SEARCH

SEARCH BY CITATION

Keywords:

  • Acidophilic microorganism;
  • Thermophile;
  • Microbial interaction

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Origins and characteristics of extremely acidic environments
  5. 3Microbial diversity in extremely acidic environments
  6. 4Mixed communities and microbial interactions in extremely acidic environments
  7. 5Outlook
  8. References

Microbial life in extremely low pH (<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 (<35°C) 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 (40–60°C) 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. 0°C) 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.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Origins and characteristics of extremely acidic environments
  5. 3Microbial diversity in extremely acidic environments
  6. 4Mixed communities and microbial interactions in extremely acidic environments
  7. 5Outlook
  8. References

Interest in the biodiversity of microorganisms which inhabit ‘extreme environments’ has increased significantly 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 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 definition of an ‘extremely acidic environment’ is open to some conjecture, for example the question of whether this should be defined 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 definition 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.

2Origins and characteristics of extremely acidic environments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Origins and characteristics of extremely acidic environments
  5. 3Microbial diversity in extremely acidic environments
  6. 4Mixed communities and microbial interactions in extremely acidic environments
  7. 5Outlook
  8. References

Extremely acidic environments may be formed by processes that are entirely natural. However, anthropogenic influences (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 acidity. These include nitrification, 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 sulfide (SO2+2H2S[RIGHTWARDS ARROW]2H2O+3S°). Oxidation of sulfur by autotrophic and heterotrophic microorganisms generates sulfuric acid (S°+H2O+1.5O2[RIGHTWARDS ARROW]H2SO4) 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 significance, however, is the generation of acidity which results from the microbial oxidation of sulfide minerals. Many metals occur as sulfides [7]; indeed, sulfides are the major mineralogical form of many commercially important metals, such as copper, lead and zinc. Iron sulfides (most notably pyrite) are the most abundant sulfide 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 sulfides are often associated with other metal sulfides 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 sulfides are also present in coal deposits (range: <1 to >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:

  • image

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 polysulfides, as well as elemental sulfur and sulfate [8]. Ferrous iron and RSCs are potential energy sources for some acidophilic chemolithotrophic prokaryotes (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 sulfide 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 sulfide minerals such as arsenopyrite (FeAsS) and realgar (AsS).

3Microbial diversity in extremely acidic environments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Origins and characteristics of extremely acidic environments
  5. 3Microbial diversity in extremely acidic environments
  6. 4Mixed communities and microbial interactions in extremely acidic environments
  7. 5Outlook
  8. References

3.1Autotrophic and heterotrophic life-styles

Most extremely acidic environments contain relatively low concentrations (<20 mg l−1) 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 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 filamentous and unicellular forms, and diatoms [12, 13]. Mesophilic acidophilic phototrophs include Euglena spp., Chlorella spp., Chlamydomonas acidophila, Ulothrix zonata and Klebsormidium fluitans. 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 pH optima for growth (ca. pH 0.7) of all known acidophilic microorganisms [18].

Table 1.  Acidophilic prokaryotic microorganisms
OrganismG+C (mol %)/phylogenetic affiliationComments
  1. aAll characterised extremely thermophilic prokaryotic acidophiles group in the order Sulfolobales within the Crenarchaeota branch of the domain Archaea.

  2. bThe moderately thermophilic acidophiles Thermoplasma and Picrophilus spp. group in the order Thermoplasmales within the Euryarchaeota branch of the domain Archaea.

  3. cThere is currently some uncertainty regarding the capacity of Sf. acidocaldarius to grow autotrophically on sulfur (see text).

Iron-oxidising prokaryotes
(a) Mesophiles
Thiobacillus ferrooxidans58–59/β-/γ-ProteobacteriaFacultative anaerobe
T. ferrooxidans’ strain m-165/γ-ProteobacteriaS° not oxidised
T. prosperus63–64/γ-ProteobacteriaHalotolerant
Leptospirillum ferrooxidans51–56/Nitrospira phylumFe2+ sole e donor
Ferromicrobium acidophilus51–55/ActinobacteriaHeterotrophic
   
(b) Moderate thermophiles
Sulfobacillus acidophilus55–57/Gram+ve divisionMay grow as autotrophs, mixotrophs of heterotrophs
S. thermosulfidooxidans48–50/Gram+ve divisionMay grow as autotrophs, mixotrophs of heterotrophs
Acidimicrobium ferrooxidans67–68.5/ActinobacteriaMay grow as autotrophs, mixotrophs of heterotrophs
L. thermoferrooxidans56/unknownAutotrophic
 
(c) Extreme thermophiles
Acidianus brierleyi31/aFacultative anaerobe
A. infernus31/aFacultative anaerobe
A. ambivalens33/aFacultative anaerobe
Metallosphaera sedula45/aObligate aerobe
Sulfurococcus yellowstonii44.5/aObligate aerobe
   
Sulfur-oxidising (non iron-oxidising) prokaryotes
(a) Mesophiles
T. thiooxidans50–52/β-/γ-ProteobacteriaAutotrophic
T. albertis61.5/unknownAutotrophic
T. acidophilus63–64/α-ProteobacteriaMixotrophic
Thiomonas cuprinus66–69/unknownMixotrophic
(S. disulfidooxidans53/Gram+ve divisionMixotrophic)
   
(b) Moderate thermophiles
T. caldus62–64/β-/γ-ProteobacteriaGrowth range 20–55°C
   
(c) Extreme thermophiles
Sulfolobus shibitae35/aMixotrophic
Sf. solfataricus34–36/aMixotrophic
Sf. hakonensis38.5/aMixotrophic
Sf. metallicus38/aAutotrophic
(Sf. acidocaldarius37/ac)
Metallosphaera prunae46/aMixotrophic
Sulfurococcus mirabilis43–46/aMixotrophic
   
Heterotrophic prokaryotes
(a) Mesophiles
Acidiphilium spp.59–70/α-ProteobacteriaSome species reduce Fe3+
Acidocella spp.59–65/α-Proteobacteria 
Acidomonas methanolica63–65/α-ProteobacteriaMethylotrophic
Acidobacterium capsulatum60/unknownCopious exopolymer
   
(b) Moderate thermophiles
Alicyclobacillus spp.51–62/Gram+ve divisionSome strains reduce Fe3+
Thermoplasma acidophilum46/bFacultative anaerobe
Th. volcanium38/bFacultative anaerobe
Picrophilus oshimae36/bStrict aerobe
P. torridus–/bStrict aerobe
   
(c) Extreme thermophiles
(Sf. acidocaldarius37/ac)
   
Other
Stygiolobus azoricus38/aObligately anaerobic and chemolithotrophic

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 filamentous 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 flagellates (Eutreptia/Bodo spp.), a ciliate (Cinetochilium sp.) and an amoeba (Vahlkampfia 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].

3.2Temperature constraints on acidophilic microorganisms

One of the more convenient ways of subdividing acidophilic microorganisms is on the basis of their response to different temperatures (e.g. [1]). Three groups have been recognised: mesophiles (Topt ca. 20–40°C), moderate thermophiles (Topt ca. 40–60°C) and extreme thermophiles (Topt >60°C; 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 disulfidooxidans, 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. disulfidooxidans 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 4° and 37°C. Although 96% of the iron-oxidising isolates and 54% of the heterotrophic isolates were classed as psychrotolerant, none was shown to be truly psychrophilic. Water samples were collected in the winter months, when temperatures ranged from 0.5 to 5°C 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 sulfide ore deposit located in the High Arctic; the mean air temperature at this site was between −15 and −20°C (range −30 to +10°C). Although autotrophic and heterotrophic carbon assimilation of microorganisms from the site were both recorded to be optimum at ca. 21°C, microbial ore dissolution at 0°C was noted to be 30% of the maximum recorded (at 21°C). 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.

3.3Response 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. 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:

  • image

is 314 kJ mol−1 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 tetrathionate 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 flexibilities 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 sulfide 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 sulfide [33]. No acidophilic archaea have been described which are capable of anaerobic growth using ferric iron as sole electron sink, or of reducing sulfate.

4Mixed communities and microbial interactions in extremely acidic environments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Origins and characteristics of extremely acidic environments
  5. 3Microbial diversity in extremely acidic environments
  6. 4Mixed communities and microbial interactions in extremely acidic environments
  7. 5Outlook
  8. References

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 (>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 filamentous and more bulky gelatinous growths within the acidic (pH 2.3) ferruginous stream that flows 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 different sizes (Fig. 1d), some of which form long filaments (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 unclassified; Fig. 1c).

image

Figure 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 off-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 different sizes and the dehydrated vestige of exopolymer. e: Scanning electron micrograph of a filamentous iron-oxidising heterotrophic bacterial isolate from the Cae Coch streamer community.

Download figure to PowerPoint

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.

image

Figure 2. Schematic representation of carbon flow and dissimilatory oxido-reduction of iron and sulfur in a extremely acidic, mineral-leaching, mesophilic environment.

Download figure to PowerPoint

4.1Competition

Acidophilic microorganisms compete for substrates, which include inorganic as well as organic electron donors. Various environmental parameters, 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 affinity for ferrous iron, tolerance of very low pH (<1.8) and greater tolerance of ferric iron, ‘L. ferrooxidans’ tends to be more effective 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 (35–40°C) temperatures (which occur, for example, during the processing of gold ores in bioreactors), whilst T. ferrooxidans is more effective in lower temperature (<25°C) situations. A recent study of the distribution of T. ferrooxidans and ‘L. ferrooxidans’ in a derelict pyrite-rich mine (Iron Mountain, California) using fluorescent 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].

4.2Predation

Grazing of mesophilic heterotrophic and chemolithotrophic bacteria by obligately acidophilic protozoa has been observed and quantified under laboratory conditions [19, 38]. A Eutreptia-like flagellate 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 flagellate within a relatively short time span [19], suggesting that these eukaryotes might be able to effect biological control of bacteria in situations (e.g. mine spoils) where the activities of the latter are detrimental to the environment.

4.3Mutualism

Interactions between acidophilic microorganisms may result in both partners gaining benefit, 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 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].

4.4Synergism

The association of two or more acidophilic microorganisms which results in their complementary activities being more efficient (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 efficient 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, high-affinity mechanism for carbon dioxide uptake, supplying fixed organic carbon to the more efficient iron-oxidiser S. thermosulfidooxidans, which has a limited ability to scavenge carbon dioxide from air.

5Outlook

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Origins and characteristics of extremely acidic environments
  5. 3Microbial diversity in extremely acidic environments
  6. 4Mixed communities and microbial interactions in extremely acidic environments
  7. 5Outlook
  8. References

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 benefits they can deliver while at the same time limiting their deleterious effects. 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.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Origins and characteristics of extremely acidic environments
  5. 3Microbial diversity in extremely acidic environments
  6. 4Mixed communities and microbial interactions in extremely acidic environments
  7. 5Outlook
  8. References
  • 1
    Norris, P.R. and Johnson, D.B. (1998) Acidophilic microorganisms. In: Extremophiles: Microbial Life in Extreme Environments (Horikoshi, K. and Grant, W.D., Eds.), pp. 133–154. Wiley, New York, NY.
  • 2
    Norris, P.R. and Ingledew, W.J. (1992) Acidophilic bacteria: adaptations and applications. In: Molecular Biology and Biotechnology of Extremophiles (Herbert, R.A. and Sharp, R.J., Eds.), pp. 115–142. Blackie, Glasgow.
  • 3
    Lane, D.J., Harrison, A.P. Jr., Stahl, D., Pace, B., Giovannoni, S.J., Olsen, G.J. and Pace, N.R. (1992) Evolutionary relationships among sulfur- and iron-oxidizing eubacteria. J. Bacteriol. 174, 269278.
  • 4
    Blake, R.C. II, Shute, E.A., Waskovsky, J. and Harrison, A.P. Jr. (1992) Respiratory components of bacteria that respire on iron. Geomicrobiol. J. 10, 173192.
  • 5
    Pronk, J.T. and Johnson, D.B. (1992) Oxidation and reduction of iron by acidophilic bacteria. Geomicrobiol. J. 10, 153171.
  • 6
    Rawlings, D.E. (1997) Biomining: Theory, Microbes and Industrial Processes, 302 pp. Springer-Verlag/Landes Bioscience, Georgetown, TX.
  • 7
    Johnson, D.B. (1995) The role of ‘iron bacteria’in the biodegradation of minerals. Biodeterior. Abst. 9, 17.
  • 8
    Sand, W., Gehrke, T., Hallmann, R. and Schippers, A. (1995) Sulfur chemistry, biofilm, and the (in)direct attack mechanism – a critical evaluation of bacterial leaching. Appl. Microbiol. Biotechnol. 43, 961966.
  • 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. 14, 103120.
  • 11
    Pronk, J.T., Meijer, W.M., Hazeu, W., van Dijken, J.P., Bos, P. and Kuenen, J.G. (1991) Growth of Thiobacillus ferrooxidans on formic acid. Appl. Environ. Microbiol. 57, 20572062.
  • 12
    Gyure, R.A., Konopka, A., Brooks, A. and Doemel, W. (1987) Algal and bacterial activities in acidic (pH 3) strip mine lakes. Appl. Environ. Microbiol. 53, 20692076.
  • 13
    Lopez-Archilla, A.I., Marin, I. and Amils, R. (1995) Microbial ecology of an acidic river: biotechnological applications. In: Biohydrometallurgical Processing II (Vargas, T., Jerez, C.A., Wiertz, J.V. and Toledo, H., Eds.), pp. 63–74. University of Chile, Santiago.
  • 14
    Brock, T.D. (1978) Thermophilic Microorganisms and Life at High Temperatures, 465 pp. Springer-Verlag, New York, NY.
  • 15
    Schleper, C., Puehler, G., Kuhlmorgen, B. and Zillig, W. (1995) Life at extremely low pH. Nature 375, 741742.
  • 16
    Johnson, D.B. and Roberto, F.F. (1997) Heterotrophic acidophiles and their roles in the bioleaching of sulfide minerals. In: Biomining: Theory, Microbes and Industrial Processes (Rawlings, D.E., Ed.), pp. 259–280. Springer-Verlag/Landes Bioscience, Georgetown, TX.
  • 17
    Norris, P.R., University of Warwick, UK, personal communication.
  • 18
    Schleper, C., Puehler, G., Kuhlmorgen, B. and Zillig, W. (1995) Life at extremely low pH. Nature 375, 741742.
  • 19
    Johnson, D.B. and Rang, L. (1993) Effects of acidophilic protozoa on populations of metal-mobilising bacteria during the leaching of pyritic coal. J. Gen. Microbiol. 139, 14171423.
  • 20
    Dufresne, S., Bousquet, J., Boissinot, M. and Guay, R. (1996) Sulfobacillus disulfidooxidans sp. nov., a new acidophilic, disulfide-oxidizing, Gram-positive, spore-forming bacterium. Int. J. Syst. Bacteriol. 46, 10561064.
  • 21
    Berthelot, D., Leduc, L.G. and Ferroni, G.D. (1994) The absence of psychrophilic Thiobacillus ferrooxidans and acidophilic heterotrophic bacteria in cold tailings effluents from a uranium mine. Can. J. Microbiol. 40, 6063.
  • 22
    Langdahl, B.R. and Ingvorsen, K. (1997) Temperature characteristics of bacterial iron solubilisation and 14C assimilation in naturally exposed sulfide ore material at Citronen Fjord, Greenland (83°N). FEMS Microbiol. Ecol. 23, 275283.
  • 23
    Johnson, D.B., McGinness, S. and Ghauri, M.A. (1993) Biogeochemical cycling of iron and sulfur in leaching environments. FEMS Microbiol. Rev. 11, 6370.
  • 24
    Brock, T.D. and Gustafson, J. (1976) Ferric iron reduction by sulfur- and iron-oxidizing bacteria. Appl. Environ. Microbiol. 32, 567571.
  • 25
    Pronk, J.T., de Bruyn, J.C., Bos, P. and Kuenen, J.G. (1992) Anaerobic growth of Thiobacillus ferrooxidans. Appl. Environ. Microbiol. 58, 22272230.
  • 26
    Bridge, T.A.M. and Johnson, D.B. (1998) Reduction of soluble iron and reductive dissolution of ferric iron-containing minerals by moderately thermophilic iron-oxidizing bacteria. Appl. Environ. Microbiol. 64, 21812186.
  • 27
    Johnson, D.B. and McGinness, S. (1991) Ferric iron reduction by acidophilic heterotrophic bacteria. Appl. Environ. Microbiol. 57, 207211.
  • 28
    Johnson, D.B., Body, D.A., Bridge, T.A.M., Bruhn, D.F. and Roberto, F.F. (1998) Biodiversity of acidophilic moderate thermophiles isolated from two sites in Yellowstone National Park, and their roles in the dissimilatory oxido-reduction of iron. In: Biodiversity, Ecology and Evolution of Thermophiles in Yellowstone National Park (Reysenbach, A.-L. and Mancinelli, R., Eds.). Plenum Press, New York, NY, in press.
  • 29
    Gyure, R.A., Konopka, A., Brooks, A. and Doemel, W. (1990) Microbial sulfate reduction in acidic (pH 3) strip-mine lakes. FEMS Microbiol. Ecol. 73, 193202.
  • 30
    Hard, B.C. and Babel, F.W. (1997) Bioremediation of acid mine water using facultatively methylotrophic metal-tolerant sulfate-reducing bacteria. Microbiol. Res. 152, 6573.
  • 31
    Segerer, A.H., Neuner, A., Kristjansson, J.K. and Stetter, K.O. (1986) Acidianus infernus gen. nov., sp. nov., and Acidianus brierleyi comb. nov.: facultatively aerobic, extremely acidophilic, thermophilic sulfur-metabolizing archaebacteria. Int. J. Syst. Bacteriol. 36, 559564.
  • 32
    Segerer, A.H., Langworthy, T.A. and Stetter, K.O. (1988) Thermoplasma acidophilum and Thermoplasma volcanium sp. nov. from solfatara fields. Syst. Appl. Microbiol. 10, 161171.
  • 33
    Segerer, A.H., Trincone, A., Gahrtz, M. and Stetter, K.O. (1991) Stygiolobus azoricus gen. nov., sp. nov. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Sulfolobales. Int. J. Syst. Bacteriol. 41, 495501.
  • 34
    Johnson, D.B. and Kelso, W.I. (1981) Extracellular polymers of acid streamers from pyritic mines. Environ. Pollut. 24, 291301.
  • 35
    Norris, P.R., Barr, D.W. and Hinson, D. (1988) Iron and mineral oxidation by acidophilic bacteria: affinities for iron and attachment to pyrite. In: Biohydrometallurgy: Proceedings of the International Symposium, Warwick 1987 (Norris, P.R. and Kelly, D.P., Eds.), pp. 43–59. Science and Technology Letters, Kew, UK.
  • 36
    Schrenk, M.O., Edwards, K.J., Goodman, R.M., Hamers, R.J. and Banfield, J.F. (1998) Distribution of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans: implications for generation of acid mine drainage. Science 279, 15191521.
  • 37
    Johnson, D.B. (1991) Biological desulfurization of coal using mixed populations of mesophilic and moderately thermophilic acidophilic bacteria. In: Processing and Utilization of High-Sulfur Coals (Dugan, P.R., Quigley, D.R. and Attia, Y.A., Eds.), pp. 567–580. Elsevier, Amsterdam.
  • 38
    McGinness, S. and Johnson, D.B. (1992) Grazing of acidophilic bacteria by a flagellate protozoan. Microbiol. Ecol. 23, 7586.
  • 39
    Hallmann, R., Friedrich, A., Koops, H.-P., Pommerening-Roser, A., Rohde, K., Zenneck, C. and Sand, W. (1992) Physiological characteristics of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans and physicochemical factors influence microbial metal leaching. Geomicrobiol. J. 10, 193206.
  • 40
    Norris, P.R. (1990) Acidophilic bacteria and their activity in mineral sulfide oxidation. In: Microbial Mineral Recovery (Ehrlich, H.L. and Brierley, C.L., Eds.), pp. 3–27. McGraw-Hill, New York, NY.
  • 41
    Clark, D.A. and Norris, P.R. (1996) Acidimicrobium ferrooxidans gen. nov., sp. nov.: mixed-culture ferrous iron oxidation with Sulfobacillus species. Microbiology 141, 785790.