基于复苏培养的海洋微生物分离新方法的机理解析及其应用

Microorganisms are ubiquitous in the ocean environment, where they play key roles in biogeochemical processes, such as carbon and nutrient cycling. However, owing to the prevailing perception that the indigenous marine bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown. Scientists have reasoned that uncultivable microorganisms might grow in pure culture if provided with the chemical components of their natural environment. However, scientists were likely to ignore the fact that that most microbial species in the biosphere that would otherwise be “culturable” may fail to grow because of their growth state in nature, such as dormancy. In our previous study, we constructed a resuscitation-culture system for the high-efficiency isolation of uncultured strains of marine sediment bacteria that are not commonly found in 16S gene clone libraries from marine environments. Here, to further understand this system, the correlation of the environmental factors and the dynamic of the active bacteria community will be analyzed. Meanwhile, the mechanism of the response of active bacteria community to environmental factors will be studied. Then, Molecular Ecological Network analysis combined with comparative genomic analysis will be conducted to explore the effect of bio-factors on culturing the uncultured. Furthermore, by simulating the main resuscitation factors, the rate of isolation of target active bacteria and the potential of this approach on mining marine microbial resources will be evaluated. Thus, this study can help to improve the utilization of the marine samples to mining marine microbial resources, especially for some rare marine samples, and the method provides a new approach to mining marine microbial resources. Also, this work will provide references for the study of the evolution and geographical distribution of marine microorganisms.

海洋中蕴藏丰富的微生物资源，但绝大多数海洋微生物难以人工培养。尽管已有的基于“模拟生境”的微生物培养方法和技术获得了部分未培养微生物，但该策略对休眠态和低丰度微生物的分离培养效果有限。我们发现，基于复苏培养的方法能够显著提高部分低丰度未培养微生物的可培养效率，但其复苏机制还不清晰。本项目以此为研究对象，首先通过考察复苏培养过程中环境因素改变与活跃微生物区系变化的相关性，结合该过程中微生物对环境变化的应答机制，筛选出潜在的复苏因子；其次，通过对培养过程中活跃微生物类群互作关系结合实验验证深入分析休眠微生物复苏的生物类环境因素；最后，在分离培养阶段，通过设计复苏环境对特定活跃微生物类群进行分离培养，并评估其培养效率、评价该方法对海洋微生物资源深度发掘的潜力。本研究将为提高未培养微生物的可培养性，针对性地培养特定微生物类群提供理论依据，也为今后海洋微生物资源的发掘提供新思路。

海洋中蕴藏丰富的微生物资源，但绝大多数海洋微生物难以人工培养。尽管已有的基于“模拟生境”的微生物培养方法和技术获得了部分未培养微生物，但该策略对休眠态和低丰度微生物的分离培养效果有限。通过项目的实施，我们发现，基于复苏培养的方法能够显著提高部分低丰度未培养微生物的可培养效率，随后对其进行深入研究发现，复苏培养过程中环境因素改变与活跃微生物区系变化存在显著相关性。结合该过程中微生物对环境变化的应答机制，筛选出潜在的复苏因子，发现短链脂肪酸在富集培养过程中可能对微生物的复苏起重要作用；其次，通过对培养过程中活跃微生物类群互作关系结合实验验证深入分析休眠微生物复苏的生物类环境因素。利用分子生态网络分析发现，硫酸盐还原细菌可以通过调节富集培养中的pH变化，影响包括海洋滑动菌目等微生物的共现性关系，进而促进其可培养；另外，在分离培养阶段，通过设计复苏环境对特定活跃微生物类群进行分离培养，并评估其培养效率、评价该方法对海洋微生物资源深度发掘的潜力。结果表明添加短链脂肪酸、生长因子等营养物质显著促进难培养微生物的分离培养，分离获得大量潜在新菌，其中包含厌氧分离一株深度进化分支的细菌，经过多相分类研究，成功建立了海洋细菌Kiritimatiellaeota门的1个新纲（Tichowtungiia classis nov.）。最后，通过对复苏培养获得的代表性类群慢生单胞菌目（Bradymonadales）细菌的生态学特性研究发现，该菌为新型捕食型细菌，且广泛分布于全球有盐环境，可能对盐环境微生物群落调控具有重要作用。本项目为提高未培养微生物的可培养性，针对性地培养特定微生物类群提供理论依据，也为今后海洋微生物资源的发掘提供新思路。