The following points highlight the six important groups of extremophiles. The groups are: 1. Acidophiles 2. Alkalophiles 3. Halophiles 4. Psychrophiles 5. Thermophiles and Hyperthermophlies 6. Barophiles.
Contents
Group # 1. Acidophiles:
Most natural environments on the earth are essentially neutral, having pH between 5 and 9. Only a few microbial species can grow at pH less than 2 or greater than 10. Microorganisms that live at low pH are called acidophiles.
Fungi as a group tend to be more acid tolerant than bacteria. Many fungi grow optimally at pH 5 or below and a few grow well at pH value as low as 2. Several species of Thiobacillus and genera of archaea including Sulfolobus and Thermoplasma are acidophilic.
This ferroxidans and Sulfolobus sp. oxidize sulfide mineral and produce sulphuric acid. The most important factor for obligate acidophily is the cytoplasmic membrane of obligatory acidophilic bacteria which actually dissolves and lyses the cell wall. This suggests that high concentration of H+ ions are needed for membrane stability.
Highly acidic environment is formed naturally from geochemical activities (such as the production of gases in hydrothermal vents and some hot springs) and from the metabolic activities of certain acidophile themselves. Acidophiles are also found in the debris left over coal mining. Interestingly, acid-loving extremophiles cannot tolerate great acidity inside their cells, where it would destroy DNA.
They survive by keeping the acid out. But the defensive molecules provide this protection as well as others that come in contact with the environment must be able to operate in extreme acidity. Indeed extremozymes (their enzymes providing adaptability) are able to work at pH below one, more acidic than even vinegar or juice of stomach. Such enzymes have been isolated from the cell wall and underlying cell membrane of some acidophiles.
(i) Physiology:
Obligate acidophiles have an optimum pH for growth which remains extremely low (1 to 4). To shield the intracellular enzymes and other components from low to medium pH, the organisms maintain a large pH gradient across the membrane. Special forms of lipids are present in their membrane which may minimize the leakage of H+ down the pH value.
For instance, the presence of certain fatty acids has been reported to provide special adaptations to growth and survival at extremely low pH. Acidophiles maintain the cytoplasmic pH around 6.5. In these organisms, the pH remains generally 1-2 which is lower in comparison to neutrophiles and alkalophiles.
In acidophiles the pH is compensated by positive inside electric potential which is opposite to that present in neutrophiles. The reversed electric potential is generated by electrogenic K+ uptake which allows the cells to extrude more H+ and thus maintain the internal pH.
(ii) Molecular Adaptation:
Most critical factor for obligate acidophily lies in the cytoplasmic membrane. When the pH is raised to neutrality, the cytoplasmic membrane of obligately acidophilic bacteria actually dissolve and the cells lyse. It is suggested that the high concentration of hydrogen ions are required for stability of membrane that allows bacteria to survive.
(iii) Applications:
Potential applications of acid-tolerant extremozymes range from catalysts for the synthesis of compounds in acidic solutions to additives for animal feed which are intended to work in animal stomach.
When added to feed, the enzymes improve the digestibility of expensive grains, therefore avoiding the need for more costly food. Rusticyanin proteins from acidophiles help in acid stability. Expression of heterogenous arsenic resistance genes in the iron-oxidizing Thiobacillus ferrooxidans has been established as biotechnological approach of bioremediation.
Group # 2. Alkalophiles:
Alkalophilies live in soils laden with carbonate and in Soda lakes, such as those found in the Rift Valley of Africa and the west U.S. The first alkalophilic bacterium was reported in year 1968.
Most alkalophilic prokaryotes studied have been aerobic non-marine bacteria and reported as Bacillus spp. Krulwich and Guffanti (1989) separated them into two broad categories: alkali-tolerant organisms (pH 7.0-9.0) (which cannot grow above pH 9.5) and alkalophilic organisms (pH 10.0- 12.0).
Most of the alkalophilic organisms are aerobic or facultative anaerobic. Some alkalophiles are Bacillus alkalophilus, B.firmus RAB, Bacillus sp. No. 8-1 and Bacillus sp. No. C-125 which bear flagella and hence are motile. The flagella induced motility is considered by a sodium motive force (smf) instead of proton motive force (pmf).
They are motile at pH 9-10.5 but no motility is seen at pH 8. The Indigo-reducing alkalophilic bacterium (Bacillus sp.) isolated from indigo ball was used to improve the indigo fermentation process. Their cell wall contains acidic compounds similar in composition to peptidoglycans.
(i) Physiology:
The cell surface of alkalophiles can maintain the neutral intracellular pH in alkaline environment of pH 10-13. The recommended concentration of NaOH for large scale fermentation is 5.2% depending upon organism. The pH should remain 8.5-11. Sodium ions (Na+) are required for growth, sporulation and also for germination. The presence of sodium ions in the surrounding environment has proved to be essential for effective solute transport through the membranes.
In the Na+ ion membrane transport system, the H+ is exchanged with Na+ by Na+/H+ antiport system, thus generating a sodium motive force (smf). This drives substrate accompanied by Na+ ions into the cell.
The incorporation of α-aminobutyrate (AIB) increased two fold as the external pH shifts from 7 to 9, and the presence of Na+ ions significantly enhance the incorporation. Molecular cloning of DNA fragments conferring alkalophily was isolated and cloned. This fragment is responsible for Na+/H+ antiport system in the alkalophily of alkalophilic microorganisms.
(ii) Molecular Adaptation:
Alkalophiles contain unusual dither lipids bonded with glycerol phosphate just like other archaea. In these lipids, long chain, branched hydrocarbons, either of the phytanly or biphytanyl type, are present.
The intracellular pH remains neutral in order to prevent alkali-labile macromolecules in the cell. The intracellular pH may vary by 1-1.5 pH units from neutrality which helps these organisms to survive in highly alkaline external environment.
(iii) Applications:
Some alkalophiles produce hydrolytic enzymes such as alkaline proteases, which function well at alkaline pH. These are used as supplements for house hold detergents.
For example an alkaline protease called subtilisin has been produced from B. subtilis which is used in detergent. The stone washed denim fabric is due to the use of these enzymes. These enzymes soften and fade fabric by degrading cellulose and releasing dyes (Table 29.1).
Group # 3. Halophiles:
Halophiles are the Gram-negative, non-spore forming, non-motile bacteria that reproduce by binary fission. They appear red pigmented due to the presence of carotenoids but sometimes they are colourless. They contain the largest plasmid so far known among all the known bacteria.
Halophiles are able to live in salty conditions through a fascinating adaptation. Because water tends to flow from the areas of high to low solute concentrations. A cell suspended in a very salty solution will lose water and become dehydrated unless its cytoplasm contains a higher concentration of salt than its environment.
Halophiles contend with this problem by producing large amounts of an internal solute or by containing a solute extracted from outside. For example, Halobacterium salinarum concentrates KCl in the interior of the cell. The enzymes in its cytoplasm will function only if a high concentration of HCl is present. But their cellular proteins contacting the environment require a high concentration of NaCl.
This group of bacteria lives in highly saline environment (> 3.5% salt concentration) such as neutral salt lakes or artificial saline source like salted food, fish, etc. Extreme halophilic organisms require at least 1.5 M (about 9%) NaCl but most of them have optimum growth at 2-4 M NaCl (12.23%). Some examples of prokaryotic extremely halophilic bacteria occurring in nature are given in Table 29.2.
(i) Physiology:
Halophilic bacteria lack peptidoglycan in cell walls and contain ether-linked lipids and archaean type RNA polymerases but Natrobacterium is extremely alkalophilic as well. Former also contains diether lipids not present in other extreme halophiles.
They are chemoorganotrophic bacteria that require amino acids, organic acids and vitamins for optimum growth. Sometimes they oxidize carbohydrates as energy source. Cytochromes a, b and c are present but membrane mediated chemiosmosis generates proton motive force. They also require sodium for Na+ ions.
Halobacterium exceptionally thrives in osmotically stressful environment and does not produce compatible solutes. Peptidoglycan is absent in their cell wall. Aspartate and glutamate (acidic amino acids) are present.
The negative charges of the carboxyl groups of these amino acids are shielded by Na+ ions. The ribosomes of Halobacterium requires high K+ ions for stability, which is a unique feature as no other group of prokaryotes requires it for internal components.
The membrane lipids of these archaea are composed of diphytanylglycerol, diether analogues of glycerophospholipids. The extreme halophiles contain high intracellular concentration of Na+ and K+ and their proteins seem to have adapted to this high salt concentration by having a higher fraction of acidic amino acid residues and a more compact packing of a polypeptide chain than protein from non-halophilic bacteria. In the halophilic bacteria generally a Na+/H+ antiporter is used to pump Na+ outwards and solute uptake has been shown to be Na+ coupled in several halobacterial species.
(ii) Molecular Adaptation:
In such bacteria K+ ions inside the cell is more than Na+ ion outside the cell which act as its solute. Hence, the cells maintain cellular integrity. Halobacteria lack peptidoglycans in their cell walls and contain ether-linked lipids and archaean type RNA polymerases which maintain the rigidity at salty conditions. These changes in cytoplasmic membrane allow such bacteria to survive.
(iii) Applications:
Certain extreme halophiles synthesize a protein called bacteriorhodopsin into their membrane. Some produce polyhydroxy alkanoates and polysaccharides, enzymes and compatible solutes. They are also used in oil recovery, cancer detection, drug screening and biodegradation of residue and toxic compounds.
Kushner (1985) defined the halobacteria based on utilization of optimum salt concentration for their growth. In this system, non-halophiles are those that grow best in media containing < 0.2 M NaCl, slight halophiles (marine bacteria) grow best at 0.2 to 0.5 M NaCl, moderate halophiles at 0.5 to 2.5 M NaCl, and extreme halophiles grow in media containing 2.5 M to 5.2 M (saturated) NaCl.
It is interesting to note that all extreme halophiles are archaea except for two species of the photosynthetic Ectothiorhodospira, one of the Acetohalobium and one actinomycete Actinopolyspora. Some actinomycete species of the genus Methanohalobium has been described. The bright red colour water of the salterns is now known to be due to the bacterioruberin pigments of the halobacteria.
The biotechnological potential of halobacteria with commercial interest is following:
(a) Bacteriorhodopsin:
The retinal proteins of halobacteria have been observed as integral proteins of the purple membrane, containing one of the proteins called bacteriorhodopsin. This protein is light-driven, proton translocator and converts sunlight to electricity.
The bacteriorhodopsin absorbs light at 570 nm. It exists in two forms. The trans configuration after excitation converted to the cis form following the absorption of light (Fig. 29.1). In this case, ATP synthesis is prevented and the electrical potential arising from the proton gradient will be the source of electricity. It is used in optical data processing and as light sensors.
A photographic film based on purple membrane displays the interesting properties as it does not require developing. Holographic films of this type are suitable for computer memory i.e. parallel processing.
Recently, biochips have been introduced in new generation of computers. In future, robots with vision may have biosensors based on this protein. Desalination of water is also demonstrated by the application of bacteriorhodopsin.
(b) Bioplastic or polyhydroxy alkanoates (PHA):
This kind of heteropolymer is biodegradable. It exhibits total resistance to water and degraded in human tissues; hence it is biocompatible. It has pharmaceutical and clinical importance, including the use in delayed drug release, bone replacement and surgical sutures. Production of PHA is always higher by using Halof. mediterranei. In addition, these halobacteria possess high genomic stability which is a pre-requisite for industrial purposes.
(c) Polysaccharides:
Microbial exopolysacchrides are used as stabilizers, thickness, gelling agents and emulsifiers in the pharmaceutical industries, paint and oil recovery, paper, textile and food industry. Halof. mediterranei produces a highly sulphated and acidic heteropolysaccharides (up to 3 g/l) which contain mannose as a major component. Such a polymer combines excellent rheological properties with a remarkable resistance to extreme of salinity, temperature and pH.
(d) Microbially enhanced oil recovery:
Residual oil in natural oil fields can be extracted by injection of pressurized water down in a new well. The bacterial biopolymers are of interest in enhanced oil recovery because of their bio-surface activity and properties of bio-emulsifiers.
(e) Cancer detection:
A protein (84 kDa) has been used from Halobacterium halobium as an antigen to detect antibodies against the human e-myc oncogene product in the sera of cancer patient suffering from pyrolytic leukaemia cell line (HL-60). The use of halobacterial antigens as probe for some types of cancer seems to be promising.
(f) Drug screening:
Plasmid, pGRB-1 of Halobacterium strain GRB-1 used in the pre- screening of new antibiotics and anti-tumor drugs affect eukaryotic type IIDNA topoisomerase and quinotone drugs which act on DNA gyrase. Such drug causes DNA cleavage of small plasmid from halophilic archaea in vivo.
(g) Liposomes:
Ether-linked lipid of the halobacteria is used in liposome preparation having great value in the cosmetic industry. Such liposomes would be more resistant to biodegradation, good shelf-life and resistance to other bacteria.
(h) Enzymes:
Proteases and amylases from Halobacterium salinarium, H. halobium, and lipases from several halobacteria have been reported. A site-specific endonuclease activity has been reported in H. halobium.
(i) Bioremediation:
Bertrand (1990) observed that the halobacterial strain EH4 isolated from a salt-mark was found to degrade alkanes and other aromatic compounds in the presence of salt.
(j) Gas vacuoles or vesicles:
Some Halobacterium spp. produce intracellular gas filled organelles called vacuoles of gas vesicles which provide buoyancy. In the future, the genes of such properties are possible to engineer in other microorganisms to produce gas vacuoles to float in water.
(k) In food:
A sauce called ‘nam pla’ is prepared in Thai from fish fermented in concentrated brine that contains a large population of halobacteria responsible for aroma production. Because they produce salt-stable extracellular proteases. It has importance in the fermentation and the flavour and aroma producing processes.
(l) Other products:
Moderate halophiles remove phosphate from saline environment. Isolation of stable antimicrobial-resistant mutants is due to the presence of cloning of the genes for over-production of interesting industrially important compounds.
Large scale cultivation of Spirulina platensis in Israel uses brackish water which is unsuitable for agriculture and the Spirulina biomass is marketed as a healthy food. Spirulina grows optimally in alkaline lakes with a salt concentration ranging from 2 to 7%.
Group # 4. Psychrophiles:
Temperature is an important environmental factor which influences the different groups of microorganisms. Different groups of microorganism based on different temperature regime are given in Fig. 29.2.
Cold environments are actually more common similar to hot environment during summer. The oceans which maintain an average temperature of 1-3°C make up our half the earth’s surface. The vast land areas of the Arctic and Antarctica are permanently frozen or unfrozen for only a few weeks in summer.
James T. Staley and his colleagues at the University of Washington have shown that microbial communities populate ice ocean water of Antarctic sea that remains frozen for much of the years. These communities include photosynthetic eukarya, notably algae and diatoms as well as variety of bacteria.
Polasomonas vacuolata obtained by Staley’s group is a prime representative of a psychrophile. Psychrotolerant can be isolated from more widely distributed habitat than psychrophiles. They can be isolated from soil, water in temperate climates as well as meat, milk and other dairy products, vegetables and fruits under refrigeration.
They grow best between 20 and 40°C but cannot grow at 0°C. After several weeks of incubation their visible growth can be observed. Its optimum temperature for growth is 4°C, and 12°C for reproduction. The cold-loving microorganisms have started to interest manufacturers who need enzymes that work at refrigerator temperature such as food processors, makers of fragrances and producers of cold-wash laundry detergents.
Some psychrophiles can be dangerous organisms for man e.g. Pseudomonas syringae, Erwinia sp., Yersinia enterocolitica, etc. Most of the foods or food products are stored at freshing temperature so that the pathogenic or saprophytic microbes cease to grow.
A majority of marine microbes is psychrophiles due to their habitat (ocean). Generally, these are Gram-negative rod shaped bacteria. Among them are pseudomonads of which P. geniculata is the most common.
The other microbes are P. putrefaciens, P.fragi and P. fluorescens, Flavo bacterium spp, Alcaligenes spp, Achromobacter and a few strains of Escherichia, Aerobacter, Aeromonas, Serratia, Proteus, Chromobacter and Vibrio are psychrophilic in nature. The common psychrophilic yeasts are species of Candida, Cryptococcus, Rhodotorula and Torulopsis.
Physiologically the Gram-negative property of the bacteria and high proportion of G+C contents are present in such microorganisms. Psychrophiles contain an increased amount of unsaturated fatty acids in their lipids. Flagellum disappears after increasing the temperature.
(i) Physiology:
Psychrophiles produce enzymes that function optimally in the cold. Its cell membrane contains high content of unsaturated fatty acid which maintains a semi-fluid state at low temperature. The lipids of some psychrophilic bacteria also contain polyunsaturated fatty acids and long chain hydrocarbons with multiple double bonds.
(ii) Molecular Adaptation:
The active transport in such organisms occurs at low temperature. It indicates that the cytoplasmic membranes of psychrophiles are constructed in such a way that low temperature does not inhibit membrane function. The membrane contains polyunsaturated fatty acids in their lipids which maintain the rigidity at low temperature and organisms thus are able to survive.
(iii) Applications:
Psychrophiles and their products have many applications as described below:
(a) Source of pharmaceuticals:
Many psychrophiles such as Streptomyces, Alteromonas, Bacillus, Micrococcus, Moraxella, Pseudomonas and Vibrio have been isolated from deep-sea sediments. They grow at temperature between – 3 and -30°C. Aquatic plants and animals are highly prone to infestation by pathogenic microorganism. An Alteromonas sp. has been reported to synthesize 2, 3-indolinedione (isatin).
This compound protects Palaeoman macrodactylus from pathogenic fungus Lagenidium callinectes. Similarly, another strain of Alteromonas sp. is intimately associated with the marine sponge Halichondria okada and produces a tetracycline alkaloid e.g. alterimide. There is wide scope for the discovery of novel biologically active compounds in marine microbiology.
An antitumor polysaccharide has been isolated as narinactin from marine actinomycetes. A mixture of protease and amylase isolated from Bacillus subtilis removes the dental plaque.
Mainly lipases are used as stereo-specific catalysts and in the biotransformations of various high value compounds such as flavouring agents and pharmaceuticals. Trehalose is formed by an enzyme trehalase present in several psychrophilic bacteria.
(b) Bacterial ice nucleating agents:
There are several uses for ice-nucleating agents (INA) produced by bacteria. They are being used in artificial snow-making, in the production of ice creams and other frozen foods.
These are also used in immunodiagnostic kits as a conjugate to antibodies and as a substitute for silver iodide in cloud seeding. Among several organisms, bacterial INAs have attracted much attention due to its ability to form ice nuclei at relatively high temperature in comparison to other sources.
(c) Fermentation industry:
Mesophilic yeasts containing unsaturated fatty acids in membranes (lipids) have been found to be resistant between -80 and -20°C. These are preferred for its storage in baking and other processing industries. Fermentation at 6-8°C reduces the inhibitory effect of ethanol on cell membrane of the yeast cells.
(d) In microbial leaching:
Currently microbial leaching operations involve oxidative solubilization of copper and uranium ores. Leaching operation in temperate countries is carried out at very low ambient temperature. Microbial leaching operation from sulfide ores is carried out at 4-37°C.
(e) In bioremediation:
Psychrophiles have ability to degrade various compounds in their natural habitat. They are used in bioremediation of several pollutants at low temperature. The bacterial strains were found to mineralize dodecane, hexadecane, naphthalene, toluene, etc.
It has been demonstrated in laboratory and field experiments using specific bacterial strains. A psychrophilic bacterium, Rhodococcus sp. strain Q15 has been studied for its ability to degrade n-alkanes and diesel fuel at low temperature.
(f) Denitrification of drinking water sources:
The presence of high nitrate concentration in water has become a major problem in many countries. The most widely used practices for removal of NO3 is the biological denitrification. Most of the denitrification processes are carried out at 10°C. The rate of denitrification in these cases can be enhanced by employing psychrophilic bacteria isolated from permanently cold habitat.
(g) Anaerobic digestion of organic wastes:
The obligate anaerobes which convert organic acids to CH4 and CO2 i.e. methanogens are highly sensitive to low temperature. The rate of methanogenesis can be increased several times by low temperature adaptation by methanogens.
The process can be made possible by selective enrichment of psychrophilic methanogens through long term laboratory trials. Methanogenium frigidum isolated from Ace lake (Antarctica) grows optimally at 15°C. This bacterium is found to produce methane from hydrogen and carbon dioxide.
Group # 5. Thermophiles and Hyperthermophlies:
Hyperthermophilic bacteria are archaea that represent the organism at the upper temperature border of life. Neutrophilic and slightly acidophilic hyperthermophiles are found in terrestrial solfataric fields, and deep oils reservoir. These exhibit specific adaptations to their environments and most of the bacteria are strictly anaerobic.
Various factors both abiotic and biotic that control the growth of all living organisms are called biotope. The moderate thermophiles are called extreme thermophiles which grow optimally between 80°C and KWC. The hyperthermophiles are unable to grow below 80°C but adapted to high temperature as they do not even grow at 80°C.
Some of the examples are given in Fig. 29.3. Thermotoga has rod shaped cells surrounded by a characteristic sheath-like structure (the ‘toga’) which balloons out at the end (A). Archaeal coccoid sulphate reducers are the members of the genus Archeoglobus (B) and Methanopyrus kandleri is a rod shaped methanogen (C).
The hyperthermophiles can grow in natural as well as in artificial environmental conditions. Natural sulphur-biotopes are usually associated with active volcanism. In such situation, soil and surface waters from S-containing acidic fields (pH 0.5-6.0) and neutral to slightly alkaline hot spring environment persists.
Well-known biotopes (a biotope has upper and lower limits for growth for each of environmental factors) of hyperthermophiles are volcanic areas such as hot springs and solfataric fields i.e. high temperature fields located within volcanic zones with much sulphur acidic soil, acidic hot springs and boiling mud.
Few of hyperthermophiles live in shallow submarine hydrothermal systems and abyysal hot vent systems called “black smokers” having temperature of about 270-380°C. The black smokers are mineral-rich hot water that makes cloud of precipitated material on mixing with sea water. Other biotopes are smouldering coal refuse piles having acidic pH and geothermally heated soil reservoirs.
Most of the hyperthermophiles are anaerobic due to low solubility of oxygen at high temperature and the presence of red gases. Anaerobic chemolithoautotrophic hyperthermophiles completely independent on sun, but they could even exist in other planets also. Hydrothermal vents in the bottom of the ocean have temperature of 350°C or greater and also show the existence of hyperthermophiles.
The recently discovered non-volcanic biotope embedded in deep geo- thermal heated oil stratification of extracted fluids evidenced for such microbial communities.
For the cultivation of such bacteria, samples are brought to the laboratory without temperature control. They are isolated by enrichment culture technique with variation in composition of substrate and control of in situ temperature. Agar is not suitable, hence more heat-stable polymer such as gellan gum or polysilicate gels are used for solidification.
Many taxonomic types of cultured hyperthermophiles are already known so far. They represent 52 species belonging to 23 genera and 11 orders of hyperthermophilic bacteria and archaea known in literature. The organisms whose optimum growth temperature is < 45°C are called thermophiles and those above 80°C are called hyperthermophiles.
(i) Physiology:
The enzymes and proteins are much more stable than the other forms and these macromolecules function at high temperature. Thermophilic proteins have different amino acid sequences that catalyse the same reaction in a mesophile which allow it to fold in a different way and thereby show heat tolerant effect. All thermophiles contain reverse gyrase, a unique type 1 DNA topoisomerase that stabilizes DNA.
Heat stability of proteins from hyperthermophiles is also due to increased number of salt bridges (bridging of charges on amino acids by Na+ or other cations) present and densely packed highly hydrophobic interior of the protein, which have membranes rich in saturated fatty acids. This allows the membrane to remain stable and function at high temperature.
Most of the hyperthermophiles are archaea which do not contain fatty acids, the lipids in their membranes but instead have hydrocarbons of various lengths composed of repeating units of 5-6 compound phytans bonded by ether linkage to glycerophosphate.
With increase in temperature of growth an increase in degree of saturation, chain length and/or iso-branching of the acyl chains are observed. Sometimes, special lipids (the sterol like hopanoids) are present in thermophiles. These may also affect an adaptation to life at high temperature by making the membrane more rigid.
(ii) Molecular Adaptation:
These bacteria contain heat-stable enzymes and proteins which regulate various macromolecular functions at high temperature. The critical amino acids substituted in one or more locations in these enzymes allow them to fold in a different manner and thereby withstand the denaturing effect of heat resulting into the survival of these organisms.
Further, the cytoplasmic membrane contains lipids rich in saturated fatty acids, thus allow the membrane to remain stable and functional at high temperature. The thermophilic archaea do not contain fatty acids in their lipids, neither its membrane has ester linkages with glycerol phosphate. This imparts more rigidity to its membrane systems.
(iii) Applications:
Most of the microorganisms that thrive above the boiling point of water belong to archaea. The enzymes of thermophiles are of great interest. Hyperthermophiles have focused on thermostable enzymes from vent. The proteins (chaperons) were also discovered.
These proteins are expressed under stress conditions and involved in protein foldings:
(a) Enzymes:
New enzymes from hyperthermophiles have reduced the number of steps needed to transform starch into fructose syrup. The amylase, glucoamylase, pullunases and glucosidases are the enzymes used in starch industry.
Pullunases are found in anaerobic bacteria. Amylases are widely used in textile, confectionery, paper, brewing, and alcohol industries. Similarly, glucosidases are used for hydrolyzing lactose syrup and mixtures to glucose and galactose.
They may have clinical applications since there is evidence of a lactase deficiency in the population which is either inherited or is the result of ageing. Glucose isomerase is widely used in the food industry which converts glucose to fructose for use as sweetner.
Due to the thermal stability of the enzymes, hyperthermophiles have been the subject of intensive investigation. Thermostable enzymes are more resistant to the denaturing activities of detergents and organic solvents. The amylases have been extracted from Pyrococcus furiosus and Pyrococcus woessei.
Enzymes have been exploited from some archaea e.g. Desulfurococcus mucosus, Staphylothermus marinus, Thermococcus celer and Thermococcus litoralis. A toga- associated amylase has also been detected from Thermotoga maritima. This enzyme is active between 70 and 100°C at pH 6. Fervidobacterium pullunolyticum has the potentiality of producing thermotolerant enzyme optima at 90°C.
Certain bacteria and archaea such as P. woesei, P. furiosus, Thermococcus litoralis, T. celer, F. pennavorans, D. mucosus, etc. are reported to produce pullunase II (amylopullunase) having 90 kDa molecular weight with temperature optima 105°C and pH 6. Some of these (P. woesei and P. furiosus) also produce glucosidases with temperature optima 110-115°C. These are useful for the bioconversion of starch into various useful products of industrial significance.
A thermostable exo-4-β-cellobiohydrolase with a half life of 70 minutes at 108°C has been isolated from Thermotoga sp. strain FjSS3- B. Similarly, thermostable xylanases have been reported from Thermotoga maritima, T. neoplolitiana, T. thermarum. P. furiosus exhibited β-xylanosidase activity. The enzymes from Thermotoga sp. are extremely stable with half-life of 8h at 90°C.
The protein hydrolyzing enzymes (proteases) have been isolated, purified and characterized from a number of thermophilic and hyperthermophilic microorganisms specially Pyrococcus, Thermococcus, Sulfolobus, Staphylothermus and Desulfurococcus.
Pyrolysin, an enzyme associated with the cell envelope which is a serine-type protease has temperature optima of 110°C and a half life of 4h at 100°C. It has been identified and characterized in P. furiosus and P. woesei. The serine- protease from Sulfurococcus mucosus exhibits its activity at 100°C.
A unique protease which hydrolyses keratin of chicken feather, hair and wool has been characterized from a bacterium F. pennavorans. A thermophilic glucose isomerase was characterized and purified from Thermotoga maritima.
Ferredoxins from Thermoplasma acidophilum, Sulfolobus acidocaldarius and Desulfurococcus mobilis have also been investigated. Hydrogenase, having a half-life of 21 h at 80-85°C has been isolated from Pyr. furiosus. A thermoactive pyruvate- ferrodoxin-oxidoreductase (POR) which catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 has been detected in D. amylolyticus, H. butylicus, Thermococcus celer, Pyrococcus woesei, P. furiosus and Thermotoga maritima.
Enzymes involved in amino acid biosynthesis such as aromatic aminotransferase from Thermococcus litoralis and Sul. solfataricus have been detected. An extremely thermostable enzyme with optimum activity at 100°C has also been detected from Methanobacterium thermoformicicum. The purified enzyme from P. woesei and P. furiosus has molecular mass of identical subunits 45 kDa each. The enzymes have heat-stability up to 70% after heat treatment at 100°C for 1 hour.
Glutamate synthetase (GS) is responsible for the synthesis of glutamine from glutamate and ammonia. The half-life of partially purified GS is 2 hours at 100°C. Two thermo-active aromatic aminotransferases from Thermococcus lithoralis has been purified and characterized, which are active at 100°C temperature. The enzyme aspartate aminotransferase transferring amino group from glutamate to oxaloacetate has been detected in Sul. solfataricus.
Taq polymerase is very important enzyme used in molecular biology for the amplification of DNA using polymerase chain reaction (PCR). This enzyme found in Thermus aquaticus is active at 80°C at pH 8.
Simpson (1990) has investigated the other DNA polymerase from Themotoga sp. Certain archaea such as Sul. acidocaldarius and Sul. solfataricus consists of DNA polymerase of a single polypeptide chain with a molecular mass of 100 kDa. The DNA polymerase from P. furiosus has also been purified.
The DNA ligase has been characterized from Thermus thermophilus. Topoisomerases type I purified from Sulfolobus acidocaldarius, Desulfurococcus amylolyticus, Thermoplasma acidophilum, Fervidobacterium islandicum, Thermotoga maritima, and Methanopyrus kandleri, while topoisomerase II has so far been isolated from Sulfolobus acidocaldarius. Thermotoga maritima contains of both gyrase and reverse gyrase enzymes. Repair of extensive DNA damage caused by ionizing-radiation at 95°C has been demonstrated in Pyrococcus furiosus.
(b) Chaperons:
The chaperons are the proteins which express under stress conditions such as elevated temperatures. They are involved in protein folding. These are detected in Su. shibate and Su. solfactaricus. It is called thermophilic factor which has 55 kDa molecular mass. Due to increase in high concentration of intracellular protein up to 105°C, this protein complex is called thermosome.
The thermosome consists of a cylindrical complex of a two stacked identical rings, each unit consists of 8 subunits around a central channel. Both subunits contain 56 and 59 kDa molecular mass. They also bind the unfolded proteins similar to chaperons. A thermostable disulfide-bond forming enzyme has been isolated, characterized and purified from Sul. solfataricus.
Group # 6. Barophiles:
Barophiles are those bacteria that grow at high pressure at 400-500 atmosphere (atm) on 2 to 3°C. Such conditions exist in deep-sea habitat about 100 metre in depth. Many are barotolerant and do not grow at pressures above 500 atm. but some live in the gut of invertebrates (amphipods and holothurians).
Photobacterium shewanella and Colwella inhabit more rapidly. Some thermophilic archaea are barophiles e.g. Purococcus spp. and Methanococcus jannaschii. Barophiles adapt the extreme pressure (200-600 bars) involving macromolecular structures in cells. Increasing pressure makes structures more compacts, and this tendency has been the principle of microscopic ordering.
(i) Physiology:
There are variations in membrane structure and function. The amount of mono- unsaturated fatty acids in the membrane increases due to increase in the pressure. The organism is thereby able to circumvent the loss of membrane fluidity imposed by increasing the pressure. As the pressure decreases, membrane fluidity presumably increases and the cells respond by decreasing the level of mono-unsaturated fatty acids.
It is evidenced that increased pressure decreases the binding capacity of enzymes for their substrates. Thus the enzymes must be folded in such a way as to minimize these pressures in barophiles. It is not known whether H+, Na+ or both are used as coupling ions in energy transduction in these organisms.
(ii) Molecular Adaptation:
In the cytoplasmic membranes of high pressure tolerant microbes, the amount of unsaturated fatty acids is more which allows the adaptative significance. Further, the adaptativity may also be due to changes in protein composition of the cell wall outer membrane called OmpH protein, a type of porin.
The porins are structural proteins meant for diffusion of organic molecules through the outer membrane and in to the periplasm. It is observed that OompH system is pressure-dependent and required for growth at high pressure.
(iii) Applications:
Barophiles are the major source of unsaturated fatty acids or polyunsaturated fatty acid. The microbial barophilism is helpful in enhancing the mining. Underground mining operations usually occur at increased pressures and temperatures and barophilic thermophiles are better adapted under such situations.
Recently, vacant salt mine area has been worked out as fermenters for the biological gasification of pretreated lignite or agricultural crops based on the involvement of extremophiles endowed with adaptation to high pressure and temperature besides salinity.