Halophilic and halotolerant microorganisms-an

FEMS Microbiology Reviews 39 (1986) 3-7 Published by Elsevier

3

FER 00020

(Halotolerance; halophilism; salt-tolerant microbes; salt-requiring microbes)

Helge Larsen Department of Biochemistry, Norwegian Institute of Technology, Universityof Trondheim, N-7034 Trondheim, Norway Received 13 March 1986 Accepted 17 March 1986

It seems appropriate to base an introduction on an illustration depicting a rough grouping of the microorganisms we are about to discuss into types on the basis of their ability to grow and proliferate at different salt concentration (Fig. 1). Salt normally means NaC1, and the distinction between 'tolerance for salt' and 'requirement for salt' should be noted [1-3]. There are several categories of halotolerant microbes (Fig. 1A): non-tolerant, those which tolerate only a small concentration of salt (about 1% (w/v); slightly tolerant, tolerating up to 6-8%; moderately tolerant, up to 18-20%; and extremely tolerant, those microbes that grow over the whole range of salt concentrations from zero up to saturation. Of considerable importance is the fact that typical spoilage bacteria, with only few exceptions, are either non-tolerant or only slightly tolerant. This is, of course, the basis for the extensive use of salt in food preservation. Most of the anaerobic spore formers, notably the clostridia, are completely inhibited at NaC1 concentrations of 5-7% (w/v), including Clostridium botulinum, and to my knowledge none Of the more halotolerant clostridia can develop at concentrations above 10% NaC1. A similar picture holds true for the majority of the Gram-negative rod-shaped bacteria. These are

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Fig. 1. Microbes grouped according to response to salt.

0168-6445/86/$03.50 © 1986 Federation of European Microbiological Societies

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Halophilic and halotolerant microorganisms-an overview and historical perspective

in the sea, e.g., salty soil, salty mud, salty food, and may also under natural conditions be exposed to a greater variation in the salt concentration than the marine microbes. Many of the moderate halophiles grow over a much wider range of salt concentrations than the marine forms; they are adapted to greater variations in the salt concentration in their environment. A large number of microbial types fall into this category, including bacteria, fungi and algae [1-3]. The moderate halophiles were for many years overshadowed by the extreme halophiles in terms of scientific interest. In recent years, however, the moderate halophiles have received more attention and the physiological and biochemical bases of some aspects of their halophilism are discussed in detail later in this publication. Finally, there is the group of extreme halophiles, including the well-known halobacteria, that is the subject of specific discussion elsewhere in this publication [7]. I would like to dwell for a moment on this group, not only for the reason that this is the group I know best myself, but also because there are a few special points I would like to make, in particular to outline how narrow-minded our approach has been to the physiology of the group until quite recently. It is well known that extremely halophilic bacteria can occur in nature in such high numbers that they impart a red colour to the environments in which they thrive. This is known from marine salterns and very salty lakes all over the world. In olden days when the production of salt from sea water was an art rather than an industry, the salt-maker would watch for the red colour to develop in the brine, and when the phenomenon occurred he knew the time had arrived to transfer his brine from the first evaporation pond, the pickle pond, to the next, the crystallisation pond, where the salt, NaC1, was to precipitate out [8]. Looking into the old literature it seems that the reddening of concentrated salt brines was not clearly recognised as a biological phenomenon, and certainly not studied as such [8]. The recognition of red-coloured extremely halophilic bacteria came from the fact that they contaminated the salt produced in the marine salterns, which was to be used as a preservative for various proteinaceous


generally completely inhibited at salt concentrations in the range 5-10% (w/v). This group comprises a number of well-known types: pseudomonads, vibrios, enterobacteria. There are, however, some striking exceptions to this rule [4]. Examples of salt-tolerant bacteria are found among the aerobic spore formers, many of which grow at salt concentrations up to 15% or even 20% (w/v). Micrococci are extreme examples of Gram-positive bacteria growing at salt concentrations up to saturation. There are also some representatives among the yeasts, the filamentous fungi and the algae that are quite salt-tolerant. When considering the halophilic microorganisms, i.e., those with a requirement of salt for growth, we can conveniently use a similar grouping (Fig. 1B). A point that could be made in this connection is that microbes that we regard as non-halophilic are often stimulated in their growth by a small concentration of salt, e.g., about 1% in the growth medium. Such an addition was not uncommonly prescribed in older formulae for culturing bacteria, and the stimulating effect was often obtained with different salts. The slight halophiles include the microbes indigenous to the marine environment. ZoBell stated in his book Marine Microbiology [5], as much as 40 years ago, that most, if not all, of the types of bacteria found in fresh water have their counterpart in the marine environment. The marine forms require salt, and optimal growth normally takes place in the presence of 2-3% NaC1. Most marine forms are, however, inhibited at only slightly higher salt concentrations. In other words, many of the marine bacteria thrive in a rather narrow range of salt concentrations. This also holds true for a number of other microbes indigenous to the sea, e.g., the phytoplankton. We know, however, that certain strains of the marine green alga, Dunaliella tertiolecta, can grow at very high concentrations of NaC1 ( > 20%, w / v ) [6]. The narrow salt range in which the marine microbes thrive is in rather striking contrast to that of many of the halophiles we assign to the group of moderate halophiles, i.e., organisms growing best somewhere in the range 5-20% ( w / v ) salt. The moderate halophiles are often found in places containing a higher salt concentration than

Another striking finding that Gibbons reported, and which was also corroborated in my own laboratory, was that the red halophiles required an extremely high concentration of Mg 2÷ for optimal growth and proliferation, namely some 10-100 fold higher than that required by 'ordinary' bacteria such as Escherichia coli [15]. Isolates from sources other than salt fish, including the Dead Sea, showed the same high requirement. I guess we all felt at the time that the very high Mg 2+ requirement was part of the halophilism story: extremely halophilic organisms were characterised by their high requirement for both NaC1 and Mg 2÷. However, our view has changed markedly in recent years. Tomlinson and Hochstein have described a halobacterium that is indeed capable of metabolising carbohydrates [16], and it is of particular interest that its carbohydrate dissimilation is not of a common kind: the organism makes use of the 'modified Entner-Doudoroff pathway' [17]. Since then, many isolates have been reported as being carbohydrate-utilisers, so that today one has the feeling that strains of halobacteria that do not utilise carbohydrates are more the exception than the rule [18,19]. Some isolates can even use glucose as the sole source of carbon and energy [20]. As to the Mg 2+ requirement, we have the extremely interesting recent reports about the alkaliphilic halobacteria (the haloalkaliphiles) which thrive at a pH so high that the concentration of soluble Mg 2÷ in the medium is very low [21,22]. Thus, we have come to realise in recent years that a variety of physiological types of halobacteria exists. Those of us working on the physiology and biochemistry of the halobacteria in the early days had isolated all our organisms from salt fish in peptone/tryptone media, conditions which favoured the development of amino-acid utilisers, altogether rather special physiological forms. We did not think much about the possibility that there could be other physiological types of halobacteria that might even be dominant in nature under special conditions, e.g., alkaline saline brines. In a review on halophilism written almost 20 years ago [23] I emphasized the fact that to our knowledge at that time, two distinct types of microbes, and only two, were extremely halophilic,


products. The bacteria occur in such salt in enormous numbers (105-106 viable bacteria g-1 salt) [1] and, given the right conditions of temperature and humidity, the bacteria will start to grow and multiply in the salted products, in extreme cases revealing themselves as a red-coloured slimy mass, causing also the most appalling smell [1]. From the beginning of this century there are reports from France on 'le rouge de la morue' and 'la rouille des salaison', from Germany on 'der rote Hund', from English-speaking countries on 'the pink' and 'the red heat', notably from Canada where there was an extensive salt fish industry, and likewise from the Scandinavian countries where the phenomenon was referred to as 'rSdmidd'--'red mites'. It was not until about 1920 that a reliable bacteriological picture started to emerge, from the work of Klebahn in Germany [9] and that of Harrison and Kennedy in Canada [10], and it was much later before insights began to be gained into the physiology and biochemistry of these organisms. One name should be highlighted in this connection, namely that of N.E. Gibbons in Canada, who really started the modern development. Before the war he was a bacteriologist at the Atlantic Fisheries Experimental Station in Halifax, and came into close contact with the extensive salt-fish industry on the East Coast of Canada. He isolated from salt fish and solar salt a number of strains of red-coloured, extremely halophilic bacteria which he then characterised [11], but is was not until after the war, when he had taken up a position at the National Research Council in Ottawa, that Gibbons started to look into the physiology and biochemistry of these organisms. His work on various aspects of the halophilic properties of these organisms has laid the foundations of our present knowledge. From the studies of Gibbons and others, including observations in my own laboratory on strains isolated from salt fish, it has emerged that these red-coloured extremely haiophilic bacteria preferred proteins and amino acids for growth, rather than carbohydrates [12]. They utilised carbohydrates only slightly [13] if at all, so the preference for proteins and amino acids was listed as a characteristic of this group of organisms [14].

kinds. These microbes included glucose-fermenting bacteria, denitrifying bacteria, sulphuroxidising bacteria and cellulose-decomposing bacteria [27]. Unfortunately, Volcani did not follow up his preliminary observations. He did not isolate these microbes, or characterise them sufficiently for us to know for certain whether the organisms he observed were merely halotolerant, or truly halophilic, or indeed whether or not they were indigenous to the Dead Sea. It was not until quite recently that microbiologists have started to take a new and serious interest in the Dead Sea [28,29] and also other hypersaline ecosystems in that area [30,31]. In recent years the extremely halophilic microbes, notably the halobacteria, have become favourite model organisms for studies in molecular biology, although often not so much for the purpose of gaining an understanding of the molecular basis for their ability to cope with the salty environment, as for the purpose of studying rather basic phenomena in molecular biology and evolution. It suffices here to mention the purple membrane and the archaebacterial traits of the halobacteria. However, much has still to be learned about the microbes living in saline environments and their relationship to those environments. There is no doubt this is an interesting and rewarding field of research for microbial ecologists and ecophysiologists.

REFERENCES [1] Larsen, H. (1962) Halophilism, in The Bacteria (Gunsalus, I.C. and Stanier, R.Y., Eds.) Vol. IV, pp. 297-342. Academic Press, New York. [2] Kushner, D.J. (1978) Life in high salt and solute concentrations: halophilic bacteria, in Microbial Life in Extreme Environments (Kushner, D.J., Ed.), pp. 317-368. Academic Press, London. [3] Brown, A.D. (1983) Halophilic prokaryotes, in Encyclopedia of Plant Physiology (Lange, O.L., Nobel, P.S., Osmond, C.B. and Ziegler, H., Eds.), Vol. 12C, pp. 137-162. Springer-Verlag, Berlin. [4] Foda, I.O. and Vaughn, R.H. (1950) Salt tolerance in the genus Aerobacter. Food Technol. 4, 182-188. [5] ZoBell, C.E. (1946) Marine Microbiology. Chronica Botanica, Waltham, MA. [6] Brown, A.D. and Borowitzka, L.J. (1979) Halotolerance of Dunaliella, in Biochemistry and Physiology of Protozoa


namely the halobacteria and the halococci. Today we have included them as two distinct genera in the family Halobacteriaceae [18]. Viewed with the eyes of the traditional bacteriologist, these two types of bacteria were quite different from each other, and the question was raised at the time why just these two types of bacteria were selected by nature for extreme halophilism. One property they had in common was the very high G + C content of their DNA. Could it be that that had something to do with their extreme halophilism? I guess we tend to smile at this naive argument today, but at that time the G + C concept seemed just as exciting as the discussion on the ribosomal R N A nucleotide catalogues is today. Developments in recent years have also shown that extreme halophilism is not limited to the classical red halophiles. Phototrophic bacteria of the genus Ectothiorhodospira have been firmly established to be extremely halophilic [24]. Furthermore, an actinomycete [25] and some cyanobacteria also fit our definition [2]. In Moscow, at the Institute of Microbiology of the USSR Academy of Sciences, G. Zavarzin and his wife, T. Zhilina, have for some time been studying halophilic methane bacteria, and have very recently reported ([26], personal communication) that they have isolated a methanogen that looks like a methanosarcina with flattened cells, requiting a minimum of 15% salt and having an optimum at 25%: in other words, an extremely halophilic methane bacterium. Accordingly, the discovery of new extremely halophilic microbes is not yet over. On the contrary, exciting new discoveries of extreme halophiles, may be ahead of us now that we are ridding ourselves of the idea that they are only found on salt fish and in the aerobic part of the water column of extremely salty lakes and ponds, and can only be grown heterotrophically on yeast a u t o l y s a t e / p e p t o n e / t r y p t o n e media. B.E. Volcani, when he did his famous work on the microbiology of the Dead Sea more than 40 years ago, deliberately sought different kinds of microbes by using selective enrichment culture techniques. He reported the occurrence in his crude enrichment cultures in Dead Sea water and 25% NaC1 of a variety of microbes of very different

[20] Rodriguez-Valera, F., Ruiz-Berragnero, F. and RamosCormenzana, A. (1980) Isolation of extremely halophilic bacteria able to grow in defined inorganic media with single carbon source. J. Gen. Microbiol. 119, 535-538. [21] Tindall, B.J., Mills, A.M. and Grant, W.D. (1980) An alkalophilic red halophilic bacterium with a low magnesium requirement from a Kenyan soda lake. J. Gen. Microbiol. 116, 257-260. [22] Soliman, G.S.H. and Trfiper, H.G., (1982) Halobacterium pharaonis sp. nov., a new extremely haloalkaliphilie archaebacterium with low magnesium requirement. Zentralbl. Bakteriol. Abt. I Orig. C 3, 318-329. [23] Larsen, H. (1967) Biochemical aspects of extreme halophilism, in Advances in Microbial Physiology (Rose, A.H. and Wilkinson, J.F., Eds.) Vol. 1, pp. 97-132. Academic Press, London. [24] Trfiper, H.G. and Imhoff, J.F. (1981) The Genus Ectothiorhodospira, in The Prokaryotes (Staff, M.P., Stolp, H., Tr~per, H.G., Balows, A. and Schlegel, H.G., Eds.) Vol. I, pp. 274-278. Springer-Verlag, Berlin. [25] Gochnauer, M.B., Leppard, G.G., Komaratat, P., Kates, M., Novitsky, T. and Kushner, D.J. (1975) Isolation and characterization of Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete. Can. J. Microbiol. 21, 1500-1511. [26] Zhilina, T.N. and Zavarzin, G.A. (1985) New methanogenie bacteria. Priroda (Moscow) 7, 103-105 (in Russian). [27] Volcani, B.E. (1944) The microorganisms of the Dead Sea, in Papers Collected to Commemorate the 70tb Anniversary of Dr. Chaim Weizmann, pp. 71-85. Daniel Sieff Research Institute, Rehovot, Palestine. [28] Oren, A. (1981) Approaches to the microbial ecology of the Dead Sea. Kieler Meeresforsch. Sonderh. 5, 416-424. [29] Oren, A. (1986) The ecology and taxonomy of anaerobic halophilic eubacteria. FEMS Microbiol. Rev. 39, 23-29. [30] Cohen, Y. Aizenshtat, Z., Stoler, A. and Jorgensen, B.B. (1980) The microbial geochemistry of Solar Lake, Sinai, in Biogeochemistry of Ancient and Modem Environments (Ralph, J.B., Trudinger, P.A. and Walter, M.R., Eds.) pp. 167-172. Springer-Verlag, Berlin. [31] Kessel, M., Cohen, Y., Walsby, A.E. (1985) Structure and physiology of square-shaped and other halophilic bacteria from the Gavisb Sabka, in Hypersaline Ecosystems (Friedeman, G.M. and Krumbein, W.E., Eds.) pp. 267-287. Springer-Verlag, Berlin.


(Levandowsky, M. and Hutner, S.H., Eds.) Vol. 1, pp. 139-190. Academic Press, New York. [7] Grant, W.D. and Ross, H.N.M. (1986) The ecology and taxonomy of halobacteria. FEMS Microbiol. Rev. 39, 9-15. [8] Baas-Besking, UG.M. (1931) Historical notes on salt and salt-manufacture. Scient. Monthly 32, 434-446. [9] Klebahn, H. (1919) Die Sch'-~idlingedes Klippfisches. Ein Beitrag zur Kennmis der salzliebenden Organismen, in Mitt. Inst. Ailg. Bot. Hamburg. Vol. 4, pp. 11-69. Otto Meissner, Hamburg. [10] Harrison, F.C. and Kennedy, M.E. (1922) The red discolouration of cured codfish. Trans. R. Soc. Canada, Sect. V, 16, 101-152. [11] Gibbons, N.E. (1937) Studies on salt fish, 1. Bacteria associated with the reddening of salt fish. J. Biol. Board Canada 3, 70-76. [12] Gibbons, N.E. (1969)Isolation, growth and requirement of halophilic bacteria, in Methods in Microbiology (Norris, J.R. and Ribbons, D.W., Eds.) Vol. 3B, pp. 169-183. Academic Press, London. [13] Gochnauer, M.B. and Kushner, D.J. (1969) Growth and nutrition of extremely balophilic bacteria. Can. J. Microbiol. 15, 1157-1165. [14] Gibbons, N.E. (1974) Family V. Halobacteriaceae ram. nov., in Bergey's Manual of Determinative Bacteriology (Buchanan, R.E. and Gibbons, N.E., 'Eds.) 8th ed., pp. 269-273. Williams and Wilkins, Baltimore, MD. [15] Brown, H.J. and Gibbons, N.E. (1955) The effect of magnesium, potassium, and iron on the growth and morphology of red halophilic bacteria. Can. J. Microbiol. 1, 486-494. [16] Tomlinson, G.A. and Hochstein, L.I. (1976) Halobacterium saccharovorum sp. nov., a carbohydratemetabolizing, extremely halophilic bacterium. Can. J. Microbiol. 22, 587-591. [17] Tomlinson, G.A., Koch, T.K. and Hochstein, L.I. (1974) The metabolism of carbohydrates by extremely halophilic bacteria: glucose metabolism via a modified EntnerDoudoroff pathway. Can. J. Microbiol. 20; 1085-1091. [18] Larsen, H. (1984) Family V. Halobacteriaeeae Gibbons 1974, in Bergey's Manual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G., Eds.) Vol. 1, pp. 261-267. Williams and Wilkins, Baltimore, MD. [19] Kushner, D.J. (1985) The Halobacteriaceae, in The Bacteria (Woese, C.R. and Wolfe, R.S., Eds.) Vol. VIII, pp. 171-214. Academic Press, New York.