理化因子对谷皮菱形藻细胞密度及中性脂含量的影响

Effects of physical and chemical factors on the growth and neutral lipid content of Nitzschia palea

以高脂微藻谷皮菱形藻(Nitzschia palea NY025)为实验材料,探讨了利用光密度法和尼罗红荧光染色法测定细胞生长和细胞中性脂含量的可行性,进而研究了温度、光强及培养基中N、P、Si含量对藻细胞生长和中性脂积累的影响。结果表明:(1)谷皮菱形藻在675nm处存在最大吸收峰,细胞密度与OD675之间存在良好的线性关系,利用光密度法和尼罗红荧光染色法表征谷皮菱形藻生物量和中性脂含量操作简单,适用于高通量样品的测定;(2)谷皮菱形藻在20℃,光强160μmol m-2s-1时生长最快,在20℃,光强200μmol m-2s-1时,有利于中性脂积累;(3)培养基中N、P、Si浓度分别为80、120、100 mg/L时,有利于谷皮菱形藻细胞生长,其中,N元素影响最大,其次是P、Si,且N、P、Si三因子以及交互作用N×P与P×Si对藻株生长作用均为显著。培养基中N、P、Si浓度分别为80、120、50 mg/L时,利于中性脂积累,其中,N元素影响最大,其次是Si、P,且因子N、Si及交互作用N×P、N×Si作用均为显著;(4)可采用两步培养法,先使谷皮菱形藻细胞大量增殖,而后适当改变培养条件,以增加脂质合成。

An algal strain, Nitzschia palea NY025, was chosen as experimental materials for its high-lipid content. The feasibility of optical density method used for cell growth determination was investigated first. Then Nile red fluorescence was used for determining the relative neutral lipid contents because neutral lipid content was a key standard of choosing algae in biodiesel production. Furthermore, the effects of temperature, light intensity, nitrogen, phosphorus and silicon on the cell growth and neutral lipid contents of N. palea were investigated. The results showed that： （1） The maximum absorption of N. palea existed at 675nm, and the absorption showed a good linear relationship with cell density. Therefore, the growth of N. palea could be obtained by measuring OD675. Optical density method and Nile red fluorescence possessed advantage of simple operation, so they were suitable for high-throughput sample analysis. （2） The optimum temperature was 20℃ for increasing the cell density and neutral lipid content of N. palea. The finest light intensity for the growth of N. palea was 160 μmol m-2s-1, while the best light intensity for neutral lipid accumulation was 200 μmol m-2s-1. It is suggested that increasing light intensity appropriately might increase neutral lipid contents in algal cells. （3） Under the optimum temperature and light intensity, the orthogonal experiments of nitrogen, phosphorus and silicon were designed to research into their effects on the growth and neutral lipid contents of N. palea. The influence of nitrogen on the growth of N. palea was the most significant, then the factor phosphorus and the last one was factor silicon. The growth of N. palea was influenced significantly by factors nitrogen, phosphorus, silicon and interaction factors nitrogen with phosphorus, nitrogen with silicon. The optimum growth rate was obtained when the content of nitrogen, phosphorus and silicon were 80 mg/L, 120 mg/L and 100 mg/L, respectively. The influence of factor nitrogen on neutral lipid accumulation was the most significant, next was factor silicon and the last one was factor phosphorus. Neutral lipids accumulation was influenced significantly by factors nitrogen, silicon and interaction factors nitrogen with phosphorus, nitrogen with silicon. The maximum neutral lipid accumulation was obtained when the concentration of nitrogen, phosphorus and silicon were 80 mg/L, 120 mg/L and 50 mg/L, respectively. （4） In conclusion, the lipid synthesis can be improved by two-step method. First the cell density of N. Palea was increased, and after gathering plenty of cells the culture conditions changed to increase neutral lipid contents.