Identifying Hidden Viable Bacterial Taxa in Tropical Forest Soils Using Amplicon Sequencing of Enrichment Cultures

SIMPLE SUMMARY: The study of a microbial community nowadays mostly relies on environmental DNA (eDNA)-based amplicon sequencing. However, some studies report that this method is not able to capture all bacterial taxa in the community. This study presents an enrichment culture-based amplicon sequencing method to estimate the proportion of culturable bacteria in soil. A bacterial community derived from this method was compared with those derived from culture-independent methods (eDNA-based amplicon sequencing). This study revealed that the majority of cultured bacteria were rare or completely absent in the community detected by the culture-independent method. Nevertheless, the dominant bacterial Operational Taxonomic Units (OTUs) were also observed, as 8 out of the 30 most frequently detected bacteria from eDNA were found in the enrichment cultures. The method proposed in this study could extend bacterial community’s information derived from the culture-independent method. Furthermore, the enrichment culture-based amplicon sequencing method could be a promising tool for quick screening of a culturable bacterial community and its associated function for various applications. ABSTRACT: This study aims to estimate the proportion and diversity of soil bacteria derived from eDNA-based and culture-based methods. Specifically, we used Illumina Miseq to sequence and characterize the bacterial communities from (i) DNA extracted directly from forest soil and (ii) DNA extracted from a mixture of bacterial colonies obtained by enrichment cultures on agar plates of the same forest soil samples. The amplicon sequencing of enrichment cultures allowed us to rapidly screen a culturable community in an environmental sample. In comparison with an eDNA community (based on a 97% sequence similarity threshold), the fact that enrichment cultures could capture both rare and abundant bacterial taxa in forest soil samples was demonstrated. Enrichment culture and eDNA communities shared 2% of OTUs detected in total community, whereas 88% of enrichment cultures community (15% of total community) could not be detected by eDNA. The enrichment culture-based methods observed 17% of the bacteria in total community. FAPROTAX functional prediction showed that the rare and unique taxa, which were detected with the enrichment cultures, have potential to perform important functions in soil systems. We suggest that enrichment culture-based amplicon sequencing could be a beneficial approach to evaluate a cultured bacterial community. Combining this approach together with the eDNA method could provide more comprehensive information of a bacterial community. We expected that more unique cultured taxa could be detected if further studies used both selective and non-selective culture media to enrich bacteria at the first step.