Effects of Spatial and Temporal Variability on Bacterial Diversity and Community Composition in Roof-Harvested Rainwater across Central Virginia

Increasing global water scarcity has sparked interest in more sustainable water sources such as rainwater harvesting (RWH). Because these systems utilize roof-runoff for collection, they can easily be contaminated with environmental bacteria from animal feces and leaf litter, potentially degrading water quality. Thus, questions remain about the safety of harvested rainwater for human use due to the limited understanding of the microbial communities contained within it. To date, there is little published research on the microbiomes of rainwater harvesting systems (RWHS) within the continental United States. This study aimed to characterize the bacterial diversity and community composition of RWHS across Central Virginia. Water samples were collected from ten systems located in four cities spanning rural farmland, small towns, and urban areas. Samples were filtered through 0.22 μm membranes to capture resident bacteria and analyzed using Nanopore sequencing of the 16S rRNA gene. A total of 80 samples were analyzed, including one sample from each system during summer 2023 and winter 2023 (n = 20), and three samples per system during summer 2024 and winter 2024 (n = 60). Alpha diversity did not differ significantly between seasons or broader geographic locations but varied significantly between individual tanks. Beta diversity analyses indicated that site-specific factors had a stronger influence on microbial community composition over either seasonal or regional differences. These findings highlight the role of localized environmental conditions in shaping rainwater microbiomes and suggest that site-specific characteristics are the primary drivers of microbial community structure in harvested rainwater systems.