), detailed
in A. ferrooxidans still occurs, were determined as approx. studies would be necessary. Another very likely explana-
1 mol l 1 for zinc(II), copper(II) and nickel(II), tion for poor bioleaching performance is the relatively
500 mmol l 1 for cadmium(II) and 80 mmol l 1 for high starting pH of 4
5 in bioreactors. Therefore, opti-
arsenic(III) (Dopson et al. 2003). These concentrations, mization steps will include acidification of the growth
which are 65
4 g l 1 for zinc and 63
5 g l 1 for copper, medium with sulphuric acid prior to inoculation in order
were far from being reached in shaking-flask and bioreac- to generate the required low-pH environment, possibly
tor experiments in this study. However, the solutions between pH 1
6–1
8. According to Bingham et al. (1996)
contained a mixture of toxic metals that could have this will support the occurrence of dissolved iron species.
caused stress and could have reduced the activity of bac- Ferric iron naturally forms red-brownish precipitates,
teria. For Acidiphilium, the minimal inhibitory concentra- like the iron-oxy-hydroxy-sulphate mineral schwertman-
tions for zinc and copper, at which metabolic activity still nite, which mainly precipitates between pH 2
8–4
6
occurs, are lower (15–30 mmol l 1 and 8–150 mmol l 1 (Bingham et al. 1996). It has extensively been shown that

Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology 1527
Bioleaching of fine-grained waste C. Klink et al.

Table 3 Analyses of the metal-containing solution after bioleaching Theisen sludge shows only slightly different bioleaching
of Theisen sludge in bioreactors with a solid load of 4% behaviour after autoclaving as compared to tyndalliza-
Acidithiobacillus tion (data not shown). To accelerate experiments, auto-
Element ferrooxidans Ram6F Nochten2 AT+Fe(II) claving is proposed to be the method of choice for
1
working in small scale. However, any sterilization pro-
Concentrations [mg l ] according to ICP-AES
cess is technically neither necessary not feasible for
Zn 1900 3150 1530
S 2550 2900 1900
pilot scale operations.
Pb 3
00 2
90 2
80 That tyndallization was effective can be deduced from
Fe 700 50 120 the fact that all tyndallized controls appeared to be sterile.
Cu 20 62 0
45 Only in nontyndallized controls, microbial growth
Sb 6
30 7
60 1
20 occurred. The isolate, which was classified as A. ferrooxi-
As 1
5 2
0 0
30 dans Hel18, is supposed to be adapted to high heavy
Mn 6
10 7
20 5
0
metal and organics concentrations in Theisen sludge.
Mo 0
01 0
02 0
06
Concentrations [lg l 1] according to ICP-MS
Therefore, stirred-tank bioreactor operation with this
Co 790 990 640 strain is under investigation. It has been described that
Ge 380 440 91 the choice of the bioleaching consortium is very impor-
Re 256 390 223 tant in stirred-tank reactors (Bryan et al. 2011). As rela-
Ag 1
0 1
4 <0
9 tively homogeneous conditions are present in stirred-tank
Inoculated reactors: A. ferrooxidans Ram6F and Nochten2.
operations, the communities should be composed of a
Control: tyndallized Theisen sludge in 9K25 (AT+Fe(II)). stable mixture of at least one iron- and one sulphur-oxi-
dizer, and mixotrophic or heterotrophic acidophiles
schwertmannite can effectively be applied as sorbent for (Okibe et al. 2003). Consequently, mixed-species consor-
arsenate removal from acidic mine waters (Paikaray et al. tia perform better in stirred-tank bioleaching experi-
2011; HoungAloune et al. 2014). The immobilization of ments, although working with pure cultures might be
arsenic by sorption to iron hydroxides and the adsorption more reliable, because it is difficult to monitor the precise
behaviour of antimony have also been described (Kolbe composition of the bioleaching experiment at any time.
et al. 2011; Muehe et al. 2013). Therefore, schwertman- Further optimization of the Theisen sludge bioleaching
nite and other iron hydroxides control the occurrence of process could possibly be achieved by using for example,
arsenic in solution. Antimony can also be removed by Leptospirillum ferrooxidans, which is able to grow at lower
sorption. The affinity of Sb(V) and Sb(III) towards iron pH values (Sand et al. 1992). Furthermore, a semi-con-
oxides depends on the antimony species, the pH of the tinuous operation in serially connected vessels instead of
liquid, and the characteristics of the iron oxides. Thus, Sb batch cultivation might be an option (Bombacher et al.
(V) adsorption onto the iron oxides is enhanced at acidic 1998).
conditions (Guo et al. 2014). With regard to the anti-
mony species, our results show that the oxidation of any
reduced antimony species (possibly as sulphides in the Acknowledgements
solid material) seems to be fast and complete under the Sebastian Eisen was funded by BMBF r4 project ‘Theisen-
given conditions. However, the sorption onto precipitated schlamm’ (project number: 033R137). Simone Schopf
iron hydroxides is not completely efficient for arsenic as was financed by the Dr.-Erich-Kr€ uger Foundation
well as for antimony. (‘BHMZ’). The authors thank Nadja Eisen for discussion
To discriminate between microbially enhanced and of T-RFLP results, Beate Erler for technical support, and
abiotic leaching, a sterilization process for Theisen Paul David Ronning for proofreading and linguistic revi-
sludge was necessary in order to prevent microbial sion.
activity in sterile controls. A commonly used method
for sterilization or germ reduction is heat-treatment
either by pressure and steam (autoclaving) or by tyn- Conflict of Interest
dallization in an oven. A logical argument against heat None declared.
treatment is the potential formation of passivation lay-
ers or the oxidation of metal sulphides. Therefore,
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1530 Journal of Applied Microbiology 120, 1520--1530 © 2016 The Society for Applied Microbiology