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FEMS Microbiology Ecology 27 (1998) 307^317


Biodiversity and ecology of acidophilic microorganisms

D. Barrie Johnson *
School of Biological Sciences, University of Wales, Bangor LL57 2UW, UK

Received 7 April 1998; received in revised form 21 July 1998; accepted 23 July 1998


Microbial life in extremely low pH ( 6 3) natural and man-made environments may be considerably diverse. Prokaryotic
acidophiles (eubacteria and archaea) have been the focus of much of the research activity in this area, primarily because of the
importance of these microorganisms in biotechnology (predominantly the commercial biological processing of metal ores) and
in environmental pollution (genesis of `acid mine drainage'); however, obligately acidophilic eukaryotes (fungi, yeasts, algae
and protozoa) are also known, and may form stable microbial communities with prokaryotes, particularly in lower
temperature ( 6 35³C) environments. Primary production in acidophilic environments is mediated by chemolitho-autotrophic
prokaryotes (iron and sulfur oxidisers), and may be supplemented by phototrophic acidophiles (predominantly eukaryotic
microalgae) in illuminated sites. The most thermophilic acidophiles are archaea (Crenarchaeota) whilst in moderately thermal
(40^60³C) acidic environments archaea (Euryarchaeota) and bacteria (mostly Gram-positives) may co-exist. Lower
temperature (mesophilic) extremely acidic environments tend to be dominated by Gram-negative bacteria, and there is recent
evidence that mineral oxidation may be accelerated by acidophilic bacteria at very low (ca. 0³C) environments. Whilst most
acidophiles have conventionally been considered to be obligately aerobic, there is increasing evidence that many isolates are
facultative anaerobes, and are able to couple the oxidation of organic or inorganic electron donors to the reduction of ferric
iron. A variety of interactions have been demonstrated to occur between acidophilic microorganisms, as in other environments ;
these include competition, predation, mutualism and synergy. Mixed cultures of acidophiles are frequently more robust and
efficient (e.g. in oxidising sulfide minerals) than corresponding pure cultures. In view of the continuing expansion of microbial
mineral processing (`biomining') as a cost-effective and environmentally sensitive method of metal extraction, and the ongoing
concern of pollution from abandoned mine sites, acidophilic microbiology will continue to be of considerable research interest
well into the new millennium. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
All rights reserved.

Keywords : Acidophilic microorganism; Thermophile; Microbial interaction

1. Introduction signi¢cantly over the past 25 years. There are many

reasons for this: some fundamental (e.g. the notion
Interest in the biodiversity of microorganisms that these environments were far more widespread
which inhabit `extreme environments' has increased during the early life of our planet and that organisms
isolated from these sites are representative of archaic
* Tel.: +44 (1248) 382358; Fax: +44 (1248) 370731; life forms), and some more applied (e.g. the increas-
E-mail: ing use of extremophiles as living organisms or as

0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 7 9 - 8

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308 D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317

sources of enzymes and other cell products in a vari- ity. These include nitri¢cation, and the formation
ety of industrial and biotechnological operations). and accumulation of organic acids either during fer-
Environments which are characterised by high levels mentation or as products of aerobic metabolism.
of acidity fall into this general category. The exact Most pertinent, however, to the genesis of extremely
de¢nition of an `extremely acidic environment' is acidic environments are the microbial dissimilatory
open to some conjecture, for example the question oxidation of elemental sulfur, reduced sulfur com-
of whether this should be de¢ned merely in terms of pounds (RSCs), and ferrous iron. Elemental sulfur
measured pH or of titratable acidity. The description may occur in geothermal areas (e.g. around the mar-
of an `extreme acidophile', however, is more gener- gins of fumaroles) where it forms by the condensa-
ally agreed upon, as a microorganism which has a tion of sulfur dioxide and hydrogen sul¢de
pH optimum for growth at (or below) pH 3.0 [1]. (SO2 +2H2 SC2H2 O+3S³). Oxidation of sulfur by
This de¢nition excludes many fungi and yeasts autotrophic and heterotrophic microorganisms gen-
which, although often tolerant of extreme acidity, erates sulfuric acid (S³+H2 O+1.5O2 CH2 SO4 )
have pH optima nearer to neutrality. This brief re- which, if not neutralised by carbonates or other basic
view article will focus on current knowledge of the minerals present, can result in a dramatic lowering of
biodiversity of extreme acidophiles, and on how pH within microsites or on the macro scale. Of
these microrganisms interact in situ. More detailed greater environmental signi¢cance, however, is the
reviews of other aspects of acidophilic microbiology generation of acidity which results from the micro-
may be found in articles by Norris and Johnson [1], bial oxidation of sul¢de minerals. Many metals occur
a general overview of acidophilic microbiology ; Nor- as sul¢des [7]; indeed, sul¢des are the major minera-
ris and Ingledew [2], which focuses on microbial logical form of many commercially important met-
adaptation to extremely acidic environments; Lane als, such as copper, lead and zinc. Iron sul¢des (most
et al. [3], which examines evolutionary relationships notably pyrite) are the most abundant sul¢de miner-
between acidophilic iron- and sulfur-oxidising bacte- als. In the past, pyrite has been mined (for its sulfur,
ria; Blake et al. [4], which describes the respiratory rather than for its iron content) but this is no longer
components of iron-oxidising acidophiles; and commercially viable. However, iron sul¢des are
Pronk and Johnson [5], which considers the role of often associated with other metal sul¢des in ore de-
acidophilic bacteria in the dissimilatory oxido-reduc- posits, and as such are inadvertently processed
tion of iron. A recent text, edited by Rawlings [6], during the mining operation, ending up as waste
includes descriptions of the physiologies of acido- materials (in mineral tailings etc.). Pyrite and other
philic bacteria and archaea that are involved in com- iron sul¢des are also present in coal deposits (range:
mercial ore processing (`biomining'), as well as de- 6 1 to s 20%) and, inevitably, in coal spoils.
tailed accounts of the application of the process. The mechanisms involved in the oxidation of py-
rite have been subject to considerable debate (e.g.
[8,9]). Current consensus is that ferric iron acts as
2. Origins and characteristics of extremely acidic the major oxidant of the mineral, as:
FeS2 ‡ 6Fe…H2 O†6 ‡ 3H2 O!Fe2‡ ‡ S2 O23
3 ‡
Extremely acidic environments may be formed by
processes that are entirely natural. However, anthro- 6Fe…H2 O†2‡ ‡
6 ‡ 6H :
pogenic in£uences (both direct and indirect) have
become increasingly important in creating such envi- The fate of the thiosulfate formed depends on en-
ronments, particularly since the onset of the indus- vironmental pH; in circum-neutral environments this
trial revolution. Indeed, the majority of extremely reduced sulfur compound (RSC) is chemically stable,
acidic sites now in existence worldwide have their but in acidic liquors it hydrolyses to form a variety
origin in one particular human activity, the mining of polysul¢des, as well as elemental sulfur and sul-
of metals and coal. fate [8]. Ferrous iron and RSCs are potential energy
A variety of microbial activities generate net acid- sources for some acidophilic chemolithotrophic pro-

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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317 309

karyotes (described below). The regeneration of the of some of these prokaryotes, most notably the iron/
ferric iron oxidant may be brought about biologi- sulfur-oxidising bacterium Thiobacillus ferrooxidans
cally or abiotically; however, oxygen is required in [10]. Most iron- and sulfur-oxidising acidophiles are
both cases, so that the continued oxidation of pyrite regarded as autotrophic, though the ability to assim-
requires the provision of both air and water. This ilate organic carbon has been demonstrated with
criterion is met when coal spoils and mineral wastes some of these (e.g. utilisation of formic acid by T.
are stored on the land surface, and when water ac- ferrooxidans [11]). Other prokaryotes which catalyse
cumulates in exposed deep mine shafts following the the dissimilatory oxidation of iron and/or RSCs are
cessation of active mining. either mixotrophic (i.e. may assimilate organic and
One other important physico-chemical feature of inorganic carbon) or else are obligately heterotro-
extremely acidic environments is that concentrations phic.
of soluble metals tend to be much greater than in In those extremely acidic environments that are
neighbouring areas of higher pH. While the solubil- illuminated, primary production may also be medi-
ities of metal oxyanions (such as molybdate) tend to ated by phototrophic acidophiles. The majority of
be lower in acidic than in neutral solutions, those of these are eukaryotic microalgae, and include ¢lamen-
cationic metals (such as aluminium and many heavy tous and unicellular forms, and diatoms [12,13].
metals) are generally much greater. The types and Mesophilic acidophilic phototrophs include Euglena
concentrations of heavy metals present in any partic- spp., Chlorella spp., Chlamydomonas acidophila, Ulo-
ular extremely acidic environment are much dictated thrix zonata and Klebsormidium £uitans. The unicel-
by the local geochemistry; metals may originate di- lular rhodophyte Galdieria sulphuraria (formerly Cy-
rectly from the oxidation of sul¢de minerals (various anidium caldarium) has been isolated from
chalcophilic metals) or from the accelerated mineral geothermal acidic springs and streams in Yellow-
weathering which occurs under conditions of extreme stone National Park and elsewhere [14]. This moder-
acidity (e.g. aluminium from the weathering of clay ate thermophile may grow as a heterotroph in the
minerals). Elevated concentrations of soluble metal- absence of light (as may Euglena spp.) and has been
loid elements may also occur in extremely acidic en- reported to grow at pH values around zero [15].
vironments, of which the most important (from the Heterotrophic microorganisms may readily be iso-
point of view of ecotoxicology) is arsenic, which oc- lated from most extremely acidic environments.
curs in several sul¢de minerals such as arsenopyrite Many are adept scavengers and rely to a greater or
(FeAsS) and realgar (AsS). lesser extent on carbon originating as leakage or lysis
products from chemolithotrophic acidophiles. Obli-
gately acidophilic heterotrophs include archaea, bac-
3. Microbial diversity in extremely acidic teria, fungi, yeasts and protozoa. Some prokaryotic
environments acidophilic heterotrophs have a direct role in the dis-
similatory oxido-reduction of iron [5]. These include
3.1. Autotrophic and heterotrophic life-styles the iron-oxidiser `Ferromicrobium acidophilus' [16]
which appears to use the energy from iron-oxidation
Most extremely acidic environments contain rela- to support growth, and various Acidiphilium-like iso-
tively low concentrations ( 6 20 mg l31 ) of dissolved lates which can use ferric iron as terminal electron
organic carbon, and may therefore be classed as oli- acceptor (see below). Many acidophilic archaea (Ta-
gotrophic. Primary production in sites which do not ble 1) are obligate heterotrophs, including Sulfolobus
receive sunlight (e.g. abandoned deep mines) is based acidocaldarius; early reports of this archaean being a
exclusively on chemolitho-autotrophy, and is inexor- facultative chemolithotroph are now thought to be
ably linked to the oxidation of ferrous iron and re- due to the inadvertent use of mixed cultures of Sf.
duced sulfur compounds. Chemolithotrophic acido- acidocaldarius and another extreme thermophile (pos-
philes have been, and continue to be, the main focus sibly Sulfolobus metallicus [17]). The two character-
of research in this area of microbiology, and much is ised species of the moderately thermophilic heterotro-
known of the detailed physiology and biochemistry phic archaean Picrophilus have the lowest recorded

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310 D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317

Table 1
Acidophilic prokaryotic microorganisms
Organism G+C (mol %)/phylogenetic a¤liation Comments
Iron-oxidising prokaryotes
(a) Mesophiles
Thiobacillus ferrooxidans 58^59/L-/Q-Proteobacteria Facultative anaerobe
`T. ferrooxidans' strain m-1 65/Q-Proteobacteria S³ not oxidised
T. prosperus 63^64/Q-Proteobacteria Halotolerant
`Leptospirillum ferrooxidans' 51^56/Nitrospira phylum Fe2‡ sole e3 donor
`Ferromicrobium acidophilus' 51^55/Actinobacteria Heterotrophic

(b) Moderate thermophiles

Sulfobacillus acidophilus 55^57/Gram+ve division May grow as autotrophs, mixotrophs of heterotrophs
S. thermosul¢dooxidans 48^50/Gram+ve division May grow as autotrophs, mixotrophs of heterotrophs
Acidimicrobium ferrooxidans 67^68.5/Actinobacteria May grow as autotrophs, mixotrophs of heterotrophs
`L. thermoferrooxidans' 56/unknown Autotrophic

(c) Extreme thermophiles

Acidianus brierleyi 31/a Facultative anaerobe
A. infernus 31/a Facultative anaerobe
A. ambivalens 33/a Facultative anaerobe
Metallosphaera sedula 45/a Obligate aerobe
Sulfurococcus yellowstonii 44.5/a Obligate aerobe

Sulfur-oxidising (non iron-oxidising) prokaryotes

(a) Mesophiles
T. thiooxidans 50^52/L-/Q-Proteobacteria Autotrophic
T. albertis 61.5/unknown Autotrophic
T. acidophilus 63^64/K-Proteobacteria Mixotrophic
Thiomonas cuprinus 66^69/unknown Mixotrophic
(S. disul¢dooxidans 53/Gram+ve division Mixotrophic)

(b) Moderate thermophiles

T. caldus 62^64/L-/Q-Proteobacteria Growth range 20^55³C

(c) Extreme thermophiles

Sulfolobus shibitae 35/a Mixotrophic
Sf. solfataricus 34^36/a Mixotrophic
Sf. hakonensis 38.5/a Mixotrophic
Sf. metallicus 38/a Autotrophic
(Sf. acidocaldarius 37/a c
Metallosphaera prunae 46/a Mixotrophic
Sulfurococcus mirabilis 43^46/a Mixotrophic

Heterotrophic prokaryotes
(a) Mesophiles
Acidiphilium spp. 59^70/K-Proteobacteria Some species reduce Fe3‡
Acidocella spp. 59^65/K-Proteobacteria
Acidomonas methanolica 63^65/K-Proteobacteria Methylotrophic
Acidobacterium capsulatum 60/unknown Copious exopolymer

(b) Moderate thermophiles

Alicyclobacillus spp. 51^62/Gram+ve division Some strains reduce Fe3‡

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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317 311

Table 1 (continued)
Acidophilic prokaryotic microorganisms
Organism G+C (mol %)/phylogenetic a¤liation Comments
Thermoplasma acidophilum 46/ Facultative anaerobe
Th. volcanium 38/b Facultative anaerobe
Picrophilus oshimae 36/b Strict aerobe
P. torridus ^/b Strict aerobe

(c) Extreme thermophiles

(Sf. acidocaldarius 37/a c

Stygiolobus azoricus 38/a Obligately anaerobic and chemolithotrophic
All characterised extremely thermophilic prokaryotic acidophiles group in the order Sulfolobales within the Crenarchaeota branch of the
domain Archaea.
The moderately thermophilic acidophiles Thermoplasma and Picrophilus spp. group in the order Thermoplasmales within the Euryarchaeota
branch of the domain Archaea.
There is currently some uncertainty regarding the capacity of Sf. acidocaldarius to grow autotrophically on sulfur (see text).

pH optima for growth (ca. pH 0.7) of all known 40^60³C) and extreme thermophiles (Topt s 60³C;
acidophilic microorganisms [18]. Table 1). The last group is made up exclusively of
A number of eukaryotes have also been reported archaea, while moderately thermophilic acidophilic
to inhabit extremely acidic environments. Rhodotor- prokaryotes include archaea and eubacteria (the ma-
ula spp. are frequently encountered (and readily iso- jority of which are Gram-positive). In contrast, mes-
lated) yeasts in acid mine drainage waters, and iso- ophilic acidophiles (autotrophs and heterotrophs)
lates belonging to other genera (e.g. Candida, are dominantly rod-shaped, Gram-negative eubacte-
Cryptococcus) have also been described [13]. Among ria. Exceptions to this general trend include `F. acid-
the ¢lamentous fungi which have been isolated from ophilus' which, on the basis of 16S rDNA base se-
acidic sites are some of the most acidophilic of all quence analysis, is located within the Actinobacteria
microorganisms; Acontium cylatium, Trichosporon [16], and Sulfobacillus disul¢dooxidans, a mesophilic
cerebriae and a Cephalosporium sp. have all been spore-forming Gram-positive eubacterium which has
reported to grow at ca. pH 0 [15]. Protozoa are fre- been reported to use pyrite and elemental sulfur as
quently encountered in acidic mineral leaching and sole energy sources or to grow heterotrophically on
related environments. A laboratory study of three various organic substrates [20]. However, there is
£agellates (Eutreptia/Bodo spp.), a ciliate (Cinetochi- some uncertainty regarding the capacity of S. disul-
lium sp.) and an amoeba (Vahlkamp¢a sp.) showed ¢dooxidans to grow chemolithotrophically, and the
that all were obligately acidophilic (growing in media isolate is, in fact, more closely related to the obli-
poised at pH 1.6 and above) and that they grazed gately heterotrophic Alicyclobacillus spp. than to the
mineral-oxidising (and other) acidophilic bacteria iron/sulfur-oxidising Sulfobacillus spp. Relatively few
[19]. studies have focused on psychrophilic and psychro-
tolerant acidophiles, even though many extremely
3.2. Temperature constraints on acidophilic acidic, low-temperature sites are known, such as
microorganisms subterranean mine waters in the mid-high latitudes.
Berthelot et al. [21] isolated acidophilic bacteria
One of the more convenient ways of subdividing from water draining a uranium mine in Ontario,
acidophilic microorganisms is on the basis of and studied their ability to grow at between 4³ and
their response to di¡erent temperatures (e.g. [1]). 37³C. Although 96% of the iron-oxidising isolates
Three groups have been recognised: mesophiles and 54% of the heterotrophic isolates were classed
(Topt ca. 20^40³C), moderate thermophiles (Topt ca. as psychrotolerant, none was shown to be truly psy-

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312 D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317

chrophilic. Water samples were collected in the win- lower concentrations than either iron or sulfate.
ter months, when temperatures ranged from 0.5 to Growth-coupled anaerobic respiration has fairly re-
5³C and it is conceivable that the higher summer cently been demonstrated with a number of acido-
temperatures experienced at the mine may have pre- philic prokaryotes, as described below. In contrast,
cluded the establishment of psychrophilic strains. no fermentative acidophiles have been described.
More recently, Langdahl and Ingvorsen [22] re- Fermentative metabolisms that produce small molec-
ported the presence of Thiobacillus-like and hetero- ular mass organic acids as end products might not be
trophic acidophiles in an exposed sul¢de ore deposit anticipated in view of the well-documented sensitiv-
located in the High Arctic; the mean air temperature ities of acidophilic microorganisms to these metabo-
at this site was between 315 and 320³C (range 330 lites [2].
to +10³C). Although autotrophic and heterotrophic The redox potential of the ferrous/ferric iron cou-
carbon assimilation of microorganisms from the site ple (+770 mV at pH 2) implies that, for organisms
were both recorded to be optimum at ca. 21³C, mi- for which ferrous iron is the only known energy
crobial ore dissolution at 0³C was noted to be 30% source (`Leptospirillum ferrooxidans' and `T. ferroox-
of the maximum recorded (at 21³C). There is likely idans' strain m-1) oxygen is, on a thermodynamic
to be an important biotechnological niche (e.g. in in basis, the only feasible electron acceptor (i.e. these
situ mining) for mineral-mobilising acidophilic bac- bacteria are necessarily obligate aerobes). However,
teria which are active at very low temperatures. those chemolithotrophic and mixotrophic acido-
philes which can use elemental sulfur and RSCs as
3.3. Response of acidophilic microorganisms to electron donors (Thiobacillus spp., Sulfobacillus spp.
molecular oxygen and a number of acidophilic archaea) can, in theory,
couple their oxidation to the reduction of ferric iron;
As with other environments, those characterised e.g. the free energy of the reaction:
by extreme acidity have zones and microsites which
vary in concentrations of dissolved oxygen [23]. Ob- S ‡ 6Fe3‡ ‡ 4H2 O!HSO3
4 ‡ 6Fe

‡ 7H‡
ligately and facultatively anaerobic acidophiles might
be predicted to exploit anoxic and microaerobic sites. is 314 kJ mol31 at pH 2 [24]. Brock and Gustafson
However, most acidophilic microorganisms that have [24] demonstrated that cell suspensions of both Thio-
been isolated are described as obligate aerobes. Of bacillus thiooxidans and T. ferrooxidans could cou-
the possible metabolic strategies open to acidophiles, ple the anaerobic oxidation of elemental sulfur to the
anaerobic respiration based on the reduction of fer- reduction of ferric iron, but did not demonstrate that
ric iron and sulfate would appear to be attractive this was an energy-transducing reaction which could
propositions, as both tend to be abundant in ex- support growth of the organisms. This question was
tremely acidic environments (Section 2). In contrast, later resolved (in the case of T. ferrooxidans) by the
nitrate tends to be present in very small amounts in work of Pronk et al. [25] who demonstrated unequiv-
these environments, though the use of explosives in ocally that this most well-known of all acidophiles is,
mining can greatly increase local concentrations of in fact, a facultative anaerobe. Bridge and Johnson
NO3 3 . Manganese tends to be present predominantly [26] have demonstrated that moderately thermophilic
in its most reduced form (Mn2‡ ) and, again, at much Sulfobacillus spp. can couple the oxidation of tetra-

Fig. 1. Acid streamer growths in an abandoned pyrite mine (Cae Coch), located in North Wales, and composite microorganisms. a: Mi-
crobial stalactite (`pipes') growths (ca. 1 m long) on a wooden roof support structure. b: Streamer growths in the acidic (pH 2.3) stream
(ca. 1.5 m wide) running through the mine. c: Orange/bronze colonies of iron-oxidising bacteria (ca. 5 mm diameter) and o¡-white colo-
nies of heterotrophic acidophiles on ferrous iron overlay medium inoculated with disrupted acid streamers. d: Scanning electron micro-
graph of an acid streamer fragment from the Cae Coch mine, showing rod-shaped bacteria of di¡erent sizes and the dehydrated vestige
of exopolymer. e: Scanning electron micrograph of a ¢lamentous iron-oxidising heterotrophic bacterial isolate from the Cae Coch stream-
er community.

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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317 313

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314 D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317

thionate to the reduction of ferric iron when grown 4. Mixed communities and microbial interactions in
under anoxic conditions, though growth of the cul- extremely acidic environments
tures was not monitored. In the same report it was
shown that the same Sulfobacillus spp. and Acidimi- Acidophilic microorganisms exist as mixed popu-
crobium ferrooxidans (all of which possess consider- lations in both natural and man-made environments.
able metabolic £exibilities in terms of energy and While in many situations their presence is evidenced
carbon acquisition) can grow anaerobically on glyc- more by products of their metabolism (of which the
erol using ferric iron as sole electron acceptor. Ferric deposition of ferric iron-rich ochre deposits are the
iron reduction has also been demonstrated with a most obvious) rather than by accumulation of bio-
number of mesophilic heterotrophic bacteria and mass, in others the reverse is true. The latter is seen
the mixotroph Thiobacillus acidophilus [27], and most dramatically in the formation of gelatinous
with some Alicyclobacillus-like thermophilic acido- macro structures, generally referred to as `acid
philes [28]. Anaerobic growth coupled to iron reduc- streamers'. These appear to be widely distributed
tion was demonstrated in one strain (SJH) of Acid- around acidic mine sites throughout the world, and
iphilium [27]. However, attempts to demonstrate that are most readily observed in subterranean locations.
acidophilic bacteria can couple the oxidation of or- One such site is an abandoned ( s 70 years) pyrite
ganic substrates to the reduction of elemental sulfur mine (`Cae Coch') located in the Conwy Valley,
have not been successful (D.B. Johnson, unpublished North Wales. The estimated biovolume of the
data). streamers within Cae Coch is in excess of 100 m3 ;
The reduction of sulfate to sul¢de has been dem- these occur as long ¢lamentous and more bulky ge-
onstrated as occurring in extremely acidic environ- latinous growths within the acidic (pH 2.3) ferrugi-
ments (e.g. [22,29]), though attempts at isolating nous stream that £ows through the mine, and as long
truly acidophilic (or acid-tolerant) sulfate-reducing (up to 1 m) microbial stalactite-type growths (`pipes')
bacteria (SRB) have generally met with failure. In which hang from the wetter parts of the roof struc-
some cases, this may be explained by the use of in- ture, particularly in the vicinity of wooden roof sup-
appropriate substrates (generally organic acids, such ports (Fig. 1a and b). Microscopic examination of
as lactate, which exist predominantly as undissoci- the streamers has shown that they are composed of
ated lipophilic acids at the pH range normally used rod-shaped bacteria of di¡erent sizes (Fig. 1d), some
for culturing acidophiles). Greater success with of which form long ¢laments (Fig. 1e), embedded in
growing SRB at low pH has been obtained with a glycocalyx which varies in composition from zone
the use of non-ionic substrates, such as glycerol to zone [34]. In addition, protozoa and rotifera have
and methanol [22,30]. been observed grazing the constituent streamer bac-
Among the acidophilic archaea, several genera are teria. The bacterial community of the Cae Coch
obligate aerobes (Picrophilus, Sulfolobus, Metallo- streamers includes a variety of chemolithotrophic
sphaera and Sulfurococcus), two genera are faculta- iron-oxidisers (T. ferrooxidans and `L. ferrooxidans')
tive anaerobes (Thermoplasma and Acidianus) and a and heterotrophs (Acidiphilium spp., `F. acidophilus',
single genus/species is obligately anaerobic (Stygiolo- and others as yet unclassi¢ed; Fig. 1c).
bus azoricus). Acidianus spp. and St. azoricus share A variety of interactions which occur between
the common trait of growing chemolithotrophically acidophilic microorganisms in their natural environ-
under anoxic conditions, using hydrogen as electron ments have been described (e.g. Fig. 2), several of
donor and elemental sulfur as electron acceptor which have been studied under laboratory condi-
[31,32]. In contrast, Thermoplasma spp. use organic tions. Among these interactions are the following.
substrates as electron donors, though sulfur is again
used as electron acceptor, being reduced to hydrogen 4.1. Competition
sul¢de [33]. No acidophilic archaea have been de-
scribed which are capable of anaerobic growth using Acidophilic microorganisms compete for sub-
ferric iron as sole electron sink, or of reducing sul- strates, which include inorganic as well as organic
fate. electron donors. Various environmental parameters,

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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317 315

(which occur, for example, during the processing of

gold ores in bioreactors), whilst T. ferrooxidans is
more e¡ective in lower temperature ( 6 25³C) situa-
tions. A recent study of the distribution of T. fer-
rooxidans and `L. ferrooxidans' in a derelict pyrite-
rich mine (Iron Mountain, California) using £uores-
cent in situ hybridisation, indicated that the latter
acidophile had the dominant role in pyrite oxidation
and acid genesis at the site [36]; similar results have
also been found during mixed culture leaching of
pyritic coal under laboratory conditions [37].

4.2. Predation

Grazing of mesophilic heterotrophic and chemo-

lithotrophic bacteria by obligately acidophilic proto-
zoa has been observed and quanti¢ed under labora-
tory conditions [19,38]. A Eutreptia-like £agellate
was found to graze T. ferrooxidans in preference to
`L. ferrooxidans' in cultures containing both chemo-
lithotrophs. Numbers of mineral-oxidising acido-
philes in a coal-desulfurisation pilot plant were
Fig. 2. Schematic representation of carbon £ow and dissimilatory
found to be dramatically lowered by an acidophilic
oxido-reduction of iron and sulfur in a extremely acidic, mineral- £agellate within a relatively short time span [19],
leaching, mesophilic environment. suggesting that these eukaryotes might be able to
e¡ect biological control of bacteria in situations
(e.g. mine spoils) where the activities of the latter
such as temperature, pH, concentrations of sub- are detrimental to the environment.
strates and of dissolved metals, have great bearing
on which particular organism(s) is(are) most success- 4.3. Mutualism
ful in any situation. Research has tended to focus on
competition between mesophilic iron-oxidising che- Interactions between acidophilic microorganisms
molithotrophs (T. ferrooxidans and `L. ferrooxidans') may result in both partners gaining bene¢t, as illus-
for ferrous iron and mineral oxidation (e.g. [35]). trated by feedback reactions which occur between
Because of its greater a¤nity for ferrous iron, toler- chemolithotrophic and heterotrophic acidophiles.
ance of very low pH ( 6 1.8) and greater tolerance of `L. ferrooxidans', and to a lesser extent T. ferroox-
ferric iron, `L. ferrooxidans' tends to be more e¡ec- idans, are both sensitive to organic acids and other
tive when leaching ores (e.g. gold concentrates) small molecular mass organic compounds. Actively
which are rich in pyrite, or in environments where metabolising and resting iron-oxidisers release these
ferrous iron concentrations and/or pH are low. In materials into culture media, where they may accu-
contrast, the faster growth rate of T. ferrooxidans mulate to levels at which there is inhibition of bac-
generally results in this iron-oxidiser dominating sit- terial growth. Heterotrophic bacteria can remove this
uations (such as enrichment cultures used frequently inhibition by metabolising the organic materials; this
to isolate iron-oxidising acidophiles) where ferrous has been postulated as the reason why co-cultures of
iron concentrations are relatively high and/or pH is iron-oxidising and heterotrophic acidophiles often
greater than ca. 2. The greater thermo-tolerance of display enhanced mineral leaching compared with
`L. ferrooxidans' also gives it a competitive advant- pure cultures [39], and why both `L. ferrooxidans'
age at slightly elevated (35^40³C) temperatures and T. ferrooxidans remain viable for longer periods

FEMSEC 965 14-12-98 Cyaan Magenta Geel Zwart

316 D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317

in resting cultures which contain either Acidiphilium other hand, the same microorganisms are responsible
spp. or `F. acidophilus' [16]. Another example of mu- for generating acidic metalliferous wastes which
tualism between acidophiles is the cycling of iron cause widespread environmental pollution. Clearly
between ferrous-oxidising chemolithotrophs (using there is a need to harness the positive aspects of
iron as electron donor) and ferric-reducing hetero- these microorganisms to accentuate the net bene¢ts
trophs (using iron as electron acceptor) in situations they can deliver while at the same time limiting their
where dissolved oxygen concentrations vary spatially deleterious e¡ects. To achieve this goal, it will be
or temporally [23]. necessary to extend our understanding of fundamen-
tal (e.g. biochemistry, molecular biology) as well as
4.4. Synergism applied (e.g. bioengineering) aspects of acidophilic
The association of two or more acidophilic micro-
organisms which results in their complementary ac-
tivities being more e¤cient (e.g. in terms of product
formation) than by either organism alone, has been
described on several occasions, mostly in the context [1] Norris, P.R. and Johnson, D.B. (1998) Acidophilic microor-
of enhanced mineral oxidation by mixed popula- ganisms. In: Extremophiles: Microbial Life in Extreme Envi-
tions. Co-cultures of `L. ferrooxidans' and the sul- ronments (Horikoshi, K. and Grant, W.D., Eds.), pp. 133^
fur-oxidisers T. thiooxidans or Thiobacillus caldus (a 154. Wiley, New York, NY.
[2] Norris, P.R. and Ingledew, W.J. (1992) Acidophilic bacteria :
moderate thermophile) have been shown to cause
adaptations and applications. In: Molecular Biology and Bio-
more e¤cient dissolution of chalcopyrite than the technology of Extremophiles (Herbert, R.A. and Sharp, R.J.,
pure culture alone [40]. Mixed cultures of `F. acid- Eds.), pp. 115^142. Blackie, Glasgow.
ophilus' and T. thiooxidans (or the mixotroph Thio- [3] Lane, D.J., Harrison, A.P. Jr., Stahl, D., Pace, B., Giovanno-
bacillus acidophilus) have been shown to oxidise py- ni, S.J., Olsen, G.J. and Pace, N.R. (1992) Evolutionary rela-
tionships among sulfur- and iron-oxidizing eubacteria. J. Bac-
rite, while no dissolution was observed in pure
teriol. 174, 269^278.
cultures of these acidophiles (P. Bacelar-Nicolau [4] Blake, R.C. II, Shute, E.A., Waskovsky, J. and Harrison, A.P.
and D.B. Johnson, unpublished data). Synergy be- Jr. (1992) Respiratory components of bacteria that respire on
tween the moderately thermophilic iron-oxidising iron. Geomicrobiol. J. 10, 173^192.
bacteria Sulfobacillus spp. and A. ferrooxidans results [5] Pronk, J.T. and Johnson, D.B. (1992) Oxidation and reduc-
tion of iron by acidophilic bacteria. Geomicrobiol. J. 10, 153^
in the mixed cultures displaying rapid oxidation of
ferrous iron without the need for extraneous organic [6] Rawlings, D.E. (1997) Biomining: Theory, Microbes and In-
materials or enhanced levels of carbon dioxide, as dustrial Processes, 302 pp. Springer-Verlag/Landes Bioscience,
has been reported for some pure cultures [41]. This Georgetown, TX.
was accounted for by A. ferrooxidans, which has a [7] Johnson, D.B. (1995) The role of `iron bacteria'in the biode-
gradation of minerals. Biodeterior. Abst. 9, 1^7.
slower rate of iron oxidation but an inducible, high-
[8] Sand, W., Gehrke, T., Hallmann, R. and Schippers, A. (1995)
a¤nity mechanism for carbon dioxide uptake, sup- Sulfur chemistry, bio¢lm, and the (in)direct attack mechanism
plying ¢xed organic carbon to the more e¤cient ^ a critical evaluation of bacterial leaching. Appl. Microbiol.
iron-oxidiser S. thermosul¢dooxidans, which has a Biotechnol. 43, 961^966.
limited ability to scavenge carbon dioxide from air. [9] Evangelou, V.P. (1995) Pyrite Oxidation and its Control, 275
pp. CRC Press, New York, NY.
[10] Leduc, L.G. and Ferroni, G.D. (1994) The chemolithotrophic
bacterium Thiobacillus ferrooxidans. FEMS Microbiol. Rev.
5. Outlook 14, 103^120.
[11] Pronk, J.T., Meijer, W.M., Hazeu, W., van Dijken, J.P., Bos,
Exploitation of acidophilic microorganisms for the P. and Kuenen, J.G. (1991) Growth of Thiobacillus ferroox-
idans on formic acid. Appl. Environ. Microbiol. 57, 2057^
processing of ores of gold, copper and other metals
(`biomining') has developed into one of the major [12] Gyure, R.A., Konopka, A., Brooks, A. and Doemel, W.
areas of biotechnology [6], with an estimated market (1987) Algal and bacterial activities in acidic (pH 3) strip
value for 1998 of over 10 billion US dollars. On the mine lakes. Appl. Environ. Microbiol. 53, 2069^2076.

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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317 317

[13] Lopez-Archilla, A.I., Marin, I. and Amils, R. (1995) Micro- in Yellowstone National Park (Reysenbach, A.-L. and Man-
bial ecology of an acidic river : biotechnological applications. cinelli, R., Eds.). Plenum Press, New York, NY, in press.
In : Biohydrometallurgical Processing II (Vargas, T., Jerez, [29] Gyure, R.A., Konopka, A., Brooks, A. and Doemel, W.
C.A., Wiertz, J.V. and Toledo, H., Eds.), pp. 63^74. Univer- (1990) Microbial sulfate reduction in acidic (pH 3) strip-
sity of Chile, Santiago. mine lakes. FEMS Microbiol. Ecol. 73, 193^202.
[14] Brock, T.D. (1978) Thermophilic Microorganisms and Life at [30] Hard, B.C. and Babel, F.W. (1997) Bioremediation of acid
High Temperatures, 465 pp. Springer-Verlag, New York, NY. mine water using facultatively methylotrophic metal-tolerant
[15] Schleper, C., Puehler, G., Kuhlmorgen, B. and Zillig, W. sulfate-reducing bacteria. Microbiol. Res. 152, 65^73.
(1995) Life at extremely low pH. Nature 375, 741^742. [31] Segerer, A.H., Neuner, A., Kristjansson, J.K. and Stetter,
[16] Johnson, D.B. and Roberto, F.F. (1997) Heterotrophic acid- K.O. (1986) Acidianus infernus gen. nov., sp. nov., and Acid-
ophiles and their roles in the bioleaching of sul¢de minerals. ianus brierleyi comb. nov.: facultatively aerobic, extremely
In : Biomining: Theory, Microbes and Industrial Processes acidophilic, thermophilic sulfur-metabolizing archaebacteria.
(Rawlings, D.E., Ed.), pp. 259^280. Springer-Verlag/Landes Int. J. Syst. Bacteriol. 36, 559^564.
Bioscience, Georgetown, TX. [32] Segerer, A.H., Langworthy, T.A. and Stetter, K.O. (1988)
[17] Norris, P.R., University of Warwick, UK, personal commu- Thermoplasma acidophilum and Thermoplasma volcanium sp.
nication. nov. from solfatara ¢elds. Syst. Appl. Microbiol. 10, 161^171.
[18] Schleper, C., Puehler, G., Kuhlmorgen, B. and Zillig, W. [33] Segerer, A.H., Trincone, A., Gahrtz, M. and Stetter, K.O.
(1995) Life at extremely low pH. Nature 375, 741^742. (1991) Stygiolobus azoricus gen. nov., sp. nov. represents a
[19] Johnson, D.B. and Rang, L. (1993) E¡ects of acidophilic pro- novel genus of anaerobic, extremely thermoacidophilic arch-
tozoa on populations of metal-mobilising bacteria during the aebacteria of the order Sulfolobales. Int. J. Syst. Bacteriol. 41,
leaching of pyritic coal. J. Gen. Microbiol. 139, 1417^1423. 495^501.
[20] Dufresne, S., Bousquet, J., Boissinot, M. and Guay, R. (1996) [34] Johnson, D.B. and Kelso, W.I. (1981) Extracellular polymers
Sulfobacillus disul¢dooxidans sp. nov., a new acidophilic, di- of acid streamers from pyritic mines. Environ. Pollut. 24, 291^
sul¢de-oxidizing, Gram-positive, spore-forming bacterium. 301.
Int. J. Syst. Bacteriol. 46, 1056^1064. [35] Norris, P.R., Barr, D.W. and Hinson, D. (1988) Iron and
[21] Berthelot, D., Leduc, L.G. and Ferroni, G.D. (1994) The ab- mineral oxidation by acidophilic bacteria : a¤nities for iron
sence of psychrophilic Thiobacillus ferrooxidans and acido- and attachment to pyrite. In: Biohydrometallurgy: Proceed-
philic heterotrophic bacteria in cold tailings e¥uents from a ings of the International Symposium, Warwick 1987 (Norris,
uranium mine. Can. J. Microbiol. 40, 60^63. P.R. and Kelly, D.P., Eds.), pp. 43^59. Science and Technol-
[22] Langdahl, B.R. and Ingvorsen, K. (1997) Temperature char- ogy Letters, Kew, UK.
acteristics of bacterial iron solubilisation and 14 C assimilation [36] Schrenk, M.O., Edwards, K.J., Goodman, R.M., Hamers,
in naturally exposed sul¢de ore material at Citronen Fjord, R.J. and Ban¢eld, J.F. (1998) Distribution of Thiobacillus fer-
Greenland (83³N). FEMS Microbiol. Ecol. 23, 275^283. rooxidans and Leptospirillum ferrooxidans: implications for
[23] Johnson, D.B., McGinness, S. and Ghauri, M.A. (1993) Bio- generation of acid mine drainage. Science 279, 1519^1521.
geochemical cycling of iron and sulfur in leaching environ- [37] Johnson, D.B. (1991) Biological desulfurization of coal using
ments. FEMS Microbiol. Rev. 11, 63^70. mixed populations of mesophilic and moderately thermophilic
[24] Brock, T.D. and Gustafson, J. (1976) Ferric iron reduction by acidophilic bacteria. In: Processing and Utilization of High-
sulfur- and iron-oxidizing bacteria. Appl. Environ. Microbiol. Sulfur Coals (Dugan, P.R., Quigley, D.R. and Attia, Y.A.,
32, 567^571. Eds.), pp. 567^580. Elsevier, Amsterdam.
[25] Pronk, J.T., de Bruyn, J.C., Bos, P. and Kuenen, J.G. (1992) [38] McGinness, S. and Johnson, D.B. (1992) Grazing of acido-
Anaerobic growth of Thiobacillus ferrooxidans. Appl. Environ. philic bacteria by a £agellate protozoan. Microbiol. Ecol. 23,
Microbiol. 58, 2227^2230. 75^86.
[26] Bridge, T.A.M. and Johnson, D.B. (1998) Reduction of solu- [39] Hallmann, R., Friedrich, A., Koops, H.-P., Pommerening-
ble iron and reductive dissolution of ferric iron-containing Roser, A., Rohde, K., Zenneck, C. and Sand, W. (1992) Phys-
minerals by moderately thermophilic iron-oxidizing bacteria. iological characteristics of Thiobacillus ferrooxidans and Lep-
Appl. Environ. Microbiol. 64, 2181^2186. tospirillum ferrooxidans and physicochemical factors in£uence
[27] Johnson, D.B. and McGinness, S. (1991) Ferric iron reduction microbial metal leaching. Geomicrobiol. J. 10, 193^206.
by acidophilic heterotrophic bacteria. Appl. Environ. Micro- [40] Norris, P.R. (1990) Acidophilic bacteria and their activity in
biol. 57, 207^211. mineral sul¢de oxidation. In: Microbial Mineral Recovery
[28] Johnson, D.B., Body, D.A., Bridge, T.A.M., Bruhn, D.F. and (Ehrlich, H.L. and Brierley, C.L., Eds.), pp. 3^27. McGraw-
Roberto, F.F. (1998) Biodiversity of acidophilic moderate Hill, New York, NY.
thermophiles isolated from two sites in Yellowstone National [41] Clark, D.A. and Norris, P.R. (1996) Acidimicrobium ferroox-
Park, and their roles in the dissimilatory oxido-reduction of idans gen. nov., sp. nov.: mixed-culture ferrous iron oxidation
iron. In: Biodiversity, Ecology and Evolution of Thermophiles with Sulfobacillus species. Microbiology 141, 785^790.

FEMSEC 965 14-12-98 Cyaan Magenta Geel Zwart