Toxicity Effects of Copper and Zinc on the Photosynthetic Efficiency and Oxidative Stress-Related Parameters of the Green Alga Chlorella vulgaris Beijerinck
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Published:
Aug 1, 2019
Keywords:
Chlorella vulgaris Heavy metal Physiology Toxicology
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Abstract
Microalgae are widely used as a model for ecotoxicological assays. The present study investigated the physiological responses of Chlorella vulgaris to five days-exposure at different concentrations of copper or zinc (control, 125, 250, 500 and 1,000 µM). Both heavy metals showed dose-dependent cellular accumulation. Decreased maximum quantum efficiency of photosystem II (Fv/Fm) was influenced by both heavy metal concentration and time of exposure. A reduction in Fv/Fm indicated that photodamage occurred from day 3 after exposure. Other toxicity symptoms included chlorophyll degradation and an increase in reactive oxygen species (ROS). While exposure to both heavy metals resulted in a decrease in chlorophyll a content to a similar extent, an increase in ROS was detected only in 1,000 µM copper, suggesting stronger toxicity effects of copper ด แรcompared to zinc. Nevertheless, an increase in lipid peroxidation was not detected, indicating that ROS produced in 1,000 µM copper was not sufficient to induce disintegration of membrane lipids via the oxidation process. Proline, an amino acid with various putative protective functions against stress, exhibited a rapid increase depending on heavy metal concentration and time of exposure. These results provide a set of effective biomarkers for heavy metal contamination using C. vulgaris as a bioindicator.
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Phetchuay, P., Peerakietkhajorn, S., Duangpan, S., & Buapet, P. (2019). Toxicity Effects of Copper and Zinc on the Photosynthetic Efficiency and Oxidative Stress-Related Parameters of the Green Alga Chlorella vulgaris Beijerinck. Journal of Fisheries and Environment, 43(2), 14–26. Retrieved from https://li01.tci-thaijo.org/index.php/JFE/article/view/173171
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References
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7. Chekroun, K.B. and M. Baghour. 2013. The role of algae in phytoremediation of heavy metals: A review. Journal of Materials and Environmental Science 4: 873-880.
8. Choudhary, M., U.K. Jetley, M.A. Knan, S. Zutshi and T. Fatma. 2007. Effect of heavy metal stress on proline, malondialdehyde, and superoxide dismutase activity in the cyanobacterium Spirulina platensis-S5. Ecotoxicology and Environmental Safety 36: 1-6.
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14. Franklin, R.E., L. Duis, B.R. Smith, R. Brown and J.E. Toler. 2003. Elemental concentrations in soils of South Carolina. Soil Science 168: 280–291.
15. Gilroy, S., N. Susuki, G. Miller, H. Choi, M. Toyota. and A.R. Deyireddy. 2014. A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling. Trends in Plant Science 19: 623–630.
16. Grotz, N. and M.L. Guerinot. 2006. Molecular aspects of Cu, Fe and Zn homeostasis in plants. Biochimica et Biophysica Acta 1763: 595-608.
17. Grima, E.M., E.H. Belarbi, F.G.A. Fernández, A.R. Medina and Y. Chisti. 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnology Advances 20: 491-515.
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22. Johnson, H.L., J.L. Stauber, M. Adams and D.F. Jolley. 2007. Copper and zinc tolerance of two tropical microalgae after copper acclimation. Environmental Toxicology 22: 234-244.
23. Kaciene, G., J. Zaltauskaite, E. Milce and R. Juknys. 2015. Role of oxidative stress on growth responses of spring barley exposed to different environmental stressors. Journal of Plant Ecology 8: 605-616.
24. Kebeish, R., Y.E. Ayouty and A. Husain. 2014. Effect of copper on growth, bioactive metabolites, antioxidant enzymes and photosynthesis-related gene transcription in Chlorella vulgaris. World Journal of Biology and Biological Sciences 2: 34-43.
25. Kowalewska, G. 1999. Phytoplankton the main factor responsible for transport of polynuclear aromatic hydrocarbons from water to sediments in the Southern Baltic ecosystem (extended abstract). Journal of Marine Science and Engineering 56: 219–222.
26. Kumar, D., L.K. Pandey and J.P. Gaur. 2016. Metal sorption by algal biomass: From batch to continuous system. Algal Research 18: 95–109.
27. Liang, X., L. Zang, S.K. Natarajan and D.F. Becker. 2013. Proline mechanisms of stress survival. Antioxidants & Redox Signaling 19: 1-14.
28. Lu, L., H. Ding, Y. Wu and W. Zhang. 2015. The combined and second exposure effect of copper (II) and chlortetracycline on fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa. Environmental Toxicology and Pharmacology 40: 140- 148.
29. Mehta, S.K. and J.P. Gaur. 1999. Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris. New Phytologist Journals 143: 253-259.
30. Oukarroum, A., F. Perreault and R. Popovic. 2012. Interactive effects of temperature and copper on photosystem II photochemistry in Chlorella vulgaris. Journal of Photochemistry and Photobiology B: Biology 110: 9-14.
31. Oves, M., K.M. Saghir, Q.A. Huda, F.M. Nadeen and T. Almeelbi. 2016. Heavy metals: biological importance and detoxification strategies. Journal of Bioremediation and Biodegradation 7: 334-344.
32. Penarrubia, L., P. Romero, A.C. Segui, A.A. Bordersia, J. Moreno and A. Sanz. 2015. Temporal aspects of copper homeostasis and its crosstalk with hormones. Frontiers in Plant Science 6: 1-18.
33. Pinto, E., K. Siguad, M.A.S. Leito, O.K. Okamoto, D. Morse and P. Colepicolo. 2003. Heavy metal–induced oxidative stress in algae. Journal of Phycology 39: 1008–1018.
34. Ravet, K. and M. Pilon. 2013. Copper and iron homeostasis in plants: the challenges of oxidative stress. Antioxidants & Redox Signaling 19: 1-14.
35. Rodrigues, M.S., L.S. Ferreira, J.C.M. Carvalho, A. Lodi, E. Finocchio and A. Converti. 2012. Metal biosorption onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris: multi-metal systems. Journal of Hazardous Materials 217–218: 246–255.
36. Saijo, Y. 1975. A method for determination of chlorophyll. Japanese Journal of Limnology 36: 103-109.
37. Salt, D.E., M. Blaylock and P.B.A. Kamar. 1995. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology 13: 468-474.
38. Saavedra, R., R. Munoz, M.E. Taboada, M. Vega and S. Bolado. 2018. Comparative uptake study of arsenic, boron, copper, manganese and zinc from water by different green microalgae. Bioresource Technolgy 263: 49-57.
39. Saradhi, A.P.P. 1991. Proline accumulation under heavy metal stress. Journal of Plant Physiology 138: 554-558.
40. Schat, H., S.S. Sharma and R. Vooijs. 1997. Heavy metal-induced accumulation of free prolinein metal-tolerant and a nontolerant ecotype of Silence vulgaris. Physiologia Plantatum 101: 477-487.
41. Schutzendubel, A. and A. Polle. 2003. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. Journal of Experimental Botany 53: 1351-1365.
42. Senevirathne, M., S.H. Kim, N. Siriwardhana, J.H. Ha and Y.J. Jeon. 2006. Antioxidant potential of Ecklonia cavaon reactive oxygen species scavenging, metal chelating, reducing power and lipid peroxidation inhibition. Food Science and Technology International 12: 12-27.
43. Shama, S.D. and R.N. Chopra. 1987. Effect of lead nitrate and lead acetate on growth of the moss Semibarbula orienianum (Web) at many growths in vitro. Journal Plant Physiology 129: 242-249.
44. Sharma, P., A.B. Jha, R.S. Dubey and M. Pessarali. 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of Botany 23: 2-28.
45. Singh, R., R. Chadetrik, R. Kumar, K. Bishnoi, D. Bhatia, A. Kumar, N.R. Bishnoi and N. Singh. 2010. Biosorption optimization of lead (II), cadmium (II) and copper (II) using response surface methodology and amicability in isotherms and thermodynamics modeling. Journal of Hazardous Materials 174: 623-634.
46. Siripornadulsil, S., S. Traina, D.P.S. Verma and R.T. Sayre. 2002. Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. Plant Cell 14: 2837-2847.
47. Smironoff, N. and Q.J. Cumbes. 1989. Hydroxyl radicals scavenging activity of compatible solutes. Phytochemistry 28: 1057-1060.
48. StatSoft. 2011. STATISTICA (data analysis software system), version 10. http://www.statsoft.com. Cited 18 July 2018.
49. Stiborova, M. 1988. Cd2+ ions copper the quaternary structure of ribulose-1,5-bisphosphate carboxylase from barley leaves. Biochemie und Physiologie Pflanzen 183: 371-378.
50. Sokolnik, A.Z., H. Asard, K.G. Koplinska and R.J. Gorecki. 2009. Cadmium and zinc- mediated oxidative burst in tobacco BY-2 cell suspension cultures. Acta Physiologiae Plantarum 31: 43-49.
51. Sun, X., Y. Zhong, Z. Huang and Y. Yang. 2014. Selenium accumulation in unicellular green alga Chlorella vulgaris and its effects on antioxidant enzymes and content of photosynthetic pigments. PLoS ONE 9: 1-8.
52. Terry, P.A. and W. Stone. 2002. Biosorption of cadmium and copper contaminated water by Scenedesmus abundans. Chemosphere 47: 249-255.
53. Tripathi, B.N. and J.P. Guar. 2004. Relationship between copper- and zinc-induced oxidative stress and proline accumulation in Scenedesmus sp. Planta Toronto 219: 397-404.
54. Tripathi, B.N., S.K. Mehta, A. Amar and J.P. Gaur. 2006. Oxidative stress in Scenedesmus sp. during short- and long-term exposure to Cu2+ and Zn2+. Chemosphere 62: 538- 544.
55. Ullah, A., S. Heng, M.F.H. Munis, S. Fahad and X. Yang. 2015. Phytoremediation of heavy metals assisted by plant growth promoting (PGP) bacteria: A review. Environmental and Experimental Botany 117: 28-40.
56. Veenstra, J.N., D. Sanders. and S. Ahn. 1999. Impact of chromium and copper on fixed film biological systems. Journal of Environmental Engineering 125: 522–531.
57. Vert, G., N. Gratz, F. Dedaldechamp, F. Gaymard, M.L. Guerino, J.F. Briat and C. Curie. 2002. IRT1, an Arabidopsis transporter essential for iron uptake from the soil and plant growth. Plant Cell 14: 1223-1233.
58. Wang, L., Y. Li, P. Chen, M. Min, Y. Chen, J. Zhu and R.R. Ruan. 2010. Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresource Technology 101: 2623-2628.
59. Wintermans, J.F.G.M. and A. DeMots. 1965. Spectrophotometric characteristics of chlorophyll a and b and their pheophytins in ethanol. Biochimica et Biophysica Acta 109: 448-453.
60. Woodrow, L. 2014. ANOVA, ANCOVA and MANOVA. In: Writing about Quantitative Research in Applied Linguistics, pp. 57-69. Palgrave Macmillan, London.
61. Wu, J.T., S.J. Chang and T.L. Chou. 1998a. Intracellular proline accumulation in some algae exposed to copper and cadmium. Botanical Bulletin- Academia Sinica Taipei 36: 89-93.
62. Wu, J.T., M.T. Hsieh. and L.C. Know. 1998b. Role of proline accumulation in response to toxic copper in Chlorella sp. (Chlorophyceae) cell. Journal of Phycology 34: 113- 117.
63. Zeraatkar, A.K., H. Ahmadzadeh, A.F. Talebi, N.R. Moheimani and M.P. McHenry. 2016. Potential use of algae for heavy metal bioremediation, a critical review. Journal of Environmental Management 122: 132-144.
64. Zhou, G.J., P.Q. Peng, L.J. Zhang and G.G. Ying. 2012. Biosorption of zinc and copper from aqueous solutions by two freshwater green microalgae Chlorella pyrenoidosa and Scenedesmus obliquus. Environmental Science and Pollution Research 19: 2918-2929.
2. Ambikapathy, J., J.S. Marshell, C.H. Hocast and A.R. Haedham. 2002. The Role of Proline in Osmoregulation in Phytophthora nicotianae. Fungal Genetics & Biology 35: 287–299.
3. Ashraf, M. and M.R. Foolad. 2007. Roles of glycine betaine and proline in improving plant abiotic stress tolerance. Environmental and Experimental Botany 59: 206-216.
4. Bates, L.S., R.P. Waldren and I.D. Teare. 1973. Rapid determination of free proline for water-stress studies. Plant and Soil 39: 205-207.
5. Burleigh, G.H., B.K. Kristensen and I.E. Bechmenn. 2003. A plasma membrane zinc transporter from Medicago truncatula is up-regulated in roots by Zn fertilization, yet down-regulated by arbuscular mycorrhizal colonization. Plant Molecular Biology 52: 1077-1088.
6. Costa, G. and J.L. Morel. 1994. Water relation, gas exchange and amino acid content in Cd-treated lettuce. Plant Physiology Biochemistry 32: 560-571.
7. Chekroun, K.B. and M. Baghour. 2013. The role of algae in phytoremediation of heavy metals: A review. Journal of Materials and Environmental Science 4: 873-880.
8. Choudhary, M., U.K. Jetley, M.A. Knan, S. Zutshi and T. Fatma. 2007. Effect of heavy metal stress on proline, malondialdehyde, and superoxide dismutase activity in the cyanobacterium Spirulina platensis-S5. Ecotoxicology and Environmental Safety 36: 1-6.
9. Dobermann, A. and J. Fairhurst. 2000. Rice: Nutrient disorders and nutrient management. Philiines: Potash & Phosphate Institute of Canada (IC) and International Rice Research Institute. 1029 pp.
10. Elbalz, A.-Y., G. Rosenfeld, E. Shinder, A. Futerman, T. Geiger and M. Schudiner. 2014. A dynamic interface between vacuoles and mitochondria in yeast. Developmental Cell 30: 95–102.
11. Enany, A.E. and A.A. Issa. 2001. Proline alleviates heavy metal stress in Scenedesmus armatus. Folia Microbiologica 46: 227-230.
12. Fathi, A.A., A.M. El-Shahed, M.A. Shoulkamy, H.A. Ibraheim and O.M. Abdel Rahman. 2008. Response of Nile water phytoplankton to the toxicity cobalt, copper and zinc. Research Journal of Environmental Toxicology 2: 67-76.
13. Filippis, L.F. and C.K. Pallaghy. 1976. The effect of sub-lethal concentrations of mercury and zinc on Chlorella: II. photosynthesis and pigment composition. Zeitschrift für Pflanzenphysiologie 78: 314-322.
14. Franklin, R.E., L. Duis, B.R. Smith, R. Brown and J.E. Toler. 2003. Elemental concentrations in soils of South Carolina. Soil Science 168: 280–291.
15. Gilroy, S., N. Susuki, G. Miller, H. Choi, M. Toyota. and A.R. Deyireddy. 2014. A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling. Trends in Plant Science 19: 623–630.
16. Grotz, N. and M.L. Guerinot. 2006. Molecular aspects of Cu, Fe and Zn homeostasis in plants. Biochimica et Biophysica Acta 1763: 595-608.
17. Grima, E.M., E.H. Belarbi, F.G.A. Fernández, A.R. Medina and Y. Chisti. 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnology Advances 20: 491-515.
18. Hafeez, B., Y.M. Khanif and M. Saleem. 2013. Role of zinc in plant nutrition a review. American Journal of Experimental Agriculture 3: 374-391.
19. Harris, P.O. and G.J. Ramelow. 1989. Binding of metal ions by particulate biomass derived from Chlorella vulgaris and Scenedesmus quadricauda. Biophysica Acta 19: 1-9.
20. Halliwell, B. and J.M.C. Gutteridge. 1999. Free radicals in biology and medicine. Oxford University Press, New York. 936 pp.
21. Hayat, S., Q. Hayat, M.N. Alyemeni, A.S. Wani, J. Pichtel and A. Ahmad. 2012. Role of proline under changing environments. Plant Signaling and Behavior 7: 1559-1578.
22. Johnson, H.L., J.L. Stauber, M. Adams and D.F. Jolley. 2007. Copper and zinc tolerance of two tropical microalgae after copper acclimation. Environmental Toxicology 22: 234-244.
23. Kaciene, G., J. Zaltauskaite, E. Milce and R. Juknys. 2015. Role of oxidative stress on growth responses of spring barley exposed to different environmental stressors. Journal of Plant Ecology 8: 605-616.
24. Kebeish, R., Y.E. Ayouty and A. Husain. 2014. Effect of copper on growth, bioactive metabolites, antioxidant enzymes and photosynthesis-related gene transcription in Chlorella vulgaris. World Journal of Biology and Biological Sciences 2: 34-43.
25. Kowalewska, G. 1999. Phytoplankton the main factor responsible for transport of polynuclear aromatic hydrocarbons from water to sediments in the Southern Baltic ecosystem (extended abstract). Journal of Marine Science and Engineering 56: 219–222.
26. Kumar, D., L.K. Pandey and J.P. Gaur. 2016. Metal sorption by algal biomass: From batch to continuous system. Algal Research 18: 95–109.
27. Liang, X., L. Zang, S.K. Natarajan and D.F. Becker. 2013. Proline mechanisms of stress survival. Antioxidants & Redox Signaling 19: 1-14.
28. Lu, L., H. Ding, Y. Wu and W. Zhang. 2015. The combined and second exposure effect of copper (II) and chlortetracycline on fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa. Environmental Toxicology and Pharmacology 40: 140- 148.
29. Mehta, S.K. and J.P. Gaur. 1999. Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris. New Phytologist Journals 143: 253-259.
30. Oukarroum, A., F. Perreault and R. Popovic. 2012. Interactive effects of temperature and copper on photosystem II photochemistry in Chlorella vulgaris. Journal of Photochemistry and Photobiology B: Biology 110: 9-14.
31. Oves, M., K.M. Saghir, Q.A. Huda, F.M. Nadeen and T. Almeelbi. 2016. Heavy metals: biological importance and detoxification strategies. Journal of Bioremediation and Biodegradation 7: 334-344.
32. Penarrubia, L., P. Romero, A.C. Segui, A.A. Bordersia, J. Moreno and A. Sanz. 2015. Temporal aspects of copper homeostasis and its crosstalk with hormones. Frontiers in Plant Science 6: 1-18.
33. Pinto, E., K. Siguad, M.A.S. Leito, O.K. Okamoto, D. Morse and P. Colepicolo. 2003. Heavy metal–induced oxidative stress in algae. Journal of Phycology 39: 1008–1018.
34. Ravet, K. and M. Pilon. 2013. Copper and iron homeostasis in plants: the challenges of oxidative stress. Antioxidants & Redox Signaling 19: 1-14.
35. Rodrigues, M.S., L.S. Ferreira, J.C.M. Carvalho, A. Lodi, E. Finocchio and A. Converti. 2012. Metal biosorption onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris: multi-metal systems. Journal of Hazardous Materials 217–218: 246–255.
36. Saijo, Y. 1975. A method for determination of chlorophyll. Japanese Journal of Limnology 36: 103-109.
37. Salt, D.E., M. Blaylock and P.B.A. Kamar. 1995. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology 13: 468-474.
38. Saavedra, R., R. Munoz, M.E. Taboada, M. Vega and S. Bolado. 2018. Comparative uptake study of arsenic, boron, copper, manganese and zinc from water by different green microalgae. Bioresource Technolgy 263: 49-57.
39. Saradhi, A.P.P. 1991. Proline accumulation under heavy metal stress. Journal of Plant Physiology 138: 554-558.
40. Schat, H., S.S. Sharma and R. Vooijs. 1997. Heavy metal-induced accumulation of free prolinein metal-tolerant and a nontolerant ecotype of Silence vulgaris. Physiologia Plantatum 101: 477-487.
41. Schutzendubel, A. and A. Polle. 2003. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. Journal of Experimental Botany 53: 1351-1365.
42. Senevirathne, M., S.H. Kim, N. Siriwardhana, J.H. Ha and Y.J. Jeon. 2006. Antioxidant potential of Ecklonia cavaon reactive oxygen species scavenging, metal chelating, reducing power and lipid peroxidation inhibition. Food Science and Technology International 12: 12-27.
43. Shama, S.D. and R.N. Chopra. 1987. Effect of lead nitrate and lead acetate on growth of the moss Semibarbula orienianum (Web) at many growths in vitro. Journal Plant Physiology 129: 242-249.
44. Sharma, P., A.B. Jha, R.S. Dubey and M. Pessarali. 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of Botany 23: 2-28.
45. Singh, R., R. Chadetrik, R. Kumar, K. Bishnoi, D. Bhatia, A. Kumar, N.R. Bishnoi and N. Singh. 2010. Biosorption optimization of lead (II), cadmium (II) and copper (II) using response surface methodology and amicability in isotherms and thermodynamics modeling. Journal of Hazardous Materials 174: 623-634.
46. Siripornadulsil, S., S. Traina, D.P.S. Verma and R.T. Sayre. 2002. Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. Plant Cell 14: 2837-2847.
47. Smironoff, N. and Q.J. Cumbes. 1989. Hydroxyl radicals scavenging activity of compatible solutes. Phytochemistry 28: 1057-1060.
48. StatSoft. 2011. STATISTICA (data analysis software system), version 10. http://www.statsoft.com. Cited 18 July 2018.
49. Stiborova, M. 1988. Cd2+ ions copper the quaternary structure of ribulose-1,5-bisphosphate carboxylase from barley leaves. Biochemie und Physiologie Pflanzen 183: 371-378.
50. Sokolnik, A.Z., H. Asard, K.G. Koplinska and R.J. Gorecki. 2009. Cadmium and zinc- mediated oxidative burst in tobacco BY-2 cell suspension cultures. Acta Physiologiae Plantarum 31: 43-49.
51. Sun, X., Y. Zhong, Z. Huang and Y. Yang. 2014. Selenium accumulation in unicellular green alga Chlorella vulgaris and its effects on antioxidant enzymes and content of photosynthetic pigments. PLoS ONE 9: 1-8.
52. Terry, P.A. and W. Stone. 2002. Biosorption of cadmium and copper contaminated water by Scenedesmus abundans. Chemosphere 47: 249-255.
53. Tripathi, B.N. and J.P. Guar. 2004. Relationship between copper- and zinc-induced oxidative stress and proline accumulation in Scenedesmus sp. Planta Toronto 219: 397-404.
54. Tripathi, B.N., S.K. Mehta, A. Amar and J.P. Gaur. 2006. Oxidative stress in Scenedesmus sp. during short- and long-term exposure to Cu2+ and Zn2+. Chemosphere 62: 538- 544.
55. Ullah, A., S. Heng, M.F.H. Munis, S. Fahad and X. Yang. 2015. Phytoremediation of heavy metals assisted by plant growth promoting (PGP) bacteria: A review. Environmental and Experimental Botany 117: 28-40.
56. Veenstra, J.N., D. Sanders. and S. Ahn. 1999. Impact of chromium and copper on fixed film biological systems. Journal of Environmental Engineering 125: 522–531.
57. Vert, G., N. Gratz, F. Dedaldechamp, F. Gaymard, M.L. Guerino, J.F. Briat and C. Curie. 2002. IRT1, an Arabidopsis transporter essential for iron uptake from the soil and plant growth. Plant Cell 14: 1223-1233.
58. Wang, L., Y. Li, P. Chen, M. Min, Y. Chen, J. Zhu and R.R. Ruan. 2010. Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresource Technology 101: 2623-2628.
59. Wintermans, J.F.G.M. and A. DeMots. 1965. Spectrophotometric characteristics of chlorophyll a and b and their pheophytins in ethanol. Biochimica et Biophysica Acta 109: 448-453.
60. Woodrow, L. 2014. ANOVA, ANCOVA and MANOVA. In: Writing about Quantitative Research in Applied Linguistics, pp. 57-69. Palgrave Macmillan, London.
61. Wu, J.T., S.J. Chang and T.L. Chou. 1998a. Intracellular proline accumulation in some algae exposed to copper and cadmium. Botanical Bulletin- Academia Sinica Taipei 36: 89-93.
62. Wu, J.T., M.T. Hsieh. and L.C. Know. 1998b. Role of proline accumulation in response to toxic copper in Chlorella sp. (Chlorophyceae) cell. Journal of Phycology 34: 113- 117.
63. Zeraatkar, A.K., H. Ahmadzadeh, A.F. Talebi, N.R. Moheimani and M.P. McHenry. 2016. Potential use of algae for heavy metal bioremediation, a critical review. Journal of Environmental Management 122: 132-144.
64. Zhou, G.J., P.Q. Peng, L.J. Zhang and G.G. Ying. 2012. Biosorption of zinc and copper from aqueous solutions by two freshwater green microalgae Chlorella pyrenoidosa and Scenedesmus obliquus. Environmental Science and Pollution Research 19: 2918-2929.