Halophiles are microorganisms that live in hypersaline environments that have salt concentrations ranging from 15 % to saturation. All three domains of life, the Archaea, the Bacteria and the Eukarya have halophilic representatives (Fig. 12.1). These organisms are abundant in hypersaline environments forming populations so dense that the red color associated with hypersaline lakes and ponds is due to the pigmentation of the halophilic Archaea and the eukaryote Dunaliella (Fig. 12.2). Although these lakes and ponds contain populations of halophilic bacteria and other members of the Eukarya they go unnoticed to the naked eye because they are not pigmented.
Halophiles fall into two categories, extreme halophiles requiring 15 % salt to saturation, or moderately halophilic organisms living in saline environments ranging from seawater to 15 %. Organisms that are halotolerant prefer non-saline environments, but can grow from essentially no salt to nearly 10 % salt.
R.L. Mancinelli, Microbial Life in Brines, Evaporites and Saline Sediments: The Search for Life on Mars. In: Water on Mars and Life, Tetsuya Tokano (ed.), Adv. Astrobiol. Biogeophys., pp. 277-297 (2005)
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12.1.1 The Place of Halophiles in the World
Halophiles are everywhere in the world where there is salt. They represent a physiologically, evolutionarily, and ecologically diverse group of organisms. Most halophiles are found interspersed among non-halophiles in the phylogenetic tree (Fig. 12.1). They encompass heterotrophs, autotrophs, and some possess light harvesting pigments either for photosynthesis, or for energy production via rhodopsin. They live in cold, or hot environments, wet environments (e.g. lakes and ponds), dry environments (e.g. soils and salt crusts), alkaline as well as neutral environments. They can be aerobes, anaerobes, or facultative anaerobes. Some have true cell walls (Bacteria and Eukarya) and some do not (Archaea). They even differ with respect to their modes of osmotic adaptation. The one characteristic they have in common is their ability to live in hypersaline environments.
12.1.2 The Halophilic Archaea
Within the domain Archaea halophily can be found in the Halobacteriaceae, the Methanospirillaceae and the Mahanosarcinaceae. Unlike the Methanospirillaceae and the Mahanosarcinaceae which have few halophilic representatives, all members of the family Halobacteriaceae are extreme halophiles. Many new species of halophilic Archaea have been isolated and characterized during the past 25 years representing diverse morphologies and physiologies. The order Halobacteriales  forms a branch within the Euryarchaeota. Halobacteriales consists of one family, the Halobacteriaceae that is divided into 15 genera with 44 species.
The Halobacteriaceae are basically aerobic heterotrophs. They are all chemoheterotrophs, and some are photoheterotrophs using bacteriarhodopsin to generate energy. Autotrophic growth, however, has never been demonstrated. Denitrification, that is anaerobic respiration, the reduction of nitrate to either N2O or N2 occurs in several species [e.g. 71, 48]. In comparison, the Methanospirillaceae and the Mahanosarcinaceae are anaerobic methanogens that use methylated amines or methanol for substrates.
The morphology of the organisms in the Halobacteriaceae includes rods, cocci, pleomorphic cells, square cells, as well as triangular and trapezoid shaped cells. These shapes are determined by the properties of the organism's cell wall and membrane. The unusual shapes such as triangles, and squares are possible because the cells do not possess significant turgor pressure , so shapes that may not be feasible for other types of organisms are feasible for these halophiles.
Most Archeael halophiles do not possess a true rigid cell wall, but rather what is called an S-layer that consists of large subunits of a large glycoprotein that depends on a high salt concentration for structural stability. When these organisms are placed in a hypotonic solution they lyse, but lysis is due to denaturation of the glycoproteins rather than osmotic pressure . The exceptions are members of the genera Halococcus and Natronococcus who do possess a rigid cell wall that does not depend on high salt for structural stability. Halococcus has a wall consisting of a heteropolysaccaride , while Natronococcus has a wall made of repeating units of poly(L-glutamine) . None of the Archaeal halophiles possess the classical cell wall components such as D-amino acids and teichoic acid.
The cell membranes of the Archaeal halophiles are composed of lipids and proteins. However, unlike other microbes they are primarily composed of branched C20 and C25 carbon chains bound to glycerol by ether bonds that form polar and neutral lipids only found in the Archaea .
Classification of the genera and species of the Halophilic Archaea is based primarily on cellular morphology, growth characteristics, membrane lipid composition, and 16S rDNA sequence data [e.g. 84].
Halophiles are spread throughout the phyla and orders, within the domain Bacteria. Halophilic Bacteria vary widely in their physiological properties, and include aerobic and anaerobic chemoheterotrophs, photoautotrophs and photoheterotrophs, as well as chemolithotrophs [80, 81].
Oxygenic photosynthetic Bacteria, i.e. the Cyanobacteria, are present in hypersaline environments. Cyanobacteria inhabiting microbial mats in salterns containing up to 25 % salt  and in evaporitic salts  are extreme halophiles. Examples of halophilic cyanobacteria include the species of Halospirulina , Microcoleus Cyanothese, Aphanothece, Chroococcidiopsis, and Myxobactron . The taxonomy of the cyanobacteria is not well defined, resulting in diverse physiological types grouped together in one genus. Thus, not all species of the above named cyanobacteria are halophilic.
Halophilic anoxygenic photosyntesizers are sulfur Bacteria. They include two genera and range from the extremely halophilic genus Halorhodospira to the moderately halophilic Ectothiorhodospira [51, 108, 112].
Unlike the halophilic Archaea, the halophilic and halotolerant Bacteria are metabolically diverse. The physiological types include aerobic and anaerobic heterotrophs, oxygenic and anoxygenic photoautotrophs, photoheterotrophs and chemolithotrophs.
The Halomonadaceae family of the Bacteria, made up of the genera Halomonas and Chromobacter, are metabolically versatile aerobic moderate halophiles that have few non-halophiles among them . Most of the halophilic anaerobic Bacteria form a phylogenetically coherent group within the order Haloanaerobiales .
Much less is known about the details of the cellular structures of the halophilic and halotolerant Bacteria than the Archaeal halophiles. However, the halophilic Bacteria have much in common with the non-halophilic Bacteria. Their cell wall appears to contain peptidoglycan much like the non-halophilic Bacteria. The cell wall of most halophilic Bacteria that have been studied contain some type of hydrophobic protein . The cell membranes are made of proteins and lipids as are non-halophilic Bacteria. They differ from their non-halophilic counterparts in that the membrane is regulated by the outside salt concentration to adjust ion permeability and the activity of the integral proteins .
The halophilic Eukarya are not as diverse as the Bacteria, and represent a small fraction of the domain Eukarya. The few types that do exist, however, contribute significantly to the biomass of hypersaline environments. Species of the green algae Dunaliella and Picosystis salinarum can grow in environments ranging from slightly brackish to saturated brines . While most diatoms cannot grow in hypersaline environments, a few, mostly members of the genera Amnphora, Nitzschia and Entomoneis can grow in saline environments containing 15 % salt . Protozoa also have members that are halophilic, most notably several genera of the ciliates, sarcodines and zooflagellates. Certain groups of fungi also thrive in hypersaline environments, and contribute significantly to the biomass of these environments .
Relatively little is known about the specifics of the physiology of the halophilic and halotolerant Eukarya. They do, however, encompass photoautotrophs, as well as heterotrophs.
The Eukarya in general have more complex cellular structures than either the Archaea, or the Bacteria. Dunaliella do not possess a rigid cell wall allowing them to swell and shrink as an immediate reaction to changes in the salinity of their environment. The cytoplasmic membrane can rapidly either take up, or efflux large molecules such as dextrans . It is thought that this ability is what enables the organisms to swell, or shrink in response to rapid changes in salt concentration in the environment. Dunaliella contain P-carotene in the inter-thylakoid space within the chloroplast. Sterols are a major component of the lipids in the fungi.
12.1.5 Halophily and Osmophily - Is There a Difference?
Osmophily refers to the osmotic aspects of life at high salt concentrations, especially turgor pressure, cellular dehydration and desiccation. Halophily refers to the ionic requirements for life at high salt concentrations. Although these phenomena are physiologically distinct, they are environmentally linked because increasing salt concentrations lead to increases in osmolarity. Because of the environmental link between osmolarity and salt concentration, a halophile must be able to physiologically compensate for the osmolarity that accompanies its optimum salt concentration requirement. Under normal growth conditions the osmotic aspect of the high salt requirement cannot be clearly separated from the solute aspect, making it difficult to study the physiology of osmophily separately from halophily.
Many microorganisms respond to increases in osmolarity by accumulating substances, termed osmotica, in their cytosol. Osmotica protect organisms from cytoplasmic dehydration and desiccation [9, 116]. With the exception of the Halobacteriaceae and the Haloanaerobiales, which use K+ as their osmoticum , glycine-betaine is the most common osmoticum in most prokaryotes [32, 64, 67] including the cyanobacterium Synechococcus (Nageli) [69, 93; reviewed in 50]. Studies of the osmoticum in halophilic eukaryotes has centered around Dunaliella and to a lesser extent Asteromonas species. It appears that halophilic eukaryotes use glycerol as their osmoticum while maintaining very low intercellular ionic concentrations [e.g. 5].
The osmoticum used by halophiles belong to different chemical classes with little structural similarity. The general properties they have in common are that they are very soluble, have no net charge, and exhibit very limited interaction with proteins. These compounds are all strong water structure formers and as such they are excluded from the hydration shell of proteins. This exclusion most likely defines their function as effective stabilizers of the hydration shell of proteins and other cytoplasmic structural elements [34, 109]. The phenomenon of preferential hydration of proteins favors a more compact protein conformation. It also opposes an increase in surface area and, because unfolding usually results in surface area increase, it favors the native structure of the protein. It is therefore primarily free water, as opposed to bound water, that responds to osmotic changes in the environment, and the compatibility of osmotic solutes is based on the fact that they specifically adjust the osmotic equilibrium of the free water fraction. Exclusion of compatible solutes from the hydration sphere of proteins is consistent with a decrease in entropy of the system (higher ordering). This entropically unfavorable condition in turn causes minimization of the excluded volume and subsequently stabilizes the conformation of a protein .
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