| The rapid increase in fossil fuel consumption leads to the danger of global energy depletion.The processing and use of fossil fuels also produce a large amount of greenhouse gases,which accelerates the process of global warming.Biomass energy is renewable energy obtained from organisms,which not only serves to overcome the problem of energy exhaustion,but also consumes carbon dioxide in the production process and reduces the environmental burden.It is an excellent potential substitute for fossil fuel energy.Microalgae and engineered microalgae are the third and fourth generation producers for biofuel in reseach and development stage.Among them,Botryococcus braunii has high lipid content and can produce extracellular hydrocarbons which can be directly utilized,thus is considered as a biomass energy producer with great potential.However,compared with other algae,B.braunii grows slowly,resulting in high culture cost and difficulty in commercialization.This dissertation focuses on how to improve the biomass and lipid production of B.braunii,so as to reduce the production and cultivation cost and to provide data supporting for the commercial development of B.braunii.The major results of the study in this dissertation are as follows:1.Selection and optimization of nutrient conditions:among the three nitrogen sources,including NaNO3,CH4N2O,and NH4Cl.NaNO3 was the best nitrogen source for the dry weight and pigment production of B.braunii,and CH4N2O achieved the highest lipid yield but with white floccule observed in the algal culture.In a comprehensive comparison,NH4Cl was the worst nitrogen source for the cultivation of B.braunii.Among 4 media of BG-11,f/2,modified Chu,and SE,the yields of total lipids,total hydrocarbons,carotenoids,and chlorophyll a in f/2 medium were the highest,reaching 5.76 mg/(L·d)、2.87 mg/(L·d)、3.41×10-2 mg/(L·d)and 3.27×10-2 mg/(L·d),respectively.The ratio of "total lipid output/nutrient input" in f/2 medium was 1.21,which was 26.9 times of that in BG11(the ratio was 0.045,as the lowest in four media).By further adjusting the concentrations of trace metal elements and vitamins,it was found that when the concentration of trace metal elements increased to 4 times of that in f/2 medium,the dry weight of B.braunii increased by 10.2%compared with original f/2 medium.At the same time,when the concentrations of trace metal elements and vitamins were both increased to 4 folds of that in original f/2 medium,the lipid yield increased by 18.2%accordingly.2.Optimization of light conditions:this study explored the effects of light cycle,light intensity,the combination or alternation of monochromatic red and blue light with a white light on B.braunii.The experimental results showed that:B.braunii adapted to long term illumination.When the light cycle was 24:0(light:dark),the dry weight and lipid yield of B.braunii reached the highest values.However,for the sake of ecological and economic benefits,the light cycle of 16:8 was proposed to cultivate B.braunii.When the light intensity was 40μmol m-2·s-1,the dry weight and lipid yield of B.braunii were extremely low.When the light intensity was increased to 75 μmol m-2·s-1,both the dry weight and lipid yield reached the highest values,and the further increased light intensity over 75 μmol m-2·s-1 could not significantly increase the dry weight and lipid yield any more.So,light intensity of 75 μmol m-2·s-1 was suggested for B.brautii cultivation.With the constant total illumination time but decreased illumination time of white light and increased illumination time of the combined monochromatic red and blue light,the biocomponent yields of B.braunii were significantly promoted at the same illumination intensity.Among these treatments,the one with illumination of the combined monochromatic red and blue light only(the illumination time of white light was decreased to zero),significantly improved biocomponent yields of B.braunii.The lipid yield of B.braunii under the combined monochromatic light of 3R4B increased by 32.4%compared with the control(white light).The hydrocarbon yield under 1R6B,the protein yield under 5R2B,and the polysaccharide yield under 1R6B was 46.8%,56.3%,and 70.1%higher than that of the control(white light),respectively.3.Selection of plant growth regulators:the effects of GA3,NAA,ETH,and 6-BA on B.braunii were examined in this study.The experimental results showed that the effects of plant growth regulators were closely related to their addition time.Addition of NAA at the 10th day since the initiation of algal cultivation,the increase in dry weight and lipid yield of B.braunii was observed.With addition of NAA at the 20th day,dry weight and lipid yield were increased by 15.3%and 35.9%compared with the control,respectively.Addition of 6-BA at the 20th day resulted in 14.1%increase in dry weight compared with the control.Moreover,6-BA addtion at different cultivation time could always increase the lipid yield,for example,the lipid yield with 6-BA addition at the 10th day was increased by 47.2%compared with the control.Furthermore,6-BA addition could also help B.braunii to increase the hydrocarbon yield.4.Orthogonal experiment of culture conditions for B.braunii:the effects of four factors,including two nutrient elements(trace metal elements and vitamins),light spectrum,and plant growth regulator of 6-BA,were tested by orthogonal experiment based on f/2 medium for B.braunii.The influence on the dry weight and hydrocarbon yield of B.braunii was observed as follows:light spectrum>trace metal element>vitamin>6-BA,and all factors except for 6BA behaved with extreme significance.The influence on lipid yield was as follows:light spectrum>trace metal element>6-BA>vitamin,and light spectrum and trace metal element were extremely significant factors.A treatment combination with two folds concentration of trace metal element and 4 folds vitamin concentration in original f/2 medium,light spectrum of 3R4B,2.5 mg/L 6-BA,was beneficial to the production of dry weight,lipid and hydrocarbon of B.braunii.Another treatment combination with two folds concentration of trace metal element and 6 folds vitamin concentration in original f/2 medium,light spectrum of 0R7B,10.0 mg/L 6-BA,could promote the pigment accumulation of B.braunii.5.Analysis of fatty acid composition and biodiesel performance of B.braunii:the fatty acid composition of B.braunii was analyzed by GC-MS,the result of which indicated that major fatty acids of B.braunii include C16:0,C18:3,C18:2,C18:1,C18:0,and C20:5.C18:1 was the majority and accounted for a proportion of more than 75%,followed by C20:5 and C16:0.Under different light spectrum,the fatty acid composition of B.braunii changed correspondingly.The light spectrum of 3R3B and 4R3B was beneficial to increase the proportion of C18:1 of B.braunii,monochromatic red light(7R0B)led to the increased proportions of C18:3 and C18:2,while the white light and monochromatic blue light(0R7B)were beneficial to the accumulation of C20:5.The changes in fatty acid composition led to the variation of biodiesel performance of B.braunii.The biodiesel produced by B.braunii had the best performance under the light spectrum of 3R4B,in which,the CN value of biodesel of B.braunii reached 46.67 and the CFPP was-10.80℃.In conclusion,the production performance and fatty acid composition of B.braunii could be adjusted by different culture conditions,thereby further improving the economic values. |
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