Schwertmannite formation at cel junctions 
by a new filament-forming Fe(I)-oxidizing 
isolate afiliated with the novel genus
Authors: Jiro F. Mori, Shipeng Lu, Mathias Händel, 
Kai Uwe Totsche, Thomas R. Neu, Vasile Vlad Iancu, 
Nicolae Tarcea, Jürgen Popp, and Kirsten Küsel
This is a postprint of an article that originaly appeared in Microbiology on January 2016. 
Mori, Jiro F. , Shipeng Lu, Mathias Händel, Kai Uwe Totsche, Thomas R. Neu, Vasile Vlad 
Iancu, Nicolae Tarcea, Jürgen Popp, and Kirsten Küsel. "Schwertmannite formation at cel 
junctions by a new filament-forming Fe(I)-oxidizing isolate afiliated with the novel genus." 
Microbiology 162, no. 1 (January 2016): 62-71. DOI: 10.1099/mic.0.000205.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Schwertmannite formation at cel junctions by a
new filament-forming Fe(I)-oxidizing isolate
affiliated with the novel genusAcidithrix
Jiro F. Mori,1Shipeng Lu,1,23Mathias Ha¨ ndel,3Kai Uwe Totsche,3
Thomas R. Neu,4Vasile Vlad Iancu,5Nicolae Tarcea,5Ju¨rgen Popp5,6
and Kirsten Ku¨sel1,2
Corespondence
Shipeng Lu
shipeng.lu@montana.edu
1Aquatic Geomicrobiology, Friedrich Schiler University Jena, Jena, Germany
2The German Centre for Integrative Biodiversity Research (iDiv) Hale–Jena–Leipzig,
Leipzig, Germany
3Hydrogeology, Friedrich Schiler University Jena, Jena, Germany
4Department of River Ecology, Helmholtz Centre for Environmental Research-UFZ,
Magdeburg, Germany
5Institute of Physical Chemistry and Abbe School of Photonics, Friedrich Schiler University Jena,
Jena, Germany
6Institute of Photonic Technology, Jena, Germany
A new acidophilic iron-oxidizing strain (C25) belonging to the novel genusAcidithrixwas isolated
from pelagic iron-rich aggregates (‘iron snow’) colected below the redoxcline of an acidic lignite
mine lake. Strain C25 catalysed the oxidation of ferous iron [Fe(I)] under oxic conditions at 258C
at a rate of 3.8 mM Fe(I) day21in synthetic medium and 3.0 mM Fe(I) day21in sterilized lake
water in the presence of yeast extract, producing the rust-coloured, poorly crystaline mineral
schwertmannite [Fe(II) oxyhydroxylsulfate]. During growth, rod-shaped cels of strain C25 formed
long filaments, and then aggregated and degraded into shorter fragments, building large cel–
mineral aggregates in the late stationary phase. Scanning electron microscopy analysis of cels
during the early growth phase revealed that Fe(II)-minerals were formed as single needles on the
cel surface, whereas the typical pincushion-like schwertmannite was observed during later growth
phases at junctions between the cels, leaving major parts of the cel not encrusted. This directed
mechanism of biomineralization at specific locations on the cel surface has not been reported from
other acidophilic iron-oxidizing bacteria. Strain C25 was also capable of reducing Fe(II) under
micro-oxic conditions which led to a dissolution of the Fe(II)-minerals. Thus, strain C25 appeared
to have ecological relevance both for the formation and transformation of the pelagic iron-rich
aggregates at oxic/anoxic transition zones in the acidic lignite mine lake.
Received 28 July 2015
Revised 13 October 2015
Accepted 23 October 2015
INTRODUCTION
Iron-oxidizing bacteria (FeOB) mediate the oxidation of
ferrous iron [Fe(I)] to ferric iron [Fe(II)] to conserve
energy for growth (Colmer & Hinkle, 1947). Biogenic
Fe(II) subsequently hydrolyses and precipitates from sol-
ution forming various Fe(II) oxides when the pH exceeds
2 (Kappler & Straub, 2005). Due to the low rates of chemi-
cal Fe(I) oxidation under acidic conditions, acidophilic
aerobic FeOB are the main drivers for Fe(I) oxidation,
especialy in acid mine drainage impacted sites (e.g. Hal-
berget al., 2006; Leduc & Ferroni, 1994; Lo´pez-Archila
et al., 2001; Tysonet al., 2004). When the pH increases,
FeOB face the problem of disposing their poorly soluble
3Present address:Department of Chemical and Biological Engineering,
Center for Biofilm Engineering, Montana State University, 313 EPS
Building, Bozeman, MT 59717, USA.
Abbreviations: ARDRA, amplified rDNA restriction analysis; EPS,
extracelular polymeric substance; FeOB, iron-oxidizing bacteria; SEM,
scanning electron microscopy.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
sequence ofAcidithrix ferooxidansstrain C25 is LN866582.
One supplementary figure is available with the online Supplementary
Material.
Microbiology(2015),00,1–10 DOI10.1099/mic.0.000205
000205G 2015 The Authors Printed in Great Britain 1
Microbiology micromic000205.3d 30/11/2015 20:38:2

Macherey-Nagel) and were screened by amplified rDNA restriction
analysis (ARDRA) with restriction enzymesHhaI andBsuRI (HaeII)
(Fermentas). Representative sequences were chosen for sequencing
(Macrogen). The raw sequences were processed in Geneious 4.6.1
(Biomaters) for trimming and assembling, folowed byBLASThom-
ology search (Johnsonet al., 2008) and phylogenetic characterization
byARB6.0.2 (Ludwiget al., 2004).
Characterization of strain C25.Growth of strain C25 was tested in
APPW+YE medium at diferent temperatures and initial pH values.
Rates of Fe(I) oxidation were determined both in APPW+YE liquid
medium and in filter-sterilized lake water colected in June 2014 from
*50 cm depth. FeSO4was added to APPW+YE medium and to lake
water [v0.2 mM Fe(I)] as Fe(I) source to reach a final concen-
tration of 25 mM. Aliquots of 5 ml pre-cultures growing in
APPW+YE medium were added at the same time to either 100 ml
medium or lake water in glass botles closed with a sterile coton
stopper and incubated at 15 or 25uC for 30 days. Potential chemical
Fe(I) oxidation was determined in abiotic controls and in inoculated
medium amended with 0.1 mM sodium azide. Al treatments were
prepared in triplicates. Aliquots of 0.2 ml were obtained with sterile
pipetes every 2–3 days for measurements of pH (pH 330+ SenTix;
WTW) and Fe(I) using the phenanthroline method (Tamura et al.,
1974). After 10 days of incubation, 0.2 g YE was added to each botle
of lake water as additional carbon source. Rates were calculated using
the linear decrease of the Fe(I) concentration.
The capacity for dissimilatory Fe(II) reduction of strain C25 was
tested in FeSO4-supplemented APPW+YE medium at 25uC after
Fe(I) was depleted and rust-coloured precipitates were formed. Then
glucose was added to reach a final concentration of 5 mM. When no
de-colouration was detected after 2 days, rubber stoppers were
removed for*2 min every other day to maintain micro-oxic con-
ditions. Reduction of Fe(II) was identified by de-colouration of the
medium and increase of Fe(I) concentration.
Atempts were also made to amplify form I (cbbL) and form I (cbbM)
(large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase,
found in autotrophic bacteria) genes for evidence of the ability of
strain C25 to fix carbon dioxide using primer sets described by
Alfreideret al.(2003, 2009).
Microscopic observation of cel –mineral aggregation. The
morphology of the isolate and its association with the Fe(II)-minerals
was observed under light and fluorescent microscopy (Axioplan,).
Bacterial cels taken from liquid culture were stained with SYTO 13
(Life Technologies) on glass slides and visualized. The light and flu-
orescent images were taken by a mono-colour camera (Axio Cam
MRm; Carl Zeiss).
The bacterial aggregates were examined by CLSM using a TCS
SP5X (Leica) equipped with a white laser source, an upright micro-
scope and|63/1.2 water immersion lens. The bacterial aggregates
were stained with SYTO 9 dye (Molecular Probes) for nucleic acid
staining. Samples were analysed by CLSM using the laser line at
Isolate WJ25 (AY495954) 
Ferithrix thermotolerans (AY140237)
Ferimicrobium acidiphilum T23 (AF251436)
Ferimicrobium sp. Mc9kl-1-4 (HM769774)
Ferimicrobium acidiphilum FeSo-D1-6-CH (FN870326)
Ferimicrobium sp. BGR 117 (GU168015)
Acidimicrobium ferooxidans DSM 10331 (NR_074390)
Clone JTC04 (AY805539)
Acidithiomicrobium sp. P2 (GQ225721)
Clone Central-Botom-cDNA clone1 (HE604007)
Clone ORCL3.9 (EF042583) 
Clone RT11-ant04-c06-S (JF737864) 
Clone LRE22B6 (HQ420111)
Clone TRA2-10 (AF047642)
Clone AMD 67 (DQ159173)
Acid streamer iron-oxidizing bacterium CS11 (AY765999)
Acidithrix ferrooxidans strain C25 (LN866582)
Clone LR AB199 (KF225655)
Clone VA2-bac e9 (JN982087)
Clone A1 a1 (HQ317068)
Clone OY07-C073 (AB552359)
Clone 20m c4 (HM745419)
Clone CN13.5m-bac c1 (KC619609)
Clone DCN-1-38 (DQ660858)
Acidithrix ferooxidans Py-F3 (KC208497)
Acid streamer iron-oxidizing bacterium KP1 (AY765991)
Clone Carn-cDNA (FR846991)
Clone TakashiA-B29 (AB254789)
0.10 
Fig. 1.16S rRNA gene phylogenetic tree showing the close relationship of strain C25 (bold) with other closely related bac-
terial isolates and clones. The tree was reconstructed using the neighbour-joining method. GenBank accession numbers for
sequences are given in parentheses.Rubrobacter radiotolerans(GenBank accession number U65647) was used as out-
group (not shown). Bar, 0.1 change per nucleotide position
;
.
Novel microbial schwertmannite formation approach
htp://mic.microbiologyresearch.org 3
Microbiology micromic000205.3d 30/11/2015 20:38:2
483 nm. Emission signals were detected from 478 to 488 nm (reflec-
tion signals from inorganic and mineral compounds) and 500 to
550 nm (SYTO 9). Optical sections were colected in thez-direction
with a step size of 0.5mm. Images were subjected to blind deconvo-
lution using Huygens 15.05 (SVI) and projected in Imaris 8.1.2
(Bitplane).
Biogenic Fe(II)-mineral identification and visualization of
cel –mineral associations.Raman spectroscopy was used to
characterize the air-dried Fe(II)-mineral phases produced by strain
C25 grown in APPW+YE medium. The Raman spectra were recor-
ded with a Jobin-Yvon Labram Raman Confocal Microscope
(Horiba). Whilst performing the measurements the microscope was
equipped with a|50/0.5 microscope objective and a 300 lines mm21
grating spectrometer with a spectral resolution of*12 cm21.
When recording of the spectra, the 532 nm laser line with a power of
50mW was employed. The samples were left out to dry at
room temperature and then Raman spectra were recorded.
The measurement time of a Raman spectrum varied from 250
to 300 s.
Scanning electron microscopy (SEM) was used to visualize the cel–
mineral associations. Droplets of the sample suspensions were put on
silicon wafers and subjected to air drying. High-resolution secondary
electron images were recorded with an ULTRA PLUS field emission
scanning electron microscope (Carl Zeiss).
RESULTS AND DISCUSSION
Placing strain C25 into the novel genusAcidithrix
From a total of 61 isolates obtained from iron snow colected
in the central basin of the lake, eight isolates were 100% iden-
tical based on ARDRA and sequencing results. One represen-
tative strain, strain C25, formed rust-coloured colonies within
1 week on FeO solid medium containing TSB. Colonies were
circular, raised and produced rusty precipitates on the plate
with a slightly brown centre. Cels of strain C25 were rod-
shaped with a length of 1.5–2mm and a width of 0.5mm,
stained Gram-positive, and were not observed to form
Table 1.List of bacterial isolates and clones closely related to strain C25 =
Sampling site Site
pH
Site tem-
perature
(8C)
Isolate/clone
designation
GenBank
accession
no.
Similarity to
strain C25 (%)
Reference
Acidic lignite mine lake 77, Germany 2.8 11.5–13 Acidithrix ferrooxidans
C25
LN866582 NA This study
Acid copper mine Mynydd Parys, UK 2.5 11.3 Acidithrix ferrooxidans
Py-F3, DSM 28176T
KC208497 100 Jones & Johnson
(2015); Kayet al.
(2013)
Acidic, iron-rich spa water in Trefriw
Wel Spa, UK
2.7 10.1 Acid streamer iron-oxi-
dizing bacterium CS11
AY765999 100 Halberget al.(2006)
Acid copper mine Mynydd Parys, UK 2.4–
2.7
8.8–12.4 Acid streamer iron-oxi-
dizing bacterium KP1
AY765991 100 Halberget al.(2006)
Acidic lignite mine lake 77, Germany 4.0 9.8 Clone Central-Botom-
cDNA 1
HE604007 100 Luet al.(2013)
Acidic river, Rio Tinto, Spain 3.2 NA Clone RT11-ant04-c06-
S
JF737864 100 Garcı´a-Moyanoet al.
(2012)
Acidic coal mine drainage in the Lower
Red Eyes spring, USA
3.0 14–24 Clone LRE22B6 HQ420111 100 Brownet al.(2011)
Acid mine drainage from Iron Moun-
tain, USA
2.5 20 Clone TRA2-10 AF047642 99.9 Edwardset al.(1999)
Copper mine drainage in the Iberian
Pyrite Belt, Spain
2.7–
2.8
NA Clone ORCL3.9 EF042583 99.7 Roweet al.(2007)
Acid mine Los Rueldos, Spain ,2 ,13 Clone LR AB 199 KF225655 99.6 Me´ndez-Garcı´aet al.
(2014)
Carbondale constructed wetland system
treating acid mine drainage, USA
2.0–
3.9
NA Clone AMD 67 DQ159173 98.3 Nicomratet al.(2008)
Volcanic ash deposit from Miyake-jima,
Japan
3.4 12.7 Clone OY07-C073 AB552359 98.1 Fujimuraet al.(2012)
Acidic river, Rio Agrio, Argentina 1.0 59 Clone VA2-bac e9 JN982087 98.0 Urbietaet al.(2012)
Acidic pit lake, Lake Concepcion, Spain 2.5–
3.5
12–13 Clone CN13.5m-bac c1 KC619609 97.2 Santofimiaet al.
(2013)
Acid mine effluent from La Zarza-Per-
runal, Spain
3.1 25.6 Clone 20m c4 HM745419 97.1 Gonza´lez-Torilet al.
(2011)
Acidic hot springs, Iceland 2 45–50 Acidimicrobium ferroox-
idansDSM 10331T
NR_074390 91.9 Clark & Norris (1996)
NA, not available in references.
J. F. Mori and others
4 Microbiology00
Microbiology micromic000205.3d 30/11/2015 20:38:3
endospores. No growth was observed in liquid FeO medium
without YE. However, beter growth was observed in
APPW+YE medium supplemented with FeSO4. Cels couldgrow in iron-free medium with a reduced growth rate.
The 16S rRNA gene of strain C25 places it within the family
Acidimicrobiaceaein the phylumActinobacteria(Fig. 1). The
16S rRNA gene of strain C25 was only 91.9% identical to
the moderate thermophileAcidimicrobium ferrooxidans
DSM 10331T (GenBank accession number NR_074390)
(Clark & Norris, 1996), but 100% identical to a high fraction
of clones obtained from RNA extracted from samples of the
same site in 2011 (Clone Central-Botom-cDNA 1 in
Table 1) (Luet al., 2013). Strain C25 also shared 100% 16S
rRNA gene identity with the FeOB strain CS11 (GenBank
accession number AY765999), strain KP1 (GenBank accession
number AY765991) andAcidithrix ferrooxidansstrain PY-F3T
(GenBank accession number KC208497) (Fig. 1, Table 1) that
was recently isolated from an acidic, metal-rich water in north
Wales in the UK (Jones & Johnson, 2015).
Strain C25 was able to grow and oxidize Fe(I) at pH 2.5–
6.5 in the temperature range from 15 to 33uC, but not at
37uC, with an optimum rate of oxidation observed
between 25 and 30uC (Table 2). However, during growth
the medium pH subsequently declined due to the release
of protons that occurs when ferric iron hydrolyses water
and produces Fe(II) oxide hydroxides. The final pH
remained stable between 2.2 and 2.3 after 14 days of incu-
bation regardless of initial pH values (Table 2).
During the initial phase of Fe(I) oxidation by strain C25, a lag
phase of*5 days was observed prior to the onset of Fe(I) oxi-
dation when grown at 25uC (Fig. 2a). During this 5 day
period, the medium pH increased slightly by*0.3 pH units
before Fe(I) was consumed at a rate of 2.94+0.27 mM
day21and rust-coloured precipitates formed. However, no
second lag phase was observed when this culture was resupple-
mented with FeSO4after Fe(I) depletion. Folowing resupple-mentation with FeSO4, the rates of Fe(I) oxidation increasedto 3.77+0.25 mM day21(Fig. 2a). Cels cultivated at 15uC
showed a slower rate of Fe(I) oxidation (1.02+0.07 mM
day21)witha3daylagphase(Fig.2b). WhenstrainC25
was cultivated in sterile-filtered lake water with a pH of 2.6
supplemented with FeSO4and YE, the observed ratesof Fe(I) oxidation were approximately 3.01+0.40
and 0.83+0.11 mM day21at 25 and 15uC, respectively
(Fig. 2c, d). Neither cel growth nor Fe(I) oxidation was
observed in any control cultures.
Dissimilatory Fe(II) reduction was observed on strain C25
within 5 days under micro-oxic condition after an adjust-
ment of the headspace and the addition of 5 mM glucose.
Al Fe(II)-minerals generated under previous oxic con-
ditions disappeared until no rust colour could be observed.
Bacterial cels regenerated filaments during Fe(II)
reduction (Fig. S1, available in the online Supplementary
Material). Similarly, strain Py-F3Tcan catalyse the dissim-
ilatory reduction of solid-phase Fe(II) under micro-oxic
conditions (Jones & Johnson, 2015) as wel as many
other acidophilicActinobacteria(Bridge & Johnson, 1998;
Itohet al., 2011; Johnsonet al., 2003, 2009).
Strain C25 showed high morphological and physiological
similarities to the strain Py-F3T. However, strain C25 has
diferent physiological characteristics compared with the
newAcidithrix-type strain. Strain C25 could not grow at
pH 2.0, but was shown to tolerate higher pH values than
strain Py-F3T. In addition, strain C25 was unable to
reduce solid-phase Fe(II) under strictly anoxic conditions,
in contrast to strain Py-F3T. The genome sequence of strain
Py-F3Tencodes two subunits for a type I ribulose 1,5-
bisphosphate carboxylase/oxygenase and several enzymes
required for carbon fixation via the Calvin–Benson–Bas-
sham cycle (Eisenet al., 2015). However, PCR products
of thecbbLgene in strain C25 were only detected as a
very weak band, thus we could not confirm the presence
of genes encoding ribulose 1,5-bisphosphate carboxylase/
oxygenase in strain C25 (data not shown).
Novel iron precipitation approach of strain C25
A novel iron precipitation approach was identified with
cels of strain C25. Schwertmannite was identified as the
sole product of Fe(I) oxidation by strain C25 using
Raman spectroscopy (Fig. 3). The peaks exhibiting at
296, 318, 351, 424, 545, 718, and 986 cm21were close to
the reference peak patern of schwertmannite (294, 318,
350, 421, 544, 715 and 981 cm21).
Fluorescence microscopic examination showed that single
cels of strain C25 formed long filaments (up to 400mm)
during the active phase of Fe(I) oxidation (Fig. 4a). No
EPS-like matrix was confirmed by SEM or fluorescence
Table 2.Growth experiments of strain C25 in APPW+YE liquid medium at diferent cultivation temperatures and with diferent
initial pH values >
Temperature (pH 2.5) Starting pH of medium (258C)
158C 258C 308C 338C 378C 2.0 2.5 4.5 6.5
+ ++ ++ +/2 – – (2.0)* ++(2.2–2.3)* ++(2.2–2.3)* ++(2.2–2.3)*
*Final pH of media at day 14 of incubation in triplicates.
Novel microbial schwertmannite formation approach
htp://mic.microbiologyresearch.org 5
Microbiology micromic000205.3d 30/11/2015 20:38:3
microscopy. Eventualy the entangled filaments were
associated with rust-coloured schwertmannite (Fig. 4b).
In the later stationary phase, long filaments broke into
smaler fragments and single cels leading to the formation
of greater cel–mineral aggregates (Fig. 4c–e). Filaments
were also observed when cels were inoculated in Fe(I)-
free medium. Interestingly, diferent stages of schwertman-
nite aggregation could be clearly diferentiated using SEM
(Fig. 5). Initialy, cels growing for 3–4 days displayed
needle-shaped whiskers formed on the cel surface, which
implies al cel-oxidized iron is cel wal-associated
(Fig. 5b). After 1 week, filamentous cels featured ‘matured’
spheres with numerous peripheral needles atached to the
cel surface only at junctions between the cels, leaving
major parts of the cel surface free of encrustation
0 5 10 15 20 25
2.0
2.5
3.0
0
5
10
15
20
25
30
0 5 10 15 20 25
2.0
2.2
2.4
2.6
2.8
3.0
pH
 
pH
 
0
5
10
15
20
25
30
Fe
(II)
 co
nc
ent
rat
io
n (
mM
)
Fe
(II)
 co
nc
ent
rat
io
n (
mM
)
Time (days) Time (days) 
(a) 
(c) 
(b) 
(d) 
Fig. 2.Ferous iron oxidation by strain C25 in (a, b) synthetic medium APPW+YE and (c, d) lake water at 25 (a, c) and 158C
(b, d), respectively. Additional ferous iron was added at day 14 of incubation in APPW+YE at 258C (a, arowed point). Strain C25
cultured in APPW+YE medium (X) or lake water (#). APPW+YE medium (&) or lake water (%) without bacterial inoculation.
Strain C25 cultured in APPW+YE medium(m) or lake water (D) with 0.1 mM sodium azide. Data represent mean¡SD;n53 <.
0 200
296
318
351424
545 718
986
Schwertmannite reference
400 600 800
Wavenumber (cm–1)
Strain C25 aggregate
1000 1200 1400
Ra
ma
n in
te
nsit
y
Fig. 3.Raman spectra of the Fe(II)-mineral produced by strain
C25 in APPW+YE medium (black line) and authentic schwert-
mannite standard (blue line).
J. F. Mori and others
6 Microbiology00
Microbiology micromic000205.3d 30/11/2015 20:38:3
(Fig. 5c). Finaly, ‘matured’ schwertmannite clumped
together with other neighbouring minerals atached to
cel surfaces, leading to the formation of coiled-cel fila-
ments. During the formation of these large cel–mineral
assemblages, the long cel filaments appeared to break
into shorter fragments. This directed mineral formation
alowed parts of the cel surface to remain free of encrusta-
tion, enabling them to retain access to dissolved Fe(I) and
other nutrients (Fig. 5d). This wel-coordinated mechan-
ism for biomineralization controled by strain C25 was dis-
tinct from other known Fe(I) oxidation approaches under
low pH conditions. Acidophilic bacteria have to maintain
their cytoplasm at neutral pH by reversing their membrane
potential. Some researchers suggest a positive charge on the
cel surfaces (Hedrichet al., 2011; Norriset al., 1992),
whilst others suggest a negative surface charge
(a) (b)
(c) (d)
(e)
Fig. 4.Formation of long filaments of strain C25 during diferent growth phases. (a–d) Cels of strain C25 in APPW+YE
medium were stained with SYTO 13 (green, nucleic acids). Images were taken at day 4 (a), 7 (b), 12 (c) and 30 (d) of culti-
vation. (e) Three-dimensional volume view of strain C25 cels which an form iron-rich aggregate, stained with SYTO 9 (green,
nucleic acid; grey, reflection). Bar, 10mm.
Novel microbial schwertmannite formation approach
htp://mic.microbiologyresearch.org 7
Microbiology micromic000205.3d 30/11/2015 20:38:3
(Baker-Austin & Dopson, 2007). Schwertmannite at the
given pH has a positive charge and schwertmannite
needle formation was observed at the sites of cel-to-cel
connections of strain C25, suggesting a heterogeneous
distribution of surface charge or an accumulation of
specific anionic compounds at the polar end of the cels
might be involved in the directed localization of the
Fe(II)-minerals at this specific position. The biominerali-
zation approaches of otherActinobacteriaspecies have
not been wel characterized. Strain Py-F3Thas only been
shown to initiate schwertmannite precipitation (Jones &
Johnson, 2015) without further details. Acidophiles,
includingLeptospirilumandFerroplasma, capable of grow-
ing in extremely acidic (pHv2) environments, are not
susceptible to iron encrustation (Druschelet al., 2004;
Tysonet al., 2004). Other acidophilic FeOB, such asFerro-
vum myxofaciensandAcidithiobacilus ferrooxidans, which
are known to grow in less extreme environments, are able
to deposit Fe(II)-minerals into their EPS as a mechanism
to avoid encrustation by the Fe(II)-minerals (Frankel &
Bazylinski, 2003; Hedrichet al., 2011).
Ecological significances of strain C25
Filamentous bacteria with a similar morphology to strain
C25 are present in iron snow as revealed by CLSM imaging
in a previous study (Luet al., 2013). The oxic/anoxic
(a) (b)
(c) (d)
Fig. 5.Scanning electron micrographs of strain C25 and Fe(II)-minerals formed by the oxidation of Fe(I) by stain C25.(a) A
single cel of strain C25.(b) The initial phase of Fe(II)-mineral precipitation on the cel filaments of strain C25.(c) Precipitation
of pincushion-like Fe(II)-mineral spheres at the joint part of cel filaments. (d) A cel–Fe(II)-mineral aggregate formed by strain
C25 cels and pincushion-like Fe(II)-mineral spheres in the later stationary growth phase. Bar, 1mm.
Anoxic
Oxic
Fe2+ O2
Strain C25
Schwertmannite Fe3+
Fe2+Redox-
cline
EPS EPSproducers
Fig. 6.Putative schematic model depicting the significant role of
strain C25 in iron snow formation in the redoxcline of acidic iron-
rich lakes.
J. F. Mori and others
8 Microbiology00
Microbiology micromic000205.3d 30/11/2015 20:38:3
transition in the redoxcline is idealy suited for micro-
organisms with the ability to oxidize Fe(I) and reduce
Fe(II). Strain C25 and other related species make up the
largest fraction of the metabolicaly active bacteria in the
iron snow, folowed by the EPS-formingFerrovumspecies
(Luet al., 2013). Thus, we suggest that strain C25 is
involved in early stages of iron snow formation via Fe(I)
oxidation and subsequent large cel aggregate formation
associated with the pincushion-like mineral schwertman-
nite (Fig. 6). EPS released byFerrovumspecies should
favour the cohesiveness and additional growth of these
pelagic aggregates, atract heterotrophic micro-organisms,
and aid in the prevention of complete iron encrustation.
These pelagic aggregates, reaching a mean size between
60 and*240mm (Reicheet al., 2011), wil begin to sink
through the redoxcline. When the oxygen concentration
declines, strain C25 could exploit the adsorbed organic
compounds as a carbon source under micro-oxic con-
ditions and trigger the utilization of schwertmannite as
an alternative electron acceptor. This reductive dissolution
ultimately results in a reduced size that would enable the
iron snow to remain in the redoxcline for an extended
period of time prior to sinking to the sediment. The
capacity of strain C25 to serve both halves of the iron
cycle provides a unique insight into the ecological relevance
and importance in linking the redoxcline with the sediment
by providing a mechanism for removal of iron, microbial
cels and organic carbon from the water column, and sub-
sequent accumulation in the sediment.
ACKNOWLEDGEMENTS
J. M. was supported by the graduate research training group ‘Altera-
tion and element mobility at the microbe–mineral interface’
(GRK1257), which is part of the Jena School for Microbial Communi-
cation and funded by the German Research Foundation (Deutsche
Forschungsgemeinschaft). S. L. was supported by the German
Centre for Integrative Biodiversity Research (iDiv) Hale–Jena–Leip-
zig and Deutsche Forschungsgemeinschaft. We thank Maren Sickin-
ger, Dr Juanjuan Wang and Dr Rebecca Cooper for technical
assistance, discussions and manuscript proofreading.
REFERENCES
Alfreider, A., Vogt, C., Hofmann, D. & Babel, W. (2003).Diversity of
ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes
from groundwater and aquifer microorganisms.Microb Ecol 45,
317–328.
Alfreider, A., Vogt, C., Geiger-Kaiser, M. & Psenner, R. (2009).
Distribution and diversity of autotrophic bacteria in groundwater
systems based on the analysis of RubisCO genotypes.Syst Appl
Microbiol32, 140–150.
Baker-Austin, C. & Dopson, M. (2007).Life in acid: pH homeostasis
in acidophiles.Trends Microbiol15, 165–171.
Bond, P. L., Smriga, S. P. & Banfield, J. F. (2000).Phylogeny of
microorganisms populating a thick, subaerial, predominantly
lithotrophic biofilm at an extreme acid mine drainage site.Appl
Environ Microbiol66, 3842–3849.
Bridge, T. A. M. & Johnson, D. B. (1998).Reduction of soluble iron
and reductive dissolution of ferric iron-containing minerals by
moderately thermophilic iron-oxidizing bacteria. Appl Environ
Microbiol64, 2181–2186.
Brown, J. F., Jones, D. S., Mils, D. B., Macalady, J. L. & Burgos, W. D.
(2011).Application of a depositionalfaciesmodel to an acid mine
drainage site.Appl Environ Microbiol77, 545–554.
Ciobota˘ , V., Lu, S., Tarcea, N., Ro¨sch, P., Ku¨ sel, K. & Popp, J. (2013).
Quantification of the inorganic phase of the pelagic aggregates from
an iron contaminated lake by means of Raman spectroscopy.Vib
Spectrosc68, 212–219.
Clark, D. A. & Norris, P. R. (1996).Acidimicrobium ferrooxidansgen.
nov. sp. nov.: mixed-culture ferrous iron oxidation withSulfobacilus
species.Microbiology142, 785–790.
Colmer, A. R. & Hinkle, M. E. (1947).The role of microorganisms in
acid mine drainage: a preliminary report.Science106, 253–256.
Druschel, G. K., Baker, B. J., Gihring, T. M. & Banfield, J. F. (2004).
Acid mine drainage biogeochemistry at Iron Mountain, California.
Geochem Trans5, 13–32.
Edwards, K. J., Goebel, B. M., Rodgers, T. M., Schrenk, M. O.,
Gihring, T. M., Cardona, M. M., Hu, B., McGuire, M. M., Hamers, R. J.,
Katrina, J. & Edwards Bret, M. (1999).Goebel Geomicrobiology of
pyrite (FeS2) dissolution: case study at Iron Mountain, California.
Geomicrobiol J16, 155–179.
Eisen, S., Poehlein, A., Johnson, D. B., Daniel, R., Schlo¨mann,M.&
Mu¨ hling, M. (2015).Genome sequence of the acidophilic ferrous iron-
oxidizing isolateAcidithrix ferrooxidansstrain Py-F3, the proposed
type strain of the novel actinobacterial genusAcidithrix.Genome
Announc3, e00382–e00315.
Falaga´n, C., Sa´nchez-Espan˜a, J. & Johnson, D. B. (2014).New
insights into the biogeochemistry of extremely acidic environments
revealed by a combined cultivation-based and culture-independent
study of two stratified pit lakes.FEMS Microbiol Ecol87, 231–243.
Frankel, R. B. & Bazylinski, D. A. (2003).Biologicaly induced
mineralization by bacteria.Rev Mineral Geochem54, 95–114.
Fujimura, R., Sato, Y., Nishizawa, T., Nanba, K., Oshima, K., Hatori,
M., Kamijo, T. & Ohta, H. (2012). Analysis of early bacterial
communities on volcanic deposits on the island of Miyake (Miyake-
jima), Japan: a 6-year study at a fixed site.Microbes Environ27, 19–29.
Garcı´a-Moyano, A., Gonza´lez-Toril, E., Aguilera, A´. & Amils, R.
(2012).Comparative microbial ecology study of the sediments and
the water column of the Rı´o Tinto, an extreme acidic environment.
FEMS Microbiol Ecol81, 303–314.
Geler, W., Klapper, H. & Schultze, M. (1998). Natural and
anthropogenic sulforic acidification of lakes. InAcid Mining Lakes:
Acid Mine Drainage, Limnology and Reclamation, pp. 3–14. Edited
by W. Geler, H. Klapper & W. Salomons. Berlin: Springer.
Gonza´lez-Toril, E., Aguilera, A., Souza-Egipsy, V., Lo´pez Pamo, E.,
Sa´nchez Espan˜a, J. & Amils, R. (2011).Geomicrobiology of La
Zarza-Perrunal acid mine effluent (Iberian Pyritic Belt, Spain).Appl
Environ Microbiol77, 2685–2694.
Grossart, H. P. & Ploug, H. (2000).Bacterial production and growth
efficiencies: direct measurements on riverine aggregates.Limnol
Oceanogr45, 436–445.
Halberg, K. B., Coupland, K., Kimura, S. & Johnson, D. B. (2006).
Macroscopic streamer growths in acidic, metal-rich mine waters in
north Wales consist of novel and remarkably simple bacterial
communities.Appl Environ Microbiol72, 2022–2030.
Hedrich, S., Lu¨ nsdorf, H., Kleeberg, R., Heide, G., Seifert, J. &
Schlo¨mann, M. (2011). Schwertmannite formation adjacent to
bacterial cels in a mine water treatment plant and in pure cultures
ofFerrovum myxofaciens.Environ Sci Technol45, 7685–7692.
Novel microbial schwertmannite formation approach
htp://mic.microbiologyresearch.org 9
Microbiology micromic000205.3d 30/11/2015 20:38:4
Itoh, T., Yamanoi, K., Kudo, T., Ohkuma, M. & Takashina, T. (2011).
Aciditerrimonas ferrireducensgen. nov., sp. nov., an iron-reducing
thermoacidophilic actinobacterium isolated from a solfataric field.
Int J Syst Evol Microbiol61, 1281–1285.
Johnson, D. B. & Halberg, K. B. (2007).Techniques for detecting
and identifying acidophilic mineral-oxidizing microorganisms. In
Biomining, pp. 237–261. Edited by D. E. Rawlings & D. B. Johnson.
Berlin: Springer.
Johnson, D. B., Okibe, N. & Roberto, F. F. (2003).Novel thermo-
acidophilic bacteria isolated from geothermal sites in Yelowstone
National Park: physiological and phylogenetic characteristics.Arch
Microbiol180, 60–68.
Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S.
& Madden, T. L. (2008).NCBI blast: a beter web interface.Nucleic
Acids Res36, W5–W9.
Johnson, D. B., Bacelar-Nicolau, P., Okibe, N., Thomas, A. &
Halberg, K. B. (2009).Ferrimicrobium acidiphilumgen. nov. sp.
nov. andFerrithrix thermotoleransgen. nov., sp. nov.: heterotrophic,
iron-oxidizing, extremely acidophilic actinobacteria.Int J Syst Evol
Microbiol59, 1082–1089.
Jones, R. M. & Johnson, D. B. (2015).Acidithrix ferrooxidansgen. nov.
sp. nov., a filamentous and obligately heterotrophic, acidophilic
member of the Actinobacteriathat catalyzes dissimilatory oxido-
reduction of iron.Res Microbiol166, 111–120.
Kappler, A. & Straub, K. L. (2005).Geomicrobiological cycling of iron.
Rev Mineral Geochem59, 85–108.
Kay, C. M., Rowe, O. F., Roccheti, L., Coupland, K., Halberg, K. B. &
Johnson, D. B. (2013).Evolution of microbial ‘streamer’ growths in
an acidic, metal-contaminated stream draining an abandoned
underground copper mine.In Life3, 189–210.
Klapper, H. & Schultze, M. (1995).Geogenicaly acidified mining
lakes - living conditions and possibilities of restoration.Int Rev
Hydrobiol80, 639–653.
Ku¨ sel, K. (2003).Microbial cycling of iron and sulfur in acidic coal
mining lake sediments.Water Air Soil Polut Focus3, 67–90.
Lane, D. J. (1991).16S/23S rRNA sequencing. InNucleic Acid
Techniques in Bacterial Systematics, pp. 115–175. Edited by E.
Stackebrandt & M. Goodfelow. New York: Wiley.
Leduc, L. G. & Ferroni, G. D. (1994).The chemolithotrophic
bacteriumThiobacilus ferrooxidans.FEMS Microbiol Rev14, 103–119.
Lo´pez-Archila, A. I., Marin, I. & Amils, R. (2001). Microbial
community composition and ecology of an acidic aquatic
environment: the Tinto River, Spain.Microb Ecol41, 20–35.
Lo´pez-Archila, A. I., Ge´rard, E., Moreira, D. & Lo´pez-Garcı´a, P.
(2004).Macrofilamentous microbial communities in the metal-rich
and acidic River Tinto, Spain.FEMS Microbiol Let235, 221–228.
Lu, S., Gischkat, S., Reiche, M., Akob, D. M., Halberg, K. B. & Ku¨ sel,
K. (2010).Ecophysiology of Fe-cycling bacteria in acidic sediments.
Appl Environ Microbiol76, 8174–8183.
Lu, S., Chourey, K., Reiche, M., Nietzsche, S., Shah, M. B., Neu, T. R.,
Hetich, R. L. & Ku¨ sel, K. (2013).Insights into the structure and
metabolic function of microbes that shape pelagic iron-rich
aggregates (iron snow).Appl Environ Microbiol79, 4272–4281.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H.,
Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors
(2004).ARB: a software environment for sequence data.Nucleic
Acids Res32, 1363–1371.
Luef, B., Aspetsberger, F., Hein, T., Huber, F. & Peduzzi, P. (2007).
Impact of hydrology on free-living and particle-associated
microorganisms in a river floodplain system (Danube, Austria).Freshw
Biol52, 1043–1057.
Me´ndez-Garcı´a, C., Mesa, V., Sprenger, R. R., Richter, M., Diez, M. S.,
Solano, J., Bargiela, R., Golyshina, O. V., Manteca, A´. & other authors
(2014).Microbial stratification in low pH oxic and suboxic macroscopic
growths along an acid mine drainage.ISME J8, 1259–1274.
Neubauer, S. C., Emerson, D. & Megonigal, J. P. (2002).Life at the
energetic edge: kinetics of circumneutral iron oxidation by lithotrophic
iron-oxidizing bacteria isolated from the wetland-plant rhizosphere.
Appl Environ Microbiol68, 3988–3995.
Nicomrat, D., Dick, W. A., Dopson, M. & Tuovinen, O. H. (2008).
Bacterial phylogenetic diversity in a constructed wetland system
treating acid coal mine drainage.Soil Biol Biochem40, 312–321.
Nixdorf, B., Hemm, M., Schlundt, A., Kapfer, M. & Krumbeck, H.
(2001).Tagebauseen in Deutschland – ein U¨berblick [Mining Lakes in
Germany – An Overview]. Berlin: UBA Texte.
Norris, P., Ingledew, W., Herbert, R. & Sharp, R. (1992).Acidophilic
bacteria: adaptations and applications. InMolecular Biology and
Biotechnology of Extremophiles, pp. 115–142. Edited by R. A.
Herbert & R. J. Sharp. Glasgow: Blackie.
Paikaray, S., Go¨tlicher, J. & Peifer, S. (2011).Removal of As(II)
from acidic waters using schwertmannite: surface speciation and
efect of synthesis pathway.Chem Geol283, 134–142.
Ploug, H., Grossart, H. P., Azam, F. & Jorgensen, B. B. (1999).
Photosynthesis, respiration, and carbon turnover in sinking
marine snow from surface waters of Southern California Bight:
implications for the carbon cycle in the ocean.Mar Ecol Prog Ser179, 1–11.
Reiche, M., Lu, S., Ciobota, V., Neu, T. R., Nietzsche, S., Ro¨sch, P.,
Popp, J. & Ku¨ sel, K. (2011).Pelagic boundary conditions afect the
biological formation of iron-rich particles (iron snow) and their
microbial communities.Limnol Oceanogr56, 1386–1398.
Rowe, O. F., Sa´nchez-Espan˜a, J., Halberg, K. B. & Johnson, D. B.
(2007).Microbial communities and geochemical dynamics in an
extremely acidic, metal-rich stream at an abandoned sulfide mine
(Huelva, Spain) underpinned by two functional primary production
systems.Environ Microbiol9, 1761–1771.
Santofimia, E., Gonza´lez-Toril, E., Lo´pez-Pamo, E., Gomariz, M.,
Amils, R. & Aguilera, A. (2013). Microbial diversity and its
relationship to physicochemical characteristics of the water in two
extreme acidic pit lakes from the Iberian pyrite belt (SW Spain).
PLoS One8, e66746.
Tamura, H., Goto, K., Yotsuyanagi, T. & Nagayama, M. (1974).
Spectrophotometric determination of iron(I) with 1,10-
phenanthroline in the presence of large amounts of iron(II).Talanta
21,314–318.
Tischler, J. S., Jwair, R. J., Gelhaar, N., Drechsel, A., Skirl, A. -M.,
Wiacek, C., Janneck, E. & Schlo¨mann, M. (2013). New cultivation
medium for FerrovumandGalionela-related strains.J Microbiol
Methods95, 138–144.
Tyson, G. W., Chapman, J., Hugenholtz, P., Alen, E. E., Ram, R. J.,
Richardson, P. M., Solovyev, V. V., Rubin, E. M., Rokhsar, D. S. &
Banfield, J. F. (2004).Community structure and metabolism through
reconstruction of microbial genomes from the environment.Nature
428,37–43.
Urbieta, M. S., Gonza´lez Toril, E., Aguilera, A., Giaveno, M. A. &
Donati, E. (2012).First prokaryotic biodiversity assessment using
molecular techniques of an acidic river in Neuque´n, Argentina.
Microb Ecol64, 91–104.
Wakao, N., Tachibana, H., Tanaka, Y., Sakurai, Y. & Shiota, H. (1985).
Morphological and physiological characteristics of streamers in acid
mine drainage water from a pyritic mine.J Gen Appl Microbiol31,
17–28.
Edited by: C. Dahl
J. F. Mori and others
10 Microbiology00
Microbiology micromic000205.3d 30/11/2015 20:38:4

Ordering reprints for Microbiology Society journals
As a result of declining reprint orders and feedback from many authors who tel us they
have no use for reprints,Microbiology Society no longer provides free reprints to corre-
sponding authors; instead, corresponding authors wil receive two emails:
i) An email including a link to download the published PDF of their paper. You
can forward this link to co-authors or others, and they can also use it to
download the published PDF. The link can be used up to 25 times. This
email wil be sent out at around the time your article is published online.
i) An email including a link to the Microbiology Society Reprint Service. You
can forward this email to your co-authors if you wish, so that they can
order their own reprints directly, or to your finance or purchasing department,
if orders are placed centraly. This email wil be sent out at around the time
that your article is finalized for printing.
When you click on the link in this second email, you wil be taken to an order page to
place your reprint order. Like most online ordering sites, it is necessary to set up an
account and provide a delivery address while placing your order, if you do not already
have an account. Once an account and delivery address have been set up, these details
wil be stored by the system for use with future orders. Payments can be made by credit
card, PayPal or purchase order.
As reprint orders are despatched by courier, there is a charge for postage and packing.
SUMMARYNYou can create or update your reprint account at any time at
htp:/microbiologysociety-reprints.charlesworth.com/
NYou wil be sent an email when the reprints of this paper are
ready for ordering
NYou cannot order reprints of this paper before this email has
been sent, as your paper wil not be in the system
NReprints can be ordered at any time after publication
NYou wil also receive an email with a link to download the PDF of
your published paper
The reprint ordering details wil be emailed to the author listed as the coresponding
author on the journal’s manuscript submission system. If your paper has been published
(the final version, not the publish-ahead-of-print version) but you have not received this
notification, emailreprints@microbiologysociety.orgquoting the journal, paper number
and publication details.
If you have any questions or comments about the reprint-ordering system or about the
link to your published paper, emailreprints@microbiologysociety.org