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Biomolecular Engineering 22 (2005) 141–146

Physiological responses of Dunaliella salina and

Dunaliella tertiolecta to copper toxicity

K. Nikookar, A. Moradshahi *, L. Hosseini

Department of Biology, College of Sciences, Shiraz University, Shiraz 71454, Islamic Republic of Iran

Abstract

Species differences in heavy metal tolerance were investigated by comparing the responses of Dunaliella tertiolecta and Dunaliella

salina to elevated concentrations of CuCl2. Although both species showed reduced cell number ml�1 of algal culture, D. salina was more

affected by increase in CuCl2. This reflects higher sensitivity of D. salina to CuCl2 compared to D. tertiolecta. Total chlorophyll in terms

of mg ml�1 was higher in D. tertiolecta at all tested CuCl2 levels, but in terms of mg cell�1 no significant difference was observed between

the two species. Total carotenoids in mg cell�1 increased with increase in CuCl2 in both species and it was about five times higher in D.

salina at all CuCl2 concentrations. While both species showed significant increase in lipid peroxidation at elevated CuCl2, the

malondialdehyde content of D. salina cells was about three times higher at most CuCl2 concentrations. Although ascorbate peroxidase

(APX) activity increased with increase in CuCl2 levels in both species, higher activity was observed in D. tertiolecta at all tested CuCl2concentrations. Cu content of D. salina cells was higher than D. tertiolecta which may be due to larger volume of D. salina cells.

In conclusion, since hydroxyl radical (HO�) produced from H2O2 by Cu2+ (Haber–Weiss cycle) is involved in lipid peroxidation,

higher ascorbate peroxidase activity in D. tertiolecta may partly account for lower sensitivity of this species to CuCl2 compared to

D. salina.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Dunaliella; Pigments; Lipid peroxidation; Ascorbate peroxidase; CuCl2

1. Introduction

Oxidative stress occurs when green algae are exposed to

adverse environmental conditions such as heavy metal stress

[1–3]. Copper (Cu) is a micronutrient essential for activity of

several enzymes and is a component of electron transport

chain in chloroplast and mitochondria [4,5]. Although Cu is

usually present at low concentrations in surface waters,

levels are elevated by industrial effluents and wastes,

agricultural runoff, sewage treatment plants, mining opera-

tions and urban runoff [6,7]. At elevated concentrations, Cu

is highly toxic to plants, algae and other organisms [6,8–10].

Copper exerts its toxicity mainly through increased

production of reactive oxygen species (ROS) via its effect

on photosynthesis [11,12]. The ROS induces membrane

* Corresponding author. Tel.: +98 711 2280916; fax: +98 711 2280926.

E-mail address: moradshahi@biology.susc.ac.ir (A. Moradshahi).

1389-0344/$ – see front matter # 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bioeng.2005.07.001

lipid peroxidation that results in disintegration of the

lamellar system and increased unstacking of thylakoids

[5,8,10,13]. In addition, excess Cu decreases the photo-

synthetic pigments by affecting both their synthesis and

degradation [10,14] and causes rubisco and phosphoenol-

pyruvate carboxylase inactivation by interaction with

SH-groups [15].

Analysis of water samples collected from Maharlu

salt lake in Iran showed the presence of high concen-

trations of several heavy metals including Cu, Zn and

Cd. Several species of unicellular green algae Dunaliella

co-exist in Maharlu salt lake [16]. Since species

differences in heavy metal tolerance have been reported

in plants and algae [17], to assess sensitivity of different

species of Dunaliella to heavy metals, the effect of CuCl2on growth, pigmentation and ascorbate peroxidase (APX)

activity of Dunaliella salina and Dunaliella tertiolecta,

isolated from Maharlu salt lake, are investigated and

compared.

K. Nikookar et al. / Biomolecular Engineering 22 (2005) 141–146142

2. Materials and methods

2.1. Purification and growth of the algae

D. tertiolecta was isolated from water samples collected

from Maharlu salt lake located 30 km southeast of Shiraz,

Iran. Single colonies were derived from individual cells by

repeated subculturing on agar plates as described by Lers

et al. [18] and Powtongsook et al. [19]. Each colony was

transferred to liquid nutrient medium [20] and D. tertiolecta

was identified as described by Preisig [21]. Purified D.

tertiolecta and D. salina which was previously isolated and

purified from Maharlu salt lake [16], were cultured in 5-L

flasks containing 3 L sterilized liquid nutrient media [20].

The flasks with a cell density of 105 cell ml�1 were

incubated in a growth chamber at 22 8C under continuous

illumination provided by cool white fluorescent lamps at an

intensity of 5000 Lux. At late logarithmic phase, each

culture was divided into twelve 250-ml parts and each part

together with 1 ml of proper concentration of CuCl2 was

added to 500-ml sterilized Erlenmeyer flasks. The flasks

were kept under the same condition as described above and

after 24 h samples were taken from each flask for analysis of

CuCl2 effects on D. salina and D. tertiolecta. All the

experiments were carried out in triplicates. Each value

represents the mean � standard error at p < 0.05 calculated

using one way ANOVA.

2.2. Determinations

For growth measurement, cell number was determined

using a haemocytometer or a Coulter counter model ZBI

with a 100-mm orifice. Total chlorophyll and total

carotenoids were extracted from the algal pellet with

100% acetone. The extract was centrifuged at 5000 � g for

10 min and total chlorophyll and total carotenoids were

determined as described by Eijckehoff et al. [22]. Lipid

peroxidation was determined by measuring the amount of

malondialdehyde (MDA) by the thiobarbituric acid (TBA)

reaction described by Dhindsa and Matowe [23]. About 108

cells were pelleted by centrifugation of appropriate volume

of algal culture and then homogenized for 30 s with a

sonicator model W-220F in 5 ml of 0.1% TCA in ice bath.

Equal volumes of homogenate and 0.5% TBA in 20% TCA

solution were mixed and incubated at 95 8C for 30 min and

quickly cooled in an ice-bath. The cooled mixture was

centrifuged at 10,000 � g for 10 min and the absorbance of

the supernatant was recorded at 532 nm and corrected for

non-specific turbidity by subtracting the absorbance at

600 nm. The MDA concentration was determined using

the extinction coefficient of 155 mM�1 cm�1. For APX

determination algal cells were pelleted by centrifugation and

homogenized in 10 ml of grinding medium as described

before [16]. The homogenate was centrifuged at 5000 � g

for 5 min and APX activity in the supernatant was measured

by monitoring the decrease in A290 for one min [24].

The 1-ml reaction mixture contained 100 mM potassium

phosphate buffer (pH 7.0), 0.22 mM ascorbate, 0.3 mM

H2O2 and enzyme extract. For Cu analysis, cells were

pelleted by centrifugation and washed with isotonic

solution. The cells were digested with concentrated HNO3

and Cu concentration was determined by atomic absorption

spectrometer (Varian SpectrAA-220).

3. Results

3.1. Effects of CuCl2 on cell number

Table 1 shows the effects of different concentrations of

CuCl2 on growth of D. salina and D. tertiolecta sampled 24,

48, and 72 h after CuCl2 treatments. At 1 mMCuCl2 (control

cultures), cell number increased with time in both species

with D. tertiolecta having higher potential for cell division

and therefore increase in cell number. At 5, 10, and 20 mM

CuCl2, decrease in cell number with increase in incubation

time occurred in both species but the decrease was more

pronounced in D. salina compared to D. tertiolecta. Same

was true for the effects of CuCl2 on cell number at each

incubation period; i.e. decrease in cell number with increase

in CuCl2 concentration and higher sensitivity of D. salina to

CuCl2.

3.2. Effects of CuCl2 on pigments content

In both species, total carotenoids expressed as mg ml�1

were not affected significantly by increase in CuCl2concentration (Table 2). When expressed in terms of

pg cell�1, increase in CuCl2 increased total carotenoids in

both species. Total carotenoid content in mg ml�1 and in

pg cell�1 were about three and five times higher in D. salina

compared to D. tertiolecta at all CuCl2 concentrations

tested, respectively.

As shown in Table 3, in D. salina total chlorophyll

expressed as mg ml�1 decreased with increase in CuCl2,

whereas in D. tertiolecta total chlorophyll was essentially

unaffected by CuCl2. At all CuCl2 concentrations, total

chlorophyll was higher in D. tertiolecta compared to

D. salina. When expressed in term of pg cell�1, increase in

CuCl2, increased total chlorophyll in both species. The

chlorophyll content per cell in D. salina and D. tertiolecta

were not significantly different at all tested CuCl2concentrations.

3.3. Effects of CuCl2 on APX activity and lipid

peroxidation

Although APX activity increased with increase in CuCl2concentrations in both species, higher activity was observed

in D. tertiolecta compared to D. salina (Table 4). At 20 mM

CuCl2, APX activity was 45% higher inD. tertiolecta than in

D. salina.

K. Nikookar et al. / Biomolecular Engineering 22 (2005) 141–146 143

Table 2

Effects of CuCl2 on total carotenoids in D. salina and D. tertiolecta expressed as mg ml�1 and pg cell�1

Species CuCl2 concentration (mM)

mg ml�1 pg cell�1

1 5 10 20 1 5 10 20

D. salina 11.5 � 2.5 12.2 � 2 13.7 � 0.6 9.3 � 1.6 7 � 0.9 10.4 � 1.1 15.9 � 0.9 16.2 � 2.7

(100) (106) (119) (81) (100) (149) (227) (231)

(100) (100) (100) (100) (100) (100) (100) (100)

D. tertiolecta 3.8 � 0.1 4 � 0.9 3.4 � 0.4 3.8 � 0.07 1.5 � 0.07 2.1 � 0.5 2.7 � 0.4 3.5 � 0.5

(100) (105) (102) (100) (100) (140) (180) (233)

(33) (32) (28) (40) (21) (20) (17) (22)

Each value is mean � S.E. In each column, bold figures in parenthesis show percent total carotenoids ofD. tertiolecta relative toD. salina. In each row figures in

parenthesis show percent total carotenoids at 5, 10, and 20 mM CuCl2 relative to control (1 mM CuCl2).

Table 1

Algal cell number as affected by different concentrations of CuCl2 and incubation period

Species Incubation time (h) CuCl2 concentration (mM)

1 5 10 20

D. salina 24 16.3 � 1.8 11.6 � 1.9 8.6 � 1.88 5.7 � 0.02

(100) (71) (53) (35)

(100) (100) (100) (100)48 20.8 � 1.9 8.6 � .72 6.8 � 0.7 5.3 � 1.9

(100) (41) (33) (25)

(128) (74) (79) (93)72 22.5 � 1.2 7.3 � 0.7 6.3 � 0.7 4 � 0.3

(100) (32) (28) (18)

(138) (63) (73) (80)

D. tertiolecta 24 24.8 � 1.8 18.9 � 0.3 14.5 � 0.4 10.7 � 1.6

(100) (76) (58) (43)

(100) (100) (100) (100)48 28.5 � 1.2 15.5 � 1.3 12.5 � 1.2 10.5 � 1.2

(100) (54) (44) (37)

(115) (82) (86) (98)72 35.5 � 1.6 13.8 � 1.9 10.7 � 0.2 9.9 � 0.9

(100) (39) (33) (28)

(143) (73) (81) (93)

Each figure shows number of cells ml�1 � 105. Each value is mean � S.E. In each column, bold figures in parenthesis show percent cell number at 48 and

72 h relative to 24 h after incubation with CuCl2. In each row figures in parenthesis show percent cell number at 5, 10, and 20 mM CuCl2 relative to control

(1 mM CuCl2).

Lipid peroxidation was determined by evaluating MDA

contents of the cells (Table 5). Increase in CuCl2concentration caused significant increase in MDA contents

of the cells in both species. Although percent increase in

Table 3

Effects of CuCl2 on total chlorophyll expressed as mg ml�1 and pg cell�1

Species CuCl2 concentration (mM)

mg ml�1

1 5 10 20

D. salina 4.8 � 2.2 3.6 � 1.5 3.7 � 0.7 2.7 �(100) (75) (77) (56)

D. tertiolecta 5.8 � 0.9 5.8 � 1.7 5.6 � 0.2 6.3 �(100) (100) (92) (108)

Each value is mean � S.E. Figures in parenthesis are percent total chlorophyll a

MDA at 10 and 20 mM CuCl2 relative to control (1 mM)

were comparable in both species, the MDA contents of

D. salina cells were about three times higher at most CuCl2concentrations.

pg cell�1

1 5 10 20

0.2 2.9 � 1.2 3.1 � 0.9 4.3 � 1.2 4.7 � 0.2

(100) (107) (148) (162)

0.3 2.3 � 0.2 3.1 � 09 3.8 � 0.3 5.8 � 1.2

(100) (135) (165) (252)

t 5, 10, and 20 mM CuCl2 relative to control (1 mM CuCl2).

K. Nikookar et al. / Biomolecular Engineering 22 (2005) 141–146144

Table 4

APX activity as affected by different concentrations of CuCl2 expressed as DO.D. min�1 106 cell�1

Species CuCl2 concentration (mM)

1 5 10 20

D. salina 0.028 � 0.01 0.044 � 0.007 0.053 � 0.005 0.056 � 0.002

(100) (157) (189) (201)

D. tertiolecta 0.047 � 0.03 0.061 � 0.01 0.095 � 0.02 0.081 � 0.01

(100) (129) (202) (172)

Each value is mean � S.E. Figures in parenthesis are percent APX activity at 5, 10, and 20 mM CuCl2 relative to control (1 mM CuCl2).

Table 5

Effects of CuCl2 on lipid peroxidation expressed as nmol MDA 109 cells�1

Species CuCl2 concentration (mM)

1 5 10 20

D. salina 0.6 � 0.2 2.4 � 0.5 1.4 � 0.05 2.0 � 0.8

(100) (400) (232) (334)

(100) (100) (100) (100)

D. tertiolecta 0.2 � 0.08 0.39 � 0.1 0.5 � 0.02 0.6 � 0.1

(100) (195) (250) (300)

(33) (16) (30) (30)

Each value is mean � S.E. In each column, bold figures in parenthesis show

percent lipid peroxidation in D. tertiolecta relative to D. salina. In each row

figures in parenthesis show percent lipid peroxidation at 5, 10, and 20 mM

CuCl2 relative to control (1 mM CuCl2).

Table 6

Cu content ofD. salina andD. tertiolecta cells (mg Cu 109 cells�1) supplied

with 1 (control), 5, and 10 mM CuCl2 for 24 h

Species CuCl2 concentration (mM)

1 5 10

D. salina 68.5 � 14.03 217.3 � 41.22 558.2 � 78

D. tertiolecta 59.5 � 6.82 168.0. � 36.7 349.2 � 045.5

Each value is mean � S.E.

3.4. Cu content of algal cells

As shown in Table 6, Cu content of both D. salina and

D. tertiolecta cells increased with increase in Cu concentra-

tions of culture media. The D. salina cells contained higher

amount of Cu compared to D. tertiolecta. When grown in

culture media with 10 mM CuCl2, 558 and 342 mg Cu per

109 cells were recorded in D. salina and D. tertiolecta,

respectively.

4. Discussion

Various factors such as annual precipitation and evapora-

tion affectCu content ofMaharlu salt lake.Averageof 6.7 mM

Cuwas recorded in water samples taken from the lake during

2002–2004 which is in the range of the concentrations used

in the present experiment. Heavy metals and other environ-

mental stresses limit growth and reproduction of the algae and

other organisms living in the Maharlu salt lake. The toxic

effect of heavy metals appears to be partly related to the

production of ROS [11]. Increase in the levels of low

molecular weight antioxidants such as carotenoids, and

antioxidant enzymes such as APX are among protective

mechanisms that serve to remove ROS [6,10]. In this work,

carotenoids content, expressed as mg cell�1, increased by

elevatedCuCl2 concentration in both species, being about five

fold higher in D. salina (Table 2). In terms of mg ml�1,

increase in total carotenoids due to elevated CuCl2 was offset

bydecrease in cell number (Tables 1 and 2). In addition to total

carotenoids, APX activity also increased in both species with

increase in CuCl2. Higher APX activity in D. tertiolectamay

partly account for the relative tolerance of this species to

CuCl2 as is evident by smaller reduction in cell number at

elevated CuCl2 concentrations (Tables 1 and 3). There are

reports concerning copper-induced activation of the anti-

oxidant systems in plants and green algae [10,12,25]. In

Lemna trisulca Cu concentrations up to 10 mM increased

carotenoids content of the fronds while significant decrease

in the pigments concentrations occurred at 25 and 50 mM

Cu [9]. It has been shown that copper toxicity activates APX

in sunflower plants and bean seedlings grown hydroponi-

cally [25,26]. Activation of APX has also been reported in

green microalgae Selenastrum capricornutum and macro-

algae Enteromorpha compressa L. [12,27]. Ceratophyllum

demersum, a free floating macrophyte, treated with 2 and

4 mM Cu showed significant increase in the activity of

antioxidant enzymes APX, catalase and superoxide dis-

mutase [10].

The principal mechanism of copper toxicity involves the

Haber–Weiss reaction, producing hydroxyl radicals from

hydrogen peroxide [11]. Hydroxyl radicals are highly

reactive and are able to oxidize and break apart biological

macromolecules such as lipids, proteins and nucleic acids

which result in cellular death [6]. Indeed, the metal induced

membrane lipid peroxidation is mostly attributed to

increased production of free radicals [28]. Enhanced lipid

peroxidation due to Cu-induced oxidative stress in aquatic

plants is reported by Devi and Prasad [10] Our results

indicate that excess Cu generates oxidative stress as is

evident from increased lipid peroxidation (Table 5). Since

APX removes hydrogen peroxide, higher APX activity in D.

tertiolecta may partly account for lower degree of lipid

peroxidation in this species compared to D. salina.

Although decrease in chlorophyll content under oxidative

stress has been reported in plants [10,29], increase in total

chlorophyll in terms of pg cell�1 was observed in both

K. Nikookar et al. / Biomolecular Engineering 22 (2005) 141–146 145

species at elevated CuCl2 (Table 4). When expressed as

mg ml�1, total chlorophyll in D. salina decreased with

increase in CuCl2 which is due to large decrease in cell

number ml�1 at elevated CuCl2 concentrations (Tables 1

and 4). In D. tertiolecta increase in total chlorophyll cell�1

was just offset by decrease in total chlorophyll ml�1 of algal

culture; therefore, total chlorophyll remained essentially

constant at all tested CuCl2 concentrations. In barley leaves,

copper inhibited pigment accumulation and retarded

chlorophyll integration into the photosystems [30]. When

effect of Ni2+ on chlorophyll biosynthesis was studied by

Manankina et al. [31], it was found that 10�4 M Ni2+

stimulated chlorophyll biosynthesis while higher concentra-

tions (10�3 M) markedly inhibited chlorophyll accumula-

tions. Low concentrations of cobalt caused significant

increase in chlorophyll a and carotenoids in Nitzchia

perminuta while higher concentrations reduced pigments

content [32]. Nyitrai et al. [33] studied the effects of low-dose

stressors Cd, Pb, Ni and Ti on the development of chloroplast

in maize and bean seedlings. Stimulation of chlorophyll

synthesis was observed during all of the treatments. It was

assumed that the low-dose stressors generate non-specific

reactions in plants which may involve changes of the

hormonal balance. Pigments production is also influenced

by light intensity. As response mechanism to oxidative

damage, total carotenoid production is enhanced at high

light intensities; to capture more light energy, chlorophyll

production is increased at lower intensities [34,35].

Bioaccumulation of heavy metals in aquatic ecosystems

have been reported by several investigators [9,10]. Although

higher amount of Cu was found in D. salina cells (Table 6),

larger cell volume of D. salina [36] may offset the

differences in Cu content between D. tertiolecta and D.

salina. In conclusion, compared to D. salina, higher

tolerance of D. tertiolecta to CuCl2 is partly explained by

higher APX activity in this species. Hydroxyl radical (HO�),

the most reactive ROS, is produced from H2O2 by Cu2+

(Haber–Weiss cycle). This ROS is involved in peroxide

radicals (ROO�) production which is intermediate in the

chain reactions of lipid peroxidation [11]. It is probable that

higher APX activity in D. tertiolecta which removes H2O2,

is partly responsible for reduced lipid peroxidation and

lower sensitivity of this species to elevated CuCl2.

Acknowledgement

The authors would like to thank the Shiraz University

Research Council for supporting this research.

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