Mixotrophic denitrification with Arundo donax straw and elemental sulfur in constructed wetlands: enhanced nitrogen removal, carbon-sulfur interactions, and greenhouse gas mitigation.

Surface flow constructed wetlands (SFCWs) treating low carbon-to-nitrogen ratio wastewater are often limited by insufficient electron donor availability, leading to inefficient denitrification and greenhouse gas emissions. This study evaluated mixotrophic SFCWs integrated with Arundo donax straw and elemental sulfur (S0) over 160 days. The total nitrogen (TN) removal rate of 794.45 mg N/(m2·d) was achieved, exceeding those of the straw-only and S0-only SFCWs by 24.16% and 213.15%, respectively. Sequential operation further reduced effluent TN to 3.07 ± 0.16 mg/L (78.90% removal). The global warming potential per unit of nitrogen removed was 24.75% lower than that of the heterotrophic SFCW. Metagenomics indicated that straw supplementation enriched heterotrophic genera (e.g., Trichlorobacter and Dechloromonas) and favored their co-occurrence with sulfate-reducing bacteria (e.g., Desulfobulbus), suggesting a potential microbial link between straw-derived carbon metabolism and sulfur transformation. Moreover, genes encoding pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (KOR) were enriched following straw supplementation, suggesting an enhanced metabolic potential for reduced ferredoxin (Fdred) generation. This candidate Fdred-associated route may provide a low-potential electron transfer pathway complementary to NADH-dependent processes. Non-targeted metabolomics further revealed the enrichment of sulfur-containing metabolites (e.g., octyl hydrogen sulfate and sulfated phenolics) and the quinone menadione, providing complementary evidence for sulfur transformation and redox mediator-associated electron transfer potential, respectively. These findings provide new insights into carbon, sulfur, and nitrogen metabolism and inform the design of wetland systems that combine advanced nitrogen removal with reduced operational greenhouse gas intensity.