南极乔治王岛冰锥洞微生物培养探索

Cultivation of microbes in habitat of cryoconite at King George Island of Antarctica

摘要:

【目的】南极洲具备独特的环境和相对的生物地理隔离，南极洲各类生境中蕴藏了大量尚未培养和难培养的微生物，也是新颖微生物物种的重要来源之一。本研究以南极冰锥洞这类特殊生境为研究对象，通过培养条件的多样化提升南极微生物的培养率和多样性，揭示南极冰锥洞可培养微生物类群多样性，为该环境可培养微生物功能研究奠定基础，也为南极极端环境未培养微生物的培养方法提供借鉴。【方法】通过采用不同培养基添加复苏促进因子(resuscitation promoting factor, Rpf)的方式，提高南极柯林斯冰盖冰锥洞生境中微生物的可培养率，探究该生境中微生物的多样性。采用4种不同营养水平的培养基，平行添加Rpf进行菌株培养，经分离纯化与16S rRNA基因鉴定，分析冰锥洞可培养微生物的多样性及培养条件对多样性的影响。【结果】本研究共分离培养细菌407株，涵盖5个门、18个科、29个属，其中：放线菌门(Actinomycetota)为优势门，占72.73%；微杆菌科(Microbacteriaceae)为优势科，占69.78%；Lacisediminihabitans属为优势属，占45.70%。从培养基效果分析，培养出菌株数从多到少依次为：R2A培养基(188株)>1/2 R2A+Rpf培养基(144株)>1/2 R2A培养基(46株)>R2A+Rpf培养基(14株)>TSB+Rpf培养基(9株)>TSB培养基(3株)=LB培养基(3株)。而LB+Rpf培养基未培养出微生物。按照文献建议的16S rRNA基因序列相似性98.65%以下为潜在新种依据，分离得到的菌株中共有69株为潜在新菌，涵盖19个潜在新种，分离潜在新种率较高的培养基为R2A培养基、1/2 R2A培养基以及1/2 R2A+Rpf培养基。【结论】本研究共采用8种不同的培养基组合，从南极冰锥洞样品中获得了一定量的可培养细菌，提示了不同培养基添加复苏促进因子对菌株生长的促进作用，并获得多个潜在新种。分离获得的菌株具有较高的多样性，为今后利用不同培养基策略分离极地特殊环境未培养微生物带来启发。

Abstract:

[Objective] Antarctica is a unique habitat harboring diverse microorganisms because of its extreme environmental conditions. The aim of this study is to promote the growth of microorganisms that are difficult to culture by changing the culture conditions to isolate lesser-known microorganisms. This study provides a better understanding of the unique microbial groups and their diversity in Antarctic cryoconites and provides guidance for the development of culture methods for uncultured microorganisms in the extreme environments of Antarctica. [Methods] We added resuscitation-promoting factor (Rpf) in different media to improve the culture efficiency of microorganisms from the cryoconite of the Collins Glacier, Antarctica, and explore the diversity of microorganisms in this habitat. Four media with different nutrient levels were used for strain culture, and Rpf was added in parallels. After isolation, purification, and 16S rRNA sequencing, the diversity of culturable microorganisms in the cryoconite and the influence of culture conditions on the diversity were analyzed. [Results] A total of 407 bacterial strains were isolated and cultured, covering 29 genera, 18 families of 5 phyla. Actinomycetota, Microbacteriaceae, and Lacisediminihabitans were the dominant phylum, family, and genus with the relative abundance of 72.73%, 69.78%, and 45.70%, respectively. According to the number of strains cultured, different media followed the trend of R2A medium (188 strains)>1/2 R2A+Rpf medium (144 strains)>1/2 R2A medium (46 strains)>R2A+Rpf medium (14 strains)>TSB+Rpf medium (9 strains)>TSB medium (3 strains)=LB medium (3 strains). No microorganism was cultured in LB+Rpf medium. According to the threshold of 16S rRNA sequence similarity below 98.65%, which indicates potential new species, 69 out of the isolated strains belonged to 19 potential new species. The media with high isolation rates of potential new species were R2A, 1/2 R2A, and 1/2 R2A+Rpf. [Conclusion] Eight different media were used to recover bacteria from the Antarctic cryoconite samples, and the addition of Rpf can generally promote the growth of strains. A relatively diverse collection of strains including several putative novel species has been isolated, which inspires the future use of different media for the isolation of microorganisms difficult to culture in extreme polar environments.