Anaerobic ammonia oxidation (anammox) is a well-developed biotechnology for treating high-strength ammonium wastewaters. Recently, partial denitrification has been considered as an alternative to supply anammox with the required nitrite. In this study, a process of sulfide-driven partial denitrification and anammox (SPDA) was developed and operated continuously in an upflow anaerobic sludge blanket (UASB) reactor for 392 days. This reactor was fed with synthetic wastewater containing 100 mgN/L nitrate, 80 mgN/L ammonium and 20–80 mgS/L sulfide. After 160 days of operation, the reactor reached stable performance, and the nitrogen removal efficiency and rate were maintained at 80% and 0.29 kgN/(m³•d), respectively. The estimated nitrogen removal via anammox and sulfide-driven denitrification were 87.2% and 12.8%. Additional batch experiments were conducted to investigate the effects of sulfide on anammox and the mechanisms of nitrogen removal in the SPDA system. The following results were obtained: (1) sulfide had an inhibitory effect on the specific anammox activity with IC₅₀ of 9.7 mgS-H₂S/L. (2) The rapid oxidation of sulfide by sulfur-oxidizing bacteria (SOB) could relieve the toxic effects of sulfide on the anammox in the SPDA system. (3) Sulfide bio-oxidation was a two-step reaction with biologically produced elemental sulfur (BPS⁰) as the intermediate, and the second step using BPS⁰ as the electron donor, can efficiently produce nitrite via partial denitrification (NO₃⁻ → NO₂⁻) as a supply for anammox. Finally, a high-throughput sequencing analysis identified Thiobacillus and Sulfurimonas as the dominant genera of SOB in the SPDA system, and Candidatus Kuenenia as the dominant anammox bacteria. Overall, this research gives the foundation for the practical application of sulfide-driven partial denitrification and anammox process in the future.

Sulfide-oxidizing autotrophic denitrification (SOAD) implemented in a moving-bed biofilm reactor (MBBR) is a promising alternative to conventional heterotrophic denitrification in mainstream biological nitrogen removal. The sulfide-oxidation intermediate – elemental sulfur – is crucial for the kinetic and microbial properties of the sulfur-oxidizing bacterial communities, but its role is yet to be studied in depth. Hence, to investigate the performance and microbial communities of the aforementioned new biosystem, we operated for a long term a laboratory-scale (700 d) SOAD MBBR to treat synthetic saline domestic sewage, with an increase of the surface loading rate from 8 to 50 mg N/(m²·h) achieved by shortening the hydraulic retention time from 12 h to 2 h. The specific reaction rates of the reactor were eventually increased up to 0.37 kg N/(m³·d) and 0.73 kg S/(m³·d) for nitrate reduction and sulfide oxidation with no significant sulfur elemental accumulation. Two sulfur-oxidizing bacterial (SOB) clades, Sox-independent SOB (SOB_I) and Sox-dependent SOB (SOB_II), were responsible for indirect two-step sulfur oxidation (S²⁻→S⁰→SO₄ ²⁻) and direct one-step sulfur oxidation (S²⁻→SO₄ ²⁻), respectively. The SOB_II biomass-specific electron transfer capacity could be around 2.5 times greater than that of SOB_I (38 mmol e⁻/(gSOB_II·d) versus 15 mmol e⁻/(gSOB_I·d)), possibly resulting in the selection of SOB_II over SOB_I under stress conditions (such as a shorter HRT). Further studies on the methods and mechanism of selecting of SOB_II over SOB_I in biofilm reactors are recommended. Overall, the findings shed light on the design and operation of MBBR-based SOAD processes for mainstream biological denitrification.

This study investigated the feasibility of a new biological nitrogen removal process that integrates sulfur-driven autotrophic denitratation (NO₃ ⁻→NO₂ ⁻) and anaerobic ammonium oxidation (Anammox) for simultaneous removal of nitrate and ammonium from industrial wastewater. The proposed sulfur(thiosulfate)-driven denitratation and Anammox process was developed in two phases: First, the thiosulfate-driven denitratation was established in the UASB inoculated with activated sludge and fed with ammonium, nitrate and thiosulfate for 52 days until the nitrite level in the effluent reached 32.1 mg N/L. Second, enriched Anammox biomass was introduced to the UASB to develop the integrated thiosulfate-driven denitratation and Anammox (TDDA) bioprocess (53–212 d). Results showed that nitrate and ammonium could be efficiently removed from synthetic wastewater by the integrated TDDA system at a total nitrogen (TN) removal efficiency of 82.5 ± 1.8% with an influent NH₄ ⁺-N of 101.2 ± 2.2 mgN/L, NO₃ ⁻-N of 101.1 ± 1.5 mgN/L and thiosulfate of 202.5 ± 3.2 mg S/L. It was estimated that Anammox and autotrophic denitritation (NO₂ ⁻→N₂) contributed to about 90% and 10% of the TN removal respectively at stable operation. The established TDDA system was further supported by high-throughput sequencing analysis that sulfur-oxidizing bacteria (e.g., Thiobacillus and Sulfurimonas) coexisted with Anammox bacteria (e.g., Ca. Kuenenia and Ca. Anammoxoglobus) in this syntrophic biocenosis. Additionally, batch experiments were conducted to reveal the kinetic rates and to reconcile the stoichiometry of the electron donor/acceptor couples of the TDDA process. The results unraveled the mechanisms in the new bioprocess: i) sulfite and elemental sulfur (S⁰) were initially generated from branched thiosulfate; ii) oxidation of sulfite and elemental sulfur coupled with fast and slow denitratation; iii) nitrite produced from denitratation together with ammonium were effectively converted to dinitrogen gas via Anammox.