Improving Astaxanthin Production By Using The Synergistic Effects Between The Metabolic Activities Of Haematococcus Pluvialis And Phaffia Rhodozyma

In the present studies, the metabolic fluxes within the cells of H. pluvialis and P.rhodozyma have been analyzed using the method of metabolic engineering.The resultsshows: P. rhodozyma assimilates glucose via Embeden-Meyerhof-Parnas(EMP)pathway, in which, the reaction from pyruvate to acetyl-CoA is inefficient and about20.2%(the stable stage) to 48.6%(the growth stage) pyruvate is excreted;In contrast,H. pluvialis catabolizes glucose along Pentose Phosphate(PP) pathway, and pyruvateis one of the substrates for the synthesis of isopentenyl-pyrophosphate(IPP), aprecursor of astaxanthin. 1% pyruvate fulx increases, 0.8% IPP rises.Therefore, thereexists a potential complementary effect between the two species on metabolicactivities.Based on the metabolic fulx analysis(MFA), H. pluvialis and P. rhodozyma weremix-cultivated to make use of the synergistic effects between them. Pyruvate and CO₂produced by P. rhodozyma and O₂ generated by H. pluvialis were utilized each otheras substrates by the two microorganisms. Consequently, the carbon fluxes towardastaxanthin synthesis within the cells of H. pluvialis and P. rhodozyma were 4 and 2.2times higher than those of the pure cultures, which promoted cell growth andastaxanthin formation. As a result, biomass and astaxanthin concentrations raisedremarkably.A series of single-factor and multi-factor orthogonal experiments were carriedout to optimize the culture conditions, indicating that optimal medium compositionand controlling parameters for cell growth and astaxanthin synthesis were different.From a point view of higher astaxanthin production, the optimal culture conditions areas follow: best medium composition: Glucose 3g/l, NaNO₃ 250mg/l, Phosphate125mg/l, CaCl₂·2H₂O 10mg/l, MgSO4·7H2O 30mg/l, FeSO₄·7H₂O 5mg/l;pH: 6,temperature: 28℃,aeration(rotation speed): 110rpm, light intensity: 120μmolphotons/m2·s。For further increased level of astaxanthin production, a two-step (stage) culturewas then carried out: in the first stage, the mixed culture was conducted on thewell-defined medium under optimal conditions to achieve a higher cell concentration;in the second stage, the culture was controlled in a way to stimulate astaxanthinsynthesis by adding glucose, sodium chloride as well as elevating the light intensity.As a result, astaxanthin production increased evidently and the maximum astaxanthinyield of 62.1mg/l was achieved. Therefore, two-step mixed culture technique providesa new technical platform for commercial production of natural astaxanthin.