A targeted liquid cultivation method for previously uncultured non-colony forming microbes

Eun-Young Seo; Dawoon Jung; Slava S Epstein; Weiyan Zhang; Jeffrey S Owen; Hiroaki Baba; Akina Yamamoto; Mifuyu Harada; Yutaka Nakashimada; Setsu Kato; Yoshiteru Aoi; Shan He

doi:10.3389/fmicb.2023.1194466

A targeted liquid cultivation method for previously uncultured non-colony forming microbes

Front Microbiol. 2023 Jun 9:14:1194466. doi: 10.3389/fmicb.2023.1194466. eCollection 2023.

Authors

Eun-Young Seo^#^{1

2

3}, Dawoon Jung^#^{1

2

3}, Slava S Epstein⁴, Weiyan Zhang¹, Jeffrey S Owen⁵, Hiroaki Baba⁶, Akina Yamamoto⁶, Mifuyu Harada⁶, Yutaka Nakashimada^{3

6}, Setsu Kato^{3

6}, Yoshiteru Aoi^#^{3

6}, Shan He^#^{1

2}

Affiliations

¹ Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, China.
² Ningbo Institute of Marine Medicine, Peking University, Ningbo, China.
³ Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashihiroshima, Japan.
⁴ Department of Biology, Northeastern University, Boston, MA, United States.
⁵ Department of Environmental Science, Hankuk University of Foreign Studies, Yongin, Republic of Korea.
⁶ Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima, Japan.

^# Contributed equally.

Abstract

A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium. The NCFLC was applied to marine samples from a coastal intertidal zone and soil samples from a forest area, and the results were compared with those from the standard direct plating method (SDP). The NCFLC yielded fastidious bacteria from marine samples such as Acidobacteriota, Epsilonproteobacteria, Oligoflexia, and Verrucomicrobiota. Furthermore, 62% of the isolated strains were potential new species, whereas only 10% were novel species from SDP. From soil samples, isolates belonging to Acidobacteriota and Armatimonadota (which are known as rare species among identified isolates) were exclusively isolated by NCFLC. Colony formation capabilities of isolates cultivated by NCFLC were tested using solid agar plates, among which approximately one-third of the isolates were non-colony-forming, approximately half-formed micro-colonies, and only a minority could form ordinary size colonies. This indicates that the majority of the strains cultivated by NCFLC were previously uncultured microbial species unavailable using the SDP method. The NCFCL method described here can serve as a new approach to accessing the hidden microbial dark matter.

Keywords: dilution-to-extinction; liquid cultivation; microbial dark matter; non-colony-forming; uncultured microbes.

Grants and funding

This work was funded by the National Natural Science Foundation of China (42176101), the Ningbo Key Science and Technology Development Program (2021Z046), the National 111 Project of China (D16013), and the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund. This work also was supported by the Japanese Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 22H04887, 20H05587, and 17F17098. DJ was personally supported by the JSPS as an International Research Fellow of the JSPS.