溶藻菌 CZBC1 的发酵工艺验证及发酵产物对铜绿微囊藻的溶藻效果

Fermentation-process verification of algicidal bacterium Bacillus cereus CZBC1 and algicidal effect of its fermentation products on Microcystis aeruginosa

  • 摘要: 为推进溶藻菌——蜡样芽孢杆菌 (Bacillus cereus) CZBC1的产业化应用,本研究利用2 L与50 L发酵罐系统,对实验室摇瓶获得的最优发酵工艺进行放大验证,通过比较2种规模下菌体浓度、细胞形态演变及过程参数的动态变化评估工艺重现性,并系统评估不同发酵产物及剂量对铜绿微囊藻 (Microcystis aeruginosa) 的溶藻效果。结果显示,溶藻菌CZBC1 50 L发酵罐的发酵参数为:麸皮和糖蜜6.84 g·L−1,酵母膏3.36 g·L−1,氯化钠 (NaCl) 1 g·L−1,磷酸二氢钾 (KH2PO4) 0.2 g·L−1,硫酸镁 (MgSO4) 0.2 g·L−1,氯化铁 (FeCl3) 0.1 g·L−1,3.08% (φ) 硫酸锰 (MnSO4) 1 mL·L−1,碳酸钙 (CaCO3) 0.1 g·L−1。装液量70% (φ),接种量1% (φ),温度30 ℃,转速500 r·min−1,通气量0.6 (V·V−1·min−1),罐压0.03~0.05 MPa,pH 6.5,发酵24 h,芽孢率可达90%以上。2 L罐菌体浓度于第24小时达峰值4.12×109 CFU·mL−1,芽孢率于第36小时超90%;50 L罐菌体浓度于第16小时达峰值3.72×109 CFU·mL−1,芽孢率于第24小时达90%以上,放大培养显著加快了菌体增殖与芽孢分化。溶藻实验表明,发酵原液与未活化芽孢菌粉无溶藻活性;上清液、活化菌粉及营养体均能高效溶藻,第10天溶藻率分别达97.45%、86.73%和97.10%,提示溶藻活性主要依赖于胞外代谢物及菌体代谢活跃状态。剂量效应显示,在初始铜绿微囊藻细胞浓度为8.31×106个·mL−1时,活化菌粉有效起始浓度不低于105 CFU·mL−1,升至106 CFU·mL−1时溶藻率达99.01%,展现出优异的溶藻潜力。综上,研究证实CZBC1菌株及其发酵工艺在蓝藻水华生物防控中具有良好的产业化应用前景。

     

    Abstract: To facilitate the industrial application of the algicidal bacterium Bacillus cereus CZBC1, this study employed 2 L and 50 L fermenter systems to scale up and validate the optimal fermentation process previously established in shake flask experiments, and assessed process reproducibility by comparing the dynamic changes in cell density, morphological evolution and process parameters under the two scales, and systematically evaluated the algicidal efficacy of different fermentation products and their dosages against Microcystis aeruginosa. The optimal fermentation parameters for CZBC1 in the 50 L fermenter were determined as follows: wheat bran and molasses (combined) at 6.84 g·L−1, yeast extract 3.36 g·L−1, NaCl 1 g·L−1, KH2PO4 0.2 g·L−1, MgSO4 0.2 g·L−1, FeCl3 0.1 g·L−1, 3.08% (φ) MnSO4 solution 1 mL·L−1, and CaCO3 0.1 g·L−1. Fermentation was carried out with a filling volume of 70% (φ), an inoculum size of 1% (φ), a temperature of 30 °C, an agitation speed of 500 r·min−1, an aeration rate of 0.6 (V·V−1·min), a fermenter pressure of 0.03–0.05 MPa, and a controlled pH of 6.5. After 24 h of fermentation, the sporulation rate exceeded 90%. In the 2 L fermenter, the cell concentration reached a peak of 4.12×109 CFU·mL−1 at 24 h, with a sporulation rate exceeding 90% at 36 h; in the 50 L fermenter, the peak cell concentration of 3.72×109 CFU·mL−1 was achieved at 16 h, and the sporulation rate exceeded 90% by 24 h, indicating that scale-up significantly accelerated both cell proliferation and sporulation. Algicidal assays revealed significant differences in the efficacy of the various fermentation products: the crude fermentation broth and non-activated spore powder exhibited no algicidal activity, whereas the supernatant, activated spore powder, and vegetative cells all showed potent algicidal effects, achieving 10-day algicidal rates of 97.45%, 86.73%, and 97.10%, respectively, suggesting that the algicidal activity is mainly dependent on extracellular metabolites and the metabolically active state of the cells. Furthermore, dose-response analysis demonstrated that, in an experimental system with an initial M. aeruginosa concentration of 8.31×105 cell·mL−1, the effective threshold concentration of activated spore powder was no less than 105 CFU·mL−1; when the concentration increased to 106 CFU·mL−1, the algicidal rate reached 99.01%, highlighting its excellent potential for cyanobacterial bloom control. In summary, these findings confirm the promising application prospects of the CZBC1 strain and its fermentation process in the biological control of cyanobacterial blooms.

     

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