| Natural astaxanthin, a kind of red keto-carotenoid and super antioxidant, has great economic value and broad market prospects. The unicellular green alga Haematococcus pluvialis can accumulate large amounts of astaxanthin under various environmental stress conditions, such as high light, high salinity, and deprivation of nutrients, either singly or in combination. Cultivation of Haematococcus pluvialis has been the commercial way to produce astaxanthin. A two-stage cultivation of Haematococcus pluvialis to produce astaxanthin has been widely adopted. The productivity of conventional autotrophic cultivation is very low and the annual output is in short supply. The growth condition for these two stages are totally different, in the first stage the green algae are in the form of vegetative flagella cells and are sensitive to the environmental condition. The cultivation conditions are so rigorous that the growth is limited in this stage. Controlling of the light-intensity and nutrients in the culture is fine work. In the second stage the algae are in the form of cyst and start to accumulate astaxanthin under the stress conditions like high light and nutrients depletion. In order to explore the efficient cultivation of this algae, previous study often focus on the optimization of photo-bioreactor and the culture conditions for these two stages.In this study a new mold named heterotrophy-dilution-photoinduction cultivation was introduced, firstly this algae were cultivated in the heterotrophic mold because in this condition the cells were kept in the vegetative form for a longer time and cell multiplications was enhanced. So the productivity of cell biomass was increased accordingly. As mentioned, the conditions for astaxanhin accumulation and cell division were different, so after the heterotrophic cultivation the cells were transferred to the photo-induction cultivation for astaxanthin accumulation, it had been found that the results fluctuated with the states of the heterotrophically cultivated cells. So after heterotrophic cultivation an acclimation process was adopted. In this course, the photosynthetic apparatus in the algal cells were rebuilt and the photosynthetic protein were activated. Meanwhile in the second phase, the mechanism for astaxanthin accumulation by Haematococcus pluvialis under the conditions like high light, nitrogen depletion is not very clear, because in the conventional photo-autotrophic cultivation, the cells were not in significantly different, while in the newly studied cultivation mold, the cells undergo the different cultivation conditions and subjected to acute changes, so it’s more suitable for the study of mechanism. Additionally, in heterotrophic cultivation, there were different forms of cells which were appropriate for the mechanism study, during the whole heterotrophic cultivation stages the largest individual cell dry weight was more than 3 ng. This research aims to solve the problems existing in conventional phototrophic cultivation mold and study the mechanism in transforming of the algal cells, finally built a new mold for Haematococcus pluvialis growth and astaxanthin accumulation.(1) Study of the conditions for heterotrophic cultivation of this algae. Firstly three kinds of culture mold were compared, the mixtrophic cultivation had the highest cell growth rate in the optimized conditions. The applicable acetate content was 1.5 g L-1. After that, the heterotrophic cultivation was conducted in the 5L fermenter and the time course was studied. It had been found that the carbon source inhibition effect induced the cells to form cyst. During this process the cell dry weight increased dramatically and the composition of the algae cells changed a lot. The content of protein decreased from 45.67±2.65% to 14.16±0.51%. And the carbohydrate increased from 12.45±0.72% to 45.44±1.64%, lipid content increased from 17.35±1.00% to 30.50±1.10%.(2) Optimization of the conditions for photo-induction of this algae.20 mM sodium acetate and 2 mM nitrogen were selected for the induction and enhancing the cells survival rate. pH was maintained by HEPES (40 mM), Tricine or Tris(HCl) buffer at 7～9. Light played an important role in the induction of this algae. Light intensity of 400 μmol photons m-2 s-1 was suitable for the induction and light quality study showed that the white light was the best light source for induction and the blue light was appropriate for astaxanthin accumulation. Continues light enhanced the astaxanthin accumulation, the productivity in the treatment of 12h:12h light cycle was lower by 18.9%. Artificial light had the similar effect on the photo-induction which was illustrated in the indoor or outdoor experiments. Light intensity and initial cell density affected the photo induction together. Light interception received by individual cell determined the results of the photo-induction. The aeration rate of 0.1～0.6 vvm had no influence on the pH and the culture results so 0.2 vvm was finally applied.(3) Strategy of photo-acclimation. The cells with different states in the heterotrophic phase was obtained and the photo-acclimation mechanism was studied. The study was conducted through detecting the change of intracellular protein involved in photosynthesis, etc., it can be seen that the content of photosynthesis relating D1 protein increased to restore and regenerate, indicating the repair of photosynthetic system in the cells under stress conditions. While photosynthesis protein PsbO and PetC activity declined gradually showing the defense mechanism of algal cells under the adverse conditions and promoting the production of astaxanthin against external stress. With the study of different cells and in order to promote the photosynthetic system repair, the photo-induction conditions were optimized by changing the initial cultivation conditions, such as setting lower light intensity and providing with carbon and nitrogen source, these can recover the algal photosynthesis system significantly, and increase the production rate of the astaxanthin.100 μmol photons m-2 s-1 was suitable for the photo-acclimation and then the Fv/Fm which indicating the potential of photosynthesis increased from 0.6 to 0.8 after one day acclimation.(4) Scaling up of different types of photo-bioreactors for the photo-induction process. In this study three kinds of photo bioreactors (raceway pond, flat plate bioreactor and bubbling column bioreactor) had been tested and the study showed that the bubbling column photo-bioreactors were suitable and applicable for the photo-induction progress among different types of reactors.1L bubbling column was scaled to 70L.15L flat plate bioreactor was scaled to 300L which was optimized to a bioreactor with built-in light source. And the performance of this new bioreactor almost eliminated the amplification effect. The scaling up in raceway pond was also successful.(5) Modelling of photo-induction. Different climate condition affected the photo-induction, the new strategy was applied in Lhasa and Kunming city, compared to the results in Jiaxing city, the productivity was 2 times higher and astaxanthin content was exceed 4%. After that the model between the results and total light amounts and average temperature during the photo-induction was built. The astaxanthin content and total light amounts fit with the Logistic equation. And the theoretic content of astaxanthin and average temperature fit with Gauss equation. When the average temperature attained 22.7℃, the volumetric content got the highest value.In conclusion, a new paradigm for the astaxanthin production from Haematococcus pluvialis has been developed. Cells in different states had been photo-induced successfully and the mechanism of cells from heterotrophic cultivation subjected to photo-induction under stress conditions was studied. An induction system had been established for the heterotrophic grown cells and the key factors were studied, the optimization of photo-induction process were conducted and the industrialization of scalable photo-bioreactors were discussed. |
PDF Full Text Request