基于介电电泳原理的深海古菌富集及高通量培养方法优化

Based on dielectrophoresis and dielectric properties of different microbes, microfluidic chip of interdigitated array microelectrodes was designed and used for marine microorganisms’ separation and enrichment. Community structure and diversity of microbes, adsorbed by electrode on the microfluidic chip under different conditions, was analyzed by Illumina Solexa high-throughput sequencing on V4 region of the 16S rRNA gene, to determine the conditions of effective enrichment for deep-sea archaea. A simple high-frequency vibration generator was successfully manufactured to generate uniform alginate beads (about 30μm), which coat single-cell of microbes. The high-throughput culture technology processes were improved after integrating continuous flow culture, high-speed cell sorter and 96-well enrichment culture techniques, which providing technical support for new microbial resources acquisition.

本项目基于介电电泳原理及不同微生物类群介电特性的差异，设计制作了阵列叉指电极微流控芯片，并利用该芯片对深海沉积物来源的微生物进行分离富集。以16S rRNA基因V4区为分子靶标，采用Illumina Solexa高通量测序分析不同介电电泳条件下被电极吸附的微生物多样性及群落结构的变化规律，以确定针对深海古菌的有效富集条件。同时，本项目利用自制简易的高频振动发生器生成大小均一（约30μm）的单细胞海藻酸钙包埋小球，整合流动培养、流式分选及96孔板加富培养技术，改善了该高通量培养技术流程并为新型菌株资源的获取提供了技术保障。

针对大多数微生物尚不能在实验室条件下培养的现状，本项目针对深海沉积物样品，优化了沉积物菌体细胞洗脱，建立了高通量单细胞培养方法。基于介电电泳原理及不同微生物类群介电特性的差异，设计了阵列叉指电极微流控芯片，用于古菌细菌的分离富集，但针对沉积物样品，该方法并未表现出显著的古菌富集效果。利用流式细胞技术，基于菌体细胞大小胞内颗粒度等自身属性，建立了分区富集的分选方法及高通量单细胞培养方法，并利用该方法获得了一批新型菌株。对这些菌株的深入研究，有利于我们深入认识深海微生物资源的多样性、生理代谢特征、生态功能以及环境适应机制。