Overlooked nitrogen-cycling microorganisms in biological wastewater treatment

Highlights

• AOA and comammox bacteria can be more abundant and active than AOB/NOB at WWTPs.

• Coupled DNRA/anammox and NO_x-DAMO/anammox/comammox processes are demonstrated.

• Substrate level, SRT and stressors determine the niches of overlooked microbes.

• Applications of overlooked microbes in enhancing nitrogen removal are promising.

Abstract

Nitrogen-cycling microorganisms play key roles at the intersection of microbiology and wastewater engineering. In addition to the well-studied ammonia oxidizing bacteria, nitrite oxidizing bacteria, heterotrophic denitrifiers, and anammox bacteria, there are some other N-cycling microorganisms that are less abundant but functionally important in wastewater nitrogen removal. These microbes include, but not limited to ammonia oxidizing archaea (AOA), complete ammonia oxidation (comammox) bacteria, dissimilatory nitrate reduction to ammonia (DNRA) bacteria, and nitrate/nitrite-dependent anaerobic methane oxidizing (NO_x-DAMO) microorganisms. In the past decade, the development of high-throughput molecular technologies has enabled the detection, quantification, and characterization of these minor populations. The aim of this review is therefore to synthesize the current knowledge on the distribution, ecological niche, and kinetic properties of these “overlooked” N-cycling microbes at wastewater treatment plants. Their potential applications in novel wastewater nitrogen removal processes are also discussed. A comprehensive understanding of these overlooked N-cycling microbes from microbiology, ecology, and engineering perspectives will facilitate the design and operation of more efficient and sustainable biological nitrogen removal processes.

Graphical abstract

Keywords

Ammonia oxidizing archaea (AOA) / Complete ammonia oxidizing (comammox) bacteria / Dissimilatory nitrate reduction to ammonium (DNRA) bacteria / Nitrate/nitrite-dependent anaerobic methane oxidizing (NO_x-DAMO) microorganisms / Engineering application

1 Introduction

Reactive nitrogen is an indispensable nutrient for life, but excess emissions of reactive N species will cause air, soil and water pollution (Stein and Klotz, 2016). Nitrification-denitrification based nitrogen removal processes have been widely applied at wastewater treatment plants (WWTPs), where ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and heterotrophic denitrifiers are dominant microbes that have been extensively studied (Wiesmann, 1994). In recent years, considerable research efforts have been devoted to the autotrophic nitrogen removal by anaerobic ammonium oxidation (anammox) bacteria (Kartal et al., 2010). As a result, relevant review articles summarizing the abundances and activities of these key functional microorganisms have been published (Kartal et al., 2010; Lu et al., 2014; Soliman and Eldyasti, 2018). However, there are diverse groups of previously “overlooked” N-cycling microorganisms that may also play important roles in wastewater nitrogen removal, despite of their relatively low abundances in the community. These microorganisms include, but not limited to ammonia oxidizing archaea (AOA), the recently discovered complete ammonia oxidizing (comammox) bacteria, dissimilatory nitrate reduction to ammonia (DNRA) bacteria, and nitrate/nitrite-dependent anaerobic methane oxidizing (NO_x-DAMO) archaea and bacteria (Fig. 1) (Tiedje, 1988; Park et al., 2006; Ettwig et al., 2010; Haroon et al., 2013; Daims et al., 2015; Van Kessel et al., 2015).

Fig.1 Overlooked microorganisms in the wastewater microbial nitrogen cycle.

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With the rapid development of high-throughput molecular biology approaches, e.g., metagenomics and transcriptomics methods, the distribution, phylogeny, and metabolic properties of the previously overlooked N-cycling microorganisms are increasingly studied in a more comprehensive manner. For example, AOA and comammox bacteria are the new members of autotrophic nitrifiers discovered by genomic sequencing rather than traditional culture-based method. Their functions in natural and engineered environments are also increasingly studied based on omics approaches (Palomo et al., 2018). It is previously believed that DNRA bacteria made limited contributions to nitrogen removal at WWTPs. However, recent studies based on metagenomics revealed their adverse roles in maintaining anammox process stability (Keren et al., 2020). NO_x-DAMO microorganisms are widespread in natural habitats but less detectable at WWTPs (Van Kessel et al., 2018; Ren et al., 2020). However, they could be the key microbes facilitating energy-neutral biological nitrogen treatment using methane as the electron donor. Although great efforts have been made in recent years to characterize these previously overlooked N-cycling microorganisms, critical knowledge gaps remain in their in situ activities and metabolic interactions with other microorganisms during biological nitrogen removal.

In this review, we synthesized the current knowledge on the distribution, ecological niche, and kinetic properties of overlooked N-cycling microorganisms, including AOA, comammox bacteria, DNRA bacteria and NO_x-DAMO microorganisms in wastewater treatment. Their potential applications in novel wastewater nitrogen removal processes, such as partial nitration/denitrification, partial nitration/anammox, partial denitritation/anammox, NO_x-DAMO/anammox, and NO_x-DAMO/comammox systems are also discussed. More insights into these previously overlooked microorganisms will contribute to a better understanding of their roles in wastewater N cycle, interactions with conventional N-cycling microorganisms in community assembly and functioning, and facilitate the development of sustainable wastewater nitrogen removal techniques in the future.

2 Ammonia oxidizing microorganisms other than canonical AOB and NOB

2.1 Ammonia oxidizing archaea (AOA)

AOA belonging to Thaumarchaeota are prevalent in diverse environments, e.g., marine, hot spring, coral, and soils (Hatzenpichler, 2012). They possess the unique 16S rRNA and amoA genes that distinguish them from AOB (Könneke et al., 2005), which can be further divided into group I.1a (Nitrosopumilus), I.1a associated (Nitrosotalea), I.1b (Nitrososphaera) and ThAOA (Nitrosocaldus) (Table 1). AOA are much smaller, and have higher ammonia and oxygen affinity (${\mathrm{K}}_{{\mathrm{m},\mathrm{N}\mathrm{H}}_3}=0.003-4.4\mathrm{\mu }\mathrm{m}\mathrm{o}\mathrm{l}/\mathrm{L},{\mathrm{K}}_{{\mathrm{m},\mathrm{O}}_2}=2.01-10.38\mathrm{\mu }\mathrm{m}\mathrm{o}\mathrm{l}/\mathrm{L}$) and lower ammonia utilization rate than AOB (Table 2) (Martens-Habbena et al., 2009; Kits et al., 2017). They can grow in acidic/alkaline, hot/cold, and eutrophic/oligotrophic environments (Hatzenpichler, 2012). The monolayer cell membrane of AOA contains glycerol dialkyl glycerol tetraethers, forming stable ether bonds that are beneficial to their adaptations to extreme environments (Schouten et al., 2013).

The presence of AOA at WWTPs has been widely reported (Sauder et al., 2012; Pan et al., 2018). Their relative abundances varied from 2.99 × 10² to 8.65 × 10⁸amoA gene copies/g sludge, accounting for 0.07%–96.04% of the total ammonia oxidizers (Fig. 2) (Park et al., 2006; Limpiyakorn et al., 2011; Gao et al., 2014; Yin et al., 2018). DNA-SIP was always used to clarify the contribution of AOA and AOB, which demonstrated that AOA were significantly dominant in winter despite numeral relationship (Pan et al., 2018). These AOA populations are mostly associated with group I.1a and group I.1b (Park et al., 2006). Factors determining the partitioning of AOA and AOB in wastewater nitrification reactors include substrate concentrations, solids retention time (SRT), temperature, and organics/toxicants. The following conditions favor the existence of AOA: 1) Nutrition levels (e.g., broad ammonia, diverse organics): The abundances ofAOA were found negatively correlated with ammonium concentration in the effluent of domestic WWTPs (Li et al., 2018). When treating landfill leachate with a high ammonia concentration (118±15 mgN/L), AOA could outcompete AOB and comammox bacteria at relatively low DO (≤1.5 mg/L) (Yang et al., 2020). Otherwise, AOA were abundant in some other industrial WWTPs (e.g., petroleum refinery), since they could utilize organics such as malate and succinate as alternative electron donors (Sauder et al., 2017). Another study demonstrated the effectiveness of adding N-acyl-l-homoserine lactones in upregulating amoA genes and the nitrification activity of AOA (Gao et al., 2019). 2) Operation parameters (e.g., low dissolved oxygen (DO), long SRT): AOA could co-exist with anaerobic microorganisms under hypoxic conditions (DO<0.1 mg/L), e.g., heterotrophic denitrifiers and anammox bacteria (AnAOB) (Straka et al., 2019). Longer SRT is usually required to sustain more AOA due to their relatively low growth rate (m_max = 0.010–0.050 /h). This explains why AOA exceed AOB as the dominant nitrifiers more frequently in biofilm systems than activated sludge (up to 1–2 orders higher than AOB) (Chen et al., 2017). 3) Environmental factors (e.g., high salinity, low temperature, sensitive to toxicants): AOA can also outcompete AOB in treating wastewater with 0.25%–3.5% salinity (1.6–81.1 times more abundant) (Wu et al., 2020). In winter, Candidatus Nitrosocosmicus exaquare are the typical AOA population at WWTPs, which could be 52.95 times more abundant than AOB (Pan et al., 2018; Fan et al., 2019). AOA encode copper-based ammonia oxidation and respiration pathways, making them more copper-dependent than AOB and sensitive to the existence of metal-complexation organics in most industrial wastewater (Stahl and De La Torre, 2012; Gwak et al., 2020). For example, AOA could be strongly inhibited by 0.5 mmol/L cysteine (62.86%–98.90%), tryptone (70.75%–100%), and histidine (94.33%–96.51%) (Gwak et al., 2020). Otherwise, AOA are generally more sensitive to some toxicants than AOB, e.g., the NO-scavenger PTIO and short-chain alkynes (Beeckman et al., 2018; Wright et al., 2020). Nevertheless, AOA could be more tolerant to antibiotics (e.g., streptomycin, kanamycin, ampicillin, and carbenicillin) than AOB, and the presence of spiramycin selected AOA as the dominant ammonia oxidizers (Zhang et al., 2015).

Tab.1 Basic characteristics of the four overlooked nitrogen-cycling microorganisms

Microbes	Reaction equation	Key characteristics	Representative species at WWTPs	Reference
AOA	NH4+ + 1.5O2 = NO2− + H2O+ 2H+ (DG°′ = - 274.7 kJ/mol)	Aerobic, Thaumarchaeota	Nitrosopumilus maritimus Candidatus Nitrosocosmicus exaquare Candidatus Nitrosotenuis cloacae	Martens-Habbena et al., 2009
Comammox bacteria	NH4+ + 2O2 = NO3− + H2O+ 2H+ (DG°′ = - 348.9 kJ/mol)	Aerobic, Nitrospira lineage II Mixotrophic (e.g., formate, urea)	Nitrospira inopinata Canditatus Nitrospira nitrosa/ nitrificans/ kreftii	Daims et al., 2015; Van Kessel et al., 2015
DNRA bacteria	C6H12O6 + 3 NO3− + 6H+ = 6 CO2 + 3NH4+ + H2O (DG°′ = - 623 kJ/mol NO3−-N)	Respiratory/Fermentative Electron donors: organics, H2, HS− Enzymes: Nar/Nir, or Nap/Nrf	Wolinella succinogenes Serratia marcescens Salmonella typhimurium Escherichia coli	Tiedje, 1988; Holmes et al., 2019; Pandey et al., 2020
N-DAMO archaea	4NO3− + CH4 = 4NO2− + CO2 + 2H2O (DG°′ = - 503 kJ/mol CH4)	Reverse methanogenesis, ANME-2D lineage Enzymes: NarGH, MCR	Candidatus Methanoperedens nitroreducens	Haroon et al., 2013
n-DAMO bacteria	8NO2− + 3CH4 + 8H+ = 4N2 + 3CO2 + 10H2O (DG°′ = - 928 kJ/mol CH4)	Inter-aerobic pathway, NC10 phylum Enzymes: NirSJFD/GH/L, pMMO	Candidatus Methylomirabilis oxyfera/sinica/ limnetica/ lanthanidiphila	Ettwig et al., 2010; He et al., 2016; Graf et al., 2018

Tab.2 Kinetic parameters of the conventional and overlooked nitrogen-cycling microorganisms

Microbes	Electron donor/acceptor	Half saturation constant of electron donors Km,d (µmol/L)	Half saturation constant of electron acceptors Km,a (µmol/L)	Maximum specific growth rate µmax (/h)	Cell yield (mg Protein /mol NH3 or NO2−)	Reference
Ammonia oxidizers	NH4+-N/O2 (bacteria)	1.9–200	6.9–17.4	0.007–0.088	250	Laanbroek et al., 1994; Jiang and Bakken, 1999; Lawson and Lücker, 2018
	NH4+-N/O2 (archaea)	0.003–4.4	2.01–10.38	0.010–0.050	298.4–304.3	Martens-Habbena et al., 2009; Qin et al., 2014; Kits et al., 2017; Lawson and Lücker, 2018
Nitrite oxidizers	NO2−-N/O2 (Nitrobacter)	49–1380	5.31–165.63	0.0115–0.125	83–108	Laanbroek et al., 1994; Blackburne et al., 2007; Nowka et al., 2015; Lawson and Lücker, 2018
	NO2−-N/O2 (Nitrospira)	9–27	2.19–5.94	0.027–0.083	120–213
Comammox bacteria	NH4+-N/O2	0.049, 0.040	NA	0.0061	394.7	Kits et al., 2017; Sakoula et al., 2020
	NO2−-N/O2	449.2, 12.5	NA	NA	NA
DNRA bacteria	Organic/ (NO3−, NO2−)	NA	NA	0.11–0.5	NA	Simon, 2002
	Inorganics/(NO3−, NO2−)	NA	NA	0.091	NA
N-DAMO archaea n-DAMO bacteria	CH4/NO3−	500	150±28.6	NA	73–85*
	CH4/NO2−	2.6–5.9, 91.8±5	4.28–7	0.0015–0.0583	NA	He et al., 2013; Winkler et al., 2015; Guerrero-Cruz et al., 2019; Lu et al., 2019
Anammox bacteria	NH4+-N/ NO2−-N	2.54–673.90	0.22–361.35	0.0022–0.014	30–70#	Ali et al., 2015; Zhang and Okabe, 2020

Note: NA: not available, * unit: mmol C/mol CH4, # unit: mmol C/mol NH4+-N.

Fig.2 Abundances of AOA, comammox bacteria, DNRA bacteria and NOx-DAMO microorganisms in wastewater treatment. Data are collected from published results, representing samples from municipal WWTPs, industrial WWTPs, recirculating aquaculture systems, and drinking water treatment systems.

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2.2 Complete ammonia oxidizing (comammox) bacteria

Comammox bacteria oxidize ammonia to nitrate via nitrite in a single microorganism. Thus far, all discovered comammox bacteria belong to Nitrospira sublineage II, and their 16S rRNA gene sequences are highly similar to those of canonical NOB-Nitrospira (Daims et al., 2015). Their amoA genes are different from those in AOB, but more identical to the particulate methane monooxygenase gene pmoA (Palomo et al., 2018). Based on the amoA gene sequences, comammox bacteria are further divided into clade A and B (Daims et al., 2015; Pjevac et al., 2017), which differ in substrate transport, energy transfer, and adaptability to changing environment (Palomo et al., 2018). The representative clade A populations include Nitrospira inopinata, the only isolated comammox bacterium so far, and enriched cultures of Candidatus Nitrospira nitrosa, Candidatus Nitrospira nitrificans, and Candidatus Nitrospira kreftii, while clade B populations still lack representative cultures (Daims et al., 2015; Van Kessel et al., 2015; Sakoula et al., 2020).

Comammox bacteria are ubiquitous at WWTPs, accounting for 0.02%–3% of the total bacteria community in nitrifying activated sludge, and 1.8%–19.4% of the biofilm communities in rotating biological contactors (Fig. 2) (Annavajhala et al., 2018; Cotto et al., 2020; Spasov et al., 2020). However, unlike AOA, comammox bacteria were not detected in reactors receiving high concentrations of ammonia, e.g., side-stream partial nitritation/anammox systems (influent ammonium>400 mgN/L) (Cotto et al., 2020). Compared to nitrification processes governed by AOB and NOB, comammox-dominated nitrification possesses advantage in maintaining performance efficiency and stability at extremely low ammonia and DO concentrations, having less N₂O emissions, and might better cooperate with anaerobic bacteria (e.g., AnAOB) in facilitating total nitrogen removal. Some study revealed that currently genome-sequenced comammox species lack NO reductases, thus merely produced N₂O abiotically (0.07%±0.006%), which was 1–2 orders lower than AOB (0.1%–8%) (Kits et al., 2019; Prosser et al., 2020).

Environmental factors shaping the ecological niches of comammox bacteria in wastewater nitrogen removal systems include: 1) Nutrition levels (e.g., low ammonia, simple organics): comammox bacteria are prevalent in natural and engineered systems with relatively low ammonium concentrations (Pjevac et al., 2017; Fowler et al., 2018). The ammonia affinity of comammox bacteria (${\mathrm{K}}_{{\mathrm{m},\mathrm{N}\mathrm{H}}_3}$ = 0.049–0.040 µmol/L) is 1–3 magnitudes lower than AOB (Table 2) (Kits et al., 2017; Sakoula et al., 2020). As a result, drinking water treatment plants and recirculating aquaculture systems provide optimal conditions for comammox bacteria, where their relative abundances can be 2.1–60.7 times higher than AOA and AOB (Bartelme et al., 2017; Wang et al., 2017; Fowler et al., 2018). Comammox bacteria clade A usually dominate in these systems (mostly Candidatus Nitrospira nitrosa), whereas clade B are more frequently detected in rapid sand filters (up to 75% of the total nitrifying community) (Camejo et al., 2017; Palomo et al., 2018; Roots et al., 2019). In addition, mixotrophic growth of comammox bacteria on simple inorganic and organic substances (e.g., H₂, formate, and urea) other than ammonia and nitrite further improves their survival and persistence in broad environments (Palomo et al., 2018; Han et al., 2019). 2) Operation parameters (e.g., high SRT and low DO): Surveys on full-scale WWTPs revealed that longer SRT (activated sludge>12.5 d, or biofilm) and lower DO (0.2–1.0 mg/L) favored comammox bacteria over canonical nitrifiers (Roots et al., 2019; Cotto et al., 2020). This can be explained by their relatively low growth rate (µ_max = 0.0061 /h) (Table 2) and high oxygen affinity (Camejo et al., 2017; Kits et al., 2017; Roots et al., 2019). Molecularly, the latter is attributable to the bd-type terminal oxidase and the oxygen-sensitive carbon fixation pathway in comammox bacteria. 3) Environmental factors (e.g., metal deficient): Compared with AOB and NOB, comammox bacteria can better survive in copper and iron deficient conditions, because they possess enzymes to maintain copper homeostasis (CopABCD) and the high iron affinity of cytochrome c biogenesis system I (Palomo et al., 2018).

3 Overlooked nitrate/nitrite reducing microbes other than heterotrophic denitrifiers

3.1 Dissimilatory nitrate reduction to ammonia (DNRA) bacteria

DNRA bacteria reduce nitrate to ammonia via nitrite in a single microorganism, with organics or inorganics as electron donors. They can be heterotrophic, autotrophic, anaerobic or aerobic (Tiedje, 1988; Pandey et al., 2020). Heterotrophic DNRA bacteria break down large molecule organics and are represented by Aerobacter aerogenes, Citrobacter freundii, Enterobacter spp., and Denitrovibrio spp. Autotrophic DNRA bacteria are less abundant, using hydrogen and sulfide as electron donors, such as Desulfovibrio gigas and Veillonella alcalescens (Holmes et al., 2019). Based on the means of ATP synthesis, DNRA bacteria are further classified into fermentative and respiratory, where the former derives energy directly from substrate-level phosphorylation and the energy for the latter is from oxidative phosphorylation (Pandey et al., 2020). Escherichia coli are representative fermentative DNRA bacteria possessing periplasmic nitrate reductase (Nap), respiratory nitrite reductase (Nrf), respiratory nitrate reductase (Nar), and cytoplasmic nitrite reductase (Nir) systems to support growth under low and high nitrate conditions (Pandey et al., 2020). Wolinella succinogenes is a typical strain of respiratory DNRA, utilizing non-fermentative organics or inorganics (e.g., formate or H₂) as electron donors, and can grow with low nitrate as they possess the high affinity Nap/Nrf system (Simon, 2002). nrfA gene serves as the molecular biomarker for DNRA bacteria and its abundances positively correlated with DNRA activity in situ (Pandey et al., 2020).

DNRA bacteria account for 0.2%–4% of total communities at full-scale municipal WWTPs across the world (Fig. 2). DNRA process is not trivial but can contribute significantly to nitrogen metabolism (9.5–15.7 nmol N/g/L) in activated sludge, granular and biofilm reactors (Chen et al., 2020; Wang et al., 2020c). They were found more abundant in systems operated under anaerobic, high C/N and high sulfide conditions, e.g., anaerobic digesters, biofloc aquaculture ponds, sulfate removing reactors (Tiedje, 1988; Akunna et al., 1993). With the upgrade of WWTPs, especially the nutrient removal processes, the ratios of DNRA/denitrifying bacteria detected in the receiving water bodies increased from 1.097 to 1.928 (Wang et al., 2020b). DNRA bacteria are important functional groups in simultaneous nitrification and denitrification processes, accounting for 37.15%–46.01% of total OTUs related to nitrogen removal (Xiang et al., 2020).

For a long time, scientists have been interested in the coexistence and competition between heterotrophic denitrifiers and DNRA bacteria. Factors influencing the relative abundances of the two populations at wastewater bioreactors mainly include: 1) Nutrition levels (e.g., electron acceptor availability, type of electron donor): From the perspective of electron acceptor, DNRA bacteria transfer more electrons per mole of nitrate reduced, and have higher affinity for nitrate (µ_max/K_s,NO3-: 26.2 µmol/L/h versus 8.6 µmol/L/h) but lower affinity for acetate (5.2 mmol/L/h versus 8.6 Van Denmol/L/h) than denitrifiers (Van Den Berg et al., 2016). Hence, DNRA bacteria have competitive advantages over heterotrophic denitrifers in nitrate limiting but electron donor abundant conditions (typically acetate/nitrate ratio higher than 1.5). The genome of Shewanella loihica strain PV-4 encodes the complete set of conventional denitrification and DNRA pathways, and relatively high C/N ratios upregulated their DNRA functional genes (e.g., nrfA) (Yoon et al., 2015). In another study, higher lactate/NO₃^- ratio (2.97) also promoted the co-existence of fermentative and respiratory DNRA, and enhanced their activities (Van Den Berg et al., 2017). Meta-analysis also suggested that DNRA rate was positively correlated with C/NO₃⁻ (Van Den Berg et al., 2016; Pandey et al., 2020). Additionally, nitrate other than nitrite serving as the electron acceptor favors DNRA process over denitrification (Kraft et al., 2014). Type of electron donor also was another important factor. Compared with heterotrophic denitrifiers, fermentative DNRA bacteria prefer to use less oxidative and labile organic carbon sources (Tiedje, 1988; Van Den Berg et al., 2017). L-sorbose or D-cellobiose enriched denitrifiers (e.g., Klebsiella), while D-glucose, D-fructose and citrate enriched DNRA bacteria (e.g., Escherichia, Sulfurospirillum) (Carlson et al., 2020). In a microbial community with both DNRA and denitrifying bacteria, DNRA process dominated when glucose and glycerol served as the carbon sources (nitrate removal rate: 2.7–10.1 mgN/g MLVSS/h), while denitrification mainly occurred when acetic acid and lactic acid were supplied (nitrate removal rate: 23.8–27.8 mgN/g MLVSS/h) (Akunna et al., 1993). Both autotrophic denitrifiers and respiratory DNRA bacteria can use inorganic compounds as electron donors (e.g., H₂, S²⁻, Fe²⁺), but FeS usually enriched denitrifiers, and H₂S favored the growth of respiratory DNRA bacteria (Yin et al., 2015; Pandey et al., 2020). 2) Environmental factors (e.g., high temperature and pH): Higher diversity and activity of DNRA bacteria were observed in WWTPs operated at higher temperature despite variations in treatment configurations (Wang et al., 2020c). In Shewanella loihica, higher pH can regulate DNRA process, as the NrfA protein has pH optima at 8.0, while denitrification was observed in acid conditions (Kim et al., 2017).

3.2 Nitrate/nitrite-dependent anaerobic methane oxidizing (NO_x-DAMO) microbes

NO_x-DAMO process is driven by a microbial consortium containing DAMO archaea and bacteria that can reduce nitrate to dinitrogen with methane as the electron donor (Ettwig et al., 2010; Haroon et al., 2013). N-DAMO archaea are members of the ANME-2D lineage capable of reducing nitrate to nitrite, with Candidatus Methanoperedens nitroreducens as the representative population (Haroon et al., 2013). Nitrate reduction is catalyzed by a pseudo-periplasm Nar-like protein complex, obtaining electrons from reverse methanogenesis (Arshad et al., 2015). Subsequently, nitrite is reduced to dinitrogen gas by n-DAMO bacteria belonging to NC10 phylum (e.g., Candidatus Methylomirabilis oxyfera, Candidatus Methylomirabilis sinica, and Candidatus Methylomirabilis limnetica) (Ettwig et al., 2010; He et al., 2016; Graf et al., 2018). Methylcoenzyme M reductase gene mcrA and particulate methane oxygenase gene pmoA are the biomarkers for DAMO archaea and bacteria, respectively (Luesken et al., 2011; Haroon et al., 2013).

NO_x-DAMO microorganisms are widely present in full-scale anaerobic reactors (Luesken et al., 2011), and have been enriched in the laboratory (Hu et al., 2014; Kampman et al., 2014). N-DAMO archaea are less abundant than n-DAMO bacteria, due to the lack of lanthanide in wastewater, a special element required by methanol dehydrogenase in N-DAMO archaea (Ren et al., 2020). Overall, n-DAMO bacteria are present at 10⁶–10⁸ 16S rRNA copies/g sludge at WWTPs (Fig. 2), with an averaged activity of 1.61–22.31 µmol CH₄/g sludge/d due to the shortage of nitrite and relatively short SRT of mainstream treatment processes (Hu and Ma, 2016; Meng et al., 2021). However, on account of the fact that methane can be produced in situ at WWTPs, this process still holds promise in future sustainable N removal techniques. NO_x-DAMO bioreactors have been established and studied more intensively at laboratory-scale. By combining anammox and NO_x-DAMO processes in a laboratory-scale membrane biofilm reactor (MBfR), enhanced total nitrogen (TN) removal rate of 0.28 kg N/m³/d was achieved (Xie et al., 2018). Laboratory-scale membrane granular sludge reactor (MGSR) with NO_x-DAMO/anammox consortium demonstrated high nitrogen removal capacity too (16.53 kg N/m³/d) (Fan et al., 2020).

The ecological niches of NO_x-DAMO microorganisms in wastewater nitrogen removal reactors are influenced by: 1) Nutrition levels (e.g., substrate level, trace element): The activity of n-DAMO bacteria increased with elevated methane concentration, whereas they can not tolerate high nitrite (${\mathrm{K}}_{\mathrm{I},{\mathrm{N}\mathrm{O}}_2^{-}}$ = 4.1±0.5 mmol/L) (He et al., 2013). Methanol and sodium acetate as additional electron donors can stimulate the activity of n-DAMO bacteria by up to 1.9 times (Ma et al., 2017). The nitrate affinities of N-DAMO archaea are similar to those of heterotrophic denitrifiers, but the nitrite affinities of n-DAMO bacteria are three orders of magnitude lower than those of denitrifiers (Table 2) (Guerrero-Cruz et al., 2019; Lu et al., 2019). In addition, trace amount of iron (20 µmol/L) and copper (10 µmol/L) promoted the growth of n-DAMO bacteria (He et al., 2015a). 2) Operation and environmental conditions (e.g., SRT, temperature and oxygen): With relatively lower growth rate than heterotrophic denitrifiers, NO_x-DAMO microorganisms can be readily washed out and a longer SRT or attached growth is generally required (Fan et al., 2020). Although n-DAMO bacteria can produce O₂ internally, the exposure to 2% oxygen reduced their nitrite reduction activity by 45%, indicating their sensitivity to oxygen (Luesken et al., 2012). In addition, mesophilic (35°C), neutral, and non-saline conditions are in favor of n-DAMO bacteria (He et al., 2015b).

4 Engineering applications of overlooked N-cycling microbes

Although overlooked N-cycling microorganisms make up rarely more than 1% of the microbial communities, they play important roles in sustaining community stability of wastewater nitrogen removal processes, helping them to cope with adverse conditions (e.g., cold, high salinity, copper deficient), and/or reducing energy consumption and greenhouse gas emissions.

4.1 AOA: ammonia oxidation under adverse conditions

Due to their relatively high ammonia/oxygen affinity (Table 2) and tolerance to low temperature, high salinity, acidity and alkalinity, AOA are more promising in treating low strength ammonia wastewater under harsh conditions. For example, when treating saline wastewater, AOA can contribute to 94.9% and 48.2% of the overall ammonia oxidation at 10°C and 35°C, respectively (Lin et al., 2020). The much higher ammonia and oxygen affinities of AOA than AOB promoted their closer metabolic interactions with AnAOB under nutrient- and oxygen-limited conditions, where the r-strategists NOB-Nitrobacter can be suppressed too. When treating low strength nitrogen sewage, AOA successfully cooperated with AnAOB in removing 90% of ammonium in a single-stage SBR (Pan et al., 2016). Reactors with efficient sludge retention (e.g., biofilm reactors, MbfR, sequencing batch biofilm reactors) were always used to sustain biomass of AOA (Straka et al., 2019; Wang et al., 2020a). For example, MBfR operated in a counter-diffusion mode, rather than diffusion better facilitate the AOA/AnAOB cooperation, as demonstrated in (Fig. 3(a)) (Straka et al., 2019). Theoretically, the TN removal by AOA-AnAOB consortia in MBfR can be up to 91.5%, and the coculture is more stable than AOB-anammox under fluctuated DO and HRT (Liu et al., 2016).

Fig.3 Potential applications of AOA (a), comammox bacteria (b), DNRA bacteria (c) and NOx-DAMO microorganisms (d) in novel wastewater nitrogen removal processes.

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4.2 Comammox bacteria: nitrification at low N loading and low DO

Although raw domestic wastewater contains 20–50 mg TKN/L, the influent to biological nitrogen removal (BNR) systems could be as low as 9.86–14 mgN/L (Wang and Chen, 2016; Wang et al., 2018). It is highly possible that comammox bacteria can replace conventional AOB, AOA, and NOB as the dominant nitrifying bacteria under low DO conditions. Traditional nitrification process requires extensive aeration (DO>2 mg/L), but comammox dominated nitrification reactor could be operated at DO<1 mg/L and in the biofilm mode (Roots et al., 2019; Cotto et al., 2020). Simultaneous nitrification-denitrification (DO<0.75 mg/L) can also occur in these systems, saving energy needed for aeration and internal recirculation (Holman and Wareham, 2005; Insel, 2007).

The unbalanced ammonia oxidation and nitrite oxidation activities of comammox bacteria lead to transient accumulation of nitrite, an important intermediate involved in diverse pathways including denitrification, DNRA, and anammox (Daims et al., 2015; Gottshall et al., 2020). With elevated ammonia addition from 0.01 to 1 mmol/L, transient nitrite accumulation up to 32.67% of converted ammonia was observed in comammox bacteria (Daims et al., 2015). Relatively high ammonium, limited oxygen supply, and addition of inhibitors (e.g., NH₂OH) further enhance the nitritation activity (Kits et al., 2019; Wang et al., 2020d). Practically, comammox bacteria performing nitritation can be coupled with denitrifiers (i.e., nitritation-denitritation, or nitrite shunt) or AnAOB (partial nitritation-anammox, PN/A) to remove total nitrogen. In the attached growth mode, comammox bacteria on biofilm or granule surface oxidize ammonia to nitrite under hypoxic conditions, which is secreted and metabolized by AnAOB due to the relative higher nitrite affinity (Fig. 3(b), Table 2). A synthetic community comprising of AnAOB (Candidatus Brocadia anammoxidans) and comammox bacteria (Nitrospira inopinata) showed excellent nitrogen removal without nitrite accumulation (Gottshall et al., 2020). However, it should be cautious that comammox bacteria may replace AnAOB as the sole N-cycling microorganism in the integrated system, performing complete nitrification under low nitrogen concentrations and resulting in process failure (Roots et al., 2019). Additionally, comammox bacteria can also survive under metal-insufficient conditions (e.g, copper and iron), which future expands their ecological niches at WWTPs (Palomo et al., 2018; Koch et al., 2019).

4.3 DNRA bacteria: coupling with anammox bacteria for enhanced N removal

One of the disadvantages of anammox is its inherent accumulation of nitrate (87% of influent nitrogen). DNRA coupled with anammox enhance total nitrogen removal by minimizing nitrate accumulation (Castro-Barros et al., 2017). DNRA bacteria were found coexisted with AnAOB in nearly all anammox systems, providing NO₂⁻/NH₄⁺ for AnAOB (Fig. 3(c)) (Park et al., 2017). Typically, fermentative DNRA bacteria (e.g. Chloroflexi) utilize large molecule organics excreted by AnAOB in the form of extracellular polymeric substances (EPS) (Lawson et al., 2017). AnAOB also perform DNRA using small organic acids (e.g. formate, acetate, propionate) and inorganics (e.g, ferrous iron and hydrogen) (Castro-Barros et al., 2017). Thermodynamically, partial DNRA-AnAOB (1984 kJ/acetate) could surpass heterotrophic denitrifiers (796.8 kJ/acetate) in carbon source limiting conditions. In the presence of high ammonium and sufficient inorganic carbon, the coupled partial DNRA-anammox process is in fact accomplished mainly by AnAOB rather than the mixed fermentative DNRA bacteria and AnAOB, representing a more sustainable autotrophic nitrogen removal technique (Castro-Barros et al., 2017). Autotrophic DNRA, e.g., Fe(II)-dependent DNRA coupled with anammox can achieve a total nitrogen removal rate of 0.23±0.01 kg N/m³/d by controlling the EDTA-2Na/Fe(II) ratio and pH (Li et al., 2020). Notably, although heterotrophic/autotrophic DNRA bacteria can provide nitrite for AnAOB growth, they also compete with AnAOB for some metabolic necessities, such as amino acids and vitamins (e.g. VB12) (Keren et al., 2020). A recently study proposed another potential application of DNRA bacteria, i.e., nitrate recovery by bioelectrochemical ammonification mediated by the electroactive DNRA bacteria Geobacter sp. (Wan et al., 2020).

4.4 NO_x-DAMO microorganisms: sustainable nitrogen removal with methane

Applications of NO_x-DAMO microorganisms in wastewater nitrogen removal are under rapid development due to their advantages in energy conservation, greenhouse gas mitigation, and sludge reduction (Van Kessel et al., 2018). Nitrate produced by AnAOB could be reduced by N-DAMO archaea, recirculating nitrite for AnAOB and n-DAMO bacteria (Fig. 3(d)). MBfR represents an optimal configuration facilitating the co-existence of NO_x-DAMO microorganisms and AnAOB, resulting in a nitrogen removal rate of up to 190 mgN/L/d (Shi et al., 2013). In the recommended A-B process for achieving carbon neutral at WWTPs, the coupled NO_x-DAMO and anammox process is promising in stage B because methane generated in stage A can be directly used without external addition of organic carbon sources. NO_x-DAMO process enables a reduction in greenhouse gas emissions by at least one fifth compared with traditional treatment (Ma et al., 2017).

The coupled NO_x-DAMO and comammox process was recently proposed as another sustainable nitrogen removal technology (Ren et al., 2020). Comammox bacteria supply electron acceptors (nitrate and nitrite) to DAMO archaea/bacteria under low N-loading and low DO conditions. Notably, the overdosed methane can be oxidized by ammonia monooxygenase in comammox bacteria, mitigating the accidental release of the potent greenhouse gas. However, the coupled process has not been demonstrated experimentally.

5 Conclusions and perspectives

This review summarized the distribution, ecological niche, kinetic properties and potential applications of the overlooked AOA, comammox bacteria, DNRA bacteria, and NO_x-DAMO microorganisms in biological wastewater treatment processes. AOA and comammox bacteria are K-strategy nitrifiers growing slower than conventional AOB/NOB, but are better adapted to oligotrophic and adverse conditions. DNRA and NO_x-DAMO microorganisms could surpass heterotrophic denitrifiers as the key nitrate reducers depending on substrate levels and operating parameters of bioreactors. Coupling them with AnAOB has been proven effective in enhancing total nitrogen removal, reducing costs and greenhouse gas emissions.

The following areas should be given priority in future studies on these overlooked microorganisms: 1) distribution pattern, in situ activities, and adaption strategies of overlooked microorganisms in traditional and novel nitrogen removal process should be evaluated more comprehensively. Efficient and accurate methods that can distinguish the contribution of specific microorganisms from similar function microbes (e.g., AOA/AOB/comammox bacteria, denitrifiers/DNRA bacteria) should be developed; 2) complex interactions (e.g., substrate cooperation/competition, toxicant detoxification) with other microorganisms in BNR systems, and the appropriate models describing these interactions could be constructed; 3) potentials and limitations of these microorganisms in more sustainable nitrogen removal techniques (e.g., coupled AnAOB, comammox, and/or NO_x-DAMO) could be assessed from engineering application points of view.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 41701269), the National Key R&D Program of China (No. 2019YFC0408800), and the Fundamental Research Funds for the Central Universities (No. 2020FZZX001-06).

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