16S rRNA gene copy number variation and sink biomass shape source contributions in community-wide microbial source tracking

Community-wide microbial source tracking (MST) based on 16 S rRNA gene amplicon sequencing is widely used to track fecal contamination, yet its outputs are influenced by technical and ecological factors that complicate interpretation. Here, we tested how 16 S rRNA gene copy number variation (16 S GCN) and sink microbial load shape inferred fecal source contributions using a spike–dilution experiment. River water was diluted to generate three background microbial load levels and then spiked with cow or pig fecal slurry across a concentration gradient. Total bacterial load was quantified by flow cytometry, and 16 S rRNA gene amplicon sequencing data were processed with and without 16 S GCN correction. Source contributions were computed using FEAST. Estimated fecal-source contributions increased with fecal input and were higher under lower background microbial load, indicating that proportional source estimates can vary with sink biomass even under comparable spike-in levels. Copy number correction consistently reduced inferred fecal source contributions and decreased the relative abundance of several feces-derived genera with high predicted 16 S GCN values. Moreover, the magnitude of correction-induced changes in host-source contributions was significantly associated with the community’s GCN profile, including feces-weighted GCN. Together, these results indicate that 16 S-based community-wide MST is sensitive to both background microbial biomass and variation in 16 S GCN across community members, highlighting the need to interpret proportional source fractions along with independent measurements of sink microbial load and careful consideration of 16 S GCN variation. This study provides practical considerations for improving the robustness and comparability of fecal source apportionment in environmental contamination monitoring.