1. 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

2. Content 1. Background 2. Materials and Methods
3. Results and discussion
4. Conclusions

3. Content 1. Background 2. Materials and Methods
3. Results and discussion
4. Conclusions

4. 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

5. 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

6. Autotrophic cultivation of S. obliquus
FAME productivity:
N starvation conditions > N sufficient conditions;
N-&P > N-&P-lim > N-&P-
Bioresource Technology , 152: 241–246

7. 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

8. 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

9. Content 1. Background 2. Materials and Methods
3. Results and discussion
4. Conclusions

10. 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)

11. Calculation of biomass productivity and fatty acid productivity

12. Calculation of fatty acid yield

13. Content 1. Background 2. Materials and Methods
3. Results and discussion
4. Conclusions

14. 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

15. 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.

16. 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

17. 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

18. 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.

19. 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

21. 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.

22. Content 1. Background 2. Materials and Methods
3. Results and discussion
4. Conclusions

23. 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.

24. Thank You For
Your Attention!