| The low lipid content, slow growth rate and less biomass were the mainbarriers for the wild microalgae strains. Hence, in this paper, microalgae wasinduced by60Co-γ ray mutation breeding technique and the mutant strainscontaining rich lipid were screened. The screening method of microalgae with richlipid by60Co-γ mutation in the best condition of mutagenic dose was illustrated.The polyphasic taxonomy of mutagen was analyzed in detail, which could help usmake clear its species. The optimal conditions of growth and lipid accumulationwere ascertained to establish the effective operation and control strategies of lipidaccumulation. The mechanism of the mutant strain with elevated lipid content wasdiscussed after multi-scale comparisons of the mutant strain and wild strain, whichcould provide the theoretical and technical guidance for large-scale cultivation ofmicroalgae and the development of biodiesel production.The wild microalgae, Scenedesmus.MC-1, was induced by60Co-γ mutation.After optimization of the dose range, the best dose of60Co-γ was100Gy and arich-lipid microalgae Scenedesmus.Z-4was screened. The lipid content andbiomass of Z-4were29%and3.0g/L, respectively, which were about60%and30%more than the corresponding parameters of wild MC-1—18%and2.3g/L. It wasfound in this experiment that60Co-γ of less doses gave a positive influence on thegrowth and lipid accumulation of microalgae cells.Mutagen Z-4could grow and accumulate with many kinds of carbon sources,such as glucose, fructose, acetate and so on. The18S rDNA sequence of microalgaedid not change with a length of1292bp before and after mutation. According to theanalysis of phylogenetic tree, Z-4had a homology up to100%with Scenedesmus sp.R-16and Scenedesmus abundans in the18S rDNA sequence, which belongs toScenedesmus. The wild strain Z-4could accumulate lipid efficiently when glucosewas served as substrate. The optimal conditions of growth and lipid production weredetermined in batch tests, when glucose was10g/L, NaNO3was0.8g/L, K2HPO4was0.04g/L, pH was at7.0-7.5, and the light-dark cycle was12:12, and themaximum lipid content and biomass could reach up to38.5%and3.9g/L,respectively.The differences of the mutant strain and wild strain were showed as follows:1）The morphology of mutagen Z-4has changed via mutagenesis, with largerbodies and rough surface. According to the analysis of surface functional groups,the oxygenic functional groups C-OH and C=O increased, which was mainly causedby more extracellular polysaccharide substance by60Co-γ mutation. 2）The physiological and biochemical characteristics have changedsignificantly, such as the intracellular matter content, growth rate, lipid-productionrate and pH tolerance, which had a positive effect on its growth and lipidaccumulation. Moreover, mutagen Z-4can produce lipid effectively when the cheapsubstrate acetate was used, which had a greater practical significance to reduceproduction cost and accelerate the pace of industrialization for biodiesel technique.3）The gene differences were analyzed for the key genes of two enzymes,acetyl-CoA carboxylase (ACCase) and phosphoenolpyruvate carboxylase(PEPCase),in the course of lipid synthesis with PCR-RFLP. The electrophoretograms of thedesired genes for two microalgae strains were different after amplification andrestriction enzyme digestion, suggesting that the sequences of mutagen Z-4havechanged near the cleavage sites. According to whole-genome sequencing of twodesired genes, the point mutations were found at the318bp and456bp of ACCasegene and the117bp of PEPCase gene, which perhaps affected the activity of thetwo enzymes, enhancing the synthesis of lipid for mutant strain Z-4. |
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