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Abstract: The area of the Dead Sea, bordering Jordan and Israel, contains many
springs with different physical and chemical properties. Hot springs are found on
the eastern shore of the lake: the springs of Zara (the ancient Callirrhoe) with tem-
peratures up to 59 °C and those of Zerka Ma’in, 5 km inland. The latter springs,
mentioned by Josephus (1st century C.E.), yield fresh water with a low sulfide
content, near-neutral pH and temperatures up to 63 °C. Although the growth of
green microorganisms was already noted in 1807 by the German explorer Ulrich
Jasper Seetzen, the microflora of the springs has remained unexplored. In 2005
we began a series of surveys characterizing the microbial diversity of the springs.
The sources of the Zerka Ma’in springs and their outflow channels are covered by
green to orange mats, containing a diverse community of unicellular and filamen-
tous cyanobacteria. Conspicuous types include Thermosynechococcus, unicellular
Gloeocapsa types, and Spirulina-like filaments. Large colonies of Scytonema were
also found. Several representative types of Thermosynechococcus, Gloeocapsa,
and Mastigocladus/Fischerella were isolated and cultured. Amplification of cyano-
bacterial 16S rRNA genes from DNA extracted from the mats showed a high di-
versity of Thermosynechococcus in the springs. Low concentrations of ammonium
and nitrate in the spring water and the presence of heterocystous cyanobacteria
indicated that biological nitrogen fixation may be important in the spring ecosys-
* corresponding author
tem. nifH genes related to those of Fischerella, Phormidium sp., and Lyngbya sp.
were amplified from the community DNA. Laboratory experiments with hetero-
cystous isolates showed the occurrence of nitrogen fixation (acetylene reduction)
at 52 °C but not at 63 °C. However, the possibility that nitrogen is fixed in situ at
63 °C cannot be excluded as reverse transcription PCR with mRNA isolated from
the site yielded an amplified gene with 100 % homology to the nifH gene found in
a filamentous cyanobacterial culture obtained from the site, and low but significant
rates of acetylene reduction were found during in situ incubations.
Key words: cyanobacteria, 16S rRNA gene sequences, hot springs, nitrogenase,
thermophilic
Introduction
The Dead Sea Rift, within the Syrian-African Rift Valley, is rich in thermal
springs. Some are freshwater springs, others are saline or hypersaline. Ex-
amples are Hamat Gader (up to 52 °C) and the hot springs of Tiberias (up
to 60 °C). The area of the Dead Sea, on the border between Jordan and
Israel, contains many springs which differ in their physical and chemical
properties. The eastern bank of the Dead Sea (Jordan) is especially rich in
thermal springs. These include the springs of Zara on the Dead Sea shore
(the ancient Callirrhoe; Donner 1963) with temperatures up to 59 °C, and
the hot springs of Zerka Ma’in, 5 km inland, up to 63 °C (Abu Ajamieh
Fig. 1. Map showing the location of the Zerka Ma’in and the Zara hot springs east
of the Dead Sea and sampling sites A1, A2, B1, and B2.
The cyanobacterial community of hot springs 111
Table 1. Chemical and physical characteristics of water samples collected at the dif-
ferent sampling sites of the Zerka Ma’in springs, as analysed at the Department of
Earth and Environmental Sciences, Yarmouk University.
Sampling site
A1 A2 B1 B2
Temperature (°C) 63 62 58 58
pH 6.69 6.62 6.38 6.81
H2S (mM) 0.3 0.3 0.03 NA
Conductivity (µS cm–1) 2140 4180 2460 2470
Total dissolved salts (ppm) 1306 1267 1428 1445
Alkalinity (ppm) 120 110 130 130
Hardness (ppm) 560 520 540 580
[Cl–] (ppm) 860.1 810.4 900.9 790.6
[NO3–] (ppm) 0 1.02 0.72 0
[SO42–] (ppm) 197 196 210 211
[HCO3–] (ppm) 2391 ND ND ND
[Mg2+] (ppm) 90 91 93 92
[Ca2+] (ppm) 185 186 193 195
[K+] (ppm) 33 35 37 38
[Na+] (ppm) 86.3 85.6 78.5 78.2
[Sr2+] (ppm) 3.31 ND ND ND
1Asdetermined by I. Gavrieli, The Geological Survey of Israel.
ND = not determined.
1 Pomati et al. 2004. 2 F. Goh, University of New South Wales, personal communi-
cation. 3 Nübel et al. 1997. 4 Mazard et al. 2004.
The cyanobacterial community of hot springs 113
cDNA was further amplified using the nifHF and the nifHR primers. The
same primer was used to screen our cultures for the nifH gene.
Nitrogen fixation activity was assessed using the acetylene reduction as-
say (Stewart et al. 1967). Subcultures of 32 mL or 11 mL in case of in situ
experiments were placed in rubber-stopped glass bottles, 72 mL and 25 mL
respectively. Acetylene (8 mL and 2 mL respectively) was injected into the
head space. Control experiments included systems without acetylene and
sterile medium with acetylene. To preserve the ethylene/acetylene ratio ob-
tained immediately after the in situ incubation was terminated, 2.5 mL of
gas from the head-space were transferred to 4 mL vacuum tubes (Vacuette,
Greiner Bio-One) containing saturated NaCl solution. Acetylene reduction
was assessed by injecting 1 mL of gas taken from the head space into a gas
chromatograph equipped with a flame ionization detector (FID-GC SRI
310). To evaluate various factors which affect the N2 fixation abilities of our
isolates, we tested activity in: 1) cultures which were initially grown in N-
free BG-11 medium (BG-110), transferred into fresh BG-110 medium and
incubated at 31, 45, 53 and 63 ºC under constant light for 24 h; 2) cultures
initially grown in BG-11 medium, transferred to BG-110 two days prior to
acetylene reduction assay, and incubated at 45 ºC under constant light and
under a 12 h dark/light regime for 48 h; 3) cultures initially grown in BG-
11 medium, washed with and transferred to BG-110 immediately prior to
acetylene reduction assay initiation and incubated at 48 ºC under a 12 h
dark/light regime for 100 h. Cyanobacterial mats were collected from site
A1 and from two nearby streams running at a temperature of 51 ºC and
39 ºC. Duplicate samples were incubated in situ at their respective sampling
sites under various conditions: 1) in pond/stream water under constant
light; 2) dark incubation in bottles covered with aluminum foil; 3) in BG-
110 medium under constant light. Incubation time ranged between 1:45–
2:45 hours.
The Bunsen gas solubility coefficient for ethylene was calculated for
each of the various incubation temperatures according to Breithbart et
al. (2004). N2 fixation rates were calculated according to Capone (1993); a
ratio of 4:1 was used to convert reduced acetylene to nitrogen fixed accord-
ing to Staal et al. (2001) and Jensen & Cox (1983). Chlorophyll was ex-
tracted either by 90 % methanol at 80 ºC (experiments 1 and 2) or by 90 %
acetone (experiment 3).
Results
The microbial mats at sites A1 and A2 (Fig. 1) were dark-green in colour.
At sites B1 and B2 and along the channel connecting them, the dominant
colour was orange, with prominent patches of green material being present
as well. Microscopic examination shows a highly diverse community of cy-
114 D. Ionescu et al.
A B
C D
E F
G H
Fig. 2. Cyanobacteria observed in the Zerka Ma’in springs (a–d) and strains cul-
tured from the samples (e–h). A – unicellular cyanobacteria from site A1, B – thin
filamentous organisms from site A2, C – spirally wound filaments from site B, D
– unicellular, Gloeocapsa-like cells from site B, E – filamentous organisms cultured
from site B1, F – Fischerella/Mastigocladus-like filaments, G – cultured Gloeo-
capsa-like cells, H – heterocystous filaments. Scale bars = 10 µm.
The cyanobacterial community of hot springs 115
Fig. 3. Phylogenetic tree showing cyanobacterial 16S rRNA gene sequences ampli-
fied from the Zerka Ma’in springs and from enrichment cultures obtained from
these springs. The sequences were aligned using the ClustalW algorithm and were
plotted as a tree using the Neighbour Joining algorithm. Escherichia coli K12 was
The cyanobacterial community of hot springs 117
Fig. 4. Phylogenetic tree showing nifH gene sequences obtained from enrichment
cultures from the Zerka Ma’in springs, as well as from cDNA collected from site
A1 at 63 °C. The sequences were aligned using the ClustalW algorithm and were
plotted as a tree using the Neighbour Joining algorithm. Methanothermobacter
thermautotrophicus was used as outgroup. The enrichment cultures are marked as
temporary Bridging the Rift Culture Collection number (tBTRCCn), and Gen-
Bank accession numbers are given after the strain designations. Sequences derived
from environmental mRNA amplification are labeled as clones.
used as outgroup. The enrichment cultures are marked as temporary Bridging the
Rift Culture Collection number (tBTRCCn). Selected GenBank accession num-
bers are given after the strain designations. Environmental sequences are labeled
as clones.
118 D. Ionescu et al.
60μm
Fig. 5. Morphological changes upon transfer of isolate tBTRCCn 101 from high-
nitrate medium to nitrogen starvation and vice-versa. A – Thick, branched fila-
ments in nitrate rich culture, B – Branching of thin filaments 24 h after transfer to
BG-110 medium at 48 ºC, C – Branching of thin filaments 24 h after transfer to BG-
110 medium at 53 ºC, D – Culture with mixed types of filaments 72 h after transfer
to BG-110 medium at 48 ºC, E – Culture with mixed types of filaments 48 h after
transfer back to nitrate rich medium. Scale bars = 10 µm.
Discussion
Unicellular cyanobacteria generally dominate hot spring communities at
temperatures approaching the upper temperature limit for photosynthesis
of 74 ºC (Castenholz 1969a, Brock 1978). Site 1A, which has the high-
est temperature of the Zerka Ma’in thermal springs area, shows a fairly
diverse community of organisms belonging to the Thermosynechococ-
cus clade, as observed microscopically (Fig. 2a) as well as by phylogenetic
analyses of samples (Fig. 3). Hot springs at lower temperatures are gen-
erally dominated by filamentous cyanobacteria (Copeland 1936, Casten-
holz 1969a, 1984, Brock 1978), and the same is true for site B of the Zerka
Ma’in springs (Fig. 2c, 2d). These include yet uncultured spiral cyanobacte-
ria (Fig. 2d). Similar tightly coiled filaments were previously reported from
Mammoth Hot Springs, Yellowstone (Spirulina cf. labyrinthiformis), where
they occurred at an upper temperature limit of 51–52 ºC (Castenholz
1977, Weller et al. 1992). Phylogenetic analyses of environmental samples
thus far failed to reveal any Spirulina-like 16S rRNA gene sequences; how-
ever, some of our clones clustered with Limnothrix, a genus that includes
a spiral organism (Limnothrix chlorospira), but does not have any known
thermophilic species. The nature of the spiral organism in the Zerka Ma’in
springs remains to be ascertained.
In previous descriptions of hyperthermal springs, including the Zara
springs in Jordan (Deranieh 1990, Weller et al. 1992), orange mats were
often reported to contain green anoxygenic photosynthetic, Chloroflexus-
like organisms. We detected the fatty acids 15:0 and 17:0 in the orange mate-
rial, found also in Chloroflexus mats at Yellowstone (Zeng et al. 1992). How-
ever, a microscopic examination of orange mats in the Zerka Ma’in springs
showed prominent presence of a unicellular Gloeocapsa-like cyanobacte-
rium (Fig. 2d), with carotenoids probably contributing most to the colour
of the mats. These unicellular organisms have been obtained in culture, and
their 16S rRNA gene sequence shows up to 99 % similarity with the recently
isolated Chroogloeocystis siderophila. This organism was originally found
in iron-rich thermal environments in Yellowstone, and requires as much as
30 µM iron for optimal growth (Brown et al. 2005). Chemical analyses of
the Zerka Ma’in spring waters showed far lower iron concentrations, rang-
ing from 0.4 µM (Khoury et al. 1984) to 2.2–3.6 µM (Rimawi & Salameh
1988). At this stage there is no evidence as to the use of siderophores and no
measurements of internal iron concentration have been done.
The occurrence of heterocysts in thermophilic filamentous cyanobacteria
has been previously documented in Fischerella/Mastigocladus spp.
(Nierzwicki-Bauer et al. 1984, Stevens et al. 1985). The isolation of
heterocystous Fischerella/Mastigocladus-like filamentous cyanobacteria
from the Zerka Ma’in spring (Fig. 2f, 2h, 5) suggests that nitrogen fixation
may occur in these springs. Although recent studies show nitrogen fixation
120 D. Ionescu et al.
1969b, 1972). These two types could also be distinguished by the nature of
their branching. A third type (EHN), which incorporates morphological
and physiological traits found in the first two types, was described by
Melick et al. (1991). The latter undergoes morphological changes similar
to the Mastigocladus-like cyanobacteria from Zerka Ma’in; however,
the trigger for these changes is different. Young cultures of EHN strain
are characterized by thin, non-branching filaments, while older cultures
resemble the conventional Mastigocladus morphology. The EHN strain
is incapable of forming heterocysts and fixing nitrogen, and nitrogen
starvation does not induce a morphology change (Melick et al. 1991). The
Zerka Ma’in organism appears to represent a fourth type, and therefore a
more detailed characterization is required.
In conclusion, the diverse cyanobacterial community of Zerka Ma’in
consists of organisms similar to those previously described in such springs,
as well as novel types. Our findings suggest a higher upper temperature
boundary (63 ºC) for cyanobacterial nitrogen fixation than previously
published (55 ºC). The physiology and ecology of nitrogen fixation in
these springs, including phenomena of light inhibition and oxygen toxicity,
remain to be further explored.
Acknowledgements
We thank the Bridging the Rift Foundation for financial support. O.L. was financially
supported by an Israeli Ministry of Sciences Fellowship for Women in Science.
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Danny Ionescu
Prof. Aharon Oren
The Hebrew University of Jerusalem
Department of Plant and Environmental Sciences
Institute of Life Sciences, and
The Moshe Shilo Minerva Center for Marine Biogeochemistry
IL-91904 Jerusalem, Israel
E-mail: orena@cc.huji.ac.il
ionescu@pob.huji.ac.il
Orly Levitan
Dr. Ilana Berman-Frank
Bar-Ilan University
The Mina and Everard Goodman Faculty of Life Sciences
IL-52900 Ramat Gan, Israel
E-mail: orlylevitan@biu.013.net.il
irfrank@mail.biu.ac.il