EFFECTS OF SALINITY AND LIGHT INTENSITY ON

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EFFECTS OF SALINITY AND LIGHT INTENSITY ON
Egypt. J. Exp. Biol. (Bot.), 11(1): 21 – 29 (2015)
© The Egyptian Soc iety of Experim ent al Biology
RESEARCH ARTICLE
J el a n Mof eed
EFFEC T S OF S AL IN IT Y AN D L IGH T IN T EN SIT Y ON PR OD U CT ION OF ß C AR OT ENE AN D GLY C EROL FR O M T H E H AL OT OLER AN T ALG A D U N A LIELLA
SA L IN A ( C HLOR OPH Y T A) IS OL AT ED F R O M Z AR AN IK N AT UR E R E SER VE ,
N ORT H SIN AI, ( EGY PT )
ABSTRACT:
The hal otolerant green alga Dunaliella salina
(isolate SEA003) recently isolat ed from salt
march at Zaranik nature reserve, N orth Sinai ,
(Egypt) was cultivat ed in t he artificial
seawater m edium (ASW) with different
salinity levels (0.5, 1, 2, 3, and 4 M), under
continuous illumination of two different li ght
intensities: 50 and 200 µmol m - ²s - 1 , duri ng 30
day of incubation. The m aximum growth
density as number of cell was recorded in
m edia cont ained 2M NaCl under the high li ght
intensity, while Chlorophyll a giving its
maximum val ues at 1 M salinity under high
light intensity. Anent values of β carotene
produced by Dunalie lla salina reveal ed that, 2
M salinity had the superiority i n enhancing β
carotene production during the 30 days of this
study, especially under high light intensity. In
contrast
with
β
carotene
productivity,
Dunalie lla sa lina gave its maximum glycerol
values at the hi gh salinity (4M). This result
reveal ed that, the algal productivit y can be
controlled, as well as directed to produce a
particular substance (such as carotene and
glycerol) on a wide scale by controlling the
growth conditions. Adjusting salinity and li ght
intensity is li kely the best strategies to
achieve optimal β-carotene or glycerol
production i n mass cultures.
KEY WO RDS :
Dunalie lla, ß-carotene, glycerol, salinity, li ght
intensity
CO RRES PO NDENCE:
J el a n Mof eed
Suez University, Faculty of Fish Resources,
Departm ent of Aquatic Environment, SuezEgypt.
E-mail: [email protected]
ART ICLE CODE : 03.02.15
ISSN: 1687-7497
On Line ISSN:
INTRO DU CTIO N:
Th e
halotolerant,
uni cellular,
bi flagellat e alga Dunalie lla is distingui shed
m orphologicall y by absence of ri gid cell w all
(Ben-Am otz, 2009; Tran et a l., 2013; Rad et
al., 2015), how ev er it shows a rem arkable
degr ee of adaptation to a variet y of salt
conc entrations (from 0. 1 to 5.0 M NaCl) in
the aquati c environm ents (Alizadeh et al. ,
2015). Thus, this organism i s unique i n its
ability to adapt i n harsh environm ental
conditi ons. Duna lie lla speci es has th e a bility
to
accum ul ate
significant
amounts
of
carot en oids (Hadi et a l., 200 8; Minga zzini et
al., 20 15), proteins, vitamins (G hoshal et al. ,
2002) and it was also recomm ended as a
good source for gl ycerol production (B en Am otz, 2003) under st ress con ditions.
Current ly, the best comm ercial source of
natural β -caroten e (with economic val ue)
am ong all organisms in the world is
Dunalie lla salina (B en-Amotz, 1995; Rad et
al., 2015), which cont ains β -carotene up to
14% of its dry weight ( Aasen et al., 1969).
Th er efor e, producti on of β -carot ene by
m ass cultivation of D. sa lin a has b ei ng
m asterful in m any count ries includi ng China,
USA and Australia (Rad et al., 2015).
Th e m echanism by which Dunalie lla
cell adapt ed to bro ad s ali nity range b as ed
on the ability of the algal c ell to change its
internal
concentration
of
carotenoi ds
es pecial ly β-carotene and glycerol (Raja et
al., 20 07). W here, significant amounts of β carot en e accumulated i n the plastid as
dropl ets to prev ent the photo-dam ag e of
chlorophyll under stress conditions "include
hi gh tem perature, light intensities, salinity
and deficiency of nutrient" (Ben-A motz and
Shaish, 1992; Ben-Amotz, 2003). Betacarot en e accum ulates within the lipid
gl obules in the spac es b etw een thyl akoi ds
located in chloropl ast (Ben-Am otz, 2003).
Thus, it has b een prop os ed that th e incr eas e
in f atty acid content und er str ess condi tions
can b e partially attributed to the increase in
β-carotene (Mend oza et a l., 1999).
It is of interest to m enti on that, liver
en zym es oxidized β-carot en e com pound
produci ng vit am in A, whi ch pl ays a vi tal role
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Egypt. J. Exp. Biol. (Bot.), 11(1): 21 – 29 (2015)
40% of the c el l dry w eight) by Du na lie lla
in increasing the effi ci ency of the immunity
(Ben-Am otz, 1990). Nowadays, the pric e of
system and
act as anti oxidants agents
gl ycerol is dep ended on th e pri ce of crud e
b eside of that it is necessary for m any
oi l, where it is m ostly produc ed from
im portant physiological functions (Mishra
petrochemical sources. Ther efor e, rec ently
and Jha, 2 011). H ence, D una lie lla's βadvancing al gal biotech nology and m ass
caroten e can be used as h ealth dietary
culture cultivation of Duna lie lla sp. for
suppl em ents for both hum an and ani mals
gl ycerol and carot enoid production and other
(Pulz and G ross, 2004), cosm etic and also in
bi otechnological
purpos es i s of
great
sev eral promi si ng pharmaceutical industri es
interest.
Th
e
abilit
y
to
induc
e,
m
odify
and
(Borowi tzka, 1999; Chidam bara-Murthy et
scale up Dun aliella sp. to produce a s eries
a l.,
2005),
reducing
the
ri sk
of
of uncommon carotenoids of high nutritional
cardiovascular diseas es (Törnwal l et al.,
and medi cal valu e and glyc erol, also opens a
2004) and controlli ng cholesterol. In addition
new field in the ar ea of Duna lie lla
β-carotene can prev ent or even i nhibit
bi otechnology. It is nec essary to menti on
various types of t um ors ( Hallm ann, 2007;
that, am ong diff erent strains of D. sa lina,
Chattopadhya y et a l., 2 008). It is well known
ther e is no predictable unique co ndition for
that, all the β-carot ene's t herapeut ic effects
reaching
the m axim um
β-carotene
or
related to its protecti ve ability agai nst
gl
ycerol
cont
ents
per
unit
tim
e
and
per
unit
potenti all y
harmful
free
radicals
and
volume for al l strai ns. Wher e, ther e ar e great
stimul atory affects to the immunit y system
physiological variations in response to
(G otz et a l., 1999). Although, nat ural β di fferent inductive growth factors.
caroten e ca n be obt ained from m any other
sources like fruits, green l eafy v eg et ables
Th er efore, it was very im portant to
and fungi (Pulz and G ross, 2004) or ev en
determine th e optim al conditions of salinity
synthetically produc ed (Mayer a nd Isler,
and irradiance enhancing the production of
1971), the Dun aliella' s β-carotene has m ore
both β- carot en e and glyc erol f rom this strain
therapeutic
effects,
wher e
conc er ning
of D. salina, rec ently isolated from salt
isom ers com position, the synthetic β m arch at Zaranik nature r eserve, N orth
caroten e o nly contains al l -trans i som ers.
Sinai, (E gypt), as well as det erm ine its
W hil e, natural β -carotene pr esent in fruits
product ivity at differ ent stress conditions for
and vegetables compos ed m ainl y of a
m ass cultivation.
mixture from 9-cis and trans β-caroten e
(Ben-Amotz and Shaish, 1992) and it is
M ATERIAL AND M ETHO DS:
highly difficult in purificati on and obtain ed by
Th e m arine microal ga D unalie lla salina
relati vely low conc entrati on. It is well known
(Eukaryota,
Viridiplantae,
C hlorophyta,
that, the 9-ci s isom er is more efficient as
Chl orophyc ea e,
Chlamydom onadales,
antioxidant t han all -trans, wher e it is
Dunal iel laceae,
Duna lie lla,
Speci es:
D.
signifi cantl y m ore s oluble in lipid and thus i t
sa lina) isolated from sal t march at Zaranik
m ay be more eff ectivel y accum ul ated in
nature r eserve, North Si nai , (Egypt), was
hum an tissue (B en-Am otz et al., 1989).
generousl y donated by do ctor: Eltanahy E.
Henc e, the high er the 9-ci s conc entrati on,
G., from the C ulture Coll ection of Botany
the hi gh er is the anticanc er a nd antioxidant
Department , Faculty of Scienc e, Mansour a
efficiency (Hu et a l., 2008). In com parison,
Uni versi ty, Egypt. The t est isolate was
among all nat ural sources, Dunalie lla sa lina
identified by m orphological and genetic
produc e th e gr eat est am ount of 9 -ci s isomer
anal ys es. Duna lie lla salin a i solat e SEA003
(m ore than 50% of all i ts isom ers). In
was d eposited i n the GenBank und er th e
addition, a Duna lie lla cell can accum ulat e β acc essi on n um ber JX220893.1 (Eltanahy et
caroten e, thousands of t im es m ore than a
al., 2015).
carrot cell (Klausner, 1986). In summary,
Duna lie lla s alin a is consider ed as th e b est G rowth Conditions:
known β -caroten e natural source (BenDuna lie lla s alina was cultivated in t he
Am otz, 2003; Rad et al., 2015), because i t
arti ficial seawat er m edium (ASW) which
has hi gh commerci al value wh er e the
cont ained: 4.5 m M MgCl 2 . 6 H 2 O, 3 mM
cultivation of microalgae and purification
CaC l 2 . 2H 2 O , 1.5 m M N aVO 3 , 5 mM KNO 3 ,
wit h organic solvents i s eco nom icall y cheap.
0.13 m M K 2 HPO 4 , 0.5 m M MgSO 4 . 7 H 2 O,
Indeed, the natural β -carot en e h as a m arket
0.02 mM ED TA, 0.02m M FeCl 3 , 1 mg l - 1 trace
price approxim ately double or tri ple m ore
el em ent stock with 10 m M MnCl 2 .4H 2 O,
than that of the synth et ic products (Pulz and
CuS O 4 . 5H 2 O , 25 m M NaHCO 3 , 50 m M
G ross, 2004).
H 3 BO 3 ,
2m M
NaMoO 4 .2H 2 O,
0.8
mM
O n the ot her hand, glyc erol i s a vital
ZnSO 4 .7H 2 O, 0.2 m M CoCl 2 .6H 2 O. Different
comm ercial
organic
chem ical
act
in
NaC l conc entrati ons (0.5, 1, 2, 3 and 4 M)
osm oregulation besid e using in
food,
wer e added to on e l itter of m edium . The p H
cosm etic and pharm aceutical industries, as
value w as adjust ed at 7.5 by addition of 40
well as it is also used as ant i -drying ag ent
m M of Tris -buffer. All stock solutions wer e
(Shariati, 2003). Under stress ci rcumstances
st erilized s eparat el y for 30 minutes and
of high sali nit y, glycerol accum ulat ed (up to
pool ed asept ically, in order to avoid
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Mofeed J, Effects of Salinity and Light Intensity on Production Of ß-Carotene and Glycerol Dunaliella Salina
precipi tati on of certain com pounds (Hejazi et
a l., 2003). After autoclaving, t he c ulture
flasks wer e lef t to cool at room temperatur e
for one day to al low for pH stabilizati on at
7.5 (Olmos et al., 2000). To ac hieve an
initial cel l density of 4000 cel l ml - 1 , 10 ml of
acti vely growing veg etative inocul um s of
Duna lie lla s alina c ells wer e inoc ulated into
steri lized fl asks containi ng 250 ml of ASW
m edium. Flasks wer e incubat ed at 28 ± 1° C
und er cont i nuous il luminat ion (24 h light) of
two different l ight intensi ties: 50 µm ol m - ²s - 1
(low light intensity) and 200 µm ol m - ²s - 1
(high
light
int ensit y)
by
cool -white
fluorescent lam ps (Philips, 50 w). To ensur e
a uniform i llumi nation of the cells, the m edia
wer e sha ked m anually twice a day. The
exp erim ent w as perform ed in triplicat e for 30
days, and all exp erim ents w er e repeat ed at
least twi ce.
G rowth Estimation:
Cell num ber w as d et ermined by di rect
counti ng
und er
a
light
microscope
(m agnificati on × 40) by usi ng a 0.1 mm deep
counti ng cham ber "haem ocytom et er" (Jam es
and Al -Khars, 1990). O nce in ev ery 5 days
the s am ples w er e col lected a nd cell counting
was perform ed. Since the alga is motil e, one
drop of Lugol’s iodi ne solut ion was ad ded to
arrest the m otilit y.
Estim ation of Chlorophyll a:
Chlorophyll extraction was carried out
according to Pistal and Lel e (2005). Four ml
aliquots sample from eac h cul ture m edium of
Duna lie lla salina cultures w er e centrifuged
at 5,000 rpm f or15 min. The pr ecipitated
p ell ets wer e was hed wi th distill ed wat er,
then susp ended i n an aceton e/w at er solut ion
(80:20 v/v) for 30 mi n. after that it was
thoroughly vortexed for 5 m in. to ensur e
com pl ete extraction and t hen cent rifuged
again for 5 m in at 5000 rpm to extract
pigm ents until t he pell ets turned cl ear/white.
Th e am ount of ext racted pi gm ents in the
solvent
p hase
was
sp ectrophotometric
quantified using method d escri bed by
Lichtenthaler and W ellburn (1985) :
-1
Chl a (µg m l ) = 11.75 (A 6 6 2 ) – 2.35 (A 6 4 5 )
Determ ination of β-C arotene Content:
Content s of β -carot ene in ea ch sam ple
wer e
d et ermined
spectrophotometrically.
Four ml from the a qu eous pha se of each
Duna lie lla culture m edium was centrifuged at
4000 rpm for 15 min, discardi ng supernatant
and then, each pr ecipitated pellet was add ed
to 4 mL (75%) aceton e soluti on, shack ed
until separati on. The abov e proc ess es
repeat ed till the ext ract turned to whit e, then
1/10 volume of 60% KO H was added at
49°C, to 10 ml volum etri c flask, the
sup ernatant
without
chlorophyll
was
transferred an d 2ml aceton e was add ed to
the vol um e. After det ermining the A 4 5 3 value,
the conc entration (A 4 5 3 ) of the β -caroten e
ISSN: 1687-7497
21
solution could b e fou nd from the standar d
curve. Standard curv e w as prepar ed by
di ssol ving
differ ent
conc entrati ons
of
st andard β -carot en e in pur e ac eto ne and
m easured at
453
nm
w hich is t he
characteristic absorpt ion for β -caroten e.
G raph was constructed by pl otti ng t he carot en e conc entrati ons on X a xis and O D
values
on
Y-a xi s
(Semenenko
and
Abdullaev, 1980).
Determi nati on of G lycerol Content:
Four ml of the growth culture of D.
sa lina was c entrifugated at 1 500 rpm for 10
min at room temperature, and th en th e
precipit ated pellets wer e wash ed t wice in a
solution of 1.5 M NaCl and 5 mm phosph at e
buffer at pH 7.5. O ne ml of peri odat e
rea gent and 2.5 ml of acetylaceton e r eag ent
wer e added to 200 µl of precipitated pellets,.
Th e mi xture was incubat ed at 4 5 o C for 20
min.
Aft er
that,
optical
density
was
determined at 410 nm . Results wer e
com pared w ith the standard curve prepar ed
by using known amount of glycerol (Chitl aru
and P ick, 1991).
Statistical Anal ysis:
Th e d ata obtain ed ar e r epr es ented as
Mean ± Standard d eviati on (m ean ± SD).
Th e si gnificance of the di fferenc e betw een
the grou ps was calcul ated by one-w ay
anal ysis of variance (ANO VA) using the
SPSS-PC
com puter
software
packag e
version 10.
RESULTS:
The
effect
of
different
salt
concentrations on cell number of Dunaliella
salina under bot h high (200 µmol m - ²s - 1 ) and
low (50 µmol m - ²s - 1 ) light intensities were
st udied. Inspection of Fig.1 revealed that,
growth density as number of cell, reached its
maximum (8.97 cell X10 6 ml - 1 ) after 30 days
of incubation in medi a contained 2M NaCl
under the high light intensity. Meanwhil e, t he
sam e sampl e under l ow light intensity gave
lower cell number (2.68 cell X10 6 ml - 1 ),
however the maximum density gained under
the low light intensity was considered duri ng
entire peri od of i nvesti gation. In contrast to
the above, low cell density were recorded in
0.5 M salinity, follow ed by that values
recorded within 1M and 4M respectively.
Concerning the incubation peri od, it is
noticeabl e that, the first ten days had
convergent the l owest cell number (Fig. 1) in
all param eters. G enerally, compared w ith t he
other salt concentrations, the 2 M salinity
supported the Dunaliella growth during t he
entire peri od of i nvestigation under the two
light intensities after the first t en days. Also it
is of interest to mention that, 2M salinity
significantly+ enhanced growth of D. salina
over the other NaCl concentrations under
hi gh light intensity duri ng the incubati on
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Egypt. J. Exp. Biol. (Bot.), 11(1): 21 – 29 (2015)
period, however i n case of low li ght intensity;
the growt h was higher but with val ues closely
to that recorded in 3 M salinity.
1M
2M
3M
1M
2M
3M
4M
Low light intensity
2.5
2
4M
mg l
-1
0.5 M
0.5 M
High light intensity
10
1.5
1
6
0.5
4
0
6
cell X10 ml
-1
8
5
2
10
15
20
25
30
Time (days)
0
15
20
25
Fig. 2. Effect of different salinity concentrations on
chlorophyll "a" (mg l-1) in Dunaliella salina under high
and low light intensity during 30 days
30
Time (days)
0.5 M
3
1M
2M
3M
4M
Low light intensity
2
6
cell X10 ml
-1
2.5
1.5
1
Time (day)
0.5
0
5
10
15
20
25
30
-0.5
Time (days)
Fig. 1. Effect of different salinity concentrations on
Dunaliella salina cell number (cell X106 ml-1 ) under
high and low light intensity during 30 days
A glance on figure 2 reveal that
Chlorophyll "a" giving its maximum values in
1 M salinity under both high and low li ght
intensity (0. 26 - 4.88 mg l - 1 - 0.07 -2.2 mg l - 1 ,
respectively),
followed
by
that
values
recorded at 2 and 3 M of salinity respectively
during the entire period of investigation. In
this context, the high intensity of li ght
enhanced chl orophyll "a" production more
than the low light intensity. However, both 0.5
and 4 M NaCl suppressed the chlorophyll a
production in tested Dunaliella salina. It is
noticeable that, generally the chlorophyll "a "
values under the hi gh light intensity were
higher t han that recorded under low li ght
intensity.
Anent values of β carotene produced
by Dunalie lla sa lina the results showed that,
2 M sali nity had t he superiority in enhanci ng
β carotene production during the 30 days of
this study, giving si gnificant high values
especially under high light intensity (2.41 34.62 mg l -1 ) f ollowed by that val ues recorded
with 3M salinity (1.41- 18.91 mg l - 1 ) and then
1M (0.77- 17.02 mg l - 1 ) under the sam e
conditi ons (Fig. 3). Neverthel ess, generally
low light intensit y enhanced β carotene
productivity at all NaCl concent rations but
with lower rate from that recorded at high
light intensity, however at low i ntensity t he
difference b et ween the recorded values with
all NaCl concentrations were convergent,
where it produced 14.2, 12.48, 10. 03, and
9.92 mg l - 1 after 30 days with 2M, 3M, 1M and
4M, respectively.
0.5 M
40
1M
1M
2M
3M
3M
4M
30
25
20
15
10
5
0
5
-5
10
15
20
25
30
Time (days)
0.5 M
0.5 M
2M
High light intensity
35
-1
10
mg l
5
-2
1M
2M
3M
4M
4M
Low light intensity
High light intensity
6
16
14
5
-1
4
mg l
10
mg l
-1
12
3
8
6
4
2
2
0
1
5
10
15
20
25
30
Time (days)
0
5
10
15
20
Time (days)
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30
Fig. 3. Effect of different salinity concentrations on βcarotene (mg l-1) yield of Dunaliella salina under high
and low light intensity during 30 days
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Mofeed J, Effects of Salinity and Light Intensity on Production Of ß-Carotene and Glycerol Dunaliella Salina
0.5 M
12
1M
2M
3M
2M
3M
4M
8
6
4
2
0
5
0.5 M
1M
High Light intensity
10
% of carot./chloro.
In contrast with β carotene productivity,
Dunalie lla sa lina gave its maximum glycerol
values (48.49 - 31.7 mg l - 1 under bot h, high
and low li ght intensity, respecti vely) within
the high salinity (4M). Compared with this
high values (4.06- 48.49 mg l - 1 ) of glycerol
relat ed to high sali nity concentration, the
recorded values in 0.5M (0.12 - 2.91 mg l - 1 )
are very l ow. Again, comparabl e t o β
carotene, the m aximum values of glycerol
were recorded under high light intensity
especially
withi n
the
highest
NaCl
concentrations (Fig. 4).
10
15
4M
20
25
30
Time (days)
High light intensity
60
0.5 M
-1
4M
8
10
0
5
10
15
20
25
30
Time (days)
0.5 M
1M
2M
3M
4M
% of carot./chloro.
mg l
3M
9
30
20
7
6
5
4
3
2
Low light intensity
1
0
30
5
10
15
20
25
30
Time (days)
25
mg l-1
2M
10
40
35
1M
Low Light intensity
50
Fig. 5. Ratio of β-carotene : chlorophyll "a" in Dunaliella
salina under high and low light intensity during 30
days
20
15
10
0.5 M
1M
2M
3M
4M
High Light intensity
5
6
0
15
20
25
30
Time (days)
Fig. 4. Effect of different salinity concentrations on glycerol
(mg l-1) yield of Dunaliella salina under high and low
light intensity during 30 days
The consi derable variation between β
carotene,
Chlorophyll
a
and
glycerol
production within the different tested salinity
concentrations
during
the
period
of
investigati on, refl ect the importance of
focusing on the ratios between β carotene
and both of Chlorophyll a and glycerol. As
regard in figure 5, the β carotene/Chlorophyll
ratio under both high and low light i ntensiti es
following the sam e approach of high values,
where it fluctuated from 9.82 to 5.49 under
high light i ntensity and from 9.66 to 6 under
low li ght intensity with high salinity (2, 3, and
4 M).
How ever,
within l ower
salinity
concentrations (0.5 and 1 M) it tended to give
lower values. Nevertheless, the situation was
reversed in case of β carot ene/Glycerol ratio,
where the cited results revealed that low
NaCl concentrations gai ned the m aximum β
carotene/Glycerol ratio (Fig. 6). Meanwhile,
minimum val ues were related to the high
NaCl concentrations, under both, high and
low light intensity, duri ng the i nvestigation
period.
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% of carot./glyc.
10
4
3
2
1
0
5
10
15
20
25
30
Time (days)
0.5 M
3.5
1M
2M
3M
4M
Low Light intensity
3
% of carot./glyc.
5
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
Time (days)
Fig. 6. Ratio of β-carotene : glycerol in Dunaliella salina
under high and low light intensity during 30 days
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Egypt. J. Exp. Biol. (Bot.), 11(1): 21 – 29 (2015)
DISC USSIO N:
O n the contrary to other chlorophyta,
Dunalie lla's cells have no rigi d cell w all, but
only enclosed by an el astic plasma membrane
covered by a m ucus surface layer, which
facilitates rapid swelling or shrinking when
subjected
to
hypotonic
or
hypertonic
condition, (Ben-Amotz, 2003), t hen it rapidly
responds to any change in osmotic pressure
by changing t he vol um e of the cell (R ad et
a l., 2015). Hence, Dunaliella sp ecies can
physi cally resi st 3 t o 4 fold decreases or
increases i n osmotic pressure (which know as
osmoregulation).
Nevertheless,
more
hypoosmotic stress will lead to bursti ng of the
cell, while the hyperosmotic stress causes
irreversible shrinkage ( B en-Amotz and Avron,
1992; Pri eto et al. , 2011; Suong et al. , 2014;
Mingazzini et al., 2015). In the present study,
the m aximum cell number (8.97 - 4.05 - 2.68 2.42 cell X 10 6 ml - 1 ) of the recent ly i solated
strain of Dunalie lla sa lina recorded within
m edia contains 2M NaCl under both high (200
µmol m - ²s - 1 ) and low (50 µmol m - ²s - 1 ) li ght
intensity followed by that r ecorded in 3M
NaCl concentration during entire p eri od of
investigati on. Meanwhil e, the lower salinity
(0.5 and 1 M) responsible for the minimum
growth, which reflected that l ow salinity, was
inappropriate for the growth of this strain of
D. salina. This agreed with Hadi et al. (2008)
results, who reported that the optimal growth
of Dunalie lla sa lina was obtained at 2 M
NaCl.
Chlorophyll i s the pigm ent of lif e in the
photosynthetic organisms, therefore this
study interested with t he influence of li ght
intensity and salinity on chl orophyll "a"
production. The cited results revealed that,
Chlorophyll a giving its maximum values
within 1 M salinity under both, high and low
light
i ntensity.
However
0.5
M
NaCl
suppressed the chlorophyll a production in
Dunalie lla salina. Previous studies have
descri bed that, salinity exerted inhi bit ory
effects on the activities of plastid's thylakoid
m embrane and some solubl e enzym es of
Dunalie lla (Finel et al., 1984). Rad et al.
(2015) also reported t hat, hypo-osmotic
shocks stim ulate a tem porary inhi bition of
photosynthesis
and
then
the
main
photosynthetic pigment (chlorophyll), while
hyper-osmotic shock induce a subst antial
inhibition of photosynthesi s, but stimulate the
glycerol synthesis. Since li ght intensit y is a
major contributi ng f actor for the pigm ent
production, the present study demonstrated
that, high light i ntensity enhance chl orophyll
a production than the low light intensity, the
result which agreed with the results obtained
by Li u and Shen (2004) in their study on D.
salina.
β-carotene
considered
as
extraphotosynthetic product, stored and accumulated
ISSN: 1687-7497
to protect cells agai nst chlorophyll catalyzed
single reacti ve oxygen and possibly other
excited chlorophyll damaging agents, and
also to protect the cell against the deleterious
ef fects of high r adiation intensity by acti ng
as a light filter or a screen to absorb
excessi ve irradiation (Ben-Amot z and A vron,
1992; Ben-Amot z, 2003 & 2009), where the
possible reasons for increasing the synthesis
of β-carotene in Dunaliella seems to be
photo-protection
through
its
absorption
properties. The oily nature of the cup shaped
chloroplast i s most efficient for this purpose.
Prieto et al. (2011) also indicated that, β carotene has various roles include acti ng as
pro-vitamin "A", absorbi ng light energy,
antioxi dation activity, oxygen transport and
general colorizing agent for many organisms.
Maximizing the production of β-carotene
per unit time in mass culture of the halotolerant green algae Dunaliella, have been
experim ented by several strategi es. These
strategi es based on the hypothesi s that,
severe physiological growth param eters, such
as high li ght int ensity (Coesel et al. , 2008),
hi gh salinity (Hadi et al., 2008), tem perat ure
(Ben-Amotz, 2009; Mingazzini et al., 2015),
and nutrient defi ci ency (Shariati, 2003; Aghaii
and Shariati, 2007) i nduce the rate of
synthesis and the accumul ati on of significant
am ounts of β-carotene (up to 14% of dry
wei ght).
Anent,
values
of
β-carotene
produced by the tested strain of Dunaliella
salina i n thi s study reveal that, 2 M salinity
had the superiority in enhancing β carotene
production during the 30 days of thi s study,
givi ng significant high values especi ally under
hi gh light intensity. Pisal and Lel e (2005) also
indicated t hat β-carotene can be greatly
enhanced at higher irradiation. Hence, it was
thought desirable t o explore the possi bility of
using higher light stress for carotenogenesis.
O n the other hand, salinity affected the β
carotene production as shown in cited results.
Ben-Amotz (2009) i ndicated that, t he highest
accumul ation of anti-oxidant vitamin A (βcarotene) in Dunalie lla sa lina was observed
when it subj ected to high intensity of light in
m edia containing high salinity (30%).
Besi de, β-carotene and under stress
conditi ons of salinity, Dunaliella species
possess the ability to accumulate significant
am ounts of glycerol (Hadi et al., 2008;
Hossei ni Tafreshi and Shariati, 2014). The
m echani sm by which Dunalie lla cells can
balance the ext racellular osmotic stress by
wide range of NaCl (from 50 mM up to 5 M
NaCl) based on the ability of t he microalga to
modify its intracell ular gl ycerol concentration
(Shariati, 2003, Raj a et al., 2007; Hadi et al.,
2008). In fact, when Dunalie lla c ells grown at
hi gh salinity, t he intracellular concentration of
glycerol i ncreased up to 50% and is sufficient
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Mofeed J, Effects of Salinity and Light Intensity on Production Of ß-Carotene and Glycerol Dunaliella Salina
to account for essentially all of t he osmotic
pressure required t o balance the extracellular
osmolarity,
while
the
i nternal
NaCl
concentrations remaini ng very low (Hosseini
Tafreshi and S hariati, 2014). Som e studi es
have confirm ed that, m easurements of
freezing point depressi on in cell sap of D.
salina indicated that, the i nternal osmotic
concentration is higher t han that exp ected
due to the presenc e of t he i ntracellular
glycerol (Li u and Shen, 2004; C hen et al.,
2011),
the
phenom ena
known
as
osmoregulation. I n this condition, glycerol
acts as a ‘compatible solute’ that protects
enzym es
against
both
i nhibition
and
inactivation (Hosseini Tafreshi and Shari ati,
2014). The m aximum values of glycerol in the
present study related to high salinity (4M)
under both, high and low light intensity.
G enerall y, the concentration of glycerol in
Dunalie lla sa lina increase with increasing
salinity, whi ch are consistent with the
previous hypothesis.
Glycerol produced in Dunaliella ei ther
by photosynthetic carbon di oxide fixation or
by degradati on of starch. The contribution of
these tw o m et abolic pathways to glycerol
synthesis mainl y depends on t he degree of
salinity stress, the availability of light and the
reserved starch. Hossei ni Tafreshi and
Shari ati (2014) reported that, both the
glycerol
synthesis
under
hypertonic
conditions and its elimination under hypotonic
condition occur in the dark or light (Rad et
a l., 2015). I n the dark, Dunaliella produces
glycerol by degradati on of starch, and the
capacity of the cells to recover from
hyperosmotic shock depends on the am ount
of reserved starch. However, i n the li ght the
hyperosmotic shock greatly encourage the
production rate of glycerol and also stimul ate
starch degradation, w hich indicate that, in the
light bot h the t wo metabolic pathways have
significant contributions in the production of
glycerol (Hossei ni Tafreshi and Shari ati,
25
2014; Tran et al., 2014). This hypothesis
greatly support ed the results of this study
where, t he maximum values of glycerol w ere
recorded under high light intensity especially
within high salinity concentrations. Meanwhile
the m inimum concentrations were obtained
under low Light i ntensity.
The β carotene/ Chlorophyll ratio under
both, hi gh and l ow light intensity following the
sam e approach of hi gh values, w hich reflect
that β carotene production increase at that
concentration. However, within lower salinity
concentrations (0.5 and 1 M) it tended to give
low values proving that t he production of β
carotene was rel ated to salinity stress.
O n the other side, in case of β carotene
/Glycerol ratio, w here the cited results reveal
that, low NaCl concentrations gained t he
maximum
β
carotene/Glycerol
ratio.
Meanwhile, minimum values were rel ated to
the high NaCl concentrati ons under both, high
and low light i ntensity duri ng the investigation
period, which reflect that the cell has shift ed
its activit y to produce gl ycerol by increase in
sali nity level. Therefore logically, from a
comm ercial point of view, the best strains of
D. salina for use in mass cultivation should
have the m aximum specific growth rate and
the highest β-carotene and/or glycerol
content per unit tim e and culture vol um e
under optimized conditions (Priet o et al.,
2011).
CO NCLUSION:
In this context, the algal productivity
can be controlled as w ell as directed to
produce a particular subst ance (such as
carotene and glycerol) on a wide scale by
controlling the growth conditions. Adjusting
sali nity and li ght i ntensity is likely the ideal
strategi es to achieve optimal β-carotene or
glycerol producti on in mass cultures of
Dunalie lla salina.
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‫ﺗﺄﺛﯿﺮ اﻟﻤﻠﻮﺣﺔ وﺷﺪة اﻻﺿﺎءة ﻋﻠﻲ اﻧﺘﺎج ﻛﻼ ﻣﻦ ‪ -β‬ﻛﺎروﺗﯿﻦ واﻟﺠﻠﺴﺮول ﻣﻦ طﺤﻠﺐ اﻟﺪوﻧﯿﺎﻟﯿﻼ‬
‫ﺳﺎﻟﯿﻨﺎ اﻟﻤﻘﺎوم ﻟﻠﻤﻠﻮﺣﺔ )اﻟﺴﻼﻟﺔ اﻟﻤﻌﺰوﻟﺔ ﻣﻦ ﻣﺤﻤﯿﺔ اﻟﺰراﻧﯿﻖ‪ ،‬ﺷﻤﺎل ﺳﯿﻨﺎء‪ ،‬ﻣﺼﺮ(‬
‫ﺟﯿﻼن ﻣﻔﯿﺪ‬
‫ﺟﺎﻣﻌﺔ اﻟﺴﻮﻳﺲ‪ ،‬ﻛﻠﯿﺔ اﻟﺜﺮوة اﻟﺴﻤﻜﯿﺔ‪ ،‬ﻗﺴﻢ اﻟﺒﯿﺌﺔ اﻟﻤﺎﺋﯿﺔ‪ ،‬اﻟﺴﻮﻳﺲ‪ ،‬ﻣﺼﺮ‬
‫ﻳﻌﺘﺒﺮ طﺤﻠﺐ ‪ Dunaliella‬اﻟﻤﻘﺎوم ﻟﻠﻤﻠﻮﺣﺔ اﺣﺪ اﻟﻤﺼﺎدر‬
‫اﻟﻄﺒﯿﻌﯿﺔ اﻟﮫﺎﻣﺔ ﻹﻧﺘﺎج ﻣﺮﻛﺒﺎت اﻟﺒﯿﺘﺎ‪ -‬ﻛﺎروﺗﯿﻦ واﻟﺠﻠﯿﺴﺮول ھﺬا‬
‫ﺑﺎﻹﺿﺎﻓﺔ اﻟﻲ ﻣﺮﻛﺒﺎت ﺣﯿﻮﻳﺔ اﺧﺮي‪ .‬ﻟﺬا ﻓﻘﺪ ﻋﻨﯿﺖ ھﺬة‬
‫اﻟﺪراﺳﺔ ﺑﺘﺤﺪﻳﺪ ﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ و ﻛﺬا ﺷﺪة اﻹﺿﺎءة اﻟﺘﻲ‬
‫ﺗﻌﺰز إﻧﺘﺎﺟﯿﺔ ﻛﻼ ﻣﻦ اﻟﺒﯿﺘﺎ‪ -‬ﻛﺎروﺗﯿﻦ و اﻟﺠﻠﯿﺴﺮول ﻣﻦ ﺳﻼﻟﺔ‬
‫ﻟﻄﺤﻠﺐ ‪ Dunaliella salina‬ﺗﻢ ﻋﺰﻟﮫﺎ ﺣﺪﻳﺜﺎ )‪(isolate SEA003‬‬
‫ﻣﻦ ﻣﻼﺣﺎت ﺑﻤﺤﻤﯿﺔ اﻟﺰراﻧﯿﻖ‪ -‬ﺷﻤﺎل ﺳﯿﻨﺎء –ﻣﺼﺮ‪ .‬ﺗﻀﻤﻨﺖ‬
‫اﻟﻨﺘﺎﺋﺞ اﻟﻤﺘﺤﺼﻞ ﻋﻠﯿﮫﺎ ﻛﻼ ﻣﻦ ﻋﺪد اﻟﺨﻼﻳﺎ و ﻧﺴﺒﺔ ﻛﻠﻮروﻓﯿﻞ أ‬
‫و ﻛﺬاﻟﻚ ﺗﺮﻛﯿﺰ اﻟﺒﯿﺘﺎ‪ -‬ﻛﺎروﺗﯿﻦ و اﻟﺠﻠﯿﺴﺮول ﺧﻼل ‪ 30‬ﻳﻮم ﻣﻦ‬
‫اﻟﺰراﻋﺔ‪ .‬أﺑﺮزت اﻟﻨﺘﺎﺋﺞ ان أﻋﻠﻲ ﻛﺜﺎﻓﺔ ﻟﻠﻨﻤﻮ )ﻛﻌﺪد ﺧﻼﻳﺎ( ﻗﺪ‬
‫ﺳﺠﻠﺖ ﻋﻨﺪ درﺟﺔ ﻣﻠﻮﺣﺔ ‪ 2M‬ﺗﺤﺖ ﺷﺪة اﻻﺿﺎءة اﻟﻌﺎﻟﯿﺔ ﺑﯿﻨﻤﺎ‬
‫ﺳﺠﻞ أﻋﻠﻲ ﻣﻌﺪل ﻻﻧﺘﺎج اﻟﻜﻠﻮروﻓﯿﻞ ﻋﻨﺪ درﺟﺔ اﻟﻤﻠﻮﺣﺔ ‪1M‬‬
‫ﻋﻨﺪ ﻧﻔﺲ ﺷﺪة اﻻﺿﺎءة ﺗﻠﯿﮫﺎ اﻟﻘﯿﻢ اﻟﻤﺴﺠﻠﺔ ﻋﻨﺪ ‪ 2M‬ﺛﻢ ‪3M‬‬
‫‪ .‬ﻓﻲ ﺣﯿﻦ أﻋﻄﺖ ھﺬة اﻟﺴﻼﻟﺔ أﻋﻠﻲ إﻧﺘﺎﺟﯿﺔ ﻟﻠﺒﯿﺘﺎ ‪ -‬ﻛﺎروﺗﯿﻦ‬
‫ﻋﻨﺪ درﺟﺔ ﻣﻠﻮﺣﺔ ‪ 2M‬ﺗﻠﯿﮫﺎ اﻧﺘﺎﺟﯿﺘﮫﺎ ﻋﻨﺪ ‪3M‬ﺛﻢ ‪4M‬ﻋﻨﺪ‬
‫ﺷﺪة اﻻﺿﺎءة اﻟﻌﺎﻟﯿﺔ ‪ .‬وﻋﻠﻲ اﻟﻨﻘﯿﺾ ﻓﻘﺪ ﺳﺠﻠﺖ أﻋﻠﻲ ﻗﯿﻢ‬
‫‪http://my.ejmanger.com/ejeb/‬‬
‫ﻣﻦ اﻟﺠﻠﺴﺮول ﻟﮫﺬة اﻟﺴﻼﻟﺔ ﻋﻨﺪ درﺟﺔ اﻟﻤﻠﻮﺣﺔ اﻟﻘﺼﻮي‬
‫)‪ .(4M‬و ﺑﮫﺬا ﻳﻤﻜﻦ اﻟﺠﺰم أﻧﺔ ﻳﻤﻜﻦ ﺗﻮﺟﯿﺔ اﻧﺘﺎﺟﯿﺔ اﻟﻄﺤﺎﻟﺐ‬
‫ﺑﺼﻔﺔ ﻋﺎﻣﺔ و ھﺬا اﻟﺠﻨﺲ ﺑﺼﻔﺔ ﺧﺎﺻﺔ ﻻﻧﺘﺎج ﻣﻮاد ﻣﻄﻠﻮﺑﺔ‬
‫أﻣﺜﻠﺔ اﻟﺒﯿﺘﺎ‪ -‬ﻛﺎروﺗﯿﻦ واﻟﺠﻠﯿﺴﺮول ﻋﻠﻰ ﻧﻄﺎق واﺳﻊ ﻣﻦ ﺧﻼل‬
‫اﻟﺘﺤﻜﻢ ﻓﻲ ظﺮوف اﻟﻨﻤﻮ‪ .‬وﻻ ﻳﻐﯿﺐ ﻋﻦ أذھﺎﻧﻨﺎ أن اﻟﻤﻨﺎطﻖ ذات‬
‫اﻟﻤﻠﻮﺣﺔ اﻟﻌﺎﻟﯿﺔ ھﻲ ﻣﻨﺎطﻖ ﻓﻘﯿﺮة اﻹﻧﺘﺎج ﻋﻠﻲ ﺟﻤﯿﻊ اﻷﺻﻌﺪة‬
‫و ﻏﯿﺮ ﻣﺴﺘﻐﻠﺔ و ﺑﺬاﻟﻚ ﻧﻜﻮن ﻗﺪ ﺗﻤﻜﻨﺎ ﻣﻦ اﻹﺳﺘﻔﺎدة ﻣﻨﮫﺎ ﻋﻦ‬
‫طﺮﻳﻖ إﻧﺘﺎج ﻣﻮاد ﺣﯿﻮﻳﺔ ھﺎﻣﺔ ﻣﻦ ﺧﻼل إﺳﺘﻐﻼل ھﺬا اﻟﺠﻨﺲ‬
‫ﻣﻦ اﻟﻄﺤﺎﻟﺐ اﻟﻤﻘﺎوﻣﺔ ﻟﻠﻤﻠﻮﺣﺔ‪.‬‬
‫اﻟﻤﺤﻜﻤﻮن ‪:‬‬
‫أ‪.‬د‪ .‬ﻣﺤﻤﺪ اﻷﻧﻮر ﻋﺜﻤﺎن‬
‫أ‪.‬د‪ .‬ﻋﻔﺖ ﻓﮫﻤﻲ ﺷﺒﺎﻧﻪ‬
‫‪On Line ISSN: 2090 - 0503‬‬
‫ﻗﺴﻢ اﻟﻨﺒﺎت‪ ،‬ﻋﻠﻮم طﻨﻄﺎ‬
‫ﻗﺴﻢ اﻟﻨﺒﺎت‪ ،‬ﻋﻠﻮم اﻟﻘﺎھﺮة‬
‫‪ISSN: 1687-7497‬‬

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