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Industrial and environmental applications of halophilic

microorganismsAharon Oren

a

aDepartment of Plant and Environmental Sciences, The Institute of Life Sciences, and

t he Moshe Shil o Miner va Cent er f or Marine Biogeochemistry, The Hebrew Universit y ofJerusalem , 91904 Jerusalem, Israel

Version of recor d f i rst publ ished: 14 Jun 2010.

To cit e t his art icle: Aharon Oren (2010): Industrial and environmental applications of halophil ic microorganisms,Environm ent al Technology, 31:8-9, 825-834

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Environmental Technology

Vol. 31, Nos. 89, JulyAugust 2010, 825834

ISSN 0959-3330 print/ISSN 1479-487X online 2010 Taylor & FrancisDOI: 10.1080/09593330903370026http://www.informaworld.com

Industrial and environmental applications of halophilic microorganisms

Aharon Oren*Department of Plant and Environmental Sciences, The Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine

Biogeochemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

TaylorandFrancis

(Received 6 August 2009; Accepted 28 September 2009)10.1080/09593330903370026

In comparison with the thermophilic and the alkaliphilic extremophiles, halophilic microorganisms have as yet foundrelatively few biotechnological applications. Halophiles are involved in centuries-old processes such as themanufacturing of solar salt from seawater and the production of traditional fermented foods. Two biotechnological

processes involving halophiles are highly successful: the production of

-carotene by the green alga Dunaliella

andthe production of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), used as a stabilizer for enzymesand now also applied in cosmetic products, from moderately halophilic bacteria. The potential use of

bacteriorhodopsin, the retinal protein proton pump ofHalobacterium

, in optoelectronic devices and photochemicalprocesses is being explored, and may well lead to commercial applications in the near future. Demand for salt-tolerantenzymes in current manufacturing or related processes is limited. Other possible uses of halophilic microorganismssuch as treatment of saline and hypersaline wastewaters, and the production of exopolysaccharides, poly-

-hydroxyalkanoate bioplastics and biofuel are being investigated, but no large-scale applications have yet beenreported.

Keywords: halophilic;

-carotene; ectoine; bacteriorhodopsin; enzymes

Introduction

In comparison to other groups of extremophiles, andespecially the thermophilic and the alkaliphilic prokary-otes that are extensively used for the production of valu-able enzymes, the extremely and moderately halophilicmicroorganisms are to some extent a neglected group

when considering the number of their biotechnologicalapplications. This is even more true when consideringthe great diversity of halophiles: they are found in allthree domains of life, Archaea, Bacteria and Eucarya,and they contain representatives of many different

physiological types, adapted to a wide range of saltconcentrations up to salt saturation.

Earlier reviews that discussed the possible applica-tions of halophiles in biotechnological and environ-mental processes [17] provided long lists of potentialuses that have been and are still being explored.However, the number of applications of halophilesthat are currently exploited is very small indeed. The

true success stories of halophile biotechnology arethe commercial production of

-carotene by strains ofthe unicellular green alga Dunaliella

and the produc-tion of ectoine, synthesized by many moderately halo-

philic bacteria to provide osmotic balance to the cells,and its application as a stabilizing agent for sensitive

enzymes and in cosmetic preparations. Many otherproducts synthesized by halophiles or processesperformed by them may have potential applications,but these have not yet led to commercially viableoperations.

Applications (current and potential) of halophilicmicroorganisms can be divided into a number ofcategories:

(1) Centuries-old processes such as the manufactur-ing of solar salt from seawater and the produc-tion of traditional fermented foods. Such

processes existed long before the nature of themicroorganisms involved became known, andlittle if anything is done to control these micro-organisms to improve the production processes.

(2) Utilization of the salt tolerance of halophilicmicroorganisms and of enzymes produced

by them to catalyze processes in high salt

environments.(3) Exploitation of the properties of specificcompounds produced by certain types of halo-

philes that enable them to withstand the high saltconcentrations in their medium (ectoine, glyc-erol and others).

*Email: orena@cc.huji.ac.il

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(4) Applications of unique compounds made bysome halophiles, not directly connected withtheir life in high salt environments. The primeexample is bacteriorhodopsin in the purplemembrane ofHalobacterium salinarum

, whichis not essential for its growth, and is stable and

active also in the absence of salt.(5) Industrial uses of compounds present in halo-philes as well as in many non-halophiliccounterparts. Compounds such as

-carotene,poly-

-hydroxyalkanoate, exopolysaccharides,etc. found in some halophiles are also made bymany other microorganisms. Sometimes there isa clear advantage to using halophiles for their

production (e.g.,

-carotene production from

Dunaliella

); in most other cases, however, thesuperiority of the halophiles as producers ofsuch compounds is yet to be proven.

This short paper does not claim to be a comprehen-sive review of all biotechnological and environmentalapplications of halophiles, now and in the future.Instead it intends to survey some processes involvinghalophiles that are already applied and to criticallyexamine a few others that are of special interest andmay be exploited in the near future.

Halophilic microorganisms and the production

of solar salt

Salt making by evaporation of seawater in shallow pondsin coastal areas in the tropics and subtropics is a tech-

nology that has existed for thousands of years [8]. Whenthe brine approaches saturation and salt starts crystalliz-ing, the brines become coloured red. Three types ofhalophilic microorganisms contribute to the colour:extremely halophilic Archaea (family Halobacteri-aceae

) that contain 50-carbon carotenoids (bacterioru-berin and derivatives) and sometimes also the retinalprotein bacteriorhodopsin (see below), the

-carotene-rich unicellular green flagellate alga Dunaliella salina

and the red halophilic bacterium Salinibacter ruber

,which contains a unique C40-carotenoid acyl glycoside.In the less saline ponds where the earlier evaporation

stages take place, dense microbial mats develop on thebottom, composed of different types of cyanobacteriaand many other kinds of microorganisms.

The importance of these biota for the salt makingprocess started to be recognized in the 1970s. The under-standing of the roles that the different types of microbes

play in determining the quality and quantity of the saltharvested led to the development of biological manage-ment practices for the operation of solar salterns [912].The planktonic communities of red microorganisms inthe crystallizer ponds are generally considered beneficial

to the salt production process. The carotenoid pigmentsabsorb light energy, and thereby raise the water temper-ature, leading to increased evaporation rates. There areeven reports showing that the halophilic Archaea may bedirectly involved in the crystallization of halite. There-fore, oligotrophic ponds that do not support adequate

development of red microbial communities are oftenfertilized. The benthic microbial mats in the earlier evap-oration ponds are also to some extent desirable, as theyeffectively seal off the bottom of the ponds, preventingleakage of brine. However, when grown excessively, thecyanobacteria (especially the unicellularAphanothecehalophytica

commonly found in salterns) can producelarge amounts of polysaccharide slime, and when thismaterial reaches the crystallizer ponds it results in theformation of poor quality salt [13].

Overall, the exact link between the biota of the salt-erns and the quality of the salt produced is still incom-

pletely understood. The size and quality of the salt

crystals formed in solar saltern crystallizer pondsworldwide is highly variable. At some sites large solidhalite crystals precipitate that are easy to process andyield a high-quality product, while elsewhere crystalsare soft, with a high content of entrapped mother liquor,and these are difficult to harvest and to purify. In allcases where the raw material is seawater of nearly iden-tical composition, biological processes in the evapora-tion and/or crystallizer ponds may well be responsiblefor the differences. Although it is clear that excessive

production of polysaccharide slime by the cyanobacte-ria may lead to a deterioration of the quality of the salt

precipitated in the crystallizers, the nature of the miss-ing link between solar salt quality and saltern pondmicrobiology is still largely unknown.

Halophiles and fermented foods

Large amounts of salt are used in the preparation ofcertain types of traditionally fermented foods. Such saltyfood products are especially popular in the Far-East.Examples are jeotgal, a traditional Korean fermentedseafood, the Japanese fugunoko nukazuke prepared byfermentation of salted puffer fish ovaries in rice bran,and nam-pla, a Thai fish sauce. The latter product is

made by adding two parts of fish and one part of marinesalts. The mixture is covered with concentrated brine(2530% NaCl) and left to ferment for about a year.

It is surprising that relatively little is known about themicroorganisms present during the preparation of thesefood products and about the roles they play during the

production process. In some cases the salt concentrationduring the fermentation process is sufficiently high forthe development of Archaea of the familyHalobacteri-aceae

. The first halophilic archaeon obtained from Thaifish sauce (nam pla) was an isolate resembling

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Halobacterium salinarum

[14], and two new species,

Halococcus thailandensis

and Natrinema gari

, wererecently isolated [15,16]. Halalkalicoccus jeotgali

is anovel isolate obtained from shrimp jeotgal [17]. Archaeal

proteases of halophilic Archaea probably take part in thefermentation process, and the Archaea may also contrib-

ute to the aroma of the sauce. Fermentations that involvelower salt concentrations generally give rise to the devel-opment of species of Tetragenococcus

, Halobacillus

,

Lentibacillus

, Halomonas

and other bacterial genera.Although many species of bacteria have been isolatedfrom such fermentations, the involvement of each organ-ism is still far from clear [18], but also here proteases are

probably involved [19]. A recent culture-independentstudy of the microbial communities involved in themanufacturing of kimchi, a traditional Korean food

based on fermented vegetables, showed the presence ofa highly diverse community of halophilic Archaea, lacticacid bacteria and other representatives of the domain

Bacteria, as well as different yeasts [20].

Production of

-carotene byDunaliella

Cultivation of the green algaeDunaliella salina

andD.bardawil

for the production of

-carotene is the majorsuccess story of halophile biotechnology [2123]. Thefirst pilot plant for mass culture ofDunaliella

was set upin the mid-1960s in the Ukraine, and since thencommercialDunaliella

growing operations were set upin different countries.

The pigment

-carotene is in high demand as anantioxidant, as a source of pro-vitamin A (retinol) andas a food colouring agent. Its antioxidant activitiesmake it popular for use in health food. While

-caroteneis present in many algae and higher plants and can also

be synthesized chemically, the chemical product differsfrom that ofDunaliella

: the synthetic form is all-

trans

-carotene, while the alga produces a high percentageof 9-

cis

-carotene. This is a more effective quencher ofsinglet oxygen and other free radicals than the all-

trans

form. However, not all authorities are equallyconvinced that the action of

-carotene in the humandiet is always beneficial.

D. salina

andD. bardawil

produce large amounts of

-carotene when grown under suitable conditions. Thepigment is found concentrated in small globulesbetween the thylakoids of the cells single chloroplast.The main environmental conditions that stimulate accu-mulation of the pigment are high light intensities, highsalinity and nutrient limitation; the slower the cellsgrow in the presence of high irradiation levels, the more

pigment is formed. Some strains may then contain morethan 10% of their dry weight as

-carotene. Much infor-mation has accumulated on the mode and control of the

biosynthesis of carotenoids by the alga [24].

Different technologies are used to grow

-carotene-rich Dunaliella

biomass in countries includingAustralia, USA, China and Israel, and these vary fromcultivation in large lagoons to intensive growth systemsat high cell densities under carefully controlled condi-tions. In extensive open pond systems no mixing is

applied, and the growth conditions are poorlycontrolled. Intensive cultivation ofDunaliella

is a high-technology operation, in which all parameters arecontrolled. In 3000 m

2

shallow (20 cm deep) paddle-wheel driven raceway ponds an average yield of 200 mg

-carotene per m

2

per day can be obtained [25]. To opti-mize growth and carotene production, nutrient levelsand pH should be carefully controlled. It is often advan-tageous to operate the system in two stages. First, alarge biomass is produced by addition of high nutrientlevels. Under these conditions the cells produce onlylittle

-carotene. In the second stage, nitrate limitationis induced to stimulate carotenogenesis [26]. Predatory

ciliates sometimes cause losses in such outdoor masscultures ofDunaliella

, and different strategies weredeveloped to minimize the problem. Finally, super-intensive cultivation systems in closed bioreactors havealso been developed, which enable high cell densitieswith a high

-carotene content greatly enriched in the 9-

cis

-carotene isomer [27,28].To harvest the cells and to further process them for

extraction and purification of the

-carotene, differentprocedures have been devised. In an extensive process,flocculation and surface adsorption are used. Inorganicor organic flocculants such as alum (aluminum sulfate),ferric chloride, ferric sulfate, lime or polysaccharidesare employed. In intensive operations centrifuges aregenerally used to harvest the cells. The pellet material isfurther purified before marketing. The supernatantwater, rich in salts, glycerol and many other compounds,can be recycled following purification by an oxidation

pond. In recent years other methods of harvesting weretested on an experimental scale, in which the pigmentwas extracted from the cells without killing them, so thatthe cells can be reused for the production of more

-carotene in a new growth cycle. Solvents such as dode-cane can be used in such microbial milking protocols[2931]. A two-phase growth system has been set up

consisting of two volumes ofDunaliella

culture in brineand one volume of dodecane. Yields were relativelylow, as only about 8% of the pigment reached the dode-cane layer [32].

A transgenic system was developed in which

-carotene is produced in Halomonas elongata

thatexpresses carotene production genes from Pantoeaagglomerans

(

Enterobacteriaceae

). High yields of

-carotene could thus be obtained in cells growing at2% salt; at 15% salt only little pigment was produced[33].

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Ectoine a multi-purpose osmotic solute

Most halophilic and halotolerant microorganismsproduce or accumulate small organic compounds intra-cellularly to provide osmotic balance with their hyper-saline environment. Thus, these organisms can excludesalt from their cytoplasm, abolishing the need to adapt

their proteins to the presence of high salt concentra-tions. A great variety of such osmotic, compatiblesolutes have been identified in the microbial world.Among the best known are glycine betaine, simplesugars such as sucrose and trehalose, and differentamino acid derivatives. Some of these osmotic soluteshave found applications in biotechnology [34].

One of the most common osmotic solutes in thedomain Bacteria is ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid). It was firstdiscovered in the haloalkaliphilic photosynthetic sulfur

bacterium Ectothiorhodospira halochloris

, but later agreat variety of halophilic and halotolerant bacteria

were found to produce this compound, often togetherwith its 5-hydroxy derivative. Ectoine can protect manyunstable enzymes and also nucleic acids against thedetrimental action of high salinity, thermal denatur-ation, desiccation and freezing, thereby increasing shelflife and activity of enzyme preparations [3538]. Forexample, ectoine inhibits spontaneous conversion oftrypsinogen to trypsin and of trypsin-catalyzed conver-sion of chymotrypsinogen to chymotrypsin, and it alsostabilizes the activity of trypsin and chymotrypsin [38].Such compatible solutes are sometimes termed molec-ular chaperones. Their mode of action is still far from

being completely understood.In recent years additional properties of interest werefound for ectoine. It is claimed that it counteracts effectsof ultraviolet UV-A-induced and accelerated skinageing, and therefore the cosmetics industry started toadd ectoine to dermatological cosmetic preparations asmoisturizers in cosmetics for the care of aged, dry orirritated skin [39]. The compound stimulates theimmune system of the Langerhans cells and the forma-tion of heat shock proteins, and reduces the formation ofsunburn cells in the skin following UV radiation[40,41]. Ectoine also inhibits aggregation and neurotox-icity of Alzheimers

-amyloid [42], and recently a clin-

ical trial was initiated to test its efficacy in inhalationsagainst bronchial asthma [43].Ectoine is commercially produced by extracting the

compound from halophilic bacteria. Industrial processesfor mass production of ectoine and hydroxyectoine weredeveloped using Halomonas elongata

and Marinococ-cus

M52, respectively. The procedure is based onbacterial milking [44,45]: the bacteria are grown to ahigh cell density in a high salt medium, so that theyaccumulate massive amounts of ectoine intracellularly.

Then an osmotic down-shock is applied. The bacteriareact by secreting most of the ectoine to the surroundingmedium, from which the compound can be collected bycrossflow filtration techniques and purified. Salt is thenadded to the bacteria, and these readapt to the high salin-ity by producing more ectoine, so that the milking

procedure can be repeated. Another possible strategy forectoine production is the use of leaky mutants thatdo not efficiently retain the compatible solutes insidethe cell, so that the compound can be continuouslyharvested from the medium. It is also possible to expressthe genes for ectoine production inEscherichia coli

orin other non-halophilic bacteria and to use such recom-

binant bacteria as a source for the compound. AnE. coli

strain harbouring the ectoine production genes from

Marinococcus halophilus

, and which expresses aspar-tate kinase from Corynebacterium glutamicum

, ensur-ing a steady supply of precursor molecules, showedexcellent production of ectoine by cells growing at 3

5% salt [46]. A transgenic E. coli

with the ectoineoperon of Chromohalobacter salexigens

expressedunder control of the tet

promoter excreted ectoine,which accumulated in the medium at concentrations upto 6 g l

1

[47].Attempts were also made to increase the salt toler-

ance of plants by expression of the genes for ectoineproduction. Tobacco plants transformed with the ecto-ine genes ofHalomonas elongata

using anAgrobacte-rium

-mediated gene delivery system showed improvedroot function and photosynthesis at increased salinity[48].

Glycerol production byDunaliella

Another compatible solute of interest is glycerol [34].Glycerol is produced by the alga Dunaliella

. Cellsgrown in near-saturated NaCl solutions may containover 67 M intracellular glycerol. Mass cultivation of

Dunaliella

for commercial production of glycerol hasbeen attempted in the past and processes for the indus-trial extraction and purification were tested [49].However, because of the low price of glycerol produced

by other methods (as a by-product of the manufacturingof animal and vegetable oils), and because of the high

cost of the harvesting of the cells, no commerciallyfeasible process was developed; this in contrast to theabove-described production of

-carotene, which is amuch more expensive product.

Bacteriorhodopsin, a photochemical material for

bioelectronics and other applications

Bacteriorhodopsin is a 25-kDa protein that carriesa retinal group bound by means of a Schiff-base to

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lysine-216. Its function was discovered in the early1970s during studies of the purple membrane, patchesof membrane that contain only bacteriorhodopsin andlipids, found within the cell membrane ofHalobacte-rium salinarum

. It serves as a light-driven proton pump.Upon excitation by light of a suitable wavelength

(maximum absorption at 570 nm in the ground state (theB state)), a complex photocycle is initiated in whichthe Schiff base is deprotonated and reprotonated and theretinal changes from its all-

trans

conformation to the13-

cis

form and back. Each of the intermediates has itscharacteristic absorption spectrum. Thus, the M inter-mediate (13-

cis

, deprotonated) is yellow, with a maxi-mum absorbance at 412 nm. The protonation/deprotonation reactions are so arranged that the protonis taken up from the cytoplasmic side and released to theoutside of the cell, so that light absorption results in thegeneration of a proton gradient that can be used foradenosine triphosphate (ATP) generation.

Bacteriorhodopsin is a highly unusual protein withinthe proteome ofHalobacterium

. Nearly all its proteinsrequire high salt concentrations for structural stabilityand activity. Bacteriorhodopsin, however, is stable inthe absence of salts, retains its photochemical propertiesover long periods, functions between 0 and 45

C in thepH range 111 and tolerates temperatures of over 80Cin water and up to 140C when dry. It is stable to expo-sure to sunlight for years, and it resists digestion bymost proteases. Furthermore, it can easily be immobi-lized on glass plates or embedded in polymers [50].

Bacteriorhodopsin is commercially manufactured inthe form of purple membrane patches prepared from

Halobacterium salinarum. Genetically altered protein isavailable as well (see below).

Although the first patents relating to potentialbiotechnological applications of bacteriorhodopsinwere issued soon after the discovery of the function ofthe molecule, and hundreds of patents now exist thatdescribe different possible uses of the molecule, no truecommercial applications yet exist to our knowledge.There are, however, plenty of ideas for such applica-tions [5060], and these may well lead to biotechnolog-ical uses in the near future.

Some of these potential uses are based on the

conversion of light energy into chemical energy, possi-ble applications being ATP generation, conversion ofsunlight into electricity and desalination of seawater.Others exploit the properties of its photocycle, espe-cially the conversion of the B state (absorbance maxi-mum 570 nm) to the M state (420 nm) and vice versa.Using site-directed mutagenesis, the molecule can beoptimized for such uses; for example, replacement ofAsp by Asn at position 96 prolongs the lifetime of theM intermediate a thousand-fold [5860]. A few possibleapplications are as follows:

(1) Holographic storage in which interferencepatterns are registered as purple or yellow areas.As the transitions are reversible, a bacterio-rhodopsin holographic matrix can be usedrepeatedly [53,55].

(2) Construction of bioelectronic elements of

computer memories and information processingunits. Optical switching is based on the BMand MB transitions. A high density of infor-mation storage is possible, and both permanentoptical image storage, data storage and transientoptical image storage (data processing) by

bacteriorhodopsin are possible.(3) Ultrafast light detection, construction of artifi-

cial retinas, detection of motion [56].(4) Nanotechnology applications such as the

construction of molecular transistors, molecularmotors, artificial retinas and molecular sensors[57].

Whether one or more of these ideas, or possibly others,for the use of bacteriorhodopsin will in the future leadto the development of commercial applications remainsto be seen.

Treatment of saline wastewaters using halophilic

microorganisms

Some industrial processes generate highly saline waste-water. One such process is the above-discussed produc-tion of-carotene fromDunaliella. After collection ofthe cells by centrifugation, hypersaline wastewater

remains, rich in organic material, including a highconcentration of glycerol. Other industries that have tocope with high salt wastewaters are pickling plants andtanneries. Wastewater brines are also produced duringoil and gas recovery processes.

Several processes have been proposed for thebiological treatment of such wastewaters to removeorganic carbon and toxic compounds. In several

Dunaliella growth facilities the wastewater is treated inoxidation ponds and recycled, and optimization studieshave been made [61].

For the biological treatment of industrial wastewa-

ters with salt concentrations up to 10%, such as thebrines generated by the pickling industry, aerobic treat-ment systems have been developed based on aerated

percolators or rotating discs to improve aeration andmixing [6273]. Most systems described below exist aslaboratory-scale models only. Such simulations gener-ally have shown satisfactory results at salt concentra-tions up to around 6%, but at higher salinities thesystems performed less well. In some studies a cultureofHalobacterium salinarum was added to improvedegradation [62,65]; whether this organism did indeed

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830 A. Oren

contribute to the performance of the system is to bedoubted asHalobacterium requires at least 15% salt forgrowth and lyses at lower concentrations. Bacteria suchasHalomonas spp. andBacillus spp. are more likely to

be active in such processes [72,73]. Sequencing batchreactors have been proposed for the purification of

tannery soak liquor and similar saline wastewaters withup to 6% salt [6870].Anaerobic biodegradation processes have also been

proposed for the treatment of saline and hypersalinewastewater. To remove nitrate from brines, a membrane

bioreactor was constructed based on denitrification bythe archaeon Haloferax denitrificans [74], and use of

Haloferax mediterranei has been proposed for bioreme-diation of nitrate and nitrite in saline groundwater incoastal areas [75]. Anaerobic reactors based on fermen-tation and methanogenesis have also been developedfor chemical wastewaters with up to 10% salt [76,77].The halophilic fermentative bacterium Halanaerobium

lacusrosei was successfully used in an anaerobicpacked bed reactor operating at salt concentrations up to10% [78,79].

Enzymes from halophilic microorganisms

In comparison to the extensive use of extremozymesfrom thermophilic and alkaliphilic Bacteria andArchaea, very few halophilic enzymes have thus farfound applications in industry and biotechnology. In

part, this is due to the limited demand for salt-tolerantenzymes in current manufacturing or related processes[80].

Many enzymatic activities of halophilic Archaeahave been characterized, including enzymes of potentialapplicative interest, such as amylases, proteases andnucleases. Many halophilic enzymes also function atelevated temperatures. However, no commercial appli-cations have yet been developed for such enzymes.Still, some archaeal enzymes are of potential interest,such as the amylase ofHaloarcula sp. that functionsoptimally at 4.3 M salt at 50C, and is stable in benzene,toluene and chloroform [81].

Halophilic Bacteria are metabolically more versatilethan the Archaea, and their enzymatic activities are

more diverse. Moreover, most haloarchaeal enzymesrequire at least 1015% salt both for stability and activ-ity, while bacterial enzymes generally do not show sucha strict salt requirement. Extensive research has there-fore been done on the properties of the enzymes fromhalophilic and halotolerant Bacteria and their possibleapplications [82]. An example from the recent literatureis the extracellular lipase from a moderately halophilicSalinivibrio sp., active at 50C [83]. One of the fewhalophilic enzymes that have been applied in industrial

processes is the nuclease H of Micrococcus varians

subsp. halophilus, used in the commercial productionof flavoring agent 5-guanylic acid (5-GMP). Thisenzyme degrades RNA at 60C and 12% salt [84].

Poly--hydroxyalkanoate production

by halophilic bacteria

Poly--hydroxyalkanoate (PHA), a polymer containing-hydroxybutyrate and -hydroxyvalerate units, is accu-mulated by many prokaryotes, Bacteria as well asArchaea, as a storage polymer. It is used for the produc-tion of biodegradable plastics (biological polyesters)with properties resembling that of polypropylene. Thetechnology for the manufacture of poly--hydroxyal-kanoate-derived plastics (Biopol) was developed byICI in the UK, using polymer produced by Cupravidusnecator(formerly named Wautersia eutropha,Ralstoniaeutropha andAlcaligenes eutrophus).

Some halophilic Archaea and Bacteria also produce

PHA. The archaeon Haloferax mediterranei can accu-mulate the compound to up to 38% of its dry weight,the highest concentrations being measured in cellsgrown at 150 g l1 salt under phosphate limitation.Such an archaeal producer of the compound may havedistinct advantages. H. mediterranei grows in simplemedia with sugars or starch as cheap carbon sources.Growth is rapid, and at the high salt concentration ofthe medium there is little danger of contamination.Moreover, the cells lyse in the absence of salt, releas-ing the polymer, so that downstream processing and

purification of the product should be relatively simple[8588].

Another halophilic candidate for PHA production isHalomonas boliviensis (Gammaproteobacteria) [89,90].It can accumulate the compound to up to 88% of its dryweight. Acetate, butyrate or sucrose can be used ascarbon sources.

In spite of the obvious potential thatHaloferax andHalomonas spp. may have as industrial producers ofPHA, no attempts have yet been made to use theseorganisms for the commercial production of biologi-cally degradable plastics.

Exopolysaccharides from halophiles

Bacterial extracellular polysaccharides have founddifferent applications as gelling agents and emulsifiers,and they are also used in microbially enhanced oilrecovery. Several halophilic microorganisms producesuch exopolysaccharides in copious amounts, and there-fore their commercial exploitation has been considered.

The archaeon Haloferax mediterranei, mentionedabove as a potential producer of PHA, also excreteslarge amounts of anionic exopolysaccharides. Thesulfated acidic heteropolysaccharide of Haloferax

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species has a high viscosity at low concentrations, itsrheological properties are excellent and it is resistant toextremes of pH and temperature. It was therefore

proposed to explore its use to enhance oil recovery fromlow productivity oil wells. In addition, the archaealmembrane lipids may act as surfactants, improving the

oil carrying properties of the water. The idea has not yetled to the commercial exploitation of the archaealexopolysaccharides.

Among the halophilic representatives of the Bacteria,theHalomonas species (H. maura,H. eurihalina) showsconsiderable promise as a producer of large amounts ofan extracellular polyanionic polysaccharide, a potentemulsifying agent that exhibits a pseudoplastic behav-iour [91]. TheH. maura exopolysaccharide (mauran)has also been shown to be an immunomodulator [9294].

Aphanothece halophytica, a halophilic unicellularcyanobacterium found in the benthic cyanobacterialmats of solar salterns and in many other hypersaline

lakes, is also known for its massive synthesis ofpolysaccharides. In solar salterns excessive polysaccha-ride production can have a negative impact on the salt

production process, as explained above. However, arecent report on the immunomodulating properties ofthe sulfated polysaccharide ofA. halophytica is ofspecial interest: when administered orally in mice, itsignificantly inhibited pneumonia induced by influenzavirus H1N1 [95].

Biofuels from halophiles

In times in which fossil fuels are getting depleted andthe world is searching for alternative sources of energy,

biofuel is a fashionable alternative. Although halophilicmicroorganisms may not be the most obvious sourcefrom which such fuels may be commercially produced,they still may be of interest.

The halophilic algaDunaliella, discussed above as acommercial source of -carotene and as a potentialsource of glycerol production, may also be consideredas the raw material for biofuel production. Catalytic

pyrolysis ofDunaliella cell material at 200240Cproduces an oil-like substance soluble in benzene. Theoverall process proved to be exothermic, so that most of

the thermal energy needed to initiate the reaction maybe regained. Up to 75% of the cell material in an algae-seawater slurry could be converted to extractable oil[9698].

Dunaliella is easy to grow, and salt water andsunlight are widely available. However, the harvestingof the microalgae is an expensive process, and the ques-tion is therefore whether the amount of biofuel

produced will offset the cost of production and harvest-ing of the biomass and its processing by pyrolysis togenerate oil.

Final comments

The above survey shows that thus far the halophilicmicroorganisms have found relatively few commer-cially viable applications. With the exception of -carotene production byDunaliella and ectoine synthesisusing Halomonas and other moderately halophilic

Bacteria, most other potential applications suggestedare no more than ideas only, waiting to be exploited.The list of possible applications presented in thesections above is by no means exhaustive and additionalideas have been presented in the literature, such as, forexample, production of liposomes for the cosmeticsindustry and exploitation ofHalobacterium gas vesiclesin biotechnological processes. Many patents have beenissued for these and other applications, but the ideas arestill to be implemented in commercial ventures.

With all the advantages listed for the use of halo-philes in industrial processes, there are disadvantagesas well. For mass cultivation of aerobic bacteria, the

low solubility of gases in concentrated brines mayseverely limit oxygen supply to the cultures. Also theaggressive nature of the salts should be taken intoaccount during the construction of reactors with metal

parts exposed to the medium. It is possible to buildcorrosion-resistant bioreactors suitable for high saltmedia, but their cost is significantly higher than that ofconventional fermenters.

The tremendous diversity of halophilic microorgan-isms found in nature is still far from being fully exploited.Approaches derived from genomics and proteomics haveopened new possibilities, and genetic systems are now

also available for a number of halophiles. Therefore, itmay be expected that novel applications will be devel-oped in the future for the highly diverse and versatileworld of halophilic microorganisms.

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