Perfluoroalkyl substance (PFAS) are persistent and ecotoxic chemicals in aquatic environments. To date, little is known about measures for strengthening PFAS removal in constructed wetlands (CWs). In this study, the removal of the target PFAS perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) was thoroughly investigated in vertical subsurface flow CWs, especially the role of iron oxide and iron-reducing bacteria (FeRB) in the substrate. The PFOA and PFOS removal in the effluent was improved by 33.0% and 40.8%, respectively, in CWs with iron oxide and FeRB. Higher concentration of fluorine ion (0.22 mg/L) and less concentration of acetate (7.5 mg/L) were also detected. The PFOA and PFOS contents were lower in iron oxide based CWs, especially at a substrate depth with poor oxygen (40-50 cm), and resulting in a PFOS content considerably (86.7%) lower than that in the control. Almost 5.07% of PFOA and 24.6% of PFOS can be removed via substrate adsorption. Microbial metagenome analysis revealed an impact of iron oxide on the increased proportion of Proteobacteria, especially β-Proteobacteria. The increased proportions of microbes or enzymes related to Fe reduction or oxidation, e.g., c-type cytochrome, reductase (NADH and NADPH), hydrogenase, ferredoxin oxidoreductase, and genes for PilA, MtrC, MtrD, MtrE, MshA, and FccA were detected, which coincided with the microbial electron transfer and Fe cycle results. Meanwhile, the enhanced functions of carbohydrate metabolism and greater proportions of genes that encoded the key enzymes involved in acetate decarboxylation (M00357: ackA, pta, and acs; Methanosarcina and Methanothrix) played a potential role in PFAS removal. Given the increasing concerns related to PFAS, this study provides an effective and ecological in situ bioremediation method for PFAS removal
