Analysis On Hydrocarbon Components, Optimisation Culture Conditions And Comparison Of Cellular Morphology Structures In Different Growth Phases Of Botryococcus Braunii

Botryococcus braunii is characterised by a conspicuous ability to synthesis and accumulate hydrocarbons. B. braunii is regarded as a potential source of renewable fuel because of its ability to produce large amounts of hydrocarbons which constitute up to 25%~40% of the dry cell mass of the green active-state colonies. Hydrocarbon contents are affected by factors such as alage strains, culture conditions and growth stages. In the present, the observed slow growth rate and biomass of the alga is a consequence of a limited supply of B. braunii. It is important to establish the optimal nutritional requirements and culture conditions for producing B. braunii hydrocarbons. In addition, only a few reports exist on variations in cellular ultrastructure characters during cultures. In the present work, the hydrocarbon component of B.braunii (FACHB 357) was analyzed, and the optimal nutritional requirements, especially nitrogen sources and culture conditions for B. braunii growth was established and discussed. The cellular structure and ultrastructure of B. braunii at different growth phases were studied. Main methods and results are as follows:1. Analysis on hydrocarbon component of B. brauniiThe lipid and hydrocarbon contents were determined and the hydrocarbon components were analyzed by GC-MS. The fat content was found to be 34.9% (w/w). Hydrocarbon recovery was 30.7%. The hydrocarbon components include n-alkanes,isoparaffin and a large amount of alkene. It may belong to the B race on the basis of the characteristic hydrocarbon they produce2. Effect of different nitrogen sources for B. braunii growthThe experiment was carried with different nitrogen sources such as potassium nitrate, sodium nitrite and ammonium chloride to study their effects. The algal growth was measured by algal optical density(OD680). Growth was determined turbidometrically at 680 nm every two days.The growth curve was derived from algal optical density for 32 days. B. braunii could grow well when nitrate is utilized as nitrogen source over a range of 1~8 mM. With 2 mM nitrate, the maximum OD680 of 0.184 was obtained during cultures. The proliferous rate was high(0.0417) .The growth of B. braunii was up to 6mM sodium nitrite as sole nitrogen source, and showed that 2mM nitrite was the suitable consentration for growth. The maximum OD680 of 0.169 was obtained and the specific growth rate during exponential phase was 0.0361 d–1. At a concentration of 0.5mM was the optimal ammonium chloride for obtaining biomass growth. The maximum OD680 of 0.114 was obtained. The proliferous rate was 0.0160d–1. A little increase in the biomass is observed as a result of use of ammonium chloride as sole nitrogen source. B. braunii grew better in the presence of potassium nitrate compared to sodium nitrate. The optimal potassium nitrate for obtaining biomass growth should be established further as result of interaction effects among culture conditions.3. Overall optimization culture nutritional conditions of the B. brauniiBased on the public literatures, the main culture nutritional conditions of the B. braunii may are carbonate, nitrate, phosphate, temperature(T) and light intensity(P). The carbonate sources, nitrate and phosphate are one-to-one correspondence to NaHCO3,KNO3,K2HPO4. According to the five main culture nutritional conditions, a two-level full factor experiment using biomass as response factor combined with response surface methodology(RSM) was made. The factorial design shows that all the five nutrition factors selected have some effect on biomass yield. The sequence according to the effect of the response results is as follows: T> K2HPO4>P> KNO3> NaHCO3. Among them the factors of T, P, K2HPO4 and NaHCO3 can produce positive effect, but the factor of KNO3 produces negative effect. The significant test of the principal /interactive factors analysis convey that KNO3, K2HPO4, T, P, T*NaHCO3 are significant factors while NaHCO3 have no significant influence on biomass. The final optimum conditions of sodium bicarbonate, potassium nitrate, dihydrogen potassium phosphate, temperature and light intensity are 0.21g/L, 0.525g/L, 0.105g/L and 3750lux, respectively. There are significant differences between it and the modified Chu13 culture medium. Then it need to be validated by further experiments.4. Variations in cellular structure and ultrastructure of B. braunii in different growth phases.To achieve variations in the physiological state of B. braunii during the cultures, the cellular structure and ultrastructure investigated of B.braunii at different growth stages were compared by light microscopy and transmission electron microscopy. The results show that in the stationary phase the algae cell turn pale yellow from green color of the active state and the refringent material attaching cells to each other turn red-orange from colorless. The diameters of colonies shifted to higher ones then decreased again along the growth phases. Many free unicells surrounded by several outer layers present in the medium at the stationary stage. Ultrastructural studies reveal that the number of the outer cell wall layers increases firstly and then decreases during the cultures. The maximum abundance in hydrocarbon droplets associated with the exponential stage. Along the growth phases, the chloroplast size decreases and It's tructure is considerably altered: gradual thylakoid lamellars disorganization and large increase in intraplastidial starch size. The cells with large nucleus often are found in the exponential phase. The mophology and arrangement of tubular cristae of mitochondria has large variations at different growth stages. An increase in vacuoles size was observed as early as the exponential phase. The number and size of Golgi bodys is larger in the exponential phase than ones in other phases. The Golgi cisternaes which were swollen and stacked loosely often present in the phase. The variations in algae color, hydrocarbon abundance, colony size and successive outer cell wall layer are discussed in terms of their morphological and structural change at the different growth stages. and the propagation and taxonomy of B. braunii are further Clarified.This research accumulates experiences for a further research of Botryococcus braunii(FACHB 357).