低生物可利用铁情况下硇洲岛潮汐带沉积物中细菌多样性及分布规律

Iron is a micronutrient that is essential for a range of important enzymatic processes in most organisms.It is present in the aerobic environment as insoluble iron(III) minerals.Iron deficiency of ocean sediments is not triggered by total iron, but triggered by low bioavailable iron. So microbia product siderophores to meet the demand of iron.Based on the previous research work, this project intends to study the bacteria (including actinomycetes) diversity, diversity of producting siderophore bacteria and diversity of bacteria which growth depend on producting siderophore bacteria in the same sample from intertidal sediments of Naozhou Island applying 454 sequencing technology, pure culture method, co-culture method and the phylogenetic analysis based on 16S rRNA gene sequence.It has important academic significance that know about the diversity and distribution regularity of producting siderophore bacteria and bacteria which growth depend on producting siderophore bacteria under the condition of serious lack of bioavailable iron in ocean.We will have an overall understanding about diversity of bacteria from intertidal sediments of Naozhou Island by polyphasic taxonomy and published of potential new taxa to uncultured bacteria isolations.It can provide experimental basis and material for carrying out bacteria evolution and adaptation mechanism under the condition of serious lack of bioavailable iron in ocean and lay a foundation for development and utilization of potential new taxa and uncultrue marine microoganism resources.

在生物当中，铁是一系列催化反应中必不可少的微量元素。在有氧环境中，铁以不溶三价铁存在。海洋沉积物中铁的缺乏不是由低总铁引起，而是由低的生物可利用铁引起，微生物产生铁载体满足对铁的需求。在前期研究工作基础之上，本项目拟应用454测序技术、纯培养法、共培养法和基于16S rRNA基因序列的系统发育分析方法对硇洲岛潮汐带同一沉积物样品中细菌（包括放线菌）多样性，产铁载体细菌的多样性，依赖产铁载体细菌生长的细菌多样性进行研究。从而了解在海洋严重缺生物可利用铁的情况下，产铁载体细菌及其促生细菌多样性和分布规律，具有重要学术意义。对分离出的“未培养微生物”，中的潜在新类群进行多相分类并有效发表，可以全面地认识硇洲岛潮汐带沉积物细菌的多样性，为开展在缺生物可利用铁情况下细菌进化与适应机理等提供实验依据与材料，并为下一步开发利用潜在新类群、“未培养微生物”等海洋微生物资源奠定基础。

本课题应用454测序技术、纯培养法、共培养法和基于16S rRNA基因序列的系统发育分析方法对硇洲岛潮汐带沉积物样品中细菌（包括放线菌）多样性，产铁载体细菌的多样性，依赖产铁载体细菌生长的细菌多样性进行研究。454测序技术结果显示从样品中共获得OTU（operational taxonomic unit） 62万多条，所有这些OTU属于原核微生物的42个门（phylum），其中放线菌门、拟杆菌门、厚壁菌门、变形菌门、浮霉菌门、酸杆菌门为主要优势类群；在所有OTU中Proteobacteria 占到33万多条，超过50%。用6种培养基对沉积物中细菌进行分离，共分离到645株纯培养物，系统发育结果表明沉积物中可培养的微生物分属于4门、28科，34属，在645株菌中，Proteobacteria占45%，Firmicutes占34%，Actinobacteria占12%。共培养结果表明共分离纯化出529株细菌，分属于3门、15科。全部1174株分离菌株系统发育表明，178株细菌为潜在新物种，并对其中6株进行了多相分类研究。对纯培养得到的645株细菌进行产铁载体筛选，显示产铁载体菌平均百分比约为65%，有约35%的细菌用CAS法没有检测出产铁载体。对共培养得到的529株细菌进行产铁载体筛选，发现22个属378个菌株在CAS平板检测上不形成黄色晕圈，可能依赖其它产铁载体菌株生长。通过本项目系统了解硇洲岛潮汐带沉积物中原核生物多样性，产铁载体细菌多样性、产铁载体细菌促生微生物多样性为开发利用本地微生物资源打下基础。本项目获得的产铁载体细菌和不产铁载体细菌为进一步研究微生物外源铁载体的欺诈行为及开展在缺生物可利用铁情况下细菌铁载体欺诈行为进化与适应机理等提供实验依据与材料，具有重要意义。