Organic sulfur-driven denitrification pretreatment for enhancing autotrophic nitrogen removals from thiourea-containing wastewater: performance and microbial mechanisms

Abstract

Thiourea (CH₄N₂S) is a widely used industrial reagent and is frequently detected in both sewage and industrial wastewater. However, treating thiourea-containing wastewater remains challenging due to its toxicity, high ammonium concentration, and low C/N ratio. In this study, a novel integrated autotrophic-heterotrophic denitrification (IAHD)- completely autotrophic nitrogen removal over nitrite (CANON) process was developed. The degradation pathway of toxic compounds, nitrogen, and sulfur release and transformation, as well as variations in functional genes were comprehensively examined. The results show that by incorporating an IAHD unit, prior to CANON, toxic thiourea was effectively degraded by the recycled nitrate from CANON. The released sulfur and organic carbon served as electron donors facilitating efficient NO₃^--N reduction. The optimal thiourea/NO₃^--N ratio for IAHD operation was determined to be 4:1 (m:m), achieving NO₃^- and thiourea removal efficiencies of 90 % and 99 %, respectively. Additionally, NH₄⁺-N and SO₄^2--S concentrations increased by 199.9 mg/L and 201.9 mg/L, respectively. Approximately 53.3 % of thiourea was converted into high-molecular-weight biological metabolites in the IAHD unit, which were subsequently and completely degraded in the CANON unit, where a robust nitrite-shunt and anammox process occurred. 16S rRNA amplicon sequencing revealed that Thiobacillus (with a relative abundance of 39.9 %) was the dominant genera in the IAHD unit, followed by Arenimonas (10.8 %) and norank_o_1013-28-CG33 (12.4 %), indicating that sulfur autotrophic denitrification was the primary pathway for thiourea degradation. Metagenomic analysis further confirmed that thiourea, acting as an electron donor, stimulated the expression of key functional genes involved in denitrification, sulfur oxidation, dissimilatory nitrate reduction, hydrolytic oxidation, and amino acid synthesis and transport pathways. These processes contributed to the active biological transformation of carbon, nitrogen and sulfur in the IAHD unit. This study demonstrates that implementing a prior autotrophic-heterotrophic denitrification unit effectively degrades toxic thiourea, thereby ensuring the subsequent nitrogen removal performance of CANON. This approach offers a new paradigm for the treatment of thiourea-containing wastewater, promoting a more efficient and low-carbon process.