Department of Chemical Engineering and Environmental Technology, Faculty of Chemistry, University of Oviedo, C/ Julián Clavería s/n, 33071, Oviedo, Spain.
Appl Microbiol Biotechnol. 2012 Dec;96(6):1465-77. doi: 10.1007/s00253-012-4254-2. Epub 2012 Jul 10.
Physiological heterogeneity constitutes a critical parameter in biotechnological systems since both metabolite yield and productivity are often hampered by the presence of undesired physiological cell subpopulations. In the present study, the physiological status and functionality of Pseudomonas taetrolens cells were monitored by multiparameter flow cytometry during fermentative lactobionic acid production at the shake-flask and bioreactor scale. In shake-flask fermentation, the onset of the lactobionic acid production phase was accompanied by a progressive loss of cellular metabolic activity, membrane polarization, and membrane integrity concomitantly to acidification. In fact, population dynamics has shown the prevalence of damaged and dead subpopulations when submitted to a pH < 4 from 16 h onwards. Furthermore, fluorescence-activated cell sorting revealed that these sublethally injured cells were nonculturable. In contrast, P. taetrolens cells exhibited a robust physiological status during bioreactor cultivations performed with a pH-shifted strategy at 6.5, remaining predominantly healthy and metabolically active (>96 %) as well as maintaining bioconversion efficiency throughout the course of the fermentation. Additionally, an assessment of the seed culture's physiological robustness was carried out in order to determine the best seed culture age. Results showed that bioreactor culture performance, growth, and lactobionic acid production efficiency were strongly dependent on the physiological heterogeneity displayed by the seed culture. This study provides the most suitable criteria for optimizing lactobionic acid production efficiency through a novel flow cytometric-based approach based on the physiological status of P. taetrolens. It also constitutes a valuable, broad-ranging methodology for the enhancement of microbial bioprocesses involved in the production of secondary metabolites.
生理异质性是生物技术系统中的一个关键参数，因为代谢产物的产率和生产力经常受到不理想的生理细胞亚群的存在的阻碍。在本研究中，通过多参数流式细胞术监测了发酵乳糖酸生产过程中摇瓶和生物反应器规模下的 Pseudomonas taetrolens 细胞的生理状态和功能。在摇瓶发酵中，乳糖酸生产阶段的开始伴随着细胞代谢活性、膜极化和膜完整性的逐渐丧失，同时伴随着酸化。事实上，群体动力学表明，当 pH<4 时，从 16 小时开始，受损和死亡的亚群占主导地位。此外，荧光激活细胞分选显示，这些亚致死损伤的细胞不可培养。相比之下，当采用 pH 偏移策略在 6.5 下进行生物反应器培养时，P. taetrolens 细胞表现出强大的生理状态，保持主要的健康和代谢活性（>96%），并在整个发酵过程中保持生物转化效率。此外，还评估了种子培养的生理稳健性，以确定最佳的种子培养龄。结果表明，生物反应器培养性能、生长和乳糖酸生产效率强烈依赖于种子培养所表现出的生理异质性。本研究通过基于 P. taetrolens 生理状态的新型流式细胞术方法，为优化乳糖酸生产效率提供了最合适的标准。它还为涉及次生代谢产物生产的微生物生物过程的增强提供了一种有价值的、广泛的方法。
