In this article we will discuss about:- 1. Meaning of Thermophiles 2. Environments Suitable for Growth of Thermophiles 3. Molecular Adaptations 4. Biotechnological Applications.
Meaning of Thermophiles:
The thermophiles are the microorganisms that grow at high temperature of 55°C or more (min. 45°C, optimum between 55-65°C, maximum 80°C). Some micro-organisms grow even at more high temperature, the optimum between 80°C and about 113°C, and are called hyper-thermophiles.
The later usually do not grow well below 55°C. Thermus aquaticus, Thermoplasma acidophilum, Bacillus stearothermophilus, etc. are the examples of thermophiles, whereas Pyrococcus abyssi and Pyrodictium occultum exemplify hyper-thermophiles.
Environments Suitable for Growth of Thermophiles:
Temperatures suitable for growth of thermophiles and hyper-thermophiles are found in nature only in certain areas. For convenience, soils subject to full sunlight are often heated to temperatures above 50°C at midday, and some soils may become warmed to even 70°C, although a few centimeters under the soil-surface the temperature is much lower.
Compost piles and silage, where the materials ferment, possess temperatures up to 70°C. However, the most extensive and extreme high-temperature habitats occurring in nature are in association with volcanic phenomena. These include, in particular, hot springs.
Most hot springs have temperatures at or near boiling. Steam vents (fumaroles) may contain temp, to the level of 150-500°C. Hydrothermal vents present in sea-bottom have temperatures of 350°C or greater.
Hot spring are found throughout the world, but are especially concentrated in the Western United States, central America, central Africa, New Zealand, Indonesia, Italy, Japan, and Iceland. The world’s largest single concentration of hot spring is in Yellowstone National Park, Wyoming (USA).
Thermophiles and Hyper-Thermophiles in Hot Springs and Hydrothermal Vents:
Many hot springs are at the boiling point and in them a variety of hyperthennophiles are typically present. The growth of such microorganisms can be studied by immersing microscope slides into the spring and retrieving them after a few days. Microscopic viewing of the slides reveals colonies of prokaryotes (Fig. 19.17) that have developed form single prokaryotic cells that attached to and grew on the glass surface.
The boiling water overflows the edges of the hot spring and flows away from the source; it gradually cools, setting up a thermal gradient. Along this gradient, various thermophiles (e.g., cyanobacterium Synechoccus) grow, with different species growing in different temperature ranges.
Large number of sulfur-oxidising chemo-lithotrophie hyperthermophiles are preset in and around sulfide- emitting hydrothermal vents. Samples collected near such vents have yielded cultures of Thiobacillus, Thiomicrospira, and Beggiatoa. At certain vent sites of the deep sea superheated hydrothermal fluid is emitted at temperatures upto 350°C.
This superheated but not boiling water (at a depth of 2600 m in sea, water does not boil due to huge hydrostatic pressures until it reaches a temperature of 450°C) could theoretically be a habitat for hyperthermophilic bacteria.
The hydrothermal fluid emitted from black smokers (a deep-sea hydrothermal vent emitting superheated 250-400°C water and minerals) contains abundant metal sulfides, especially iron sulfides, and cools quickly as it mixes with cold sea water.
The precipitated metal sulfides form a tower referred to as a “chimney”. It is found that the walls of smoker chimneys teem with hyperthermophilic prokaryotes such as Methanopyrus. The most thermophilic of all prokaryotes, species of Pyrolobus and Pyrodictium also reside in smoker chimney walls.
Molecular Adaptations to Thermophiles:
Following are the factors that help thermophiles and hyperthermophiles thrive at high temperatures:
1. Enzymes and other proteins of thermophiles often differ to some extent in their amino acid sequence from enzymes that catalyze the same reaction in mesophiles. It appears that a critical amino acid substitution in only a few locations in the enzyme of thermophiles allows it to fold in way that makes them much more stable to heat and, as a result, they function optimally at high temperatures.
2. Thermophiles typically possess lipids rich in saturated fatty acids in their cytoplasmic membranes thus allowing the membranes to remain stable and functional at high temperatures. Saturated fatty acids form a stronger hydrophobic environment than do unsaturated fatty acids. Such a hydrophobic environment helps for the membrane stability in thermophiles.
3. Heat stability of proteins in hyperthermophiles is also improved as a result of an increased number of ionic bonds between the positive and negative charges of various amino acids. This makes densely packed highly hydrophobic interiors of the proteins, which naturally resist unfolding of proteins in the aqueous cytoplasm.
Also, the solutes di-inositol phosphate, diglycerol phosphate, and manosylglycerate that are produced in high amounts in the cytoplasm of certain hyperthermophiles help stabilise their proteins against thermal degradation.
4. Since most of the hyperthermophiles are archaebacteria, they do not possess fatty acids at all in their membranes, but instead contain lipids having “branched” C40 hydrocarbon chains composed of repeating units of the five-carbon compound, called isoprene, bonded by ether-linkage (-O-).
The ether-linkages provide more stability to membranes against thermal breakage and the branching of the hydrocarbon chains decrease membrane fluidity. Therefore, the membranes of hyperthermophiles are stable under high temperature conditions.
Additionally, the overall structure of cytoplasmic membranes of hyperthermophiles forms a lipid monolayer and this structure is undoubtedly much more heat stable in comparison to lipid bilayer of species of bacteria and eukaryotes.
Biotechnological Applications of Thermophiles:
Thermophilic and hyperthermophilic microorganisms offer some major advantages for industrial and biotechnological processes, many of which run more rapidly and efficiently at high temperatures. Extremozymes and molecular chaperons obtained from hyperthermophiles are the examples.
The term ‘extremozyme’ has been coined to describe enzymes that function at some environmental extreme such as high temperatures and low pH.
Since most of the industrial processes operate best at high temperatures, extremozymes from hyperthermophiles are widely used in both industry and research, and such extremozymes are proteases, amylases, glucose isomerase pullulanases, xylanases, glucoamylases, Tag and Pfu DNA polymerases, glutamate synthetase (GS), and aminotransferases.
Proteases (protein hydrolysing enzymes) have been isolated, purified and characterised from Pyrococcus, Thermococcus , Sulfolobus, Staphylothermus, and Desulfurococcus. Pyrolysin, a serine-type protease, has temperature optima of 110°C and a half-life of 4 hour at 100°C. The serine-protease from Sulfurococcus mucosus exhibits its activity at 100°C.
Amylases have been extracted from Pyrococcus furiosus and P. woessei, and are widely used in textile, confectionary, brewing, paper, and alcohol industries.
(iii) Glucose isomerase:
Glucose isomerase is widely used in the food industry. This enzyme converts glucose to fructose for use as sweetener.
Pullulanase II (amylopullulonase), obtained from Pyrococcus woessei, P. furiosus, Thermococcus lithoralis, T. celer, Fervidobacterium pennavorans, and Desulfurococcus mucosus, has temperature optima 105°C and is useful for the bioconversion of starch into various useful products of industrial significance.
(v) Taq and Pfu DNA polymerases:
Taq and Pfu DNA polymerases are used in molecular biology for the amplification of DNA using polymerase chain reaction (PCR). Taq polymerase found in Thermus aquaticus is active at 80°C at pH 8.
(vi) Glutamase synthetase:
Glutamase synthetase (GS) is active at 100°C and is used for the synthesis of glutamine from glutamate and ammonia.
Two thermo-active aromatic aminotransferases has been purified and characterised from Thermococcus lithoralis, and are active at 100°C. The enzyme aspartate aminotransferase that transfers amino group from glutamate to oxaloacetate has been isolated from Sulfolobus solfataricus.
2. Molecular Chaperons:
Molecular chaperons are a type of proteins that express under stress conditions such as elevated temperatures and are involved in protein folding. They do not become part of the assembled proteins, but only assist in the folding process. Molecular chaperons seem to be both extremely widespread and their sequences highly conserved.
Indeed, one important function of molecular chaperons is to prevent improper aggregation of proteins. Molecular chaperons, in addition to folding newly synthesised proteins, can also refold proteins that have partially denatured in the cell before proteases recognise them as improperly folded and destroy them (Fig. 19.18). Molecular chaperons have been isolate from Sulfolobus shibate and S. solfataricus.