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.

In this study a University of Cape Town (UCT) Integrated Fixed Film Activated Sludge (IFAS) Membrane BioReactor (MBR) wastewater treatment plant was monitored in terms of nitrous oxide (N₂O) emissions. The short term effect on the N₂O emission due to the influent carbon-to-nitrogen (C/N) ratio variation (C/N ratios of 2, 5 and 10 gCOD/gN) was evaluated. Since in a previous study, the effect of the C/N ratio was studied in the same system without biofilm (UCT-MBR configuration) the main aim here was to investigate the role of biofilms on N₂O emissions. Under all the investigated C/N ratios, the N₂O fluxes and the average emission factors were lower than that of previous studies with no biofilm presence. The total average N₂O emission was 0.5% of the influent nitrogen with biofilm (IFAS system) and 3.5% without biofilm. This result emphasizes the potential role of the biofilms in attenuating the N₂O emissions especially in the case of stress conditions (i.e., low C/N influent ratios). An increase of N₂O flux from the anoxic reactor (till 28 mgN₂O m^-2h^-1) occurred at the lowest influent C/N tested (2 gCOD/gN - phase III). At C/N equal to 2 gCOD/gN the anoxic reactor was the main source of N₂O, contributing 45% of all produced N₂O. This result was attributed to an incomplete denitrification caused by a lack of organic carbon and a slight increase of dissolved oxygen concentration.