Environmental Microbiology (2007) 9(2), 298–307
Microbial communities in a porphyry copper tailings
impoundment and their impact on the geochemical
dynamics of the mine waste
Nouhou Diaby,1* Bernhard Dold,1
Hans-Rudolf Pfeifer,1 Christof Holliger,2
D. Barrie Johnson3 and Kevin B. Hallberg3
1
Centre d’Analyse Minérale, Faculty of Geosciences and
Environment, University of Lausanne, BFSH2, CH-1015
Lausanne, Switzerland.
2
Laboratory of Environmental Biotechnology, Swiss
Federal Institute of Technology, Lausanne, EPFL
Ecublens, CH-1015 Lausanne, Switzerland.
3
School of Biological Sciences, University of Wales,
Bangor, LL57 2UW, UK.
Summary
The distribution and diversity of acidophilic bacteria
of a tailings impoundment at the La Andina copper
mine, Chile, was examined. The tailings have low
sulfide (1.7% pyrite equivalent) and carbonate (1.4%
calcite equivalent) contents and are stratified into
three distinct zones: a surface (0-70-80 cm) ‘oxidation
zone’ characterized by low-pH (2.5–4), a ‘neutraliza-
tion zone’ (70–80 to 300–400 cm) and an unaltered
‘primary zone’ below 400 cm. A combined cultivation-
dependent and biomolecular approach (terminal
restriction enzyme fragment length polymorphism
and 16S rRNA clone library analysis) was used to
characterize the indigenous prokaryotic communities
in the mine tailings. Total cell counts showed that the
microbial biomass was greatest in the top 125 cm of
the tailings. The largest numbers of bacteria (109 g-1
dry weight of tailings) were found at the oxidation
front (the junction between the oxidation and neutral-
ization zones), where sulfide minerals and oxygen
were both present. The dominant iron-/sulfur-
oxidizing bacteria identified at the oxidation front
included bacteria of the genus Leptospirillum
(detected by molecular methods), and Gram-positive
iron-oxidizing acidophiles related to Sulfobacillus
(identified both by molecular and cultivation
methods). Acidithiobacillus ferrooxidans was also
Received 17 February, 2006; accepted 8 August, 2006.
*For correspondence. E-mail Nouhou.Diaby@unil.ch; Tel.
(+41) 21 692 43 21; Fax (+41) 21 692 43 15.
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd
doi:10.1111/j.1462-2920.2006.01138.
detected, albeit in relatively small numbers. Het-
erotrophic acidophiles related to Acidobacterium
capsulatum were found by molecular methods, while
another Acidobacterium-like bacterium and an
Acidiphilium sp. were isolated from oxidation zone
samples. A conceptual model was developed, based
on microbiological and geochemical data derived
from the tailings, to account for the biogeochemical
evolution of the Piuquenes tailings impoundment.
Introduction
Pollution caused by acidic, metal-rich effluents, generally
referred to as acid mine drainage (AMD; or acid rock
drainage, ARD), is a major environmental problem in
various parts of the world. Acid mine drainage forms when
sulfide minerals in rocks and mine wastes are oxidized on
exposure to oxygen and water (Nordstrom and Alpers,
1999). Rates of sulfide mineral oxidation are generally
slow in unaltered (massive) rock assemblages, but are
greatly accelerated as a result of mining activities, as a
result of disaggregation of rocks and coal strata resulting
in far greater mineral surface areas being exposed to
oxygen and water.
Following exploration, ores are extracted (from under-
ground or open pit mines), crushed and milled to reduce
grain size. The fine grains are then mixed with water and
chemical reagents to separate economically important
minerals from waste minerals (tailings). Up to 80–99% of
the crushed ore is typically dumped as tailings wastes.
Tailings from porphyry copper mines typically contain
0.4–4% sulfur, mainly as pyrite (FeS2; Dold and Fontboté,
2001). These waste materials are transported in suspen-
sion for final deposition in impoundments, and sulfide
mineral oxidation is limited as long as the tailings remain
water-saturated. However, once operations cease, water
levels in tailings impoundments will fall (unless manage-
ment practices are adopted to prevent this) and unsatur-
ated zones (containing both oxygenated water and
atmospheric oxygen) form, facilitating the oxidation of
sulfide minerals and resulting in the formation of AMD
(Dold and Fontboté, 2001). The two main oxidants of
pyrite in nature are oxygen and ferric iron, and the relative
importance of these depends on ambient pH. At low pH
 Microbial communities and geochemistry of mine tailings 299
(< 3) pyrite oxidation by ferric iron is 10–100 times faster
than by oxygen (Ritchie, 1994), although at near neutral
pH values, oxygen is a more important pyrite oxidant
(Evangelou, 1995). This is related to the solubility of ferric
iron, which is highly pH-dependent. The abiotic oxidation
of ferrous iron is also highly pH-dependent, and proceeds
very slowly (even in oxygen-saturated solutions) at
pH < 3.5 (Stumm and Morgan, 1981). However, some
acidophilic bacteria and archaea are able to catalyse
ferrous iron oxidation in low pH environments, regenerat-
ing ferric iron, and some of these (and others) also gen-
erate sulfuric acid via oxidation of elemental sulfur and
reduced inorganic sulfur compounds (Baker and Banfield,
2003; Johnson and Hallberg, 2003). For this reason, both
iron- and sulfur-oxidizing prokaryotes are important in
accelerating the rate of production of AMD. Several
studies have analysed geochemical parameters in tailings
and the evolution of secondary mineralization (e.g. Nord-
strom and Alpers, 1999; Dold and Fontboté, 2001) and
other studies have focused on the role of microorganisms
in generating acidic, metal-rich waters in abandoned
mines and mine wastes (e.g. Bryner et al., 1967; Blowes
et al., 1998; Baker and Banfield, 2003; Johnson and Hall-
berg, 2003). However, understanding the interaction
between indigenous microorganisms and geochemical
parameters is a key factor for devising improved strate-
gies for managing mine wastes, and preventing the for-
mation of AMD.
In this study, the indigenous microbial communities in
the Piuquenes tailings impoundment at the La Andina
mine, Chile was studied using a combined cultivation-
based and cultivation-independent approach. Microbial
communities in samples taken at different depths in the
tailings impoundment were investigated, and related to
the geochemical parameters of the mine waste obtained
in other studies.
Results
Distribution of microbes in the stratified tailings
impoundment
Total cell counts showed that the majority of microbes
were found in the oxidation zone of the tailings impound-
ment (Fig. 1). The greatest number of the cells (> 109 g-1
dry tailings) was detected at the oxidation front (65–75 cm
of depth). Counts of culturable microbes were lower but
generally reflected those of the total cell counts. Total
numbers of colony-forming units (cfu) were greater in the
oxidation zone and in the 125 cm sample than at lower
depths (varying between 104 and 106 per gram dry weight
of tailings; Table 1 and Fig. 1), where they represented up
to 0.25% of the total counts. Iron-oxidizing acidophiles
were isolated only from samples taken from the oxidation
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 298–307
Microbial numbers (g–1 dry tailings)
10 2 10 5 10 8
0
100
200
300
Plate counts
400 Total cell counts
Oxidation front
Fig. 1. Depth-related changes in microbial numbers in the
Piuquenes tailings impoundment.
zone, while heterotrophic acidophiles and acidophilic
sulfur-oxidizers were also isolated from the 125 cm
sample, located within the neutralization zone. Sulfate-
reducing bacteria (SRB) were also isolated from the oxi-
dation zone (although not from the surface sample) and,
again, from the 125 cm depth tailings sample (Table 1).
Identification of bacterial isolates
Based on their colony morphological characteristics, a
total of eight different acidophiles were isolated from tail-
ings samples taken from between 0 and 70 cm depth.
Following purification of these isolates, and screening of
amplified 16S rRNA genes by restriction fragment length
polymorphism (RFLP), it was concluded that the eight
colony variants probably represented four distinct micro-
bial species, and one each of these representative iso-
lates (coded CH1, CH2, CH3 and AP3IO) were selected
for phylogenetic analysis. The 16S rRNA gene of iron-
oxidizing isolate AP3IO was found to have > 99%
sequence identity to Acidithiobacillus ferrooxidans phylo-
genetic group II (Karavaiko et al., 2003), and 98.5% to the
type strain (ATCC 23270) of this acidophile. Isolate CH2,
also an iron-oxidizer, was shown to be closely related to
iron/sulfur-oxidizers of the phylogenetic group Firmicutes
(low G + C Gram-positive bacteria). The 16S rRNA
sequence was remotely related (92.9% identity) to genes
from the Gram-positive iron-oxidizing acidophiles SLC66
(Johnson et al., 2001) and Y0010 (Johnson et al., 2003).
The closest described bacterium to CH2 was Sulfobacil-
lus thermosulfidooxidans (Kovalenko and Malakova,
300 N. Diaby et al.
Table 1. Numbers of culturable bacteria in the tailings profile.

Depth Iron-oxidizing Heterotrophic Sulfate-reducing Sulfur-oxidizing

(cm) acidophiles acidophiles prokaryotes acidophiles Total

5 1.36 ¥ 105 1.25 ¥ 106 <10 6.25 ¥ 103 1.39 ¥ 106

50 2.17 ¥ 103 2.17 ¥ 103 7.17 ¥ 103 <10 1.15 ¥ 104
70 4.35 ¥ 103 1.96 ¥ 104 3.41 ¥ 106 <10 3.43 ¥ 106
125 < 10 2.28 ¥ 105 1.82 ¥ 106 1.06 ¥ 103 2.05 ¥ 106
260 <10 <10 <10 <10 <10
395 <10 <10 <10 <10 <10

Counts of iron-oxidizing acidophiles were obtained from Feo solid media, heterotrophs from Yeo, sulfate-reducing prokaryotes from agar-gelled
DSM medium 63, and sulfur-oxidizers from FeSo plates. All counts given are of colony-forming units (cfu) g-1 dry weight of tailings, and the lower
limit of detection is 10 cfu g-1.

1984) with which it shared only 80% 16S rRNA gene
sequence identity.
Of the two heterotrophic bacteria isolated from the Piu-
quenes tailings, isolate CH1 had a 16S rRNA gene
sequence identity of 95.9% with the soil isolate Ellin 310
and 93.9% with Acidobacterium capsulatum (Kishimoto
et al., 1991). In contrast, CH3 had a 16S rRNA gene
sequence very close (99.4% identical) to Acidiphilium sp.
C-1 and 97.9% identical to the type strain of Acidiphilium
acidophilum (ATCC 27807; Hiraishi et al., 1998).
Community composition of the unculturable Piuquenes
tailings microbes
T-RFs, indicating that there was more bacterial diversity
in this subsample than in the other (Fig. 2B). From a 16S
rRNA gene clone library of the second subsample, six
different restriction enzyme patterns were obtained from
14 clones. A representative of each RFLP profile (coded
ND2 to ND7) was selected and cloned. The sequences
of clones ND2 and ND4 were identical to each other, and
were most closely related to bacteria detected in acidic
soil samples from Yellowstone National Park (Norris
et al., 2002; Fig. 3). These two clones were only remotely
(89%) related to classified acidophilic bacteria of the
genus Sulfobacillus. A second clone obtained from this
library, ND6, had a 16S rRNA gene very close (> 99%)

Extraction of DNA and polymerase chain reaction (PCR)

amplification of bacterial 16S rRNA genes was successful
100
Relative abundance (%)

only with samples taken at the oxidation front (one sub-

A
sample at 65–70 cm and a second subsample taken from
80
70 to 75 cm). Quantifiable amounts of DNA were not
obtained from any other tailings samples, possibly due to 60
the lower number (by a factor of 10) of microbes in these
40
samples compared with those at the oxidation front.
Amplification of 16S rRNA genes using archaeal-specific 20
primers was unsuccessful for all samples.
Terminal restriction fragment length polymorphism was 0
used as the first step to characterize the microbial com- 75 227 368 369 373
Relative abundance (%)

munities in the tailings profile. The first oxidation front 20

B
subsample (65–70 cm) showed the predominance
15
(~80%) of a T-RF of 369 bp (Fig. 2A). A total of nine
clones out of 10 from a gene library of 16S rRNA genes 10
amplified from this zone were analysed by RFLP and
found to contain the same cloned gene. The 16S rRNA 5
gene from a single representative clone, ND1, was
sequenced. This clone was similar (95% identity) to bac- 0
teria belonging to Leptospirillum group 3 (Fig. 3), includ- 90 134 197 225 343 361 366 367 368 369
ing clone AW11 (Druschel et al., 2004) and RCP2-12 Terminal restriction fragment size
(Brofft et al., 2002). This group of microbes was repre- (nucleotides)
sented only by environmental clones until the recent iso-
lation of “Leptospirillum ferrodiazotrophum” from that Fig. 2. Terminal restriction fragment length polymorphism analysis
of bacteria present in tailings samples taken from 65 to 70 cm (A)
same site (Tyson et al., 2005). The second subsample and 70–75 cm (B). The labelled PCR products were digested with
from the oxidation front (70–75 cm) yielded several the restriction enzyme HhaI prior to electrophoresis.

© 2006 The Authors

Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 298–307
 Microbial communities and geochemistry of mine tailings 301

ND2
ND4
Uncultured Gram+ acidophile YNPRH70A (AF465653)
Thermal soil bacterium YNPFFP9 (AF391988)
Sulfobacillus acidophilus DSM 10332 (AB089842)
Sb. thermosulfidooxidans strain G2 (AY140233)
Iron-oxidizing acidophile Y0010 (AY140235)
Iron-oxidizing acidophile SLC66 (AY040739)
CH2
Alicyclobacillus disulfidooxidans (U34974)
Acidiphilium sp (D30769)
CH3
Acidiphilium acidophilum (D86511)
Acidiphilium cryptum (D30733)
Acidithiobacillus ferrooxidans strain TFY (AF465608)
AP3IO
Acidithiobacillus ferrooxidans (AJ278723)
Leptospirillum ferriphilum strain Warwick (AF356831)
Leptospirillum sp. DSM 2391 (AJ237903)
ND6
Leptospirillum ferrooxidans strain BTC2 (AF356833)
uncultured bacterium clone AW11 (AF543503)
uncultured bacterium clone RCP2-12 (AF523925)
ND1
uncultured Acidobacterium YNPRH72A (AF465659)
CH1
uncultured bacterium RCP2-4 (AF523897)
ND3
ND5
ND7
Acidobacterium capsulatum (D26171)
uncultured eubacterium clone WR896 (AJ292801)
Bacterium Ellin310 (AF498692)
uncultured eubacterium WD247 (AJ292581)
Acidobacteriaceae isolate WJ7 (AY096034)

0.1

Fig. 3. Bacterial phylogenetic tree, based on 16S rRNA gene sequences, of bacteria detected as clones (ND1 to ND7) and isolates
(CH1, CH2, CH3 and AP3IO) in the Piuquenes tailings impoundment oxidation front. Database accession numbers of the gene sequences
from other bacteria are given in parentheses. The scale bar represents 0.1 nucleotide substitutions per site.

to the type strain of Leptospirillum ferriphilum (ATCC the subsamples of the oxidation front (Fig. 2). This indi-
49881; Coram and Rawlings, 2002). cates that the microbes represented by these clones were
Heterotrophic bacteria were also identified in the the dominant microbes in this environmental sample.
second subsample of the oxidation zone by molecular
analyses. Clones ND3 and ND5 (each sharing an identi-
Table 2. Comparison of calculated terminal restriction enzyme frag-
cal gene sequence) had 16S rRNA gene sequences ment (T-RF) length of the clones from Piuquenes tailings samples
related (98%) to an uncultured bacterium, clone RCP2-4, with those observed by T-RFLP analysis of the same samples.
found in an AMD-impacted wetland (Brofft et al., 2002),
and to isolate WJ7 from a wetland constructed to treat Calculated T-RFs Observed T-RFs
Clone (nucleotides) (nucleotides)
AMD (Hallberg and Johnson, 2003). ND7 was related to
the soil bacterium Ellin 310 (Sait et al., 2002) at 97% ND1 371 366, 367, 368, 369
ND2 228 225
(Fig. 3). These clones shared a gene sequence identity of ND3 93 90
95% and 96%, respectively, to the tailings isolate CH1. ND4 228 225
The T-RFs calculated from each of the cloned gene ND5 93 90
ND6 375 366, 367, 368, 369
sequences (Table 2) closely matched, to within one to five ND7 93 90
nucleotides, T-RFs found during the T-RFLP analysis of

© 2006 The Authors

Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 298–307
302 N. Diaby et al.
Discussion
This work is, to our knowledge, among the first to have
described the microbiology of mine tailings using a com-
bination of cultivation and molecular microbial ecology
techniques. The results of all methods used in this study
indicate that the greatest concentration of bacteria were
located in the oxidation zone of the tailings (0–75 cm
depth) and large numbers were present in the first portion
of the neutralization zone (to 125 cm). That the greatest
number of microbes detected were in this zone may be
due to the fact that the oxygen content in pore air is 16.4%
on the surface and decreased to 0% below the oxidation
zone (Dold et al., 2005). Furthermore, the tailings above
this zone were depleted in metal sulfides, and thus lacked
a major source of electron donors. Anaerobes (SRB) were
detected below the oxidation zone. Some of the acido-
philic bacteria isolated from Piuquenes tailings are
autotrophic, and synthesize biomass by utilizing CO2 as
carbon source. They obtain energy for carbon fixation and
growth by oxidation of ferrous iron and/or sulfur, derived
from sulfide minerals present in the tailings. These in turn
support the growth of heterotrophic acidophiles and SRB
that live on the dissolved organic carbon (exudates,
lysates, etc.) derived from the primary producers
(Johnson, 1998).
Three different species of iron-oxidizing bacteria were
found in the oxidation zone of the tailings deposit using
cultivation and/or biomolecular techniques. The iron- and
sulfur-oxidizing acidophile At. ferrooxidans was isolated
from tailings samples on solid media. This is the most
widely studied of all acidophiles, and is readily isolated
from acidic sulfide-rich environments by enrichment or
plating (Hallberg and Johnson, 2001). However, the fact
that it was not detected in T-RFLP profiles suggests that it
is not as significant, numerically, as other bacteria in the
Piuquenes tailings. A similar situation was the case with
the Gram-positive isolate CH2, a Firmicute distantly
related to unclassified iron-oxidizing bacteria isolated
from weathering sulfidic regoliths and from a geothermic
site in Yellowstone National Park (Johnson et al., 2001;
2003). In contrast, Leptospirillum spp. were detected in
the tailings samples by biomolecular methods, but were
not isolated on solid media. Two Leptospirillum-like bac-
teria were detected in clone libraries, one highly related
(> 99% gene 16S rRNA gene sequence identity) to
L. ferriphilum, and another more distantly related (95%
identity) to “L. ferrodiazotrophum”. These two species are
similar in many respects (both are known to use only
ferrous iron as energy source, are obligate aerobes and
extreme acidophiles), although a differentiating feature is
that “L. ferrodiazotrophum” is able to fix nitrogen while
L. ferriphilum is not (Tyson et al., 2005). Interestingly, both
of these Leptospirillum spp. (although not L. ferrooxidans)
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 298–307
were detected at Iron Mountain, California. Within the
abandoned mine L. ferriphilum was found to be the more
numerically abundant bacterium, and it was hypothesized
that the key role of “L. ferrodiazotrophum” was to provide
fixed nitrogen to L. ferriphilum (the major primary pro-
ducer) in this nitrogen-depleted environment (Tyson et al.,
2005). It is quite possible that a similar scenario exists in
the Piuquenes tailings deposits. More definitively, the bio-
molecular data indicated that the dominant mineral-
oxidizing prokaryotes in the tailings deposits were
Leptospirillum spp., rather than other iron-oxidizing
bacteria or archaea. Previous work has shown that
L. ferrooxidans has a higher affinity for ferrous iron, and is
more tolerant of ferric iron, than is At. ferrooxidans (Norris
et al., 1988), although no corresponding data have been
published for other Leptospirillum spp. Other work has
also shown that Leptospirillum is more significant than
At. ferrooxidans in mineral leaching environments
(Rawlings et al., 1999).
Heterotrophic acidophiles were also detected in the
Piuquenes tailings impoundment. The two isolates
obtained were both related to known genera of acido-
philes (Acidiphilium and Acidobacterium), while the three
bacteria identified in clone libraries were all members of
the Acidobacteriaceae. Acidiphilium spp. are widely dis-
tributed in mineral leaching environments (Hallberg and
Johnson, 2001) while relatively few Acidobacteriaceae
have been cultivated, even though they are known to be
widely distributed in acidic and non-acidic environments
(Hugenholtz et al., 1998). While there is no evidence for
the direct participation of any of these bacteria in sulfide
mineral dissolution, the closest cultivated relative of
isolate CH3, A. acidophilum, is the only known species of
Acidiphilium to grow autotrophically on sulfur (generating
sulfuric acid), and both Acidiphilium spp. and
Acidobacterium-like isolates are able to accelerate the
reductive dissolution of ferric iron minerals, such as schw-
ertmannite (Bridge and Johnson, 2000; Coupland, 2006).
No physiological traits can be ascribed with any cer-
tainty to the other bacterium detected in the tailings by
gene cloning (represented by clones ND2 and ND4) as
this microorganism is only very remotely related to the
iron- and sulfur-oxidizing acidophiles of the genus
Sulfobacillus. The most closely related microbes to ND2/
ND4 have only been detected in other studies by molecu-
lar means.
The other important group of prokaryotes detected
(although not identified) within the tailings deposit were
dissimilatory sulfate reducers. In some tailings samples,
these were more numerous than the other physiological
groups of bacteria detected, particularly at the oxidation
front and below. Interestingly, although most sulfate-
reducing prokaryotes are obligate anaerobes and highly
sensitive to acidity, they were present in significant
© 2006 The Authors
 Microbial communities and geochemistry of mine tailings 303
2+
Fe → Fe
3+ CO2
Leptospirillum spp.;
At. ferrooxidans; Pyrite oxidation
2+
Gram-positive Fe -
oxidizers 3+
FeS2 + 6Fe + 3H2O → 7Fe
2+
+ 2S2O3 + 6H
3+ 2+
Fe → Fe
Acidiphilium spp., DOC
Acidobacteriacea
2+
Plume of Fe
Fig. 4. Model of the microbial impact on geochemical dynamics observed in the Piuquenes tailings impoundment (Dold and Fontboté, 2001).
DOC, dissolved organic carbon.
numbers within the oxidation zone of the tailings impound-
ment (although not in the surface samples) as well as in
the neutralization zone.
Other research carried out at the La Andina mine has
described the geochemistry of the tailings (Dold and Font-
boté, 2001; Dold et al., 2005). Within the oxidation zone,
ferric iron is the dominant iron species (with large concen-
trations of this species present at the oxidation front), and
sulfide minerals are depleted compared with lower zones.
Below the oxidation front, there is a dramatic reduction in
the measured redox potential (of about 300 mV) corre-
sponding to the appearance of ferrous iron as the domi-
nant soluble iron species; sulfate concentrations also
decrease from about 24 g l-1 in the oxidation zone to
about 15 g l-1 below the oxidation front. Dissolved organic
carbon, in the form of small molecular weight aliphatic
acids (formic, acetic and pyruvic), has also been detected
within the tailings, at a maximum concentration of about
10 mg l-1 at 265 cm depth (Dold et al., 2005).
Combining the microbiological results from the current
work with geochemical data from other reports, a model to
explain the observed changes within the Piuquenes tail-
ings impoundment has been proposed (Fig. 4). As the
water level with the impoundment falls, ingress of oxygen
promotes the oxidative dissolution of pyrite and other
sulfide minerals, primarily by Leptospirillum spp. Other,
iron- and sulfur-oxidizing, acidophiles (Sulfobacillus spp.
and At. ferrooxidans) contribute to this process by gener-
ating sulfuric acid, as well as by oxidizing ferrous iron.
Lysates and exudates (dissolved organic carbon)
from autotrophic acidophiles support the growth of
heterotrophic acidophiles (iron-reducing Acidiphilium and
Acidobacterium-like bacteria, and sulfate-reducing
prokaryotes) while oxidation products of the primary pro-
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 298–307
Oxidation
Zone
2- 2-
S2O3 → SO4
Acidithiobacillus spp.
2-
+
Oxidation
Front
Neutralization
DOC
2-
SO4 → HS
- Zone
SRB
ducers (ferric iron and sulfate) act as terminal electron
acceptors for the heterotrophs where oxygen is limiting or
absent. Below the oxidation front, dissimilatory reduction
of ferric iron and sulfate are considered to be the domi-
nant geochemical processes, both of which are ultimately
limited by the availability of these oxidized species or by
electron donors. The oxidation front will continue to
migrate downwards, depending on the rate at which the
water table in the tailings falls. Ultimately, if and when the
impoundment is fully drained, the tailings could become
essentially fully oxidized, resulting in the dissolution of
vast quantities of waste sulfide minerals from the mining
operation and generation of acidic, metal-rich effluents.
Experimental procedures
Area of study
The La Andina mine is a part of the Rio Blanco-Los Bronces
ore body, which is a giant copper-molybdenum porphyry
system with > 50 ¥ 106 tonnes ore containing 1.0–1.5 wt% Cu
(Serrano et al., 1996). It is located 50 km north-east of San-
tiago in the west flank of the central Chilean Andes (Fig. 5).
The mined ore is processed by an alkaline circuit using lime
for the flotation (pH 10.5). The Piuquenes tailings impound-
ment, where sampling was carried out in November 2002,
was in operation from 1970 to 1980, and contains an average
of 0.22% copper. It is located at an altitude of 2150 m above
sea level in the N-S trending valley of the Rio Blanco River,
and has an alpine climate. The impoundment has a surface
area of 83.7 ha, a volume of 24 ¥ 106 m3, and contains an
estimated 37 ¥ 106 tones of tailings. These have a low-sulfide
content (1.7 wt% pyrite equivalent; mainly as pyrite with
traces of chalcopyrite, bornite, chalcocite and covelite), and a
low-carbonate content (1.4 wt% calcite equivalent; mainly as
calcite, siderite and ankerite; Dold and Fontboté, 2001). The
depth profile of Piuquenes tailings sediment shows three
304 N. Diaby et al.
Dam
Oxidation zone
Neutralization zone
Primary zone
Fig. 5. Location and vertical and horizontal profile of the Piuquenes tailings impoundment at the La Andina mine, Chile.
zones: a low pH (2–4) orange coloured ‘oxidation zone’ from
0 to 70–80 cm, a ‘neutralization zone’ from 70–80 to 300–
400 cm and a ‘primary zone’ of unaltered tailings (Dold and
Fontboté, 2001).
Sampling
Core samples of mineral tailings were obtained by driving a
metallic tube into the impoundment using percussion soil
sampling equipment. The samples were taken from a single
vertical profile in order to study the pattern of the microbial
populations in relation to the oxidation of tailings and depth-
related variations of geochemical parameters described by
Dold and Fontboté (2001). Samples for cultivation studies
were put into sterile Falcon tubes (Semadeni SA) and main-
tained at 4°C. Samples for DNA extraction were also col-
lected in Falcon tubes, but were plunged, on site, into a dry
ice-ethanol bath for flash-freezing. They were then stored in
at -70°C until processing. Samples for total cell counts were
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 298–307
0m
Oxidation zone
0.75 m
Neutralization zone
Oxidation
4m front
Primary zone
57 m
0m
57 m
fixed on site in a freshly prepared solution containing 3%
(w/v) paraformaldehyde (PFA) in 10 ¥ PBS (phosphate buff-
ered saline: 1 ¥ PBS = 150 mM NaCl, 10 mM Na2HPO4,
3 mM NaH2PO4, pH 7.2 and filtered through 0.2 mm cellulose
nitrate membranes) and held at 4°C for 24 h. The samples
were then centrifuged (4500 g, 5 min), washed three times
with 1 ¥ PBS and resuspended in 2 ml of 50% (v/v) absolute
ethanol and 50% 1 ¥ PBS. They were stored at -70°C until
processing. Three subsamples were taken at each depth.
Total cell counts
For total cell counts, tailings samples fixed in PFA were
stained with SYBR Green II (Zarda et al., 1997). Samples
(60 ml) were mixed with 940 ml of 0.1% sodium pyrophos-
phate in distilled water, and subjected to mild sonication using
a Sonoplus HD2070 (Bandelin, Germany). Aliquots of 10 ml of
the sonicated samples were spotted in gelatine-coated slides
(black, eight wells, Cel-line, Erie Scientific, USA) and dried at
© 2006 The Authors
 Microbial communities and geochemistry of mine tailings 305
42°C for 30 min. Next, cells were dehydrated by immersing
slides in 50%, 80% and 96% (v/v) ethanol solutions for 3 min
each, and then stained with 10 ml of SYBR Green II (10-3
dilution of the stock in TE pH 8) for 15 min in the dark, rinsed
with distilled water and air-dried in the dark. Stained slides
were mounted with Citifluor AF1 (Citifluor, London, UK) and
observed with a Nikon Eclipse E-800 epifluorescent micro-
scope at 1000¥ magnification with immersion oil. For each
sample, three replicate subsamples were counted.
Viable cell counts
Most solid media used for cultivation and isolation of bacteria
were ‘overlay media’ that facilitate the growth of the majority
of autotrophic and heterotrophic acidophiles (Johnson, 1995;
Hallberg and Johnson, 2003). These included: ferrous iron
overlay plates (Feo), ferrous iron/thiosulfate overlay plates
(FeThio), yeast extract overlay plates (Yeo); ferrous iron/
tetrathionate overlay plate (FeSo). Neutrophilic sulfate-
reducing bacteria (nSRB) were enumerated using DSM
medium 63 (http://www.dsmz.de/microorganisms/media_list.
php) solidified with 1.5% (w/v) agar. Plate inocula were pre-
pared by placing 1 g of moist tailings sample into 10 ml of
basal salt solution (adjusted to pH 4 with H2SO4; Johnson,
1995) and shaken vigorously to dislodge microorganisms.
Aliquots of 100 ml of these suspensions were spread on the
various solid media, and plates were incubated at 30°C.
Anaerobic conditions for SRB were generated using the
using the AnaeroGenTM AN25 system (Oxoid, UK). After
8 weeks, plates were examined and colonies were grouped
using differences in their morphological characteristics
(Johnson et al., 2005). Representative colonies of each
group were subcultivated on respective solid overlay (or
liquid) media, prior to identification via analysis of their 16S
rRNA genes.
DNA extraction
DNA from tailings samples (0.25–1 g) was extracted using a
MoBio Ultra Clean Soil DNA extraction kit in the following the
manufacturer’s instructions (with inclusion of the PCR Inhibi-
tor Removal Solution). The resultant DNA solutions were
stored at -20°C until used for molecular analysis.
16S rRNA gene amplification
16S rRNA genes of microbial isolates and DNA extracted
from tailings samples were amplified by the PCR using
eubacterial primers 8f and 1492r (Lane, 1991) and archaeal
primers Arch21f and 1492r (DeLong, 1992). Reactions (25 ml)
contained 1 ¥ PCR buffer, 200 mM dNTP, 0.25 mM each of the
forward and reverse primers, 0.5 U Taq polymerase and 1 ml
of DNA. Polymerase chain reaction consisted of a denaturing
step at 95°C for 5 min before 30 cycles as follows: 95°C for
30 s, 56°C for 30 s and 72°C for 1.5 min, and a final exten-
sion step for 10 min at 72°C.
Terminal restriction enzyme fragment length
polymorphism analysis
For T-RFLP analysis, PCR was carried out as above, but
using the 8f primer labelled with either the fluorochrome
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 298–307
hexachlorofluorescein (Hex) coupled with the 534r primer
(Muyzer et al., 1993), or with the 8f primer labelled with Cy5
and the 1492r primer. The amount of template DNA in the
reaction was 2 ng (DNA concentration determined using the
Picogreen assay, Molecular Probes). The PCR reactions
were carried out in triplicate and pooled. Following confirma-
tion of successful PCR reaction by agarose gel electrophore-
sis, the PCR products were purified with the QIAquick PCR
Purification Kit (QIAGEN).
Purified PCR products were digested with the restriction
enzyme HhaI at 37°C for 4 h. Hex-labelled PCR products
(1.0 ml of the digestion reaction) were mixed with 0.5 ml of
GS-500-ROX size standard (Applied Biosystems) in 8.5 ml of
formamide, incubated at 95°C for 2–3 min and immediately
placed on ice for 5 min. The entire 10 ml was analysed using
the ABI Prism 3100 system. The sizes of the resulting terminal
restriction enzyme fragments (T-RFs), labelled with Cy5, were
determined by capillary electrophoresis using a CEQ8000
Genetic Analysis System (Beckman Coulter, UK) and com-
parison to the mobility of the size standard fragments. In both
cases, peak areas (related directly to fluorescence of the
peak) for each T-RF relative to the total peak areas were used
to determine the relative abundance of individual microorgan-
isms within the community under investigation.
Phylogenetic analysis
Amplified 16S rRNA genes of the microbial isolates obtained
in this study were sequenced directly from PCR products
using the 8f primer, following the ABI Prism BigDye Termina-
tor cycle-sequencing reaction kit protocol and the ABI Prism
3100 system. Polymerase chain reaction-amplified 16S rRNA
genes of DNA extracted from tailings samples were cloned
into the pGEMT-Easy vector (Promega) following manufac-
turer’s instructions. Restriction enzyme fragment length poly-
morphism was used to group cloned genes (using the vector
specific primers T7 and SP6), and plasmids from unique
clones thus identified were purified using MiniPrep Qiagen
DNA purification kits, ahead of sequencing. The gene
sequences of the isolates and clones described here have
been deposited in GenBank under the Accession numbers
DQ355183–DQ355191.
The various genes obtained were compared with those
available in GenBank by BLAST searches (Altschul et al.,
1997) to find the nearest relatives and percentage of gene
sequence identity. A phylogenetic tree was constructed by
making an alignment of about 500 nucleotides of the gene
sequences with ClustalX (Thompson et al., 1997). This align-
ment was used to make a phylogenetic tree by neighbour-
joining (Saitou and Nei, 1987) and the confidence in the
branching order was tested by Bootstrap analysis (100 trials)
using algorithms in ClustalX. The tree was viewed using
TreeView software (Page, 1996).
Acknowledgements
We would like to thank CODELCO, Division Andina (espe-
cially Luis Serrano and Ricardo Vargas) for making possible
access to the Piuquenes tailings impoundment, Professor
Jacques Wiertz and all his staff, Department of Mining Engi-
306 N. Diaby et al.
neering, University of Santiago, Chile for their logistical
support in Santiago de Chile. This work was carried out with
the financial support of the Centre d’Analyse Minérale (CAM),
University of Lausanne, the Laboratory of Environmental Bio-
technology, Swiss Federal Institute of Technology Lausanne
(EPFL), the Commission for Research Partnership with
Developing Country (KFPE), Bern, the Augustin Lombard
Foundation, Geneva, and the Ernst and Lucie Schmidheiny
Foundation, Geneva.
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