| Astaxanthin, an excellent antioxidant, has various important biological functions including UV light protection, immune response, anti-inflammatory, anti-cancer and pigmentation, therefore it shows applications in cosmetic, pharmaceutical, nutraceutical and aquaculture industries. However, astaxanthin cannot be synthesized by animals and humans themselves. Instead, Haematococcus pluvialis (H. pluvialis), a type of freshwater green alga, has become one of the potent sources for natural astaxanthin production. Yet the industrial large-scale production of H. pluvialis is still a challenging task because of the intrinsic shortcomings of natural H. pluvialis such as slow growth rate, susceptibility to contamination and low biomass concentration. In this thesis work, the atmospheric pressure argon dielectric barrier discharge (DBD) which is an effective tool to obtain desired mutants was employed to induce mutant algal strains with enhanced production of astaxanthin. The mutagenesis effects were studied for the purpose of improving the efficiency of DBD based biotechnology for mutation breeding. In order to acquire higher astaxanthin productivity from H. pluvialis, it is critical not only to develop efficient mutagenesis techniques, but also to establish rapid and effective screening methods which are highly demanded in current research and application practice.The main contents and results of this work are summarized in the following:1. Study on the DBD optimal mutagenesis conditions for acquiring H. pluvialis with improved production of astaxanthin. In this work, the optimal conditions for the DBD-caused mutation were first determined based on the lethality measurement and then various algal mutants were acquired by DBD treatment.2. Study on the methods for rapid screening astaxanthin-hyperproducing H. pluvialis mutants through microspectroscopy. We develop the new approach to screening the astaxanthin-hyperproducing strains based on FTIR and Raman microspectroscopy. In this way we have achieved rapid and quantitative analysis of the algal cells in terms of astaxanthin, P-carotene, proteins, lipids and carbohydrates on the micro-scaled level. In particular, we have found that the ratio of the infrared absorption band at 1740 cm-1 to the band at 1156 cm1 can be utilized for identifying astaxanthin-hyperproducing strains at their early stage of growth. Especially, with MCR treatmenof spectroscopy data, the high chemical contrast in single cells can be obtained via Raman imaging, which can identify the individual pure components including astaxanthin, P-carotene, and chlorophyll. As for the analysis of bulk samples, the method of PCA shows the good ability for distinguishing different algal strains. Meanwhile, the near-infrared spectroscopy (NIRS) in combination with biochemical assay was also employed for evaluation of the H. pluvialis mutants. The partial least squares (PLS) models of total biomass, astaxanthin content and astaxanthin expressed as a percentage of dry weight (DW) were developed. Furthermore, the PLS models were employed to evaluate H. pluvialis mutants.3. Study on the characteristics of astaxanthin-hyperproducing mutants of H. pluvialis. For analysis of the DBD-induced algal mutants, genetic variation was checked by random amplification of polymorphic DNA (RAPD), and the pigment pattern in response to inhibitors (Diphenylamine and Nicotine) of carotenoid biosynthetic pathway of H. pluvialis was characterized by high-pressure liquid chromatography. Furthermore, photosynthetic activity and gene expression level were also examined by pulse amplitude modulated fluorometry (PAMF) and quantitative real-time PCR, respectively. Our results confirmed that the enhanced astaxanthin accumulation in the mutants was associated with the increase expression levels of lycopene cyclase (LCY) gene which was involved in the conversion of lycopene to β-carotene.4.Study on the biological effect of H. pluvialis induced by DBD exposure. In this study, the H. pluvialis cells upon DBD exposure were detected by biochemical assay. And it demastrate that the elevated level of ROS in algal cells plays the key role in the caused biological effect. |
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