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Fatty acid productivity of Scenedesmus obliquus under nitrogen starvation in mixotrophic cultivation exceeds the combination of autotrophic and heterotrophic cultivations Xiao-Fei SHEN (Ph.D. Candidate) Department of Chemistry University of Science and Technology of China [email protected] Content 1. Background 2. Materials and Methods 3. Results and discussion 4. Conclusions Content 1. Background 2. Materials and Methods 3. Results and discussion 4. Conclusions 1.Background-Comercial production obstacle Background Nutrient, light, water, carbon source Select species Cultivation Harvest Oil extraction and transesterification Biodiesel Lipid productivity Biomass productivity Lipid content University of Science and Technology of China Nitrogen starvation Lipid content Biomass productivity Lipid productivity decrease Role of phosphorus The effect of P concentration on biomass and lipid productivities of microalgae under nitrogen starvation conditions University of Science and Technology of China -1 -1 FAME productivity (mg L day ) Autotrophic cultivation of S. obliquus 28 N &P N &P-lim N &P- 24 N-&P N-&P-lim N-&P- 20 FAME productivity: 16 N starvation conditions > N sufficient 12 conditions; 8 N-&P > N-&P-lim > N-&P- 4 0 4th 8th 12th Time (days) 4th 16th Bioresource Technology . 2014, 152: 241–246 Heterotrophic cultivation of S. obliquus with acetate 55.9mg/L/d Both the highest FAME productivity and FAME yield were also obtained under N-&P. FAME productivity under nitrogen starved conditions increased fourfold than that under nitrogen sufficient conditions. Applied Energy. 2015, 158: 348–354 Whether oil production from mixotrophic culture can exceed the sum of those from autotrophic and heterotrophic culture? Contradictory conclusions from previous studies Influence factors? Algae species and carbon source Nitrogen starvation can significantly increase the lipid content and the lipid productivity of microalgae in both autotrophic and heterotrophic systems Mixotrophic cultivation The cell concentration and lipid production of Scenedesmus obliquus The lipid production of Chlorella vulgaris under mixotrophic Chlorella sp. was even higher Acetate mixotrophic cultivation was lower than under than the sum of those from photoautotrophic Nitrogen starvation heterotrophic cultivation due to the unsatisfactory and heterotrophic culture. lipid content (13.8%) of mixotrophic culture Content 1. Background 2. Materials and Methods 3. Results and discussion 4. Conclusions Materials and Methods Algae species: Axenic Scenedesmus obliquus NIES-2280 Basic medium: BG-11 medium 5 g·L-1 sodium acetate was added as organic carbon source Nitrogen source was removed from the media Phosphorus concentration: 40 mg·L-1 (Sufficient) Proteomics analysis: the isobaric tags for the relative and absolute quantitation technique (iTRAQ) Calculation of biomass productivity and fatty acid productivity Biomass productivity was calculated based on: 1 −1 Biomass productivity 𝑚𝑔 · 𝐿 2 ·𝑑 −1 = 𝐵𝑖𝑜𝑚𝑎𝑠𝑠 𝑚𝑔 ·𝐿−1 𝑡1 −𝐵𝑖𝑜𝑚𝑎𝑠𝑠 𝑚𝑔 ·𝐿−1 𝑡0 𝑐𝑢𝑙𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑑 1 FA (fatty acid) productivity was calculated based on: 2 FA productivity 𝑚𝑔 · 𝐿−1 · 𝑑−1 𝐵𝑖𝑜𝑚𝑎𝑠𝑠 𝑚𝑔 · 𝐿−1 × 𝐹𝐴 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑚𝑔 · 𝑔−1 𝑡1 − 𝐵𝑖𝑜𝑚𝑎𝑠𝑠 𝑚𝑔 · 𝐿−1 × 𝐹𝐴 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑚𝑔 · 𝑔−1 = 𝑐𝑢𝑙𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑑 3 𝑡0 Calculation of fatty acid yield 1 The fatty acid yield was calculated based on COD coefficient (the amount of O2 2 needed to oxide the COD, O2 g/ COD g). COD coefficient of acetate and fatty acids 3 (CxHyOz) could be calculated by Eq. (1): 4 5 6 𝐶𝑂𝐷 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝐶𝑥 𝐻𝑦 𝑂𝑧 = 2𝑥+𝑦 2−𝑧 ×16 12𝑥+𝑦+16𝑧 (1) Then the fatty acid yield could be obtained by Eq. (2): 𝐶𝑂𝐷 𝑓𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑 ×𝑀𝑎𝑠𝑠 𝑓𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑 𝑔 𝑎𝑐𝑒𝑡𝑎𝑡𝑒 ×𝑀𝑎𝑠𝑠 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 𝑎𝑐𝑒𝑡𝑎𝑡𝑒 𝑔 Fatty acid yield = 𝐶𝑂𝐷 (2) Content 1. Background 2. Materials and Methods 3. Results and discussion 4. Conclusions During the whole cultivation period, the biomass productivities of the mixotrophic culture exceeded the combination of the autotrophic and heterotrophic cultures. These two absorption curves are very similar, and no significant difference was found between the assimilation rates from the heterotrophic and mixotrophic cultures 1. At the end of the experiment, the fatty acid contents of the autotrophic, heterotrophic, and mixotrophic cultures were 19.7, 46.7, and 58.3%, respectively. 2. The highest fatty acid content was obtained in the mixotrophic culture. 3. The fatty acid content of all the three groups rose steadily and significantly. C18:1 was the most predominant composition, accounting for 52.8% and 66% of the total fatty acids in the mixotrophic and heterotrophic cultures, respectively. The highest unsaturated fatty acid content was obtained in mixotrophic culture The protein contents of all the three systems decreased significantly during the cultivation period The lowest protein content was obtained in the mixotrophic culture; it declined sharply from 55.6% to 9.1% during the nine-day cultivation period. No significant difference was observed between the results for the heterotrophic and mixotrophic cultures Fatty acid productivity (mg/L/d) Fatty acid yield Autotrophic 14.7±2.1 ----- Mixotrophic 118.4±6.4 0.45±0.04* Heterotrophic 57.5±5.3 0.23±0.02 Note: the fatty acid produced through autotrophic process has been deducted. More assimilated acetate is directed to lipid synthesis rather than protein and starch accumulation with the presence of light and supply of CO2. Auto- Hetero- Mixo- Proteomics analysis 1,065 proteins were identified Isobaric tags for the relative and absolute quantitation technique (iTRAQ) Compare the expression levels of proteins from mixotrophic and heterotrophic cultures Identify key proteins of lipid synthesis 299 proteins had significant changes in the expression level Some proteins participated in growth and lipid synthesis processes Accession Biological process Protein name Peptides (95%) Fold change CV Expression A8IWA6 Growth Glutamate synthase, NADH-dependent 15 1.71 0.10 Up-regulated A8HNQ7 Lipid metabolism Thioredoxin reductase 1 2.42 0.37 Up-regulated A8IRQ1 Lipid metabolism Ribose-5-phosphate isomerase 7 1.77 0.01 Up-regulated A8JGJ6 Lipid metabolism Mg protoporphyrin IX S-adenosyl methionine O-methyl transferase 2 1.77 0.04 Up-regulated A8J2S0 Small molecular metabolism Citrate synthase 3 2.74 0.10 Up-regulated A8J0R7 Generation of precursor metabolites and energy Isocitrate dehydrogenase 1 2.60 0.14 Up-regulated A0A0D2K714 Carbohydrate metabolism Pyruvate kinase 15 2.17 0.09 Up-regulated B6E5W6 Carbohydrate metabolism Glucose-6-phosphate isomerase 9 1.71 0.10 Up-regulated A0A0D2NR30 TCA cycle The activity of the TCA cycle is improved in mixotrophic culture, Catabolic process Glycerol-3-phosphate dehydrogenase resulting in more fatty acid synthesis in2S. obliquus2.52cells. 0.23 Up-regulated A8I8Z4 Lipid metabolism Ribosomal protein 2 0.59 0.10 Down-regulated A8IKQ0 Lipid metabolism Fructose-1,6-bisphosphatase 4 0.52 0.22 Down-regulated D8TTF7 Lipid metabolism Plastid acyl-ACP desaturase 2 0.15 0.22 Down-regulated A0A0D2JX51 Small molecular metabolism Malate dehydrogenase 16 0.65 0.19 Down-regulated Q4U1D9 Biosynthetic process Soluble starch synthase III 3 0.58 0.10 Down-regulated Q8VXQ9 Carbohydrate metabolism Glyceraldehyde-3-phosphate dehydrogenase A, chloroplast 20 0.24 0.48 Down-regulated Content 1. Background 2. Materials and Methods 3. Results and discussion 4. Conclusions Conclusions Under nitrogen starvation, the biomass and biodiesel productivities of mixotrophic S. obliquus exceeded the combination of autotrophic and heterotrophic cells when using acetate as carbon source. The fatty acid yield from mixotrophic culture (0.45) was almost two times greater than for heterotrophic culture (0.23). Proteomics analysis revealed that the activity of the TCA cycle was improved in mixotrophic culture when compared with heterotrophic culture.