Environmental DNA-Based Bacterial Community Characteristics in Rural Greywater: A Case Study from Eastern China

Rural greywater management is a critical global challenge due to the lack of centralized treatment in dispersed communities. This study aimed to characterize the pollution characteristics and bacterial community structure of samples from four greywater collection tanks in eastern China using high-throughput sequencing and absolute quantification of the 16S rRNA gene. Pollution characteristics showed spatial heterogeneity: chemical oxygen demand ranged from 19.8 to 272.5 mg/L, total nitrogen from 8.6 to 16.4 mg/L, and dissolved oxygen from 1.3 to 5.3 mg/L. Dissolved greenhouse gases also varied, with N₂O reaching 103.6 ppmv and CH₄ up to 50.4 ppmv. Based on the estimated absolute abundance of 16S rRNA gene copies, we found that the bacterial communities were dominated by Pseudomonadota, Actinomycetota, Bacteroidota, and Bacillota. Key genera such as Acinetobacter, Pseudomonas, and unclassified Enterobacteriaceae were positively correlated with nitrate, suggesting their potential association with denitrification and potential N₂O production. The methanotrophic genus Methyloparacoccus was enriched in a tank with high dissolved organic carbon. Co-occurrence network analysis revealed that core taxa like unclassified Paracoccaceae and Limnohabitans function as module hubs, maintaining community stability. These findings reveal associations between bacterial taxa, pollutant transformation, and greenhouse gas emissions in rural greywater and provide fundamental insights to support the development of low-carbon, resource-oriented treatment technologies.