Lipid Composition And The Compound Specific Isotopic Ratio Of Fatty Acids In Phytoplankton

In a series of incubation experiments, mircoalgal lipid compositions and the compound carbon isotopic ratios of fatty acids in membrane and intracellular components have been investigated following three experimental stages: 12:12 h light:dark (L:D) cycle light incubation; 0:24 h L:D dark incubation; oxic/anoxic dark degradation. Through the investigation, the lipid production, growth and degradation during the life of microalgal cells have been studied. Also via this study, the fatty acid distribution and its compound carbon isotopic ratios in membrane and intracellular components of microalgae have been explored. The conclusions are demonstrated as followed:1 The lipid compositions of microalgae at different growth stageThe major fatty acids of Platymonas subcordiforus contain16:4,16:0,18:2,18:1,18:0 and 20:5, while those of Isochrysis 3012, Gymnodinium sp., Skeletonema costatum and Heterosigma akashiwo Hada include 14:0,16:1,16:0,18:4,18:2,18:1,20:5(Gymnodinium sp. does not include 20:5 but 22:6) at different growth stage. Comparing to those of logarithmic-growth stage, the proportions of polyunsaturated fatty acids have been increased by more than 10 % in the stationary phase, while the contents of saturated fatty acids have decreased by more than 20%. However, there is no significant variation for the proportions of most microalgal monounsaturated fatty acids at different growth stage. Moreover, the proportion of 18:4 in the stationary phase is higher than that of logarithmic-growth stage, while the content of 16:0 has decreased about 8-26%.In addition, the sterol species of microalgae are distinct in logarithmic-growth and stationary phase. In the logarithmic-growth stage, the major detected sterols of Platymonas subcordiforus in logarithmic-growth phase are including 27â–³⁵ (1.4 %),28â–³⁵,24(4.5 %),28â–³⁵(7.0 %),29â–³^5,22(1.2 %),29â–³⁵, Dimethyl (3.9 %) and 29â–³⁵,24(1.3 %); Isochrysis 3012 includes 27â–³⁵ (2.8 %),28â–³⁵ (2.1 %),29â–³^5,22 (2.4 %),29â–³⁵, Dimethyl (24.3 %); Heterosigma akashiwo Hada involves 27â–³⁵ (3.3 %),28â–³⁵ (2.2 %),29â–³⁵, Dimethyl (24.3 %) and 29â–³⁵,24 (3.1 %); the dominating sterols in Skeletonema costatum contain 27â–³⁵ (11.2 %),28â–³⁵ (19.1 %) and 29â–³⁵, Dimethyl (10.4 % ). However, in the stationary phase, there are only 27â–³⁵ (1.4 %),28â–³⁵ (36.9 %) and 29â–³⁵, Ethyl(2.2 %) in Platymonas subcordiforus, 28â–³⁵(3.6 %),29â–³^5,22(14.3 %),29â–³⁵,24(11.8 %),30â–³22(8.9 %) and 31â–³⁵(35.1 %) in Isochrysis 3012, 28â–³⁵ (3.4 %) and 29â–³⁵, Ethyl (55.7 %) in Heterosigma akashiwo Hada, 27â–³⁵ (21.2 %),28â–³⁵ (28.9 %),29â–³⁵, Ethyl (11.9 %) and 29â–³⁵,24 (11.8 %) in Skeletonema costatum. In addition, the variation of alcohols between different growth stage is similar to that of sterols.2 The production, growth and degradation of microalgal lipids during the cell life During the light and dark incubation, the concentrations of microalgal lipids markedly change with time. In the light culturing experiments of Isochrysis 3012, the concentrations of 16:1, 16:0 and 20:5 increase with time, but with a dramatic accretion in stationary phase (8.3, 2.7 and 2.7μg/ml cell, respectively), while that of 18:0 quickly increases in logarithmic-growth phase (from 0.2μg/ml cell to 0.9μg/ml), but with no obvious increment in stationary phase. And for Gymnodinium sp., 16:0, 18:4, 18:0 and 22:6 steadily increase their concentrations with time, but 18:0 stops gaining from the ninth day and keeps at about 0.8μg/ml cell, while others build up continually but slowly after the twelfth day, and at the end of the light incubation, their concentrations are respectively 3.3, 3.0 and 3.0μg/ml cell. In addition, the phytol shows a quick accumulation in logarithmic-growth phase (0.4 and 0.3μg/ml cell, respectively in Isochrysis 3012 and Gymnodinium sp.) while most of the sterols are built up in stationary phase.While in the dark incubation, the concentrations of most fatty acids rapidly decrease with time, except 18:0 which has little change with time. The 16:1, 16:0 and 20:5 in Isochrysis 3012 respectively reduce their concentrations from 10.4, 5.5, and 5.6μg/ml cell to 0.3, 1.0 and 0.1μg/ml cell while those of 16:0, 18:4 and 22:6 in Gymnodinium sp. change from 3.3, 3.0 and 2.2μg/ml cell to 0.7, 0.1 and 0.0μg/ml cell. Besides, the major sterol 24-Methylcholesta -5,22-Dien-3β-ol in Gymnodinium sp. is significantly degraded from the second day in dark incubation, and at the end of dark experiment its concentration is only 0.1μg/ml cell. Interestingly, the 24-Ethylcholest- 5, 22-Dien-3β-οl and 23, 24- Diethylcholesta- 5, 22- Dien- 3β-ol in Isochrysis 3012 have exhibited an increasing trend with time. Their concentrations respectively increase from 0.8 and 1.7μg/ml cell to 1.3 and 3.5μg/ml cell in the 2- 10 days.3 The degradation of microalgal lipids under different redox conditionsMicroalgal lipids have showed different degradation profiles under oxic/anoxic conditions. The decomposition of 16:0 and 18:0 are similar under the oxic and anoxic conditions, while 20:5 and all the studied sterols degrade a little faster under anoxic than oxic conditions. In addition, the 16:0 and 20:5 in Isochrysis 3012 have higher degradation rate than 16:0 and 18:0 under both oxic and anoxic conditions.4 the fatty acid composition and its compound carbon isotopic ratio in membrane and intracellular components of microalgaeThe membrane and intracellular fatty acids in cells of Isochrysis 3012 and Gymnodinium sp. are separated by silica gel column. Variations of membrane vs intracellular lipids during the whole culturing time indicate their different functions during cell growth and metabolism. During light incubation, membrane fatty acids are more abundant in early logarithmic-growth stage (100.0 % and 86.7 %, respectively), while intracellular lipids are more plentiful in late stationary stage (56.6 % and 29.3 %, respectively). Compound-specific fatty acids in intracellular and membrane components have also showed distinct distribution at different growth stage. In logarithmic-growth phase, 20:5 (28.9 %) is the most abundant fatty acid in membrane fatty acids of Isochrysis 3012, while 18:4 (23.9 %) is the most plentiful one in Gymnodinium sp. However, in stationary phase, 22:6 becomes the dominating fatty acid in membrane fatty acids of Isochrysis 3012, but the 18:4 (14.6%) in membrane fatty acids of Gymnodinium sp. is still the primary one. In addition, a decrease inδ13C of membrane and intracellular fatty acids has been observed from logarithmic to stationary phase: a depletion of 2-5‰inδ13C has occurred for the membrane fatty acids in Isochrysis 3012 expect 20:5,while theδ13C ratios for 16:0 in membrane lipids and 14:0 for intracellular lipids have depleted about 10‰. However, theδ13C of the same fatty acids in membrane and intracellular components of microalgae are similar.During dark incubation, the proportions of intracellular fatty acids have clearly decreased: the contents of 14:0, 16:0 and 20:5 in Isochrysis 3012 have reduced about 7 % while those of 20:5 and 22:6 in Gymnodinium sp have respectively dropped by 4.6 % and 8.6 %. At the same time, there is a positive shift in theδ13C of most membrane and intracellular fatty acids. In detail, theδ13C ratios of membrane fatty acids in Isochrysis 3012 have enriched about 0.5-2.3‰, while those of intracellular 16:1 and 16:0 respectively increased 1.3‰and 3.9‰; Although there is small enrichment for theδ13C ratios of membrane fatty acids in Gymnodinium sp., a significant increase of 5.6‰in theδ13C ratio of intracellular 16:1 has been observed.The fatty acid compositions in membrane and intracellular components vary in oxic and anoxic conditions. All the detected fatty acids are membrane fatty acids in Isochrysis 3012 under oxic conditions while the proportions of membrane and intracellular fatty acids are respectively 63.8 % and 36.2 % in Gymnodinium sp. Under anoxic conditions, the contents of membrane and intracellular fatty acids in Isochrysis 3012 are respectively 87.6 % and 12.4 %, of which saturated fatty acids are most plentiful in membrane fatty acids while 16:0 and 18:0 are the only intracellular fatty acids. And for Gymnodinium sp., the membrane and intracellular fatty acid proportions are respectively 3.9 % and 96.1 %. 14:0, 16:0 and 18:0 are the most abundant fatty acids in membrane components while 18:0 is the sole fatty acid in intracellular fatty acids.