水-沉积物界面细菌种群协作规律及可培养性研究

The development of molecular ecology revealed unprecedent environmental microbial diversity, but the isolation and cultivation of strains are still crucial for in-depth study on the related biological mechanisms and products. Natural microorganisms survive on population collaboration, thus explaination on this rule and the further improvement of bacterial culturability are an essential and ageless task to impel. Water-sediment interface is a symbol habitat for microbial pollution collaboration and processes a large quantity of uncutured or viable but non-culture bacteria. On the base of years of study on the bacteria at water-sediment interface and their function on phosphorus cycling, we found that the effect of population interaction is an important factor for the bacterial culturing. Using interfacial sediments as environmental materials, this project plans to identify the succession of culture-independent bacterial community by molecular ecological methods; further using the original nutrient element from habitat, interspecific compatibility construction, metabolic networks and simulated experiment, to improve the isolation conditions and culture the uncultured or viable but non-culture bacteria; comparing to the the succession of culture-independent bacterial community, to determine the regulation of alived population dynamics; make further efforts to establish cultivation strategies of uncultivable bacteria mainly based on the population collaboration, to explore their survival mechanism and improve the efficiency of bacterial culturability, develop new bacterial resources, and make progress of microbial taxonomy status in the long run.

分子生态学的发展揭示了环境中前所未有的微生物多样性，但微生物菌种资源的获得，始终是深入生物学机理研究、代谢产物拓新的关键。自然界微生物以种群协作形式赋存，如何揭开这一规律，提高菌物可培养性，是微生物学发展中一个关键的瓶颈问题，需耐心攻克。水-沉积物界面是微生物种群协作形式的良好体现，并蕴藏大量未培养微生物。本项目基于多年对水-沉积物界面微生物与磷物质循环研究，拟以界面沉积物为对象，分子生态学方法鉴定不同条件下不依赖于培养的细菌种群演替规律；改善分离培养条件，采用原始生境因子、种间配伍、代谢网络构建、模拟反应等方法，并结合微毛细管等技术，筛选难培养或活的非可培养菌；比较不依赖于培养的细菌种群演替，确立活菌种群的消长规律和协作方式；重点从种群协作、种间依赖上，建立未培养微生物的培养策略，提高细菌可培养效率，阐释细菌种群协作的规律和相关机制，开发细菌种质资源，丰富系统分类现状。

微生物菌种实物资源的获得，是继分子生态学研究之后，微生物学得以深入探索和应用发展的必要前提。目前能够分离得到的微生物只是环境中实际微生物总量的极少数部分，其中种群协作的隔离是限制其可培养性的关键因素。本项目以太湖界面沉积物为材料，探索细菌种群协作规律和改进分离培养方法，以提高环境微生物可培养效率。基于常规PCR和PMA-PCR，16S rRNA基因克隆文库、高通量测序和传统分离培养的研究，鉴定了界面沉积物中细菌的可培养性，结果表明Steroidobacter、Denitratisoma等为活的不可培养菌属。种群协作对细菌可培养性影响的研究结果表明，在属分类水平上，Microbacterium、Arthrobacter的培养更依赖于种群协作，Kocuria的培养性可在cAMP或H3BO3作用下得以提高；Pedobacter、Exiguobacterium的培养依赖于C4-HSL作用下的协作；Brevundimonas的培养性提高可得益于C4-HSL或H3BO3，等。合适浓度的cAMP、C4-HSL或 H3BO3等信号物质，可分别提高上述对应物种的可培养性；另外原始营养因子保留可使可培养细菌的种类数量提升到1.6倍，结冷胶培养基凝固剂改善可明显增加可培养菌属的种类。以改进的分离培养方法，开发出6株微生物新种质资源，如Nocardioides taihuensis sp. nov.（CGMCC 4.7318T）、Longivirga aurantiacus gen. nov. sp. nov.（CGMCC 4.7317T）等。研究结果为理解环境中细菌种群协作规律和生存机制提供参考和依据，并提高菌物可培养效率，利于种质资源开发。