Research in Microbiology 154 (2003) 466–473

www.elsevier.com/locate/resmic

Mini-review

The microbiology of acidic mine waters

D. Barrie Johnson ∗ , Kevin B. Hallberg
School of Biological Sciences, University of Wales, Bangor, LL57 2UW, UK
Received 21 March 2003; accepted 23 April 2003

Abstract
Acidic, metal-rich waters generated by the microbially accelerated dissolution of pyrite and other sulfide minerals, are frequently
encountered in derelict mine sites, including many that have been long-abandoned. While these waters are major causes of environmental
pollution and are toxic to the majority of prokaryotic and eukaryotic organisms, some life forms (mostly bacteria and archaea) thrive within
them. “Acidophiles” comprise a surprisingly wide diversity (in terms of both physiology and phylogeny) of microorganisms. This article
reviews current knowledge of the distribution and biodiversity of this group of extremophiles.
 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Acidophile; Acid mine drainage; Acidithiobacillus; Acidiphilium; Acid streamers; Ferroplasma; Leptospirillum; Iron; Mines; Sulfur

1. Acidic mine waters: origin and chemistry Whilst sulfide mineral oxidation may be abiotic, the rate of
reaction is far greater, by many orders of magnitude, in the
In all but the most arid areas of the planet, deep mining presence of certain lithotrophic (“rock eating”) prokaryotes.
and surface (opencast) mining of metal ores and coal For acid-stable sulfides, such as pyrite, these microorgan-
results in the contamination of ground and/or surface waters. isms accelerate the oxidative dissolution of the mineral by
The nature and extent of this contamination is highly re-generating ferric iron (reaction 2):
variable, depending on such factors as the nature of the
4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2 O. (2)
ore body and associated geological strata, climate and
mining engineering constraints. The most well documented Reaction 1 is abiotic (and may occur in anaerobic as well
type of water pollution associated with mining is that as aerobic environments) while oxygen-requiring reaction
which results from the accelerated oxidative dissolution of 2 is primarily biological in acidic (pH < 4) waters. Some
exposed minerals, principally sulfides, giving rise to acidic, bacteria and archaea can also oxidize thiosulfate, the initial
metal-enriched waters generally referred to as “acid mine sulfur by-product of pyrite oxidation (reaction 1), and other
drainage” (AMD) or “acid rock drainage” (ARD). reduced inorganic sulfur compounds (RISCs) and elemental
The origin and nature of AMD has been described in sulfur, producing sulfuric acid (reaction 3):
detail elsewhere (e.g., [20,31]). Briefly, minerals such as +
S2 O2− 2−
3 + 2O2 + H2 O → 2H + 2SO4 . (3)
pyrite (FeS2 ; the most abundant of all sulfide minerals) are
chemically (and biologically) stable in situations where both Further acidity results from the hydrolysis of ferric iron
oxygen and water are excluded. However, upon exposure (reaction 4):
to both moisture and air (e.g., following the fracturing and Fe3+ + 3H2 O → Fe(OH)3 + 3H+ . (4)
exposure of sulfide-containing mineral ores and sulfidic
coals) sulfide minerals will oxidize spontaneously, with Proton acidity resulting from reactions 3 and 4 may be
either molecular oxygen or ferric iron acting as the oxidant counter-balanced by alkalinity resulting from the dissolu-
(e.g., reaction 1): tion of basic (chiefly carbonate) minerals in the ore body or
+ mine spoil, though this may be an important neutralization
FeS2 + 6Fe3+ + 3H2 O → 7Fe2+ + S2 O2−
3 + 6H . (1) mechanism only in the short term. Water draining mines and
mine spoils are therefore often acidic, and frequently con-
* Corresponding author. tain elevated concentrations (relative to unpolluted streams)
E-mail address: d.b.johnson@bangor.ac.uk (D.B. Johnson). of aluminum and various heavy metals, such as copper,
0923-2508/$ – see front matter  2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
doi:10.1016/S0923-2508(03)00114-1
 D.B. Johnson, K.B. Hallberg / Research in Microbiology 154 (2003) 466–473 467

(a) (b)

Fig. 1. (a) An acidic (pH 2.3) iron-rich stream within an abandoned pyrite mine, north Wales. The stream is ramified with growths of “acid streamers”,
(b) destruction of trees caused by the AMD stream on leaving the mine.

Table 1
Physico-chemical and microbiological characteristics of acid mine drainage from various sites worldwide
Storwartz Ynysarwed, Bullhouse, Wheal Jane, Killingdal King’s mine Parys mine, Rio Tinto, Iron
mine, Walesb Englandb Englandb mine dump, stream, Walesb Spaind Mountain,
Norwaya Norwaya Norwayc Californiae
pH 6.5 6.2 5.9 3.4 2.77 2.75 2.5 2.2 0.5 – 1
Eh (mV) – +214 +257 +462 – – +685 +450
Fetotal 1.60 160 61 290 265 172 650 2.3 × 103 13–19 × 103
Fe2+ – 140 45 250 – – 650 1.5 × 103 13–19 × 103
Al 0.03 – 1.2 27 38.3 22.5 70 – 1.4–6.7 × 103
Mn 1.35 – 15 8 4.24 0.78 10 – 17–120
Cu 0.06 – – 1.2 5.65 15.8 40 109 120–650
Zn 2.13 – – 132 61.1 25.4 60 225 700–2600
Sulfate 151 464 – 400 1219 668 1550 10 × 103 20–108 × 103
Fe-oxidizers <102 <102 40 3.0 × 104 1.3 × 102 1.0 × 103 1.3 × 103 – –
(moderate
acidophiles)
Fe-oxidizers <102 <102 <102 1.0 × 103 2.1 × 105 5.6 × 104 3.5 × 103 1.3 × 106 –
(extreme
acidophiles)
S-oxidizers <102 <102 <102 <102 <102 <102 <102 6.1 × 106 –
(extreme
acidophiles)
Heterotrophic 7.0 × 102 <102 <102 3.0 × 102 2.4 × 105 1.6 × 104 2.0 × 103 – –
acidophiles
Note. All concentrations of metals and sulfate are in mg/l and acidophiles are #/ml. –, not determined. Data are from: (a) Johnson et al., unpublished; (b) [23];
(c) [22]; (d) [28]; and (e) [32].

zinc and manganese. These metals originate from dissolved nating primarily from the oxidation of arsenopyrite, FeAsS,
sulfides, and other associated minerals, and are held in so- or arsenic containing pyrite) may also be present at greatly
lution because of their greater solubility in acidic liquors. elevated concentrations in AMD. While both As(III) and
However, iron, which may be present in either (or both) As(V) may be present, the more oxidized state (e.g., ar-
ferrous and ferric forms (depending on water pH) is, in senate) tends to be more prevalent in AMD. In contrast,
most cases, the dominant metal present in AMD, soluble the concentrations of dissolved organic matter in AMD
ferric iron imparting the characteristic red color to ex- tends to be relatively low (<20 mg/l), favoring oligotrophic
tremely acidic mine waters (Fig. 1) while orange-yellow rather than copiotrophic heterotrophs. The chemical compo-
ferric iron-rich sediments (“ochre” or “yellow boy”) are sitions of several representative AMD waters are given in
common in higher pH streams. The metalloid arsenic (origi- Table 1.
468 D.B. Johnson, K.B. Hallberg / Research in Microbiology 154 (2003) 466–473

2. Acidophilic bacteria and archaea (Cu2 S), are acid-soluble and can be oxidized by prokaryotes
such as Acidithiobacillus thiooxidans and Metallosphaera
Prokaryotic microorganisms that are metabolically active sedula that generate sulfuric acid.
in extremely acidic environments (often described as having In contrast, iron-reducing bacteria have been shown to
a pH < 3) are widely distributed in the domains Bacteria and accelerate the reductive dissolution of ferric iron-containing
Archaea. The physiological and phylogenetic diversity of minerals, such as jarosites and goethite. These include het-
acidophilic prokaryotes has been reviewed elsewhere (e.g., erotrophic mesophilic bacteria, such as Acidiphilium spp.,
[16]). Acidophiles have been categorized using a number that do not oxidize ferrous iron [5], and moderate ther-
of different criteria, such as temperature (as mesophiles, mophiles, such as Sulfobacillus spp., that either oxidize
moderate thermophiles and thermophiles) and pH optima, ferrous iron or reduce ferric iron, depending on prevail-
and also on the basis of cellular carbon acquisition; au- ing concentrations of dissolved oxygen [4]. Interestingly,
totrophs assimilate CO2 , heterotrophs assimilate organic all strains of Acidiphilium spp. appear to be able to cat-
carbon and mixotrophs use both. In general, the most ex-
tremely thermophilic acidophiles are archaea rather than
bacteria (Fig. 2), which might be expected since archaea Table 2
Acidophilic prokaryotic microorganisms
frequently inhabit the most extreme niches on our planet.
Thermo-acidophilic prokaryotes are particularly abundant in Mineral-degrading acidophiles Thermal Phylogenetic
classification* affiliation
geothermal areas (e.g., [6]) and in anthropogenic environ-
ments such as self-heating coal spoils (Section 3.4). 1a. Iron-oxidizers
Leptospirillum ferrooxidans Meso Nitrospira
Another useful subdivision of acidophiles differentiates
L. ferriphilum Meso Nitrospira
those that can solubilize minerals and those that cannot. L. thermoferrooxidans Mod Thermo Nitrospira
The first group includes prokaryotes that accelerate mineral “Thiobacillus ferrooxidans” m-1 Meso β-Proteobacteria
dissolution by an oxidative route, and those that use a re- “Ferrimicrobium acidiphilum” Meso Actinobacteria
ductive mechanism. The role of iron- and sulfur-oxidizing Ferroplasma acidiphilum Meso Thermoplasmales
“Fp. acidarmanus” Meso Thermoplasmales
bacteria and archaea in the dissolution of sulfidic miner-
als is well documented, and is utilized in “biomining” [34] 1b. Sulfur-oxidizers
Acidithiobacillus thiooxidans Meso β/γ-Proteobacteria
where their activities facilitate the extraction of gold (which At. caldus Mod Thermo β/γ-Proteobacteria
occurs as fine-grain occlusions in refractory ores) or the Thiomonas cuprina Meso β-Proteobacteria
solubilization of base metals (such as copper and cobalt). Hydrogenobacter acidophilus Mod Thermo Aquifacales**
Sulfide minerals differ in their susceptibility to oxidation. Metallosphaera spp. Ext Thermo Sulfolobales
Some (such as pyrite) are oxidized by ferric iron (as above), Sulfolobus spp. Ext Thermo Sulfolobales
and acidophilic iron-oxidizing prokaryotes (e.g., Leptospir- 1c. Iron- and sulfur-oxidizers
Acidithiobacillus ferrooxidans Meso β/γ-Proteobacteria
illum ferrooxidans, Acidithiobacillus (“At.”) ferrooxidans,
Acidianus spp. Ext Thermo Sulfolobales
Acidimicrobium (“Am.”) ferrooxidans, Sulfolobus metalli- Sulfolobus metallicus Ext Thermo Sulfolobales
cus) have a central role in their dissolution in environmental 1d. Iron-reducers
and commercial contexts. Other sulfides, such as chalcocite Acidiphilium spp. Meso α-Proteobacteria
1e. Iron-oxidizers/reducers
Acidimicrobium ferrooxidans Meso Actinobacteria
1f. Iron-oxidizers/reducers
and sulfur-oxidizers
Sulfobacillus spp. Meso and Firmicutes
Mod Thermo
2. Heterotrophic acidophiles
(non mineral-degrading)
Acidocella spp. Meso α-Proteobacteria
Acidisphaera rubrifaciens Meso α-Proteobacteria
Acidobacterium capsulatum Meso Acidobacterium
Acidomonas methanolica Meso α-Proteobacteria
Alicyclobacillus spp. Meso Firmicutes
Picrophilus spp. Mod Thermo Thermoplasmales
Thermoplasma spp. Mod Thermo Thermoplasmales
3. Obligate anaerobes
Stygiolobus azoricus Ext Thermo Sulfolobales
Acidilobus aceticus Ext Thermo Sulfolobales
Fig. 2. Temperature and pH optima of acidophilic prokaryotes. Key: * Meso—mesophiles (Toptimum < 40 ◦ C); Mod Thermo—moderate ther-
!, iron-oxidizing bacteria; (") iron-oxidizing archaea; (1) sulfur-oxidizing mophiles (Toptimum 40–60 ◦ C); Ext Thermo—extreme thermophiles
bacteria; (2) sulfur-oxidizing archaea; (P) heterotrophic bacteria; (Q) het- (Toptimum > 60 ◦ C).
erotrophic archaea. ** Inferred ability to oxidize minerals (via production of sulfuric acid).
 D.B. Johnson, K.B. Hallberg / Research in Microbiology 154 (2003) 466–473 469

Fig. 3. Block diagram showing the distribution of different genera of acidophilic microorganisms in acidic mine waters (location given in Table 1). Solid blocks
indicate the presence while empty blocks indicate that a given acidophile was not detected in the mine water.

alyze the dissimilatory reduction of ferric iron to ferrous, concentrations may be <10% saturation. Novel unicellular
though in some strains this is constitutive while in others iron-oxidizing lithotrophic bacteria have recently been iso-
it is inducible [21]. Other acidophilic microorganisms have lated from iron-rich groundwaters and the roots of aquatic
not been shown to have a direct role in mineral breakdown. macrophytes [30]. Again, these are “gradient” organisms
These include photosynthetic eukaryotes, such as Cyanid- and, since they grow best at pH’s ca. 5.8–7.0, they are not
ium caldarium (a moderately thermophilic rhodophyte) and acidophiles and will not be considered further in this review.
the mesophile Euglena mutabilis, both of which can have Further evidence of the importance of previously un-
an important role in primary production in acidic environ- known bacteria mediating chemical changes (ferrous iron-
ments that receive light. Some heterotrophic prokaryotes and possibly sulfur-oxidation) in neutral pH and moder-
(such as Acidocella and Thermoplasma) may contribute to ately acidic mine waters has emerged from work carried
mineral dissolution indirectly by forming commensal asso- out with AMD discharges in the UK and Norway [17].
ciations with iron- and sulfur-oxidizers, whereby they utilize Bacteria have been isolated, either directly from AMD or
organic compounds (cell exudates and lysates) originating following enrichment in liquid cultures, that oxidize fer-
from the autotrophic primary producers, thereby essentially rous iron at pH 3–5. Three phylogenetically-distinct groups
“detoxifying” the environment for the more sensitive (e.g., of these “moderate acidophiles” have been described: one
to organic acids) latter group [19]. group is most closely related to the neutrophilic acetogen
Table 2 lists the various species of acidophilic microor- Frateuria aurantia (a γ-proteobacterium), a second ap-
ganisms that can grow in extremely acidic (pH < 3) waters. pears to consist of novel Thiomonas spp. (β-Proteobacteria)
Not all of these organisms have been officially classified (as and the third group is phylogenetically highly related to
indicted by names in quotation marks) and others require re- the sulfur-oxidizer Halothiobacillus neapolitanus (also a
classification, such as Thiobacillus ferrooxidans m-1, which γ-proteobacterium). Interestingly, none of these genera has
is phylogenetically distinct from bona fide Thiobacillus and previously been reported to include iron-oxidizing species.
Acidithiobacillus spp. Surveys of various mine waters have indicated that mod-
erately acidophilic iron-oxidizers are widely distributed in
all but the most acidic mine waters (Table 1 and Fig. 3).
Oxidation of ferrous iron by these bacteria, and subsequent
3. Acidophilic prokaryotes indigenous to mine waters
hydrolysis of ferric iron, causes AMD pH to fall, possibly
to the level at which the more familiar extreme acidophiles
3.1. Moderately acidic and circum-neutral pH mine waters (Section 3.2) become active.

Neutrophilic iron-oxidizing and iron-depositing bacteria 3.2. Extremely acidic mine waters
were amongst the first prokaryotes to be observed and de-
scribed. Stalked (e.g., Gallionella ferruginea) and filamen- The microbiology of extremely acidic (pH < 3) streams
tous (e.g., Leptothrix discophora) “iron bacteria” are, due and rivers draining mining-impacted areas has been exam-
to their characteristic accumulation of ferric iron deposits, ined by relatively few research groups. The Afon Goch
amongst the most obvious microorganisms in freshwater en- (a stream draining the former Parys copper mine in north
vironments. Due to the rapid abiotic oxidation of ferrous iron Wales; pH 2.2–2.8) was found to contain about 103 /ml of
in aerated, circum-neutral pH waters, these bacteria tend to both At. ferrooxidans and L. ferrooxidans [39]. The relative
grow in the oxic/anoxic interface where dissolved oxygen numbers of these iron-oxidizers changed with distance from
470 D.B. Johnson, K.B. Hallberg / Research in Microbiology 154 (2003) 466–473

the discharge adit, and appeared to correlate with changing as neutrophilic heterotrophs), some of which produce co-
concentrations of ferrous iron; L. ferrooxidans has a higher pious quantities of exopolymers while others are filamen-
affinity for Fe(II) and these bacteria were particularly nu- tous. Analysis of streamers from Parys mountain and Cae
merous when ferrous iron concentrations were <10 mg/l. In Coch (AMD pH 2.7–3.05) using FISH has indicated that
contrast, At. ferrooxidans was noted to be the most numer- they were composed predominantly of β-Proteobacteria,
ous iron-oxidizer present in AMD draining the abandoned with smaller numbers of “Ferrimicrobium”-like bacteria
Cae Coch pyrite mine in north Wales, where ferrous iron and γ-Proteobacteria (Kimura et al., unpublished). Sulfate-
concentrations were often >500 mg/l and the pH was 2.3– reducing bacteria (SRB) have also been isolated from enrich-
2.5 [29]. At. ferrooxidans-like bacteria were also found to be ment cultures of acid streamers [37]. In contrast, streamers
the dominant iron-oxidizer in a stream draining the former and slimes from the hyperacidic mine waters at Iron Moun-
King’s copper mine in Roeros, Norway [22]; moderately- tain, California (described in Section 3.3), appear to be very
acidophilic (Thiomonas-like) isolates have also been found different from those in cooler, higher pH waters.
in this AMD, though in far smaller numbers than the ex-
treme acidophiles. Numbers of heterotrophic acidophiles in 3.3. Hyperacidic mine waters
the same steam were similar to those of At. ferrooxidans;
five different genera/species of obligate heterotrophs were The Richmond mine at Iron Mountain, California, con-
isolated from the King’s mine AMD, including Acidiphilium sists of a pyrite-rich ore body that was mined periodically
cryptum, Acidiphilium rubrum, and apparently novel species for gold, silver, copper, zinc and pyrite until the 1960s, when
of Acidocella, Acidisphaera and Acidiphilium. mining ceased. In some areas of the mine, the AMD is the
The Rio Tinto, which flows from the Peňa de Hierro in most acidic water recorded, with a pH as low as ∼ −4 [32].
southwestern Spain and enters the Atlantic Ocean at Huelva, In a series of papers, the microbiology of the AMD in the
is a 100 km-long extremely acidic (pH 2.0–2.5), iron- (ca. Richmond mine has been explored using mainly molecu-
1–10 g/l) and sulfate- (ca. 12–27 g/l) rich river, and is prob- lar techniques. Initial studies using FISH focused on the
ably the most famous water body of its kind. Mining at Rio microbes commonly associated with AMD, namely At. fer-
Tinto has been in place for over five thousand years, and con- rooxidans and L. ferrooxidans. In samples taken in January,
tinues (albeit on a relatively small scale) to the present day. 1997, both species were found in free-flowing mine water,
Interestingly, the river Tinto is acidic and metal-impacted mine water associated with the pyritic sediment and in slime
even prior to flowing through the mine site, presumably (streamer) material in the mine [36]. Interestingly, in waters
due natural exposure of the Iberian pyritic belt close to its of pH < 1.3 and with a temperature >30 ◦ C, L. ferrooxi-
source. Microbial communities have been studied by Amils dans was found to constitute ∼10% of the total population
et al. [28] and acidophilic bacteria related to L. ferroox- whilst At. ferrooxidans was not detected. Neither of these
idans, At. ferrooxidans, At. thiooxidans and Acidiphilium two species dominated the total microbial population in the
were identified. A combination of denaturing gradient gel Richmond mine, eukaryotes were generally minor compo-
electrophoresis (DGGE) and fluorescent in situ hybridiza- nents, but occasionally constituted up to 25% of the total
tion (FISH) confirmed the presence of these acidophiles population, and archaea made up less than 5%. The majority
(∼80% of total cells) and also showed that Ferroplasma of the bacteria detected with the eubacterial probe, however,
acidiphilum/Thermoplasma acidophilum-like archaea were were unidentified at that time.
also present in the Tinto (Ricardo Amils, personal commu- Seasonal variations in the microbial populations at Iron
nication). Mountain were also followed using FISH [10]. Again, in
In the Lusatia region of eastern Germany, there are an es- January bacteria dominated the microbial population, and
timated 200 acidic (pH < 3) lakes of a surface area >1 ha, L. ferrooxidans outnumbered At. ferrooxidans. During the
which have arisen as a result of the infill of voids follow- summer, however, archaea increased to about 50% of the to-
ing opencast mining of open lignite coal. A large amount of tal population, with a concomitant fall in bacterial numbers.
research has been carried out on the geochemistry, microbi- This increase in archaea was correlated with the low rain-
ology and remediation strategies of these water bodies [13]. fall period in California, which was reflected by an increase
Photosynthetic (e.g., Ochromonas, Chlamydomonas) and in the conductivity of the mine water samples. Through en-
lithotrophic (e.g., Acidithiobacillus spp.) as well as het- richment with pyrite as energy source, a culture medium of
erotrophic (e.g., Acidiphilium and “Ferrimicrobium” spp.) pH 1.0 and mine water collected in July 1997 as inoculum,
microorganisms have been identified in these acid min- a culture developed that was dominated by archaea [11].
ing lakes. Sulfate-reducing bacteria belonging to the genus Amplification of the 16S rRNA gene and sequence analysis
Desulfobacter have been detected in mine lakes that have showed that this microbe, called “Ferroplasma (Fp.) acidar-
been amended with organic carbon [33]. manus”, was highly related (>98% sequence identity) to Fp.
The most dramatic evidence of microbial life in metal- acidiphilum, an archaeon that had been isolated from a bi-
rich, acidic waters lies in the often voluminous “acid stream- oleaching reactor [15]. Using the sequence data obtained, a
ers” growths (e.g., [9]). These are mixed communities of probe was designed that targeted “Fp. acidarmanus” and it
autotrophic and heterotrophic acidophilic bacteria (as well was shown to be the dominant archaeon at Iron Mountain.
 D.B. Johnson, K.B. Hallberg / Research in Microbiology 154 (2003) 466–473 471

utilized and, in general, confirmed results found by the gene

library studies, i.e., in some slime material the group 3 lep-
tospirilli were the predominant microbe present (accounting
for nearly 100% of the eubacteria), while in other samples
“Fp. acidarmanus” dominated (∼100% of the total micro-
bial population). Interestingly, the use of probes targeting
other acidophiles revealed the dominance of Sulfobacillus
species in some slime samples. The δ-Proteobacteria and
“Ferrimicrobium acidiphilum” were detected only at low
levels, or not at all. Other acidophiles that were not detected
by FISH included At. caldus and heterotrophic Acidiphilium
spp. That the streamer (and slime) material in Iron Moun-
tain differs dramatically from the streamers found in Parys
mountain and Cae Coch (see Section 3.2) is not too sur-
prising given the dramatic differences in physico-chemical
parameters of the three sites (see Table 1).
Fig. 4. The three phylogenetic groups of Leptospirillum. Group 1 contains
the type species (T ) of L. ferrooxidans, group 2 consist of, amongst other
3.4. Mineral tailings, spoils and sediments
strains, the type species (T ) of L. ferriphilum and group 3 consists solely of
environmental clones. The bar shows 0.05 inferred nucleotide substitutions
per 100 nucleotides for the horizontal branch lengths. The source of waters draining working and abandoned
mines may be underground workings, and/or surface de-
The above studies focused on the microbial population posits of waste materials (spoil heaps). Tailings (waste
that included planktonic cells, cells attached to fine grained fine-grain minerals from ore processing by grinding and
pyrite particles on the floor of the mine and on slime ma- flotation) are frequently stored under water, in order to
terial in the mine water, and specifically targeted relatively reduce exposure to oxygen and the ensuing oxidative disso-
few microbial species. 16S rRNA gene libraries made from lution of acid-generating sulfidic minerals. There are several
DNA prepared from two different slime samples, a slime reports on the microbiology of these ecosystems, includ-
on the surface of the pyrite on the tunnel floor and “snot- ing pioneering work by Tom Brock’s research team in the
tites” (slime growths hanging from the roof of the mine USA (e.g., [8]). Coal spoil heaps are susceptible to ignite
tunnel) revealed a greater degree of microbial diversity at spontaneously, and may smolder for many years. One of
Iron Mountain [3]. Although it is difficult to draw firm con- the first moderately thermophilic acidophiles to be discov-
clusions about the relative abundance of microbes based on ered (Thermoplasma acidophilum) was isolated from such
such an approach, it was found that the two gene libraries a site [8]. Schippers et al. [35] examined vertical hetero-
produced were dominated (75 to 77%) by bacteria that were geneity (from 0–5 m) of microbial populations in uranium
related to Leptospirillum spp. However, sequencing of the spoil heaps in Germany. Iron- and sulfur-oxidizing bacteria
representative genes showed that, while a few were related to were detected, but their numbers declined dramatically be-
L. ferrooxidans, the major clone (BA29, representing ∼70% low 1.5–2 m depth, corresponding with a marked decrease
of clones in the library) of each library was only remotely in oxygen concentrations. Earlier, Goebel and Stackebrandt
related to L. ferrooxidans and a second, recently desig- [14] had analyzed microbial diversity in a chalcopyrite over-
nated species [7], Leptospirillum ferriphilum (Fig. 4). The burden heap in Australia, using a molecular approach (DNA
sequence of this clone also revealed that the probe (LF581) extraction, construction of a clone library and analysis of
used in the earlier studies at Iron Mountain would not detect 16S rRNA genes). Iron/sulfur-oxidizing bacteria (L. ferroox-
group 3 leptospirilli. Other bacterial genes amplified from idans, At. ferrooxidans, At. thiooxidans, At. caldus) and
the slime library were nearly identical to that from “Ferrimi- heterotrophic (Acidiphilium spp.) acidophiles were identi-
crobium acidiphilum” and another that was more remotely fied using this approach.
related to “Fm. acidiphilum”, but more closely related to Iron- and sulfur-oxidizing acidophiles have also been
clones from an acidic peat bog. Two other clones were ob- found in mine tailings (including samples taken below the
tained that belonged to the δ-Proteobacteria (which includes water table) by a number of research groups (e.g., [38,40]).
sulfate- and metal-reducing bacteria) but were not related to Aerobic Acidiphilium-like heterotrophic acidophiles have
any known bacteria in this group. The “snottite” library also also been isolated and characterized from mine tailings [1].
included 16S rRNA genes from archaea that were similar Fortin and Beveridge [12] detected up to 106 sulfate reduc-
to Thermoplasma acidophilum and one that was identical to ing bacteria/g (dry wt) in mineral tailings at a copper/zinc
F. acidiphilum/“Fp. acidarmanus”. mine in Ontario, Canada. Bacteria resembling spore-forming
Quantitative studies on these same slime samples and Desulfotomaculum spp. and vibrioid, motile Desulfovibrio
streamer material were carried out using FISH [2]. Using were observed, though these were not formally identified.
the new sequence data, additional 16S rRNA probes were Interestingly, SRB were found in oxygen-containing as well
472 D.B. Johnson, K.B. Hallberg / Research in Microbiology 154 (2003) 466–473

as anaerobic zones in the tailings, and also in acidic (pH 2.5) redox reactions that these microbes carry out to achieve a
as well as circum-neutral pH samples. desired remediation goal (e.g., iron oxidation for iron re-
Anaerobic acid-tolerant bacteria have also been isolated moval from AMD, and sulfate reduction for chalcophilic
from sediments in AMD streams and lakes. Küsel et al. [25] metal removal and alkalinity generation). The topic of re-
isolated an iron-reducing bacterium from anoxic acidic mediation of AMD pollution has been thoroughly reviewed
(pH 3.2) sediment from a mining lake that coupled the elsewhere [41].
oxidation of glucose to the reduction of iron(III). The isolate The improvement of solid media [18] and the recent
was found by 16S rRNA gene sequence analysis to be a application of molecular techniques to the study of AMD
strain of Acidiphilium cryptum, and later work confirmed has led to a thorough re-evaluation of the microbial diver-
that, like other Acidiphilium species, this isolate was more sity of this extreme environment. Only until about a decade
effective at reducing iron when small amounts of oxygen ago, relatively few acidophiles were recognized, and en-
were present [5,24]. Sen and Johnson [37] obtained mixed richment/cultivation based studies have led to erroneous
cultures containing SRB from Afon Goch (Parys copper conclusions about the significance of At. ferrooxidans and
mine) that were able to reduce sulfate in batch culture At. thiooxidans in AMD. Recent advances in molecular biol-
at pH > 2.4. Two SRB isolates were obtained, both of ogy allow the positive identification of microbes to be made
which formed endospores and were shown to be related to easily through 16S rRNA gene sequence analysis, rather
Desulfosporosinus orientis (∼95% identity of 16S rRNA than simply making a presumptive identification of a mi-
genes). One of the isolates grew as distinctly large (>5 µm crobe based on physiological characteristics (many traits
long) motile cells, and appeared to ferment mannitol as are now known to be shared amongst various groups of
well as oxidize glycerol (Sen et al., unpublished data). acidophiles). Continued media development guided by in-
Küsel et al. [26] also isolated a fermentative anaerobe formation gathered through cultivation-independent (molec-
from AMD sediment. This isolate, which was also capable ular) studies of AMD will lead to the ability to cultivate even
of acetogenic growth on hydrogen, was found to very more acidophiles, allowing a greater understanding of these
closely related (99.6% sequence similarity of its 16S rRNA important microbes.
gene) to Clostridium scatologenes. In other work, Küsel
et al. [27] carried out a series of mesocosm experiments
using sediment samples taken from a coal mining-impacted Acknowledgement
acid lake in Lusatia. Bacteria that were able to couple the
oxidation of elemental sulfur to the reduction of ferric iron We would like to thank the BBSRC (grant ref. 5/BRM18412),
were estimated to be 1% of the total microbial population. In U.K., for financial support under the DTI LINK scheme.
addition, a SRB was isolated from a sample with an in situ
pH of 5.2. Phylogenetic analysis revealed that this was again
most closely related to the spore-former Desulfosporosinus References
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