Classification of bacteria based on their salinity tolerance according with criteria proposed by Kushner and Kamekura .
Hypersaline environments are those with salt concentrations 9–10 times higher (30–35% of NaCl) than sea water (3.5% of NaCl). At high concentrations of soluble salts, cytoplasm—mainly of bacteria and archaea—is exposed to high ionic strength and achieves osmotic equilibrium by maintaining a cytoplasmic salt concentration similar to that of the surrounding media. Halophilic enzymes are extremozymes produced by halophilic microorganisms; they have similar characteristics to regular enzymes but different properties, mainly structural. Among these properties is a high requirement of salt for biological functions. Furthermore, the discovery of enzymes capable of degrading biopolymers offer a new perspective in the treatment of residues from oil deposits, under typically high conditions of salt and temperature, while giving valuable information on heterotrophic processes in saline environments.
- synthesis intracellular compounds
Extreme environments involve a wide range of extreme conditions (pH, temperature, pressure, light intensity, oxygen, nutrient conditions, heavy metals, and salinity). Hypersaline environments are those with salt concentrations 9–10 times higher (30–35% of NaCl) than sea water (3.5% of NaCl). These sites are widely distributed around the world and can harbor microorganisms from three different life domains (archaea, bacteria, and eukaryota); together, these microorganisms are known as halophiles, which survive or even thrive in saline environments .
2. Classification of halophiles and halophile environments
Nowadays, several classifications of halophiles have been suggested; the classification proposed by Ollivier et al.  considers those microorganisms capable of growing in salt concentrations ≥150 g L−1 (15% w/v, 2.5 M) as halophiles. Another classification considers the optimum growth salinity as follows: mild halophiles (1–6%, w/v NaCl), moderate halophiles (7–15%) and extreme halophiles (15–30%) . On the other hand, Ventosa and Arahal  defines halophiles as organisms that have an optimal growth above 3% salt concentration; if the optimal growth occurs between 3 and 15% salt, they are regarded as moderate halophiles; and when it occurs above 15% and up to halite saturation (34%), they are regarded as extreme halophiles. In addition, DasSarma and DasSarma  described halophiles as those organisms that thrive from sea salinity (~0.6 M) up to saturation salinity (>5 M NaCl). However, the most complete and widely used classification scheme was proposed by Kushner and Kamekura , in which halophilic microorganisms are separated into six groups based on their salt requirement and tolerance (Table 1): non-halophiles are those that have optimal growth in culture media containing less than 0.2 M NaCl; slight halophiles (marine bacteria) grow best in media with 0.2–0.5 M NaCl; moderate halophiles grow best with 0.5–2.5 M NaCl; borderline extreme halophile that growth best at 2.5–4.0 M; extreme halophiles show optimal growth in culture media containing NaCl concentration between 4 and 5.9 M; and finally halo-tolerant microorganisms, which are non-halophiles that can tolerate high salt concentrations but do not require salt to survive; any microorganism viable at 2.5 M of NaCl is considered extremely halotolerant. Archaea and bacteria are the most widely distributed organisms in hypersaline environments , especially in those in which salinities exceed 1.5 M (about 10%). In recent years, halophilic organisms are mainly isolated from saline environments, such as salt lakes, marine solar salterns, saline soils, and marine sediments (see Table 1). However, halophile bacteria have also been isolated from some non-common places, for example, textile effluents, halophytes, mine tailings as well as processed foods (Table 1).
2.1. Hypersaline environments
Hypersaline environments are extreme habitats with limited microbial diversity as result of high salt concentrations and other environmental factors. Nowadays, most environmental studies have been carried out on aquatic habitats, such as saline lakes and solar salterns used for the production of salt for commercial purposes . Nevertheless, halophilic bacteria can be found in other habitats including saline soils, salted foods and other products, hides, and deep-sea brine pools [7, 9, 10, 11]. Depending on whether they originated or not from seawater, hypersaline environments are classified as thalassohaline and athalassohaline, respectively.
2.1.1. Thalassohaline environments
The thalassohaline environments are saline environments of marine origin, which contain the following ions: Cl−1, Na+, Mg2+, SO42−, K+, Ca2+, Br−, HCO3−, and F− . Some examples of thalassohaline as explained as follows.
22.214.171.124. Solar salterns
These sites have a similar composition to seawater and they are used for salt production by evaporation. They generally consist of several ponds interconnected to form the so-called multipond system. Seawater is pumped or allowed to flow into the first ponds, and as a consequence of solar evaporation, the concentration of salts increases slightly and the water is moved to the next ponds, where it will concentrate further. Finally, in the last pond (called crystallizer), common salt is precipitated . Many studies have focused on the isolation of bacteria harbored in hypersaline environments, identifying the following major groups: Bacteroidetes , Firmicutes [13, 14, 15], ϒ-Proteobacteria [13, 16, 17], and ϒ-Proteobacteria being the most abundant.
Saline soils are those with an electrical conductivity (EC) higher than 4 dS mL−1, approximately 40 mM NaCl . Nowadays, salinized areas are increasing at rate of 10% annually for various reasons, including low precipitation, high surface evaporation, weathering of native rocks, irrigation with saline water, and poor cultural species practices . Several studies on hypersaline soils mainly isolated moderate halophiles and non-halophilic bacteria affiliated with different genera of the following taxonomic groups: Firmicutes [20, 21, 22], actinobacteria [23, 24], and proteobacteria [25, 26].
126.96.36.199. Great Salt Lake
In 1957, a rock-filled railroad causeway was completed across the lake, dividing it into a northern and a southern basin. The northern arm presented high salinity (33%), while the southern arm separated by a semipermeable rock causeway contains a moderate concentration of salt (12%) . A number of studies on the isolation of bacteria from the sediment from Great Salt Lake have been carried out [28, 29].
2.1.2. Athalassohaline environments
These are environments that do not have a marine origin and their ionic proportions are quite different from that of the dissolved salts in seawater . They reflect the composition of the surrounding geology, topography, and climate conditions, often particularly influenced by the dissolution of mineral deposits .
188.8.131.52. Dead Sea
The Dead Sea, which is actually an inland lake, is famous for being so saline that people can float with ease on its surface. The site is composed mainly of divalent ions like Mg2+ . The Dead Sea is a hypersaline lake with 34% salinity, and its name is due to the lack of any living macroscopic creatures. The lake consists of a deeper northern basin and a shallow southern basin, which has been recently dried up and used for commercial mineral production . The water level is dependent on the balance between amount of freshwater inflow and evaporation . The Jordan River is the main source of freshwater inflow, in addition to several water springs and the complex system of underwater springs, which has been recently discovered . Metagenomic studies demonstrated the presence of Halobacterium-like sequences and Mg2+ transport-related proteins, suggesting a potential adaptation to the high magnesium concentration by Dead Sea halophiles . In addition, metagenomic sequence analysis and amino acid profiling also demonstrated the presence of halophiles never previously isolated or sequenced in the Dead Sea [35, 36]. It was recently discovered that the Dead Sea harbors some bacteria with biotechnological properties, such as
184.108.40.206. Soda lakes
Athalassohaline alkaline salt lakes (or soda lakes), rich in NaCl, NaHCO3, and Na2CO3, are usually formed by dissolution of rocks that are low in magnesium and calcium, which would otherwise cause carbonate to precipitate [37, 38]. The most studied lakes are those in the East African Rift Valley, continental Russia, and the USA. In addition to being able to tolerate high pH values and elevated salinities, microbes inhabiting soda lakes have to cope with low availability of NH4+, caused by weak dissociation of ammonia at high pH. An accumulation of stressful but volatile NH3 may occur in enclosed alkaline-saline systems such as sea ice or locally in soda lakes . Several studies about bacteria isolation from soda lakes from different continents are available [39, 40, 41, 42, 43, 44, 45, 46, 47].
Halophilic microorganisms have several biotechnological applications, such as
2.2. Biology and adaptation of halophilic bacteria
The first chemical stress encountered during the evolution of life on earth may have been salt stress. Thus, from the beginning, organisms must have evolved strategies and effective mechanisms for the stabilization of protoplasmic structures and ion regulation . At high concentrations of soluble salts, cytoplasm—mainly of bacteria and archaea—is exposed to high ionic strength and achieves osmotic equilibrium by maintaining a cytoplasmic salt concentration similar to that of the surrounding media. This can affect microbes via two primary mechanisms: osmotic effect and specific ion effects. Soluble salts increase the osmotic potential (more negative) of the soil water, drawing water out of cells which may kill microbes and roots through plasmolysis [49, 50].
To thrive in the hypersaline environment, halophiles have two main adaptation mechanisms to prevent NaCl from diffusing into the cells. The first mechanism is accumulation of inorganic ions (mainly KCl) for balancing osmotic pressure. This mechanism is mainly utilized by aerobic and extremely halophilic archaea and some anaerobic halophilic bacteria [32, 49, 51]. In contrast, most halophilic bacteria accumulate water soluble organic compounds of low molecular weight, which are referred to as compatible solutes or osmolytes, to maintain low intracellular salt concentration [52, 53, 54].
2.2.1. Salt-in mechanisms
As mentioned above, microorganisms that grow optimally in the presence of extremely high salinities (up to 5 M NaCl), accumulate intracellular potassium and chloride ions in concentrations higher than the external NaCl concentration to maintain a turgor pressure. This so-called “salt-in” strategy is observed in
2.2.2. Synthesis of intracellular compounds
As explained above, microorganisms have the ability to adapt to or tolerate stress caused by salinity by accumulating osmolytes, also known as compatible solutes. The compatible solute strategy is broadly known in domain archaea, bacteria, as well as eukarya. Organisms accumulate organic solutes by uptake from the environment or
Organic solutes act as stabilizers for biological structures and allow the cells to adapt not only to salts but also to heat, desiccation, cold, or even freezing conditions . Many halophilic bacteria accumulate ectoine or hydroxyectoine as the predominant compatible solutes. Other intracellular compatible solutes include amino acids, glycine betaine and other compounds accumulated in small amounts .
Mei et al.  describe the physiology of a
The most common inorganic solutes used as osmolytes by salinity tolerant microbes are potassium cations, while proline and glycine betaine are the main organic osmolytes . However, the synthesis of these compounds requires high amounts of energy [50, 66]. Given these high energetic requirements, there are few reports of halophilic microorganisms that can produce compatible solutes to mitigate the stress by variable concentrations of salts. The capacity of two halophilic strains is noteworthy:
2.3. Production of extremozymes
Halophilic enzymes are extremozymes produced by halophilic microorganisms; they have similar characteristics to regular enzymes but different properties, mainly structural. Among these properties is a high requirement of salt for biological functions. In recent years, different studies have focused on the detection of halophiles in saline environments in order to isolate and characterize new enzymatic activities. This resulted in several halophile hydrolases being described, including amylases, lipases, and proteases. Furthermore, the discovery of enzymes capable of degrading biopolymers offer a new perspective in the treatment of residues from oil deposits, under typically high conditions of salt and temperature, while giving valuable information on heterotrophic processes in saline environments.
2.3.1. Extremozymes-producing halophiles
Nowadays, investigation on the production of extremozymes from different bacterial genus and halophilic archaea has intensified. This interest is due to their capacity to efficiently catalyze a process and show optimal activities at different salt concentrations. Halophiles are the most probable source of extremozymes, since they are also capable of tolerating alkaline pH and high temperatures, as reported by several authors [68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79].
Most of the evaluation studies on the enzymatic capacities of halophiles begin with the isolation of these microorganisms from environments considered extreme due to specific characteristics such as high salt concentrations, high pH values, and extreme temperature conditions. Sánchez-Porro et al.  report the isolation of moderately halophile strains from water and salterns in different areas of southern Spain: Almería (Cabo de Gata), Cádiz (San Vicente and San Fernando), and Huelva (Isla Bacuta, Río Tinto and Isla Cristina). Isolates have been identified as members of the genera
In 2007, Vidyasagar et al.  isolated the extreme halophile
Rohban et al.  studied extremophiles in Howz Soltan, a hypersaline lake located in central Iran. The organisms successfully isolated produced a wide variety of extracellular enzymes, where 84.4% had lipase activity, 76.6% amylase, 43.2% protease, 41.1% inulinase, 39.8% xylanase, 29.4% cellulase, 14.2% DNase, and 12.1% pectinase. Halophile strains were identified as members of the following genera:
In 2011, Perez et al.  reported the isolation and purification of a lipase obtained from the
In agreement with previous studies from 2003, 2007, and 2009, Shahbazi and Karbalaei-Heidari  reported the capacity of
During 2013, studies were made on enzyme-producing halophilic archaea capable of synthesizing two new alcohol-dehydrogenases, amylase and a thermostable halo-alkaliphile α-amylase; the producing organisms were identified as
The bacteria isolated from a saline lake in Iran produced lipases, these bacteria belonging to the genera
A halo-alkaliphile, thermostable extracellular protease was reported by Selim et al.  produced by
Gupta et al.  reported a halo-alkaliphile isolated from a soil sample collected from the Sambhar Lake in Rajasthan, northern India, which produced an extracellular alkaline protease; the results of the analysis of gene 16S rRNA showed a 98% match with
Dumorné et al.  stated that that the halophiles
2.4. Physicochemical parameters and kinetic properties of extremozymes from halophilic microorganisms
Halophilic enzymes have specific mechanisms for solubility at high salt concentrations, such as a highly negative superficial charge given by carboxylic groups that depend on high salt concentrations to remain soluble. Halophilic archaea are known to secrete active proteases at high concentrations of NaCl (4 M), and to accumulate high concentrations of KCl in their cytoplasm in order to face osmotic stress, while maintaining the conformation of their proteins. The study made by Akolkar and Desai  suggests that proteases from haloarchaea may be active and stable in the presence of osmolytes different from NaCl/KCl at different degrees, as shown by the kinetics and thermodynamic analyses of casein hydrolysis produced by
The affinity of an enzyme to hydrolyze a substrate is determined by the Michaelis constant (
|Category||Salt tolerance (M)||Example||Isolation site||References|
|Slight halophile||0.2–0.5||Textile mill effluent|||
|Tissues of the halophyte |||
|Moderate halophile||0.5–2.5||Marine sediments|||
|Root of |||
|Stems of |||
|Root of |||
|Textile effluent drain|||
|Candidatus ||Hypersaline soda lakes|||
|Rhizosphere of saline-tolerant pokkali rice|||
|Marine saltern located in Huelva, Spain|||
|Root nodules of |||
|Leaves of |||
|Marine solar saltern|||
|Borderline extreme halophile||2.5–4.0||Marine solar saltern|||
|Hypersaline sediment of the Great Salt Lake|||
|Aran-Bidgol salt lake, Iran|||
|Sediments of the hypersaline lake Sehline Sebkha|||
|Under the salt crust of El-Jerid hypersaline lake in southern Tunisia|||
|Korean fermented food (kimchi)|||
|Marine solar saltern|||
|Wetland in Iran|||
|Marine solar saltern|||
|Extreme halophile||4–5.9||Saltern crystallizer ponds in Alicante and Mallorca, Spain|||
|Mud of the hypersaline Lake Aran-Bidgol, Iran|||
|Hypersaline soda lake|||
|Halo-tolerant||A non-halophile that tolerant salt; if it is viable 2.5 M, in is considered extremely halotolerant||Sediment from a salt lake|||
|Halophytic plant (|||
|Traditional Korean fermented seafood|||
|Edge of mine tailings|||
|Acid curd cheese called Quargel|||
Nowadays, recombinant DNA techniques and genetic engineering are used to obtain customized extremozymes to be used for specific purposes, greatly improving their catalytic ability, as demonstrated by Kui et al.  with the expression of genes from extremozyme β-1,4-xylanase, which was cloned from
2.5. Halophile extremozyme applications
As mentioned above, halophiles are good sources of several extremozymes, and among them hydrolases have been the most studied, mainly amylases, proteases, lipases, xylanases, cellulases, and DNases. Some extremozymes from halophiles exhibit extraordinary biochemical properties, which show the potential for industrial applications. It has been demonstrated that extremozymes derived from halophiles are able to function under harsh conditions and remain stable and active with different properties than conventional enzymes, offering opportunities in several applications such as environmental bioremediation, food processing, and residual water treatment. Recent research points out the application of halophilic extremozymes in the production of biofuels. Since several halophiles are also alkaliphiles, their enzymes are of interest for the textile and detergent industries, and some have been explored as raw materials in the production of commercial enzymes, particularly proteases and amylases [5, 100, 101, 102, 103, 104, 105].
Halophile microorganisms have the ability to adapt to or tolerate stress caused by salinity by accumulating osmolytes. Halophilic microorganisms have several biotechnological applications, in recent years, uses of halophilic microorganisms have significantly increased. Many enzymes, stabilizers, and valuable compounds from halophiles may present advantages for the development of biotechnological production processes. Halophiles are the most probable source of extremozymes, since them also capable of tolerating alkaline pH and high temperatures.
The authors thank the support granted by PROFAPI-ITSON Projects 2018-1076 and 2018-1169 for the realization of the present investigation. MS Vásquez-Murrieta appreciates the scholarships of Comisión de Operación y Fomento de Actividades Académicas (COFAA), Estímulos al Desempeño de los Investigadores (EDI-IPN), and Sistema Nacional de Investigadores (SNI-CONACyT). B Román-Ponce thanks for the Postdoctoral fellowships awarded by Consejo Nacional de Ciencia y Tecnología (CONACyT).