Mixotrophic partial denitrification coupled with anammox achieves robust nitrogen removal under organic-rich conditions: Insights into metabolic adaptations of sulfur-oxidizing and anammox bacteria.

Abstract

Sulfur-driven partial denitrification coupled with anammox (SPDA) process is a promising sustainable nitrogen removal pathway. However, its microbial adaptability and metabolic mechanism under long-term organic-rich conditions remain poorly understood. In this study, a single-stage mixotrophic partial denitrification-anammox (MPDA) system, driven by both thiosulfate and organic carbon from real domestic wastewater, was established and operated for 292 days. The system demonstrated excellent and robust performance, achieving average ammonia and total nitrogen removal efficiencies of 93.3% and 90.9%, respectively, despite fluctuating organic loading rates (0.35-0.45 kg COD/m³/d). 15 N isotopic tracing revealed that the PDA processes driven by thiosulfate and organic carbon contributed 51.3% and 27.7% of nitrogen removal, respectively. DNA-SIP experiments identified key facultative mixotrophic sulfur-oxidizing bacteria (SOB) (e.g., Thiobacillus and norank_f_Hydrogenophilaceae) as primary contributors to nitrite supply. Notably, the core anammox bacteria, Candidatus Brocadia, exhibited a positive correlation with organic load and was actively labeled by organic 13 C-carbon, suggesting previously overlooked organoautotrophic activity. Metagenomic analysis revealed that Thiobacillaceae employed the Calvin cycle for carbon fixation and possessed a complete pentose phosphate pathway, while Brocadiaceae utilized the Wood-Ljungdahl pathway alongside a near-complete TCA cycle and robust glycogen metabolism to support their mixotrophic capabilities. This study provides novel insights into the metabolic plasticity and synergistic interactions between SOB and anammox bacteria, demonstrating the feasibility and mechanism of the MPDA process for efficient nitrogen removal in organic-laden wastewater.

Keywords

Identifiers

DOI

10.1016/j.watres.2026.125599

PubMed

41740290

Journal

Water research

Year

2026