Research Of Cultural Conditions Of Transgenic Marine Chlorella Sp. MACC/C95 With Phytase Gene

Chlorella Sp.MACC/C95,a species of marine microalgae,is of substantial interest in aquaculture for its good nutritive characteristics.For providing valuable feed resources,a transgenic marine Chlorella sp.MACC/C95 with phytase gene had been conducted in our lab. In order to improve the dry cell biomass productivity and the intracellular phytase specific activity of the transgenic marine Chlorella sp.,a series of researches were carried out from the basic cultures of small bottles,high density cultures of the photobioreactors to the heterotrophic cultivations.The main results are summarized as following.1.The results of experiments showed that the optimal photoperiod of Light:Dack for the transgenic marine ChlorelIa sp.was 12h:12h.Under the optimal concentrations of Zn²⁺ 0.1μmol/L,Mo⁶⁺0.015μmol/L,Co²⁺ 0.01μmol/L,Cu²⁺ 0.015μmol/L and Mn²⁺ 0.8μmol/L,the growth of the transgenic marine Chlorella sp.arrived to its maxium and a high cell biomass productivity was obtained,and the highest activity of phytase in the cells of transgenic marine Chlorella sp.was obtained by the optimal concentrations of Zn²⁺ 0.1μmol/L,Mo⁶⁺ 0.0μmol/L, Co²⁺ 0.04μmol/L,Cu²⁺ 0.015μmol/L and Mn²⁺ 0.4μmol/L.Therefore,a high cell biomass productivity and high activity of phytase in the cells of transgenic marine Chlorella sp.could been obtained under the optimal concentrations of those micro-elements.2.The transgenic marine Chlorella sp.was cultured successfule in a 20L photobioreactor, under the conditions of temperature 20℃,light 3750 lx and areation rate 0.1 vvm,the cell biomass productivity and the activity of phytase in the cells of transgenic marine Chlorella sp. arrived 0.378 g/L and 1113.52 U/mg,respectively.By the fed-batch culture mode with constant and various feeding rate in the 20L photobioreactor,the activity of phytase in the cells of transgenic marine Chlorella sp.arrived 1066.78 U/mg and 881.07U/mg at day 6, which was 1.34 and 1.11 times of the control,and the cell biomass productivity and the activity of phytase in the cells of transgenic marine Chlorella sp.were 1.98,2.01 times and 1.64,1.91 times of that in 6L photobioreactor,respectively.The contents of ash and mineral element between the cells of transgenic marine Chlorella sp.and the non-transgenic Chlorella sp.also were compared.The results showed that the ash content was lower in the cells of transgenic marine Chlorella sp.,and which was about 3.5%of the cell dry weight.Without the contents of Ca and Na,the other mineral element contents in the cells of transgenic marine Chlorella sp.were lower than that of the control.3.When glucose and yeast powder were used as organic carbon and nitrogen resources, the level of the biomass productivity and the expression of the phytase of the transgenic marine Chlorella sp.could be increased significantly.Under the conditions of heterotrophic and mixotrophic,the cell biomass productivity and the activity of phytase in the cells of transgenic marine Chlorella sp.arrived 1.041-1.12 g/L and 1277.13-1306.78 U/mg,which were 5.24-5.63 and 1.98-2.02 times of that in the autotrophic control group,respectively. However,the phytase activity in the cells of transgenic marine Chlorella sp.was inhibited when glucoe as single organic carbon resource.When yeast powder was used as single organic nitrogen resource,the cell biomass productivity and the expression level of the phytase of transgenic marine Chlorella sp.could significantly be increased.And under the heterotrophic cultural conditions of illumination and darkness,the dry cell biomass productivity and the specific activity of phytase in the cells arrived 0.76 g/L and 0.57 g/L,1357.32 U/mg and 1338.31 U/mg,which were 3.81 and 2.85 times,2.10 and 2.07 times of that in control group of autotrophic cultivation,respectively. The content of protein,chlorophyll andβ-carotene in the cells could be increased by the cultural condition of illuming heterotrophic cultivation,but the expression of the phytase in the cells was inhibited.