Extremophilic microbes: Diversity and perspectives
T. Satyanarayana1,*, Chandralata Raghukumar2 and S. Shivaji3
1 2

Department of Microbiology, University of Delhi South Campus, New Delhi 110 021, India Biological Oceanography Division, National Institute of Oceanography, Dona Paula, Goa 403 004, India 3 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

A variety of microbes inhabit extreme environments. Extreme is a relative term, which is viewed compared to what is normal for human beings. Extreme environments include high temperature, pH, pressure, salt concentration, and low temperature, pH, nutrient concentration and water availability, and also conditions having high levels of radiation, harmful heavy metals and toxic compounds (organic solvents). Culture-dependent and culture-independent (molecular) methods have been employed for understanding the diversity of microbes in these environments. Extensive global research efforts have revealed the novel diversity of extremophilic microbes. These organisms have evolved several structural and chemical adaptations, which allow them to survive and grow in extreme environments. The enzymes of these microbes, which function in extreme environments (extremozymes), have several biotechnological applications. Antibiotics, compatible solutes and other compounds obtainable from these microbes are also finding a variety of uses. MODERATE environments are important to sustain life. Moderate means environments with pH near neutral, temperature between 20 and 40°C, air pressure 1 atm and adequate levels of available water, nutrients and salts. Many extreme environments, such as acidic or hot springs, saline and/or alkaline lakes, deserts and the ocean beds are also found in nature, which are too harsh for normal life to exist. Any environmental condition that can be perceived as beyond the normal acceptable range is an extreme condition. A variety of microbes, however, survive and grow in such environments. These organisms, known as extremophiles, not only tolerate specific extreme condition(s), but usually require these for survival and growth. Most extremophiles are found in microbial world. The range of environmental extremes tolerated by microbes is much broader than other life forms. The limits of growth and reproduction of microbes are, –12° to more than +100°C, pH 0 to 13, hydrostatic pressures up to 1400 atm and salt concentrations of saturated brines. Besides natural extreme environments, there are manmade extreme conditions such as coolhouses, steamheated buildings and acid mine waters.
*For correspondence. (e-mail: tsnarayana@vsnl.net) 78

Life in extreme environments has been studied intensively focusing attention on the diversity of organisms and molecular and regulatory mechanisms involved. The products obtainable from extremophiles such as proteins, enzymes (extremozymes) and compatible solutes are of great interest to biotechnology. This field of research has also attracted attention because of its impact on the possible existence of life on other planets. The progress achieved in research on extremophiles/ thermophiles is discussed in international conferences held every alternate year in different countries. The last conference on extremophiles was held in USA in 2004, and that on thermophiles in England in 2003, and the forthcoming one is due in Australia in 2005. In this article, an attempt has been made to review the diversity of microorganisms that occur in extreme environments, their adaptations and potential biotechnological applications. The ‘Great Plate Count Anomaly’ wherein only a miniscule fraction of microorganisms (<5%) are detected using the culturedependent methods as compared to in situ detection methods and culture independent methods, such as the ‘rRNA approach’ is now well known. The latter approach has indeed revealed a greater degree of biodiversity but suffers from the fact that other characteristics of living organisms cannot be ascertained. Therefore, there is a need to have methods, which not only reflect the phylogeny of the bacteria, but also their biotechnological potential and this indeed is possible using the ‘metagenome’ approach discussed elsewhere in this special section. Metagenome represents the genomes of the total microbiota found in nature, and involves cloning of total DNA isolated from an environmental sample in a bacterial artifical chromosome (BAC) vector and expressing the genes in a suitable host. Thus the metagenome approach, apart from providing phylogenetic data based on 16S rRNA gene sequence analysis and enumerating the extent of microbial diversity, also provides an access to genetic information of uncultured microorganisms. In fact, metagenome libraries could form the basis for identification of novel genes from uncultured microorganisms. Recognizing the potential of this approach, molecular biologists and chemists have undertaken a project to establish the metagenome of ‘Whole earth’ (soil) to catalogue genes for useful natural products such as enzymes, antibiotics, immunosupCURRENT SCIENCE, VOL. 89, NO. 1, 10 JULY 2005

but the flux of DOC in lake water is similar to that in seawater. which have scant and unpredictable rainfall and poorly developed soils. the temperature is above 60°C. at places with boiling water (e. New Zealand. Industrial processes including cement manufacture. Thermal vent sites have recently been found in the Indian Ocean. 1. increase in pH is due to microbial ammonification and sulphate reduction. Therefore. Open oceans and seas are more homogeneous low nutrient (oligotrophic) environments than the soils. lignite or sulphur mines. VOL.1 and 1.g. Desert soils in particular. Solfatara fields. the refuse piles are self-heating and provide the high-temperature environment required to sustain thermophiles. Waters are richer in polymeric material than low molecular compounds. keeping water in liquid state and managing the decrease in solubility of oxygen. the microbes CURRENT SCIENCE. other environmental parameters such as pH. support phototrophic algae. The best known and well-studied geothermal areas are in North America (Yellowstone National Park). glaciers. Most of the acidic pyrite areas have been created by mining and are commonly formed around coal. food processing and paper and pulp manufacture produce man-made unstable alkaline environments. mining. 89. Geothermal areas are characterized by high or low pH. and the ferrous iron in the latter to ferric form. available energy sources. The primary areas with pH lower than 3 are those where relatively large amounts of sulphur or pyrite are exposed to oxygen. Italy and the Soviet Union. disposal of blast furnace slag. The illuminated regions. Indian Sambhar Lake). In alkaline environments such as soils. little liquid water comes out to the surface. All such areas have very high sulphide concentrations. In all acidic niches. cold environments can be divided into two categories: psychrophilic (permanently cold) and psychrotrophic (seasonally cold or where temperature fluxes into mesophilic range) environments. on mountains where snow or ice melts. and by water derived from leached silicate minerals. Both these processes occur abiotically. 79 Extreme environments Low temperature (cold) environments prevail in fresh and marine waters.g. majority of them are anaerobes. With increase in temperature. Fresh waters contain relatively high concentrations of organic matter. Iceland. The best studied and most stable alkaline environments are soda lakes and soda deserts (e. cold environment dominates the biosphere. polar and high alpine soils and waters. The pH of these environments fluctuates due to their limited buffering capacity and therefore. and it is kept constant by continual volcanic activity. sun-heated litter and soil or sediments reaching 70°C. Deep water contains less DOC than the surface water. the temperature of the atmosphere is consistently <5°C. acidic hot springs and boiling mud pots. the temperature decreases progressively and temperatures below –40°C have been recorded. As the altitude increases. Manikaran. ionic strength and nutrients influence the diversity of thermophilic microbial populations. At altitudes >3000 m. where hydrostatic pressure keeps the water in liquid state. a variety of high temperature. The temperature is cold. Low temperatures are characteristic of mountains where snow or ice remains year-round. alkalitolerant microbes are more abundant in these habitats than alkaliphiles. Hot water springs are situated throughout the length and breadth of India. According to Morita1. Both sulphur and pyrite are oxidized abiotically through an exothermic reaction where the former is oxidized to sulphuric acid. the acidity is mostly due to sulphuric acid. two major problems are encountered. Due to spontaneous combustion. These fields are located within active volcanic zones that are termed ‘high temperature fields’. saw dust and coal refuse piles.SPECIAL SECTION: MICROBIAL DIVERSITY pressants and anticancer agents. but are increased 106 times through the activity of acidophiles. which are at 5°C or colder. 10 JULY 2005 . and are quite toxic due to high concentrations of heavy metals. growing above boiling point of water have been isolated from hydrothermal vents. Himachal Pradesh). however. These include volcanic and geothermal areas with temperatures often greater than boiling. with sulphur acidic soils. called oligotrophic. The major man-made cold environments are refrigerators and freezers of industrial and domestic food storage. Oceans represent 71% of earth’s surface and 90% by volume. Open ocean water contains the least organic material with dissolved organic carbon (DOC) between 0. Soils are far more heterogeneous low-nutrient environments. Thus. and the hot springs are often associated with steam holes called fumaroles. Fresh water alkaline hot springs and geysers with neutral/alkaline pH are located outside the volcanically active zones. These are characterized by the presence of large amounts of Na2CO3 but are significantly depleted in Mg2+ and Ca2+ due to their precipitation as carbonates. represent very low-nutrient environments. East African Rift valley. natural and man-made habitats exist. NO. and biological self-heated environments such as compost. In thermal springs. hay. These are very low in organic matter. Thus it may not be too long before this approach is also customized to hunt for genes of interest in metagenomes from various extreme environments. part of the year. electroplating. Such soils are generally low in organic matter and available water and range from acidic to strongly alkaline on the surface. Soils and oceans represent typical low nutrient environments. plants and animals. and pH values as low as 1. Although habitats with elevated temperatures are not as widespread as temperate or cold habitats.0 mg carbon l–1. The salinity ranges from 5% (w/v) to saturation (33%). Because of elevated temperatures. such as mining outflows and tailings dams. characterize other types of geothermal areas. Japan. Besides temperature. The nutrient levels are very low in certain freshwater lakes.

Besides natural hypersaline natural lakes. 3. resulting in the formation of microbial films and layers. VOL. 10 JULY 2005 .5 0 18 Subglacial ice 4* + + 0 0 0 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 NA Lake sediment 5* 0 + 0 0 0 + 0 0 + 0 + 0 0 0 0 + + + + 0 NA Saline lake sediment 6* + + + +. Environments with high hydrostatic pressures are typically found in deep sea and deep oil or sulphur wells. Subglacial ice75. Some microbes are very resistant to drying. Many hypersaline bodies are derived from the evaporation of seawater.5 0 0 0 23 2* 4 0. see the respective publication. The two largest and best-studied hypersaline lakes are the Great Salt Lake (USA) and the Dead Sea in the Middle East.5 0 0 2 0 2 0 0 6. Almost all barophiles isolated to date have been recovered from the deep sea below a depth of approximately 2000 m. data not available. Saline lake sediment77. 2. solid surfaces play an important role and have strong effect on colonization.5 41 16 2 5. 5. under each class. several of which are stratified with respect to salinity. A number of alkaline hypersaline soda brines also exist. 80 CURRENT SCIENCE. and 200 to 400 bar of hydrostatic pressure. including Wadi Natrum (Egypt). where seawater penetrates through seepage or via narrow inlets from the sea.SPECIAL SECTION: MICROBIAL DIVERSITY In habitats with low nutrients. about 100 odd new species of Gram-negative and Gram-positive bacteria from various habitats ranging Table 1. numerous artificial solar lakes have been constructed for producing sea salts. Environments containing high concentrations of organic solvents are those polluted with petroleum or synthetic organic solvents. For details of the identity of the clones up to the genera level. Continental shelf73. +. Microbes may be exposed to intense sources of radiation in the form of γ-irradiation as a means of sterilization or by being in close proximity to nuclear reactors. while the latter is slightly acidic. Many evaporation ponds are found near coastal areas. Desiccation results in extreme osmotic stress and low aw values.g. Although the oceans are the largest saline body of water. The aw (water activity) of 0. 89. 4. bacteria in the family Deinococcaceae. Great Basin lakes (USA). Values indicate percentage of clones. hypersaline environments are generally defined as those containing salt concentrations in excess of seawater (3. and Sambhar Lake (Rajasthan. indicates presence but % not known and NA. This is believed to stem from their thick cell walls. Comparison of the bacterial diversity in different habitats of Antarctica as determined by the rRNA approach Continental shelf sediment Cyanobacterial mat sample 3* 5 9 1 2 0 0 0 15 1 0 9 0 34 5 0 0 0 0 0.85 is considered as the limit. Bacteria adapted to such an extreme environment are able to grow around or beyond 100°C.5 mol l–1 NaCl. A common phenomenon in hypersaline environments is the production of gradients of salinity due to evaporation of seawater. Soda brines lack magnesium and calcium divalent cations because of their low solubility at alkaline pH. Mat sample74.5% total dissolved salts). with salt concentration of about 3–3. which are called thalassic. Diversity of microbes in extreme environments and their adaptations Psychrophiles As of now.5 0. Lake sediment76. High-pressure condition is also met within soils where factors such as high temperature. 6. 16 0 0 0 0 36 0 + 0 + + + 0 0 + 0 5 + Bacterial class α-Proteobacteria β-Proteobacteria ã-Proteobacteria ä-Proteobacteria å-Proteobacteria Acidobacteria Gemmatimonadetes Bacteroidetes Actinobacteria Chloroflexi Chlamydiae Nitrospira Clostridium–Bacillus Cytophaga-Flavobacteria Spirochaetales Green non sulphur Planctomycetales Cyanobacteria OP11 Group/Others Uncultured Archaeae 1* 4 1 41 4 4 0 0 0 3 0 2 0 0 15 0 1 1. the former is slightly alkaline. NO. which help in protecting membrane integrity. Lake Magadi (Kenya). below which microbes tolerant to high osmotic pressures are found to grow only in concentrated syrups. e. Hypersaline evaporation ponds have also been found in Antarctica.5 4 5 0 2 0 6 1. 1. India). high salinity and nutrient limitation may exert further stress on living species.

89. sandstone. The strain identification number and 16S rRNA gene accession number are given within brackets. This suggests that members in the domain Archaea are also capable of psychrophilic life style and require further in- Psychrobacter aquaticus (CMS 56T) (AJ584833) 100 68 100 98 Psychrobacter vallis (CMS 39T) (AJ584832) Psychrobacter psychrophilus (CMS 30T) (AJ584831) Psychrobacter proteolyticus (DSM 13887T) (AJ272303) Sphingobacterium antarcticum (6B1y) (AJ576248) Pseudomonas antarctica (CMS 35T) (AJ537601) 100 100 100 97 71 100 Pseudomonas meridiana (CMS 38T) (AJ537602) Pseudomonas proteolytica (CMS 64T) (AJ537603) Pseudomonas brennerii (CFML 97-391T) (AF268968) Halomonas glaciei (DD 39T) (AJ431369) Halomonas variabilis (DSM 3051T) (M93357) 100 87 100 100 100 Planomicrobium psychrophilus (CMS 53orT) (AJ314746) Planomicrobium mcmeekinii(ATCC 700539T) (AF041791) Planococcus maitriensis (S1) (AJ544622) Planococcus antarcticus (CMS 26orT) (AJ314745) Sporosarcina psychrophilus (IAM 12468T) (D16277) Sporosarcina mcmurdoensis (CMS 21wT) (AJ514408) Arthrobacter flavus (CMS 19yT) (AJ242532) Arthrobacter agilis (DSM 20550T) (X80748) 85 100 99 100 99 100 Kocuria rosea (DSM 20447T) (X87756) Leifsonia rubra (CMS 76rT) (AJ438585) 100 100 100 Leifsonia aurea (CMS 81T) (AJ438586) Leifsonia poae (VKM Ac1401) (AF116342) Pseudonocardia antarctica (DVS 5a1T) (AJ576010) 100 Pseudonocardia alni (IMSNU 20049T) (AJ252823) Sphingobacterium multivorum (IAM 15026) (AB100739) Arthrobacter roseus (CMS 90rT) (AJ278870) Kocuria polaris (CMS 76orT) (AJ278868) Figure 1. Alteromonas. anoxic hypolimnion of Ace Lake. A comparison of the diversity of bacteria in different habitats of Antarctica is given in Table 1 and a phylogenetic tree suggesting the relationships among psychrophilic bacteria of Antarctica is shown in Figure 1. 10 JULY 2005 81 . Polaromonas. Psychroserpens. fresh water and marine lakes. Methanococcoides burtonii was isolated from perennially cold. Shewanella. CURRENT SCIENCE. Methanogenium. UPGMA phylogenetic tree showing the relationships between psychrophilic bacteria from Antarctica isolated by Indian scientists (underlined) based on 16S rRNA gene sequence analysis. Photobacterium. Leifsonia aurea3. Octandecabacter. Arthobacter. Methanococcoides. The psychrophilic and barophilic bacteria. NO. Various species within the genera Alcaligenes. belong to γ-Proteobacteria. which have been cultivated. For the first time. VOL. Methanosarcina. A psychrophilic and slightly halophilic methanogen.SPECIAL SECTION: MICROBIAL DIVERSITY from soil. Bacteroides. 1. Aquaspirillum. Brevibacterium. Colwellia. Sporosarcina macmurdoensis4 and Kocuria polaris5 have been reported from Antarctica. Gelidibacter. The genus Moritella appears to be composed of psychrophiles only. Moritella. Shewanella and Vibrio have been reported to be psychrophilic2. Microbacterium. Bacillus. sea ice and oceans have been reported. Micrococcus. Phormidium. Photobacterium. Antarctica6. Polaribacter. Moritella and Alteromonas haloplanktis.

CURRENT SCIENCE. synthesize cold shock and cold-acclimation proteins. pusillus). at82 tempts have been made to understand sensing temperature and transduction of signals for specific induction of the genes. Studies on the molecular basis of cold adoption. Most of these snow algae are psychrotrophs. Thermoplasma acidophilum). Myceliophthora thermophila. thermophilum. Bacillus psychrophilus. Most of the organisms belonged to the genera Arthrobacter. The presence of cold active enzymes12–14 and the ability to support transcription and translation in psychrophiles were demonstrated at low temperatures15. which could serve as cryoprotectants both for the organisms and their enzymes23. Studies have also revealed the presence of certain genes which were active at low temperature17.18. so as to modulate membrane fluidity. many of the isolates are psychrotrophs. The incidence of psychrophiles in the atmosphere. where the temperatures are between –20 and –40°C. needs confirmation. Thermoascus aurantiacus. green or yellow. Snow algal flagellates include. and capability to sense temperature8. T. Many of the psychrophiles isolated from soils of these caves resembled Arthrobacter glacialis. Brock24 suggested a definition: ‘a thermophile is an organism capable of living at temperatures at or near the maximum for the taxonomic group of which it is a part’. Dactylomyces thermophilus. emersonii. VOL. Thermodesulfobacterium commune. Scytalidium thermophilum. Therefore. thermophilus. Geobacillus stearothermophilus. biotin and niacin1. it appears that psychrophiles have evolved from their mesophilic counterparts inhabiting permanently cold environments2. T. Recent studies of low-temperatureinduced changes in the composition of LPS highlight the importance of LPS in cold adaptation22.11. therefore. such as Trichosporon pullulans. C. Several bacteria and archaebacteria. which function as transcriptional enhancers and RNA-binding proteins. Bacterial growth could occur in clouds. Lapland. subtilis and some species of Arthrobacter and Corynebacterium. B. A. Thermophiles Microbes capable of growth at high temperatures are a wide variety of prokaryotes as well as eukaryotes. Pseudomonas and Flavobacterium.9. 1. As much as 30% of the marine picoplankton from both polar and temperate coastal waters are Archaea and the majority is associated with the Crenarchaeota. have also opened up a number of avenues with respect to our understanding of optimization of biological processes required for cell growth and survival. genes essential for survival at low temperatures were identified. More importantly. Psychrotrophs isolated from food and dairy products include mesophilic bacteria such as Bacillus megaterium. psychrotrophs and psychrophiles. pichinchae. Thielavia terrestris). Euryarchaeota. Sea-ice microbes produce polymeric substances. psychrophiles have increased levels of unsaturated fatty acids that further increase with reduction in temperature. Differential phosphorylation of membrane proteins19. Bacteria procured from an altitude of 7000 m were typical soil forms and over the ocean may be marine. Ascomycetes (Chaetomium thermophile. Talaromyces thermophilus.SPECIAL SECTION: MICROBIAL DIVERSITY vestigations. Basidiomycetes (Phanerochaete chrysosporium) and Hyphomycetes (Acremonium alabamensis. Thermoactinomyces vulgaris. soils. Their adaptation to low temperature is dependent on a number of survival strategies such as the ability to modulate membrane fluidity. Clostridium thermohydrosulfuricum. and Cyanidium caldarium) and protozoa (Cothuria sp. which are capable of growth at elevated temperatures have been classified into moderate (Bacillus caldolyticus. is to understand and unravel the physiological and molecular basis of survival and growth of psychrophiles. C. C. rubroleosa. In any cold environment. ability to regulate gene expression at low temperatures. Oxytricha falla. Mougeotia sp. Naegleria fowleri) grow at high temperatures. 10 JULY 2005 . This definition has the advantage of emphasizing the taxonomic distinctions of thermophily in different groups of organisms. Gram-positive bacteria and proteobacteria2. and this may be responsible for the occurrence of relatively high levels of cobalamin. Psychrotrophic bacteria have been reported in permanently cold caves in the Arctic. nesting materials of birds. In response to sudden changes in environmental temperature (cold shock). could serve as excellent model systems to understand the molecular basis of low temperature adaptation. A phylogenetic analysis of SSU rRNA sequences of the cultivated psychrophiles suggested five major lineages of cultivated obligate psychrophilic bacteria and archaea: Crenarchaeota. with the Arthrobacter predominating. NO. Sulfolobus acidocaldarius. Furthermore. Numerous bacteria can be isolated from troposphere and stratosphere. however. Thermomicrobium roseum. Cercosulcifer hamathensis. The challenge. R. apart from unravelling the molecular mechanisms involved. wood chips and many other sources25–27. Chloromonas brevispina. polyptera and Chlamydomonas nivalis 7. extreme (Thermus aquaticus. Melanocarpus albomyces. ability to carry out biochemical reactions at low temperatures. A few thermophilic fungi belonging to Zygomycetes (Rhizomucor miehei. an important strategy for cold adoption. Cyclidium citrullus. Thermoanaerobacter ethanolicus. the Alps and Romania. the Pyrenees. In addition. 89. Carotenoids are also shown to regulate membrane fluidity due to their temperature-dependent synthesis10. Some algae (Achanthes exigua. Thermomyces lanuginosus. Unlike mesophilic bacteria. Algae are found where snow melts and snow surface is red. Aquaspirillum arcticum and Arthrobacter globiformis. Malbranchea cinnamomea) have been isolated from composts. Tetrahymena pyriformis.20 and probably LPS21 have also been implicated in temperature sensing. Psychrophilic bacteria have the unique ability to survive and grow at low temperatures and thus. Flexibacter–Cytophaga– Bacterioides group.16.

A higher content of arginine and lower content of lysine have been reported for thermostable proteins. Pyrobaculum islandicum. 10 JULY 2005 The hyperthermophilic extreme acidophiles. 1. the cells have a positive inside membrane potential caused by amino acid side chains of proteins and phosphorylated groups of nucleic acids and metabolic intermediates. terrestrial hot springs. The first of the phylotypes was suggested to co-branch with Shewanella.7) Picrophilus oshimae and P. T. submarine hydrothermal vents and geothermally heated oil reserves and oil wells. Methanococcus vulcanicus. CURRENT SCIENCE. It also overexpresses a potent cytoplasmic membrane H+-ATPase to facilitate efflux from the cell. have been isolated from warm springs and hot springs. and non-sporing species of 83 . Pyrolobus fumarii. Kashefi and Lovely36 have recently reported isolation of an archaeal strain 121 that reduced Fe(III) to Fe(II) from a water sample of Mothra hydrothermal vent field (Northern Pacific field) with upper temperature limit of 121°C. ferrooxidans has been attributed to a high degree of inherent secondary structure and hydrophobic environment in which it is located in the cell. Archaeoglobus profundus. VOL. Thermococcus littoralis. alkaliphilic Gram-positive and endospore-forming Bacillus spp. 2–3. The membrane lipids of thermophiles contain more saturated and straight chain fatty acids than mesophiles.SPECIAL SECTION: MICROBIAL DIVERSITY Dictyoglomus thermophilum. Sulfurococcus mirabilis. 89.g. are likely to play a role in stabilizing and refolding proteins as they begin to denature. thiooxidans and Leptospirillum ferrooxidans are encountered in acidic mine drainage waters and mineral processing bioreactors. Pyrococcus furiosus. and these may protect DNA. a type1 DNA topoisomerase that causes positive supercoiling and therefore. Alkaliphiles From neutral soils. Other microbes that occur in hot environments include Metallosphaera that oxidizes sulphidic ores and Stygiolobus sp. may stabilize the DNA. while Bacillus acidocaldarius. The acid stability of rusticyanin (acid-stable electron carrier) of T. In Dunaliella acidophila. Sufurococcus. Proton movement into the cell is minimized by an intracellular net positive charge. The diversity of bacteria of a hot spring in Bukreshwar (West Bengal..0.3-diphosphoglycerate. nucleic acids and proteins are generally susceptible to heat and therefore. Heat shock proteins. the low intracellular pH leads to protonation of titrable groups and produces net intracellular positive charge. Thermotoga mritima) and hyperthermophiles (Methanoccus jannaschii. A relatively low level of positive charge has been linked to acid stability of secreted proteins. Pyrodictium occultum. that act as titrable groups. there is no single factor that enables all thermophiles to grow at elevated temperatures. The most extreme acidophiles (pH optimum 0. Most hyperthermophiles are anaerobic and many are chemolithotrophs. Methanopyrus kandleri. 0–3. with pH optima for growth at or below 3. Thiobacillus caldus was isolated from hot acidic soils. Certain properties of proteins such as a higher degree of structure in hydrophobic cores. Protein stability may also be assisted by polyamines. which is the highest upper temperature limit reported for any microbe till date. the surface charge and inside membrane potential are positive. Many archaeal species contain a paracrystalline surface layer (S-layer) with protein or glycoprotein and this is likely to function as an external protective barrier. The red alga Cyanidium caldarium (pH opt. in solfataras of Yellowstone National Park. by minimizing electrostatic repulsion and protein folding. Extremely thermophilic Geobacillus thermooleovorans strains were isolated from hot spring34 and paper pulp35 samples. Histone-like proteins that bind DNA have been identified in hyperthermophiles. Thermoplasma acidophilum was isolated from self-heating coal refuse piles. Acidophiles Acidophiles maintain their internal pH close to neutral and therefore.. such as thermopsin (a protease of Sulfolobus acidocaldarius) and an α-amylase of Alicyclobacillus acidocaldarius. an iron reducer. Besides. torridus were isolated from two solfataric locations in northern Japan. Mesophilic and sulphur-oxidizing acidophiles such as Thiobacillus ferrooxidans. sun-heated soils. As a result. cyanobacteria and green non-sulphur and lowGC Gram-positive bacteria from 16S rDNA clones. has also been isolated from solfataric fields. Sulfolobus. For any microbe. NO. Desulfurolobus and Acidianus produce sulphuric acid from the oxidation of elemental sulphur or sulphidic ores. which is expected to reduce influx of protons into the cells. The sediment samples were shown to contain γ-proteobacteria. Nannoarchaeum equitans) – based on their optimum temperature requirements28–32. Acidimicrobium ferroxidans and Sulfobacillus sp. lipids. Acidianus infernos. chaperones. 45°C) was recovered from cooler streams and springs in Yellowstone National Park. There are no particular carbon-use or energy generation pathways that are exclusively linked to growth at high temperatures. hyperthermophiles have a reverse gyrase. proline residues with fewer degrees of freedom). Thermoplasma volcanicum that grows at pH 2 and 55°C. India) was recently assessed by a culture-independent approach33. These have been isolated from composts. which reduces elemental sulphur. This allows thermophiles to grow at higher temperatures by providing the right degree of fluidity needed for membrane function. The green alga Dunaliella acidophila is adapted to pH range. Ignicoccus islandicum. accumulation of intracellular potassium and solutes such as 2. is also known. an increased number of hydrogen bonds and salt bridges and a higher proportion of thermophilic amino acids (e. maintain a large chemical proton gradient across the membrane.

and soils in Tibet. The peptidoglycan 84 layer in alkaliphiles has a higher cross-linking rate at higher pH values. Chemosynthetic exo. and alkalitolerant fungi have been isolated. Besides controlling protons.and endo-symbiotic bacteria provide the main source of food for several specialized vent animals. Soda lakes often exhibit blooms of phototrophs such as Cyanospira (Anabaenopsis) sp. while Ancyclobacterium sp. Aeromonas.SPECIAL SECTION: MICROBIAL DIVERSITY Pseudomonas. protein and gene expression has been investigated. The photosynthetic yield of Spirulina surpasses that of others in the terrestrial environment. Alkaliphilic spirochetes such as Spirochaeta alcalica and haloalkalophilic S. The increase in unsaturation produces more fluid membrane and counteracts the effects of the increase in viscosity caused by CURRENT SCIENCE. East African lakes contain Spirulina platensis and other lakes harbour Spirulina maxima. Colwellia and Motiella. Barophilic bacteria have been isolated from several locations and identified41. Lorenz and Molitoris46 cultivated a few marine yeasts at 20–40 MPa pressure. where vent fluids mix with cold bottom seawater. India and Russia harbour different populations of prokaryotes. NO. Non-sporulating filamentous fungi and yeasts have been isolated from deep-sea sediments at 0. Several alkaliphilic. Barophiles Organisms growing at high pressures.1 MPa45. the relative amount of monounsaturation and polyunsaturation increases in the membrane. was described from Kraft paper and board process effluents. rich in methylamine-utilizing methanogens such as Methanohalophilus. Micrococcus.1 Mpa) with almost same frequency as that of facultative psychrophilic bacteria. Vibrio. Several filamentous fungi were isolated from deep-sea calcareous sediments at 10 MPa pressure that corresponds to 1000–3000 m depth. Moderately haloalkaliphilic methanotrophs such as Methylobacter alcaliphilus and Methylomicrobium alcaliphilum were reported from Lake Khadyn and Kenyan soda lake sediment. Thermoanerobacter sp. Psychrophilic deep-sea.500 m depth and cultured at >100 Mpa at 2°C and 40 Mpa above 100°C40. ATP synthases in alkaliphilic Bacillus species have been demonstrated to be exclusively proton translocating. 89. Significant blooms of red pigmented Ectothiorhodospira mobilis and E. Black anoxic lake sediments. Paracoccus. Filamentous fungi and actinomycetes were isolated at 1 bar (0. The coexistence of Archaea was shown along with Pseudomonas in Mariana Trench41. exhibited increased proportion of unsaturated fatty acid in the cell membrane47. Corynebacterium and Actinopolyspora. it is possible that some of these have acquired barotolerance44. also occur. Electrogenic proton extrusion is mediated by respiratory chain activity and protons are transported back into the cells via antiporters. The intracellular pH regulation is dependent on the presence of sodium. Owens Lake in California. Highly saline and alkaline environments such as those observed in Lake Magadi in Rift Valley. 10 JULY 2005 . The lakes are often coloured red due to large numbers of haloalkalophilic archaea such as Natronobacterium pharaonis. Microbial communities are either free-living populations associated with discharged vent fluids or those occurring as microbial mats on surfaces that are in direct contact with the discharged vent fluids. several saline soda lakes. VOL. thermophilic and non-extremophilic microbes were also isolated from these sediments. Shewanella. There is a plethora of microbes that inhabit hydrothermal vents. Photobacterium. The effect of pressure on cell membrane. which is exchanged from cytoplasm into the medium by H+/Na+ antiporters. Chlorococcum sp. Sodium-dependent ATP synthases have not been identified in alkaliphiles. Pakistan. Deep-sea hydrothermal communities are centered around interfacial zone. this may provide shielding effect by ‘tightening’ the cell wall. The combined action of antiporters coupled with respiration provides the cell with a means of controlling its internal pH. low and high temperatures. They play an important role in sulphur cycle in these lakes by utilizing H2S as an electron donor in photosynthesis. Alkaliphilic Exiguobacterium aurantiacum was described from man-made alkaline environment such as potato processing waste. The bacteria often form dense mats and are ‘grazing grounds’ for some of the vent animals. but not for ATP synthesis. Na+-coupled solute symporter and Na+-driven flagella rotation ensure a net sodium balance37–39. and Thermopallium natronophilum were recovered from lake sediments. 1. respectively.42. A barotolerant Alteromonas sp. In response to high pressure. while maintaining adequate Na+ levels through symport and flagella rotation. Deep-sea bacteria have been isolated from the deepest part of the ocean at 10. Anaerobic alkalithermophiles Clostridium. which contribute to its rich biodiversity. Most of the barophilic and barotolerant bacteria belong to γ-Proteobacteria43. Calcium springs in Oman yielded aerobic species of Bacillus. low and high organic nutrients are expected to thrive under deep-sea conditions.. Sodium gradient is used to energize solute transport and flagella movement. which are efficient at transporting H+ into the cell at the expense of Na+ export from it. Flavobacterium. gregoryi and Natronococcus occultus. extremely barophilic bacteria that belong to γ-proteobacteria include. Pseudomonas and members of enterobacteria. the Wadi Natrum Lake in Egypt. Na+-dependent pH homeostasis requires reentry of Na+ into the cell. respectively. N. asiatica have been isolated from water of lake Magadi and lake Khadyn. Gram-positive endospore-forming Bacillus sp. An alkaliphilic sulphate-reducing bacterium Desulfonatronovibrio hydrogenovorans was reported from Lake Magadi. and Pleurocapsa sp. vacuolata have been found together with cyanobacteria in soda lakes. Further. most of these were common terrestrial fungi and therefore. Alkaliphiles maintain a neutral or slightly alkaline cytoplasm.

Salted foods enrich proteolytic Halobacterium salinarum. Halococcus. In solar salterns. A pressure-regulated operon was identified in a barophilic bacterium DB6705. M. as prevails in the deep sea. Oligotrophic bacterium Sphingomonas sp. Another cyanobacterium reported from the Great Salt Lake is. Dactylococcopsis salina. Halophilic bacterial and archaeal diversity was comprehensively reviewed by Grant et al.5 M NaCl). Phototrophic bacteria occur beneath the cyanobacterial layers in anaerobic but lighted zones in hypersaline microbial mats.. Halobaculum.2–16. furiosus and Thermus aquaticus. Cladosporium glycolicum was found growing on submerged wood in the Great Salt Lake. In hypersaline lakes. Proteins of halobacteria are either resistant to high salt concentrations or require salts for activity. Salinococcus and Bacillus have been isolated from saline soils and salterns.8 mg dissolved organic carbon per liter. Stachybotrys chartarum. Protozoa such as Porodon utahensis and Fabrea salina have been described from saline environments. Green algae predominantly use polyols as compatible solutes (e. strain RB2256 isolated from the Resurrection Bay. P. Alaska 85 . Raghavan and Furtado52 have recently reported a population of 5–7 × 103 extreme archaeal halophiles per g of offshore marine sediments from the west coast of India using an MPN method.SPECIAL SECTION: MICROBIAL DIVERSITY high pressure. Eurotium amstelodami. Photobacterium SS9 expressed two outer membrane proteins (porins) under different pressures49. green algae of the genus Dunaliella (D. This strain appeared to produce different DNA-binding proteins at different pressures48. Halotolerant yeast Debaryomyces hansenii was isolated from seawater. which was cloned and sequenced. These cytoplasmic membrane proteins are thought to be pressure sensors controlled by membrane fluidity. Alteromonas. Barophiles are extremely sensitive to UV light and therefore. O. Pseudomonas and Vibrio. bacteriorhodopsin. Halophilic fungi such as Polypaecilum pisce and Basipetospora halophila were isolated from salted fish. glycerol in D. for growth. Haloferax. Some diatoms accumulate proline and oligosaccharides for osmoregulation. However. Phormidium ambiguum. Halorubrum. Filamentous Microcoleus chthonoplastes. Marinomonas. species of Acinetobacter. salina have also been described from the green second layer of mats in hypersaline lakes. Although sodium also accumulates in molar range. salina). Ascomycotina (Chaetomium aureum. In natural ecosystems. Buchalo et al. and their proportion is dependent on the ability of an individual to dominate in a particular environment. Neutral hypersaline waters all over the world harbour halobacteria belonging to the archaeal genera. suggesting the apparent importance of membrane components in high-pressure adaptation50. Halobacteria produce buoyant gas vesicles like many aquatic bacteria. An ORF encodes CydD protein that is required for cytochrome–bd complex in the aerobic respiratory chain. The intracellular salt concentration in halobacteria is very high and generally organic compatible solutes are not accumulated. Halobacterium. but similar species of genera Marinococcus.49 have reported 26 fungal species representing 13 genera of Zygomycotina (Absidia glauca). the diversity of cyanobacteria of hypersaline environment has not been studied extensively. halophilus have been isolated from hypersaline lakes. Oscillatoria neglecta. flavigenum. Halophiles At moderately high salinities (1–3. Thielavia terricola) and mitosporic fungi (Acremonium persicinum. NO. This uses glycine betaine as the major compatible solute. including alkaline saline environments. Deleya. Emericella nidulans.g. viridis) are ubiquitous. A variety of species of diatoms such as Amphora coffeaeformis and Nitzschia and Navicula have been found at 2 M NaCl. Thiocapsa halophila synthesizes glycine betaine and N-acetylglutaminylglutamine amide for osmoprotection. was shown to possess a pressure regulated operon. as seen in DNA polymerases of hyperthermophiles Pyrococcus strain ES4. Halobacteria have purple membrane that contains crystalline lattice of chromoprotein. the thermal inactivation of which is reduced by hydrostatic pressure. Gymnoascella marismortui. A moderately barophilic Shewanella sp. C. Halophilic methanogens such as Methanohalophilus mahii.51. which acts as a lightdependent transmembrane proton pump. The membrane potential generated is used to drive ATP synthesis and supports a period of phototrophic growth. Pressure may stabilize proteins and retard thermal denaturation. Aerobic Gram-negative organotrophic bacteria that are abundant in brines of medium salinity are. the ratio of cytoplasmic potassium to sodium is high. 10 JULY 2005 Oligotrophs/oligophiles Oligotroph is an organism that is capable of growth in a medium containing 0. taken up from the medium or synthesized from choline. Sporosarcina. aerobic heterotrophs are not as common as Gram-negative bacteria. Ulocladium chlamydosporum) from the Dead Sea. Many hypersaline environments exhibit methanogenesis. Flavobacterium. VOL. limnetica and O. marine stromatolites and solar ponds. Unicellular Aphanothece halophytica grows over a wide range of salt concentrations. 1. salina. cyanobacteria predominate planktonic biomass. The moderately halophilic Chlorobium limnicola takes up glycine betaine from the environment and synthesizes trehalose for use as an osmolyte. oligotrophs and eutrophs (copiotrophs) coexist. D. 89. parva. CURRENT SCIENCE. require dark or light-reduced environment. Haloterrigena and Halorubrum. Low molecular-weight substances like methylamines are the likely major substrates for these methanogens. evestigatum and M. Haloarcula. Potassium ions are accumulated internally up to 5 mol l–1 concentration. D.

relatively small size and genome size). the total market for alkaline proteases in Japan was around 15. DNA polymerases have been obtained from Thermococcus littoralis. This bacterial strain was shown to grow at 2% n-butanol. the identification of novel compounds and pathways. VOL. Thus. Pseudomonas sp. igneus were isolated from hydrothermal vents. Increase in lipoprotein content may mechanically strengthen the outer membrane structure.g. while Methanopyrus kandleri. Ancalomicrobium. M. In P. pH 4–5) methanogens have also been reported. and a long time to lose53. Alkaliphilic (Methanosalus zhilinae. However. e. Dictyoglomus thermophilum in Kluyveromyces lactis and Trichoderma reesei. hydrocarbons and heavy metals). 1. on exposure to γ-irradiation. SB1 from sediment and water samples of Mandovi estuary in Goa that was tolerant to a wide range of organic solvents. P. Oligotrophy appears to be a growth strategy that is not inviolable but may take a long time to adopt. Caulobacter. glucoamylase and glucose isomerase that are active in a narrow pH range for the cost-effective starch hydrolysis and these can now be obtained from extremophiles34. and endolithic microbes that live in rocks). xerophiles and xerotolerants (surviving very low-water activity. shared properties similar to Sphingomonas (e. (P. Pyrococcus woesii and P. alkaline proteases in detergents. Sardesai and Bhosle58 isolated Bacillus sp.g.56 and Nakajima et al. Toluene-tolerant Pseudomonas putida IH-2000 was isolated from soil in Japan by Inoue and Horikoshi55. pH 8. with 30% of the total worldwide production for detergents. In Deinococcus radiodurans. It is a huge market. the cell viability is not affected. The likely potential has been increasing exponentially with the isolation of new microbial strains.5–10 copies of the chromosome depending on growth rate). Methanococcoides burtonii and Methanogenium frigidum were isolated from Antarctica. Thermus aquaticus. Methanogens can be isolated from diverse range of salinities. putida. in spite of the severe damage to DNA. The immobilized thermostable chaperonins CpkA and CpkB of hyperthermophlic archaeon Thermococcus kodakaraensis KOD1 were reported to be useful in enzyme stabilizaCURRENT SCIENCE. fungi such as Xeromyces bisporus. Gram-negative bacteria generally show higher tolerance for organic solvents than Gram-positive forms. While investigating the diversity of these bacteria in Leh soils. oligotrophs would ideally have large surface area to volume ratio. Prosthecomicrobium. furiosus for application in polymerase chain reaction (PCR).SPECIAL SECTION: MICROBIAL DIVERSITY retained its ultramicrosize irrespective of the growth phase. Development of an ideal starch saccharification process involves use of thermostable enzymes such as α-amylase that does not require Ca2+ for its activity/stability. NO. Some hyperthermophilic Archaea such as Thermococcus littoralis and Pyrococcus furiosus are also known to survive high levels of γ-irradiation.g. Pramanik et al.3) and acidophilic (Methanosarcina sp. Alkali and thermostable xylanases are useful in pre-bleaching of pulps in order to reduce chlorine requirement in pulp bleaching64–67. Bergquist et al. Characteristics that are considered to be important for oligotrophic microbes include a substrate uptake system that is able to acquire nutrients from its surroundings. and the molecular and biochemical characterization of cellular components.69 have attempted to clone and express two xylanases from an extreme thermophile. toxitolerants (those tolerant to organic solvents. or carbon concentration. Thermotoga maritime. which is likely to help in improving organic solvent tolerance. A number of microbes that are adapted to low nutrient environments produce appendages to enhance their surfaces. 89.g. Antarctic lakes and hydrothermal vents. Examples of extremozymes that are now commercially used include. This bacterium is also resistant to mutagenic chemicals.000 million yens. carbon source. The ability of D. solvent tolerance is considered to be due to efflux pumps that remove the solvent from bacterial cell membranes. putida. but the growth was severely retarded at 3% level. chlorophilus and P.68. jannaschi and M.57 obtained several toluene-tolerant microbes. single copy of the rRNA operon.59–63. isolated from the Resurrection Bay. syringe) show high solvent tolerance levels and this is probably based on the cell surface properties of such species78. 10 JULY 2005 Other extremophilic microbes Methanogenic Archaea are often considered extremophiles. high-affinity uptake systems with broad substrate specificities and an inherent resistance to environmental stresses (e.2–10. Hyphomicrobium. amylopullulanase. e. Labrys and Stella.g. radiodurans to use its genome multiplicity to repair DNA damage appears to be the most fundamental reason for the extraordinary radioresistance of this species. Other microbes that may be considered extremophiles include. DNA is severely damaged and the intact chromosomal DNA replaces the fragmented DNA. and is also multigenomic (e. Another oligotroph Cycloclasticus oligotrophicus RB1. heat.54 have recently reported oligophiles to be abundant in nature. Later Aono et al. 86 . Radiation resistance Radiation resistance can occur due to prevention or efficient repair of damage. In 1994.. UV irradiation and desiccation. hydrogen peroxide and ethanol). Extremophiles in biotechnology A major impetus that has driven extensive and intensive research efforts on extremophiles during the last decades is the potential biotechnological applications associated with these microbes and their products. Methanohalobium avestigatum was recovered from hypersaline Sivash Lake. 2.

screening antitumour drugs Liposomes for drug delivery and cosmetic packaging Heating oil Protein and cell protectants in a variety of industrial uses (e. β-carotene and cell extracts (e. CURRENT SCIENCE. amylopullulanase. lipases. waste treatment. glucoamylase. dehydrogenases) Various enzymes (e. and waste gas desulfurization Waste treatment and methane production Molecular biology Detergents Cheese manufacture Contact-lens cleaning solutions. dietary supplements. and proteases α-Amylases. ice cream. freezing. amylases. nucleases.g. heating) Various industrial uses (e. proteases) γ-Linoleic acid. 10 JULY 2005 87 Potential applications of extremophiles in biotechnology* Use DNA amplification by PCR Ligase chain reaction (LCR) Diagnostics Dairy products Baking and brewing and amino acid production from keratin Starch processing. hypersaline waste brines contaminated with a wide range or organics) Surfactants for pharmaceuticals Detergents Gelatin removal on X-ray film Hide dehairing Foodstuffs. Spirulina and Dunaliella) Microorganisms Microorganisms Membranes Alkaliphiles Proteases. and glucose and fructose for sweeteners Chemical synthesis Paper bleaching Pharmaceutical Molecular sieves Surfactants for oil recovery Bioleaching. oxidases) Methanogens Halophiles Bacteriorhodopsin Polyhydroxyalkanoates Rheological polymers Eukaryotic homologues (e. bioremediation. and feedstock Fermenting fish sauces and modifying food textures and flavours Waste transformation and degradation (e. myc oncogene product) Lipids Lipids Compatible solutes Various enzymes (e. and pharmaceuticals Pulp bleaching Fine papers. cellulases. α-glucosidase. and amylases Lipases and proteases Proteases Polyunsaturated fatty acids Various enzymes β-Galactosidase Ice nucleating proteins Ice minus microorganisms Various enzymes (e.g. 1.g.g. meat tenderizing Food additives.g. coal. pullulanase. lipases and pullulanases Proteases Elastases. Source Thermophiles DNA polymerase DNA ligase Alkaline phosphatase Proteases and lipases Lipases.g. dietary supplements Modifying flavours Lactose hydrolysis in milk products Artificial snow. pullulanase.g. other freezing applications in the food industry Frost protectants for sensitive plants Biotransformations Bioremediation. and degumming Oil recovery Antibiotics Recovery of metals and desulfurication of coal Organic acids and solvents Bioconversion of water insoluble compounds (e. chemicals. food colouring. keritinases Cyclodextrins Xylanases and proteases Pectinases Alkaliphilic halophiles Various microorganisms Acidophiles Sulphur-oxidizing microorganisms Microorganisms Organic solvent tolerant microbes Radiation-resistant microbes Oligotrophs/oligophiles Barophiles *Modified from Cavicchioli and Thomas50. amylopullulanase and xylose/glucose isomerases Alcohol dehydrogenase Xylanases Antibiotics S-layer proteins and lipids Oil degrading microorganisms Sulphur oxidizing microorganisms Thermophilic consortia Psychrophiles Alkaline phosphatase Proteases. xylanases. NO. VOL.SPECIAL SECTION: MICROBIAL DIVERSITY Table 2. environmental biosensors Methane production Optical switches and photocurrent generators in bioelectronics Medical plastics Oil recovery Cancer detection.g. 89.g. sterols). biosurfactants Degradation of organopollutants in radioactive mixed-waste environments Bioassay of assimilable organic carbon in drinking water Microbially enhanced oil recovery process . flavouring agents) Health foods. cellulases.

and Moyer. This suggests potential use of genetically engineered extremophilic microbes in bioremediation of waste sites contaminated with a variety of organopollutants plus radionuclides and heavy metals50. S. M. N. 2003.. Jagannadham. Prabahar. 131–145.. Matsumoto. Carotenoids of an Antarctic psychrotolerant bacterium Sphingobacterium antarcticus and a mesophilic bacterium Sphingobacterium multivorum. 15. 3. K. 2003. and Shivaji. Evol. Franzmann. nov. S. Adaptation to low temperature and regulation of gene expression in Antarctic psychrophilic bacteria. vol. and Rohde. NO. origin of..... 313–317. 183–187. pp. 1994. S. Polar Biol. 2.. K. S. structure and interaction of the pigment with synthetic membrane. S. 423–435. Prakash. 15. I. The recombinant strain expressed toluene dioxygenase that enabled oxidation of toluene... Microbiol. K. Y. vol. 1918–1923. Academic Press. 10. S. R. Narayanan. Shivaji. 2003. 1998. from a cyanobacterial mat sample from a pond in Macmurdo Dry valleys. 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