HalophilesShiladitya DasSarma, University of Maryland, Baltimore, Maryland, USA

Priya DasSarma, University of Maryland, Baltimore, Maryland, USA

Halophiles are salt-loving organisms that flourish in saline

environments and can be classified as slightly, moderately

or extremely halophilic, depending on their requirement

for sodium chloride. Although most marine organisms are

slight halophiles, moderate and extreme halophiles are

generally more specialised microbes, which inhabit

hypersaline environments with salinity higher than in

the sea. Hypersaline environments are found all over the

world, in arid, coastal, and deep-sea locations, under-

ground salt mines, and artificial salterns. Halophilic

microorganisms include a variety of heterotrophic, pho-

totrophic, and methanogenic archaea, photosynthetic,

lithotrophic, and heterotrophic bacteria, and photo-

synthetic and heterotrophic eukaryotes. Examples of

well-adapted and widely distributed extremely halophilic

microorganisms include archaea for example, Halo-

bacterium sp. NRC-1, cyanobacteria such as Aphanothece

halophytica, and the green alga Dunaliella salina. Multi-

cellular halophilic eukaryotic organisms include brine

shrimp and the larvae of brine flies. Halophilic organisms

either accumulate internal organic compatible solutes to

balance the osmotic stress of the environment or produce

acidic proteins to increase solvation and improve function

in high salinity.

Introduction

Although salts are required for all life forms, halophiles(from the Greek, hal, meaning sea or salt, and philos,meaning loving) are distinguished by their requirement ofhigh salinity conditions for growth. They may be classifiedaccording to the degree of their salt requirement: slight

halophiles grow optimally at 0.2–0.85M (1–5%) sodiumchloride (NaCl); moderate halophiles grow optimally at0.85–3.4M (5–20%) NaCl; and extreme halophiles growoptimally at 3.4–5.1M (20–30%) NaCl. In contrast, non-halophiles grow optimally in less than 0.2M NaCl con-centrations. Halotolerant organisms can grow in eitherhigh salinity or in the absence of a high concentration ofsalt. Many halophiles and halotolerant microorganismscan grow over a wide range of salt concentrations, withrequirement or tolerance for salts sometimes dependent onenvironmental and nutritional factors.High osmolarity in hypersaline conditions is deleterious

to most cells since water is lost to the external medium. Toprevent loss of cellular water, halophiles generally accu-mulate high solute concentrations within the cytoplasm(Roberts, 2005; Yancey, 2005). When an isoosmotic bal-ance with the medium is achieved, cell volume is main-tained. The compatible solutes or osmolytes whichaccumulate in halophiles are generally amino acids, sugarsand polyols, which do not interfere with intracellular pro-cesses and have no net charge at physiological pH. Halo-tolerant yeasts and green algae accumulate polyols,whereas most halophilic and halotolerant bacteria accu-mulate zwitterionic species (containing both positive andnegative charges), such as glycine betaine and ectoine.Compatible solute accumulation may occur by bio-synthesis, de novo or from storage material, or by directuptake from the medium. A major exception is foundamong the haloarchaea and some extremely halophilicbacteria, which accumulate potassium chloride (KCl)equal to the external concentration of NaCl. Theseorganismsproduce acidic proteins that can function in highsalinity by remaining solvated and reducing aggregation,precipitation and denaturation (Madern et al., 2000).

Hypersaline Environments

Though the oceans are, by far, the largest body of salinewater, constituting approximately 99% of the biosphere,hypersaline environments are generally defined as thosecontaining salt concentrations in excess of seawater (3.5%total dissolved salts).Most hypersaline bodies are thalassic

Advanced article

Article Contents

. Introduction

. Hypersaline Environments

. Prokaryotic Halophiles

. Halophilic Archaea

. Eukaryotic Halophiles

. Biotechnology

. Conclusions and Future Prospects

Online posting date: 15th March 2012

eLS subject area: Microbiology

How to cite:DasSarma, Shiladitya; and DasSarma, Priya (March 2012) Halophiles.In: eLS. John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0000394.pub3

eLS & 2012, John Wiley & Sons, Ltd. www.els.net 1

(from the Greek, thalassa, sea), as they derive from theevaporation of seawater and retain the relative proportionof salts in the sea. A great diversity of microbial life isobserved in thalassic brine upward from marine salinity(� 0.6M) to approximately 3–3.5M NaCl, at which pointonly a few extreme halophiles can survive, for exampleHalobacterium, Dunaliella, and a few bacterial species.Athalassic waters are those in which the salts are in non-marine proportions, and are less common.

Two large and well-studied hypersaline lakes are theGreat Salt Lake (Figure 1), in thewesternUnited States, andthe Dead Sea, in the Middle East. The Great Salt Lake islarger (� 4000 km2) and shallower (on average 10m deep),and is thalassic. The Dead Sea is smaller (400 km2) anddeeper (300m), and is athalassic, with a relatively highconcentration of magnesium salts. Both of these lakes areclose to neutral pH, although the former is slightly alkalinewhereas the latter is slightly acidic. Compared to smallerhypersaline ponds, the composition of larger lakes remainfairly constant as a result of their size; recently, however,human activities have had significant effects on the chem-istry and biology of many. For example, a railroad cause-way built in 1959 divided theGreat Salt Lake into northernand southern sections, leading to dilution of the southernsection, which receives the greatest inflow of freshwaterfrom streams, and the concentration of the northern sec-tion to nearly saturating salinity (Gwynn, 2002). Diversionof incoming freshwater streams for irrigation in the DeadSea basin in recent years has also had a significant impacton its size and salinity, and has resulted in concern aboutthe lake’s long-term viability (Abu Ghazleh et al., 2011).

Many small evaporation ponds or sabkhas are foundnear coastal areas, where seawater penetrates throughseepage or via narrow inlets from the sea. Notable amongthese are Solar Lake and other ponds near the Red Seacoast, Guerrero Negro on the Baja California coast, LakeSivash near the Black Sea and Shark Bay in WesternAustralia. Hypersaline evaporation ponds have also beenfound in Antarctica (e.g. Deep Lake, Organic Lake andLake Suribati), and also in the Atacama Desert region of

Chile, which are located in some of the driest areas of theworld (Javor, 1989).A number of alkaline hypersaline soda brines exist,

including theWadiNatrun lakes of Egypt, LakeMagadi inKenya, and the Great Basin lakes of the western UnitedStates (Mono Lake, Owens Lake, Searles Lake and BigSoda Lake), several of which are intermittently dry (Javor,1989). Soda brines lack magnesium and calcium divalentcations because of their low solubility at alkaline pH.Manysmaller hypersaline pools represent especially dynamicenvironments, experiencing significant seasonal variationsin size, salinity and temperature.In addition to natural hypersaline lakes, numerous

artificial solar salterns have been constructed for the pro-duction of sea salts. These usually consist of a series ofshallow evaporation ponds connected by pipes and canals,with brine being directed into smaller ponds as salinityincreases through evaporation. During the process ofevaporation, sequential precipitation of salts, calciumcarbonate, calcium sulfate (gypsum) and NaCl (halite)occurs. After precipitation of NaCl, the potassium andmagnesium chloride and sulfate may be harvested, or theremaining brines (called ‘bitterns’) returned to the sea.Hypersaline environments also occur in subterranean

evaporite deposits and deep-sea basins created by saltdomes. Deep-sea brines are relatively stable as a result oftheir higher density than surrounding seawater and theyhave been found in the Red Sea, Gulf of Mexico andMediterranean Sea. Recent studies have shown evidencefor microbial activity in some deep-sea hypersaline basins,including one containing 5Mmagnesium chloride (MgCl2),and in subsurface halite deposits over 10 million years old(Antunes et al., 2011; van derWielen et al., 2005; Fish et al.,2002). Sulfur cycling and methanogenesis were shown todrive microbial colonisation in a Mediterranean deephypersaline basin (Borin et al., 2009). Recent evidence forsubterranean brines has also been discovered on Mars,where seasonal flows from the sides of craterswere detectedby an orbiting satellite (McEwen et al., 2011).Evaporation of hypersaline brines is frequently

observed, leading to a gradient of salinity, which inturn leads to sequential blooms of diverse microbialspecies adapted to different ranges of salinity (Figure 2).In solar salterns, as brine is concentrated from 1M NaClto approximately 3.5M, dense algal populations aresupported, on which brine shrimp and larvae of brineflies feed. Protozoa are also found, as are yeasts andfungi. Microbial mats, containing predominantly photo-synthetic unicellular and filamentous cyanobacteria, pur-ple and green sulfur and nonsulfur bacteria coverthe bottom of many hypersaline ponds (Caumette, 1993;Figure3). In the anoxic zones of themats and in the sedimentbelow, a variety of sulfur oxidising, sulfate reducing,homoacetogenic, methanogenic, and heterotrophic bac-teria and archaea occur. From approximately 4M NaClto saturation (45.1M NaCl), halophilic archaea dominatethe brine pools and most other microbial activityceases.

Figure 1 Bloom of halophilic microorganisms. Dense growth of halophilic

microorganisms in hypersaline environments leads to reddening of the

brine. Photo Dr. S. DasSarma.

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Prokaryotic Halophiles

A great diversity of prokaryotic halophiles exists in natureand they have been studied by both culturing and non-culturing techniques, with sequence-based approachesbecoming increasingly popular for phylogenetic andtaxonomic classification. An approach used for classifyingcultured halophiles is by analysis of the nucleotidesequence of multiple genes, a method called multilocussequence typing (MLST) (Papke et al., 2011; de la Habaet al., 2011). For noncultured halophiles, environmentalmetagenomic studies using sequencing and phylogeneticanalysis of 16S ribosomal ribonucleic acid (rRNA) genesare beginning to expand the breadth of diversity knownamong halophiles.

Halophilic Archaea

These extreme halophiles grow best at the highest salinities(3.4–5.1M NaCl) (Figure 2), forming dense blooms (up to

108 cellsmL2l), and resulting in the red colour of manybrines (Figure 1). Common species of halophilic archaea,also called haloarchaea, are rods, cocci or disk-shaped,although triangular and even square-shaped species exist.Many are pleiomorphic, especially when the ionic con-ditions of the media are altered, and most lyse below1–1.5M NaCl. Haloarchaea are members of the archaealdomain (they are also called halophilic archaea, and for-merly halobacteria). They have recently been proposed tobe composed of two families (Minegishi et al., 2011), andinclude about three dozen genera. A recent metagenomicstudy of a solar saltern showed the occurrence of a majornew phylotype, called nanohaloarchaea, with small cells(50.8 um) (Narasingarao et al., 2011). Other nonculturebased studies have suggested that novel species similar tohaloarchaea may occur in the human gastrointestinal tract(Oxley et al., 2010).The genomes of haloarchaea have been of significant

interest due to their evolutionary position and physio-logical capability to grow at the most extreme hypersalineconditions. The first haloarchaeal genome completed was

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microorganisms depicted are Synechococcus sp. PCC7002 (PR-6), a slightly halotolerant cyanobacterium, Fabrea salina (Fs), a moderately halophilic

protozoan, Dunaliella salina (Ds), a halophilic green algae, Aphanothece halophytica (Ah), an extremely halophilic cyanobacterium and Halobacterium sp. (H),

an extremely halophilic archaeon. The salinity of seawater and the hatch range for brine shrimp are noted.

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Halophiles

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for Halobacterium sp. NRC-1, which has a 2.57Mb GC-rich genome (DasSarma, 2004). The genomes of over adozen haloarchaeal species have now been completelysequenced and analysed using bioinformatic tools, show-ing both the conserved information transfer genes anddiversemetabolic genes (Capes et al., 2011;Anderson et al.,2011).

Early microbiological analysis was conducted on severalclosely related Halobacterium strains isolated in the mid-twentieth century from salted fish and meat from Europeand North America (DasSarma and DasSarma, 2008;DasSarma et al., 2010b, a). These are amino acid-utilisingfacultative aerobes, which require a number of growthfactors and slightly elevated temperatures (38–458C) foroptimal growth. Most have distinctive features such as gasvesicles, purple membrane and red-orange carotenoids.Many are facultative anaerobes and grow utilising respir-ation of dimethoxylsulfoxide and trimethylamine N-oxide(a saltwater fish osmolyte) (Yancey, 2005), fermentation ofdifferent sugars, breakdown of arginine and light energy,mediated by retinal pigments.

Some haloarchaeal species utilise carbohydrates, forexample Haloarcula marismortui, Haloarcula vallismortisand Haloferax volcanii from the Dead Sea, Haloferaxmediterranei and Halorubrum saccharovorum from salt-erns, and Halorubrum lacusprofundi, a psychrotrophicspecies fromDeepLake,Antarctica.Glucose is oxidised bya modified Entner–Doudoroff pathway and the resultingpyruvate is further oxidised by pyruvate oxidoreductaseand the tricarboxylic acid cycle. H. marismortui and H.volcanii have been reported to use a novel pathway forcentral metabolism, where acetyl-CoA is oxidised toglyoxylate via the key intermediate methylaspartate(Khomyakova et al., 2011). Several haloarchaea are cap-able of growth on single carbon sources such as sugars,glycerol and acetate, and recent genomic studies are pro-viding additional insights into metabolic pathways (Satoand Atomi, 2011). Some haloarchaea, such as H. medi-terranei accumulate polyhydroxyalkanoates, when grownin carbon excess conditions, or respire anaerobically vianitrate reductase (Han et al., 2010; Bonete et al., 2008).

Some halophilic species are polyextremophilic and aretolerant of additional extremes (Bowers et al., 2006). Forexample, the Antarctic species, H. lacusprofundi, is cold-adapted and can grow down to228C, and the Red Sea hotbrine species,Halorhabdus tiamatea, is thermotolerant andcan tolerate up to 55–608C. The alkaliphiles, Natronomo-nas pharaonis from Wadi Natrun and Natronococcusoccultus from LakeMagadi, have pH optima in the 9.5–10range and do not grow below pH 8.5. Slight acidophiles,such asHalarchaeum acidiphilum is able to grow at 4.0–6.0.

The intracellular salt concentration of haloarchaea hasbeen found to be extremely high,withK+ ions accumulatedinternally to near 5M (Roberts, 2005). The concentration ofNa+ ions appears to be in the molar range, with the K+/Na+ ratio in the cytoplasm over 100. The K+ gradient ismaintained by a combination of an electrogenic Na+/H+

antiporter and two K+ uniporters (Figure 4). Amino acid

uptake is carried out by a Na+/amino acid symporter.See also: Sodium, Calcium and Potassium ChannelsProteins of haloarchaea are either resistant to high salt

concentrations or require salt for activity. Both genomesequencing and proteome analysis have shown that theycontain an excess proportion of acidic to basic amino acids,a feature likely to be required for protein activity at highsalinity (Joo and Kim, 2005; DasSarma, 2004) (Figure 5).This characteristic is shared with proteins from somehalophilic bacteria, for example Salinibacter ruber (whichalso grows in crystalliser ponds). Surface negative chargesare thought to be important for solvation of halophilicproteins, and to prevent the denaturation, aggregation andprecipitation, which usually result when nonhalophilicproteins are exposed to high salt concentrations. Thestructures of haloarchaeal glucose dehydrogenase, malatedehydrogenase, dihydrofolate reductase and large ribo-somal subunit have been determined by X-ray crystal-lography (Britton et al., 2006; Ban et al., 2000). See also:Protein StabilityA well-studied feature of haloarchaea is the purple

membrane, found in specialised regions of the cell mem-brane, which contains a two-dimensional crystalline latticeof a single chromoprotein, bacteriorhodopsin. Bacter-iorhodopsin contains a proteinmoiety, bacterio-opsin, anda covalently bound chromophore, retinal, and acts as alight-dependent transmembrane proton pump (Lanyi,2004) (Figure 4). The membrane potential generated can beused to drive adenosine triphosphate (ATP) synthesis andsupport a period of phototrophic growth. Bacter-iorhodopsin is induced by low oxygen tension and highlight intensity and can cover more than 50% of the surfaceof cells. The structures of bacteriorhodopsin and sensoryrhodopsin have been solved through electron and X-raydiffraction. Although originally thought to be unique tohaloarchaea, similar photosensory receptors and light-driven pumps have been discovered in a wide rangeof microorganisms, including both bacteria and fungi.See also: Archaeal Cells; RhodopsinHaloarchaea produce large quantities of red-orange

carotenoids. Carotenoids have been shown to be necessaryfor stimulating an active photorepair system for repair ofthymine dimers resulting from ultraviolet radiation(Crowley et al., 2006). The most abundant carotenoids areC-50 bacterioruberins, although smaller amounts of bio-synthetic intermediates such as b-carotene and lycopeneare also present. Retinal is produced by oxidative cleavageof b-carotene, a step which requires molecular oxygen.Several retinal proteins, in addition to bacteriorhodopsin,are also produced by haloarchaea, including halorho-dopsin, which is an inwardly directed light-driven chloridepump, and two sensory rhodopsins, which mediate thephototactic response (swimming toward green light andaway from blue and ultraviolet light) (DasSarma et al.,2001).Haloarchaea often produce buoyant gas vesicles, which

are hollow, rigid proteinaceous subcellular structures sur-rounding a gas-filled space. The function of gas vesicles for

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Figure 4 Integrated view of the biology of the extremely halophilic archaeon Halobacterium derived from its genome sequence. Many informational and operational processes revealed from the genome

sequence are shown. Transporters in the membrane are highlighted, including light-driven proton and chloride pumps, bacteriorhodopsin (BR) and halorhodopsin (HR), and the sodium/proton antiporter

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haloarchaea, organisms whose primary metabolism isaerobic, and that live in environments in which the solu-bility of molecular oxygen is low (due to high salinity andelevated temperatures), is to enable the cells to float to themore oxygenated surface layers. This also increases theavailability of light for purple membrane-mediated pho-tophosphorylation. See also: Bacterial and ArchaealInclusions

Haloarchaeal mutants, which have lost the ability toproduce purple membrane, gas vesicles, or carotenoids,occur spontaneously at a high-frequency. Genetic analysishas shown that recombinational events promoted bytransposable insertion sequences are responsible for theobserved genetic instabilities (DasSarma, 1989). Extra-chromosomal replicons, the structure and function ofwhich have been revealed through genome sequencing, arecommonly found to be reservoirs of insertion sequences(Ng et al., 1998). Several of these replicons contain essentialgenes and have been proposed to function as requiredminichromosomes rather than dispensable plasmids. Thefinding of plasmids and insertion sequences has aided thedevelopment of genetic tools for analysis andmanipulationof these organisms.

Haloarchaea are members of the archaeal domain andare phylogenetically distinct from both bacteria andeukaryotes. As such, they exhibit characteristic features,including eukaryotic-like deoxyribonucleic acid (DNA)replication, transcription, and translation machinery,branched-chain, ether-linked archaeal lipids, anda cell wallS-layer composedof a glycoprotein, similar to that found insome bacteria. See also: Archaeal Cell Walls

Cyanobacteria

Cyanobacteria dominate the planktonic biomass and formmicrobial mats in many hypersaline lakes. The top brownlayer of saline microbial mats contains a common uni-cellular cyanobacterial species, Aphanothece halophytica

(Figure 3). This species can grow over a wide range of saltconcentrations, from2 to 5MNaCl, is an extreme halophilewith a salt optimum of 3.5M, which lyses in distilledwater. It uses glycine betaine as its major compatible solute(Figure 6), which it can either take up from the mediumor synthesise from choline. A. halophytica and similarunicellular cyanobacteria have been described from GreatSalt Lake, Dead Sea, Solar Lake and artificial solar ponds.Another planktonic cyanobacterium reported from GreatSalt Lake is Dactylococcopsis salina. A thermophilic spe-cies has been identified in a geothermal seawater lagoon insouthwest Iceland (Banerjee et al., 2009).Avarietyoffilamentous cyanobacteria, for example in the

order Oscillatoriales, such as Oscillatoria neglecta, Oscilla-toria limnetica and Phormidium ambiguum, develop in thegreen second layer of mats in hypersaline lakes (Fourcanset al., 2004; Figure 3). These are more moderate halophiles,usually growing optimally at 1–2.5M NaCl, which formheterocysts and fix nitrogen. Another common species inthe same family isColeofasciculus chthonoplastes (formerlyMicrocoleus chthonoplastes). See also: Cyanobacteria

Other phototrophic bacteria

Some phototrophic bacteria occur beneath the cyano-bacterial layers in anaerobic, but lighted zones in hyper-salinemicrobial mats (Fourcans et al., 2004; Figure 3). Theyusually grow anaerobically by anoxygenic photosynthesis,although many also have the capacity to grow aerobicallyas heterotrophs. They can use reduced sulfur (hydrogensulfide, elemental sulfur), organic compounds, or hydrogenas electron donors. They include green and purple sulfurand nonsulfur bacteria and are characterised by their use ofbacteriochlorophyll pigments. The green sulfur bacteria,such as the slight to moderately halophilic Chlorobiumlimicola and Chlorobium phaeobacteroides, deposit elem-ental sulfur granules outside their cells and are capable ofnitrogen fixation. C. limicola can take up glycine betaine

(a) (b)

Figure 5 Extremely halophilic archaeal (a) and human (b) protein-DNA complexes. Models of similar transcription initiation complexes, showing the

protein surface charges (red for acidic or negative and blue for basic or positive), surrounding the DNA double helix. The haloarchaeal proteins are acidic

whereas the human proteins are basic. Adapted from DasSarma et al. (2006) & S. DasSarma.

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from the environment and synthesises trehalose for use asosmolytes. The moderately halophilic, anoxygenic, pho-totrophic, filamentous green nonsulfur bacteria such asChloroflexus aurantiacus can also be slightly thermophilic.See also: Green Sulfur Bacteria

Halophilic purple sulfur bacteria include Halochroma-tium glycolicum, which grows photoorganotrophicallyusing glycolate and glycerol, Halochromatium salexigens,and Thiocystis violascens. They synthesise N-acet-ylglutaminylglutamine amide as a minor component oftheir compatible solute and use sucrose and glycine betainefrom their environment. Thiocapsa roseopersicina andThiohalocapsa halophila, from Guerrero Negro both syn-thesise sucrose and take up glycine betaine from theenvironment. T. halophila also synthesises glycine betaineand N-acetylglutaminylglutamine amide for osmoprotec-tion. The purple nonsulfur bacteria Rhodothalassiumsalexigens from evaporated seawater pools and Rhodovi-brio salinarum from a saltern both use glycine betaine andectoine as osmolytes (Figure 6).

The purple sulfur bacteria, such as Ectothiorhodospiraspecies, dominate alkaline soda lakes (Ollivier et al., 1994).The moderate halophile Ectothiorhodospira mobilis isa strict anaerobe and uses carboxamines as compatiblesolutes and the osmolyte N-a-carbamoyl-L-glutamine-1-amide to counter osmotic stress. The extreme halophile,Halorhodospira halochloris, isolated from Wadi Natrun,was the first bacterium shown to synthesise and accumulateectoine, a cyclic aminoacid,which it uses alongwith glycinebetaine and trehalose as a compatible solute.

Sulfur oxidising bacteria

Below the cyanobacteria and the phototrophic bacteria inmicrobial mats, halophilic, filamentous, carbon dioxide-fixing bacteria oxidise hydrogen sulfide (and elementalsulfur) to sulfate (Figure 3). Examples include filamentousBeggiatoa alba from Guerrero Negro and Beggiatoa lepti-formis from Solar Lake, and the unicellular proteobacter-ium Halothiobacillus halophilus from Lake O’Grady inWestern Australia.

Anaerobic bacteria and archaea

Alarge variety of facultative and strictly anaerobic bacteriaand archaea inhabit the bottom layers of microbial matcommunities and sediment in hypersaline lakes (Ollivieret al., 1994, Figure 3). These include fermentative bacteria,homoacetogenic bacteria, sulfate-reducing bacteria andmethanogenic archaea. Fermentative anaerobic bacteria,which grow at saturated NaCl concentrations have beendescribed. One example is Halanaerobacter chitinivorans,isolated from a saltern, which is capable of fermentingchitin from brine shrimp and brine flies. Another moremoderate halophilic isolate, Halocella cellulolsilytica, fer-ments carbohydrates including cellulose. Sporohalobacterlortetii and Orenia marismortui are sporogenous and fer-ment carbohydrates.Several homoacetogens, strict anaerobes which produce

acetate from oxidation of sugars or amines, have beendescribed. For example, Halanaerobium saccharolyticumferments carbohydrates and N-acetylglucosamine and cangrow at a wide range of NaCl concentrations. Theextremely halophilicAcetohalobium arabaticum (with a salttolerance of 1–4.5M NaCl), isolated from Lake Sivash,grows on glycine betaine and trimethylamine and canreduce carbon dioxide to acetate. It is a likely competitor ofsulfate-reducing bacteria for hydrogen.Sulfate-reducing bacteria use sulfate as the terminal

electron acceptor, although many can also utilise othersulfur compounds, nitrate and fumarate. They differ intheir ability to oxidise different compounds, though mostuse low-molecular weight organic compounds like lactate,pyruvate, ethanol, and volatile fatty acids, or hydrogen aselectron donors. A few can use carbon dioxide as their solecarbon source. Although many slightly halophilic sulfatereducers have been isolated, mostly from marine environ-ments, relatively few of these can survive at an extremelyhigh salinity. Desulfohalobium retbaense, isolated fromLake Retba, Senegal, and Desulfovibrio halophilus, fromSolar Lake, are two moderately halophilic species whichcan grow at up to 4M NaCl, but only relatively slowly.Another isolate, from the deep-sea hypersaline pools in theRed Sea, is similar toD. halophilus. The osmoregulation ofsulfate-reducing bacteria likely occurs by accumulation ofsalts internally.Among anaerobic archaea, halophilic methanogens are

generallymethylotrophic and are strict anaerobes. Several,mostly moderate halophilic methanogens have been iden-tified, including Methanohalophilus halophilus from amicrobial mat, Methanohalophilus mahii from Great SaltLake and Methanohalophilus portucalensis from a Portu-guese saltern. The slight halophile, Methanosalsum zhili-nae, is also an alkaliphile and a slight thermophile. Theextremely halophilic methanogen, Methanohalobiumevestigatum, with a NaCl optimum of 4.5M, is also athermophile with a temperature optimum of 508C.Methanogenesis has been reported from moderately toextremely high salinity deep-sea brine pools in the Gulfof Mexico and Mediterranean Sea. Methanogens use

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Figure 6 Structure of three common compatible solutes in halophiles.

Zwitterionic forms of glycine betaine and ectoine, and the neutral glycerol

are commonly found in halophilic microorganisms and help to balance the

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b-amino acids (b-glutamine, N-a-acetyl-b-lysine) as com-patible solutes and play an important role in the anaerobicdegradation of glycine betaine in their environments. Theirintracellular salt concentration is somewhat higher than inmost bacteria (approximately 0.6M KCl) but is signifi-cantly lower than that found in extremely halophilicarchaea. Recently, a community of the halophilic archaeahas been reported from sulfide and sulfur-rich springs,including one described species, Haloferax sulfurifontis(Elshahed et al., 2004). See also: Euryarchaeota; Metha-nogenesis Biochemistry; Methanogenesis: Ecology

Aerobic and facultative anaerobicGram-negative bacteria

Many moderately halophilic, heterotrophic Gram-negative bacteria belonging to the Halomonas and Chro-mohalobacter genera have been described. Other generawith halophilic representatives include Salinivibrio,Arhodomonas, Pseudomonas, Flavobacterium, Alcaligenes,Alteromonas,Acinetobacter andSpirochaeta.Most of theseare heterotrophs, and include the nitrate reducing Arho-domonas aquaeolei, isolated from subterranean brineassociated with an oil field, and Chromohalobacter mar-ismortui, from the Dead Sea. Others include Chromohalo-bacter beijerinckii, from salted beans preserved in brine,Pseudomonas halophila, from Great Salt Lake and Salini-vibrio costicola, originally isolated from Australian bacon.SeveralHalomonas species are capable of nitrate reduction,including Halomonas elongata, isolated from a solar salt-ern, and Halomonas halodenitrificans, isolated from meatcuring brines. Others include Halomonas eurihalina, isol-ated from saline soil, which produces an extracellularpolysaccharide, Halomonas halodurans, from estuarinewaters, which is capable of degrading aromatic com-pounds, Halomonas pantelleriensis, which grows at a pHoptimum of 9, and Halomonas subglaciescola, frombeneath the ice of Organic Lake in Antarctica. Theseorganisms primarily use glycine betaine or ectoine ascompatible solutes. Genes for uptake of glycine betainefrom the medium and its synthesis from choline, and syn-thesis of ectoine have been cloned fromHalomonas speciesand some other halophiles (Bestvater et al., 2008; Canovaset al., 2000).

Among spirochetes, themoderate halophile,Spirochaetahalophila, found in Solar Lake, is a chemolithotroph cap-able of iron andmanganese oxidisation. The flavobacteria,Psychroflexus gondwanensis and Salegentibacter salegens,are psychrotolerant halophiles isolated from AntarcticLakes. See also: Spirochaetes

Gram-positive bacteria

This group includes moderately halophilic species of thegenera Nesterenkonia, Tetragenococcus, Salinicoccus,Bacillus, Halobacillus and Marinococcus (Ventosa et al.,1998). They include cocci such as Nesterenkonia halobia,isolated fromsalterns,which produce yellow-red carotenoid

pigments, Nesterenkonia lacusekhoensis, from the hypersa-line Ekho Lake in East Antarctica, Tetragenococcus halo-philus, from fermented soy sauces, squid liver sauce andbrine for curing anchovies, which is capable of lactic acidfermentation, and severalSalinicoccus species from salterns.Other examples include Gracilibacillus dipsosauri, from thenasal cavity of a desert iguana, Bacillus haloalkaliphilus,fromWadi Natrun andVirgibacillus halodenitrificans, froma solar saltern in southern France.Halobacillus litoralis andHalobacillus trueperi were isolated from Great Salt Lake.Halobacillus halophila is an endospore forming bacterium,from which the compatible solute N-e-acetyllysine wasoriginally isolated. Many of these organisms use proline,ectoine, or N-acetylated diamino acids, which they arecapable of synthesising, as compatible solutes. See also:Gram-type Positive BacteriaHalophilic actinomycetes from saline soils include

Actinopolyspora halophila, which grows best at moderateNaCl concentrations, and is one of the few heterotrophicbacteria which can synthesise the compatible solute glycinebetaine.

Eukaryotic Halophiles

Algae

At moderately high salinities (1–3.5M NaCl) dense popu-lations (4105 per mL) of green algae are supported (Javor,1989). Dunaliella species, for example Dunaliella salina,Dunaliella parva and Dunaliella viridis, are ubiquitous andare the main source of food for brine shrimp and the larvaeof brine flies. Most species of green algae are moderatehalophiles, with only a few extremely halophilic species, forexample Dunaliella salina and Asteromonas gracilis, whichare capable of slow growth at up to saturated NaCl con-centrations (Figure 2).The algae predominantly use polyols as compatible

solutes.Dunaliella salina synthesises glycerol in response toosmotic stress (Chen and Jiang, 2009) (Figure 6). Thecytoplasmic concentration of glycerol can reach 7M whengrown in medium containing 5M NaCl and can constituteover 50% of the dry weight of cells. However, the intra-cellular sodium concentration has been reported to be lessthan 100mM over a wide range of external salt concen-trations. During moderate dilution stress glycerol does notleak out of cells, but is instead metabolised and trans-formed into osmotically inactive reserve material.Diatoms are algae surrounded by silica cell walls and are

commonly found, but rarely abundant in hypersalineenvironments. A variety of species have been found atapproximately 2M NaCl, and the upper limit for diatomgrowth is reported to be approximately 3M NaCl.Examples of common diatoms include Amphora coffeae-formis and Nitzschia and Navicula species. Though osmo-regulation has not been extensively studied in diatoms,accumulation of proline and oligosaccharides has beenreported in some species. See also: Diatoms

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Protozoa

A large variety of protozoa exist in hypersaline environ-ments, but few have been extensively described. Onemoderately halophilic ciliate, Fabrea salina, has been isol-ated from several saline lakes from Africa to Australia.Although in freshwater, protozoa are known to regulateosmotic pressure with contractile vacuoles that expelwater, their mechanism of osmoregulation in hypersalinebrine has not yet been investigated. See also: ProtozoanDiversity and Biogeography; Protozoan Ecology

Fungi

Yeasts and filamentous fungi are well adapted to toleratehypersaline environments. They grow best under aerobicconditions on carbohydrates at moderate temperaturesand acidic to neutral pH. Debaryomyces hansenii, a halo-tolerant yeast, isolated from seawater can grow aerobicallyin salinities of up to 4.5M NaCl, and has been studiedextensively by genome sequencing and transcriptomicanalysis (Gonzalez et al., 2009). It produces glycerol as acompatible solute during logarithmic phase and arabitol instationary phase. Hortaea werneckii, a melanised fungus,was isolated from hypersaline waters of solar salterns andits osmoresponsive genes have been identified by tran-scriptomic methods (Lenassi et al., 2007). It is one of themost salt tolerant fungi known, growing at a wide range ofsalinity up to saturating NaCl concentration. Xerophilic,halophilic fungi, for example Polypaecilum pisce have alsobeen isolated from salted fish. See also: Fungal Ecology

Multicellular eukaryotes

There are a surprising number of invertebrates that cansurvive in hypersaline environments (Javor, 1989). Insectssuccessful in hypersaline environments include brine flies,such as Ephydra cinerea, and brine shrimp, Artemia fran-ciscana and related species. Other invertebrates includerotifers such as Keratella quadrata, flat worms suchas Macrostomum species, copepods such as Robertsoniasalsa, and ostracods such as Cyprideis torosa. A varietyof obligate and facultative halophytic plants, for exampleAtriplex halimus, Mesembryanthemum crystallinum andLaguncularia mangrove species can also survive in soilswith moderately high salinity. Few vertebrates can survivehypersaline conditions, with Tilapia species observed inmoderately high salinity. Many hypersaline environmentssupport a wide diversity of birds, one of the most spec-tacular of which is the pink flamingo. This bird is bornwhite and obtains its colour from the pigments of halo-philic microorganisms in its food.

Biotechnology

Although traditional commercial uses of halophiles havebeen in the food and nutraceutical industries (e.g. fer-mentation of soy and fish sauces, b-carotene production),

recent trends are to exploit the many novel and uniquemolecules (e.g. enzymes, bacteriorhodopsins, etc.) in theseorganisms, for molecular biotechnological applications(DasSarma et al., 2010a; Oren, 2010). A most usefulhalophilic biomolecule type is their retinal-containingchromoproteins, which are being used for diverse appli-cations, such as in biocomputing (Birge, 1995), as a light-sensitive neurological probe (Gradinaru et al., 2009), andfor treatment of blindness from retinitis pigmentosa(Busskamp et al., 2010). Halophilic microorganisms alsoproduce many stable enzymes (including hydrolyticenzymes such asDNases, lipases, amylases, gelatinases andproteases) capable of functioning under conditions, whichwould lead to precipitation or denaturation of most otherproteins. Compatible solutes of halophilic bacteria areused in cosmetics and improving hydration propertiesgenerally (Bestvater et al., 2008). Halophilic proteins alsocompete effectively with salts for hydration, a propertywhich may result in their functioning in low water activityenvironments, including organic solvents. Biodegradablepolymers such as polyhydroxyalkanoates are produced inlarge quantities by some halophilic microbes (Han et al.,2010), and have both medical and other specialised appli-cations. A variety of other novel halophilic biomoleculesalso have been targeted for commercial applications, forexample, gas vesicles for bioengineering floating particles,pigments for food colouring and metabolites as stressprotectants.Halophiles are becoming increasingly valuable for

bioremediation since many industrial processes releasesalts as contaminated brine effluent into the environment.Another growing area for applications of halophiles is inthe development of sustainable bioenergy technology toaddress concerns about petroleum shortage and globalwarming. See also: Bioremediation

Conclusions and Future Prospects

Halophiles are an interesting class of extremophilicorganisms which have adapted to harsh, hypersaline con-ditions. They can compete successfully for water and resistthe denaturing effects of salts. A wide variety of micro-organisms are halophilic, including extremely halophilicand methanogenic archaea, cyanobacteria, and green andpurple bacteria, sulfur oxidising bacteria, anaerobic fer-mentative, homoacetogenic, sulfate reducing bacteria, andGram-negative and Gram-positive heterotrophic bacteria.In addition, some algae, protozoa, fungi and a few higherorganisms have adapted to life in high salinity.Adaptation to hypersaline conditions is interesting from

anevolutionary standpoint.The earliest prokaryotic fossilsfound in ancient stromatolites (whichmaybemore than 3.5billion years old), are very similar in appearance to themicrobial mats found in modern hypersaline ponds. Thehalophilic andmethanogenic archaea are bothmembers ofthe archaeal branch of the phylogenetic tree which likelyappeared very early in evolution. These findings, and the

Halophiles

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likelihood for concentration of brines during prebioticevolution, suggest that adaptation to salts may have beenamong the earliest evolutionary inventions (Dundas,1998). Future studies will provide greater insights intothese fascinating evolutionary questions.

The diversity of microorganisms in hypersaline envir-onments is also of growing interest in research and edu-cation. Few hypersaline environments have been carefullysurveyed using molecular methods. Recent findings ofbacterial and archaeal metabolic activities suggest thatthese environments harbour diverse communities ofmicrobes, many of which have not been cultured. Meta-genomic studies of hypersaline environments are beginningto yield interesting results, for example the discovery of atiny ‘nano’ species that is smaller than most free-livingspecies. The possibility that extreme halophiles may sur-vive on other planets, such as some recently reported salineenvironment onMars, is intriguing. Because of such novelcharacteristics and their relative safety, some halophileshave become popular for educational uses in schools andcolleges (DasSarma, 2006, 2007).

The molecular mechanisms used for adaptation tohypersaline conditions are now being investigated usingmodern technologies. Powerful tools for genetic andgenomic analysis of halophiles, including DNA micro-arrays, gene knockouts, and proteomics, are now routinelyused (e.g. seeDasSarma et al., 2006). Halophilic organismscan exclude salts by synthesis of an equally high concen-tration of uncharged compatible solutes or osmolytes, andcontain stable macromolecules which can withstand thedenaturing effects of salts. The genes and proteins involvedin synthesis and accumulation of compatible solutes andtheir regulation are being explored. For archaea, recentstructure-function studies are aimed at determining howtheir highly acidic proteins can function in high salinity(Bergqvist et al., 2003; Britton et al., 2006).

Finally, as a result of natural and man-made environ-mental changes, desiccated and saline environments are onthe increase globally, and are likely to have significanteffects on the biosphere in the twenty-first century. As aresult, a better understanding of halophiles is needed toaddress future challenges for conservation of ecologicallysensitive areas, aswell as to engineer plants and fish to growin more saline environments, and exploit halophilicmicrobes for many other opportunities in biotechnology.

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Harbor, NY: Cold Spring Harbor Laboratory Press.

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Adaptation to Life at High Salt Concentrations in Archaea,

Bacteria, and Eukarya. Dordrecht, Netherlands: Springer.

Vreeland RH and Hochstein LI (eds) (1993) The Biology of

Halophilic Bacteria. Boca Raton, FL: CRC Press, Inc.

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