Ecological Indicators 45 (2014) 103–109

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Ecological Indicators
journal homepage: www.elsevier.com/locate/ecolind

MT-like proteins: Potential bio-indicators of Chlorella vulgaris for zinc

contamination in water environment
Hong Yang a,b , Zhi-Yong Huang a,∗ , Jian Li a , Yue Hu a
a
College of Bioengineering, Jimei University, Xiamen 361021, China
b
Management Office of Scientific Research, Longyan University, Longyan 364012, China

a r t i c l e i n f o a b s t r a c t

Article history: Chlorella vulgaris has been usually used to monitor the toxicity of Zn in water environment, but the bio-
Received 4 September 2013 chemical responses of the algae to Zn2+ at different levels remain unknown to date. In the present study,
Received in revised form 17 March 2014 the growth inhibition, the antioxidant enzymes, the subcellular Zn and the induced Zn-metallothionein-
Accepted 20 March 2014
like (Zn-MT-like) proteins in C. vulgaris exposed to 0, 5, 10, 20, 50 and 100 ␮mol l−1 of Zn2+ were
investigated. Results showed that the growth of C. vulgaris was significantly inhibited (p < 0.05) at Zn2+
Keywords:
level above 5 ␮mol l−1 . Compared with the control group, the activities of peroxidase (GSH-Px), superox-
C. vulgaris
ide dismutase (SOD) and catalase mostly increased with the rises of Zn2+ concentrations ranging from 5 to
Metallothionein-like protein
Bio-indicator
100 ␮mol l−1 , except for GSH-Px with a lower value in the 100 ␮mol l−1 group compared with that of the
Zinc 50 ␮mol l−1 group, and SOD with no significant difference at the 5, 10 and 100 ␮mol l−1 groups compared
Water environment with that of the control group. Contrarily, the activities of guaiacol peroxidase significantly decreased
(p < 0.05) at Zn2+ concentrations from 5 to 100 ␮mol l−1 compared with that of the control group. After
72 h exposure, Zn in the algal cells was observed to mainly distribute in the form of heat stable fractions,
in which Zn-binding metallothionein-like (MT-like) proteins were largely biosynthesized. In addition,
the amounts of Zn-MT-like proteins induced in the algae significantly increased (p < 0.05) with the rises
of Zn2+ concentrations at the assay levels. The results indicated that the activities of antioxidant enzymes
and the induction of Zn-MT-like proteins in C. vulgaris may play important roles in dealing with the tox-
icity of Zn2+ to the algal cells, and the induction of Zn-MT-like proteins in C. vulgaris can be used as a
potential bio-indicator to monitor the contamination of Zn2+ in water environment.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Chlorella vulgaris (C. vulgaris), displaying high growth rates
under autotrophic and heterotrophic conditions, is a genus of uni-
cellular green algae, and has been widely used to monitor the
contamination of heavy metals in water environment because of
the algal strong bio-sorption abilities (Kajan et al., 1992). However,
some biochemical responses of C. vulgaris to heavy metals remain
unknown, in which some heavy metals pose favorable effects on the
algal growth at certain concentrations, while many of them present
adverse effects even at very low concentrations (Bajguz, 2011;
Huang et al., 2009; Muyssen and Janssen, 2001; Wang and Wang,
2011; Wang et al., 2011). Zinc (Zn) is one of the most important
metals participating in various biological functions in microalgae,
∗ Corresponding author. Tel.: +86 592 6181102; fax: +86 592 6180470.
E-mail address: zhyhuang@jmu.edu.cn (Z.-Y. Huang).
but the metal may also cause severe intracellular damage by inhib-
iting algal metabolic processes at high concentrations (Price and
Morel, 1994). For example, the median lethal concentrations (LC50 )
of Zn2+ to C. vulgaris ranged from 2.4 mg l−1 to 100 mg l−1 (Maeda
et al., 1990; Rachlin and Farran, 1974). But the toxicity of Zn2+ to C.
vulgaris may also depend on the metal species, the exposure dura-
tion and the testing medium characteristics (Muyssen and Janssen,
2001; Yeh and Chang, 2012). Hence, it is important to investigate
the biochemical responses of C. vulgaris to Zn2+ in water environ-
ment.
Many researches have demonstrated that heavy metals may
induce the production of reactive oxygen species (ROS) in
autotrophic C. vulgaris (Tripathi and Gaur, 2004). To prevent the
ROS damages, various anti-oxidative enzymes may be induced and
activated in the algal cells, among which superoxide dismutase
(SOD), catalase (CAT), peroxidase (POD) and glutathione peroxi-
dase (GSH-Px) are usually involved (Valavanidis et al., 2006). For
example, the activity of SOD in C. vulgaris exposed to 100 ␮mol l−1
Cd2+ obviously increased, while the activity of CAT decreased in the

http://dx.doi.org/10.1016/j.ecolind.2014.03.017
1470-160X/© 2014 Elsevier Ltd. All rights reserved.
104 H. Yang et al. / Ecological Indicators 45 (2014) 103–109
same testing medium (Niczyporuk et al., 2012). Cu2+ at the con-
centration of 0.5 ␮g ml−1 was also able to decrease the CAT activity
in C. vulgaris (Mallick, 2004). However, Pb2+ was able to increase
the activities of SOD, CAT and GSH-Px in C. vulgaris (Bajguz, 2010).
Therefore, the activities of anti-oxidative enzymes in C. vulgaris
may vary with different kinds of stress metals and different metal
concentrations. Similarly, Zn2+ is also able to cause an oxidative
damage to C. vulgaris through increasing the levels of ROS species
such as the superoxide radical (• O2 − ), hydroxyl radical (• OH), and
the hydrogen peroxide (H2 O2 ) (Andrade et al., 2006; Mittler, 2002;
White and Jahnke, 2002). However, there is insufficient scientific
data to interpret the biochemical responses of the algae to the
oxidative damage of Zn2+ .
Phytochelatins (PCs), with a general structure of (␥-Glu-Cys)n-
Gly (n = 2–11), are well-known polypeptides induced in C. vulgaris
to sequester the influx heavy metals in cells. They play impor-
tant roles on the detoxification of heavy metals to the algae
(Bajguz, 2002; Gekeler et al., 1988). In addition, metallothionein-
like (MT-like) proteins have also been found in C. vulgaris capable
of detoxifying the toxicities of heavy metals such as Cd2+ and Zn2+
(Huang et al., 2009; Yoshida et al., 2006). Therefore, the induction
of PCs and MT-like proteins in micro-algae may depend on the algal
species and the kinds of metals.
C. vulgaris has been widely used to monitor the toxicity of Zn
in water environment (Kajan et al., 1992; Muyssen and Janssen,
2001). However, the study on the biochemical responses of C. vul-
garis exposed to Zn2+ is rarely conducted. In the present study, some
biochemical parameters, including the growth rates, the activities
of anti-oxidative enzymes, the distributions of subcellular Zn, and
the induction of Zn-binding MT-like proteins, in C. vulgaris exposed
to Zn2+ at different concentrations are to be measured. The bio-
chemical responses of the algae in dealing with the toxicity of Zn2+
are to be investigated.
2. Materials and methods
2.1. Cultivation and biomass test
C. vulgaris was donated from Fisheries College, Jimei Univer-
sity, China. The algae were cultured and amplified in Erlenmeyer
flasks with equal volumes (v/v) of the inoculums and the culture
medium consisting of 1.0 g l−1 NaHCO3 , 0.5 g l−1 KNO3 , 0.5 g l−1
MgSO4 ·7H2 O, 0.02 g l−1 K2 HPO4 , 2 ␮g l−1 vitaminB12 and 0.6 mg l−1
vitamin B1 in sterilized seawater based on a f/2 nutritional com-
position (Guillard and Ryther, 1962). The cultivation temperature
maintained at 26 ± 1 ◦ C with white fluorescent illumination by a
cycle of 12 h light (300 ␮E m−2 s−1 ) per 24 h in a PGX-280B illumi-
nation cultivation cabinet (Ningbo, China). Filtered air was bubbled
into the algal suspension. For the convenience, the relationship
between the spectrophotometrical absorbance at 686 nm (A686 )
(measured with LabTech-2100 spectrophotometer, Beijing, China)
and the cell densities (counted by B2221P microscope, Shanghai,
China) in the algal suspension were established. The initial cell den-
sities were about 8 × 106 cells ml−1 . During the exponential growth
phase, the regression equation between the cell densities (y) and
A686 (x) was established as follows:
y = 2 × 107 x + 889331, R2 = 0.997
2.2. Growth inhibition test
Based on the Zn criterion (15.4 ␮mol l−1 ) of seawater quality
standard (Level II) set in China (GB 3097-1997) and the contamina-
tion of Zn in seawater reported recently (Pan and Wang, 2012), the
growth inhibition of C. vulgaris were tested at the concentrations of
Zn2+ ranging from 0 to 100 ␮mol l−1 . Each of 200 ml of algal suspen-
sion, at mid-exponential growth phase, was added with ZnCl2 stock
solution at the concentrations of 0, 5, 10, 20, 50 and 100 ␮mol l−1
Zn2+ . The algal suspensions with the addition of Zn2+ were labeled
as Zn groups accordingly and the algal suspension without adding
Zn2+ was set as control group. Three replicates for each group
were carried out. Because the concentration of Zn2+ in the con-
trol groups mainly from seawater was only 0.48 ␮mol l−1 based on
our measurement, Zn2+ levels in each of Zn groups were tagged
based on the concentrations of Zn2+ added in the corresponding
algal suspensions. All the assay groups were incubated at the same
cultivation conditions as described above, except for shaking five
times per day by hand instead of automatically bubbling in filtered
air. Cell densities (cells ml−1 ) for each algal suspension were calcu-
lated by measuring the A686 values at the time intervals of 0, 12,
24, 48, 72, 96 and 120 h. After 120 h exposure, the growth rates
based on the initial and final cell densities were calculated accord-
ing to the method described in our previous report (Huang et al.,
2009).
2.3. Measurement of antioxidant enzymes
2.3.1. Activities of superoxide dismutase (SOD)
After exposure for 72 h, each of 40 ml algal suspension was
centrifuged at 4000 × g for 10 min. The pellets were respectively
washed with 10 mmol ml−1 EDTA and ultrapure-water to remove
Zn2+ absorbed on the surfaces of algal cells. The cells re-suspended
with ice-chilled 10 mmol l−1 Tris–HCl (pH 8.0) containing 2% (w/v)
of polyvinyl polypyrrolidone (PVP) were homogenized with ultra-
sonic disintegrator (Scientz-IID, Ningbo, China); and then, the
homogenates were centrifuged at 10,000 × g for 20 min (4 ◦ C). By
means of pyrogallol auto-oxidation, the activities of SOD in the
supernatants (defined as enzyme extracts) were measured accord-
ing to the method of Marklund and Marklund (1974). Briefly, the
inhibition rates of SOD in the enzyme extracts were estimated by
detecting the spectrophotometrical absorbance at 325 nm in the
assay mixture containing 2.8 ml of 50 mmol l−1 Tris–HCl (pH 8.0),
0.1 ml of 5 mmol l−1 pyrogallol and 0.1 ml of the enzyme extract.
The enzyme causing 50% inhibition of the pyrogallol auto-oxidation
rate was defined as one unit of SOD activity, expressed as the units
per 108 cells of C. vulgaris. The procedural blanks, by replacing the
enzyme extracts as the homogenized medium, i.e., 10 mmol l−1
Tris–HCl (pH 8.0) containing 2% (w/v) of PVP, were run for back-
ground subtraction.
2.3.2. Activities of glutathione peroxidase (GSH-Px)
The activities of GSH-Px were estimated based on a modi-
fied method of Rotruck et al. (1973). Briefly, 0.5 ml of enzyme
extract, as described above except for the use of 10 mmol l−1 phos-
phate buffer (pH 6.2) in algal cell homogenization, was mixed
with 0.35 ml of reaction solution consisting of 1 mmol l−1 GSH,
0.34 mmol l−1 EDTA, 25 mmol l−1 sodium azide and 0.5 mol l−1
phosphate buffer. After incubating at 37 ◦ C for 5 min, 0.175 ml of
1.25 mmol l−1 H2 O2 was added, and the mixture was incubated
again at 30 ◦ C for 5 min. The reaction was stopped by adding
3.25 ml of 10% (w/v) trichloroacetic acid, and then the mixture
was centrifuged at 4000 × g for 10 min. 2 ml of the supernatant
was respectively mixed with 2 ml of 0.5 mol l−1 disodium hydro-
gen phosphate, 0.15 ml of 6 mol l−1 NaOH and 0.3 ml of 1 mmol l−1
5,5 -dithio-bis(2-nitrobenzoic acid) (in 1% sodium citrate). After
reacting for 3 min, the mixture was measured at 412 nm. The GSH-
Px activity was expressed as the micromole of oxidized glutathione
per 1010 cells and per minute. The procedural blanks were run for
background subtraction.
 H. Yang et al. / Ecological Indicators 45 (2014) 103–109
2.3.3. Activities of catalase (CAT)
The activities of CAT were measured based on the method of
Fossati et al. (1980). Briefly, 0.2 ml of CAT enzyme extract, by using
10 mmol l−1 phosphate buffer (pH 7.0) in algal cell homogeniza-
tion, was mixed with 0.5 ml of 100 mmol l−1 phosphate buffer (pH
7.0) and 0.1 ml of 150 mmol l−1 H2 O2 . The reaction was initiated by
the addition of H2 O2 at 25 ◦ C, and the decreases of spectrophoto-
metrical absorbance as a consequence of H2 O2 decomposition were
measured at 240 nm (A240 nm ). One unit of CAT activity was defined
by decreasing 0.1 U of A240 nm value per minute, and the CAT activ-
ities in algae were expressed as the enzyme units per 109 cells. The
procedural blanks were run for background subtraction.
2.3.4. Activities of guaiacol peroxidase (POD)
The activities of POD were estimated according to the method
of Andrade et al. (2006). Briefly, an assay solution was prior pre-
pared by mixing 50 ml of 100 mmol l−1 phosphate buffer (pH 6.0),
19 ␮l of 30% H2 O2 and 28 ␮l of guaiacol. Then, 3 ml of the assay
solution was mixed with 1 ml of POD enzyme extracted by homog-
enizing the algal cells with 50 mmol l−1 phosphate buffer (pH 6.5).
The formation rate of oxidized guaiacol was recorded by monitor-
ing the spectrophotometrical absorbance at 470 nm (A470 nm ). One
unit of the POD activity was defined by the increase of 0.01 U of
A470 nm value per minute, and the POD activities were expressed as
the enzyme units per 108 cells. The procedural blanks were run for
background subtraction.
2.4. Measurement of total cellular Zn and subcellular Zn
concentrations
After 72 h exposure, the cells in 160 ml of algal suspen-
sion exposed to Zn2+ at the tagged concentrations of 0, 5 and
100 ␮mol l−1 were harvested. After washed as described above,
the cells were homogenized with 10 mmol l−1 of Tris–HCl buffer
(containing 2 mmol l−1 ␤-mercaptoethanol, pH 7.8) for the mea-
surement of total cellular Zn and subcellular Zn.
Total cellular Zn was measured with ICP-MS based on the
method as described in our previous report (Huang et al., 2009). The
adding standard assays were carried out for the quality assurance
of Zn measurement, and the recoveries ranging from 80% to 102%
were achieved. The total cellular Zn was expressed as the amounts
(nmol) of Zn per 109 cells.
The fractionation of subcellular Zn was carried out by differ-
ential centrifugation procedures based on the previous reports
(Redeker et al., 2007; Wallace et al., 1998, 2003). Briefly, 4 ml of
the algal homogenate was centrifuged at 2000 × g for 15 min (4 ◦ C)
for fractionating the organelles and proteins in supernatants and
the cellular debris and granules in pellets. The pellets were re-
suspended in 0.4 ml of ultrapure-water and heated at 100 ◦ C for
2 min, followed by adding 0.4 ml of 1 mol l−1 NaOH and incubat-
ing at 65 ◦ C for 60 min. After centrifuging at 10,000 × g for 30 min
(4 ◦ C), the metal granules in pellets and the cellular debris in super-
natants were separated. The supernatants containing organelles
and proteins in the previous procedure were ultra-centrifuged
at 100,000 × g for 60 min (4 ◦ C) to obtain organelles (lysosomes,
mitochondria and microsomes) in pellets, and cytoplasm frac-
tions in supernatants. The supernatants containing cytoplasm
were subsequently heated at 80 ◦ C for 10 min, and then cooled
in ice for 60 min for centrifuging at 21,000 × g (30 min, 4 ◦ C) to
separate the heat-denatured fractions (HDF) in pellets and the
heat-stable fractions (HSF) containing MT-like proteins in super-
natants. After digested with 65% HNO3 , Zn amounts in each of the
pellets and supernatants as described above were measured with
ICP-MS.
105
2.5. Measurement of Zn-binding metallothionein-like (MT-like)
proteins
For the measurement of Zn-binding MT-like proteins, each of
100 ml algal suspensions after 72 h exposure was centrifuged, and
the cells were washed as described above. Then, the cells were
homogenized with 10 mmol l−1 Tris-HCl (containing 2 mmol l−1 ␤-
mercaptoethanol, pH 7.8), and the homogenate was adjusted to
5 ml with the Tris-HCl buffer and filtered through 0.45 ␮m mem-
brane for MT measurement. Zn-binding MT-like proteins in C.
vulgaris were measured according to the method of Huang et al.
(2009). Briefly, an Agilent 1260 chromatographic system with a size
exclusion chromatographic column (7.8 mm i.d × 300 mm, 6 ␮m)
was used. The filtered homogenates were separated by eluting the
column with 10 mmol l−1 Tris–HCl (pH 7.6). The flow rate was set as
1 ml min−1 and the injection volume was 20 ␮l. The combination of
HPLC and ICP-MS was accomplished by connecting the outlet of the
UV detector of HPLC with the inlet of ICP nebulizer using a Teflon
tubing. The standard reference material of Zn-MTs induced from
rabbit liver (Lugu Biotechnology Corporation, Hunan, China) was
run at the same chromatographic conditions to identify the peaks of
MT-like proteins. The signals of spectrophotometrical absorbance
at 254 nm and 66 Zn were simultaneously monitored. The retention
time of standard reference material of Zn-MTs and the Zn-MT-
like proteins extracted from C. vulgaris was 6.81 min and 6.87 min,
respectively. The amounts of Zn-MT-like proteins were expressed
as the intensities (counts) of 66 Zn detected per 107 algal cells.
2.6. Data analysis
Data are presented as mean ± standard deviation for three repli-
cates. The effects of Zn2+ concentrations and exposure time on the
growths of C. vulgaris were statistically analyzed with a repeated
measures method in which Mauchly’s test was used for spheric-
ity. In addition, the activities of antioxidant enzymes in the algae
exposed to Zn2+ at different concentrations were compared with
the method of one-way ANOVA. The software of SPSS 19.0 for Win-
dows was used for all the data analysis as described above.
3. Results
3.1. Growth inhibition of C. vulgaris exposed to Zn2+
Fig. 1a showed the growth of C. vulgaris exposed to Zn2+ at
concentrations ranging from 0 to 100 ␮mol l−1 during 120 h cul-
tivation. By the Mauchly’s test, the sphericity hypothesis could not
be refused (p = 0.323), and the test showed that the concentrations
of Zn2+ played significantly inhibiting effects (p < 0.05) on the algal
growth. Therefore, the multivariate test was as follows for investi-
gating the inhibiting effects of Zn2+ on the algal growth. The results
showed that the algal growth were significantly inhibited (p < 0.05)
at Zn2+ concentrations more than 5 ␮mol l−1 , and no significant
difference (p > 0.05) of the inhibitions was observed at Zn2+ con-
centrations ranging from 10 to 100 ␮mol l−1 . Compared with the
control group, the algal biomass in the groups of 5 ␮mol l−1 and
10 ␮mol l−1 respectively decreased by 11.0% and 46.3% after 72 h.
Data in Fig. 1b showed that the values of growth rates after five
days’ exposure were negative for most of the Zn groups except for
the group of 5 ␮mol l−1 . With a positive value of growth rate, the
algal growth in 5 ␮mol l−1 group showed no significant difference
(p > 0.05) compared with that of control group, but a slight inhibi-
tion could be observed because the growth rate was a bit lower than
that of the control group. In addition to the growth inhibition by
Zn2+ at different levels, the algal growths were also significantly
inhibited (p < 0.05) with the exposure time based on the test of
106 H. Yang et al. / Ecological Indicators 45 (2014) 103–109

2.2x10 7 (a)
(b)
0.3
7
2.0x10

7 0.2
1.8x10
-1
0 mol L
Cell densities/ml

Growth rate (day )

7

-1
1.6x10 -1 0.1
5 mol L
-1
7
10 mol L
1.4x10 -1
20 mol L 0.0
-1
1.2x10
7 50 mol L
-1
100 mol L -0.1
7
1.0x10

6
-0.2
8.0x10

6
6.0x10 -0.3
0 12 24 48 72 96 120 0 5 10 20 50 100
Time course (h) 2+ -1
Concentrations of Zn (µmol l )

Fig. 1. Biomass versus time–course (a) and growth rates (b) of C. vulgaris exposed to different concentrations of Zn2+ for 5 days.

repeated measures ANOVA. For example, the algal biomass in the
group of 50 ␮mol l−1 decreased by 43% in the exposure duration
from day 1 to day 5.
3.2. Activities of antioxidant enzymes in C. vulgaris exposed to
Zn2+
The activities of antioxidant enzymes including GSH-Px, SOD,
CAT and POD in C. vulgaris exposed to Zn2+ at different concen-
trations for 72 h were shown in Fig. 2. The activities of GSH-Px in
all Zn2+ groups were significantly higher (p < 0.05) than that in the
control group, and the activities significantly increased (p < 0.05)
with the rises of Zn2+ concentrations ranging from 5 to 50 ␮mol l−1 ,
except for the 10 and 20 ␮mol l−1 groups with similar GSH-Px
activities. But the activity of GSH-Px in 100 ␮mol l−1 group was
significantly lower (p < 0.05) than that of the 50 ␮mol l−1 group. For
example, the activities of GSH-Px in the algae exposed to Zn2+ at 10,
50 and 100 ␮mol l−1 were respectively 0.3, 1.3 and 1.2-folds higher
than that of the control group. For the activities of SOD, no signifi-
cant difference (p > 0.05) was observed among the Zn groups at 5, 10
and 100 ␮mol l−1 and the control group; while the SOD activities
significantly increased (p < 0.05) at the Zn2+ concentrations of 20
and 50 ␮mol l−1 . And a significant decrease (p < 0.05) of SOD activ-
ity also occurred at 100 ␮mol l−1 group compared with that of the
50 ␮mol l−1 group. Results in Fig. 2 showed that the activities of CAT
significantly increased (p < 0.05) with the Zn2+ concentrations ran-
ging from 5 to 100 ␮mol l−1 , but no significant difference (p > 0.05)
was respectively observed in the groups of 5 and 10 ␮mol l−1 , and in
the groups of 20 and 50 ␮mol l−1 . Contrarily, significant decreases
(p < 0.05) of POD activities were observed with the rises of Zn2+
concentrations, especially at Zn2+ levels above 10 ␮mol l−1 . For
example, the POD activities in the groups of 5, 10 and 100 ␮mol l−1
decreased by 16%, 87% and 97% compared with that of the control
group. With no significant difference at Zn2+ concentrations from
10 to 100 ␮mol l−1 , the POD activities kept at low levels and were
significantly lower (p < 0.05) than those of the 5 ␮mol l−1 group and
the control group.
per 109 cells) of the control group, but 5-fold of cellular Zn increased
in the 100 ␮mol l−1 group.
Distributions of subcellular Zn in C. vulgaris exposed to
5 ␮mol l−1 and 100 ␮mol l−1 Zn2+ were shown in Table 1. For the
quality control of Zn measurement
 in subcellular fractionation, the
sum of zinc amounts ( Zn) in all five subcellular fractions was
compared with the total cellular Zn directly measured. As shown in
Table 1, the accurate measurement of subcellular Zn in the present
experiment was achieved with  the recoveries of 80%, 102% and
102% calculated by the ratios of Zn/total cellular Zn in 0, 5 and
100 ␮mol l−1 groups, respectively. Data in Table 1 showed that the
amounts of Zn distributing in each of the subcellular fractions in
5 ␮mol l−1 group were slightly higher than those of the control
group, but no significant difference (p > 0.05) of the distributions
was observed between the two groups except for Zn level in HSF
with 5.05 nmol per 109 cells in 5 ␮mol l−1 group which was signif-
icantly higher (p < 0.05) compared with that of the control group.
Especially, the total amount of Zn in the detoxified fractions (includ-
ing debris, granules and HSF), which accounted for 69.7% of the
total cellular Zn in 5 ␮mol l−1 group, was not significantly different
(p > 0.05) compared with that (72.3%) of the control group. How-
ever, Zn levels in all the subcellular fractions in 100 ␮mol l−1 group
were significantly higher (p < 0.05) than those of the control group
and the 5 ␮mol l−1 group, respectively. In addition, Zn amount in
the detoxified fractions accounted for 80% of the total cellular Zn in
the 100 ␮mol l−1 group, which was respectively higher than those
in the 5 ␮mol l−1 group and the control group.
3.4. Induction of MT-like proteins in C. vulgaris
The induction of Zn-MT-like proteins in C. vulgaris exposed to
Zn2+ at different concentrations was shown in Fig. 3. Result showed
that the amounts of Zn-MT-like proteins in the algae significantly
increased (p < 0.05) with the rises of Zn2+ concentrations. With the
highest level in 100 ␮mol l−1 group, the amount of Zn-MT-like pro-
teins induced in C. vulgaris was 1.65-fold higher than that of the
control group.

3.3. Total cellular Zn and subcellular Zn concentrations 4. Discussion

After 72 h exposure, the amounts of total cellular Zn were Zn is one of the most important essential metals participating
11.6 and 55.9 nmol l−1 per 109 cells in 5 and 100 ␮mol l−1 groups, in various biological functions in C. vulgaris, but the excess of Zn
respectively. No significant increase (p > 0.05) of cellular Zn was may cause severe intracellular damage by inhibiting algal metabolic
observed in the 5 ␮mol l−1 group compared with that (9.3 nmol l−1 processes. Results in Fig. 1 showed that C. vulgaris was sensitive to
 H. Yang et al. / Ecological Indicators 45 (2014) 103–109 107

d 14
3.5 b
e SOD
GSH-Px b
Activities of GSH-Px (Unit/10 cells)

12
3.0

Activities of SOD (Unit/10 cells)

10

10
2.5

8
c a
2.0 c 8
b ab
a
6 a
1.5 a

1.0 4

0.5 2

0.0 0
0 5 10 20 50 100 0 5 10 20 50 100
2+ -1 2+ -1
Zn concentrations (µmol l ) Zn concentrations (µmol l )

3.5
1.6
d
CAT a POD
3.0 1.4
b

Activities of POD (Unit/10 cells)

1.2
Activities of CAT (Unit/10 )
9

2.5
c c 8 1.0
2.0 b b
0.8
a
1.5 0.6

1.0 0.4
c
0.2 c
0.5 c c
0.0
0.0
0 5 10 20 50 100
0 5 10 20 50 100 2+ -1
2+
Zn concentrations (µmol l )
-1 Zn concentrations (µmol l )

Fig. 2. Effects of Zn2+ on the activities of antioxidant enzymes in C. vulgaris exposed to different concentrations of Zn2+ for 72 h.

Zn2+ because more than 5 ␮mol l−1 of Zn2+ led to serious inhibition
for the algal growth. But great differences of the growth inhibi-
tion have been observed for C. vulgaris under the stress of Zn2+
based on previous reports. For example, the autotrophic cells of C.
vulgaris were able to tolerate Zn2+ at concentrations from 400 to
800 ␮mol l−1 (Ikegami et al., 2005). However, it was also reported
that 1.6 ␮mol l−1 of Zn2+ would cause 50% death of C. vulgaris after
72 h exposure (Muyssen and Janssen, 2001). Except for the metal
concentrations, it has been demonstrated that many factors, such
as the initial cell densities (Lee et al., 2011), the cell growth rates
(Muyssen and Janssen, 2001), the culture medium composition
(Yeh and Chang, 2012), as well as the assay environment (Taghavi
et al., 2013), may greatly influence the testing results of growth
inhibition. For example, the sensitivity of micro-algae to metal
toxicity increased with the decreases of the initial cellular densities
(Moreno-Garrido et al., 2000). Therefore, the lowest initial cellular
density of 104 –105 cells ml−1 has been recommend for the tests of
growth inhibition of micro-algae to toxic substances such as heavy
metals (OECD, 1984; Wong and Couture, 1986). For the accuracy
and reliability, an initial cellular density of 8 × 106 cells ml−1 was
used for the tests of growth inhibition in the present experiment.
Significant inhibition (p < 0.05) was observed on the growth of C.
vulgaris under Zn2+ stress at above 5 ␮mol l−1 . In addition, the algal
growths were significantly inhibited (p < 0.05) with the extension
of exposure time.
Oxidation damage caused by Zn2+ stress is one of the severe
problems on the algal growth (Andrade et al., 2006; Mittler, 2002;
White and Jahnke, 2002). For protecting against the potential ROS

Table 1
Distribution of subcellular Zn in C. vulgaris exposed to 0, 5, 100 ␮mol l−1 Zn2+ for 72 h (nmol/109 cells).

Zn2+ (␮mol l−1 ) Subcellular fractions

Granules Debris Organelles HDF HSF

0 1.28 ± 0.02 a
0.58 ± 0.11 a
1.63 ± 0.40 a
0.44 ± 0.11 a
3.54 ± 0.01 a
5 1.42 ± 0.03 a 1.75 ± 1.13 a 2.64 ± 0.69 a 0.93 ± 0.41 a 5.05 ± 0.39 b
100 2.76 ± 0.04 b 3.75 ± 1.03 b 6.02 ± 0.75 b 5.15 ± 0.53 b 39.6 ± 5.4 c

HDF, heat-denatured fractions; HSF, heat-stable fractions.

Different letters of a, b and c indicate the significant differences of Zn concentrations in the same subcellular fractions among different testing groups (p < 0.05).
108
4
2.5x10
Intensities of Zn (Counts/10 cells)
4
2.0x10
7
4
1.5x10 c 2011). Although the total cellular Zn in 100 ␮mol l−1 group was
b 3.8-fold higher than that in 5 ␮mol l−1 group as shown in Table 1,
2+
4
1.0x10
a 5 and 100 ␮mol l−1 groups, respectively. Results in Table 1 also
3
5.0x10
0.0
0 5 10
2+
Zn concentrations (µmol l )
Fig. 3. Induction of Zn-MT-like proteins in C. vulgaris exposed to different concen-
trations of Zn2+ for 72 h.
damage, a series of antioxidant enzymes and antioxidant sub-
stances in algae may be induced and activated, among which the
antioxidant enzymes such as GSH-Px, SOD, CAT and POD are usually
involved (Artetxe et al., 2002). The antioxidant enzymes induced by
the stress of heavy metals, such as Cd2+ , Cu2+ , Cr (VI) and Pb2+ in
microalgae have been well studied and reported (Rai et al., 2013;
Tripathi et al., 2006). But there is little scientific data about the
activities of the antioxidant enzymes in C. vulgaris under Zn2+
stress. As shown in Fig. 2, the stresses of Zn2+ could significantly
increase (p < 0.05) the activities of GSH-Px, SOD and CAT except
for the SOD activities with the significant increases only at Zn2+
concentrations of 20 and 50 ␮mol l−1 , respectively. SOD, located in
cytosol, nucleus and mitochondrial matrix, is a category of metal-
loenzymes, catalyzing the dismutation reaction of • O2 − to O2 and
H2 O2 which was subsequently reduced to H2 O by GSH-Px in cytosol
or by CAT in peroxisomes (Liang, 2011), and playing an initial role
on removing the ROS products (Kamiński et al., 2012; Nishikawa
et al., 2009). In the present study, the increase of SOD, GSH-PX and
CAT activities in C. vulgaris was one of the important biochemi-
cal responses of the algae in detoxifying the stress of Zn2+ . But the
decreases of GSH-Px and SOD at 100 ␮mol l−1 as described above
indicated that the oxidation damage of Zn2+ at high levels might
exceed the protective thresholds of the algal antioxidant enzyme
systems, resulting in serious growth inhibition for the algae. Similar
to the antioxidant enzymes of SOD, GSH-Px and CAT, POD should
also present the abilities to eliminate H2 O2 for protecting the cell
growth (Ali and Alqurainy, 2006), but the activities of POD surpris-
ingly decreased with the rises of Zn2+ concentrations as shown in
Fig. 2. The decreases of POD activities with the rises of Zn2+ concen-
trations in C. vulgaris remained unknown, but similar phenomenon
has also been observed in Zygophyllum coccineum exposed to heavy
metals based on the previous report (Morsy et al., 2012).
Subcellular fractionation of metals has been widely used to
reveal the strategies of internal compartmentalization of toxic
metals in organisms (Rosabal et al., 2012; Vijver et al., 2004; Wang
and Rainbow, 2006). However, little study has been carried out to
investigate the distribution of subcellular Zn as well as the synthesis
of Zn-binding proteins in C. vulgaris exposed to Zn2+ (Lavoie et al.,
2009; Miao and Wang, 2007). In the present study, the distribu-
tions of Zn in the partitioned subcellular fractions of algae showed
that no significant difference (p > 0.05) of the distributions of Zn
in all the subcellular fractions in 5 ␮mol l−1 group was observed
compared with those of the control group, except for a significant
increase (p < 0.05) of Zn level in HSF. However, zinc levels in all
H. Yang et al. / Ecological Indicators 45 (2014) 103–109
f subcellular fractions in 100 ␮mol l−1 group significantly increased,
and Zn mainly distributed in heat stable fractions (as shown in
Table 1). Debris, granules and HSF are considered to be the main
e
biological fractions playing important roles on the compartmental-
ization and detoxification of heavy metals in algae, while organelles
d
and HDF are sensitive fractions to heavy metals (Wang and Wang,
the proportions of Zn levels in debris, granules and HSF fractions
similarly accounted for 70% and 80% of the total Zn amounts in
showed that Zn in the detoxified metal fractions mainly derived
from the heat stable fractions. For example, Zn amount in HSF in
100 ␮mol l−1 group accounted for more than 85% of that in the
heat stable fractions. Therefore, HSF played an important role on
compartmenting and detoxifying Zn toxicity in the cells of C. vul-
20 50 100 garis.
-1
Phytochelatins induced in C. vulgaris are considered to be the
main peptides for sequestering heavy metals in algal cells, which
is usually used to explain the detoxification of heavy metals to
the algae (Akhter et al., 2012; Zhang et al., 2008). However, the
induction of metallothionein-like proteins has also been observed
in green algae under the stress of heavy metals (Yoshida et al.,
2006). For example, an apo-MT-like protein with 6152 Da has been
separated from C. vulgaris exposed to Zn2+ and Cd2+ in our pre-
vious study (Huang et al., 2009). In the present study, Zn-MT-like
proteins were readily induced in C. vulgaris when the algae were
exposed to Zn2+ at various concentrations. As shown in Fig. 3, the
amounts of Zn-MT-like proteins significantly increased (p < 0.05)
with the increases of Zn2+ concentrations. The significant induc-
tion of Zn-MT-like proteins in C. vulgaris was consistent with the
distributions of subcellular Zn in heat stable fractions especially in
the species of HSF, indicating that MT-like proteins might be the
main constituent in C. vulgaris to compartmentalize Zn in the algal
cytoplasm, and played important roles on sequestering and detox-
ifying Zn in the algal cells. The result suggests that the induction
of Zn-MT-like proteins in C. vulgaris may be used as the potential
indicators to reflect the pollution levels of Zn2+ in water environ-
ment.
5. Conclusion
The growth of C. vulgaris was significantly inhibited (p < 0.05) by
Zn2+ at above 5 ␮mol l−1 . After 72 h exposure, the activities of SOD,
GSH-Px and CAT in the algae mostly increased with the rises of Zn
concentrations from 5 to 50 ␮mol l−1 , but the POD activities signifi-
cantly decreased (p < 0.05) with the increases of Zn concentrations.
Large amounts of cellular Zn in C. vulgaris were compartmental-
ized in heat stable fractions, in which Zn-MT-like proteins were
induced. In addition, the amounts of Zn-MT-like proteins in the
algal cells significantly increased (p < 0.05) with the rises of Zn2+
concentrations in exposure medium.
Acknowledgements
The work was supported by grants from the Natural Science
Foundation of Fujian Province of China (2012J01046), the Sci-
ence and Technology Planning Project of Fujian Province, China
(2012Y0052), the Science and Technology Planning Project of Xia-
men, China (3502Z20113024), the Foundation of the Key Project
Laboratory of Urban Environment and Health, Institute of Urban
Environment, Chinese Academy of Sciences (KLUEH201303), and
the Foundation for Innovative Research Team of Jimei University
(2010A007).
 H. Yang et al. / Ecological Indicators 45 (2014) 103–109
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