微生物“暗物质”在微氧条件下的可培养性及代谢特征的多组学研究

Microbial dark matters (MDMs) are the microbial majorities that have not been isolated in the laboratory so far. Many of them exhibit significant great scientific and applicable values. Direct and indirect evidences from recent studies have shown that concentration of atmospheric oxygen during cultivation could be a fundamental factor for successful isolation of many aerobes. Therefore, we propose the hypothesis that low-level oxygen, especially for oxygen less than 1%, is at least one of the prerequisites for cultivation and isolation of many aerobic MDMs. In this project, we plan to study on two types of microbial communities with many MDMs, i.e., activated sludge and marine sediment in the mangrove zone. On one hand, iChip cultivation technique will be applied to evaluate the cultivability of certain samples under different oxygen concentrations and the mechanisms of typical oxygen adaption will be characterized on genetic and physiological levels with genomic and transcriptomic investigations, respectively. On the other hand, oxygen adaptions and metabolic characters under different oxygen concentrations for MDMs with high taxonomic ranks will be revealed on community-level by metagenomic and metatranscriptomic approaches. These characteristics will be adopted for designing cultivation conditions for certain MDMs with high taxonomic ranks. The project is aim to understand the effect of oxygen on the cultivation of MDMs and to illustrate the physiological mechanisms behind it, as well as to uncover the enigma of the uncultivable nature of most microbes with novel ideas and techniques.

微生物暗物质（MDMs)，即迄今为止未被分离培养出的大多数微生物，具有重要的基础研究意义与应用价值。一些直接和间接证据表明，氧和活性氧类物质的浓度是影响许多好氧型微生物无法被培养的主要原因。因此，申请人提出微氧条件，特别是低于1%的氧浓度，是许多好氧MDMs可被实验室分离培养的必要条件之一的假设。本课题将以两类典型样品，即活性污泥和红树林区沉积物作为研究对象，一方面采用iChip培养技术，考察在不同氧水平下可培养微生物的类群，并对典型低氧适应型微生物进行基因组和转录组分析，从分子遗传和生理水平考察适应机制；另一方面基于二代测序的宏基因组和宏转录组方法，分析微氧条件下高分类阶元MDMs在群落水平的低氧适应与代谢特征，并将分析结论应用于特定高分类阶元MDMs的富集和分离。本课题旨在系统阐明氧浓度对好氧MDMs培养的影响及其生理机制，为解决环境中重要MDMs的分离和利用提供新的思路与尝试。

本项目通过比较特定环境样本中（主要是活性污泥和土壤样品）的细菌，特别是被称为微生物“暗物质”的未培养细菌在好氧、厌氧和微氧条件下的可培养性与代谢活性。研究结果明确了微氧条件是许多特定细菌类群能够在培养平板上生长并形成菌落的必要条件，通过转录水平的分析支持了微氧条件是某些未培养细菌门类存活生长所必需的；在项目执行中，基于测序技术，我们建立了一个用于高通量评估培养平板上生长出来的微生物类群的方法，并基于此方法评价了不同氧气条件和培养基的培养组细菌组成，通过实验定量评估了所谓“绝大多数微生物无法培养”这一经典观点；基于该方法，我们成功分离到了一株为潜在新门的细菌EBPR01，证明了此方法对于指导分离微生物暗物质的有效性；另外，我们还通过对平板培养组样品的直接宏基因组测序，获得了大量来自活性污泥体系的微生物基因组，目前正在利用这些基因组的信息和机器学习方法，研究包括培养平板上的细菌对微氧条件的适应性的机制。本项目的相关研究成果，将为从高通量测序和组学手段出发，解决未培养微生物的培养问题提供新的思路。