geomicrobiology of extremely acidic subsurface environments

M IN I R E V I EW

Geomicrobiology of extremely acidic subsurface environments

David Barrie Johnson

School of Biological Sciences, Bangor University, Bangor, UK

Correspondence: David Barrie Johnson,

School of Biological Sciences, Bangor

University, Bangor LL57 2UW, UK. Tel.:

+44 1248 382358; fax: +44 1248 370731;

e-mail: [email protected]

Received 13 October 2011; revised 16

December 2011; accepted 16 December

2011.

DOI: 10.1111/j.1574-6941.2011.01293.x

Editor: Tillmann Luders

Keywords

acidophiles; Acidithiobacillus; Ferroplasma;

‘Ferrovum’; pyrite; subsurface.

Abstract

Extreme acidophiles (microorganisms with pH optima of < 3) can colonize

and exploit subterranean environments, such as abandoned metal sulfide

mines, that have the potential for developing widespread or isolated pockets of

acidity. Although acidophiles can utilize a wide range of electron donors, inor-

ganic materials (reduced sulfur, ferrous iron, and possibly hydrogen) are often

the most abundant sources of energy for acidophiles in the subsurface. The

diversity and interactions of acidophilic microbial communities in two aban-

doned sulfide mineral mines (in Iron Mountain, California, and the Harz

mountains in Germany) and a sulfidic cave (Frasissi, Italy) are reviewed. In

addition, the contrasting geomicrobiology of two abandoned sulfide mineral

mines in north Wales is described. Both are extremely acidic (pH~2) and low-

temperature (8–9 °C) sites, but one (Cae Coch) is essentially a dry mine with

isolated pockets of water, while the other (Mynydd Parys) contains a vast

underground lake that was partially drained several years ago. The microbial

communities in these two mines exhibit different relative abundances and often

different species of archaea and bacteria. Wooden pit props, submerged in the

underground lake, act as a slow-release source of organic carbon in the subter-

ranean Mynydd Parys lake, supporting a microbial community that is more

enriched with heterotrophic microorganisms.

Introduction: phylogenetic andmetabolic diversity of acidophilicmicroorganisms

Acidophiles are organisms (chiefly microorganisms) that

can grow in low pH environments. The generally

accepted consensus is that these may be categorized as:

(1) extreme acidophiles, which have pH optima for

growth of < 3; (2) moderate acidophiles, which have pH

growth optima of 3–5; and (3) acid-tolerant organisms,

which have pH growth optima of > 5 but which are also

metabolically active in acidic environments. This article

focuses on how extreme acidophiles may colonize and

exploit the subsurface and gives examples where the bio-

diversity of acidophilic microorganisms and interactions

between them have been studied in four abandoned deep

mines and one cave.

Extreme acidophiles are widely distributed throughout

the three Domains of life (Johnson, 2009), and it appears

that the ability to grow at low pH has arisen indepen-

dently rather than having evolved from a single acido-

philic common ancestor. Within the Eukarya, examples

of extreme acidophiles include species of fungi, algae,

protozoa, and rotifera. Numerous species of acidophilic

Crenarchaeota and fewer species of Euryarchaeota have

been described. Extreme acidophiles are also widely dis-

tributed within the domain Bacteria, including members

of the phyla Proteobacteria (alpha, beta, and gamma clas-

ses), Nitrospirae, Firmicutes, Actinobacteria, and Acidobac-

teria.

Taken as a whole, extreme acidophiles display a wide

range of metabolic activities, including the abilities to use

solar or chemical energy, to utilize either inorganic or

organic carbon as their major (or sole) source of carbon,

and to grow in the presence or absence of oxygen (John-

son & Hallberg, 2009). Acidophiles vary widely in their

temperature ranges and optima, from psychrotolerant

(mostly bacteria) species that grow at temperatures as low

as ~5 °C to thermophilic (exclusively archaea) species

that can grow at up to ~80 °C. One of the key generic

FEMS Microbiol Ecol && (2011) 1–11 ª 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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characteristics that differentiates extreme acidophiles from

other prokaryotes is the widespread ability to use inor-

ganic materials, such as ferrous iron, reduced sulfur, and

hydrogen, as electron donors (Schippers et al., 2010).

While many extreme acidophiles use small molecular

weight organic electron donors (obligate or facultative

heterotrophs), chemoautotrophic bacteria and archaea

are, by far, the most widely studied acidophiles and are

often the most numerous microorganisms in low pH

environments, because inorganic energy sources (e.g. sul-

fide minerals) are often far more abundant than metabo-

lizable organic carbon. Indeed, many extremely

acidophilic environments are oligotrophic with respect to

their organic matter contents, containing < 10 mg L�1 of

dissolved organic carbon (DOC).

The vast majority of known acidophiles use oxygen as

terminal electron acceptor, which is hardly surprising as

most classified species have been isolated or enriched

under aerobic conditions. However, it is becoming appar-

ent that iron (III) can be used by many phylogenetically

diverse acidophilic bacteria and archaea as an alternative

electron acceptor under conditions of oxygen limitation

or anoxia. In many ways, this is not surprising because

the Eh of the ferrous/ferric couple at low pH (usually

quoted as + 0.77 V) is not much less electropositive than

that of the oxygen/water couple (1.12 V at pH 2), so the

metabolic ‘penalty’ for using ferric iron as an alternative

electron acceptor to oxygen at extremely low pH is not

that great. In addition, the far greater bioavailability of

iron (III) at extremely low pH, because of its abundance

and far greater solubility, makes it far more accessible to

iron-reducing acidophiles than to their neutrophilic

counterparts. Ferric iron respiration has been reported

for all iron-oxidizing bacteria that can use an alternative

electron donor (e.g. reduced sulfur) to ferrous iron and

also for many heterotrophic bacteria that do not oxidize

iron (Coupland & Johnson, 2008). In contrast to dissimi-

latory iron reduction, few acidophilic bacteria and

archaea that grow via dissimilatory reduction of sulfate

and/or elemental sulfur have been described, the excep-

tions being the thermo-acidophilic crenarchaeotes Acidi-

anus, Stygiolobus, Sulfurisphaera, and the moderately

thermophilic euryarchaeote Thermoplasma, all of which

have been reported to use elemental sulfur as terminal

electron acceptor. It has also been claimed that some

strains of Acidithiobacillus (At.) ferrooxidans can grow by

coupling the oxidation of hydrogen to the reduction of

elemental sulfur (Ohmura et al., 2002). The dearth of

acidophilic (as opposed to acid-tolerant) sulfate-reducing

bacteria (SRB) is, however, rather surprising, given the

facts that (1) sulfate is usually present in elevated concen-

trations (and is often the major anion) in extremely

acidic environments and (2) the sulfate/sulfide couple is

more electropositive at low pH (Eh of + 75 mV at pH 2)

than at circum-neutral pH (�220 mV), which makes it,

like iron (III), a more favorable electron acceptor at low

pH, in energetic terms. The probable reasons for very few

acidophilic SRB having been described are related to

techniques used for their enrichment and isolation, where

factors such as the pH-related toxicities of commonly

used enrichment substrates (e.g. lactic acid) as well as

metabolic waste products (sulfide, which is present as

H2S at low pH, and acetic acid produced by the ‘incom-

plete oxidizers’) have been overlooked. There have, how-

ever, been some reports of novel species of SRB that are

metabolically active at pH � 3 (e.g. Kimura et al., 2006;

Jameson et al., 2010; Nancucheo & Johnson, 2012). Inter-

estingly, all of these are Firmicutes and include some Des-

ulfosporosinus spp. and other apparently novel genera of

SRB.

While anaerobic respiration is not that uncommon

among acidophiles, fermentative metabolism appears to

be very rare. Again, part of the reason for this is that

small molecular organic weight organic acids excreted as

fermentative waste products exist predominantly as their

undissociated forms in extremely acidic liquors (i.e. at

pH below their pKa values) and as such are generically

highly toxic to acidophilic prokaryotes (Norris & Ingle-

dew, 1992).

Potential energy sources for acidophilicmicroorganisms in subsurfaceenvironments

Most of the information that has been published on aci-

dophilic life in the subsurface has come from research

carried out in abandoned deep mines and caves, rather

than drill cores taken from the Earth’s crust. Given suit-

able conditions – most notably of geology and hydrology

– acidic conditions can form and develop, because of

ongoing microbial activity, within such subterranean

environments. Various energy sources that are used by

acidophiles can be present in abandoned mines and,

occasionally, in caves. These include:

Sulfide minerals and hydrogen sulfide

The former are particularly important in many aban-

doned metal mines, principally because many base metals

(e.g. copper, lead and zinc) that are mined in opencast or

deep mining operations occur primarily as sulfide miner-

als within often vast ore bodies. Refractory gold deposits

are often intimately associated with sulfide minerals

(chiefly pyrite and arsenopyrite), and iron sulfides are

also found associated with coal deposits. The reduced

sulfur moiety in these minerals represents a potential

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Ecol && (2011) 1–11Published by Blackwell Publishing Ltd. All rights reserved

2 D.B. Johnson

microbial energy source, as does (in those minerals where

it occurs) ferrous iron. The mechanisms involved in the

dissolution of pyrite and other sulfide minerals have been

the subject of a considerable amount of research

(reviewed in Rohwerder et al., 2003). While the primary

attack on the mineral in low pH environments is essen-

tially abiotic [mediated by iron (III)] and does not

involve molecular oxygen, regeneration of ferric iron and

oxidation of the reduced sulfur oxyanions produced are

both microbially mediated and oxygen-consuming

processes.

Hydrogen

Although hydrogen has received relatively little attention

as an energy source used by acidophiles, the potential for

hydrogen genesis in acidic subsurface environments is

considerable. For example, abandoned metallic structures

in disused mines will react with acid produced from the

oxidative dissolution of sulfide minerals to generate

molecular hydrogen. Likewise, hydrogen in the subsurface

can be generated by reactions between basalts and water

(Stevens & McKinley, 1995) and radiolysis (Blair et al.,

2007). The ability to use hydrogen as an electron donor

has been reported for At. ferrooxidans (Drobner et al.,

1990) and has also been observed in a number of other

species of acidophilic bacteria (e.g. Acidithiobacillus caldus

and some Sulfobacillus spp.; S. Hedrich and D.B. Johnson,

unpublished data).

Organic materials

In general, extremely acidic environments tend to contain

relatively small concentrations of DOC that can be uti-

lized by heterotrophic and mixotrophic acidophiles.

Potential extraneous sources of organic matter in caves

and deep mines include bat guano and wooden structures

(pit props) used to support mine roofs. Autochthonous

carbon derives from primary producers (chemoautotro-

phic acidophiles) as exudates and products of cell lysates

(e.g. Nancucheo & Johnson, 2010). These bacteria and

archaea form the base of an acidophilic food web that

can include simple animal life forms, as well as heterotro-

phic prokaryotic and eukaryotic microorganisms, in sub-

terranean environments (Johnson, 2009).

Examples of acidophilic life insubterranean environments

Examples of acidophilic microbial communities that have

been studied in subterranean (abandoned metal mines

and, in one case, a cave system; Table 1) environments

are described below.

Richmond mine, Iron Mountain, California

The Richmond mine at Iron Mountain is probably the

most extensively studied of all extremely acidic sites. This

was formerly an important copper mine, and also a

source of gold, silver, and zinc, and of pyrite (for sulfuric

acid production). The site operated intermittently as a

deep mine and opencast operation until 1962. Microbial

activity, stimulated by oxygen accessed via shafts and adits

within the deep mine, has resulted in the oxidative disso-

lution of pyrite and other sulfide minerals occurring at

greatly accelerated rates within Iron Mountain and causes

the temperatures within the mine to be much higher than

ambient (Nordstrom et al., 2000). Even more noteworthy

are the levels of acidity that have developed in water

bodies within the mine. These were reported by Nord-

strom et al. to include some registering negative pH val-

ues (as low as �3.6) making these the most acidic natural

water bodies yet described. The extreme acidity results

from the acid-generating oxidative dissolution of pyrite,

coupled with concentration of hydronium ions because of

water evaporation. The formation of basic efflorescent

iron minerals such as copiapite and jarosite also contrib-

utes to the acidity of water pools within the mine.

The microbiology of the water bodies and of the mac-

roscopic slime and streamer growths within Iron Moun-

tain has been elucidated by Jill Banfield and colleagues at

UC Berkeley, California. The more acidic and higher

Table 1. Physico-chemical data and dominant prokaryotes identified in some extremely acidic subterranean environments (ND, not determined)

Richmond mine

(Iron mountain) Frasissi cave

Drei Kronen und

Ehrt mine Cae Coch mine

Mynydd Parys

mine subterranean lake

pH < 0–2 0–1 2.6 1.8–2.3 2.3

Temperature (˚C) 35–45 13 15 8–9 8–9

Soluble Fe (g L�1) 2.7–141 < 0.1 3.3 1–30 0.6

DOC (mg L�1) ND ND ND 3–24 4–5

Dominant prokaryotes Leptospirillum spp.

‘Fp. acidarmanus’

At. thiooxidans

(Sulfobacillus/

Acidimicrobium spp.)

Leptospirillum sp.

‘Ferrovum’ sp.

At. ferrivorans

‘Fv. myxofaciens’

At. ferrooxidans/ferrivorans

Acidobacteriaceae

(Methanogenic archaea)


Geomicrobiology of extremely acidic subsurface environments 3

temperature sites have been shown to be dominated by

Leptospirillum spp. and a novel Ferroplasma (Fp.) sp.

(‘Ferroplasma acidarmanus’; Edwards et al., 2000). Two

distinct Leptospirillum spp. were detected: one (‘Group

II’) that is closely related to the characterized species

Leptospirillum ferriphilum and the other (‘Group III’) that

was isolated and named as “Leptospirillum ferrodiazotrop-

hum” (Tyson et al., 2005). All Leptosprillum spp. so far

described are obligate autotrophs that appear to grow

only by coupling the oxidation of ferrous iron to the

reduction of molecular oxygen. The ‘Group II’ Leptospiril-

lum in Iron Mountain (in contrast with many other

L. ferriphilum isolates) appeared not to be able to fix

dinitrogen, a trait that was confirmed for the ‘L. ferro-

diazotrophum’ isolate, and which is also known in strains

of ‘Group I’ leptospirilli (Leptospirillum ferrooxidans,

which was not found in Iron Mountain). ‘Ferroplasma

acidarmanus’ is heterotrophic and the most acidophilic of

all known iron-oxidizing prokaryotes, capable of growth

at pH 0 (Edwards et al., 2000). Many other acidophilic

prokaryotes detected within Iron Mountain are also obli-

gate or facultative heterotrophs, such as “Ferrimicrobium

Fm. acidiphilum” and Sulfobacillus spp. (Bond et al.,

2000). In contrast, the most well studied of all iron-/sul-

fur-oxidizing acidophiles, At. ferrooxidans, was only found

in cooler (20–30 °C) and less acidic (pH 2–3) waters in

the mine that were peripheral to the ore body (Edwards

et al., 1999). The major primary producers (carbon-fix-

ers) within Iron Mountain therefore appear to be bacteria

(Leptospirillum spp.) that use energy from oxidizing fer-

rous iron, derived from residual pyrite within the mine,

and one of these (‘L. ferrodiazotrophum’) appears also to

be the major nitrogen fixer in this ecosystem. The princi-

pal prokaryotes involved in mineral dissolution within

this abandoned mine are the archaeon ‘F. acidarmanus’

and ‘Group II’ Leptospirillum. These observations corre-

late well with the recorded characteristics of both L. ferr-

iphilum and Ferroplasma spp. in being extreme

acidophiles, capable of growth at pH < 1, that grow in

moderately thermal (30–50 °C) environments (Coram &

Rawlings, 2002; Dopson et al., 2004), these being condi-

tions that prevail within the Richmond mine. Elegant

work carried out by the Banfield group on the genomics

(Tyson et al., 2004) and proteomics (Ram et al., 2005) of

biofilm communities within Iron Mountain has provided

revealing insights into the nature of extremophile com-

munities and interactions (Denef et al., 2010). One of the

particularly interesting findings revealed by community

genomic analysis was the presence of novel archaeal lin-

eages that were identified as very small (< 0.45 lm diam-

eter) pleomorphic cells within biofilms and designated as

ARMAN (Archaeal Richmond Mine Acidophilic Nanoor-

ganisms; Baker et al., 2006).

The Frasissi cave system, Italy

The Grotta Grande del Vento-Grotta del Flume (Fras-

issi) cave complex, Italy, is an example of a sulfidic

cave formed when sulfide-rich groundwaters contact

oxygen within subterranean carbonate rock strata. Sulfu-

ric acid formed by (microbiological) oxidation of the

sulfide results in dissolution of the carbonate minerals,

a process that can lead to the development of extensive

cave systems, such as the Lechugilla cave in New Mex-

ico, which contains an estimated 201 km of passages.

Although the acidity generated is often neutralized by

the carbonate minerals, particularly in the stream waters

on the cave floors, areas of extreme acidity may

develop. Such is the case with the Frasissi cave, where

viscous biofilms of extremely low pH (0–1) have been

found (Vlasceanu et al., 2000; Macalady et al., 2007,

2008). Molecular analysis of these low pH pendulous

biofilms (‘snotites’) has revealed that they have very

limited biodiversity and are dominated by bacteria

related to the mesophilic sulfur-oxidizing autotroph

Acidithiobacillus thiooxidans. Other bacteria identified

include bacteria related to Gram-positive Sulfobacillus

and Acidimicrobium spp., and archaea related to Ferropl-

asma (Macalady et al., 2007). Interestingly, although

Sulfobacillus spp., like Acidithiobacillus spp., can use

reduced sulfur species as electron donors, those Acidi-

microbium and Ferroplasma spp. that have been charac-

terized are known to oxidize ferrous iron but not

sulfur, raising the probability that the latter two species

in Frasissi (where ferrous iron is not a significant source

of energy) represent new phenotypes. Other microorgan-

isms detected within the slime growths include protists

and filamentous fungi, as well as bacteria of the pro-

posed ‘TM6’ lineage.

Drei Kronen und Ehrt pyrite mine, Harz

Mountains, Germany

An abandoned pyrite mine in central Germany was

reported by Ziegler et al. (2009) to contain small micro-

bial stalactite-like structures attached to the mine ceiling.

Molecular analysis of the microbial growths showed that

they were dominated by Leptospirillum- and ‘Ferrovum’

(Fv.)-like bacteria (both autotrophic acidophiles that

appear to use only ferrous iron as electron donor) with

apparently smaller numbers of heterotrophic (Acidiphili-

um-like) bacteria. More recently, archaeal sequences clo-

sely related to the ARMAN group detected in Iron

Mountain and also to uncultivated members of the Ther-

moplasmatales have been detected in the biofilm commu-

nities in this abandoned mine (S. Ziegler, pers.

commun.).


4 D.B. Johnson

Low-temperature (10 °C), extremelyacidic subterranean environments: theabandoned Cae Coch and Mynydd Parysmines, North Wales

The geobiology of two other abandoned sulfide ore

mines, both based in north Wales, has been extensively

studied over the past 30 years. Both mines contrast with

the Richmond mine in being at constant low tempera-

ture (8–9 °C) and have water bodies of pH (2–2.5) sim-

ilar to that reported for the Drei Kronen und Ehrt

pyrite mine (Table 1). A major difference between these

two abandoned mines is, however, that one (the Cae

Coch ‘sulfur’ mine) is mostly dry, with water accumula-

tion being mostly restricted to several small pools and a

single drainage stream, while the other (Mynydd Parys

copper mine) is a flooded deep mine that has been

partly dewatered. These contrasting conditions have

facilitated the development of very different indigenous

microbial communities.

Cae Coch ‘sulfur’ mine

Cae Coch (which translates from Welsh as ‘red field’) is a

small pyrite mine located in an elevated position above

the Conwy Valley in northwest Wales (Johnson et al.,

1979). It was actively mined in the 19th century and

again between 1914 and 1918, where the fine-grade pyrite

ore was sourced for sulfur in the manufacture of explo-

sives. Although there was some further exploratory work

at the mine during 1940, no ore was extracted, and since

that time, the site has been abandoned.

The pyrite ore body at Cae Coch is about 1–1.5 m

thick and has been intruded, at an angle of about 30°,between flaggy black bituminous shales and an altered

dolerite sill (Ball & Bland, 1985). Although much of the

ore body has been removed, the mine ceiling and the

large pillars supporting the ceiling structure are composed

of pyrite. Rainwater (c. 1.5 m year�1) percolating through

the thin surface soil and the rock overburden flows

through cracks and fissures in the residual pyrite intru-

sion where it emerges as highly acidic water droplets that

are red colored, because of the presence of soluble (ferric)

iron (Fig. 1, and Supporting information, Fig. S1a).

Water issuing from larger fissures causes occasional pools

to form deeper (~200 m) within the mine (Fig. 2) and is

the source of a stream that runs through and drains the

mine. Wherever water collects within the Cae Coch mine,

it is colonized by acidophilic microbial communities that

grow as a variety of macroscopic structures, ranging from

biofilms (c. 0.5–2 cm thick) on inclined wall surfaces, to

microbial stalactites (some over 1 m long; Fig. 2), and to

‘streamer’ growths that completely fill the drainage

stream. The volume of the streamer and slimes growths

within Cae Coch has been estimated to exceed 100 m3,

making this abandoned mine the site of the largest accu-

mulation of macroscopic acidophilic biomass yet

described.

Research on the microbiology of the Cae Coch mine in

the 1970s (e.g. Johnson et al., 1979) was limited by the

lack of appropriate techniques then available to cultivate

acidophilic microorganisms and also predated the advent

of molecular tools for elucidating microbial community

composition. However, the site has been revisited and

2

A

3

3+

Fe +

“Fv. myxofaciens” (At. ferrivorans)

Drainage stream/acid streamers

Roof water droplets

At. ferrivorans (L. ferrooxidans)

DOC

cidiphilium spp.

CO2

CO2

DOC

Fe +

Fissured pyrite ore

FeS2 + 8H2O + 14Fe

2SO42- + 16H+ + 15Fe2+

Surface water flow

Surface soil/vegetation

Fig. 1. Schematic of the biogeochemistry of

the abandoned Cae Coch mine, with the

dominant bacteria involved in transformations

of iron and carbon highlighted (modified from

Kimura et al., 2011).



extensively researched in the past decade using new and

improved cultivation approaches (e.g. the use of selective

and efficient solid media; Johnson & Hallberg, 2007) in

tandem with PCR-based techniques [terminal restriction

enzyme fragment length polymorphism (T-RFLP) and

clone libraries] and fluorescent in situ hybridization

(FISH) (Kimura et al., 2011). From these studies, it has

become apparent that there is a clear differentiation

between the microbial communities involved in the oxi-

dative dissolution of the residual pyrite in the ceiling of

Cae Coch (which are responsible for generating the

acidic, metal-rich conditions) and those which form the

macroscopic streamer/slime structures in the pools and

stream on the mine floor.

Accessing the interstitial water in the pyritic mine ceil-

ing is not feasible, given the precarious structural state of

the mine. However, the red-colored (pH 2.0) water drop-

lets that were found directly on the ceiling lower surface

and on the thin inorganic stalactites that developed from

these iron-rich liquors were considered to be representa-

tive of this interstitial water from which they originated.

Both cultivation-dependent and cultivation-independent

analyses supported the conclusion that the microbial

community of the roof/ceiling droplets (the primary min-

eral degraders) was dominated by two species of iron-oxi-

dizing acidophilic bacteria: Acidithiobacillus ferrivorans

(Hallberg et al., 2010) accounting for ~95% of the bacte-

ria present and L. ferrooxidans the remaining 5% (Kimura

et al., 2011). No archaea were detected by FISH, and ar-

chaeal 16S rRNA genes could not be amplified from

water droplet samples. Like At. ferrooxidans, At. ferrivo-

rans is an obligately autotrophic, facultatively anaerobic

acidophile that uses ferrous iron and reduced sulfur as

electron donors. However, unlike At. ferrooxidans, At.

ferrivorans is psychrotolerant and grows at temperatures

as low as 4 °C, a trait that probably underpins its success-

ful colonization and exploitation of the pyrite ore body

in the low-temperature Cae Coch mine.

The streamer and slime microbial communities in Cae

Coch were shown to be far more biodiverse than the

mineral-degrading microbial communities and also varied

between locations (e.g. from pool to pool, and in differ-

ent stratified layers within the drainage stream). The

dominant prokaryotes (up to 94% by FISH analysis) pres-

ent were mostly Betaproteobacteria, and in the majority of

cases, this bacterial class was represented by a single spe-

cies (‘Ferrovum myxofaciens’) although bacteria closely

related to the neutrophile Gallionella ferruginea were also

abundant in one microbial stalactite analyzed. ‘Fv. myxo-

faciens’ appears, like Leptospirillum spp., to use only fer-

rous iron as an electron donor, and like At. ferrivorans, it

is psychrotolerant (D.B. Johnson et al., unpublished

data). It is particularly adept at producing EPS, causing

cultures to form streamer-like growths in liquid media.

Smaller numbers of Alphaproteobacteria (which included

Acidiphilium spp. and a novel Sphingomonas isolate),

Gammaproteobacteria (At. ferrivorans and bacteria related

to the unclassified species ‘WJ2’), Acidimicrobiaceae (e.g.

Ferrimicrobium-like bacteria), L. ferrooxidans, and Firmi-

cutes (distantly related to Sulfobacillus spp.) were also

encountered in the macroscopic growths although some

of these appeared to be site specific. Archaea were again

not detected by FISH in streamer/slime samples, although

archaeal 16S rRNA genes were occasionally amplified

using DNA extracted from streamer growths. In addition,

archaea were detected by FISH and by gene amplification

in one pool located deep within the mine that had a

much greater concentration of dissolved solutes (and

lower pH) than the other water bodies sampled (Table 1).

Screening of archaeal clone libraries obtained from this

pool and a streamer sample revealed the presence of only

one operational taxonomic unit, corresponding to a eury-

archaeote most closely related (92% 16S rRNA gene

sequence similarity) to a clone found in Iron Mountain

and more distantly (89% 6S rRNA gene sequence similar-

ity) to Ferroplasma, Thermoplasma, and Picrophilus, all

three of which are heterotrophic acidophiles.

Fig. 2. Acid waters and microbial growth in the abandoned Cae

Coch ‘sulfur’ mine: (top) an acidic iron-rich pool and macroscopic

streamers; (bottom) microbial stalactites on a semi-vertical mine wall

face.


6 D.B. Johnson

The collated geomicrobial data from the Cae Coch

mine have facilitated a re-appraisal of the diversity of

microbial communities in highly acidic, low-temperature

subterranean environments. In the complete absence of

light, all primary production is necessarily mediated by

chemoautotrophic bacteria. The residual, partly excavated,

and exposed pyrite (FeS2) ore body is the energy source

that fuels carbon dioxide fixation. On the basis of the

known physiological characteristics of the primary pro-

ducers identified, it appears that ferrous iron rather than

reduced sulfur is acting as the major energy resource in

this environment. Two of the three characterized primary

producers (‘Fv. myxofaciens’ and L. ferrooxidans) are

known to use ferrous iron as sole electron donor, and the

closest cultivated relative of the Gallionella-like clone also

oxidizes iron, but not reduced sulfur. In contrast, At. ferr-

ivorans, implicated as the major microorganism involved

in mineral dissolution in Cae Coch, can oxidize both fer-

rous iron and reduced sulfur, but like At. ferrooxidans,

strains of this acidophile oxidize iron preferentially, utiliz-

ing sulfur compounds only when the ferrous iron is

depleted. Concentrations of ferrous iron found in the

roof droplets (~570 mg L�1) and in the stream and pool

waters (~200–12 000 mg L�1) suggest that this energy

source does not become depleted in Cae Coch. There are

several mechanisms by which ferrous iron may be gener-

ated in acidic environments, including (1) as a result of

attack by iron (III) on pyrite [Eqn. (1)]; (2) from the

reaction between iron (III) iron and thiosulfate formed in

reaction (1) [Eqn. (2)], and (3) by microbial dissimila-

tory reduction, for example, by Acidiphilium spp. [Eqn.

(3)], where ‘e�’ represents a generic electron donor):

FeS2 þ 6Fe3þ þ 3H2O ! 7Fe2þ þ S2O2�3 þ 6Hþ (1)

S2O2�3 þ 8Fe3þ þ 5H2O ! 8Fe2þ þ 2SO2�

4 þ 10Hþ (2)

Fe3þ þ e� ! Fe2þ (3)

It is interesting in this respect to note that no acidophilic

autotrophic bacteria that oxidize reduced sulfur but not

ferrous iron (e.g. At. thiooxidans) were detected within

the Cae Coch ecosystem.

Much of the metabolizable organic carbon in Cae Coch

is thought to originate from the iron-oxidizing chemo-

autotrophs, as other sources (bat guano, pit props, etc.)

are relatively rare and mostly remote from the water

bodies in the mine. Interestingly, the largest concentra-

tions of DOC (24 mg L�1) were found in the ceiling

droplets where the microbial community was restricted to

autotrophic acidophiles (which was probably due to the

particularly harsh chemical environment (31 g L�1 of

ferric iron) of these droplets; acidophilic heterotrophic

bacteria tend to be more sensitive to soluble ferric iron

than autotrophic species). Much smaller DOC concentra-

tions (3–12 mg L�1) were recorded where heterotrophic

acidophiles were also part of the microbial communities

(Kimura et al., 2011).

Mynydd Parys copper mine

Mynydd Parys (‘Parys mountain’), located in the north-

east corner of Anglesey in north Wales, is an ancient site

of copper mining, dating back to the Bronze Age. In the

18th century, Mynydd Parys was the world’s largest cop-

per mine, but its importance declined in the 19th cen-

tury, with extractive mining ending at around 1880. The

site was first excavated as an underground mine, and later

as an opencast operation, and extensive underground

mine workings are still in place below the two opencast

voids. The mineralization at Mynydd Parys is associated

with a late Ordivician submarine volcanic event involving

the extrusion of silicic lavas and pyroclastics, in an

ancient ‘black smoker’, with pyrite, chalcopyrite (CuFeS2),

sphalerite (ZnS), and galena (PbS) as the major sulfide

minerals present.

Copper continued to be produced at Mynydd Parys

long after active mining came to an end. This was essen-

tially via in situ bioleaching of the residual exposed ore

body, in which the deep mine workings were allowed to

flood and periodically drained to facilitate extraction of

solubilized copper by cementation. This continued until

about 1950, when the site was abandoned and the valves

used to drain the mine were closed, resulting in the

buildup of an underground lake within the hollowed-out

mine, which remained undisturbed for over 50 years.

Concern about the state of the concrete wall in which the

valves were embedded and the risk that corrosion and

catastrophic failure of this dam would cause flooding and

severe pollution of a nearby coastal town resulted in the

mine being actively dewatered (Coupland & Johnson,

2004). Between April and July in 2003, c. 274 000 m3 of

acidic, metal-rich water (Table 2) was pumped out of the

underground mine and allowed to flow, untreated, into

the Irish Sea. Once the underground lake level had fallen

sufficiently, the concrete dam was breached and the over-

flow from the now more shallow underground lake was

allowed to flow through it unimpeded.

The dewatering event presented an opportunity not

only to record the biodiversity of the low-temperature,

extremely acidic underground lake water, but also to

sample newly accessible sites within the drained area of

the mine. Using a combination of cultivation-dependent

and cultivation-independent techniques, Coupland &

Johnson (2004) found that the subterranean water con-

tained ~5 9 105 cells mL�1 (corresponding to about

27.5 kg of microbial biomass in the total drained water),



comprising autotrophic and heterotrophic acidophilic

bacteria and archaea. T-RFLP analysis of bacterial 16S

rRNA genes indicated that the dominant bacteria present

were iron-oxidizing acidithiobacilli. Whether At. ferrooxi-

dans or At. ferrivorans was not ascertained, but given the

low temperature of the subterranean lake the latter, is

more likely to have been more dominant. L. ferrooxidans,

Fm. acidiphilum (a heterotrophic iron-oxidizer), and bac-

teria related to Gallionella, as well as heterotrophic acido-

philes (Acidiphilium, Acidobacterium and Acidisphaera),

were also detected in T-RFLP profiles and, in some cases,

isolated on solid media. The predominance of iron oxi-

dizers over sulfur-oxidizing bacteria suggested, as with

Cae Coch, a primary role for ferrous iron as an energy

source in the subterranean Parys lake. No archaea were

isolated from the drained water, but analysis of archaeal

clone libraries revealed a very different scenario to Cae

Coch, with ~95% of clones being related to methanogenic

archaea and only ~5% to Thermoplasma/Ferroplasma-like

archaea (Coupland, 2005).

The newly accessible depths within Mynydd Parys were

explored, as soon as deemed safe, by a group of speleolo-

gists (the Parys Underground Group). Among the interest-

ing features they noted was the presence of large, two-

dimensional gelatinous materials, which they referred to

as ‘drapes’ hanging from previously inundated pit props

(Fig. S1c). Shortly after the mine was dewatered, micro-

bial stalactites and streamer-like materials were found to

be growing from the adit roofs and in newly formed

pools (Fig. S1b and d). T-RFLP analysis of bacterial 16S

rRNA genes obtained from the ‘drapes’ showed that they

were very different in species composition to the stream-

ers (Fig. 3) and also to the drained water. Bacteria related

to heterotrophic, rather than autotrophic, acidophiles

were identified in clone libraries of the ‘drapes’ (Table 3)

and included two species (Ferrimicrobium and Acidimicro-

bium) that are known to both oxidize and reduce iron

and another (Acidobacterium) known to catalyze the

dissimilatory reduction of iron (III) (Coupland & John-

son, 2008). In contrast, T-RFLP analysis of archaeal genes

showed that there was considerable similarity between the

archaeal communities in the ‘drapes’ and streamers

within Mynydd Parys (Fig. 3), and the relatively large

number of restriction digest fragments indicated a signifi-

cant diversity of archaea in these macroscopic growths.

As with the subterranean lake water, clone library analysis

indicated that most of the archaea present were related to

methanogens (Coupland, 2005).

The data obtained on the microbial communities in

the subterranean lake at Mynydd Parys revealed a con-

trasting picture to that found within the ‘dry’ Cae Coch

mine. A schematic summary of the major biogeochemical

transformations considered to be occurring before the

dewatering event (and presumably still occurring in the

remaining underground lake) is shown in Fig. 4. Limited

oxygen ingress (by diffusion) facilitates ferrous iron oxi-

dation by bacteria such as Leptospirillum, while reduction

of the ferric iron produced by reaction with sulfide miner-

als [as Eqn. (1)] or by dissimilatory reduction catalyzed by

heterotrophic (e.g. Acidobacterium spp.) or autotrophic

Table 2. Geochemical composition of the underground lake water

within the Mynydd Parys mine removed during its partial drainage in

2003

Analyte

Mean concentration in

underground lake (mg L�1)

Total amount in

drained water (tonnes)

pH 2.35

Iron* 600 180

Copper 30 7.5

Zinc 50 14.8

Aluminum 60 15.3

Manganese 10 3.1

*~90% Fe2+.

61 75 88 93 9417

819

4

204

206

299

361

367

440

451

525

530

571

10

20

30

40

50

60

70

80

Drapes

35 fm streamer

45 fm streamer

65 71 73 9712

513

516

717

819

023

428

028

328

531

131

443

243

550

451

051

452

153

153

354

457

9

Drapes

35 fm streamer

45 fm streamer

0

5

10

15

20

25

30

35

40

0

45

Drapes

35 fm streamer

45 fm streamer

Rel

ativ

e p

eak

area

(%

)

T-RF length (nt)

Fig. 3. T-RFLP profiles of 16S rRNA genes of macroscopic acidophile

communities in the dewatered zone of the Mynydd Parys mine: (top)

bacteria, digested with CfoI; (bottom) archaea, digested with MspI.

The streamer samples were taken from 35 and 45 fathoms (fm;

equivalent to 64–82 m) below the land surface.


8 D.B. Johnson

(e.g. Acidithiobacillus spp.) bacteria [Eqn. (3)] results in

the dominant form of iron in the subterranean lake being

ferrous. The submerged pit props appear to be a signifi-

cant carbon source for heterotrophic bacteria in the

Mynydd Parys lake, more so because of the limited activi-

ties of the iron-oxidizing autotrophs because of restricted

oxygen availability. Elsewhere, mine timber has been

reported to support methane formation in enrichment cul-

tures (Kruger et al., 2008). Some components of the DOC

(and possibly hydrogen) are also assumed to be used by

methanogens in the subterranean lake. Elevated concentra-

tions of soluble sulfate (~2 g L�1) and the prevailing

anaerobic conditions (indicated by the presence of metha-

nogens and confirmed by measurements of dissolved oxy-

gen in the drained water) would appear to be conducive

to SRB. However, such bacteria were not detected in the

subterranean lake in Mynydd Parys, although acidophilic

SRB have previously been detected in, and isolated from,

mine environments (e.g. Rowe et al., 2007). One possible

reason for this is that suitable electron donors for SRB

were not present within Mynydd Parys. Acidophilic SRB

have been observed to be associated with phototrophic ac-

idophiles (i.e. in surface environments) and have also been

shown to use organic substrates derived from these

eukaryotic algae as electron donors (e.g. Nancucheo &

Johnson, 2011).

Unanswered questions, newopportunities, and future directions

There remain many unanswered questions relating to the

acidophilic microbiology of subsurface environments, as

Table 3. Identities and relative abundances of clones identified in a 16S rRNA gene library of DNA extracted from macroscopic ‘drapes’ within

the freshly dewatered zone of the Mynydd Parys underground mine

Clone code No. of clones* T-RF (nt)† Nearest relatives (GenBank Accession No.) Identity (%)

KCDC12 3 361 Actinomycete clone RCP1-56 (AF523907) 98.7

‘Ferrimicrobium acidiphilum’ (AF251436) 90.9

KCDC13 8 93 Acidobacteriaceae clone C47.14PG (AF431496) 94.9

Acidobacterium capsulatum (D26171) 90.4

KCDC26 4 367 Actinomycete clone RCP1-37 (AF523912) 94.6

Acidimicrobium ferrooxidans (U75647) 87.6

KCDC27 6 525 DGGE band C4 (AJ517308) 98.6

Actinobacteria clone AKYG619 (AY921831) 89.2

KCDC28 7 194 Acidobacteriaceae clone RCP2-4 (AF523897) 99.8

Acidobacteriaceae isolate WJ7 (AY096034) 94.2

KCDC34 3 530 Acidobacteriaceae clone WS089 (AY174195) 89.7

KCDC35 1 204 Acidisphaera rubrifaciens (D86512) 95.1

KCDC37 8 61 c-proteobacterium WJ2 (AY096032) 99.0

*Of 40 clones sequenced.

†Amplified 16S rRNA genes digested with CfoI.

Fig. 4. Schematic of the biogeochemistry of the underground lake at the abandoned Mynydd Parys mine. Ferric iron formed by microbial

oxidation of ferrous iron in the lake surface waters is reduced back to ferrous by reactions involving residual sulfide minerals on the shaft and

adit walls, and by heterotrophic and autotrophic acidophiles that use ferric iron as a terminal electron acceptor. DOC originated from residual

wooden mine support structures (and from autotrophic iron- and sulfur-oxidizing bacteria) is used as electron donor by heterotrophic bacteria

and methanogenic archaea.



well as indications that these sites contain many species

of bacteria and archaea that are still to be described. The

importance of hydrogen as an electron donor for acido-

philes in the subsurface (and elsewhere) remains entirely

unknown, although the presence of hydrogenase genes in

the genomes of many acidophiles that have been

sequenced, and the demonstration that at least some

known species can grow autotrophically on hydrogen in

acidic liquors, suggests that hydrogen can be an impor-

tant electron donor for acidophiles.

Analysis of the biogeochemistry of the Cae Coch mine

has revealed key roles for bacteria (At. ferrivorans and

‘Fv. myxofaciens’) that have only recently been described.

Presumptive acidophilic methanogens are present in

macroscopic growths and the subterranean lake within

Mynydd Parys. Although methanogens have previously

been detected in extremely acidic environments (e.g.

Sanz et al., 2011), strains that grow optimally at pH < 3

have not been isolated or characterized. Perhaps, most

intriguing of all are the (presumptive) acidophilic nano-

archaea that have been found in Iron Mountain

and more recently in the Drei Kronen und Ehrt pyrite

mine.

The Iberian Pyrite Belt Subsurface Life Detection project

is timetabled to commence in late 2011. This is planned

to drill into the earth’s crust to a depth of 1000 m

below sea level within the Iberian Pyrite Belt in southern

Spain, the site of the most famous of all extremely acidic

water bodies (the Rio Tinto). This project will provide

answers to questions relating to how deep acidophilic

microbial communities can extend into the lithosphere,

how their diversities change with depth, and how acido-

philes live and interact with each other in the deep sub-

surface.

Acknowledgements

The author wishes to express his immense gratitude to

Dr David Jenkins, a geochemist, caving and mining

enthusiast, and former mentor and colleague at Bangor

University, for not only opening his eyes to life in the

acidic subsurface many years ago, but also in his contin-

ued exploration of the depths of Mynydd Parys and pro-

viding some of the microbial materials described in

this manuscript. Thanks are also due to Drs Sakurako

Kimura and Christopher Bryan (Cae Coch), Kris

Coupland (Mynydd Parys), and Kevin Hallberg (both

sites).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Geochemical and microbial features within (a)

the abandoned Cae Coch ‘sulfur’, and (b–d) the dewa-

tered-zone of the Mynydd Parys mine: (a) inorganic

(ferric iron) stalactites forming on a pyritic mine ceiling;

(b) an acidic, ferrous iron-rich pool; (c) microbial

‘drapes’ at 45 fathoms below the land surface; (d) micro-

bial stalactites hanging from the mine ceiling. Images (b–d) are courtesy of Dr David Jenkins.

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.



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