Influences Of Cultural Conditions On The Growth And Accumulation Of Ceramide In Emiliania Huxleyi

Ceramide,as an intracellular signal molecule,plays an important role in regulating cell differentiation,proliferation,apoptosis and aging.As a novel biologically active substance,the unique physiological function and drug effect of ceramide makes it be a kind of high added value and has huge market potential of active material with a wide range of applications in pharmaceutical health care products,food,cosmetic and chemical engineering.There is very small amount of ceramide in cells（0.01%-0.2%biomass dry weight）,and they are mainly derived from plants including rice,wheat,corn and konjac.Thus the low yield is the bottleneck for the development of ceramide functional products.Compared with terrestrial plant,microalge have many advantages for ceramide production,such as easily cultivation,short life cycle and high lipid content.Emiliania huxleyi is a marine eukaryotic microalgae.It can produce abundant secondary metabolism active substances.Recently,an unusual ceramide from E.huxleyi was isolated and analyzed,implicating a novel structure with highly bioactive.However,low biomass concentration and ceramide production in E.huxleyi restricts the application of ceramide.Therefore,it is the key to enhance the algal biomass and the level of ceramide synthesis.Culturing microalgae in mixtrophic or heterotrophic conditions could potentially yield higher biomass concentration and algal products of high value.Up to now,E.huxleyi has not been reported to possess the ability to grow mixotrophically and heterotrophically,and how the relationship between biomass and yield of ceramide under different culture conditions remains unknown.In this paper,E.huxleyi BOF92 was used as the material,characteristics of photoautotrophy,mixotrophy and heterotrophy were studied.Meanwhile parameters of cell growth,biochemical components and cell structure were examined.The effects of culture conditions on the growth and ceramide content of E.huxleyi were also investigated.The main results are as follows:1.Effects of sources and concentration of organic nutrient on the growth of E.huxleyi were investigated.The results showed that E.huxleyi could grow under heterotrophic condition with short time light activation,but the growth rate was low.It could not be cultured under chemoheterotrophic and photoheterotrophic conditions.It could grow mixotrophically and the utilization of organic nutrient sources was selective.The maximum cell density of E.huxleyi was1.43 folds of that obtained with glycine and 1.6 times of that with serine.2.The contents of biochemical components under different trophic modes were different significantly.Compared with photoautotrophic condition,the content of soluble protein obviously increased under mixotrophic conditions,whereas the content of soluble carbohydrate decreased and the content of total lipid maintained 15.97 pg·cell^-1and16.42 pg·cell^-11 respctively.Under mixtrophic condition,the content of Chla and Chlc were 1.43 folds and 2.58 folds higher than those obtained under phototrophic respectively,whereas the content of carotenoid reduced dramatically.3.ELSD-HPLC method was developed to measure the concentration of ceramide in E.huxleyi.There was no great difference both under photoautotrophic and mixotrophic culture condition.Along with the culture,the content both of total lipid and ceramide increased significantly and reached to the maximum until stable stage.The most cell density was obtained at 6.29×10⁶cells·mL^-11 under mixotrophic condition and the production of ceramide can reach to 393.5μg·L^-1,which was 1.38 folds higher than that of the photoautotrophic condition.4.Nile red staining method detecting the relative content of lipid of E.huxleyi was established.The optimal measurement conditions were:excitation wavelength 491 nm,emission wavelength555 nm,dyeing concentration 1.6μg·mL^-1,the cell concentration（1.6-1.9）×10⁴ cells·mL^-1,DMSO concentration 45%,pretreatment temperature 37.5℃.5.Based on the f/2 medium,single-factor experiments and response surface methodology were applied for the optimization of growth,the content of lipid and ceramide in E.huxleyi.The results showed that both of the biomass and ceramide yield were increased obviously under the recombinant medium of 0.011 mM Fe³⁺+3 mM NaHCO₃+5 mM Serine.The optimized culture conditions were:inoculation cell density 4.5×10⁵ cells·mL^-1,light intensity 20μmol·m^-2·s^-11 and batch culture.The cultures could be harvested after inoculation 4-5 days,with ceramide production of 0.454 mg·L^-11 and was 1.59 times in the control.