# River Microbiomes as Sentinels of National‐Scale Freshwater Ecosystems

## TL;DR

River biofilms can serve as sensitive indicators of freshwater health, offering a scalable approach to monitor ecological changes using microbial communities.

## Contribution

This study provides the first national-scale assessment of river biofilm bacterial communities using 16S rRNA sequencing and identifies ecological thresholds through threshold indicator analyses.

## Key findings

- River biofilm communities are structured around a consistent core microbiome and are strongly influenced by alkalinity, dissolved oxygen, nitrate-N, and temperature.
- Network analysis revealed modular organization with keystone taxa that likely contribute to community stability.
- Threshold indicator analyses identified taxa with ecological breakpoints, suggesting potential for early detection of environmental changes.

## Abstract

Freshwaters face increasing pressures from chemical, hydrological and climatic changes, yet tools for assessing their condition remain limited. River biofilms, composed of diverse microbial communities, integrate environmental signals over space and time, making them sensitive indicators of river health. Using 16S rRNA gene sequencing of more than 1600 biofilms collected across a national river network, we quantified bacterial diversity and community composition and applied network analysis to identify ecologically cohesive sub‐communities with keystone taxa underpinning community stability. Alkalinity, dissolved oxygen, nitrate‐nitrogen and temperature were among the principal gradients shaping community composition. Threshold indicator analyses identified taxa with breakpoints along these gradients, revealing interpretable ecological thresholds. Our results demonstrate the potential for microbiome‐based monitoring frameworks that could complement existing biotic indices, enabling early detection of ecological changes and supporting the integration of genomic indicators into routine ecosystem assessment. This scalable approach offers a powerful strategy for managing freshwaters under accelerating anthropogenic pressures.

River biofilms are complex microbial assemblages that underpin aquatic food webs and play a central role in biogeochemical cycling. By responding to environmental signals over space and time, biofilm microbial communities can act as sensitive indicators of river health. We present the first national‐scale assessment of river biofilm bacterial communities in England, integrating 16S rRNA gene sequencing from > 1600 biofilms with high‐resolution water chemistry data spanning the river network. Biofilm communities were highly diverse yet structured around a consistent core microbiome. Network analysis revealed a modular organisation with keystone taxa likely contributing to community stability. Community composition was strongly structured by alkalinity, dissolved oxygen, nitrate‐N and temperature, and threshold indicator analyses identified taxa exhibiting clear ecological breakpoints along these gradients. Together, these results demonstrate the ecological significance of river biofilms and establish a scalable framework for incorporating microbial indicators into freshwater monitoring. This approach offers opportunities for earlier detection of ecological disruption, improved insight into ecosystem resilience and strengthened freshwater management under intensifying anthropogenic pressures.