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Abstract Development of novel technologies for the remediation of industrial and hazardous wastes is increasing rapidly. Particular attention is being focused upon the usage of biological treatment systems. Aerobic and anaerobic microbial treatment processes have been successfully employed in the destruction and/or removal of organic compounds, inorganics and metals. Cyanide is a known toxic chemical produced through anthropogenic activities and industries that use ore leaching, electroplating, steal production, plastics, and synthetic fibers. In this study, the cyanobacterial cyanase enzyme was targeted to cytosol and overexpressed in Chlamydomonas reinhardtii via Agrobacterium tumefaciens mediated coculivation transformation method in order to establish a synthetic pathway for cyanide degradation. Transgenic C. reinhardtii algae containing the necessary genes for the novel pathway were generated. Variable amounts of RNA were detected by qRT-PCR. Simultaneous over-expression of cyanase would ultimately result in the formation of CO2 and NH3 from cyanide. Summary 133 Biochemical and physiological analyses were performed under cyanide stress conditions using C. reinhardtii transgenic for evaluating the impact of the novel pathway in algae: A) CYN-overexpressor and wild type C. reinhardtii were allowed to grow under different concentrations of cyanide (25, 50, 100,150 and 200 mg/L). CYN transgenic strain showed the specific activity of cyanase enzyme, which was obviously more than their corresponding activities in wild species. The study demonstrated that the ability of Cr.CYN genetically modified strains to grow at high concentrations of cyanide compared to the unmodified strain, especially at 150 and 200 mg/L. B) The study showed that cyanide had an inhibitory effect on mean growth rate, relative growth rate, generation time and number of recycling of both wild and transgenic samples as compared to the control. C) To evaluate the percentage of removal of cyanide in both microalgae, transgenic type and wild type was treated with different concentration of cyanide (25, 50, 100, 150 and 200 mg/L), the modified Summary 134 Cr.CYN strains showed high cyanide removal efficiency compared to the unmodified strain. At the lowest concentration of cyanide 25 mg/L, the percentage of cyanide removal was reached 100% after 24 hours for the genetically modified alga, compared to the wild type reached 100% after 48 hours. The non-modified alga was unable to grow under high concentration of cyanide 200 mg/L. D) The study showed the ability of genetically modified alga to produce high level of ammonia compared to wild type. E) In addition to the decrease in the pigment content of the wild type was highly observed as compared with the genetically modified organism. The percentage of reduction in chlorophyll a was reached to 37.3% in the transgenic alga compared to 75.82% in the non-transgenic alga. Also, the percentage of reduction in chlorophyll b was reached 35.75% in the transgenic alga compared to 81.36% in the wild type. F) The study showed increases in cyanase activity as concentration of cyanide increase especially, in case of transgenic type. The maximum activity was indicated in presence of 100 mg/l cyanide it Summary 135 reached eight folds more than wild type activity at the same cyanide concentration. G) Also, some factors are taken in consideration like different pH, contact time. Moreover, percentage of cyanide removal by transgenic type two folds more than wild type at awide range of pH. Results of this study provide an effective eco-friendly phycoremediation system for cyanide detoxification using transgenic micro algae. |