Journal of Environmental Chemical Engineering 9 (2021) 105583

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Journal of Environmental Chemical Engineering

journal homepage: www.elsevier.com/locate/jece

Recent developments of the mainstream anammox processes: Challenges

and opportunities
Hoang Phuc Trinh a, 1, Sang-Hoon Lee a, 1, Garam Jeong b, Hyeokjun Yoon b, Hee-Deung Park a, *
a
School of Civil, Environmental and Architectural Engineering, Korea University, Seoul, South Korea
b
Biological and Genetic Resources Assessment Division, National Institute of Biological Resources, Incheon, South Korea

A R T I C L E I N F O A B S T R A C T

Editor: Dr. Z. Wen Anaerobic ammonium oxidation (anammox) processes are gaining attention owing to their advantages over the
conventional biological nitrogen removal processes. Although technologies related to sidestream anammox
Keywords: processes are being adopted as the latest advanced solutions, the application of these processes to mainstream
Anaerobic ammonium oxidation (anammox) wastewater is still under investigation. Here, we have comprehensively reviewed the challenges and opportu­
Mainstream applications
nities of mainstream anammox processes. Mainstream processes face a variety of challenges that limit their stable
Autotrophic nitrogen removal
operation such as the difficulty of out-selection of nitrite-oxidizing bacteria, high organic carbon to nitrogen (C/
Deammonification
N) ratio, retention of anammox bacteria, and the influence of high concentrations of ammonia and nitrite
compounds. For widespread applications of mainstream anammox processes in full-scale reactors in wastewater
treatment plants (WWTPs), efficient strategies are necessary to manage high carbon to nitrogen ratios, improve
performance in low-intensity wastewater, and retain anammox bacteria. Key elements in the design, operation,
and maintenance of mainstream anammox processes in full-scale WWTPs are also suggested. Overall, this review
provides a critical perspective on the challenges and opportunities associated with mainstream anammox pro­
cesses, which will be of use to researchers and engineers in future.

1. Introduction substantially limited by high operational costs (mostly owing to aera­

As a result of the increasing demand of water for life and production,
more than 730 million tons of sewage along with other discharges are
generated and released into water bodies every year, and 80% of these
discharges are left untreated, which is a serious challenge for sustainable
development [1]. Wastewater treatment is mostly based on physical,
chemical, and biological processes [2]. In general, treatments based on
biological processes, which rely on the metabolic capability of micro­
organisms provide a more sustainable performance and have lower
operational costs compared to physical and chemical processes [2,3].
Nutrients such as nitrogen and phosphorus cause eutrophication in the
aquatic environment, which hinder the quality of domestic and indus­
trial water supplies [4]. The demand for nitrogen removal during
wastewater treatment has increased, especially for treatment of organic
wastewater with high ammonia concentrations. Accordingly, biological
nitrogen removal processes based on nitrification ((Eqs. (1, 2)), and
denitrification ((Eq. (3)) are conventionally employed in most waste­
water treatment plants (WWTPs). However, these technologies are
tion), generation of secondary pollutants, and the need to supply carbon
resources [5,6]. To address these limitations, researchers are increas­
ingly focusing on anaerobic ammonium oxidation (anammox) processes
(Eq. (4)), mainly because of their energy- and cost-saving properties
associated with the reduction of aeration (0.9 k Wh kg–1 N against
3.5 k Wh kg–1 N) and excess sludge treatment [5], as well as the lack of
carbon supply (0 kgCH3OH kg− 1 N against 3 kgCH3OH kg− 1 N) [7–9]
(Fig. 1). In addition, by operating a completely autotrophic
nitrogen-removal over nitrite (CANON) reactor, not only the settling of
biomass was improved, but the aeration energy for oxygen supply was
reduced by 63% compared to conventional nitrification-denitrification
nitrogen removal systems [9]. A DEMON system was evaluated to
consume 25% less energy cost than conventional nitrogen removal
systems [9] (0.75 €/kg of N removed was estimated for a partial nitri­
fication anammox system [10]). Moreover, by adjusting the WWTP
using the anammox process the methanol supply was reduced by 64%,
thus saving up to € 380, 000 every year [6]. Therefore, anammox pro­
cesses in ammonium (NH+ 4 )-rich influent wastewater treatment have

* Correspondence to: School of Civil, Environmental and Architectural Engineering, Korea University, Anam-Ro 145, Seongbuk-Gu, Seoul 02841, South Korea.
E-mail address: heedeung@korea.ac.kr (H.-D. Park).
1
Both authors contributed equally to this work.

https://doi.org/10.1016/j.jece.2021.105583
Received 22 February 2021; Received in revised form 9 April 2021; Accepted 24 April 2021
Available online 30 April 2021
2213-3437/© 2021 Elsevier Ltd. All rights reserved.
H.P. Trinh et al.
become more promising areas of research, and the prospects for their
wide applications are increasing [11].
NH+ -
4 + 1.5O2 → NO2 + H2O + 2H
+
NO-2 + 0.5O2 → NO-3
NO-3 → NO-2 → NO → N2O → N2
NH+ + 1.32NO-2
+ 0.066HCO-3
+ 0.13H → 1.02N2 + +
0.26NO-3
4
0.066CH2O0.5N0.15 + 2.03H2O
Some studies have reported the removal of NH+
and generation of
4 studies.
nitrogen gas (N2 gas) via an unknown mechanism during the anaerobic
remineralization of lake sediments and organic matter [12,13]. These
processes began to receive attention from the academic world when the
first anammox bacteria were reported in 1999 [14]. Anaerobic
ammonium-oxidizing bacteria (AnAOB) are chemolithoautotrophs that
can adopt NH+ -
4 and nitrite (NO2) as a primary electron donor and ter­
minal electron acceptor, respectively [10,15,16]. Anammox processes
normally occur under two sequential pathways. First, under aerobic
conditions, partial nitritation oxidizes half of the influent ammonium to
nitrite through the metabolism of autotrophic ammonium-oxidizing
microorganisms with dominant AOB. Such microbes include Nitro­
somonas eutropha, N. europaea, and Nitrosospira [9]. Second, under
anaerobic conditions, anammox processes convert ammonium and ni­
trite into nitrogen gas by the action of anammox bacteria [9]. These
anammox bacteria are gram-negative bacteria that contain high lipid
content and are known to exhibit red color owing to their unique cy­
tochrome during the enrichment culture. To date, a total of five anam­
mox bacterial genera (Brocadia, Kuenenia, Anammoxoglobus, Jettenia,
and Scalindua) [17] have been reported, and a new bacterium, named
Anammoximicrobium moscowii belonging to the phylum Planctomycetes,
recently have been proposed [18].
Although the anammox processes for the treatment of sidestream
wastewater have been actively adopted worldwide, those for the treat­
ment of mainstream wastewater have been mostly conducted at labo­
ratory scales, and are limited in their full-scale applications [19]. In a
review by Cho et al. [20], high NO-2 concentration, high salinity, sul­
fides, toxic metal elements, and toxic organic compounds were pre­
sented as potential inhibitors that could damage anammox bacteria and
adversely affect the nitrogen removal performance. However, for
mainstream wastewater treatment, high chemical oxygen demand to
nitrogen (COD/N) ratios may cause heterotrophic bacteria to
Fig. 1. Nitrogen cycle in autotrophic nitrogen removal technologies and conventional nitrification/denitrification technologies [55].
Journal of Environmental Chemical Engineering 9 (2021) 105583
outcompete anammox bacteria, and nitritation (i.e., conversion of NH+ 4
into NO-2) processes are difficult to maintain through the selective
retention of ammonium-oxidizing bacteria (AOB) against
(1)
nitrite-oxidizing bacteria (NOB) [21]. Furthermore, several other chal­
(2) lenges exist, including the efficient retention of anammox bacteria under
conditions of low nitrogen concentration, negative effects of
(3) low-temperature wastewater on microbial communities, performance of
practical applications in different areas, high effluent concentration of
+
NO-3, and organic residues in mainstream treatment [22]. Therefore, this
(4)
research subject requires more careful consideration and intensive
In this study, we primarily focused on providing a comprehensive
review of the technical challenges faced during the application of
mainstream anammox processes in full-scale WWTPs, including (i) dif­
ficulties in NOB out-selection, (ii) high organic carbon to nitrogen (C/N)
ratios, (iii) retention of anammox bacteria, and (iv) the influence of NH+
4
and NO-2-N concentrations. Furthermore, in this manuscript, we discuss
the potential applications of anammox processes in mainstream waste­
water treatment such as (i) management of high C/N ratio to maxi­
mizing the recovery of C based energy, (ii) improvement in the
performance of low-strength wastewater, (iii) effective retention of
anammox bacteria, and (iv) practical applications and mechanisms of
anammox process in full-scale WWTPs. Accordingly, this review pro­
vides a novel aspect of the applications of the mainstream anammox
processes along with its operational conditions and design factors, as
well as the potential of its full-scale operation for future development.
2. Challenges in mainstream anammox processes
Nitritation is the step that involves the microbial oxidation of NH+
4 to
NO-2, and it is the common stage for anammox processes. In a previous
study [23], AOB enrichment and NOB washout were achieved during
the nitritation step by applying selective pressure on two groups of
bacteria. The strategies for controlling nitritation include providing
low-dissolved oxygen (DO) (0.3–1.5 mgO2 L− 1) [24], high-temperature
(35 ◦ C), short sludge retention time (SRT) (2–3 days) [25], control of
aeration time [26], and sludge treatment based on free ammonia (FA)
and free nitrous acid (FNA) [27]. Nevertheless, for the practical appli­
cation of mainstream wastewater treatment, several critical issues
should be addressed (Fig. 2).
2
H.P. Trinh et al.
Fig. 2. Challenges for mainstream anammox processes application.
2.1. The out-selection of NOB
The nitritation step significantly affects the successful operation of
anammox applications [28]. Therefore, the balancing of the different
functions of the microbial groups is important [22]. The most chal­
lenging task is considered to be the complete out-selection of NOBs, such
as under low-strength nitrogen and low-temperature conditions, espe­
cially in the case of the frequently varied NH+ 4 -N feeding that triggers
over-aeration [22]. In the sidestream deammonification with high NH+
FA and FNA were adopted as parameters for controlling the nitritation
process [29,30]; however, this strategy no longer guarantees successful
mainstream processes under low-nitrogen concentrations [19].
Previously, it was reported that under FA concentration of
0.1–1.0 mg L–1, the NO-2 oxidation by NOB was inhibited, while the NH+
oxidation by AOB was inhibited at 10–150 mg L–1 [31]. Moreover, the
selective inhibition of NOB via NO-2 accumulation could occur at an FA
concentration of 1.0–10 mg L–1 [31]. Balmelle et al. [32] reported that
the AOB activity was reduced by 40% at an FA concentration of
25 mg L–1, whereas Kim et al. [33] exhibited reduced NOB activity
(greater than 50%) at an FA concentration of 0.7 mg L–1. An FA level of
10.6 mgNH3 L–1 was recommended to maintain the stable NO-2 accu­
mulation rate of 84% [34]. Owing to the presence of Nitrous acid
(HNO2), nitrifying organisms began to be inhibited by FNA concentra­
tions of 0.22–2.8 mg L–1 [31]. Wang et al. [35] reported that FNA was
substantially more toxic to NOB than AOB under anoxic conditions.
Although pH control is an alternative, it is not an economically
feasible approach to nitritation owing to the expensive cost of chemicals.
In general, an effective strategy for controlling nitritation is to adjust the
operation temperature higher than 25 ◦ C, to grew AOB relatively faster
than NOB [36]. However, this strategy is also not feasible for practical
full-scale applications. Until now, most conventional engineering
Journal of Environmental Chemical Engineering 9 (2021) 105583
strategies to nitritation were based on controlling the DO of the bio­
reactors, which involves operating DO at set-points of 0.5–0.9 mg L–1,
and then reducing DO levels to 0.06 mg L–1 to determine the NH+ 4 or
NO-2 reduction slopes at the decreased DO level [37]. However, the
reliability of equipment, such as the air blower, is critical for the stable
operation of the process [38]. In general, the influence of wastewater,
reactor configurations, and their operations on the optimum microbial
community, as well as the selection of microbial communities in the
4, mainstream process require further investigation.
2.2. High ratio of organic carbon to nitrogen
The C/N ratio of conventional domestic influent wastewater is
4
excessively high (ranging from 10 to 12 [39,40]); therefore, it is
generally unsuitable for mainstream nitritation/anammox processes
[22]. The optimum ratio for a partial nitritation/anammox (PN/A)
treatment is usually approximately 2–5 [41], or even lower than 0.5 [39,
42]. The high concentration of organic matter in the influent wastewater
adversely affects the growth of anammox bacteria [43]; however,
determining the exact reasons for this phenomenon remains a significant
challenge. The suggested mechanisms for the inhibitory effect of organic
carbon on the anammox process are “out-competition and inhibition”
and ‘‘metabolic pathway conversion inhibition” [44]. First, Chamchoi
et al. [45] and Güven et al. [46] reported that under high COD con­
centrations, the heterotrophic bacteria can grow faster than anammox
bacteria because they compete for space and nutrients each other, sug­
gesting that the activity of anammox bacteria decreases owing to spatial
and nutritional limitations. Chen et al. [47] also demonstrated that the
growth of denitrifying bacteria is rapidly stimulated by the addition of
glucose in an anammox reactor, whereas that of anammox bacteria is
unaffected by glucose supplementation [46]. Second, several
3
H.P. Trinh et al. Journal of Environmental Chemical Engineering 9 (2021) 105583

researchers [46,48,49] observed reduced anammox activity in the ni­ efficiency (NRE), for treating mainstream and sidestream wastewater
trogen removal process, and this indicates that anammox bacteria under different C/N ratios are compared in Fig. 3. At low C/N ratios
-
metabolized organic carbons rather than NH+ 4 and NO2 as substrates. (0.0–1.0), anammox processes exhibited high NRE in both mainstream
Interestingly, when acetate was supplemented during the reactor oper­ and sidestream wastewater treatment (71–96%). However, the NRE
ation as a substrate, anammox bacteria revealed different metabolic gradually decreased in the sidestream anammox processes (58.0–85.4%)
properties for “metabolic pathway conversion inhibition” [46]. when the C/N ratio increased over 1.0, while that in the mainstream
Several recent nitritation/anammox reactors have been operated in anammox processes decreased abruptly (4–77.3%). In general, the NREs
mainstream wastewater treatment under different organic C/N ratios, in the mainstream anammox systems are not stable compared to those in
and the challenges faced during the operation period are summarized in the sidestream anammox systems. Therefore, further investigations on
Table 1. Studies have reported that a C/N ratio of 2.7–2.9 triggered a the practical applications and control strategies for mainstream anam­
reduction in both AOB and anammox activities [24]. In contrast, under mox processes are difficult.
the increased C/N ratio conditions from 1.1 to 1.5, the nitrogen removal
process improved because of the increased number of anammox bacte­ 2.3. Retention of anammox bacteria
ria, while, their grow rate is decreased under 2.0 of C/N ratio. These
results indicate that these phenomena could be attributed to the limited For the successful start-up and process stability of anammox pro­
production of NO-2 [50]. Moreover, AOB can be washed out from the cesses, an biomass retention mechanism should be efficiently controlled
reactor at a C/N ratio of 0.8 [25]. Even in the study conducted by Chen to maintain a favorable microbial community owing to the relatively
et al. [51], when the C/N ratio of influent wastewater increased from 0.5 lower growth rates of autotrophs [50]. The accurate doubling time of
to 0.75, the efficiency of nitrogen removal from the nitritation/a­ anammox bacteria remains unclear owing to the lack of a pure culture to
nammox process decreased from 79% to 52%. Based on the operational date [39]. The reported doubling time of anammox bacteria varies be­
data observed in the laboratory-scale reactors (Table 2), the perfor­ tween studies, with a typical range of 10–12 days [8,52]. However,
mance of the anammox processes, in terms of nitrogen removal depending on the species and growth conditions, or even at the
pilot/full-scale at low-temperatures, the observed doubling times can
Table 1 increase to 25–30 days [53,54]. Furthermore, because the concentration
Performance of anammox process for treating mainstream-like wastewater of NH+ 4 is negligible in municipal wastewater, the start-up and mainte­
under different organic carbon to nitrogen ratio. nance of anammox activity remains a challenge [19]. Consequently, to
Reactor C/N Inf. TN NRE (%) Problems Reference efficiently handle the retention of anammox biomass in the mainstream
type (mgCOD (mgN L–1) nitritation/anammox process, an extended SRT should be applied,
mg–1N) especially at temperatures below 20 ◦ C [22,39].
SBR 0.6–2.0 22.0±5.9 70.0–40.0 COD [24] Brocadia, Kuenenia, and Anammoxoglobus species are reported to be
increased, distributed in not only the natural environment but also wastewater
the diffusion treatment facilities currently in operation [55]. Among them, Brocadia
limitation
species are the most commonly observed in wastewater treatment fa­
impacted
AnAOB more cilities, both in the sidestream and mainstream anammox processes
than NOB, (including moving bed biofilm reactor (MBBR), as well as in simulta­
reduced on neous nitrification and anammox reactors, denitrification reactors,
both the AOB sequencing batch reactors (SBRs), up-flow anaerobic sludge blanket
and AnAOB
activities
(UASB) reactors, membrane bioreactors, etc.) [56–61].
SBR 1.1–2.0 59.3 30.8–73.5 AnAOB [50] Interestingly, although the Brocadia species was the first anammox
activity and species to be reported [62], the Kuenenia species is known to be the most
abundance extensively studied anammox species in terms of cell structure, physi­
increased
ology, biochemistry, and comparative genomics research [63]. In
2.0–2.5 59.3 73.5–77.3 AnAOB [50]
activity particular, the key proteins involved in the genome of Kuenenia stutt­
stopped gartiensis (e.g., hydrazine synthase, hydrazine dehydrogenase, hydrox­
increasing as ylamine dehydrogenase, and an S-layer protein with an encoded
increasing hypothesized anammox pathway) have been experimentally validated
amounts of
nitrogen was
in recent studies [64–66]. The most interesting feature of the anammox
removed species was the experimental verification of a key intermediate hydra­
UASB 1.0# 45.9 88.4 Lower carbon [104] zine produced from HNO2 [67]; this suggests that the Kuenenia species
resulted in might include novel metabolic pathways for nitrogen.
high removal
Furthermore, some studies have reported that Kuenenia species,
rate
NRBC 0.5–0.75 100.0–200.0 79.0–52.0 Decrease in [51] which are known to live in freshwater environments [68], are enriched
nitrogen from wastewater treatment facilities operated by the anammox pro­
removal cesses. For example, Akaboci et al. [69] reported that Kuenenia species
efficiency are predominant in the process of operating a granular one-stage partial
MBBR 3.2# 21.8±5.2 73.0–91.0 No [57]
interruption
nitritation/anammox SRT at a low-temperature (≤ 15 ◦ C), thus sug­
because gesting that these species could play a crucial role in anammox pro­
nitrogen load cesses. Similarly, Gu et al. [70] reported that during the operation of A-B
was less processes, which consist of an anaerobic fixed-bed reactor and SBR,
H–MBBR 3.2# 21.8±5.2 63.0 Less stable in [57]
Kuenenia species were enriched in the biofilm of the MBBR system.
removing
nitrogen due Interestingly, Yang et al. [71] reported that in the process of altering
to increase in partial nitritation/anammox processes from the sidestream to the
nitrate mainstream, the Kuenenia species has been predominated instead of the
produced Brocadia species. In addition to the Brocadia species, the development of
Note: Inf. TN, Influent Nitrogen Concentration; NRE, Nitrogen Removal Effi­ the Kuenenia species in WWTPs has been widely reported [14,18,48,
ciency; “#” indicates estimated number based on the given information. 72–75].

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H.P. Trinh et al. Journal of Environmental Chemical Engineering 9 (2021) 105583

Table 2
Performance of anammox process for treating wastewater under different operational conditions.
Reactor Temperature HRT C/N (mgCOD Inf. TN (mgN NLR (mgN L–1 NRE (%) Major Anammox species (Candidatus genus) Reference
type (oC) (h) mg–1N) L–1) d–1)

H–MBBR 15.0 13.0# 3.2 21.8 109.0# 63.0 Brocadia [57]

MBBR 9.0# 73.0–91.0
MBBR 30.0 24.0# 0.7 862.0 1450.0 82.0 Kuenenia stuttgartiensis [79]
MBfR 20.8 4.0 – 50.0 310.0# 91.7–94.7 Brocadiae [127]
MBR 30.0 40.0# – – 1000.0 80.0–85.0 Brocadia sp. 40, Brocadia Caroliniensis [60]
SBR 25.0 11.3# 0.6 22.0 46.8 70.0 – [24]
2.0 40.0
SBR 30.0 6.0# 1.1 59.3# 126.3 30.8 – [50]
1.5 113.8 63.1
2.0 114.0 73.5
2.5 122.5 77.3
SBR 35.0 6.0 – 68.0# 272.0# 92.0 Kuenenia (61%), Brocadia anammoxidans [128]
(32%), Brocadia fulgida (6%)
SBR 35.0 0.8 – 200.0 800.0# 75.0# – [100]
SBR 24.0 31.2# 1.4# 291.0 223.0# 80.0 Brocadia Scalindua [56]
SBR 20.0 6.4 – 337.5# 1200.0–1500.0 60.0 Brocadia fulgida [129]
SBR 15.0 24.0# – 590.0 775.0# 36.5# Brocadia fulgida [101]
SBR 25.0 42.5# – 740.0 533.0# 95.0 Brocadia fulgida [130]
SBR 15.0 14.8# – 800.0# 1230.0–1340.0 70.5# Kuenenia [89]
SBR 30.3 28.8# – 938.0 840.0# 51.0# Brocadia fulgida [101]
SBR 35.0 24.0 – – 380.0# 85.0 Brocadia sp. 40 [61]
SBR 15.0 24.0# – – 300.0 16.7# Kuenenia stuttgartiensis [80]
UASB 28.5 4.6 1.0 45.9 400.0 88.4 – [104]
UASB 30.0 0.1 0.5 51.41# – 92.3 – [131]
16.0 0.3 78.5
UASB 11.0 – 1.3 70.0# 1800.0 82.0 Brocadia anammoxidans [58]
ABR 30.1 72# 0.6–0.8 114.8# 38.27# 89.0–96.0 – [47]
1.2–2.3 4.0
ABR 30.0 – 1.4 178.1# – 58.0 – [47]
1.3 193.7# 63.0
SBAR 10.0 7.2 – 160.0 530.0 48.0 Brocadia fulgida [21]
20.0 85.0
15.0 73.0
CSTF 20.0 – – 100.0# 2150.0 74.0 Brocadia fulgida [15]
30.0 2090.0 78.0
CSTR 25.0 12.0 – 250.0 2500.0 77.3 Kuenenia [129]
SNAD 22.6–28.6 4.0# 2.4 57.7# 346.2# 85.4 Kuenenia Brocadia [59]
CANON 35.0 7.0 0.3 246.0 843.0# 85.0 Scalindua [132]
1.3 68.1
NRBC 35.0 8.0 0.8 250.0 690.0 52.0 – [51]
13.0 0.5 200.0 690.0 79.0
SHARON 29.0 28.2# – 657.0 560.0# 92.0 – [53]

Note: Temp., Temperature; HRT, Hydraulic Retention Time; Inf. TN, Influent Nitrogen Concentration; NLR, Nitrogen Loading Rate; NRE, Nitrogen Removal Efficiency;
“#” indicates estimated number based on the given information.

Other species belonging to anammox bacteria, such as the Anam­
moxoglobus, Jettenia, and Scalindua species, are known to be primarily
distributed in landfill leachate anaerobic treatment systems, freshwater,
and marine ecosystems. As such, they are limitedly observed in sewage
treatment systems [48,55,76,77].
2.4. Effect of nitrogen concentration
Various loading rates of nitrogen and influent nitrogen concentra­
tions in previous studies can significantly affect the nitrogen removal
rate (NRR), dominant bacterial species, growth capability, and optimal
potential of long-term anammox processes [78], and they are presented
in Table 2 and Fig. 4. Generally, in the range of nitrogen loading rates
(NLR; 300–900 mg L–1 d–1), NRE was higher under the sidestream
anammox processes (68–94%) than that under the mainstream anam­
mox processes (52–96% or even lower than 60%). Interestingly, when
the NLR continued to increase up to 1000–2500 mgN L–1 d–1, the
sidestream anammox processes still maintained their high performance
(60–85%) in the nitrogen removal process. Fig. 4 also demonstrated that
the NRE for mainstream anammox systems was unstable, although these
systems were operated under the same NLR conditions.
NH+4 is considered to be the major form of nitrogen source in the
influent wastewater for most WWTPs [79], and although it is a
significant part of the autotrophic nitrogen removal process, the high
concentration of NH+ 4 adversely affects anammox bacteria [11]. A low
concentration of NH+ 4 related to high cell density would lead to
decreased anammox bacterial activity [14]. Moreover, the feeding of
-
insufficient amounts of NH+ 4 and NO2 would stimulate the growth of
unwanted bacteria, thereby interfering with anammox activity [14].
Because NO-2 is an important substrate of anammox bacteria, its optimal
concentrations would be necessary for the anammox processes. How­
ever, a high concentration of HNO2 also inhibits anammox activity [11].
Unlike AOB and NOB, as mentioned in Section 2.1, the inhibitory con­
centrations of FA and FNA on the growth of anammox bacteria were
reported to be above 50 mg L–1 and 5 μg L–1, respectively [44].
A critical concentration of FA was observed at a range of
35–40 mg L–1 [80]. In contrast, at a lower FA concentration of
13 mg L–1, the inhibition of anammox activity was reported in a previ­
ous study [81]. In the results obtained by Bae et al. [82], a specific
anammox activity (SAA) decrease of 21.2% was observed when the FA
increased drastically from 1.1 to 21.5 mg L–1 (total nitrogen concen­
tration was 160 mg L–1). Waki et al. [81] reported that FA concentra­
tions of 13–90 mg L–1 could adversely impact the performance of
anammox processes. However, FA concentrations below 13–15 mg L–1
were not considered relevant to the anammox processes [83]; even an
FA concentration of approximately 20 mg L–1 has been reported to exert

5
H.P. Trinh et al. Journal of Environmental Chemical Engineering 9 (2021) 105583

Fig. 3. Performance of anammox process for treating mainstream- (hollow circle) and sidestream-like (solid triangle) wastewater under different C/N ratio values.

Fig. 4. Comparison of nitrogen loading rate in the performance of anammox process for treating mainstream- (hollow circle) and sidestream-like (solid triangle)
wastewater under different nitrogen loading rates.

no effect on the stability of the process [80].
Anammox bacteria are more sensitive to FNA concentrations than FA
[84]. Under lower pH values (pH < 7.1), the toxic effect of NO-2-N was
most significantly triggered by FNA [85]. Previous studies [16,62,80]
reported that the critical NO-2-N concentration was in the range of
100–350 mg L–1, while Niu et al. [86] inferred that a high removal ef­
ficiency was realized for FA < 5 mg L–1 and FNA < 0.002 mg L–1. In
addition, the inhibitory influence of FNA was observed at a concentra­
tion of approximately 213 g L–1 [44,80]. The most significant decrease
in SAA was reported by Bae et al. [82]: the FNA increased from 0.05 to
2.4 mg L–1. However, Isaka et al. [84] determined that the anammox
bacterial activity was inhibited by the higher than 280 mg L–1 of NO-2-N.
Similarly, Strous et al. [87] also determined that the anammox activity
was completely inhibited when the concentration of NO-2-N exceeded

6
H.P. Trinh et al.
100 mg L–1. Generally, for the stable anammox system operation, TN,
FA, and FNA concentrations should be below 320 mg L–1 [86],
15 mg L–1 [88], and 1.5 μg L–1 [88], respectively.
3. Opportunities for the application of mainstream anammox
processes
Although anammox processes encounter several challenges that
must be addressed, they provide the interesting possibility of trans­
forming most of the biodegradable organic matter arriving in the
WWTPs into biogas (i.e., methanization), as they do not require biode­
gradable COD for denitrification [39]. This application can save energy
based on the demand on many WWTPs, as well as reduce operating costs
[57]. According to the important issues already mentioned, proposed
solutions have been continuously investigated to improve and extend
these processes in their wide applications.
3.1. Management of high C/N ratio for maximizing C energy recovery
Anaerobic digestion processes are conventionally used to convert
COD to recoverable energy [89]. The maximum production of CH4 from
biodegradable COD is approximately 0.35 Nm3CH4 kg–1COD [39],
which can be used to operate heat facilities, motors and provide income
for the industry generating the wastewater as well as WWTPs [90].
However, this direct application is potentially inhibited by low-strength
and low-temperature water, and the recovery of chemical energy is a
serious challenge [39]. The general concept is demonstrated in the A-B
processes (Fig. 5) based on the modification presented by Li et al. [22]
and Xu et al. [19], which was designed to maximize the capturing ca­
pacity of carbon resources from wastewater before they pass through
autotrophic metabolisms (e.g., nitritation/anammox). The “A” process
involves biodegradation of COD and methanization (e.g., organic mat­
ters and biogas) through several techniques such as the anaerobic pro­
cess, high rate activated sludge (HRAS), integrated fixed-film activated
sludge (IFAS), enhanced biological phosphorus removal (EBPR), chem­
ically enhanced primary treatment (CEBT), and bioelectrochemical
system. Because anammox processes do not require organics for deni­
trification, organic mass (COD) are transformed into methane for energy
production, which could increase methane production by 47%, and
cause energy reduction for air supply by 50% in comparison with con­
ventional biological nitrogen removal techniques [91]. The “B” process
is specifically contributed to treat the remaining nutrients (nitrogen)
passing through the autotrophic metabolic pathway.
For the anaerobic process, a UASB reactor was operated at 20 ◦ C to
pretreat the primary wastewater, and then filtered using a textile filter
Fig. 5. Flow scheme of the “A-B processes” for COD capture prior to mainstream nitritation/anammox applications.
7
Journal of Environmental Chemical Engineering 9 (2021) 105583
with a pore size of 20 µm to extract soluble COD (sCOD) and NH+ 4
concentration in an influent ranged of 44–88 mg(sCOD) L–1 and 35–50
mgN d–1, respectively, for the followed deammonification processes,
such as IFAS reactor or MBBR, which achieved an NRE and NRR of
70 ± 4% and 55 ± 6 gN m–3 d–1, respectively, by combining the deni­
trification and anammox processes [92]. A high capacity for obtaining
COD with high food-to-microorganism (F/M) ratios at a relatively short
HRT and low SRT has been demonstrated via HRAS processes. The
influent sCOD, colloidal matter, and particulate in the form of sludge
were concentrated by the high-rate aerobic system HRAS processes,
which could then be utilized for energy recovery via anaerobic digestion
[93]. Wett et al. [37] successfully applied a full-scale HRAS to remove
COD before it was adopted in mainstream aerobic reactors that were
strengthened with AOB, and anammox bacteria obtained from a side­
stream DEMON reactor. A hydrocyclone classifier was also used to retain
anammox granules by selecting a high-density sludge fraction, while
intermittent aeration was adopted to repress the NOB activity [37].
Moreover, by operating full-scale HRAS processes with 1 h of HRT and
0.3 days of SRT to obtain a high Biochemical Oxygen Demand (BOD)
removal efficiency (~90%) and treat the effluent with a single-stage
nitritation/anammox system at 19 ◦ C over 10 months, approximately
46% of total NRE was achieved [94].
The EBPR process was applied for the simultaneous removal of
organic carbon and phosphorus from domestic wastewater. A system
comprising two SBRs was adopted in the treatment of practical domestic
wastewater by integrating enhanced biological phosphorus removal
with nitratation/anammox [95]. Under intermittent anaerobic-aerobic
conditions, phosphorus was removed in the first SBR with a short SRT
of 2–3 days to prevent nitrification; then, the nitratation/anammox SBR
was responsible for treating the remaining NH+ 4 with a highest NRR of
0.12 kgN m–3 d–1 [95]. The CEPT process removes particulate and
colloidal matter via coagulation and flocculation by adding chemicals
(e.g., polymers and metal salts) to primary clarifiers [22,96]. However,
the CEPT might be ineffective in removing sCOD, and this could affect
the nitritation/anammox performance. Therefore, certain factors (e.g.,
the raw wastewater characteristics and sCOD from the primary clarifers)
should be thoroughly evaluated before designing CEPT as a pretreat­
ment. The bioelectrochemical system behaves as a pretreatment alter­
native to directly generate electricity or enhance nitrogen removal
through current generation [97]. More than 90% of the suspended solids
and 75% of the total COD were treated by a microbial fuel cell system.
Then, the remaining nitrogen at the anode effluent was treated by
nitritation–anammox in the cathode [98,99].
H.P. Trinh et al.
3.2. Performance improvement of low-strength wastewater
Both the anammox and partial nitritation processes are significantly
influenced by the conditions of the low-strength wastewater treatment.
In fact, the nitritation step is considered as one of the primary processes,
because this step selects and grows the AOB effectively, outcompetes the
-
NOB, and obtains the oxidation of NH+ 4 to NO2 at approximately 50%.
However, the treatment of these types of wastewater is very difficult,
especially in low-temperature regions [39]. Recently, most of the
autotrophic nitrogen removal systems reported in the literature were
operated at temperature conditions of over 25 ◦ C and influent nitrogen
concentrations of more than 100 mgN L–1 [79]. Although the concen­
tration ratio of AOB to NOB in the sludge is relatively constant, the
reduction of temperature could be more beneficial to the highly
competitive NOB, because of their higher activity than AOB and their
higher production of NO-3 [100]. A significantly reduced SAA has been
observed when the operational temperature was decreased below 25 ◦ C
[101]. Moreover, the virtually negligible FA could inhibit anammox
processes in the case of low NH+ 4 concentration, usually approximately
or less than 50 mg L–1 [36]. Therefore, the analysis of low-temperature
and low-nitrogen concentrations in wastewater application opens up
new avenues for applying anammox processes in designing
energy-saving WWTPs.
Currently, there increase an interest in anammox research under
low-temperature and low-nitrogen conditions [100]. The Brocadia spe­
cies (e.g., Brocadia sinica and Brocadia fulgida) is usually involved in
processes operated at a temperature range of 5–6 ◦ C. Kuenenia stuttgar­
tiensis was also determined in a continuous bioreactor system that
operated at temperatures lower than 20 ◦ C [100,102]. Lotti et al. [103]
reported that an adapted anammox sludge exhibits a higher specific
anammox rate at a lower temperature. At 15 ◦ C, the PN/A process was
stably operated on pretreated municipal wastewater with a nitrogen
influent concentration of 21 ± 5 mgNH+ 4 -N L , and it gained an NRR of
–1
30 mgN L–1 d–1 in the range of conventional nutrient removal systems
[104]. By controlling DO up to 2.5 mgO2 L–1 for the repression of the
NOB activity, this could lead the efficient suppression of the nitritation
at both 20 and 15 ◦ C, thereby also leading the sufficient NRRs of 0.44
and 0.40 gNTotal L–1 d–1 with optimal NREs of 86% and 73%, respec­
tively. Moreover, under the same conditions with 15 ◦ C, an estimated
growth rate of 0.017 d− 1 was observed, fulfilling a decisive prerequisite
for the application of the processes in mainstream WWTPs [105]. In a
mainstream IFAS deammonification process, 10% of the primary
effluent wastewater before the biological phosphorus and COD removal
stages was directly diverted to the deammonification reactor to raise the
influent C/N ratio from 2.3 to 3.1 g(sCOD) g–1NH+ 4 -N. In this system,
72% of TN removal was maintained during the decline in temperature
from 20 ◦ C to 12 ◦ C, and the anammox metabolism routed about 53%
of nitrogen removal, and it exhibited no negative impact on anammox
activity owing to the increased carbon in the influent wastewater [106].
In a hybrid PN/A MBBR system with an NH+ 4 concentration of 23 mg L ,
–1
the AOB and NOB were enriched preferentially in the flocs, while the
anammox bacteria were enriched in the biofilm. The actual growth rates
of both AOB and NOB decreased, and the washout of NOB from the flocs
was observed, whereas the minor NOB fraction remained in the biofilm
inhibited by the decreased DO concentration (from 1.2 to 0.17 mg L–1)
[107]. To date, it was suggested that the anammox population has been
activated under conditions of low nitrogen concentration and
low-temperature, and this has become a promising scenario for appli­
cation in mainstream WWTPs [43,103].
3.3. Effective retention of anammox bacteria
The growth of anammox bacteria is significantly slow, and the op­
timum anammox biomass retention is essential for its application in
different wastewaters, especially under low-temperature conditions [43,
80]. Efforts on research and applications in lab-scale and full-scale
8
Journal of Environmental Chemical Engineering 9 (2021) 105583
mainstream wastewater to achieve high biomass retention have been
reported, which involves the use of membrane bioreactors, biofilm
technology, SBR, MBBR, hydrocyclone and screen, screening or sieving,
and rotating drum screen. Fernández et al. [39] reported that the most
widely studied and implemented mechanisms for obtaining high SRT of
anammox biomass rely on the formation of biofilms, whose high density
provides a significantly high retention of biomass in the reactors.
Although membrane bioreactors have been adopted for research at
laboratory scales to obtain the total retention of biomass [80], they are
rarely employed in full-scale practical applications [39]. Biofilm tech­
nology is being adopted in some of the recent applications of municipal
wastewater treatment by the PN/A system [80]. In addition, the SBR has
been employed for anammox reactors and can be used in combination
with biofilm systems as it allows very high SRTs close to the total
retention of biomass [39,50,108]. Recent studies have focused on
municipal water treatment via the SBR system [21,50,108] with the
expectation of practically applying the anammox technology in the
future owing to its high biomass retention efficiency and the operational
flexibility of bioreactors. In addition, anammox immobilized cells have
been adopted in previous studies for synthetic wastewater owing to their
high biomass content, prevention of washout biomass, and lower
toxicity to microorganisms [20]. By operating a hybrid PN/A MBBR over
a broad simulated SRT ranged from 6.8–24.5 days, the direct competi­
tion for NO-2-N between NOB and AnAOB (retained in the biofilm and
behaving as a “NO-2-N-sink”) triggered a difference in the actual growth
rates of AOB and NOB (i.e., μNOB < μAOB in flocs) and allowed for the
selective NOB washout while retaining AOB [107]. However, studies on
their practical application in wastewater and improvement of polymeric
gel disposal to the environment need further consideration.
Other approaches for securing effective retention for anammox are
hydrocyclone and screen. Waste sludge in the sidestream deammonifi­
cation was returned to the mainstream deammonification processes for
the separation of anammox aggregates by hydrocyclones [37]. By
returning anammox aggregates from cyclone separation, the SRT of
anammox bacteria in the system can be considerably prolonged [19].
Larger aggregates from the underflow stream of a hydrocyclone
demonstrated that they exhibited significantly higher anammox activity
than the smaller ones [109]. In general, anammox aggregates are larger
but less compressible than AOB and NOB [19]. Moreover, based on these
findings, screening or sieving has been developed for the selective sep­
aration and retention of anammox aggregates [110]. This approach re­
lies on the essential concept that NOB prefers to grow in flocs, while
anammox bacteria exist in granules [111]. By using a vibrating screen
with more moderate normal forces (by water sprinklers) and additional
tangential forces (by applying mechanical vibration) to allow for a
gentle separation of granules entrapped in flocs and shearing of the
surface of granules, a better balancing retention efficiency of anammox
bacteria granules (41% of the anammox activity) and washout of NOB
(92% activity washout) were recorded, which resulted in an enhanced
NOB out-selection (AOB/NOB ratio of 2.3) and a total NRE of 70%
(effluent total nitrogen concentration < 10 mgN L–1) with the COD/N
ratio of influent wastewater at 1.4 [111]. For the mainstream deam­
monification full-scale reactor in the Strass WWTP (Austria), superior
anammox bacteria retention efficiencies, achieved by the rotating drum
screen (η = 72%) over the cyclone (η = 42%), triggered a prospected
increase in capacity by 80–90% and enhanced the success of mainstream
deammonification by decreasing its dependency on nitrite residuals
[112]. Research on the operation and choice of physical selectors is
required to directly determine the operational strategy and footprint
necessary to obtain higher anammox bacteria retention and NOB
out-selection in mainstream deammonification, as well as to reduce the
demand for tight aerobic SRT control [112].
3.4. Commercialized processes for mainstream treatment
Owing to the lack of pure cultures, stringency of operational
H.P. Trinh et al. Journal of Environmental Chemical Engineering 9 (2021) 105583

conditions and low growth rate of anammox bacteria, the practical
application of anammox processes is limited by its research progress
[113]. However, the application of anammox to nitrogen removal pro­
cesses would replace the conventional denitrification step, thereby
saving half of the nitrification aeration costs, and thus facilitating in the
reduction of operational costs [114]. Owing to continuous and suc­
cessful studies, the first full-scale granular anammox reactor with
loading rate of up to 750 kgN d–1 was introduced for sludge treatment in
the municipal wastewater treatment plant of Rotterdam, the
Netherlands in 2002, and it symbolically represented the beginning of
the commercial application of anammox processes [8]. Moreover, the
one-stage partial nitritation/anammox reactor (4 m3), which was used
for the supplementation of the actual effluent to the A-stage of the above
WWTP, was operated separately at temperature conditions of summer
(23.2 ± 1.3 ◦ C) and winter (13.4 ± 1.1 ◦ C), and it exhibited total NRR
values of 0.223 ± 0.029 and 0.097 ± 0.016kgN m3 d–1, respectively [94,
115]. Some studies have reported that heterotrophic biomass was effi­
ciently washed out of the system, while anammox bacteria grew under
mainstream WWTP conditions, and new granules were enriched and
efficiently retained in the system [94]. These results indicate that the
proposed reactor with granular sludge has a high propensity to be suc­
cessfully applied for the autotrophic nitrogen removal from the main­
stream of WWTPs [115]. Successful full-scale anammox application on
high nitrogen concentration wastewater aided this accomplishment of
mainstream wastewater treatment with several efficient techniques for
anammox enrichment and full-scale application in practical treatment
(Fig. 6).
A full-scale one-stage PN/A MBBR process in Klagshamn WWTP,
Malmö, southern Sweden, which was designed to treat up to 200 kgN
d–1, was started up without inoculum within 111 days [116]. Heated
dilution water has been reported as a prerequisite for the rapid start-up,
and it provided significant results, such that the NRR reached 1.8 gN m3
L–1 and the nitrogen removal was greater than 80% at 35 ◦ C [116].
Another typical full-scale enhanced nitrogen removal via the in situ
enrichment of anammox bacteria for mainstream wastewater was suc­
cessfully applied in the Xi’an municipal WWTP located in Shanxi

Fig. 6. Efficient approaches for anammox enrichment and full-scale application in practical treatment. (a) Hydrocyclones in combination with the mainstream and
sidestream system; (b) hydrocyclones for enriched biomass in SBR; (c) chemical supply for the improvement of processes; (d) dilution of water and heat exchange for
the improvement of growth rate; (e) moving carriers for the enhancement of efficiency; and (f) robust separation for retaining biomass.

9
H.P. Trinh et al.
province, China, with a capacity of 250, 000 m3 d–1. In addition, this was
an anaerobic-anoxic-oxic (A2O) (HRT 2.0 h, 2.6 h and 6.4 h respec­
tively) process and it was upgraded by adding moving carriers
(approximately 25% filling) to the anoxic zone, and equipping sub­
mersible mixers and guide walls to ensure complete interaction between
wastewater and the carriers, as well as installing intercept screens in the
outlet to prevent loss of the suspended carriers [117]. In approximately
2 years of continuous operation, when the influent COD/TN ratio varied
from 1.2 to 7.9, nitrogen was efficiently removed with an average
effluent total inorganic nitrogen (TIN) concentration of 7.6 mg L–1. In
addition, by batch tests, 15N-stable isotope tracing tests and mass bal­
ance analysis determined that NO-2 could be produced via denitration,
and its adoption by anammox bacteria along with 15.9% of nitrogen loss
was likely attributed to the anammox processes being carried out by the
anoxic–carrier biofilm [117,118]. Moreover, metagenome sequencing
analysis showed that the relative abundance of anammox bacteria in the
anoxic-carrier biofilms was significantly higher than that of the reported
level in the flocculent sludge of conventional WWTPs [118].
By augmenting AOB and anammox biomass from sidestream SBR,
preserve anammox bacteria with hydrocyclone, and inhibiting NOB by
controlling aeration, as indicated by the higher concentrations of nitrite
rather than nitrate in the effluent and quantified by aerobic activity tests
(approximately 75% inhibition), the Strass WWTP (Austria) achieved
stable mainstream anammox technologies [37,119,120]. Similar to the
Strass WWTP, the demonstration projects at Glarnerland WWTP, where
the anammox activity in the mainstream was enhanced by transferring a
major part of the produced waste sludge in the sidestream, realized the
feasibility of the deammonification concept when achieving an
ammonia-removal efficiency greater 90% (maximum load of 200 kg
NH+ 4 -N) [121]. However, investigations were conducted to understand if
anammox can complement the conventional nitrification/deni­
trification processes in a temperate zone at the Marselisborg WWTP
(Aarhus, Denmark) by feeding excess sludge from a sidestream tank to
mainstream tanks. It was found that after more than 3 years of opera­
tion, approximately 1% of NRE was contributed by anammox with the
conversion rates being 800–900 times lower than that achieved by
deammonification through 15N-labeling experiments at different sets of
temperature (10, 20, and 30 ◦ C) [122]. This observation could be
explained by the lack of adaptation to lower temperatures as well as the
small abundance of anammox bacteria in the reactors, due to which the
anammox processes were excluded by the dominant nitrification [122].
The measurements of 15N-labeling rate also proved the obvious depen­
dence of anammox activities on temperature (7.7 ± 0.6 and 72.9 ± 7.2
μmol gVSS h–1 at 10 ◦ C and 30 ◦ C, respectively) [122]. Another project
applied by two-stage mainstream deammonification processes without
bioaugmentation for nitrogen polishing was conducted at Chesapeake
Elizabeth WWTP (VA, USA) [123]. Partially nitrified effluent waste­
water was operated as the first-stage activated sludge process by con­
-
trolling the ratio of NH+ 4 to NO2 and nitrate (NOx-N), followed by
anammox MBBR as the second stage [123]. The results indicated that
the system was partially successful in NOB outcompeting, which adopts
transient aeration with a high DO set-point of 1.0 mg/L and short SRT of
approximately 0.1 d at 20 ◦ C. In addition, the performance of anammox
MBBR polishing was stable, although the nitrogen removal was limited
by the influent COD/NH4 ratio of 8:11 [123]. In a full-scale anammox
mainstream technology, Changi Water Reclamation Plant (200,000 m3
d–1), Singapore, intermittent aeration as a spatial fashion (controlling
DO at 1.5–2.0 mgO2 L–1 by ceramic) was applied to oxidize approxi­
-
mately 72% NH+ 4 to NO2 and then removed by anammox processes in
the anoxic zone (the DO dropped to below 0.1 mgO2 L–1 within 1 min
when aeration was stopped) [124]. In the ANITA™ Mox processes, a
single-stage deammonification process utilizing MBBR technology was
successfully applied to treat mainstream effluent with low sCOD at
65 mg L–1 content using the IFAS configuration (controlled DO at
0.2–0.8 mg L–1) after the HRAS process [125]. A unique feature of IFAS
ANITA™ Mox is its easy and robust separation system (i.e., media
10
Journal of Environmental Chemical Engineering 9 (2021) 105583
sieves) that retains anammox biomass in the reactor for mainstream
applications [125]. Preliminary results indicate that this application
seems to be efficient in suppressing the NOB activity while gaining
N-removal rate up to 0.2–0.3 kgN m3 d–1 at 16–18 ◦ C, which is higher
than what is commonly achieved in conventional biological nitrogen
removal activated sludge systems [125].
Up to date, Aqualia (AquELAN® with granular biomass), Paques
(ANNAMOX® with granular biomass), Veolia (ANITA® Mox with MBBR
and IFAS), DC water (DEMON® with granular biomass) and DEMON
GmbH are leading industrial corporations which have made efforts to
develop marketable products for the practical application of anammox
processes [126]. Applications of this eco-friendly and economic pro­
cesses are expected to become a commercial technique for mainstream
wastewater treatment in the near future.
4. Future prospects of mainstream anammox application
Over the past decade, new discoveries and experiments have
significantly improved our understanding of mainstream anammox
processes. However, the full-scale application of these processes requires
technical solutions to address immediate challenges. First, such tech­
nologies must solve the problem of applying anammox processes to low-
concentration nitrogen wastewater. The partial nitrification/anammox
process can be considered to operate under stable conditions in
municipal wastewater with low-concentration nitrogen inflows. Second,
the removal of NOB from the treatment system is also a significant
operational challenge. Alternating aeration is a useful strategy applied
to mainstream anammox processes for NOB inhibition as it is difficult to
restore activity in a limited time while increasing anammox activity
during the transition from anoxic to aerobic, even in low temperate
conditions. In addition, FA and FNA have recently been proposed for
NOB selection in mainstream processes due to a more potent biocidal
effect on NOB compared to AOB and AnAOB. Third, organic carbon
should be minimized in the anammox reactor, as organic matter nega­
tively affects the anammox reaction. The A-B process was developed to
maximize the capture capacity of carbon sources from influent waste­
water to manage high C/N ratios before the anammox reaction takes
place. The anaerobic reactor of the A-B process not only helps to reduce
organic carbon content but also contributes to the production of biogas
by anaerobic digesters. Methane in biogas can be utilized to produce
heat and/or electric energy, which is generally used for the operation of
wastewater treatment plants. The supply of NH4Cl can improve low-
intensity influents as well as reduce the C/N ratio. However, this
chemical method must be carefully evaluated based on the operational
expenses for gaining stability. Finally, the biggest challenge is main­
taining a very slow growth of AnAOB in mainstream anammox pro­
cesses. The effective maintenance of AnAOB can be ensured by applying
several methods that have been proven useful in recent years. According
to laboratory-based studies, full-scale reactors, membrane bioreactors,
MBBR, and SBR systems were successful in holding high concentrations
of AnAOB, and they provided operational flexibility. Considering the
economic way to keep AnAOB in the mainstream anammox processes,
physical devices such as hydro-cyclones, sieving equipment, and me­
chanical vibrating screens can be selected. Based on the suggested ap­
proaches, companies can develop marketable products and secure
sludges enriched with AnAOB for practical applications to mainstream
anammox processes.
5. Conclusions
In this review, we have provided the challenges and opportunities of
mainstream anammox processes by analyzing their experimental results
and associated performances as reported in recent publications. Four
major challenges have been identified for the application of the anam­
mox process to mainstream wastewater: (1) low concentration of
ammonia in the influent, (2) the difficulty of NOB out-selection, (3) high
H.P. Trinh et al.
organic carbon to nitrogen ratio, and (4) retention of slow-growing
AnAOB. Possible and efficient strategies have been proposed to over­
come these four challenges. Partial nitritation and subsequent anam­
mox, aeration control, removal of organic carbon before anammox
reaction, and physical separation of AnAOB have been suggested to
overcome the main challenges. Discussion of the main challenges and
strategies to solve these challenges would be useful in establishing future
research directions for the development of mainstream anammox pro­
cesses. Currently, there are limited cases in which mainstream anammox
processes have been applied at full scale. However, as analyzed in this
review, if new processes are developed considering the solutions to the
factors that hinder the application of mainstream anammox, it is ex­
pected that the mainstream anammox processes will be popular in the
near future.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was supported by a grant from the National Institute of
Biological Resources (NIBR), funded by the Ministry of Environment
(MOE) of South Korea (NIBR202019102) and the National Research
Foundation of Korea (NRF) (NRF-2020R1A6A1A03045059).
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