Development Of Mutation Breeding Of Microalgae And High-osmotic Stress Cultivation Of Chlorella Protothecoides

Biodiesel has been recently recognized as a kind of renewable and pollution-free energies. Currently, the cost for biodiesel production from lipids extracted from microalgae biomass still is rather high, it is difficult to be realized commercially at the industrial scale. In order to increase the microalgae cell concentration and the oil content in microalgae cells with low oil extraction cost, the main aims of this thesis are to develop a novel and high efficient procedure for mutation and seed breeding of microalgae, and to establish a high-osmotic stress cultivation of Chlorella protothecoides to accumulate lipids in cells. The results are showed as follows:Initially, low field nuclear magnetic resonance (LF-NMR) was applied for lipid content determination. Four quantitative methods for lipid content in microalgae cells which include LF-NMR and conventional methods like Nile red stain, Solhex extract and Chromatography-Mass Spectrometer (GC-MS) were summarized based on their characteristics. Additionally, several mutation treatments, including atmospheric and room temperature plasma (ARTP), UV radiation and UV radiation combined with LiCl, were tested to treat microalgae cells, the results showed that ARTP has higher positive rate of C. protothecoides cells. Based on above results, therefore, a novel mutation breeding procedure for microalgae’s cells was successfully developed. Namely, the microalgae’s cells was treated by ARTP at first, then spread on plates containing orlista. After incubation of 7 d,108 high-yield mutants were selected from 3098 colonies using Nile red stain. The selected mutants were heterotrophically cultured in shaking flasks. Then the lipid contents of mutants were rapidly analyzed using LF-NMR to screen high-yield mutant. Eventually, a high-lipid mutant, D4G5, was obtained, which has of oil-accumulating ability and higher cell density in shaking flask culture compared with original algae strain.(2) High osmotic stress culture to improve lipid accumulation in C. protothecoides cells was developed. Sodium chloride was selected among 4 osmotic stress agents tested here, and then the two-phase cultivation mode was established for C. protothecoides culture. At the stationary phase of C. protothecoides cells, the sodium chloride was added into broth, resulting in 80% higher of fatty acid content in microalgae than that of microalgae grown under the normal osmotic condition.(3) Mechanism of high osmotic stress leading fatty acid accumulation in C. protothecoides cells was preliminarily elucidated.i. At the cellular level, concentrated NaCl can stimulate cells to produce massive radical oxygen species (ROS), which decrease pyruvate kinase’s activity, however, and enhance acetyl Co A carboxylase’s activity. Due to being one of key enzymes through fatty acid biosynthesis pathway, acetyl CoA carboxylase can improve fatty acid production in cells.ii. Upon the transcriptomic data, it found that the main genes encoding key enzymes in fatty acid biosynthetic pathway did not significantly up-regulated, which is not proportional to fatty acid level. The possible reasons would be that there is a cascade signal transduction in cells, which will be further investigated.In summary, a high efficient and simply mutation breeding for C. protothecoides was developed, and a two-phase culture for C. protothecoides culture to effectively accumulate fatty acids in cells was conducted as well.