Carbon–sulfur regulation for deep nitrogen removal in Arundo donax straw/elemental sulfur-amended surface flow constructed wetlands: Metagenomic and metabolomic insights

1:1 A. donax /S 0 achieved 77.6 % TIN removal with low net SO2–4 accumulation. • A. donax /S 0 imbalance was associated with donor limitation or sulfide/DNRA risks. • Vertical profiles showed lower-layer carbon release and NO–3-N reduction. • The 1:1 ratio showed greater carbon oxidation and sulfur transformation potential. • Multi-omics suggested associations between carbon–sulfur metabolism and N removal. Plant-derived carbon can sustain heterotrophic denitrification in low carbon-to-nitrogen (C/N) constructed wetlands, but how the relative loading of elemental sulfur (S 0 ) and solid carbon regulates mixotrophic nitrogen removal remains unclear. In this study, six surface flow constructed wetlands (SFCWs) amended with different Arundo donax straw/S 0 ratios were operated for 96 days to treat simulated secondary effluent. Under the tested conditions, the 1:1 ratio (450 g/450 g) showed the best performance, with a stable-period total inorganic nitrogen (TIN) removal efficiency of 77.6 ± 5.7 % and a relatively low net SO 4 2– accumulation per unit TIN removed (1.47 mg SO 4 2– /mg N). Insufficient S 0 weakened late-stage NO 3 – -N removal; the excess S 0 configuration showed HS – /S 2– accumulation and was associated with a higher risk of dissimilatory nitrate reduction to ammonium without improving nitrogen removal; and carbon-limited configurations showed insufficient electron donor availability. Vertical profiles showed that carbon release and NO 3 – -N reduction were concentrated in the lower layers. Metagenomic analysis showed that the 1:1 configuration enriched representative functional taxa, including hydrolytic/fermentative bacteria ( Clostridium ), sulfur-transforming bacteria ( Sulfuritalea , Thiobacillus ), and nitrogen reduction-related taxa ( Azonexus ). Functional gene abundances suggested greater potential for anaerobic carbon oxidation and sulfur transformation in the 1:1 configuration. Metabolomic analysis indicated substrate-associated changes in sulfur-related metabolites and in pathways related to nicotinamide adenine dinucleotide and coenzyme A. Network analysis further showed that carbon metabolism and sulfur transformation were closely associated with nitrogen removal performance. These results provide new insights into substrate regulation of mixotrophic denitrification in SFCWs treating low C/N wastewater. Download: Download high-res image (198KB) Download: Download full-size image