FEMS Microbiology Reviews 39 (1986) 17-22 17 Published by Elsevier

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, University of 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, fall- ing 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 taxo- nomic groups found are the ones that also pre- dominate in ocean waters, such as representatives of the genera Vibrio, Pseudomonas and Flavobac- terium. However, in hypersaline soils, the taxo- nomic groups present are those typical of normal soils, such as Pseudomonas, Bacillus and Gram- positive 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 char- acteristics of the hypersaline environments drasti- cally affect the types of halophilic bacteria pre- sent, and that the halophilic character has arisen in many phylogenetic groups of eubacteria.

The organisms included in the category de- scribed 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]. How- ever, 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 re- quirements for optimal growth.

For many years, moderately halophilic eubac- teria 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. HALOPHILIC EUBACTERIA IN A MULTI- 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 con- ditions. Since the ponds communicate only with others of very similar concentration, contamina-

tion with allochthonous microorganisms is insig- nificant. 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) salin- ity, and just as significant numbers of halobacteria begin to appear, they decrease, virtually disap- pearing 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 salini- ties 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

N ~ 12

Santa Pola

Mediterranean Sea

I k m I 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].

3 2

H cells/rnl

2.10 = -

4

2 2 2 1

1 3 1 I ) 2 1

1 1

20 13.7

10 30 40 50 Tota( salts % 6 20.5 27 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 by Vibrio, A c i n e t o b a c t e r

and Flavobac t e r ium . Gram-posi t ive cocci were also relatively abundant . Usually, the same groups, and even the same proportions, are found in the ocean [6], with the sole exception of the Gram-

19

positive cocci, a l though these are present in marine sediments [7].

The distribution of these taxonomic groups across the salinity range is not homogeneous [5]. Vibr io representatives were much more abundant at lower salinities, whilst Gram-posit ive cocci seemed to prefer the higher part of the range, making the proport ions of taxonomic groups found at the lower salinities even more similar to the typical seawater population.

4. H Y P E R S A L I N E SOILS

Hypersaline soils are widely distributed around the world, and they usually support populat ions 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 concentrat ion of NaC1 in the soil moisture ranging f rom 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, a l though the salt con- centrat ion 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] Alicante saltern [5] Huelva saltern a (Chesapeake Bay) (Mediterranean) (Atlantic)

Group % Group % Group

Vibrio 56 Vibrio 16 Pseudomonas 18 Pseudomonas,

A lteromonas, 54 A lcaligenes,

Flavobacterium 10 Flavobacterium 9 Spirillum A cinetobacter 5 A chromobacter Chromobacterium 1 Hyphomicrobium 6 Gram + cocci 11 Cytophaga Gram + rods 3

Microcyclas A ctinomycetes 0.5 Enterobacteriaceae 0.5

Vibrio Pseudomonas, Alteromonas, A Icaligenes. Flavobacterium Acinetobacter

Gram + cocci Unidentified Gram - rods

27

32

2 6

3

30

a Marquez, M.C., Ventosa, A. and Ruiz-Berraquero, unpublished data.

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 Total number 10-15% Salts 15-25% Salts 25$ NaCI

saturation of strains

Pseudomonas, Alteromonas, Alcaligenes 66 (42.6%) 109 (54%) 219 (59.7) 394

Vibrio 52 (33.4%) 35 (17.3%) 26 (7%) 113 Gram-positivecocci 12 (7.8%) 17 (8.4%) 51 (14%) 80 Flavobacterium 12 (7.8%) 24(11.9%) 32 (8.7%) 68 Acinetobacter 4 (2.6%) 10 (4.9%) 19 (5.2%) 33 Gram-posit iverods 3 (1.9%) 7 (3.5%) 15 (4.1%) 25 Chromobacterium 0 0 5 (1.3%) 5 Enterobacteriaceae 4 (2.6%) 0 0 4 Actinornycetes 2 (1.3%) 0 0 2

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 mod- erate halophiles isolated from water; these grew

6 -

~6 5 -

(J

g E3

! ,A 8,

2 ,A B, ,A B, ,A B, ,A 8,

I 3 4 5

Fig. 3. Histogram showing the viable bacterial counts of plant- free 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.

only rarely with only 0.9% (w/v) salts in the medium. This may be a consequence of the het- erogeneity 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-

Table 3

Proportions of taxonomic groups mal and hypersaline soils [6,8]

of eubacteria found in nor-

Normal soils % (Range) Hypersaline soils %

Bacillus 7-67 Pseudomonas 22 A rthrobacter 5 -60 Bacillus 19 A ctinomycetes 1 O- 33 A Icaligenes 11 A grobacterium 1- 20 Micrococcus 8 Pseudomonas 3- 5 A rthrobacter 6 A Icaligenes 2-12 Planococcus 5 Flavobacterium 2-10 Staphylococcus 4 Corynebacterium < 5 Actinomycetes 3 Micrococcus < 5 Vibrio 3 Staphylococcus < 5 Flaoobacterium 3 Xanthomonas < 5 Corynebacteriurn 2 Mycobacteriurn < 5 Acinetobacter 1

Halobacterium 1 Unidentified 12

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 con- centrated by filtration and then plated onto a specific medium for halophilic bacteria, many dif- ferent 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 compet- ing with marine bacteria, particularly since in the ocean, growth rates are generally limited by nutri- ent concentrations rather than by the physiologi- cal response to salt. Nevertheless, it is still difficult to understand how the halophilic character of these organisms can be preserved over the predict- ably 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

21

Table 4

Proportions of taxonomic groups of halophilic eubacteria iso- lated by enrichment from the sea a

Group %

A lt eromonas 31 Alcaligenes 20 Vibrio 20 Flavobacterium 16 Micrococcus 5 A cinetobacter 3 Unidentified 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 con- centration 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 pre- dominate at about 15% salts, when marine bacteria decreased greatly. Non-halophilic bacteria disap- peared 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 hyper- saline water reservoirs by the deposition of evaporites [15]. The abundance of this type of rock leaves no doubt that hypersaline environ- ments have been widely distributed during past and present times, particularly those with inter- mediate 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.

ACKNOWLEDGEMENTS

The author express his thanks to Dr. A Ventosa for furnishing unpublished data and for valuable discussions.

REFERENCES

[1] Forsyth, M.P. and Kushner, D.J. (1970) Nutrition and distribution of salt response in populations of moderately halophilic bacteria. Can. J. Microbiol. 16, 253-261.

[2] Kushner, D.J. (1978) Life in high salt and solute con- centrations: halophilic bacteria, in Microbial Life in Ex- treme Environments (Kushner, D.J., Ed.) Academic Press, New York.

[3] Rodriguez-Valera. F., Ruiz-Berraquero, F. and Ramos- Cormenzana, A. (1981) Characteristics of the hetero- trophic bacterial populations in hypersaline environments

of different salt concentrations. Microbial Ecol. 7, 235-243.

[4] Ventosa, A., Quesada, E., Rodriguez-Valera, F., Ruiz- Berraquero, F. and Ramos-Cormenzana, A. (1982) Numerical taxonomy of moderately halophilic gram-nega- tive rods. J. Gen. Microbiol. 128, 1959-1969.

[5] Rodriguez-Valera, F., Ventosa, A., Juez, G. and Imhoff, J.F. (1985) Variation of environmental features and mi- crobial populations with salt concentrations in a multi- pond saltern. Microbial Ecol. 11, 107-115.

[6] Atlas, R.M. (1981) Microbial Ecology, Addison-Wesley, Philippines.

[7] Moriarty, D.J.W. and Hayward, A.C. (1982) Ultrastruc- ture of bacteria and the proportions of Gram-negative bacteria in marine sediments. Microbial Ecol. 8, 1-12.

[8] Quesada, E., Ventosa, A., Rodriguez-Valera, F. and Ramos-Cormenzana, A. (1982) Types and properties of some bacteria isolated from hypersaline soils, J. Appl. Bacteriol. 53, 155-161.

[9] Rodriguez-Valera, F., Ruiz-Berraquero, F. and Ramos- Cormenzana, A. (1979) Isolation of extreme halophiles from seawater. Appl. Environ. Microbiol. 38, 164-165.

[10] Ventosa, A., Rodriguez-Valera, F., Poindexter, J.S. and Reznikoff, W.S. (1984) Selection of moderately halophilic bacteria by gradual salinity increases. Can. J. Microbiol. 30, 1279-1282.

[11] Forsyth, M.P., Shindler, D.B., Gouchnauer, M.B. and Kushner, D.J. (1971) Salt tolerance of intertidal marine bacteria. Can. J. Microbiol. 17, 825-828.

[12] Rodriguez-Valera, F., Ventosa, A., Quesada, E. and Ruiz- Berrequero, F. (1982) Some physiological features of Halococcus at low salt concentrations. FEMS Microbiol. Lett. 15, 249-252.

[13] MacLeod, R.A. (1965) The question of the existence of specific marine bacteria. Bacteriol. Rev. 29, 9-23.

[14] Imhoff, J.F. and Rodriguez-Valera, F. (1984) Betaln is the main compatible solute of halophilic eubacteria. J. Bacteriol. 160, 339-343.

[15] Blatt, M., Middleton, G. and Murray, R. (1980) Origin of sedimentary rocks, 2nd ed., Prentice-Hall, Englewood Cliffs, NJ.

[16] Kirkland, D.W. and Evans, R. (1981) Source-rock poten- tial of evaporitic environment. Am. Assoc. Petrol. Geol. Bull. 62, 181-190.