SRP471510 PRJNA1039741 Effects of biochar layer position on treatment performance and microbial community in subsurface flow constructed wetlands for removal of cadmium and lead Cd and Pb were considered to be highly toxic to most organisms and can contaminate the food chain. The removal path of Cd and Pb along with the microbial community structure were investigated in the case of constructed wetlands (CWs) with different spatial distribution of biochar. The biochar as a layer at the bottom of CW (BCW) system exhibited maximum Cd and Pb removal efficiencies of 96.64%-98.55% and 97.16%-98.88%. Biochar characterization showed that -COOH, -C=O and -C-OH groups may form complexes with Cd and Pb. CO32- and OH- can also react with Cd and Pb to form Cd/PbCO3 and Cd/Pb(OH)2 precipitates. Compared with original soil (OS), BCW resulted in an increase at the phylum level in the relative proportions of Proteobacteria, Firmicutes, Acidobacteriota, Verrucomicrobiota, Desulfobacterota, Armatimonadota, Bacteroidota, Patescibacteria, Basidiomycota and the genus level were Burkholderia-Caballeronia-Paraburkholderia, Citrifermentans, norank_f__norank_o__Chthonomonadales, Cellulomonas, Geothrix, Terracidiphilus, unclassified_f__Gallionellaceae, Microbacterium, Vanrija, Apiotrichum, Saitozyma Fusarium. Cd and Pb were positively correlated with Verrucomicrobiota and Basidiomycota norank_f__Methylacidiphilaceae Meyerozyma and Vanrija. This work highlighting the value of biochar layer position within the soil substrate to improve the CW performance.