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Nitrite Oxidation in Wastewater Treatment: Microbial Adaptation
and Suppression Challenges
Zicheng Su, Tao Liu, Jianhua Guo, and Min Zheng*
Cite This: https://doi.org/10.1021/acs.est.3c00636
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ABSTRACT: Microbial nitrite oxidation is the primary pathway that generates nitrate in wastewater treatment systems and can be
performed by a variety of microbes: namely, nitrite-oxidizing bacteria (NOB). Since NOB were first isolated 130 years ago, the
understanding of the phylogenetical and physiological diversities of NOB has been gradually deepened. In recent endeavors of
advanced biological nitrogen removal, NOB have been more considered as a troublesome disruptor, and strategies on NOB
suppression often fail in practice after long-term operation due to the growth of specific NOB that are able to adapt to even harsh
conditions. In line with a review of the history of currently known NOB genera, a phylogenetic tree is constructed to exhibit a wide
range of NOB in dierent phyla. In addition, the growth behavior and metabolic performance of dierent NOB strains are
summarized. These specific features of various NOB (e.g., high oxygen anity of Nitrospira, tolerance to chemical inhibitors of
Nitrobacter and Candidatus Nitrotoga, and preference to high temperature of Nitrolancea) highlight the dierentiation of the NOB
ecological niche in biological nitrogen processes and potentially support their adaptation to dierent suppression strategies (e.g., low
dissolved oxygen, chemical treatment, and high temperature). This review implicates the acquired physiological characteristics of
NOB to their emergence from a genomic and ecological perspective and emphasizes the importance of understanding physiological
characterization and genomic information in future wastewater treatment studies.
KEYWORDS: microbial nitrification, nitrite oxidation, nitrite-oxidizing bacteria (NOB), suppression, kinetics; short-cut nitrogen removal
1. INTRODUCTION
Microbial nitrite oxidation to nitrate is performed by a group
of microorganisms, named after their major function as nitrite-
oxidizing bacteria (NOB). As one of the primary producers of
nitrate, NOB are widespread in natural environments, such as
in soil,
1,2
ocean,
3,4
freshwater,
5
and hot springs.
6−8
Members of
NOB are diverse, which have been found spanning 4 phyla and
12 genera (including candidate genera) with considerable
functional and physiological diversities.
NOB also play an essential role in biological nitrogen
removal processes within modern wastewater treatment plants
(WWTPs).
9−11
Biological nitrogen removal was initially
proposed in the middle of the twentieth century in response
to the growing issue of eutrophication. Until now,
nitrification−denitrification has still been the most widely
adopted nitrogen removal process. In this process, nitrite
generated from ammonia oxidation is subsequently oxidized to
nitrate by NOB, which is then anoxically reduced back to
nitrite and further to dinitrogen gas by denitrifying micro-
organisms. An obvious issue associated with the conventional
nitrification−denitrification is the transformation between
nitrite and nitrate, which leads to significant dissipation of
energy (from nitrite to nitrate) and organic carbon (from
nitrate to nitrite).
12
Received: January 24, 2023
Revised: August 8, 2023
Accepted: August 9, 2023
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© XXXX The Authors. Published by
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https://doi.org/10.1021/acs.est.3c00636
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In recent years, increasing attention has been paid to
upgrading traditional WWTPs with the aim of reducing
operational costs and carbon footprints. These advanced
biological nitrogen removal processes are usually associated
with the fate of nitrite. Unlike the traditional nitrification−
denitrification pattern, researchers are endeavoring to develop
a more energy-ecient and more sustainable flow of nitrogen
in WWTPs, that is, to directly reduce nitrite to nitrogen gas,
bypassing nitrate. For example, partial nitritation and anammox
(PN/A, or deammonification) and nitrite shunt are the two
most widely known state-of-the-art shortcut nitrogen removal
techniques, which could significantly save energy and organic
carbon sources compared to the traditional nitrification−
denitrification process.
13,14
With that, nitrite accumulation
becomes a critical prerequisite, while as an oxidizer of nitrite,
NOB are considered as the enemy to the shortcut nitrogen
removal systems. Indeed, the NOB suppression can be
achieved easily in high-strength wastewater due to the in situ
formed free ammonia (FA) and free nitrous acid (FNA).
However, in mainstream systems where the nitrogen is low
(40−60 mg N/L), stable NOB suppression is a known
challenge, which is a hot research topic and also the focus of
the present review. To suppress the NOB in low-strength
wastewater, various control strategies have been proposed. For
example, lowering the dissolved oxygen (DO) concentration is
a very typical approach, as it manipulates the competition
between AOB and NOB.
15
Shortening the solid retention time
(SRT) is another strategy to wash out NOB due to the higher
growth rates of AOB compared to those of NOB.
16
Moreover,
the ex situ treatment of mainstream sludge has been proven
eective as well. About 20−30% of mainstream sludge is
centrifuged and treated by dierent methods on a daily basis,
e.g., FA and FNA, which is shown capable of selectively
suppressing NOB in the mainstream treatment.
17
However,
most of these strategies have only been shown to inhibit NOB
in a relatively short period, and the long-term operation under
practically relevant conditions usually results in the emergence
and adaptation of specific nitrite oxidizers.
18
This is intrinsi-
cally because of distinct physiological characteristics of diverse
nitrite oxidizers, which may pose a fundamental challenge to
next-generation shortcut nitrogen removal processes.
Previous reviews focused either more on the fundamentals of
NOB or summarizing approaches to suppress NOB from
practical engineering aspects. For instance, Daims et al.
19
delved into the biological mechanisms and ecological
interaction of NOB, while Cao et al.
20
discussed key microbes
and interactions in PN/A and suggested critical factors for the
process control, including operational parameters and
bioreactor selection. Additionally, Wang et al.
12
models from
a kinetic perspective for sidestream and mainstream PN/A
processes and identifies suitable operational windows. This
review aims to build a bridge between these two aspects.
Specifically, the integration of perspectives from micro-
biologists and wastewater engineers will be beneficial to shed
light on the question of why NOB suppression in engineered
wastewater systems is dicult. Moreover, the review will
facilitate the development of more practical solutions for NOB
suppression in municipal WWTPs based on the most recent
fundamental knowledge, thus underpinning the paradigm shift
to carbon-neutral and energy-positive wastewater management
in the future.
2. HISTORY OF NOB DISCOVERY
The advancement in cultivation technology, molecular
methods, and bioinformatics has broadened the diversity of
NOB. To date, all known chemolithoautotrophic nitrite
oxidizers are bacteria spanning 4 phyla including Pseudomo-
nadota (also known as Proteobacteria), Nitrospirota (also
known as Nitrospirae), Nitrospinota (also known as Nitro-
spinae), and Chloroflexota (also known as Chloroflexi), which
Figure 1. Historic discovery of NOB and complete ammonia oxidation (comammox) genera. The red line indicates the increase in the total
number of NOB/comammox genera identified. The colored background of the genera names represents the phylum to which each NOB/
comammox belongs to. The phylogenetic tree is a pruned tree from a previous “tree of life” study,
21
and branches with colored contour depict the
phylum. The discovery of NOB genera was first reported in the following studies: (a) ref 22, (b) ref 23, (c) ref 24, (d) ref 1, (e) ref 25, (f) refs 26
and 27, (g) ref 28 (h) ref 29, and (i) ref 30.
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B
consist of 7 genera and 5 candidate genera (without isolates
obtained) (Figure 1).
Nitrobacter (Nb.) winogradsky was the first NOB isolate
obtained by serial dilution, which is a significant milestone in
the history of NOB discovery.
31
Nb. winogradsky grows
aerobically with nitrite as an electron donor and can also
alternatively live on simple organic compounds (e.g., pyruvate,
formate, acetate, and yeast extract-peptone).
32−34
After about a
century, Nitrococcus and Nitrospina were identified in marine
ecosystems in 1971, with two strains isolated from surface
samples of South Pacific waters and South Atlantic waters,
respectively.
23
Unlike Nitrobacter, they were both obligate
chemoautotrophic NOB, as no growth was observed in organic
media. Until 1986, a new genus of NOB Nitrospira was
discovered, and Nitrospira (Ns.) marina was isolated from a
water sample at a depth of 206 m from the Gulf of Maine in
the Atlantic Ocean.
24
In the following more than a century,
until now, the genus Nitrospira has been found to be the most
ubiquitous NOB in natural and engineered ecosystems. A
phylogenetic analysis of 16S rRNA genes indicates that
Nitrospira can be classified into at least six sublineages,
exhibiting a greater phylogenetic diversity than other NOB
genera.
19
Within sublineage II, a number of Nitrospira species
including the representative isolate Ns. inopinata were shown
to be able to perform complete ammonia oxidation
(comammox).
26,27
This discovery is a breakthrough that
fundamentally changed the previous perception by demon-
strating that complete nitrification can be carried out by a
single microorganism rather than multiple microorganisms in
concert. Additionally, Nitrospina belonging to a distinct novel
phylum Nitrospinota was recently discovered based on an in-
depth evaluation of the draft genome of Nitrospina (Nn.)
gracilis.
35
It is considered to be an important NOB population
in marine environments.
30,36,37
The knowledge of NOB diversity keeps evolving, and more
novel NOB have come to light based on the improvement of
culture-independent approaches (e.g., meta-omics). The novel
NOB genus Candidatus (Ca.) Nitrotoga belonging to β-
Proteobacteria was discovered in 2007 based on the enrich-
ment from permafrost-aected soil in Siberian Arctic.
1
Members of the genus Ca. Nitrotoga can generally adapt to
the cold environment (<10 °C) except for Ca. Nitrotoga (Nt.)
fabula.
38
In contrast, the genus Nitrolancea positioning in the
Chloroflexota phylum seems to be thermophilic, which was
initially discovered in a nitrifying bioreactor at an elevated
temperature of 37 °C.
25
More recently, two draft genomes of
putative thermophilic NOB were retrieved from a metagenome
yielded from the Yellowstone Hot Spring enrichment,
provisionally named “Ca. Nitrocaldera robusta” and “Ca.
Nitrotheca patiens”.
8
Both candidate genera belong to the
phylum Chloroflexota and expectedly prefer a thermophilic
lifestyle. In the Nitrospinae phylum, two new marine genera, “
Ca.Nitrohelix vancouverensis” and “Ca. Nitronauta litoralis”
were recovered by using cell sorting, activity screening, and
incubation.
30
These studies exemplify an ecient protocol
enabling physiological investigation rather than traditional
cultivation. A recent work using metagenome-assembled
genomes and single-cell amplified genomes also proposed a
provisional genus “Candidatus Nitromaritima” in a deep-
branching lineage under Nitrospinota.
28
These results
collectively indicate that applications of culture-independent
approaches will likely further enlarge the pool of NOB than we
expected before.
According to the 16S rRNA phylogeny (Figure 2a), NOB is
distributed in at least four deep branches. The phylogenetic
distance between NOB groups is large, with a 16S rRNA
sequence identity of 62.4−92.2% between genera. The
phylogeny of the key marker gene nxrA (alpha subunit of
nitrite oxidoreductase) of NOB shows a congruent pattern
(Figure 2b), which can be divided into three main clusters and
likely correlated with the dierent features, for example, energy
eciency (see more discussions in Section 4.1) and the locale
in the cell membrane. Specifically, the cytoplasmic nitrite
oxidoreductase (NXR) is represented by Nitrobacter and
Nitrolancea, and the periplasmic NXR is represented by
Nitrospira and Nitrospina. The recent identification of Ca.
Nitrotoga has revealed novel sub-branches of periplasm NXR
(Figure 2b), which is associated with a unique soluble NXR
periplasmic holoenzyme.
39
Analogous to the 16S rRNA
phylogeny, the three branches of nxrA are divergent from
each other, indicating at least three evolution origins.
39,40
The metabolic versatility of NOB has been revealed
previously, showing their additional capability of performing
for example urea hydrolysis and using formate,
41
hydrogen,
42
Figure 2. Phylogenetic tree of currently known NOB and their nxrA. (a, left) 16S rRNA phylogenomic tree of NOB. Colored backgrounds indicate
the NOB found in WWTPs. Light blue and light orange indicate not validly published nomenclature, or no isolates were reported so far. (b, right)
Phylogenetic tree of NOB nxrA. Colored backgrounds indicate the types of nxrA. Black dots at the branch nodes indicate bootstrap values (based
on 1000 iterations) >75%. On both sides, an asterisk indicates phototrophic NOB.
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C
sulfide,
4
and cyanate
43,44
as substrates for their growth (Figure
3). Such highly functional diversity supports the survival of
NOB in dierent natural and engineered systems but adds
diculty to suppressing the nitrite oxidation in wastewater
treatment using a single strategy. Moreover, these diverse
functions are widely possessed by dierent NOB, while the
species within the same genus may show dierent functions. As
the nitrifying community in wastewater-engineered systems is
mostly revealed by 16S rRNA amplicon sequencing, the
resolution is inadequate to show the microbial community
Figure 3. Functional genes in NOB Nitrospira, comammox Nitrospira,Nitrobacter,Ca. Nitrotoga, and Nitrolancea based on mapped reads to the
annotated ORFs. Twelve representative genomes that contained at least one of the related genes are displayed. Metagenemarks
45
was used to
predict the genes of each genome, and functional annotation referred to KEGG,
46
COG,
47
and Uniprot
48
databases. Notably, due to dierences in
MAG quality and limitations of databases we used, it is possible that some genomes missed functional genes. As gene expression is regulated by
various complex factors, such as environmental conditions, nutritional status, and intra- and extracellular signals, gene annotation only represents
the potential for functional expression of these genes and cannot ensure that they will be expressed in the actual environment, which requires
further investigation.
Table 1. Physiological Features of 22 Isolated/Enriched NOB Cultures, Including Optimum Temperature, Mean (±SD) Half-
Saturation Constants (Km), and Maximum Nitrite Oxidation Activities (Vmax)
a
organism name sample source optimum temp
(°C) Km(μM NO2−)Vmax (μmol NO2−/(mg protein
h)) source
Nitrospira moscoviensis corroded iron pipe of a heating system 39 9 ±3 18 ±156,85
Nitrospira defluvii enrichment from activated sludge sample 32 9 ±3 48 ±256,84,86
Nitrospira sp. NJ1 WWTP 31 10 ±2 31 ±587
Nitrospira sp. ND1 WWTP ND 6 ±1 45 ±788
Nitrospira sp. Ecomares 2.1 moving-bed filter of an aquaculture
system 28−30 54.0 ±11.9 21.4 ±1.2 89
Nitrospira sp. BS10 WWTP 28 27 ±11 20 ±256
Nitrospira inopinata hot water pipe from an oil exploration
well 37 372 ±55 16.9 56,69
Nitrobacter hamburgensis
X14 soil ND 544 ±55 64 ±156,90,91
Nitrobacter winogradskyi soil 32 309 ±92 78 ±534,56,91,92
Nitrobacter vulgaris sewage ND 49 ±11 164 ±984,93
Nitrobacter sp. Nb-311A surface waters of the Atlantic Ocean ND 27.6 ±6.7 95.2 ±7.0 89
Ca. Nitrotoga sp. AM1 sand of eelgrass zone 16 24.7 ±9.8 ND 55
Ca. Nitrotoga arctica permafrost soil 10/13−19 58 ±28 26 ±356,80
Ca. Nitrotoga sp. HAM-1 WWTP ND ND ND 83
Ca. Nitrotoga fabula KNB WWTP 24−28 89.3 ±3.9 27.6 ±8.4 38
Ca. Nitrotoga sp. HW29 recirculation aquaculture system 22 ND ND 94
Nitrospina watsonii 347 Black Sea, 100m depth 28 18.7 ±2.1 36.9 ±2.2 23,89,95
Nitrococcus mobiliz surface water of the South Pacific Ocean 25−30 119.7 ±34.0 141.0 ±10.6 23,89
Nitrolancea hollandica laboratory-scale bioreactor 40 1000 NA
25,96
Ca. Nitrohelix vancouverensis sandy coastal surface sediment samples ND 8.7 ±2.5 ND 30
Ca. Nitronauta litoralis sandy coastal surface sediment samples ND 16.2 ±1.6 ND 30
Nitrospina gracilis 3/211 surface water of the Atlantic Ocean 25−30 20.1 ±2.1 41.4 ±9.4 23
a
ND means not determined.
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D
structure at the species level. In specific circumstances,
especially when the NOB control failed and the adaptation
of NOB is observed, using advanced metagenomic sequencing
will be conducive to providing more accurate information in
high resolution.
Both phylogenetic trees based on 16S rRNA and the nxrA
gene are rooted at midpoints. In the 16S rRNA gene tree (left),
there are 4 major branches in accordance to phyla level,
namely Pseudomonadota (Proteobacteria), Chloroflexota,
Nitrospiraota, and Nitrospinota. In contrast, the nxrA
phylogenetic tree is mainly divided into three major branches.
These include cytoplasmic nxrA represented by Nitrococcus,
Nitrobacter, and Nitrolancea, periplasmic nxrA represented by
Nitrospira and Nitrospina, and a middle branch mainly
represented by Ca. Nitrotoga yet to be clarified. The major
branches of the trees are composed of highly divergent
sequences, suggesting significant evolutionary divergence
between lineages. Upon closer inspection, the topology of
the branching patterns has similarities between the trees. For
example, in both trees, Nitrospira,Nitrospina, and other
Nitrospinota-aliated genera still cluster together (although
Nitrospira seems more independent), with only minor
dierences in their branching patterns or sequence divergence.
Moreover, Nitrococcus,Nitrobacter, and Thiocapsa also remain
relatively close to each other. These clades likely represent
closely related lineages that have undergone relatively recent
evolutionary diversification. However, the overall patterns of
evolutionary relationships among the 16S rRNA and nxrA
genes are not necessarily alike; therefore, it is necessary to
choose the appropriate gene when using genetic analysis to
interpret NOB evolutionary relationships.
3. PRIMARY NOB IN GLOBAL FULL-SCALE WWTPS
Nitrification converts ammonia (NH3) to nitrite (NO2−) and
then to nitrate (NO3−), which is generally carried out by
ammonia-oxidizing bacteria (AOB)/ammonia-oxidizing arch-
aea (AOA), NOB, and comammox bacteria in wastewater
treatment systems. NOB Nitrobacter cells were frequently
detected together with Nitrospira, which were believed to
dominate nitrification in wastewater treatment systems.
49,50
In
recent years, with more accessible sequencing and quantifica-
tion technologies, there has been an increasing amount of
evidence highlighting the prevalence of Nitrospira in activated
sludge of WWTPs.
51
A systematic global-sampling eort of
∼1200 activated sludge samples from 269 WWTPs demon-
strated that Nitrospira were the dominant NOB in most
examined WWTPs.
52
Similarly, other studies also showed that
sublineages I and II of the genus Nitrospira were ubiquitously
detected in WWTPs.
50,53
It is therefore tempting to assume
that the operational conditions in conventional activated
sludge processes are favorable to Nitrospira in comparison to
those of other NOB genera. The dominance of Nitrospira is
likely due to the physiological features and metabolic versatility
of this genus, as elaborated below.
3.1. Physiological Features. Table 1 summarizes the key
physiological features of the most kinetically characterized
NOB, including the optimum temperature, half-saturation
nitrite constant (Km), and maximum nitrite oxidation rate
(Vmax). These characterization studies unanimously reveal that
the nitrite anity constant of Nitrospira (Km= 6−54 μM
NO2−, excluding comammox Nitrospira) is much lower than
the counterpart of other NOBs found in wastewater treatment.
Since the in situ nitrite concentration is generally below 50 μM
NO2−in widely installed continuous flow activated sludge
processes, Nitrospira may have a greater resilience and adaptive
ability over other NOB genera. For example, Nitrospira
outcompeted Nitrobacter when the nitrite concentration was
controlled below 3 mg NO2−-N/L.
54
However, it should be
noted that the sequencing batch reactor (SBR), a configuration
widely applied in small-/medium-scale WWTPs, often
transiently accumulates nitrite in the aerobic period. The
increased nitrite concentration can provide opportunities for
the growth of other NOB such as Ca. Nitrotoga, which have a
slightly higher Kmvalue in the range 24−86.5 μM NO2−.
38,55,56
By using 16S rRNA gene specific PCR and FISH, Lucker et al.
verified the presence of Nitrotoga-like bacteria in 11 of 15 full-
scale SBRs surveyed, in which the nitrite level could reach as
high as 0.48 mg/L NO2−within a typical cycle.
10
Nitrospira generally has a slow growth rate. For example, the
generation times for two Nitrospira representatives, Ns.
moscoviensis (sublineage II) and Ns. defluvii (sublineage I),
were determined to be 32 and 37 h, respectively,
56
which were
longer than the doubling time of 13 h for Nb. vulgaris and 26 h
for Nb. winogradskyi. This indicates that the SRT applied in
many WWTPs (∼15 days) is important to the retention of
Nitrospira. In agreement with this hypothesis, a study adopting
dierent SRTs reported that Nitrospira greatly outnumbered
Nitrobacter at a longer SRT of 40 days.
57
Attributed to the slow
growth rate, biofilms provide a more suitable niche for the
growth of Nitrospira. In a full-scale hybrid biofilm and activated
sludge reactor, the metagenomic approach revealed that the
biofilm had significantly higher abundances of Nitrospira
compared to the suspended sludge.
58
In addition to the
canonical Nitrospira, researchers highlighted that the comam-
mox Nitrospira also prefer to grow in biofilms with long SRT.
For example, comammox Nitrospira were found dominating
the biofilm in a rotating biological contactor in WWTP,
59
and
a survey on 14 full-scale nitrogen removal systems also revealed
that the long SRT (>10 days) and attached growth phase were
significantly correlated to the prevailing comammox Nitro-
spira.
60
Our recent studies also showed the prevalence of
comammox Nitrospira in nitrification biofilms attached to
sponge and plastic carriers.
61,62
Although oxygen is also essential to NOB growth, the
oxygen anity was rarely quantified due to the mass transfer
resistance among cell aggregates. In engineered systems,
researchers usually use apparent half-saturation constants for
oxygen (KO) to describe the oxygen anity of NOB, which
typically ranges from 0.06 to 1.0 mg O2/L for NOB in
WWTPs.
63−67
Since DO concentrations in nitrifying tanks are
usually in the range of 1−3 mg O2/L, which is higher than
most of the apparent KOvalues reported, the oxygen may not
be the factor aecting the competition between Nitrospira and
other NOB. However, this is only applicable to the NOB
community in flocs, while in biofilms, the DO level may play
an important role because of the higher apparent KOdue to
mass transfer resistance.
68
From the above discussion, the low
in situ nitrite concentration and the long SRT are likely to
jointly contribute to the dominance of Nitrospira in full-scale
WWTPs.
3.2. Metabolic Versatility. Recent findings have suggested
that the prevalence of Nitrospira in wastewater treatment
systems could also be related to their metabolic versatility.
First, some Nitrospira members are capable of utilizing
ammonia as an energy source (i.e., comammox Nitrospira),
which is the main form of nitrogen in raw wastewater. In
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particular, comammox Nitrospira exhibit a higher anity for
ammonia compared to most other canonical ammonia
oxidizers.
69
Field investigations have shown that comammox
Nitrospira are present in nearly all wastewater treatment
processes.
70−72
Due to the high anity for ammonia, the
systems with comammox Nitrospira may have higher ammonia
removal eciencies compared to nitrogen removal systems
where they are absent.
60
Still, more studies should be dedicated
to further quantifying the contribution of comammox Nitro-
spira to ammonia and nitrite removal in WWTPs.
Second, Nitrospira members have versatile metabolic
pathways, such as the metabolism of urea, cyanate, and
hydrogen. Among all, the degradation of urea is more relevant
to wastewater treatment, as over 80% of ammonia nitrogen in
domestic wastewater is from urine.
73
It has been demonstrated
that Ns. moscoviensis belonging to Nitrospira sublineage II can
cleave urea to ammonia and CO2,
41
and the produced
ammonia can be further supplied to ammonia oxidizers.
Indeed, the operon encoding the functional urease (ure) exists
in the genomes of many other species of Nitrospira such as Ns.
sp. BS10
74
and Ns. sp. ND1,
75
illustrating the ecological
importance of urea to Nitrospira. Additionally and intriguingly,
three novel comammox species were selectively enriched in a
urine-fed reactor.
76
Similar to other comammox, Ns. inopinata,
Ca. Ns. nitrosa, and Ca. Ns. nitrificans, all these comammox
Nitrospira genomes contained a complete urea utilization
pathway. The slow release of ammonia from urea hydrolysis
might contribute to the prevalence of comammox Nitrospira in
the urine-treatment reactor. Although some AOB and AOA
also have ureolytic activities,
77−79
the successful enrichment
from the urine-fed reactor indicates that urea is an important
factor to the selection of comammox Nitrospira, which requires
further investigations.
3.3. Impact of Seasonal Temperature Variation. From
a global perspective, seasonal temperature variation is an
important factor shaping the microbial communities in full-
scale WWTPs. In particular, some full-scale WWTPs are
located in the temperate zone, which generally exhibits annual
average temperatures lower than those of tropical and
subtropical regions. Various Ca. Nitrotoga strains were
discovered from the surface layer of permafrost soil with an
extreme temperature range (−48 to 18 °C) and permanently
frozen sediments.
80
Despite the extremely low temperature
where they were found, the optimal temperatures of seven Ca.
Nitrotoga strains were above 10 °C, and five among them were
above 20 °C. The result suggested that Ca. Nitrotoga are
psychrotolerant rather than psychrophilic, which might adapt
to the low temperature when the soil froze more than 3000
years ago and survive as a frozen “living fossil”.
80
Because of
the psychrotolerant feature, Ca. Nitrotoga may become the
primary NOB in wastewater-engineered systems with low
temperatures. For example, Ca. Nitrotoga were observed to be
the transiently dominant NOB in cold seasons at some
WWTPs.
11,81
An investigation recognized Ca. Nitrotoga as the
key NOB in full-scale WWTPs, with Ca. Nitrotoga detected in
WWTPs between 7 and 16 °C.
10
In this study, Ca. Nitrotoga
was the only detectable NOB in two full-scale WWTPs from
Germany at temperatures of 16 and 9 °C, respectively. A
recent study showed Ca. Nitrotoga co-occurred with Nitrospira
only in an SBR reactor operated at low temperatures (4−14
°C), while it was almost not detected in a similar reactor but at
elevated temperatures (22−34 °C). This together indicates
that Ca. Nitrotoga may have a specific temperature preference
and highlights the importance of operating temperature as a
factor in the selection of NOB communities in wastewater
treatment processes.
82
Indeed, the optimal temperature for the
growth in the Ca. Nitrotoga genus is obviously lower than that
of Nitrospira (Table 1), which supports the competitive
advantages of Ca. Nitrotoga over Nitrospira at low temper-
atures.
83
In a coculture of Ns. defluvii and Ca. Nt. sp. BS, the
microbial community shifted at 17 °C, where Ca. Nt. sp. BS
became the dominant NOB.
84
Laboratory bioreactor and
coincubation studies also confirmed that temperature is a
deciding factor aecting niche occupation of Nitrotoga-like
bacteria in activated sludge.
1,82
4. NOB SUPPRESSION AND ADAPTATION IN
ADVANCED NITROGEN REMOVAL PROCESSES
In traditional wastewater treatment, aeration for nitrification
consumes significant energy for the oxidation of nitrite to
nitrate, while the reduction of nitrate to nitrite requires
organics for heterotrophic denitrification or potentially
through endogenous denitrification.
97
Therefore, increasing
attention has been drawn to advanced nitrogen removal via the
nitrite pathway, which is a cost-eective alternative and also
known as shortcut biological nitrogen removal. The shortcut
nitrogen removal technologies mainly include the nitrite shunt
and PN/A (or deammonification), which can be achieved in
both two-stage (i.e., oxidation and reduction processes in two
bioreactors) and one-stage (i.e., oxidation and reduction
processes in one bioreactor) configurations. In theory,
ammonia is first converted to nitrite, which will be removed
by heterotrophic denitrifiers in the nitrite shunt and by
anammox bacteria in the PN/A process. Compared with
Table 2. Dominant NOB under Dierent Control Strategies
strategy reactor type NOB DO
(mg O2/L) SRT (days) harsh treatment ref
oxygen-based control UMABR Nitrospira 0.6 ±0.1 infinite 110
oxygen-based control MBBR Nitrospira 0.15−0.18 uncontrolled 111
oxygen-based control MBBR Nitrospira and
Nitrobacter <0.03 uncontrolled 112
oxygen-based control SBR Nitrospira 0.2−6.5 uncontrolled to
14.4 ±264
FNA-based sidestream
treatment SBR Ca. Nitrotoga >1 mg HNO2-N/L FNA 113
FA-based sludge treatment SBR Ca. Nitrotoga 1.5−2.0 15 354 ±58 mg N/L FA 114
FNA-based sidestream
treatment anoxic/oxic (An/O)
reactor Ca. Nitrotoga 15 1.87 mg N/L FNA 115
ex situ control with harsh
treatments SBR Nitrospira and
Nitrobacter 1.5−2.0 15 4.23 mg N/L FNA and sequentially 210
mg N/L FA 103
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conventional nitrification and denitrification, the nitrite shunt
process reduces the aeration requirement by 25%, organic
carbon consumption by 40%, and sludge production by up to
55%,
98
while these benefits are further enhanced in the PN/A
process achieving the reduction of aeration, organic carbon
consumption, and sludge production by nearly 60%, 100%, and
80%, respectively.
99,100
Despite these remarkable benefits, a critical challenge
associated with the success of nitrite shunt and PN/A is the
suppression of NOB. Generally, strategies to minimize NOB
activity and achieve PN are developed based on three
principles: (1) dierent kinetics of AOB and NOB, (2)
dierent resilience of AOB and NOB to harsh treatment, and
(3) the competition for nitrite by other microorganisms (e.g.,
anammox bacteria) (Table 2). Accordingly, the suppression of
NOB can be realized by in situ control and ex situ treatment.
For the in situ control, strategies adopting low DO, high
residual ammonium, short SRT, and combining anammox
bacteria to compete for nitrite are proposed as favorable
strategies to selectively inhibit NOB. Apart from that, recently,
a novel approach was developed by forming an in situ acidic
condition in promoting the stable nitrite accumulation, or
partial nitritation.
101,102
Regarding the ex situ control, biocidal
treatments using such as FA,
103
FNA,
104
sulfide,
105
ultra-
sound,
106,107
and light irradiation
108
were found to be ecient
for the selective inhibition of NOB. Both in situ control and ex
situ treatment can be practical. While the in situ strategies are
generally easier to implement, the ex situ treatment most times
requires additional capital costs for a separate treatment tank.
However, the previous economic analysis showed that the
capital cost of the ex situ treatment by FNA was only
approximately 5% of the total reduced cost by replacing
nitrification-denitrification with the PN/A process.
109
Nonetheless, the stable NOB suppression in long-term
operation remains challenging under mainstream conditions
with low ammonium concentration and seasonally varying
temperatures. From a kinetic point of view, the development of
NOB control strategies can be found in a recent review.
12
Herein, a microbial perspective of NOB suppression and
adaptation to dierent treatment processes is detailed, since
dierent types of NOB (i.e., Nitrospira,Nitrobacter,Nitro-
lancea, and Ca. Nitrotoga) appear to be adapted to dierent
control strategies, leading to the failure of nitrogen removal via
the nitrite pathway (Figure 4).
4.1. Nitrospira Surviving in Low-DO and Low-Nitrite
Systems. Low-DO control is undoubtedly the most
recognized strategy for NOB inhibition and is widely applied
in shortcut nitrogen removal processes. In comparison to the
two-stage configuration, the one-stage shortcut nitrogen
removal processes require more strict DO control strategies
because the residual oxygen may pose inhibitory eects to
anaerobic microbes such as anammox bacteria and denitrifiers
growing in the same niche. Therefore, the supplied oxygen in
theory should be sucient only to support the ammonia
oxidation while limiting the subsequent nitrite oxidation by
NOB and any potential inhibitory impacts on anammox
bacteria or denitrifiers. In previous reports, measures such as
transient anoxia, DO-based aeration, and intermittent aeration
have been proven to be eective in achieving shortcut nitrogen
removal performance.
116−118
However, Nitrospira has stubbornly appeared in many one-
stage shortcut nitrogen removal processes, likely because the
Nitrospira sublineage I, as one of the most abundant Nitrospira
branches in WWTPs, has an even higher anity for oxygen
(0.09 ±0.02 mg O2/L) than most of AOB.
64
For example, Liu
and Wang
57
showed that Nitrospira gradually became
predominant, leading to the failure of nitritation in a long-
term reactor operated at low DO (≤0.5 mg O2/L). The
complete nitrification was achieved at a low DO of 0.3 ±0.14
mg O2/L with Nitrospira abundance of up to 2.64 ×106cells/
mL, Liu et al.
119
also detected Nitrospira under extremely
hypoxic conditions (0.02−0.10 O2mg/L) in a one-stage PN/A
process.
The dierent oxygen anities of the NOB are likely related
to electron transfer and terminal oxidases. Specifically, aerobic
respiration relies on the respiratory chain, where electrons
generated during nitrite oxidation flow from NXR to
cytochrome cand then to the terminal oxidase.
38
Three
distinct types of terminal oxidase have been well-studied and
present in the respiratory chain of dierent NOB.
120−122
The
aa3-type heme-copper oxidase (A-class HCO) is commonly
found in Nitrobacter,
123,124
which shows a lower anity for
oxygen. Nitrospira contain a putative cytochrome bd-like
terminal oxidase, which could also receive electrons derived
from nitrite or low-potential donors like organic carbon.
125,126
The cbb3-type terminal oxidase, a member of the C-class HCO
possessing an extremely high anity for oxygen,
38
is used by
NOB species to adapt to O2concentrations at nanomolar
concentration, e.g., Nn. gracilis
35
and Ca. Nt. fabula.
39
This
thus supports the occurrence of Ca. Nitrotoga and Nitrospira in
a low-oxygen nitrifying bioreactor.
127
Figure 4. Conceptual diagram describing the adaptation of NOB to dierent treatment processes in the shortcut nitrogen removal processes.
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Previous studies also reported the dominance of comammox
Nitrospira in a mainstream nitrifying moving bed biofilm
reactor (MBBR)
128
and nitrogen removal system based on an
anammox process,
129
both of which were under low-DO
conditions. However, a recent full-scale survey suggested that
the presence of comammox is not significantly related to DO
levels.
60
In addition, comammox Nitrospira were successfully
enriched from an MBBR with a sucient DO supply,
62
and the
apparent KOvalue of this comammox-dominated culture was
determined to be 2.8 ±0.4 mg O2/L, indicating that this
cluster of comammox Nitrospira may not have a strong anity
for oxygen. However, the study has its limitations on the mixed
culture. Moreover, the eciency of oxygen mass transfer in
biofilms can also aect the measurement of the apparent
anity for oxygen.
Another strategy to suppress NOB in one-stage nitrogen
removal processes is a low in situ nitrite concentration. Ideally,
nitrite produced by AOB in a one-stage nitrite shunt or PN/A
is immediately consumed by denitrifiers and anammox
bacteria, leading to a gradual washout of NOB due to failure
in the competition for nitrite. However, the nitrite uptake
competency depends on the nitrite anities of NOB and other
nitrite scavengers, which can vary between genera or even
species levels. As shown in Figure 3b, the nxr genes can be
divided into at least two major types, based on their subcellular
locus at periplasm or cytoplasm.
19
In particular, Nitrospira and
Nitrospina harbor the periplasmic NXR,
130
while cytoplasmic
NXR mainly occurs in Nitrobacter,Nitrolancea, and Nitrococcus.
Theoretically, the periplasmic NXR energetically prevails over
cytoplasmic NXR,
131,132
as nitrite oxidation takes place outside
the cytoplasm and the liberated protons directly contribute to
the proton motive force (PMF). This could become the
greater energic advantage of NOB using periplasmic NXR than
cytoplasmic NXR, as nitrite oxidation only yields low energy
(△G°′ =−74 kJ/mol NO2−).
19,130
In agreement with this
fundamental knowledge, previous studies observed that the
cytoplasmic group usually dominates over the periplasmic
group in environments with a relatively higher level of nitrite,
and vice versa for low concentration of nitrite.
25,56
These two
dierent types of NXR are likely associated with dierent
anities for nitrite, which therefore dierentiate the growing
niche of dierent NOB. Members of the Nitrospira with
periplasmic NXR have a lower Kmfor nitrite than other NOB
populations or even anammox bacteria (Table 1). This may
explain why Nitrospira have a stronger endurance to low levels
of nitrite and can compete for nitrite against denitrifiers and
anammox bacteria in the one-stage shortcut nitrogen removal
process.
4.2. Nitrobacter and Ca. Nitrotoga Tolerating Ex Situ
Harsh Treatment. In recent years, a number of studies have
achieved NOB suppression based on FNA (HNO2)/FA
(NH3) inhibitory and biocidal eects,
133
by sidestream sludge
treatment,
134−136
or ex situ treatment. Under acidic conditions,
nitrite can form FNA (HNO2⇌H++ NO2−), which is an
inhibitor of nitrite oxidizers,
17,137
and free ammonia is the un-
ionized form of ammonium that can form under alkaline
conditions.
138
Generally, FNA causes biocidal eects likely due
to the oxidative damage by various reactive nitrogen and
oxygen species dissociated from FNA, which can lead to
oxidative damage to cellular proteins, cell membrane, and
nucleic acids.
133
Dierently, the inhibitory eects of FA are
attributed to the passive diusion of FA molecules into cells,
causing proton imbalance or potassium deficiency.
103,104
All of
these strategies assume that NOB are more sensitive to harsh
conditions than AOB, thus being selectively inhibited;
Nitrospira is especially sensitive to these harsh conditions.
However, several strains of NOB show a certain robustness
against these harsh treatments. As observed in the alternating
treatment by FNA and FA, a clear shift in microbial
community indicated that Nitrobacter may be tolerant to the
FNA treatment.
103
FNA treatment often comes with a low pH,
while to date, only a Nitrobacter strain has been reported to be
acidophilic NOB that can oxidize nitrite at a pH as low as 3.5.
2
In the natural environment, Nitrobacter have been also
detected in large numbers of acidic habitats such as acidic
soil.
2
However, the mechanism of acidophilic Nitrobacter
tolerating the acidic environment remains unknown.
Another recently recognized NOB, Ca. Nitrotoga, have been
shown to have a high tolerance to both FNA and FA treatment
as well.
113
It was reported that FNA ranging from 0 to 1.37 mg
HNO2-N/L was unable to inactivate nitrite oxidation due to
the emergence of Ca. Nitrotoga (from 0% to 4.51%).
115
Likewise, in an activated sludge system regularly exposed to
∼220 mg NH3-N/L FA, the dominant NOB shifted from
Nitrospira (from 61.12% to 2.18% of the nitrifiers) to Ca.
Nitrotoga (from 4.6% to 85.25% of the nitrifiers).
134
The
proliferation of Ca. Nitrotoga was also observed in a recent
study, in which alternating FNA and FA treatment and low
DO control were both applied.
113
All of these results imply
that Ca. Nitrotoga could be a critical challenge to the shortcut
nitrogen removal that is achieved based on sidestream
inactivation using FNA/FA. The mechanism for the tolerance
of Ca. Nitrotoga to FNA (weakly acidic conditions) and FA
(weakly alkaline conditions) is a fundamental question worth
more exploration in the future.
As is known, the NOB suppression is easy in treating high-
strength wastewater during which the entire system is
continuously exposed to the in situ high FNA/FA. However,
in the ex situ sludge treatment by FNA/FA to achieve NOB
suppression in low-strength wastewater, only 20−30% of
mainstream sludge is generally treated every day, which will
provide a certain feasibility for NOB to adapt to the treatment
in long-term operation, as observed in many aforementioned
studies.
102,109
Based on the learning from the NOB
suppression in high-strength wastewater, researchers hypothe-
sized that an in situ harsh treatment of mainstream sludge (e.g.,
in situ high FNA) might enable robust NOB suppression as the
whole bulk of the sludge is continuously exposed to the harsh
condition continuously. Drawing upon this hypothesis, a
robust nitritation process and stable NOB suppression have
been reported recently in an in situ acidic reactor, where the
operating pH was controlled at 5−6.
101
Of note, the low pH
was not achieved by adding acids but was self-sustained by the
microbial ammonia oxidation (NH4++ 1.5 O2→NO2−+ 2 H+
+ H2O), which produces protons. Operation using real sewage
demonstrated stable suppression of NOB in the long term
because AOB can produce nitrite and protons to form in situ
FNA (NO2−+ H+↔HNO2) at a ppm level, as an inhibitor to
NOB.
139,140
As the entire system was exposed to high FNA
continuously, the NOB suppression was very stable, similar to
the principle of NOB suppression in high-strength wastewater
treatment. Interestingly, the novel AOB Ca. Nitrosoglobus were
enriched within these acidic systems, which exhibited extreme
tolerance to acid and FNA,
141
while no known NOB was able
to survive under such harsh conditions (i.e., FNA > 1 mg
HNO2-N/L).
142
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It is reasonable to speculate that Nitrobacter and Ca.
Nitrotoga can be as phylogenetically complex as Nitrospira,
with some branches being tolerant to the harsh treatment and
some not. However, in comparison to Nitrospira, under-
standing the diversities of Nitrobacter and Ca. Nitrotoga is to be
advanced, and the mechanisms behind their tolerance against
FNA or FA are to be further explored. The above experimental
evidence also suggests that it would be extremely challenging
to suppress NOB growth using a single strategy.
132,143
4.3. Nitrolancea Proliferating with High Temperature
and Short SRT. Due to the slow growth rate of NOB and
dierent responses to temperature variation, short SRT and
higher temperatures are proposed to suppress NOB activity.
These strategies have been used in the classical SHARON
(single reactor system for high activity ammonium removal
over nitrite) process, one of the earliest processes designed to
treat high-strength wastewater.
133
The SHARON reactors
employed high temperatures (30−40 °C) and short SRT
(∼1.5 days). In spite of many successful demonstrations, the
new NOB genus Nitrolancea has been identified in engineered
systems with elevated ammonium or nitrite concentrations.
Nitrolancea (Nl.)hollandica was first isolated from a bioreactor
with 428 mM of ammonia to achieve partial nitrification but
ended up with nitrite oxidation. In this case, only a small
amount of Nitrobacter cells were detected, without other
known NOB, indicating the major contribution from an
unknown NOB. The obtained isolate Nitrolancea (Nl.)
hollandica can tolerate a broad temperature range of 25−63
°C and nitrite up to 75 mM, with an optimum temperature of
40 °C.
25
More importantly, a maximal specific growth rate of
0.019 h−1was observed for Nl. hollandica,
25
which is several
times higher than that of other known NOB, suggesting that to
wash out Nl. Hollandica, an even shorter SRT may be needed.
Recently, Spieck et al. obtained four more Nitrolancea strains
from a centrate treatment reactor, all of which have similar
physiological features including thermotolerance, the capability
to grow at high nitrite concentrations, and a fast-growing
pattern.
96
To date, limited studies have reported the presence
of Nitrolancea in reactors; similar 16S rRNA sequences were
found in a partial nitrification reactor
144
and FNA-treated
wastewater.
145
5. CONCLUSIONS AND FUTURE PERSPECTIVES
The discovery of a novel NOB is a key path to broaden our
knowledge for understanding microbial nitrite oxidation in
wastewater treatment. In recent years, novel NOB have come
to light with dierentiated physiology features and metabolic
versatility, resulting in robust adaptability of the NOB to
dierent environments. It can be foreseen that more unknown
NOB species will be discovered in the future. A basic approach
to study novel NOB in previous studies is based on
cultivation/isolation and characterization, which is time-
consuming and labor-intensive. In many studies focusing on
wastewater treatment, 16S rRNA gene amplicon sequencing
has been applied, but the resolution is too low to distinguish
species or reveal their functions. Considering that activated
sludge systems are significantly complex, in situ investigation of
NOB in wastewater treatment processes can be an eclectic
solution. Given the emergence of advanced detection methods,
there should be a tendency for researchers to incorporate
advanced detection techniques with traditional experimental
approaches. For example, metagenomic sequencing is now
intensely used to unravel functional NOB communities in
environmental samples, and this can be more explored in
wastewater samples to identify new NOB. In particular, this
approach can also be used in advanced nitrogen removal
processes that require NOB suppression yet face challenges.
Genomic features of these adapted NOB in shortcut nitrogen
removal can be an important indicator of their physiological
eects, like the nitrite and oxygen anities, as mentioned
above. In parallel, metatranscriptomics can be used to identify
gene expressions in the coculture of NOB and other nitrifiers,
which helps to reveal the synergy and other physiological
eects with their partners compared to single culture and the
NOB physiology, such as the mechanism of the tolerance to
FNA and anity for oxygen. Moreover, many previous studies
have focused on the development of new control strategies
while relatively missing the characterization of abundant NOB
when the process performance fails. To better suppress NOB
in such systems, more attention should be paid to profiling the
physiological characteristics of the NOB, especially at species
levels.
The discovery of a novel NOB also provides opportunities to
suppress or promote nitrite oxidation in wastewater treatment.
Taking the genus Nitrospira as an example, it is now gradually
realized that the functional diversity within genera is non-
negligible, exhibiting diverse physiological features. Among this
genus, comammox Nitrospira feature an extremely high anity
to ammonia and may outcompete other ammonia-oxidizers
under oligotrophic conditions.
69
While being detected in
various wastewater treatment units, comammox Nitrospira was
believed to damage the shortcut nitrogen removal via the
nitrite pathway, as ammonia is oxidized straight away to nitrate
by this unique single microorganism. However, more recent
studies have indicated that ammonia oxidation can be
interrupted at nitrite by comammox bacteria, which have
been successfully coupled with anammox bacteria in dierent
configurations.
134,135
In two very recent reports, comammox
Nitrospira were found to be the main ammonia-oxidizing
community (accounting for 89.2 ±7.9% in total prokaryotic
amoA copies) in a PN/A process at low ammonium loading,
135
and in a symbiotic association of coincubating comammox
Nitrospira and anammox bacteria enabled a sustained nitrogen
loss.
134
The new knowledge highlights a potentially positive
role of comammox Nitrospira in shortcut nitrogen removal
processes, in addition to producing less greenhouse gas N2O
and leading to low residual ammonium in the final
euent.
136,146,147
More specifically, this review also elaborates
that some members in the genera of Ca. Nitrotoga,Nitrobacter,
and Nitrolancea can become important nitrite oxidizers under
specific conditions, such as cold temperature, acidic pH, and
thermophilic processes. With new insights into subdivided
NOB species, there is thus a need to re-evaluate the regarding
contribution to nitrite oxidation especially when corresponding
strategies are being designed to suppress a specific NOB in
advanced nitrogen removal processes. To resolve the puzzle of
mechanisms that are not yet clear, cultivating and character-
ization of these new NOB would be the prerequisites, and new
information needs to be bridged to detailed operation of
engineering systems.
Finally, the review also highlighted the challenge of NOB
suppression using a single strategy. This is primarily because
while using a single strategy to suppress NOB, some specific
NOB that are resistant to this applied strategy may gain a
competitive edge over others, leading to the failure of
suppression in long-term operation. Due to the high diversity
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I
of NOB, it appears that there is no “one-size-fits-all” solution.
However, it is also unlikely that a specific NOB strain can
survive under all of the dierent strategies. For example,
although Nitrospira can survive under low-DO and low-nitrite
conditions, they are very sensitive to harsh treatment by FNA
and FA. Adversely, Nitrobacter are relatively tolerant to FNA
and FA treatment, while they do not usually survive under low-
oxygen conditions. This knowledge suggests that the
integration of multiple strategies may be necessary to
eectively inhibit NOB in long-term operation, which can be
applied either alternatively or concurrently. For instance, the
alternation of ex situ FNA and FNA treatment on a monthly
basis has shown to be more eective in maintaining NOB
suppression than applying each of them individually;
103
a
combined strategy including low DO, ex situ treatment by FA,
and in situ competition by anammox bacteria has been applied
to stably inhibit NOB growth, while without any of the three
factors, NOB adaptation was observed.
148
In addition to the
combined strategies, another potential option to suppress
NOB is to achieve in situ harsh treatment, inspired by the
learning from high-strength wastewater treatment. Due to the
low nitrogen concentration, achieving high in situ FNA as that
in high-strength wastewater needs to lower the pH to 5−6.
Fortunately, this can be achieved by the newly discovered
acidic-tolerant AOB “Ca. Nitrosoglobus”, which generates
protons and nitrite forming in situ high FNA without any
chemical input.
139
As the entire system is continuously
exposed to the in situ high FNA, NOB adaptation is unlikely
to be achieved. Notably, partial denitrification and anammox
(PD/A) could be also a feasible alternative to skirt NOB
suppression, which has been the challenging step in PN/A.
149
PD/A includes nitrification (NH4+→NO3−), partial
denitrification (NO3−→NO2−), and anammox and exhibits
higher stability compared to PN/A,
150,151
which can be a
compromise if PN/A fails.
■AUTHOR INFORMATION
Corresponding Author
Min Zheng −Australian Centre for Water and Environmental
Biotechnology, The University of Queensland, St. Lucia,
Queensland 4072, Australia; orcid.org/0000-0001-9148-
7544; Email: m.zheng@uq.edu.au
Authors
Zicheng Su −Australian Centre for Water and Environmental
Biotechnology, The University of Queensland, St. Lucia,
Queensland 4072, Australia; orcid.org/0000-0002-
3189-6212
Tao Liu −Australian Centre for Water and Environmental
Biotechnology, The University of Queensland, St. Lucia,
Queensland 4072, Australia
Jianhua Guo −Australian Centre for Water and
Environmental Biotechnology, The University of Queensland,
St. Lucia, Queensland 4072, Australia; orcid.org/0000-
0002-4732-9175
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.est.3c00636
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This study was supported by the Australian Research Council
(ARC) Discovery Project DP230101340. The authors thank
Dr. Yue Zheng, Dr. Zhiyao Wang, and Professor Zhiguo Yuan
for a review and helpful discussions. M.Z. is the recipient of an
Advance Queensland Industry Research Fellowship and an
ARC Industry Fellowship (IE230100245). T.L. is an ARC
Discovery Early Career Researcher Award (DECRA) Fellow
(DE220101310). Z.S. acknowledges the China Scholarship
Council (CSC) and the University of Queensland (UQ) for
scholarship support.
■REFERENCES
(1) Alawi, M.; Lipski, A.; Sanders, T.; Eva-Maria-Pfeiffer; Spieck, E.
Cultivation of a Novel Cold-Adapted Nitrite Oxidizing Betaproteo-
bacterium from the Siberian Arctic. Isme J. 2007,1(3), 256−264.
(2) Hankinson, T.; Schmidt, E. An Acidophilic and a Neutrophilic
Nitrobacter Strain Isolated from the Numerically Predominant
Nitrite-Oxidizing Population of an Acid Forest Soil. Appl. Environ.
Microbiol. 1988,54 (6), 1536−1540.
(3) Fuessel, J.; Lam, P.; Lavik, G.; Jensen, M. M.; Holtappels, M.;
Guenter, M.; Kuypers, M. M. M. Nitrite Oxidation in the Namibian
Oxygen Minimum Zone. Isme J. 2012,6(6), 1200−1209.
(4) Fuessel, J.; Lucker, S.; Yilmaz, P.; Nowka, B.; van Kessel, M. A.
H. J.; Bourceau, P.; Hach, P. F.; Littmann, S.; Berg, J.; Spieck, E.;
Daims, H.; Kuypers, M. M. M.; Lam, P. Adaptability as the Key to
Success for the Ubiquitous Marine Nitrite Oxidizer Nitrococcus. Sci.
Adv. 2017,3(11), No. e1700807.
(5) Black, E. M.; Just, C. L. The Genomic Potentials of NOB and
Comammox Nitrospira in River Sediment Are Impacted by Native
Freshwater Mussels. Front. Microbiol. 2018,9, 2061.
(6) Edwards, T. A.; Calica, N. A.; Huang, D. A.; Manoharan, N.;
Hou, W.; Huang, L.; Panosyan, H.; Dong, H.; Hedlund, B. P.
Cultivation and Characterization of Thermophilic Nitrospira Species
from Geothermal Springs in the US Great Basin, China, and Armenia.
Fems Microbiol. Ecol. 2013,85 (2), 283−292.
(7) Lebedeva, E. V.; Off, S.; Zumbraegel, S.; Kruse, M.; Shagzhina,
A.; Lucker, S.; Maixner, F.; Lipski, A.; Daims, H.; Spieck, E. Isolation
and Characterization of a Moderately Thermophilic Nitrite-Oxidizing
Bacterium from a Geothermal Spring. Fems Microbiol. Ecol. 2011,75
(2), 195−204.
(8) Spieck, E.; Spohn, M.; Wendt, K.; Bock, E.; Shively, J.; Frank, J.;
Indenbirken, D.; Alawi, M.; Lucker, S.; Hupeden, J. Extremophilic
Nitrite-Oxidizing Chloroflexi from Yellowstone Hot Springs. ISME J.
2020,14 (2), 364−379.
(9) Juretschko, S.; Timmermann, G.; Schmid, M.; Schleifer, K.-H.;
Pommerening-Röser, A.; Koops, H.-P.; Wagner, M. Combined
Molecular and Conventional Analyses of Nitrifying Bacterium
Diversity in Activated Sludge: Nitrosococcus Mobilis and Nitro-
spira-Like Bacteria as Dominant Populations. Appl. Environ. Microbiol.
1998,64 (8), 3042−3051.
(10) Lucker, S.; Schwarz, J.; Gruber-Dorninger, C.; Spieck, E.;
Wagner, M.; Daims, H. Nitrotoga -like Bacteria Are Previously
Unrecognized Key Nitrite Oxidizers in Full-Scale Wastewater
Treatment Plants. ISME J. 2015,9(3), 708−720.
(11) Saunders, A. M.; Albertsen, M.; Vollertsen, J.; Nielsen, P. H.
The Activated Sludge Ecosystem Contains a Core Community of
Abundant Organisms. ISME J. 2016,10 (1), 11−20.
(12) Wang, Z.; Zheng, M.; Duan, H.; Yuan, Z.; Hu, S. A 20-Year
Journey of Partial Nitritation and Anammox (PN/A): From
Sidestream toward Mainstream. Environ. Sci. Technol. 2022,56, 7522.
(13) Turk, O.; Mavinic, D. S. Preliminary Assessment of a Shortcut
in Nitrogen Removal from Wastewater. Can. J. Civ. Eng. 1986,13 (6),
600−605.
(14) Lackner, S.; Gilbert, E. M.; Vlaeminck, S. E.; Joss, A.; Horn, H.;
van Loosdrecht, M. C. M. Full-Scale Partial Nitritation/Anammox
Experiences −An Application Survey. Water Res. 2014,55, 292−303.
Environmental Science & Technology pubs.acs.org/est Critical Review
https://doi.org/10.1021/acs.est.3c00636
Environ. Sci. Technol. XXXX, XXX, XXX−XXX
J
(15) Zheng, M.; Li, H.; Duan, H.; Liu, T.; Wang, Z.; Zhao, J.; Hu,
Z.; Watts, S.; Meng, J.; Liu, P.; Rattier, M.; Larsen, E.; Guo, J.; Dwyer,
J.; Van Den Akker, B.; Lloyd, J.; Hu, S.; Yuan, Z. One-year stable
pilot-scale operation demonstrates high flexibility of mainstream
anammox application. Water Res. X 2023,19, 100166.
(16) Hellinga, C.; Schellen, A. A. J. C.; Mulder, J. W.; van
Loosdrecht, M. C. M.; Heijnen, J. J. The sharon process: an
innovative method for nitrogen removal from ammonium-rich waste
water. Water Sci. Technol. 1998,37 (9), 135−142.
(17) Hu, Z.; Liu, T.; Wang, Z.; Meng, J.; Zheng, M. 2023. Toward
energy neutrality: novel wastewater treatment incorporating acid-
ophilic ammonia oxidation. Environ. Sci. Technol. 2023,57 (11),
4522−4532.
(18) Agrawal, S.; Seuntjens, D.; De Cocker, P.; Lackner, S.;
Vlaeminck, S. E. Success of Mainstream Partial Nitritation/Anammox
Demands Integration of Engineering, Microbiome and Modeling
Insights. Curr. Opin. Biotechnol. 2018,50, 214−221.
(19) Daims, H.; Luecker, S.; Wagner, M. A New Perspective on
Microbes Formerly Known as Nitrite-Oxidizing Bacteria. Trends
Microbiol. 2016,24 (9), 699−712.
(20) Cao, Y.; van Loosdrecht, M. C. M.; Daigger, G. T. Mainstream
Partial Nitritation−Anammox in Municipal Wastewater Treatment:
Status, Bottlenecks, and Further Studies. Appl. Microbiol. Biotechnol.
2017,101 (4), 1365−1383.
(21) Hug, L. A.; Baker, B. J.; Anantharaman, K.; Brown, C. T.;
Probst, A. J.; Castelle, C. J.; Butterfield, C. N.; Hernsdorf, A. W.;
Amano, Y.; Ise, K.; Suzuki, Y.; Dudek, N.; Relman, D. A.; Finstad, K.
M.; Amundson, R.; Thomas, B. C.; Banfield, J. F. A New View of the
Tree of Life. Nat. Microbiol. 2016,1(5), 1−6.
(22) Vinogradskij, S. N. Recherches sur les organismes de la
nitrification; Impr Charaire: 1889.
(23) Watson, S. W.; Waterbury, J. B. Characteristics of Two Marine
Nitrite Oxidizing Bacteria, Nitrospina Gracilis Nov. Gen. Nov. Sp. and
Nitrococcus Mobilis Nov. Gen. Nov. Sp. Arch. Fur Mikrobiol. 1971,
77 (3), 203−230.
(24) Watson, S.; Bock, E.; Valois, F.; Waterbury, J.; Schlosser, U.
Nitrospira-Marina Gen-Nov Sp-Nov - a Chemolithotrophic Nitrite-
Oxidizing Bacterium. Arch. Microbiol. 1986,144 (1), 1−7.
(25) Sorokin, D. Y.; Luecker, S.; Vejmelkova, D.; Kostrikina, N. A.;
Kleerebezem, R.; Rijpstra, W. I. C.; Damste, J. S. S.; Le Paslier, D.;
Muyzer, G.; Wagner, M.; van Loosdrecht, M. C. M.; Daims, H.
Nitrification Expanded: Discovery, Physiology and Genomics of a
Nitrite-Oxidizing Bacterium from the Phylum Chloroflexi. Isme J.
2012,6(12), 2245−2256.
(26) van Kessel, M. A. H. J.; Speth, D. R.; Albertsen, M.; Nielsen, P.
H.; Op den Camp, H. J. M.; Kartal, B.; Jetten, M. S. M.; Lucker, S.
Complete Nitrification by a Single Microorganism. Nature 2015,528
(7583), 555−559.
(27) Daims, H.; Lebedeva, E. V.; Pjevac, P.; Han, P.; Herbold, C.;
Albertsen, M.; Jehmlich, N.; Palatinszky, M.; Vierheilig, J.; Bulaev, A.;
Kirkegaard, R. H.; von Bergen, M.; Rattei, T.; Bendinger, B.; Nielsen,
P. H.; Wagner, M. Complete Nitrification by Nitrospira Bacteria.
Nature 2015,528 (7583), 504−509.
(28) Ngugi, D. K.; Blom, J.; Stepanauskas, R.; Stingl, U.
Diversification and Niche Adaptations of Nitrospina-like Bacteria in
the Polyextreme Interfaces of Red Sea Brines. Isme J. 2016,10 (6),
1383−1399.
(29) Spieck, E.; Spohn, M.; Wendt, K.; Bock, E.; Shively, J.; Frank,
J.; Indenbirken, D.; Alawi, M.; Lucker, S.; Hupeden, J. Extremophilic
Nitrite-Oxidizing Chloroflexi from Yellowstone Hot Springs. ISME J.
2020,14 (2), 364−379.
(30) Mueller, A. J.; Jung, M.-Y.; Strachan, C. R.; Herbold, C. W.;
Kirkegaard, R. H.; Wagner, M.; Daims, H. Genomic and Kinetic
Analysis of Novel Nitrospinae Enriched by Cell Sorting. ISME J.
2021,15 (3), 732−745.
(31) Winogradsky, S. Contributions a La Morphologie Des
Organisms de La Nitrification. Arkhiv Biol. Nauk St Petersbourg
1892,1, 87−137.
(32) Bock, E. Growth of Nitrobacter in Presence of Organic-Matter
0.2. Chemo-Organotrophic Growth of Nitrobacter-Agilis. Arch.
Microbiol. 1976,108 (3), 305−312.
(33) Steinmuller, W.; Bock, E. Enzymatic Studies on Autotrophi-
cally, Mixotrophically and Heterotrophically Grown Nitrobacter-
Agilis with Special Reference to Nitrite Oxidase. Arch. Microbiol.
1977,115 (1), 51−54.
(34) Sundermeyer, H.; Bock, E. Energy-Metabolism of Autotroph-
ically and Heterotrophically Grown Cells of Nitrobacter-Winograd-
skyi. Arch. Microbiol. 1981,130 (3), 250−254.
(35) Lucker, S.; Nowka, B.; Rattei, T.; Spieck, E.; Daims, H. The
Genome of Nitrospina Gracilis Illuminates the Metabolism and
Evolution of the Major Marine Nitrite Oxidizer. Front. Microbiol.
2013,4, 27.
(36) Beman, J. M.; Leilei Shih, J.; Popp, B. N. Nitrite Oxidation in
the Upper Water Column and Oxygen Minimum Zone of the Eastern
Tropical North Pacific Ocean. ISME J. 2013,7(11), 2192−2205.
(37) Sun, X.; Kop, L. F. M.; Lau, M. C. Y.; Frank, J.; Jayakumar, A.;
Lucker, S.; Ward, B. B. Uncultured Nitrospina -like Species Are Major
Nitrite Oxidizing Bacteria in Oxygen Minimum Zones. ISME J. 2019,
13 (10), 2391−2402.
(38) Kitzinger, K.; Koch, H.; Lucker, S.; Sedlacek, C. J.; Herbold, C.;
Schwarz, J.; Daebeler, A.; Mueller, A. J.; Lukumbuzya, M.; Romano,
S.; Leisch, N.; Karst, S. M.; Kirkegaard, R.; Albertsen, M.; Nielsen, P.
H.; Wagner, M.; Daims, H. Characterization of the First “Candidatus
Nitrotoga” Isolate Reveals Metabolic Versatility and Separate
Evolution of Widespread Nitrite-Oxidizing Bacteria. mBio 2018,9
(4), e01186−18.
(39) Boddicker, A. M.; Mosier, A. C. Genomic Profiling of Four
Cultivated Candidatus Nitrotoga Spp. Predicts Broad Metabolic
Potential and Environmental Distribution. Isme J. 2018,12 (12),
2864−2882.
(40) Hemp, J.; Lucker, S.; Schott, J.; Pace, L. A.; Johnson, J. E.;
Schink, B.; Daims, H.; Fischer, W. W. Genomics of a Phototrophic
Nitrite Oxidizer: Insights into the Evolution of Photosynthesis and
Nitrification. ISME J. 2016,10 (11), 2669−2678.
(41) Koch, H.; Lucker, S.; Albertsen, M.; Kitzinger, K.; Herbold, C.;
Spieck, E.; Nielsen, P. H.; Wagner, M.; Daims, H. Expanded
Metabolic Versatility of Ubiquitous Nitrite-Oxidizing Bacteria from
the Genus Nitrospira. Proc. Natl. Acad. Sci. U. S. A. 2015,112 (36),
11371−11376.
(42) Koch, H.; Galushko, A.; Albertsen, M.; Schintlmeister, A.;
Gruber-Dorninger, C.; Lucker, S.; Pelletier, E.; Le Paslier, D.; Spieck,
E.; Richter, A.; Nielsen, P. H.; Wagner, M.; Daims, H. Growth of
Nitrite-Oxidizing Bacteria by Aerobic Hydrogen Oxidation. Science
2014,345, 1052−1054.
(43) Kitzinger, K.; Marchant, H. K.; Bristow, L. A.; Herbold, C. W.;
Padilla, C. C.; Kidane, A. T.; Littmann, S.; Daims, H.; Pjevac, P.;
Stewart, F. J.; Wagner, M.; Kuypers, M. M. M. Single Cell Analyses
Reveal Contrasting Life Strategies of the Two Main Nitrifiers in the
Ocean. Nat. Commun. 2020,11 (1), 767.
(44) Palatinszky, M.; Herbold, C.; Jehmlich, N.; Pogoda, M.; Han,
P.; von Bergen, M.; Lagkouvardos, I.; Karst, S. M.; Galushko, A.;
Koch, H.; Berry, D.; Daims, H.; Wagner, M. Cyanate as an Energy
Source for Nitrifiers. Nature 2015,524 (7563), 105−108.
(45) Zhu, W.; Lomsadze, A.; Borodovsky, M. Ab Initio Gene
Identification in Metagenomic Sequences. Nucleic Acids Res. 2010,38
(12), No. e132.
(46) Kanehisa, M.; Sato, Y.; Kawashima, M.; Furumichi, M.; Tanabe,
M. KEGG as a Reference Resource for Gene and Protein Annotation.
Nucleic Acids Res. 2016,44 (D1), D457−462.
(47) Galperin, M. Y.; Wolf, Y. I.; Makarova, K. S.; Vera Alvarez, R.;
Landsman, D.; Koonin, E. V. COG Database Update: Focus on
Microbial Diversity, Model Organisms, and Widespread Pathogens.
Nucleic Acids Res. 2021,49 (D1), D274−D281.
(48) The UniProt Consortium. UniProt: The Universal Protein
Knowledgebase in 2023. Nucleic Acids Res. 2023,51 (D1), D523−
D531.
Environmental Science & Technology pubs.acs.org/est Critical Review
https://doi.org/10.1021/acs.est.3c00636
Environ. Sci. Technol. XXXX, XXX, XXX−XXX
K
(49) Wagner, M.; Koops, H.-P.; Flood, J.; Amann, R.; Rath, G. In
Situ Analysis of Nitrifying Bacteria in Sewage Treatment Plants. Water
Sci. Technol. 1996,34 (1-2), 237.
(50) Daims, H.; Nielsen, J. L.; Nielsen, P. H.; Schleifer, K. H.;
Wagner, M. In Situ Characterization of Nitrospira-like Nitrite
Oxidizing Bacteria Active in Wastewater Treatment Plants. Appl.
Environ. Microbiol. 2001,67 (11), 5273−5284.
(51) Zhang, T.; Shao, M.-F.; Ye, L. 454 Pyrosequencing Reveals
Bacterial Diversity of Activated Sludge from 14 Sewage Treatment
Plants. ISME J. 2012,6(6), 1137−1147.
(52) Wu, L.; Ning, D.; Zhang, B.; Li, Y.; Zhang, P.; Shan, X.; Zhang,
Q.; Brown, M. R.; Li, Z.; Van Nostrand, J. D.; Ling, F.; Xiao, N.;
Zhang, Y.; Vierheilig, J.; Wells, G. F.; Yang, Y.; Deng, Y.; Tu, Q.;
Wang, A.; Zhang, T.; He, Z.; Keller, J.; Nielsen, P. H.; Alvarez, P. J. J.;
Criddle, C. S.; Wagner, M.; Tiedje, J. M.; He, Q.; Curtis, T. P.; Stahl,
D. A.; Alvarez-Cohen, L.; Rittmann, B. E.; Wen, X.; Zhou, J. Global
Diversity and Biogeography of Bacterial Communities in Wastewater
Treatment Plants. Nat. Microbiol. 2019,4(7), 1183−1195.
(53) Gruber-Dorninger, C.; Pester, M.; Kitzinger, K.; Savio, D. F.;
Loy, A.; Rattei, T.; Wagner, M.; Daims, H. Functionally Relevant
Diversity of Closely Related Nitrospira in Activated Sludge. Isme J.
2015,9(3), 643−655.
(54) Tangkitjawisut, W.; Limpiyakorn, T.; Powtongsook, S.;
Pornkulwat, P.; Suwannasilp, B. B. Differences in Nitrite-Oxidizing
Communities and Kinetics in a Brackish Environment after
Enrichment at Low and High Nitrite Concentrations. J. Environ. Sci.
2016,42, 41−49.
(55) Ishii, K.; Fujitani, H.; Soh, K.; Nakagawa, T.; Takahashi, R.;
Tsuneda, S. Enrichment and Physiological Characterization of a Cold-
Adapted Nitrite-Oxidizing Nitrotoga Sp from an Eelgrass Sediment.
Appl. Environ. Microbiol. 2017,83 (14), e00549−17.
(56) Nowka, B.; Daims, H.; Spieck, E. Comparison of Oxidation
Kinetics of Nitrite-Oxidizing Bacteria: Nitrite Availability as a Key
Factor in Niche Differentiation. Appl. Environ. Microbiol. 2015,81 (2),
745−753.
(57) Liu, G.; Wang, J. Long-Term Low DO Enriches and Shifts
Nitrifier Community in Activated Sludge. Environ. Sci. Technol. 2013,
47 (10), 5109−5117.
(58) Chao, Y.; Mao, Y.; Yu, K.; Zhang, T. Novel Nitrifiers and
Comammox in a Full-Scale Hybrid Biofilm and Activated Sludge
Reactor Revealed by Metagenomic Approach. Appl. Microbiol.
Biotechnol. 2016,100 (18), 8225−8237.
(59) Spasov, E.; Tsuji, J. M.; Hug, L. A.; Doxey, A. C.; Sauder, L. A.;
Parker, W. J.; Neufeld, J. D. High Functional Diversity among
Nitrospira Populations That Dominate Rotating Biological Contactor
Microbial Communities in a Municipal Wastewater Treatment Plant.
Isme J. 2020,14 (7), 1857−1872.
(60) Cotto, I.; Dai, Z.; Huo, L.; Anderson, C. L.; Vilardi, K. J.; Ijaz,
U.; Khunjar, W.; Wilson, C.; De Clippeleir, H.; Gilmore, K.; Bailey,
E.; Pinto, A. J. Long Solids Retention Times and Attached Growth
Phase Favor Prevalence of Comammox Bacteria in Nitrogen Removal
Systems. Water Res. 2020,169, No. 115268.
(61) Huang, T.; Xia, J.; Liu, T.; Su, Z.; Guan, Y.; Guo, J.; Wang, C.;
Zheng, M. Comammox Nitrospira Bacteria Are Dominant Ammonia
Oxidizers in Mainstream Nitrification Bioreactors Emended with
Sponge Carriers. Environ. Sci. Technol. 2022,56 (17), 12584−12591.
(62) Zhao, J.; Zheng, M.; Su, Z.; Liu, T.; Li, J.; Guo, J.; Yuan, Z.; Hu,
S. Selective Enrichment of Comammox Nitrospira in a Moving Bed
Biofilm Reactor with Sufficient Oxygen Supply. Environ. Sci. Technol.
2022,56 (18), 13338−13346.
(63) Laanbroek, H. J.; Gerards, S. Competition for Limiting
Amounts of Oxygen between Nitrosomonas Europaea and Nitro-
bacter Winogradskyi Grown in Mixed Continuous Cultures. Arch.
Microbiol. 1993,159 (5), 453−459.
(64) Law, Y.; Matysik, A.; Chen, X.; Thi, S. S.; Nguyen, T. Q. N.;
Qiu, G.; Natarajan, G.; Williams, R. B. H.; Ni, B.-J.; Seviour, T. W.;
Wuertz, S. High Dissolved Oxygen Selection against Nitrospira
Sublineage I in Full-Scale Activated Sludge. Environ. Sci. Technol.
2019,53 (14), 8157−8166.
(65) Manser, R.; Gujer, W.; Siegrist, H. Consequences of Mass
Transfer Effects on the Kinetics of Nitrifiers. Water Res. 2005,39
(19), 4633−4642.
(66) Picioreanu, C.; Pérez, J.; van Loosdrecht, M. C. M. Impact of
Cell Cluster Size on Apparent Half-Saturation Coefficients for
Oxygen in Nitrifying Sludge and Biofilms. Water Res. 2016,106,
371−382.
(67) Moussa, M. S.; Hooijmans, C. M.; Lubberding, H. J.; Gijzen, H.
J.; van Loosdrecht, M. C. M. Modelling Nitrification, Heterotrophic
Growth and Predation in Activated Sludge. Water Res. 2005,39 (20),
5080−5098.
(68) Lackner, S.; Terada, A.; Horn, H.; Henze, M.; Smets, B. F.
Nitritation Performance in Membrane-Aerated Biofilm Reactors
Differs from Conventional Biofilm Systems. Water Res. 2010,44
(20), 6073−6084.
(69) Kits, K. D.; Sedlacek, C. J.; Lebedeva, E. V.; Han, P.; Bulaev, A.;
Pjevac, P.; Daebeler, A.; Romano, S.; Albertsen, M.; Stein, L. Y.;
Daims, H.; Wagner, M. Kinetic Analysis of a Complete Nitrifier
Reveals an Oligotrophic Lifestyle. Nature 2017,549 (7671), 269−
272.
(70) Annavajhala, M. K.; Kapoor, V.; Santo-Domingo, J.; Chandran,
K. Comammox Functionality Identified in Diverse Engineered
Biological Wastewater Treatment Systems. Environ. Sci. Technol.
Lett. 2018,5(2), 110−116.
(71) Wang, Y.; Ma, L.; Mao, Y.; Jiang, X.; Xia, Y.; Yu, K.; Li, B.;
Zhang, T. Comammox in Drinking Water Systems. Water Res. 2017,
116, 332−341.
(72) Yang, Y.; Daims, H.; Liu, Y.; Herbold, C. W.; Pjevac, P.; Lin, J.-
G.; Li, M.; Gu, J.-D. Activity and Metabolic Versatility of Complete
Ammonia Oxidizers in Full-Scale Wastewater Treatment Systems.
mBio 2020,11 (2), 1 DOI: 10.1128/mBio.03175-19.
(73) Udert, K. M.; Larsen, T. A.; Biebow, M.; Gujer, W. Urea
Hydrolysis and Precipitation Dynamics in a Urine-Collecting System.
Water Res. 2003,37 (11), 2571−2582.
(74) Sakoula, D.; Nowka, B.; Spieck, E.; Daims, H.; Lucker, S. The
Draft Genome Sequence of “Nitrospira Lenta” Strain BS10, a Nitrite
Oxidizing Bacterium Isolated from Activated Sludge. Stand. Genomic
Sci. 2018,13 (1), 32.
(75) Ushiki, N.; Fujitani, H.; Shimada, Y.; Morohoshi, T.; Sekiguchi,
Y.; Tsuneda, S. Genomic Analysis of Two Phylogenetically Distinct
Nitrospira Species Reveals Their Genomic Plasticity and Functional
Diversity. Front. Microbiol. 2018,8, 2637 DOI: 10.3389/
fmicb.2017.02637.
(76) Li, J.; Hua, Z.-S.; Liu, T.; Wang, C.; Li, J.; Bai, G.; Lucker, S.;
Jetten, M. S. M.; Zheng, M.; Guo, J. Selective Enrichment and
Metagenomic Analysis of Three Novel Comammox Nitrospira in a
Urine-Fed Membrane Bioreactor. ISME Commun. 2021,1(1), 1−8.
(77) Alonso-Sáez, L.; Waller, A. S.; Mende, D. R.; Bakker, K.;
Farnelid, H.; Yager, P. L.; Lovejoy, C.; Tremblay, J.-E.; Potvin, M.;
Heinrich, F.; Estrada, M.; Riemann, L.; Bork, P.; Pedrós-Alió, C.;
Bertilsson, S. Role for Urea in Nitrification by Polar Marine Archaea.
Proc. Natl. Acad. Sci. U. S. A. 2012,109 (44), 17989−17994.
(78) Kitzinger, K.; Padilla, C. C.; Marchant, H. K.; Hach, P. F.;
Herbold, C. W.; Kidane, A. T.; Könneke, M.; Littmann, S.;
Mooshammer, M.; Niggemann, J.; Petrov, S.; Richter, A.; Stewart,
F. J.; Wagner, M.; Kuypers, M. M. M.; Bristow, L. A. Cyanate and
Urea Are Substrates for Nitrification by Thaumarchaeota in the
Marine Environment. Nat. Microbiol. 2019,4(2), 234−243.
(79) Tourna, M.; Stieglmeier, M.; Spang, A.; Könneke, M.;
Schintlmeister, A.; Urich, T.; Engel, M.; Schloter, M.; Wagner, M.;
Richter, A.; Schleper, C. Nitrososphaera Viennensis, an Ammonia
Oxidizing Archaeon from Soil. Proc. Natl. Acad. Sci. U. S. A. 2011,108
(20), 8420−8425.
(80) Keuter, S.; Koch, H.; Sass, K.; Wegen, S.; Lee, N.; Lucker, S.;
Spieck, E. Some like It Cold: The Cellular Organization and
Physiological Limits of Cold-Tolerant Nitrite-Oxidizing Nitrotoga.
Environ. Microbiol. 2022,24 (4), 2059−2077.
(81) Keene, N. A.; Reusser, S. R.; Scarborough, M. J.; Grooms, A. L.;
Seib, M.; Domingo, J. S.; Noguera, D. R. Pilot Plant Demonstration of
Environmental Science & Technology pubs.acs.org/est Critical Review
https://doi.org/10.1021/acs.est.3c00636
Environ. Sci. Technol. XXXX, XXX, XXX−XXX
L
Stable and Efficient High Rate Biological Nutrient Removal with Low
Dissolved Oxygen Conditions. Water Res. 2017,121, 72−85.
(82) Liu, Y.; Li, S.; Ni, G.; Duan, H.; Huang, X.; Yuan, Z.; Zheng, M.
Temperature Variations Shape Niche Occupation of Nitrotoga-like
Bacteria in Activated Sludge. ACS EST Water 2021,1(1), 167−174.
(83) Alawi, M.; Off, S.; Kaya, M.; Spieck, E. Temperature Influences
the Population Structure of Nitrite-Oxidizing Bacteria in Activated
Sludge. Environ. Microbiol. Rep. 2009,1(3), 184−190.
(84) Wegen, S.; Nowka, B.; Spieck, E. Low Temperature and
Neutral PH Define “Candidatus Nitrotoga Sp.” as a Competitive
Nitrite Oxidizer in Coculture with Nitrospira Defluvii. Appl. Environ.
Microbiol. 2019,85 (9), e02569-18 DOI: 10.1128/AEM.02569-18.
(85) Ehrich, S.; Behrens, D.; Lebedeva, E.; Ludwig, W.; Bock, E. A
New Obligately Chemolithoautotrophic, Nitrite-Oxidizing Bacter-
ium,Nitrospira Moscoviensis Sp. Nov. and Its Phylogenetic Relation-
ship. Arch. Microbiol. 1995,164 (1), 16−23.
(86) Spieck, E.; Hartwig, C.; McCormack, I.; Maixner, F.; Wagner,
M.; Lipski, A.; Daims, H. Selective Enrichment and Molecular
Characterization of a Previously Uncultured Nitrospira-like Bacterium
from Activated Sludge. Environ. Microbiol. 2006,8(3), 405−415.
(87) Ushiki, N.; Fujitani, H.; Aoi, Y.; Tsuneda, S. Isolation of
Nitrospira Belonging to Sublineage II from a Wastewater Treatment
Plant. Microbes Environ. 2013,28 (3), 346−353.
(88) Fujitani, H.; Ushiki, N.; Tsuneda, S.; Aoi, Y. Isolation of
Sublineage I Nitrospira by a Novel Cultivation Strategy. Environ.
Microbiol. 2014,16 (10), 3030−3040.
(89) Jacob, J.; Nowka, B.; Merten, V.; Sanders, T.; Spieck, E.;
Daehnke, K. Oxidation Kinetics and Inverse Isotope Effect of Marine
Nitrite-Oxidizing Isolates. Aquat. Microb. Ecol. 2017,80 (3), 289−
300.
(90) Laanbroek, H.; Bodelier, P.; Gerards, S. Oxygen-Consumption
Kinetics of Nitrosomonas-Europaea and Nitrobacter-Hamburgensis
Grown in Mixed Continuous Cultures at Different Oxygen
Concentrations. Arch. Microbiol. 1994,161 (2), 156−162.
(91) Both, G. J.; Gerards, S.; Laanbroek, H. J. Kinetics of Nitrite
Oxidation in Two Nitrobacter Species Grown in Nitrite-Limited
Chemostats. Arch. Microbiol. 1992,157 (5), 436−441.
(92) Laudelout, H.; Vantichelen, L. Kinetics of the Nitrite Oxidation
by Nitrobacter-Winogradskyi. J. Bacteriol. 1960,79 (1), 39−42.
(93) Bock, E.; Koops, H.-P.; Möller, U. C.; Rudert, M. A New
Facultatively Nitrite Oxidizing Bacterium, Nitrobacter Vulgaris Sp.
Nov. Arch. Microbiol. 1990,153 (2), 105−110.
(94) Hupeden, J.; Wegen, S.; Off, S.; Luecker, S.; Bedarf, Y.; Daims,
H.; Kuehn, C.; Spieck, E. Relative Abundance of Nitrotoga Spp. in a
Biofilter of a Cold-Freshwater Aquaculture Plant Appears To Be
Stimulated by Slightly Acidic PH. Appl. Environ. Microbiol. 2016,82
(6), 1838−1845.
(95) Spieck, E.; Keuter, S.; Wenzel, T.; Bock, E.; Ludwig, W.
Characterization of a New Marine Nitrite Oxidizing Bacterium,
Nitrospina Watsonii Sp Nov., a Member of the Newly Proposed
Phylum “Nitrospinae. Syst. Appl. Microbiol. 2014,37 (3), 170−176.
(96) Spieck, E.; Sass, K.; Keuter, S.; Hirschmann, S.; Spohn, M.;
Indenbirken, D.; Kop, L. F. M.; Luecker, S.; Giaveno, A. Defining
Culture Conditions for the Hidden Nitrite-Oxidizing Bacterium
Nitrolancea. Front. Microbiol. 2020,11, 1522.
(97) Ji, J.; Peng, Y.; Wang, B.; Wang, S. Achievement of High Nitrite
Accumulation via Endogenous Partial Denitrification (EPD).
Bioresour. Technol. 2017,224, 140−146.
(98) Antileo, C.; Medina, H.; Bornhardt, C.; Munoz, C.; Jaramillo
Montoya, F.; Proal Najera, J. B. Actuators Monitoring System for
Real-Time Control of Nitrification−Denitrification via Nitrite on
Long Term Operation. Chem. Eng. J. 2013,223, 467−478.
(99) Jetten, M. S. M.; Horn, S. J.; van Loosdrecht, M. C. M.
Towards a More Sustainable Municipal Wastewater Treatment
System. Water Sci. Technol. 1997,35 (9), 171−180.
(100) Wett, B. Development and Implementation of a Robust
Deammonification Process. Water Sci. Technol. J. Int. Assoc. Water
Pollut. Res. 2007,56 (7), 81−88.
(101) Li, J.; Xu, K.; Liu, T.; Bai, G.; Liu, Y.; Wang, C.; Zheng, M.
Achieving Stable Partial Nitritation in an Acidic Nitrifying Bioreactor.
Environ. Sci. Technol. 2020,54 (1), 456−463.
(102) Wang, Q.; Ye, L.; Jiang, G.; Hu, S.; Yuan, Z. Side-Stream
Sludge Treatment Using Free Nitrous Acid Selectively Eliminates
Nitrite Oxidizing Bacteria and Achieves the Nitrite Pathway. Water
Res. 2014,55, 245−255.
(103) Duan, H.; Ye, L.; Lu, X.; Yuan, Z. Overcoming Nitrite
Oxidizing Bacteria Adaptation through Alternating Sludge Treatment
with Free Nitrous Acid and Free Ammonia. Environ. Sci. Technol.
2019,53 (4), 1937−1946.
(104) Wang, D.; Wang, Q.; Laloo, A.; Xu, Y.; Bond, P. L.; Yuan, Z.
Achieving Stable Nitritation for Mainstream Deammonification by
Combining Free Nitrous Acid-Based Sludge Treatment and Oxygen
Limitation. Sci. Rep. 2016,6, 25547.
(105) Delgado Vela, J.; Dick, G. J.; Love, N. G. Sulfide Inhibition of
Nitrite Oxidation in Activated Sludge Depends on Microbial
Community Composition. Water Res. 2018,138, 241−249.
(106) Huang, S.; Zhu, Y.; Lian, J.; Liu, Z.; Zhang, L.; Tian, S.
Enhancement in the Partial Nitrification of Wastewater Sludge via
Lowintensity Ultrasound: Effects on Rapid Start-up and Temperature
Resilience. Bioresour. Technol. 2019,294, No. 122196.
(107) Huang, S.; Zhu, Y.; Zhang, G.; Lian, J.; Liu, Z.; Zhang, L.;
Tian, S. Effects of Low-Intensity Ultrasound on Nitrite Accumulation
and Microbial Characteristics during Partial Nitrification. Sci. Total
Environ. 2020,705, No. 135985.
(108) Wang, L.; Qiu, S.; Guo, J.; Ge, S. Light Irradiation Enables
Rapid Start-Up of Nitritation through Suppressing NxrB Gene
Expression and Stimulating Ammonia-Oxidizing Bacteria. Environ.
Sci. Technol. 2021,55 (19), 13297−13305.
(109) Duan, H.; Wang, Q.; Erler, D. V.; Ye, L.; Yuan, Z. Effects of
Free Nitrous Acid Treatment Conditions on the Nitrite Pathway
Performance in Mainstream Wastewater Treatment. Sci. Total
Environ. 2018,644, 360−370.
(110) Li, X.; Sun, S.; Badgley, B. D.; Sung, S.; Zhang, H.; He, Z.
Nitrogen Removal by Granular Nitritation−Anammox in an Upflow
Membrane-Aerated Biofilm Reactor. Water Res. 2016,94, 23−31.
(111) Laureni, M.; Falås, P.; Robin, O.; Wick, A.; Weissbrodt, D. G.;
Nielsen, J. L.; Ternes, T. A.; Morgenroth, E.; Joss, A. Mainstream
Partial Nitritation and Anammox: Long-Term Process Stability and
Effluent Quality at Low Temperatures. Water Res. 2016,101, 628−
639.
(112) Li, J.; Peng, Y.; Zhang, L.; Liu, J.; Wang, X.; Gao, R.; Pang, L.;
Zhou, Y. Quantify the Contribution of Anammox for Enhanced
Nitrogen Removal through Metagenomic Analysis and Mass Balance
in an Anoxic Moving Bed Biofilm Reactor. Water Res. 2019,160,
178−187.
(113) Zheng, M.; Li, S.; Ni, G.; Xia, J.; Hu, S.; Yuan, Z.; Liu, Y.;
Huang, X. Critical Factors Facilitating Candidatus Nitrotoga To Be
Prevalent Nitrite-Oxidizing Bacteria in Activated Sludge. Environ. Sci.
Technol. 2020,54, 15414.
(114) Wang, Z.; Zheng, M.; Hu, Z.; Duan, H.; De Clippeleir, H.; Al-
Omari, A.; Hu, S.; Yuan, Z. Unravelling Adaptation of Nitrite-
Oxidizing Bacteria in Mainstream PN/A Process: Mechanisms and
Counter-Strategies. Water Res. 2021,200, No. 117239.
(115) Ma, B.; Yang, L.; Wang, Q.; Yuan, Z.; Wang, Y.; Peng, Y.
Inactivation and Adaptation of Ammonia-Oxidizing Bacteria and
Nitrite-Oxidizing Bacteria When Exposed to Free Nitrous Acid.
Bioresour. Technol. 2017,245, 1266−1270.
(116) Ma, Y.; Domingo-Félez, C.; Plósz, B. Gy.; Smets, B. F.
Intermittent Aeration Suppresses Nitrite-Oxidizing Bacteria in
Membrane-Aerated Biofilms: A Model-Based Explanation. Environ.
Sci. Technol. 2017,51 (11), 6146−6155.
(117) Ge, S.; Peng, Y.; Qiu, S.; Zhu, A.; Ren, N. Complete Nitrogen
Removal from Municipal Wastewater via Partial Nitrification by
Appropriately Alternating Anoxic/Aerobic Conditions in a Continu-
ous Plug-Flow Step Feed Process. Water Res. 2014,55, 95−105.
(118) Pellicer-Nacher, C.; Sun, S.; Lackner, S.; Terada, A.; Schreiber,
F.; Zhou, Q.; Smets, B. F. Sequential Aeration of Membrane-Aerated
Environmental Science & Technology pubs.acs.org/est Critical Review
https://doi.org/10.1021/acs.est.3c00636
Environ. Sci. Technol. XXXX, XXX, XXX−XXX
M
Biofilm Reactors for High-Rate Autotrophic Nitrogen Removal:
Experimental Demonstration. Environ. Sci. Technol. 2010,44 (19),
7628−7634.
(119) Liu, T.; Hu, S.; Yuan, Z.; Guo, J. High-Level Nitrogen
Removal by Simultaneous Partial Nitritation, Anammox and Nitrite/
Nitrate-Dependent Anaerobic Methane Oxidation. Water Res. 2019,
166, No. 115057.
(120) Gong, X.; Garcia-Robledo, E.; Schramm, A.; Revsbech, N. P.
Respiratory Kinetics of Marine Bacteria Exposed to Decreasing
Oxygen Concentrations. Appl. Environ. Microbiol. 2016,82 (5),
1412−1422.
(121) Pitcher, R. S.; Watmough, N. J. The Bacterial Cytochrome
Cbb3 Oxidases. Biochim. Biophys. Acta BBA - Bioenerg. 2004,1655,
388−399.
(122) Ushiki, N.; Jinno, M.; Fujitani, H.; Suenaga, T.; Terada, A.;
Tsuneda, S. Nitrite Oxidation Kinetics of Two Nitrospira Strains: The
Quest for Competition and Ecological Niche Differentiation. J. Biosci.
Bioeng. 2017,123 (5), 581−589.
(123) Calhoun, M. W.; Thomas, J. W.; Gennis, R. B. The
Cytochrome Oxidase Superfamily of Redox-Driven Proton Pumps.
Trends Biochem. Sci. 1994,19 (8), 325−330.
(124) Starkenburg, S. R.; Larimer, F. W.; Stein, L. Y.; Klotz, M. G.;
Chain, P. S. G.; Sayavedra-Soto, L. A.; Poret-Peterson, A. T.; Gentry,
M. E.; Arp, D. J.; Ward, B.; Bottomley, P. J. Complete Genome
Sequence of Nitrobacter Hamburgensis X14 and Comparative
Genomic Analysis of Species within the Genus Nitrobactei. Appl.
Environ. Microbiol. 2008,74 (9), 2852−2863.
(125) Borisov, V. B.; Gennis, R. B.; Hemp, J.; Verkhovsky, M. I. The
Cytochrome Bd Respiratory Oxygen Reductases. Biochim. Biophys.
Acta-Bioenerg. 2011,1807 (11), 1398−1413.
(126) Mundinger, A. B.; Lawson, C. E.; Jetten, M. S. M.; Koch, H.;
Lucker, S. Cultivation and Transcriptional Analysis of a Canonical
Nitrospira Under Stable Growth Conditions. Front. Microbiol. 2019,
10, 1 DOI: 10.3389/fmicb.2019.01325.
(127) Park, H.-D.; Noguera, D. R. Nitrospira Community
Composition in Nitrifying Reactors Operated with Two Different
Dissolved Oxygen Levels. J. Microbiol. Biotechnol. 2008,18 (8), 1470−
1474.
(128) Roots, P.; Wang, Y.; Rosenthal, A. F.; Griffin, J. S.; Sabba, F.;
Petrovich, M.; Yang, F.; Kozak, J. A.; Zhang, H.; Wells, G. F.
Comammox Nitrospira Are the Dominant Ammonia Oxidizers in a
Mainstream Low Dissolved Oxygen Nitrification Reactor. Water Res.
2019,157, 396−405.
(129) Shao, Y.-H.; Wu, J.-H. Comammox Nitrospira Species
Dominate in an Efficient Partial Nitrification−Anammox Bioreactor
for Treating Ammonium at Low Loadings. Environ. Sci. Technol. 2021,
55 (3), 2087−2098.
(130) Lucker, S.; Wagner, M.; Maixner, F.; Pelletier, E.; Koch, H.;
Vacherie, B.; Rattei, T.; Damste, J. S. S.; Spieck, E.; Le Paslier, D.;
Daims, H. A Nitrospira Metagenome Illuminates the Physiology and
Evolution of Globally Important Nitrite-Oxidizing Bacteria. Proc. Natl.
Acad. Sci. U. S. A. 2010,107 (30), 13479−13484.
(131) Meincke, M.; Bock, E.; Kastrau, D.; Kroneck, P. Nitrite
Oxidoreductase from Nitrobacter-Hamburgensis - Redox Centers and
Their Catalytic Role. Arch. Microbiol. 1992,158 (2), 127−131.
(132) Spieck, E.; Aamand, J.; Bartosch, S.; Bock, E. Immunocy-
tochemical Detection and Location of the Membrane-Bound Nitrite
Oxidoreductase in Cells of Nitrobacter and Nitrospira. Fems Microbiol.
Lett. 1996,139 (1), 71−76.
(133) Duan, H.; Gao, S.; Li, X.; Ab Hamid, N. H.; Jiang, G.; Zheng,
M.; Bai, X.; Bond, P. L.; Lu, X.; Chislett, M. M.; Hu, S.; Ye, L.; Yuan,
Z. Improving Wastewater Management Using Free Nitrous Acid
(FNA). Water Res. 2020,171, No. 115382.
(134) Li, S.; Duan, H.; Zhang, Y.; Huang, X.; Yuan, Z.; Liu, Y.;
Zheng, M. Adaptation of Nitrifying Community in Activated Sludge
to Free Ammonia Inhibition and Inactivation. Sci. Total Environ.
2020,728, No. 138713.
(135) Wang, Z.; Zheng, M.; Xue, Y.; Xia, J.; Zhong, H.; Ni, G.; Liu,
Y.; Yuan, Z.; Hu, S. Free Ammonia Shock Treatment Eliminates
Nitrite-Oxidizing Bacterial Activity for Mainstream Biofilm Nitritation
Process. Chem. Eng. J. 2020,393, No. 124682.
(136) Zheng, M.; Wang, Z.; Meng, J.; Hu, Z.; Liu, Y.; Yuan, Z.; Hu,
S. Inactivation Kinetics of Nitrite-Oxidizing Bacteria by Free Nitrous
Acid. Sci. Total Environ. 2021,752, No. 141876.
(137) Anthonisen, A. C.; Loehr, R. C.; Prakasam, T. B. S.; Srinath, E.
G. Inhibition of Nitrification by Ammonia and Nitrous Acid. J. Water
Pollut. Control Fed. 1976,48 (5), 835−852.
(138) Liu, Y.; Ngo, H. H.; Guo, W.; Peng, L.; Wang, D.; Ni, B. The
Roles of Free Ammonia (FA) in Biological Wastewater Treatment
Processes: A Review. Environ. Int. 2019,123, 10−19.
(139) Wang, Z.; Zheng, M.; Meng, J.; Hu, Z.; Ni, G.; Guerrero
Calderon, A.; Li, H.; De Clippeleir, H.; Al-Omari, A.; Hu, S.; Yuan, Z.
Robust Nitritation Sustained by Acid-Tolerant Ammonia-Oxidizing
Bacteria. Environ. Sci. Technol. 2021,55 (3), 2048−2056.
(140) Wang, Z.; Zheng, M.; Duan, H.; Ni, G.; Yu, W.; Liu, Y.; Yuan,
Z.; Hu, S. Acidic Aerobic Digestion of Anaerobically-Digested Sludge
Enabled by a Novel Ammonia-Oxidizing Bacterium. Water Res. 2021,
194, No. 116962.
(141) Wang, Z.; Ni, G.; Maulani, N.; Xia, J.; De Clippeleir, H.; Hu,
S.; Yuan, Z.; Zheng, M. Stoichiometric and Kinetic Characterization
of an Acid-Tolerant Ammonia Oxidizer ‘Candidatus Nitrosoglobus.’.
Water Res. 2021,196, No. 117026.
(142) Meng, J.; Hu, Z.; Wang, Z.; Hu, S.; Liu, Y.; Guo, H.; Li, J.;
Yuan, Z.; Zheng, M. Determining Factors for Nitrite Accumulation in
an Acidic Nitrifying System: Influent Ammonium Concentration,
Operational PH, and Ammonia-Oxidizing Community. Environ. Sci.
Technol. 2022,56 (16), 11578−11588.
(143) Duan, H.; Watts, S.; Zheng, M.; Wang, Z.; Zhao, J.; Li, H.;
Liu, P.; Dwyer, J.; McPhee, P.; Rattier, M.; Larsen, E.; Yuan, Z.; Hu, S.
Achieving Robust Mainstream Nitrite Shunt at Pilot-Scale with
Integrated Sidestream Sludge Treatment and Step-Feed. Water Res.
2022,223, No. 119034.
(144) Yu, H.; Tian, Z.; Zuo, J.; Song, Y. Enhanced Nitrite
Accumulation under Mainstream Conditions by a Combination of
Free Ammonia-Based Sludge Treatment and Low Dissolved Oxygen:
Reactor Performance and Microbiome Analysis. RSC Adv. 2020,10
(4), 2049−2059.
(145) Wang, B.; Wang, Z.; Wang, S.; Qiao, X.; Gong, X.; Gong, Q.;
Liu, X.; Peng, Y. Recovering Partial Nitritation in a PN/A System
during Mainstream Wastewater Treatment by Reviving AOB Activity
after Thoroughly Inhibiting AOB and NOB with Free Nitrous Acid.
Environ. Int. 2020,139, No. 105684.
(146) Gottshall, E. Y.; Bryson, S. J.; Cogert, K. I.; Landreau, M.;
Sedlacek, C. J.; Stahl, D. A.; Daims, H.; Winkler, M. Sustained
Nitrogen Loss in a Symbiotic Association of Comammox Nitrospira
and Anammox Bacteria. Water Res. 2021,202, No. 117426.
(147) Kits, K. D.; Jung, M.-Y.; Vierheilig, J.; Pjevac, P.; Sedlacek, C.
J.; Liu, S.; Herbold, C.; Stein, L. Y.; Richter, A.; Wissel, H.;
Brueggemann, N.; Wagner, M.; Daims, H. Low Yield and Abiotic
Origin of N2O Formed by the Complete Nitrifier Nitrospira
Inopinata. Nat. Commun. 2019,10, 1836.
(148) Wang, Z.; Zheng, M.; Hu, Z.; Duan, H.; De Clippeleir, H.; Al-
Omari, A.; Hu, S.; Yuan, Z. Unravelling Adaptation of Nitrite-
Oxidizing Bacteria in Mainstream PN/A Process: Mechanisms and
Counter-Strategies. Water Res. 2021,200, No. 117239.
(149) Du, R.; Peng, Y.; Cao, S.; Wang, S.; Wu, C. Advanced
Nitrogen Removal from Wastewater by Combining Anammox with
Partial Denitrification. Bioresour. Technol. 2015,179, 497−504.
(150) Cao, S.; Du, R.; Peng, Y.; Li, B.; Wang, S. Novel Two Stage
Partial Denitrification (PD)-Anammox Process for Tertiary Nitrogen
Removal from Low Carbon/Nitrogen (C/N) Municipal Sewage.
Chem. Eng. J. 2019,362, 107−115.
(151) Du, R.; Peng, Y.; Ji, J.; Shi, L.; Gao, R.; Li, X. Partial
Denitrification Providing Nitrite: Opportunities of Extending
Application for Anammox. Environ. Int. 2019,131, No. 105001.
Environmental Science & Technology pubs.acs.org/est Critical Review
https://doi.org/10.1021/acs.est.3c00636
Environ. Sci. Technol. XXXX, XXX, XXX−XXX
N
