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

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.