不同类型含磷营养物质对微小亚历山大藻(Alexandrium minutum)生长和毒素产生的影响

EFFECTS OF DIFFERENT PHOSPHORUS SUBSTRATES ON GROWTH AND TOXIN GENERATION OF ALEXANDRIUM MINUTUM

采用显微镜细胞计数和麻痹性贝毒的高效液相色谱分析方法,通过对比磷酸二氢钾、磷酸甘油和三磷酸腺苷(ATP)等含磷营养物质对微小亚历山大藻生长和毒素产生的影响,研究了微小亚历山大藻对不同类型含磷营养物质的利用情况。结果表明,在磷限制条件下,加入磷酸二氢钾、磷酸甘油和ATP均能促进微小亚历山大藻的生长,所获得的最大藻细胞密度分别为22700、28400和27800藻细胞/ml,对数期生长速率分别为0.12/d、0.14/d和0.15/d,溶解态有机磷对藻生长的促进作用稍好于溶解态无机磷。三种含磷营养物质对毒素产生的效应没有明显差别,稳定期藻细胞毒素含量分别为22.87、23.98和21.80fmol/cell。磷限制对毒素组成的影响很大,磷限制条件下膝沟藻毒素GTX1/4的含量占绝对优势,GTX2/3含量很少。在添加含磷营养物质后,GTX1/4所占比例降低,GTX2/3比例增加,说明磷限制显著改变了微小亚历山大藻的毒素组成。对藻体碱性磷酸酶活性分析的结果显示,磷限制组具有最高的碱性磷酸酶活性,添加有机磷化合物实验组的碱性磷酸酶活性显著高于添加无机磷化合物的实验组。研究结果表明,微小亚历山大藻可以利用实验中采用的无机磷化合物和有机磷化合物,但其利用溶解态有机磷化合物的机制尚不清楚,可能是通过诱导合成碱性磷酸酶来水解培养基中的溶解态含磷有机物,从而释放无机磷酸盐用于藻细胞的生长。

Effects of three different phosphorus substrates, KH2PO4, glycerophosphate and ATP, on the growth and toxin generation of Alexandrium minutum were studied. A. minutum cultured in 500ml flasks containing modified f/2 medium with only 1μmol/L dissolve inorganic phosphorus （DIP） and no silicate was used in the experiment. The batch culture of the algae were maintained at （21 ±0.5）℃ in 14L： 10D pattern （light intensity 4000 lx）. On the 21st day, when original phosphorus in the culture medium was depleted, three additional phosphorus substrates, i.e. KH2PO4, glycerophosphate and ATP, were added to make three separate groups, respectively. And the concentration of phosphorus added was 10μmol/L. A control group was set with no phosphorus added. The results showed that： The adding of all the three different phosphorus substrates could promote the growth of A. minutum under phosphorus-depleted condition. In stationary phase, the maximum cell concentrations reached 22700, 28400, and 27800 cells/ml, respectively, much higher than the concentration of the control group. After the addition of phosphorus substrates, the toxin contents per single cell of the three groups first decreased from the same value of 18.92 to 12.86, 10.55 and 8.90 fmol/cell, and then increased gradually to 22.87, 23.98 and 21.80 fmol/cell, respectively. In contrast, the toxin contents in the control group increased gradually to 37.16 fmol/cell till the 31st day. The toxin composition could be affected by the supply of phosphorus. GTX1 and GTX4 became dominant in the control group when the algae were limited by phosphorus. After phosphorus substrates being added, the ratio of GTX1/4 decreased, while that of GTX2/3 increased. On the contrary, no clear difference was observed in the substrate-added groups in perspectives of affecting toxin level, and toxin composition in A. minutum. Analytical results of alkaline phosphatase （AP） activity showed that the groups added with dissolved organic phosphorus （DOP） had higher activity than that added with DIP. However, the control group had the highest activity among all the groups. Therefore, AP activity can be used as an indicator for phosphorus limitation for A. minutun. One day after adding 10μmol/L of those three phosphorus substances, the total concentration of phosphorus in A. minutum cultures decreased so quickly that only 51%, 36% and 47% of KH2PO4, glycerophosphate and ATP, respectively, had left in A. minutum cultures. It is clear that A. minutum can use both DOP and DIP as the phosphorus sources for growth and toxin generation during which AP might play an important role in DOP utilization by the algae.