Enrichment of different taxa of the enigmatic candidate phyla radiation bacteria using a novel picolitre droplet technique.

ABSTRACT

The candidate phyla radiation (CPR) represents a distinct monophyletic clade and constitutes a major portion of the tree of life. Extensive efforts have focused on deciphering the functional diversity of its members, primarily using sequencing-based techniques. However, cultivation success remains scarce, presenting a significant challenge, particularly in CPR-dominated groundwater microbiomes characterized by low biomass. Here, we employ an advanced high-throughput droplet microfluidics technique to enrich CPR taxa from groundwater. Utilizing a low-volume filtration approach, we successfully harvested a microbiome resembling the original groundwater microbial community. We assessed CPR enrichment in droplet and aqueous bulk cultivation for 30 days using a novel CPR-specific primer to rapidly track the CPR fraction through the cultivation attempts. The combination of soil extract and microbial-derived necromass provided the most supportive conditions for CPR enrichment. Employing these supplemented conditions, droplet cultivation proved superior to bulk cultivation, resulting in up to a 13-fold CPR enrichment compared to a 1- to 2-fold increase in bulk cultivation. Amplicon sequencing revealed 10 significantly enriched CPR orders. The highest enrichment in CPRs was observed for some unknown members of the Parcubacteria order, Cand. Jorgensenbacteria, and unclassified UBA9983. Furthermore, we identified co-enriched putative host taxa, which may guide more targeted CPR isolation approaches in subsequent investigations.