一株嗜碱盐单胞菌ZB109的生长特性及四氢嘧啶发酵条件的优化

Halomonas alkaliphila ZB109: growth characterization and optimization of fermentation conditions for ectoine accumulation

摘要:

【背景】 西藏扎布耶盐碱湖水体富含高浓度CO₃^2-、HCO₃^-和Na⁺，可能栖息具有潜在应用价值的盐单胞菌(Halomonassp.)菌株。【目的】 筛选积聚相容溶质四氢嘧啶的盐单胞菌菌株，并分析四氢嘧啶积聚的影响因素和最优发酵条件。【方法】 采用Horikoshi-I培养基分离扎布耶盐碱湖水样中嗜碱盐且能高效积聚四氢嘧啶的盐单胞菌株，并明确生理生化特性和分类学地位。基于单因素条件、Plackett-Burman、Box-Behnken试验和HPLC检测，分析菌株胞内四氢嘧啶积聚量的关键影响因素并优化其发酵条件。【结果】 筛选获得扎布耶盐碱湖盐单胞菌共计10株(分属5个种)，优势种是嗜碱盐单胞菌(H. alkaliphila，4株，占40%)。H. alkaliphila ZB109的最佳生长盐度范围为0.5-2.5 mol/L，最适生长pH值8.0-10.0，四氢嘧啶的初始积聚量为303.62 mg/L。菌株ZB109的菌落圆形，边缘光滑，呈黄色，革兰氏染色阴性，显微形态呈短杆状、周身纤毛。NaCl、Mg²⁺和底物l-谷氨酸钠浓度是菌株ZB109积聚四氢嘧啶的关键影响因素。采用响应面法优化培养条件，菌株ZB109单批次摇瓶发酵48 h的四氢嘧啶积聚量可达696.313 mg/L。【结论】 菌株ZB109兼具耐盐和耐碱的生长特性，相较于其他分离盐单胞菌分离株，四氢嘧啶的积聚潜力更大，可为后续四氢嘧啶的大规模发酵生产提供良好的菌种种质资源。

Abstract:

[Background] Zabuye Salt Lake with high concentrations of CO₃^2-, HCO₃^-, and Na⁺ may harbor Halomonas strains with potential applications. [Objective] To screen the Halomonas strains capable of accumulating the compatible solute ectoine, analyze the factors influencing ectoine accumulation, and optimize the fermentation conditions for ectoine accumulation in these strains. [Methods] The Horikoshi-I medium was used to isolate the strains with salinity tolerance and capable of efficiently accumulating ectoine from the water samples collected from Zabuye Salt Lake. The strains were then characterized in terms of biochemical properties and taxonomic status. Single factor tests, Plackett-Burman design, Box-Behnken design, and HPLC were employed to analyze the key factors influencing the intracellular ectoine accumulation and optimize the fermentation conditions. [Results] A total of 10 strains belonging to 5 species of Halomonas were isolated from Zabuye Salt Lake, and the dominant species was H. alkaliphila (4 strains, 40%). H. alkaliphila ZB109 presented good growth within the salt range of 0.5-2.5 mol/L and pH 8.0-10.0, with the initial ectoine accumulation of 303.62 mg/L. The colonies of ZB109 were round, yellow, with smooth edge. ZB109 was a Gram-negative bacterial strain with short rod cells growing cilia around. NaCl, Mg²⁺, and the concentration of sodium l-glutamate (substrate) were the key factors influencing the accumulation of ectoine by strain ZB109. After the fermentation conditions were optimized by response surface methodology, strain ZB109 was able to accumulate ectoine up to 696.313 mg/L (48 h) in a single batch of shake flask fermentation. [Conclusion] Strain ZB109 with both salt and alkali tolerance demonstrates stronger potential in the accumulation of ectoine than other wild-type strains of Halomonas. It serves as an elite strain for the large-scale production of ectoine by fermentation.