Bull Environ Contam Toxicol (2014) 93:405–409 DOI 10.1007/s00128-014-1363-x
Effects of Cadmium and Copper Biosorption on Chlorella vulgaris Fabiano C. P. de Abreu • Pe´ricles N. M. da Costa • Ariadne M. Brondi Eduardo J. Pilau • Fa´bio C. Gozzo • Marcos N. Eberlin • Marcello G. Trevisan • Jerusa S. Garcia
•
Received: 18 November 2013 / Accepted: 16 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Changes in protein levels and lipid compositions in algal cells indicate the severity of stress related to toxic concentrations of heavy metals. In this study, the effects of exposure to cadmium and copper on Chlorella vulgaris and its capacity to remove metals were evaluated. The data revealed ion removal activity by microalgae under all treatments and different levels of protein expression after 48 h of exposure. Furthermore, we analyzed lipids contents to characterize them. Keywords Metal contamination Protein Lipids MALDI Q-TOF MS Heavy metals are accumulating in the environment as a result of increasing industrial activities, which causes serious environmental pollution. Several species of microalgae have been observed to develop resistance to a specific type of metal ion in natural waters, and they have attracted considerable attention for their capacity to remove heavy metals. The toxicity of a metal appears to be related to its interactions with the surface of the cell or to its intracellular accumulation in microalgae. Many heavy F. C. P. de Abreu P. N. M. da Costa A. M. Brondi M. G. Trevisan J. S. Garcia (&) LACFar, Institute of Chemistry, Federal University of Alfenas – UNIFAL-MG, Alfenas, Minas Gerais 37130-000, Brazil e-mail: [email protected] E. J. Pilau F. C. Gozzo M. N. Eberlin Institute of Chemistry, University of Campinas - UNICAMP, Caixa-Postal: 6154, Campinas, Sa˜o Paulo 13084-862, Brazil M. G. Trevisan National Institute of Science and Technology of Bioanalytical INCTBio, State University of Campinas, Campinas, Sa˜o Paulo 13084-653, Brazil
metal ions have a direct influence on various physiological and biochemical processes of microalgae, such as photosynthesis, growth inhibition, membrane injury, respiration, nutrient uptake, etc. (Arunakumara and Zhang 2008) At low concentrations, cooper is an essential micronutrient for several physiological processes, but at high concentrations, it can become a toxic metal similar to cadmium, which is an extremely toxic element that can induce several symptoms of phytotoxicity and affect the uptake of nutrients (Quian et al. 2009). These metals can induce alterations in proteins, DNA and cellular lipids by producing reactive oxygen species (Ercal et al. 2001). In this study, the green microalgae Chlorella vulgaris was selected to evaluate the effect of cadmium and copper contamination on its growth and the potential for metal removal through toxicity tests. This species was chosen due to its capacity to interact with several important heavy metal ions in aqueous solution and its ease in culturing under laboratory conditions (Khummongkol et al. 1982). Additionally, we aim to analyze the protein content after exposure to metals ions and characterize the lipids present in the microalgae.
Materials and Methods Chlorella vulgaris was cultured in Bold’s Basal Medium, pH 6.6, at 24 ± 2°C under a light intensity of 180 lmol photons m-2 s-1 using fluorescent light employing 12/10 h light/dark cycle. The cell growth was measured by counting the number of cells (Nageotte, Hausser Scientific) under a microscope (Eclipse 50i, Nikon). To observe the effects of Cd2? and Cu2? on the growth of C. vulgaris, microalgae culture was exposed to four different concentrations of Cd2? (26.6; 44.4; 106.7 e 213.5 lmol L-1) and Cu2? (2.83; 5.6; 15.7 e 47.2 lmol L-1) in order to evaluate
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the effective concentration that inhibits 50 % of microalgae growth (EC50).These experiments (n = 3) were performed using glass flasks that contained 150 mL of the medium with initial inoculums of 105 cel mL-1. The number of microalgae cells was evaluated after 48 h of exposure, and the suppression of cell growth was determined by comparing the number of cells obtained in metal treatments related to a control culture (without metal contamination). The culture was centrifuged at 4,000 rpm at 4°C for 15 min and the supernatant (culture medium) and pellet (microalgae biomass) were stored for subsequent analysis. Three replicates were performed for each treatment. In the C. vulgaris bioaccumulation studies, the amounts of Ca2?, Cd2?, Cu2?, Fe2? and Mn2? present in both culture medium and biomasses (around 50 mg) were quantified before and after 48 h of exposure to contamination. The quantification was performed using a Shimadzu AA-7000 flame and graphite furnace atomic absorption spectrometer (FAAS and GFAAS, respectively). The operating parameters of analytical instrumentation were performed according manufacturer recommendations. The determinations of Cu2?, Cd2? and Mn2? in samples (n = 3) were conducted using GFAAS, whereas the quantification of Ca2? and Fe2? were performed by FAAS. For determination of metal ions in supernatant, the samples obtained were only acidified with HNO3 (final concentration of 2 % v/v), while the biomass samples were decomposed using a 12:2 (v/v) mixture of HNO3 and H2O2 and subjected to a water bath at 110°C for 5 min. Subsequently, the biomass samples were placed in an ultrasonicator (Quimis, Q-335D) for 15 min at 50 Hz and 378 W. After, the biomass samples were subjected to a water bath at 110°C for 40 min. Finally, these samples were adjusted to 1 mL using deionized water. The protein extraction procedure was based on that reported by Garcia et al. (2009). Microalgae biomass was frozen in N2 and manually milled. The proteins were extracted by adding buffer (7 mol L-1 urea, 2 mol L-1 thiourea, 2 % (w/v) CHAPS and 4 % w/v DTT) at 25°C for 5 min. The ratio of protein to buffer used was 1:10 (w/v). The samples were subsequently vortex mixed for 3 min. The samples were also submitted to sonication at for 5 min (three cycles were employed). Finally, the remaining insoluble material was removed by centrifugation at 6,000g for 5 min at 4°C. The total protein content was determined according to the Bradford method using bovine albumin as a standard. The measurements (n = 3) were performed at 595 nm using a spectrophotometer (Spectrum, model SP 2000). The lipids from C. Vulgaris that were subjected to different conditions were extracted according to the recommendations of Blight and Dyer (Bligh and Dyer 1959). For extraction, approximately 30 mg (fresh weight) of
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microalgae were vortex mixed for 2 min with 0.45 mL of deionized water and 1.7 mL of chloroform:methanol (1:2, v/v), n = 4. Subsequently, the samples were centrifuged at 6,000g for 10 min at 25°C. The lower organic phases were collected and evaporated to dryness under nitrogen, and the total lipid contents were resuspended with 30 lL of nhexane (Correˆa et al. 2011). Then, 2 lL of the n-hexane phase were placed on the MALDI plate and maintained at 25°C until the solvent completely evaporated. The MALDI matrix (1 lL) was added to the sample, and it was also dried. The matrix (10 mg mL-1 of a-CHCA) was prepared in 50:50 H2O/MeCN with 0.1 % (v/v) H3PO4. The MALDI Q-TOF mass spectra were acquired using a Waters Synapt HDMS (Waters, Manchester, UK) mass spectrometer. The MALDI(?)-QTOF-MS analysis was performed using reflectron V-mode and a 200-Hz solid state (Nd:YAG) laser. The typical operating conditions were as follows: laser energy, 250 a.u.; sample plate, 20 V; and Trap and Transfer collision energies of 6 and 4 V, respectively. Spectra were recorded over the mass range from m/z 700 to 1,000. To classify the microalgae samples, principal component analysis (PCA) was performed. For the PCA, the MS datasets from the samples were organized into a matrix using the XS (Extended Statistics) module of the MarkerLynx (Waters, USA) software package. The data were truncated at 0.1 Da of peak separation and a threshold of 50 counts of intensity, and then the data were exported to Matlab. The PCA of the data was conducted in MatLab version 7.1 (The Mathworks Inc., USA) employing the PLS_toolbox (EigenVector Co., USA).
Results and Discussion The severity of growth inhibition increased with increasing concentrations of Cd2? and Cu2?. In fact, the effective concentration of a heavy metal that causes 50 % inhibition of microbial growth at 48 h is widely used as an index of toxicity (Regaldo et al. 2013). The EC 50 observed for Cd2? was 44.4 lmol L-1 or 5.0 mg L-1 and to Cu2? was 15.6 lmol L-1 or 1.0 mg L-1. Table 1 presents the concentrations of metals determined in the culture media (before the insertion of Cd2? and Cu2? contamination and after 48 h) and the results of metal exposure to the microalgae biomass. It is important to emphasize that metal mass balance found in all samples are satisfactory. The concentrations of metal ions decrease after exposure in all treatments, and its consequent increase in the biomass were observed. These data demonstrate the bioaccumulation activity by C. vulgaris. For the removal of Cd2?, approximately 44 % of this analyte (336 lg) initially present in culture medium (765 lg) was accumulated in the microalgae biomass. C. vulgaris can remove and accumulate
Bull Environ Contam Toxicol (2014) 93:405–409 Table 1 Metal determination (average ± standard deviation, n = 3) by atomic absorption spectrometry in different samples
Metal ion
Cd2?
407
Sample
Treatment Control
Cd2?*
Cu2?**
–a
5.10 ± 0.01
–a
–
a
2.86 ± 0.02
–a
–
a
336 ± 3
–a
Microalgae (lg mg )
–
a
3.60 ± 0.01
–a
Initial conc. in medium (mg L-1)
0.32 ± 0.01
0.31 ± 0.04
1.41 ± 0.02
Final conc. in medium (mg L-1)
0.23 ± 0.01
0.21 ± 0.01
0.91 ± 0.02
Initial conc. in medium (mg L-1) -1
Final conc. in medium (mg L ) Mass removed from medium (lg) -1
Cu2?
Ca2?
* Cd2? at 5 mg L-1 and ** Cu2? at 1 mg L-1 a
Fe2?
\LOQ
LOQðCu2þ Þ = 0.003 mg L-1; LOQðCa2þ Þ = 0.07 mg L-1; LOQðFe2þ Þ = 0.10 mg L-1; LOQðMn2þ Þ = 0.01 mg L
-1
14 ± 2 0.30 ± 0.01
0.45 ± 0.01
Initial conc. in medium (mg L-1)
6.80 ± 0.01
6.78 ± 0.01
6.82 ± 0.02
Final conc. in medium (mg L-1)
1.26 ± 0.01
1.26 ± 0.02
1.28 ± 0.01
Mass removed from medium (lg) Microalgae (lg mg-1)
831 ± 2 3.40 ± 0.02
828 ± 3 3.30 ± 0.02
831 ± 3 2.50 ± 0.01
Initial conc. in medium (mg L-1)
0.99 ± 0.01
1.00 ± 0.01
1.02 ± 0.01
Final conc. in medium (mg L-1)
0.55 ± 0.02
0.59 ± 0.02
0.57 ± 0.04
Mass removed from medium (lg)
66 ± 3
-1
-1
LOQðCd2þ Þ = 0.031 mg L ;
Mass removed from medium (lg) Microalgae (lg mg-1)
Mn2?
15 ± 2
75 ± 3 1.00 ± 0.01
62 ± 3
68 ± 6 5.80 ± 0.04
Microalgae (lg mg )
8.0 ± 0.01
8.0 ± 0.02
Initial conc. in medium (mg L-1)
0.40 ± 0.01
0.40 ± 0.01
0.42 ± 0.02
Final conc. in medium (mg L-1)
0.14 ± 0.01
0.16 ± 0.01
0.15 ± 0.01
Mass removed from medium (lg)
39 ± 2
Microalgae (lg mg-1)
1.22 ± 0.03
Cd2? because its exterior surface contains proteins and carbohydrates that are capable of reacting with metal ions (Ruangsomboon and Wongrat 2006). Note that cadmium is a non-essential metal that has a high toxicity, and it can substitute for other metal ions (primarily Zn2?, Cu2? and Ca2?) in metalloenzymes and exhibits a very strong affinity to biological structures that contain-SH groups (Garcia et al. 2009). In addition, copper is an essential micronutrient for algal growth and also plays a vital role as an enzymatic cofactor and electron carrier in the photosynthetic and respiratory processes. Approximately 35 % of the Cu2? (75 lg) that was initially present in the culture medium (211 lg) was detected in the microalgae biomass. According to Sabatini et al. (2009), the first responses of the algae to diminish the toxic effect of copper is to bind this ion to the cellular wall, which prevents its entrance to the cell and reduces its transport through the cell membrane. However, Ruangsomboon and Wongrat (2006) reported that after 48 h of exposure time, microalgae such as C. vulgaris are able to accumulate metal ions via intracellular uptake. The low removal of Cd2? and Cu2? can also be related to the low cell density used to remove the initial metal concentration in the medium. Additionally, cadmium is not an essential element to the survival of microalgae; therefore, a higher cell density is required for a greater effectiveness of removal. The bioaccumulation of Ca2?, Fe2? and Mn2? by C. vulgaris is also presented in
37 ± 2 0.84 ± 0.01
41 ± 3 1.18 ± 0.01
Table 1. In general, the levels of these analytes decrease in the culture medium from all treatments after 48 h, and they were not influenced due to the metal ion contamination. This fact suggests that these micronutrients are essential for the maintenance of microalgae development. Calcium is a macronutrient and activates several enzymes, and it participates in regulating the cell membrane permeability and neutralizing toxic acids. Iron is important for protein synthesis, electron transport, and the formation of chlorophyll. Finally, manganese is involved in the activation of enzymes, electron transport during photosynthesis, the formation of chlorophyll and the development of chloroplasts. A significant reduction of Mn2? level in the biomass was observed (1.22–0.84 lg mg-1, around 30 %) compared to control in the Cd2? treatment. The same was checked for Ca2? and Fe2? levels in the biomass (3.4–2.5 and 8.0–5.8 lg mg-1, respectively, around 27 %) in Cu2? treatment. These results suggest that Cd2? and Cu2? might compete among the various metal ions in solution for microalgae surface binding sites depending on their specific features, which is a common event, influencing the rate of ion bioaccumulation (Monteiro et al. 2011). The effect of the bioaccumulation of metal ions by C. vulgaris was also investigated through protein and lipid analyses. The protein levels observed in the control, cadmium and copper treatments were 70.1 ± 0.1; 61.1 ± 0.2 and 58.0 ± 0.1 lg g-1 (fresh weight), respectively. According
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Fig. 1 MALDI(?)-QTOF mass spectra of lipids from C. Vulgaris submitted at different treatments: a control, b Cd2? at 5 mg L-1, c Cu2? at 1 mg L-1 and d PCA plot for the MALDI(?)-QTOF data: (filled diamond) control, (1) Cd2? and (filled triangle) Cu2?
Lei et al. (2007), C. vulgaris have ca. 256 lg g-1 of protein (dry weight), but their buffer extraction and culture conditions employed were different than those adopted in this work. Metal toxicity can inhibit protein activity or disrupt their structures (Garcia et al. 2009). In this sense, a reduction of 13 % and 17 % of the protein levels in the C. vulgaris biomass under the Cd2? and Cu2? treatments, respectively, was observed. This result can be related to the adverse effects on the development of microalgae subjected to such a condition, which reflects the protein content and a possible down-regulation occurrence. Different detoxification processes performed by microalgae consist of those involving peptides, metallothioneins, primarily the post-transcriptionally synthesized class III metallothioneins, or phytochelatin. The synthesis of phytochelatins can be induced by Cd2? and Cu2? and several other heavy metals (Simmons et al. 2009). Zheng et al. (2011) performed a comparative study on the different cell disruption of lipids from C. vulgaris and determined the fatty acid composition using gas chromatography and mass spectrometry. Although the contents of unsaturated and saturated fatty acids (C14–C20) were available in this cited work, determining the influence of metals contamination on the compositions of lipids in C. vulgaris was not performed. Therefore, the extraction of lipids from the metals contamination and control treatments was performed, and the lipids
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were characterized. The MALDI(?) Q-TOF MS (Fig. 1) shows that the lipids of C. vulgaris display a unique spectrum that functions as a fingerprint for the rapid and clear identification of this microalgae. The major lipid ions for C. vulgaris were m/z 608.32, 609.31, 750.61, 778.60, 871.62 and 937.68. Different classes of lipids, such as monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylinositol and others, were observed (Ferreira et al. 2010; He et al. 2011). Note that the control sample (Fig. 1a) shows a different profile than the contamination samples (Fig. 1b, c). The same ions or the same classes of lipids were observed; however, the intensity of the ions was different, which indicates that metal contamination significantly changes the quantities of lipids in C. vulgaris according the PCA analysis (Fig. 1d). The PCA scores plot clearly shows splitting of the control and contaminated samples into three distinct groups. In the MALDI(?) Q-TOF MS results, PC1 and PC2 were observed to account for 58.16 % and 32.64 % of the total variance, respectively. Therefore, the total variance explained by the PCs is 90.8 % at a confidence level of 95 %. Toxicity tests are useful tools for determining the effects of pollutants on cell growth and viability. Developing more sensitive tests is especially important for evaluating the potential impact of pollution on aquatic ecosystems. An excessive concentration of Cd2? or Cu2? (at 5 and
Bull Environ Contam Toxicol (2014) 93:405–409
1 mg L-1, respectively) in culture medium markedly inhibits the growth of C. vulgaris, and its effects on the protein levels and contents of lipids were observed. The present study confirmed that C. vulgaris is an effective biomaterial for detoxifying and remediating free metal ions from waters. Acknowledgments The authors acknowledge to CNPq, CAPES (Process Casadinho/PROCAD 552387/2011-8) and FAPEMIG (Process APQ-00410-09) for financial support and Prof. Pio Colepicollo (University of Sa˜o Paulo, Brazil) for providing microalgae culture.
References Arunakumara KKIU, Zhang X (2008) Heavy metal bioaccumulation and toxicity with special reference to microalgae. J Ocean Univ Chin 7:60–64 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 Correˆa SC et al (2011) Evaluation of dehydrated marolo (Annona crassiflora) flour and carpels by freeze-drying and convective hot-air drying. Food Res Int 44:2385–2390 Ercal N, Gurer-Orhan H, Aykin-Burns N (2001) Toxic metals and oxidative stress part I: mechanisms involved in metal induced oxidative damage. Curr Top Med Chem 6:529–539 Ferreira CR et al (2010) Single embryo and oocyte lipid fingerprinting by mass spectrometry. J Lipid Res 51:1218–1227 Garcia JS et al (2009) Evaluation of metal-ion stress in sunflower (Helianthus annuus L.) leaves through proteomic changes. Metallomics 1:107–113 He H, Rodgers RP, Marshall AG, Hsu CS (2011) Algae polar lipids characterized by online liquid chromatography coupled with
409 hybrid linear quadrupole ion Trap/Fourier transform ion cyclotron resonance mass spectrometry. Energy Fuels 25:4770–4775 Khummongkol D, Canterford G, Fryer C (1982) Accumulation of heavy metals in unicellular algae. Biotechnol Bioeng 24:2646–2660 Lei A, Hu Z, Wong Y, Tam NF (2007) Removal of Xuoranthene and pyrene by different microalgal species. Bioresour Technol 98:273–278 Monteiro MC, Castro PML, Malcata FX (2011) Capacity of simultaneous removal of zinc and cadmium from contaminated media, by two microalgae isolated from a polluted site. Environ Chem Lett 9:511–517 Quian H, Li J, Sun L, Chen W, Sheng GD, Liu W, Fu Z (2009) Combined effect of copper and cadmium on Chlorella vulgaris growth and photosynthesis-related gene transcription. Aquat Toxicol 94:56–61 Regaldo et al (2013) Bioaccumulation and toxicity of copper and lead in Chlorella vulgaris. J Algal Biomass Utln 4:59–66 Ruangsomboon S, Wongrat L (2006) Bioaccumulation of cadmium in an experimental aquatic food chain involving phytoplankton (Chlorella vulgaris), zooplankton (Moina macrocopa), and the predatory catfish Clarias macrocephalus 9 C. Gariepinus. Aquat Toxicol 78:15–20 Sabatini SE et al (2009) Oxidative stress and antioxidant defenses in two green microalgae exposed to copper. Ecotox Environ Safe 72:1200–1206 Simmons DBD, Hayward AR, Hutchinson TC, Emery RJN (2009) Identification and quantification of glutathione and phytochelatins from C. vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction. Anal Bional Chem 395:809–817 Zheng H, Yin J, Gao Z, Huang H, Ji X, Dou C (2011) Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves. Appl Biochem Biotechnol 164:1215–1224
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Effects of Cadmium and Copper Biosorption on Chlorella vulgaris Fabiano C. P. de Abreu • Pe´ricles N. M. da Costa • Ariadne M. Brondi Eduardo J. Pilau • Fa´bio C. Gozzo • Marcos N. Eberlin • Marcello G. Trevisan • Jerusa S. Garcia
•
Received: 18 November 2013 / Accepted: 16 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Changes in protein levels and lipid compositions in algal cells indicate the severity of stress related to toxic concentrations of heavy metals. In this study, the effects of exposure to cadmium and copper on Chlorella vulgaris and its capacity to remove metals were evaluated. The data revealed ion removal activity by microalgae under all treatments and different levels of protein expression after 48 h of exposure. Furthermore, we analyzed lipids contents to characterize them. Keywords Metal contamination Protein Lipids MALDI Q-TOF MS Heavy metals are accumulating in the environment as a result of increasing industrial activities, which causes serious environmental pollution. Several species of microalgae have been observed to develop resistance to a specific type of metal ion in natural waters, and they have attracted considerable attention for their capacity to remove heavy metals. The toxicity of a metal appears to be related to its interactions with the surface of the cell or to its intracellular accumulation in microalgae. Many heavy F. C. P. de Abreu P. N. M. da Costa A. M. Brondi M. G. Trevisan J. S. Garcia (&) LACFar, Institute of Chemistry, Federal University of Alfenas – UNIFAL-MG, Alfenas, Minas Gerais 37130-000, Brazil e-mail: [email protected] E. J. Pilau F. C. Gozzo M. N. Eberlin Institute of Chemistry, University of Campinas - UNICAMP, Caixa-Postal: 6154, Campinas, Sa˜o Paulo 13084-862, Brazil M. G. Trevisan National Institute of Science and Technology of Bioanalytical INCTBio, State University of Campinas, Campinas, Sa˜o Paulo 13084-653, Brazil
metal ions have a direct influence on various physiological and biochemical processes of microalgae, such as photosynthesis, growth inhibition, membrane injury, respiration, nutrient uptake, etc. (Arunakumara and Zhang 2008) At low concentrations, cooper is an essential micronutrient for several physiological processes, but at high concentrations, it can become a toxic metal similar to cadmium, which is an extremely toxic element that can induce several symptoms of phytotoxicity and affect the uptake of nutrients (Quian et al. 2009). These metals can induce alterations in proteins, DNA and cellular lipids by producing reactive oxygen species (Ercal et al. 2001). In this study, the green microalgae Chlorella vulgaris was selected to evaluate the effect of cadmium and copper contamination on its growth and the potential for metal removal through toxicity tests. This species was chosen due to its capacity to interact with several important heavy metal ions in aqueous solution and its ease in culturing under laboratory conditions (Khummongkol et al. 1982). Additionally, we aim to analyze the protein content after exposure to metals ions and characterize the lipids present in the microalgae.
Materials and Methods Chlorella vulgaris was cultured in Bold’s Basal Medium, pH 6.6, at 24 ± 2°C under a light intensity of 180 lmol photons m-2 s-1 using fluorescent light employing 12/10 h light/dark cycle. The cell growth was measured by counting the number of cells (Nageotte, Hausser Scientific) under a microscope (Eclipse 50i, Nikon). To observe the effects of Cd2? and Cu2? on the growth of C. vulgaris, microalgae culture was exposed to four different concentrations of Cd2? (26.6; 44.4; 106.7 e 213.5 lmol L-1) and Cu2? (2.83; 5.6; 15.7 e 47.2 lmol L-1) in order to evaluate
123
406
the effective concentration that inhibits 50 % of microalgae growth (EC50).These experiments (n = 3) were performed using glass flasks that contained 150 mL of the medium with initial inoculums of 105 cel mL-1. The number of microalgae cells was evaluated after 48 h of exposure, and the suppression of cell growth was determined by comparing the number of cells obtained in metal treatments related to a control culture (without metal contamination). The culture was centrifuged at 4,000 rpm at 4°C for 15 min and the supernatant (culture medium) and pellet (microalgae biomass) were stored for subsequent analysis. Three replicates were performed for each treatment. In the C. vulgaris bioaccumulation studies, the amounts of Ca2?, Cd2?, Cu2?, Fe2? and Mn2? present in both culture medium and biomasses (around 50 mg) were quantified before and after 48 h of exposure to contamination. The quantification was performed using a Shimadzu AA-7000 flame and graphite furnace atomic absorption spectrometer (FAAS and GFAAS, respectively). The operating parameters of analytical instrumentation were performed according manufacturer recommendations. The determinations of Cu2?, Cd2? and Mn2? in samples (n = 3) were conducted using GFAAS, whereas the quantification of Ca2? and Fe2? were performed by FAAS. For determination of metal ions in supernatant, the samples obtained were only acidified with HNO3 (final concentration of 2 % v/v), while the biomass samples were decomposed using a 12:2 (v/v) mixture of HNO3 and H2O2 and subjected to a water bath at 110°C for 5 min. Subsequently, the biomass samples were placed in an ultrasonicator (Quimis, Q-335D) for 15 min at 50 Hz and 378 W. After, the biomass samples were subjected to a water bath at 110°C for 40 min. Finally, these samples were adjusted to 1 mL using deionized water. The protein extraction procedure was based on that reported by Garcia et al. (2009). Microalgae biomass was frozen in N2 and manually milled. The proteins were extracted by adding buffer (7 mol L-1 urea, 2 mol L-1 thiourea, 2 % (w/v) CHAPS and 4 % w/v DTT) at 25°C for 5 min. The ratio of protein to buffer used was 1:10 (w/v). The samples were subsequently vortex mixed for 3 min. The samples were also submitted to sonication at for 5 min (three cycles were employed). Finally, the remaining insoluble material was removed by centrifugation at 6,000g for 5 min at 4°C. The total protein content was determined according to the Bradford method using bovine albumin as a standard. The measurements (n = 3) were performed at 595 nm using a spectrophotometer (Spectrum, model SP 2000). The lipids from C. Vulgaris that were subjected to different conditions were extracted according to the recommendations of Blight and Dyer (Bligh and Dyer 1959). For extraction, approximately 30 mg (fresh weight) of
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Bull Environ Contam Toxicol (2014) 93:405–409
microalgae were vortex mixed for 2 min with 0.45 mL of deionized water and 1.7 mL of chloroform:methanol (1:2, v/v), n = 4. Subsequently, the samples were centrifuged at 6,000g for 10 min at 25°C. The lower organic phases were collected and evaporated to dryness under nitrogen, and the total lipid contents were resuspended with 30 lL of nhexane (Correˆa et al. 2011). Then, 2 lL of the n-hexane phase were placed on the MALDI plate and maintained at 25°C until the solvent completely evaporated. The MALDI matrix (1 lL) was added to the sample, and it was also dried. The matrix (10 mg mL-1 of a-CHCA) was prepared in 50:50 H2O/MeCN with 0.1 % (v/v) H3PO4. The MALDI Q-TOF mass spectra were acquired using a Waters Synapt HDMS (Waters, Manchester, UK) mass spectrometer. The MALDI(?)-QTOF-MS analysis was performed using reflectron V-mode and a 200-Hz solid state (Nd:YAG) laser. The typical operating conditions were as follows: laser energy, 250 a.u.; sample plate, 20 V; and Trap and Transfer collision energies of 6 and 4 V, respectively. Spectra were recorded over the mass range from m/z 700 to 1,000. To classify the microalgae samples, principal component analysis (PCA) was performed. For the PCA, the MS datasets from the samples were organized into a matrix using the XS (Extended Statistics) module of the MarkerLynx (Waters, USA) software package. The data were truncated at 0.1 Da of peak separation and a threshold of 50 counts of intensity, and then the data were exported to Matlab. The PCA of the data was conducted in MatLab version 7.1 (The Mathworks Inc., USA) employing the PLS_toolbox (EigenVector Co., USA).
Results and Discussion The severity of growth inhibition increased with increasing concentrations of Cd2? and Cu2?. In fact, the effective concentration of a heavy metal that causes 50 % inhibition of microbial growth at 48 h is widely used as an index of toxicity (Regaldo et al. 2013). The EC 50 observed for Cd2? was 44.4 lmol L-1 or 5.0 mg L-1 and to Cu2? was 15.6 lmol L-1 or 1.0 mg L-1. Table 1 presents the concentrations of metals determined in the culture media (before the insertion of Cd2? and Cu2? contamination and after 48 h) and the results of metal exposure to the microalgae biomass. It is important to emphasize that metal mass balance found in all samples are satisfactory. The concentrations of metal ions decrease after exposure in all treatments, and its consequent increase in the biomass were observed. These data demonstrate the bioaccumulation activity by C. vulgaris. For the removal of Cd2?, approximately 44 % of this analyte (336 lg) initially present in culture medium (765 lg) was accumulated in the microalgae biomass. C. vulgaris can remove and accumulate
Bull Environ Contam Toxicol (2014) 93:405–409 Table 1 Metal determination (average ± standard deviation, n = 3) by atomic absorption spectrometry in different samples
Metal ion
Cd2?
407
Sample
Treatment Control
Cd2?*
Cu2?**
–a
5.10 ± 0.01
–a
–
a
2.86 ± 0.02
–a
–
a
336 ± 3
–a
Microalgae (lg mg )
–
a
3.60 ± 0.01
–a
Initial conc. in medium (mg L-1)
0.32 ± 0.01
0.31 ± 0.04
1.41 ± 0.02
Final conc. in medium (mg L-1)
0.23 ± 0.01
0.21 ± 0.01
0.91 ± 0.02
Initial conc. in medium (mg L-1) -1
Final conc. in medium (mg L ) Mass removed from medium (lg) -1
Cu2?
Ca2?
* Cd2? at 5 mg L-1 and ** Cu2? at 1 mg L-1 a
Fe2?
\LOQ
LOQðCu2þ Þ = 0.003 mg L-1; LOQðCa2þ Þ = 0.07 mg L-1; LOQðFe2þ Þ = 0.10 mg L-1; LOQðMn2þ Þ = 0.01 mg L
-1
14 ± 2 0.30 ± 0.01
0.45 ± 0.01
Initial conc. in medium (mg L-1)
6.80 ± 0.01
6.78 ± 0.01
6.82 ± 0.02
Final conc. in medium (mg L-1)
1.26 ± 0.01
1.26 ± 0.02
1.28 ± 0.01
Mass removed from medium (lg) Microalgae (lg mg-1)
831 ± 2 3.40 ± 0.02
828 ± 3 3.30 ± 0.02
831 ± 3 2.50 ± 0.01
Initial conc. in medium (mg L-1)
0.99 ± 0.01
1.00 ± 0.01
1.02 ± 0.01
Final conc. in medium (mg L-1)
0.55 ± 0.02
0.59 ± 0.02
0.57 ± 0.04
Mass removed from medium (lg)
66 ± 3
-1
-1
LOQðCd2þ Þ = 0.031 mg L ;
Mass removed from medium (lg) Microalgae (lg mg-1)
Mn2?
15 ± 2
75 ± 3 1.00 ± 0.01
62 ± 3
68 ± 6 5.80 ± 0.04
Microalgae (lg mg )
8.0 ± 0.01
8.0 ± 0.02
Initial conc. in medium (mg L-1)
0.40 ± 0.01
0.40 ± 0.01
0.42 ± 0.02
Final conc. in medium (mg L-1)
0.14 ± 0.01
0.16 ± 0.01
0.15 ± 0.01
Mass removed from medium (lg)
39 ± 2
Microalgae (lg mg-1)
1.22 ± 0.03
Cd2? because its exterior surface contains proteins and carbohydrates that are capable of reacting with metal ions (Ruangsomboon and Wongrat 2006). Note that cadmium is a non-essential metal that has a high toxicity, and it can substitute for other metal ions (primarily Zn2?, Cu2? and Ca2?) in metalloenzymes and exhibits a very strong affinity to biological structures that contain-SH groups (Garcia et al. 2009). In addition, copper is an essential micronutrient for algal growth and also plays a vital role as an enzymatic cofactor and electron carrier in the photosynthetic and respiratory processes. Approximately 35 % of the Cu2? (75 lg) that was initially present in the culture medium (211 lg) was detected in the microalgae biomass. According to Sabatini et al. (2009), the first responses of the algae to diminish the toxic effect of copper is to bind this ion to the cellular wall, which prevents its entrance to the cell and reduces its transport through the cell membrane. However, Ruangsomboon and Wongrat (2006) reported that after 48 h of exposure time, microalgae such as C. vulgaris are able to accumulate metal ions via intracellular uptake. The low removal of Cd2? and Cu2? can also be related to the low cell density used to remove the initial metal concentration in the medium. Additionally, cadmium is not an essential element to the survival of microalgae; therefore, a higher cell density is required for a greater effectiveness of removal. The bioaccumulation of Ca2?, Fe2? and Mn2? by C. vulgaris is also presented in
37 ± 2 0.84 ± 0.01
41 ± 3 1.18 ± 0.01
Table 1. In general, the levels of these analytes decrease in the culture medium from all treatments after 48 h, and they were not influenced due to the metal ion contamination. This fact suggests that these micronutrients are essential for the maintenance of microalgae development. Calcium is a macronutrient and activates several enzymes, and it participates in regulating the cell membrane permeability and neutralizing toxic acids. Iron is important for protein synthesis, electron transport, and the formation of chlorophyll. Finally, manganese is involved in the activation of enzymes, electron transport during photosynthesis, the formation of chlorophyll and the development of chloroplasts. A significant reduction of Mn2? level in the biomass was observed (1.22–0.84 lg mg-1, around 30 %) compared to control in the Cd2? treatment. The same was checked for Ca2? and Fe2? levels in the biomass (3.4–2.5 and 8.0–5.8 lg mg-1, respectively, around 27 %) in Cu2? treatment. These results suggest that Cd2? and Cu2? might compete among the various metal ions in solution for microalgae surface binding sites depending on their specific features, which is a common event, influencing the rate of ion bioaccumulation (Monteiro et al. 2011). The effect of the bioaccumulation of metal ions by C. vulgaris was also investigated through protein and lipid analyses. The protein levels observed in the control, cadmium and copper treatments were 70.1 ± 0.1; 61.1 ± 0.2 and 58.0 ± 0.1 lg g-1 (fresh weight), respectively. According
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408
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Fig. 1 MALDI(?)-QTOF mass spectra of lipids from C. Vulgaris submitted at different treatments: a control, b Cd2? at 5 mg L-1, c Cu2? at 1 mg L-1 and d PCA plot for the MALDI(?)-QTOF data: (filled diamond) control, (1) Cd2? and (filled triangle) Cu2?
Lei et al. (2007), C. vulgaris have ca. 256 lg g-1 of protein (dry weight), but their buffer extraction and culture conditions employed were different than those adopted in this work. Metal toxicity can inhibit protein activity or disrupt their structures (Garcia et al. 2009). In this sense, a reduction of 13 % and 17 % of the protein levels in the C. vulgaris biomass under the Cd2? and Cu2? treatments, respectively, was observed. This result can be related to the adverse effects on the development of microalgae subjected to such a condition, which reflects the protein content and a possible down-regulation occurrence. Different detoxification processes performed by microalgae consist of those involving peptides, metallothioneins, primarily the post-transcriptionally synthesized class III metallothioneins, or phytochelatin. The synthesis of phytochelatins can be induced by Cd2? and Cu2? and several other heavy metals (Simmons et al. 2009). Zheng et al. (2011) performed a comparative study on the different cell disruption of lipids from C. vulgaris and determined the fatty acid composition using gas chromatography and mass spectrometry. Although the contents of unsaturated and saturated fatty acids (C14–C20) were available in this cited work, determining the influence of metals contamination on the compositions of lipids in C. vulgaris was not performed. Therefore, the extraction of lipids from the metals contamination and control treatments was performed, and the lipids
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were characterized. The MALDI(?) Q-TOF MS (Fig. 1) shows that the lipids of C. vulgaris display a unique spectrum that functions as a fingerprint for the rapid and clear identification of this microalgae. The major lipid ions for C. vulgaris were m/z 608.32, 609.31, 750.61, 778.60, 871.62 and 937.68. Different classes of lipids, such as monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylinositol and others, were observed (Ferreira et al. 2010; He et al. 2011). Note that the control sample (Fig. 1a) shows a different profile than the contamination samples (Fig. 1b, c). The same ions or the same classes of lipids were observed; however, the intensity of the ions was different, which indicates that metal contamination significantly changes the quantities of lipids in C. vulgaris according the PCA analysis (Fig. 1d). The PCA scores plot clearly shows splitting of the control and contaminated samples into three distinct groups. In the MALDI(?) Q-TOF MS results, PC1 and PC2 were observed to account for 58.16 % and 32.64 % of the total variance, respectively. Therefore, the total variance explained by the PCs is 90.8 % at a confidence level of 95 %. Toxicity tests are useful tools for determining the effects of pollutants on cell growth and viability. Developing more sensitive tests is especially important for evaluating the potential impact of pollution on aquatic ecosystems. An excessive concentration of Cd2? or Cu2? (at 5 and
Bull Environ Contam Toxicol (2014) 93:405–409
1 mg L-1, respectively) in culture medium markedly inhibits the growth of C. vulgaris, and its effects on the protein levels and contents of lipids were observed. The present study confirmed that C. vulgaris is an effective biomaterial for detoxifying and remediating free metal ions from waters. Acknowledgments The authors acknowledge to CNPq, CAPES (Process Casadinho/PROCAD 552387/2011-8) and FAPEMIG (Process APQ-00410-09) for financial support and Prof. Pio Colepicollo (University of Sa˜o Paulo, Brazil) for providing microalgae culture.
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