Screening, Identification Of Lipid-rich Microalgal Germplasms And Breeding Of New Varieties

A new and renewable clean energy was urgent to be searched because of the increasing depletion of fossil fuels and enhance of environmental stress. The photo synthetic efficiency of unicellular microalgae is several times of that of lipid palm, rice and other crops. The microalgae have been considered to be a ideal source of biodiesel for its fast growth and high lipid content. Because the key technologies of high-density and low-cost cultivation have not yet been resolved, the industrialization cost of the energy algae was very high. Therefore, screening new algae species with high yield and high lipid content and exploring low-cost culture condition are the key factors of the energy algae industry, which is a hotspot in the world. This study was designed to screen algae with high biomass production and lipid content, establish a core germplasm collection for energy microalgae, optimize the culture condition of preponderant algae and reduce the cost of industrial cultivation And the methods cultivating new lipid algae was the basic of algae utilization and biomass energy research.In this test, microalgae lipid can be qualitatively and quantitatively analyzed by Nile Red fluorescence staining method, through fluorospectro photometer and multifunctional ELIASA. The detection conditions was optimized:Nile Red dye concentration 1μg/ml, the dyeing time 6 min, DMSO concentration 20%, and tested algae density OD600=0.12～0.52. Large-scale primary screening of lip id-rich microalgae was researched by the multi-functional ELIASA. At the same time, lipid accumulation of the same algae at different culture condition and different develop period was observed by fluorospectro photometer.With highoil content and high productivity for the breeding goal,496 microalgae were screened preliminarily by Nile Red fluorescence staining method in our laboratory. And 40 strains with relatively high oil content were obtained.Lipid fluorescence intensity, maximum fluorescence intensity and the average growth rate of microalgae were compared at the same density (OD=0.35) by the dynamic growth and lipid accumulation. Preliminarily, microalgae named La4-37, La2-14, SA2-1, SA2-17 and SA2-8, which had high lipid content and high productivity, were the potential biodiesel feedstock. Algae named La4-37 was chosen to be identified. The cell morphologic observation indicated that the cell of La4-37 was spherical and sporogony, chromatoplast was single, green and goblet. The cell size of La4-37 was 3-10μm.1756bp DNA sequences of La4-37 were obtained by amplified 18S rDNA. The result of cell morphologic observation and 18S rDNA evaluation was indicated that La4-37 was indentified as Chlorella sorokiniana, Chlorella, Chlorellaceae, Trebouxiophycea, Chlorophyta. The growth and lipid accumulation of La4-37 were observed. As a result, a transient lipid accumulation at lag phase, but abundant lipid accumulation at the late and stationary phase of exponential growth. Finally, the biomass (1.072g/l), the average growth rate (0.26 d-1) and lipid fluorescence intensity (8.71×10-5/cell) of La4-37 were detected. Compared to other oil-producing microalgae, La4-37 with high lipid content and high biomass and high growth rate was a potential energy microalgae.Environmental condition would affect the microalgae growth and lipid production seriously. Growth and lipid accumulation of La4-37 indifferent nitrogen concentrations and different pH was tested. The results were showed that the lipid accumulation of La4-37 increased and the growth rate decreased with the reduction of nitrogen concentration The results of pH gradient test indicated that La4-37 grew best at pH 7～8, but couldn't grow barely at low pH (4-5). In addition, La4-37 could grow to a certain extent (the average growth rate 0.2 d-1) at pH 12. The lipid accumulation of La4-37 was increased with the increase of pH. When the pH was 12, the relative lipid content was the highest.In order to obtain algal strains with higher oil content and growth rate, La4-37 was treated with ultraviolet irradiation.296 mutagenic microalgae were obtained after preliminary screening. Two mutagenic microalgae M077 and M040 with relatively lipid content were isolated through the lipid fluorescence detection and the lipid fluorescence intensity at the same algal density by Nile Red fluorescence staining methodThe growth and lipid dynamic fluorescence intensity of mutagenic microalgae was showed that the growth cycle of mutant strains and the period of lipid accumulation were the same. When the mutagenic microalgae grew to the stationary phase, the lipid accumulation had improved greatly (the lipid fluorescence intensity of M077 and M040 was 6.2 and 1.7 times as that of La4-37, respectively). But, the biomasses of mutagenic microalgae were decreased. The biomass of M077 was 49% of La4-37, and the biomass of M040 was 93% of La4-37.