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In the mining-impacted Rio Tinto, Spain, Fe-cycling microorganisms influence the transport of heavy metals (HMs) into the Atlantic Ocean. However, it remains largely unknown how spatial and temporal hydrogeochemical gradients along the Rio Tinto shape the composition of Fe-cycling microbial communities and how this in turn affects HM mobility. Using a combination of DNA- and RNA-based 16S rRNA (gene) amplicon sequencing and hydrogeochemical analyses, we explored the impact of pH, Fe(III), Fe(II), and Cl2 on Fecycling microorganisms. We showed that the water column at the acidic (pH 2.2) middle course of the river was colonized by Fe(II) oxidizers affiliated with Acidithiobacillus and Leptospirillum. At the upper estuary, daily fluctuations of pH (2.7 to 3.7) and Cl2 (6.9 to 16.6 g/L) contributed to the establishment of a unique microbial community, including Fe(II) oxidizers belonging to Acidihalobacter, Marinobacter, and Mariprofundus, identified at this site. Furthermore, DNA- and RNA-based profiles of the benthic community suggested that acidophilic and neutrophilic Fe(II) oxidizers (e.g., Acidihalobacter, Marinobacter, and Mariprofundus), Fe(III) reducers (e.g., Thermoanaerobaculum), and sulfate-reducing bacteria drive the Fe cycle in the estuarine sediments. RNA-based relative abundances of Leptospirillum at the middle course as well as abundances of Acidihalobacter and Mariprofundus at the upper estuary were higher than DNA-based results, suggesting a potentially higher level of activity of these taxa. Based on our findings, we propose a model of how tidal water affects the composition and activity of the Fe-cycling taxa, playing an important role in the transport of HMs (e.g., As, Cd, Cr, and Pb) along the Rio Tinto. [ABSTRACT FROM AUTHOR]