| 其他摘要 | Astaxanthin has being widely used as food, feed and drugs, due to its superior performance as coloration feed additives and antioxidant activities. Microalga Haematococcus pluvialis (Chlorohyceae) is by far the most safe, efficient source of astaxanthin, therefore to culture this microalga for astaxanthin production has a very high economic and social benefits. Cultivation of H. pluvialis for astaxanthin production has a long history; however, the technology in industrial scale still remains at the level of 20 years ago. China is an emerging market for astaxanthin of H. pluvialis and the industrialization process stands in a stage of rapid development, but the production technologies lag behind foreign manufacturers. Therefore, production technologies of H. pluvialis, of a high level and with independent intellectual property rights, will be required.
Previously, we observed special characteristics in the morphology, biochemistry and cell transformation of H. pluvialis red zoospores. Based on this observation, we recognize the red zoospores may play an important role in its life cycle, and an in-depth study about the development process of red zoospores may help to build a novel cultivation mode. In this study, the effects of environmental factors related to encystment and germination, i.e. nitrate concentration, light intensity and initial cell density, on the germination rate and synchronization level of H. pluvialis hematocysts were investigated firstly, which may help to build an artificial control method for synchronous germination of hematocysts and synchronous encystment of red zoospores. Then, we try to clarify the metabolic patterns of cellular fat, protein, carbohydrate and pigments during the formation process of red zoospores, via a multiple observations of microscopic examination and physiological and biochemical study. Cultures, which contained synchronous red zoospores, were prepared through the aforesaid cell cycle synchronization method, and latterly were studied to evaluate tolerance to adverse environment, via analysis of photosynthesis, survival rate and growth under high light and/or adverse temperature. Finally, a novel mode for growing H. pluvialis, namely regeneration cultivation mode, was proposed, and the applications of this mode in outdoor and indoor conditions were evaluated in different scales. The achieved main results were as follows:
Firstly, an artificial control method for synchronous germination of hematocysts and synchronous encystment of red zoospores was developed. A 90% of hematocysts germination rate at 48 h and a 100% of red zoospores encystment rate were achieved at optimum conditions. The aforementioned efficiencies could be achieved in continues several generations. The results also suggested that absorbing a certain amount of nitrogen was required to induce hematocysts germination, which could be adjusted by light intensity. The transformation between germination of hematocysts and encystment of red zoospores was controlled by interaction of light intensity and nitrogen consumption. The results also suggested that the germination rate was negatively associated with the maturation degree of hematocysts, which could be simply reflected in ratio of total carotenoids to total chlorophyll (Car / Chl).
Secondly, both carbohydrate and fat were equally important as main carbon reserves of H. pluvialis hematocysts. Mobilization of carbohydrate firstly appeared to meet energy requirement for formation of sporangium, and then the mobilization of fat appeared subsequently to satisfy energy requirement for releasing red zoospores and to provide precursors for synthesizing proteins and structure lipids. The mobilization of fat could be delayed, but cannot be replaced, by photosynthesis. The results suggested the photosynthesis could be enhanced with increasing of chlorophyll and lutein, which was a product of degradation of astaxanthin, to provide fatty acid as energy for germination. Both sun-shading effect of astaxanthin and xanthophyll cycle play important functions to protect sporangium and red zoospores during germination of H. pluvialis hematocysts.
Thirdly, a novel regeneration cultivation mode to grow H. pluvialis with hematocysts as inoculums was proposed, in which the reproduction of red zoospores by hematocysts germination and the encystment of daughter cells sequentially took place in a same cultivation condition. The newly formed hematocysts retain ability to germination, and thus could be used as inoculums for next batch of cultivation. Moreover, conditions of high light and nitrogen limitation were critical to success of this cultivation mode.
Fourthly, red zoospores were more resistant than green zoospores to high light, which could be owe to sun-shading and ROS scavenging effect of cellular astaxanthin, and ROS consumption via active synthesis of astaxanthin, as well as the ability to transform into palmella, which had even higher resistant. However, a further study was required to clarify the distribution among different protection strategies, including photochemical quenching, non-photochemical quenching and scavenging enzyme system. Our results also suggested the H. pluvialis could be resistant to long period of frozen stress, but can not resistant to high temperature stress, especially for its sporangium and red zoospores.
Lastly, H. pluvialis biomass with high content of astaxanthin was achieved through this proposed regeneration cultivation mode in different scales of outdoor and indoor cultivations. The best biomass productivity and astaxanthin productivity from indoor cultivation were 0.498 g/L/d and 24.1 mg/L/d, respectively. The best biomass productivity and astaxanthin productivity from indoor cultivation were 0.327 g/L/d and 11.5 mg/L/d, respectively. The process for re-encystment was controlled by specific nitrogen input (SNI), which was adjusted by interaction of nitrate concentration, light intensity and cell density. Moreover, there is a positive correlation between the astaxanthin productivity and interaction of light intensity and cell density. Also, the results suggested the light energy efficiency is at least 50% less for outdoor condition than indoor condition. |
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