Advancements in Microbial Nitrogen Pathways for Sustainable Wastewater Treatment

Abstract

Over the past few decades, the discovery of novel microbial processes, biochemical reactions, and previously uncharacterized microorganisms has significantly enhanced our understanding of nitrogen (N) cycling across terrestrial and aquatic ecosystems, including engineered environments such as wastewater treatment systems. These scientific advancements are catalyzing a paradigm shift toward treatment strategies that are not only energy-efficient and cost-effective, but also environmentally sustainable, with the added benefit of mitigating greenhouse gas emissions. The current review highlights recent breakthroughs in microbial N cycling, with particular emphasis on their practical applications in wastewater treatment. Emerging processes, such as nitrous oxide (N₂O) mitigation, electro-anammox, ferric iron-dependent ammonium oxidation (Feammox), and complete ammonia oxidation (comammox), offer promising strategies for sustainable and low-energy N removal. Nevertheless, a significant challenge persists in translating these laboratory-scale innovations into full-scale, real-world applications, especially within decentralized treatment infrastructures. Bridging this gap is essential for realizing robust, low-carbon, and sustainable wastewater management systems in the decades to come.

1. Introduction

Nitrogen (N) is an essential element for the survival and growth of all living organisms. Its transformation and bioavailability are governed by a complex network of biogeochemical processes collectively referred to as the N cycle [1,2]. This cycle encompasses a range of microbial pathways that facilitate the conversion of N among gaseous, inorganic, and organic forms [1,3]. Microorganisms, therefore, play a pivotal role in regulating N dynamics across terrestrial, aquatic, and atmospheric ecosystems. However, anthropogenic activities, particularly those associated with agricultural intensification, have significantly disrupted the natural N cycle [4,5,6]. The widespread application of synthetic fertilizers, primarily produced via the Haber-Bosch process, which converts atmospheric N₂ into reactive ammonium (NH₄⁺), has greatly increased the global reservoir of bioavailable N [3,7,8]. This anthropogenic enrichment has precipitated a range of environmental challenges, including groundwater contamination, eutrophication of aquatic systems, elevated N concentrations in wastewater, and enhanced emissions of greenhouse gases (GHGs) [7,9,10,11].

Wastewater treatment systems play a vital role in reducing anthropogenic impacts on the N cycle by alleviating nitrogenous compounds from effluents prior to their discharge into natural ecosystems. This removal is achieved through an integration of biological, chemical, and physical processes [12,13]. Traditionally, biological N removal has been dominated by nitrification and denitrification, which are key processes in activated sludge systems. However, these conventional approaches are highly energy-intensive, with aeration required to supply oxygen (O₂) for the oxidation of NH₄⁺ to nitrite (NO₂⁻) and subsequently to nitrate (NO₃⁻), accounting for approximately 50% of total energy consumption [5,14]. Furthermore, the activated sludge process requires the supplementation of an external organic C source to facilitate microbial denitrification (conversion of NO₃⁻ to N₂), which substantially elevates operational expenditures. In addition, these conventional systems are a significant contributor to N₂O emissions [15], a potent GHG with a 273-times higher concentration than that of CO₂, and a significant agent of stratospheric ozone depletion [4,16]. These constraints highlight the urgent need for advanced N removal technologies that are both energy- and carbon-efficient, environmentally sustainable, and capable of substantially minimizing N₂O emissions, thereby transcending the limitations of traditional nitrification-denitrification frameworks.

Recent advancements in microbial N cycling (Figure 1) have significantly deepened our understanding of the diversity, functionality, and ecological role of N-transforming microorganisms. These discoveries have revealed novel microbial taxa, biochemical reactions, and metabolic pathways with promising potential for application in wastewater treatment. Although several reviews have addressed microbial N cycling, most have primarily concentrated on well-established processes, with limited emphasis on emerging innovations. For instance, Kuypers et al. [1] provided a comprehensive overview of the biological N cycle in natural environments, emphasizing biochemical mechanisms and ecological interactions, but without a specific focus on wastewater treatment. Similarly, Shaaban [10] reviewed novel microbial-based approaches to mitigate N₂O emissions in arid soils, but highlighted a lack of information on N₂O reduction within engineered wastewater systems. Other in-depth reviews have explored existing N recovery and removal technologies and the associated microbial ecology [17], but have not integrated the dimension of microbial N₂O emission and their mitigation. Moreover, recent assessments of nutrient removal and recovery technologies have evaluated aspects such as technology readiness levels, energy requirements, and environmental footprints, but have largely explored the innovative microbial processes that could drive next-generation N removal solutions [18].

Considering these developments, the present review focuses on emerging trends in microbial N cycling with direct implications for wastewater treatment. Its primary objective is to provide a comprehensive yet concise synthesis of innovative N-cycling processes currently under investigation. These include advanced strategies for partial nitritation-anammox (PN/A), complete ammonia oxidation (comammox), extracellular electron transfer (EET)-dependent anammox (electro-anammox), and ferric Fe-dependent NH₄⁺ oxidation (Feammox). Additionally, the review explores recent insights into N₂O emissions and associated mitigation strategies. By offering an updated perspective on microbial N cycle in wastewater systems, this work highlights the biotechnological potential and applications of newly identified microorganisms and processes. Through the identification of novel pathways and cutting-edge technologies, this review aims to inform future research directions and facilitate the integration of these processes into both centralized and decentralized treatment infrastructures. Ultimately, it contributes to the overarching goal of developing energy-efficient, low-emission, and sustainable wastewater treatment solutions that address the environmental impacts of the disturbed N cycles.

Review Methodology for Literature Survey

This review was developed through a comprehensive survey of peer-reviewed literature and relevant technical reports published between 2000 and 2025. The primary databases consulted included Web of Science, Scopus, and Google Scholar, using combinations of keywords such as “anammox,” “partial nitritation/anammox (PN/A),” “NOB suppression,” “mainstream,” “sidestream,” “domestic wastewater,” and “high-strength wastewater.” Preference was given to recent studies (2015–2025) that provided mechanistic insights or practical evaluations of anammox and PN/A systems under both sidestream (high-ammonium) and mainstream (low-strength domestic) wastewater conditions. This review emphasizes key advances in process optimization, microbial ecology, and engineering applications that have shaped the evolution of PN/A technologies. Some niche or site-specific case studies and non-English publications were excluded to maintain focus on the most influential and widely cited contributions.

2. Implications of Complete Ammonia (NH₃) Oxidation Bacterial Community in Wastewater Treatment

Nitrification is usually recognized as a two-step microbial process. In this process, NH₃-oxidizing archaea (AOA) and bacteria (AOB) catalyze the oxidation of NH₄⁺ to NO₂⁻, which is subsequently oxidized to NO₃⁻ by NO₂⁻-oxidizing bacteria (NOB) [19,20]. However, this long-standing paradigm has been fundamentally redefined following the discovery of complete ammonia oxidizers (comammox), microorganisms capable of performing both steps of nitrification within a single organism [21]. Comammox bacteria, classified under the genus Nitrospira, exhibit distinct metabolic and physiological characteristics that enable the autonomous conversion of NH₄⁺ to NO₃⁻. These bacterial species have been revealed as prevalent and frequently dominant populations in various wastewater treatment systems [22], highlighting their ecological and functional significance in N removal processes.

The comammox process offers several advantages over anammox-based and conventional nitrification-denitrification systems, such as partial denitrification/anammox (PD/A) and partial nitritation/anammox (PN/A). Its principal benefits include the following: (i) enhanced energy efficiency, (ii) adaptability to oligotrophic and microaerophilic environments, (iii) high biomass yield, (iv) reduced N₂O emissions, and (v) strong substrate affinity for NH₄⁺ [23]. Although PN/A and PD/A processes simplify N removal by reducing the number of biological transformation steps compared to conventional systems, they demand precise regulation of NO₂⁻ concentrations to sustain anammox activity and mitigate NO₃⁻ accumulation. This requirement necessitates stringent control of carbon dosing and aeration, especially in PD/A configurations, which can complicate reactor operation and compromise system stability. In contrast, although the comammox process does not decrease the number of N transformation steps, it facilitates process control by eliminating the necessity to manage multiple specialized microbial consortia and maintain a narrow NO₂⁻ concentration range. This intrinsic operational simplicity minimizes the risk of NO₂⁻ accumulation and enhances overall process resilience. Furthermore, comammox bacteria exhibit the capacity to thrive across a wide range of oxygen concentrations [24], thereby offering improved flexibility and stability under the fluctuating oxygen levels and loading conditions commonly encountered in wastewater treatment plants (WWTPs).

Despite only a limited number of comammox isolates and enrichment cultures obtained to date, their affinity for oxygen is widely considered as a key factor underpinning their competitive advantage over conventional nitrifiers. Genomic and ecophysiological analysis indicates that comammox bacteria are well adapted to micro-aerophilic environments [23]. The occurrence of extreme-affinity O₂-binding proteins, such as bd-type terminal oxidases, in their genomics further enhances their ability to persist and function efficiently under O₂-limited conditions [25]. Nevertheless, these intrinsic O₂ affinities have not yet been directly quantified through pure-culture studies of Nitrospira species [19].

A more comprehensive understanding of the physiological constraints and oxygen kinetics of comammox bacteria is therefore essential for elucidating their competitive dynamics within mixed nitrifying communities. Such insights will be instrumental in guiding the design and optimization of reactor configurations and operational protocols for next-generation wastewater treatment systems [19].

3. Synergistic Potential of Comammox-Anammox Systems for Energy-Efficient Nitrogen Removal

The potential of comammox bacteria in the N elimination process is remarkably noteworthy within the context of next-generation wastewater treatment systems, where energy efficiency and GHG mitigation are key priorities [26]. Despite their widespread occurrence in WWTPs, the oligotrophic growth strategy of comammox bacteria suggests that they may be outcompeted by other ammonia oxidizers under nutrient-rich conditions [27]. Therefore, operational parameters and reactor configurations should be designed to simulate conditions conducive to the proliferation of these oligotrophic microorganisms. Specifically, maintaining high solids retention times (SRTs), low NH₄⁺ concentrations, and low dissolved oxygen (DO) levels can provide a competitive edge to comammox bacteria over canonical ammonia oxidizers [27]. While comammox bacteria offer a promising route for energy-efficient NH₄⁺ oxidation, they are not sufficient as a standalone approach for complete N removal. Integration with other biological processes, such as autotrophic denitrification or anammox, is essential to achieve carbon-neutral N elimination and to convert the metabolic intermediates into inert N gas (N₂). Cooperative interactions between anammox and comammox microorganisms have been demonstrated in both laboratory-scale experiments and full-scale in side-stream WWTPs [24,28], although these systems were not originally designed to enrich mixed comammox–anammox consortia. This cooperation represents a potential evolutionary and operational advancement over the conventional partial PN/A process. Compared with traditional mainstream PN/A systems, which rely on partial nitritation by canonical AOB followed by anammox conversion of NO₂⁻ to N₂, the comammox-anammox route represents an emerging configuration. This approach is still in the exploratory and pilot-scale stage; its operational stability and energy balance relative to PN/A remain hypotheses under investigation. Preliminary data from lab reactors indicate lower aeration demand and reduced N₂O emissions, but full-scale validation is not yet available [28,29]. Unlike canonical ammonia oxidizers, comammox Nitrospira cannot utilize NO₂⁻ as their sole substrate because their genomes lack assimilatory NO₂⁻ reductase genes [25]. Consequently, enriching comammox within PN/A systems may reduce competition for NO₂⁻ and limit unwanted NO₃⁻ accumulation typically caused by NOB activity. Moreover, the low DO concentrations that favor comammox proliferation also help suppress NOB growth, providing an additional operational benefit for PN/A system optimization [30].

Recent investigations have explored the sustainability and energy efficiency of comammox-driven N elimination, mainly when coupled with anammox processes. Comammox-anammox systems functioning under low NH₄⁺ concentrations and extremely low DO levels (0.05 mg L⁻¹) have accomplished high N elimination efficiencies [24,29,31]. These systems demonstrate significant energy savings by minimizing aeration demands while maintaining stable N₂ production. In addition to their energy advantages, comammox-anammox systems exhibit substantially lower N₂O emissions compared with conventional nitrification-based treatments [32,33]. For instance, enrichment of comammox Nitrospira populations has been shown to drastically decrease N₂O emissions related to the nitrification process, emphasizing their importance in climate-resilient N removal [34]. Although the comammox-anammox pathway is highly promising for the elimination of N, it may even produce minor amounts of NO₃⁻ as a byproduct. Nonetheless, research suggests that under low DO concentrations, interactions between anammox and comammox microbes favor partial nitritation over complete nitrification, thus limiting NO₃⁻ accumulation [35]. Furthermore, recent innovations, for instance, embedding synthetic anammox–comammox consortia in hydrogel matrices, have accomplished entire N elimination with minimal NO₃⁻ production [26]. Even when NO₃⁻ is generated, its concentrations in comammox-anammox systems are generally comparable to or lower than those produced in traditional PN/A configurations that rely solely on ammonia-oxidizing bacteria. This reduction may result from dissimilatory NO₃⁻ reduction to NH₄⁺ (DNRA) activity within anammox consortia and/or the contribution of heterotrophic denitrifiers [26]. Collectively, these findings highlight the distinct advantages of comammox-anammox systems, offering a balanced, low-emission, and energy-efficient alternative that may outperform PN/A under low-DO and low-ammonium conditions, although this remains a working hypothesis requiring further pilot-scale verification.

4. Advancing Comammox-Anammox Integration in Wastewater Treatment

The integration of anammox and comammox bacterial communities has shown promising results in controlled laboratory studies; however, it has yet to be fully realized at practical or industrial scales. Future research should be conducted to synthesize the anammox–comammox consortia and the evaluation of reactor parameters, such as sequential batch operations and environmental conditions, that promote the formation of anammox-comammox granules. In a granular sludge setup, comammox-associated bacteria are likely to colonize the outer aerobic zone, though anammox bacteria preferentially inhabit the inner layer, O₂-limited regions. This spatial stratification encourages synergistic interactions between the two microbial communities, enhancing overall N removal efficiency. Given the extended solids retention times (SRTs) required to enrich both anammox and comammox populations [27], granular biofilm configurations are particularly well-suited for sustaining these communities over long operational periods.

Comammox bacteria have demonstrated significantly higher activity in biofilm environments compared to suspended growth systems, as observed in groundwater wells [36,37], drinking water purification tanks [38], and full-scale WWTPs [39]. Biofilm-based operational media improve the persistence and mobility of comammox communities by providing protection against chemical and mechanical disturbances. Their favorable growth conditions are generally associated with slow proliferation, substrate-influx-limited development, and complex spatial organization, such as colony clustering within microbial biofilms and aggregates [21]. Granular sludge systems further support comammox activity by improving mass transfer rates through optimized hydrodynamics and elevated surface area-to-volume ratios, which are vital for active O₂ diffusion and substrate uptake [40]. However, these systems often require sophisticated operational management, including controlled mixing to maintain granule integrity and precise aeration regulation. Although biofilm enhancement proposes substantial benefits for comammox-based N removal, fully harnessing their potential necessitates advanced enrichment strategies and further investigation into optimal environmental and operational conditions. Developing reliable approaches to enhance comammox abundance in bioreactors and other engineered systems is vital to expand their practical applications. The comammox communities display low species and strain diversity both in N removal environments, suggesting specialized adaptations to wastewater ecosystems [41]. So far, only a limited number of comammox strains have been successfully cultured in laboratory settings. Enrichment is often achieved by supplying minimal substrate concentrations, which suppress faster-growing competitors and favor comammox proliferation [36]. For instance, Sakoula et al. [23] reported up to 90% enrichment of Candidatus Nitrospira kreftii under substrate-limited conditions. However, other studies indicate that low NH₄⁺ concentrations and extended SRT alone do not fully account for comammox dominance [42]. In addition, the use of alternative substrates, such as urea, has been shown to influence species composition, underscoring the complexity of factors governing comammox community dynamics. [42]. These discoveries underline the necessity for more adapted cultivation and enrichment strategies to support comammox species. Based on these advantages, the development of enriched comammox cultures and granular biofilm technologies holds promise for advancing their application in energy-efficient N removal. The primary aim is to control the micro-aerophilic characteristics of comammox species to attain energy efficiency for the complete elimination of N in various systems. Such innovations not only increase the feasibility of wastewater treatment but also enhance operational efficiency through the integration of strong and adaptable sustainable biological solutions.

5. Emerging Insights into Anammox-Based N Removal for Domestic Wastewater Treatment

In the N cycle, anammox facilitates the direct conversion of NH₄⁺ to N₂ using NO₂⁻ as the electron acceptor [43,44]. In contrast to conventional nitrification and denitrification, both of which are energy-intensive, anammox offers a more energy-efficient and environmentally sustainable pathway for N elimination from wastewater treatment systems. This process can reduce energy consumption by up to 60%, eliminate the requirement of external C substrates, decrease excessive sludge production by approximately 80%, and significantly mitigate or even eliminate N₂O emissions [5]. These benefits highlight the robust potential of anammox as a crucial constituent for accomplishing energy-neutral wastewater treatment [45]. Anammox has been effectively implemented at full scale, particularly for the treatment of NH₄⁺-rich wastewater such as industrial effluents and side streams [46]. Additionally, research has focused on integrating anammox into municipal wastewater treatment through PN/A processes. Triumphant full-scale mainstream anammox implications have been documented worldwide [47]. Despite these promising developments, the widespread implementation of anammox under mainstream conditions continues to face several technical and operational challenges. Traditional PN/A has matured to full-scale implementation for sidestream and some mainstream applications, yet its success still depends on tight aeration control and NOB suppression. Emerging variations, such as electro-anammox and hybrid PN/A–BES systems, represent conceptual extensions rather than replacements. Their comparative advantages in energy consumption and N₂O mitigation are hypothesized based on laboratory data [48,49] and require long-term pilot confirmation. One of the most critical issues is the effective suppression of NOB, whose activity results in undesirable NO₃⁻) accumulation in the effluent and diminished anammox efficiency by competing for NO₂⁻. Addressing this challenge is essential for optimizing the performance and stability of mainstream PN/A systems.

Over the past two decades, substantial progress has been made in understanding and mitigating these limitations. Wang et al. [50] comprehensively reviewed the recent advances, emphasizing adaptation strategies and mechanisms for NOB suppression in both side-stream and mainstream PN/A configurations. The primary approaches for NOB control in a PN/A system involve decreasing O₂ availability, applying free nitrous acid (FNA) and free ammonia (FA) inhibition approaches, and using the advantages of acidic partial nitritation [50]. Although these strategies differ in their implementation, they share a common objective, which is maintaining the overall rate of NOB growth at zero or below from a kinetic standpoint [50]. Regulating DO at low levels effectively restricts the aerobic metabolism essential for NOB growth. Likewise, the FA/FNA inhibition strategy, though less potent under mainstream conditions because of the typically lower substrate quantity, has been enhanced through advanced modifications, for example, ex situ treatment steps, which significantly improve its practical applicability [50]. Furthermore, recent breakthroughs in acidic partial nitritation have revealed promising novel avenues for NOB suppression. The identification of acid-tolerant AOB, for instance Candidatus nitrosoglobus, in naturally acidic and artificial ecosystems, indicates that these microorganisms may sustain activity under such conditions which are inhibitory to conventional NOB, thus presenting novel opportunities for stable partial nitritation [50]. Looking ahead, the potential of NOB suppression in PN/A systems looks highly encouraging, yet it will depend on the integration of newly discovered bacterial taxa with improved reactor design and process control strategies. Such synergistic integration is expected to further optimize system resilience, ultimately leading to robust and adaptive mainstream wastewater treatment processes capable of enduring environmental and operational fluctuations. A comparative summary of anammox-based and related N-removal processes has been provided in

Table 1. Comparative summary of anammox-based and related N-removal processes.

Process	Operating DO (mg L−1)	Temperature Window (°C)	C/N Requirement	Main Bottleneck	Demonstrated Scale	Typical N-Removal Rate (kg N m−3 d−1)	Typical N2O Factor (% of Influent N)	Indicative Energy Cost (kWh kg−1 N Removed)	References
Mainstream PN/A	0.15–0.5	10–30	Low (autotrophic)	NOB suppression; low-T activity of anammox	Pilot to full	0.05–0.25	0.1–0.5	0.2–0.4	[45,46]
Comammox–anammox	0.05–0.3	10–30	Low (autotrophic)	Slow comammox growth; coexistence balance	Lab to pilot	0.02–0.15	≤0.3	0.3–0.5	[27,47,51]
Electro-anammox	0 (anoxic cathode)	15–35	None (autotrophic, electrons via anode)	Electrode stability; biofilm conductivity	Lab	0.05–0.20	<0.1	0.1–0.3	[52,53]
Feammox	0 (strict anaerobic)	20–40	None (autotrophic)	Low rate; Fe(III) supply; microbial uncertainty	Lab	0.001–0.01	Not reported	≈0 (exothermic, no aeration)	[54,55,56]

Notes: Values are indicative averages from reported ranges across laboratory, pilot, and full-scale studies. DO: dissolved Oxygen; C/N: carbon-to-nitrogen ratio; PN/A: partial nitritation/anammox; NOB: nitrite-oxidizing bacteria.

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6. Domestic Wastewater Treatment and Challenges Associated with PN/A

Implementing PN/A for domestic wastewater treatment faces multiple challenges beyond the suppression of NOB. These include maintaining stable NO₂⁻ production, reduced performance under low temperature and low NH₄⁺ levels, and the inherently slow growth rate of anammox bacteria [57]. To address these limitations, recent research has explored the integration of anammox with novel microbial pathways and organisms, such as AOA, comammox, and N-DAMO [26,58,59,60,61,62]. These approaches utilize diverse microbial consortia to generate NO₂⁻ for the anammox process more efficiently and stably than conventional AOB. Unlike AOB, these emerging microbial groups are often better adapted to the low-temperature, low NH₄⁺ conditions typical of domestic wastewater. Furthermore, they can proliferate under micro-aerophilic or anaerobic environments, lowering the O₂ demand of the system. This not only enhances energy efficiency but also suppresses NOB proliferation. Consequently, integrating such microbial processes can improve the effectiveness and resistance of PN/A systems, enabling stable functioning across variable wastewater conditions while promoting more energy-efficient and sustainable N removal. Despite their potential, these innovative combinations are not yet ready to substitute for traditional PN/A systems. Continued investigation is essential to deepen understanding of microbial interactions and to engineer cooperative consortia suitable for large-scale applications. Among recent advancements, electro-anammox has evolved as a promising alternative for traditional PN/A [48]. This process harnesses the extracellular electron transfer (EET) potential of anammox bacterial community to oxidize NH₄⁺ to N₂, utilizing an anode with a dignified capability as the electron acceptor in a bio-electrochemical system (BES) [48]. Electro-anammox offers several advantages over conventional PN/A and PD/A systems, making it promising as an eco-friendly and less energy-consuming option for N-rich wastewater treatment.

A major advantage of electro-anammox is the complete removal of aeration, a major energy consumer in conventional PN/A systems. By removing the need for partial nitritation, electro-anammox significantly reduces running expenses and consumes less energy. Moreover, it operates effectively at low voltage [48], permitting it can be driven by renewable energy sources, for instance, wind or solar [63]. The process also enables energy recovery, as NH₄⁺ oxidation can produce H₂ gas at the cathode of a microbial electrolysis cell [63], which facilitates simultaneous wastewater treatment and energy generation. Importantly, since NO₂⁻ is not an electron acceptor in this system, no NO₃⁻ is formed, eliminating the need for downstream treatment and avoiding NOB competition [48,49]. Nevertheless, electro-anammox still faces key challenges related to N removal rates, scale-up feasibility, and the stable treatment of municipal wastewater. These limitations stem largely from BES constraints and the complexity of maintaining anammox biofilms on electrodes. To address these issues, integrating electro-anammox with conventional PN/A systems has been proposed. By incorporating anammox biofilm-coated electrodes into granular PN/A reactors under microoxic conditions, multiple NH₄⁺ oxidation pathways could operate simultaneously within a single reactor, improving N removal efficiency.

In an electro-anammox reactor, only the electrode-attached anammox bacterial community participates in NH₄⁺ oxidation, leading to decay of suspended biomass without an electron acceptor. Resulting organic byproducts can inhibit reactor performance. However, coupling electro-anammox with PN/A would enable parallel NH₄⁺ elimination by electrode biofilms and suspended PN/A granules, supporting a more diverse and robust bacterial community. Though some aeration would still be required, the oxygen demand would be greatly reduced, minimizing NO₂⁻ overproduction and suppressing NOB activities compared with those typical of PN/A. Comparative assessments of PD/A, PN/A, and electro-anammox highlight discrepancies in scalability, resource recovery, energy consumption, and treatment efficiency. Electro-anammox is particularly promising for minimizing energy use due to its low-voltage operation and compatibility with renewable energy integration. It efficiently converts NH₄⁺ directly to N₂ without generating NO₃⁻, yet its N removal rate remains lower than that of mature PD/A and PN/A systems, which can handle higher loadings. Hence, while electro-anammox is conceptually innovative and may enhance sustainability, these claims are presently speculative. Further comparative trials with mainstream PN/A are needed to quantify its real-world stability, kinetics, and life-cycle energy savings. The utilization of resources in electro-anammox is further enhanced by hydrogen gas recovery, a valuable byproduct absent from other anammox-based systems. However, scaling electro-anammox to full-size municipal applications remains difficult, while PN/A and PD/A have already been successfully implemented at large scales. Hybrid systems combining electro-anammox with PN/A could overcome these limitations, integrating the high efficiency of PN/A with the energy-saving and resource-recovery potential of BES technologies. Overall, while emerging anammox-based techniques correspond to a significant step towards sustainable wastewater treatment, translating lab innovations to full-scale implications remains the next frontier. PN/A continues to serve as a standard for energy-efficient N elimination and may be combined into existing infrastructure, as shown in full-scale provisions, for instance, Xi’an and Changi [47,64]. Further investigations should emphasize enhancing microbial synergies, improving reactor configurations, scaling up electro-anammox systems, and integrating renewable energy inputs to realize the full potential of anammox-driven wastewater treatment for a low-carbon future.

7. Feammox Pathways for the Treatment of Wastewater

The Feammox pathway represents a newly identified coupling between the N and Fe biogeochemical cycles, wherein anaerobic oxidation of NH₄⁺ is linked to the reduction of ferric iron [Fe(III)], resulting in the formation of NO₂⁻, NO₃⁻, or N₂ [61,62,65,66]. Unlike mainstream PN/A, which has reached pilot-to-full-scale maturity in several wastewater settings, Feammox remains confined to laboratory microcosms and bench-scale soil reactors. Mechanistic understanding, including microbial identity, electron-transfer pathways, and product stoichiometry (NO₂⁻, N₂, or NH₂OH), is still under active investigation. Consequently, Feammox should be regarded as a conceptual biogeochemical process rather than a treatment technology. This process has been detected across various environments, including paddy fields, freshwater, sediments, forest soils, as well as wastewater systems, demonstrating its broad ecological importance [61,62]. Feammox offers a potentially sustainable, low-energy pathway for anaerobic N removal, particularly suited to municipal wastewater with low C/N ratios. Feammox differs fundamentally from mainstream PN/A, as it proceeds under strictly anaerobic conditions and couples NH₄⁺ oxidation to Fe(III) reduction rather than nitrite. However, unlike PN/A, Feammox remains at an early experimental stage with no demonstrated engineering configuration; energy-neutral performance and microbial mechanisms are still hypothetical. Recent investigations have increasingly focused on the feasibility of applying Feammox in engineered wastewater systems, highlighting its mechanisms, applications, challenges, and prospects [56,61,62]. Despite this growing focus, the direct application of Feammox in wastewater systems persists in its infancy. A variety of experimental attempts have been made without fully elucidating the underlying mechanisms. Current understanding of the microorganisms responsible, the molecular pathways involved, and the metabolic and genetic foundations of NH₄⁺ oxidation coupled to Fe(III) reduction remains limited. Furthermore, the exact products and intermediates of the Feammox reaction are still debated, as various studies have reported N₂, NO₂⁻, or NO₃⁻ as terminal products [61,62]. It also remains unclear whether Feammox generates N₂O, emphasizing the need for future research to evaluate potential N₂O emissions and their implications for the greenhouse gas balance.

A major challenge in Feammox research lies in the complicated and highly diverse bacterial diversity within natural and engineered systems [56,61,62]. This diversity complicates the isolation, enrichment, and functional classification of the microorganisms truly responsible for Feammox activity. Numerous investigations have revealed electroactive bacteria, such as Shewanella and Geobacter species, within Feammox-associated communities [56]. Even though these genera are recognized for their Fe-reducing capacities, they lack the canonical enzymatic system for NH₄⁺ oxidation both under aerobic and anaerobic conditions. This ambiguity raises questions regarding whether Feammox observed in mixed cultures is a distinct biological process or a result of overlapping pathways involving traditional nitrification/denitrification and heterotrophic Fe reduction. So far, only a single enrichment culture, i.e., Acidimicrobiaceae species A6, has been reported as a genuine Feammox performer [67]. Nevertheless, the absence of confirmed enzymes or functional genes linked to extracellular electron transfer (EET) and NH₄⁺ oxidation in this microorganism highlights the urgent need for deeper investigations, both at the genetic and molecular levels. While Feammox is proposed as a potential low-energy complement to PN/A, this proposition remains a hypothesis until kinetic parameters and reactor feasibility are experimentally confirmed. Although Feammox offers intriguing theoretical potential for energy-neutral nitrogen removal, it is not deployment-ready. Its practical scalability remains an open question, given uncertainties surrounding reactor design, Fe(III) supply and regeneration, and long-term performance. Thus, Feammox should not be interpreted as equivalent in technological maturity to PN/A, but rather as an emerging research frontier requiring fundamental validation before any engineering application can be envisioned. Feasibility assessments must address several key questions: Is the process sustainable under operational conditions? Can the required Fe(III) be regenerated or recycled efficiently, given that Feammox consumes large quantities of iron relative to the amount of NH₄⁺ removed? Moreover, the environmental economics and applications of large-scale Fe supplements need to be examined to ensure practical scalability.

Critical knowledge gaps in microbiological, molecular, and biochemical understanding currently limit the advancement of Feammox applications. Future research should focus on isolating, identifying, enriching, and characterizing the core microbial taxa responsible for Feammox, as well as elucidating the genes, enzymes, and electron transfer pathways involved. Experimental designs should incorporate appropriate control groups that obviously distinguish Feammox activities from other N and Fe cycling pathways, including assimilatory NH₄⁺ uptake. Establishing this mechanistic foundation will be pivotal for establishing the Feammox process and combining it into viable, low-carbon wastewater treatment approaches. Future research should prioritize isolation of key microorganisms, quantification of iron-cycle coupling efficiency, and evaluation of Fe source sustainability to determine whether Feammox could ever transition from concept to controllable engineering process.

8. Understanding and Mitigating N₂O Emissions in Wastewater Systems

8.1. Biology of N₂O Production and Consumption

Current wastewater treatment facilities are major contributors to GHG emissions, specifically N₂O (Figure 1). These emissions result from the net balance between microbial N₂O production and consumption, processes driven by a complex network of microbial groups (Figure 2). The primary N₂O producers include AOMs [36], denitrifying glycogen-accumulating organisms (DGAOs) [50]. denitrifying polyphosphate-accumulating organisms (DPAOs) [68], and various autotrophic denitrifying bacterial communities. Many heterotrophic denitrifying bacteria (HDB) exhibit dual functionality; they can act as both a source and sink of N₂O, whereas non-denitrifying N₂O-reducing bacteria (ND-N₂ORB) act solely as N₂O consumers.

A major constraint in utilizing HDB as an effective sink for N₂O lies in regulating their governing denitrification phenotypes. Certain HDB preferentially use N₂O as an electron acceptor over NO₃⁻, making them effective N₂O consumers under optimal conditions [69,70]. However, their N₂O-reducing performance is strongly constrained by the availability and composition of organic carbon sources [71]. Similarly, some ND-N₂ORB that lack the NO₂⁻ reductase (nir) gene may be ineffective because abiotic reactions can reform N₂O [72]. The expression of N₂O reductase (nosZ), which encodes the enzyme catalyzing N₂O reduction to N₂, is highly sensitive to environmental factors such as O₂, NO₂⁻, and NO₃⁻ [73,74]. Because N₂O concentrations in wastewater bioreactors are typically low and N₂O reductase is strongly inhibited by oxygen, developing high N₂O-affinity [75] and O₂-tolerant HDB and ND-N₂ORB [76] are critical for establishing robust microbial sinks.

The physiological diversity and environmental sensitivity of microbial N₂O producers and consumers provide valuable insights for devising N₂O mitigation strategies in wastewater management. Key approaches include:

• Minimizing the activity of dominant N₂O-producing microorganisms, particularly AOMs and denitrifying PAOs/GAOs.
• Enhancing the activity and abundance of ND-N₂ORB, especially those with oxygen-tolerant and high-affinity Nos systems.
• Improving the N₂O sink potential of HDB through selective enrichment, adaptive evolution, or bioengineering strategies.

A more comprehensive understanding of the regulatory and metabolic traits governing N₂O production and reduction, coupled with the discovery and characterization of novel N₂O-reducing microorganisms, will be essential for designing next-generation wastewater treatment systems that minimize or eliminate N₂O emissions.

8.2. Process-Level Mitigation in Reactors

A variety of strategies have been proposed to mitigate N₂O emissions in engineered wastewater systems. Traditional PN/A systems primarily aim at nitrogen removal, whereas the strategies summarized here extend treatment goals toward explicit N₂O mitigation and recovery. These technologies (biofilter off-gas polishing, MABR, and CANDO) are at varying readiness levels, from pilot to early demonstration, and their advantages over PN/A in reducing net greenhouse-gas footprints remain hypotheses supported by short-term case studies. Effective mitigation technologies aim to create favorable microenvironments that enhance the activity of N₂ORB, enabling them to function efficiently as biological N₂O sinks.

One promising approach involves the use of “sensu lato biofilms”, particularly trickling biofilters devised to remedy N₂O-comprising exhaust gas. A pilot-scale biofilter system has successfully demonstrated a substantial reduction in N₂O emissions [77,78]. Since the majority of N₂O emissions from WWTPs occur via exhaust gas rather than through liquid effluent [79]. The deployment of such biofilters represents a viable approach to minimizing gaseous N₂O emissions. N₂O accumulation often results from oxygen inhibition of N₂O reductase [80]. However, within the anoxic microzones of thick biofilms, N₂ORB can avoid this inhibition and maintain active N₂O reduction. Gel immobilization of N₂ORB has been shown to further sustain their sink activity (Suenaga et al., 2018a) [81]. Inside the gel matrix, N₂ORB remains metabolically active even under high bulk oxygen concentrations, effectively mitigating oxygen sensitivity while maintaining high N₂O reduction capacity [81,82].

Providing sufficient organic carbon is crucial for stimulating the activities of HDB, which can act as effective N₂O sinks under appropriate conditions. The membrane-aerated biofilm reactors (MABRs) offer a particularly advantageous design for this purpose. In the MABR, oxygen is delivered counter-currently from the bottom of the biofilm, minimizing oxygen intrusion into the carbon-rich layers [83]. This configuration prevents excessive oxidation of organic carbon and promotes its utilization for N₂O reduction within the mid-layers of the biofilm, where N₂ORB are active. A pilot-scale MABR has confirmed N₂O emission factors significantly lower than the Intergovernmental Panel on Climate Change (IPCC) default value of 1.6% [84], highlighting its potential as a cost-effective and energy-efficient technology for mitigating N₂O emissions from WWTPs.

8.3. Post-Treatment, Off-Gas Capture, and Resource Recovery

While mitigation technologies reduce N₂O release, N₂O recovery systems focus on capturing and repurposing this potent greenhouse gas. The coupled aerobic-anoxic nitrous decomposition operation (CANDO) process exemplifies this approach. It exploits the metabolic capabilities of denitrifying polyphosphate-accumulating organisms (DPAOs) and denitrifying glycogen-accumulating organisms (DGAOs) to generate N₂O. These microorganisms produce polyhydroxyalkanoates (PHAs) in the absence of O₂ and later use them as electron donors in the presence of O₂ to convert NO₂⁻ to N₂O [85]. Although gaining higher N₂O yields (60–70%), CANDO faces key challenges related to NO₂⁻ accumulation and effective recovery of soluble N₂O. An alternative novel N₂O recovery method involves photocatalytic hybrid systems, such as the Thiobacillus denitrificans-CdS composite, which performs light-driven NO₃⁻ reduction to N₂O with yields exceeding 96% [86]. Although promising, this technology remains at an early stage and requires further optimization for scalability and operational stability. Once captured, N₂O can be utilized in multiple industrial applications [87]. Practical onsite applications include co-combustion of N₂O with methane (CH₄) for energy recovery [88]. Other uses include serving as a catalyst in food containers, an oxidizing agent in chemical production, or a combustion enhancer in engines [86,88]. However, challenges in N₂O storage and transport necessitate a focus on localized or onsite utilization to minimize risks and logistical complexity.

Despite encouraging progress, most N₂O recovery concepts remain speculative beyond pilot-scale testing, and their comparative performance relative to PN/A-based mitigation must be validated through integrated life-cycle and energy analyses [89]. Achieving consistent NO₂⁻ accumulation, efficient N₂O capture, and sustainable energy recovery are essential to prevent unintended N₂O emissions. Future research should prioritize:

(i)

Optimization of system design to enhance process stability, efficiency, and adaptability across variable wastewater types, including leachate, manure, and nightsoil [89].

(ii)

Reduction in operational complexity and costs, particularly by refining environmental conditions necessary for the metabolic activities of DGAOs and DPAOs.

(iii)

Integration of CANDO with existing wastewater infrastructure, allowing for hybrid systems that combine N₂O mitigation and recovery in a single treatment framework.

Addressing these challenges will be critical to realizing the scalability, energy efficiency, and sustainability of N₂O recovery technologies. Continued interdisciplinary research, spanning microbial ecology, reactor engineering, and process modeling, will accelerate the translation of N₂O management strategies from concept to full-scale implementation, contributing to the long-term goal of climate-resilient and low-emission wastewater treatment systems. Comparative data on these approaches are summarized in

Table 2. Representative N₂O mitigation and recovery technologies.

Technology	Mechanism	Technology Readiness Level (TRL)	Key Limitations	References
Biofilter off-gas polishing	Biological oxidation of N2O in exhaust gas via nitrifying/denitrifying biofilms	7–8 (pilot to full)	Requires gas capture; sensitive to humidity and NO2−/O2 ratios	[90,91,92]
MABR (Membrane-Aerated Biofilm Reactor)	Stratified O2 delivery through membranes forming aerobic-anoxic gradients, reducing N2O intermediates	7 (pilot to full)	Membrane fouling: control of DO flux; capital cost	[93,94,95]
CANDO/CANDO+ (Coupled Aerobic-Anoxic Nitrous Decomposition and Recovery)	Two-stage process: biological NO2− → N2O generation and subsequent thermal or catalytic conversion to N2 with energy recovery	6–7 (pilot)	Control of NO2− accumulation; N2O capture and storage logistics	[96,97]
Catalytic/Photocatalytic N2O reduction	Abiotic conversion of N2O to N2 using metal or light-activated catalysts	3–4 (lab)	Catalyst deactivation; energy input requirement	[98,99]
Biotic N2O sink bioaugmentation	Enrichment of high-affinity N2O-reducing bacteria for in situ reduction of N2O to N2	4–6 (lab to pilot)	Maintaining active populations; substrate competition	[100,101,102]

Notes: MABR: membrane-aerated biofilm reactor; CANDO/CANDO+: coupled aerobic-anoxic nitrous decomposition (and recovery); TRL: technology readiness level; DO: dissolved oxygen.

.

9. Future Directions for N₂O Mitigation and Technology Integration

Despite the development of numerous strategies aimed at mitigating N₂O emissions [89], no universally applicable solution exists across all operational contexts. Consequently, the selection of an appropriate mitigation approach must be tailored to specific system characteristics, environmental conditions, and economic considerations.

Future research should clearly differentiate between validated mainstream PN/A practices and emerging alternatives (comammox-anammox, electro-anammox, Feammox, and CANDO). Many reported advantages, such as enhanced stability or lower energy demand, should be considered working hypotheses until substantiated by pilot or full-scale operational data. One promising approach involves the catalytic combustion of N₂O with CH₄, which enables simultaneous N₂O abatement and energy recovery. However, the efficacy of this method depends on the efficient and leak-free transfer and collection of gaseous N₂O. Any unintended leakage during transport could offset the intended emission reduction by introducing additional GHGs. Therefore, the successful maturation and large-scale deployment of N₂O recovery technologies are contingent upon the development of robust downstream processes for gas handling, purification, and utilization.

Recent advances in omics-based analyses and cultivation techniques have greatly expanded the catalog of known N₂ORB. Newly identified strains exhibit high N₂O affinity [75], enhanced oxygen tolerance [76,81,82], and the capacity to thrive across a broad range of environmental conditions [103]. This physiological versatility presents new opportunities for bioaugmentation and cross-system applications, wherein N₂ORB populations from engineered environments (e.g., anaerobic digestate) can be transferred into agricultural soils, and vice versa, to establish stable microbial sinks for N₂O mitigation [103,104]. Looking forward, in-depth investigations into the ecophysiology and metabolic plasticity of N₂ORB, combined with strategic technology transfer between engineered and natural systems, will be essential for developing resilient and sustainable N₂O management strategies. The Integration of these biological solutions with process engineering innovations will ultimately accelerate the transition toward climate-resilient, low-emission wastewater and agricultural systems.

10. Quantitative Comparison of Emerging and Conventional N Removal Processes

Conventional nitrification-denitrification systems are highly energy-consuming. For example, in mainstream municipal treatment, energy demands of approximately 0.5–1.5 kWh kg⁻¹ N removed (equivalent to ~1.8 MJ m⁻³ influent) have been reported, with a large share attributed to aeration and external carbon addition [105]. In contrast, side-stream autotrophic processes such as PN/A typically require 0.8–2.0 kWh kg⁻¹ N removed, with significant reductions in aeration (~60%) and C dosing (~100%) compared to conventional systems [106]. Studies of comammox bacteria, e.g., Nitrospira inopinata, demonstrate much lower N₂O emissions than canonical AOB: emission yields were shown to be minimal and predominantly abiotic in origin [33]. Such low-emission nitrifier populations are promising for designing low-C N removal systems. Additionally, comammox-dominated reactors have reported high NH₄⁺–N removal efficiencies (~98%) and dissolved N₂O emissions much lower than those of activated-sludge controls [33]. Collectively, these data show that integrating novel microbial processes (PN/A, comammox) within wastewater systems can substantially reduce both energy input and GHG output compared to traditional nitrification-denitrification, thus supporting the goal of decentralized, energy-efficient, low-C wastewater treatment.

11. Conclusions

The transformative potential of innovative microbial pathways in advancing wastewater treatment toward greater sustainability and energy efficiency is becoming increasingly evident. Recent advances in uncovering novel N transformation pathways highlight the importance of interdisciplinary collaboration and the translation of emerging microbiological and engineering innovations into practical applications. Although modifying existing centralized wastewater infrastructure poses significant challenges, many of the novel processes reviewed here could be effectively deployed in decentralized phase I/phase II configurations (Figure 3), which emphasize modularity and operational flexibility. Among emerging strategies, mainstream anaerobic treatment coupled with PN/A in a phase I/phase II stage configuration appears to offer the optimal supportable path for N elimination. This approach effectively balances resource efficiency with climate mitigation potential. However, realizing its full potential will require continued technological development and validation through field-scale implementation. Such systems are particularly suitable for decentralized or small-scale applications, where their adaptability and lower capital investment requirements make them ideal for diverse geographic and socioeconomic contexts. Combining these novel N-cycling processes into phase I/phase II systems represents a paradigm shift toward low-energy, low-emission, and high-efficiency wastewater treatment that simultaneously improves effluent quality and reduces GHG emissions. Future progress in N removal will depend on a deeper understanding of nitrogen-transforming microorganisms and their ecological interactions. As emphasized throughout this review, the success of emerging biotechnologies is closely linked to microbial discovery and functional characterization. Expanding cultivation and enrichment efforts, particularly those targeting extremophiles and acidophilic microorganisms, will enrich the microbial toolkit available for process engineering. For example, acid-tolerant partial nitritation bacteria could provide effective control of NOB in PN/A systems, thereby improving process stability under fluctuating operational conditions.

Equally critical is the scaling-up of laboratory and pilot-scale technologies to real-world implications. Pilot and full-scale demonstrations are fundamental for validating process performance, assessing operational robustness, and refining system configurations for seamless integration with existing infrastructure. In this context, exploring alternative electron acceptors and donors may reveal novel biochemical routes for N elimination, enhancing process versatility and resilience across a range of wastewater compositions.

In parallel, sustained efforts must be directed toward assessing and minimizing the environmental footprint of these novel technologies, particularly regarding GHG emissions and energy consumption. Integrating these processes into adaptable, modular, and scalable phase I/phase II treatment frameworks provides a pathway toward wastewater systems that not only meet stringent environmental regulations but also align with broader global sustainability objectives.

Collectively, these interdisciplinary efforts will foster the next generation of climate-resilient wastewater treatment technologies, enabling effective nitrogen removal, reduced emissions, and enhanced water quality across diverse environmental and economic landscapes.