Screening And Assessment,Strategies For High-yield Oil Of Oleaginous Microalgae And Their Differentiations In Physiology And Biochemistry

Oleaginous microalgae are considered as an optimal feedstock for biodiesel production. With the growing energy crisis, research on biodiesel production from microalgae has been the concern of governments and researchers worldwide. To enhance lipid productivity is one of the most critical problems to realize microalgal biodiesel industrialization. Due to the limitation of both of oleaginous microalgal species and experimental condition, some of previous studies have less guiding significance. Moreover, studies on natural oleaginous microalgae (non-model microalgae) are limited. To overcome these shortcomings are exactly the key solving steps for theoretical bottleneck and lipid productivity improvement. This study mainly focused on "oleaginous microalgal screening","effects of high light intensity, initial nitrogen supply, urea on the growth and neutral lipid accumulation of oleaginous microalgae","regulations and differences in physiology and biochemistry of two natural microalgal species under various intial nitrogen supply","growth, carbon flow distribution, and photosynthetic performance of oleaginous microalgae in the process of lipid accumulationand","assessment, and economic analyses of oleaginous microalgae in large-scale cultivation". The main results are listed as follows:1. Twenty strains of freshwater and marine microalgae, including17strains of Chlorophytes,2strains of Eustigmatophytes, and1strain of Bacillariophytes, were investigated in this study. By comparing their biomass concentration, total lipid content, β-carotene content and total lipid yield, six strains of oleaginous microalgae which had over2.0g L-1of lipid yield were selected. These were E. vischeri, S. deserticola, C. tatrense, N. oculata, C. ellipsoideum, and C. nivale. After testing by prediction model of the quality of biodiesel, their lipids complied with the standards set by China (BD100), USA (ASTM6751), and EU (EN14214). Among them, the lipid yield of E. vischeri could reach up to4.0g L-1. Simultaneously, it could accumulate2.39%DW β-carotene. This microalga was considered to have extremely high developing value. 2. By analyzing the level of biomass concentration, neutral lipid content, and neutral lipid yield, we investigated effects of high light intensity, initial nitrogen supply, and urea on growth and lipid accumulation in six strains of oleaginous microalgae. The results indicated that these three kinds of strategies of high-yield oil (high light intensity, initial nitrogen supply, and urea) could improve the neutral lipid yield, but a close relationship exists between lipid production, microalgal species, and individual strategies. The neutral lipid yield of microalgae after optimization were listed follows:E. vischeri(4.9g L-1, high light+urea+low nitrogen supply), S. deserticola (4.1g L-1, high light+nitrate+high nitrogen supply), C. ellipsoideum (3.8g L-1, high light+urea+low nitrogen supply), N. oculata (3.0g L-1, high light+nitrate+high nitrogen supply), C. nivale (2.7g L-1, high light+nitrate+low nitrogen supply), C. tatrense(2.1g L-1, high light+nitrate+high nitrogen supply). Considering the changes in biomass concentration and neutral lipid content, we found three patterns of response to low nitrogen supply. These were type-I (decrease in biomass concentration and increase in lipid content), type-â…¡ (reduction in both biomass concentration and lipid content), and type-â…¢ (enhancement of both biomass concentration and lipid content). Type-â…¢ microalgae could be the potential candidate for the large-scale oil production.3. The growth, biochemical composition, nitrogen uptake, photosynthesis performance and respiration of two kinds of natural oleaginous microalgae, S. deserticola (type-I) and R. vischeri (type-â…¢), under3.5mM,5.9mM,8.8mM, and17.6mM of initial nitrogen supply were studied. The main results were listed as follows:(1) Growth characteristic:the optimal nitrogen concentration for cell division of two microalgal strains was8.8mM. However, the optimal nitrogen concentration for biomass accumulation was17.6mM(S. deserticola) and5.9mM(E. vischeri), respectively. With the initial nitrogen supply decreased (17.6mMâ†'3.5mM), cell weight of S. deserticola reduced, while cell weight of E. vischeriincreased.(2) NO3-uptake and utilization:S. deserticola and E. vischeri had the similar nitrogen uptake rate. Nevertheless, the protein synthesis rate and yield in E. vischeri were higher than that of S. deserticola, respectively.(3) Biochemical composition:with the cultivation time, the carbohydrate and protein content in S. deserticola and E. vischeri showed a decreased tendency, while total lipid content showed an increased tendency. High nitrogen supply could accelerate protein synthesis, but low nitrogen supply was beneficial to the synthesis of carbohydrate and lipid in S. deserticola and E. vischeri. The synthesis rate of neutral lipid and content in S. deserticola was significant higher than that of E. vischeri. However, E. vischeri obtained higher carbohydrate content than S. deserticola. In the middle and later periods of culture, S. deserticola could accumulate large amount of canthaxanthin, the content of which was negative positive correlation with nitrogen concentration. However, K vischeri could accumulate large amount of P-carotene, the content of which was positive correlation with nitrogen concentration. All of other pigments, including chrolophyll and carotenoid, showed a great decrease tendency in content.(4) Photosynthetic performance and respiration:with the cultivation time, the respiration rate and photosynthetic efficiency of S. deserticola and K vischeri, expressed as Fv/Fm, rETR and Pn, showed a decreased tendency. E. vischeri exhibited less non-photochemical quenching and higher photosynthetic efficiency than S. deserticola.5. A strain of oleaginous microalgae JNU13, which was isolated from a tropical lake in Guangdong province, was used as experimental material in this study. By analyzing18S rRNA gene sequence and cellular ultrastructure, this microalgal strain was identified to be Chlorella vulgaris JNU13. The growth, carbon flow distribution and photosynthetic performance were investigated. The main results were listed as follows:(1) in the early stage of cultivation, lipid accumulation and cell division of Chlorella vulgaris JNU13could be synchronized. During the middle and late stages of cultivation, cell division suspended, the increase in lipid content became the main reason for biomass concentration increase;(2) in the early stage of cultivation, most of the CO2fixed by microalgae were used for the synthesis of carbohydrate. However, during the middle and late stages of cultivation, the fixed carbon was mainly used for the synthesis of lipid;(3) the decreased photosynthetic efficiency had obvious feedback inhibition on lipid accumulation of Chlorella vulgaris JNU13. The energy distribution between photosystems has been changed in the process of lipid accumulation (reduce in the energy distribution of photosystem I, no obvious change in the energy distribution of photosystem II). The activity of z-scheme electron transport chain related to oxygen release decreased. Alternative electron transport chain could offere energy for the growth of Chlorella vulgaris JNU13. High photosynthetic efficiency was the main reason that high nitrogen supply treatments could obtain higher lipid content than low nitrogen supply treatment.4. The large-scale cultivation of S. deserticola and N. oculata were conducted in a self-designed flat glass photobioreactor (24L, light path:4.0cm). Potential lipid productivity and cost of these two strains of microalgae were investigated. The results showed that biomass concentration, lipid content and lipid yield of S. deserticola and N. oculata cultured in flat glass photobioreactor were remarkable low compared to that of bubble column photoreactor (0.3L, light path:3.0cm). The areal biomass productivity of S. deserticola and N. oculata were16.43g m2day-1and17.08g m2day-1, respectively. The areal lipid productivity of S. deserticola and N. oculata were7.64g m2day-1and5.48g m2day-1. The total costs of S. deserticola and N. oculata were Y1868.2kg-1and Y1949.9kg-1, respectively. Under the laboratory conditions, high agitation, aeration and light cost resulted in high total cost of microalgal biomass.