| ?-carotene is an important antioxidant and natural pigment.It is widely used as nutraceuticals,food dyes,feed additives and other products.?-carotene is mainly composed of 9-cis and all-trans isomers,and the higher ratio of 9-cis/all-trans,the higher antioxidant and anti-cancer activity.Compared with chemical synthesized products(only containing all-trans structure),natural ?-carotene(containing 9-cis/all-trans structure)products is more favourable.Dunaliella saliva can accumulate large amounts of ?-carotene,among which high light intensity is the most important stress to promote ?-carotene accumulation.Therefore,a precise light control model is in urgent demand toguide the large-scale ?-carotene production process,but it is in lack by far.At the same time,an in-depth understanding of the changes in the metabolic pathways of ?-carotene under stressed conditions,and the relationship with changes in the photosynthetic system can provide important theoretical basis for enhancing the accumulation of ?-carotene in D.salina.To achieve this goal,this thesis takes D.salina as the research object,and an online monitoring platform of microalgae,Algal station,was used to accurately determine the effects of light cycle,illumination mode,light intensity,flash frequency,as well as carbon source and other elements in culture medium,on D.salina cell growth,?-carotene accumulation and photosynthetic activity.The obtained results are as below:1.Optimizing the growth and ?-carotene accumulation medium of D.salina.Using bicarbonate as carbon source,the concentration of Ca2+,Mg2+ and trace elements is optimized,it was found that the ?-carotene content can be enhanced by changing the content of Fe3+ and Co2+ in the medium.Under the optimized conditions,the maximum biomass concentration was 0.71 g·L-1,with corresponding ?-carotene content of 4.76%,and ?-carotene yield could reach up to 32.0 mg·L-1.At the same time,a real-time online detection platform was used to monitor the changes of growth,?-carotene accumulation and the maximum chemical quantum yield of photosystem ?(Fv/Fm)respectively under the conditions of two carbon sources(NaHCO3 and CO2)in D.salina.The results show that using NaHCO3 as a carbon source not only could reduce the cost of ?-carotene production from D.saliva,but also could increase the production of ?-carotene significantly,and it was found that the value of Fv/Fm in NaHCO3 culture was lower than that in CO2 culture,which proved that high concentrations of NaHCO3 can be used as an important stress factor for ?-carotene accumulation.2.The influence of light mode and photoperiod on the growth and ?-carotene accumulation in D.salina was studied.The light control platform in Algal Station,an online monitoring system for microalgae,was used to simulate outdoor sunlight exposure mode("sine wave")and traditional laboratory light mode "Block wave",and "continuous light" were conducted for comparative experiments,and the changes of chlorophyll fluorescence parameters under different light modes were monitored real-time online,and the effects of different light modes on the growth,?-carotene accumulation and photosynthetic characteristics in D.salina were investigated.The results showed that the maximum ?-carotene yield(67.8 mg L-1)was obtained under the "sine" mode compared with other lighting modes in D.salina.Also,the biomass loss at night was lowest(4.15%)under "sine" mode,and continuous monitoring of Fv/Fm reveals that there was a strong correlation between the periodic fluctuation pattern of D.salina Fv/Fm and ?-carotene accumulation under different illumination modes,thus indicating that such illuminating strategy can promote the content of ?-carotene more than continuous strong light illumination.Moreover,at a strong flash frequency of 50Hz,the photosynthesis efficiency of D.salina cells increased by increasing the electron transfer rate(rETR)and reducing the heat dissipation capacity(NPQ)of the photosynthetic system,thereby accumulating a higher ?carotene(4.82%)content.3.The influence of the light intensity in the PBRs microenvironment on the growth and ?carotene accumulation in D.salina was studied.The light attenuation model of D.salina in a flat-plate reactor was established,and the light intensity during the ?-carotene induction process was controlled through the Algal Station system precisely.The extinction coefficient and scattering coefficient of D.salina cells at different growth stages were calculated through the light attenuation model,and the relationship between them and the average light intensity was determined.In addition,by controlling the average light intensity and induction time,the correlation between the average number of photons received per cell(APRPC)in the microenvironment and ?-carotene accumulation was analyzed.The results indicated that the Cornet attenuation model can accurately simulate the light attenuation of D.salina at different growth stages.It was found that once APRPC reached 0.7?mol photons cell-1,?-carotene accumulation was triggered,and became saturated at 9.9?mol photons cell-1(7.24%),indicating that the precise control of ?-carotene accumulation can be achieved by controlling the microenvironment light intensity of the cells in the reactor precisely.Finally,a dynamic model of D.salina cell growth and ?-carotene accumulation based on the influence of multiple factors such as micro-environment light intensity was established,and the the model was verified under given culture conditions,showing accurate prediction.4.Further exploration of the relationship between D.salina ?-carotene and photosynthetic system,as well as the synthesis mechanism of ?-carotene under stress conditions was conducted.Cell growth,Fv/Fm,heat dissipation capacity(NPQ),intracellular reactive oxygen species(ROS)content,H2O2 expression,and ?-carotene content in cells were detected under different stress conditions(salt stress,nitrogen stress,and high light intensity).The results showed that the accumulation of ?-carotene under the above-mentioned stress conditions were all caused by the cells being subjected to oxidative stress.By adding different concentrations of exogenous H2O2,the effect of H2O2 on ?-carotene accumulation was further verified.By studying the effect of ROS on the ?-carotene accumulation in D.salina at the transcription level,the molecular pathway of ?-carotene accumulation in D.salina was further verified.The results showed that Fv/Fm was negatively correlated with ?-carotene accumulation(r=-0.924),and heat dissipation capacity(NPQ)was positively correlated with ?-carotene accumulation(r=0.895)under different stress conditions,the addition of exogenous reactive oxygen species can increase the accumulation of ?-carotene in cells significantly.Transcriptomics data showed that by adding exogenous H2O2,phytoene synthase(PSY),dehydrogenase(PDS)and ?-carotene dehydrogenase(ZDS)as rate-limiting enzymes in the ?-carotene accumulation pathway were up-regulated due to slowed down starch synthesis and accelerated fatty acids degradation,thus leading to excessive accumulation of ?-carotene.This study analyzed the influence of light on the accumulation of ?-carotene in D.salina accurately,and provided a theoretical basis for establishing a controllable culture system to achieve efficient and stable accumulation of ?-carotene in D.salina.The technical means used in this study are expected to be applied in practical process of ?-carotene production in D.salina,thus promoting its industrial development.In addition,these technical means and research methods also provided a model for accurately studying the process of various carotenoids accumulation in other microalgae species. |
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