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

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 b

LBA-Laboratory of Biology of Algae, Dept. of Biology, University of Rome “Tor Vergata”, via Cracovia 1, 00133 Rome, Italy 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 solutions 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 decreased for higher initial metal concentrations. • Intracellular metal accumulation occurred in polyphosphate bodies.

a r t i c l e

i n f o

Article history: Received 23 January 2017 Received in revised form 24 May 2017 Accepted 24 May 2017 Available online xxxx Editor: D. Barcelo Keywords: Metal uptake Desmodesmus sp. Chlorella vulgaris Biosorption Polyphosphate bodies

a b s t r a c t The concentration of metal ions in aqueous media is a major environmental problem due to their persistence and non-biodegradability that poses hazards to the ecosystem and human health. In this study, the effect of Cu and Ni on the growth of two green microalgal strains, Chlorella vulgaris and Desmodesmus sp., was evaluated along with the removal capacity from single metal solutions (12 days exposure; metal concentration range: 1.9– 11.9 mg L−1). Microalgal growth showed to decrease at increasing metal concentrations, but promising metal removal 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 removal efficiency reached 32% for C. vulgaris and 39% for Desmodesmus sp. In addition, Desmodesmus sp. growth and metal removal were evaluated employing bimetallic solutions. In these tests, the removal efficiency for Cu was higher than that of Ni for all the mix solutions tested with a maximum of 95%, while Ni-removal reached 90% only for the lowest concentrations tested. Results revealed that the biosorption of both metals reached maximum removal levels within the fourth day of incubation (with metal uptakes of 67 mgCu gDW−1 and 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 cultures. Our results indicate the potential of employing green microalgae for bioremediation of metal-polluted waters, 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: [email protected] (L. Rugnini), [email protected] (G. Costa), [email protected] (R. Congestri), [email protected] (L. Bruno).

http://dx.doi.org/10.1016/j.scitotenv.2017.05.222 0048-9697/© 2017 Elsevier B.V. All rights reserved.

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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 several 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 worldwide 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 enrichment 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 precipitation or oxidation and solvent extraction, prove often ineffective or require high energy input, capital investment and operational costs (Chan et al., 2013; Gupta and Rastogi, 2008). On the other hand, recently, several types of both living and non-living microorganisms including yeast, bacteria, algae and fungi, have shown high binding and uptake capacities 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 increasing interest for their great potential to be used for different biotechnological 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 surface 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 bioaccumulation 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 remove 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 accumulation such as polyphosphates bodies (or granules) was also microscopically 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 outflow 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, indicating 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 supplemented with different concentrations of copper (Cu) or nickel (Ni). The Cu stock solution (1000 mg L−1) was prepared from copper sulphate (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, SigmaAldrich 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 Spectrophotometer) 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 derived from triplicate measures of OD560 of the cultures, as shown in Eq. (1) (Jiang et al., 2011): μ¼

ln N2 − ln N1 t 2 −t 1

ð1Þ

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 determine Cu and Ni concentrations. The Cu or Ni removal efficiency (E, %) from the aqueous medium was calculated using Eq. (2) (Zhou et al., 2012):

E¼


C 0 −C f  100 C0

ð2Þ

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, following Eq. (3) (Yang et al., 2015):

q¼

V  C 0 −C f M


ð3Þ

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 considered. 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 between 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

ð4Þ

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 mechanism, 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-2phenylindole 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 significant at p b 0.05.

Mix 1 Mix 2 Mix 3

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 Chen, 2014). In the present work, the growth of Desmodesmus sp. and C. vulgaris was monitored in presence of different concentrations of Cu and Ni by measuring the OD and the chlorophyll a (Chla a) content. 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 concentration of Ni tested (11.4 mg L−1), μ was only 18% lower than the control. Additionally, two-way RM ANOVA indicated that the Chl a content decreased significantly (p b 0.0001) with increasing Cu concentrations. 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% reduction) after 12 days of incubation (Fig. 1) and for all the tested Cu concentrations, 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 several 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., 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 increasing 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 decreased 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 attributed 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.

0 Cu (mg L 2.0 6.0 11.9

Table 1 Metal concentrations in the mixed solutions. Cu (mg L−1)

Ni (mg L−1)

1.6 9.8 18.9

1.3 7.4 13.8

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Desmodesmus sp. μ (day−1)

C. vulgaris

0.0854 ± 0.0002

0.1115 ± 0.0055

0.0781 ± 0.0008 0.0718 ± 0.0007 0.0321 ± 0.0003

0.1090 ± 0.0019 0.1048 ± 0.0018 0.0977 ± 0.0069

0.0793 ± 0.0003 0.0729 ± 0.0007 0.0702 ± 0.0002

0.0944 ± 0.0049 0.0869 ± 0.0038 0.0834 ± 0.0040

−1

)

Ni (mg L−1) 1.9 5.7 11.4

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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 be noticed that the total amount of Cu and Ni removed by both strains increased for increasing initial metal concentrations, in agreement with data reported by different authors (e.g. Monteiro et al., 2009). According to a two-way ANOVA analysis, Cu removal was significantly higher than that of Ni in both strains (p b 0.05). The maximum levels of Cu removal from the liquid solution attained for an initial Cu concentration of 11.9 mg L− 1, were 43% and 39% for Desmodesmus sp. and C. vulgaris, respectively. The corresponding metal uptakes were 33.4 mgCu gDW− 1 and 37.0 mgCu gDW−1 for Desmodesmus sp. and C. vulgaris. However, the removal efficiency and metal uptake of Cu by Desmodesmus sp. were two times greater than C. vulgaris at the lowest concentration tested (1.9 mg L−1). Zhou et al. (2012) reported higher

Table 3 Copper and Nickel removal (E) and metal uptake (q) of Desmodesmus p. and C. vulgaris after 12 days of incubation. Cu

Desmodesmus sp.

C. vulgaris

Ni

E C0 (mg L−1) (%)

q (mgCu gDW−1)

E q C0 (mg L−1) (%) (mgNi gDW−1)

2.0 6.0 11.9 2.0 6.0 11.9

2.9 9.3 33.4 1.5 / 37.0

1.9 5.7 11.4 1.9 5.7 11.4

33 32 43 13 / 39

30 39 / 6.0 32 39

2.6 17 / 0.7 9.3 30.6

removal efficiency values than those obtained here. After 8 days of culturing, the microalgae Chlorella pyrenoidosa and Scenedesmus obliquus removed 79.3–90.9% and 75.9–91.4% of copper, respectively, but the initial concentration of the metal ranged from 0.2 to 2.0 mg L−1, 10 to 6 times less than the concentrations tested here. The majority of the studies on copper removal were carried out with C. vulgaris in various forms (non-living, living, free, and immobilized). The metal uptake capacity ranged from 1.8 (Dönmez et al., 1999) to 48.17 mg gDW−1 (Romera et al., 2007) in non-living forms and from 3.63 (Tien et al., 2005) to 63.08 mg gDW−1 in living cells (Mehta and Gaur, 2001). When exposed to 1.9 mg L−1 of Ni, Desmodesmus sp. removed 30% showing a metal uptake of 2.6 mg gDW −1, while C. vulgaris removed only 6% of the initial 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 reported elsewhere using different biosorption materials. Rani et al. (2013) achieved a maximum biosorption capacity of 26.1 mg gDW−1 for Ni ions (C0 = 20 mg L−1) from aqueous solutions using the fungus Aspergillus fischeri as a biosorbent, while in this study C. vulgaris sorbed 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 capacity for Cu biosorption by Streptomyces lunalinharesii of 11.53 mg gDW−1, while in this study Desmodesmus sp. showed a maximum Cu-uptake three times greater than yeast (33.4 mg gDW−1). According to the present study, Desmodesmus sp. and C. vulgaris were found to present good accumulation properties for copper and nickel, when exposed separately to these metals, exhibiting a slightly inhibited growth at the highest concentrations tested, demonstrating the efficiency 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 indicated that the two strains of microalgae employed presented a similar removal efficiency for both Cu and Ni at the highest concentrations tested; however, the removal efficiency of Desmodesmus sp. was significantly higher at the lower concentrations applied (p b 0.05, via Student's ttest). 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 removal capacity than those grown in otherwise clean environments (Chong et al., 2000). Cyanobacteria, diatoms and green algae are abundant 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 artificial 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 reported 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, following a linear pattern; in this condition, the amount of biomass obtained 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 period, the amount of metal ions removed by the microalgal biomass did not significantly change with time, but some fluctuations were observed; these may be probably attributed to membrane transport of metal ions, their uptake and/or excretion by metabolically (energetically) driven processes (Garnham et al., 1992; Markou et al., 2015).

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Bioaccumulation is often related to the activation of a defence mechanism by which microorganisms handle metabolically the presence of a toxic metal, involving metallothioneins, phytochelatins and the sequestration 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 concentrations (Monteiro et al., 2009). Our data showed that the uptake of Cu increased 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 characterized by more than one toxic or potentially toxic substance (Kumar et al., 2015). Therefore, the examination of the effects of multi-metal solutions is more representative of actual environmental problems than single metal studies (Monteiro et al., 2009). Microalgae have been reported to tolerate multiple heavy metal species, but there are scarce reports 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 presence 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 conditions (e.g. 90 vs 43% for the highest concentrations). In addition the removal 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% respectively), but was similar or slightly lower for the mixtures with higher concentrations compared to the single metal solutions. These results suggest 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

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.

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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 concentrations. A similar effect of synergic biosorption in the presence of a binary 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 exposed 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 described 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 capacity 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 copresence 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.

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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-molecularweight thiols (e.g., cysteine) (Arunakumara and Xuecheng, 2008; Monteiro et al., 2009). In addition Cu may enhance the activities of antioxidant enzymes and compete with Ni to bind with membrane proteins 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 interaction 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 exchangeable with the protons bound to the active sites of the biosorbent. Data obtained herein seems to confirm this preferential binding possibly due to the relative strength, competition for some common adsorption 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 maximum sorption capacity for each metal was lower when the biomass

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was used to treat the multi-metal solutions, suggesting in this case antagonistic effects between metals in the solutions. The potential of living charophytes for phytoremediation of Cd, Zn and Pb was assessed in different studies (e.g. Gomes and Asaeda, 2009, 2013). The results obtained 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: phosphate 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

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.

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showed no visible staining of PolyP bodies, confirming the evidence that these granules are involved in P and metal storage. Dwivedi (2012) reported 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 presence of Pb or Cu (10 mg L−1) showed expanded thylakoids and an increase 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 treatment 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 resulted 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 lowest concentration tested to b 40% at higher concentrations (maximum uptake of 17.76 ± 0.23 mg gDW−1). In agreement with previous studies, 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 understand the detailed mechanisms influencing the binding capacity, synergistic 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). References Abdel-Ghani, N.T., El-Chaghaby, G.A., 2014. Biosorption for metal ions removal from aqueous solutions: a review of recent studies. Int. J. Latest Res. Sci. Technol. 3 (1), 24–42. Ajayan, K.V., Selvaraju, M., Thirugnanamoorthy, K., 2011. Growth and heavy metals accumulation potential of microalgae grown in sewage wastewater and petrochemical effluents. Pak. J. Biol. Sci. 14, 805–811. Aksu, Z., Dönmez, G., 2006. Binary biosorption of cadmium(II) and nickel(II) onto dried Chlorella vulgaris: co-ion effect on monocomponent isotherm parameters. Process Biochem. 41 (4), 860–868. Anastopoulos, I., Kyzas, G.Z., 2015. Progress in batch biosorption of heavy metals onto algae. J. Mol. Liq. 209, 77–86. Arunakumara, K.K.I.U., Xuecheng, Z., 2008. Heavy metal bioaccumulation and toxicity with special reference to microalgae. J. Ocean Univ. China 7 (1), 60–64. Bačkor, M., Pawlik-Skowrońska, B., Budová, J., Skowroński, T., 2007. Response to copper and cadmium stress in wild-type and copper tolerant strains of the lichen alga Trebouxia erici: metal accumulation, toxicity and non-protein thiols. Plant Growth Regul. 52 (1), 17–27. Bruno, L., Di Pippo, F., Antonaroli, S., Gismondi, A., Valentini, C., Albertano, P., 2012. Characterization for biofilm-forming cyanobacteria for biomass and lipid production. OF Applied Microbiology]–>J. Appl. Microbiol. 113 (5):1052–1064. http://dx.doi.org/ 10.1111/j.1365-2672.2012.05416. Bux, F., Chisti, Y. (Eds.), 2016. Algae Biotechnology Products and Processes. Springer. Castenholz, R.W., 2001. Phylum BX. Cyanobacteria. oxygenic photosynthetic bacteria. Bergey's Manual of Systematic Bacteriology, second ed. Springer, New York, pp. 473–487. Chan, A., Salsali, H., McBean, E., 2013. Heavy metal removal (copper and zinc) in secondary effluent from wastewater treatment plants by microalgae. ACS Sustain. Chem. Eng. 2, 130–137. Chong, A.M.Y., Wong, Y.S., Tam, N.F.Y., 2000. Performance of different microalgal species in removing nickel and zinc from industrial wastewater. Chemosphere 41 (1-2), 251–257. Clemens, S., Persoh, D., 2009. Multi-tasking phytochelatin synthases. Plant Sci. 177 (4), 266–271.

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