BioRisk 3: 69-82 (2009) owt Me LP ae ae
doi: 10.3897/biorisk.3.29 RESEARCH ARTICLE BioRisk

www.pensoftonline.net/biorisk Biodiversity & Ecosystem Risk Assessment

Morphological, phylogenetic and
physiological diversity of cyanobacteria
in the hot springs of Zerka Ma’in, Jordan

Aharon Oren'’, Danny Ionescu'??, Muna Y. Hindiyeh**, Hanan I. Malkawi*?

| The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel2. The Bridging the Rift Foundation
3 The Ruppin Academic Center, Emek-Hefer, Israel; current address: Max Planck Institute for Marine Micro-
biology, Bremen, Germany 4 The German Jordanian University, Amman, Jordan § Department of Biological
Sciences, Yarmouk University, Irbid, Jordan

Corresponding author: Aharon Oren (orena@cc.huji.ac.il)

Academic editors: L./. Musselman, FE Krupp | Received 9 April 2009 | Accepted 22 November 2009 | Published 28 December 2009

Citation: Oren A, Ionescu D, Hindiyeh MY, Malkawi HI (2009) Morphological, phylogenetic and physiological diver-
sity of cyanobacteria in the hot springs of Zerka Main, Jordan. In: Krupp F, Musselman LJ, Kotb MMA, Weidig I (Eds)
Environment, Biodiversity and Conservation in the Middle East. Proceedings of the First Middle Eastern Biodiversity
Congress, Aqaba, Jordan, 20—23 October 2008. BioRisk 3: 69-82. doi: 10.3897/biorisk.3.29

Abstract

The freshwater thermal springs of Zerka Main, located in Jordan in the mountains of Moab east of the
Dead Sea, are densely inhabited by cyanobacteria up to the highest temperature of 63 °C. We have inves-
tigated the cyanobacterial diversity of these springs and their outflow channels by microscopic examina-
tion, culture-dependent and culture-independent phylogenetic analysis, and by physiological studies of
selected isolates of special interest. Both unicellular and filamentous types of cyanobacteria are present,
and we identified morphological types such as Thermosynechococcus, Chroogloeocystis, Fischerella (Mastigo-
cladus), Scytonema (occurring as large masses at lower temperatures), and others. Although morphologi-
cally similar cyanobacteria have been identified in hot springs world-wide, the Zerka Ma’in strains were
phylogenetically distinct based on 16S rRNA gene sequence analysis. Considerable diversity was detected
also in the gene sequences of nifH (nitrogenase reductase), encoding one of the key enzymes involved in
nitrogen fixation. Nitrogen fixation in a Mastigocladus isolate obtained from the springs was investigated
in further depth. The heterocystous strain could fix nitrogen (as assayed by acetylene reduction) at tem-

peratures up to 53°C.

Keywords
Cyanobacteria, Jordan, Zerka Main, thermophilic cyanobacteria, biodiversity, 16S rRNA phylogeny, ni-

trogen fixation

Copyright A. Oren. et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

70 Aharon Oren et al. / BioRisk 3: 69-82 (2009)

Introduction

The hot springs (up to 63 °C) of Zerka Main, located in Jordan in the mountains of
Moab near the north-eastern end of the Dead Sea (Fig. 1) were first mentioned by the
Jewish-Roman historiographer Flavius Josephus (37 to c. 100 C.E.): “In the ravine which
encloses the town [Herod’s fortress Machaerus; the present day ruins of Makaur] on the
north, there is a place called Baaras. ... In this same region flow hot springs, in taste wide-
ly differing from each other, some being bitter, while others have no lack of sweetness.”

The first explorer to reach the Zerka Main hot springs was the German Ulrich
Jasper Seetzen (1767-1811), who visited the site just over 200 years ago. In the report
of his visit to the site in 1807 he mentioned the presence of green slimy material that
consists of microscopic algae: “These springs are about two hours distant from the
Dead Sea, to which the track from here appears to be very difficult. In the water grew a
green slimy small alga” (Seetzen 1854; translation A.O.). It is curious that none of the
later explorers who visited the site in the course of the 19" century and the first years
of the 20" century mentioned the so prominent green growth in the waters of the hot
springs (Figs 2A-C). However, a few observations on the microbial mats in the springs
were published by the German geologist Max Blanckenhorn, who surveyed the area
in 1908: “An algologist could find here, as well as in the other hot sulfur springs of
Palestine, a wonderful area for observations and collection. ... There where the water
was particularly hot, blue-green Cyanophyceae appeared to dominate. For the rest,
the whole bottom of the stream and the rocks present in it are covered by green mats.
These felt-like mats are often small in the form of a sponge or pillow with a dark-green,
somewhat wrinkled skin ...” (Blanckenhorn 1912; translation A.O.).

The cyanobacterial flora of the nearby hot springs of the Zara area near the shore
of the Dead Sea, the site of the ancient Kallirrhoé (Donner 1963), was surveyed in
1936 (Frémy and Rayss 1938; Rayss 1944). However, no studies of the cyanobacteria
of Zerka Main have been conducted until 2005. The peace treaty between Jordan and

SS
Zerka Ma'in spring area

~
a

ba

aw
" \; \ A=

Zara spring area |)

i Tyke

1
fee Ss

Figure I. Satellite image of the Dead Sea and the location of the Zerka Main spring area.

Morphological, phylogenetic and physiological diversity of cyanobacteria in the hot springs... 71

Israel of 1994, and the establishment of the Bridging the Rift Foundation in the year
2000, promoting peace in the Middle East through science, enabled us to perform the
first biological surveys to characterize the highly interesting microbial communities of
Zerka Main and to discover some of its many interesting and sometimes unique fea-
tures. Sponsored by the Bridging the Rift Foundation, our team of Jordanian and Israeli
scientists has made a number of sampling trips to the site in 2005 to 2007 (Ionescu et al.
2007, 2009; Oren et al. 2008). We here present some of the results of our cyanobacterial
diversity studies of the Zerka Ma’in hot springs, based both on microscopical characteri-
zation of the organisms present and on molecular, 16S rRNA gene-based techniques.

Materials and methods
Sample collection, cyanobacterial cultivation and identification

Cyanobacterial mats were collected from the Zerka Main springs on December 14,
2005, November 16, 2006 and July 3, 2007. Samples were transferred to glass vials

£ ria gi ed 5s ai

= ar B ~
9, = *
ss rs

Figure 2. Views of the thermal spring area of Zerka Main. Panel A shows one of the springs in Area A

indicated in Fig. 1, Panel D provides an overview of the waterfalls with hot water of about 58 °C to 60 °C
in Area B.

FAD. Aharon Oren et al. / BioRisk 3: 69-82 (2009)

and tubes and used for microscopic identification of the organisms present, isolation
of cyanobacterial strains, and molecular studies. Most experimental work referred to
below was done based on the July 2007 samples. Site A (Figs 1, 2A) consists of two
pools, the upper one flowing into a lower one through a pipe. The temperature of the
upper one was 63 °C during all sampling trips. The lower pool ranged between 62 °C
and 63 °C. The upper pool is shallow (ca 50 cm) and surrounded by rock walls. Green
mats were found on the rocks, at and slightly above the water air interface. Submerged
rocks are covered by mats as well. The lower pool is deeper, larger and surrounded
by vegetation. Site B (Fig. 1B) is located ca 500 m west of site A. Green mats are
found on the channels’ earth banks and on submerged rocks. An orange mat is often
found beneath the green mat. Site C (Fig. 1C) is a stream located 25 m above site
A. The main stream is a combination of two smaller ones at a temperature of 25 °C
and 51 °C. Over a distance of 25 m from the confluence point the temperature of
the stream reaches 39 °C. The water from the springs at site B flows through a 50 m
channel that ends in a waterfall (Fig. 1D). The temperature of the water was 59 °C
along the entire channel.

Samples were examined and photographed in a Zeiss Axiovert 135 TV micro-
scope equipped with phase-contrast optics. Morphological types were identified to
the genus level on the basis of the identification systems proposed by Geitler (1932)
and the “form-genus” approach of Castenholz (2001). When relevant, names in com-
mon use but without standing in the botanical and bacteriological nomenclature
were used as well (e.g. Zhermosynechococcus). We have isolated representative types
of filamentous and unicellular cyanobacteria by enrichment and direct isolation on
agar plates of growth medium BG-11, using incubation temperatures of 45 °C and
55 °C. Cultures are maintained on agar slides in the laboratory of A.O.; the filamen-
tous Mastigocladus-like strain nBTRCC 101 has been submitted for deposition in the
UTEX - The culture collection of algae at the University of Texas at Austin (tempo-
rary accession number: ZZ867).

Sequencing and analysis of cyanobacterial 16S rRNA genes

For molecular 16S rRNA-sequence based analysis, samples were placed in 15 ml ster-
ile tubes containing 2 ml of lysis buffer (100 mM Tris-HCI, 50 mM EDTA, 10 mM
NaCl, 1% SDS, pH 8), followed by extraction with phenol-chloroform-isoamy] al-
cohol (25:24:1). After washing the extracts with chloroform-isoamyl alcohol (24:1),
DNA was precipitated with cold ethanol, washed with ice-cold 70% ethanol, and
resuspended in water. Fragments of the 16S rRNA gene were amplified by PCR, us-
ing cyanobacteria-specific primers as specified in Ionescu et al. (2009). The primer
sets 29F — 809R and 740F — 1494R were used for cultures, while 106f and 781R
(Niibel et al. 1997; Ionescu et al. 2009) were used for environmental samples. Ampli-
cons originating from environmental sequences were cloned using the InsTAclone kit
(K1214, Fermentas, Lithuania) and verified using colony PCR. Successful reactions

Morphological, phylogenetic and physiological diversity of cyanobacteria in the hot springs... 73

were sent for cloning and sequencing at the Genome Sequencing Center at Washing-
ton University, St. Louis, MO. To obtain reliable results each clone was sequenced in
both directions. Each individual sequence used for the phylogenetic analysis is the
result of two aligned sequences from the same clone. All sequences were compared
to the NCBI nr databases using the NetBlast application (available from NCBI). The
top five hits as well as some additional relevant sequences were used for phyloge-
netic analysis. Sequences were aligned using the Muscle 3.6 software (Edgar 2004).
Phylogenetic trees were constructed using the MEGA 4.0 (Tamura et al. 2007). The
Distance Matrix was calculated using the Jukes-Cantor algorithm and the trees were
constructed using the Minimum Evolution method. Validity of tree topology was
evaluated using the bootstrap method (100 replicates). Environmental 16S rRNA
gene sequences from Zerka Main are available from the GenBank at accession num-

bers EU326950-327016.

Nitrogen fixation studies

To assess the importance of nitrogen fixation to the cyanobacteria in the Zerka Main
springs, we used the acetylene reduction test to quantify the nitrogenase activity of the
community. Biomass was collected from the major spring of Area A (63°C) and from
two nearby streams with temperatures of 51 °C and 39 °C. Acetylene reduction tests
were performed in situ in the light and in the dark for 1:45-2:45 hours, incubation
times being limited due to logistic constraints. Full details of the experimental condi-
tions were given by Ionescu et al. (2009).

Results
Physical and chemical properties of the samples collected

The major springs that issue at sites A and B as indicated in Fig. 1 had a temperature
of 62 °C to 63°C, independent of the season of sampling. Most samples collected
from the springs and their outflow channels had temperatures between 63 and 51 °C.
During our last sampling trip (June 2007) we found a channel located about 100 m
north of the major springs of site A, which had not been surveyed previously. Its water
temperature was 39°C.

The waters of the springs differed little in chemical properties. The total dissolved
salts concentrations ranged between 1,267 and 1,445 mg/L, with an alkalinity of 110-
130 mg/L. A typical analysis (water from site A pictured in Fig. 2A) gave (mg/L):
CI, 810; SOs 196; Ca**, 186; Mg’*, 91; Na‘, 86; K*, 35. Up to 0.3 mM sulfide was
measured in the water sampled near the sources. The pH ranged from 6.4 to 6.8. More
detailed chemical analyses have been reported elsewhere (Abu Ajamiech 1980, 1989;
Rimawi and Salameh 1988; Ionescu et al. 2009).

74 Aharon Oren et al. / BioRisk 3: 69-82 (2009)

Microscopical observations of the spring samples and the cyanobacterial
cultures obtained

Microscopical examination of samples collected from the springs and their outflow
channels showed a dominance of unicellular cyanobacteria. Figure 3 presents a repre-
sentative selection of organisms encountered at temperatures between 58 °C and 63°C.
At the highest temperatures the cyanobacterial mats were dark green in colour (Figs 2A,
B). Here unicellular 7hermosynechococcus-type cyanobacteria dominated (Fig. 3A). As
water temperature decreases downstream the outflow channels, additional types of cy-
anobacteria started to appear, as shown in Figs 3B and 3C. Occasionally tightly wound,
thin Spirulina-like filaments were encountered (Oren et al. 2008). Thus far phyloge-
netic analyses of environmental samples (see below) did not yield any Spirulina-like
16S rRNA gene sequences; however, some of our clones clustered with Limnothrix, a
genus that includes a (non-thermophilic) spiral organism (Limnothrix chlorospira).

The area of the outflow channels in Area B above the waterfalls (Fig. 2D) had mats
coloured in part orange, and here we mainly found small Gloeocapsa-like unicellular
cyanobacteria. More extensive illustrations of the morphological types of cyanobacteria
found in the Zerka Main spring area were published elsewhere (Ionescu et al. 2009).

Of special interest is the profuse growth of masses of the branching hetero-
cystous cyanobacterium Scytonema observed along some of the outflow channels in
the spring area above the waterfalls. The Scytonema colonies consist of blackish to
dark-green material attached to the rocks (Fig. 4). The colonies are not in direct con-
tact with the hot spring waters, but they are continuously sprayed by small droplets
of water from the stream.

We succeeded in growing Chroogloeocystis and Mastigocladus/ Fischerella types from

samples collected at different places of the Zerka Main thermal area. Some of the iso-

Figure 3. Microphotographs of unicellular cyanobacteria from the Zerka Main hot springs. Different

types of unicellular cyanobacteria are shown, collected from the Zerka Main hot springs in November
2006 at temperatures between 58 °C and 63°C.

Morphological, phylogenetic and physiological diversity of cyanobacteria in the hot springs... 75

lated strains resembled morphologies seen in the field-collected material; others were
of types not observed by direct examination of the samples (Ionescu et al. 2007, 2009).
The cultures are referred to by accession numbers that start with tBTRCCn (see also
Fig. 5). Unfortunately we did not yet succeed in obtaining 7hermosynechococcus-like
organisms from the Zerka Ma’in springs in culture.

16S rRNA gene-based phylogenetic diversity of cyanobacteria

Figure 5 presents the phylogenetic relationships of selected environmental 16S rRNA
gene sequences obtained from the Zerka Ma’in springs, indicating the temperature
from which the different sequences were recovered, and the sequences of the cyano-
bacteria grown from the springs as indicated by their tB TRCCn numbers. Only part
of the sequences obtained is shown, and similar related sequences are clustered to-

gether. For example, the upper box in Fig. 5 (“Thermosynechococcus-like clones”) is
based on 46 distinct and different sequences amplified from the environmental DNA.
The Zhermosynechococcus cluster appears to be particularly diverse in the springs (Oren
et al. 2008; Ionescu et al. 2009). More extensive phylogenetic trees were given in
Oren et al. (2008).

Figure 4. Growth of Scytonema in the Zerka Ma‘in area. The picture shows growth of large colonies of
Scytonema on rocks sprayed by water from a thermal stream, and microphotographs of Scytonema fila-
ments showing the thick sheath and heterocysts (arrows).

76 Aharon Oren et al. / BioRisk 3: 69-82 (2009)

& Thermosynechococcus-like clones (over 51 °C)
99

bi Thermosynechococcus elongaus BP| BA000039
al. Thermosynechococcus-like clones (over 58 °C
ie == Thermosynechococcus-like clones (over 58 °C

60
od J hermosynechococcus-like clones (39 °C

72 Synechococcus elongatus PCC 6301 AP00823 |
P oe Prochlorococcus marinus MIT 9312 CPO001 I |
Halomicronema excentricum TFEP| AF320093
26 Staniera cyanosphera AF|3293 |
22 Limnothrix redekei AJ344559
a2 Dermocarpella incrassata AJ344559
be Leptolyngbia sp. AY23959|
35 Pseudodanabaena tremula UTCC 471 AF218371

tBTRCCn 301 DQ471441

94 Uncultured Oscillatoria from the Philippines DQI31173
83| ' Fischerella major NIES 692 AB093487 (Mastigocladus laminosus)
6 99|- Uncultured bacterium clone B95 AF407731

95) tBTRCCn 403 DQ471444
Noctoc sp. PC 7120AF317631
38 Microcoleus sp. PCC 7420 X70770

j
54 991 # tBTRCCn 28 DQ471449
Chroogloeocystis siderophila 5.2 sc AY38079 |
43 Chroococcidiopsis sp. BB823 AJ344553
34 99 Chroococcidiopsis sp. PCC743 | ABO74506
99" Chroococcidiopsis thermalis AB039005
Clone A32
Clone A34

Clone DI55
{2 — Clone DI73
Clone D7
Clone A33
Clone A36
Clone A54
Clone D93
Clone D9I
Clone D89
Clone D90
a 9¢ Clone DI75
90 Clone D183
Clone A39
Clone A35
Clone A38
83 Uncultured Antarctic bacterium AF076 164

3 tBTRCCn 407 DQ71447

96 Filamentous-like clones (58 °C

i | Se ;
y Filamentous-like clones (63 °C

= “Sl Filamentous-like clones (39 °C & 51 °C
817 Oscillatoria sp. J24 AF263344
sate Cyanobacterium sp. OS type | L04709
tBTRCCn 102 DQ471443
8B'] tBTRCCn 302 DQ471445
47

BIR n 208 DO4/1446

39 °C (A) & 58°C (D)

18

59 _Filamentous-like clones (39 °C
Synechococcus sp. C9 AF 132773

Oscillatoria limnetica AJ007908
Escherichia coli U00096

0.02

Figure 5. Phylogenetic tree of Zerka Main cyanobacteria, based on 16S rRNA gene sequences. The
minimum evolution phylogenetic tree is based on 16S rRNA gene sequences of cyanobacterial isolates ob-
tained from the Zerka Ma’in springs (indicated by tB TRCCn numbers) and on cyanobacterial sequences
recovered by PCR amplification from DNA extracted from biomass collected between December 2005
and June 2007 from the thermal springs and outflow channels. The temperature of the waters from which

the 16S rRNA genes were recovered is indicated.

Morphological, phylogenetic and physiological diversity of cyanobacteria in the hot springs... 77

Nitrogen fixation studies on the thermophilic cyanobacteria

Chemical analyses of the Zerka Ma’in spring waters (Abu Ajamieh 1980, 1989) show
that the concentrations of nitrogen compounds (ammonium, nitrate) are negligible.
Therefore the ability to fix gaseous nitrogen will be advantageous to the cyanobacteria
that colonise the springs. The observation of heterocysts in the Scytonema colonies
bordering some of the outflow channels (Fig. 4) suggests that nitrogen fixation by the
local cyanobacterial communities may indeed occur.

We detected significant rates of acetylene reduction at all sites sampled, including
the 63 °C site. The highest calculated nitrogenase activity (0.0025—0.012 nmol N pg
chlorophyll! h') was obtained at 51 °C in the dark. At 63 °C we measured rates of
0.001—0.0025 nmol N pg chlorophyll" h', rates that are low, but significantly higher
than background values. Acetylene reduction rates in the light were 30% to 50% lower
than those measured in the dark.

The fact that light inhibited nitrogenase activity suggests that the activity is prob-
ably located in oxygenic phototrophs, the nitrogenase of which is inhibited by pho-
tosynthetically produced oxygen. To test whether mRNA transcripts of the nifH (ni-
trogenase reductase) gene may be present in the Zerka Main cyanobacterial commu-
nity at the highest im situ temperature of 63°C, we fixed samples in liquid nitrogen;
prepared cDNA from the RNA isolated from the community, and amplified gene
sequences using nifH-specific PCR primers from this cDNA. Using this procedure
we isolated a gene identical to a nifH gene found in a filamentous cyanobacterial cul-
ture obtained from the site. A full account of these experiments was given by Ionescu
et al. (2009).

Gene sequences of cyanobacterial nifH genes were recovered both from the
community DNA and from selected isolates obtained from the spring. Sequenc-
es of nifH obtained from the environmental DNA were related to those from
Fischerella, Phormidium, and Lyngbya spp. (Ionescu et al. 2009). We also sequenced
the nifH gene of a heterocystous isolate related to Mastigocladus/ Fischerella (strain
nBTRCC 101).

Nitrogen fixation by this isolate is now being investigated in further depth. Op-
timal rates of acetylene reduction were measured at 45 °C (up to 24.5 nmol N ug
chlorophyll! day’). The maximum temperature for nitrogen fixation in this strain was
found to be 52°C to 53°C. When grown under light/dark cycles, acetylene reduction
rates were higher than under constant light. When a culture grown in nitrate-rich
medium was transferred to nitrogen-depleted medium, formation of heterocysts was
induced, and acetylene reduction activity started after 48 hours. Quantitative PCR
analysis showed expression of the 7ifH gene to be subject to a circadian rhythm. The
nature of the phenomenon is currently under investigation.

78 Aharon Oren et al. / BioRisk 3: 69-82 (2009)

Discussion

At the highest temperatures (up to 63 °C), unicellular cyanobacteria dominated in the
Zerka Main springs area. As water temperature decreases downstream the outflow
channels, additional types of cyanobacteria started to appear, including filamentous
cyanobacteria belonging to the Mastigocladus-Fischerella group, known from thermal
springs worldwide (Brock 1978; Castenholz 1969, 1973; Ward and Castenholz 2000).
Spirulina labyrinthiformis was earlier reported as the dominant organism in material
collected by A. Aaronson from a 52°C spring of Zerka Main (Rayss 1944). Aaron-
son had joined Blanckenhorn during his above-mentioned 1908 survey of the area
(Blanckenhorn 1912), but no further information is available on the exact site and date
of collection and no further details have been reported.

The profuse growth of masses of the branching heterocystous cyanobacterium Scy-
tonema observed along some of the outflow channels in the spring area above the water-
falls (Fig. 4) is of special interest. It is well possible that these are the “felt-like mats ....
in the form of a sponge or pillow with a dark-green, somewhat wrinkled skin”, to which
Blanckenhorn referred in the quotation given above. Growth of Scytonema was also
reported from the nearby hot springs of Zara that were surveyed for cyanobacteria and
microalgae in 1936 (Frémy and Rayss 1938). The filaments of Scytonema are surrounded
by a thick, dark brown, layered sheath that has a high content of scytonemin, a dimeric
indole alkaloid synthesised from aromatic amino acid residues, which absorbs UV-A
radiation (Castenholz and Garcia-Pichel 2000). Qualitative and quantitative informa-
tion about the content of sctyonemin and other UV-absorbing pigments in the mate-
rial from Zerka Main has been provided elsewhere (Oren et al. 2008). Scytonemin has
its absorbance maximum at 384 nm, with a broad absorption band. Thus the cells are
effectively protected against UV-induced cell damage. It remains to be determined, to
what extent this property is of importance to the physiology of Scytonema at the Zerka
Main site. At its location at about 250 m below mean sea level the local level of UV
radiation is lower than at higher altitudes, and hardly any traces of other UV-absorbing
compounds such as mycosporine-like amino acids could be detected in any other types
of cyanobacteria found so abundantly in and around the springs (Oren et al. 2008).
Literature data also suggest that the scytonemin content of cyanobacteria that produce
the compound may be regulated by factors not directly connected with the light inten-
sity and light quality found in their environment (Castenholz and Garcia-Pichel 2000).
It should be noted that Scytonema is not a thermophile, and at Zerka Main its colonies
are exposed to ambient air temperatures rather than to the temperatures of the thermal
spring water (Fig. 4). Surveys of springs in Yellowstone National Park, USA (where UV
levels are high at an elevation of > 2000 m above sea level) showed 55°C to be the up-
per limit for growth of sheathed, scytonemin-containing species of cyanobacteria such
as Pleurocapsa and Calothrix (Wickstrom and Castenholz 1978).

The isolation of a unicellular cyanobacterium with a 16S rRNA gene with 99%
similarity with Chroogloeocystis siderophila, an organism originally found in iron-rich

Morphological, phylogenetic and physiological diversity of cyanobacteria in the hot springs... 79

thermal environments in Yellowstone and requiring high iron concentrations for
growth (Brown et al. 2005), is remarkable. The Zerka Main waters do not have a high
iron content.

Based on the information presented in the tree shown in Fig. 5, a number of inter-
esting conclusions can be drawn: (1) All sequences recovered from the cyanobacteria
of Zerka Ma‘in appear to be unique, and none of the sequences found was identical to
any sequence found in the GenBank database. (2) Some of the Zerka Ma’in organisms
have close relatives in other thermal springs worldwide, but there are other types as
well that have not been reported from elsewhere. (3) None of the sequences found in
our cultures were retrieved directly from the environmental DNA as well. This holds
also true for the two cultures of heterocystous cyanobacteria affiliated with the genera
Fischerella and Mastigocladus we have obtained and studied for their nitrogen fixation
properties (see below). No related sequences were yet detected among the environ-
mental 16S rRNA gene fragments cloned from the DNA isolated from the site. (4) In
many cases sequences found in the lower temperature waters show phylotypes distinct
from those present at the higher temperature sites. (5) The sequences in the large box
(A32 to A38), which appear to have no equivalent elsewhere, are of special interest. We
have no cultured representative of this group yet, so no information is available about
the morphology of the organisms that harbor these sequences. Sequences belonging to
this group have been retrieved both from 58°C thermal waters and from a cooler site
where we measured 39 °C.

Chemical analyses of the Zerka Ma’in spring waters (Abu Ajamieh 1980, 1989)
show that the concentrations of nitrogen compounds (ammonium, nitrate) are negli-
gible. Therefore studies of the nitrogen fixation potential of the cyanobacterial com-
munity in the springs were initiated. The finding of nitrogenase at 63 °C was somewhat
surprising, as a temperature of 55 °C was generally considered to be the upper limit of
nitrogen fixation by cyanobacteria (Mastigocladus) in hot spring environments (Fogg
1952, Stewart 1970, Wickstrom 1980). However, the recent finding of transcripts of
nif genes derived from Synechococcus ecotypes in Octopus Spring, Yellowstone Na-
tional Park, USA, at temperatures up to 63.4°C (Steunou et al. 2006) suggests that
the upper temperature limit of cyanobacterial nitrogen fixation may be higher than
previously assumed.

It is intriguing that the unique environment of the Zerka Main hot springs has not
been surveyed before by biologists. To our knowledge this is the only site in the Mid-
dle East where thermal waters of such high temperatures flow undisturbed and enable
the development of a diverse community of phototrophic and other microorganisms
adapted to life at temperatures up to 63°C. ‘The hot springs of Tiberias, Israel, used as
a thermal spa since Roman times, reach temperatures very similar to those of Zerka
Main. Some exploration of the cyanobacteria present at the site has been done in the
past (Dor 1967). However, these springs do not currently flow freely outdoors, so that
thermophilic cyanobacteria and other microorganisms adapted to life at high tempera-
tures have little opportunity to develop.

80 Aharon Oren et al. / BioRisk 3: 69-82 (2009)

Conclusions

The thermal springs of Zerka Ma’in, Jordan, are inhabited by a great diversity of ther-
mophilic unicellular and filamentous cyanobacteria including Thermosynechococcus,
Chroogloeocystis, Fischerella (Mastigocladus), and Scytonema (occurring as large masses
at lower temperatures). Based on 16S rRNA gene sequence analysis, the Zerka Main
strains were phylogenetically distinct from morphologically similar cyanobacteria
found in hot springs world-wide. Low rates of nitrogen fixation were detected up to
63°C, the highest temperature recorded in the springs.

Acknowledgements

We thank the Bridging the Rift Foundation for coordinating the surveys of the Jor-
danian hot springs and for financial support. We are further grateful to the organizers
of the First Middle Eastern Biodiversity Congress, held in Aqaba, Jordan, in October
2008, for giving us the opportunity to present our results at the meeting.

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