Using reclaimed water as a resource for landscape water replenishment may alleviate the major problems of water resource shortages and water environment pollution. However, the safety of the reclaimed water and the risk of eutrophication caused by the reclaimed water replenishment are unclear to the public and to the research community. This study aimed to reveal the differences between natural water and reclaimed water and to discuss the rationality of reclaimed water replenishment from the perspective of microorganisms. The microbial community structures in natural water, reclaimed water and natural biofilms were analyzed, and the community succession was clarified along the ecological niches, water resources, fluidity and time using 16S rRNA gene amplicon sequencing. Primary biofilms without the original community were added to study the formation of microbial community structures under reclaimed water acclimation. The results showed that the difference caused by ecological niches was more than those caused by the fluidity of water and different water resources. No significant difference caused by the addition of reclaimed water was found in the microbial diversity and community structure. Based on the results of microbial analysis, reclaimed water replenishment is a feasible solution that can be used for supplying river water.
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