利用改良培养基探究西太平洋海水可培养细菌多样性

Exploring the diversity of cultivated bacteria in the Western Pacific waters through improved culture media

摘要:

[目的] 西太平洋复杂的海洋生态环境孕育了其独特的生物群落，蕴含着种类丰富的海洋微生物资源。本研究基于分离培养技术探究了西太平洋海域不同水深细菌的多样性，并尝试通过改良培养基提高海洋细菌可培养性。[方法] 采用改良的2216E固体培养基（IMA）、R2A固体培养基（R2A）、MBM固体培养基（MBM）、TCBS固体培养基（TCBS）和改良的2216E液体富集培养基（IMB）5种不同培养基进行微生物培养，通过菌株分离纯化、16S rRNA基因序列鉴定，分析西太平洋表层至6000 m水深可培养细菌的多样性以及不同培养基在分离培养异养细菌方面的优势。[结果] 本研究共获得1293株异养细菌，分属于4门7纲14目26科52属119种，其中变形菌门（Proteobacteria）为主导类群。纲水平上，γ-变形菌纲（Gammaproteobacteria）、α-变形菌纲（Alphaproteobacteria）和放线菌纲（Actinobacteria_c）为优势菌群。5种培养基所获得的最优势门都为变形菌门，最优势纲都为γ-变形菌纲，除TCBS培养基优势目是弧菌目（Vibrionales），最优势目都为交替单胞菌目（Alteromonadales）。此外，5种培养基在各分类水平上均体现出不同的选择性。5种培养基在种水平上可培养细菌多样性由高到低依次为R2A、IMA、MBM、TCBS以及IMB。分离自R2A的特有属数目最多，可达10个。随水深增加，可培养异养微生物属的数量呈减少趋势。分得菌株中共有68株为潜在新菌，新菌率在IMA、R2A和MBM中相对较高。[结论] 本研究用5种不同培养基从西太平洋海水中获得大量可培养细菌，具有较高的多样性，同时揭示了不同培养基对可培养海洋细菌的选择性。本研究为进一步的生态学研究和分子生物学研究等提供了宝贵的种质资源，也为未来利用改良培养基分离难培养海洋微生物带来启发。

Abstract:

[Objective] The complex marine environment of the Western Pacific has unique ecosystems and biological communities, and contains abundant marine microbial resources. The community structure of bacteria in different water depths in the Western Pacific was studied based on isolation culture technique. We tried to improve the capturability of marine bacteria by modifying the culture medium. [Methods] We used five different culture media in this study: improved 2216E agar medium (IMA), R2A agar medium (R2A), MBM agar medium (MBM), thiosulfate citrate bile salts sucrose agar medium (TCBS) and improved 2216E liquid enriched medium (IMB). Through strain isolation and 16S rRNA gene sequence identification, the community structure and diversity of culturable bacteria from the surface layer to 6000 meters water depth of the Western Pacific were analyzed. We compared the similarities and differences of bacterial diversity in different culture media. [Results] In this study, we isolated and identified 1293 strains in total, which affiliated to four phyla, seven classes, 14 orders, 26 families, 52 genera and 119 species. Proteobacteria were the dominant group recovered from the seawaters. At the class level, γ-Proteobacteria, α-Proteobacteria, and Actinobacteria_c were the dominant microflora. The most dominant phyla obtained from the five cultures were all Proteobacteria, and the most dominant class was γ-Proteobacteria. Except for the TCBS in which the dominant order was Vibrionales, the most dominant order in other four media was Alteromonadales. In addition, the five media exhibited different selectivity at each taxonomic level. The diversity of the cultivable bacteria in the five culture media from high to low was R2A, IMA, MBM, TCBS and IMB. The most unique genera (up to 10) were isolated from R2A. With the increase of water depth, the number of cultivable heterotrophic microorganisms showed a decreasing trend. A total of 68 strains were potential new bacteria among all the isolates, and the rates of new bacteria were relatively high among IMA, R2A and MBM. [Conclusion] In this study, cultivable bacteria from the seawater of the Western Pacific were studied by using five different culture media, showing a high diversity and revealing the selectivity of different media for cultivable marine bacteria. This research has provided valuable microbial resources for other ecological and molecular studies, and it has also brought more inspiration for using improved culture medium to isolate uncultured marine microorganisms in the future.