RESEARCH ARTICLE

Bioweathering of Kupferschiefer black shale (Fore-Sudetic

Monocline, SW Poland) by indigenous bacteria: implication for
dissolution and precipitation of minerals in deep underground
mine
Renata Matlakowska1, Aleksandra Skłodowska1 & Krzysztof Nejbert2
1
Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw, Warsaw, Poland; and 2Institute of Geochemistry,
Mineralogy and Petrology, Faculty of Geology, University of Warsaw, Warsaw, Poland

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Correspondence: Renata Matlakowska,
Laboratory of Environmental Pollution
Analysis, Faculty of Biology, University of
Warsaw, Miecznikowa 1, 02-096 Warsaw,
Poland. Tel./fax: +48 22 5541219; e-mail:
rmatlakowska@biol.uw.edu.pl
Received 6 October 2011; revised 18 January
2012; accepted 30 January 2012.
Final version published online 28 February
2012.
DOI: 10.1111/j.1574-6941.2012.01326.x
Editor: Christian Griebler
MICROBIOLOGY ECOLOGY
Abstract
The Upper Permian polymetallic, organic-rich Kupferschiefer black shale in the
Fore-Sudetic Monocline is acknowledged to be one of the largest Cu-Ag depos-
its in the world. Here we report the results of the first study of bioweathering
of this sedimentary rock by indigenous heterotrophic bacteria. Experiments
were performed under laboratory conditions, employing both petrological and
microbiological methods, which permitted the monitoring and visualization of
geomicrobiological processes. The results demonstrate that bacteria play a
prominent role in the weathering of black shale and in the biogeochemical
cycles of elements occurring in this rock. It was shown that bacteria directly
interact with black shale organic matter to produce a widespread biofilm on
the Kupferschiefer shale surface. As a result of bacterial activity, the formation

of pits, bioweathering of ore and rock-forming minerals, the mobilization of

Keywords
biogeochemical cycles; biomineralization;
outer membrane vesicles; ancient organic
matter; mineral dissolution.
elements and secondary mineral precipitation were observed. The chemistry
of the secondary minerals unequivocally demonstrates the mobilization of
elements from minerals comprising Kupferschiefer. The redistribution of P, Al,
Si, Ca, Mg, K, Fe, S, Cu and Pb was confirmed. The presence of bacterial outer
membrane vesicles on the surface of black shale was observed for the first time.
Biomineralization reactions occurred in both the membrane vesicles and the
bacterial cells.

Kupferschiefer contains the largest Cu-Ag concentrations

Introduction
The biotransformation of organic-rich sedimentary rocks
plays a fundamental role in the biogeochemical cycles of
elements (Petsch et al., 2005; Ehrlich & Newman, 2008).
This phenomenon involves a series of interactions
between the components of rocks and microorganisms
that lead to rock deterioration and the redistribution of
elements. These complex natural processes are of great
environmental significance and are also of potential use
in environmental biotechnology (Ehrlich & Newman,
2008).
The present study examined the bioweathering of
Kupferschiefer black shale from the Fore-Sudetic Mono-
cline (SW Poland). The highly mineralized Upper Permian
in the world (Speczik, 1995; Oszczepalski, 1999;
Piestrzyński & Wodzicki, 2000). The Kupferschiefer
horizon is developed as a dark grey organic matter-rich
black shale that is highly enriched in ore minerals
(sulphides and sulphosalts). This deposit differs from
known deposits which are mostly acidic in that it is
characterized by a neutral or slightly alkaline pH reach-
ing 8.5. More than 100 Cu, Ag, Fe, Ni, Co, Mo, Hg,
Bi, Au, Pt and Pd ore minerals have been recognized in
this shale (Speczik, 1994, 1995). These ore minerals
together with organic matter and rock-forming minerals
such as aluminosilicates, carbonates and detrital quartz
are dispersed within a clayey matrix (Speczik, 1994,
1995).

FEMS Microbiol Ecol 81 (2012) 99–110 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
100
Kupferschiefer black shale is significantly enriched in
organic matter (OM). The OM content ranges from 1 to
30 wt%, with an estimated average of around 6 wt%
(Speczik & Püttmann, 1987; Püttmann et al., 1988, 1991;
Speczik, 1994; Oszczepalski, 1999; Bechtel et al., 2000,
2001). The organic matter is of marine origin and is rep-
resented mainly by witrinite, liptinite and inertinite
(Speczik & Püttmann, 1987; Sawłowicz, 1989; Speczik,
1994). The OM extracted with chloroform occurs as
long-chain saturated and unsaturated hydrocarbons, poly-
cyclic aromatic hydrocarbons and acid esters (Skłodowska
et al., 2005). Metalloporphyrins have also been detected
in black shale ore (Sawłowicz, 1985).
We recently analyzed the biotransformation of Kupfers-
chiefer black shale by a community of eight bacterial
strains isolated from this sedimentary rock (Matlakowska
et al., 2010; Matlakowska & Skłodowska, 2011). Of these
strains, five belong to the Gammaproteobacteria (Pseudo-
monas sp., Acinetobacter sp.), one to the Firmicutes (Bacil-
lus sp.) and two to the Actinobacteria (Microbacterium
sp.) (Matlakowska & Skłodowska, 2009). It was confirmed
that these indigenous bacteria can use black shale organic
matter as their sole carbon and energy source. These iso-
lates are characterized by high resistance to metals and
metalloids, the presence of esterase, lipase and dioxygenase
activities, assimilation of organic acids, degradation of
phenanthrene, anthracene and pyrene, as well as sidero-
phore production (Matlakowska & Skłodowska, 2009).
In the present study, we monitored and visualized the
interactions of microorganisms with black shale as well as
the microtextural and geochemical changes of this rock
during bacterial weathering. Experiments were performed
to examine (1) the pattern of black shale colonization by
bacteria, (2) the microtopography of the surface of thin
shale sections following treatment with bacteria, (3) the
mineral composition of black shale after bacterial treat-
ment, (4) the nature of the elements mobilized by cul-
tures after bacterial treatment and secondary precipitates,
and (5) the microscopic structure of the bacterial biofilm
growing on the black shale surface. These experiments
were extended with a geochemical characterization of
untreated black shale.
Materials and methods
Geological materials
Samples of highly mineralized Kupferschiefer black shale
were taken from the underground Lubin Mine below
600 m (Fore-Sudetic Monocline, SW, Poland). Two
types of specimen were used: (1) sections of Kupfers-
chiefer shale pieces mounted in aluminium rings using
Araldite 2020 (Huntsman Advanced Materials, Switzerland)
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
R. Matlakowska et al.
in which one side of the specimen was finely polished
using a 1- and 0.25-lm abrasive diamond (the short-
ened names polished sections or sections were used in
the manuscript) and (2) crushed shale with diameters
of 2.0–3.0 mm. The polished sections of the Kupfers-
chiefer black shale were used for the monitoring of the
bioweathering on the surface of black shale and crushed
samples were used for the analyses of element mobiliza-
tion.
Microorganisms
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All bacterial strains were originally isolated from Kupfers-
chiefer black shale (Lubin Mine) and have been described
previously (Matlakowska & Skłodowska, 2009) (NCBI
GenBank accession numbers of isolates in brackets). The
mixed culture of microorganisms contained equal num-
bers of Microbacterium sp. LM1 (EU821337), Microbacte-
rium sp. LM2 (EU821338), Acinetobacter sp. LM3
(EU821339), Bacillus sp. LM4 (EU821340), Pseudomonas
sp. LM5 (EU821341), Pseudomonas sp. LM6 (EU821342),
Pseudomonas sp. LM7 (EU821343) and Pseudomonas sp.
LM8 (EU821344). The number of bacteria in each culture
was calculated using a Bürker chamber under light
microscopy.
Growth media
Luria–Bertani medium (LB; Sambrook & Russell, 2001)
was used for inoculum preparation. The mineral salts
medium used in experiments to examine shale biowea-
thering contained the following masses of salts per litre of
demineralized water: 800 mg of K2HPO4, 200 mg of
KH2PO4·2H2O, 1000 mg of (NH4)2SO4, 200 mg of
MgSO4·7H2O, 100 mg of CaCl2·2H2O, pH 7.0 (Hartmans
et al., 1989).
Bioweathering experiments
To visualize the bioweathering of black shale by bacteria
under polarized reflected light and by scanning electron
microscopy (SEM), polished sections of the black shale
were used. The sections were washed in distilled water,
ethyl alcohol and sterile distilled water. The sterile sec-
tions were placed in 100-mL flasks with bacterial cultures
(preparation of bacterial cultures below). Sterile sections
placed in sterile mineral medium acted as control. All
experiments were prepared in triplicate and incubated
at 28 °C under aerobic conditions for 60 days without
shaking. The volume changes due to evaporation were
compensated by the addition of sterile distilled water.
Sections of black shale colonized over 30 or 60 days
were rinsed in growth medium, stained with DAPI
FEMS Microbiol Ecol 81 (2012) 99–110
Bioweathering of Kupferschiefer black shale
(4′,6-diamidino-2-phenylindole), and the surface viewed
by epifluorescence microscopy.
The batch culture experiment with crushed shale was
carried out in 100-mL flasks containing 10 g of sterilized
crushed shale ore suspended in 100 mL of bacterial cul-
ture (preparation of bacterial culture below). Black shale
ore samples were sterilized by heating three times, for a
period of 1 h at 100 °C, on three successive days. This
method of sterilization was used to minimize the destruc-
tion of organic compounds. Sterile mineral medium sup-
plemented with black shale without added bacteria was
used as control. All cultures with crushed shale were pre-
pared in triplicate and maintained under aerobic condi-
tions, on a rotary shaker (150 r.p.m.) at 22 °C for
60 days. As a control for transmission electron micros-
copy analysis, the bacteria were cultivated on mineral
medium with glucose (20 mM).
To prepare the bacterial culture in mineral medium
(pH 7.0) the cells from an exponentially growing bacterial
culture cultivated in LB medium were harvested by cen-
trifugation (10 000 g, 4 °C, 10 min) and washed three
times in mineral salts medium. The pelleted cells, resus-
pended in mineral salts medium, were used to inoculate
the mineral medium (the final number of bacterial cells
was 8 9 106 mL 1).
Microscopic analyses of rock
The identification of ore minerals in the Kupferschiefer
shale and observation of the early stages of bacterial colo-
nization on the surface of polished sections of shale were
performed using standard petrographical microscopy,
working in reflected-light geometry (ECLIPSE E600W
POL microscope, Nikon Group, Japan). This microscope
also permitted illumination by UV light (EX 400-440,
DM 455, BA 750) for the identification of organic matter
in Kupferschiefer shale, and for documenting the distri-
bution of bacterial colonies in the early and final stages
of colonization.
X-ray powder diffraction (XRD) analyses
XRD analyses of powdered black shale were performed
using a Philips X’Pert PRO diffractometer (PANalytical B.
V., the Netherlands). Shale samples were manually
ground to a powder in an agate mortar, then mounted
on a standard metal holder and irradiated with CoKa
radiation. Data were collected over the range from 2.505°
to 95.996°2Θ in a step-scan mode employing a step size
of 0.001 2Θ and count duration of 1 s per step. The min-
eralogy of the polished shale surfaces, before and after
bacterial colonization, was compared using a Bruker AXS
D8 Discover diffractometer (Bruker AXS, Germany).
FEMS Microbiol Ecol 81 (2012) 99–110
101
Element concentration analysis
The chemical composition of the aqueous phase of batch
cultures containing crushed shale at the end of the experi-
ment was determined by inductively coupled plasma emis-
sion spectrometry (ICP-ES) using an Optima 5300 DV
spectrometer (Perkin-Elmer). All analyses were performed
in triplicate. The sample preparation procedure comprised
centrifugation of the culture suspension (10 000 g at 4 °C,
10 min), filtration (pore size 0.22 lm) and digestion of
the supernatants with 65% nitric acid at 22 °C.
Whole rock analyses of the Kupferschiefer shale were
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performed by the ACME Analytical Laboratories Ltd.
Vancouver, Canada (http://www.acmelab.com). The sam-
ples for analysis were ground using a Retsch Planetary
Ball Mill PM100 to a fraction size below 200 mesh., and
then sent to ACME Laboratories Ltd. as powder ready for
analysis. Standard ACME procedures for the determina-
tion of chemical composition were applied. The major
and trace elements were analyzed by ICP-ES, and by
inductively coupled plasma mass spectrometry (ICP-MS),
respectively. Samples for both methods were prepared by
fusion of 0.2 g of the shale with LiBO2 followed by diges-
tion in dilute nitric acid. The reference materials STD
SO-18, STD DS7 (both are in-house reference materials
of ACME Analytical Laboratories) and STD OREAS45PA
(Ore Research and Exploration PTY LTD, Australia) (lat-
ter two for base metal content) were used for the ICP-ES
and ICP-MS analyses to check the accuracy and precision
of the results. The mean detection limit for base metal
contents ranged from 0.01 to 0.1 mg kg 1, whereas that
for Au was close to 0.05 lg kg 1. The total carbon and
sulphur concentrations were determined by infrared
absorption spectroscopy using the Leco method (Leco
Application Notes No. ASTM E1915). The mean detec-
tion limit for this method is 0.02 wt%. All analyses were
performed in duplicate.
SEM with energy dispersive X-ray spectroscopy
(EDS)
The imaging of the Kupferschiefer samples, before and
after treatment with bacteria, was also performed using
the back scattered (BSE) and secondary electrons (SE)
techniques. These analyses were performed using a JEOL
JSM-6380LA scanning electron microscope (JEOL Co.
Japan). The chemical composition of ore minerals in the
shale was determined using a CAMECA SX100 micro-
probe (CAMECA, Gennevilliers, France). X-ray microa-
nalyses were performed by wavelength dispersive X-ray
(WDS) spectrometry with an accelerating voltage of
15 kV and a beam-current intensity of close to 20 nA.
A defocused beam of 2 lm and count durations of 20 s
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
102 R. Matlakowska et al.

for the peak and 10 s for the background were applied.
Synthetic and natural materials supplied by CAMECA
and/or SPI Inc. (West Chester, PA) were used as calibra-
tion standards. The matrix effect (ZAF correction) was
carried out using standard PAP procedures (Pouchou &
Pichoir, 1985).
In addition, after treatment with bacteria, the black
shale ore surface was fixed in formaldehyde vapour, gold-
coated and observed using a scanning electron micro-
scope (Leo 1430VP; LEO Electron Microscopy Inc.).
highly mineralized shales with clearly visible tiny inclu-
sions, aggregates and veinlets of ore minerals (Supporting
Information, Fig. S1). XRD analyses revealed that the
shale consisted of detrital quartz (SiO2), clay minerals,
feldspar [(K,Na)AlSi3O8–CaAl2Si2O8], dolomite [CaMg
(CO3)2], calcite (CaCO3), bornite (Cu5FeS4) and pyrite
(FeS2). Illite [(K,H3O)(Al,Mg,Fe)2(Si,Al)4O10[(OH)2,(H2O)]
constituted a major component of the clayey background
of the shale. Small volumes of kaolinite [Al2(Si2O5)
(OH)4] and chlorite [(Mg,Fe)3(Si,Al)4O10(OH)2·(Mg,
Fe)3(OH)6] were observed during the SEM analysis.
The ore minerals in the examined samples consisted of

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Transmission electron microscopy (TEM) with
EDS
Bacterial cells were fixed with 3% glutaraldehyde in
sodium cacodylate buffer and then treated with osmium
tetroxide for 4 h. Following dehydration using increasing
concentrations of ethanol, the cells were embedded in
Epon and ultrathin sections were prepared. Microscopic
observations of the bacterial cells were performed using a
JEM 1400 electron microscope (JEOL Co. Japan)
equipped with an energy-dispersive full-range X-ray
microanalysis system (EDS INCA Energy TEM, Oxford
Instruments, UK) with accelerating voltage of 120 kV,
tomographic holder and a high resolution digital camera
(CCD MORADA; SiS-Olympus, Germany).
Results
Geochemical and petrographic characterization
of black shale
The examined shale samples showed no traces of later
alteration and they strictly corresponded to reduced facies
of the Kupferschiefer. All samples represented laminated,
bornite, chalcocite (Cu2S), digenite (Cu9S5), chalcopyrite
(CuFeS2), pyrite, sphalerite (ZnS), galena (PbS), covellite
(CuS), and minerals belonging to the tennantite-tetrahe-
drite series [(Cu, Fe)12As4S13–(Cu, Fe)12Sb4S13] (Fig. S2).
As mentioned above, most ore minerals occurred as
dispersed inclusions in a clayey matrix, and their sizes
ranged from submicroscopic to 50 lm. Thin bornite and/
or bornite-chalcopyrite veinlets, parallel to the Kupfers-
chiefer lamination, and large aggregates of sulphides
(50 lm to 1 mm) were also frequently observed (Figs S1a
and S2a).
The total carbon concentration in the examined
Kupferschiefer samples reached up to 9.68 wt% (Table 1).
Three types of organic matter (OM) were observed
during the petrographical study: structured macerals,
amorphic organic matter together with solid bitumens,
and inclusions of liquid hydrocarbons within calcite and
dolomite aggregates (Fig. S3). The amorphic organic
matter and solid bitumens occurred as a disseminated
submicroscopic intergrowth within the clayey matrix
of the shale and their presence was indicated by a sub-
tle yellow-brown colour under UV illumination (Fig. S3a,
b,c).

Table 1. Chemical composition of examined Kupferschiefer black shale from Lubin Mine

Element (wt%) Element (mg kg 1) Element (mg kg 1) Element (mg kg 1)

SiO2 41.0 Cu > 10 000 Be 7 La 25.2

Al2O3 16.5 Ag > 100 Rb 213 Ce 49.6
Fe2O3 3.7 Sc 15 Sr 92 Pr 6.15
MgO 3.4 V 1246 Cs 55 Nd 24.6
CaO 3.3 Co 388 Ba 242 Sm 4.8
Na2O 0.3 Ni 479 Ga 22 Eu 0.9
K2O 5.0 Zn 194 Sn 6 Gd 4.3
TiO2 0.7 Zr 126 Tl 0.2 Tb 0.6
P2O5 0.1 Nb 15.4 Pb 1957 Dy 3.4
MnO 0.1 Mo 260 Bi 0.6 Y 18.2
Cr2O2 0.04 Cd 1.1 As 398 Ho 0.6
Tot C 9.7 Hf 3.9 Se 34 Er 1.9
Tot S 2.9 Ta 1.1 Hg 1.3 Tm 0.3
LOI 18.8 Au < 0.5 Th 11 Yb 1.8
Total 92.9 W 2.7 U 23 Lu 0.3

LOI, loss on ignition.

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Bioweathering of Kupferschiefer black shale
Among the structured OM, microscopically visible thin
ribbons oriented parallel to the lamination of the
Kupferschiefer black shale were dominant (Fig. S3a,b).
Their yellow-brown colour upon illumination with UV
resembled highly degraded microbial mats as described by
Schieber (1986), Pacton et al. (2009) and Kazimierczak &
Kremer (2009).
The concentrations of major and trace elements in the
black shale are presented in Table 1. The proportions of
SiO2, Al2O3, Fe2O3, MgO and CaO reached up to 41, 16.5,
3.7, 3.4 and 3.3 wt%, respectively. Substantial amounts of
K2O (5.0 wt%) and S (2.9 wt%) and a low amount of
P2O5 (0.1 wt%) were also detected. The analyzed black
shale had also extremely high Cu and Ag contents, exceed-
ing 104 mg kg 1 and 100 mg kg 1, respectively (Table 1).
In a previous study, the concentrations of Cu and Ag
found in the black shale profile ranged up to 10 wt% and
up to 100 mg kg 1, respectively (Piestrzyński & Wodzicki,
2000). High concentrations of Co (388 mg kg 1), Ni
(479 mg kg 1), Zn (194 mg kg 1), Mo (260 mg kg 1),
Pb (1957 mg kg 1) and As (398 mg kg 1) were also evi-
dent. The contents of Hg, Se, U, Bi and Cd were consider-
ably lower (below 40 mg kg 1) (Table 1).
Colonization of black shale by bacteria and
biofilm formation
The experiments using polished sections of Kupferschiefer
shale as a substrate incubated with indigenous bacteria
revealed a specific affinity of these microorganisms for col-
onization. Compared with the control shale section incu-
bated in sterile mineral medium (Fig. 1a), after 30 days of
incubation with bacteria, microorganisms appeared along
Fig. 1. Colonization of surfaces of the
Kupferschiefer shale observed after 30 days:
(a) epifluorescence micrograph of control
section incubated in sterile mineral medium;
(b) epifluorescence micrograph of sections
treated with bacteria showing the distribution
of DAPI-stained microorganisms on the OM-
rich laminae; (c) partial colonization of bornite
aggregate by microorganisms (reflected light
micrograph). Black arrows indicate fine-
grained secondary minerals; (d) image as in
(c), biofilm and DAPI-stained microorganisms
(epifluorescence micrograph). Bn, bornite; OM,
organic matter; BF, biofilm.
FEMS Microbiol Ecol 81 (2012) 99–110
103
fine, yellow laminae within the shale that were rich in OM
(Fig. 1b), indicating a selective microbial attachment to
the shale surface. At the end of the experiment, bacteria
had colonized other areas of the shale including ore min-
erals and rock-forming minerals (Figs 1c,d and 2a,b). Fig-
ure 1c and d shows the same area of black shale observed
under reflected light and epifluorescence microscopy,
respectively, clearly documenting the presence of the
microbial biofilm on the surface of bornite. On uncolon-
ized areas of the bornite surface, dispersed fine-grained
secondary minerals had developed (Fig. 1c).
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Bioweathering of black shale
The microtopography of polished sections of black shale
after bacterial treatment was examined using SEM. The
formation of pits, typical of bioweathering, in close prox-
imity to bacterial biofilm, was observed in some regions
of the shale (Fig. 2). These pits were clearly visible on the
surface of sulphides (Fig. 2a and c) and carbonates (cal-
cite, dolomite) (Fig. 2a). An enhanced relief between
minerals forming the clayey matrix was also observed
during SEM investigations. On the surface of control sec-
tions of black shale incubated in sterile mineral medium
these changes were not observed (Fig. 2d).
Reflected light images of polished sections of black
shale revealed changes in the appearance of the ore min-
erals after bacterial treatment (Figs 3 and S4). Bornite,
pyrite, chalcopyrite, galena, chalcocite and covellite
appeared partly covered by a transparent layer (Figs 3b
and S4). Under reflected light this layer on the sulphide
aggregate caused chromatic dispersion of light. This was
manifested by a spectrum of colours resembling a rainbow.
(a) (b)
(c) (d)
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104
(a)
(b)
(c)
(d)
Fig. 2. SEM images of biofilm (a,b) and numerous pits (a–c) on the
surface of the Kupferschiefer black shale after 60 days of bacterial
treatment and sterile control section (d). Arrows indicate pits on the
shale surface. BF, biofilm.
In the sterile control samples these changes to the shale
surface were not observed (Fig. 3c and d).
XRD analyses of polished sections and crushed shale
following bacterial treatment did not reveal any signifi-
cant changes in the mineralogical composition compared
with untreated and sterile control samples. The XRD data
only showed minerals that were present in amounts
exceeding a few vol% of the sample.
The mobilization of elements from black shale
during bacterial treatment
Colonization of the black shale by microorganisms caused
the release of elements into the aqueous phase of the bac-
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
R. Matlakowska et al.
terial culture. Figure 4 presents the concentrations of 19
elements in the aqueous phase of the bacterial culture
and of the sterile control containing crushed shale after
60 days. The role of bacteria in mobilization of K, P, Cu
and As was clearly evident. The concentrations of other
elements such as Na, S, Ca, Mg, Mo and Si were lower in
the bacterial culture than in the sterile control, where
only chemical weathering was observed. The concentra-
tions of Sr, Al, V, Mn, Cd, Zn, Co, Ag and Pb in the
aqueous phase of both the bacterial culture and the con-
trol were below 0.25 mg L 1. Bacterial growth and activ-
ity also caused alkalization of the growth medium at the
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end of experiment (Fig. 4).
Secondary precipitates on the bioweathered
shale surfaces
SEM-EDS examination of polished sections of the Kup-
ferschiefer shale identified the development of secondary
phases on the black shale due to microbial action. Repre-
sentative images of minerals precipitated during bacterial
growth are shown in Fig. 5. The secondary biominerals
appeared irregularly dispersed on the shale surfaces
(Fig. 5a and c). In many places these minerals occurred
within (Fig. 5b) and/or on the top of the biofilm (Fig. 5b
and d), with large crystals frequently covered by biofilm
(Fig. 5a,b,f). Most of the secondary minerals were identi-
fied as phosphates rich in Pb, Si, Al, Ca and Cu (Fig. 5a–
c), as Ca-rich carbonates, sometimes containing Pb
(Fig. 5e and f), and as sulphates (gypsum [CaSO4·2
(H2O)]) (Fig. 5b). Sites covered by thin SiO2 group min-
erals were common (Fig. 5c,d,f), which indicates extensive
Si mobility. A massive accumulation of Cu-sulphides was
identified at the interface between dolomite, calcite aggre-
gates and the clayey matrix (Fig. 5g and h). The Cu/S
ratio of these sulphides was close to 1.7. In the sterile
control, only the precipitation of Ca-rich phosphates was
observed (data not shown).
Bacterial biofilm and membrane vesicles
Ultrathin cross- and longitudinal sections of biofilm and
bacterial cells grown on the surface of black shale were
examined using transmission electron microscopy (Figs 6
and S5). Numerous outer membrane vesicles (OMVs) 50
–500 nm in diameter appeared to be the main compo-
nents of the biofilm matrices (Fig. 6a and b). Blebbing
cells as well as single and grouped vesicles were visible
(Fig. 6a and c). Some of the cells and membrane vesicles
were surrounded by a matrix of extracellular material
(Figs 6B and S5c,d). Observation of the ultrastructure of
bacterial cells revealed peripherally located spaces with
high electron density (Figs 6c and S5a,b). In contrast,
FEMS Microbiol Ecol 81 (2012) 99–110
Bioweathering of Kupferschiefer black shale
Fig. 3. Reflected light images of the ore
mineral surfaces: (a,b) the surface of black
shale at the beginning of the experiment (a)
and after 60 days of bacterial treatment (b);
(c,d) control samples of black shale before (c)
and after 60 days in sterile mineral medium
(d). Black arrows indicate fine-grained
secondary minerals. Bn, bornite; Gn, galena;
Cc, chalcocite; Cv, covellite; Py, pyrite; Sp,
sphalerite.
cells from a control culture grown in mineral media with
glucose had a typical shape and ultrastructure and did
not produce OMVs (Fig. 6d).
Intracellular biomineralization was clearly visible in the
bacterial cells as well as in OMVs (Fig. 7). EDS micro-
analysis of the extracellular matrix and of electron dense
bodies in bacterial cells and OMVs revealed the presence
of P, Mg, Si, Al and Ca (Fig. 7). The same qualitative
chemical composition was found in all cases. No inclu-
sions were observed in bacteria cultured in mineral med-
ium with glucose (control) (Fig. 6d). The background
composition in the culture liquid is shown in spectrum 6
(Fig. 7).
Discussion
The Upper Permian Kupferschiefer black shale located in
the Fore-Sudetic Monocline is a complex and multi-com-
ponent sedimentary rock. To improve our understanding
of the role of bacteria in bioweathering of this rock, we
monitored and visualized the interactions between micro-
organisms and three main components of black shale:
(1) organic matter, (2) minerals and (3) mobilized
elements. To visualize the effects of these interactions,
standard petrological methods of rock analysis were com-
bined with microbiological studies. It was demonstrated
that bacteria directly interact with black shale organic
matter, develop a biofilm on its surface, and influence the
rock-forming and ore minerals.
The selective affinity of bacteria for OM was demon-
strated by the localization of the biofilm along thin
organic-rich strips oriented parallel to the lamination of
the black shale (Fig. 1). The areas of OM concentration
FEMS Microbiol Ecol 81 (2012) 99–110
105
(a) (b)
(c) (d)
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were the sites where biofilm growth and bioweathering
processes occurred. These results are in agreement with
those obtained by Petsch et al. (2001), who showed the
selective colonization of New Albany Shale by bacteria
that were attached to organic-rich laminas.
Growth and activity of indigenous bacteria affected the
microtopography and mineralogy of the black shale sur-
face as well as the chemistry of the aqueous phase of
cultures. The role of indigenous bacteria in rock deterio-
ration and the geochemical cycles of P, Al, Si, Ca, Mg, K,
Fe, S, Cu and Pb during Kupferschiefer black shale bio-
weathering were evident. However, the concentrations of
the elements presented in Fig. 4 do not reflect the real
redistributions caused by bacterial action. The identified
variations in the chemistry were a consequence of two
processes: the mobilization of an element by the direct
and indirect interaction of the bacteria with components
of the Kupferschiefer, and the crystallization of the
secondary minerals that substantially lowered the concen-
tration of the element in the aqueous phase (Fig. 5).
The mobilization of organic phosphorus and its immo-
bilization in inorganic secondary minerals and in bacterial
cells and OMVs was confirmed (Figs 4, 5 and 7). The for-
mation of phosphate minerals is a very common process
observed in sedimentary environments (Ehrlich & Newman,
2008). The main sources of phosphorus in black shale are
detrital apatite [Ca5(PO4)3(OH,F,Cl)] and thin biogenic
carbonate-fluorapatite [Ca5(PO4,CO3)3F] that are dis-
persed within the clayey matrix, and phosphorus bound
to the OM, which is the most accessible form for bacteria.
It is known that organic matter represents a major source
of phosphorus for bacteria and also for the production of
phosphate minerals (Ehrlich & Newman, 2008). These
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
106 R. Matlakowska et al.

licates as well as the biogenic silica associated with the

K
organic matter are potential sources of this element. The
P high potassium mobility (Fig. 4) suggests that a major
source of silica was probably the clayey matrix of the
Na Bacteria
Control
shale, where the predominant component illite was bio-
S weathered during degradation of the dispersed organic
matter. The expected increase in the concentration of Al
0 50 100 150 200 250 300 350 400
Concentration (mg l–1)
in the bacterial culture supernatant was not observed,
probably due to the de novo crystallization of Al-rich
Ca phosphates (Fig. 5a,b).
Mg
The secondary sulphides growing at the interface
between carbonate aggregates and the clayey matrix

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Mo (Fig. 5g) were responsible for a local pH reduction that
Si was effective in promoting the extensive dissolution of
carbonates (Anderson, 1983). This dissolution, docu-
Cu Bacteria
Control mented by SEM investigation (Fig. 2a,b), was confirmed
As by Ca mobilization and enhanced alkalinity measured at
0 2 4 6 8 10 12 14 16 the end of the experiment (Fig. 4). Crystallization of sec-
Concentration (mg l–1) ondary copper sulphides recognized in this study resem-
bles deposition of Cu-sulphides underneath a water table,
Sr close to the interface of oxidized/reduced zones in the
weathered Cu-rich deposits (Guilbert & Park, 1986). The
Al
composition of these secondary sulphides, also described
V as supergene accumulations, is dominated by chalcocite
Mn
group minerals with a Cu/S ratio of 1.12–2.0. The origin
pH of supergene sulphides in the deposit can be connected
Bacteria 8.35
Cd
Control 7.83 either with microbial activity or crystallization during
Zn true abiotic mineralization processes (Brimhall et al.,
1985; Ague & Brimhall, 1989). The lack of sulphate-
Co
reducing bacteria in the community used in the experi-
Ag Bacteria ment support the second (abiotic) mechanism. The Cu2+
Control
Pb
mobilized during bioweathering of the Kupferschiefer
may replace Fe in primary Fe-Cu sulphides such as chal-
0.00 0.05 0.10 0.15 0.20 0.25
copyrite, bornite and pyrite. The crystallization of the sec-
Concentration (mg l–1)
ondary sulphides reflect the local changes of pH/Eh
Fig. 4. Mobilization of elements from black shale to the aqueous conditions, probably determined by irregular distribution
phase of the bacterial culture and the sterile control after 60 days of of organic matter and Ca-Mg carbonates within the Kup-
incubation. Table – pH concentration in the aqueous phase at the ferschiefer (Figs 1a and S1, S3).
end of the experiment (60 days). Bars represent the standard The other possible mechanisms of the observed process
deviation of measurements. of secondary copper sulphides precipitation may indi-
rectly involve bacterial activity. Such a phenomenon was
described in the Lower Williams Lake tailings basin, Elliot
results are in agreement with those of our recent study Lake in Canada. The authors suggested that ‘conditions
describing the mobilization of phosphorus from organic promoting the reprecipitation of secondary pyrite can be
matter extracted from black shale and the intracellular described as neutral to weakly alkaline and reducing
accumulation of phosphorus by bacteria during growth assisted by microbial activity’ (Paktung & Dave, 2002). In
(Matlakowska & Skłodowska, 2011). our study, such conditions may have been created in the
The mobility of Si observed during the batch experi- pits or within the biofilm observed on the surface of
ment (Fig. 4) and the extensive growth of the SiO2 group Kupferschiefer black shale (Fig. 2a–c). There probably
of secondary minerals on the polished sections of exist microniches created by bacterial activity, character-
Kupferschiefer (Fig. 5c,d,f) unequivocally documented ized by microaerobic conditions, which together with sol-
primary dissolution of the silica-rich minerals in the stud- ubilized organic matter are known to be strong reducing
ied black shale sample. The detrital quartz and aluminosi- factors.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 81 (2012) 99–110
Published by Blackwell Publishing Ltd. All rights reserved
Bioweathering of Kupferschiefer black shale 107

(a) (b)

(c) (d)

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(e) (f)
Fig. 5. Biomineralization on the surface of the
Kupferschiefer black shale. SEM images after
60 days of bacterial treatment: (a) sheaf-like
aggregate of Al-rich phosphate growing on
the biofilm – for details see magnified region
(inset); (b) biofilm covering black shale
containing numerous bacteria and biominerals
– gypsum and phosphate rich in Ca, Pb, Cu
and Si; (c,d) two generations of the
biominerals – Ca and Si phosphate and fine-
grained SiO2 (for details see d); (e) aggregate (g) (h)
of Pb-rich carbonate minerals; (f) calcite
covered by biofilm; (g) secondary sulphide
aggregates; (h) EDS spectra of secondary
sulphide aggregates (EDS spectrum 1) and
dolomite (EDS spectrum 2) presented on (g).
Cal, calcite; Dol, dolomite; Gp, gypsum; BF,
biofilm; CM, carbonate minerals; PM,
phosphate minerals; SIM, fine-grained SiO2.

Secondary sulphides and the increased concentrations
of Cu, Zn, Pb and As in the aqueous phase of the bacte-
rial culture (Fig. 4) were evidence of the alteration of pri-
mary sulphides and sulphosalts (Fig. S2a,f). The
dissolution of the sulphides was concurrent with the crys-
tallization of the secondary sulphates (Fig. 5b), which,
together with crystallization of the Ca-phosphates (Fig. 5b
and c), effectively lowered the concentration of Ca in the
culture supernatant (Fig. 4). Crystallization of the second-
ary carbonates (Fig. 5e and f) also enhanced this effect.
Alterations of the primary minerals, including sulp-
hides, may have occurred during indirect interaction of
the organic compounds (e.g. siderophores or other bacte-
rial metabolites) with these minerals, leading to their par-
tial dissolution and to surface pitting. As mentioned in
the introduction, all of the microorganisms used in this
study are able to produce siderophores, which not only
facilitates the mobilization of ferric ions from insoluble
compounds but may also strongly influence mineral dis-
solution and element mobilization (Ehrlich & Newman,
2008). In our studies, indirect mobilization of elements
from the black shale by bacterial metabolites were
observed, too (unpublished data).
The geochemical cycles of P, Ca, Mg and Al were also
influenced by biomineralization within the bacterial cells
and OMVs, as well as the accumulation of elements in
the biofilm matrix (Fig. 7). The present study is the first
to show that (1) bacteria colonize black shale, creating a
biofilm containing OMVs, and (2) biomineralization
occurs in these OMVs. These results provide new insight
into the activities of bacterial membrane vesicles, but fur-
ther studies are required to characterize their role in the

FEMS Microbiol Ecol 81 (2012) 99–110 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
108 R. Matlakowska et al.

(a) (b)

(c) (d)

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Fig. 6. Ultrathin section TEM images of
bacterial cells growing on the surface of
polished sections of black shale (a–c) and in
mineral medium with glucose (control) (d).
Arrows indicate outer membrane vesicles.

(a)

(b)

Fig. 7. EDS analyses of biofilm matrix (spectra

1 and 2) and secondary minerals occurring in
bacterial cells (spectrum 3) and membrane
vesicles (spectra 4 and 5). Images a and b
show different regions of the samples.
Spectrum 6 shows the background chemical
composition.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 81 (2012) 99–110
Published by Blackwell Publishing Ltd. All rights reserved
Bioweathering of Kupferschiefer black shale
biomineralization process. Besides the biodegradation of
persistent ancient organic matter, the production of
OMVs can an adaptation mechanism of indigenous
microorganisms to extreme conditions prevailing in black
shale. A possible benefit of the production of OMVs, and
especially the biomineralization of Al in OMVs, is the
increased surface area available for metal adsorption and
neutralization of the toxic effects of high concentrations
of elements such as copper, arsenic or silver. On the
other hand, the ability of bacteria to immobilize Al and
Si and other elements has important environmental
implications for their transfer from the aqueous phase to
the sediment because after bacterial death the cell-bound
elements may develop as mineral microdeposits.
Taken together, the results of present study increase
our knowledge about black shale bioweathering and con-
firm the influence of bacteria on the geochemical cycles
of elements in ancient sedimentary rock. The main phe-
nomena observed in this study were: (1) the mobilization
of dozens of elements from black shale, (2) the redistribu-
tion of phosphorus from organic compounds into inor-
ganic compounds, (3) the mobilization of Al, Si, Ca and
Mg from rock, forming secondary minerals, and their
immobilization in phosphates and sulphates, (4) the
mobilization of Cu and Pb from primary sulphides and
sulphosalts and their precipitation as secondary sulphides,
phosphates and carbonates, and (v) the redistribution of
S from primary sulphides to sulphates and secondary
sulphides. The role of biofilms, including OMVs, in the
redistribution of elements is also noteworthy.
The biogeochemical processes described in this report
and studied under controlled laboratory conditions are
assumed to proceed in the natural environment in deep
underground mines. The black shale horizon, cropped
out during exploitation in the underground Lubin Mine
and other mines located on the Fore-Sudetic Monocline,
forms a huge surface exposed to the activity of oxygen
and microorganisms. Evidence of this activity is found in
the numerous mineral precipitates observed on the sur-
face of black shale as well as the high concentrations of
elements in leakage water and bottom sediments within
this mine. These processes are also observed on outcrops
of the Cu-rich deposits in the North Sudetic Basin.
All of the bacterial strains isolated from Lubin Mine
are widespread in nature and can be recovered from dif-
ferent environments. However, the indigenous bacteria
present in Lubin Mine exhibit a range of physiological
properties clearly related to the geochemical parameters
of their environment. The origin of microorganisms iso-
lated from the mine is unknown. It is supposed some of
these microorganisms could be brought in with water, air
from the ventilation system or even by people working in
the mine. However, our studies showed a strong adapta-
FEMS Microbiol Ecol 81 (2012) 99–110
109
tion of the microorganisms to the specific and extreme
underground conditions developed during almost
50 years of exploitation and confirmed their role in
changes of the native environment.
Acknowledgements
This work was supported by a research grant from the
Ministry of Science and Higher Education (Poland) (No.
N304068635). Electron microscopic observations were
performed in the Laboratory of Electron Microscopy at
the Nencki Institute of Experimental Biology (Warsaw,
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Poland). The equipment of this laboratory was installed
within a project sponsored by the EU Structural Fund:
Centre of Advanced Technology BIM – Equipment pur-
chase for the Laboratory of Biological and Medical Imag-
ing. We gratefully acknowledge the help of Dr John
Gittins with English language correction.
Authors’ contributions
Renata Matlakowska: 60%; Aleksandra Skłodowska: 10%;
Krzysztof Nejbert: 30%.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. The distribution of ore minerals and organic
matter within the examined Kupferschiefer black shale
(SEM images).
Fig. S2. Mineralogy of the sulphides present in samples
of Kupferschiefer black shale.
Fig. S3. Distribution of organic matter (OM) within the
Kupferschiefer shale.
Fig. S4. Reflected light images of the ore mineral surfaces.
Fig. S5. Ultrathin section TEM images of bacterial cells
(a–c) and membrane vesicles (d) occurring on the surface
of polished sections of Kupferschiefer black shale.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
FEMS Microbiol Ecol 81 (2012) 99–110