Introduction

The extremophiles are those microorganisms whose optimal growth conditions are found outside of the normal environment. It is now well recognized that many parts of the world contain extreme environment such as polar regions, acidic and alkalophilic springs and cold pressurized depths of the ocean are colonized by microbes which are specially adapted to these exceptional environments. The restricted ranges of microbes have the ability to inhabit in extreme environments. Extremeis defined as the fact that microbes not only survive but actually grow in some of unusual environment on earth. It has stimulated scientific curiosity about the mechanisms permitting the survival and growth in such surroundings. These special organisms might provide a valuable resource for the exploitation of new chemical and biotechnological processes.

Most of the extremophiles are prokaryotes and archaea. They produce enzymes, antibiotics, etc. which have biotechnological importance. Thermostable enzymes for specific applications in industries are more robust than their low temperature relates and may show enhanced resistance to organic solvents. In addition, they remove and recover metals and degrade toxic pollutants.

This chapter describes the important groups of extremophiles such as acidophiles, alkaliphiles, halophiles, thermophiles and hyperthermophiles, psychrophiles and barophilic.

Acidophiles

Most natural environments on the earth are essentially neutral, having pH between 5 and 9. 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 is needed for membrane stability.

The highly acidic environment is formed naturally from geochemical activities such as the production of gases in hydrothermal vents and some hot springsand 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. 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, their enzymes providing adaptabilityare 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.

Physiology

The presence of certain fatty acids has been reported to provide special adaptations to growth and survival at extreme low pH. Acidophiles maintain the cytoplasm pHin 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 electrogenic potassium uptake which allows the cells to extrude more hydrogen and thus maintain the internal

Adaptation

 Most critical factor for obligate acidophily lies in the cytoplasmic brane. 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 s are required for stability of membrane that allows bacteria to survive.

Alkalophiles

Alkalophilies live in soils laden with carbonate and in Soda lakes, such as those found in the Rift Valley of Africa. Most alkalophilic prokaryotes studied have been aerobic non-marine bacteria and reported as Bacillus sp. Krulwich and Guffanti (1989) separated them into two broad categories: alkali-tolerant organisms pH 7.0-9.0which cannot grow above pH 9.5and alkalophilic organisms pH10 -12. Most of the alkalophilic organisms are aerobic or facultative anaerobic. The flagella induced motility is considered by a sodium motive force instead of proton motive force. 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.

Physiology

The cell surface of alkalophiles can maintain the neutral intracellular pH in alkaline environment of pH 10-13. The presence of sodium ions in the surrounding environment has proved to be essential for effective solute transport through the membranes. In the sodium ion membrane transport system, the hydrogen is exchanged with sodium hydrogen antiport system, thus generating a sodium motive force. This drives substrate accompanied by sodiumins into the cell. The incorporation of a-aminobutyrate increasedtwo-fold as the external PH shifts from 7 to 9 and the presence of sodium ions significantly enhance the incorporation. This fragment is responsible for sodium hydrogen antiport system in the alkalophily of alkalophilic microorganisms.

Adaptation

Alkalophiles contain unusual diether lipids bonded with glycerol phosphate just like other archaea. In these lipids, long chain, branched hydrocarbons, either of the phytanyl or bi phytanyl 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.

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 colorless. 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. Halophiles contend with this problem by producing large amounts of an internal solute or by containing a solute extracted from outside. Thehalobacterium salinarum concentrates KCI in the interior of the cell. The enzymes in its cytoplasm will function only if a high concentration of HCI 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 about 12.23% NaCl.

Physiology

Halophilic bacteria lack peptidoglycan in cell walls and contain ether-linked lipids and Archaean type RNA polymerases but Nitrobacterium 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.  The aspartate and glutamate amino acids are present. The negative charges of the carboxyl groups of these amino acids are shielded by sodium ions. The ribosomes of Halobacterium requires high potassium ions for stability, which 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 sodium and potassiumand 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 -halophilic bacteria. In the halophilic bacteria generally a sodium hydrogen antiporter is used to pump Na outwards and solute uptake has been shown to be Nacoupled in several halo bacterial species.

Adaptation

In such bacteria potassium ions inside the cell is more than Na ion side the cell which act as its solute. Hence, the cells maintain cellular integrity halo bacteria peptidoglycans in their cell walls and contain ether-linked lipids and archaeon type RNA polymerases which maintain the rigidity at salty conditions. These changes in cytoplasmic membrane allow such bacteria to survive.

Psychrophiles

Temperature is an important environmental factor which influences the different groups of organisms. Different groups of microorganisms based on different temperature regime are givenCold environments are actually more common similar to hot environment during summer. Theoceans which maintain an average temperature of 1-3°C make up our half the earth's surface. These communities include photosynthetic eukaryote, notably algae and diatoms as well as varietyof bacteria. They can be isolated from soil, water in temperate climates as well as meat, milk and other dairyproducts, 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 eg. Pseudomonas syringe and Yersinia enterocolitica, etc. Most of the foods or food products are stored at fleshing temperatureso 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. geniculate is the most common. The other microbes are P. putrefacient, P fragile and P. fluorescens, Flavobacterium sp. Alcaligenes sp. Achromobactersp.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+Ccontents are present in such microorganisms. Psychrophiles contain an increased amount of unsaturated fatty acids in their lipids.

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.

 Adaptation

The active transport in such organisms occurs at low temperature 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 maintains the rigidity at low temperature and organisms thus are able

Thermophiles

Hyper thermophilic bacteria are Archaea that represent the organism at the upper temperature border of life (Stetter, 1992). 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. The moderate thermophiles are called extreme thermophiles which grow optimally between 80°C and 100°C. The hyperthermophiles are unable to grow below 80°C but adapted to high temperature as they do not even grow at 80°C. Thermotoga has rod shaped cells surrounded by a characteristic sheath-like structure which balloons out at the end. Archaeal coccoid sulphate reducers are the members of the genus Archeoglobus and Methanopyrus is a rod-shapedmethanogen. 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 Sulphur containing acidic fields pH 0.5-6.0 and neutral to slightly alkaline hot spring environment persists. The well-known biotopes has upper and lower limits for growth for each of environmental factors of hyperthermophiles are volcanic areas such as hot springs andhigh temperature fields located within volcanic zones with much sulphur acidic soil, acidic hot springs and boiling mud.

Hyperthermophiles

They live in shallow submarine hydrothermal systems and abyssal 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 smoldering coal refuse piles having acidic pH and geothermally heated soil reservoirs (Fuchs, 1992). 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 plants 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.The cultivation of suchbacteria, 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 polyciliate gels are used for solidification. Many taxonomic types of cultured hyperthermophiles are already known so far. The organisms whose optimum growth temperature is 45°C are called thermophilesand those above 80°C are called hyperthermophiles.

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 catalase 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 of  topoisomerase that stabilizes DNA.

The heat stability of proteins from hyperthermophiles is also due to increased number of salt bridges 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 iso-branching of the acyl chains are observed.

 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. 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,

Barophiles

Barophiles are those bacteria that grow at high pressure at 400-500 atmosphere on 2- 3°C. Such conditions exist in deep-sea habitat about 100 meter in depth. Many are bar tolerant and do not grow at pressures above 500 atm. Photobacterium Shawnelle and Colwell inhabit more rapidly. The thermophilic archaea are barophiles such as Purococcus sp. and Methanococcus sp. Barophiles adapt the extreme pressure 200-600 bars involving macromolecular structures in cells.

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.

Adaptation

In the cytoplasmic membranes of high-pressure tolerant microbes, the amount of unsaturated fatty acids is more which allows the adaptative significance. The adaptivity may be due to changes in protein composition of the cell wall outer membrane calledH 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 SH system is pressure-dependent and required for growth at high pressure.

Conclusion

The potential applications of acid-tolerant extremozymes range from catalyststhe synthesis of compounds in acidic solutions to additives for animal feed which are intended work in animal stomach. When added to feed, the enzymes improve the digestibility of expensive grains, therefore avoiding the need for more costly food. Thiobacillus ferrooxidans has been established as biotechnological approach of bioremediation

Alkalophiles produce hydrolytic enzymes such as alkaline proteases, which function well at alkaline pH. These are used as supplements for house hold detergents. An alkaline protease called subtilisin has been produced from Bacillus 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.

Halophiles are used in oil recovery, cancer detection, drug screening and biodegradation of residue and toxic compounds.Psychrophiles are used in source of pharmaceuticals and denitrifying of drinking water sources. Thermophiles enzymes are most important used in molecular biology for the amplification of DNA using polymerase chain reaction.

Barophiles are the major source of unsaturated fatty acids or polyunsaturated rated fatty acid. The microbial barophilism is helpful in enhancing the mining. The underground mining operations usually occur at increased pressures and temperatures and barophilic thermophiles are better adapted under such situations,

References

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