Broad Dissemination of Plasmids across Groundwater-Fed Rapid Sand Filter Microbiomes

Biological rapid sand filtration is a commonly employed method for the removal of organic and inorganic impurities in water which relies on the degradative properties of microorganisms for the removal of diverse contaminants, but their bioremediation capabilities vary greatly across waterworks. Bioaugmentation efforts with degradation-proficient bacteria have proven difficult due to the inability of the exogenous microbes to stably colonize the sand filters. Plasmids are extrachromosomal DNA elements that can often transfer between bacteria and facilitate the flow of genetic information across microbiomes, yet their ability to spread within rapid sand filters has remained unknown. Here, we examine the permissiveness of rapid sand filter communities toward four environmentally transmissible plasmids, RP4, RSF1010, pKJK5, and TOL (pWWO), using a dual-fluorescence bioreporter platform combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results reveal that plasmids can transfer at high frequencies and across distantly related taxa from rapid sand filter communities, emphasizing their potential suitability for introducing bioremediation determinants in the microbiomes of underperforming water purification plants. IMPORTANCE The supply of clean water for human consumption is being challenged by the appearance of anthropogenic pollutants in groundwater ecosystems. Because many plasmids can transfer horizontally between members of bacterial communities, they comprise promising vectors for the dissemination of pollutant-degrading genetic determinants within water purification plants. However, their ability to spread within groundwater-fed rapid sand filters has not been explored. Here, we investigate the transfer dynamics of four transmissible plasmids across rapid sand filter communities originating from three different waterworks in Denmark. Our results revealed a significant ability of natural plasmids to transfer at high frequencies and across distantly related taxa in the absence of plasmid selection, indicating their potential suitability as vectors for the spread of bioremediation determinants in water purification plants. Future work is required to assess the biotechnological applicability and long-term maintenance of exogenous plasmids within sand filter communities.