Reclaimed water influences bacterioplankton and bacteriobenthos communities differently in river networks

Reclaimed water reuse is a promising strategy for addressing water scarcity; however, its potential ecological impact remains largely unknown. In particular, the differential effects of reclaimed water on microbial communities in various habitats remain poorly understood. Here, we aimed to elucidate the distinct effects of reclaimed water on bacterioplankton and bacteriobenthos communities in reclaimed water-receiving river networks from multiple perspectives, including community structure, co-occurrence patterns, assembly mechanisms, and nitrogen cycle function. Significant differences in microbial composition were observed between the plankton and benthic habitats, and the average numbers of amplicon sequence variants (ASVs) that originated from the wastewater treatment plants (WWTP) sites were 310.0 and 613.3, respectively, indicating a stronger association between WWTP and benthic habitats. Random forest and network co-occurrence analyses identified the genus Clostridium_sensu_stricto as a biomarker and key module hub. The assembly of bacteriobenthos communities was driven primarily by deterministic processes (58.74% for River-S and 58.94% for WWTP-S), whereas for bacterioplankton communities, this proportion was reduced to 18.02% (River-W) and 19.09% (WWTP-W). The qPCR revealed a large difference in abundance between the N cycling related genes of bacteriobenthos (average 2.47 × 10⁶ copies/ng) and bacterioplankton (average 3.11 × 10³ copies/ng) communities, and different interaction patterns with functional genes. Variance partitioning analysis (VPA) indicated that nitrogen was the most important pollutant, affecting the structure and ecological functions of microbial communities. Moreover, pathway analysis suggested that the reuse of reclaimed water may have enhanced the N-cycling functions of microbial communities and the emission of nitrous oxide.