The Fed-batch Of Chattonella Marina (Hongkong Strain) And Purification Of Hemolytic Toxins From The Alga

Hemolytic toxin is produced by ichthyotoxic algae, which is considered to beone of the major causes of fish mortality. Blooms of ichthyotoxic algae have broughthuge losses of fish farming. The poisoning mechanism and chemical structure of thehemolytic toxin are still unclear. In this paper, batch culture of Chattonella marina(Hongkong strain) was studied under room condition. Separation and purification ofhemolytic toxin from the alga cell were also studied.The results are as follows.(1) The maximum cell density in C. marina cell cycle was studied when the cellswere incubated in different incubation-culture medium volume ratio of1%,10%,15%,20%,25%and50%. Except the initial incubation volume of50%,cell incubated in incubation volume of1%to50%all get to the maximumcell density on the19thday of incubation. Considering various factors, indoorbatch culture C. marina cell choose ratio of15%to25%are optimal for thegowth of the cell can achieve better results.(2) In the time of adding medium experiment, adding algal culture media on day12th and14th， the maximum cell density of C. marina can reached1.11×104cells/mL and1.31×104cells/mL，respectively. Therefor, it is better to addmedia in the first14days.(3) When adding update rate of20%,30%,40%culture medium, cell density ofC. marina increased as the ratio increased. However, when the culturemedium update rate was50%, the cell density decreased. The culturemedium update rate of40%benefit the growth of C. marina with the highestcell density of1.17×104cells/mL. So adding update rate of40%culturemedium will be get higher cell density.(4) The levels of nitrate, ammonium, phosphate and silicate in the secondaryutilizated culture water were higher than fresh sea water except nitrite. Therewas no siginificant difference of maximum cell density in the late logarithmic phase between secondary utilizated culture water and fresh seawater (P>0.05). Howevey, hemolytic activity of C. marina varied. Thelargest hemolytic percentage of34.94%was found when adding80%secondary utilizated culture water into fresh sea water. Therefore we canknow the nutrient concentrations affect the hemolytic activity on algal cells.(5) When algae mud stored at4℃,-20℃,-80℃experimental conditions,stored at4℃algae mud hemolytic toxin activity started to decrease aboutat the first7days, which was extracted by blood hemolysate buffer.Additionally,-20℃and-80℃extraction hemolytic activity was almostunchanged. As to stored temperature, buffer extract stored at-80℃hashigher hemolytic activity than at-20℃. It is optimal for the alge mud to bestored in buffer at-80℃.(6) Five compounds were isolated and purified from C. marina. According to thedifferent physical and chemical constants and spectroscopic characterizationmethods, four compounds were identified as palmitic acid (Z-3), palmitoleicacid (Z-4), myristic acid (Z-6)3fatty acids classes and sitosterol (Z-1).(7) Comparing the four extraction methods, hemolytic activity from high to lowis buffer extaction, methanol extraction, water extraction and alcoholprecipitation, and finally dialysis extraction.