Plant litter promotes nitrate-sulfate co-removal in constructed wetlands: DOM dynamics and metagenomic insights into carbon, nitrogen, and sulfur transformations.

Plant litter is a low-cost carbon source for constructed wetlands (CWs), but its effectiveness and associated microbial mechanisms in nitrate-sulfate co-contaminated wastewater remains insufficiently understood. This study evaluated cattail and reed litters as in situ carbon sources by integrating substrate characterization, dissolved organic matter (DOM) spectroscopy, microbial community analysis, and metagenomic annotation. Both litter-amended systems effectively promoted simultaneous nitrogen and sulfate removal. The cattail-amended system achieved total nitrogen (TN) removal efficiencies of 73.48%-93.82% and a maximum SO42- removal efficiency of 58.27%, while the reed-amended system achieved 61.10%-91.73% TN removal and 52.90% maximum SO42- removal. DOM optical indices and PARAFAC analysis showed a gradual shift from protein-like to microbial humic-like fluorescence in the cattail-amended system, whereas microbial humic-like fluorescence dominated earlier in the reed-amended system. Metagenomic analysis revealed differences in carbon, nitrogen, and sulfur metabolic potentials. The cattail-amended system showed higher abundances of PPP-related genes associated with NADPH-generating reactions and several denitrification- and sulfur-oxidation-related genes, including narGHI, nirS/K, nosZ, sox, sqr, and fccA/B. The reed-amended system showed higher abundances of selected EMP- and TCA-associated genes and several reductive sulfur genes, including dsr, phs, and ttrABC. The detection of doxD and ttrABC further suggested potential thiosulfate-tetrathionate transformation. Overall, plant litter amendment effectively supported nitrate-sulfate co-removal, while integrated DOM, microbial, and metagenomic analyses revealed different metabolic potentials under cattail and reed amendment.