Strong catchment-specific structuring of Swedish wastewater microbiomes in a paired two-timepoint metagenomic survey

Wastewater microbial communities integrate signals from human populations, environmental inputs, and sewer infrastructure, but the extent to which these communities vary between wastewater catchments compared with individual sampling occasions remains incompletely understood.

We analyzed influent wastewater from 16 wastewater treatment plant sites across Sweden, collected at two paired within-year timepoints, Week 3 and Week 21, using shotgun metagenomic sequencing and compositional data analysis.

Classified genus-level profiles were dominated by bacteria (96.54%), with smaller contributions from viruses (2.03%), eukaryota (1.05%) and archaea (0.40%). Genus-level alpha diversity increased between the two sampled timepoints, with median within-site changes of +10 genera in richness and +0.21 in Shannon diversity (p < 0.003). In contrast, overall community composition was primarily structured by wastewater treatment plant site: site explained 58.3% of total variance (p = 0.0003), whereas sampling timepoint explained 3.7% and was not significant (p = 0.106). Within-site compositional change between the two timepoints was nevertheless evident (p = 4.8 × 10^-4), but the magnitude and direction of change varied across sites, indicating heterogeneous local shifts rather than a synchronized national temporal pattern. Genera detected in at least 75% of sites at both sampled timepoints accounted for most classified community abundance, whereas most measured within-site Aitchison turnover was accounted for by genera outside the high-prevalence shared fraction. Geographic distance and the number of connected inhabitants showed no significant association with genus-level community composition.

These findings indicate that Swedish influent wastewater microbiomes are strongly catchment-specific across paired sampling timepoints and support the use of site-specific reference profiles when interpreting wastewater metagenomic data. Denser temporal sampling and additional catchment metadata will be needed to assess seasonality, long-term stability, and the local drivers of wastewater microbiome variation.