Methodological Study On Preparation And Evaluation Of Botryococcus Protoplast

Biofuel is a safe, environment-friendly and renewable energy, which is the alternative offossil fuel. Many species of Botryococcus is rich in hydrocarbons, which attracts extensiveattention. Because of its low growth rate and volatility of hydrocarbons content, thedevelopment and utilization of Botryococcus as biofuel feedstock is limited. In this paper, onthe basis of the systematic comparison of the cell growth and energy compounds(hydrocarbons and fatty acids) content in Botryococcus sudeticus UTEX2629andBotryococcus sp. CCMP2742, Botryococcus UTEX2629was selected for optimizing theconditions of protoplast preparation and the quantitative detecting method of protoplastpreparation rate, meanwhile a new system was established for the evaluation and preparationof Botryococcus protoplast. The main results were as follows:1. Botryococcus UTEX2629was unicellular and grew well autotrophically in ModifiedBold3N Medium. The maximal cell concentration was1.0×107/mL, and the average specificgrowth rate before the19th day was0.25d-1. Botryococcus CCMP2742in colonies grew wellautotrophically in DV-Y Medium while2g/L KNO3as nitrogen source was added. Themaximal cell concentration of the green cells was7.5×106/mL, and the average specificgrowth rate before the19th day was0.17d-1.2. The energy compounds (fatty acids and hydrocarbons) composition and content inlogarithmic phase cells were measured by GC-MS, the results showed that the biofuel contentof Botryococcus UTEX2629was increasing with the growth of cells as OD690. In latelogarithmic phase (OD690=1.5), the highest contents of the fatty acids and hydrocarbons were5.13%dry weight and5.48‰dry weight, respectively. The fatty acids content of CCMP2742was increasing with the cell growth, but the hydrocarbons content was decreased after aperiod of rising time with the growth of cells. In late logarithmic phase (OD690=1.3), themaximal content of the fatty acids reached11.97%dry weight. In late logarithmic phase(OD690=1.1), the maximal content of the hydrocarbons reached7.72‰dry weight.3. Three quantitative detecting methods of Botryococcus protoplast preparation rate weresystematic compared, which used Botryococcus UTEX2629cells in the middle logarithmicphase as the material. The results showed that the variation trend and most value of protoplast preparation rate detected by these three methods were almost the same. The absolute countingmethod by flow cytometry and absolute counting method with cell wall staining by flowcytometry were more intelligent, fast and efficient than the traditional method with amicroscope. But operation process of cell wall staining was complicated, and this methodneeded a more advanced laser device. After the overall consideration, absolute countingmethod by flow cytometry was realized as the best quantitative detecting method of protoplastpreparation rate, which was fast (in20seconds), accurate (cell concentration below108/mLcan be accurate counted), sensitive (cell size larger than0.5μm can be detected).4. The conditions of protoplast preparation were systematic optimized after thecomposition, dosage of complex enzymes and enzymlysis time were systematic compared.The results showed that the optimal concentration of cellulase, pectinase and macerozymewas6.66%（w/v）,6.66%（w/v）and13.32%（w/v）in complex enzymes, and the appropriateenzymolysis time was12h. Under this optimal condition, the maximal protoplast preparationrate was21.41%. The results indicated a new method and material was established andsupplied for the genetic transformation of Botryococcus.