Research Journal of Biotechnology Vol.

15 (5) May (2020)

Res. J. Biotech
Review Paper:
Osmoadaptation in halophilic bacteria and archaea
Mukhtar Salma1,2*, Malik Kauser Abdulla1 and Mehnaz Samina1
1. School of Life Sciences, Forman Christian College (A Chartered University), Ferozepur Road, Lahore 54600, PAKISTAN
2. School of Biological Sciences, University of the Punjab, Lahore, PAKISTAN
*salmamukhtar85@gmail.com

Abstract which constitutes approximately 99% of the biosphere.

Microbial diversity characterized from saline Based on their origin, most of the hypersaline bodies are
environments shows a great adaptation ability to grow classified into thalassic environments with salt concentration
under a wide range of salt concentrations. Halophiles similar to sea water. These environments were usually
derived from evaporation of sea water. They have Na+ and
are salt loving organisms that grow in salt affected
Cl- dominating ions and the pH is near neutral to slightly
environments and can tolerate a wide range of salt alkaline. Hypersaline thalassic bodies such as brines, salt
concentrations. According to their salt requirements, lakes often display bright red colour due to red pigments of
they can be categorized into slight, moderate and haloarchaea.3
extreme halophiles. These organisms have a unique
ability to survive and grow at high salt concentration Two large and best-studied hypersaline lakes are the Great
and thus could serve as tremendous model systems to Salt Lake in the USA and the Dead Sea in the Middle East.
understand the molecular basis of high salt adaptation. A great diversity of microbial life is observed in such
environments with salinity upto 3-3.5M salt concentrations.
The physiology of the halophiles is affected by change Hypersaline lakes and ponds of Mediterranean salterns in
France contain saline water (3.1-3.6M salt concentrations)
in salt concentration, pH, growth temperature and
from where sodium chloride precipitates in the form of halite
nature of available nutrients. Halophilic bacteria and crystals.4 In Pakistan, Khewra Salt Mine also provides a rich
archaea have evolved two strategies to deal with high and extensive habitat for halophytes and halophile
osmotic stress. Most of the bacteria use ‘compatible microorganisms. Rhizosphere microbiome of halophytes
solute strategy’ in which they accumulate different contains a wide diversity of halotolerant and halophilic
organic compounds like proline, ectoine, glycine microorganisms.5 These habitats have harsh environment
betaine and glutamine. Halophilic archaea and conditions such as high salinity, low oxygen availability,
anaerobic bacteria cope with high salt stress by ‘salt in high temperature, high light intensity and extremely
strategy’ in which they maintain their internal alkalinity.
environment by accumulation of high concentration
KCl ions. Halophiles have evolved two basic strategies to survive in
salt stress conditions. In ‘compatible solute’ strategy, most
Keywords: Halophilic bacteria, Haloarchaea, of the halophilic bacteria cope with their external
Osmoregulation, Compatible solutes, Inorganic ions. environments by accumulating small, highly water-soluble
organic compounds like glycine, betaine, proline, glutamine,
potassium, glutamic acid and ectoine. In ‘salt in’ strategy,
Introduction bacterial cells maintain internal environment by
Halophiles represent an interesting class of extremophiles. accumulating high concentrations of potassium chloride.1,3
They are salt-loving organisms that grow in saline Halophilic bacteria can also be used in food (fermentation of
environments. Halophilic organisms can grow in hypersaline soy and fish sauces) and nutraceutical industries (β-carotene
environments which require at least 0.2M NaCl for their production).4,6 Mostly halophilic bacteria have ability to
growth. They can be classified as slightly, moderately or degrade certain toxic compounds and have role in
extremely halophilic depending on their requirement for bioremediation of heavy metals.7 Halophiles can also be
sodium chloride. Slight halophiles are microorganisms used in biofuel production to overcome the expected
which can grow at 0.2-0.85M NaCl (1-5%) concentrations, shortage of petroleum and concerns regarding global
moderate halophiles that grow optimally at 0.85-3.4M NaCl warming.8 Halophiles have enzymes (extremozymes) that
(5-20%) concentrations and extreme halophiles which can enable these organisms to function in such extreme
grow optimally at 3.4-5.1M NaCl (20-30%) concentrations. environments.9,10 In this review, we mainly focused on the
Halotolerant organisms have ability to grow in the saline osmoregulation of halophilic microorganisms specially
environments but they can also grow in the absence of a high
bacteria and archaea. Different strategies used in
concentration of salt.1,2 osmoadaptation of halophiles were explained thoroughly.
Hypersaline environment is usually described as that
contains significant salt concentrations with salinity level
Halophilic bacteria
more than ocean water (3.5% of total dissolved salts). Mostly halophilic bacteria are cocci, rod, triangular and even
Oceans are the largest body of saline water in the world square-shaped. Some strains are pleiomorphic especially

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when the ionic conditions of the media are altered and most hydrogen sulfide to form sulfates. For example,
lie below the NaCl level of 1M/L. The physiology of the Achromatium volutans, a filamentous bacterium was
moderate and extreme halophilic bacteria is affected by isolated from solar lakes and Thiobacillus halophilus a
change in salt concentration, growth temperature and nature halophilic chemoauthotrpohic bacterium was isolated from
of available nutrients.11-13 Extremely halophilic bacteria the hypersaline lake, Australia.20
generally grow slowly. Halophilic bacteria and archaea can
be identified commonly by phenotypic characterization as Haloarchaea
well as 16S rRNA gene sequences.14 Moderately halophilic The extremely halophilic archaea are members of the class
bacteria are dominant in mostly hypersaline environments Halobacteria.21 They are cocci, rods or a multitude of
and they constitute a major proportion of total microbial involution forms from disks to triangle. They grow in the
population in the hypersaline environments and play a major presence of amino acids and salt with at least 1.5M NaCl
ecological role. (9%) concentration and lack muramic acid containing
peptidoglycan in the cell envelope. They are mostly
Currently, a large number of bacterial species related to pigmented bacteria (orange to red) due to presence of
different bacterial phyla especially gram-positive showed carotenoids.22 They have bacterioruberins, a light-sensitive
moderately halophilic response. Moderately halophilic protein which provides chemical energy for the cell by using
bacteria include members of Proteobacteria (Halomonas, sunlight to pump out of the cell.
Chromohalobacter, Pseudomonas, Marinobacter,
Rhodospirillum, Aeromonas, Alteromonas, Rhodovibrio, The intracellular enzymes of halobacteria have a
Halovibrio and Alcaligenes), Firmicutes (Halobacillus, requirement for high levels of KCl with 3M-5M
Virgibacillus, Oceanobacillus, Staphylococcus, concentration. They have many antibiotic resistant genes to
Gracilibacillus, Clostridium, Pontibacillus, Sporosarcina survive in hypersaline environments such as salt mines, salt
and Planococcus), Actinobacteria (Kocuria, Streptomyces lakes, soda lakes, salterns and marine environments.
and Rubrobacter), Actinomycetes (Nocardia, Nocardiopsis, Haloarchaeal genera Halorubrum, Halobacterium,
Streptomonospora, Actinopolyspora and Nesterenkonia) and Halococcus, Haloferex, Natronococcus and Haloarcula
Bacteriodetes (Flavobacterium, Salinibacter and belonged to the Halobacteriaceae. Intracellular gas vesicles
Polaribacter). are synthesized by several halophilic bacteria and archaea.
These vesicles are usually filled by different gases and
Mostly extreme halophilic bacteria contain a variety of provide buoyance and enable cells to regulate their position
carotenoids as carotenoids help membrane stabilization in in the aquatic environments.1,6
Thermus thermophiles, Ruberobacter radiotolerans and
help to tolerate the high osmotic stress in Halobacterim Halobacterium salinarum contains fibrillary structures
spp.13-15 Many gram-positive bacteria isolated from different (fibrocrystalline bodies) which represent the presence of
saline environments (salt lakes, salt mines and salt marshes) cytoskeleton-like organelle in haloarchaeal cells.23 Some
also have carotenoids which indicate the crucial role of anaerobic archaeal genera especially methanogens have
carotenoids in osmotolerance of these bacteria.16 been reported from the hypersaline environments. They use
methyl related substrates for their growth and have been
Cyanobacteria are characterized by the presence of isolated from the Great Salt Lake and solar salterns.
chlorophyll and phycobilin pigments. They are Methanohalophilus and Methanohalobium, extreme
photosynthetic bacteria (planktonic biomass) and form halophilic and thermophilic methanogen, generally grow
microbial mats in many hypersaline lakes.17 Aphanothece with optimum 4.4M NaCl concentrations and 50℃. These
halophytica is an extreme halophile form of brown layer of bacteria have intra-cellular high salt concentration.24
microbial mat on the water surface. Mostly cyanobacteria Methanogenesis is a process of organic matter degradation
use glycine betaine as the major compatible solute which by bacteria and archaea especially in marine sediments.
they take from the medium or synthesize from choline.18 During this process, bacteria produce methane by sulfate
Many genera related to halophilic cyanobacteria have been reduction pathway as well as acetate pathway.25
described from the Dead Sea, the Great Salt Lake, Solar
Lake and other salt lakes and ponds but the cyanobacterial Osmoregulation by halophilic bacteria and
diversity has not been studied extensively from the archaea
hypersaline environments.19
Water is one of the most important prime elements
responsible for life. Living organisms have ability to adapt
Moderately halophilic purple sulfur bacteria like
to changes in external environment (osmotic pressure) to
Chromatium spp. have ability to store sulfur granules inside
survive under harsh conditions.26 In exposure to hypersaline
cells and they grow phototrophically by using glycerol or
environments, water usually moves out of the microbial cells
glycolate. Rhodospirilum salexigens (purple non-sulfur
along the osmotic gradient and it causes dehydration of the
bacterium) can use glycine, betaine or ectoine as osmolytes.
cytoplasm. Halophilic bacteria and archaea have adapted
Sulfur oxidizing bacteria are halophilic gram-positive,
genetic and physiological modifications to survive in
filamentous CO2 fixing bacteria. They oxidize sulfur and
extreme (hypersaline) environments. They can be classified

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according to their salt dependency or salt requirement. osmoadaptation (Fig. 2). Cyanobacteria and Proteobacteria
Therefore halophiles have adapted different mechanisms of have ability to synthesize glucosyl glycerol or use it from the
osmoadaptation to survive in hypersaline environments.27,28 medium in salt stress environments.38-40
Halophilic bacteria and archaea generally use two
mechanisms of adaptation to hypersaline environments: (1) Zwitter solutes: Halophilic bacteria have some neutral
the accumulation of compatible solutes (osmolytes) to the amino acids which are used as compatible solutes in salt
imposed osmotic pressure, (2) the maintenance of high stress conditions. But most of the neutral amino acids are
intracellular ionic (K+) concentrations and adaptation of utilized at high concentrations due to lack of availability of
entire intracellular enzymatic machinery to function in these amino acids (Fig. 2). As free polar amino acids are used
hypersaline conditions (Fig. 1 and 2). as intermediates in protein synthesis process, bacterial cells
accumulate or synthesize some zwitterionic molecules
Saum and Müller29 explained different mechanisms used for derived from amino acids as osmolytes.41,42
osmoregulation of Halobacillus spp. as shown in fig. 1.
Mostly halophilic bacteria and archaea have two component Betaine: Betaine is a natural compound having a negatively
regulatory systems. They have osmosensor and charged ions carboxylate group and a positively charged
osmoregulator proteins to recognize osmotic imbalance. phosphonium ions or ammonium ions. It is used as osmolyte
Regulator proteins bind DNA and regulate gene expression for the protection of cells to cope high osmotic stress, high
and sensor proteins (histidine kinases) detect the signals. salinity and high temperature.43 Different intracellular
This system controls the changes in osmotic pressure on both enzymes are involved for the accumulation of betaine. They
sides of the cell membrane.30 maintain internal balance by regulation of water inside the
cells and thus protect the cells from dehydration (Fig. 2).
Compatible solute strategy: Mostly halophilic bacteria
balance their cytoplasm with the high salt concentration of Different halophilic bacteria (Halomonas, Virgibacillus,
the medium by the accumulation of osmolytes (compatible Oceanobacillus, Kocuria and Polaribacter) can synthesize
solutes). These compatible solutes are excreted in the betaine from glycine with the primary amine methylated to
cytoplasm either by the bacterial cell itself or can be taken form a quaternary amine.44,45 Betaine concentration inside
from the medium. Osmolytes can be classified into two main the cells increases with increase in salt concentrations. Some
groups; organic solutes (trehalose, ectoine, betaine, proline), methanogens (Methanohalophilus and Methanohalobium)
inorganic solutes (K+, Mg2+, Na+).31,32 Mostly bacteria lack can accumulate and also synthesize betaine as an osmolyte
intracellular systems for active transport of water to cope either by methylation of glycine or oxidation of choline.24,42
with external osmotic stress. Therefore, they balance internal
environment by transport or synthesis of a group of organic Mostly halotolerant and halophilic bacteria use two
solutes (compatible solutes) without affecting metabolic metabolic pathways to synthesize betaine. Gram-positive
function of the cell.33,34 Compatible solutes can be classified bacteria (Virgibacillus, Oceanobacillus and Staphylococcus)
according to their chemical nature: (1) anionic solutes, (2) synthesize betaine by oxidation of choline with a single
zwitterionic solutes and (3) non-charged solutes (Fig. 2). enzyme (choline oxidase).46 In gram-negative bacteria (E.
coli), it is coded by bet operon with four genes and can occur
Organic anions: Mostly halophilic bacteria have high as membrane associated system. The bet operon includes
intracellular K+ and have negative potential inside the cell. betA gene (choline dehydrogenase), betB (betaine aldehyde
Organic anions (negatively charged solutes) are used to dehydrogenase), betI (putative regulator) and betT (choline
balance internal environment to survive at high salt transporter).47 These enzymes belong to family of
concentrations (Fig. 2). Some halophilic bacteria such as oxireductases. Choline is changed into an intermediate
Halomonas usually synthesize ectoine and Halobacterium compound (glycine betaine aldehyde) by choline
can synthesize L-glutamate as an osmolytes.35 These organic dehydrogenase and finally betaine (osmolyte) is produced by
anionic solutes have a carboxylate group which supplies the a catalytic enzyme (Fig. 1).
negative charge or may contain sulfate or phosphate groups.3
Methanogens and halobacteria increase their glutamate Sometimes microorganisms prefer to accumulate the betaine
levels with increasing external salt concentrations. The as synthesizing it. This process is because of the limited
negatively charged glutamates are accumulated at availability of precursors (substrates) rather than the result
concentrations comparable to the intracellular K+ of a low expression level of the genes. Some extreme
concentration.36,37 halophiles like methanogens (Methanohalophilus and
Methanohalobium) and haloalkalophilic sulfur bacteria like
Polyols: The polyol compounds like sorbitol, arabitol, Ectothiorhodospira usually synthesize betaine by
mannitol and glycerol are used as compatible solutes by dimethylglycine and sarcosine. This process involves two
halotolerant bacteria, algae, plants and some fungi.28 enzymes, dimethylglycine methlytransferase and sarcosine
Halotolerant bacteria included Bacillus, Pseudomonas, methyltransferase. Concentration levels of K+ ion and
Aeromonas, Stenotrophomonas and Zymomonas using sarcosine usually regulate the biosynthesis of betaine inside
sorbitol, mannitol and glucose (osmolytes) for the cell.48

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Ectoine: Ectoine, a cyclic tetrahydropyrimidine is used as Some Proteobacteria and marine Cyanobacteria are known
an osmolyte in halotolerant and halophilic bacteria (Fig. 1 to accumulate sucrose as an osmolyte in osmotic stress
and 2). It is either accumulated from the external medium or conditions.28,59 Cyanobacteria catalyzed sucrose by two
synthesized by bacterium itself. It was detected in a pathways, one with sucrose-6-phosphate synthase (SPS) and
halophilic, phototrophic bacterium, Halorhodospora the other involved sucrose-6-phosphate phosphatase (SPP).
halochloris isolated from the hypersaline Mono Lake.49
Recently, ectoine has been detected in a number of Proteins (Amino acids and peptides): Proline, acetylated
halotolerant and halophilic bacteria related to different glutamine dipeptide and carboxamine are the main
phylogenetic groups (Halomonas, Oceanobacillus, osmolytes from the class proteins (Fig. 2). They are mostly
Marinococcus and Nesterenkonia).34,36 Some detected from marine phototrophic bacteria
methylotrophic bacteria Methylophaga and Methylarcula (Ectothiorhodospira mobilus) and halophilic purple sulfur
can also synthesize ectoine to balance their intracellular bacteria. Protein solute molecules included Carbamoyl-L-
environment under the osmotic stress.24 glutamine 1-amide detected from the halophilic marine
phototrophic bacteria and a dipeptide N-acetyl glutaminyl
The biosynthesis and regulation of ectoine has been studied glutamine amide is usually synthesized by halophilic purple
in a large number of halophilic bacteria especially with detail sulfur bacteria.60,61
in Halomonas and Oceanobacillus.35,50,51 Aspartate
aldehyde is used as a precursor molecule in the biosynthesis Salt-in strategy: This strategy is used by two
of ectoine. This molecule is converted into 2, 4- phylogenetically unrelated groups: anaerobic halophilic
diaminobutyric acid and finally as a result of acetylation, bacteria (Haloanaerobiales) and aerobic halophilic archaea
ectoine is formed.28 The biosynthesis of ectoine is involved (Halobacteriales). These groups use inorganic ions (K+,
by the enzymatic action of three genes: ectA, ectB and ectC. Mg2+, Na+) to cope with external osmotic stress (high salt
Diaminobutyric acid acetyltransferase is encoded by the concentrations). All intracellular machinery is adapted to the
gene ectA, diaminobutyric acid aminotransferase is encoded high salt concentration in the external environment of the
by ectB and ectoine synthase is encoded by ectC.52,53 The cell (Fig. 1 and 2).1,43 The composition and concentration of
enzymes involved in biosynthesis of ectoine were these inorganic ions usually vary among different species
functionally characterized in Halomonas elongata. Activity and even within the same species by means of synthesis or
of diaminobutyric acid acetyltransferase and transport. KCl is generally used to balance intracellular salt
aminotransferase is regulated by intracellular K+ ion concentrations in the family Halobacteriaceae.
concentration.
In halophilic archaea, all the intracellular machinery
Non-charged Solutes: Some polar and non-charged organic (enzymes and other cell proteins) are adapted to the presence
molecules have been detected as compatible solutes in of high salt concentrations in the external medium.62,63
halotolerant and halophilic bacteria (Fig. 2). Some halophilic Different enzymes with unique characteristics are involved
bacteria form soil and marine environments accumulate in balancing of internal environment of the cell. These
glycerol as an osmolyte to protect cells in salt stress enzymes have been structurally modified with excess of
conditions.20,33 Glycerol has also been detected in some acidic amino acids and low content of hydrophobic amino
eukaryotes like halotolerant yeast Debaryomyces hatnsenii acids.
and Hortea werneckii under osmotic stress environments.54
Negatively charged derivatives of both myo-inositol and Halophilic enzymes and other proteins from the haloarchaea
glycerol are accumulated by some haloarchaeal strains.22 maintain their proper conformation and can function only in
the presence of high salt. Therefore halophilic archaea and
Carbohydrates: Some sugar molecules are also used as bacteria can flourish only in the presence of high salt.64-66
osmolytes for balancing of intracellular environment in salt Halophilic enzymes from the haloarchaea are defined either
stress conditions. They are usually reducing in nature and according to the salt tolerance of the organism from which
react with surface proteins of the cell. Some non-charged they are detected or with respect to their own salt
organic molecules like glycerol bind with reactive end of the requirements for activity, stability or solubility.
sugar to overcome the reducing nature of the sugar
molecules (Fig. 2). Conclusion
Microorganisms living under saline environments have
Different sugars with neutral molecules like glycerol or special genetic and physiological characteristics to survive
glyceramide have been detected from halotolerant and under harsh conditions. Halophiles can grow in wide range
halophilic bacteria.55 Trehalose is a disaccharide sugar of salt concentrations and can be classified into slight,
molecule used as a compatible solute identified in a variety moderate and extreme halophiles according to their salt
of organisms like bacteria, fungi and plants.56 It helps cells requirements. Halotolerant microorganisms have ability to
to cope with desiccation, heat, cold and hypersaline grow either under hypersaline conditions or even in the
environments. It is also used as a signaling molecule and absence of high salt concentrations. These organisms usually
energy source in different metabolic pathways.57,58 use two basic mechanisms for adaptation to high salt

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concentrations. Most of the bacteria use ‘compatible solute compounds like proline, ectoine, glycine betaine and
strategy’ in which they accumulate different organic glutamine.

Figure 1: Overview of osmoregulation in halophilic bacteria and archaea

Figure 2: Different strategies used for osmoregulation in halophilic bacteria and archaea

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Halophilic archaea and anaerobic bacteria cope with high 13. Amoozegar M.A. et al, Oceanobacillus halophilus sp. nov., a
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