Variations Of Lipid Accumulation And Key Gene Expressions Of Dunaliella Tertiolecta Under Stress Conditions

Due to the gradual shortage of fossil resources combined with environment pollution caused by its usage in large scale, development of renewable and environment-friendly energy has become an important research field. Microalgae have a strong ability of carbon fixation, high growth rate and short cell cycle. Biodiesel based on microalgae is renewable and could release less toxic substances into the environment. Consequently, research of microalgae biodiesel has aroused extensive interest over the world.In the present study, we adopted Dunaliella tertiolecta as the main research object and investigated the optimal conditions of ultrasonic-intensified, microwave-assisted and the combined methods using response surface methodology and orthogonal test design. Plackett-Burman Design（PBD） was adopted to screen out the critical factors from seven nutritional elements including NaNO₃, NaH₂PO₄?2H₂O, NaHCO₃, NaCl, Fe-EDTA,MgSO4?7H2O and CaCl2?2H2O. We optimized the critical factors by response surface methodology in order to obtain the maximum lipid productivity. In addition, it was also investigated by gas chromatography that the response of algal fatty acid metabolism to the screened main influential ingredients. Neutral lipid induced by different conditions was analyzed by flow cytometry（FCM）, and also observed by fluorescence microscope. The isozyme activity of malic enzyme in different salinities was investigated by using isozyme electrophoresis. The transcript levels of ACL and ACS in D. tertiolecta in response to nitrogen/phosphorous deficiency and salinity were analyzed by qRT-PCR.The optimal condition of microwave-assisted extraction was microwave power 471.17 W, extraction time 162.12 s, liquid-solid ratio 100.23ml/g. Under the optimized conditions, the predicted extraction ratio was 56.7189%, and the validated value was 57.02 ±1.04%. The optimal condition of ultrasonic-assisted extraction was ultrasonic power 369.22 W, extraction time 5.24 min, liquid-solid ratio 124.91ml/g. Under the premium conditions, the predicted extraction rate was 46.5445% and the validated value was 45.94 ±1.22%. When the two techniques were utilized in combination, the optimized condition was ultrasonic power 320 W, ultrasonic time 4 min, microwave power 280 W, microwave time 120 s and liquid-solid ratio 100 ml/g and extraction rate was 49.97±0.87%%. By comparing the R values, the influential powers of the five factors were ranged in the order as follows liquid-solid ratio>microwave power>microwave time> ultrasonic time>ultrasonic power.Plackett-Burman Design（PBD） was adopted to screen out the critical factors from seven nutritional elements. And then Box-behnken Design（BBD） uses critical factors to get the optimal nutritional conditions for achieving maximum lipid productivity. The results showed that: there are NaNO₃, NaCl, NaHCO₃, NaH₂PO₄?2H₂O four critical factors; GC analysis showed different influences of them on fatty acid methyl esters and NaCl had the most effects on fatty acid composition of D.tertiolecta; NaCl 87.69（g/L）, Na NO₃ 1.77（g/L）, NaH₂PO₄?2H₂O 1.92（g/L）, NaHCO₃ 5.57（g/L） are the optimal culture conditions. The theoretical lipid productivity is 12.925 mg/L/day. Under the optimal conditions, lipid productivity in the verification experiment was 12.576±0.12 mg/L/day.cDNAs of the small and large subunits of ACL（DtACLA and DtACLB） and their genomic sequences were isolated from Dunaliella tertiolecta using RT-PCR, RACEs and genomic walking techniques. The expressions of genes DtACLA, DtACLB and ACS were fairly consistent with lipid accumulation in response to nitrogen deficiency, suggesting that both subunits of ACL from D. tertiolecta contribute to the active site of ACL and the ACL activity was related to the rate of lipid synthesis in D. tertiolecta.Given the results above, it could be concluded that microwave possessed a better extractive ability than ultrasound. The main fatty acids in Dunaliella tertiolecta are C16 and C18 series, indicating they are ideal for biodiesel production. Nitrogen /phosphorous starvation and low salinity could induce more neutral lipid content. And moreover, nitrogen starvation is more efficient in contrast with the other two methods. However, algal photosynthesis was inhibited by N/P-starvation. There was a correlation between activities of ME isoenzyme and neutral lipid accumulation. N-starvation and low salinity enhanced activities of ACL and ACS, which were advantageous for lipid accumulation.