305
FEMS Microbiology Ecology 45 (1987) 305-312
Published by Elsevier
FEC 00135
Low-molecular mass carbohydrate accumulation in cyanobacteria
from a marine microbial mat in response to salt
Lucas J. Stal a and Robert
H. Reed b
a Geomicrobiology Diuision, Uniuersity of Oldenburg, D-2900 Oldenburg. F. R. G. and h Department of Biological Sciences,
University of Dundee, Dundee DDI 4HN, U.K.
Received 20 March 1987
Revision received 2 June 1987
Accepted 4 June 1987
Key words:
Cyanobacteria;
Microbial
mat; Osmotic adjustment;
Trehalose; Sucrose
1. SUMMARY
Forty seven strains of cyanobacteria,
all isolated from microbial mats of intertidal
sediments
of the island of Mellum (North Sea), were analyzed
for the presence of organic osmotica. The cyanobacteria examined belonged to taxonomically
different groups and were classified
according
to
their salt optimum
and salt tolerance
as either
freshwater, brackish or marine. Except betaine, all
organic
osmotica
known
to occur in cyanobacteria, were found. The results showed no clear
correlation
between
the chemical nature of the
organic solute and the salt optimum or salt tolerance of the cyanobacteria
examined,
indicating
that these solutes are not specific to this marine
habitat.
All strains
belonging
to the Nostoc/
Anabuena-group
accumulated
sucrose as the sole
organic
osmoticum.
The marine,
heterocystous
Culothrix sp. accumulated
trehalose. All strains of
the
LPP-group
(Lyngbyu,
Plectonemu,
Correspondence to: L.J. Stal, Geomicrobiology Division, University of Oldenburg, P.O. Box 2503, D-2900 Oldenburg, F.R.G.
0168-6496/87/$03.50
Carbohydrates;
Glucosylglycerol;
Phormidium) accumulated
glucosylglycerol
as sole
or primary organic solute. Some LPP-strains
accumulated a disaccharide
as a secundary solute, e.g.
sucrose or trehalose. Gloeocupsu, Synechocystis and
Spirulinu
accumulated
glucosylglycerol.
Two
marine Oscillutoria accumulated
trehalose, whereas
a freshwater
Oscillatoriu with a broad salinity
tolerance, accumulated
sucrose.
Analysis of field samples of the microbial mats
demonstrated
the presence of glycerol, glucosylglycerol, sucrose and trehalose. The relative abundance of the different compounds
was related to
the species composition
as could be predicted
from laboratory
observations.
These data suggest
that these carbohydrates
have a function in maintaining osmotic balance in the organisms
within
the microbial mat.
2. INTRODUCTION
Cyanobacteria
occur in a variety of environments, ranging from freshwater to hypersaline
systems [l]. Moreover, these organisms occur in environments
which show dramatic changes in salin-
0 1987 Federation of European Microbiological Societies
306
ity. Intertidal
sediments
are exposed to seawater
each tidal cycle, whereas supralittoral
sediments
are only rarely exposed to seawater. If exposed to
the air, evaporation
will result in increasing salinity, whereas rainfall may reduce the salinity almost to freshwater
conditions.
Such dramatic
changes in salinity are particularly
important
in
the microbial
mats of the temperate
North Sea
region [2].
Bacteria and algae which are exposed to salt
must accumulate osmotically-active
compatible
solutes to generate positive hydrostatic (turgor) pressure. Previously, several papers have dealt with the
mechanism
of osmotic adjustment
in cyanobacteria in response to salt stress (see [3] for references). Several low-molecular
carbohydrates
and
quaternary
ammonium
compounds
have been
found
in osmotically-significant
amounts
in
laboratory isolates of cyanobacteria.
These include
the disaccharides
sucrose [4,5] and trehalose [6]
and the heteroside
@a-D-ghCOpyraUOSyl-(l-2)glycerol (glucosylglycerol)
[7] and the quaternary
ammonium
compounds
tri-methyl glycine (glycine
betaine) [8,9] and &i-methyl glutamate (glutamate
betaine) [lo].
Mackay
et al. [ll] examined
28 strains
of
cyanobacteria
from a diverse range of freshwater,
marine and hypersaline
environments.
According
to their upper salinity tolerance, they divided these
strains in 3 groups. ‘Freshwater’
organisms
grew
in BG-11 medium with up to 4.5% NaCl added
but not in a seawater-based
medium (medium M).
The strains that grew well in medium
M were
divided in two groups. Group I grew in medium
M with total NaCl up to a concentration
of 11%
(‘marine’ strains) and group II grew with 13% or
more
NaCl
(‘hypersaline’
strains).
Only
the
‘marine’ strains accumulated
the heteroside glucosylglycerol, whereas the other strains accumulated
simple sugars or quaternary
ammonium
compounds [lo]. These authors therefore
concluded
from these studies that the chemical nature of the
organic solute could be used as a taxonomical
marker
to distinguish
marine
strains.
Subsequently, Mackay et al. [lo] extended their study to
a total of 36 strains and found that none of the
glucosylglycerol
accumulating
strains
produced
additional
solutes (i.e. trehalose,
sucrose or be-
taine). This was taken as evidence for the absence
in marine cyanobacteria
of the biochemical
pathways required to produce these osmotica.
In contrast,
Reed and co-workers
found no
close correlation between habitat and the chemical
nature of the organic osmoticum [9,12]. They suggested that glucosylglycerol
accumulation
is not
unique to marine cyanobacteria
but that the upper
salt tolerance limit might depend, in part at least,
on the type of solute produced
[12]. At high
concentrations,
sucrose inhibits
enzymatic
reactions in vitro [13,14] suggesting that sucrose-accumulating
strains may be less halotolerant
than
other isolates [3].
Some cyanobacteria
have been found to accumulate sucrose [15] or trehalose [16] in addition to
glucosylglycerol.
The ratio in which sucrose and
glucosylglycerol
occurred in some strains of Synechocystis appeared to depend on temperature
and
salinity with greatest amounts of sucrose in cells
grown at high temperature
in hyposaline
media
[17]. This has demonstrated
that not all glucosylglycerol-accumulating
cyanobacteria
are devoid of
the biochemical
pathways required to synthesize
other organic solutes.
Taken together, these studies suggest that no
unique organic solute occurs in marine cyanobacteria. Among the strains tested so far, there is a
tendency for marine isolates to accumulate
glucosylglycerol,
whereas sucrose and trehalose more
often occur in stenohaline,
salt-sensitive
freshwater strains [3]. Betaines seem to be restricted to
hypersaline
cyanobacteria
[10,18]. This class of
compatible
solute is also accumulated
in other
extreme
halophilic
phototrophic
bacteria,
e.g.
Ectothiorhodospira
halochloris [19].
The cyanobacteria
tested thus far originated
from a range of different habitats and locations,
isolated
using a variety of culture
media and
maintenance
regimes. In many cases, isolates obtained from culture collections
are poorly documented, with no clear indication
of the original
habitat of the strain [9,11] adding a further problem to any interpretation
of results. Therefore, it
seemed worthwhile to examine a variety of different strains isolated from a single marine environment using a single set of culture conditions.
Previously,
the isolation
and salt-tolerance
char-
307
acteristics of more than 60 cyanobacteria
from a
marine microbial mat have been reported [20]. All
cyanobacteria
were isolated from intertidal
sediments of the island of Mellum (North Sea), representing
all of the major cyanobacterial
genera
found at this location.
The purpose of this study was to examine the
organic osmotica
in these strains to determine
whether the type of organic solute is linked to the
degree of halotolerance
of each isolate in culture.
3. MATERIAL
AND
METHODS
3.1. Organisms and growth conditions
The organisms used in this study are listed in
Table 1. The strains have been described
in a
previous paper [20]. The strain numbers refer to
the collection
of the Geomicrobiology
Group of
Oldenburg
University.
In most cases the generic
assignments
were made according to Rippka and
coworkers
[21]. However,
we have assigned
Anabaena and Nostoc to a single group. Also we
have not distinguished
LPP groups A and B. Table 1 only refers to 22 of the 47 strains tested, as
before [20]. Since many of the isolates carried
under different numbers in the collection do not
differ significantly
from the strains listed here, the
present work supports
the proposal
that these
organisms are multiple isolates of the same species.
However, the strains listed in Table 1 are different
in one or more respects [20].
All strains were routinely grown at their optimum salinity. The media used were the freshwater
medium
BG-11, the artificial
seawater medium
ASN III and a 1 : 1 mixture of both media [21].
To examine
the organic
osmotica
in these
cyanobacteria,
the cultures were grown both at the
lower and upper limit of salinity
tolerance,
as
determined
previously
[20]: organic osmotica are
thus recognised
as solutes that occur in greater
amounts
in cyanobacteria
grown at the upper
limit of salinity tolerance. Media with higher salinities than ASN III were obtained by increasing the
concentration
of NaCI: all other constituents
were
maintained
constant.
The cyanobacteria
were
grown in 2-liter Erlenmeyer
flasks, containing
1
liter of medium. The flasks were incubated
in a
Table
1
Cyanobacteria
Strain
No.
and growth
medium
Assignment
Medium
63
Gloeocapsa
sp.
86
Synechocystis
85
Spirulinu
08
Oscrllatorra
sp.
ASN III
23
sp.
sp.
ASN III
61
Oscillatoria
Oscillatoria
03
LPP
ASN III
06
LPP
ASN
III
05
LPP
ASN
III/2
11
LPP
ASN
III
19
LPP
BG-I 1
ASN III/2
sp.
sp.
ASN III/Z
BG-I 1
ASN III/2
ASN III
32
LPP
72
LPP
BG-11
75
LPP
ASN III/2
81
LPP
ASN III
48a
Calothrix
28
Nostoc/Anabaena
Nostoc/Anabaena
Nostoc/Anabaena
Nostoc/Anabaena
Nostoc/Anabaena
Nostoc/Anabaena
56
51
57
59
69
sp.
ASN III
ASN III/Z
ASN III/2
BG-11
BG-11
BG-11
ASN III/2
Gallenkamp
illuminated
shaking
incubator
at
20” C and 100 rpm. Incident
light intensity
was
135.10”
Q. rnp2. s-’ (400-750 nm) as measured
with a Techtum
QSM-2500
quantaspectrometer
(Sweden) which equalled ‘2.5 klux. In contrast,
Oscillatoria sp. strain 08 and strain 23 were grown
in small
50-ml Erlenmeyer
flasks, containing
30
ml of medium
at a light intensity
of 70.10”
Q.me2.s-’
(1.3 klux). Sufficient
cell material
was obtained
by combining
several cultures of
each isolate of Oscillatoria.
3.2. Field samples
Samples of the microbial
mats were taken in
September 1986. The samples were taken from the
west bank of the island of Mellum (North Sea).
Six sampling sites were chosen. The sampling sites
were located on a transect from the Mean High
Waterlevel at Spring Tide (M.H.W.S.) (Site 1) to
Mean Low Waterlevel at Neap Tide (M.L.W.N.)
308
(Site 6). The mats along this transect showed
changing populations of cyanobacteria. An extensive description of these mats was given previously
[22]. The sites included old, established mat systems (Site 1) as well as sediment which was freshly
colonized in spring 1986 (Site 6). The cyanobacterial mat from site 1 was a 0.5-l mm thick,
rigid layer almost devoid of trapped sand granules. Site 2, in contrast, was characterized by mats
of 2-3 mm thickness and contained a lot of sand
trapped by the cyanobacterial trichomes. Site 3
was very similar to site 2 but differed in the
thickness of the cyanobacterial layer (l-2 mm).
The mats of the sites 1-3 were all typically
dominated by Microcoleus chthonoplastes. The sites
4-6 were characterized by thin layers of cyanobacteria (0.5-l
mm), loosely associated with the
sediment. The species composition in these mats
changed from A4. chthonoplastes as the dominant
organism (site 4) to Oscillatoria sp. (sites 5 and 6)
(see Table 5).
The mat was sampled by peeling off the mat
(established mats), carefully removing any of the
anoxic layers beneath the cyanobacterial mat as
well as green algae above the cyanobacterial layers.
Non-established mats were sampled by scraping
off the cyanobacterial layer. The mats were then
frozen at - 20 o C until analyzed.
3.3. Analysis
of low-M,
carbohydrates
and
quaternaty ammonium compounds
The cultures were harvested by centrifugation
and rinsed in distilled water prior to freeze-drying.
Freeze-dried cultures and frozen mat samples were
transferred to hot 80% ethanol and extracted overnight at 40°C. Low-M, carbohydrates were determined as the trimethylsilyl derivatives using
gas-liquid chromatography [6]. Quaternary ammonium compounds were analyzed using ‘H
nuclear magnetic resonance (NMR) spectroscopy
1231.
3.4. Determination of chlorophyll a
Chlorophyll a and pheophytin a content of
extracts of the mat samples were determined
according to [24]. The pigments were determined
in the same extract as that used for the
carbohydrate determination. Because during the
extraction procedure, part of the chlorophyll will
be converted to pheophytin, the sum of chlorophyll a plus pheophytin a was used for the calculations.
4. RESULTS
AND DISCUSSION
The salinity tolerance of each isolate was
defined as the upper and lower salinities at which
sustained growth was obtained, while the salinity
optimum was defined as the salinity at which the
fastest growth rate occurred. If the growth of a
culture was unaffected over a range of salt concentrations, the lower salinity was regarded as the
optimum. Some cultures showed equal growth
rates in freshwater as well as in full-strength
seawater. Such cultures apparently did not require
elevated salt concentrations for optimal growth
and, in accordance with Rippka et al. [21], were
defined as freshwater strains.
Table 2 lists the strains with their respective
salinity tolerance and the organic osmotica accumulated at the upper salinity limit for growth.
Quaternary
ammonium compounds (betaines)
were not detected in any of the strains (detection
limit 0.01 mg [23]). This was in agreement with the
limited salt tolerance of the strains examined. To
date, only halotolerant hypersaline cyanobacteria
have been found to accumulate betaines [3,10,18]
and no strain showed good growth above doublestrength seawater during the present study.
All of the LPP-strains accumulated osmotically-significant
amounts of glucosylglycerol.
Gloeocapsa, Synechocystis and Spirulina also accumulated glucosylglycerol as the sole organic solute
and showed good growth from freshwater up to
full-strength
seawater
(30%0).
Synechocystis
showed optimum growth in freshwater and was
therefore
described
as a freshwater
cyanobacterium. In contrast Gloeocapsa and Spirulina
required elevated salt concentrations
and were
described as brackish since growth was optimum
at half-strength seawater (15%0). Of the 9 LPPstrains, 6 grew well up to double-strength seawater
(57%0). Two strains were assigned as freshwater
organisms showing optimal growth in BG 11
medium. Freshwater strain 19, however, tolerated
309
Table 2
Principal organic solutes and salt tolerances of 22 strains of
cyanobacteria isolated from microbial mats of marine intertidal sediments
Strain
Organic solute
Salt tolerance (L)
. ,
Gloeocapsn 63
glucosylglycerol
brackish
O-30
glucosylglycerol
fresh
O-30
glucosylgJycero1
brackish
O-30
trehalose
trehalose
sucrose
marine
marine
fresh
15-30
glucosylglycerol
glucosylgfycerol
glucosylglycerol
glucosylglycerol
glucosylgfycerol
glucosylglycerol
glucosylglycerol/
trehalose
ghrcosylglycerol/
sucrose
glucosylglycerol
marine
marine
brackish
marine
fresh
brackish
o-57
o-57
15-57
15-30
o-57
15-30
brackish
marine
15-57
15-57
trehalose
sucrose
sucrose
sucrose
sucrose
sucrose
sucrose
marine
brackish
brackish
fresh
fresh
fresh
brackish
15-30
15-30
O-30
O-30
o-15
O-30
o-15
Synechocystis
Spin&m
Oscillatoria
Oscillatorio
Osicllatoria
LPP
LPP
LPP
LPP
LPP
LPP
LPP
86
85
08
23
61
03
06
05
11
19
32
72
LPP 75
LPP 81
Colothrix 48a
Nostoc/Anabaena
Nostoc/Anabaena
Nostoc/Anabaena
Nostoc/Anabaena
Nostoc/Anabaenn
Nostoc/Anobaena
28
56
51
57
59
69
fresh
o-57
O-30
up to double-strength seawater. Two LPP-strains
accumulated a second solute in addition to glucosylglycerol. Thus strain 72 and 75 accumulated
trehalose and sucrose, respectively as secondary
osmotica. These results support the proposal that
glucosylgycerol-accumulating
strains do not lack
pathways for the synthesis of trehalose or sucrose,
in contrast to the hypothesis of Mackay et al.
[lO,ll]. This was also shown for several Synechocystis strains, which accumulated sucrose in addition to glucosylglycerol [15,17] and the secondary solute trehalose in Spirulina platensis [16].
Two strains of Oscillatoria sp. did not grow at
all in freshwater. Strain 23 only grew in fullstrength seawater. The strains 23 and 08 were
morphologically almost identical. They differed by
the ability of strain 23 to fix N, aerobically [25].
Both of these marine strains of Oscillatoria accumulated trehalose as sole organic osmoticum, suggesting that marine cyanobacteria are not characterized by the accumulation of glucosylglycerol
as the sole organic osmoticum. Moreover, Calothrix sp. strain 48a also did not grow in freshwater
and showed optimal growth in full-strength
seawater. This organism was morphologically
rather similar to the Oscillatoria. Calothrix, however, is a heterocystous, nitrogen-fixing organism.
Also this organism accumulated trehalose. Oscillatoria sp. strain 61 differed morphologically from
the other oscillatorians. This strain was considered
as a freshwater organism, according to the salinity
optimum for growth but showed sustained growth
up to double-strength seawater. Growth was not
inhibited up to double-strength seawater although
the organism accumulated sucrose as sole organic
osmoticum, in contrast to the hypothesis that
sucrose may limit the upper salinity limit for
growth [3].
All strains of the Nostoc/Anabaena
group accumulated sucrose including strains with elevated
salt requirements for growth. This is in agreement
with the data existing so far for this group of
cyanobacteria [9,26] and suggests that the occurrence of sucrose in this group may be a taxonomic
feature, independent of halotolerance and salt requirements.
These data show: (1) that the designation of
cyanobacteria as marine or freshwater organisms
cannot be linked simply to the chemical nature of
the organic osmoticum, (2) that no clear correlation exists between the lower and upper salinity
tolerance of the cyanobacteria and the class of
organic accumulated solute, (3) that certain glucosylglycerol-accumulating
cyanobacteria may synthesize either trehalose or sucrose as a secondary
solute in response to salt stress, clearly indicating
that these organisms possess the biochemical pathways to produce such substances.
To establish whether these organic solutes are
also found in cyanobacteria growing in the marine
environment, samples were collected from the same
microbial mats from which the cyanobacteria were
isolated originally. Tables 3 and 4 summarize the
observations on the low-M, carbohydrates ex-
310
Table
Although osmotic adjustment
has been studied in
only a few members of this group of organisms,
no species has yet been shown to accumulate
glycerol
[27]. In addition,
the production
of
glycerol from the hydrolysis
of glucosylglycerol
during sampling is unlikely, because glucose was
not detected in significant
quantities
(Tables 3
and 4).
In autumn, the mats are often covered by dense
populations
of the green alga Enteromorpha
sp.
Although care was taken to remove this alga as far
as possible, the occurrence of sucrose in the samples may, in part at least, be explained
by the
presence of this organism. The alga collected from
the mats separately,
contained
this disaccharide
(Table 4) and sucrose is known to be involved in
the osmotic adjustment
processes of Enteromorpha
3
Low-M,
carbohydrates
Mellum
(North
pled, representing
Amounts
in samples
Sea) intertidal
of microbial
sediments.
mats in different
from
Six sites were sam-
stages of development
are given in mg carbohydrate/kg
Carbohydrate
mats
dry sediment.
Site
1
2
3
5
6
49
21
16
45
17
111
390
64
107
185
13
88
Sucrose
21
7
12
88
4
117
Trehalose
96
17
53
114
19
151
Glucose
12
0
2
0
0
0
Glycerol
Glucosylglycerol
4
tracted from the mats. These extracts were not
examined
for betaines, because these substances
did not occur in any of the cyanobaterial
cultures
in the laboratory.
All cyanobacterial
low-M,
carbohydrate
osmotica were found in extracts of
the microbial mats i.e. glucosylglycerol,
trehalose
and sucrose. In addition, appreciable
amounts of
glycerol were found. We do not know which
organisms
in the mat were responsible
for the
observed glycerol. It is known that some unicellular green algae accumulate
glycerol. In particular
Chlamydomonas
spp. and Dunaliella
spp. are
known to produce high amounts
of glycerol in
saline media [27]. However, these unicellular
green
algae have not been observed in these North Sea
mats although other unicellular
green algae have
been observed in low numbers
([2], unpublished
observations).
We have observed that, in autumn,
high numbers of diatoms may occur in the mat.
[=I.
The isolation of a large number of cyanobacteria from one environment
gives the impression
of a highly diverse population
of cyanobacteria.
This is only partly true. Detailed microscopic observations
over several years have revealed that
only two species were of quantitative
importance
in the mat. The dominant
cyanobacteria
were
Oscillatoria from the type of strain 23 and Microcoleus chthonoplastes (strain 11). M. chthonoplastes
is the mat-builder
and responsible
for the tough
and leathery structure of established
mat systems
[22]. On the other hand, the pioneer organism is
Oscillatoria, found to be especially abundant
in
freshly colonized
sediments.
Depending
on the
season and the type of mat sampled, usually mixtures of both species are found [22]. However, no
Table 4
See legend of Table 3
Amounts
given.
in mg carbohydrate/mg
Enteromorpha
sp. was
chlorophyll
sampled
n +pheophytin
a. Also the carbohydrate
content
of extracts
of Enteromorpha
from the top of the mat from site 1.
sp.
Carbohydrate
Enteromorpha
Glycerol
0.6
3.3
4.3
Glucosylglycerol
0.2
26.0
19.1
Sucrose
3.2
1.4
1.4
Site
1
2
3
4
5
6
3.0
26
8.6
134
20.0
85
9.8
110
1.6
39
1.9
141
9.5
182
Trehalose
0
4.5
3.6
7.1
54
Glucose
0.4
0.6
0
0.3
0
0
0
sp. are
311
heterocystous
cyanobacteria
have ever been observed in the mat while Gloeocapsa, Synechocystis,
Spirulina and Oscillatoria from the type of strain
61, and several Phormidium-like
species are found,
but only in low numbers.
The composition
of the population
has been
shown to be correlated with nitrogen fixation [29].
Oscillatoria sp. is an aerobic nitrogen fixer whereas
M. chthonoplastes is not [20]. The species composition at the different sites is presented in Table 5.
Due to the fact that the two dominant
cyanobacterial species in this environment
contain different organic solutes the relative abundance
of
both species in a sample correlated with the ratio
of glucosylglycerol
and trehalose (Table 5). It is
suggested
that the relative abundance
of both
species in this environment
can be estimated
by
quantifying
the major organic solutes in mat samples.
The data presented
here unequivocally
show
that glucosylglycerol
is not unique as an osmoticum in marine cyanobacteria.
Whether
organic
solutes are linked to specific taxonomic groups of
cyanobacteria
remains uncertain
and requires a
thorough taxonomic
study in which osmotic adjustment should be considered.
ACKNOWLEDGEMENTS
We wish to thank K. Heselmeyer and H. Ufken
for their assistance in analyzing the field samples
and W.E. Krumbein
for his comments
on the
manuscript.
Thanks are also due to J.A. Chudek
for the ‘H NMR analyses.
Financial
support by a travel grant from the
Deutsche
Forschungsgemeinschaft
(Sta 234/2-l)
to LJS is greatly acknowledged.
Table 5
REFERENCES
Species composition of the microbial mat
Only the relative frequencies are given. Ratios of glucosylglycerol to trehalose were calculated on the basis of pigment
content of the mat (see Table 4).
111Carr, N.G. and Whitton, B.A. (1982). The Biology of
Site
Glucosylglycerol
trehalose
Cyanobacteria
PI
1
5.8
M. chthonoplastes
Oscillatoria sp.
++++
++
2
5.3
M. chthonoplastes
Oscillatoria sp.
Gloeocapsa sp.
++++
++
+
[41
[51
3
2.8
M. chthonoplastes
Oscrllatoria sp.
++++
++
4
1.6
M. chthonoplmtes
Oscillatoria sp.
Gloeocapsa sp.
Spin&to sp.
++++
++
+
+
M. chthonoplastes
Oscillatoria sp.
Gloeocapsa sp.
Spirulina
+++
++++
+
+
M. chthonoplastes
Oscillatoria sp.
Spidina
sp.
Synechocystis sp.
++
++++
+
+
5
6
1.0
0.6
+ + + + = dominant species;
+ + + and + + =
organisms present; + = only few organisms present.
(31
[61
[71
181
[9]
[lo]
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