| Astaxanthin is a high-value additive ketocarotenoid applied in feed, nutraceutical, food and cosmetics industries. Haematococcus pluvialis has the ability to accumulate it when was exposed to environmental or nutritional stresses. mutation breeding technique was applied to obtain astaxanthin-overproducing strain in the experiment.The initial strain of Haematococcus pluvialis was treated with UV, NTG, EMS and mutants were screened byβ-lonone and nicotine. According to the principle of lethal rate from 85% to 90%, the optimum doses were determined. Under the certain cell density(OD674=0.030～0.035), the optimum doses were UV 28min; NTG 3.67×10-3 moL/L, EMS 0.41moL/L;β-lonone 2.8×10-4 mol/L , nicotine 9.6×10-4 mol/L.Through single mutagenesis, four mutants were found for higher astaxanthin production and content. The astaxanthin production and content of mutants were enhanced, UV-N2 27.1%, 14.4%; UV-β5 29.4%, 35.2%; EMS-N5 11.9%, 7.8%; NTG-β6 15.9%, 20.3% . Compared with four mutants, UV was more effective, so UV-N2, UV-β5(by UV irradiation) were selected as the starting strains for combined mutagenesis by EMS and NTG treatment. Finally, an astaxanthin-overproducting mutant UN-β5-6 was obtained with a perfect transmissibility. The productivity of astaxanthin of UN-β5-6 amounted to 29.453 mg/L and 14.913 mg/gDCW, which were 17.9% and 20.8% higher compared with the parent, 49.2% and 60.4% higher than that of the initial strain, respectively.The specific growth rate and specific astaxanthin accumulation rate were calculated of the initial strain and the mutant(UN-β5-6) in two different growth stages. The results showed that in green stage , the specific growth rate of the mutant was much lower than that of the initial strain, but reversely in red stage. On the other hand, the specific astaxanthin accumulation rate of the mutant was far greater than that of the initial strain. Identification of the changes on the Chla/Chlb and absorption, fluorescence spectra of photosynthetic pigments extracted by 95% ethanol(EX=337nm), and fluorescence spectra of cell suspensions at room temperature(EX=490nm) showed that in green stage, Chla/Chlb of mutant was smaller than initial strain, but reversely in red stage. The result suggested the proportion of light harvesting chlorophyll a/b protein complexes (LHC) increased of the mutant in the green stage, but reversely in red stage. Namely, the composition of photosystem changed. Associated with the changes of spectra, the conclusion was made that photosystem II activity of mutant decreased, simultaneity, an increase of photosystem I activity. That is to say the gene modified as well as controlling external conditions which lead to inhibiting PSâ…¡activity or improving PSâ… activity both can promote the synthesis of astaxanthin.
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