Optimization Of Breeding For The Oleaginous Micro Algae And Genetic Engineering Of Cyanobacteria Producing Polyunsaturate Fatty Acids

Microalgae have the characteristics of oil-rich,rapidly reproduce and short growth cycle,which can be used as substitute of functional food and feed additives.However,there are many scientific and technical problems during the time of culture,such as low biomass and polyunsaturated fatty acids concentration,difficult to scale up,high energy consumption,high fresh water consumption and so on,which lead to high cost of microalgae biomass and component and restrict its commercialization.Therefore,this thesis regards Isochrysis.zhanjiangensis,Thalassiosira.pseudonana and Synechococcus sp.PCC 7002 as the research object,studied the optimization of algal cultivation,design of photobioreactor,harvesting of microalgae by bio-flocculation,reuse of flocculation supernatant medium,and genetically engineered cyanobactium producing polyunsaturated fatty acids.(1)Optimization culture of I.zhanjiangensis and T.pseudonana.This thesis explores effects of trophic modes,light intensity and NaNO3 concentration on the growth and lipid accumulation as well as the relationship between nitrogen consumption and growth in the course of culture.The results show that microalgae consumed more nitrogen while it grows faster.Compared to the conditions of photoautotrophic and photoheterotrophic cultivation,more nitrogen was consumed to meet the growth needs under mixotrophic cultivation conditions.There was more protein accumulation under the condition of sufficient nitrogen and mixotrophic cultivation,whereas more oil accumulation under the condition of low NaNO3 concentration.Considering the factors such as lipid productivity and saving resources,the best lipid productivity of 80.06 mg/L/d for I.zhanjiangensis was obtained under the mixotrophic cultivation when the light intensity is 100 ?mol/m2/s and the NaNO3 concentration is 375 mg/L.The highest biomass productivity of 344.40 mg/L/d was achieved with the mixotrothic culture systems operated at the light intensity of 100 ?mol/m2/s and NaNO3 concentration of 375 mg/L following the semi-continuous mode with the renewal rate of 35%and period of 1 d.Moreover,the PUFA was accounted for 30.82%of the total fatty acid content at this time.Therefore,it is a suitable condition for producing microalgal oil as a nutritional substitute.The highest lipid productivity of 52.47 mg/L/d corresponding to the biomass productivity of 227.18 mg/L/d for T.pseudonana,was achieved under the semi-continuous mode with the renewal rate of 25%and period of 1 d when NaNO3 concentration of 375 mg/L with light intensity of 200 ?mol/m2/s by photoautotrophic cultivation.(2)Design and experiment of photobioreactor for microalgae cultivation.Column and flat plate photobioreactors were designed for efficient and low-cost cultivation of microalgae.Compared with the column photobioreactor,higher biomass productivity of 228.89 mg/L/d and lipid productivity of 83.34 mg/L/d can be obtained when microalgae cultured in flat plate photobioreactor,indicating that the flat plate photobioreactor is beneficial to the growth and lipid accumulation of microalgae.The biomass productivity(342.83 mg/L/d),lipid productivity(77.96 mg/L/d)and the fatty acid composition were similar to that of the Erlenmeyer flask when the microalgae I.zhanjiangensis was cultured in a flat plate photobioreactor under semi-continuous mode.Compared with the results from the Erlenmeyer flask,the microalgae T.pseudonana also achieved similar results of biomass productivity(170.00 mg/L/d)and lipid productivity(41.48 mg/L/d)in flat plate photobioreactor under batch mode.Moreover,theflat plate photobioreactor has the advantage of larger light specific surface area,easy release of dissolved oxygen and low cost,which is benefical to scale up simply by increasing the length of the reactor,so it is suitable for large-scale cultivation of microalgae outdoors.(3)Harvesting of microalgae and reuse of flocculation supernatant.The results show that the highest flocculation efficiency was only 40%achieved at pH=12 when alkalinity-induced flocculation was performed,whereas the flocculation efficiency of self-flocculating microalgae T.pseudonana is over 80%at pH=10 for 1 h.The highest flocculation efficiency was up to 90%when the mixing ratio of algae was 2:1,induced by increasing the medium pH value to 10 for 3 h.Satisfactory results were also found with chitosan at an adjusted pH of 5 using concentrations at 25 mg/L,after testing a flocculant range of 5-30 mg/L.The reuse of flocculating supernatant which was added nutrients can reduce the consumption of fresh water.Furthermore,the biomass productivity of high pH induced bioflocculation supernatant decreased obviously after two cycles of reused,whereas the reused chitosan supernatant maintained an approximate biomass productivity of 246.67 mg/L/d and lipid productivity of 84.90 mg/L/d after three cycles of reused.(4)The cyanobacterial fatty acid desaturase genes expressed in Synechococcus sp.PCC 7002.In order to increase the total unsaturated fatty acid content,genetic engineering was used to modify fatty acid metabolism.The recombinant vectors Syd6D,Syd15D,Syd6Dd15D,pSDPcE6-glnA,pSDTpD5D-glnA,and pSDPcE6TpD5D-glnA were constructed.Then the recombinant vectors Syd6D,Syd15D and Syd6Dd15D expressing the ?15 fatty acid desaturase and?6 fatty acid desaturase genes were transferred by homologous recombination into Synechococcus sp.PCC 7002,resulting in relatively high accumulation of a-linolenic acid(ALA),y-linolenic acid(GLA),and stearidonic acid(SDA).Overexpression of the ?5 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of ALA compared with the wild-type while ?6 desaturase gene expression produced both GLA and SDA.Co-expression of ?15 and A6 fatty acid desaturase genes resulted in low-level accumulation of GLA but much larger amounts of SDA,accounting for as much to 11.64%of the total fatty acid content.