Selenium-induced Changes in Activities of Antioxidant Enzymes and
Contents of Photosynthetic Pigments in Spirulina platensis
Tian-Feng Chen1, Wen-Jie Zheng2*, Yum-Shing Wong1 and Fang Yang2
(1 Department of Biology, The Chinese University of Hong Kong, Hong Kong SAR;
2 Department of Chemistry, Jinan University, Guangzhou, Guangdong 510632, China)
Supported by the National Natural Science Foundation of China and Guangdong Province.
*Author for correspondence.
Tel: +86 (0)20 8522 6360;
Fax: +86 (0)20 8522 1697;
E-mail: <tzhwj@jnu.edu.cn> or <ctf12343@yahoo.com.cn>.
Abstract
Spirulina platensis (S. platensis) exposed to various selenium (Se) concentrations (0, 10, 20, 40,
80, 150, 175, 200, 250 mg/L) accumulated high amount of Se in a dose- and time- dependent
manner. Under low Se concentrations (≤150 mg/L), Se induced increases in biomass
concentration, contents of photosynthetic pigments, and activities of guaiacol peroxidase (GPX),
superoxide dismutase (SOD), catalase (CAT) and Gua-dep peroxidases (POD), which indicated
that antioxidant enzymes played an important role in protecting cells from Se stress. Higher Se
concentrations (≥ 175 mg/L) led to higher Se accumulation and increases in activities of GPX,
SOD, CAT and POD, but also induced lipid peroxidation (LPO) coupled with potassium leakage
and decreases in biomass concentration and contents of photosynthetic pigment. The results
indicated that increases in activities of the antioxidant enzymes were not sufficient to protect cell
membrane against Se stress. Time-dependent variations in the activities of antioxidant enzymes,
contents of chlorophyll a and carotenoid and the LPO level were also investigated under
representative Se concentrations of 40 and 200 mg/L. Opposite variation trends between
SOD-CAT activities, and GPX-POD-APX activities were observed during the growth cycles. The
results showed that the prevention of damage to cell membrane of S. platensis cells could be
achieved by cooperative effects of SOD-CAT and GPX-POD-APX enzymes. This study
concludes that S. platensis possessed tolerance to Se and could protect themselves from
phytotoxicity induced by Se by altering various metabolic processes.
Keywords: selenium; Spirulina platensis; accumulation; antioxidant enzymes; photosynthetic pigments.
The trace mineral selenium (Se) is an essential nutrient of fundamental importance to human
biology (Rayman 2000;Zheng et al. 2001). Both Se deficiency and toxicity occur worldwide,
depending on Se availability in the environments (Zhang et al. 2007). Since it is generally
believed that organic Se compounds are better and safer than inorganic Se as a dietary supplement,
a variety of Se-enriched biological products including garlic, yeast, and lactic acid bacteria etc.
have been commercialized. The Se supplementation using microorganisms has received much
attention in the past decade (Chasteen and Bentley 2003). Se was also found toxic at high intake
for human, animals and plants. Toxicity of Se is thought to occur due to its indiscriminative
substitution of sulfur into proteins(Brown and Shrift, 1982), the prooxidant ability to catalyze the
oxidation of thiols and simultaneously generate superoxide Stewart et al, 1999).
All aerobically growing microorganisms encounter toxic, reactive oxygen species (ROS)
(Halliwell and Gutteridge, 1999). These toxic forms of activated oxygen react with cellular
components resulting in oxidation of proteins and nucleic acids as well as lipid peroxidation,
leading further to inactivation of enzymes, disruption of membranes, mutations, and ultimately
cell death (Imloy and Linn, 1998;Halliwell and Gutteridge, 1999). Plant cell’s defence against the
damaging effects of oxidative stress involves both enzymatic and non-enzymatic components
(Candan and Tarhan, 2003). The enzymatic components may directly scavenge reactive oxygen
species (ROS) or may act by producing a non-enzymatic antioxidant. A variety of non-enzymatic
antioxidants such as carotene, ascorbate, a-tocopherol may play an important role in the cellular
response to oxidative stress by reducing certain ROS (Mager and De Kruijft, 1995). Superoxide
radicals generated are converted to H2O2 by the action of superoxide dismutase (SOD). The
accumulation of H2O2 is prevented in the cell by guaiacol peroxidase, catalase and peroxidases
(GPX, CAT and POD), or by the ascorbate-glutathione cycle where ascorbate peroxidase (APX),
reduces it to H2O (Donahue et al, 1997). Among these, the GPX level was changed in response to
various physical, chemical and biological stresses. Changes in GPX activity and isozyme profiles
have been suggested as indicators for environmental stresses (Lee, 2002). Till now, many works
have been carried out to study the physiological roles of Se in higher plants (Xue et al, 1993;
Huang et al, 1994; Hartikainen et al, 2000;Golubkina et al, 2005; Zhang et al, 2007). However,
the involvement of antioxidant system in the cell response to Se is still unclear. The question as to
how Se acts at the cellular level and how plants defend themselves against Se stress is receiving
increasing attention.
Spirulina platensis (S. platensis), a blue-green microalgae, has long been grown
photoautotrophically for production of functional food. Many works have shown that S. platensis
was a good carrier for Se accumulation. Furthermore, Se accumulation could improve the quality
of S. platensis by enhancing the production of its biomass, photosynthetic pigments and protein
concentrations (Huang et al, 2001, 2002; Li et al, 2003；Zheng et al, 2003a, 2003b; Chen et al,
2005, 2006a, 2006b). However, we also found that Se has toxic effects on the growth of S.
platensis depending on the Se levels (Chen et al, 2005, 2006a). Till now, less information on
Se-induced oxidative stress and antioxidant response in S. platensis could be obtained. The
objective of this study was to investigate the accumulation of Se in S. platensis and its effects on
the antioxidant systems (including antioxidant enzymes and non-enzyme components). This study
may offer some insight into mechanisms underlying Se tolerance and accumulation in plants and
enhance our understanding of the interaction between Se and S. platensis.
Results
Se accumulation and its effects on biomass and contents of photosynthetic pigments in
S. platensis
According to our results shown in Fig.1A, S. platensis accumulated Se efficiently during the
cultivation and biotransformed the inorganic Se to organic Se at a high rate. Se content in S.
platensis increased significantly with Se in culture media from 0 to 250 mg/L (P < 0.05). The Se
species accumulated mainly consisted of organic Se (above 70%) and inorganic Se (< 30%)
respectively, indicating that S. platensis was a good carrier for Se enrichment. Se accumulation
and biotransformation significantly (P < 0.05) affected the growth of S. platensis, which was
reflected by changes in biomass concentration. As shown in Fig.1B, Se has either stimulating or
toxic effects on S. platensis depending on the Se levels in media. The increase in biomass
concentration was obtained in cells exposed to low Se concentrations (≤150 mg/L), with the
highest one (1.69g/L) found at the Se concentration of 40 mg/L. The even higher initial Se
concentrations (≥ 175 mg/L) resulted in lower biomass concentration, which might be due to the
toxic effects of high Se stress. In particular, cultures were found to turn red due to the occurrence
of elemental Se, when S. platensis was exposed to Se at concentrations ≥ 300 mg/L and the
color would increase with Se added to the media (data not shown).
Under Se concentrations ≤150 mg/L, Se significantly (P < 0.05) elevated the levels of
photosynthetic pigments (including lutein, β-carotene, and chlorophyll a) (Fig.1C), with the
maximum ones (2.78, 2.51 and 25.1 mg/g respectively) obtained at Se concentration of 40 mg/L.
In contrast, under higher Se concentrations (≥175 mg/L), significant decline (P < 0.05) in the
overall contents of photosynthetic pigments was evident with respect to control.
Effects of Se on the activities of antioxidant enzymes, lipid peroxidation and membrane
permeability in S. platensis
The variations in activities of antioxidant enzymes, LPO level and potassium cation (K+)
concentration in S. platensis were investigated in the presence of Se ranging from 0 to 250 mg/L
(Fig. 2). In the present study, it was observed that Se accumulation resulted in hyperactivities of
GPX, SOD, CAT, Gua-dep POD. The enzyme activities continued to augment with the increase
in Se concentration in the culture media. The maximum activities were found at Se treatment of
250 mg/L as 12.9 ± 0.3; 14.7 ± 0.6; 1.43 ± 0.06; 0.25 ± 0.01 IU/mg protein respectively. A sharp
elevation in GPX and CAT activities was observed when cells were exposed to Se at 250 mg/L.
However, the APX activity remained almost similar under Se concentration at 0 - 40 mg/L, with
the maximum one found at Se 40 mg/L as 0.047 ± 0.002 IU/mg protein. Higher Se concentrations
(80 – 250 mg/L) resulted in significant decrease in APX activity (P < 0.05), which reached the
minimum (0.005 ± 0.0004 IU/mg protein) under Se treatment of 250 mg/L.
As shown in Fig. 2C, LPO level decreased with Se concentrations (0 – 40 mg/L) and reached
the minimum (4.9 ± 0.6 nmol MDA/g DW) at Se treatment of 40 mg/L, which increased
significantly (P < 0.05) with a positive correlation to Se concentration from 80 to 250 mg/L. The
maximum one (56.8 ± 1.9 nmol MDA/g DW) was obtained at Se treatment of 250 mg/L.
Potassium cation leakage was also observed in S. platensis in a concentration dependent manner
(P < 0.05) with cell disruption observed under microscopy (data not shown).
Variations in total Se contents in S. platensis with cultivation time
The representative Se treatments, 40 mg/L and 200 mg/L, were chosen for further investigation,
since significant increase in growth, photosynthetic pigment contents, and antioxidant enzyme
activities in S. platensis was observed at Se treatment of 40 mg/L, while significant decrease
obtained at Se treatment of 200 mg/L.
Variations in total Se contents in S. platensis with cultivation time under representative Se
concentrations were plotted in Fig. 3. Under Se treatment of 40 mg/L, the amount of Se
accumulated in S. platensis increased proportionally with the cultivation time, without lag time
observed. About 57.06 % (133.23 µg/g) of total Se (233.53 µg/g) was accumulated during the
initial 3 exposure days. Slower increase was found from day-4 to day-11. At higher Se treatment
of 200 mg/L, about 76.19 % (345.76 µg/g) of total Se (453.79 µg/g) was accumuled in the first 3
exposure days. The highest Se concentration (465.73 µg/g) was found in day-9, which decreased
slightly from day-10 to day-11.
Changes in contents of photosynthetic pigments, activities of antioxidant enzymes and lipid
peroxidation with cultivation time
As shown in Fig.4A, significant (P < 0.05) and continuous decrease in the Chl a content was
observed in control and cells under Se treatment of 200 mg/L, which shown a positive correlation
with the cultivation time from day-0 to day-11. However, under Se treatment of 40 mg/L, the Chl
a content reached the maximum value (29.3 mg/g) at day-3, and decreased significantly
afterwards (P < 0.05). As shown in Fig.4B, in control and cells under Se treatment of 200 mg/L,
carotenoid contents decreased up to day-7 and increased significantly afterwards (P < 0.05).
Significant (P < 0.05) and continuous increase of carotenoid contents was observed when S.
platensis was exposed to Se at 40 mg/L, which shown a positive correlation with the cell
cultivation time.
Time-dependent variations in activities of antioxidant enzymes and lipid peroxidation were
plotted in Fig. 5. The GPX activity increased sharply in day-1 under Se treatment of 40 mg/L and
up to day-3 under Se treatment of 200 mg/L, followed by significant decrease afterwards (P <
0.05). The maximum activities were found as 6.85 ± 0.6 and 14.17 ± 1.0 IU/mg protein under Se
treatments of 40 mg/L and 200 mg/L respectively (Fig. 5A). Comparing with the control, SOD
activity in cells under both Se treatments decreased up to the day-5 and then increased
significantly afterwards (P < 0.05). The CAT activity decreased sharply in the day-1 and
increased up to the day-7 and then decreased significantly afterwards (P < 0.05). The maximum
activities were found as 1.49 ± 0.05 and 1.59 ± 0.04 IU/mg protein under Se treatments of 40
mg/L and 200 mg/L respectively (Fig. 5C). The POD activity reached the maximum values (0.25
± 0.01, 0.29 ± 0.01 and 0.41 ± 0.01 IU/mg protein) in control and in cells under Se treatments of
40 mg/L and 200 mg/L in day-5 by showing a positive correlation with Se concentration and
cultivation time. Afterwards, POD activity decreased significantly (P < 0.01). Sharp increase in
APX activity were observed in the initial 3 days, with the maximum values (0.067 ± 0.006, 0.079
± 0.004 and 0.087 ± 0.004 IU/mg protein in control, Se treatments of 40 mg/L and 200 mg/L)
obtained in day-3. APX activity decreased significantly (P < 0.01) afterwards. Especially, a sharp
decrease under Se treatment of 200 mg/L was observed after the day-7.
Significant (P < 0.05) and continuous decrease in the LPO level was observed in control and
cells under Se treatment of 40 mg/L from day-1 to day-7. However, under Se treatment of 200
mg/L, LPO level remained almost similar up to day-3 and then increased sharply to day-11
afterwards (Fig. 5F).
Discussion
Se is an essential micronutrient of fundamental importance to organisms. S. platensis has been
found to possess tolerance to high levels of Se, indicating that it was a good carrier for Se
accumulation. Se has either stimulating or toxic effects on S. platensis depending on the Se level
in the culture media. According to the results obtained in this study, S. platensis could accumulate
Se efficiently during the cultivation and the accumulated amount increased with the
concentrations of Se treatments (≤150mg/L). The higher initial Se concentrations, such as ≥200
mg/L, led to high Se accumulation, but induced much lower biomass concentrations and
decreases in contents of photosynthetic pigments (Fig.1). The majority of the total Se was
accumulated rapidly in the initial 3 exposure days and most of it was transformed to organic Se
(above 70 %) (Fig.3). Occurrence of elemental Se was observed under Se concentrations ≥ 300
mg/L, which was considered as a mechanism of detoxification of excess Se by transforming it to
the insoluble elemental form (Li et al, 2003).
Se is also important for antioxidant response in organisms. Se acted as an antioxidant at low
concentrations. As a component of glutathione peroxidase, a widely recognized direct or indirect
function of Se is the removal of reactive oxygen species (Drake, 2006). In the current study, It
was found that GPX, SOD, CAT, Gua-dep POD activities increased significantly and LPO level
decreased significantly in the cells treated with Se from 0 to 40 mg/L (Fig. 2). This may be the
indirect evidence of antioxidant activity of Se in the plant cells. However, Se acts as a prooxidant
under excess concentrations. As shown in Fig.2., under Se treatments ranging from 80 to 250
mg/L, although the increase in GSH-Px, SOD, CAT and POD activities, the increase in the LPO
level, K+ leakage and decrease in contents of photosynthetic pigments were observed. These
results indicate that the increase in activities of antioxidant enzymes was not sufficient to protect
the cells from the Se-induced toxic damage. Se, with the charges of positive four and positive six,
reacts directly with cysteine clusters in the catalytic subunits of enzymes, such as protein kinase,
oxidizing the sulfhydryl groups to disulfide linkages, thereby inactivating the enzyme (Spallholz,
1994). Since oxidation of sulfhydryl groups is also associated with the production of both
superoxide and peroxide, cell damage induced by selenite may be triggered by either enzyme
inactivation or ROS production. The toxic effects of excess Se on S. platensis may be due to this
pro-oxidant mechanism.
The antioxidant responses of S. platensis against Se treatment were also found in a duration
dependent manner. Under Se concentration of 40 mg/L, Se acted as an antioxidant and had
stimulating effects on growth of S. platensis. As shown in Fig.5, from the first to the fifth
exposure day, in spite of the decrease in SOD and CAT activities, the GPX, POD, APX activities
and carotenoid concentrations increased significantly (P < 0.05). LPO levels decreased
significantly, indicating that increases of these antioxidant enzyme activities and photosynthetic
pigments could provide sufficient protection to the membrane against ROS. Afterwards, SOD and
CAT activities increased, while GPX, POD and APX activities decreased significantly. LPO
levels also decreased gradually. The results indicated that the increases in SOD and CAT
activities were sufficient to protect cell membrane lipids. At high Se concentration of 200 mg/L,
Se acted as a pro-oxidant and had toxic effects on growth of S. platensis. In spite of the decrease
in SOD and CAT activities, the GPX, POD and APX activities and carotenoid contents continued
to increase up to the day-3. LPO levels remained similar, indicating that increases of these
antioxidant enzyme activities and carotenoid concentrations could effectively inhibit the lipid
peroxidation. The significant and continuous increase in LPO level up to the day-11 indicated the
aggravating damage to S. platensis. LPO level increased in the control after the day-7, indicating
aging-evoked oxidative reactions. This reaction pattern appeared to be counteracted by increases
in SOD and CAT activities and carotenoids contents.
Opposite variation trends between SOD-CAT activities and GPX-POD-APX activities were
observed during the cultivation. The Se-induced decrease in SOD and CAT activities indicated
that lower amounts of superoxide anion radicals were produced in cells due to the higher activity
of GPX, POD and APX. It can be hypothesized that the increase in GPX, POD and APX, which
were scavengers of H2O2 and lipid hydroperoxides, resulted in reduced formation of superoxide
anion radicals through the dynamic inter-transformation among oxygen species. On the other
hand, Se increased GPX activities and enhanced the spontaneous disproportion of superoxide
radicals and, consequently, reduced the need for their scavenger SOD. These reaction patterns
might explain the indifferent response of antioxidant enzymes to toxic Se concentration (200
mg/L). Because GPX, POD and APX activities were significantly lower and markedly less
increased, it was necessary to maintain SOD and CAT activities. These results show that the
prevention of damage to cell membrane of S. platensis can be achieved by co-operative effects of
the whole system of antioxidant enzymes.
Non-enzymatic antioxidants such as carotenoids, play an important role in the cellular response
to oxidative stress by reducing ROS (Mager and De Kruijft, 1995). Under stress conditions,
accumulation of excited molecules in the pigment bed, like the chl* and singlet oxygen, might
occur (Foyer et al, 1994). These highly cytotoxic species and their deleterious products could
seriously disrupt metabolism through oxidative damage to cellular components (De Vos, 1989).
Carotenoids were found to be able to protect the photosynthetic membrane from photooxidation
by effectively scavenging singlet oxygen and the quenching triplet state of chlorophyll
(Demmig-Adams, 1990). The increase in carotenoid contents in S. platensis after day-7 in control
cells and under Se concentration of 200 mg/L may be due to these antioxidant mechanisms.
Based on the results obtained in this study, it could be concluded that S. platensis possessed
tolerance to Se and could protect themselves from toxicity of excess Se by altering various
metabolic processes with the involvement of the whole antioxidant systems.
Materials and methods
Culture of S. platensis, Se treatments and estimation of biomass concentration
The microalgal strains used in this study was S. platensis, kindly provided by Research Center of
Hydrobioloty of Jinan University (Guangzhou, China), and the axenic treatment was carried out
in our laboratory. The cultivation of S. platensis was carried out in 250 mL Erlenmeyer flasks
containing 100 mL Zarrouk medium (pH 9.0) at 30 ± 1°C with a light illumination of 88 MJ m-2
s-1 and a 14:10 h light:dark cycle. The experiments were started with a 10 % (V/V) inoculation
from the stock cultures. Se was added at the first day in form of sodium selenite (Na2SeO3) to get
different final Se concentrations (0, 10, 20, 40, 80, 150, 175, 200, 250 mg/L). All solutions and
experimental containers were autoclaved at 121°C for 15 min. The dry weight of S. platensis was
determined by drying the cells at 70°C in a vacuum oven until constant weight.
Determination of total, inorganic and organic Se concentrations
Se concentration was determined by ICP-AES method (Chen et al., 2006 b). The sample was
digested with 3 mL concentrated nitric acid and 1 ml H2O2 in a digestive stove (Qian Jian
Measuring Instrument Co., Ltd., China) at 180 °C for 3 h. The digested product was reconstituted
to 10 ml with Milli-Q H2O and used for total Se determination. For inorganic Se determination,
the dried algal samples were extracted with 15 % HCl and the extract was analyzed by ICP-AES
directly. Organic Se concentration was calculated from the difference between the total Se
concentration and the inorganic Se concentration. Determinations were made in duplicate using
external standards. The determination limit, defined as 3× standard deviation (SD) of blanks, is
0.23 ng/mL. Good recovery ranging from 94.8 ％ to 104.2％ was obtained. The linear range of
detection was from 0.23 to 1000.00 ng/mL.
Photosynthetic pigment extraction and HPLC analysis
10 mL of cell suspensions was centrifuged at 8,000 rpm (Universal 32R, Hettich Zentrifugen) for
10 min at 4°C. The pellets were resuspended in acetone, and then sonicated for 30×5 sec (4 sec
interval; 150W) with an ultrasonic cell crusher (Model Jy 98-3, China). The mixtures were then
centrifuged at 10,000 rpm (Universal 32R, Hettich Zentrifugen) for 10 min at 4°C to remove the
cell debris. The obtained extracts were analyzed by RP-HPLC (HP1100) using a C18 column
(LiChrospher®100RP-18, endcapped 5 µm) with the mobile phase consisting of dichloromethane:
acetonitrile: methanol: water (22.5: 9.5: 67.5: 0.5; v/v/v/v) (Yuan et al, 1997). The flow rate was
1.0 mL/min. Photosynthetic pigments were detected by a UV-Vis absorbance detector set at 450
nm with a reference wavelength at 730 nm.
Assay of activities of antioxidant enzymes
Cell suspensions were centrifuged and the pellets were then resuspended in a pre-chilled
phosphate buffer (pH 7.0), and disrupted by ultrasonication. The extract was centrifuged at 10
000g for 30 min and the supernatant was used for antioxidant enzyme analysis.
Glutathione peroxidase (EC 1.11.1.9) activity was modified from the method of Flohe and
Gunzler (Flohe and Gunzler, 1984). For the enzyme reaction, 0.2 mL of the supernatant was
placed into a tube and mixed with 0.4 mL GSH (reduced glutathione, Sigma product, analytical
grade), and the mixture was put into an ice bath for 30 min. Then the mixture was centrifuged for
10 mins at 3000 rpm, 0.48 ml of the supernatant was placed into a cuvette, and 2.2 ml of 0.32 M
Na2HPO4 and 0.32 ml of 1.0 mmol/L 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB, Sigma) were
added for color development. The absorbance at wavelength 412 nm was measured with a UV
500 (Unican company) spectrophotometer after 5 min. The enzyme activity was calculated as a
decrease in GSH within the reaction time as compared to that in the non-enzyme reaction.
The activity of superoxide dismutase (SOD) was assayed by measuring its ability to inhibit the
photochemical reduction of nitro blue tetrazolium (NBT) (Beauchamp and Fridovich, 1971).
Reaction mixture lacking enzyme developed the maximum color which decreased with increasing
volume of added enzyme extract. The volume of enzyme extract corresponding to 50 % inhibition
of the reaction was calculated by plotting a graph between enzyme concentration in reaction
mixture and its absorbance at 560 nm and was considered as one unit enzyme. SOD activity was
expressed as unit mg-1 protein.
The activity of catalase (CAT) was measured by monitoring H2O2 decomposition at 240 nm in
3 mL reaction mixture containing 50 mmol/L phosphate buffer (pH 7.0), 15 mmol/L H2O2, 100
mL enzyme extract and 0.1 % (V/V) Triton X-100 (Aebi, 1984). The activity was expressed in
terms of μmol of H2O2 reduced min/mg/protein.
The activity of ascorbate peroxidase (APX) was measured according to the method of Nakano
and Asada (Nakan and Asada, 1981) by estimating the rate of ascorbate oxidation (extinction
coefficient: 2.8 mM-1 cm-1). The enzyme activity was expressed in terms of μmol of ascorbate
oxidized min/mg/protein.
For the measurement of guaiacol-dependent peroxidase (EC 1.11.1.7) activity, the reaction
mixture contained 25 mmol/L phosphate buffer (pH 7.0), 0.05 % guaiacol, 10 mmol/L H2O2 and
enzyme. Activity was determined by the increase in absorbance at 470 nm due to guaiacol
oxidation (E = 26.6 mM–1 cm–1) (Hemeda and Klein, 1990).
Determination of Lipid peroxidation
LPO was estimated based on thiobarbituric acid (TBA) reactivity. Samples were evaluated for
malondialdehyde (MDA) production using a spectrophotometric assay for TBA. The extinction
coefficient of 153 mM–1 cm–1 at 532 nm for the chromophore was used to calculate the MDA-like
TBA produced (Buege and Aust, 1978). Membrane permeability was estimated in terms of K+
leakage. Potassium concentration was estimated by ICP-AES method on a Optime 2000 DV
spectrometer (Perkin Elmer company).
Statistical analysis
All experiments were performed with at least 3 replicates. Microcal origin 6.0 was used to
determine the mean value, standard deviation and significant differences amongst the treatments
at P < 0.05.
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Total Se
Organic Se
Organic Se percentage
400
75
50
200
25
Biomass concentration (g DW / L)
0
Lutein and β-carotene (mg/g DW)
100
Organic Se percentage ( % )
a
0
2.0
b
1.5
1.0
0.5
0.0
4
30
Lutein
ß -carotene
Chl a
c
20
2
10
0
Chla (mg/g DW)
Total Se and organic Se
concentration (μg/g DW)
600
0
0
10
20
40
80
150 175 200 250
Se concentration (mg/L)
Fig.1. Accumulation of Se (A) in S. platensis and its effects on the biomass
concentrations (B) and contents of photosynthetic pigments (C). Values
expressed are means ± SD of three replicates
a
SOD
GSH-Px
10
0
0.08
b
CAT
POD
APX
0.8
0.04
0.0
0.00
400
c
+
K concentration
LPO level
60
300
40
200
20
100
+
LPO level (nmol MDA / g DW)
80
APX ( IU / mg protein )
1.6
K concentration ( µmol / g DW)
GSH-Px and SOD ( IU / mg protein )
CAT and POD ( IU / mg protein )
20
0
0
10
20
40
80
150 175 200 250
0
Selenium concentration (mg/L)
Fig.2. Effects of Se on the antioxidant enzymes, LPO levels and potassium cation
concentration in S. platensis. Values expressed are means ± SD of three replicates
Se content (µg / g DW)
600
40 mg/L
200 mg/L
400
200
0
0
2
4
6
8
10
12
Time (d)
Fig.3. Variations in total Se concentration in S. platensis with cultivation time under representative Se
concentrations of 40 mg/L and 200 mg/L. Values expressed are means ± SD of five replicates
35
Chl a concentration ( mg / g DW)
a
30
25
20
15
Carotenoids concentration ( mg / g DW)
Control
40 mg/L Se
200 mg/L Se
0
2
4
6
8
10
12
10
12
6
Control
40 mg/L Se
200 mg/L Se
b
5
4
3
0
2
4
6
8
Time (d)
Fig.4. Changes in contents of photosynthetic pigments in S. platensis with cultivation time under representative
Se treatments of 40 mg/L and 200 mg/L. A, Chl a; B, Carotenoids (including lutein and β-carotene). Values
expressed are means ± SD of three replicates
20
a
Control
40 mg/L Se
200 mg/L Se
20
15
10
5
0
0
6
8
10
POD (IU / mg protein)
Control
40 mg/L Se
200 mg/L Se
1.0
0.5
0.15
0
2
4
6
e
8
10
14
12
0.05
2
4
6
Time (d)
8
10
2
4
6
d
8
10
12
Control
40 mg/L Se
200 mg/L Se
0.4
0.2
0
2
4
6
8
10
12
6
8
10
12
40
Control
40 mg/L Se
200 mg/L Se
0
0
0.0
12
0.10
0.00
16
0.6
1.5
Control
40 mg/L Se
200 mg/L Se
10
12
c
0.0
APX (IU / mg protein)
4
LPO levels (nmol MDA / g DW)
CAT (IU / mg protein)
2.0
2
b
18
SOD (IU / mg protein)
GSH-Px IU / mg protein
25
12
f
Control
40 mg/L Se
200 mg/L Se
30
20
10
0
0
2
4
Time (d)
Fig. 5. Changes in antioxidant enzyme activities and LPO level in S. platensis with cultivation time under
representative Se treatments of 40 mg/L and 200 mg/L. Values expressed are means ± SD of three replicates.