Science of the Total Environment 601–602 (2017) 959–967

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Testing of two different strains of green microalgae for Cu and Ni removal

from aqueous media
L. Rugnini a,⁎, G. Costa b, R. Congestri a, L. Bruno a
a
LBA-Laboratory of Biology of Algae, Dept. of Biology, University of Rome “Tor Vergata”, via Cracovia 1, 00133 Rome, Italy
b
Laboratory of Environmental Engineering, Dept. Civil Engineering and Computer Science Engineering, University of Rome “Tor Vergata”, via del Politecnico 1, 00133 Rome, Italy

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Green microalgae are tested to treat so-

lutions from 2 to12 mg L− 1 of Cu and/
or Ni.
• Chlorella vulgaris and Desmodesmus sp.
survived for 12 days in presence of Cu
or Ni.
• Desmodesmus sp. removed 90–94% of
Cu after 4 days from all mix solutions
tested.
• Ni removal from mix solutions de-
creased for higher initial metal concen-
trations.
• Intracellular metal accumulation oc-
curred in polyphosphate bodies.

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

Article history: The concentration of metal ions in aqueous media is a major environmental problem due to their persistence and
Received 23 January 2017 non-biodegradability that poses hazards to the ecosystem and human health. In this study, the effect of Cu and Ni
Received in revised form 24 May 2017 on the growth of two green microalgal strains, Chlorella vulgaris and Desmodesmus sp., was evaluated along with
Accepted 24 May 2017
the removal capacity from single metal solutions (12 days exposure; metal concentration range: 1.9–
Available online 3 June 2017
11.9 mg L−1). Microalgal growth showed to decrease at increasing metal concentrations, but promising metal re-
Editor: D. Barcelo moval efficiencies were recorded: up to 43% and 39% for Cu by Desmodesmus sp. and C. vulgaris, respectively, with
a sorption capacity of 33.4 mg gDW−1 for Desmodesmus sp. As for Ni, at the concentration of 5.7 mg L−1, the re-
Keywords: moval efficiency reached 32% for C. vulgaris and 39% for Desmodesmus sp. In addition, Desmodesmus sp. growth
Metal uptake and metal removal were evaluated employing bimetallic solutions. In these tests, the removal efficiency for Cu
Desmodesmus sp. was higher than that of Ni for all the mix solutions tested with a maximum of 95%, while Ni-removal reached
Chlorella vulgaris 90% only for the lowest concentrations tested. Results revealed that the biosorption of both metals reached max-
Biosorption imum removal levels within the fourth day of incubation (with metal uptakes of 67 mgCu gDW−1 and
Polyphosphate bodies
37 mgNi gDW−1). Intracellular bioaccumulation of metals in Desmodesmus sp. was evaluated by confocal laser
scanning microscopy after DAPI staining of cells exposed or not to Cu during their growth. Imaging suggested
that Cu is sequestered in polyphosphate bodies within the cells, as observable also in phosphorus deprived cul-
tures. Our results indicate the potential of employing green microalgae for bioremediation of metal-polluted wa-
ters, due to their ability to grow in the presence of high metal concentrations and to remove them efficiently.
© 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author.
E-mail addresses: Lorenza.Rugnini@uniroma2.it (L. Rugnini), costa@ing.uniroma2.it (G. Costa), roberta.congestri@uniroma2.it (R. Congestri), laura.bruno@uniroma2.it (L. Bruno).

http://dx.doi.org/10.1016/j.scitotenv.2017.05.222
0048-9697/© 2017 Elsevier B.V. All rights reserved.
960 L. Rugnini et al. / Science of the Total Environment 601–602 (2017) 959–967
1. Introduction
Metals and metalloids are natural earth crust and soil constituents.
Some metals are micronutrients necessary for living organisms (e.g.
Zn, Cu, Mn, Ni, and Co), while others have unknown biological functions
(e.g. Cd, Pb, and Hg). In many ecosystems, the soil concentration of sev-
eral of these constituents has reached toxic levels as a consequence of
human activities (Herrera-Estrella and Guevara-Garcia, 2009). Water
pollution by metals and metalloids is an environmental problem world-
wide and represents a potential risk to human and ecosystem health (Fu
and Wang, 2011; Hong et al., 2011; Mota et al., 2015). In fact, not being
susceptible to biodegradation, metals and metalloids may
bioaccumulate through the food chain (Doshi et al., 2007; Kumar
et al., 2015) determining toxic or carcinogenic effects (Fu and Wang,
2011; Gaur and Adholeya, 2004). Human activities that lead to an en-
richment of metals and metalloids in aquatic systems include discharges
of untreated effluents from mining, refining, electroplating, chemical
and metallurgical industries, while natural processes include geological
weathering (Ajayan et al., 2011; Kumar et al., 2015; Monteiro et al.,
2009). Conventional physico-chemical methods for the treatment of
wastewater containing metals such as ion exchange, chemical precipita-
tion or oxidation and solvent extraction, prove often ineffective or re-
quire high energy input, capital investment and operational costs
(Chan et al., 2013; Gupta and Rastogi, 2008). On the other hand, recent-
ly, several types of both living and non-living microorganisms including
yeast, bacteria, algae and fungi, have shown high binding and uptake ca-
pacities with regard to metals and metalloids (Abdel-Ghani and
El-Chaghaby, 2014; Kumar et al., 2015; Shanab et al., 2012), suggesting
the potential for developing biological technologies for metal removal
from aqueous media. Cyanobacteria and microalgae are raising an in-
creasing interest for their great potential to be used for different bio-
technological applications in the fields of energy, photonics and
environment (Bruno et al., 2012; De Angelis et al., 2016; Di Pippo
et al., 2013; Gismondi et al., 2016). In particular, cyanobacteria and
green microalgae naturally growing in metal contaminated waters
have proven to be the best candidates for a highly effective removal of
metals from aqueous solutions (Anastopoulos and Kyzas, 2015; Yang
et al., 2015). These photosynthetic microorganisms are relatively easy
to cultivate using wastewater as growth medium; furthermore, they
have high sorption capacities and strong metal ion sorption selectivity
(De Philippis et al., 2011; Markou et al., 2015; Micheletti et al., 2008;
Rossi et al., 2012). Some microalgae and cyanobacteria are metal stress
tolerant and possess high resistance towards metal toxicity, large sur-
face area and high binding affinity (Bux and Chisti, 2016). Metal ions
are taken up in a two-stage adsorption and bioaccumulation process.
At first, the metal ions are passively adsorbed on the cell surface (of
both living and non-living biomass) just in a few seconds or minutes;
then the ions are transported slowly inside the cell membrane and are
accumulated intracellularly. Bioaccumulation occurs only in living
cells, involving transport of metal ions across the cell membrane barrier
and subsequent binding to cytoplasmic proteins or polysaccharides, or
to specific cellular compartments such as vacuoles or polyphosphate
bodies (Dwivedi, 2012; Gupta and Rastogi, 2008; Kumar et al., 2015).
The two above described processes, involving both adsorption and bio-
accumulation by living and/or dead microorganisms, can be defined as
‘biosorption’, following Gadd (2008). Among the most abundant metals
found in industrial wastewater are copper and nickel (Markou et al.,
2015). To the best of our knowledge, only few studies have explored
the in-vivo sorption capacity of green microalgae with regard to copper
(Cu) and nickel (Ni). The main goals of this research were to test the
ability of two microalgal strains, namely Desmodesmus sp. and Chlorella
vulgaris, to grow in aqueous media enriched with Cu and Ni and to re-
move these metals from the solution. In particular, the effects of metal
concentration on biomass growth, and metal uptake by the microalgal
cultures were first analysed separately using single metal solutions
(Cu or Ni). Then, the same types of tests were performed with mixed
solutions containing both Cu and Ni, in order to assess whether the
co-presence of these metals may affect the removal ability of
Desmodesmus sp. Finally, the analysis of intracellular sites for metal ac-
cumulation such as polyphosphates bodies (or granules) was also mi-
croscopically performed.
2. Materials and methods
2.1. Microalgal strains
The green microalgae selected for performing the Cu and Ni removal
tests were Desmodesmus sp. strain VRUC281 and Chlorella vulgaris strain
CCAP 211/12. The strain of Desmodesmus sp. was isolated from the out-
flow of a secondary sedimentation tank of a municipal wastewater
treatment plant (WWTP) located south of Rome (Italy) and maintained
in the ‘Univ. Roma Tor Vergata Culture Collection’ (Castenholz, 2001).
The sludge of the secondary sedimentation tank where Desmodesmus
sp. was isolated from, contained copper, nickel, chromium, cadmium
and lead at concentrations of 509, 21, 29, 3, 71 mg/kg, respectively, indi-
cating the presence of these metals in the wastewater treated in the
plant. The CCAP 211/12 strain was obtained from the Culture Collection
of Algae and Protozoa (CCAP, Scotland). Both cultures were maintained
in BG11 medium (Rippka et al., 1979) at 18° ± 2 °C and 29 μmol photon
m−2 s−1.
2.2. Experiments performed with single metal solutions
Desmodesmus sp. and C. vulgaris strains were grown for 12 days
(chronic exposure) at 22° ± 2 °C, receiving 25 ± 0.4 μmol
photons m−2 s−1 in a 12 h light/12 h dark regime, in BG11 medium sup-
plemented with different concentrations of copper (Cu) or nickel (Ni).
The Cu stock solution (1000 mg L−1) was prepared from copper sul-
phate (CuSO4·5H2O, Sigma-Aldrich reagent grade) and added to the
growth medium to obtain three different working concentrations (2.0,
6.0 and 12.0 mg L−1). Analogously, the Ni stock solution
(1000 mg L−1) was prepared from nickel sulphate (NiSO4, Sigma-
Aldrich reagent grade) and added to the growth medium to obtain the
concentrations reported above for Cu. Experiments were carried out in
triplicate and cultures in BG11 medium were used as control. The initial
concentrations of Cu and Ni in all of the solutions prepared for the tests
were analysed by ICP-OES employing an Agilent 710-ES spectrometer
and the resulting values were 2.0, 6.0 and 11.9 mg L−1 for Cu and 1.9,
5.7 and 11.4 mg L− 1 for Ni. Growth measurements were performed
spectrophotometrically (Varian Cary 50 Bio UV–Visible Spectrophotom-
eter) by regularly monitoring the optical density (OD) at 560 nm (Leduy
and Therien, 1977; Mostafa et al., 2012). The chlorophyll a content was
determined as described by Jeffrey and Humphrey (1975). Growth rates
(μ) were calculated at the exponential phase of the growth curves de-
rived from triplicate measures of OD560 of the cultures, as shown in
Eq. (1) (Jiang et al., 2011):
ln N2 − ln N1
μ¼ ð1Þ
t 2 −t 1
where N1 and N2 represent the OD values at times t2 and t1, respectively.
After a contact time of 12 days, the cultures were centrifuged at
5000 rpm for 10 min at room temperature and the supernatants were
filtered through 0.45 μm cellulose acetate filters, acidified with nitric
acid (Sigma-Aldrich reagent grade) and analysed by ICP-OES to deter-
mine Cu and Ni concentrations. The Cu or Ni removal efficiency (E, %)
from the aqueous medium was calculated using Eq. (2) (Zhou et al.,
2012):


C 0 −C f  100
E¼ ð2Þ
C0
 L. Rugnini et al. / Science of the Total Environment 601–602 (2017) 959–967
where C0 and Cf are the initial and the final concentrations of Cu or Ni
(mg L−1) in the liquid solution, respectively. The metal uptake (q, mg
metal/g dry biomass) was calculated on the basis of the liquid solution
concentration values at the beginning and at the end of the test, follow-
ing Eq. (3) (Yang et al., 2015):

Chen, 2014). In the present work, the growth of Desmodesmus sp. and
V  C 0 −C f
q¼
M and Ni by measuring the OD and the chlorophyll a (Chla a) content.
where V is the solution volume (L) and M the dry weight of the culture
biomass (g DW) at the end of the experiment.
2.3. Experiments performed with metal solution mixtures
The biosorption ability of Desmodesmus sp. was also analysed using a
mixed solution containing both Cu and Ni. The concentration of each
metal in the solution, resulting from ICP-OES analysis, is reported in
Table 1; control cultures grown in metal-free medium were also consid-
ered. The study was performed at similar conditions to those used for
the single metal ion experiments (200 mL total volume, similar metal
concentrations). Cell growth was monitored every 2 days by measuring
OD560. The values were employed to estimate the concentration (g L−1)
of biomass dry weight (DW) on the basis of a linear relationship be-
tween OD560 and DW, see Eq. (4), obtained after extensive data analysis.
   
DW g L−1 ¼ 0:6621  OD560 þ 0:0801 R2 ¼ 0:9732
Growth rates, Cu and Ni removal efficiencies and metal uptakes
were evaluated after 4, 8 and 12 days of contact time, employing the
same methods reported for the single metal experiments.
2.4. Microscopy observation of polyphosphate granules
Polyphosphate bodies facilitate accumulation and storage of metals
such as Pb, Mg, Zn, Cu, Ni, Cd and Hg in microalgal cells (Dwivedi,
2012; Kumar et al., 2015; Wang and Dei, 2006). To study this mecha-
nism, Desmodesmus sp. was incubated in BG11 medium with or without
a phosphorus source and supplemented with 2.0 mg L−1 of Cu. After
96 h, a 15 mL culture was centrifuged (5000 rpm for 10 min) and
100 μL of cell pellet was stained with 50 μg mL−1 of 4′,6-diamidino-2-
phenylindole dihydrochloride (DAPI) for 10 min at room temperature.
Following DAPI incubation, samples were mounted on glass slides and
observed with a Confocal Laser Scanning Microscope (CLSM); stained
polyphosphate granules show a yellow fluorescence emission between
at 500 and 550 nm (Eixler et al., 2005; Hong-Hermesdorf et al., 2014;
Meza et al., 2015).
2.5. Statistical analysis
The experimental data were statistically analysed using the
GraphPad Prism software, version 7.03 (USA), via ANOVA analysis of
variance and the Student's t-test. Differences were considered signifi-
cant at p b 0.05.
Table 1
Metal concentrations in the mixed solutions.
Cu (mg L−1) Ni (mg L−1)
Mix 1 1.6 1.3
Mix 2 9.8 7.4
Mix 3 18.9 13.8
961
3. Results and discussion
3.1. Microalgal growth with single metal solutions
Few studies have focused on metal removal by living green
microalgae (Anastopoulos and Kyzas, 2015; Chan et al., 2013; He and
C. vulgaris was monitored in presence of different concentrations of Cu
ð3Þ
Data obtained revealed similar growth patterns between the two strains
as shown in Table 2. Comparison of growth rate values (Student's t-test)
indicated that the growth of Desmodesmus sp. was more affected by Cu
than Ni (p b 0.05). At the highest concentration of Cu, the growth rate of
Desmodesmus sp. was reduced by 55% while even at the highest concen-
tration of Ni tested (11.4 mg L−1), μ was only 18% lower than the con-
trol. Additionally, two-way RM ANOVA indicated that the Chl a
content decreased significantly (p b 0.0001) with increasing Cu concen-
trations. Indeed, the Chl a content decreased from 1.545 ±
0.015 μg mL−1 in the control to 0.220 ± 0.008 μg mL−1 (N85% reduc-
tion) after 12 days of incubation (Fig. 1) and for all the tested Cu concen-
trations, the Chl a content was reduced by 50% compared to the control
at day 4. Cu is an essential micronutrient that acts as a cofactor for sev-
eral biological mechanisms such as photosynthetic electron transport
and the cytochrome c oxydase, or in proteins and enzymes (De la
Cerda et al., 2008; Miazek et al., 2015). Nevertheless, an excess of Cu
can be toxic to the cells by inhibiting electron transport at P680 as
well as by inactivating some PSII reaction centres or by promoting the
generation of ROS leading to cell death (Yang et al., 2015; Mota et al.,
ð4Þ 2015). The presence of Cu or Ni did not appear to markedly inhibit the
growth of C. vulgaris (p b 0.05). However, compared to the control, the
growth rate and the Chl a content were significantly influenced by Ni
(p b 0.05), and no significant differences (p N 0.05) were detected at in-
creasing Cu concentrations, as shown by the Student's t-test. When the
Ni was higher than 1.9 mg L−1, μ decreased by 22–25% and Chl a de-
creased from 1.878 ± 0.012 μg mL−1 in the control to 1.326 ± 0.004
and 1.206 ± 0.011 μg mL−1 (about 29 and 36% less than the control)
at 5.7 and 11.4 mg L−1 of Ni, respectively. The different effect of the
two metals on the growth of Desmodesmus sp. and C. vulgaris may be at-
tributed to the intrinsic composition of the cell walls of these two
strains: proteins, carbohydrates and lipid react differently with metallic
species in a species-specific manner (Kumar et al., 2015; Monteiro et al.,
2009; Zhou et al., 2012).
3.2. Cu and Ni removal in single metal experiments
It is generally accepted that living microalgal cells may be effective in
removing metals from wastewater because of their large surface area
and high binding affinity for metal ions (Ajayan et al., 2011; Chong
et al., 2000; Guçlu and Ertan, 2012; Yan and Pan, 2002). The ability of
living cells of Desmodesmus sp. and C. vulgaris of separately removing
Table 2
Growth rates (μ) of Desmodesmus sp. and C. vulgaris incubated at different concentrations
of Cu or Ni.
Desmodesmus sp. C. vulgaris
μ (day−1)
0 0.0854 ± 0.0002 0.1115 ± 0.0055
−1
Cu (mg L )
2.0 0.0781 ± 0.0008 0.1090 ± 0.0019
6.0 0.0718 ± 0.0007 0.1048 ± 0.0018
11.9 0.0321 ± 0.0003 0.0977 ± 0.0069
Ni (mg L−1)
1.9 0.0793 ± 0.0003 0.0944 ± 0.0049
5.7 0.0729 ± 0.0007 0.0869 ± 0.0038
11.4 0.0702 ± 0.0002 0.0834 ± 0.0040
962 L. Rugnini et al. / Science of the Total Environment 601–602 (2017) 959–967

Fig. 1. Time course of Chl a content (expressed in μg mL−1) in Desmodesmus sp. at different initial concentrations of Cu (A) and Ni (B) and in C. vulgaris at different initial concentrations of
Cu (C) and Ni (D). Data are mean values ± standard deviations.

Cu or Ni, applying a contact time of 12 days, is reported in Table 3. It can removal efficiency values than those obtained here. After 8 days of cul-
be noticed that the total amount of Cu and Ni removed by both strains turing, the microalgae Chlorella pyrenoidosa and Scenedesmus obliquus
increased for increasing initial metal concentrations, in agreement removed 79.3–90.9% and 75.9–91.4% of copper, respectively, but the ini-
with data reported by different authors (e.g. Monteiro et al., 2009). Ac- tial concentration of the metal ranged from 0.2 to 2.0 mg L−1, 10 to 6
cording to a two-way ANOVA analysis, Cu removal was significantly times less than the concentrations tested here. The majority of the stud-
higher than that of Ni in both strains (p b 0.05). The maximum levels ies on copper removal were carried out with C. vulgaris in various forms
of Cu removal from the liquid solution attained for an initial Cu concen- (non-living, living, free, and immobilized). The metal uptake capacity
tration of 11.9 mg L− 1, were 43% and 39% for Desmodesmus sp. and ranged from 1.8 (Dönmez et al., 1999) to 48.17 mg gDW−1 (Romera
C. vulgaris, respectively. The corresponding metal uptakes were 33.4 et al., 2007) in non-living forms and from 3.63 (Tien et al., 2005) to
mgCu gDW− 1 and 37.0 mgCu gDW−1 for Desmodesmus sp. and 63.08 mg gDW−1 in living cells (Mehta and Gaur, 2001). When exposed
C. vulgaris. However, the removal efficiency and metal uptake of Cu by to 1.9 mg L−1 of Ni, Desmodesmus sp. removed 30% showing a metal up-
Desmodesmus sp. were two times greater than C. vulgaris at the lowest take of 2.6 mg gDW −1, while C. vulgaris removed only 6% of the initial
concentration tested (1.9 mg L−1). Zhou et al. (2012) reported higher concentration. However, for higher initial Ni concentrations the two
microalgae showed similar removal efficiencies (around 39%). The
metal uptakes obtained in this study are slightly higher than those re-
ported elsewhere using different biosorption materials. Rani et al.
Table 3 (2013) achieved a maximum biosorption capacity of 26.1 mg gDW−1
Copper and Nickel removal (E) and metal uptake (q) of Desmodesmus p. and C. vulgaris af- for Ni ions (C0 = 20 mg L−1) from aqueous solutions using the fungus
ter 12 days of incubation. Aspergillus fischeri as a biosorbent, while in this study C. vulgaris sorbed
Cu Ni
30.6 mg gDW−1 (Co = 11.4 mg L−1) and Desmodesmus sp. 17 mg gDW−1
(Co = 5.7 mg L−1) of Ni. Veneu et al. (2013) reported a maximum ca-
C0 E q C0 E q
pacity for Cu biosorption by Streptomyces lunalinharesii of
(mg L−1) (%) (mgCu (mg L−1) (%) (mgNi
gDW−1) gDW−1) 11.53 mg gDW−1, while in this study Desmodesmus sp. showed a max-
imum Cu-uptake three times greater than yeast (33.4 mg gDW−1). Ac-
Desmodesmus sp. 2.0 33 2.9 1.9 30 2.6
6.0 32 9.3 5.7 39 17 cording to the present study, Desmodesmus sp. and C. vulgaris were
11.9 43 33.4 11.4 / / found to present good accumulation properties for copper and nickel,
C. vulgaris 2.0 13 1.5 1.9 6.0 0.7 when exposed separately to these metals, exhibiting a slightly inhibited
6.0 / / 5.7 32 9.3 growth at the highest concentrations tested, demonstrating the efficien-
11.9 39 37.0 11.4 39 30.6
cy of these microalgae for metal bioremediation.
 L. Rugnini et al. / Science of the Total Environment 601–602 (2017) 959–967
3.3. Experiments performed with metal solution mixtures
The results of the tests carried out with single metal solutions indi-
cated that the two strains of microalgae employed presented a similar
removal efficiency for both Cu and Ni at the highest concentrations test-
ed; however, the removal efficiency of Desmodesmus sp. was significant-
ly higher at the lower concentrations applied (p b 0.05, via Student's t-
test). This may be related to the fact that microalgal isolates obtained
from polluted environments (such as the strain of Desmodesmus sp.
employed in this study) might be more suitable for metal removal; it
is in fact generally reported that these isolates exhibit a higher metal re-
moval capacity than those grown in otherwise clean environments
(Chong et al., 2000). Cyanobacteria, diatoms and green algae are abun-
dant in phototrophic biofilm colonizing wastewater treatment plants,
WWTPS (Congestri et al., 2005, 2006) and several studies demonstrated
the potential of using microalgae isolates from WWTPs and other artifi-
cial systems to remove contaminants (Delgadillo-Mirquez et al., 2016;
Guzzon et al., 2008; Samorì et al., 2013). On this basis, we decided to
carry out the tests with the mixture of copper and nickel with the strain
of Desmodesmus sp. Fig. 2 shows the dry weight and the Chl a content of
Desmodesmus sp. grown in the three different mixture solutions report-
ed in Table 1. Two-way RM ANOVA was performed on data concerning
the growth of Desmodesmus sp. at increasing metal concentrations and
incubation time. In Mix 1, growth was similar to control cultures, fol-
lowing a linear pattern; in this condition, the amount of biomass obtain-
ed after 12 days of incubation was 0.44 ± 0.01 g L−1, 6% less than the
control. Desmodesmus sp. growth, however, was negatively affected by
increasing metal concentrations (p b 0.0001). When the living biomass
was exposed to Mix 2 or Mix 3, the concentration of biomass (expressed
as dry mass) obtained after 4 days was about 50% of that of the control;
in addition, the Chl a content was b0.10 ± 0.06 μg mL−1 after 4 days of
incubation (compared to 6.37 ± 0.005 μg mL−1 in control cultures). As
shown in Fig. 3, Cu removal efficiency was significantly higher than that
of Ni in all the three mixed solutions (p b 0.05). Cu-removal was very
high with a maximum efficiency of 94.9%, 91.3% and 90% for Mix 1,
Mix 2 and Mix 3, respectively; instead, Ni removal was 90% only for
the lowest concentration tested (1.3 mg L−1 in Mix 1) and was b 40%
both for Mix 2 and Mix 3. In addition, the experimental results suggest
that the simultaneous biosorption of Cu and Ni in this study was fast,
since for all the tested conditions apart from the mixture presenting
the lowest concentrations (Mix 1), the maximum levels of removal
were achieved within the fourth day of incubation (Fig. 3). After this pe-
riod, the amount of metal ions removed by the microalgal biomass did
not significantly change with time, but some fluctuations were ob-
served; these may be probably attributed to membrane transport of
metal ions, their uptake and/or excretion by metabolically (energetical-
ly) driven processes (Garnham et al., 1992; Markou et al., 2015).
Fig. 2. Temporal evolution of: (A) growth of Desmodesmus sp. (gDW L−1) and (B) Chl a content (μg mL−1) at different concentrations of Cu and Ni in mixed solutions. Data are mean values
± standard deviations.
963
Bioaccumulation is often related to the activation of a defence mecha-
nism by which microorganisms handle metabolically the presence of a
toxic metal, involving metallothioneins, phytochelatins and the seques-
tration and compartmentalization into the vacuole (De Philippis et al.,
2011; Kumar et al., 2015). The amount of metal removed by microalgae
depends on its initial concentration in solution: the maximum sorption
capacity is generally reported to increase for higher metal concentra-
tions (Monteiro et al., 2009). Our data showed that the uptake of Cu in-
creased from 4.19 ± 0.19 mg gDW−1 to 66.97 ± 0.85 mg gDW−1 from
Mix 1 (1.6 mg L−1) to Mix 3 (18.9 mg L−1); Ni uptake showed a similar
pattern, with the highest uptake (17.76 ± 0.23 mg gDW−1) achieved
for Mix 3. It is interesting to note that the removal efficiency and
metal uptake trends with time were very similar for mixtures 2 and 3,
while for Mix 1 Cu and Ni uptake showed to decrease with time after
4 days, differently from the removal efficiency trends (see Fig. 3). This
is related to the fact that only for Mix 1 the algal biomass showed to
grow with time and therefore, since the final concentration of metals
in solution remained constant after 4 days, Cu and Ni uptakes decreased
with time. The fact that even though the amount of biomass increased
over time metal removal efficiency did not increase after 4 days, may
be tentatively ascribed to a reduction in effective surface due to partial
biomass aggregation (Monteiro et al., 2009; Romera et al., 2007).
Generally, wastewater discharged into freshwater bodies is charac-
terized by more than one toxic or potentially toxic substance (Kumar
et al., 2015). Therefore, the examination of the effects of multi-metal so-
lutions is more representative of actual environmental problems than
single metal studies (Monteiro et al., 2009). Microalgae have been re-
ported to tolerate multiple heavy metal species, but there are scarce re-
ports available on combined effects that are commonly summarized
into synergism, antagonism, and non-interactive or additive (Kumar
et al., 2015). Phormidium sp. showed to successfully grow in the pres-
ence of cadmium, zinc, lead, copper and nickel from 0 to 10 mg L−1,
while Scenedesmus sp. was reported to tolerate metals such as copper,
nickel, cadmium at lower concentrations (2–5 mg L−1) (Shanab et al.,
2012; Rani et al., 2013) than the ones tested herein. Fraile et al.
(2005) revealed that the presence of Cd decreased the uptake of Zn in
a competitive manner. Further, Aksu and Dönmez (2006) described
competitive adsorption of Cd and Ni in C. vulgaris.
In this study, Cu removal from the mixture solutions was notably
higher than that resulting for single metal solutions for all the tested con-
ditions (e.g. 90 vs 43% for the highest concentrations). In addition the re-
moval efficiency of Ni was also higher for Mix 1 compared to that
obtained for the Ni-only solution at a similar concentration (90 vs 30% re-
spectively), but was similar or slightly lower for the mixtures with higher
concentrations compared to the single metal solutions. These results sug-
gest the occurence of a synergistic effect for the removal of the two metals
when co-present at the lower concentrations tested (Mix 1), and a
964 L. Rugnini et al. / Science of the Total Environment 601–602 (2017) 959–967

Fig. 3. Removal efficiency (E, %) and uptake (mg gDW−1) of Cu and Ni as function of time in: Mix 1 (A and B), Mix 2 (C and D) and Mix 3 (E and F) for Desmodesmus sp. Data are mean values
± standard deviations.

synergistic effect only for Cu removal in the presence of significant Ni con-
centrations. A similar effect of synergic biosorption in the presence of a bi-
nary metal solution was also reported by Monferrán et al. (2012), who
studied the removal of chromium (Cr) and Cu by the aquatic macrophyte
Potamogeton pusillus. Data obtained revealed that when P. pusillus was ex-
posed to a solution containing 19.2 mM of Cr, removal effeciencies were
ehnanced from 26 to 65% in the presence of Cu (15.7 mM).
Metal ions have been reported to bind to chelating proteins de-
scribed as class III metallothioneins (phytochelatins-PCs or MtIII) and
enter the cell by endocytosis (Arunakumara and Xuecheng, 2008;
Kumar et al., 2015). All higher plants and most algae possess the capac-
ity to synthesize PCs which requires post-translational activation by
metals (Grill et al., 1985; Clemens and Persoh, 2009; Monteiro et al.,
2009; Kumar et al., 2015). PC synthase is activated both in the presence
of toxic metals such as Cd, Pb, Hg and even at high concentrations of
trace metals such as Cu and Zn. Increased synthesis of PCs has also
been reported in other green algae as Stigeoclonium sp., S. tenue, and
Trebouxia erici in response to excess of Cd and Cu (Bačkor et al., 2007;
Pawlik-Skowrońska, 2003, 2001). Wei et al. (2003) also reported that
the addition of Cd, Cu and Zn to microalgal assemblages resulted in an
increase of 3.6, 1.8, and 3.2 fold in PC production, respectively. The co-
presence of Cu and Ni in the mix solutions tested in this study thus
may have increased the activation of PCs compared to the single metal
solutions, thus improving their removal efficiency.
 L. Rugnini et al. / Science of the Total Environment 601–602 (2017) 959–967
As for possible explanations why this mechanism was evident only
for Cu at higher metal concentrations, it has been reported that when
the extracellular concentration of a metal ion is considerably higher
than its intracellular counterpart (such as in Mix 2 and Mix 3), metal
ions compete for binding to multivalent ion carriers or alternatively
enter the cell by active transport after binding to low-molecular-
weight thiols (e.g., cysteine) (Arunakumara and Xuecheng, 2008;
Monteiro et al., 2009). In addition Cu may enhance the activities of an-
tioxidant enzymes and compete with Ni to bind with membrane pro-
teins in order to protect the cells, similarly to what is reported for
higher plants (Wu and Zhang, 2002). Moreover, Cu was reported to be
generally preferred to Ni by Chlorella miniata and C. vulgaris due to a
stronger binding strength which favours a more covalent nature of in-
teraction between the metal ion and the ligands (Lau et al., 1999);
even Markou et al. (2015) reported higher Cu biosorption onto living
biomass of Arthrospira platensis respect to Ni. Several studies
(Micheletti et al., 2008; Kumar et al., 2015) also reported that in
multi-metal solutions containing Cu and Ni, metal affinity is Cu N Ni.
In these solutions, a drastic reduction in the sorption of Ni was observed
and all binding sites were reported to be occupied by the other metals,
pointing out a very selective affinity. Moreover, divalent Ni in water
generates very stable aqueous complexes that may become poorly ex-
changeable with the protons bound to the active sites of the biosorbent.
Data obtained herein seems to confirm this preferential binding possi-
bly due to the relative strength, competition for some common adsorp-
tion sites and PC activation. However, a comprehensive description on
the mechanisms underlying metal toxicity and removal in mixed
multi-metal solutions by microalgae is yet to be elaborated.
Bioaccumulation in multi-metal solutions has been also studied
using dried biomass of charophytes (Chara aculeolata and Nitella
opaca) exposed to Cd, Zn, Pb (Sooksawat et al., 2016). Authors reported
that compared to the biosorption in single-metal solutions, the maxi-
mum sorption capacity for each metal was lower when the biomass
Fig. 4. CLSM images of Desmodesmus sp. grown in complete BG11 medium without (control) and with Cu supplemented at 2 mg L−1 for 96 h (A and C, respectively) after DAPI staining to
evidence PolyP bodies. Colonies in B and D are from cultures grown in BG11 medium without P-source, without (B) and with Cu (D). Scale bars: A and D = 7 μm; B and C = 5 μm.
965
was used to treat the multi-metal solutions, suggesting in this case an-
tagonistic effects between metals in the solutions. The potential of living
charophytes for phytoremediation of Cd, Zn and Pb was assessed in dif-
ferent studies (e.g. Gomes and Asaeda, 2009, 2013). The results obtain-
ed in those studies showed that also calcifying macroalgae may ensure
bioaccumulation of metals, but in comparison with living microalgae,
their growth should last between 4 and 84 weeks to allow root
elongation.
3.4. Cu accumulation in polyphosphate granules
Exposure of cells to toxic metals for a long period of time was tested
in different studies to assess cell capacity to grow and incorporate the
toxic metal intracellularly (Monteiro et al., 2009). Polyphosphate is a
multi-functional compound. Its most significant functions are: phos-
phate and energy reserves, divalent cation sequestration and storage,
membrane channel formation, participation in phosphate transport,
cell envelope formation and function, control of gene activity, regulation
of enzyme activities and a role in stress response and stationary-phase
adaptation (Meza et al., 2015). Acknowledging the fact that
polyphosphate (PolyP) bodies appear to be a distinctive microalgal
trait and enable storage of certain nutrients in microalgae, Dwivedi
(2012) proposed that these granules could sequester metals thereby
performing two different functions, providing a storage pool for metals
and enabling a detoxification mechanism. CLSM analysis was performed
in order to investigate possible sites of metal accumulation in PolyP
bodies. As shown in Fig. 4, the morphological features (size and
shape) of Desmodesmus sp. remained unchanged for all the conditions
tested. PolyP granules were influenced by the presence or absence of P
and Cu. Compared to the control (Fig. 4A), PolyP granules can be clearly
observed in cultures grown in presence of both P and Cu (Fig. 4C), or
when the P-source was absent, but cells were exposed to the metal
(Fig. 4D). In cultures grown without P and Cu sources (Fig. 4B), cells
966 L. Rugnini et al. / Science of the Total Environment 601–602 (2017) 959–967
showed no visible staining of PolyP bodies, confirming the evidence that
these granules are involved in P and metal storage. Dwivedi (2012) re-
ported that Scenedesmus obliquus accumulated increased amounts of Cd
and Zn with high phosphorus concentrations, similar to the results
shown in Fig. 4C. In addition, cyanobacterial strains grown in the pres-
ence of Pb or Cu (10 mg L−1) showed expanded thylakoids and an in-
crease in inclusions/polyphosphate bodies (Maldonado et al., 2011;
Pereira et al., 2011).
4. Conclusions
Microalgae exhibit constitutive mechanisms for the removal of free
metal ions from water, thereby suggesting their applicability as a treat-
ment for metal contaminated aqueous media. Our study evaluated the
capability of two strains of green microalgae, Desmodesmus sp.
VRUC281 and C. vulgaris CCAP 211/12, to tolerate and to remove Cu or
Ni from a liquid medium at different concentrations. Both strains were
able to survive at least for 12 days in the presence of Cu or Ni and result-
ed as good biosorbents for Cu or Ni ions (up to 43% of bioremoval at
11.9 mgCu L− 1). When exposed to a mixture of these two metals,
Desmodesmus sp. growth showed a linear pattern when Cu and Ni initial
concentrations in solution were about 2.0 mg L−1 (Mix 1), but higher
concentrations (Mix 2 and Mix 3) reduced both the growth rate and
the Chl a content. However, these tests also showed that Desmodesmus
sp. was able to remove between 90 and 94% of Cu after 4 days in all the
mix solutions tested, while Ni removal decreased from 90% for the low-
est concentration tested to b 40% at higher concentrations (maximum
uptake of 17.76 ± 0.23 mg gDW−1). In agreement with previous stud-
ies, metal removal reached maximum values before four days and
polyphosphate bodies seemed to be involved in the metal sequestration
mechanism, as confirmed by DAPI staining. In conclusion, the results of
this study indicate the potential of the two strains tested of becoming an
efficient and economic biosorbent for bioremediation of metal-polluted
waters. Further studies will be necessary to evaluate the in situ ability of
the microalgae to remove metals from wastewater and to better under-
stand the detailed mechanisms influencing the binding capacity, syner-
gistic and antagonistic effects in presence of metal mixtures, the
possible involvement of phytochelatins' synthesis and the kinetics of
physical adsorption (both in living and dead microalgae).
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