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ECOLOGY AND BIODIVERSITY OF EXTREMELY ACIDOPHILIC MICROOORGANISMS
Douglas E. Rawlings
University of Stellenbosch, South Africa.
D. Barrie Johnson
University of Wales, Bangor, UK
Keywords: acidic environments, acid mine drainage, chemolithotrophs, extreme acidophiles, ferric iron respiration, iron cycling, microbial biodiversity, mineral biooxidation, iron oxidation, sulfur oxidation, thermophiles
1. Definition of Extreme Acidophily
2. Low pH environments
3. Carbon and Energy Sources of Extreme Acidophiles
4. Biodiversity of Extremely Acidophilic Bacteria
5. Acidophilic Archaea
6. Eukaryotic Acidophiles
7. Relationships Between Acidophilic Microorganisms
6. Eukaryotic Acidophiles
As well as acidophilic prokaryotes, eukaryotic life forms may also be active in environments of pH <3. Many of these are, however, acid-tolerant rather than truly acidophilic, and may grow equally well, or better, in circumneutral pH environments. Most acidophilic/acid tolerant eukaryotes are microorganisms. Notable exceptions are the angiosperms Juncus bulbosus, Phragmites australis, and some Typha spp., which may colonize metal-rich acidic waters.
6.1. Fungi and Yeasts
Many yeasts and fungi grow in acidic soils and peat bogs of pH 3–5, though fewer are active in more extremely acidic environments. Among the most frequently encountered yeasts in AMD waters are Rhodotorula, Candida, Cryptococcus, and Trichosporon spp.. Growth of a number of filamentous fungi at pH <3 has been documented. These include Mucor racemosus (pH range 2.0–9.2), Fusarium oxysporum, Oidiodendron griseum, Aspergillus spp., Penicillium spp., Sclerotium rolfsii, and Trichoderma harziunum. Other notable acidophilic fungi are (i) Acontium velatum, a copper-tolerant (160 mmol L–1) mitosporic fungus that grows between pH 0.2 and 0.7, and (ii) Scytalidium acidophilum, which also tolerates 160 mmol L–1 copper and grows at pH 0 (though not at pH >7) and optimally between pH 1 and 2. The latter mitosporic fungus has been isolated from industrial acidic waste waters, AMD draining a uranium mine, and from soil near a surface sulfur stockpile.
Protozoa are frequently encountered in acidic metal-rich waters, though there are few reports describing growth of protozoa at low pH under laboratory conditions. Most field information relates to ciliates, with Urotricha, Vorticella, and Oxytricha being the most important genera in acidic mining lakes of pH 2–3. Other protozoa that have been shown to grow in very acidic liquors (pH 1.6–2.0) include Eutreptia and Bodo (flagellates), Vahlkampfia (amoeba), and Cinetochilium (ciliate). Acidophilic/acid-tolerant protozoa are phagotrophic on a variety of acidophilic bacteria, including chemolithotrophic iron/sulfur-oxidizers and heterotrophic bacteria.
Besides chemolithotrophic prokaryotes, extremely acidic (and illuminated) waters may also be populated with microalgae as primary producers. As with other types of organisms, there are obligate acidophiles (which may grow in waters as low as pH 0.05) and acid-tolerant species. Representatives include certain chlorophytes (e.g., Dunaliella acidophila, Chlamydomonas spp.), rhodophytes (e.g., Cyanidium caldarium), chrysophytes (e.g., Ochromonas spp.), dinophytes (e.g., Gymnodinium sp.) and euglenoids (e.g. E. mutabilis). Diatoms are relatively rare in waters of pH <3.5. C. caldarium is a moderately thermophilic red algae (temperature optimum 45 °C; pH optimum 1.5) that can grow heterotrophically as well as autotrophically, and may be isolated from acidic geothermal pools and streams. Two related rhodophytes, which inhabit similar environments and which are often confused with C. caldarium, are Galdieria sulphuraria and Cyanidioschyzan merolae.
E. mutabilis is frequently considered to be a useful indicator species of AMD pollution. Together with a second Euglena spp. (E. gracilis) it may form filamentous greenish-brown streamer structures in metal-rich streams of + pH 2 (Figure 9, Section 2.2). E. gracilis tends to be the more successful species in waters containing small concentrations of heavy metals, but the more heavy metal-tolerant E. mutabilis dominates in metal-rich acidic water courses.
Multicellular animal life tends to be very rare in extremely acidic environments; the most widely reported group are rotifers. Acid tolerant rotifers have a lower limit of about pH 2.0–2.4, and often have a wide pH range for growth. The most frequently observed species in AMD include Cephalodella hoodi, Elosa worallii, Philodina roseola, and some others.
7. Relationships Between Acidophilic Microorganisms
Acidophiles display a similar range and diversity of interactions as may be found in a "nonextreme" environment. One major contrast between extremely acidic and more "normal" environments is, however, the pivotal importance of chemolithotrophy as the major form of primary production in the former.
Acidophilic bacteria and archaea compete for communal substrates and energy sources. For example, At. thiooxidans, At. ferrooxidans, At. caldus, and A. acidophilum compete for sulfur and RISCs at temperatures between ~25 and 35 °C, while at 40–50 °C, the major potential sulfur-oxidizers are At. caldus and Gram-positive Sulfobacillus spp.. Similarly, iron oxidation at mesophilic temperatures may be mediated by L. ferrooxidans, At. ferrooxidans, F. acidophilum, or Ferroplasma spp.. The most successful species in a particular situation will be dictated by various microbial physiological (e.g., growth rates and substrate affinities) and environmental parameters (e.g., pH, heavy metal concentrations and total dissolved solids).
Associations between acidophilic microorganisms in which both partners derive benefit are widespread in extremely acidic environments. Mixed cultures of the iron oxidizer L. ferrooxidans and the sulfur-oxidizing At. caldus (or At. thiooxidans) tend to be more efficient at oxidizing pyrite than pure cultures of L. ferrooxidans alone, while neither of the sulfur oxidizers can solubilize pyrite when grown in pure culture. One explanation for this is that iron oxidation by L. ferrooxidans leads to the production of RISCs that this acidophile is unable to metabolize but that serve as an energy source for sulfur-oxidizers. Oxidation of ferrous iron, as noted in Eq. (4), is a proton-consuming reaction, which may cause the solution pH to rise above the optimum range of the iron-oxidizer. By oxidizing the RISCs produced as side-products of pyrite oxidation to sulfuric acid, the sulfur-oxidizers are able to maintain a lower more optimum pH.
Another mutualistic interaction that has been described for acidophilic microorganisms occurs between autotrophic and heterotrophic acidophiles, and is based on carbon flow (Figure 13). A significant proportion (~10–15%) of the carbon fixed by iron- and sulfur-oxidizing prokaryotes is released into the environment from both viable and dead cells. Acidophilic chemolithotrophs show variable, but sometimes quite acute, sensitivity to dissolved organic carbon (particularly small molecular weight organic acids) so that metabolism of these compounds by indigenous heterotrophic acidophiles essentially "detoxifies" the solution phase for the primary producers. In addition, the end products of chemolithotrophic metabolism (ferric iron and sulfate) can act as alternative terminal electron acceptors for some heterotrophic acidophiles (and for sulfur-metabolizing At. ferrooxidans) in oxygen-depleted zones (e.g., stream sediments, submerged tailings, inner cores of mineral spoil heaps). These in turn are reduced to ferrous iron and hydrogen sulfide, which are potential energy sources for the chemolithotrophs (Figure 14). Cycling of reduced and oxidized forms of iron and sulfur in juxtaposed aerobic and anaerobic environments is a feature of extremely acidic environments.
Figure 13. Carbon flow between autotrophic and heterotrophic acidophilic microorganisms Primary production in acidic environments may be powered by sunlight (phototrophy) or chemical oxidations (chemotrophy).
Figure 14. Biotransformations of iron and sulfur in extremely acidic environments Reduced forms of these elements (ferrous iron, RISCs) act as electron donors for chemolithotrophic prokaryotes in aerobic environments, while oxidized forms (ferric iron, sulfate) may be used as electron acceptors in anaerobic and microaerobic sites. Elemental sulfur may be used as electron donor or acceptor (depending on environmental conditions) by some thermo-acidophilic archaea.
Interactions between two or more acidophiles that results in their complementary activities being more efficient than that of either microorganism acting alone has been described. An example is the oxidation of pyrite by mixed cultures of F. acidophilum and At. thiooxidans. Unlike autotrophic iron-oxidizers, F. acidophilum requires an organic carbon that At. thiooxidans may provide, while the chemolithotrophic sulfur oxidizer is dependent on the ongoing provision of RISCs produced by ferric iron attack on the sulfide mineral. Neither of these bacteria can oxidize pyrite in pure culture, but mixed cultures are highly efficient at so doing.
Ammensalism refers to the repression of one or more species by toxins produced by another, and this can again be seen with some chemolithotrophic/heterotrophic acidophilic communities. As noted in Section 3, heterotrophic acidophiles vary in the sensitivities to hydrogen ions and some heavy metals (such as ferric iron). Acidocella spp. are, in general, more sensitive to both than are Acidiphilium spp.. Therefore, end metabolic products of acidophilic iron or sulfur oxidizers can suppress populations of more rapidly growing Acidocella spp. in favor of slower growing Acidiphilium spp.
Grazing of acidophilic bacteria by acidophilic/acid-tolerant protozoa and rotifers has been observed in environmental samples, and (in the case of protozoa) has been quantified under laboratory conditions (Figure 15). Acidophilic protozoa display preferential grazing of some acidophilic bacteria over others, which appears to be related to motility, formation of filaments and flocs, and the presence of potentially unpalatable storage compounds (e.g., elemental sulfur) within the bacterial population.
Figure 15. Scanning electron micrograph of an acidophilic protozoan (Cinetochilium sp.) grazing a mixed population of iron-oxidizing and heterotrophic bacteria in a laboratory culture (pH 1.8) containing pyritic coal.
Extremely acidic environments are fairly widespread on Earth, some resulting from geothermal and others from industrial (mainly mining) activities. Iron- and sulfur-oxidizing chemolithotrophic organisms play a very important role in these environments and sulfuric acid is usually the cause of the extreme acidity. Acidic environments are sources of primary productivity (carbon dioxide fixation) based on the energy released from inorganic oxidation-reduction reactions rather than radiation energy from sunlight. The ability of ferrous iron to serve as an electron donor under aerobic conditions and ferric iron as an electron acceptor under anaerobic conditions (iron cycling) is a feature of these acid environments made possible by the solubility of ferrous and ferric iron in acid solutions. Adding to interest in this field is that some of the high temperature, iron- and sulfur-energized, acidic environments have features that are similar to those thought to prevail during the earliest stages of the development of life on Earth.
Acidophilic microorganisms are faced with several metabolic challenges arising from their environment. Cytoplasmic pH values are near-neutral that means that adaptations to membrane transport and energy production process have had to be made to allow for the steep gradient in pH across the membrane. Many organic acids are fully protonated in low pH environments and when in this form they are able to pass through the cell membrane in an uncontrolled manner. Once they enter the near-neutral pH environment of the cytoplasm, these organic acids become dissociated and for this reason, many organic acids are toxic to acidophilic bacteria unless present at suitably low concentrations (see Ions Transport and Genetics of Acidophiles). As with most environments, highly acidic environments contain a complex web of primary producers and organisms that grow in close beneficial association with or that exploit the primary producers. The unique features associated with extremely acidic environments mean that they provide a rich source of biodiversity that is different from that found elsewhere on earth.
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Acid mine drainage (AMD): The sometimes highly acidic streams that drain from mining waste or coal storage piles and that are produced by the natural oxidation of sulfur-containing minerals to sulfuric acid.
Bioleaching: The solubilization of metals as a result of microbial activity, typically the conversion of an insoluble metal sulfide to a water-soluble metal sulfate.
Biooxidation: The removal of electrons from minerals by microorganisms resulting in their oxidation.
Chemolithotroph: A carbon dioxide-fixing organism that obtains its energy by the removal of electrons from reduced inorganic substances such as ferrous iron or reduced forms of sulfur and transfers these electrons to an electron acceptor like oxygen.
Exergonic: Producing energy that is available for work.
Exopolymer: A substance that is produced outside the cell wall of some organisms and that consists largely of polysaccharide material.
Mixotroph: An organism that is able to use a combination of carbon dioxide and organic sources of carbon.
RISC: A reduced inorganic sulfur compound.
Solfatara: Geothermally heated, sulfur-rich terrestrial ecosystems (hot springs, mudpots, and fumaroles).
Goebel B.M., Norris P.R., and Burton N.P. (2000). Acidophiles in biomining. Applied Microbial Systematics (ed. Fergus G. Priest and Michael Goodfellow), pp. 293–314. Dordrecht: Kluwer. [This chapter covers the acidophiles that are used for the commercial recovery of metals from ores, including how they may be identified using molecular techniques.]
Johnson D.B. (1998). Biodiversity and ecology of acidophilic microorganisms. FEMS Microbiology Ecology 27, 307–317. [A minreview on the diversity and ecological relationships found among acidophilic bacteria and archaea.]
Rawlings D.E. (1997). Biomining: Theory, Microbes and Industrial Processes. 302 pp. Berlin: Springer-Verlag. [This multiauthor book deals with the use of acidophiles in biomining and includes a section on acidophilic microorganisms, including their physiology and diversity.]
Wächtershäuser G. (1990). Evolution of the first metabolic cycles. Proceedings of the National Academy of Science (USA) 87, 200–204. [This is an article concerning the possibility of "pyrite-pulled" early metabolic reactions. It contains references to earlier related work.]
Douglas Eric Rawlings was born in East London, South Africa in November 1950. He completed BSc Hons (1972) and PhD degrees (1976) at Rhodes University in Grahamstown, South Africa. After two years as a research officer at the Leather Industries Research Institute and four years as a lecturer at the University of the Witwatersrand, he moved to the University of Cape Town (1982) where he was promoted to an ad hominem chair as Professor of Microbiology in 1988. In July 1998, he took up his present appointment as Professor of Microbiology at the University of Stellenbosch.
The research of Professor Rawlings has mostly concerned the bacteria involved in the extraction of minerals from ores. He was involved in the initial development of a commercially successful process for the biooxidation of difficult-to-treat gold-bearing arsenopyrite ores. The bulk of his research has focused on the molecular biology of the biomining bacterium, Acidithiobacillus ferrooxidans and more recently this work has been extended to include other biomining bacteria. The findings of this research have been published in over 80 peer-reviewed journal articles and book chapters. In 1997, he edited a book entitled Biomining: Microbes, Theory and Industrial Processes. He currently serves on the editorial board of the journals Applied and Environmental Microbiology and International Deterioration and Biodegradation, is General Secretary of the Royal Society of South Africa, and has served several terms on the council of the South African Society for Microbiology. Professor Rawlings is a life fellow and a distinguished teacher of the University of Cape Town, a recipient of a silver medal from the South African Society for Microbiology, the PanLab’s award from the Society for Industrial Microbiology (USA), and is a fellow of the Royal Society of South Africa and a founder member of the South African Academy of Science.
David Barrie Johnson was born in New Tredegar, South Wales, in March 1954. He obtained a BSc (Hons) in Biochemistry and Soil Science from the University of Wales, Bangor in 1975 and a PhD (also from UWB) in 1979. He joined the academic staff at UWB as a demonstrator in 1977 and was appointment as a lecturer in 1979. In 1994, he was promoted to Senior Lecturer in the School of Biological Sciences at UWB.
Barrie Johnson's research activities have been in the field of environmental microbiology. In particular, he and his research team have been concerned with the microbiology of extremely acidic environments, both from pure and applied perspectives. His work has taken him to mining areas and geothermal sites in different parts of the world, including Yellowstone National Park, Wyoming, and the Caribbean Island of Montserrat. A major interest involves studying the biodiversity of acidophilic microorganisms, and he and his team have been very successful in isolating and characterizing novel acidophiles from natural and anthropogenic environments, in elucidating how these microorganisms with each other in nature. The applied aspects of his research include the use of acidophiles to accelerate mineral dissolution by oxidative and reductive mechanisms and investigating the use of these microorganisms to remediate acidic wastewaters. Outside of UWB, Barrie Johnson has worked at the Idaho National Engineering and Environmental Laboratory, USA and has lectured at universities in many different countries. He is currently serving on the editorial boards of Biotechnology Letters and Resource and Environmental Biotechnology. He has 90 publications as book chapters and in scientific journals.
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