Optimization Of Culture Conditions For Astaxanthin Production From Haematococcus Pluvialis

Astaxanthin has a wide application prospect in the fields of food,medicine and feed because of its strong antioxidant biological function.Therefore,it is especially important to optimize the culture conditions to improve the astaxanthin yield of H.pluvialis.In this study,two strains of H.pluvialis（FACHB-721 and Flotow ny-144）were used as the research objects.The two-step culture method was used to optimize the culture conditions and improve the yield of astaxanthin.Firstly,the regulation methods to increase the green vegetative cell density of H.pluvialis were screened through seaweed extracts,growth hormone and organic carbon sources.Then,the optimal induction conditions and medium were used to obtain the highest astaxanthin yield.The specific results are as follows:（1）Compared with the control group,the biomass of green vegetative cells cultured with 300 times of Gracilaria extract in BG11 medium increased by 20%.However,the extracts of Amphora and Chlorella significantly reduced the density of green vegetative cells.（2）The associated bacteria were obtained from H.pluvialis 144 by plate method.The results showed that the addition of the associated bacteria in the medium could significantly reduce the cell mortality of both strains of H.pluvialis under salt and high light stress conditions.The cell mortality of H.pluvialis 721 and 144 reached20%and 33.3%after growing at 5 psu salinity for 7 days,while the cell mortality of H.pluvialis 721 and 144 was 0 when supplemented with 1%salt of associated bacteria solution(OD₅₀₀=0.5).After adding salt,Fv/Fm of cells decreased significantly at 2d and 4d,indicating that salt induced stress on the cells of H.pluvialis.However,the Fv/Fm of cells treated with bacteria had no significant difference compared with the control,which further indicated that the addition of bacteria could alleviate the salt stress of cells.（3）Astaxanthin induction conditions were optimized by orthogonal design with four factors（flora,nitrogen,salt and light）.The results showed that the optimal combination was adding bacteria and salt,without nitrogen under high light,and the maximum astaxanthin yield of H.pluvialis 721 and 144 reached 85.5mg/L and77.6mg/L after 24 days of induction.It is a combination of bacteria,nitrogen,salt5 and highlights.The effect of light intensity on H.pluvialis was extremely significant,while other conditions were not.Under low light,the yield of astaxanthin was significantly lower than that of other treatments.The interaction analysis showed that the addition of bacteria had an interaction effect with light intensity,and only under high light did the bacteria have a promoting effect on the accumulation of astaxanthin.（4）The content of MDA and the relative content of three key enzymes（ACCase、PSY、BKT）revealed that the associated bacteria extended the time of astaxanthin synthesis by reducing the stress degree of cells,but did not inhibit the accumulation of astaxanthin.Under the same induction condition,the astaxanthin content of single cells containing associated bacteria eventually reached the same level as that of normal cells.Based on the protective effect of associated bacteria,survival rate and astaxanthin accumulation were increased during cell induction.（5）Based on the results of orthogonal experiment,the induction conditions of the associated bacteria were further optimized to shorten the induction time.The results showed that the astaxanthin yield reached 110mg/L after 10 days of induction under the condition of gradually increasing illumination（eventually up to 20,000 Lux）and salinity（finally up to 5）,and the astaxanthin yield was 1.88times higher than that without the addition of bacteria.（6）The pipeline-type bioreactor was used to grow H.pluvialis 144 under the above optimal conditions.The results showed that the cell density of green vegetative cells could reach 8×10⁵/m L after 14 days of culture.In the induction stage,the associated bacteria was added,and the astaxanthin yield reached 18mg/L in 1d under the Hai Nan natural sunshine（40000Lux,salinity 5）for 4h,while the astaxanthin yield could reach 161.2mg/L in 10d under the optimized system（gradually increasing illumination and finally reaching 2000Lux and gradually increasing salinity and finally reaching 5）.（7）The associated bacteria were isolated and purified,and 3 strains were obtained（Q1,Q2,Q3）.Q3 could significantly increase the survival rate of 5psu after salinity induction and identification.The 16S rDNA identification showed that Lysinibacillus macroides was the main strain to improve the resistance of H.pluvialis.