Specie Screening/Heterotrophic Culture Optimization Of Chlorella Protothecoides And Research On Mechanisms Of Lipid Accumulation Under Salt Stress

CO₂ concentration in the atmosphere is continuously increasing because of the immoderate use of fossil fuels, and the global warming is emerging as a result of greenhouse effect, making serious damage to the global environments. Besides, the energy crisis will be coming in the not too distant future for the depletion of unrenewable fossil fuel resources. The use of biotechnology to change today's world development pattern led by fossil fuels and chemical industry is becoming more and more attractive, especially using microalgae to solve the energy problem is very promising and feasible. This work mainly focused on a systematic research on algal species screening and heterotrophic culture process optimization with both high lipid content and productivity, by using the microalgae Chlorella protothecoides which can be grown autotrophically or heterotrophically. The mechanisms of lipid over-accumulation in heterotrophic culture cells of C. protothecoides under salt stress condition were systematically investigated, too.First, a rapid quantification method of cellular lipid in C. protothecoides was established using low-field nuclear magnetic resonance ?LF-NMR? technique. Results indicated that LF-NMR signals linearly correlated with cellular lipids of C. protothecoides significantly, with the correlation coefficient of linear regression ?R²? higher than 0.99 in LF-NMR signal calibration experiment. Also, the percentage differences of lipid quantification results by LF-NMR and standard GC-MS method were less than 2%, and the relative standard deviation of lipid quantification results by LF-NMR was smaller than 2%. Moreover, the LF-NMR technique was used to allow for a rapid, direct and nondestructive cellular lipid quantification, according to which a real-time and dynamic lipid concentration monitoring in heterotrophic culture process was successfully realized, valuably contributing to on-line culture process optimization.Second, the strain breeding improvement on C. protothecoides with high lipid accumulation performance was investigated. The novel mutation technique, atmospheric and room temperature plasma ?ARTP? mutation method, was employed in the mutation breeding works, and an efficient composite screening procedure comprising preliminary screening with Nile red staining and re-screening with LF-NMR technique was proposed. A total number of 3098 mutants with higher lipid content were firstly obtained using the high-throughput screening method with Nile red, in which 1003 mutants with better lipid production performance were selected as candidates for re-screening by LF-NMR, finally 8 mutants whose lipid contents were 80% higher than that of wild strain were obtained. Particularly,3 strains out of the 8 mutants had a very high lipid production ability in both stability verification experiment in shaking flask and scale-up culture experiment in a 5 L bioreactor.Obtaining the oleaginous algal strains, heterotrophic culture medium was optimized mainly focusing on the effects of major nutrients ?carbon, nitrogen and phosphorus source? on cell growth and lipid production of C. protothecoides. Results indicated that phosphorus depletion would limit biomass accumulation, but had no contribution to cellular lipid accumulation. In contrast, nitrogen depletion could significantly enhance cellular lipid accumulation, making lipid content and yield of glucose increase from 20% and 100 mg/g under sufficient nitrogen source condition to 28-30% and 130-140 mg/g, respectively. Behinds, glucose concentration also had a significant impact on cell growth and lipid production, thus maintaining suitable nutrient concentrations and proportions in culture medium was a simple and effective way to improve lipid production efficiency, then an optimal initial concentration of 50,5 and 1.0 g/L for glucose·H2O, NaNCO3 and K2HPO4 in batch culture medium was optimized, yielding a maximum specific growth rate of 0.77 h-1 and lipid yield of 104.7 mg/g; but NaNO3 concentration was decreased to 4.0 g/L in nitrogen limitation medium, under which lipid content and yiled could be improved over 45% and 35% than those obtained in sufficient nitrogen source medium.With culture medium optimization results, heterotrophic culture process of C. protothecoides for high efficiency lipid production was comprehensively optimized. In oxygen supply investigation, it was found that high oxygen supply benefits cell growth but limits lipid accumulation, thus a two-stage oxygen supply strategy was proposed in culture process, in which cell growth phase was supplied with sufficient oxygen, lipid accumulation phase was maintained with low oxygen supply. Applying this two-stage oxygen supply strategy to the fed-batch culture combining the nitrogen depletion strategy, a very high lipid content of 36.8% and lipid productivity of 175.2 mg/L/h was obtained after 192 h culture in a 5 L bioreactor, giving a final maximal biomass concentration of 91.4 g/L. Furthermore, a novel dual-mode culture coupling autotrophic process with heterotrophic process was proposed, where the CO₂-enriched off-gas from heterotrophic culture was aerated into autotrophic culture as inorganic carbon source, this made carbon source utilization more efficient and reduced CO₂ emission.Moreover, environmental factors that could improve lipid accumulation in heterotrophic culture cells of C. protothecoides were explored and investigated. Lipid accumulation could be effectively enhanced by imposing 30 g/L NaCl stress at late logarithmic growth phase, yielding a very high lipid content of 41.2% and lipid productivity of 185.8 mg/g, which was 4.1 and 1.9-fold higher than that of control, respectively. Then, the effects of salt stress and osmotic stress were comparatively investigated. The mechanisms of lipid accumulation in heterotrophic culture cells of C. protothecoides under salt stress were summarized and revealed based on the multi-level association analysis of cellular oxidative response, key enzymes activity and biochemical composition alterations. Namely, cell status and substance storage ?including energy? would shift from growth and carbohydrate storage to lipid accumulation under salt stress condition mediated by cellular excessive ROS.Finally, the effects of salt stress on lipid accumulation were further confirmed by scale-up culture in a 5 L bioreactor, and its molecular mechanisms on lipid accumulation were further investigated by de novo transcriptome sequencing and metabonomics analysis. Results showed that total 1742 genes were differentially expressed in the pairwise comparison among 3 time points of growth phase, stationary phase and salt stress phase,488 genes out of which were significantly up-regulated expressed after C. protothecoides was imposed with salt stress. Then, differentially expressed genes before and after salt stress were annotated with Gene Ontology ?GO?, Pathway and KEGG metabolic networks analysis, it was showed that cell physiology and metabolism of C. protothecoides changed significantly under salt stress condition, also supported by the analysis of intracellular metabolite content changes involved in central carbon metabolism pathways. The flux increases in fatty acid biosynthesis, carbohydrate degradation and pentose phosphate pathway were the immediate cause of lipid accumulation, however, the flux decreases in tricarboxylic acid cycle and nucleic acid metabolism pathway limited cell growth, which was the indirect cause of lipid accumulation. In general, these results systematically and comprehensively illuminated the mechanisms of lipid accumulation under salt stress at a molecular level.