大气压冷等离子体调控微生物细胞膜通透性的研究

An interested phenomenon was discovered in our previous study, that permeability of microbial cell membrane was increased, resulting in microbial growth, by cold plasma at atmospheric pressure under the specific discharge parameters. However, the mechanism of membrane permeability activated by plasma still remains unclear. Activation of ion channels is one of the reasons that the membrane permeability is enhanced. Calcium ion (Ca2+) is an essential messenger, which mediates a wide variety of cellular processes, including cell growth. It is very significant to study the regulation of calcium channel by plasma. Calcium channels are activated by membrane depolarization due to alteration of electrochemical gradient. Correspondingly, the specific activity and protein expression of Ca2+-dependent ATPase are enhanced with an immediate and transient elevation of the Ca2+ concentration in the cytosol ([Ca2+]cyt), which drives the secondary transport of metabolic products and nutrients, alleviating the inhibition of products and promoting cell growth and formation of target products. Cold plasma at atmospheric pressure could produce various reactive factors, including reactive oxygen species (ROS), ultraviolet (UV) radiation, energetic ions, and charged particles. Therefore, plasma possesses physical and chemical characteristics. Compared with traditional methods of metabolic regulation, such as the physical method or chemical method, the effect of plasma on cell membrane permeability is more notable. To explore the mechanism of cell membrane permeability regulated by cold plasma at atmospheric pressure, in this program, considerable attention is focused on the researches of the property of plasma discharge, the expressional and functional changes of calcium channels, as well as the alteration of Ca2+-dependent ATPase activity. These results will provide a theoretical basis for a novel approach of metabolic regulation, control of cold plasma at atmospheric pressure, and also lay a foundation for an equipment of in-situ enhanced microbial fermentation by cold plasma.

我们前期研究发现一个有趣现象：大气压冷等离子体在特定放电条件下，可以提高微生物细胞膜通透性，促进菌体生长。但是，等离子体提高细胞膜通透性机理尚不明确。细胞膜通透性提高机制之一是离子通道开放。钙离子是重要第二信使，因此研究等离子体对钙通道调控具有重要意义。电化学势能变化使细胞膜去极化，钙通道开放，胞内钙离子流瞬间变化，提高钙依赖ATPase离子泵跨膜动力，引起代谢产物和营养物质二次运输，解除代谢产物抑制，促进细胞生长和目标产物形成。大气压冷等离子体含有大量活性因子，同时具有物理和化学活性，与单一物理或化学调控方法相比，其调控效果更加显著。因此，本项目拟从等离子体放电性质、钙通道表达和功能差异、钙依赖ATPase活性三个方面，揭示大气压冷等离子体调控细胞膜通透性机理。该研究为建立一种新型代谢调控方法- - 大气压冷等离子体法提供理论依据，最终为建立大气压冷等离子体在线强化微生物发酵装置奠定基础。

本研究将大气压介质阻挡放电等离子体作为一种新型代谢调控方法，用于提高工业微生物细胞膜通透性，从而提高菌体生产能力。大气压介质阻挡放电等离子体预处理4 min Klebsiella pneumoniae接种体，在6%甘油发酵培养基中进行间歇发酵，最终1，3-丙二醇产量和生产强度均比对照提高56%，细胞膜通透性在发酵过程中始终高于对照组。从酵母浸粉中分离筛选出一株高产乙醇菌株DL5168，经形态学观察、生理生化学及分子生物学鉴定，确定该菌株为酵母属的酿酒酵母（Saccharomyces cerevisiae）。将该菌株用于等离子体强化葡萄糖发酵生产乙醇中。不同处理时间S. cerevisiae 接种体在发酵不同时期，细胞膜电位、膜通透性和细胞质内钙离子浓度均出现不同程度增加或减少，而且酿酒酵母乙醇生产能力也发生改变。建立响应面方法优化实验条件，并将该方法应用到优化与等离子体放电相关的实验参数中。优化条件处理酿酒酵母接种体，发酵最终菌体生物量、乙醇浓度和乙醇转化率分别比对照提高24%，42%和33%。综上所述，我们得到初步结论，大气压介质阻挡放电等离子体可以通过调控细胞膜通透性来提高工业微生物生产能力。