Breeding A Highly CO₂ Tolerent Microalga By UV-Iradiation And Analysia Of RbcL Sequence Variations Among Differernt Strains

With the global economy developed rapidily and social advancement, the demand of fossil fuel was increasingly growing,which had bring about the concentration of CO₂ increasing highly.Consequently, leaded a series of environmental problems, which seriously affected human production and life. So, controlled the CO₂ emission had important significance. CO₂-fixation by microalgae photosynthesis was a economical and effective technique. But generally the optium CO₂ concentration of microalgae was 1-5%. When the concentration of CO₂ was greater than 5%, the growth of algae would be inhibited, and the CO₂ fixation rate was relatively low. To solve this problem, this paper adopted ultravioler radiation mutagenesis technique, selected a microalgae which could effectively fix CO₂ in highly concentration of CO₂;determined the culture characteristics of the mutants,we got a greater CO₂ fixation rate than the parent strain; cloned the ribulose 1,5-bisphosphate carboxlase/oxygenase large subunit gene(rbcL) of the parent strain and mutants strains, identified the parent strain and analysised the rbcL sequence variations among different strains.1. The parent strain was treated by ultraviolet radiation, which was in 20W ultraviolet, the irradiation distance was 25cm and time was 30min. After mutagenic treatment, the parent strain was cultured by inletting 30% CO₂. Finally, we got 3 strains of mutants (named mutant B-1, B-2 and B-3), which growh rate was higher than the parent strain growth rate.2. Their genetic stability was analysised by comparing the fourth generation growth curve and the first generation growth curve.Good genetic stability was concluded. Analysised carbon content of mutants, compared with the parent strain, the carbon content of mutants B-1, B-2 and B-3 was increased by 1.72%, 1.68%, 1.41% respectively.3. Setting diffetent gradient of CO₂ concentration, temperature, pH, intensity of illumination to determinate the optimum growth conditions. Controlled the optimum culture conditions, 20% CO₂, 25℃, pH 6, 25000Lux, the CO₂ fixation rate of mutants B-1, B-2 and B-3 was 1.43g/l·d, 1.50g/l·d, 1.44g/l·d respectively, the CO₂ fixation rate of parent strain was 1.02g/l·d. Under the optimum culture conditions, studied the effect of different nitrogen sources on the CO₂ fixation rate of mutant B-2. When the nitrogen source is NH₄Cl, the growth rate of mutant B-2 was inhibited. When the concentration of NaNO₂ was greater than 150mg/l, the mutant B-2 was died. When the nitrogen source was NaNO3, the mutant B-2 can grow rapidly. When the concentration of NaNO3 was 2g/l, the maximum CO₂ fixation rate of the mutant was 1.54g/l·d.4. The ribulose 1,5-bisphosphate carboxlase/oxygenase large subunit gene of the parent strain was cloned to identificate chlorella sp.. We analysised the sequence using BLAST, phtlogenetic trees and genetic distance, ultimately determined that chlorella sp. had the closest relationship with chlorella sp.IFRPD 1018. We named chlorella sp.YANG-1. Simultaneously, we cloned ribulose 1,5-bisphosphate carboxlase/oxygenase large subunit gene(rbcL) of mutants B-1, B-2 and B-3.Under the same culture conditions, the CO₂ fixation rates of mutants B-1, B-2 and B-3 was higher than the parent strain. We analysised the differences of rbcL sequences of the parent strain and mutants. We concluded that the differences of rbcL sequences may be associated with the CO₂ fixation rate.In summary, in this paper, we got 3 strains of mutants (named mutant B-1, B-2 and B-3). Compared with the parent strain, the carbon content of mutants B-1, B-2 and B-3 were increased by 1.72%, 1.68%, 1.41% respectively. Controlled the inlet gases, 20% CO₂, the CO₂ fixation rate of parent strain and mutants B-1, B-2 and B-3 were 1.02g/l·d, 1.43g/l·d, 1.50g/l·d, 1.44g/l·d respectively. We identified the parent strain was closest with chlorella sp.IFRPD 1018, named chlorella sp.YANG-1. We analysised the differences of rbcL sequences of the parent strain and mutants. We concluded that the differences of rbcL sequences may be associated with the CO₂ fixation rate.