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 sxf0912@mail.ustc.edu.cn

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

Background 1.Background-Comercial production obstacle Select species Cultivation Oil extraction and transesterification Nutrient, light, water, carbon source Harvest Biodiesel Lipid productivity Biomass productivity Lipid content University of Science and Technology of China

Lipid productivity decrease Lipid content Biomass productivity Role of phosphorus Lipid productivity decrease Nitrogen starvation The effect of P concentration on biomass and lipid productivities of microalgae under nitrogen starvation conditions University of Science and Technology of China

Autotrophic cultivation of S. obliquus FAME productivity: N starvation conditions > N sufficient conditions; N-&P > N-&P-lim > N-&P- 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

Mixotrophic cultivation 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 Scenedesmus obliquus Acetate Nitrogen starvation The cell concentration and lipid production of mixotrophic Chlorella sp. was even higher than the sum of those from photoautotrophic and heterotrophic culture. The lipid production of Chlorella vulgaris under mixotrophic cultivation was lower than under heterotrophic cultivation due to the unsatisfactory 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

Calculation of fatty acid yield

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

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. The highest fatty acid content was obtained in the mixotrophic culture. The fatty acid content of all the three groups rose steadily and significantly.

C18:1 was the most predominant composition, accounting for 52 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. Auto- Hetero- Mixo- More assimilated acetate is directed to lipid synthesis rather than protein and starch accumulation with the presence of light and supply of CO2.

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 1,065 proteins were identified 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 A8IRQ1 Ribose-5-phosphate isomerase 7 1.77 0.01 A8JGJ6 Mg protoporphyrin IX S-adenosyl methionine O-methyl transferase 2 0.04 A8J2S0 Small molecular metabolism Citrate synthase 3 2.74 A8J0R7 Generation of precursor metabolites and energy Isocitrate dehydrogenase 2.60 0.14 A0A0D2K714 Carbohydrate metabolism Pyruvate kinase 2.17 0.09 B6E5W6 Glucose-6-phosphate isomerase 9 A0A0D2NR30 Catabolic process Glycerol-3-phosphate dehydrogenase 2.52 0.23 A8I8Z4 Ribosomal protein 0.59 Down-regulated A8IKQ0 Fructose-1,6-bisphosphatase 4 0.52 0.22 D8TTF7 Plastid acyl-ACP desaturase 0.15 A0A0D2JX51 Malate dehydrogenase 16 0.65 0.19 Q4U1D9 Biosynthetic process Soluble starch synthase III 0.58 Q8VXQ9 Glyceraldehyde-3-phosphate dehydrogenase A, chloroplast 20 0.24 0.48 TCA cycle The activity of the TCA cycle is improved in mixotrophic culture, resulting in more fatty acid synthesis in S. obliquus cells.

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.

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