Isolation And The Basic Study On Genetic Engineering Of Microalgae For Biofuels

Because of the increasing global energy demand, many countries have begun to transition to various forms of renewable energy. Compared to fossil fuels, biodiesel is more environmentally-friendly and sustainable, so it could be a viable alternative to replace conventional petroleum. However, the high cost of raw materials limits current production of biodiesel. As such, the search for an improved raw material is one of the major objectives in biodiesel research. Microalgae are photosynthetic microorganisms that convert sunlight, water, and carbon dioxide to biomass and have many advantages for the production of biodiesel compared to other oil crops. First, the cultivation of microalgae does not require large areas of arable land. Second, microalgae can grow rapidly and many microalgal species are rich in oils. Therefore, microalgae could represent an available raw material for biodiesel production. Increasing of microalgal biomass and lipid content can further improve their biofuel production, and make microalgae to be more competitive in the biofuel production, which is the main direction and the hotspot in the microalgal biofuel research.This thesis is a basic study on the genetic engineering of microalgae for biofuel, and has focused on two aspects, one is the study on the selection and culture optimization of microalga strain, another is the basic research for further genetic modification of microalgae. The main works are as follows:(1) Isolation of the microalgae with high biomass and high lipidA novel green unicellular microalga was isolated from the freshwater of the Inner Mongolia Province of China and named as CS-01. Morphological features and the phylogenetic analysis for the18S rRNA gene reveal that the isolate is a Chlorella sorokiniana strain. The strain has a fast growth rate, and can grows between pH7-10and temperatures of20-35℃with optimal conditions at pH9and30℃. A nitrogen source test reveals that this strain can grow well with nitrate and urea, but not ammonium. The strain can also use many carbon sources to grow, and has high biomass and lipid content when cultured with glucose. Due to the potential for the strain in biofuels, the strain was selected to further study in this thesis.(2) The optimization of the culture conditions of the microalgae for biofuel production Different from limited nitrogen to increase the lipid content, mixotrophy, heterotrophy and cultures with high iron concentration can improve greatly the growth rate and lipid content of the freshwater microalgae C. sorokiniana CS-01. Furthermore, most of lipids in C. sorokiniana CS-01are neutral and saturated, suggesting that the lipids are able to produce high quality biofuels. And the alga strain showed the stronger tolerance of glucose than other Chlorella species, and more iron tolerance than marine microalga C. vulgaris.(3) Expression differences of genes related with lipid biosynthesis in cells with low and high lipid content.By analyses on the relations among the changes of gene expression, cell growth and lipid content in the different culture conditions, this study found that the rbcL gene expression can be used to reflect the rate of photosynthesis. The prominent feature of mixotrophic culture is the presence of two energy sources:organic carbon sources and light, so microalgae may benefit from both photoautotrophic and heterotrophic growth. C. sorokiniana CCTCC M209220may utilize the organic carbon source first and reduce the photosynthetic efficiency. In addition, rbcL gene involved in photosynthesis still retains a small amount of expression in the log phase of heterotrophy, but is drastically reduced in the stationary phase of heterotrophy. The expression level of accD gene in heterotrophy is correlated with the concentration of residual glucose, however, the expression level of the gene can be related to the lipid content. Furthermore, except heterotrophy, the expression level of accD can be used as a remark to evaluate the lipid content. There are both of the homogeneity ACCase and heterogeneous ACCase in C. sorokiniana CS-01. But, the homogeneity ACCase has less contribution to the total lipid accumulation. Furthermore, that the high iron concentration can improve the rbcL, accD and accl gene expression may be one of the reasons for increased lipid content in the culture with high iron concentration.(4) Genefishing analysis in the cultures with low and high lipid content.Genefishing is a simple and quick method to study the expression differences on the genomic level, and can be used to quickly search these genes with obvious expression differences effected by culture conditions. The results of Genefishing showed the expression levels of most genes were increased with the increase of Fe concentrations, suggesting that Fe can enhance the expression level for some genes involved in lipid synthesis. In addition, it was found that two genes may be associated with lipid synthesis by the Genefishing analysis, the two genes also showed increase in expression with the increase of iron concentration.(5) Identification of lipid biosynthetic pathways in the microalga C. sorokiniana CS-01based on transcriptome sequencing and annotationThrough improved cDNA library preparation methods, it is the first time the whole transcriptome sequencing of the freshwater microalga C. sorokiniana CS-01was completed. In addition, the assembly, gene ontology analysis and further functional annotation of sequence were processed. Finally, a large number of genes in the microalga were obtained, and the microalga gene library was improved further. According to the presence of enzymes, the complete pathways were described from the CO2fixation to triacylglycerol (TAG) biosynthesis, including lipid biosynthesis, elongation and metabolism, indicating carbon flux goes to TAG and other competing branches for the genetic engineering of improving lipid yield in microalgae. Furthermore, identification of genes involved in cell growth and death provides a strategy for improving microalgal growth at a molecular level to finally increase total lipid yield.(6) Expression differences of whole transcriptome in cultures with different lipid contentThe transcription libraries of microalga in cultures with different lipid content were sequenced. The changes of expression level were compared according to lipid changes among the culture conditions, in order to find the key genes affecting lipid content. This study found that a lot of genes change expression levels with the changes of lipid, supporting the further study on genetic engineering of microalgae in the future. Of these genes, the functions of many genes have not yet been annotation, but they may be related to lipid synthesis due to their changes of expression correlated with lipid content. Further annotations and identifications of these genes will contribute to a more understanding of the synthesis and metabolism of lipids in the microalgae. It is expected to not yet reported more new genes related to lipid synthesis. In addition, we also found that some unknown genes with high expression levels in all cultures, their regulatory elements will be helpful to build high expression vectors in microalgae.(7) Colony-PCR for microalgaeIt is necessary to verify a large number of microalgal transformants when constructing the engineering of microalgae, but the isolations of DNA for PCR are incredibly time-consuming and tedious. However, colony-PCR is a method that utilizes a minimal quantity of cells from the medium or a single colony from a plate to amplify only the expected fragments for analysis without the need to isolate pure DNA. When comparing the efficiencies of five different lysis buffers, a Chelex-100solution was found to be superior with DNA extraction, PCR reaction, and DNA storage. The Chelex-100buffer also was not sensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically engineered transformants as well as bioprospecting of natural microalgal isolates.