ALKALIPHILES -THE EXTREMOPHILES

Alkaliphiles
Introduction:

As humans, we enjoy fairly temperate environmental conditions. , we can hang out on a pretty hot summer day, or even in cold winter evenings. But when we consider the vast ranges of environmental conditions present on Earth, humans are able to survie in limited environmental conditions. There are many environments in which life is present are unimaginable for human survival. One such environment is that of extreme pH, specifically, very basic or high pH environments (pH > 10). The organisms that thrive there are alkaliphiles.

Defination:
Alkaliphiles are extremophilic organism that are able to survie in high pH environment (pH>10)
.These organisms are further divided in to Alkalitolerant microorganism which may grow well up to pH 10 but exhibit more rapid growth below pH 9.5, Facultative Alkaliphiles that grow well at netural pH and Obligate Alkaliphiles that grow above pH 9. In addition to this because of their natural habitat many of these alkaliphiles organisms are adopted at high salt concentration n they are called as Haloalkaliphiles. They require both an alkaline pH (>pH 9) and high salinity (up to 33% [wt/vol] NaCl).
History:
The discovery of alkaliphiles was fairly recent. The use of alkaliphilic microorganisms has a long history in Japan, since from ancient times indigo has been naturally reduced under alkaline conditions in the presence of sodium carbonate. Indigo from indigo leaves is reduced by particular bacteria that grow under these highly alkaline conditions in a traditional process called indigo fermentation. The most important factor in this process is the control of the pH value. Formerly, indigo reduction was controlled only by the skill of the craftsman. Microbiological studies of the process, however, were not conducted until the rediscovery of these alkaliphiles The first paper concerning an alkaline protease was published in 1971. Genes responsible for the alkaliphily of Bacillus halodurans C-125 and Bacillus firmus OF4 have been analyzed.
Distribution and isolation
Alkaliphiles have been isolated mainly from neutral environments, sometimes even from acidic soil samples and feces. Haloalkaliphiles have been mainly found in extremely alkaline saline environments, such as the Rift Valley lakes of East Africa and the western soda lakes of the United States.
Habitat
The soda lakes in Africa are one example of a well-known environment with pH values greater than 9. The lakes are so alkaline that they could literally burn the skin right off most organism so one might guess that these waters might be devoid of fish, plants, or any life but when we take a closer look then we can see that the waters are teeming with life. Natronobacterium magadii, for example, was isolated from Lake Magadi, a soda lake located in the Rift Valley of Africa; N. magadii grows optimally at a pH of about 10. other examples include alkaline lakes of Kenya,Wadi Natrun in Egypt. Besides the soda lake alkaliphiles are also present in alkali soil. Alkali soil result from the complete oxidation of organic matter in the areas of high aeration and high temperature. Such area are seen in desert soil of western United States where the pH may be as high as 10
Alkaliphiles
Archaeal alkaliphiles
Halobacteriaceae
Halorubrum,
Natronbacterium
Natronococcus

Methanosarcinaceae
Methanohalophilus spp

Other Alkaliphiles are the members of Bacillaceae, Clostridiaceae and gama proteobacteria



Organism Ph Notes
Nitrosomonas spp 13
Nitrosobacter spp 13
Bacillus pasteurii 11 Hydrolyses urea at 10% n grow well,n have
specific req for ammonia
Bacillus sphaericus 11 don’t req ammonia
Bacillus pantothenticus 11 “
Bacillus rotans 11 “
Bacillus alkalophilous 10 it do not req ammonia nor
hydrolize urea
Bacillus circulans 11
Clostridium thermohydrosulfuricum anaerobic
Anaerobranca horikoshii. 6.9 to 10.3
Clostridium paradoxum, 7.6 and 9.8 alkali-thermophile


FUNGI
Penicillium variable 11
Fusarium Bullatum 11
Fusarium oxysporium 11

GREEN ALGAE
C.pyrenoidosa 11
C.ellipsoidea 11

BLUE GREEN ALGAE
Gloeothece linaris 10
Mycrocystis aeruginosa 10
Plectonema nostocorum 13

PROTOZOA
Eugelena gracilis 11

ARCHEON
Thermococcus alcaliphilus hyperthermophilic archaeon growing on polysulfide at alkaline pH and at temperatures between 56 and 90°C, from a shallow marine hydrothermal vents., with an optimum around 9.0. Thermococcus acidaminovorans. 9 anaerobic alkalithermophiles

Haloalkaliphiles
Natronobacterium pharaonis to be transferred to Natronomonas gen. nov. as Natronomonas pharaonis gen. nov. comb. nov.; Natronobacterium vacaolatum to be transferred to the genus Halorubrum as Halorubrum vacuolatum comb. nov.; and Natronobacterium magadii to be transferred to the genus Natrialba as Natrialba magadii.
The new nomenclacture was on the base of phylogenetic tree reconstructions,
and sequences of spacer regions between 16S and 23S rRNA genes.
OTHER ALKALIPHILES
Desulfonatronovibrio hydyogenovorans., an alkaliphilic, sulfate-reducing bacterium, from Lake Magadi. is an obligatory sodium-dependent alkaliphile,; , and the optimum pH is 9.5 to 9.7. The optimum NaCl concentration for growth is only 3% (wt/vol) (206-208).
halotolerant alkaliphilic obligate methanotrophic bacterium Methylobacter alcaliphilus The strains grow fastest at pH 9.0 to 9.5 NaCl for growth in alkaline medium.

Industrial Applications of Alkaliphilic Enzymes
Alkaliphilic enzymes have many industrial applications.
Proteases are used as detergent additives And in the hide-dehairing process.

many alkaline proteases have been produced by alkaliphilic Bacillus strains and are commercially available.
(iii) Other applications. An interesting application of alkaline protease is the use of an alkaline protease to decompose the gelatinous coating of X-ray films, from which silver was recovered.
chitinases from the alkaliphilic Nocardiopsis albus

Hide
Proteases are a group of enzymes whose catalytic function is to hydrolyze (breakdown) peptide bonds of proteins (also called proteolytic enzymes or proteinases; proteinase K is a common protease used in laboratory DNA extractions). Proteases are extracellular. They are capable of digesting insoluble polymers such as cellulose, protein, and starch. The products of digestion are then transported into the cell where they are used as nutrients for growth.
Alkaline proteases have an optimum pH greater than or equal to 9.0. They hydrolyze proteins and break them down into more soluble polypeptides or free amino acids. Alkaline proteases are the most widely used enzymes in the detergent industry. They remove stains such as grass, blood, egg, and human sweat. For an enzyme to be used as a detergent additive it must be stable and active in the presence of typical detergents ingredients, such as surfactants, builders, bleaching agents, bleach activators, fillers, fabric softeners, and various other formulation aids. While alkaliphilic bacteria are not the only source of alkaline proteases, Bacillus subtilis (neutrophilic) and Bacillus licheniformis (alkaliphilic) are major players in the detergent industry.
In addition to detergent additives, alkaline proteases are used in the hide-dehairing process, where dehairing is carried out at pH values between 8 and 10. These enzymes are also commercially available from companies.
Starch-degrading enzymes, such as amylase, hydrolyze starch to produce glucose, maltose,
Pullulanase is a good candidate for a dishwashing detergent additive. In some food industries, enzymes showing activity at lower temperatures have been requested for use in food processing. Psychrotrophic bacteria are thought to be potential producers of these enzymes. an alkalipsychrotrophic strain, Micrococcus sp. strain 207, extracellularly produced amylase and pullulanase.

Cellulases are used as laundry detergent additives.
Xylanases are a possible application in biologic debleacing processes (i.e., in pulp-milling factories)
. Since the demonstration that alkali-treated wood pulp could be biologically bleached by xylanases instead of by the usual environmentally damaging chemical process involving chlorine, the search for thermostable alkaline xylanases has been extensive
Four thermophilic alkaliphilic Bacillus strains (W1, W2 , W3, and W4) produced xylanases
Pectinases improve the production of paper by making it high-quality, nonwoody, and stronger. They are also used in waste treatment.Lipase Two bacterial strains were selected as potent producers of alkaline lipase. These were identified as Pseudomonas nitroreducens and P. fragi.
BIOCHEMISTRY, MOLECULAR BIOLOGY AND PHYSIOLOGY OF ADAPTATION
Internal pH
Most alkaliphiles have an optimal growth pH at around 10, which is the major difference from neutrophilic microorganisms. Therefore, the question arises how these alkaliphilic microorganisms can grow in such an extreme environment. Now internal cytoplasmic pH can be estimated from the pH of intracellular enzymes. For example, -galactosidase from an alkaliphile, Micrococcus sp. strain 31-2, had its optimal catalytic pH at 7.5, suggesting that the internal pH is around neutral. Another method to estimate internal pH is by measuring the distribution of weak acid inside and outside of the cell, which are not actively transported by cells. The internal pH was maintained at around 8, despite a high external pH of 8 to 11, as shown in Table1.Therefore, one of the key features in alkaliphily is associated with the cell surface, which maintains the intracellular neutral environment separated from the extracellular alkaline environment.
Acidic polymers. Since the protoplasts of alkaliphilic Bacillus strains lose their stability in alkaline environments, it has been suggested that the cell wall may play a key role in protecting the cell from alkaline environments. Components of the cell walls of several alkaliphilic Bacillus spp. have been investigated. In addition to peptidoglycan, alkaliphilic Bacillus spp. contain certain acidic polymers, such as galacturonic acid, gluconic acid, glutamic acid, aspartic acid, and phosphoric acid . The negative charges on the acidic nonpeptidoglycan components may give the cell surface its ability to adsorb sodium and hydronium ions and repulse hydroxide ions and, as a consequence, may assist cells to grow in alkaline environments.
Peptidoglycan. The peptidoglycans composition is same but have an excess of hexosamines and amino acids in the cell walls compared to that of the neutrophiles.
pH Homeostasis
Na+ Ions and Membrane Transport
Alkaliphilic microorganisms grow vigorously at pH 9 to 11 and require Na+ for growth. According to the chemiosmotic theory, the proton motive force in the cells is generated by the electron transport chain or by excreted H+ derived from ATP metabolism by ATPase. H+ is then reincorporated into the cells with cotransport of various substrates. In Na+-dependent transport systems, the H+ is exchanged with Na+ by Na+/H+ antiporter systems, thus generating a sodium motive force, sodium then re enters into the cell along with the other substrates from na+ symporter. Thus the internal ph is maintained.diagram
Cell wall :
The exterior surface of the cell are also important for maintaining a pH differential. This is supported by evidence that the protoplasts of alkaliphilic Bacillus spp are unstable in alkaline conditions. The peptidoglycan in these strains has higher cross-linking rate at higher pH values,which may provide a shielding effect by “tightening” the cell wall. Large amounts of acidic compounds, including teichuronic acid, teichoic acid, uronic acids, and acidic amino aicds, are wvident in alkaliphilic cell walls compared to the cell walls of nonalkaliphilic microorganisms. The negative charge of these acidic substances may create a more neutral layer close to the outer surface of the cell.
Genetics
How alkaliphiles adapt to their alkaline environments is one of the most interesting and challenging topics that might be clarified by genome analysis. Physical maps for alkaliphilic B. firmus OF4 is published. A physical map of B. firmus OF4 is consist of a circular chromosome of approximately 4 Mb, with an extrachromosomal element of 110 kb (49, 172). Although analysis is still in progress, several open reading frames for Na+/H+ antiporters that may play roles in pH homeostasis have been detected.
Enzymology
ALKALINE ENZYMES studies of alkaliphiles have led to the discovery of many types of enzymes that exhibit interesting properties.
Alkaline Proteases
alkaline serine protease from alkaliphilic Bacillus sp. strain 221. The optimum pH of the enzyme was 11.5, and 75% of the activity was maintained at pH 13.0. . The molecular weight of the enzyme was 30,000, Subsequently, two Bacillus strains, AB42 and PB12, were reported which also produced an alkaline protease These strains exhibited a broad pH range (pH 9.0 to 12.0), with a temperature optimum of 60°C for AB42 and 50°C for PB12. thermostable alkaline protease from a thermophilic alkaliphile, Bacillus sp. strain B18. The optimum pH and temperature were pH 12 to 13 and 85°C, Extracellular endopeptidase from a strain of Bacillus pumilus
keratinase of a feather-degrading Bacillus licheniformis strain, PWD-1. This enzyme was stable from pH 5 to 12.

Structural analysis by X-ray analysis to understand the alkaline adaptation mechanism of the enzyme .This analysis revealed a decrease in the number of negatively charged amino acids (aspartic acid and glutamic acid) and lysine residues, and an increase in arginine and neutral hydrophilic amino acids (histidine, asparagine, and glutamine) residues during the course of adaptation.
Enzyme urease from B.pasteurii optimun 9.2 ph
Ezyme pectinase ph 10to 10.5
Alkaline amylase ph 9.2 to 10.5
Alkaline catalase ph 10
Beta 1,3- gluconase ph 8.5

Significance
Alkaliphiles, have tremendous potential in many commercial applications.They are not only valuable resources for developing novel biotechnological processes, but also ideal models for investigating how biomolecules are stabilized when subjected to extreme environments. Studies of the life and ecological phenomena in extreme environments will help to gain more insights into the evolution of life and the development of special ecosystems.. These understandings will ultimately be used to improve the efficiency of , enzymes,which will heavily contribute to the development of industrial biotechnology .