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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2004/06–1–49–57
http://www.ijab.org
Production of Antioxidant by the Green Alga Dunaliella salina
HANAA H. ABD EL-BAKY1, FAROUK K. EL BAZ AND GAMAL S. EL-BAROTY†
Agricultural Division, National Research Center, Dokki, Cairo, Egypt
†Department of Biochemistry, Faculty of Agriculture, Cairo University, Egypt
1Corresponding author’s e-mail: Abdelbakyh@hotmail.com
ABSTRACT
The variation of the lipophilic (carotenoids and α-tocopherol) and hydrophilic (glutathione and ascorbic acid) antioxidant
contents, and the activities of antioxidant enzyme such superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), as
well as cellular malonaldehyde and stable radicals of D. salina in response to ultraviolet B (UV-B radiation 290-320 nm) and
secondary carotenoid induction conditions (nitrogen starvation and high NaCl concentration) were examined. The results
indicate that nitrogen deficiency combined with NaCl stress and UV-B irradiated is potential increase of both lipophilic and
hydrophilic antioxidant contents. Also, HPLC analysis of carotenoids extracts showed that D. salina accumulated significant
quantities of β–carotene and secondary carotenoids, mainly astaxanthin and zeaxanthin. Furthermore, the activity of
antioxidant enzymes CAT, SOD and POD showed a positive significant correlation with the antioxidant content and with the
exposured UV-B irradiance. Cellular malondialhyde content and quantities of alkyl radical-PER signal indicators of lipid
peroxidation were much higher in irradiant cells compared to unirradiant cells. These result revealed that D. salina had high
resistance to environmental conditions. These qualities therefore make D. salina good candidates for successful culture in
open ponds to production of useful materials, such as β–carotene, astaxanthin, zeaxanthin, ascorbic acid and α-tocopherol.
Also, it could be used to provide a rich source of such antioxidant for health foods.
Key Words: Dulinaliella salina; Nitrogen; Salt stress; Carotenoids; Tocopherols; UV-B radiation; Antioxidant enzyme
INTRODUCTION
Use of microalgae for human consumption as a source
of high value health food, functional foods and for
production of biochemical products, such as vitamins,
carotenoids, phycocyanin and polyunsaturated fatty acids
including the omega-3 fatty acids have been developed
(Richmond, 1986; Borowitzka, 1992; Pugh et al., 2001).
Among the various microalgae that have been explored for
their suitability for commercial potential: Dunaliella
species, Chlorella species and Spirulina species are three
major type that have been used successfully to produced
high concentrations of valuable compounds such as lipids,
protein and pigments (Abe et al., 1999; El-Baz et al., 2002;
Abd El-Baky et al., 2002). Dunaliella species are able to
accumulate large amount of carotenoids (Ben-Amotz et al.,
1982; El-Baz et al., 2002). D. salina contains up to 10% β-
carotene on dry weight basis when grown under stress
conditions including: high salt concentration, high light
intensity and nitrogen limitation (Ben-Amotz & Avron,
1988; El-Baz et al., 2002). β-carotene and other carotenoids
(astaxanthin & lutein) are integral part of the photosynthetic
apparatus in algae and functions as accessory pigments in
the harvesting complex and as protective agents against the
active oxygen products (AOS) that are formed from photo-
oxidation. These oxygen radicals can react with
macromolecules and lead to cellular damages (Malanga et
al., 1997). The mechanism of biological effect of
illumination (including near-UV-B) appear to involve
endogenous photosensitization and formation of AOS, such
as from singlet oxygen (1O2), superoxide radical (O-
2),
hydroxyl radical (-OH) and hydrogen peroxide (H2O2)
(Martin & Burch, 1990; Malange & Puntarulo, 1995). The
algae have developed defiance system against photo-
oxidative damage by antioxidative mechanisms to detoxify
and eliminate these highly reactive oxygen species. These
antioxidant defiance system includes hydrophobic
(carotenoids & α-tocopherol) and hydrophilic antioxidant
(ascorbic acid & glutathione) and antioxidant enzymes likes
superoxide dismutase (SOD), catalsae (CAT), glutathione
reductase (GR) ascorbic peroxidase (APX) and peroxidase
(POD) (Rao et al., 1996; Malanga et al., 1997; Rijstenbil,
2002). The author previously reported that D. salina
accumulated large amount of carotenoids, α-tocopherol and
ascorbic acid, and enhanced the activities of the antioxidant
enzymes when grown under high light intensity, in media
containing high salt concentration and/or limiting nitrogen
(El-Baz et al., 2002). These results suggested that the
adaptive response of these algae to high illumination could
depend on the activity of antioxidant enzyme and the ability
to accumulate simultaneously large quantities of
carotenoids, vitamin E and vitamin C. However, these
antioxidant products are mainly aimed at the health food
market for direct human consumption which recognized as
safe (Abe et al., 1999; El-Baz et al., 2002). Since many
authors postulated that a high intake of antioxidant
compounds might decrease the risk of cancer, aging,
inflammation, stroke disease and neurodegenerative disease
ABD EL-BAKY et al. / Int. J. Agri. Biol., Vol. 6, No. 1, 2004
50
(Parkinson’s & Alzheimer’s) in human and experimental
animals (Schwartz, 1996, Abd El-Baky et al., 2002; Abouel
Enein et al., 2003).
The aim of this work was to study the used of D.
salina
cells to production of antioxidant substances by
expose to low-dose of UV-B radiation when grown in
media containing deferent level of NaCl and nitrogen
concentrations. The profile of the activity of antioxidant
enzymes (SOD, CAT & POD), lipid peroxidation, lipid –
soluble antioxidant (vitamin E, β-carotene & other
carotenoids) and water-soluble antioxidant (ascorbic acid &
glutathione) were examined.
MATERIALS AND METHODS
Algal source. Marine microalgae Dunaliella salina was
obtained from the Culture Collection of Botany Department,
Texas University, Austin, Texas, U.S.A.
Growth conditions. Dunaliella salina was cultured in a 4 L
flask with 2.5 L of culture medium containing 8% NaCl and
5 mM nitrogen, pH 8.5 during spring season in National
Research Center (NRC). Media and nutrients were
sterilized. All glass and plastic ware were washed with 10%
HNO3 and rinsed with distilled water. The cultures were
gassed with 1.5% volume CO2 in air and were grown at 20-
25ºC. The mass culture of D. salina were carried out with
KNO3 as nitrogen source with two different concentrations
5 and 2.5 mM N, in media containing 8, 12 and 16% NaCl,
respectively. The cultures illuminated with continuous cool
white fluorescent lamps (Philips 40 W) at light intensity
levels were approximately 200 W m-2 (El-Baz et al., 2002).
An ultraviolet- B lamp (radiation 290-320 nm, UVM-57
Chromatovue) at irradiance of 0.50 W m-2 was used for
irradiated the culture for 4 h-1. All algal grown under
experimental conditions were performed in triplicate for 15
days.
Growth measurements. The growth of Dunaliella salina
was measured by dry weight methods and optical density
(O.D) as described by Payer (1971).
Harvesting. Under all experimental conditions the cells
culture at stationary-phase were harvested at 4°C by
centrifugation at 6000 x g for 15 min and frozen at -20°C.
Extraction of carotenoids and tocopherols. Carotenoids
and tocopherols were extracted from algal cells with 1: 10
(w/v) tetrahydrofuran (THF) in present 30 mg-1 of BHT (2,6
ditertra-butyl-p-cresol) and magnesium carbonate (1 g10 g-1
sample). After 24 h, the aliquot of the clear extracted
pigments was filtered and evaporated to sample value 5 mL
under a stream of nitrogen. The extracted pigments were
saponified with 25 mL of 10% methanolic potassium
hydroxide for 2 h at room temperature, then carotenoids and
tocopherols were extracted with dichloromethane. The
solvent layer was then separated by separator funnel, several
time washed with distilled water, then dried on Na2SO4, and
dryness under nitrogen (Farag et al., 1998).
Determination of algal total carotenoids. The total
carotenoids was determined by spectrophotometric method
at 450 nm, β-carotene served as a standard compound was
used for preparing the calibration curve (Semenenko &
Abdullaev, 1980).
Identification of carotenoids. Carotenoids were separated
by thermoseparation Liquid Chromatography system
consisted of a spectra system UV 2000 detector (hold at 438
nm) and Spectra System P2000 pump, on a 250x4.6 mm
(i.d) column packed with Chromosil C18 material, 5 μm
particle size and eluted with 80:10 (v/v) acetonitrile:
methanol, at flow rate of 1 mL min-1. Some available
standard carotenoids: β-carotene, zeaxanthin, lutein,
astaxanthin and cryptoxanthin (Sigma) were also run by the
same HPLC method (Honya et al., 1994; Abd El-Baky,
2003).
Determination of algal tocopherols. Tocopherols were
determined by HPLC equipped with Spectra System
UV2000 detector at 290 nm and sparated on a 250 x 4.6 nm
(i.d) column packed with Vydac and eluted with 90:10
acetonitrile: methanol (v/v) at a flow rate of 1 mL min-1.
Standard of α tocopherol (Sigma) was ran under the same
conditions (Abd El-Baky, 2003).
Extraction and determination of ascorbic acid. Ascorbic
acid (vitamin C) was extracted from the cells with 2% meta-
phosphoric acid, and determined by spectrophotometric
methods using 2, 6 di-chlorophenol indophenol dye
(Augustin et al., 1985).
Preparation of cytosolic fraction. Algae cells were excised
and homogenized using mortar and pestle in 5 mL of ice-
cold extraction buffer (250 mM sucrose & 25 mM Tris, pH
7.2). The homogenate was centrifuged at 16000 x g for 20
min at 4°C and supernatant (extracts) was used for analyses
of enzyme activities, glutathione (GSH), lipid peroxidation
and protein content.
Determination of glutathione (GSH). The GSH content of
algal cell extracts was measured by reaction with
5,5`dithiobis-2-nitrobenzoic (DTNB) reagent to give a
compound that absorbed at 412 nm (Silber et al., 1992).
GSH was expressed as μM.
Enzymes assays. The activity of cytosolic SOD (EC
1.15.1.1) was determined by photochemical method
(Ginnopolitis & Ries, 1977). Spectrophotometric method of
Chance and Maehly (1955) was used to assay the POD
activity (EC 1.11.1.7). The CAT enzyme (EC 1.11.1.6)
activity was assayed Spectrophotometrically by the
decomposition of H2O2 at 240 nm in a reaction mixture
containing of a 10 mM H2O2 and 25 mM phosphate buffer,
pH 7.0 (Hans- Luck, 1970).
Determination of lipid peroxidation products. The lipid
peroxidation products in algal cells extracts was estimated
by the formation of thiobarbaturic acid reactive substances
(TBARS) and quantified in term of malonaldhyde (MDA)
as described by Haraguchi et al. (1997). The lipid
peroxidation was expressed as micromoles of MDA. The
extinction coefficient of TBARS was taken as 1.56x10 5 at
wave length 532 nm.
PRODUCTION OF ANTIOXIDANT BY THE GREEN ALGA / Int. J. Agri. Biol., Vol. 6, No. 1, 2004
51
Detection of free radical by ESR-Spain. The algal cells
(0.2 g) from both control and treatment were lyophilized in
a speed Vac Sc 100 (savant). The powdered cells (0.05 g)
were prepared in 1,1 diphenyl-2 picrylhydrazyl (DPPH). No
increased in back ground ESR signal occurred when DPPH
alone was exposed to UV-light. ESR spectra were obtained
at room temperature using a Bruker (Karlsruhe, Germany),
Spectrometer ELEXSYS E-500, operating at 9.808 GHz
with 100 kHz modulation frequency. EPR instruments
setting for the Spin trapping experiments were: microwave
power, 20.2 mW; modulation amplitude 1.0 G; time
constant, 51 ms; receiver gain 60.0. EPR for all samples
were recorded at exactly the same spectrometer settings and
the first derivative EPR spectra were double-integrated to
obtain the area intensity.
Determination of protein. Protein content in algal buffer
extracts was assayed spectrophotometrically at 595 nm,
using comassein blue G 250 as a protein binding dye
(Bradford, 1976). Bovine serum albumin (BSA) was used as
a protein standard. Protein concentrations in the samples
were calculated from the calibration curve, in mg protein in
mL extract. Activities of SOD, CAT and POD were
normalized to protein concentration.
Statistical analysis. Data represent the means ± SD. Results
were analyzed by one- way ANOVA and Scheffe’ F-test to
identify significant differences between groups. P-values <
0.01 were considered significant. All analyses performed
using Co Stat software version 4 (Abacus Concepts, Inc.,
Berkeley, CA).
RESULTS AND DISCUSSION
Influence of growth conditions on antioxidant contents
of D. salina. The lipophilic (α-tocopherol and total
careotenoids), and hydrophilic (glutathione & ascorbic
acid) antioxidant contents were determined in algae cells
grown under all experimental conditions are presented in
Table I and II.
Lipophilic antioxidant. The cellular carotenoid contents
in irradiated cells was increased gradually by decrease
nitrogen level combined with increase NaCl concentration
in nutrient medium (Tables I, II). According to the level of
total carotenoid contents in algae cells grown under
experimental conditions, the highest level of total
carotenoids content mg g-1 cells and percentage of d.w (in
parentheses) was obtained in UV-Birradiated cells grown in
media containing 2.5 mM nitrogen and 16% NaCl (N
deficiency & NaCl stress), with value 115.2 mg g-1 cells
(11.5%). Whereas, these values were 14.5 (1.45%) and 9.2
mg g-1 cells (0.92%) in UVB-irradiated cells and
unirradiated cells grown under optimum conditions (5 mM
N and 8% NaCl), respectively. Therefore, UV-B-exposure
and the cells growth conditions affected the accumulation of
carotenoids in D. salina.
The carotenoid profiles in unirradiated and irradiated
D. salina cells grown under all experimental conditions
were verified by HPLC separation on the RP-18 silica gel
(Table I, & Fig. 1). The unirradiated D. salina culture grown
under optimum conditions was characterized by the lower
content of β-carotene as compared with other culture
exposed to UVB-radiation grown under the same
conditions. However, the results indicated a direct
relationship between β-carotene percent in irradiated cells
and increasing salinity and decrease N levels in the nutrient
medium. In general, in all treated cells, β-carotene,
astaxanthin and zeanxanthin were identified as major
carotenoids. The concentration of these carotenoids ranged
from 3.2 to 62.31, 2.13 to 21.31 and 1.41 to 14.51 mg g-1 of
cells d.w., respectively. These mean that the ratio of these
carotenoids to total caroteoids under stress condition was
Table I. Effect of UV- irradiation, nitrogen limitation and salt stress on carotenoids profile of Dunaliella salina
Total carotenoids β-carotene Astaxanthin Zeaxanthin Lutein Cryptoxanthin
Treatment mg g-1 %Ratiomg g
-1 %
% of
Total
car. mg g-1 %
% of
Total
car. mg g-1 %
% of
Total
car. mg g-1 %
% of
Total
car. mg g-1 %
% of
Total
car.
Optimum n utrients
not exposed to UV
9.2 0.92 1.0 3.2 0.32 34.78 2.13 0.213 23.1 1.41 0.14 15.3 0.92 0.092 1 0.21 0.02 2.28
Optimum n utrients
exposed to UV 5mM
Nitrogen + 8 % NaCl
14.5 1.45 1.58 5.11 0.51 35.24 3.21 0.321 22.1 2.01 0.20 13.9 1.11 0.11 7.6 0.42 0.04 2.89
2.5 mM Nitrogen +8
% NaCl
31.1 3.11 3.38 12.91 1.29 41.15 4.51 0.45 14.5 3.10 0.31 9.96 1.51 0.15 4.8 0.81 0.08 2.6
Zero Nitrogen + 8 %
NaCl
45.5 4.55 4.94 20.33 2.03 44.68 6.21 0.62 13.6 4.21 0.42 9.2 2.13 0.21 4.7 1.01 0.10 2.2
12 % NaCl + 5 mM
Nitrogen
54.1 5.41 5.88 25.45 2.54 47.04 8.95 0.89 16.5 5.31 0.53 9.8 2.31 0.23 4.27 1.15 0.11 2.12
12 % NaCl + 2.5 mM
Nitrogen
105.1 10.5 11.42 57.11 5.71 55.29 18.34 1.83 17.4 12.31 1.23 11.71 6.57 0.65 6.2 2.48 0.24 2.35
16% NaCl + 5 mM
Nitrogen
72.9 7.29 8.05 35.42 3.54 48.58 12.31 1.23 16.8 7.35 0.73 10.1 4.42 0.44 5.7 1.6 0.16 2.26
16% NaCl + 2.5 mM
Nitrogen
115.2 11.5 12.6 62.31 6.23 54.08 21.31 2.13 18.49 14.51 1.45 12.59 7.14 0.71 6.19 2.73 0.27 2.37
All values are significant at ( P< 0.5); Car. = Carotenoid
ABD EL-BAKY et al. / Int. J. Agri. Biol., Vol. 6, No. 1, 2004
52
about 20, 10 and 10, times that in cells grown under
optimum condition. However, depending on growth
conditions lutein and cryptoxanthin were detected in D.
salina. The amount of these carotenoids quantity increased
in irradiated D. salina cells gradually under a variety
conditions as a function of the N limitation and high NaCl
concentration in growth medium. For instance, the
concentration of lutein and cryptoxanthin in irradiated cells
grown in media containing 16% NaCl in present 2.5 and 5
mM N (in parentheses) were 7.14 (4.42) and 27.35 mg g-1
(16.50 mg g-1), respectively in comparison to 0.92 and 21
mg g-1 respectively, in unirradiated cells grown under
optimum conditions (5 mm N & 8% NaCl). Thus, it seems
that UV-B exposed, nitrogen limitation and high salt
concentration play an important role in controlling
carotenogenesis in D. salina. Accordingly, the production of
β-carotene, astaxanthin, zeanxanthin, lutein and
cryptoxanthin could be enhanced with cultured D. salina
under environmental stress conditions.
Algae of the genus Dunaliella
can be accumulate large
amount of carotenoids per cell reaching up to 10% of dry
weight when grown under stress conditions such as high
intensity irradiation, high salt concentration and nutrient
limitation (Ben-Amotz & Avron 1988; Bar et al., 1995; El
Baz et al., 2002). However, carotenoids play a highly
effective role in protecting photosynthesis pigment, enzyme
and membrane against photoxidative damage (Götz et al.,
1999). Under high irradiation, the photosynthetic apparatus
dose not sufficient utilizes light energy, and the excess
energy lead to the formation of free radicals rather than
active oxygen molecules (singlet oxygen). These radicals
are responsible for peroxidation reactions that destroy
various compounds of photosynthesis apparatus. Thus, the
algae such as Dunaliella and Chlorella accumulate large
amount of β-carotene, astaxanthin, and zeanxanthin for
scavenge or eliminating as well as for reducing the radicals,
reaching the cell component (Rise et al., 1994; El-Baz et al.,
2002). In this study exposed of D. salina to UV-B
irradiation caused significant increase in the amount of
carotenoids, as compared with unexposed cells. Thus, a
positive relationship between carotenoids content and relief
from UV-B inactivation was observed. Also, the results
showed that in UV-B exposed cells, the carotenoids
biosynthesis is shifted toward formation of astaxanthin and
cryptoxanthin as shown in Table II. It was shown recently
that astaxanthin and zeananthin are the most effective
protection against UV-B radiation in some microorganism,
and that it prevents radical peroxidation processes in
liposomes much better than β-carotene (Götz et al., 1999).
Furthermore, these results are evidence for a mechanism for
protecting the cells against irradiation damage. However,
Götz et al. (1999) reported that β-carotene and astaxanthin
in cyanobacterium exposed to UV-B radiation exert their
protective function as antioxidants to inactive UV-B-
induced radicals in photosynthetic membrane. On other
hand, the carotenoids in the cell membrane of microalgae
Table II. Effect of UV- irradiation, nitrogen limitation and salt stress on antioxidant substances of Dunaliella
salina
α-Tocopherol Ascorbic acid GSH
Treatment mg g-1
% Ratio mg g-1
% Ratio μM Ratio
Optimum nutrients not exposed to UV 0.368 0.036 1.0 2.7 0.27 1.0 97.12 1.0
Optimum nutrients exposed to UV 0.427 0.042 1.24 0.7.2 0.72 2.67 174.11 1.80
2.5 mM Nitrogen +8 % NaCl 0.584 0.058 1.61 11.9 1.19 4.42 185.24 1.91
Zero Nitrogen + 8 % NaCl 0.896 0.089 2.47 15.4 1.54 5.70 209.51 2.16
12 % NaCl + 5 mM Nitrogen 1.91 0.191 5.19 9.5 0.95 3.52 190.97 1.97
12 % NaCl + 2.5 mM Nitrogen 2.67 0.267 7.25 21.3 2.13 4.96 240.0 2.27
16% NaCl + 5 mM Nitrogen 2.38 0.238 6.47 13.4 1.34 7.89 220.84 2.47
16% NaCl + 2.5 mM Nitrogen 3.83 0.383 10.4 25.41 2.54 9.41 253.4 2.61
Table III. Effect of UV- irradiation, nitrogen limitation and salt stress on antioxdant enzyme system catalase,
peroxidase and superoxid dimutase of Dunaliella salina
Catalase Peroxidase Superoxide dismutase
Treatment
U mg-1
protein
Ratioa Ratiob U mg-1
protein
Ratioa Ratiob U mg-1
protein
Ratioa Ratiob
Optimum nutrients not exposed to UV 3.04 1.0 1.38 1.0 18.0 1.0
Optimum nutrients exposed to UV 20.13 6.62 1.0 9.32 6.75 1.0 20.36 1.13 1.0
2.5 mM Nitrogen +8 % NaCl 23.24 7.64 1.15 11.45 8.29 1.22 22.32 1.24 1.11
Zero Nitrogen + 8 % NaCl 26.35 8.67 1.31 13.22 9.56 1.42 26.40 1.47 1.29
12 % NaCl + 5 mM Nitrogen 27.11 8.92 1.35 15.41 11.17 1.65 29.21 1.62 1.43
12 % NaCl + 2.5 mM Nitrogen 35.62 11.7 1.77 24.11 17.47 2.59 36.41 2.02 1.79
16% NaCl + 5 mM Nitrogen 31.45 10.3 1.56 18.21 13.19 1.95 33.61 1.87 1.65
16% NaCl + 2.5 mM Nitrogen 39.55 13.0 1.96 26.41 19.14 2.83 40.11 2.23 1.97
Ratioa :Optimum nutrients not exposed to UV / treatment; Ratiob:Optimum nutrients exposed to UV / treatment; All values are significant at ( P< 0.5)
PRODUCTION OF ANTIOXIDANT BY THE GREEN ALGA / Int. J. Agri. Biol., Vol. 6, No. 1, 2004
53
could act as a filter for UV-B- radiation (Jürgens &
Weckesser, 1985).
Tocopherol. The cellular content of tocopherols in
unirradaited and irradiated D. salina
grown under all
experimental condition is show in Table II and Fig 2. The
results indicate that D. salina
responds to stress condition
by accumulated significant amount of α-tocopherol.
Tocopherol values obtained in irradiated cells grown at
16% NaCl in present of 2.5 and 5 mM N were 3.83 and
2.38 mg g-1, respectively, whereas these values were 2.67
and 1.91 mg g-1 at 12% NaCl, respectively. Therefore
changes are due to differences in the α-tocopherol
production in function of NaCl and nitrogen
concentration.
Hydrophilic antioxidants. With respect to the content of
hydrophilic antioxidants, both ascorbic acid and glutathione
were detected in D. salina.
The concentration of these
antioxidants significant increased as results to expose UV-
B, and grown under nitrogen limitation and high NaCl
concentration in growth media (Table II). Maximum values
were obtained in irradiated cells grown in medium
containing 2.5 mM nitrogen and 16% NaCl with 25.41 mg
g-1 cells d.w and 253.4 μM, respectively. Whereas,
minimum values were found in unirradiated cells grown
under optimum condition, with 2.7 mg g-1 and 97.12 μM,
respectively, compared with value 7.2 mg g-1 and 174.11
μM in irradiated cells grown under the same condition.
In general, the variation in hydrophilic and lipophilic
antioxidant content in irradiated Dunaliella cells depending
upon nitrogen and NaCl concentration. Similar finding
obtained by El-Baz et al. (2002), they reported that the
cellular antioxidant levels increased in D. salina when
grown under nitrogen limitation and high NaCl
concentration and exposure to high light intensity. Abe et al.
(1999) reported that the Trentepohlic aurea cells was
accumulated 2.1 mg β-carotene, 0.3 mg L-ascorbic acid and
2.4 mg tocopherols, respectively, g-1 d.w when the culture
exposed to high light intensity (430 μmol photon m-2 S-1).
While in T. Pseudonana, UV-B caused a significant
increase in GSH content (Rijstenbil, 2000). However, the
variations in β-carotene, vitamin C and E content of D.
tertiolecta have been shown to results concentration in
culture from nitrogen sources and media (Abalde et al.,
1991).
As a general mechanism the present results revealed
that the D.
salina exposed to UV-B radiation grown
under combined stress conditions (high NaCl
concentration, & nitrogen deficiency) is usually correlated
with accumulation of more efficient antioxidative
compounds such as ascorbic acid, glutathion, tocopherol
and carotenods (UV-B screening compounds). These
compounds can inactivate oxygen radicals, or the
effective replacement of damaged constituents can resist
UV-B stress (Götz et al., 1999). The negative effect of the
various environmental stresses is at least partially due to
the generation of active oxygen species (AOS) (Shalata &
Tal, 1998). The AOS are produced during normal aerobic
metabolism by the interaction between O2 and electrons
leaks from electron transport chains in the chloroplasts
and mitochondria (Halliwell, 1997). The AOS molecule
•OH (hydroxyl), H2O2 (hydrogen peroxide), O2
(superoxide) and 1O2 (singlet oxygen) are not controlled
by protective systems and may destroy proteins, lipid and
pigments such as chlorophyll under stress conditions.
Thus, algae increased the production of antioxidant or
elevated activities of protective enzymes to detoxify and
eliminate the highly reactive oxygen species. The
antioxidant defense system includes hydrophobic
molecules such as carotenoids and tocopherols to remove
the singlet oxygen. While the hydrophilic antioxidant
ascorbic acid and glutathione are effective chemical
scavengers of oxygen radicals (Shalate & Tal, 1998).
Activities of antioxidant enzyme system. The levels of
antioxidant enzyme activities CAT, SOD and POD in D.
salina grown under all experimental conditions are shown
in Table III. These enzyme activities in radiated D. salina
cells were enhanced under all experimental conditions.
UV-B exposure, increase the SOD, POD and CAT
activities in D. salina as compared with unexposed cells,
but the induced in SOD level was not significant
(P>0.05). The variation in these enzyme activities in all
experiment was caused by variation in cell growth
conditions. However, these enzyme activities were
Table IV. Effect of UV-irradiation, nitrogen
limitation and salt stress on lipid peroxidation of D.
salina
MAD Ratio
Treatment
mM mg-1
protein
Optimum nutrients not exposed to UV 1.04 1.00
Optimum nutrients exposed to UV 4.97 4.77
2.5 mM Nitrogen +8 % NaCl 6.70 6.42
Zero Nitrogen + 8 % NaCl 7.31 7.02
12 % NaCl + 5 mM Nitrogen 8.93 8.58
12 % NaCl + 2.5 mM Nitrogen 17.12 16.44
16% NaCl + 5 mM Nitrogen 10.50 10.08
16% NaCl + 2.5 mM Nitrogen 19.60 18.83
Table V. Effect of UV-irradiation, nitrogen limitation
and salt stress on free radical levels of D. salina
Relative free radical
Treatment
% Ratioa Ratiob
Optimum nutrients not exposed to UV 100% 1.00
Optimum nutrients exposed to UV 295.8 2.95 1.00
2.5 mM Nitrogen +8 % NaCl 213.7 2.13 0.72
Zero Nitrogen + 8 % NaCl 274.7 2.74 0.928
12 % NaCl + 5 mM Nitrogen 340.6 3.40 1.15
12 % NaCl + 2.5 mM Nitrogen 591.0 5.91 1.99
16% NaCl + 5 mM Nitrogen 82.0 0.82 0.277
16% NaCl + 2.5 mM Nitrogen 97.6 0.97 0.33
Ratioa :Optimum nutrients not exposed to UV / treatment; Ratiob:
Optimum nutrients exposed to UV / treatment
ABD EL-BAKY et al. / Int. J. Agri. Biol., Vol. 6, No. 1, 2004
54
significantly increased as results to nitrogen limitation
and increase the NaCl concentration in growth media. For
instances, irradiated cells grown under high salt
concentration (16%) combined with nitrogen limitation
(2.5 mM N), had an induce in CAT, POD and SOD
activities approximately 1.96, 2.83 and 1.97 times as high
as that in irradiated cells grown under optimum
conditions. Whilst these values were 6.62, 6.75 and 1.13
in irradiated cells grown in 5 mM N and 8% NaCl. At 2.5
mM N. with 12% and 16% NaCl (in parenthesis), the
cellular CAT, POD and SOD levels were being 1.77
(1.96), 2.59 (2.83) and 1.79 (1.97) times over than that in
irradiated cells grown under optimum condition. These
results collectively suggested that an UV-B enhanced
these enzyme activities correlated with decrease N level
and an increased in NaCl concentration in the growth
media. However, several investigator reported that the
plant have evolved a better antioxidant mechanisms
including antioxidant enzyme like SOD, CAT and POD to
increased the resistance to environmental stresses
including salt, nitrogen and carbon limitation, drought and
UV-irradiation (Rao et al., 1996; Zhang & Kirhham,
1996; Shalata & Tal, 1998; Abd El-Baky, 2003). In
which, plants or algae subject to environmental stress, the
Fig. 1. HPLC profile of carotenoids and tocopherol of Dunaliella salina expoused with UV-B. a: Control not
treated with UV-B ( Carotenoids); b:Control treated with UV-B (carotenoids);c: Standard of Carotenoids; d: Control
not treated with UV-B (Tocopherol); e: Control treated with UV-B (Tocopherol); f: Standard Alpha tocopherol
Compound (RT): Astaxanthin (3.6-4.2); Lutein (5.5-6.2); Cryptoxanthine (8.9-9.5); b-carotene (12.0- 13.5);
Zeaxanthin (7.2 - 8.2)
PRODUCTION OF ANTIOXIDANT BY THE GREEN ALGA / Int. J. Agri. Biol., Vol. 6, No. 1, 2004
55
balance between the production of AOS and the
quenching activity of antioxidants may be upset and
oxidative damage may result (Cakmak & Marschner,
1992). Rijstenbil (2002) found that exposure diatom
Thalassiosira pseudonana to UV-b and UV-A caused, a
significant increase in SOD activity. Gossett et al. (1996)
suggested that protection from oxidative damage induced
under salt stress by more active ascorbare- glutathine
cycle and a higher level of antioxidant enzymes like CAT,
SOD and POD. However, Strid (1993) and Hernandez et
al. (1995) reported that Cu, Zn-SOD and ascorbate,
peroxidase have a role in preventing NaCl or UV-B
irradiation induced oxidation stress in some plants.
Generally, enzymes and secondary compounds of
higher plants have been showed using in vitro experiments
to protect against oxidative damage by inhibiting or
quenching free radicals and reactive oxygen species. For
example, vitamin C, Vitamin E and β-carotene all have
antioxidant activity, and thus provide a cellular defense
against reactive oxygen species which damage DNA (Peto
et al., 1981; Barclay et al., 1983; Mckersie et al., 1990;
Hunter et al., 1994).
The cellular content of lipid and water-soluble
antioxidants and the antioxidant enzyme level were affected
in whole cells exposed to UV, as compared to control value.
The increase in carotenoids, α-tocopherol, ascorbic acid and
glutathion content and enzyme level of SOD, CAT and
POD in the irradiated algae reported here, suggests that the
adaptation to photooxidative stress in algae cells by
regulation the synthesis and repair of proteins and enhanced
antioxidative protection system (Malanga et al., 1997).
Lipid peroxidation. The content of MAD (a by-product of
the lipid peroxidation) (Table IV) is currently used as an
index of lipid oxidation caused by UV-B in unicellular
algae, bacterial, higher plants and animals tissues. It is also,
widely used as a biomarker for oxidation damage (Malanga
Fig. 2. Electron paramgnatic spectra (a) and free radicals level (b) of control and UV-B treated D. salina cells as
effected by nitrogen limitation and salt stress
(a) Control with UV-B
(a) Control with out UV-B
(a) 2.5 N + 16 % NaCl
(a) 2.5 mM N + 8 % NaCl
)b(
0
100
200
300
400
500
600
Relative f ree radical levels
(%)
T1T2T3T4T5T6T7T8
Treatments
Free radical
ABD EL-BAKY et al. / Int. J. Agri. Biol., Vol. 6, No. 1, 2004
56
et al., 1997; Rijstenbil, 2002). The content of MAD in non-
irradiate Dunaliella cells were 1.04 mM mg-1 protein and
significant (P<0.05) increased in irradiate cells grown under
all experimental conditions ranged from 4.97 to 19.60 mM
mg-1 protein (Table IV). The higher MAD level with values
17.12 and 19.60 mM mg-1 protein was obtained when cells
exposed to UV-B and grown in medium containing low
nitrogen level (2.5 mM) coupled with increased in NaCl
concentration 12 and 16%, respectively. The large increases
in MDA level in D. salina grown under all experimental
conditions indicate that UV-B enhanced active oxygen
production ·OH (hydroxyl radicals), which led to lipid
peroxidation process (MAD). The enhancement lipid
peroxidation in Dunaliella culture exposed to UV-B, when
grown under nitrogen limitation and salt stress conditions
has been reported by Shelly et al. (2002). In general, the
enhanced of lipid peroxidation in many microorganisms
may results from a complexity of environmental factors
including light, nutrient limitations (nitrogen or carbon) and
high solar radiance (Butow et al., 1998; Rijstenbil, 2002).
Alkyl radical. The direct detection of stable free radical in
lyophilized of all algal treated cells was measured by more
sensitive technique, PER spectrometer at room temperature.
The EPR stable free radical recorded with g-value at a
maximum absorption of 2.006 is shown in Fig. 2 and
relative % of free radical is under the pecks is shown in
Table 5. The data revealed that UV-B exposure significantly
increased radical content in the algal cells grown under
optimum conditions to 295.5% of the unexposed cells
(100%). The value quantitation of stable free radicals in
algal cells grown in nitrogen limitation 2.5 mM and zero
combined with 8% NaCl media was 213.7 and 274.7%
respectively, of the control. In contrast, at 12% NaCl, the
amount of free radicals being 3.4 and 5.9 times over than
that in the control and these values were decreased to 0.97
and 0.82 of control in algal cells grown at 16% NaCl. There
is well-supported evidence that the antioxidant compounds
found in algae cell possess free radical scavenging
properties. However, these results indicated that the
significant increase in stable free radical in algal cells after
UV-B exposure and these are in agreement with previous
reports from Malanga et al. (1997) in cells, microalgae
Chlorella vulgaris. In which, quantitation of alkyl radical
ESR signals in chloroplasts of C. vulgaris indicated that
UV-B exposure significantly increased radical content in the
membranes.
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(Received 01 November 2003; Accepted 10 December 2003)
