| The decreasing petroleum storage and environmental pollution associated with fossil fuelconsumption are two substantial issues that need good solutions for sustainable development ofhuman society. Microalgae are considered to be a potential renewable resource to producebiodiesel, and mass culture of oleaginous microalgae is an important alternative for solving theenergy problem. The objective of improving lipid content in microalgae requires a more detailedunderstanding of the culture condition, lipid synthesis, and mechanism of lipid metabolism. Nowa two-stage culture mode, with first a nutrient-sufficient biomass production phase followed by alipid induction phase under nutrient deprivation, is widely used to improve the lipid synthesis ofalgae. Nitrogen and phosphorus in the medium are main elements in the growth and lipidaccumulation of microalgae. Nutritional sufficiency such as appropriate nitrogen and phosphateis conductive to the algal growth, but the lipid accumulation of algae is mainly induced by thedeprivation of nitrogen and phosphorus. In our experiment, the effect of nitrogen and phosphoruson the growth and photosynthetic reaction, the lipid synthesis in nitrogen-deprived Isochrysisgalbana, and the proteomic variation in the process of lipid accumulation were primarilyanalyzed, and the mechanism of lipid metabolism was discussed preliminarily so as to providethe initial values and datas. The main progress are as follows:1. The growth and photosynthetic reaction of I. galbana in different nitrogen concentration wereanalyze. The results show that, the growth of I. galbana was restricted under the condition ofnitrogen-deprivation and low nitrogen (0.44mM), which was mainly reflected by the obviousreduction of cell density and chlorophyll content, dramatic decline of photosynthetic efficiency.With the rising nitrogen concentration, the growth rate of algal cells also increased gradually. At1.76mM nitrogen concentration, the growth rate of I. galbana was the highest, the cell densityand chlorophyll content had a substantial increase, and the photosynthetic efficiency also kept inhigher level. At3.52mM nitrogen concentration, the growth of I. galbana was restrained, whichwas manifested as the dramatic decline of cell density, chlorophyll content and photosyntheticefficiency. From this, nitrogen concentration has an obvious effect on the growth and photosynthetic reaction, and too low or high nitrogen concentration go against the algal growth.2. Based on the experiment of nitrogen concentration, the growth and photosynthetic reaction inphosphorus-deprived I. galbana were analyzed. The results show that, in low nitrogen (0.44mM)and normal nitrogen (0.88mM) concentration, the phosphorus-added (0.05mM) andphosphorus-deprived group in I. galbana had no obvious difference in the cell density,chlorophyll content and photosynthetic efficiency. At1.76mM nitrogen concentration, the celldensity, chlorophyll content and photosynthetic efficiency of phosphorus-deprived I. galbanawere far lower than the phosphorus-added group. At3.52mM nitrogen concentration, the celldensity and photosynthetic efficiency of phosphorus-deprived I. galbana were obviously lowerthan the phosphorus-added group, but the chlorophyll content had no dramatic differencebetween the two group. From this, phosphorus deprivation restricted the growth andphotosynthetic reaction of I. galbana, but nitrogen deficiency can cover the effect of phosphorusdeprivation on the algae. With the increasing nitrogen concentration, phosphorus deprivation hada more marked restriction on the algal growth.3. By analyzing the lipid content of I. galbana under the condition of normal nitrogenconcentration (0.88mM) and nitrogen deprivation, it was found that the protein synthesisdecreased dramatically, the growth rate and photosynthetic efficiency also declined obviously, atthe same time the content of lipid and fatty acid obviously increased in nitrogen-deprived I.galbana, of which saturated fatty acid content was obviously higher than the group of normalnitrogen. This indicates that nitrogen deprivation can restrict the lipid accumulation of I. galbana,but induce the lipid accumulation of algal cells, especially the triacylglycerol (TAG).4. The differentially co-expressed proteins in nitrogen-deprived and nitrogen-added (0.88mM) I.galbana were comparatively analyzed by using two dimensional electrophoresis (2-DE) andmatrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS).2-DE maps showed that the most significant proteomic variations happened on the2ndday, andmore proteins were expressed in N-deprived I. galbana. Forty-five protein spots showeddramatic changes in nitrogen-deprived I. galbana compared with the N-added group. Thirty-sixof the differentially expressed protein spots were successfully identified, including32up-regulated protein spots (representing23proteins) and4down-regulated protein spots(representing4proteins). The identified proteins were divided by their molecular functions intoseven categories as follows: energy production and transformation (13spots), substancemetabolism (12spots), signal transduction (1spot), molecular chaperone (2spots), transcriptionand translation (3spots), immune defense (2spots) and cytoskeleton (3spots). These alteredproteins slowed cell growth and photosynthesis of I. galbana directly or indirectly, but at the same time increased lipid accumulation. Eight key enzymes involved in lipid metabolism viadifferent pathways were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH),phosphoglycerate kinase (PGK), enolase, aspartate aminotransferase (AST), fumarate hydratase(FH), citrate synthase (CS), O-acetyl-serine lyase (OAS-L) and ATP sulfurylase (ATPS). Theresults suggested that the glycolytic pathway and citrate transport system were the main routesfor lipid anabolism in N-deprived I. galbana, and that the tricarboxylic acid (TCA) cycle,glyoxylate cycle and sulfur assimilation system were the major pathways involved in lipidcatabolism. |
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