Download The ecology and taxonomy of anaerobic halophilic eubacteria

FEMS Microbiology Reviews 39 (1986) 23-29
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FER 00023
The ecology and taxonomy of anaerobic halophilic eubacteria
(Anaerobic; halophilic; Dead Sea; Halobacteroides; Haloanaerobiaceae)
Aharon Oren
Division of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received 13 March 1986
Accepted 17 March 1986
1. SUMMARY
A number of obligately anaerobic chemoorganotrophic moderately haloEhilic bacteria
have been isolated from the bottom sediments of
the Dead Sea and the Great Salt Lake, Utah: (1)
Halobacteroides halobius, a long motile rod from
the Dead Sea, fermenting sugars to ethanol,
acetate, H 2 and CO2; (2) Clostridium lortetii, a
rod-shaped bacterium from the Dead Sea, producing endospores with attached gas vacuoles; (3) a
spore-forming motile rod-shaped bacterium, fermenting sugars, isolated from the Dead Sea; (4)
Haloanaerobium praevalens, isolated from the
Great Salt Lake, fermenting carbohydrates,
peptides, amino acids and pectin to acetate, propionate, butyrate, H 2 and CO 2.
Analysis of their 16S rRNA shows that these
organisms are related to each other, but unrelated
to any of the other subgroups of the eubacterial
kingdom, to which they belong.
Ha. praeoalens and Hb. halobius regulate their
internal osmotic pressure by the accumulation of
salt (Na +, K +, CI-) rather than by organic osmotic
solutes.
2. INTRODUCTION
Most of our knowledge about halophilic microorganisms and the mechanisms of haloadaptation
is based on studies with aerobic organisms. Aerobic
halophilic bacteria (e.g., the genus Halobacterium),
cyanobacteria, and unicellular green algae (Dunaiiella) are commonly found in hypersaline environments such as salterns, the Great Salt Lake
or the Dead Sea. However, the sediments of hypersaline water bodies are generally anaerobic,
partly as a result of biological activity in the
sediment and the overlying water, and also because of the limited solubility of oxygen in hypersaline brines. The biology of the anaerobic hypersaline environments has been relatively little
studied, even though the first bacteria ever isolated from a hypersaline environment were
anaerobic clostridia, though not halophilic ones,
causing tetanus and gas gangrene, isolated by
Lortet from Dead Sea mud towards the end of the
19th century [1]. In this paper I will review our
knowledge on the nature of those bacteria (especially eubacteria), able to grow in anaerobic hypersaline environments, their ecology, their taxonomic relationships and their mode of haloadaptation.
The archaebacterial genus Halobacterium,
though typically consisting of aerobic bacteria,
contains a number of strains capable of anaerobic
life, using different modes of energy generation in
the absence of oxygen: (1) light can serve as an
energy source for anaerobic growth in bacteriorhodopsin-containing strains [2] (on the condition
0168-6445/86/$03.50 © 1986 Federation of European Microbiological Societies
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that retinal is supplied, as its synthesis is
oxygen-dependent); (2) in certain strains, nitrate
can serve as an alternative electron acceptor in
respiration; (3) at least some strains can use
arginine as energy source for fermentation [2]; (4)
Halobacterium vallismortis has been described as a
facultative anaerobe [3], but its mode of energy
generation in the absence of oxygen has not been
elucidated.
It is unknown whether these modes of anaerobic
life in halobacteria are of ecological importance.
Likewise, we do not know if the moderately
halophilic eubacterium Vibrio costicola, which is a
facultative anaerobe, grows anaerobically in nature.
Obligately anaerobic photosynthetic bacteria of
the genus Ectothiorhodospira are common in al-
i
Fig. 1. Anaerobic halophilic bacteria. (A) Hb. halobius (B) C. lortetii. (C) Strain DY-1 (young cells). (D) Strain DY-1 (sporulating
cell). (E) Ha. praevalens. Phase-contrast micrographs.
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kaline hypersaline lakes of the Wadi Natrun
(Egypt) [4] and Kenya.
3. ANAEROBIC HALOPHILIC BACTERIA
FROM THE BOTTOM SEDIMENTS OF THE
DEAD SEA
Our interest in anaerobic halophilic bacteria
originated during a systematic survey of the biology of the Dead Sea. Until 1979, the northern
basin of the lake was 'permanently' stratified [5],
with an upper aerobic layer 40-80 m deep, separated by a thermocline and pycnocline from the
deeper brines. This lower water mass (down to a
maximal depth of 320 m) was anaerobic, as were
the bottom sediments. Anaerobic halophilic
bacteria were recovered from these sediments as
early as 1943 [6], but unfortunately these early
isolates have not been preserved. In February
1979, an overturn of the lake's water column
caused a complete mixing, and oxygen penetrated
down to the bottom. However, even today the
deeper layers of the bottom sediments remain
anaerobic.
The lower water mass that existed before 1979
contained between 0.23 and 0.56 mg of H2S per 1,
which was relatively enriched with light isotopes
of sulphur, indicating a possible involvement of
biological sulphate reduction in its formation [7].
All our attempts to isolate the organism(s) responsible for this sulphate reduction remained
unsuccessful; instead, these sediments yielded 3
novel obligately anaerobic, moderately halophilic
bacteria.
(1) A very long, slender rod-shaped Gramnegative bacterium (Fig. 1A), motile by means of
peritrichous flagella, described as Haiobacteroides
halobius gen.nov., sp.nov. [8]. The type strain,
ATCC35273, was isolated from an enrichment
culture containing 80% Dead Sea water, pyruvate,
and yeast extract. It grows with doubling times as
short as 55 rain under optimal conditions (41°C,
1.5 M NaC1). The strain requires NaCI concentrations between 1.4 and 2.8 M, tolerates MgC12
concentrations of up to 1.5 M (in addition to 1.5
M NaCI), and grows best at 37-42°C.
(2) A rod-shaped Gram-negative bacterium,
motile by means of peritrichous flagella, and producing terminal endospores with attached gas
vacuoles (Fig. 1B), described as Clostridium lortetii
sp.nov. [9]. The type strain, ATCC35059, was isolated from an enrichment culture containing 80%
Dead Sea water, lactate and yeast extract. It requires NaC1 concentrations between 1 and 2 M
and the optimum growth temperature is 37-45°C.
(3) A rod-shaped Gram-negative bacterium,
motile by means of peritrichous flagella (Fig. 1C),
and sometimes seen to produce terminal endospores, but without gas vacuoles (Fig. 1D). The
strain was designated as DY-1. Minimal doubling
times were as short as 40 min, in media containing
between 0.5 and 2 M NaCI, and at 36-45°C
(Oren, unpublished results).
4. OTHER ANAEROBIC CHEMOORGANOTROPHIC HALOPHILIC ISOLATES
A non-motile, rod-shaped, obligately anaerobic
halophile was isolated by Zeikus and co-workers
from bottom sediments in the south arm of the
Great Salt Lake, Utah (Fig. 1E). The organism
was described as Haloanaerobium praevalens
gen.nov., sp.nov. [10], the type strain being deposited as DSM2228. The bacterium may be identical to 'Bacteroides halosmophilus' isolated by
Baumgartner in 1937 from salted anchovies and
solar salt [11], but which has been lost. Ha.
praevalens is Gram-negative, grows best in NaC1
concentrations between 1 and 4 M, and at temperatures of 25-45°C.
5. DISTRIBUTION AND ECOLOGY OF
ANAEROBIC HALOPHILIC EUBACTERIA
Anaerobic sediments of hypersaline water bodies are often characterised by very high organic
matter contents, thus an abundant development of
anaerobic halophiles may be expected. However,
surprisingly few data are available on the distribution of anaerobic halophilic bacteria and on their
in situ activities.
In anaerobic sediments from the bottom and
shores of the Dead Sea, long, rod-shaped bacteria
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of the type Hb. halobius are quite abundant. By
means of serial dilutions in growth medium we
estimated 103-10 5 viable cells per g sediment [8].
N o quantitative data are available on the abundance of C. lortetii. Its possession of gas vacuoles
may be of interest from an ecological point of
view. Russian scientists, who have isolated similar
(but non-halophilic) clostridia with gas vacuoles
attached to the endospores, have speculated that
the gas vacuoles provide the endospores (which
are oxygen-resistant) with a means of dispersion
by flotation [12]. Whether this theory is relevant in
the case of the Dead Sea bacteria seems doubtful.
In laboratory cultures, vacuolated endospores
never displayed buoyancy; moreover, our strain of
C. lortetii was isolated from sediment at a depth
of 60 m, at which the pressure-sensitive gas vesicles
would have been collapsed.
N o data are available on the abundance of
sporulating anaerobes resembling strain DY-1.
Morphological types like those shown in Fig. 1D
m a y be widespread in anaerobic sediments
throughout the world: similar cells were observed
in anaerobic mud from Australia, containing 21%
NaCI (F.J. Post, Utah State University, Logan,
personal communication).
Ha. praevalens is present in Great Salt Lake
sediments in extremely high numbers: in sediments from the south arm of the lake more than
10 8 cells per ml were counted [10,13]. These sediments demonstrate high rates of microbial decomposition of compounds such as acetate, glucose,
lactate, methanol, methylmercaptan, and methionine (last 3 compounds yielding methane as a
product) [13].
The anaerobic hypersaline brines of the Orca
basin in the Gulf of Mexico, whose NaC1 concentrations exceed 5 M below a depth of 2260 m,
show high bacterial counts (1.44-2.9 × 10 5
cells/ml), a high ATP content, and high rates of
uridine uptake and breakdown and uptake of
acetate [14]. No further characterisation of the
bacteria present has been reported.
6. N U T R I T I O N A N D F E R M E N T A T I O N PATTERNS OF ANAEROBIC
HALOPHILIC
CHEMOORGANOTROPHIC EUBACTERIA
The four isolates of obligately anaerobic
halophilic bacteria described above are all chemoorganotrophs, generating energy by means of
fermentation. Minimal media that support growth
of Hb. halobius and strain DY-1 are simple: in
addition to inorganic salts they should contain a
suitable carbon and energy source (e.g., glucose),
L-leucine, L-cystein (as reducing agent) and vitamins; DY-1 requires biotin, and Hb. halobius requires biotin and p-aminobenzoate [8]. C. lortetii
probably has complex nutritional requirements,
and the minimal requirements of Ha. praevalens
have not been reported.
Hb. halobius and strain DY-1 ferment carbohydrates: both utilise glucose, fructose, sucrose
and starch as carbon and energy source [8]. Hb.
halobius in addition used galactose and pyruvate.
C. lortetii grows in a rich medium containing
L-glutamate, yeast extract, casamino acids and
nutrient broth [9], but how m a n y of these ingredients are actually required remains to be de-
Table 1
Fermentation products of anaerobic halophilic bacteria a
Amounts (mmol) are given relative to acetate ( = 100).
Acetate
Propionate
100
15
36
Isobutyrate
nbutyrate
Isovalerate
Ethanol
Formate
CO2
H2
65
15
180
194
N.D.
60
35
200
136
N.D.
110
Ha. praevalens
no glucose
+ glucose
Hb. halobius
C. lortetii
Strain DY-1
100
100
100
100
38
152
226
6
2
7
7
3
a Data are derived from [8-10] and unpublished results. N.D., not determined.
77
27
termined. The addition of glucose to this medium
stimulates growth, but glucose is udlised only at
the end of the exponential growth phase, probably
after other, more favorable substrates have been
depleted [9]. The Great Salt Lake isolate Ha.
praevalens ferments carbohydrates, peptides,
amino acids and pectin [10].
Fermentation products of the 4 isolates include
acetate, ethanol, hydrogen, CO 2 and others (Table
1).
7. THE TAXONOMIC POSITION OF ANAEROBIC H A L O P H I L I C C H E M O O R G A N O TROPHIC EUBACTERIA
The four anaerobic halophilie bacteria described all stain Gram-negative. The Gram-negative character of their cell envelope has been continued by means of electron microscopy in the
case of Hb. halobius [8]., Ha. praeoalens [10] and
C. lortetii [9]. The Gram-negative character of the
cell envelope of C. lortetii may preclude its classification in the genus Clostridium; also, data on the
structure of the 16S ribosomal RNA do not suggest that C. lortetii is closely related to the nonhalophilic clostridia (see below).
The four anaerobic halophiles share a low percentage of guanine plus cytosine in their DNA:
Ha. praeoalens has 27 mol % [10], strain DY-1 has
29.6 mol % (Oren, unpublished results); Hb.
halobius has 30.7 mol % [8]; and C. lortetii has
31.5 mol % [9].
0.3
0.4
0.5
$AB
Anaerobic halophile DY.1
I
~[~
I
t .
.
.
.
.
.
.
~lostridium Ior(etii
ATCC 35059
Halobacteroides halobius
ATCC 35273
.
Haloanaero ~J~ll~ oraevalens
OSH 2228
Fig. 2. Dendrogram showing the phylogenetic position of 4
anaerobic halophific eubacteria, based on their 16S rRNA
oligonucleotide similarity coefficients (SAB) [15]:
Comparative 16S oligonucleotide cataloguing
[15] showed that Ha. praeoalens and Hb. halobius
obviously belong to the eubacterial kingdom, but
do not show any clear relationship with any of the
recognised subgroups within the eubacteria [16],
except possibly with the spirochaetes [17]. Moreover, the two halophilic anaerobes are related to
each other [16], and a new family, the
Haloanaerobiaceae, has been proposed for these
novel organisms. The isolates C. iortetii and DY-1
also belong to this group (Fig. 2).
8. OSMOREGULATION IN ANAEROBIC
HALOPHILIC C H E M O O R G A N O T R O P H I C
EUBACTERIA
Halophilic andhalotolerant microorganisms can
be divided into two groups with respect to their
mode of osmoregulation. The extremely halophilic
archaebacteria ( Halobacterium, Halococcus ) contain high intracellular salt concentrations (mainly
KC1), roughly iso-osmotic with the external salt
solution. Halophilic unicellular green algae and
halophilic cyanobacteria accumulate different
organic osmotic solutes in their cytoplasm
(glycerol, carbohydrates, betaine), and betaine is
the main osmotic solute in halophilic eubacteria
[18]. Ha. praeoalens and Hb. halobius [19] both
contain high internal Na ÷, K ÷ and C1- concentrations, approximately balancing the osmotic pressure of the cytoplasm with that of the external
medium. No significant concentrations of organic
molecules known as osmotic solutes in other microorganisms such as polyols, betaine and different amino acids could be demonstrated in the
anaerobic halophiles.
The aerobic halophilic archaebacteria (Halobacterium, Halococcus) which balance their intracellular osmotic pressure by accumulating salts,
possess special adaptations enabling their enzymes
to be stable and active in the presence of high salt
concentrations: the proteins have a high content
of acidic amino acids, and are relatively poor in
basic and hydrophobic amino acids. Such proteins
not only function in the presence of high salt
concentrations, but they even require them for
stability and activity. The proteins from the
28
anaerobic halophilic eubacteria Hb. halobius, Ha.
praevalens and C. lortetii also contain an excess of
acidic amino acids, and a low content of basic and
hydrophobic amino acids [19]. Thus, their mode of
haloadaptation resembles that of the extremely
halophilic aerobic archaebacteria, rather than that
of the moderately halophilic aerobic eubacteria.
This paper is based on studies supported by
grants from the Israeli Ministry of Energy and
Infrastructure, the Houston Lighting and Power
Company, Dynatech R / D Company, USPH grant
No. AI 12277, and NSF grant DEB-8107061.
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9. FINAL CONCLUSIONS
It is becoming increasingly clear that even such
extreme environments as the anaerobic sediments
of hypersaline water bodies are inhabited by a
variety of microorganisms. A number of fermentative obligate anaerobes have now been isolated
and characterised. However, not all types of
anaerobic halophiles are known, since biological
sulphate reduction occurs in many anaerobic hypersaline habitats (such as the Dead Sea [7], and
the Solar Lake in Sinai, in which the deeper layers
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concentrations [20]), but no extremely halophilic
sulphate-reducing bacteria have been described.
The most salt-tolerant isolate we know of is a
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The recent discovery of halophilic methanogenic bacteria suggests that even at salinities approaching saturation, complete anaerobic degradation of organic compounds may be possible;
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such environments has not yet been studied thoroughly, and there is no doubt that a wealth of
novel and interesting microorganisms await discovery. Their isolation and characterisation should
complete our understanding of the ecology of
hypersaline water bodies.
ACKNOWLEDGEMENTS
I thank M. Kessel, B.J. Paster, M. Shilo, L.
Vlodavsky, W.R. Weisburg and C.R. Woese, who
all contributed to the data presented in this review.
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