Metal Tolerance in Halotolerant Bacteria

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The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them -W.L.Bragg

A vast variety of organisms belonging to different domains of life (Archaea, Bacteria and Eukarya) reside in extreme habitats i.e. those that lie outside the range of conditions in which most of organisms live. MacElroy in 1974 coined the term extremophiles. Such organisms were described by Kristjansson and Hreggvidsson (1995) as those which thrive beyond ‘ normal ’ environmental parameters. They may thrive either in physical extremes, like temperature, radiation, pressure, or geochemical extremes, like desiccation, salinity, pH, depletion of oxygen, extreme redox potential, or more than one extreme condition simultaneously. By studying such extremophiles and their characteristic environment new insights may be gained that are important for the study of evolutionary relationships, emergence of new species and various ecological relationships among organisms which compensate certain environmental externalities. Extremophiles isolated from diverse habitats can be studied for certain novel metabolic pathway/metabolites (enzymes) characteristic to them that may have certain bioremediation potential (Oarga, 2009).

They also produce biocatalysts with unique properties that function under extreme conditions comparable to those prevailing in various industrial processes (Niehaus et al., 1999; Kumar et al., 2011). Extremophilic bacteria follow different metabolic pathway and produce novel substances under stress (Kanekar et al., 2008).

Extremophiles are of following types: psychrophiles (thrive at low temperatures), thermophiles (high temperature), acidophiles (low pH), alkaliphiles (high pH), piezophiles (under extremes of pressure), xerophiles (desiccation), and halophiles (salinity).

Halophiles are a class of extremophiles that grow at high salt conc. The greatest part of the biosphere is saline which inhabits a large diversity of organisms. Oceans and sea water contain 35 g/L dissolved salts. Halophiles are present in all three domains of life (Archaea, Bacteria and Eukarya). Pigmentation is also seen in numerous halophiles. They even impart color to their habitat where they thrive, eg., Bacterioruberin pigment in the membrane of red halophilic archaea belonging to the family Halobacteriaceae are responsible for pigmentation of brines; presence of bacteriorhodopsin may contribute a purple color etc (Oren, 2002).

Halophiles have been classified by Kushner on the basis of their salt requirement into following categories (Table 1)

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Table 1. Classification of halophiles based on salt requirement

( Nieto 1989; Oren, 2006)

Halophiles are being explored for the following (Margesin and Schinner, 2001).

- Bacteriorhodopsin (pigment from halophiles) has applications in holography, spatial light modulators, optical computing, and optical memories.
- Compatible solutes are useful as stabilizers of biomolecules and whole cells, salt antagonists, and stress-protective agents.
- Biopolymers (biosurfactants and exopolysaccharides) are of interest for microbially enhanced oil recovery.
- Halotolerant microorganisms play an essential role in food biotechnology for the production of fermented food and food supplements

These products can be obtained from non-halophiles, but halophilic microorganisms may present advantages over the use of non-halophilic counterparts (Oren, 2002).

Few microorganisms can adapt to life over whole salt conc. range from fresh water to halite saturation like Halomonas elongate. Among the halophilic prokaryotes some are adapted to [Abbildung in dieser Leseprobe nicht enthalten]This article is not included in your organization's subscription. However, you may be able to access this article under your organization's agreement with Elsevitolerate other forms of stress in addition to salt stress i.e. thermophilic, psychrophilic, and alkaliphilic halophiles are known. Acidophilic halophiles have not been reported (Oren, 2006).

1.1 Isolation of halophiles

Halophilic microorganisms can be isolated from different saline environments and different strains even belonging to the same genus have various applications (Zhuang et al., 2010). [Abbildung in dieser Leseprobe nicht enthalten] This article is not included in your organization's subscription. However, you may be able to access this article under your organization's agreement with ElseIt is difficult to generalize and describe media and conditions suitable for all or even a group of halophiles. They largely depend on the source from where the isolation is attempted. Most halophilic bacteria generally grow best on slightly alkaline pH. The media is thus modified accordingly like seawater is being added to the media for isolation of halophiles from tidal flats. Similarly, optimum temperature for isolation and cultivation also depends on source of sample (Flannery, 1956).

Halophiles are a diverse class with respect to metabolic diversity also. They include oxygenic and anoxygenic phototrophs, aerobic heterotrophs, fermenters, denitrifiers, sulfate reducers, and methanogens. This metabolic diversity decreases with salinity (Oren, 2002).

Some studies have shown the dependence of salt requirement and tolerance on temperature as in the case of Haloferax volcanii (halophilic archaea) whose minimum and optimum salt concentrations shifted to higher values with increasing temperature. Another example is that of Halomonas halophila which requires 7.5% salt as optimum conc. for growth at 32 and 42º C ; whereas 5% salt as optimum at 22º C (Ventosa et al., 1998).

A large no. of halophilic actinomycetes have been discovered from saline soils of different locations (Solanki and Kothari, 2011). Studies concerning their biological characterization have also been done indicating that halotolerant and halophilic actinomycetes have extensive adaptability to Na+, K+ and Mg2+ (Tang et al., 2002). Their potential applications like enzyme production, antimicrobial action, bioremediate heavy metals, solvent tolerance, biosurfactant production etc. have also been explored.

Greater biocatalytic potential have been reported in halotolerant/halophilic organisms, among the various groups of extremophiles. They produce extracellular enzymes, some of which are shown to work optimally at alkaline pH, eg., Streptomyces clavuligerus strain MIT-1 is a salt tolerant and alkaliphilic organism which secretes alkaline protease. Its growth and protease production was optimum at pH 9 (Thumar and Singh, 2007; Solanki and Kothari, 2011).

1.2 Strategies to tolerate salt

All halophiles must maintain their cytoplasm isoosmotic with their surrounding medium. Biological membranes are permeable to water, and active energy-dependent inward transport of water to compensate for water lost by osmotic processes is energetically not feasible. Moreover, cells that keep a turgor need even to maintain their intracellular osmotic pressure higher than that of their environment.

There are two fundamentally different strategies used by halophilic microorganisms to balance their cytoplasm osmotically with their medium (Galinski and Trüper, 1994; Zahran, 1997).

(1) Accumulation of molar concentrations of potassium and chloride. This strategy requires extensive adaptation of the intracellular enzymatic machinery to the presence of salt, as the proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. It is called the 'high-salt-in strategy '. Microorganisms employing such strategy generally cannot survive in low salt media. This strategy is energetically less costly to the cell, but still not widely used among the different phylogenetic and physiological groups of halophiles. Examples of organisms employing this strategy are Halobacterium salinarum, Haloarcula marismortui etc .

(2) Second strategy is based on the biosynthesis and/or accumulation of organic osmotic solutes. Cells utilizing this strategy exclude salt from their cytoplasm as much as possible. The high concentrations of organic 'compatible' solutes do not greatly interfere with normal enzymatic activity. Few adaptations of the cells' proteome are therefore needed. Organisms employing this strategy can often adapt to a surprisingly broad salt concentration range. Many organic compounds (amino acids, glycine betaine, ectoine, hydroxyectoine etc.) serve as osmotic solutes in halophilic microorganisms, in both prokaryotes and eukaryotes. Most compatible solutes are based on amino acids and amino acid derivatives, sugars, or sugar alcohols, and are either uncharged or zwitterionic (Kurz, 2008; Vreeland, 1987). This 'low-salt-in strategy' of haloadaptation with accumulation of organic osmotic solutes is widespread in the small subunit rRNA sequence-based phylogenetic tree of life.

All groups of halophiles have not yet been examined for the occurrence and distribution of organic solutes (Oren, 2008). The osmotic solutes described earlier may be either produced by de novo synthesis or the organism may accumulate it from the environment (medium). The latter mechanism is preferred over de novo synthesis (Galinski and Trüper, 1994; Oren, 1999; Irwin and Baird, 2004). The intracellular accumulation of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya (Kempf and Bremer, 1998).

Ectoines constitute predominant class of osmolytes accumulated by halophiles. Eg. Halomonas elongata strain KS3 have been shown to accumulate ectoine and hydroxyectoine as compatible solutes (Ventosa et al., 2008).

Occurrence of certain solutes in halophilic/halotolerant prokaryotes is often correlated with their position in the phylogenetic tree of life, i.e. a few solutes in the domain Archaea have not yet detected elsewhere within the tree.

Halophilic methanogens like Methanohalophilus species contain, in addition to glycine betaine found widespread in nature, β-amino acids and derivatives that are rarely found in other groups: β-glutamine, β-glutamate, and Nε-acetyl-β-lysine. Sulfotrehalose has thus far been found only in a few alkaliphilic members of the Halobacteriaceae; it is accumulated in substantial concentrations (up to 1 M) in addition to KCl which serves as the main osmotic solute like in their neutrophilic relatives (Oren, 2008).

Salt-tolerant bacteria usually exhibit structural modifications to cope with salt stress. One important aspect of structural adaptations is the change in composition of the cell envelope and membranes. The stretched state of the wall and the internal osmotic pressure of bacteria are usually affected by the biophysical properties of the stress bearing peptidoglycan. Changes in composition of bacterial membranes which might be caused by environmental factors are thought to act as an adaptive response to maintain membrane stability and function. In fact, structural adaptations of membranes mainly involve alterations in the composition and synthesis of proteins, lipids and fatty acids (Zahran, 1997).

Paul et al. (2008) from India compared the genomes (genes) and proteomes (protein patterns) of halophiles and non-halophiles. From their experiments it was concluded that halophilic proteins have following features:

1. Low hydrophobicity, i.e. they are more hydrophilic than hydrophobic
2. Lots of acidic amino acids – especially aspartic acid; under-representation of cystine
3. Limited helix formation and higher occurrence of coiled structures

At the DNA level, the dinucleotide abundance profiles of halophilic genomes have some distinctive and common characteristics, i.e., specific DNA salt-adaptation signatures. Thus, it may halophiles have special membrane proteins and internal proteins that enable them to survive salty environments (Reinhardt, 2010).

1.3 Haloalkaliphiles

The group of bacteria able to grow under alkaline conditions in the presence of salt are referred as haloalkaliphiles. They have twin extremities of pH and salinity (Singh et al., 2010). These organisms require high concentrations of NaCl, high pH (8.5 - 11), and low Mg2+ (<10mM) (Jones and Grant, 1999). Tindall in 1984 first mentioned the occurrence of haloalkaliphiles. Such organisms possess special adaptation mechanisms for survival in high salinity and alkaline pH that make them interesting not only for fundamental research but also towards exploration for applications (Horikoshi, 1999; Singh, 2010). Mostly they have been isolated from alkaline soda lakes but some are being isolated from natural saline habitats as well. Limited attempts have been made to explore their enzymatic potential. Haloalkaliphilic organisms isolated from natural saline habitats of coastal Gujarat have been studied for amylase and protease production. All the isolates secreted protease in haloalkaline medium, but none secreted amylase. Higher salt was required for the enzyme secretion (Dodia et al., 2006). Salinity is also an important defining factor in the alkaline lakes.

Study of haloalkaliphilic bacteria isolated from sea water, saline soil and other saline habitats (coastal Gujarat) have indicated extracellular secretion of proteases, amylases, chitinase and lipases. The patterns revealed that the secretion and properties of extracellular enzymes could also be useful as marker to judge the microbial heterogeneity among the haloalkaliphilic bacteria. Majority of these enzymes were also thermostable in nature where salt acted as positive effectors. Further, majority of the enzymes displayed salt-dependent resistance against chemical denaturation, a rare feature among the proteins. Therefore, these enzymes could provide a unique model to study protein folding and stability under set of extreme conditions (Singh et al., 2010).

Novel members belonging to this category of organisms have also been discovered from lake in California, eg., Spirochaeta americana sp. nov ., that grows at 370C, 3% NaCl w/v and pH 9.5 (Hoover et al., 2003).

Moderately halophilic and alkalitolerant Halomonas campisalis, isolated from alkaline lonar lake (India), was able to produce enzymes amylase and lipase at alkaline pH. It showed the presence of PHA (Polyhydroxyalkanoate) within 24 h of incubation with a good yield of 75%. It is the first report on production of PHA by H. campisalis (Kanekar et al., 2008).

1.4 How they tolerate metals?

Certain transition metals like manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn) serve as essential cofactors in the physiology of all organisms. Recently it has been estimated that over half of all proteins in every organism are metalloproteins. They are essential in trace amounts, but are toxic at higher levels as they directly/indirectly compromise DNA, protein, and membrane integrity and function thereby proving lethal to the cell. This is evident from the eg. cycling in redox states of metals such as Fe and Cu and antioxidant scavenging by redoxinactive metals such as Zn can both cause oxidative damage to cell components through increased production of reactive oxygen species (Kaur et al., 2006).

Metal toxicity is being avoided/resisted by organisms by the following processes: selective uptake, trafficking, and efflux of metal ions, enzymatic conversion of metals into non- or less-toxic redox states, or sequestering toxic metal ions with ferritins and metallothioneins. These mechanisms are often regulated by free metal-ion concentration. Therefore all the other factors such as salinity, pH, temperature, and growth-medium components that can alter effective free metal ion concentration in the cell influence the metal stress response (Kaur et al., 2006).

Heavy metals influence the microbial population by affecting their growth, morphology, biochemical activities and ultimately resulting in decreased biomass and diversity. Heavy metals can damage the cell membranes, alter enzymes specificity, disrupt cellular functions and damage the structure of the DNA. Toxicity of these heavy metals occurs through the displacement of essential metals from their native binding sites or through ligand interaction. Toxicity can occur as a result of alterations in the conformational structure of the nucleic acids and proteins and interference with oxidative phosphorylation and osmotic balance (Rathnayake et al., 2009).

In Pseudomonas sp. strain 40, salinity-dependent cadmium tolerance has been reported. Poor growth at 2 mg/mL and no growth at 2.5 mg/mL of CdCl2 were observed at 1 M NaCl, whereas moderate growth was observed at 2.5 mg/mL of CdCl2 in 2-4 M NaCl. Cadmium toxicity was enhanced in presence of NaNO3 and Na2SO4. Cadmium ions react with chloride ions to form complexes whose nature depends on the chloride concentration (Ventosa et al., 1998).

Metal tolerance level of a gram-negative moderately halophilic eubacteria, Halomonas elongata and the genes responsible for the tolerance have been reported. It showed the highest resistance to cadmium (8 mM). It was found that the presence of other metals with Cd, increase Cd toxicity, and the optimum salt concentration for high Cd resistance was 10% NaCl. Characterization of genes involved in metal resistance in halophilic bacteria has been reported for the first time. Multiple resistance systems rather than a single are possibly involved in cadmium tolerance (Amer et al., 2005).

Effect of salinity and media constituents (yeast extract) on toxicity of heavy metals for certain strains has also been reported. The general trends that were observed are: on lowering the salinity the sensitivity to cadmium was enhanced and sometimes to cobalt and copper, whereas on increasing the salinity only a slight decrease in the cadmium, copper, and nickel toxicities was observed. Reducing the concentration of yeast extract (0.01% w/v) resulted in an increased sensitivity to all metals; whereas on increasing the concentration, the toxicities of nickel and zinc were only slightly lessened (Nieto et al., 1989).

Intracellular contents of magnesium and manganese contents were measured in bacteria of several halophilic levels, in Vibrio costicola, a moderately halophilic eubacterium growing in 1 M NaCI, Halobacterium volcanii, a halophilic archaebacterium growing in 2.5 M NaCl, Halobacterium cutirubrum, an extremely halophilic archaebacterium growing in 4 M NaCl, and E. coli, a non halophilic eubacterium growing in 0.17 M NaCl. Their contents varied with the growth phase, being maximal at the early log phase. The increase of magnesium and manganese contents associated with the halophilic character of the bacteria suggests that manganese and magnesium play a role in haloadaptation (Medicis et al., 1986).

The mechanism by which microorganisms resist to various metals can be either accumulation in the form of particular protein-metal association, or blockage at the level of the cell wall and the systems of membrane transportation. In experiments related to determination of MIC (minimum inhibitory concentration) of heavy metals, certain factors like, metal-binding capacity of the microorganisms, chelation to various components of the media, and formation of complexes cause a reduction in the activities of free metals thereby posing problems related to determination of actual concentration of metals that inhibits the microbial growth. Such metal resistant bacteria can be used as bio-indicators of pollution (Hassen et al., 1998)

Apart from reports studying the tolerance to toxic heavy metals and oxyanions, effect of salinity on tolerance to toxic oxyanions has also been reported in spore forming bacilli. It was shown that increase in salinity from 5% (w/v) to 15% (w/v) enhanced tolerance. One of the reasons for the high tolerance to oxyanions was the presence of Na/K in chemical structure of the oxyanions. Sodium and potassium are necessary elements for the activity of enzymes and pumps in halophiles; therefore it seems that these elements enhanced the toxic metal tolerance (Ventosa et al., 1998; Amoozegar et al., 2005).

Studies have also shown that megaplasmids of unknown function are harbored by the majority of halobacteria (Gutiérrez et al., 1986). An insight of metal tolerance may reveal some functions for some of these plasmids and, in addition, the possible heavy metal resistances could be used in halobacteria as genetic markers. Additionally, metal tolerance could be relevant to the ecology and physiology of halobacteria, since they usually grow in habitats such as solar salterns or hypersaline lake that may be polluted with heavy metals (Nieto et al., 1987).

1.5 Scope

Microorganisms play a major role in bioremediation or biotransformation processes of toxic elements, converting them to less toxic or non-toxic elements. Determining the potential of microorganisms and their tolerance against high concentrations of toxic metals will assist the selection of suitable species for bioremediation and biotransformation of toxic metals (Amoozegar et al., 2005). Metal resistant bacteria have potential application in toxic metal control in waste water treatment. As certain microorganisms are responsible for environmental metal transformation, they may serve as bioassay indicator organisms in polluted and non polluted environments (Trevors et al., 1985)

Bacterial population from natural environment can be exploited for treating heavy metal containing wastes (Sannasi et al., 2009). Treatment of industrial waste containing heavy metals by artificially mutated bacteria may be a solution for effluent disposal problems. Use of mutation in metal resistant bacteria enhances the bioremediation of heavy metals from effluents of the factories and improves the disposal problems of the waste with little expense (Shakibaie et al., 2008)

By studying metal tolerance among bacteria, those that seem to be highly sensitive to a particular metal ion can thus serve as biosensor to detect that particular metal ion in environmental sample (Rathnayake et al., 2009).

Due to the above mentioned attributes of halophiles, they can serve as model for stress response in bacteria as they can even tolerate multiple stresses of metals, alkalinity, etc.

1.6 Aims and Objectives

- Isolation, identification and characterization of halotolerant bacteria from saline soil of Khambhat (Gujarat)
- Characterization of isolates with respect to metal tolerance
- Study of organism’s response to single metal challenge
- Study of organism’s response to two or more metals at a time