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FEMS Microbiology Ecology 27 (1998) 307^317
MiniReview
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
Abstract
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
Interest in the biodiversity of microorganisms
which inhabit `extreme environments' has increased
* Tel.: +44 (1248) 382358; Fax: +44 (1248) 370731;
E-mail: [email protected]
signi¢cantly over the past 25 years. There are many
reasons for this: some fundamental (e.g. the notion
that these environments were far more widespread
during the early life of our planet and that organisms
isolated from these sites are representative of archaic
life forms), and some more applied (e.g. the increasing 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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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317
sources of enzymes and other cell products in a variety of industrial and biotechnological operations).
Environments which are characterised by high levels
of acidity fall into this general category. The exact
de¢nition of an `extremely acidic environment' is
open to some conjecture, for example the question
of whether this should be de¢ned merely in terms of
measured pH or of titratable acidity. The description
of an `extreme acidophile', however, is more generally agreed upon, as a microorganism which has a
pH optimum for growth at (or below) pH 3.0 [1].
This de¢nition excludes many fungi and yeasts
which, although often tolerant of extreme acidity,
have pH optima nearer to neutrality. This brief review article will focus on current knowledge of the
biodiversity of extreme acidophiles, and on how
these microrganisms interact in situ. More detailed
reviews of other aspects of acidophilic microbiology
may be found in articles by Norris and Johnson [1],
a general overview of acidophilic microbiology ; Norris and Ingledew [2], which focuses on microbial
adaptation to extremely acidic environments; Lane
et al. [3], which examines evolutionary relationships
between acidophilic iron- and sulfur-oxidising bacteria; Blake et al. [4], which describes the respiratory
components of iron-oxidising acidophiles; and
Pronk and Johnson [5], which considers the role of
acidophilic bacteria in the dissimilatory oxido-reduction of iron. A recent text, edited by Rawlings [6],
includes descriptions of the physiologies of acidophilic bacteria and archaea that are involved in commercial ore processing (`biomining'), as well as detailed accounts of the application of the process.
2. Origins and characteristics of extremely acidic
environments
Extremely acidic environments may be formed by
processes that are entirely natural. However, anthropogenic in£uences (both direct and indirect) have
become increasingly important in creating such environments, particularly since the onset of the industrial revolution. Indeed, the majority of extremely
acidic sites now in existence worldwide have their
origin in one particular human activity, the mining
of metals and coal.
A variety of microbial activities generate net acid-
ity. These include nitri¢cation, and the formation
and accumulation of organic acids either during fermentation or as products of aerobic metabolism.
Most pertinent, however, to the genesis of extremely
acidic environments are the microbial dissimilatory
oxidation of elemental sulfur, reduced sulfur compounds (RSCs), and ferrous iron. Elemental sulfur
may occur in geothermal areas (e.g. around the margins of fumaroles) where it forms by the condensation of sulfur dioxide and hydrogen sul¢de
(SO2 +2H2 SC2H2 O+3S³). Oxidation of sulfur by
autotrophic and heterotrophic microorganisms generates sulfuric acid (S³+H2 O+1.5O2 CH2 SO4 )
which, if not neutralised by carbonates or other basic
minerals present, can result in a dramatic lowering of
pH within microsites or on the macro scale. Of
greater environmental signi¢cance, however, is the
generation of acidity which results from the microbial oxidation of sul¢de minerals. Many metals occur
as sul¢des [7]; indeed, sul¢des are the major mineralogical form of many commercially important metals, such as copper, lead and zinc. Iron sul¢des (most
notably pyrite) are the most abundant sul¢de minerals. In the past, pyrite has been mined (for its sulfur,
rather than for its iron content) but this is no longer
commercially viable. However, iron sul¢des are
often associated with other metal sul¢des in ore deposits, and as such are inadvertently processed
during the mining operation, ending up as waste
materials (in mineral tailings etc.). Pyrite and other
iron sul¢des are also present in coal deposits (range:
6 1 to s 20%) and, inevitably, in coal spoils.
The mechanisms involved in the oxidation of pyrite have been subject to considerable debate (e.g.
[8,9]). Current consensus is that ferric iron acts as
the major oxidant of the mineral, as:
FeS2 ‡ 6Fe…H2 O†6 ‡ 3H2 O!Fe2‡ ‡ S2 O23
3 ‡
‡
6Fe…H2 O†2‡
6 ‡ 6H :
The fate of the thiosulfate formed depends on environmental pH; in circum-neutral environments this
reduced sulfur compound (RSC) is chemically stable,
but in acidic liquors it hydrolyses to form a variety
of polysul¢des, as well as elemental sulfur and sulfate [8]. Ferrous iron and RSCs are potential energy
sources for some acidophilic chemolithotrophic pro-
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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317
karyotes (described below). The regeneration of the
ferric iron oxidant may be brought about biologically or abiotically; however, oxygen is required in
both cases, so that the continued oxidation of pyrite
requires the provision of both air and water. This
criterion is met when coal spoils and mineral wastes
are stored on the land surface, and when water accumulates in exposed deep mine shafts following the
cessation of active mining.
One other important physico-chemical feature of
extremely acidic environments is that concentrations
of soluble metals tend to be much greater than in
neighbouring areas of higher pH. While the solubilities of metal oxyanions (such as molybdate) tend to
be lower in acidic than in neutral solutions, those of
cationic metals (such as aluminium and many heavy
metals) are generally much greater. The types and
concentrations of heavy metals present in any particular extremely acidic environment are much dictated
by the local geochemistry; metals may originate directly from the oxidation of sul¢de minerals (various
chalcophilic metals) or from the accelerated mineral
weathering which occurs under conditions of extreme
acidity (e.g. aluminium from the weathering of clay
minerals). Elevated concentrations of soluble metalloid elements may also occur in extremely acidic environments, of which the most important (from the
point of view of ecotoxicology) is arsenic, which occurs in several sul¢de minerals such as arsenopyrite
(FeAsS) and realgar (AsS).
3. Microbial diversity in extremely acidic
environments
3.1. Autotrophic and heterotrophic life-styles
Most extremely acidic environments contain relatively low concentrations ( 6 20 mg l31 ) of dissolved
organic carbon, and may therefore be classed as oligotrophic. Primary production in sites which do not
receive sunlight (e.g. abandoned deep mines) is based
exclusively on chemolitho-autotrophy, and is inexorably linked to the oxidation of ferrous iron and reduced sulfur compounds. Chemolithotrophic acidophiles have been, and continue to be, the main focus
of research in this area of microbiology, and much is
known of the detailed physiology and biochemistry
309
of some of these prokaryotes, most notably the iron/
sulfur-oxidising bacterium Thiobacillus ferrooxidans
[10]. Most iron- and sulfur-oxidising acidophiles are
regarded as autotrophic, though the ability to assimilate organic carbon has been demonstrated with
some of these (e.g. utilisation of formic acid by T.
ferrooxidans [11]). Other prokaryotes which catalyse
the dissimilatory oxidation of iron and/or RSCs are
either mixotrophic (i.e. may assimilate organic and
inorganic carbon) or else are obligately heterotrophic.
In those extremely acidic environments that are
illuminated, primary production may also be mediated by phototrophic acidophiles. The majority of
these are eukaryotic microalgae, and include ¢lamentous and unicellular forms, and diatoms [12,13].
Mesophilic acidophilic phototrophs include Euglena
spp., Chlorella spp., Chlamydomonas acidophila, Ulothrix zonata and Klebsormidium £uitans. The unicellular rhodophyte Galdieria sulphuraria (formerly Cyanidium caldarium) has been isolated from
geothermal acidic springs and streams in Yellowstone National Park and elsewhere [14]. This moderate thermophile may grow as a heterotroph in the
absence of light (as may Euglena spp.) and has been
reported to grow at pH values around zero [15].
Heterotrophic microorganisms may readily be isolated from most extremely acidic environments.
Many are adept scavengers and rely to a greater or
lesser extent on carbon originating as leakage or lysis
products from chemolithotrophic acidophiles. Obligately acidophilic heterotrophs include archaea, bacteria, fungi, yeasts and protozoa. Some prokaryotic
acidophilic heterotrophs have a direct role in the dissimilatory oxido-reduction of iron [5]. These include
the iron-oxidiser `Ferromicrobium acidophilus' [16]
which appears to use the energy from iron-oxidation
to support growth, and various Acidiphilium-like isolates which can use ferric iron as terminal electron
acceptor (see below). Many acidophilic archaea (Table 1) are obligate heterotrophs, including Sulfolobus
acidocaldarius; early reports of this archaean being a
facultative chemolithotroph are now thought to be
due to the inadvertent use of mixed cultures of Sf.
acidocaldarius and another extreme thermophile (possibly Sulfolobus metallicus [17]). The two characterised species of the moderately thermophilic heterotrophic archaean Picrophilus have the lowest recorded
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Table 1
Acidophilic prokaryotic microorganisms
Organism
G+C (mol %)/phylogenetic a¤liation
Comments
Iron-oxidising prokaryotes
(a) Mesophiles
Thiobacillus ferrooxidans
`T. ferrooxidans' strain m-1
T. prosperus
`Leptospirillum ferrooxidans'
`Ferromicrobium acidophilus'
58^59/L-/Q-Proteobacteria
65/Q-Proteobacteria
63^64/Q-Proteobacteria
51^56/Nitrospira phylum
51^55/Actinobacteria
Facultative anaerobe
S³ not oxidised
Halotolerant
Fe2‡ sole e3 donor
Heterotrophic
(b) Moderate thermophiles
Sulfobacillus acidophilus
S. thermosul¢dooxidans
Acidimicrobium ferrooxidans
`L. thermoferrooxidans'
55^57/Gram+ve division
48^50/Gram+ve division
67^68.5/Actinobacteria
56/unknown
May grow as autotrophs, mixotrophs of heterotrophs
May grow as autotrophs, mixotrophs of heterotrophs
May grow as autotrophs, mixotrophs of heterotrophs
Autotrophic
(c) Extreme thermophiles
Acidianus brierleyi
A. infernus
A. ambivalens
Metallosphaera sedula
Sulfurococcus yellowstonii
31/a
31/a
33/a
45/a
44.5/a
Facultative anaerobe
Facultative anaerobe
Facultative anaerobe
Obligate aerobe
Obligate aerobe
Sulfur-oxidising (non iron-oxidising) prokaryotes
(a) Mesophiles
T. thiooxidans
50^52/L-/Q-Proteobacteria
T. albertis
61.5/unknown
T. acidophilus
63^64/K-Proteobacteria
Thiomonas cuprinus
66^69/unknown
(S. disul¢dooxidans
53/Gram+ve division
Autotrophic
Autotrophic
Mixotrophic
Mixotrophic
Mixotrophic)
(b) Moderate thermophiles
T. caldus
62^64/L-/Q-Proteobacteria
Growth range 20^55³C
(c) Extreme thermophiles
Sulfolobus shibitae
Sf. solfataricus
Sf. hakonensis
Sf. metallicus
(Sf. acidocaldarius
Metallosphaera prunae
Sulfurococcus mirabilis
35/a
34^36/a
38.5/a
38/a
37/a
46/a
43^46/a
Mixotrophic
Mixotrophic
Mixotrophic
Autotrophic
c
)
Mixotrophic
Mixotrophic
Heterotrophic prokaryotes
(a) Mesophiles
Acidiphilium spp.
Acidocella spp.
Acidomonas methanolica
Acidobacterium capsulatum
59^70/K-Proteobacteria
59^65/K-Proteobacteria
63^65/K-Proteobacteria
60/unknown
Some species reduce Fe3‡
(b) Moderate thermophiles
Alicyclobacillus spp.
51^62/Gram+ve division
Some strains reduce Fe3‡
Methylotrophic
Copious exopolymer
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311
Table 1 (continued)
Acidophilic prokaryotic microorganisms
Organism
G+C (mol %)/phylogenetic a¤liation
b
Comments
Thermoplasma acidophilum
Th. volcanium
Picrophilus oshimae
P. torridus
46/
38/b
36/b
^/b
Facultative anaerobe
Facultative anaerobe
Strict aerobe
Strict aerobe
(c) Extreme thermophiles
(Sf. acidocaldarius
37/a
c
Other
Stygiolobus azoricus
38/a
Obligately anaerobic and chemolithotrophic
)
a
All characterised extremely thermophilic prokaryotic acidophiles group in the order Sulfolobales within the Crenarchaeota branch of the
domain Archaea.
b
The moderately thermophilic acidophiles Thermoplasma and Picrophilus spp. group in the order Thermoplasmales within the Euryarchaeota
branch of the domain Archaea.
c
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
acidophilic microorganisms [18].
A number of eukaryotes have also been reported
to inhabit extremely acidic environments. Rhodotorula spp. are frequently encountered (and readily isolated) yeasts in acid mine drainage waters, and isolates belonging to other genera (e.g. Candida,
Cryptococcus) have also been described [13]. Among
the ¢lamentous fungi which have been isolated from
acidic sites are some of the most acidophilic of all
microorganisms; Acontium cylatium, Trichosporon
cerebriae and a Cephalosporium sp. have all been
reported to grow at ca. pH 0 [15]. Protozoa are frequently encountered in acidic mineral leaching and
related environments. A laboratory study of three
£agellates (Eutreptia/Bodo spp.), a ciliate (Cinetochilium sp.) and an amoeba (Vahlkamp¢a sp.) showed
that all were obligately acidophilic (growing in media
poised at pH 1.6 and above) and that they grazed
mineral-oxidising (and other) acidophilic bacteria
[19].
3.2. Temperature constraints on acidophilic
microorganisms
One of the more convenient ways of subdividing
acidophilic microorganisms is on the basis of
their response to di¡erent temperatures (e.g. [1]).
Three groups have been recognised: mesophiles
(Topt ca. 20^40³C), moderate thermophiles (Topt ca.
40^60³C) and extreme thermophiles (Topt s 60³C;
Table 1). The last group is made up exclusively of
archaea, while moderately thermophilic acidophilic
prokaryotes include archaea and eubacteria (the majority of which are Gram-positive). In contrast, mesophilic acidophiles (autotrophs and heterotrophs)
are dominantly rod-shaped, Gram-negative eubacteria. Exceptions to this general trend include `F. acidophilus' which, on the basis of 16S rDNA base sequence analysis, is located within the Actinobacteria
[16], and Sulfobacillus disul¢dooxidans, a mesophilic
spore-forming Gram-positive eubacterium which has
been reported to use pyrite and elemental sulfur as
sole energy sources or to grow heterotrophically on
various organic substrates [20]. However, there is
some uncertainty regarding the capacity of S. disul¢dooxidans to grow chemolithotrophically, and the
isolate is, in fact, more closely related to the obligately heterotrophic Alicyclobacillus spp. than to the
iron/sulfur-oxidising Sulfobacillus spp. Relatively few
studies have focused on psychrophilic and psychrotolerant acidophiles, even though many extremely
acidic, low-temperature sites are known, such as
subterranean mine waters in the mid-high latitudes.
Berthelot et al. [21] isolated acidophilic bacteria
from water draining a uranium mine in Ontario,
and studied their ability to grow at between 4³ and
37³C. Although 96% of the iron-oxidising isolates
and 54% of the heterotrophic isolates were classed
as psychrotolerant, none was shown to be truly psy-
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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317
chrophilic. Water samples were collected in the winter months, when temperatures ranged from 0.5 to
5³C and it is conceivable that the higher summer
temperatures experienced at the mine may have precluded the establishment of psychrophilic strains.
More recently, Langdahl and Ingvorsen [22] reported the presence of Thiobacillus-like and heterotrophic acidophiles in an exposed sul¢de ore deposit
located in the High Arctic; the mean air temperature
at this site was between 315 and 320³C (range 330
to +10³C). Although autotrophic and heterotrophic
carbon assimilation of microorganisms from the site
were both recorded to be optimum at ca. 21³C, microbial ore dissolution at 0³C was noted to be 30%
of the maximum recorded (at 21³C). There is likely
to be an important biotechnological niche (e.g. in in
situ mining) for mineral-mobilising acidophilic bacteria which are active at very low temperatures.
3.3. Response of acidophilic microorganisms to
molecular oxygen
As with other environments, those characterised
by extreme acidity have zones and microsites which
vary in concentrations of dissolved oxygen [23]. Obligately and facultatively anaerobic acidophiles might
be predicted to exploit anoxic and microaerobic sites.
However, most acidophilic microorganisms that have
been isolated are described as obligate aerobes. Of
the possible metabolic strategies open to acidophiles,
anaerobic respiration based on the reduction of ferric iron and sulfate would appear to be attractive
propositions, as both tend to be abundant in extremely acidic environments (Section 2). In contrast,
nitrate tends to be present in very small amounts in
these environments, though the use of explosives in
mining can greatly increase local concentrations of
NO3
3 . Manganese tends to be present predominantly
in its most reduced form (Mn2‡ ) and, again, at much
lower concentrations than either iron or sulfate.
Growth-coupled anaerobic respiration has fairly recently been demonstrated with a number of acidophilic prokaryotes, as described below. In contrast,
no fermentative acidophiles have been described.
Fermentative metabolisms that produce small molecular mass organic acids as end products might not be
anticipated in view of the well-documented sensitivities of acidophilic microorganisms to these metabolites [2].
The redox potential of the ferrous/ferric iron couple (+770 mV at pH 2) implies that, for organisms
for which ferrous iron is the only known energy
source (`Leptospirillum ferrooxidans' and `T. ferrooxidans' strain m-1) oxygen is, on a thermodynamic
basis, the only feasible electron acceptor (i.e. these
bacteria are necessarily obligate aerobes). However,
those chemolithotrophic and mixotrophic acidophiles which can use elemental sulfur and RSCs as
electron donors (Thiobacillus spp., Sulfobacillus spp.
and a number of acidophilic archaea) can, in theory,
couple their oxidation to the reduction of ferric iron;
e.g. the free energy of the reaction:
2‡
‡ 7H‡
S ‡ 6Fe3‡ ‡ 4H2 O!HSO3
4 ‡ 6Fe
is 314 kJ mol31 at pH 2 [24]. Brock and Gustafson
[24] demonstrated that cell suspensions of both Thiobacillus thiooxidans and T. ferrooxidans could couple the anaerobic oxidation of elemental sulfur to the
reduction of ferric iron, but did not demonstrate that
this was an energy-transducing reaction which could
support growth of the organisms. This question was
later resolved (in the case of T. ferrooxidans) by the
work of Pronk et al. [25] who demonstrated unequivocally that this most well-known of all acidophiles is,
in fact, a facultative anaerobe. Bridge and Johnson
[26] have demonstrated that moderately thermophilic
Sulfobacillus spp. can couple the oxidation of tetra-
C
Fig. 1. Acid streamer growths in an abandoned pyrite mine (Cae Coch), located in North Wales, and composite microorganisms. a: Microbial 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 colonies of heterotrophic acidophiles on ferrous iron overlay medium inoculated with disrupted acid streamers. d: Scanning electron micrograph 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 streamer community.
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D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317
FEMSEC 965 14-12-98 Cyaan Magenta Geel Zwart
313
314
D.B. Johnson / FEMS Microbiology Ecology 27 (1998) 307^317
thionate to the reduction of ferric iron when grown
under anoxic conditions, though growth of the cultures was not monitored. In the same report it was
shown that the same Sulfobacillus spp. and Acidimicrobium ferrooxidans (all of which possess considerable metabolic £exibilities in terms of energy and
carbon acquisition) can grow anaerobically on glycerol using ferric iron as sole electron acceptor. Ferric
iron reduction has also been demonstrated with a
number of mesophilic heterotrophic bacteria and
the mixotroph Thiobacillus acidophilus [27], and
with some Alicyclobacillus-like thermophilic acidophiles [28]. Anaerobic growth coupled to iron reduction was demonstrated in one strain (SJH) of Acidiphilium [27]. However, attempts to demonstrate that
acidophilic bacteria can couple the oxidation of organic substrates to the reduction of elemental sulfur
have not been successful (D.B. Johnson, unpublished
data).
The reduction of sulfate to sul¢de has been demonstrated as occurring in extremely acidic environments (e.g. [22,29]), though attempts at isolating
truly acidophilic (or acid-tolerant) sulfate-reducing
bacteria (SRB) have generally met with failure. In
some cases, this may be explained by the use of inappropriate substrates (generally organic acids, such
as lactate, which exist predominantly as undissociated lipophilic acids at the pH range normally used
for culturing acidophiles). Greater success with
growing SRB at low pH has been obtained with
the use of non-ionic substrates, such as glycerol
and methanol [22,30].
Among the acidophilic archaea, several genera are
obligate aerobes (Picrophilus, Sulfolobus, Metallosphaera and Sulfurococcus), two genera are facultative anaerobes (Thermoplasma and Acidianus) and a
single genus/species is obligately anaerobic (Stygiolobus azoricus). Acidianus spp. and St. azoricus share
the common trait of growing chemolithotrophically
under anoxic conditions, using hydrogen as electron
donor and elemental sulfur as electron acceptor
[31,32]. In contrast, Thermoplasma spp. use organic
substrates as electron donors, though sulfur is again
used as electron acceptor, being reduced to hydrogen
sul¢de [33]. No acidophilic archaea have been described which are capable of anaerobic growth using
ferric iron as sole electron sink, or of reducing sulfate.
4. Mixed communities and microbial interactions in
extremely acidic environments
Acidophilic microorganisms exist as mixed populations in both natural and man-made environments.
While in many situations their presence is evidenced
more by products of their metabolism (of which the
deposition of ferric iron-rich ochre deposits are the
most obvious) rather than by accumulation of biomass, in others the reverse is true. The latter is seen
most dramatically in the formation of gelatinous
macro structures, generally referred to as `acid
streamers'. These appear to be widely distributed
around acidic mine sites throughout the world, and
are most readily observed in subterranean locations.
One such site is an abandoned ( s 70 years) pyrite
mine (`Cae Coch') located in the Conwy Valley,
North Wales. The estimated biovolume of the
streamers within Cae Coch is in excess of 100 m3 ;
these occur as long ¢lamentous and more bulky gelatinous growths within the acidic (pH 2.3) ferruginous stream that £ows through the mine, and as long
(up to 1 m) microbial stalactite-type growths (`pipes')
which hang from the wetter parts of the roof structure, particularly in the vicinity of wooden roof supports (Fig. 1a and b). Microscopic examination of
the streamers has shown that they are composed of
rod-shaped bacteria of di¡erent sizes (Fig. 1d), some
of which form long ¢laments (Fig. 1e), embedded in
a glycocalyx which varies in composition from zone
to zone [34]. In addition, protozoa and rotifera have
been observed grazing the constituent streamer bacteria. The bacterial community of the Cae Coch
streamers includes a variety of chemolithotrophic
iron-oxidisers (T. ferrooxidans and `L. ferrooxidans')
and heterotrophs (Acidiphilium spp., `F. acidophilus',
and others as yet unclassi¢ed; Fig. 1c).
A variety of interactions which occur between
acidophilic microorganisms in their natural environments have been described (e.g. Fig. 2), several of
which have been studied under laboratory conditions. Among these interactions are the following.
4.1. Competition
Acidophilic microorganisms compete for substrates, which include inorganic as well as organic
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) situations. A recent study of the distribution of T. ferrooxidans and `L. ferrooxidans' in a derelict pyriterich mine (Iron Mountain, California) using £uorescent 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
Fig. 2. Schematic representation of carbon £ow and dissimilatory
oxido-reduction of iron and sulfur in a extremely acidic, mineralleaching, mesophilic environment.
such as temperature, pH, concentrations of substrates and of dissolved metals, have great bearing
on which particular organism(s) is(are) most successful in any situation. Research has tended to focus on
competition between mesophilic iron-oxidising chemolithotrophs (T. ferrooxidans and `L. ferrooxidans')
for ferrous iron and mineral oxidation (e.g. [35]).
Because of its greater a¤nity for ferrous iron, tolerance of very low pH ( 6 1.8) and greater tolerance of
ferric iron, `L. ferrooxidans' tends to be more e¡ective when leaching ores (e.g. gold concentrates)
which are rich in pyrite, or in environments where
ferrous iron concentrations and/or pH are low. In
contrast, the faster growth rate of T. ferrooxidans
generally results in this iron-oxidiser dominating situations (such as enrichment cultures used frequently
to isolate iron-oxidising acidophiles) where ferrous
iron concentrations are relatively high and/or pH is
greater than ca. 2. The greater thermo-tolerance of
`L. ferrooxidans' also gives it a competitive advantage at slightly elevated (35^40³C) temperatures
Grazing of mesophilic heterotrophic and chemolithotrophic bacteria by obligately acidophilic protozoa has been observed and quanti¢ed under laboratory conditions [19,38]. A Eutreptia-like £agellate
was found to graze T. ferrooxidans in preference to
`L. ferrooxidans' in cultures containing both chemolithotrophs. Numbers of mineral-oxidising acidophiles in a coal-desulfurisation pilot plant were
found to be dramatically lowered by an acidophilic
£agellate within a relatively short time span [19],
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
are detrimental to the environment.
4.3. Mutualism
Interactions between acidophilic microorganisms
may result in both partners gaining bene¢t, as illustrated by feedback reactions which occur between
chemolithotrophic and heterotrophic acidophiles.
`L. ferrooxidans', and to a lesser extent T. ferrooxidans, are both sensitive to organic acids and other
small molecular mass organic compounds. Actively
metabolising and resting iron-oxidisers release these
materials into culture media, where they may accumulate to levels at which there is inhibition of bacterial growth. Heterotrophic bacteria can remove this
inhibition by metabolising the organic materials; this
has been postulated as the reason why co-cultures of
iron-oxidising and heterotrophic acidophiles often
display enhanced mineral leaching compared with
pure cultures [39], and why both `L. ferrooxidans'
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
spp. or `F. acidophilus' [16]. Another example of mutualism between acidophiles is the cycling of iron
between ferrous-oxidising chemolithotrophs (using
iron as electron donor) and ferric-reducing heterotrophs (using iron as electron acceptor) in situations
where dissolved oxygen concentrations vary spatially
or temporally [23].
4.4. Synergism
The association of two or more acidophilic microorganisms which results in their complementary activities 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
of enhanced mineral oxidation by mixed populations. Co-cultures of `L. ferrooxidans' and the sulfur-oxidisers T. thiooxidans or Thiobacillus caldus (a
moderate thermophile) have been shown to cause
more e¤cient dissolution of chalcopyrite than the
pure culture alone [40]. Mixed cultures of `F. acidophilus' and T. thiooxidans (or the mixotroph Thiobacillus acidophilus) have been shown to oxidise pyrite, while no dissolution was observed in pure
cultures of these acidophiles (P. Bacelar-Nicolau
and D.B. Johnson, unpublished data). Synergy between the moderately thermophilic iron-oxidising
bacteria Sulfobacillus spp. and A. ferrooxidans results
in the mixed cultures displaying rapid oxidation of
ferrous iron without the need for extraneous organic
materials or enhanced levels of carbon dioxide, as
has been reported for some pure cultures [41]. This
was accounted for by A. ferrooxidans, which has a
slower rate of iron oxidation but an inducible, higha¤nity mechanism for carbon dioxide uptake, supplying ¢xed organic carbon to the more e¤cient
iron-oxidiser S. thermosul¢dooxidans, which has a
limited ability to scavenge carbon dioxide from air.
5. Outlook
Exploitation of acidophilic microorganisms for the
processing of ores of gold, copper and other metals
(`biomining') has developed into one of the major
areas of biotechnology [6], with an estimated market
value for 1998 of over 10 billion US dollars. On the
other hand, the same microorganisms are responsible
for generating acidic metalliferous wastes which
cause widespread environmental pollution. Clearly
there is a need to harness the positive aspects of
these microorganisms to accentuate the net bene¢ts
they can deliver while at the same time limiting their
deleterious e¡ects. To achieve this goal, it will be
necessary to extend our understanding of fundamental (e.g. biochemistry, molecular biology) as well as
applied (e.g. bioengineering) aspects of acidophilic
microbiology.
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