| Biodiesel production can’t meet commercial demand due to the imbalance of biomass and oil content. To address these issues, it is imperative to explore some effective strategies for improving the lipid productive of oleaginous alga. The objectives of this study were to determine the general patterns of biomass and oil content in C. protothecoides cells by heterotrophy-dilution-photoinduction cultivation,and to further unravel lipid synthesis and regulation mechanism in cells. The main results were as follows:(1)In the experimental process, the biochemical parameters in algal cells were determined in every stage. The results clearly showed that the higher cell density was easily accomplished at the end of heterotrophic phase, the biomass yield was 12.28 ±0.06 g/L. The contents of starch, chlorophyll and protein in cells were in a low level,at 1.18%, 6.92%, 31.20%. In addition, the content of lipid was merely 15.08% of cell dry weigh(CDW). The proportions of total unsaturated fatty acids(UFAs) accounted for 82.47%; The cultures were collected by centrifugation, diluted with scale 1:6 and transferred into photo-induction medium for continued growth with illumination.Under photo-dilution-induction condition, the starch, chlorophyll and protein content gradually increased from 31.20%, 1.18%, 31.20% to 42.25%, 3.80%, 35.17% at 12 h,especially, starch accumulation occurred evidently in photoinduced cells, which represented 6.11-fold higher than that of the end of heterotrophic stage. However, the biomass decreased from 12.28 g/L to 9.05 g/L and the lipid accumulation was negligible at the first 12 h photo-induction; The biomass concentration decreased gradually to 6.23±0.11 g/L at 48 h photo-induction. And the contents of starch,chlorophyll and protein also decreased gradually. In contrast, the lipid started to accumulate and became the predominant storage compound in algal cells. The lipid content was increased from 15.08% to 50.50% of CDW by 48 h photo-induction. The maximum lipid productivity of 623 mg/L/d was accomplished. The UFAs decreased to 52.44% after a second phase of photo-induction. It was worth noting that the predominant unsaturated fatty acid(28% of total fatty acids) was oleic acid in cells,which was suggested as an excellent feedstock for conversion into bio-fuel. In addition, the increment of saturated fatty acids(SFA) in photo-induced process was due to the rapid synthesis of palmitic acid. Therefore, the major fatty acids(more than90%) were C16 and C18 in this study.(2)The objectives of this study were to further unravel lipid synthesis and regulation in cells by employing proteomics. The results clearly showed that 38 protein spots were confidently identified, among these identified protein spots, 24 were down-regulated and 14 were up-regulated in algal cells. They were assigned into11 categories. Chlorophyll a/b-binding protein(CAB)related to photosynthesis were down-regulated while the content of chlorophyll decreased. This might be due to the longer light irradiation generated oxidative damage to photosynthesis system;Interestingly, eight down-regulated proteins were identified as the same ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit(RuBisCO), this indicated that RuBisCO was not the major contributor to supply intermediates for lipid accumulation under photo-induction condition, it might come from the decomposition of some energy substances(chlorophyll, protein and starch); Nine proteins related to ATPase showed down-regulation,in these proteins,3 proteins which were located in the chloroplast demonstrated that ATP synthesis was inhibited by photo-induction. Maybe this was one of the main factors which leaded to lower biomass and the decrease of some energy substances(chlorophyll, protein and starch);In this study, the up-regulated fructose-1,6-bisphosphate aldolase(FBA) and glucose-1-phosphate adenylyltransferase(SAT) were involved in carbohydrate metabolism process and drived the carbon flow into the downstream direction of glycolysis, it provides s important precursors(pyruvic acid and acetyl CoA) for the lipid synthesis; The two up-regulated proteins, comprising the succinate dehydrogenase(SQR) and malate dehydrogenases( MDH) participated in TCA cycle.The up-regulation of this enzyme indicated that TCA cycle was significantly enhanced and a large number of FADH2 molecules were generated under photoinduction; In this study, one of the key enzymes responsible for β-oxidation of fatty acids, namely acyl-Co A dehydrogenase was down-regulated after photo-induction. The down-regulation of this enzyme indicated the reducedβ-oxidation as well as increased lipid biosynthesis in algal cells under photo-induction condition; The down-regulations of 60 s ribosomal protein L5 led to the protein synthesis function of endoplasmic reticulum decline. And it finally resulted in the reduced cell growth and division so that the biomass was dramatically decreased; It was supposed that the up-regulation of hsp70 might play an important protective role against high light intensity stress. It played an importent role of maintaining themetabolic balance in cell and provided relative steady environment to the lipid biosynthesis. |
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