Physiological and biochemical studies of copper toxicity and bio-adsorption in unicellular green algae, chlorella vulgaris

Copper toxicity to unicellular green algae Chlorella vulgaris has been investigated by studying its effect on various physiological processes. A concentration dependent reduction on growth rate, cell size, fluorescence activity, photosynthetic rate, nutrient utilization (NO3- & PO43-), protein synthesis and nitrate reductase activities was observed in different copper ion concentrations. A copper concentration of 6 ppm provoked about 50% growth reduction (EC50) which can be truly reflected by the induced lag period and retardation in the growth pattern and specific growth rate. Above the EC50 concentration, normal cell division, fluorescence activity and photosynthetic rate were seriously inhibited, these seems to the most important points of copper toxicity. Nutrient utilization was also affected by the copper ion, a 50% and 80% reduction in nitrate ion uptake was observed at 6 ppm and 8 ppm copper concentration, respectively. Internal metabolic processes such as nitrate reductase activity and protein synthesis were also under the influence of the copper ion. Being a very good copper ion absorbent. Chlorella cells were found to remove more than 35% of the copper ion from the culture medium followed by bio-accumulation inside the cells. This copper ion accumulation ability was also dependent on the external copper ion concentration. A higher copper concentration usually resulted in a large quantity of metal adsorption. The adsorption patterns were found to followed both the classical Langmuir and Freundlich adsorption isotherms. Scatchard equation transformation further proved that a combination of copper ion binding sites was presented. Under appropriate copper stress, the acid soluble thiols content in the algal cells was found to increase drastically, whereas the cellular glutathione and cysteine content decreased proportionally. This implied that phytochelatin like metal binding complex was actively bio-synthesized from glutathione and cysteine. On the other hand, the induction of metal binding complex resulted in the increase amount of acid soluble thiols. Chromatographic fractionation using gel filtration liquid chromatography, ion exchange fast protein filtration liquid chromatography, and reverse-phase high performance filtration liquid chromatography revealed the presence of the copper-binding complex with low molecular mass of about 2,300 daltons. The experiment on the inducibility of this complex further indicated that its binding affinity increased with the amount of metal presented