Study On The Effect Of Nitrogen Stress In Chlorella Vulgaris And Development Of A Stable Genetic System For This Alga

At present,the energy crisis has attracted wide attention all over the world.Therefore,it is urgent to find a new type of economic,environmentally friendly and renewable energy.In recent year,biodiesel has elicited much interest as a renewable energy source.In which,microalgae is considered as a high quality raw material for biodiesel production and is gaining more and more attention.Chlorella vulgaris has a good potential in biodiesel production.The lipid productivity of Chlorella vulgaris is closely related to the culture conditions(e.g.,nitrogen regulation),but the mechanisms such as morphological and cellular component changes of algal cells under nitrogen regulation are not well understood,and there is an urgent need to better understand the mechanisms of growth and lipid accumulation under nitrogen-regulated conditions.In addition,it is possible to genetically engineer the lipid metabolic pathway of the algal species to improve lipid yield.However,there are still few efficient and stable genetic transformation systems for this species,and there is an urgent need to establish a stable genetic transformation system in this species in order to lay the foundation for improving oil accumulation through genetic modification in the future.In this study,a dominant oleaginous C.vulgaris strain was first screened.Growth characteristics,biochemical and morphological changes triggered by nitrogen stress were then investigated in this alga.Finally,a stable genetic system based on a novel selective marker was developed in this alga.The main results can be summarized as follows:(1)The growth kinetics,photosynthetic pigment concentration and lipid biosynthesis of six C.vulgaris strains,i.e.GF-4,FACHB-8,FACHB-31,FACHB-1068,FACHB-1072 and FACHB-1227,were respectively evaluated.We found that the biomass production,biomass productivity,specific growth rate,chlorophyll a and carotenoids concentrations,lipid content and lipid productivity of FACHB-1068 were much higher compared to the other five strains.At the end of cultivation,the biomass production,lipid content and lipid productivity were 2.23 g/L、28.07%和46.38 mg L-1d-1,respectively.We finally chose FACHB-1068 as the dominant starting strain for the subsequent studies.Moreover,this strain was characterized by ITS approach and thus classified as C.vulgaris.(2)The effect of nitrogen stress strategies(including nitrogen limitation and starvation)on growth characteristics,biochemical and morphological changes were investigated in Chlorella vulgaris FACHB-1068.The results showed that nitrogen limitation(nitrate concentration<21.66 mg/L)and starvation enhanced the lipid content but generally decreased the biomass productivity,pigment concentration,and protein content in algal cells.Compared to nitrogen limitation strategy,3-day nitrogen starvation was more effective in improving lipid accumulation.Besides,nitrogen starvation caused significant changes in cell morphology,with an increase in numbers and total size of lipid droplets and starch granules.Fatty acid profile analysis showed that saturated fatty acids(C16:0,C20:0,and C18:0)accounted for the majority of the total fatty acids(～80%).Our work may contribute to a better understanding of the biochemical and morphological changes in microalgae under nitrogen stress.Besides,our work may provide valuable information on increasing the lipid productivity of oleaginous microalgae.(3)The sensitivity of the algal cells to the four antibiotics was investigated,and it was found that the algal cells were highly sensitive to balomycin,and 30 μg/mL of balomycin could completely inhibit the growth of the algal cells.Hence,paromomycin and the corresponding selective marker gene Aph Ⅷ gene was thus chose in this study.So far a novel selective marker based on Aph Ⅷ gene has been developed in C.vulgaris.In addition,the Aph Ⅷ gene(selective marker gene)and the Venus gene(reporter gene)was introduced into C.vulgaris cells using a polyethylene glycol(PEG)mediated method.PCR analysis and southern blot showed that these two gene was integrated into C.vulgaris genome.Fluorescence microscopy demonstrated that Venus was expressed as yellow fluorescence in the cytoplasm of transformed cells.Besides,we found that the Venus was also detectable in transformants after 3 passages.Overall,we have successfully developed a stable genetic system in C.vulgaris.Our study will lay foundation to increase the lipid productivity by genetic engineering strategy in future.