World

Journal

of Microbiology

8 Biotechnology

11, E-94

Biotechnological applications and potentialities of halophilic microorganisms A. Ventosa”

and J.J. Nieto

Halophilic microorganisms are found as normal inhabitants of highly saline environments and thus are considered extremophiles. They are mainly represented, but not exclusively, by the halobacteria (extremely halophilic aerobic Archaea), the moderate halophiles (Bacteria and some methanogens) and several eukaryotic algae. These extremophilic microorganisms are already used for some biotechnological processes, for example halobacteria are used for the production of bacteriorhodopsin, and the alga Dunalielh is used in the commercial production of j?carotene. Several other present or potential applications of halophiles are reviewed, including the production of polymers (polyhydroxyalcanoates and polysaccharides), enzymes, and compatible solutes, and the use of these extremophiles in enhanced oil recovery, cancer detection, drug screening and the biodegradation of residues and toxic compounds. Key words: Bacteriorhodopsin, polymers.

biotechnology,

compatible

solutes, Dunalielh, enzymes, extremophiles,

Hypersaline habitats constitute a typical example of ‘extreme’ environments in which relatively low microbial species diversity can be found (Brock 1979). The spectrum of organisms that thrive in such saline biotopes is mainly determined by parameters such as salinity, 0, solubility, ionic composition and, in some cases, temperature and pH (Rodriguez-Valera 1988). However, a variety of organisms inhabits hypersaline environments, ranging from higher organisms such as the brine shrimp (Artemia salina) and the brine fly (Ephydra), to eukaryotic, photosynthetic flagellates belonging to the genera Dunalieh and Asteromonas, cyanobacteria (Syr~&~occus) and a heterogeneous group of prokaryotes (Archaea and Bacteria) which constitute the predominant microflora (Rodriguez-Valera et al. 1985; Rodriguez-Valera 1988). The microorganisms, which are specialized for living in these extreme hypersaline environments, are designated halophiles, whereas those capable of growth in the absence of salt, but tolerant of varying concentrations, are considered to be halotolerant. Nevertheless, the concepts of halophilic and halotolerant organisms, as well as their response to salt, vary depending on the

The authors are with the Departamento Universidad de Sevilla, 41012 Sevilla. sponding author. @ 1995 Rapid

Communications

of Oxford

de Microbiologia y Parasitologia. Spain; fax: 34 5 4628162. ‘Corre-

halophiles,

halobacteria,

criteria used. Thus, Kushner (1985) defined several categories of microorganisms according to the salt concentration that was optimal for growth. In this system, non-halophiles are those that grow best in media containing < 0.2 M NaCl (some of which, the halotolerant, can tolerate higher concentrations), slight halophiles (marine bacteria) grow best with 0.2 to 0.5 M NaCI, moderate halophiles grow best with 0.5 to 2.5 M NaCl, and extreme halophiles show optimal growth in media containing 2.5 to 5.2 M (saturated) NaCl. In hypersaline habitats, especially those in which salinities can exceed 1.5 M (about IO%, w/v), two main groups of microorganisms predominate: the moderately halophilic bacteria and the extremely halophilic bacteria. The former are more abundant at intermediate salinities (1.5 to 3 M, approx. 10 to 20% salt) whereas highly saline environments (salt concentrations higher than 3 M, about 20%) are dominated by extremely halophilic Archaea, mostly halobacteria. However, there exists a saline range in which both these physiological groups of microorganisms are able to co-exist and compete (Rodriguez-Valera el al. 1985; RodriguezValera 1988). Tables I and 2 show the variety of genera to which extremely halophilic bacteria and moderate halophiles can be assigned. As can be seen, the greater diversity is found in moderate halophiles; some are methanogens which

Ltd Wo&l

Journal of Micnbiology

6 Biotechnology.

Vd II, 1995

85

A. Ventosa and 1.1. Niefo Table 1. Prokaryotlc species.

genera

that

include

extremely

presence of red-coloured slimy masses and an appalling smell. This phenomenon is caused by the growth of some halobacteria and moderate halophiles, which are proteolytitally active, and causes considerable degradation of the salted material. This typical spoilage and its prevention attracted the interest of some microbiologists towards halophiles in the early 20th Century. It was not until the studies of Klebahn (1919) that the microbiological nature of the reddening of salt ponds and salted spoiled products was demonstrated. There were subsequently several studies on halophilic microorganisms from different salt lakes (Post 1977; Rodriguez-Valera 1988; Javor 1989), beginning with that of Elazari-Volcani (1940) on the microflora of the Dead Sea. Despite these early studies on the physiology, ecology and biochemistry of the halophiles (and especially of the extremely halophilic bacteria), which revealed their highly unusual nature (Larsen 1962; Lanyi 1974; Dundas 1978; Kushner 1978), the study of halophilic organisms only really gained any momentum when it was discovered that some of them, the halobacteria, could be included within a novel phylogenetic branch of the Archaea (previously Archaebacteria) (Woese & Fox 1977; Woese 1987). The study of these microbes, as of other extremophiles, was also boosted by interest in the possibility of life on other planets on which very extreme environments are found (Brock 1979). From the early 198Os, halophilic microorganisms have been considered as a group of extremophiles with a biotechnological potential similar to that of other extremophilic microorganisms. Compared with the other Archaea, the halobacteria have some advantages in terms of their potential for genetic manipulation: they are easy to grow and maintain in the laboratory and the necessity for aseptic conditions is decreased to a minimum. These advantages are, in general, also applicable to moderately halophilic bacteria and halophilic algae. In this review, we will

halophlllc

Halobacterla

Methanogens

Bacteria

Halobacterium Haloferax Haloarcula Halococcus Natronobacteriom Natronococcus

Methanohalobium

Acetohalobium Actinopolyspora Ectothiorhodospira

belong to the domain Archaea, and a few are phototrophic, prosthecate, actinomycete or sulphate-reducing species but most are chemoorganotrophic, Gram-negative Bacteria (Ventosa 1988, 1993). In contrast, all extreme halophiles, except for two species of the photosynthetic Ectothiorhodospira, one of Acefohalobium and one of the actinomycete Acfinopolyspora, are Archaea. These are mainly, but not exclusively, represented by halobacteria; some methanogenic species of the genus Mefhanohalobium have recently been described (Kushner 1985; Ventosa 1989; ollivier et al. 1994). Although the halophiles have attracted a great deal of attention in the last two decades, because of interest in the mechanisms of adaptation to extreme hypersaline environments, man has been making use of them dating back at least 5000 years. Thus, salt makers have known since ancient times that the bright red colour of the water of the saltems, now known to be due to the bacterioruberin pigments of the halobacteria, was an indication that the concentrated seawater could be drawn off to another ditch in order to obtain the final crystallization (Baas-Be&rig 1931). Salt has been used in many human cultures for culinary purposes, both as seasoning and for preserving fish, meat and vegetable products, and for preserving raw hides (for leather industry) and human bodies (mummies in ancient Egypt). It has long been known that the spoilage of salted-preserved goods was frequently associated with the Table

2. Prokaryotic

genera

that Include

moderately

halophilic

species. Bacteria

Archaea Chemoorganotrophlc

Phototrophic Gram-negative

Methanohalophilus Halomethanococcus

Arhodomonas Chromohalobacter Deleya Dichotomicrobium Flavobacterium Haloanaerobium Halobacteroides Haloincola

Halomonas Halovibrio Paracoccus Pseudomonas Spirochaeta Sporohalobacter Vibrio Volcaniella

Grampositive Bacillus Clostridium Marinococcus Micrococcus Salinicoccus Sporosarcina

Actlnomycete

Actinopolyspora

Sulphatereducing Desulfohalobium Desulfovibrio

Rhodospirillium Ectothiorhodospira Chromatium Thiocapsa

Biotechnology of hnlophiles examine each of the major groups of halophilic microorganisms and their most interesting current or potential applications. We will focus on the biotechnological potential of each of the three major groups of halophiles with commercial interest: the halobacteria, the moderate halophiles, and the halophilic algae.

Halobacteria Bacteriorhodopsin Since the retinal proteins of halobacteria were discovered in the early 1970s as integral proteins of the purple membrane, several applications of one of them, bacteriorhodopsin, a unique, light-driven, proton translocator, have been described. The major interest for commercial purposes lies in its potential use in artificial membranes capable of converting sunlight to electricity (Prentis 1981; Rodriguez-Valera 1992). In this case, ATP synthesis would be prevented and the electrical potential arising from the proton gradient would be the source of the electricity. A bacteriorhodopsin system offers several benefits if used as an optical recording material, since it is very stable and easy to immobilize on solid substrates, producing photoelectric signals which are extremely reproducible (Konig 1988). Bacteriorhodopsin-containing purple membranes from Halobacterium halobium are being commercially produced by the Consortium fur Elektrochemische Industrie GmbH, in Germany, and used for optical data processing (optical switches, holography, information storage), non-linear optics and as light sensors. A ‘photographic’ film based on purple membranes displays interesting properties since it does not require developing and can be used several times. Holographic films of this type are suitable for computer memory, i.e. parallel processing (Rodriguez-Valera 1992). The electrical response produced as sunlight passes through bacteriorhodopsin may have applications in the development of ‘biochips’ in a new generation of computers (Hong 1986). Thus, while the retina uses a pigment called rhodopsin for the light-to-electricity conversion, such commercial sensors would make use of cheaper bacteriorhodopsin. To turn this protein into a light sensor, it is spread in a thin film sandwiched between an oxide electrode and an electrically conductive gel. The molecules of bacteriorhodopsin change shape when light strikes the film and the changing shape creates a displacement of charge which generates an electrical signal that travels through the electrode (Vsevolodov & Dyukova 1994). Use of bacteriorhodopsin has several advantages: it is one of the best understood photobiological systems; it is very stable at different temperature (0 to 45 “C) and pH values (1 to 11) (the protein layer utilized is exceptionally stable since it is kept saturated with water); its photochemical reactions are selfregenerative; and it is a protein with sufficient complexity to allow for genetic, immunological or chemical manipula-

tion (Konig 1988; Chen & Birge 1993). It is thought that, with some additional development, bacteriorhodopsinbased biosensors might be used to endow industrial robots with vision. Other potential applications of bacteriorhodopsin include its use in the desalination of salt water (Prentis 1981). Bioplasfics The polyhydroxyalkanoates (PHA) constitute a heterogeneous family of polyesters, usually employed as microbial carbon storage material, that can be used to replace thermoplastics. Poly-P-hydroxybutirate (PHB) is the most common. Bacterial PHA offer several advantages over other plastics: they are biodegradable; exhibit total resistance to water; and they are biocompatible, i.e. they are tolerated and degraded in human and animal tissues (Hrabak 1992). This latter property gives the PHA pharmaceutical and clinical importance, including use in delayed drug release, bone replacement and surgical sutures (Lafferty et al. 1988). Some halobacteria, especially Haloferax mediferranei, can produce large amounts of PHA when growing on carbohydrates such as starch or glucose (Femandez-Castillo et al. 1986; Lillo & Rodriguez-Valera 1990). Moreover, the production of PHA by this halophile offers a number of advantages over that by other PHA bacterial producers. For example, the amounts of polymer accumulated by Halof mediterrunei are as high as those produced by other microorganisms but the costs are lower; for example Halof. mediferranei can use starch as carbon source instead of dearer substrates such as glucose (Keeler 1991). The fragility of halobacterial cells when exposed to low salt concentrations allows the development of greatly simplified recovery processes and, since Halof. mediferranei is unable to degrade the intracellular PI-IA granules, greater freedom when handling cultures leads to higher yields. Another great advantage is the ease of cultivation; batches of up to 100 1 can be grown under very simple conditions (Kushner 1966). Species of the genus Haloferax also exhibit the fastest growth rates and widest physiological versatility (Rodriguez-Valera et al. 1983), permitting continuous growth conditions to be used with very little, if any, contamination. In addition, these halobacteria possess high genomic stability, which is a prerequisite for industrial processes (Lillo & RodriguezValera 1990; Rodriguez-Valera & Lillo 1992). Polysaccharides Exo-polysaccharides of microbial origin are of biotechnological interest because they can be used to modify the rheological properties of aqueous systems, enhancing the viscosity of the solutions even at low concentrations (Sutherland 1986). Furthermore, the industrial production of exo-polysaccharides is not vulnerable to crop failure, marine pollution or climatic conditions. Microbial exo-polysaccharides are currently utilized as stabilizers, thickeners, gelling agents and emulsifiers in the pharmaceutical, paint, oil recovery,

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A. Ventosa and J.]. Nieto paper, textile and food industries. Xanthan gum, the polysaccharide from Xanthomonas campestris, is the major bacterial exe-polysaccharide in commercial production (Sutherland 1983). Additional studies to find alternative sources are obviously needed. Halophiles have usually been overlooked in most screening programmes for exo-polysaccharides. Recently, however, it has been found that Halof. mediterranei produces a highly sulphated and acidic heteropolysaccharide (up to 3 g/l) which contains mannose as a major component (Anton et al. 1988). Such a polymer combines excellent rheological properties with a remarkable resistance to extremes of salinity, temperature, and pH (Anton et al. 1989). It therefore has great potential, for example as an emulsifying agent and mobility controller in the oil industry, in the clearing of oil spills, and in microbially-enhanced oil recovery processes. Microbially-enhanced Oil Recovery Residual oil in natural oil fields can be extracted by injection of pressurized water down a new well. The water displaces the oil and pushes it to the surface through one of the original oil wells. The efficiency of this process can be maximized by increasing the viscosity and decreasing the surface tension of the water used. Bacterial biopolymers are of interest in enhanced oil recovery because of their biosurface activity and bioemulsifying properties (Cooper 1986). The halobacterial membrane lipids exhibit many relevant properties; the ether-linked lipids possess very low melting points, are resistant to degradation by acids, alkalis, and heat and have an emulsifying ability, with an adequate hydrophile-lipophile balance, that produces good water-inoil emulsions (Post & Collins 1982). The conditions existing in oil deposits are often saline, strata usually being associated with such reservoirs. The use of a saline-resistant surfactant, such as the polysaccharide produced by Haloferaw mediterranei, would be very convenient in enhancing oil recovery in these circumstances. Polysaccharide-producing halobacteria might be added to the water used straight from the fermenter, since the extracellular polysaccharide would increase the water’s viscosity whilst the lipids liberated from lysed cells would act as surfactants to improve its oil-carrying properties. The surface activities of 16 halobacterial strains, including fresh isolates from the Great Salt Lake, Utah, have been measured on whole cells, growth media, and lipid extracts by Post & Al-Harjan (1988). When three of the strains were used to extract bitumen from Utah tar-sands, either as whole cells in growth medium or lysed cells in distilled water, all increased oil recovery. Cancer Detection An 84-kDa protein from Halobacterium halobium has been used as antigen to detect antibodies against the human cmyc oncogene product in the sera of cancer patients (Ben-

Mahrez et al. 1988). This was based on the demonstration that a polyclonal antiserum raised against the 84-kDa protein recognizes the c-myc protein obtained from the nuclei of a human pryelocytic leukaemia cell line (HL-60). These preliminary results were then confirmed in experiments in which the sero-positivities of colon-rectal cancer patients were determined using the archaebacterial 84-kDa protein or the human c-myc protein produced in kcherichia coli by genetic engineering as antigen (Ben-Mahrez et al. 1991). Similar percentages were obtained for the patients (57% when using the human c-myc protein and 64% with the archaebacterial protein) but there were interesting differences in the results for the healthy controls. Use of the cmyc protein indicated 17% of the controls were seropositive, whereas use of the 84-kDa protein only indicated that 1.6% were positive. This implies that the epitopes recognized by normal sera are hardly present in the halobacterial protein. Therefore, the use of halobacterial antigens as probes for some types of cancer seems to be promising. Drag Screening Halobacteriwn strain GRB-1 harbours a plasmid, pGRB-1 (Ebert et al. 1984) that can be used in the pre-screening of new antibiotics and anti-tumour drugs that affect eukaryotic type II DNA topoisomerase (epipodophyllotoxines) and quinolone drugs which act on DNA gyrase. In vivo such drugs cause DNA relaxation and DNA cleavage of small plasmids from halophilic Archaea (Sioud et al. 1987) and therefore act on Archaea as they do on Bacteria and Eukarya. The single- and double-stranded breaks in the DNA of the multicopy plasmid pGRB-I can be easily monitored in vitro, on agarose gel (Forterre 1989). Furthermore, other anti-turnour drugs, such as the anthracyclin daunorubicin, can also be detected in the same system, because they change the electrophoretic mobility of the plasmid, permitting a rapid evaluation of the degree or type of activity (Forterre et al. 1991). Liposomes An interesting potential application of the unique etherlinked lipids of the halobacteria is their use in novel types of liposomes, which have great value in the cosmetic industry. Such liposomes would be more resistant to biodegradation than those used at the moment and thus with a better shelf life, since halobacterial lipids are relatively resistant to the action of other bacteria (Galinski & Tindall 1992). Enzymes Exoenzymes with polymer-degrading capacity are of great commercial interest (see Leuschner & Antranikian 1995). There are a number of enzymes of this type, produced by some halobacteria, that have optimal activity at high salinities and could therefore be used in many harsh industrial processes where the concentrated salt solutions used would otherwise inhibit many enzymatic conversions. The intracel-

Biotechnology of kalopkiles lular concentration of salt for extreme halophiles is higher than 3.5 M, but this is mostly KCl, not NaCl (Kushner 1985). Halophilic enzymes are therefore uniquely adapted to function in conditions with low water potential. In addition, most halobacterial enzymes are considerably thermotolerant (Oren 1983), remaining stable at room temperature over long periods. The hypersaline requirement of some halophilic proteins can make the purification and handling of these enzymes difficult. For example, the enzymes cannot be subjected to ionic-adsorption chromatography and alternative procedures that are unaffected by high concentrations of salt (gel filtration, hydroxyapatite chromatography, hydrophobic and sulphate-mediated chromatography) or that take place at low salt concentrations (ion exchange chromatography) have to be used to purify them (Hochstein 1988). The few halobacterial exo-enzymes that have been reported include the amylases produced by Halob. kalobium (Good & Hartman 1970) and Hulob. sodomense (Oren 1983), the proteases from Halob. salinarium (Norberg & Hofsten 1969) and Halob. kalobium (Izotova et al. 1983), and the lipases from several halobacteria (Gonzalez & Gutierrez 1970).

A site-specific endonuclease activity has been reported in Halob. kalobium and other halobacteria (Schinzel & Burger 1986). The restriction endonuclease HacI has been isolated from ‘Halococcus acefoinfaciens’ and a corresponding production process patented (Obayasi et al. 1988). Other restrictases, from Halob. ctitirubrum (HcuI), Halob. kalobium (HklI), Halob. salinarium (HsaI), and ‘Halococcus agglomerafus’(HagI), have recently been isolated and are currently available from commercial suppliers (New York Biolabs). The utilization of halobacteria for the screening of new restrictases, as well as other enzymes for molecular biology, appears to be another promising application. Degradation of Toxic Compounds Very frequently, hypersalme environments (including hypersaline lakes and soils and salt ponds near river estuaries) are contaminated with harmful toxic compounds. Therefore, the search for microorganisms able to degrade toxic agents in the presence of high salt concentrations appears to be desirable, especially in terms of biological treatment of h’rg hl y saline industrial waste effluents. In a recent study (Bertrand et al. 1990), the halobacterial strain EH4, isolated from a salt-marsh in the south of France, was found to biodegrade alkanes (tetradecane, hexadecane, eicosane, heneicosane and pristane) and aromatic hydrocarbons (acenaphtene, phenaphtrene, anthracene and 9-methyl-anthracene). The best biodegradation (62%) was achieved on eicosane in a medium prepared with natural hypersaline water collected from a salt-marsh (3.5 M NaCl). The aromatic compounds were degraded to a lesser extent (19% to 24%). Since other halobacteria, such as those belonging to

the genus Haloferax, possess higher metabolic potential than EH4, more extensive studies focused on the capabilities of such microorganisms to degrade organic pollutants are clearly needed. Gas Vesicles Some halobacteria produce intracellular, gas-filled organelles called vacuoles or gas vesicles which provide buoyancy (Walsby 1994). Very frequently, mutants lacking gas vesicles (Vat-) are formed, resulting in orange, translucent colonies instead of the pink, opaque colonies, typical of the wild-type vacuolated (Vat+) strains. The genomic location of the gas vesicle genes of Halob. kalobium and other halobacteria, and the characterization of the mechanism of the Vat- mutations, have been reported (DasSarma et al. 1987; Pfeifer & Englert 1992). It has now been suggested that gas vesicles are synthesized by a complex pathway involving about 13 or I4 genes. Additional studies to elucidate the regulation, assembly and structure of the vesicles are now in progress. In the near future, it should be possible to genetically engineer other microorganisms to produce gas vesicles and therefore to float. This process has great potential for biotechnology. For example, the problems of sedimentation of microorganisms growing in fermenters, and the expensive agitation techniques used to prevent them could be minimized. Halobacteria in Food Although the undesired growth of halobacteria on salted food was the origin of the scientific interest in these organisms, their growth on food can sometimes be profitable. Thus, one sauce (nam pla in Thai) prepared from fish fermented in concentrated brine, traditionally used as a food condiment in South-East Asia, contains a large population of halobacteria and this has been claimed to be important in aroma production (Saisithi ef al. 1966); halobacterial populations peak in the salty liquor (4.4 to 5.1 M NaCl) after 3 weeks and then persist throughout the fermentation period of about 12 months (Thongthai & Siriwongpairat 1990). Recent studies on these nam pla halobacteria, using polar liquid analysis and DNA hybridization, have shown that they are mostly Halob. salinarium (Thongthai et al. 1992). Since these halobacteria produces salt-stable extracellular proteases, it is likely that they are indeed important in the fermentation and the flavour- and aroma-producing process.

Moderate

Halophiles

Enzymes More halophilic extra- and intra-cellular enzymes have been isolated and characterized from moderate halophiles than from the halobacteria. The exoenzymes include several amylases, nucleases, proteases and 5’-nucleotidases from several moderately halophilic bacteria. The properties

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A. Ventosa and 1.1. Nieto and functions of such enzymes have been reviewed by Kamekura (1986). Some are of considerable commercial interest, such as the n&ease H produced by Micrococcus varians subsp. halophillls. This is used for the industrial production of the flavouring agents 5’-guanylic acid and 5’-inosinic acid (Kamekura et al. 1982) in a bioreactor system with a column of flocculated cells (Onishi et al. 1991). Several halotolerant bacteria produce salt-resistant enzymes, such as the amylase produced by a Bacillus sp; this is stable at 60°C and 5 M NaCl and could be used in the treatment of effluents containing starchy or cellulosic residues (Khire & Pant 1992). Compatible Solutes Most halophilic and halotolerant bacteria accumulate intracellular concentrations of organic compounds called ‘compatible solutes’, because they are solutes responsible for the osmotic balance of the cells and are also compatible with the cellular metabolism (Galinski & Tindall 1992; Speelmans et al. 1995). These osmolytes are sugars (trehalose and sucrose), amino acids (glycine, alanine, proline, etc), polyols (glycerol, mannitol, sorbitol), betaine and ectoines (ectoine and hydroxyectoine). The main compounds produced or accumulated by halophilic eubacteria when they are grown at increasing salt concentrations are betaine and ectoines (Galinski & Tindall 1992). The compatible solutes may act as stabilizers of enzymes and whole cells. Thus, hydroxyectoine protects lactate dehydrogenase and other enzymes against high and low temperatures, salt and desiccation (Galinski & Lippert 1991). Industrial use of some of these compatible solutes, which are easily produced by biotechnological processes, is a very promising field. The solutes could be used as stabilizing compounds in enzyme technology and for cosmetics (Galinski & Tindall 1992). Enhanced Oil Recovery As well as the production of polysaccharide additives for the water used to enhance oil recovery (as described for the halobacteria), the oil industry is also interested in the in situ production of microbial ‘plugging’ agents. For this purpose, it is necessary to find microorganisms which can grow and produce extracellular polymers under the environmental conditions which occur in the oil reservoir, i.e. high salinity and temperatures, in an anaerobic environment. A pre-screening study of such microorganisms has been performed by Pfiffner et al. (1986); over 200 bacterial strains were isolated from different sources (oil wells, brine injection water, anaerobic sewage sludge, soils in the vicinity of oil wells and effluents of sandstone cores) and selected for anaerobic growth at 50°C and extracellular polysaccharide production in a sucrose mineral salts medium with NaNO, and up to 10% (w/v) NaCl. One strain, probably a Bacillus sp., grew and produced polysaccharide at salinities up to 12% (w/v) NaCl, showing optimal production under anaerobic conditions with 4% to 10% (w/v) NaCl. The polymer

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showed pseudoplastic behaviour, was resistant to shear and thermal degradation, and had a higher viscosity at dilute concentrations and elevated temperatures than xanthan gum. Moderate halophiles with such properties could probably grow in oil reservoir brines and, therefore, would be useful for in situ microbially-enhanced oil recovery processes. Recently, Quesada et al. (1993) reported the production of an extracellular polysaccharide by the moderate halophile Volcaniella eurihalina. This exopolysaccharide was optimally produced at 7.5% (w/v) salt and 32°C. Furthermore, solutions of this polysaccharide exhibited pseudoplastic behaviour with viscosities which are resistant to high ionic strength, quite thermostable, and highest at acidic pH values. Biodegradation of Residues Although the great potential of halophiles in the degradation of toxic industrial residues has already been recognized, there have been few related studies on moderate halophiles. A moderately halophilic bacterium isolated from a hypersaline spring in Utah degraded several toxic organo-phosphorus compounds using an enzyme, organophosphorus acid anhydrase, which has been purified and characterized (DeFrank & Cheng 1991). Obligately anaerobic, moderately halophilic bacteria could play an important role in the biotransformation of some unusual chemicals that can pollute hypersaline environments (Oren et al. 1992). Oren et al. (1991) reported the degradation of several nitro-substituted aromatic compounds by Haloanaerobium praevalens and Sporohalobacfer marismortui at very high rates in the presence of 13% to 14% (w/v) NaCl. A field study performed in the Great Salt Lake, Utah, yielded microbial growth at salinities up to 17.2% (w/v), after enrichment with mineral oil as substrate (Ward & Brock 1978). No growth occurred with >20% (w/v) salt nor after 5 days’ incubation with 220% (w/v) salt supplemented with hexadecane. The growth at relatively low salinities indicates the involvement of moderate halophiles rather than extremely halophilic Archaea. The isolation of microorganisms from an Antarctic saline lake that were able to degrade several hydrocarbon compounds, including hexadecane and phenanthrene, has been reported (McMeekin et al. 1993). Moderate halophiles isolated from hypersaline soil have been used for the biological treatment of hypersaline waste waters (with a phenol concentration of 1.1 mM) involving a sequencing batch reactor operated at 15% salt (Woolard & Irvine 1992). As a result of industrial activities, ecosystems are often subjected to heavy metal pollution. The responses of a large number of moderate halophiles and halobacteria, the majority of them isolated from heavy-metal-contaminated hypersaline environments, to 10 heavy metals which are common industrial pollutants has been investigated (Nieto et al. 1987, 1989). Although most of the strains were

Biotechnology of halophiles

EcoRl

Sgill /

1

Pstl

Pstl

Hindlll Figure 1. Restriction map of plasmid pMH1, showing cleavage sites for the restriction endonucleases used and the restriction fragment (Km) in which the gene that codifies for resistance to kanamycin is located.

sensitive to Hg and resistant to Pb’+ and Cr2+, their susceptibilities to the rest of the metals showed considerable variation. Some isolates demonstrated a high resistance to Co, Ni, Cd and AsO,. As a result of these studies, a range of concentrations has been proposed for defining metal resistance in halophilic bacteria (Nieto 1991). Apart from the ecological role played by halophiles in the biotransformation of heavy metals in natural environments and the possible use of metal-resistant halophilic strains in the biological treatment of polluted saline wastes, it has been claimed that these metal-resistant halophiles could be used as bioassay indicator organisms in polluted saline environments (Trevors et al. 1985). Other Potential Products Moderate halophiles could be used to remove phosphate from saline environments in a cheaper alternative to chemical approaches (Ramos-Cormenzana 1989). Increasing irrigation has led to 30% to 50% of agricultural areas being affected by salinity. The potential use of moderate halophiles in the recovery of saline soils is therefore gaining in importance. The study of the genetics of moderate halophiles is still in its infancy compared with the large amount of knowledge gained in recent years with the halobacteria. This has greatly hampered genetic manipulation of the moderate halophiles but a few studies have recently focused on the isolation and molecular characterization of autochthonous plasmids from these halophiles (Femlndez-Castillo ef al. 1992; Ventosa et al. 1994). The rates of mutation of resistance to several antimicrobial agents have been determined in several moderate halophiles, and these are useful for the isolation of stable antimicrobial-resistant mutants

(Nieto et al. 1993). Figure I shows the physical map of pMH1, the first plasmid to be reported from moderately halophilic bacteria. This, and other plasmids which are currently being studied in our laboratory, appear to have considerable potential as cloning vectors for this group of extremophiles, and should permit genetic manipulation for over-production of industrially interesting compounds. New antibiotics are also being isolated and characterized from marine bacteria. As halobacteria produce a great variety of bacteriocins, named halocins (Rodriguez-Valera et al. 1982; Meseguer et al. 1986), it would not be surprising if other obligate halophiles were also to produce antimicrobial compounds. Further studies focused on this field appear especially interesting. Although some useful plants are already suitable for widespread cultivation in brackish waters and arid saline lands where conventional crops cannot be grown, an amazing biotechnological improvement could be the transfer of genes for salt and drought tolerance from halophiles to some strains of, for example, wheat, barley and rice.

Other Halophiles The main halophilic eukaryotes of hypersaline environments are species of the unicellular photosynthetic alga Dunuliellu. Unlike other algae, this organism lacks a rigid cell wall and this permits a high response to extracellular changes in osmotic pressure. Hence, this alga is the most halotolerant eukaryotic organism known and can grow over a wide range of salt concentrations, from 0.2% to about 30% or 35% (w/v) salts. It is often found as the predominant microorganism in aquatic habitats containing up to 10% (w/v) salts. Dunaliella inhabits oceans, salt marshes, saltwater ditches close to the sea, saltems and brine lakes such as the Dead Sea in Israel and the Great Salt Lake in Utah. It is a potential producer of a range of products of great economic interest, including protein, p-carotene and glycerol. The risks of contamination are decreased to a minimum because of Dunaliellu’s extreme salt tolerance, and it grows as a monoculture in open ponds and on an industrial scale. Depending on growth conditions, the alga can contain >40% protein, 50% glycerol or 12% /j-carotene. Systems for producing high yields of biomass, rich in proteins, similar in composition to that of soya meal but with a relatively high lysine content, have been developed and the biomass used as a feedstock in mariculture (crab, shrimp, shellfish) and for livestock such as chickens. Dried Dunaliella biomass is a good feedstock as it is easily and fully digestible by animals because of the absence of cell walls. Productivity of this alga in open-ponds systems is about 50 times higher than that of fish farming and loo-fold greater than crop production (Avron & Ben-Amotz 1992). Some Dunaliella species, such as D. bardawil, can accumulate substantial quantities of p-carotene, sufficient to reach

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A.

Venlosa and1.1.Nieto

a commercial scale of operation (Ben-Amotz & Avron 1990). Natural sources of p-carotene are increasing in importance with the build-up of information relating carotenaids to preventive medicine. Natural p-carotene (as produced by Dunaliella) contains a mixture of all-trans p-carotene, together with a few other steroisomers. In contrast, synthetic p-carotene is almost exclusively composed of trans$-carotene. Recent studies have shown than the natural steroisomeric mixture is better accumulated in the animal livers and P-carotene-rich D. bardawil could be used as a chemo-preventive agent in human cancer (Nagasawa et al. 1989). B-Carotene also has major applications in the food industry, serving as natural, food-colouring agent, and as a source of provitamin A in animal feed, as an additive to cosmetics, multi-vitamin preparations and health food products. Units for the commercial cultivation of Dunaliella have been constructed in Israel, Australia and the USA (BenAmotz & Avron 1990). Depending on the salt concentration of the growth medium, Dunaliella can accumulate more than the 50% of its biomass as glycerol. This is an important commercial organic chemical, widely used in the pharmaceutical, chemical and food industries. Although it is currently produced by the petrochemical industry, using propylene as a starting material, mutants of D. parva have recently been obtained which can produce up to 90% glycerol, suggesting a future biotechnological alternative. Algal paste derived from cultivated Dwaliella could be a commercial source of other interesting compounds, including enzymes (dihydroxy-acetone reductase), fatty acids and growth factors. Lastly, liquid fuel, in the form of a mixture of hydrocarbons, has been produced from D. parua; the major nutrients (C, N) could be recycled and pyrolysis of the biomass was exothermic, enabling the recycling of an important fraction of the thermal energy (Ginzburg 1991). The salt-tolerant, red microalgae Porphyridium, which grows in up to 1 M NaCl, is a potential commercial producer of phycoerythrin, a red food pigment, and arachidonic acid, used as a human dietary supplement (Galinski & Tindall 1992). Another source of protein from microbial biomass in natural ponds is the cyanobacteria Spirdina. This halophile has an unusually high protein content (about 60%), and this has a higher amino-acid spectrum than that of plant proteins. Spirtllina has been used traditionally as a supplementary food, in the form of dried cakes, by natives around Lake Chad in Africa and Lake Texcoco, Mexico. Experimental, large-scale cultivation of Spiruhna platensis in Israel uses brackish water unsuitable for agriculture and the biomass is marketed as a health food (Galinski & Tindall 1992). Other units for large-scale production operate in Lake Texcoco, producing a single-cell-biomass for use as a feed additive for animals or as a health food. The organism has one very useful characteristic: it grows optimally in alkaline lakes with a salt concentration rang-

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ing from 2% to 7% (w/v). Few other organisms can grow in these conditions and the lake flora tends to be monospecific, permitting recovery of the Spirdina by simple filtration. Experimental studies aimed at the mass cultivation in outdoor ponds of other halotolerant cyanobacteria, such as Synechococcw elongates, are now being performed.

Acknowledgements Research in the authors’ laboratory was supported by grants from the Commission of the European Communities (Generic Project “Biotechnology of Extremophiles”, BIOCT93-02734) and Ministerio de Educaci6n y Ciencia, Spain (PB92-0670, PB93-0920 and BI0944846-CE).

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Biotechnological applications and potentialities of halophilic microorganisms.

Halophilic microorganisms are found as normal inhabitants of highly saline environments and thus are considered extremophiles. They are mainly represe...
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