Journal of General Microbiology (1980), 119, 535-538. Printed in Great Britain
535
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Isolation of Extremely Halophilic Bacteria Able to Grow
in Defined Inorganic Media with Single Carbon Sources
By F. R O D R I G U E Z - V A L E R A , l F. R U I Z - B E R R A Q U E R 0 2
A N D A. R A M O S - C O R M E N Z A N A 2 *
1 Departamento de Microbiologia, Centro de Estudios Universitarios, Alicante, Spain
2 Departamento de Microbiologia, Universidad de Granada, Granada, Spain
(Received 15 April 1980)
Pigmented pleomorphic rods morphologically similar to Halobacterium volcanii were
isolated from a salt pond near Alicante, Spain. The salt concentration range for growth
(10 to 35 %, w/v, total salts) was more moderate than that typical of Halobacterium.
Twenty-two strains were characterized as members of the genus Halobacterium, but were
distinguished from currently recognized species by the absence of complex nutrient requirements, a rapid growth rate, nutritional versatility and salt-dependent pigment production.
A typical strain (R-4) has been deposited in the Czechoslovak Collection of Microorganisms,
no. CCM 3361.
INTRODUCTION
The Halobacteriaceae are considered a fairly homogeneous group, some features of which
are their extreme requirements for salts, their pigment production and complex nutritional
requirements (Gibbons, 1974). However, this apparent homogeneity might be a reflection
of the specific methods applied for the isolation of these bacteria (Dundas, 1977). Recent
evidence suggests that the physiology of the genus Halobacterium is more complex than
considered previously. In support of this, the isolation of carbohydrate-utilizing strains
(Tomlinson & Hochstein, 1972) and strains that grow at moderate salt concentrations
(Mullakhanbhai & Larsen, 1975) are noteworthy.
In the present paper, we describe the isolation and properties of an extreme halophile able
to grow on defined inorganic media with a single carbon source, such as glucose or pyruvate.
METHODS
Source of isolates. The micro-organisms described were isolated from seawater evaporation ponds near
Alicante, Spain. Collection strains Halobacterium sulinurium CCM 2148 and H. trupanicum NCMB 767
were supplied by the Spanish Collection of Type Cultures.
Culture media. Media were composed of inorganic salts corresponding in proportions to the artificial
seawater solution of Subov (1931). Keeping these proportions, the concentrations of all salts (except CaCI,
and NaHCO, which were maintained at seawater concentrations) were increased to give the total concentration required (Rodriguez-Valera et al., 1979). The composition of a medium with 20 % salt content was
(%, w/v): NaCl, 15.6; MgC12.6H20,1.3; MgS04.7H20, 2.0; CaC12.6H20,0.1; KCI, 0.4; NaHCO,, 0.02;
NaBr, 0.05. The non-specific enrichment was done in an Erlenmeyer flask with 0.5 I of a medium with 25 %
salts and 0.1 % (w/v) yeast extract (Difco). Pure cultures were isolated with similar medium with 2 % agar
(Difco) and 1 % yeast extract. The specific enrichment was done in the same conditions with a medium (the
minima] medium) of the following composition (%, w/v): salts (as above), 20; NH,CI, 0.2; FeC1,.6H20,
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430005; KH2P04,0.05; glucose, 1.0;500 i.u. penicillin ml-l. The solid medium was of similar composition
without penicillin. For both enrichments, cultures were incubated at 38 "C with agitation by a magnetic
stirrer. Plates were incubated in sealed plastic bags at the same temperature. The pH of all media was
adjusted to 7.0with 1 M-KOH and the incubation temperature was always 38 "C.
Morphological study. Microscopic preparations were made using the technique of Dussault (1955).In vivo
observations were made in a salt solution of identical composition and concentration to that of the culture
medium, using a phase contrast Zeiss microscope.
Salt concentration range for growth. Growth was determined on solid medium having 2, 5, 10, 15, 20,25,
30 or 35 % total salts, supplemented with 1 % yeast extract (Difco), 0.5 % Casamino acids (Difco) and 0.1 %
glucose. The same medium with 20 % salts was used for the maintenance of the isolates. In liquid medium,
the growth rate was estimated by inoculating 0.1 ml of a suspension of the strain, pre-grown to visible
turbidity in 25 % salt medium (composition as above), into a spectrophotometer tube containing 3 ml
medium at the specific salt concentration. The tubes were incubated in a horizontal position with orbital
agitation (100 strokes min-l), and the absorbance at 520 nm was measured at regular intervals.
Quantitative determination of pigments. The pigments were extracted as described by Gochnauer et al.
(1972). Cells were grown in medium with 15, 20 or 25 % salts and 1 % Difco yeast extract (100 ml medium
in 500 ml Erlenmeyer flasks), incubated at 38 "C and agitated by a magnetic stirrer. When an absorbance of
about 0-7was reached, cells were harvested by centrifugation, the pigments were extracted and their spectra
were determined in a Perkin Elmer UV-visible dual beam spectrophotometer. The protein content of the
suspension was determined after Lowry.
Carbon substrate utilization. The ability to grow with a single organic compound as sole source of carbon
and energy was determined in the minimal medium by replacing glucose with the test substrate. The buffered
medium contained 0.1 M-2-N-morpholinoethanesulphonicacid (MES) and the pH was adjusted with
4 M-KOH.In liquid medium, growth was studied in 500 ml Erlenmeyer flasks with 100 ml medium agitated
by a magnetic stirrer. Residual glucose, in culture filtrates, was determined colorirnetrically with o-toluidine.
Biochemical tests. Oxidase production was determined after Gaby & Hadley (1 957). Starch hydrolysis
was examined by the method of Gibbons (1957).For the nitrate reduction test we used an Eimhjellen (1965)
medium supplemented with 0.1 % KNO, and the method of Cowan & Steel (1974). Gelatinolytic and
caseinolytic activities, and the hydrolysis of Tween 40, were determined with the modified medium of
Norberg-Hofsten (Gutierrez & Gonzalez, 1972). Indole and H,S production were determined using a
medium containing 20 % salts, 1 % yeast extract (Difco) and 1 % tryptose (Merck), with a strip of lead
acetate paper or paper impregnated with Kovacs' reagent inserted in the mouth of the tubes. Catalase was
detected by flooding the growth on plates with 3 % H z 0 2 .
RESULTS
The first evidence of the existence in salt pond samples of extremely halophilic bacteria
not requiring growth factors came from a general experiment in which colonies showing a
mixed population appeared in the non-specific enrichment with 25 % saltslyeast extract.
Two strains able to grow in the minimal medium were isolated from these plates. Subsequently, we used minimal medium to which penicillin had been added to prevent the growth
of moderately halophilic bacteria (specific enrichment, see Methods). After inoculating this
medium with samples from seawater evaporation ponds, slightly pink growth appeared
during the second or third day of incubation. When this enrichment culture was plated on
to minimal medium the colonies were of very uniform appearance, similar to the selected
colonies originally obtained from the non-specific enrichment culture. Twenty pure cultures
were isolated from the specific enrichment culture and were characterized together with the
first two selected strains. The results were similar for all isolates so only one (R-4), isolated
from the specific enrichment culture, is described further.
Morphological features. Morphologically, the isolate appeared similar to Halobacterium
volcanii (Mullakhanbhai & Larsen, 1975). The Gram-negative cells were very pleomorphic,
spherical, rectangular or disc-shaped (Fig. 1a) and tended to swell and form spheres at low
salt concentration. Reproduction was accomplished by means of a central constriction that
finally ended in a thin bridge between the daughter cells. In old cultures, cells contained gas
vesicles (Fig. 1b). No motility was observed. The colonies, on minimal medium, were opaque
and bright pink, and were about 2 mm diam. after 4 d incubation (colonies of extreme
halophiles classically described are much smaller).
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537
Fig. 1. Photomicrographs, prepared according to the technique of Dussault (1955), of Halobacterium
sp. strain R-4 from a 20 % salts complex liquid medium; (a) exponentially growing cells; (b) old
culture.
Salt concentration range for growth. In solid medium the isolate grew in the presence of
10 to 35 76 total salts (7.6 to 27 % NaCl). The growth rates in liquid medium were high at
all concentrations between 10 to 35 %, the optimum being at 20 % total salts (15.6 % NaCl);
the growth rate at this salt concentration was 0.12 h-l.
Pigment production. The intensity of the pigmentation was strongly influenced by the salt
concentration of the medium, the colonies being almost colourless at 25 % salts while at
15 % they were strongly pigmented. To examine whether this variation was due to a change
in the total amounts of carotenoids produced, the latter were extracted from cells grown
with 15, 20 or 25 % salts. There was a dramatic difference in absorption at the three carotenoid peaks, being about 20 times more with 15 % than with 25 % total salts (based on the
same amount of cell protein). The change of pigmentation was peculiar to these strains;
other Halobacterium strains isolated from the same habitat, as well as the collection strains
H. salinarium CCM 2148 and H. trapanicum NCMB 767, did not show this effect.
Carbon substrate utilization. On solid medium, all 22 strains isolated grew well and fast
(visible growth in 3 to 4 d) on glucose, sucrose, fructose, pyruvate, citrate or glutamic acid,
more slowly on mannitol, lactose, galactose or glycerol, and very slowly and poorly on
acetate. In liquid MES-buffered medium with 1 % glucose as substrate, the growth rate was
less than with 1 % yeast extract plus glucose. In both cases the glucose concentration decreased to about 0.1 % when the stationary phase was reached. In non-buffered liquid
medium the growth was less extensive but the growth rate decreased very little; in the nonbuffered medium with glucose the pH had decreased from 7 to 5-8 after 24 h incubation
at 38 "C.
Biochemical tests. All isolates were oxidase and catalase positive ; Tween 40, starch, casein
and gelatin were all hydrolysed; nitrate was reduced to nitrite; and all produced indole but
not H,S.
DISCUSSION
The features of the micro-organisms described here suggest that the type is quite
distinct from the currently recognized species of Halobacterium (Gibbons, 1974). The salt
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concentration range for growth, morphology and pigmentation are typical for the genus
Halobacterium. Our strains are similar in many respects to some newly isolated Halobacterium spp. such as H . saccharovorum Tomlinson & Hochstein 1972and H . volcanii. Halobacterium saccharovorum is able to utilize carbohydrates (in yeast extract or peptone supplemented medium) and H. volcanii has a moderate (1 5 % NaCI) salt optimum for growth and
shows the same highly pleomorphic shape and lack of motility. The main distinctive
characteristics of our isolates are the absence of complex nutrient requirements, their rapid
growth rates, nutritional versatilityand salt-dependent pigment production. These properties
offer significant advantages for studying the genetics of extremely halophilic bacteria, an important omission to date in our knowledge of these micro-organisms. Our isolates, together
with the new species cited above, provide new data concerning the genus Halobacterium,
whose taxonomy now merits major revision. A typical strain (R-4) has been deposited in the
Czechoslovak Collection of Microorganisms, no. CCM 3361.
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