Effect of temperature on opportunistic pathogen gene markers and microbial communities in long-term stored roof-harvested rainwater

Roof-harvested rainwater (RHRW) has received increasing attention in recent years as an alternative water source for domestic use, yet its biological stability during storage is not fully understood. This study investigated the effects of temperature (4 degrees C, 20 degrees C and 30 degrees C) on the microbiological characteristics of RHRW over a storage period of 60 days by targeting different microbial groups including total bacteria and fecal indictor Escherichia coil, bacterial opportunistic pathogen genera and species (Legionella spp, Legionella pneumophila, Mycobacterium spp, Mycobacterium aviwn, Pseudomonas aeruginosa), and two amoebas (Acanthamoeba and Vermantoeba vermifonnis). The rainwater chemistry demonstrated no obvious change during storage. The highest biomass was observed in RHRW stored at 30 degrees C, as measured by heterotrophic bacterial counts, adenosine triphosphate, and 16S rRNA gene numbers. Gene markers of E. coli, Legionella spp., P. aeruginosa, and V. vermiformis were detected in fresh RHRW and can persist during RHRW storage; whereas P. aeruginosa was the only species demonstrated significant regrowth at higher storage temperatures (P < 0.05). Acanthamoeba spp. was only detected in RHRW after 50 days of storage at three investigated temperatures, highlighting increased health risks in long-term stored RHRW. Bacterial community compositions were significantly different in RHRW stored at different temperatures, with increased variations among triplicate storage bottles noted at higher temperatures along with storage time. The results provide insights into RHRW storage practices in terms of mitigating microbial contamination risks.