Halophiles

Applications

 

Halophilic organisms provide an interesting source of biological tools to be applied in industry and biotechnology. The unique features of these “salt-loving” microorganisms offer many possibilities of applications in several processes, such as bioremediation, chemical reactions, biomolecules production, among many others.

 

Because of their ability to successfully grow within a high-salt concentration medium, halophiles biomass could be used to catalyse chemical reactions within a very salty environment, or even to overproduce an interest protein in such conditions, if necessary. Besides using a growth culture of cells, isolated enzymes could be applied, thanks to its remarkable properties.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Manufacturing of Salt from Seawater

Salt production from evaporation of seawater in coastal areas produces brines with salt concentrations near saturation, inducing salt crystallization. At this stage, the brines display a red vibrant colour due to the specific presence of three types of halophilic microorganisms: a β-carotene-containing unicellular alga Dunaliella salina; a red halophilic bacterium Salinibacter ruber, which accumulates an special carotenoid acyl glicoside; and extremely halophilic Archaea (family Halobacteriaceae), which contains 50-carbon carotenoids, as bacterioruberin, and often the retinal protein bacteriorhodopsin [4].

 

The presence of these organisms positively affects salt quality and quantity, including crystal sizes and consistence. Therefore, managing strategies have been created to optimise the production process since 1970s. For example, the microorganisms red pigmentations contributes to elevate the saltern's temperature, increasing evaporation and crystallization rates [4]; therefore, different microbiotas will impact salt production in a different manner. The characterization and comprehension of these population's ecology and biology is essential to understand halophiles' role during the process and, moreover, to enable man to control it in a better way.

 

 

Production of Fermented Foods

Some traditional fermented food requires large amounts of salt during its preparation, consisting in a perfect environment for halophiles' development. Salt concentration in these products may be as higher as the one required by extreme halophilic Archaea, allowing its growing and survival. Thus, it is assumed that enzymes from halophilic organisms take part actively in the fermentation process [4], although there are no studies focusing on this subject. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Catalysis of Processes in High Salt Environments

Compared to those from non-halophilic organisms, halophilic enzymes exhibit interesting properties, making them unique in what concerns to their ability to catalyse reactions under high salt concentrations. Most proteins would aggregate and precipitate when immersed in a salty medium, therefore unfolding from their 3D native structure and losing function capability [2], [3]. Halophilic organisms have evolved their proteome to be adapted to salty environments, and their strategy is based on amino acids chemical characteristics. Halophiles possess a highly acidic protein content, with low levels of hydrophobic amino acids; this leads to a  low prevalence of hydrophobic interaction between proteins and a consequent high salt need to maintain those interaction [5].

 

Such singular enymes can be used to catalyse a diverse range of processes within salt rich environment, which could never be carried out by non-halophilic enzymes. Many of the called "extremozymes" have already been used in industry, such as thermophilic and alkaliphilic proteins from extremophiles microorganisms. In comparison to the extensive application those enzymes have been used for in industry and biotechnology, just a few number of halophilic enzymes have been used so far. Nevertheless, several enzymes from halophilic organisms, including haloarchaea, have been identified and characterized, such as amylases, proteases and lipases. They exhibit a great potential for further applications due to their remarkable physicochemical properties; for example, amylase from Haloarcula sp. shows optimum activity at 4.3 M salt and 50ºC, besides its stability in benzene, toluene and chloroform [4].

 

As a great example of application for halophilic enzymes, including those from extreme halophilic Archaea, we have the treatment of saline wastewater; the effluent generated by some industrial processes contains high concentrations of salt and, often, other compouds, both organic and inorganic. The disposal of these residues is usually expensive and sometimes ineffective. To improve the treatment process, different strategies have been combined, as the addition of microorganims cultures to mechanical devices. Halobacterium salinarum, for example, was added to aerobic treatment systems as an attempt to boost degradation rates. Whether these strategies would work properly in large scale remains unknown, because only laboratory-scale models have been tested [4].

 

Another example is the bioremediation of saline groundwater in coastal aereas, sometimes rich in nitrite and nitrate. Archaeal species Haloferax denitrificans and Haloferax mediterranei, both members of Halobacterium genus, have been proposed and studied with this aim [4]. 

 

 

Production and Usage of Specific Compounds

Some compounds produced by halophiles, including extreme halophilic Archaea, have been intensively studied, and many of them have a great potential to be applied in industry or biotechnology [6]. Some of these compounds' function in halophiles cells are to protect them from radiation, to help them withstand the salty environment, and even to produce energy [6]. Below there are some examples of these compounds (not all of them are produced by haloarchaea, given that other halophilic organisms are more easily cultivated and managed and, thus,have been further explored as biofactories):

 

β-Carotene

This pigment is highly demanded from industry due to its antioxidant and food colouring properties, besides being a pro-vitamin A (retinol) compound. Halophilic algae Dunaliella salina and Dunaliella bardawil produce large quantities of this pigment when grown in optimum conditions, and they are a successfull example of halophilic biotechnology application. At industry level, production is carried out in different ways, from culture in lagoons to containment grothw systems as bioreactors. Transgenic strains have already been developed to increase β-carotene production rates [4]. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ectoine

This osmotic solute is produced by some halophilic bacteria species in order to maintain osmotic balance within their salty medium. The compound became famous and desired for cosmetics industry when it was indicated as a protector against ultraviolet UV-A effects and accelerated sking aging; ectoine also stimulates immune system of Langerhans cells and reduces generation of "sunburn cells" after UV radiation. Ectoine is massively produced through industrial processes using halophilic bacteria [4].

 

Bacteriorhodopsin

This 25 kDa chromoprotein is found embedded in the purple membrane of haloarchaea [4]; it is constituted by the protein moiety and a covalently bound retinal molecule, which is a chromophore. Bacteriorhodopsin acts as a light-dependent transmembrane proton pump: retinal molecule absorbs light energy and the protein pumps a proton across the membrane, creating a motive force that will be coupled to adenosine triphosphate (ATP) synthesis [1]. Therefore, haloarchaea may have some phototrophic growth phases, induced by low oxigen levels and high light intensity. These conditions induce bacteriorhodopsin levels at such high levels that the protein can cover more than 50% of cell surface [6].

Unlike other proteins from extreme halophilic archaea, which require very salty environments for structural stability and activity, bacteriorhodopsin is stable both in high or low salt concentrations, maintaining its photochemical properties over long periods. It also remains functional within a wide range of temperature (0 - 45ºC) and pH (1 - 11) values, and resists to sunlight exposure over years and is not digested by most proteases. Such remarkable characteristics make this biomolecule have a great biotechnological potential. Its use has been studied for several purposes, such as biocomputing, light-sensitive neurological probe, molecular transistors, and even as a treatment of some types of blindness [4], [6]. Site-directed mutagenesis approaches has also been used to optimize the molecule according to its final application [4]. The prospects for bacteriorhodopsin commercial uses are very promising.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References

 

1. Madigan, M. and Brock, T. (2012). Brock biology of microorganisms. San Francisco, Calif.: Pearson.

2. Falb, M., Müller, K., Königsmaier, L., Oberwinkler, T., Horn, P., von Gronau, S., Gonzalez, O., Pfeiffer, F., Bornberg-Bauer, E. and Oesterhelt, D. (2008). Metabolism of halophilic archaea. Extremophiles, 12(2), pp.177-196.

3. Oren, A. (2008). Microbial life at high salt concentrations: phylogenetic and metabolic diversity.Saline Systems, 4(1), p.2.

4. Oren, A. (2010). Industrial and environmental applications of halophilic microorganisms.Environmental Technology, 31(8-9), pp.825-834.

5. Tokunaga, M. (2014). [online] Development of highly soluble enzymes adopting the structural characteristics of halophilic enzymes. Available at: http://www.nisr.or.jp/englishHP/report2003/NISR03tokunaga.pdf [Accessed 24 Nov. 2014].

6. DasSarma, S. and DasSarma, P. (2001). Halophiles. eLS.