一株扎布耶盐碱湖伪盐生杜氏藻的分离鉴定及生长影响因素分析

Isolation, identification, and analysis of growth influencing factors of a Dunaliella pseudosalina strain from Zabuye Salt Lake

摘要:

【背景】杜氏藻(Dunaliella)具有独特的生理特性和丰富的胞内次级代谢物，在医药、食品、养殖业、化工、轻工等领域具有广泛的应用价值。【目的】分析影响杜氏藻生物量的生长因素和最优培养条件，以期提高杜氏藻的生物量；明确最佳培养条件下杜氏藻胞内次级代谢物的种类。【方法】采用纯培养法分离伪盐生杜氏藻(Dunaliella pseudosalina) ZBY-1，18S rRNA基因测序明确其分类学地位；采用单因素试验探究其生长最佳条件以及营养盐种类及浓度；采用正交试验优化分离藻株的培养条件；利用液相色谱-质谱联用技术(LC-MS)检测藻株ZBY-1在最佳培养条件下的次级代谢产物。【结果】藻株ZBY-1的最适生长盐度为10%，最适生长pH值为8.5，最适生长温度为25 ℃。单因素分析显示最适碳源、氮源、磷源的种类和浓度分别为NaHCO₃ 1.26、CO(NH₂)₂ 0.84、NaH₂PO₄ 0.06 g/L。优化生长条件后，藻株ZBY-1的细胞密度可达(2.57±0.12)×10⁷个/mL，较优化前提高了1.25倍。LC-MS分析表明胞内次级代谢物的种类主要是萜类、生物碱类、苯丙素类、氨基酸相关化合物等；显著富集的代谢物通路是辅助因子生物合成、植物类次级代谢物合成和ABC转运等途径。【结论】扎布耶盐碱湖的地理位置特殊，藻类资源未被深入挖掘。分离藻株ZBY-1可适应高盐碱环境，为后续胞内次级代谢物的应用开发提供一定的参考依据。

关键词:伪盐生杜氏藻;分离;生长特性;正交试验;代谢组学

Abstract:

[Background] With rich metabolites and unique physiological properties, Dunaliella has wide applications in the pharmaceutical, food, aquaculture, chemical, and light industries. [Objective] To analyze the factors affecting the biomass of Dunaliella and the optimal culture conditions with a view to increasing the biomass of Dunaliella and identify the secondary metabolites of Dunaliella under the optimal culture conditions. [Methods] D. pseudosalina ZBY-1 was isolated by the culture method and identified by 18S rRNA gene sequencing. The growth conditions (nutrients and concentrations) were optimized by single factor and orthogonal experiments. The secondary metabolites of strain ZBY-1 cultured under the optimal conditions were then analyzed by LC-MS. [Results] The strain ZBY-1 showcased the best growth performance at 10% NaCl, pH 8.5, and 25 ℃. The optimal carbon, nitrogen, and phosphorus sources and their concentrations for this strain were NaHCO₃ 1.26 g/L, CO(NH₂)₂ 0.84 g/L, and NaH₂PO₄ 0.06 g/L, respectively. The cell density of strain ZBY-1 cultured under the optimal conditions reached (2.57±0.12)×10⁷ cells/mL, which increased by 1.25 times compared with that before optimization. LC-MS results showed that secondary metabolites were mainly terpenoids, alkaloids, phenylpropanoids, and amino acid-related compounds. The enriched metabolite pathways were the cofactor biosynthesis pathway, plant-derived metabolite biosynthesis pathway, and the ABC transport pathway. [Conclusion] Zabuye Salt Lake has a special geographical location, whereas the algal resources remain to be exploited. The isolated strain ZBY-1 can adapt to the high saline-alkali environments, which provide a basis for subsequent development and application of the metabolites of this strain.

Key words:Dunaliella pseudosalina;isolation;growth characteristics;orthogonal experiment;metabolomics