Preliminary Study On The Breeding Of High-lipid Content Marine Golden Algae

The six strains of marine golden algae are obtained from the Microalgae Culture Center(MACC) in Ocean University of China are cultured with f/2medium under specific environment inwhich temperature is25°C, the salinity is31, with continuous aeration and the light intensity is100μmol·m-2s-1. The two excellent strains of microalgae are Isochrysis galbana H24andOchrosphaero neopolitana H32that have rapid growth, higher photochemical efficiency, strongvitality and the high total lipid content are selected from six strains. The total lipid content of I.galbana H24and O. neopolitana are19.54%and19.88%respectively. The effects of nutritionconcentration, nitrogen source, sodium nitrate concentration, sodium tungstate, palnt growthregulator on the chlorophyll fluorescence parameters (maximal photochemical efficiency of PSII,F_v/Fm; potential activity of PSII, F_v/F_o; actual photochemical efficiency of PSII, ΦPSII; relativeelectron transport rate, rETR; photochemical quenching, qP), cell density, chlorophyll content, totallipid content and fatty acid compositions of I. galbana H24and O. neopolitana are examined inbatch cultures by biochemical analysis and chlorophyll fluorescence analysis.The results indicate that different nutrition concentration has significant effects on thechlorophyll fluorescence parameters, growth, total lipid content and fatty acid compositions of I.galbana H24and O. neopolitana (P<0.05). The final cell density, chlorophyll content, dry weight,total lipid content and lipid productivity under2f medium are significantly higher than those inother treatments, which suggest that under this experimental condition, the most appropriatemedium for growth and lipid accumulation of the two strains of golden algae is2f. When thenutrition concentration is4f, all the contents of14:0,16:1(n-7),18:1(n-9),22:6(n-3) andmonounsaturated fatty acid(MUFA) in the two strains of golden algae are the highest, while thecontents of20:5(n-3)and polyunsaturated fatty acid(PUFA) in I. galbana H24and the contents of20:5(n-3)and saturated fatty acid(SFA) in O. neopolitana are the lowest. When the nutritionconcentration is2f, the content of SFA in I. galbana H24and the contents of16:0and PUFA are thelowest.Microalgae can absorb and utilize the nitrogen source, for example, nitrate, ammonium andurea et al, but there are some differences in the utilization degree and absorption rate. The resultsshow that nitrogen source has significant effects on the chlorophyll fluorescence parameters, growth, total lipid content and fatty acid compositions of I. galbana H24and O. neopolitana (P<0.05).When nitrogen source is ammonium nitrate or urea, the total lipid content of I. galbana H24reachesthe maximum value, but there is no significant difference between them. The highest total lipidcontent of O. neopolitana is found in urea treatment. SFA, MUFA and PUFA contents of the twostrains of golden algae have the same variation pattern with nitrogen source. The maximum value of16:0,16:1(n-7) and SFA are found in ammonium bicarbonate treatment. Sodium nitrate or urea asnitrogen source,18:1(n-9) and MUFA contents are the highest. When nitrogen source areammonium chloride and ammonium nitrate, PUFA content is the highest.The results indicate that sodium nitrate concentration has significant effects on the chlorophyllfluorescence parameters, growth, total lipid content and fatty acid compositions of I. galbana H24and O. neopolitana(P<0.05). With sodium nitrate concentration increasing, the chlorophyll contentand total lipid content show a trend that first increasing then decreasing. When the sodium nitrateconcentration is3.52mM, the chlorophyll content and total lipid content of I. galbana H24reach themaximum value. When the sodium nitrate concentration is3.52mM and7.04mM, the chlorophyllcontent and total lipid content of O. neopolitana are significantly higher than those in othertreatments, but there are no significant differences between them. As the sodium nitrateconcentration is0mM, the content of16:0and the total content of SFA and MUFA in the twostrains of marine golden algae are the maximum value, while the contents of20:5(n-3) and PUFAare the minimum value.Sodium tungstate can inhibit the activity of nitrate reductase and limit the absorption ofnitrogen. The results indicate that sodium tungstate concentration has significant effects on thechlorophyll fluorescence parameters, growth, total lipid content and fatty acid compositions of I.galbana H24and O. neopolitana(P<0.05). With sodium tungstate concentration increasing, the totallipid content shows a change trend which is firstly increasing then decreasing. When the sodiumtungstate concentration is5mM, the total lipid content of I. galbana H24and O. neopolitana are themaximum value. As the sodium tungstate concentration is5mM in I. galbana H24, the contents of16:1(n-7), SFA and MUFA are highest, while the contents of22:6(n-3) and PUFA are lowest. Thecontents of14:0and18:1(n-9) are the maximum value in the sodium tungstate concentration of8mM. When the sodium tungstate concentration is5mM, the contents of16:0,16:1(n-7) and SFA arethe highest in H32, while the contents of14:0and22:6(n-3) are the lowest. As the sodium tungstate concentration is8mM in O. neopolitana, the contents of MUFA and18:1(n-9) are the highest, whilethe contents of16:1(n-7),20:5(n-3) and PUFA are the lowest. With adding sodium tungstate, thecontent of saturated fatty acid increases, while unsaturated fatty acid content decreases in the twostrains of golden algae.IAA and2，4-D belong to plant growth regulator. They can promote growth of unicellularalgae, shorten the growth cycle and increase biomass. The results show that IAA and2,4-Dconcentration have significant effects on the chlorophyll fluorescence parameters, growth, totallipid content and fatty acid compositions of I. galbana H24and O. neopolitana(P<0.05). For I.galbana H24, the most significant promotion to the growth is observed when IAA is0.1mg/L, and0.25mg/L for O. neopolitana. When the total lipid content of I. galbana H24and O. neopolitanaare the maximum value, the IAA concentrations are0.1mg/L and0.5mg/L respectively. As the IAAconcentration is1mg/L, the contents of16:0,16:1(n-7), the total SFA and MUFA are the highest in I.galbana H24, while14:0,22:6(n-3) and PUFA contents are the lowest. The contents of14:0andSFA are the maximum value in the IAA concentration of0.5mg/L in I. galbana H24. The content ofSFA is the maximum value in the IAA concentration of0.1mg/L in O. neopolitana. Meanwhile,when the IAA concentration is0mg/L, the contents of18:1(n-9) and MUFA are the highest andPUFA content is the lowest.For I. galbana H24, the most significant promotion to the growth is observed when2，4-D is0.25mg/L, and0.5mg/L for O. neopolitana. When the total lipid content of I. galbana H24and O.neopolitana are the maximum value, the2，4-D concentrations are0.1mg/L (but there is nosignificant difference with0.25mg/L treatment) and0.25mg/L respectively. In I. galbana H24,when2，4-D concentration is0.1mg/L, SFA content is the highest and the contents of18:1(n-9),20:5(n-3) and MUFA are the lowest; As the2，4-D concentration is0.5mg/L, all the contents of16:1(n-7),18:1(n-9),20:5(n-3) and MUFA are the maximum value and PUFA content is theminimum value. For O. neopolitana H32, when2，4-D concentration is0.25mg/L, the total contentsof SFA and MUFA are the highest, while22:6(n-3) and PUFA contents are the lowest. In this study,IAA and2，4-D have no significant effect on promoting the synthesis of fatty acids in I. galbanaH24and O. neopolitana. High concentration IAA and2，4-D have significant effect on inhibitingthe growth of the two strains of golden algae.The conclusion of this study suggests the technical research line which can increase the biomass and photochemical efficiency and improve the microalgae total lipid content is that afterselecting the best nutrition concentration and nitrogen source, add the appropriate concentration ofIAA or2，4-D in the initial stage of growth and culture to the exponential growth stage (about7-9days). After a certain biomass, add sodium tungstate for nitrogen limiting process, then microalgaeoils can be accumulated largely. This method can save time and effort, lower the cost, is a fast andeasy way in providing technique and data reference in large-scale cultivating microalgae andextracting useful materials.