基于液滴阵列微流控技术的慢生长微生物的分离培养和鉴定：原理、方法和实例

Microorganisms are widely distributed in environments, and are diverse in species and metabolisms. They play essential roles in the earth's ecosystem and also contribute to biological geocyclings of elements. Currently, only a minor portion of the total microbial diversity has been cultivated at defined culture conditions. It is generally considered that cultivation of the majority of the microbial diversity in lab depends on new strategy and new technology, and the understandings of the physiology and ecology of those uncultured microbes are key to solve the problems that challenge that our human beings. This project is aimed to optimize and establish new technique for cultivation of slow growing microbes, by taking advantage of previous development of a droplet array microfluidic (DAM) system. New devices of anaerobic cultivation will be linked to the DAM system, thus the new system can be used at aerobic, microaerobic and anaerobic conditions. Soil and marine sediment samples will be used to demonstrate the efficiency and usefulness for cultivation of previously uncultured microbes with the newly established systems. Simultaneously, the metagenomes of the soil and marine sediment will be obtained and sequence. The metagenome data will be used to extract information of microbial community structure and to predict metabolic pathways, with emphasis on DNA segments/contigs that are highly similar to those of slow growing microbes. It is expected that high-level new taxa will be discovered and be described of slow growing microbes that represent novel linage of the microbial world. Genomes of selected new taxa will be sequenced, and new physiology and ecological implications of slow growing bacteria will be explored.

微生物物种丰富、代谢多样，但仅有很小一部分微生物能够在实验室培养；分离和培养更多目前尚未培养成功的未培养微生物，对于研究和认识微生物在地球生态系统和物质元素生物化学循环中的重要作用，至关重要。许多未培养微生物是那些生长速度慢、人们对其生长条件和生理需求还不清楚的慢生长微生物。本项目在实验室前期建立的液滴阵列微流控技术分离培养微生物基础上，进一步优化液滴阵列微流控技术，并与厌氧操作技术偶联，建立在好氧、微好氧和厌氧条件下培养慢生长微生物的成套技术。拟选择土壤和海洋沉积物样品为材料，展示技术的有效性和实用性。预期建立一套（好氧、微好氧、厌氧）基于液滴阵列微流控技术的慢生长微生物培养技术，从土壤和海洋沉积物样品中成功培养多个未培养微生物，并建立多个新的微生物高级分类单元，选择部分新分类单元进行基因组测序，初步探讨新分离的慢生长微生物的生理和生态学特性。

微生物物种丰富、代谢多样，但仅有很小一部分微生物能够在实验室培养；分离和培养更多目前尚未培养成功的未培养微生物，对于研究和认识微生物在地球生态系统和物质元素生物化学循环中的重要作用，至关重要。许多未培养微生物是那些生长速度慢、人们对其生长条件和生理需求还不清楚的慢生长微生物。为了丰富微生物资源，拓展对于未知微生物生理和生态学特性的认识，本项目搭建完善了基于微流控技术的慢生长微生物分离培养液滴阵列平台和微液滴分离系统，针对不同环境，建立了（好氧、微好氧和厌氧条件下）慢生长微生物培养体系及方法；利用搭建的平台及培养方法，从土壤样品、海洋样品、极端酸性环境、白蚁及小鼠肠道等环境分离获得多个慢生长及稀有微生物菌株，获得了属/种水平新物种百余株，有效发表十余种，大大提高了环境样品中可培养微生物的比例；研究了分离获得的慢生长微生物的生理生化及基因组特征，揭示了微生物的慢生长遗传机制及环境适应机制。项目执行过程中，培养博士研究生4名，在站博士后2名，形成了一支学科交叉高水平研究队伍。发表文章3篇（标注基金编号），举办国际会议2次，参加国内国际会议并作大会报告5次，研究成果推进了中-泰生物技术联合实验室项目的完成。