Key Technology Study Of Rapid Determination Of Chrysolaminarin And Production From Phaeodactylum Tricornutum By Photo-fermentation

Chrysolaminarin is a kind of?-1,3-glucan accumulated in the marine microalgae such as diatoms,chrysanthemums and xanthellae,and has multiple biological activities including anti-oxidation,anti-viral,anti-tumor,hypoglycemic and lipid-lowering and immune regulation.Additionally,previous studies have shown that algae-derived?-1,3-glucan has better biological activities than that of?-1,3-glucan from yeast and other sources.Phaeodactylum tricornutum is a special chrysolaminarin-producing marine microalgae with the advantages of fast cell growth,strong environmental adaptability under both autotrophic and mixotrophic culture conditions and high intracellular chrysolaminarin content between 12and 33 percent of dry weight.This study firstly established the rapid estimation method for detecting chrysolaminarin content in P.tricornutum and then systematically optimized the cultivating conditions of in P.tricornutum,and investigated the effects of different culture strategies such as fed-batch cultivation,light intensity supply and nutrient addition on the cell growth and chrysolaminarin accumulation of P.tricornutum,which finally developed the photo-fermentation technology for the high-efficient proudciton of chrysolaminarin in fed-batch cultures by P.tricornutum.The main research results are listed as follows:1.The extraction procedure of intracellular crude polysaccharides from P.tricornutum(the number of solvent extractions,the final concentration of TCA and whether alcohol precipitation treatment)was optimized systematically.The results showed that the optimum extraction conditions were as follows:4 times of extraction by TE buffer,final TCA concentration of 10%(m/v)and alcohol precipitation of polysaccharides for purification.Subsequently,the applicability,relevance and accuracy of the three methods,i.e.,phenol-sulfuric acid method,fluorescence detection method,and gel liquid chromatography method,for the detection of chrysolaminarin content from P.tricornutum were investigated and compared to highlight the feasibility of the established method for rapid estimation of intracellular chrysolaminarin in P.tricornutum.The aniline blue fluorescence detection method is determined to be the best detection method,the detection time is about 2 hours,and the detection limit is 4.12?g/m L.2.The effects of different culture conditions on the cell growth and chrysolaminarin accumulation of P.tricornutum in the shake flask systems were systematically investigated in the study.When sodium nitrate and glycerol(0.10 mol/L)were used as the nitrogen and carbon sources with the carbon to nitrogen ratio of 30 for the cultivation of P.tricornutum associated with the inoculation cell density of 1.0×10⁷cells/m L,the highest biomass yield,chrysolaminarin content and yield of P.tricornutum were 254.69 mg/L/d,17.22%DW and49.13 mg/L/d,respectively.Additionally,when tryptone and urea were used as mixed nitrogen sources instead of sodium nitrate and 0.10 mol/L glycerol was used as carbon source,the carbon to nitrogen ratio(C_Gly/N_T/U)of 15 and sodium silicate concentration of 15 mg/L(1/2 Si)were more suitable for P.tricornutum cultivation,and the highest biomass yield,chrysolaminarin content and yield of P.tricornutum were 282.38 mg/L/d,16.35%DW and45.33 mg/L/d,respectively.Cultivating P.tricornutum with mixed nitrogen sources can obtain higher biomass yield,while using sodium nitrate as nitrogen source can obtain higher yields of chrysolaminarin.3.In the 5 L external photo-fermentation system,the no-fed culture of P.tricornutum under certain culture conditions(tryptone and urea are used as mixed nitrogen sources(the initial carbon to nitrogen ratio(C_Gly/N_T/U)is 15 and the light intensity is 100?160?mol/m²/s)was investigated firstly.The highest biomass yield,chrysolaminarin content and yield of P.tricornutum were 315.00 mg/L/d and 13.37%DW and 50.22 mg/L/d,respectively,which were 12.77%,29.05%and 44.60%higher than the sodium nitrate group(initial C_Gly/N_{Na NO3}was 30)(p<0.05).Comparatively,in the fed-batch cultures(192 h supplemental carbon source or nitrogen source,so that the carbon-nitrogen ratio of the medium is 30),although the mixed nitrogen source did not significantly favored algal cell growth(355.56 mg/L/d)compared with that of the sodium nitrate group,the chrysolaminarin content and yield were achieved at19.19%DW and 143.77 mg/L/d,respectively,which were 89.81%and 130.40%higher than the sodium nitrate group(p<0.01).The results demonstrated that the fed-batch culture was more favorable for the accumulation of chrysolaminarin in algal cells compared with the no-fed culture mode.4.In the 5 L external photo-fermentation system,the strategy of step increase of light intensity(100?260?mol/m²/s)was utilized for algae cultivation.The results demonstrated that the largest biomass yield,chrysolaminarin content and yield were 437.50 mg/L/d,27.02%DW and 218.25 mg/L/d,respectively,which were 22.09%,26.44%and 43.95%higher than that of the two-stage dimming(100?160?mol/m²/s)strategy(p<0.01).It.Furthermore,the addition of glycerol integrated with the step increase of light intensity from 100?mol/m²/s to350?mol/m²/s was utilized to investigate their effects on the cell growth and chrysolaminarin accumulation of P.tricornutu.The results showed that the culture with two times of glycerol addition achieved the highest biomass concentration of 570.83 mg/L/d at the end of the culture without significant difference from that of the culture with eight times of glycerol additions.However,the highest content and yield of chrysolaminarin were 28.50%DW and233.26 mg/L/d,respectively,which were 12.83%and 36.99%higher than that the experimental culture with 8 times of glycerin addition(p<0.05).5.The molecular weight determination and structure identification of chrysolaminarin extracted and purified from P.tricornutum biomass were constructed in the study.The GPC analysis showed that the average molecular weight of chrysolaminarin in P.tricornutum was5.80 KDa.In the FT-IR spectrum,these was a characteristic absorption peak of?-glycosidic bond at 888.90 cm^-1,which indicated that the purified chrysolaminarin fits into the catogrory of?-type polysaccharide.Additionally,the absorption peak at 1000?1200 cm^-1demonstarted that the structure of?-glucans mainly consists of?-(1?3)linked bluconic backbone.Similarly,¹³C-NMR results showed that the chrysolaminarin in P.tricornutum belongs to the?-1,3-glucan polysaccharide family.