HALOPHILES, INDUSTRIAL APPLICATIONS
PRIYA DASSARMA , JAMES A.
COKER , VALERIE HUSE , and
SHILADITYA DASSARMA
University of Maryland,
Baltimore, Maryland
featuring these products have become commercially successful. The compatible solutes of halophiles (8) are used
extensively in the cosmetics and food industries as moisturizers and preservatives and in the educational sphere
as models for teaching (9). A number of other applications,
including several in the chemical, environmental, biofuel
pharmaceutical, and health care industries, are likely to
be available in the future.
INTRODUCTION
SPECIALIZED PRODUCTS
Salt is essential for all life forms on earth, with
excess salinity representing a common stress condition.
Halophiles are organisms that require more than 0.2 M
NaCl for their growth and can resist the effects of
osmotic stress. Halophiles are found in nearly all major
microbial clades, including prokaryotic (Bacteria and
Archaea) and eukaryotic forms. They are classified as
slight halophiles when they grow optimally at 0.2–0.85
M (2–5%) NaCl, as moderate halophiles when they grow
at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles
when they grow at 3.4–5.1 M (20–30%) NaCl (1). At
lower salinities, members of the Eukarya and Bacteria
dominate populations, while members of the Archaea
dominate at higher salinities. For the purposes of this
encyclopedia entry, we will primarily concern ourselves
with applications of moderate and extreme halophilic
microorganisms and their products. The biology of
halophiles has been extensively reviewed (1–4).
Halophiles are found in environments all over the
planet—ranging from natural brines in coastal and deep
sea pools to salt mines and solar salterns (Fig. 1). The commercial importance of halophiles has been recorded since
ancient times. For example, as a part of their payment,
Roman soldiers were given ‘salt money’ (salarium argentum) and from this practice, the English word ‘salary’
(sal = salt, ary = pertaining to or connected with) has
been derived. Intense reddening of solar salterns resulting from blooms of halophilic microorganisms was used
as a biomarker for successful salt production in ancient
cultures (e.g. China and the Middle East). Even today,
halophilic microbes are critical for salt production—their
red-purple color increases absorption of solar radiation
and speeds up the evaporation process, leading to the
increased production of crystals of appropriate size and
shape. Halophiles are also important in the food industries
where they are used in the preparation of sauces such as
soy, miso and nam pla as well as in the production of other
products such as cheeses, salt cured meats, and fish (4,5).
Modern uses of halophiles include the production of natural nutritional supplements, β-carotene, vitamins, and
other components from green algal Dunaliella species (6)
for human consumption, and for the manufacturing of
two-dimensional films of purple membrane containing the
bacteriorhodopsin protein from archaeal Halobacterium
species for biosensor applications (7). Several companies
Halophiles produce novel biomolecules that are of
commercial interest, several of which have been or are
currently being exploited. Among them, the halophilic
archaea (haloarchaea) contain retinal proteins, such as the
light-driven proton pump, bacteriorhodopsin, in the purple
membrane that allows them to grow phototrophically, and
sensory rhodopsins, which allow for phototactic responses.
Retinal proteins are being developed for a variety of
optical devises, for example, as photosensors. Halophile
compatible solutes (Fig. 2) have also been employed by
industry for a wide range of uses from feed additives to
cosmetics (8).
Retinal proteins. With roughly 80 U.S. patents pertaining to bacteriorhodopsin from Halobacterium and commercial production by several companies, it is one of the
most recognized products derived from halophiles. The
ability to convert light to chemical energy in a nonchlorophyll system was first discovered in the haloarchaea (10).
The apoprotein responsible for this, bacterio-opsin, is combined with a retinal protein to make bacteriorhodopsin,
which is then organized into a two-dimensional crystalline
array in the purple membrane of haloarchaea (Fig. 3).
Bacteriorhodopsin is the simplest biological transducer
of solar energy and uses light to establish a transmembrane gradient of proton electrochemical potential (11).
The primary structure of bacteriorhodopsin was solved in
the 1970s, making it one of the first membrane proteins
for which this was accomplished (12). Bacteriorhodopsin
has several qualities that make it useful in an industrial setting: it is very stable over a range of temperatures (0 to 45◦ C) and pH values (1–11), its reactions
are self-regenerative and can be manipulated chemically,
genetically, or immunologically (13).
If ATP synthesis is blocked, bacteriorhodopsin will
produce an electric potential from its proton gradient.
Bacteriorhodopsin in the purple membrane is extremely
stable and can be immobilized in a single layer on solid substrates and can reliably produce photoelectric signals (14).
These traits make it attractive for many industrial applications. Bacteriorhodopsin from Halobacterium species is
being marketed for light sensors, nonlinear optics, and
optical data processing (15,16). It is also being considered
for use as an erasable photochromic film. A schlieren apparatus using a bacteriorhodopsin film as an adaptive image
grid with white light illumination has been proposed to
Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. Flickinger
Copyright © 2010 John Wiley & Sons, Inc.
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HALOPHILES, INDUSTRIAL APPLICATIONS
Figure 1. Blooms of halophiles in salterns.
Different colors reflect predominance of
diverse halophiles at varying salinities.
[Image provided by Cheetham Salt Ltd, Australia].
be used at NASA’s Kennedy Space Center for the remote
detection of gas leaks (17). This type of film is appealing
since it does not require chemical development and can
be used repeatedly. Bacteriorhodopsin films also have the
potential to be used as biochips which could pass electrical
signals, thereby replacing the integrated circuits in modern computers (18). Since bacteriorhodopsin can convert
light into electrical impulses, it can also be used as a light
sensor. To do this, a thin film of the pigment is placed
between an oxide electrode and an electrically conductive
gel. As light strikes the sensor, the charge displaced by
bacteriorhodopsin is transmitted to the electrode (19). It
has even been suggested that bacteriorhodopsin could be
used to give sight to industrial robots (20).
Compatible Solutes. Halophiles prevent the loss of
cellular water in their saline environments either by accumulating osmotically balanced levels of internal salts
or by producing compatible solutes or osmolytes that
can be used to maintain the stability of biomolecules.
Most halophilic bacteria and eukaryotes exclude salts,
and instead accumulate high solute concentrations within
the cytoplasm. These osmolytes are usually amino acids
(e.g. glycine- betaine, ectoine) or sugars and polyols (e.g.
sucrose, trehalose, and glycerol) (Fig. 2) which do not
disrupt metabolic processes and have no net charge at
physiological pH. Halotolerant yeasts and green algae
accumulate polyols, while most halophilic and halotolerant
bacteria accumulate zwitterionic species. The compatible
solutes may be accumulated via biosynthesis, de novo,
or from storage material, or by direct uptake from the
medium (21).
Ectoine and its derivatives have been patented as moisturizers in cosmetics (22), and as stabilizers in polymerase
−
O
+
N
Glycine betaine
O
OH
Glycerol
HO
OH
O
OH
Ectoine
N
NH
Figure 2. Compatible solutes are produced by halophiles to counteract osmotic stress of saline environments. Several common
examples are glycine betaine, glycerol, and ectoine.
chain reactions (23). Glycine betaine, a common osmolyte,
has been proposed as a feed additive. Its biosynthetic
pathway has been characterized and expression of Ectothiorhodospira halochloris genes has been engineered in
Escherichia coli, leading to betaine accumulation and
improved salt tolerance (24). Halomonas boliviensis is
being explored for its use in the production of ectoine in
batch-fed cultures (25). The green alga, Dunaliella salina,
is a good commercial source of glycerol, as an emulsifier,
HALOPHILES, INDUSTRIAL APPLICATIONS
(a)
(b)
Figure 3. Haloarchaea
produce
purple
membrane
as
two-dimensional sheets of bacteriorhodopsin. (a) High-level
producers of purple membrane result in cells that appear purple.
(b) Two-dimensional hexagonal array of bacteriorhodopsin in
purple membrane is observed by x-ray diffraction.
softener, protective medium, and as nutrient and nutrient
supplements among other applications (26,27).
FOOD AND AGRICULTURAL APPLICATIONS
While halophiles have traditionally been important in the
production of certain fermented foods, their application in
the nutritional products industry has been more recent.
The production of some salted food products, fermented or
preserved, are often done on an artisanal scale (28). There
are also large-scale industrial productions of other products, for example, soy sauce and fish sauce that exploit the
use of the degradative powers of halophiles. Both hydrolases and isomerases from halophiles may find increased
application in the food industry. Halophiles including
Halobacteria, Halococci, and Natronococci have been isolated from various food sources including fermented foods,
and sauces including kimchi and proteases have been isolated from halophiles from sources such as Thai fish sauce
(29,30).
Nutritional Applications. The green single-celled
alga, D. salina accumulates carotenoids at high salinity, with an optimum yield of β-carotene around 24%
NaCl. It is the richest source of β-carotene known (6,31),
and is being produced commercially in large-scale outdoor facilities, which produce 30–40 g dry weight/m2 /day
over sustained periods. The optimized, higher salt concentration results in a reduction of predatory protozoal
species and noncarotenoid producing Dunaliella species.
Additional research has found that the daily collection
of algal culture and adjustment of light intensity helped
to keep the balance between cell biomass and β-carotene
accumulation (6).
β-carotene is a lipid- and oil-soluble product with industrial applications as a suspension or solution in vegetable
oil, for food coloring in margarine, baked goods, and some
prepared foods (32). Water-soluble, dispersible, and emulsion formulations or microencapsulated beadlets allow for
food applications, including addition to orange drinks,
confectionery, and other prepared foods. Nutritional supplements prepared by oil suspension encapsulation, or
3
tableting using beadlet forms further expand the possibilities. β-carotene is pro-Vitamin A, and is an excellent
source of this vitamin since it is nontoxic even in high
doses (32). In addition to β-carotene, D. salina also produces ζ -carotene and xanthophylls such as zeaxanthin,
cryptoxanthin, and lutein.
Genetic Transfer of Salt Tolerance and Resistance. The widespread use of irrigation is responsible
for salt contamination of about one-quarter to one-third of
the world’s prime agricultural land (33). Most crop plants
are highly sensitive to salt, which causes osmotic stress
and water deficit as a result of an influx of sodium ions
into cells, and greatly limits productivity. Genetic engineering of salt resistance or tolerance properties into crop
plants has been shown by the directed expression of vacuolar sodium–proton antiporters. The endogenous vacuolar
gene AtNHX1 of Arabidopsis thaliana was used to increase
salt tolerance in this model plant up to 200 mM NaCl.
Although these results represent important advances, the
level of salt tolerance is still relatively low. Tolerance in
land plants to salinity levels akin to the ocean (530 mM)
could be improved using mechanisms that have evolved in
halophilic microorganisms enabling the use of seawater for
irrigation, which could truly revolutionize agriculture and
dramatically increase the area of arable land. The level
of salt resistance in halophilic microorganisms is much
higher than in plants, with some species capable of growth
in even saturating salinity (5.3 M). Halophilic characteristics of microorganisms result from the synthesis or uptake
of compatible solutes in the cytoplasm, which operate to
balance the osmotic strength of the medium and prevent
water loss (1).
HALOPHILIC ENZYMES
The primary interest in enzymes from extremophiles
stems from their activity under unique conditions. There
has been extensive research into how key metabolic
enzymes from these organisms can be exploited for
industrial use. Halophilic enzymes are of particular
interest because they are active in environments with
low water activity. They are thought to remain active by
having a predominance of negatively charged residues
on the solvent-exposed surfaces of the protein. These
negative charges attract water molecules and thereby
keep the proteins hydrated so that they do not precipitate.
It has also been shown that the hydrogen bonds formed
between the negative side chains and the water molecules
lead to the formation of a stable hydration shell (34).
The increase in negative charges also results in an
increase in ion-pair networks in halophilic enzymes (35).
Other proposed adaptations of halophilic enzymes are the
reduction of hydrophobic surfaces and the unusually high
number of ordered side chains (36).
Salt lowers water activity (aw ) and the aw of a saturated NaCl solution (0.75) is equivalent to a 60% solution
of DMF (N,N,-dimethyl formamide) (37). Organic solvents
are often used in industrial applications and sometimes
force enzymes to bind to specific enantiomers. The selectivity resulting from the lowered aw comes at the cost
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HALOPHILES, INDUSTRIAL APPLICATIONS
of a severe reduction (99.999%) in the activity of most
enzymes (38). However, this low level of water activity is ideal for halophilic enzymes, which have enhanced
activity under these conditions. Reports have shown that
halophilic enzymes display substantial levels of activity
in organic media, potentially making them an ideal choice
for biocatalysts in the presence of organic solvents (39).
Proteases. Halophilic proteases are widely used in
the detergent and food industries. They are also used
in the baking, dairy, and leather industry as well as in
the manufacture of soy products and in the production
of aspartame. Proteolytic activity with potential industrial application has been characterized in Halobacterium
spp. (40,41), Haloferax mediteranei (42), Natrialba asiatica (43), Natrialba magadii (44), Natronococcus occultus
(45,46), and Natronomonas pharaonis (47). For example,
the protease from Halobacterium produced a series of
glycine-containing oligopeptides with yields of up to 76%
in an environment with low water activity (33% DMF
and 69% water) without degradation due to secondary
hydrolysis (48).
Glycosyl Hydrolases. Glycosyl hydrolases have been
widely used for a wide variety of purposes including the
complete or partial degradation of cellulose, agar, agarose,
lactose, and amylose. Hemi-cellulose is the second most
abundant renewable polysaccharide found in nature. The
main component of hemi-cellulose is xylan, which is composed of xylose residues linked by β-1,4 glycosidic bonds.
Complete breakdown of xylan requires both xylanase and
xylosidase activity. Both of these activities have been
recorded in Halorhabdus utahensis, which was isolated
from the Great Salt Lake, Utah. Xylanases are used in
the manufacture of coffee (49), livestock feeds (50), and
flour (51). Xylanases can also be used in place of chlorine
bleaching for the removal of residual lignin from pulp (52).
Another important glycosyl hydrolase is agarase,
catalyzing the hydrolysis of agar which is composed
of subunits of galactose that form a polymer through
alternating 1,3-linked β-D-galactopyranose and 1,4-linked
3,6-anhydro-β-L-galactopyranose. Agarases are important
in the laboratory and industrial setting for liberating
DNA and other embedded molecules from agarose, and
extracting bioactive or medicinal compounds from algae
and seaweed. Further, they produce neoagarosaccharides,
which have been shown to inhibit the growth of bacteria,
slow down the degradation of starch, and present anticancer and antioxidation activities (53). Agarases have
been purified from several salt-tolerant microbes found in
the ocean (e.g. Pseudoalteromonads, Pseudomonas, and
Vibrio) and a patent has been issued on an agarase from
a salt marsh microorganism (54).
β-Galactosidases. β-galactosidase has been widely
used as a reporter molecule for molecular biology and
for the removal of lactose from dairy products. Two
β-galactosidases from halophiles have been characterized.
The first is from Haloferax alicantei and has exhibited
optimal activity in 4 M NaCl (55). The second, a family 42
β-galactosidase from the halotolerant Antarctic isolate,
Planococcus sp. has been shown to have activity near that
of the E. coli LacZ enzyme and retain roughly 40% of its
optimal activity in 4 M KCl (56).
Amylases. Amylases are used industrially in the first
step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile
industry in the de-sizing process and added to laundry detergents. In both cases, this is done to degrade
starch added to garments. Amylases have been characterized from many halophilic strains including moderate
halophiles such as Halomonas meridiana (57) to extreme
halophiles like Haloarcula hispanica (58) and Natronococcus amylolyticus (59).
Restriction Enzymes. An industrial application for
halophilic enzymes is the use of restriction endonucleases
for molecular biology applications. Site-specific endonuclease activity was reported in the haloarchaeon Halobacterium halobium (60). Another endonuclease (HacI) has
been isolated from Halococcus acetoinfaciens and the process for its production has been patented (61). The enzymes
HcuI, HhlI, and HsaI have been isolated from Halobacterium cutirubrum, H. halobium, and Halobacterium salinarium, respectively, and produced on a commercial scale.
Esterases and Lipases. Esterases and lipases are
widely used as biocatalysts because of their ability to
produce optically pure compounds. These types of enzymes
have also been useful ingredients in laundry detergents
for the removal of oil/grease stains. An esterase from
Haloarcula marismortui has recently been purified and
characterized (62). This enzyme exhibits a preference for
short chain fatty acids and monoesters and is dependent
on the presence of salt for proper folding and activity.
ENVIRONMENTAL APPLICATIONS
About 5% of worldwide effluents are saline or hypersaline,
and halophiles that are able to grow and flourish in
these environments show potential for use in bioremediation (63). The use of halophiles for bioremediation and
biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being
widely explored. Identification of both individual and consortional groups of halophiles from saline wastewater from
paper, textile, and oil industry processes (approximately
10 barrels of saline wastewater/barrel of oil produced) has
also led to the exploration of specific enzymes that could
be exploited in treatment (64,65). Since halophiles are
diverse and widespread and the large numbers of contamination sites are often saline to hypersaline, environmental
applications of halophiles hold significant promise.
Hydrocarbon Degradation. Researchers have
isolated consortia as well as identified individual microorganisms involved in biodegradation of hydrocarbons.
Halophiles associated with waste from oil production sites
were examined, and indigenous halophilic or halotolerant
bacteria that degraded petroleum hydrocarbons in
oil-brine soil were reported. Enrichments for halophiles,
for example, Marinobacter spp., that degraded benzene,
toluene, ethylbenzene, and xylenes, and converted
benzene into CO2 was also performed (66). The results
suggested that the culture was able to mineralize benzene.
Other work on aromatic substances include studies on
Haloferax and Halorubrum species for the biotreatment
HALOPHILES, INDUSTRIAL APPLICATIONS
of highly saline industrial effluents, enriching for those
species that could aerobically use benzoic acid and other
aromatic substrates as the sole source of carbon and
energy (67,68). Another study showed that Haloferax sp.
strain D1227 from hypersaline oil brine contaminated
soil was able to utilize aromatic compounds as its sole
carbon source (69). Halomonas sp. HTB24 isolated from
hypersaline olive oil wastewater has been found to
convert tyrosol to the antioxidant hydroxytyrosol (HT)
and 3,4-dihydroxyphenylacetic acid (DHPA), which in
turn was degraded to succinate and pyruvate (70).
Some halophiles including the model haloarchaeon,
Halobacterium sp. NRC-1 produce unique gas filled
organelles, called gas vesicles. These organelles allow the
microbe to obtain vertical buoyancy. Scientists have characterized the genetic basis for gas vesicle production and
have designed a vector for bioengineering other microbes
such as those involved in hydrocarbon degradation to
float after transformation (71). When mass-produced,
these genetically engineered microbes would thus have
the dual ability to degrade hydrocarbons as well as float
to where the hydrocarbons are found. The process may
also have applications for other bioremediation as well as
fermentation processes such as those found in beer and
wine-making. Another approach to the biodegradation
of hydrocarbons under development is the utilization of
the halophiles themselves or their enzymes. Oil and salt
deposits are commonly found in conjunction with one
another. Therefore, it is plausible that some halophiles
may be able to utilize the hydrocarbons and potentially
convert them into more inert materials.
In trying to remediate hydrocarbon spills and contaminants, industry applies the use of surfactants and emulsifiers to solubilize and disperse hydrophobic compounds.
Research has found that halophiles produce biosurfactants with potential biotechnological applications, such
as to treat hydrocarbon pollution in marine aquaculture
(64,72). Using qualitative drop-collapse and emulsification
activity assays, several halophilic strains that required a
minimum of 15% w/v NaCl significantly reduced surface
tension of the growth medium. Several strains exhibited high emulsion-stabilizing capacity and also stabilized
diesel oil-in-water emulsions over a broad range of pH
conditions in up to 35% sodium chloride concentration
through multiple cycles of freezing and thawing (64).
Another study found several potentially useful facultative bacterial strains tolerant of high temperature and
salinity from an Iranian oil reservoir brine that produced
surfactants (73).
Tanning Industry. During the tanning process, the
stripped animal skins are first soaked in sodium chloride
in order to preserve them. The salt is then removed by
soaking in water at the tannery. The resulting 1–10%
w/v soaking liquor effluent is filled with organic matter and, generally, the water is removed by evaporation
and the remaining salt is unusable unless treated. If
proper treatment methods can be developed to reduce the
organic load, the salt produced through evaporation could
be reused in the skin pickling and other tannery processes.
Four strains, Pseudomonas aeruginosa, Bacillus flexus,
Exiguobacterium homiense, and Staphylococcus aureus,
5
were isolated from marine and tannery saline wastewater samples and could be used in bioremediation (63).
Halophiles have been used in several studies, examining
both aerobic and anaerobic treatment of tannery wastewater. Different waste removal properties were identified,
including the use of an aerobic sequencing batch reactor
for removal of phosphate, nitrogen, and organic load at
moderate salinity (63).
Extremely halophilic archaea produce halocins—antimicrobial chemicals released to kill or inhibit growth of
other halophilic archaea, and can be used in the tanning
industry. The tanning process involves the use of vast
quantities of crude salt that often contains inclusions of
halophiles that can, under optimum water conditions and
temperatures, be released onto the hide and bring about
damage by lipolysis. The industry has long been using bactericides, which have to some extent been losing efficacy
as the bacteria become resistant, and most often do not
work against the haloarchaea present at all. In a series of
studies of the microbial populations of salterns in Turkey,
lipase negative halocin producing strains that could potentially destroy harmful halophiles including haloarchaea
and save the hide have been identified (74).
Textile Industry. The textile industry produces a large
quantity of polluted wastewater containing azo dyes, phenol, and toxic anions. The effluent is also highly saline
with typical salt concentrations of 15–20%. Moderate
halophiles such as Salinicoccus iranensis and Halomonas
species have been isolated from these wastewaters and
are able to decolorize azo dyes and use phenol as a primary source of carbon and energy (75,76). Treatment of
organic pollutants from the textile industry has also been
reported.
Nitrate Pollution. Haloferax mediterranei from salt
ponds in Spain has been described as a denitrifier and
was shown to grow using NO3– , NO2– or NH+
4 as inorganic nitrogen sources and therefore could be used for
bioremediation of nitrate and nitrite concentrations in the
presence of high salt (77). Without the removal of these
hazardous compounds, consumers of contaminated well
water may experience problems including, but not limited
to, blue baby syndrome.
Heavy Metal Pollution. Due to the evaporitic nature
of hypersaline environments, heavy metals are frequently
found in concentrated brine. As a result, many halophiles
have developed tolerance to heavy metals. For example,
Halobacterium sp. NRC-1 was reported to be resistant to
arsenic as a result of a plasmid-encoded arsenite and antimonite extrusion system that may be potentially usable
for arsenic cleanup (78). The halophilic chromate reducing
bacterial strain Nesterenkonia sp. MF2 isolated from tannery effluents was found to tolerate 600 mM of chromate.
MF2 completely reduced 0.2 mM highly toxic and soluble Cr(VI) (as CrO24 – ) into almost nontoxic and insoluble
Cr(III) under aerobic conditions at 1.5 M NaCl. Halophilic
bacteria from the Dead Sea have also been reported to be
able to detoxify lead and cadmium (79).
Aquaculture. Though halophilic algae are useful for
the production of carotenoids, the process itself results
in the production of hypersaline brine waste. Halophilic
bacteria have been used to efficiently remove glycerol
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HALOPHILES, INDUSTRIAL APPLICATIONS
from D. salina carotenogenesis media. This reduction of
the organic matter in the effluent allows for recycling of
the treated wastewater for reutilization in the carotene
production process (80). Moreover, addition of a Halobacterium sp. to a simulated saline activated sludge culture
wastewater in an aerobic-biological reactor by fed-batch
operation resulted in significant improvements in Chemical Oxygen Demand (COD) removal efficiency (63).
BIOFUEL PRODUCTION
The potential for using hypersaline brine to grow microalgae that use carbon dioxide, sunlight, and water to create
biomass and biofuels is great. Global efforts have targeted
biofuels to constitute a substantial portion of the transport
sector in the near future. As a result, there is currently a
major effort underway for the production of monocultures
of these microbes and for the development of large-scale
culturing and harvesting techniques. Others are exploring
the use of halophilic microbes to produce oil (81). While
soybeans produce about 50 gallons of oil per acre per year,
and canola produces about 130, some algae produce about
4000 gallons per acre a year (82). Some unicellular algae
reportedly contain sufficient quantities of oil that may be
extracted, processed, and refined into transportation fuels,
using currently available technology. A major advantage of
this process is the potential for use of nonarable land and
nonpotable water, minimizing the use of fresh water and
land currently used to displace food crop cultures. Their
production is also not seasonal and they can be harvested
daily.
MEDICAL APPLICATIONS
Although uses of halophiles for medical applications have
not yet been established, certain novel features of these
microorganisms and their relative safety and scale-up
potential have provided significant impetus for future
applications. Haloarchaea have been shown to produce
biodegradable plastics, adjuvating protein and lipid vesicles, and microcidal halocins. They also have phytochemicals that mitigate the damage from reactive oxygen species
and DNA repair enzymes that protect the cells against
damaging radiation, which may be translated for medical applications. The potential for long-term impact of
halophilic products in the medical and pharmaceutical
industries is significant.
Bioplastics. Polyhydroxyalkanoates (PHA) are a heterogeneous group of polyesters which are often employed
as a mode of carbon storage by microbial cells. Halophilic
archaea produce large quantities of PHA which have been
utilized for their application as biodegradable plastics.
The most common is poly-beta-hydroxybutyrate (PHB).
PHAs can be used as a replacement for thermoplastics
and are biodegradable, biocompatible, and resistant to
water. Therefore, they are of interest to the medical and
pharmaceutical community for use in surgical sutures,
bone replacement, and delayed release medications (83).
Haloarchaea such as H. marismortui and H. mediterranei
are capable of producing large amounts of PHA using
glucose and/or starch as substrates. H. marismortui has
been shown to accumulate PHB up to 21% cellular dry
weight when grown in a minimal media supplemented
with glucose (84). H. mediterranei can accumulate about
6 g (60% of the total biomass dry weight) of PHB per liter
of culture supplemented with starch, a much cheaper
substrate (85). The vulnerability of the haloarchaeal cells
to pure water (no salt) makes isolation of PHA granules
relatively easy (86,87).
Gas Vesicles and Liposomes in Vaccine Development. The standard for vaccine design has long
included attenuated or killed pathogens in conjunction
with an exogenous adjuvant, as a means of introducing
antigens to the immune system. Unfortunately, they
include the risks of infection and/other adverse reactions.
Halobacterium sp. NRC-1 gas vesicles can be genetically
engineered to present antigens and have been found to
be self-adjuvating. Antigen presentation can therefore
be achieved using only parts of infectious molecules or
viruses, similarly resulting in long-term protective immunity (88). These vesicles are stable biological structures
resistant to degradation, devoid of nucleic acids, easy
to harvest by lysis and flotation and are nontoxic. In
preliminary studies, mice immunized with recombinant
gas vesicles expressing an simian immunodeficiency virus
(SIV) peptide elicited a strong antibody response and
immune memory (89). The potential for rapid, low-cost vaccine production, and increased safety make Halobacterium
an excellent candidate for production of a vaccine vector.
Intranasal administration of vaccines has become a
popular concept for diseases where microbes colonize
mucosal surfaces. However, the use of attenuated
pathogens for this purpose raises safety concerns mainly
because of the direct access of the pathogen to the brain.
In cases where the pathogens or molecules, including
lipopolysaccharides, are still potent, this could pose a
significant health risk. Antigens that are derived from
recombinant methods are currently the topic of interest
for new vaccine designs. However, these antigens require
adjuvants since they illicit a low immune response (90).
Archaeal polar lipids (archaeosomes), from H. salinarum,
have been used as an adjuvant and as a means to
deliver the antigen undamaged. This is in contrast to
conventional ester lipids, which require an additional
adjuvant like the cholera toxin B subunit to improve
vaccine efficacy (90). Polar lipids from Archaea have
several advantages compared to the lipids used from
bacteria. Their resilience to chemical and enzymatic
treatments along with temperature and pH make them
much easier to work with (91). In trials testing the
immune response of a nasal polar lipid vaccine in mice,
results were extremely positive and no toxicity was
observed (90). Although there are several unresolved
issues including the toxicity, efficiency in preparation,
and long-term response to intranasally administered
archaeal polar lipids, the fact that they are nonreplicating
and induces an effective immune response suggests great
promise (90).
Archaeosomes have also been prepared for intravenous
and oral vaccines, which have been tested for safety. It
HALOPHILES, INDUSTRIAL APPLICATIONS
is assumed that oral vaccines, the most attractive of
all administration methods, would likely require higher
doses because of absorption and other uptake issues (91).
However, haloarchaeal polar lipids demonstrate several
advantages to ester lipids and may be very useful for
vaccine delivery.
Halocins and Microhalocins. It has been widely
demonstrated that bacteria produce bacteriocins which
inhibit the growth of other bacteria, but there is only
limited information on the antimicrobials produced in
archaea. It is known that they display sensitivity to antibiotics in a significantly different fashion from the bacteria;
however, some haloarchaea have been shown to produce
molecules with antibiotic qualities (92). These molecules
are called halocins and microhalocins, and consist of proteins and peptides, respectively (93). They have been
hypothesized to reduce competition and assist in lysing
cells of other organisms to supplement their own nutrients.
The quantity of bacterial cells eliminated as a result
of halocin protein concentration has been studied to test
the use of halocins as antibiotics (92). In addition to having antimicrobial properties, halocin H6 can inhibit the
Na+/H+ exchanger (NHE) in mammalian cells. This property can protect tissues including the myocardium from
damage caused when blood returns after ischemic conditions. Currently, halocin H6 is the only known biological
molecule that exerts a specific inhibitory effect on the NHE
of mammalian cells (94).
Microhalocins are thought to begin as ‘‘pro-proteins’’
and are then altered into their final form, via a mechanism
that is still being investigated. Microhalocin S8, from
haloarchaeal strain S8a, for example, is also hydrophobic
making it difficult to elucidate its mode of action (92).
Several studies have examined halocin and microhalocin targets. The host organism, other haloarchaea
as well as bacteria. Both halocin, H6, from Haloferax gibbonsii (Ma2.39) and microhalocin S8 were found to retain
∼50% activity at temperatures close to 100◦ C (92). These
proteins have promising applications for the future of
antimicrobial and pharmaceutical product development.
EDUCATIONAL USES
Extreme halophiles are readily accessible for educational
applications and have been successfully marketed to college and high school students. The relative safety of many
extreme halophiles, including both halophilic archaea and
algae, has provided the impetus for development of educational products for the early education market. These
products provide students access to basic microbiological techniques, novel extreme environments, as well as
genomic and bioinformatic information.
7
microbes will continue to be tapped as a source of enzymes,
and used as producing and biodegrading organisms in
industrial settings.
Though promising in many ways, the production
of halophilic enzymes for industrial processes is still
challenging. Novel enzymes, although sufficient for their
purification and characterization on a laboratory scale,
are not easily scaled up for use on an industrial scale.
Transfer of the genes for these enzymes to other, faster
growing or metabolically diverse organisms may result in
larger yields.
Another challenge is corrosion, since high salt concentrations rapidly corrode most stainless steel vessels used
for culturing. New culturing methods include bioreactors
made of polyetherether ketone, tech glass (borosilicate
glass), and silicium nitrate, and nitrite ceramics, which
have been introduced for cultivation of halophiles that
produce poly-L-glutamic acid and poly-β-hydroxybutyric
acid (65,83).
As these challenges are met, the uses of halophiles for
industry will continue to expand. Some of the applications
on the horizon are highly specialized and potential blockbusters, that may revolutionize processes, for example,
new vaccines, high density computer storage, new therapeutic drugs, environmental bioremediation, and biofuel
production.
Acknowledgments
The Authors were supported by NSF grant MCB-0296017
and NASA grant NNX08AT70G to S. DasSarma and
the NIGMS Initiative for Minority Student Development
Grant (R25-GM55036) and Procter and Gamble to V. Huse.
We also thank Anjali DasSarma and Melinda Capes for
careful reading and editing of the manuscript.
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Please note that the abstract and keywords will not be included in the printed book, but are required for the
online presentation of this book which will be published on Wiley’s own online publishing platform.
If the abstract and keywords are not present below, please take this opportunity to add them now.
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Abstract: Halophilic (salt-loving) microorganisms have evolved physiological and genetic characteristics for success in
their harsh saline environments. These characteristics include internal compatible solutes, efficient ion pumps, UV
absorbing pigments, and acidic proteins, which help them to resist osmotic stress and the denaturing effects of salts,
as well as the accompanying intense solar radiation. Many of the same characteristics which help them to counteract
the deleterious effects of their environment also constitute valuable resources for biotechnology and nanotechnology.
Although currently established applications of halophiles are limited primarily to compatible solutes in the cosmetic
industry, and β-carotene and hydrolases in the nutritional and food industries, many other novel applications are under
development. They include specialized applications for films of bacteriorhodopsin from halophiles for a variety of optical
nanodevices, including high-capacity computer storage, and for stable enzymes from halophiles for use as industrial
catalysts, some capable of functioning in organic solvents. Metabolic activities of halophiles are also being tapped for
a variety of environmental applications, such as bioremediation of pollutants in saline wastewaters. Some subcellular
components, such as polyhydroxyalkanoate granules, gas vesicles, and phospholipid vesicles, and bioactive compounds like
halocins are being developed for medical applications. Applications of halophilic microorganisms are varied and represent
significant commercial opportunities in the chemical, environmental, biofuel, medical, and educational industries.
Keywords: halophiles; enzymes; bioremediation; Archaea; medicine; bacteriorhodopsin; protease; biodegradation; gas;
vesicle; biofuel