Microbial Growth at Extreme Salinity and Extreme pH and Applications of Extremophiles

Microbial Growth at Extreme Salinity

Microorganisms that tolerate or require high salt concentration are called halotolerant and halophilic organisms respectively. The growth of halophiles, therefore, requires at least some NaCl but the optimum varies with the organisms. The terms mild halophile and moderate halophile are used to describe halophiles with low (1-6%) and moderate (6-15%) NaCl requirements respectively. Extreme halophiles are those which require 15-30% of NaCl.

In nature, there are such many environments where the salt concentration is found to be maximum. One of the best examples of this type of environment is the salt lake. Another example is the dead sea (50-70%). At high salt concentration, the hypertonic environment tends to dehydrate non-halotolerant microorganisms. In addition affecting osmotic pressure, high salt concentration tends to denature proteins, that is they disrupt the tertiary structure of proteins which is essential for enzymatic activity.

Examples of halophilic bacteria include Halobacterium spp., Halococcus spp., Ectothiorhodospira spp.

Examples of halotolerant bacteria include Staphylococcus spp., Halomonas spp.

Molecular adaptation of halophily

Many microorganisms that survive these environments of halophily utilize several mechanism. The main mechanism of salt concentration of a balancing solute to equal the salt concentration found external to the cell. When an organism grows in a medium with a low water activity, it can obtain water from it’s environment only by increasing it’s internal solute concentration. An increase in internal solute concentration can be accomplished by either pumping inorganic ions into the cell from the environment or synthesizing on concentrating organic solutes. The solute used inside the cell for adjustment of cytoplasmic water activity must be non-inhibitory to biochemical processes within cell. Such compounds which are either synthesized or concentrated inside the cells are called compatible solutes. These substances are highly water soluble sugars, alcohol, amino acids or their derivatives or in case of extreme halophilic bacteria, K⁺ ions. The amount of compatible solute that can be made or that can be accumulated is a genetically directed characteristic. For instance, Gram positive cocci of the genus Staphylococcus, a halotolerant organism, can use the amino acid ‘proline’ as a compatible solute. Similarly, glycine betaine, a derivative of the amino acid ‘ glycine’ in which the protons on the amino group are replaced by three methyl groups which leaves permanent positive charge on N-atom increasing its overall solubility, is widely distributed as a compatible solute in many halophilic bacteria. Halophilic green algae produce mainly glycerol as a compatible solute.

Microbial Growth at Extreme p^H

Microorganisms generally cannot tolerate extreme p^H values. The p^H of an environments affects microorganisms and microbial enzymes directly and also influence the dissociation and solubility of many molecules that indirectly influence microorganisms. The p^H determines in impart the solubility of CO₂ influencing the rate of photosynthesis, the availability of required nutrients such as ammonium and phosphate and the mobility of heavy metals such as copper which are toxic to microorganisms. There are however, scidophilic and alkaliphilic organisms that can tolerate or even require extreme p^H for growth. Examples of acidic environment include acid hot spring, the GI tract, mining waste streams, acid mine waste water, etc. Some acidotolerant bacteria like Lactobacillus

and acidophile like Thibacillus, Sulfolobus create their own low p^H environment by producing acids. Lactobacillus is a mixed acid fermenter and Sulfolobus produces sulfuric acid. Bacillus acidocaldarius and Thermoplasma acidophilus are heterotrophic thermoacidophilus that live in acidic environment created by chemolithotrophic, sulfur oxidizer such as in acidic hot spring. Archae like Picrophilus are found in dry and extremely acidic soil (p^H < 0.5) solfataric gases to about 55. Similarly, many bacteria and fungi can tolerate alkaline p^H up to 9. True alcaliphiles include some Bacillus strains such as Bacillus alcalophilus and Bacillus pasteurii. Halophilic microorganisms such as Halobacterium, Natrobacterium and Natronococcus are also alcaliphiles and live in saline lake with high p^H. Clostridium paradoxum has a p^H maximum than 10 at 55.

Molecular adaption

A common feature of microorganisms that tolerate or even require p^H extreme for growth is that their cytoplasm is maintained close to neutrality because, despite the requirements of a particular organisms for specific p^H, the optimal growth p^H represents the p^H of the extracellular environment only. The intracellular p^H must remain near neutrality in order to prevent destruction of acid or alkali labile macromolecules in the cell. Furthermore, their cell wall and membrane need to be adapted to keep their integrity under the p^H extreme and performed chemo-osmotic ATP synthesis under these unusual conditions. The precise structural and biochemical adaptation remain insufficient however; a common strategy used by microorganisms to deal with high or low p^H values usually involves modification in the cell membrane. The first of these modifications is the structure of membrane compounds to allow them to be acid tolerant for acidophiles. This include the incorporation of very long chain dicarboxylic fatty acids (32-36 carbon) which make up more than 50% of the membrane fatty acid. These specialized fatty acids inhibit acid hydrolysis of the membrane.

The second modification involves control of ion transport across the membrane. But controlling ion transport, these organisms can maintain an internal p^H in the range of 5-7, even though the external p^H < 2.

Applications of Extremophiles

1.Application of extremophiles in research medicine

Extremophilic enzymes have become the model system to study enzyme evolution, enzyme stability, activity mechanism, protein structure-function relationship and biocatalysis under extreme condition. Few important applications of extremophiles in research and medicine are as follows:

a)The term extremozyme refers to the enzyme from extremophiles microorganisms. One of the prototypes of these biological proteins that has found its way into common everyday use in diagnostic research laboratories worldwide is Taq DNA polymerase. The enzyme is isolated from bacterium Thermus aquatics. This enzyme is heat stable at 95, ideal for use in polymerase chain reaction. The enzyme’s heat stability makes such reaction efficient enough to be possible on a routine base by reducing the need for adding extra polymerase due to the reaction.

More recently, other thermostable polymerase, each of with different advantages for PCR technique have become available. One of these, Pfu polymerase (isolated from hyperthermophile Pyrococcus furiosus) has a higher replication fidelity than Taq DNA polymerase. PCR technique is constantly being refined to diagnose a wide range of viral, bacterial, and fungal diseases in human and animals. Such assays allows qualification of viruses at level where they would be undetectable by other techniques as well as molecular genotyping of different strains.

b) Close control of blood glucose is essential to avoid the long-term adverse consequences of elevated blood glucose including neuropathies, blindness and other consequences. Non-invasive measurements of blood glucose have been a long standing research goal. Such a capability would immediately allow a development of variety of devices for diabetic health care including continuous painless glucose monitoring, control of an insulin pump and warning systems for hyper and hypo glycemia conditions. At present, the only reliable method to measure blood glucose is by a finger stick and subsequent glucose measurement typically by glucose oxidase. This procedure is painful and even the most complaint individuals with good understanding and motivation for glucose control are not willing to finger stick themselves more than a few times per day. A thermostable glucokinase from the thermophilic organism, Bacillus stearothermophilus, was studied for use as glucose sensor and glucose assay is quick by this method.

The enzyme glucodehydrogenase from the thermoacidophilic archaeon, Thermoplasma acidophilus, is also used for glucose sensing.

c) Measurement of sodium and potassium in the blood are routine part of clinic blood analysis. It would be valuable to have simple methods for rapid testing especially for potassium which is measured during hypertensive screening. A variety of fluorescence probes have been developed that respond to sodium or potassium. However, measurements of these ions in the blood are particularly different and the sensing of sodium using the enzyme pyruvate kinase from Bacillus acidocaldarius overcomes this problem.

2.Application of extremophiles in industries

In particular enzymes from thermophiles and hyperthermophiles possess a great potential for biotechnological applications due to their higher resistance not only to temperature but also to chemicals, organic solvents and extreme p^H values. Both extremophiles derived enzymes and the whole microorganisms are exploitable. Thermophiles have yielded stable α-amylase for starch hydrolysis, xylanase for paper bleaching and protease for food processing, baking, brewing and detergent purpose. Cellulose can be used for the treatment of juices, colour-brightening in detergents and treating cellulose containing biomass and probes to improve their digestibility and nutritional quality. Alcaliphilic Bacillus strains have been isolated to produce enzymes appropriate for laundary and dish-washing detergents. Fabric processing can also benefit from extremophiles enzymes. A thermostable and alcalistable protease enzyme is used to improve wool properties and improve dye penetration.

Psychrophiles are a potential source of suitable enzymes for use in biosensors as well as polyunsaturated fatty acids for the pharmaceutical industries. Similarly, halophiles are being exploited as the source of carotene compatible solutes, glycerols, and surfactants for pharmaceutical use. Extremophilic microorganisms may also comprise a large reservoir of the novel therapeutic agent. For example extremophiles extracts were found to have activity against some Candida and Aspergillus species. Similarly, iron binding antifungal compounds called ‘pyochelin’ was isolated from novel species of Pseudomonas. Dried Dunaliella, a halophile is being investigated as a food supplement with anti-oxidant properties.

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