The implementation of mainstream anaerobic ammonium oxidation (anammox) can facilitate the realization of carbon-neutral wastewater treatment. However, this technology remains challenging owing to the inability to stably provide nitrite. In this study, we developed a novel nitrification and elemental sulfur-based partial autotrophic denitrification/anammox (NS⁰DA) process for mainstream nitrogen removal. The NS⁰DA system consists of a nitrification reactor and a combined elemental sulfur-based denitrification and anammox (S⁰DA) reactor. Each reactor was independently initiated and optimized before being integrated. At mainstream nitrogen levels (48.5 ± 1.7 mg NH₄⁺-N/L) and 25 °C, the NS⁰DA system achieved 89.1 ± 5.7 % total nitrogen (TN) removal efficiency, with an effluent TN concentration of 5.4 ± 2.8 mg N/L. The system exhibited a low N₂O emission factor (0.23 %), significantly lower than other anammox-based systems. The S⁰DA reactor achieved a nitrogen removal rate of 0.53 kg N/(m³·d) with a short hydraulic retention time (2 h). Anammox accounted for 87.3 ± 7.0 % of the TN removal in the S⁰DA reactor. Isotope experiments and kinetic analysis revealed the cooperation between anammox and denitrification for nitrogen removal. Polysulfides formed in the S⁰DA reactor enhanced the utilization rate of elemental sulfur. High-throughput sequencing identified Thiobacillus and Candidatus Brocadia as the dominant genera of sulfur oxidation and anammox, respectively. The nitrogen and sulfur metabolic pathways were further verified through metagenomic analysis. Overall, the NS⁰DA process provides a stable and efficient nitrogen removal process, minimizing oxygen demand, eliminating organic carbon requirements, and reducing N₂O emissions compared to conventional nitrification/denitrification. This approach offers a promising solution for mainstream nitrogen removal in wastewater treatment.

Exploring alternative water sources and improving the efficiency of energy uses are crucial approaches to strengthening the water-energy securities and achieving carbon mitigations in sub(tropical) coastal cities. Seawater use for toilet flushing and district cooling systems is reportedly practical for achieving multiaspect benefits in Hong Kong. However, the currently followed practices are yet to be systematically evaluated for scale expansions and system adaptation in other coastal cities. The significance of using seawater to enhance local water-energy securities and carbon mitigations in urban areas remains unknown. Herein, we developed a high-resolution scheme to quantify the effects of the large-scale urban use of seawater on a city’s reliance on non-local and non-natural water and energy supplies and its carbon mitigation goals. We applied the developed scheme in Hong Kong, Jeddah, and Miami to assess diverse climates and urban characteristics. The annual water and energy saving potentials were found to be 16-28% and 3-11% of the annual freshwater and electricity consumption, respectively. Life cycle carbon mitigations were accomplished in the compact cities of Hong Kong and Miami (2.3 and 4.6% of the cities’ mitigation goals, respectively) but not in a sprawled city like Jeddah. Moreover, our results suggest that district-level decisions could result in optimal outcomes supporting seawater use in urban areas.

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.

Biochemical oxidation and reduction are the principle of biological water and wastewater treatment, in which electron donor and/or acceptor shall be provided. Elemental sulfur (S⁰) as a non-toxic and easily available material with low price, possesses both reductive and oxidative characteristics, suggesting that it is a suitable material for water and wastewater treatment. Recent advanced understanding of S⁰-respiring microorganisms and their metabolism further stimulated the development of S⁰-based technologies. As such, S⁰-based biotechnologies have emerged as cost-effective and attractive alternatives to conventional biological methods for water and wastewater treatment. For instance, S⁰-driven autotrophic denitrification substantially lower the operational cost for nitrogen removal from water and wastewater, compared to the conventional process with exogenous carbon source supplementation. The introduction of S⁰ can also avoid secondary pollution commonly caused by overdose of organic carbon. S⁰ reduction processes cost-effectively mineralize organic matter with low sludge production. Biological sulfide production using S⁰ as electron acceptor is also an attractive technology for metal-laden wastewater treatment, e.g. acid mine drainage. This paper outlines an overview of the fundamentals, characteristics and advances of the S⁰-based biotechnologies and highlights the functional S⁰-related microorganisms. In particular, the mechanisms of microorganisms accessing insoluble S⁰ and feasibility to improve S⁰ bio-utilization efficiency are critically discussed. Additionally, the research knowledge gaps, current process limitations, and required further developments are identified and discussed.

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.

Stabilization of waste activated sludge (WAS) is an essential step for the disposal or reuse. In this study, WAS stabilization via electrochemical pretreatment (EPT) at 0–15V was evaluated for simultaneous dewaterability enhancement, pathogen removal and H₂S mitigation. The mechanism underlying EPT was investigated and discussed based on the changes in the physicochemical (e.g., particle size, zeta potential, hydrophobicity and extracellular polymeric substances) and biological characteristics (i.e. cell morphology, and distribution and percentages of live/dead cells) of WAS with different EPT voltages. The results revealed that EPT disintegrated WAS flocs and disrupted the cell walls leading to a reduction in particle size (by up to 50%), increased release of extracellular and intracellular substances (by up to 4 times) to facilitate WAS stabilization. With EPT at 15V, the capillary suction time of WAS decreased by 42%, and the concentrations of E. coli and indicator pathogens (Salmonella spp. and Streptococcus faecalis) fell by nearly 5 log₁₀ reaching U.S. EPA hygienization levels. Furthermore, EPT at 12V or higher suppressed the amounts of dissolved sulfide and H₂S_(g) produced from the WAS under anaerobic conditions by over 99%. This study demonstrates the feasibility of EPT for simultaneous WAS dewaterability enhancement, pathogen inactivation and H₂S mitigation, providing a one-step alternative for sludge stabilization.

Recovering materials with high value and increasing market demand from sewage and/or sludge is becoming more attractive than recovering traditional resources such as nutrients and biogas. Sulfated polysaccharides (SPs) are valuable and scarce raw materials that can only be produced from marine algae and a few types of animal tissues. This study evaluated if SPs are present in activated sludge obtained from saline sewage with a high level of sulfates present. The presence of SPs-containing extracellular polymeric substances (EPS) was confirmed and quantified for both sludge from lab-scale reactors and full-scale plants for the first time. SPs in the sludge of a lab-scale reactor operated under alternating aerobic/anoxic conditions with 500 mg/L sulfate in the influent (which is typical of Hong Kong saline sewage) reached 342.8 ± 0.3% mg/gVSS, and sludge taken from a full-scale saline wastewater treatment plant (WWTP) contained 418.1 ± 0.4% mg/gVSS of SPs. Purity of the extracted SPs was comparable to that of commercial industrial-grade products. Key bioactivities of SPs (i.e. fucoidan, carrageenan and heparin), namely anti-angiogenesis, anticoagulant and antioxidant, were confirmed after extraction and purification. Interestingly, operating conditions had a strong influence on the contents and types of SPs synthesized in sludge as well as its bioactivities. Although the detailed synthetic pathways of SPs in activated sludge remain unclear, the current study has made a first attempt to recover a high–value scarce resource from biological wastewater treatment.

Recently, the Sulfate reduction Autotrophic denitrification Nitrification Integrated (SANI^®) process was developed for the removal of organics and nitrogen with sludge minimization in the treatment of saline sewage (with a Sulfate-to-COD ratio > 0.5 mg SO₄ ^2--S/mg COD) generated from seawater used for toilet flushing or salt water intrusion. Previously investigated in lab- and pilot-scale, this process has now been scaled up to a 800-1000 m³/d full-scale demonstration plant. In this paper, the design and operating parameters of the SANI demo plant built in Hong Kong are analyzed. After a 4-month start-up period, a stable sulfur cycle-based biological nitrogen removal system having a hydraulic retention time (HRT) of 12.5 h was developed, thereby reducing the amount of space needed by 30-40% compared with conventional activated sludge (CAS) plants in Hong Kong. The demo plant satisfactorily met the local effluent discharge limits during both the summer and winter periods. In winter (sewage temperature of 21 ± 1 °C), the maximum volumetric loading rates for organic conversion, nitrification, and denitrification were 2 kg COD/(m³·d), 0.39 kg N/(m³·d), and 0.35 kg N/(m³·d), respectively. The biological sludge production rate of SANI process was 0.35 ± 0.08 g TSS_produced/g BOD₅ (or 0.19 ± 0.05 g TSS/g COD), which is 60-70% lower than that of the CAS process in Hong Kong. While further process optimization is possible, this study demonstrates the SANI process can be potentially implemented for the treatment of saline sewage.

Sulfate-reducing bacteria (SRB) can beneficially be applied to domestic wastewater treatment. In general, formed sulfide will stay in liquid phase, resulting in an elevated sulfide content, which might have inhibiting effects on the SRB. To study effects of environmental conditions on the SRB resistance against sulfide, two sequencing batch reactors fed with artificial domestic wastewater were operated at sulfate-reducing conditions. Required sulfide concentration within the reactor was achieved by adding 400 or 800 mg COD/L (acetate and propionate), the latter resulting in proportionally more sulfide production. Batch tests revealed that sulfide inhibited the rate of sulfate reduction by 50% at a concentration of 200 mg/L sulfide for biomass from the reactor fed with 400 mg COD/L. After adaptation to a feed of 800 mg COD/L, resulting in higher sulfide exposures, sulfide was less inhibitive to SRB. Complete COD removal was achieved in the reactor fed with 800 mg COD/L, and the SRB population changed from one (Desulfotalea arctica) to two (Desulfobacter postgatei and Desulfocapsa sulfexigens) dominant species. Results indicate that SRB are capable of adapting to higher sulfide exposure. Therefore, the SRB can also be applied to treat wastewater with higher COD levels, blackwater for instance.