Investigation On The Effect And Their Mechanisim Of Different Induction Condition On Astaxanthin Production In Haematococcus Pluvialis LUGU

Astaxanthin.has been applied widely in various commercial fields with their powerful natural antioxidant activity.Haematococcus pluvialis used as major source of natural astaxanthin has been received more and more attention recently.In this study,the optimal medium for the culture and induction of H.pluvialis LUGU was selected respectively.Then the effect of butyl hydroxyanisole(BHA)and fulvic acid(FA)on algal biomass and astaxanthin production were investigated individually and synergistically.Astaxanthin content was also measured to determine the appropriate time node for adding BHA to obtain a higher induction efficiency.What's more,seven astaxanthin biosynthetic genes,namely,isopentenyl diphosphate isomerase(ipi),phytoene synthase(psy),phytoene desaturase(pds),lycopene ?-cyclase gene(lyc),??carotenoid oxygenase gene(bktl),carotenoid ketolase genes(bkt2),and carotenoid hydroxylase gene(chy)were cloned and the expression patterns were detected by RT-PCR.Finally,the composition and content of fatty acid were evaluated,and the expression profiles of five fatty acid synthetic genes during astaxanthin acculation was analyzed.They offer insight into the molecular and physiological basis of astaxanthin accumulation as well as the relationship between astaxanthin and fatty acid synthesis.The concrete results are summarized as follows:(1)The optimum growth and induction medium for H.pluvialis LUGU was determined according to the biomass of algal cell and astaxanthin accumulation respectively.The maximum biomass concentration(1.38 g L-1)was obtained in the BBM culture medium,with 1.26,1.20 and 1.21,1.58 and 1.21 times than MAV,MCM,SE,KUh1 and Cz-M1,respectively.So BBM was determined as the basis medium for the culture of H.pluvialis LUGU.Then the maximum astaxanthin production was 28 mg g-1 under inducing conditions with SE medium,increased by 60.83%,66.07%than BG11 and BBM,respectively.(2)The biomass of H.pluvialis LUGU was 73.24%and 318%under 2 mg L-1 BHA and 5 mg L-1 FA addition,respectively,and the maximum production in astaxanthin was 2.03 and 1.62 higher than control.On the other hand,the maximum production 20.96 mg g-1 was obtained by the synergetic effect by 2 mg L-1 BHA and 5 mg L-1 FA.Although the production was 79.30%higher than control,lower than that of BHA treated group.So 2 mg L'1 BHA is the best inductor for astaxanthin.(3)The response of biomass and astaxanthin to the optimum BHA treatment was measured at different growth stages,including at the initial phase,the mid-exponential growth phase,the late-exponential growth phase,and the stationary phase.In general,the group in late-exponential growth phase obtained the highest biomass concentration of 0.75 g L-1 on day 9,which is approximately 103%,79%,and 25%of the concentration other treatments,respectively.Meanwhile,the maximum production 29.3 mg g-1 DW was obtained was gained in on day 13,which is only slightly higher than the 29.03 mg g-1 production in the late-exponential cells,but much higher than others(2.93-and 1.74-fold,respectively).So the cells under the late-exponential growth condition is more suitable for astaxanthin production.(4)The enzyme genes in astaxanthin synthesis were cloned successfully and the transcript levels of seven astaxanthin biosynthetic genes were monitored and compared by qRT-PCR.The results showed that the maximum expression level of the ipi,psy,pds,lcy,chy,bktl and bkt2 on day 3,5,7,7,3,3 and 3,with 8.98,10.06,3.89,9.78,10.75,39.99 and 33.25 times than control,respectively.However,the production of astaxanthin was increased greatly on day 5.Therefore,the transcriptional up-regulation of them provide rate-limiting steps for astaxanthin biosynthesis in Haematococcus pluvialis LUGU with BHA induction.(5)According to the various composition and content during induction period,the highest content of fatty acid was gained on day 13,when the maximum production of astaxanthin was obtained.However,the content of saturated fatty acids in control was 64.52%while there was no saturated fatty acids dedected in the experimental group on the day 5.But monounsaturated fatty acid was not detected in control and reached 49.68%in the experimental group on day 5.So the results show that the astaxanthin ester synthesis need monounsaturated fatty acids.