Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period.

Suhyun Kim,Md Rashedul Islam,Jang-Cheon Cho,Ilnam Kang

doi:10.3389/fmicb.2021.700637

Suhyun Kim, Md Rashedul Islam + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2021.700637

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Abstract

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3–20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

Highlights

Driven by the recent development of next- and thirdgeneration sequencing technologies, myriads of single-cell amplified genomes and metagenome-assembled genomes have been retrieved from diverse environments using multi-omics approaches (Zhang et al, 2010)
Many abundant freshwater bacterial lineages have been isolated from Lake Soyang during the highthroughput dilution-to-extinction culturing (HTC) campaigns, our research has mainly focused on the stable cultivation of acI bacterial cultures, as the initial HTC cultures of acI bacteria were not revived from frozen glycerol stocks
Our results indicated that the bacterial community composition of Lake Soyang was similar to that of many lacustrine environments worldwide (Newton et al, 2011; Jezbera et al, 2012; Okazaki et al, 2021)

Summary

Introduction

Driven by the recent development of next- and thirdgeneration sequencing technologies, myriads of single-cell amplified genomes and metagenome-assembled genomes have been retrieved from diverse environments using multi-omics approaches (Zhang et al, 2010). These innovative cultureindependent approaches have enabled the discovery of genomic information from uncultured bacteria and archaea, which resulted in the creation of the “microbial dark matter” concept (Rinke et al, 2013; Williams and Embley, 2014). The success of HTC has been attributed to the physical isolation of microorganisms in low-volume growth chambers (microtiter wells) enriched with specific microorganisms under culture conditions mimicking those of their natural habitats (e.g., natural seawater medium)

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