FEMS Microbiology Reviews 39 (1986) 3-7
Published by Elsevier
3
FER 00020
Halophilic and halotolerant microorganisms-an overview
and historical perspective
(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
Growth
~'~'l
J~
l no~~S,:"~
ght~ eatr ~eX~~
10
20
30 g NaCI
20
30 g NaCI
no
B. Requirement
Growth
10
Fig. 1. Microbes grouped according to response to salt.
0168-6445/86/$03.50 © 1986 Federation of European Microbiological Societies
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
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
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].
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,
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
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.
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