| Botryococcus braunii can synthesize and store large amounts of hydrocarbons in thegrowth process. The total hydrocarbon content is generally more than30%of cell dryweight, up to86%. B. braunii exists in three different races: A, B, and L. The races can bedifferentiated generally on the basis of the characteristic hydrocarbons they produce. In thepresent, the main factors limit the practical application of B.braunii are can not get highbiomass and hydrocarbon content in a short time. N is an essential element of algae growth,and nitrogen deficiency can stimulate lipid accumulation of hydrocarbons. In this study, theB. braunii, Abt02strain,was isolated from Fuxian lake in Yunnan, China. The strain wassubjected to several analyses, including cytological observation, phylogenetic comparisonwith31other B. braunii strains from three races (A,B,and L) based on their18S rDNAsequences, and hydrocarbon composition analysis by gas chromatography massspectrometry (GC-MS). Ascertained the race and hydrocarbons of Abt02strain. Changes inphysiological characteristics and expression analysis of the genes related hydrocarbonsynthesis were also studied under nitrogen deficiency culture conditions.Cells of B.braunii were width of9.61±3.03μm, length of14.27±1.09μm. Thecolony color in the stationary phase is orangebrownish. The result of GC-MS showed theB.braunii Abt02containing the specific hydrocarbon of B race—squalene, the content ofsqualene and its derivatives reached more than80%, which concluded that B.braunii Abt02belonging to B race. Phylogenetic tree based on18S rDNA sequences of strains that knownchemical race showed that, B.braunii Abt02gathered in the cluster of B race, andminimum genetic distance with the strains (Ayame, Bot25, Bot30-1, Bot60) belonging toB are0.03.There is no significant logarithmic phase under nitrogen deficiency condition, and theaverage growth rate is very slow. However, the content of carotenoids has an increasing trend with cultured time under nitrogen deficiency conditions, while there is no obviousincrease under the normal culture conditions.On the45th day of N deficiency condition theratio of carotenoids and chlorophyll is0.74more than the0.16of normal condition.Withthe culture time of N deficiency, the colony became loose and cells start to free away,thenthere are more and more single cells in the medium. The algal cells are highly resistant tonitrogen deficiency. Add BG-11into the midium after a long time (about three months) inthe N deficiency condition, the algal cells restore viability and began to split after5days.However, the cells began to decline: contents exudated and cells became transparent undernormal culture condition.The hydrocarbon content under N deficiency condition is higher than normalcondition at the same time, on the45th day of N deficiency condition the hydrocarboncontent is56.89%more than the43.75%of normal condition. There is no significantdifference in the hydrocarbon component between the N deficiency and normalcondition,but the content of squalene and its derivatives under nitrogen deficiency developan increasing trend more than normal condition.The result of fluorescence quantitativeanalysis showed the expression of squalene synthase gene under N deficiency condition ishigher than normal culture conditions. The expression level of genes (DXS、MCS、DLS)which participating in the rate-limiting step of the hydrocarbon synthesis are also higherthan normal culture conditions. It is the high expression of these genes under high nitrogendeficiency conditions prompted massive accumulation of hydrocarbons. This is thefoundation of using genetic engineering to achieve high hydrocarbon-containing algaestrains. |
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