FEMS Microbiology Reviews 39 (1986) 17-22
Published by Elsevier
17
FER 00022
The ecology and taxonomy of aerobic chemoorganotrophic
halophilic eubacteria
(Halophiles; halophilic eubacteria; salterns; Vibrio; Pseudomonas; Bacillus; moderate halophiles)
Francisco Rodriguez-Valera
Department of Microbiology, Faculty of Medicine, Universityof Alicante, Alicante, Spain
Received 13 March 1986
Accepted 17 March 1986
1. SUMMARY
2. INTRODUCTION
There exists a wide diversity of halophilic
eubacteria with chemoorganotrophic-aerobic
metabolism. Most of them have a more moderate
salt response than halophilic archaebacteria, falling into the category of moderately halophilic
bacteria. Although mostly isolated from salted
food, their natural habitats are hypersaline waters
of intermediate levels of salt concentration, and
hypersaline soils. In hypersaline waters, the taxonomic groups found are the ones that also predominate in ocean waters, such as representatives
of the genera Vibrio, Pseudomonas and Flavobacterium. However, in hypersaline soils, the taxonomic groups present are those typical of normal
soils, such as Pseudomonas, Bacillus and Grampositive cocci. The halophilic bacteria from soils
are also more resistant to exposure to low salt
concentrations than the organisms isolated from
waters. Therefore, it seems that the general characteristics of the hypersaline environments drastically affect the types of halophilic bacteria present, and that the halophilic character has arisen
in many phylogenetic groups of eubacteria.
The organisms included in the category described by the title are a heterogeneous group
characterized by growth over a wide range of salt
concentrations. This range is difficult to define
because of its variability depending on growth
conditions (temperature and nutrients) [1]. However, these organisms show, in general, optimum
growth at concentrations between 0.5-2.5 M NaC1
[2]. Hence, they have been usually designated as
moderately halophilic bacteria, while the halophilic
archaebacteria (halobacteria) have higher salt requirements for optimal growth.
For many years, moderately halophilic eubacteria were isolated from the same type of samples
as the halobacteria: salted food, marine salt and
very saline lakes. In a recent review [2] all the
organisms listed as 'moderately halophilic bacteria
under recent or current investigation' had been
isolated from salted food or salt of different
origins. Obviously, the systematic isolation of these
organisms from natural environments is required.
We know that the aerobic halophilic eubacteria
are a diverse group with representatives of most of
the genera that can be found in the ocean, and
they can be isolated in large numbers from hyper-
0168-6445/86/$03.50 © 1986 Federation of European Microbiological Societies
18
saline waters of intermediate salt concentrations
and from hypersaline soils.
3. H A L O P H I L I C E U B A C T E R I A IN A M U L T I POND SALTERN
The multi-pond saltern is a very useful system
for studying hypersaline waters of intermediate
concentration [3,4]. Fig. 1 shows the map of a
saltern near Alicante that we studied. As shown in
the figure, this structure generates a gradient of
salt concentrations in which there are individual
ponds with a range of salt concentrations which is
kept approximately constant. Thus, the microbial
populations that develop can be considered as
being kept under relatively constant salinity conditions. Since the ponds communicate only with
others of very similar concentration, contamina-
N
~
tion with allochthonous microorganisms is insignificant. Fig. 2 shows the numbers of aerobic
halophilic eubacteria and archaebacteria present
at different salt concentrations. The halophilic
eubacteria predominate from 10-25% (w/v) salinity, and just as significant numbers of halobacteria
begin to appear, they decrease, virtually disappearing at over 30% salinity [4,5]. There seems to
be very little overlap between the environments
occupied by the two major groups of halophilic
prokaryotes.
The taxonomic groups of aerobic eubacteria
found in two multi-pond salterns are shown in
Table 1, and their distribution at different salinities in Table 2. First of all, the similarities between
the two salterns are very apparent, in spite of the
fact that one was located on the Mediterranean
coast and the other on the Atlantic coast of Spain.
The most abundant groups were Pseudomonas and
12
Santa Pola
Mediterranean Sea
I
I km
I
Fig. 1. Diagram of a solar saltern located on the Mediterranean coast, 22 km south of Alicante (Spain). The numbers show the total
salts content of the waters of the ponds (% w/v) during sampling carried out in the summer of 1979. The arrows indicate the
direction of the flow of seawater from the Mediterranean Sea [4].
19
positive cocci, although these are present in marine
sediments [7].
The distribution of these taxonomic groups
across the salinity range is not homogeneous [5].
V i b r i o representatives were m u c h more a b u n d a n t
at lower salinities, whilst Gram-positive cocci
seemed to prefer the higher part of the range,
making the proportions of taxonomic groups f o u n d
at the lower salinities even more similar to the
typical seawater population.
1 1
3
H
cells/rnl
2
2.10 = 4
22
2
1
1
1
10
6
20
13.7
3
I
30
20.5
) 2
40
27
1
50 Tota( salts %
3 4 3 Na C1%
Fig. 2. Distribution of halophilic eubacteria (empty bars) and
halophilic archaebaeteria (hatched bars) through the gradient
of salinities in a multi-pond saltern. The values under the bars
represent the salt concentration of the pond; numbers on top
refer to the number of ponds at every concentration sampled.
The decrease of numbers of halobacteria over 45% salts does
not correspond with microscopic examination data that give
even higher numbers in those ponds [3].
related genera, followed b y Vibrio, A c i n e t o b a c t e r
and F l a v o b a c t e r i u m . Gram-positive cocci were also
relatively abundant. Usually, the same groups,
and even the same proportions, are f o u n d in the
ocean [6], with the sole exception of the G r a m -
4. H Y P E R S A L I N E S O I L S
Hypersaline soils are widely distributed around
the world, and they usually support populations of
halophilic plants that can stand extremely high
NaC1 concentrations. Quesada et al. [8] studied
the microflora of a variety of hypersaline soils
with different salinities and plant coverages, the
concentration of NaC1 in the soil moisture ranging
f r o m 5-10.7% ( w / v ) .
In those soils, quite high bacterial counts were
obtained as shown in Fig. 3 (up to a 10 6 cells per g
soil), fairly high counts for any type of soil. The
highest counts were always obtained on media
containing 10% ( w / v ) salts, although the salt concentration of the medium did not markedly affect
the viable counts up to 20% ( w / v ) (media ranging
Table 1
Relative abundance of taxonomic eubacterial groups in the sea and two solar salterns
Sea [6]
(Chesapeake Bay)
Alicante saltern [5]
(Mediterranean)
Huelva saltern a
(Atlantic)
Group
%
Group
%
Group
Vibrio
Pseudomonas
56
Vibrio
Pseudomonas,
A lteromonas,
A lcaligenes,
Flavobacterium
A cinetobacter
Chromobacterium
16
Vibrio
Pseudomonas,
Alteromonas,
A Icaligenes.
Flavobacterium
Acinetobacter
27
Gram + cocci
Unidentified
Gram - rods
3
Flavobacterium
Spirillum
A chromobacter
Hyphomicrobium
Cytophaga
Microcyclas
18
10
6
Gram + cocci
Gram + rods
A ctinomycetes
Enterobacteriaceae
a Marquez, M.C., Ventosa, A. and Ruiz-Berraquero, unpublished data.
54
9
5
1
11
3
0.5
0.5
32
2
6
30
20
Table 2
Taxonomic distribution of 724 heterotrophic halophilic eubacteria isolated from the ponds of a solar saltern [5]
Numbers in brackets are percentages of each group in the salt range considered.
Taxonomic group
No. of strains isolated from ponds with
10-15% Salts
Pseudomonas, Alteromonas,
Alcaligenes
Vibrio
15-25% Salts
66 (42.6%)
52 (33.4%)
12 (7.8%)
12 (7.8%)
4 (2.6%)
3 (1.9%)
0
4 (2.6%)
2 (1.3%)
Gram-positivecocci
Flavobacterium
Acinetobacter
Gram-positiverods
Chromobacterium
Enterobacteriaceae
Actinornycetes
109 (54%)
35 (17.3%)
17 (8.4%)
24(11.9%)
10 (4.9%)
7 (3.5%)
0
0
0
from 0.9-25% salt content were utilized).
The majority of the organisms isolated in this
study grew at 0.9-20% (w/v) salts, and optimally
at 5-10% (w/v), i.e., they were moderately
halophilic. However, they differed from the moderate halophiles isolated from water; these grew
6-
25$ NaCI
saturation
219 (59.7)
26 (7%)
51 (14%)
32 (8.7%)
19 (5.2%)
15 (4.1%)
5 (1.3%)
0
0
Total
number
of
strains
394
113
80
68
33
25
5
4
2
only rarely with only 0.9% (w/v) salts in the
medium. This may be a consequence of the heterogeneity of the soil habitat where the salinity
can obviously change markedly with distance and
time.
The organisms isolated from these hypersaline
soils correspond to taxonomic groups different
from those found in hypersaline waters. Although,
as shown in Table 3, the proportions of taxonomic
groups found in those soils were similar to the
ones found in normal soils, e.g., Vibrio spp. ap-
~6 5 Table 3
(J
Proportions of taxonomic groups of eubacteria found in normal and hypersaline soils [6,8]
Normal soils
g
E3
!
,A B,
,A 8,
,A B,
,A B,
,A 8,
I
2
3
4
5
Fig. 3. Histogram showing the viable bacterial counts of plantfree soil (A) and rhizosphere soil (B) obtained from 5 samples
(1-5) of hypersaline soils containing from 3 to 6.5% CI-.
Counts represent average values obtained on 3 nutrient-rich
media containing 5% salts; values were very similar in the 3
media used.
Bacillus
A rthrobacter
A ctinomycetes
A grobacterium
Pseudomonas
A Icaligenes
Flavobacterium
Corynebacterium
Micrococcus
Staphylococcus
Xanthomonas
Mycobacteriurn
% (Range)
7-67
5 -60
1O- 33
1 - 20
3- 5
2-12
2-10
<5
<5
<5
<5
<5
Hypersaline soils
%
Pseudomonas
Bacillus
A Icaligenes
Micrococcus
A rthrobacter
Planococcus
Staphylococcus
Actinomycetes
Vibrio
Flaoobacterium
Corynebacteriurn
Acinetobacter
Halobacterium
22
19
11
8
6
5
4
3
3
3
2
1
1
Unidentified
12
21
pear in very low numbers, whereas Gram-positive
rods that are virtually absent in hypersaline waters
appeared here in important numbers.
All these facts seem to reflect a separate origin
for halophilic eubacteria from soil and water, since
it appears that halophilic bacteria in water are
marine bacteria adapted to living at higher salt
concentrations, whilst the ones in soils are related
to normal soil bacteria. However, there seems to
be a very general preference amongst all these
organisms to grow optimally at around 10% (w/v)
salt concentration, in spite of their taxonomic and
ecological diversity.
5. HALOPHILIC BACTERIA IN THE OCEAN
If a large enough sample of seawater is concentrated by filtration and then plated onto a
specific medium for halophilic bacteria, many different types of microorganism can be isolated.
Halophilic eubacteria belonging to a variety of
taxonomic groups, halophilic archaebacteria of the
genus Halococcus, and even halophilic Dunaliella,
are present in seawater from geographical sites as
different as the Mediterranean Sea and the Atlantic
Ocean. They are present in extremely low, but still
significant, numbers [9,10,11]. Most halophilic
eubacteria can grow, although sometimes slowly,
at seawater salt concentrations, and Halococcus
spp. can survive for long periods in seawater [12].
However, it is difficult to interpret the presence
of such organisms in the sea from an ecological
perspective, particularly when such organisms are
isolated from geographical sites whose shores lack
extensive hypersaline sites. It is possible that
halophilic eubacteria have limited success competing with marine bacteria, particularly since in the
ocean, growth rates are generally limited by nutrient concentrations rather than by the physiological response to salt. Nevertheless, it is still difficult
to understand how the halophilic character of
these organisms can be preserved over the predictably very long periods in which they would exist
far from hypersaline waters.
Table 4 shows various taxonomic groups of
halophilic eubacteria that were isolated from
Atlantic Ocean water samples. It is remarkable
Table 4
Proportions of taxonomic groups of halophilic eubacteria isolated by enrichment from the sea a
Group
%
A lt eromonas
Alcaligenes
Vibrio
Flavobacterium
Micrococcus
A cinetobacter
Unidentified
31
20
20
16
5
3
5
a Ventosa, A., unpublished data.
that most of the groups that could be found in
hypersaline waters could also be isolated from
those samples. In any case, these data leave little
doubt about the origin of halophilic populations
in hypersaline waters derived from evaporation of
seawater. In an experiment carried out by Ventosa
et al. [10], the concentration of salts of a culture,
inoculated by a concentrated seawater sample,
was increased within a month from seawater concentration to 27% (w/v) salinity. The results
showed what might happen to the composition of
a population of bacteria of the marine habitat
when the salt concentration increases due to
evaporation. Moderate halophiles started to predominate at about 15% salts, when marine bacteria
decreased greatly. Non-halophilic bacteria disappeared much earlier (around 6-10% salts).
6. CONCLUSIONS
The data shown here clearly support the idea
that the halophilic character is widespread among
eubacteria of widely differing phylogenetic origins.
Could this mean that, in terms of evolution, the
distance between soil or marine bacteria and their
halophilic counterparts is relatively short? What
we know about the physiology of marine and
moderately halophilic bacteria suggests that the
difference between them is basically a shift to a
higher salt requirement. Both have salt-dependent
cell envelopes and transport systems [13] and
probably utilize the same compatible solutes [14].
Therefore, it is possible that a relatively low num-
22
ber of mutational changes, perhaps even at the
level of regulatory mechanisms, could transform a
marine Vibrio, for example, to a moderately
halophilic Vibrio. The study of the sequences of
phylogenetic probes such as the 5S rRNA in these
two kinds of organisms could help in answering
this question.
Geologists can easily identify ancient hypersaline water reservoirs by the deposition of
evaporites [15]. The abundance of this type of
rock leaves no doubt that hypersaline environments have been widely distributed during past
and present times, particularly those with intermediate salt concentrations such as are required
for the deposition of gypsum [16]. It is clear that
the ecological niche of the halophilic eubacteria
has been important enough for long and complex
processes of adaptation.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
ACKNOWLEDGEMENTS
The author express his thanks to Dr. A Ventosa
for furnishing unpublished data and for valuable
discussions.
[11]
[12]
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[13]
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New York.
[3] Rodriguez-Valera. F., Ruiz-Berraquero, F. and RamosCormenzana, A. (1981) Characteristics of the heterotrophic bacterial populations in hypersaline environments
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