# Multi-Metagenome Analysis Unravels Community Collapse After Sampling and Hints the Cultivation Strategy of CPR Bacteria in Groundwater

## TL;DR

This study explores how CPR bacteria in groundwater change after sampling and suggests strategies to cultivate them, which could help understand their roles and characteristics.

## Contribution

The study introduces a multi-metagenome approach to understand CPR bacteria's ecological roles and proposes actionable cultivation strategies.

## Key findings

- CPR communities in groundwater collapse rapidly after sampling due to competition with non-CPR bacteria.
- Ampicillin helps stabilize CPR communities by suppressing competitors.
- Long-term enrichment revealed CPR bacteria's resilience and identified potential symbiotic partners for co-culture trials.

## Abstract

Groundwater harbors phylogenetically diverse Candidate Phyla Radiation (CPR) bacteria, representing an ideal ecosystem for studying this microbial dark matter. However, no CPR strains have been successfully isolated from groundwater, severely limiting further research. This study employed a multi-metagenome approach, integrating time-resolved sampling, antibiotic/nutrient interventions, and microbial correlation networks to unravel CPR ecological roles in groundwater and provide insights into their subsequent cultivation. Through 36 metagenomes from a groundwater system containing at least 68 CPR phyla, we revealed the time-sensitive collapse of CPR communities: total abundance plummeted from 7.9% to 0.15% within 48 h post-sampling, driven by competition with rapidly dividing non-CPR bacteria, such as members of Pseudomonadota. Ampicillin (100 mg/L) stabilized CPR communities by suppressing competitors, whereas low-nutrient conditions paradoxically reversed this effect. Long-term enrichment (14 months) recovered 63 CPR phyla (0.35% abundance), revealing their survival resilience despite nutrient deprivation. Correlation networks prioritized Actinomyces, a novel Acidimicrobiaceae genus, Aestuariivirga, Baekduia and Caedimonadaceae as potential CPR partners, providing actionable targets for co-culture trials. Here, we propose actionable recommendations spanning groundwater sampling, activation status, identification of CPR symbiotic partners, and optimization of culture conditions, which bypass traditional blind cultivation and are critical for future efforts to cultivate CPR bacterial strains from groundwater. Cultivating CPR bacteria will contribute to clarifying their diversity, ecological roles, evolutionary mechanisms, metabolic pathways, and genetic potential.