journal of environmental sciences 124 (2023) 146–155

Available online at www.sciencedirect.com

www.elsevier.com/locate/jes

Impact of soluble organic matter and particulate

organic matter on anammox system: Performance,
microbial community and N2 O production

Yingying He 1, Hongyu Mao 1, Jacek Makinia 2, Jakub Drewnowski 2,

Bing Wu 1, Jun Xu 1, Li Xie 1,∗, Xi Lu 1,∗
1 Key Laboratory of Yangtze Water Environment, Ministry of Education, Tongji University, Shanghai 200092, China
2 Faculty of Civil and Environmental Engineering, Gdansk University of Technology, Gdansk 80-233, Poland

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the effects of soluble readily biodegradable COD (sCOD) and particulate slowly
Received 4 November 2021 biodegradable COD (pCOD) on anammox process were investigated. The results of the long-
Accepted 7 November 2021 term experiment indicated that a low sCOD/N ratio of 0.5 could accelerate the anammox
and denitrification activity, to reach as high as 84.9%±2.8% TN removal efficiency. Partial
denitrification-anammox (PDN/anammox) and denitrification were proposed as the major
Keywords: pathways for nitrogen removal, accounting for 91.3% and 8.7% of the TN removal, respec-
Anammox tively. Anammox bacteria could remain active with high abundance of anammox genes to
Partial denitrification maintain its dominance. Candidatus Kuenenia and Thauera were the predominant genera in
Microbial community structure the presence of organic matter. Compared with sCOD, batch experiments showed that the
Carbon composition introduction of pCOD had a negative effect on nitrogen removal. The contribution of den-
Nitrous oxide production itrification to nitrogen removal decreased from approximately 14% to 3% with increasing
percentage of pCOD. In addition, the analysis result of the process data using an optimized
ASM1 model indicated that high percentage of pCOD resulted in serious N2 O emission (the
peak value up to 0.25 mg N/L), which was likely due to limited mass diffusion and insufficient
available carbon sources for denitrification. However, a high sCOD/N ratio was beneficial for
alleviating N2 O accumulation.
© 2022 The Research Center for Eco-Environmental Sciences, Chinese Academy of
Sciences. Published by Elsevier B.V.

wastewater treatment plants. However, the anammox bac-

Introduction teria are sensitive to numerous factors, including the pres-
ence of organic matter (Pijuan et al., 2020). It was reported
Anammox is considered as a promising method for the nitro-
that high organic matter concentration could cause low ni-
gen removal from domestic wastewater, in which anammox
trogen removal efficiency and even lead to destruction of the
microorganisms convert ammonium and nitrite into nitrogen
system performance, which resulted from excessive multi-
gas and nitrate (Guo et al., 2022). As a cost-effective and sus-
plication of heterotrophic bacteria compared with anammox
tainable way for biological nitrogen removal from wastewa-
bacteria (Chen et al., 2016). Recently, it has been discovered
ter, the anammox process has been successfully applied in
that low COD addition was advantageous in anammox sys-

∗
Corresponding authors.
E-mails: sally.xieli@tongji.edu.cn (L. Xie), luxi953@outlook.com (X. Lu).

https://doi.org/10.1016/j.jes.2021.11.007
1001-0742/© 2022 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
 journal of environmental sciences 124 (2023) 146–155
tems (He et al., 2018; Wang et al., 2016a; Zhang et al., 2020),
while PDN/anammox has been proposed as a feasible path-
way when treating wastewater with a low COD/N ratio sewage.
For example, Li et al. (2020a) increased the nitrogen removal
efficiency by 17% by applying partial denitrification/anammox
(PDN/anammox) in an anaerobic/anoxic/oxic (AAO) system at
low COD/N ratio (2.7 ± 0.4) sewage. Despite promising results,
the knowledge of the interactions of functional bacteria in the
overall nitrogen removal chain remains unclear, which needs
further investigation.
Particulate slowly biodegradable COD (pCOD) is also an im-
portant fraction of the organic matter in domestic wastewa-
ter. The pCOD needs to be hydrolyzed extracellularly to solu-
ble readily biodegradable COD (sCOD) before it can be utilized
by the microorganisms (de Kreuk et al., 2010). For example,
Shi et al. (2020) discovered that starch could be mostly con-
verted to acetate and dissolved saccharide to promote PDN.
Thus, considering the large amount of pCOD (approximately
50% of TCOD) in real wastewater, it was necessary to identify
the effect of carbon source on the anammox system perfor-
mance. Moreover, a few studies showed that Candidatus Broca-
dia acted as the predominant anammox bacteria in anammox
bioreactors with organic matter addition (Sheng et al., 2018;
Wang et al., 2019). However, Wang et al. (2018) reported a sig-
nificant abundance of Candidatus Kuenenia revealing positive
correlations with some heterotrophic bacteria and survived
on organic compounds. Therefore, the population structure of
anammox reactors controlling the relative importance of den-
itrification and anammox processes is essential to determine.
Additionally, N2 O has recently received widespread atten-
tion due to its high global warming potential (298 higher than
CO2 ) recently, and denitrification has been identified as one of
most important contributors to N2 O production in biological
nitrogen removal systems (Frank et al., 2012). Heterotrophic
denitrification could be stimulated by organic matter addition
and thereby caused variable N2 O production in the anammox
systems. Zhang et al. (2016) demonstrated that the coupling
of anammox and denitrification accounted for N2 O accumu-
lation in the biofilters at the COD/N ratio of 1.0 with sodium
acetate addition, reaching the highest accumulation rate of
N2 O (8.0 ± 0.4 g/m3 /hr). However, the effects of soluble organic
matter (sCOD) and pCOD on N2 O emissions have not been in-
vestigated yet. So far, mathematical models have been consid-
ered as a promising tool for illustrating the dynamic trends of
N2 O production. The extended ASM1 combining NH2 OH oxi-
dation by AOB, autotrophic denitrification by AOB, as well as
heterotrophic denitrification by heterotrophs, was developed
and applied successfully to analyze N2 O production under dif-
ferent conditions (Lu et al., 2018; Al-Hazmi et al.,2021), which
can be used as a guiding tool to further control N2 O production
in deammonification systems.
The effects of COD/N on recent anammox studies were
characterized by applying simple organic matter. However,
pCOD couldn’t be neglected in practice. It would be useful
for assessing the effect of sCOD and pCOD on nitrogen re-
moval and N2 O production, which gave precise information
for practical application in wastewater treatment. The objec-
tives of this study are to evaluate the following issues: (1) ni-
trogen removal performance and metabolic mechanism at a
low sCOD/N ratio (sCOD/N=0.5) in a long-term cultivation ex-
147
periment; (2) microbial community shift in response to sCOD
addition; (3) the effect of sCOD/COD ratio (sCOD%) and corre-
sponding COD/N ratio on anammox system and N2 O produc-
tion in the presence of pCOD in the short-term experiments.
1. Materials and methods
1.1. Reactor set up and operation
A laboratory-scale sequencing batch reactor with an effective
volume of 3 L (200 mm in diameter and 160 mm in height)
was used in this study. The bioreactor was operated at 30
°C with a water bath temperature control system. The pH
was controlled in the range 7.5-7.9, and no external oxygen
was provided. Anaerobic sludge and anammox sludge were
taken from a full-scale municipal wastewater treatment plant
(Shenyang, China) and a laboratory-scale up-flow anaerobic
sludge bed (Suzhou, China), respectively, and mixed by a me-
chanical stirrer with a volume ratio of 3:1. The mixed liquid
suspended solids (MLSS) and mixed liquid volatile suspended
solids (MLVSS) were approximately 10.2 g/L and 4.2 g/L, respec-
tively.
Synthetic water consisted of NH4 + -N, NO2 − -N, and trace
element solution (details are shown in Appendix A Table S1).
The hydraulic residence time (HRT) of the one-stage bioreactor
was maintained at 24 hr. The reactor started up successfully at
influent NH4 + -N and NO2 − -N concentrations of 40 mg/L and
50 mg/L and then the nitrogen loading was enhanced by im-
proving the influent NH4 + -N and NO2 − -N concentrations of
90 mg/L and 110 mg/L during phase I. To investigate the long-
term effect of sodium acetate as organic matter in the anam-
mox system, the sCOD/N ratio was controlled at 0.5 during
phase II.
1.2. Batch tests (sCOD% and COD/N ratio)
To study the effect of different COD/N ratios (i.e., 0.5, 1 and 2)
and corresponding sCOD% (i.e., 100%, 50%, 30% and 15%) on
the nitrogen removal performance in the mainstream anam-
mox system, batch experiments were carried out with the
biomass from the bioreactor. The influent NH4 + -N and NO2 − -
N concentrations were 40 mg/L and 50 mg/L, respectively.
Sodium acetate and starch were chosen as sCOD and pCOD,
respectively. Each batch experiment lasted for 240 min. Dur-
ing the reaction phase, a mixed liquor sample was collected to
determine the concentration of NH4 + -N, NO2 − -N, and NO3 − -N
every 30 min.
1.3. Analytical methods
1.3.1. Chemical analysis
MLSS and MLVSS were determined according to the APHA
standard methods (APHA, 2012). Regarding chemical analy-
sis, samples were collected from the reactors and the super-
natants were filtered through a 0.45 μm filter membrane. The
concentrations of NH4 + -N, NO2 − -N and NO3 − -N were mea-
sured using Hach DR6000 spectrophotometer (Hach Co., Love-
land, Co., USA). The pH was measured using an online pH
manufacturer.
148 journal of environmental sciences 124 (2023) 146–155
1.3.2. Calculation methods
The total nitrogen removal efficiency was calculated using
Eq. (1):
−
Total nitrogen removal efficiency = (NH+
4 − Ninf + NO2 − Ninf
+ NO− + − − μL of TransStart FastPfu DNA Polymerase, 10 ng of template
3 − Ninf − NH4 − Neff − NO2 − Neff − NO3 − Neff )/
 DNA, and 20 μL of ddH2 O. PCR reactions were performed in
+
(NH 4 − Ninf + NO− −
2 − Ninf + NO3 − Ninf × 100%
where NH4 + -Ninf , NO2 − -Ninf , NO3 − -Ninf , NH4 + -Neff , NO2 − -Neff ,
and NO3 − -Neff are the influent and effluent concentrations of
nitrogen species.
The sCOD/N ratio was calculated using Eq. (2):
sCOD/N = COD/N × sCOD%
1.4. Microbial analysis
1.4.1. DNA extraction and qPCR
The obtained sludges of the bioreactor at the end of phase I
(265 days) and II (320 days) were frozen at −24 °C after cen-
trifugation (1000 r/min for 10 min). Total community genomic
DNA was extracted following the procedures recommended
by the E.Z.N.A.® soil DNA Kit (Omega Bio-Tek, Norcross, GA,
USA). The DNA concentration and purity were determined by
a NanoDrop 2000 UV-vis spectrophotometer (NanoDrop Tech-
nologies, CA, USA).
The abundance of anammox bacterial 16S rRNA gene
anammox genes encoding, hydrazine synthase (hzs) and hy-
drazine oxidoreductase (hzo), and denitrification genes encod-
ing respiratory NO3 − -N reductase (narG), periplasmic NO3 − -N
reductase (narA), NO2 − -N reductase (nirS and nirK) and nitrous
reductase (nosZ) in the two samples were quantified by qPCR
with the extracted DNA as template. The primer pairs and pro-
tocols for qPCR are shown in Appendix A Table S2. The SYBR®
Green qPCR experiments were carried out in quadruplicate
with the ABI 7300 sequence detection system (ABI, USA). The
qPCR reactor mixture (20 μL) contained 10 μL of 2X Taq Plus
Master Mix (2X) (Vazyme, China), 0.8 μL of forward and reverse
primer (5 μmol/L), 1 μL of template DNA, and 7.4 μL of ddH2 O.
The reaction program was as follows: initial denaturation at
95°C for 5 min, denaturing at 95°C for 30 sec, followed by 35
cycles of annealing for 30 sec and extension at 72°C for 1 min.
The annealing temperatures of hzsB, hzo, hzsA, narA, nirS, nosZ,
narG, and nirK were 50, 50, 55, 55, 55, 55, 58, and 58°C, respec-
tively. Then the abundance of each gene was normalized to
DNA mass (copies/μL DNA). The efficiency of the qPCR assays
ranged from 90% to 105%, and the R2 value was more than 0.98.
1.4.2. High-throughput sequencing and bioinformatics analy-
sis
To determine the bacterial community structure of the
SBR at two phases, the hypervariable region V3-V4 of
the bacterial 16S rRNA gene was amplified with primer
pairs 515F (5-GTGCCAGCMGCCGCGG-3 ) and 806R (5 -
GGACTACHVGGGTWTCTAAT-3 ) using an Applied Biosys-
tems GeneAmp® 9700 PCR thermocycler (Thermo Fisher
Scientific, MA, USA). Nested PCR (Cai et al., 2014) was
performed for the anammox 16S rRNA gene with initial
primer pairs 46f (5-GGATTAGGCATGCAAGTC-3 ) and 1390r
(5-GACGGGCGGTGTACAA-3 ), followed by second primer
pairs 368F (5-TTCGCAATGCCCGAAAGG-3 ) and 820R (5 -
AAAACCCCTCTACTTAGTGCCC-3 ). The PCR mixtures con-
tained 4 μL of TransStart FastPfu buffer, 2 μL dNTPs (2.5
mmol/L), 0.8 μL of forward and reverse primer (5 μmol/L), 0.4
(1)
triplicate. The PCR product was extracted from 2% agarose
gel and purified using the AxyPrepTM DNA Gel Extraction
Kit (Axygen Biosciences, Union City, CA, USA) according
to the manufacturer’s instructions and quantified using a
QuantusTM Fluorometer (Promega, USA). Purified amplicons
were pooled in equimolar amounts and paired-end se-
quenced on an Illumina MiSeq PE300 platform (Illumina, San
(2) Diego, USA) according to the standard protocols by Majorbio
Bio-Pharm Technology Co. Ltd. (Shanghai, China).
1.4.3. Alpha diversity analysis
The microbial diversity of each single sample was estimated
by alpha diversity analysis. Species richness and diversity
statistics, including the Coverage, Chao, ACE, and Shannon in-
dices, were calculated according to the OTU results. Then, all
the effective sequences without primers were submitted for
downstream analysis (Xu et al., 2020).
1.5. N2 O production and modelling approach
Four batch experiments (1) sCOD%=30%, COD/N=0.5; (2)
sCOD%=15%, COD/N=1.0; (3) sCOD%=50%, COD/N=0.5; (4)
sCOD%=50%, COD/N=2.0, were selected for N2 O production
measurement, which were evaluated by the extended ASM1
(Lu et al., 2018). N2 O concentrations in the liquid phase
were continuously measured by a Clark-type N2 O microsen-
sor (Unisense Environment A/S, Denmark) and logged every 20
sec. The measured concentrations of NH4 + -N, NO2 − -N, NO3 − -
N and N2 O-N were used by model predictions. The extended
ASM1 was implemented in the GPS-X 6.4 simulation platform
(Hydromantis, Canada), which used a special utility called
“Model Developer”. The definition of the state variables, sto-
ichiometric matrix and process rate equations are presented
in Appendix A Tables S3, S4, S5, respectively. In this study, the
adjusted kinetic parameters were in the range of the litera-
ture (ηgHET1 = 0.029-0.8, ηgHET2 = 0.016-0.16, ηgHET3 = 0.075-
0.81, KNO2 ,HET = 0.06-8 mg N/L, KN 2 O ,HET , = 0.005-0.05 mg N/L,
ηgAOB1 = 0.0015-0.45, ηgAOB2 = 0.006-0.72 in which ηgHET1 , ηgHET2
and ηgHET3 represent heterotrophic anoxic reduction factors;
KNO2,HET represents nitrite half-saturation coefficient for het-
erotrophs; KN2O,HET represents N2 O half-saturation coefficient
for heterotrophs; ηgAOB1 and ηgAOB2 represent ammonia oxida-
tion factors).
2. Results and discussion
2.1. Nitrogen removal performance with/without sCOD
Fig. 1 shows the nitrogen removal performance in the biore-
actor over the operational time of 336 days. The whole pe-
riod was divided into two phases based on the sCOD addi-
 journal of environmental sciences 124 (2023) 146–155
Fig. 1 – Nitrogen removal performance of the anammox reactor of different operational condition: influent and effluent
characteristics of NH4 + -N, NO2 − -N and NO3 − -N with the corresponding total nitrogen removal efficiency.
tion: phase I (day 0-265) without sCOD and phase II (day 266-
336) with sCOD/N ratio of 0.5 (97.5 mg COD/L). In phase I, the
anammox system was initiated successfully at two different
nitrogen concentrations of 90 (day 0-149) and 200 mg N/L (day
150-265), respectively. Low effluent NH4 + -N (7.9±6.8 mg/L)
and NO2 − -N (2.5±4.6 mg/L) concentrations were obtained af-
ter adaptation. However, increasing the nitrogen loading led
to NO3 − -N accumulation, which increased from 24.9±6.4 to
58.0±11.1 mg/L. The TN removal efficiency was maintained at
70% at the same time. The long-term effect of a low sCOD/N
ratio of 0.5 on the anammox process was investigated in phase
II. Both the effluent NH4 + -N and NO2 − -N concentrations were
almost close to 0 mg/L. Moreover, a decrease in the NO3 − -N
concentration (30.9±7.9 mg/L) was achieved obviously, corre-
sponding to the TN removal efficiency as high as 84.9%±2.8%.
To calculate the relative contribution of PDN/anammox
and denitrification, the stoichiometric relationships according
to the following formulas were used for the calculation of the
nitrogen removal pathway (Zhan et al., 2020): (i) the molar ra-
tio of NO3 − -N/ NO2 − -N in PDN is 1:1; (ii) the molar ratio of
NH4 + -N/ NO2 − -N in the anammox process is 1:1.32; (iii) the
molar ratio of NO3 − -N/N2 in denitrification is 1:0.47.
− − −
NH+ +
4 +1.32NO2 +0.066HCO3 +0.13H → 1.02N2 +0.26NO3
+0.066CH2 O0.5 N0.15 +2.03H2 O (3)
NO− +
3 +1.08CH3 OH+H → 0.065C5 H7 O2 N + 0.47N2 +0.76CO2
+2.44H2 O (4)
The contribution of nitrification to nitrogen removal was
negligible because the DO concentration in this reactor was
controlled under 0.4 mg/L (Zhang et al., 2019a). It will be
proved by microbial community (Section 1.3). Therefore, the
subsequent decrease in NH4 + -N with sCOD addition was
mainly due to the enhanced anammox activity. However, the
additive NO2 − -N was insufficient for the decrease in NH4 + -
N by the anammox activity in phase II as theoretically 1.32
149
g of NO2 − -N is required to remove 1 g of NH4 + -N. It is likely
that produced NO2 − -N from NO3 − -N by PDN was consumed
by active anammox bacteria. Moreover, in the presence of or-
ganic matter, the produced NO2 − -N from NO3 − -N for anam-
mox didn’t fit the decrease in NO3 − -N based on the molar ratio
of NO3 − -N / NO2 − -N in PDN, while NO2 − -N accumulation was
not observed, showing that NO3 − -N reduction to N2 occurred
in this system. Thus, PDN/anammox and denitrification are
proposed as the major pathways for nitrogen removal. The rel-
ative contributions of PDN/anammox and denitrification are
shown in Fig. 2. With sCOD addition, anammox accounted for
100% and 91.3% on nitrogen removal in phases I and II, respec-
tively, implying its leading role in the system. Overall, a high
TN removal efficiency was achieved by the low sCOD/N ratio
via PDN/anammox and denitrification processes.
2.2. Nitrogen removal metabolic pathway
Fig. 3a shows the abundances of functional genes related to
anammox and denitrification in the different phases. The in-
troduction of sCOD can contribute to 184%, 247% and 161%
higher relative abundances of napA, nirS and nosZ, respectively,
which involved in denitrification. Moreover, the expression of
anammox-related functional genes, including hzs and hzo also
showed an increase, especially hzo (increased by 175%). To elu-
cidate the involved relationship between the functional genes
and nitrogen removal, the metabolic pathway for nitrogen re-
moval in the presence of sCOD was proposed.
Fig. 3b shows the strengthened metabolic pathways by red
lines according to the variation in the functional genes. Both
napA and narG were responsible for PDN (from NO3 − -N to
NO2 − -N). The obvious up-regulation of the napA further con-
firmed the occurrence of PDN for enhanced nitrogen removal.
On the other hand, the enhancement of nirS and nosZ showed
good agreement with the improvement of denitrification. Both
nirS and nirK could reduce NO2 − -N to NO (Shu et al., 2015).
The nirS might have a particular advantage in NO2 − -N reduc-
150 journal of environmental sciences 124 (2023) 146–155
Fig. 2 – The contribution of PDN/anammox and denitrification to TN removal at the nitrogen concentration of 200 mgN/L in
two phases.
Fig. 3 – (a) Quantitative analysis of nitrogen
transformation-related genes at different times of the
long-term experiment. (b) Proposed metabolic pathways for
nitrogen transformation in the presence of organic matter
(The significantly enhanced pathways represented the
increase by more than 150% in the corresponding genes).
tion than nirK based on the higher abundance (nirS: 5.6 × 1010
copies/g vs. nirK: 1.6 × 108 copies/g), which was also observed
in other studies (Chen et al., 2019; Cui et al., 2020). In addition,
the increasing abundance of anammox-related hzo related to
anammox was in accordance with efficient nitrogen removal
performance by the anammox activity.
Furthermore, with the enhanced denitrification activity,
the abundance of genes related to NO2 − -N reduction (the sum
of the relative abundance of nirS and nirK) was approximately
Table 1 – Richness and diversity of sludge at different
phases.
Samples shannon ace chao coverage
Day 265 4.34 732.85 724.31 1.00
Day 320 3.66 602.87 601.58 1.00
10-fold greater than that related to NO3 − -N reduction (the
sum of the relative abundance of narG and napA) in the sys-
tem, implying a higher NO2 − -N reduction rate. In addition, the
abundance of the anammox gene was approximately 468-fold
greater than that of the denitrification gene with sCOD addi-
tion, which indicated that the anammox activity was dom-
inant in this system. These results further suggested that
anammox significantly contributed to nitrogen removal re-
gardless of induction of denitrification with sCOD addition.
2.3. Microbial community shift
A shift in the microbial community in the long-term exper-
iment was identified by high-throughput sequencing to fur-
ther investigate the effect of sCOD on the anammox system.
Table 1 shows the richness and diversity measures of the bac-
terial community from the two samples. Both Chao and Ace
indices decreased, which indicated that species richness de-
creased. Additionally, the microbial diversity estimated by the
Shannon indices also lowered. It confirmed that even a low
sCOD/N ratio could affect the microbial communities.
The relative taxonomic abundance of species is shown in
Fig. 4a at the phylum level. It was shown that the sCOD did
not affect the main phyla in the anammox system, including
Proteobacteria, Planctomycetes, and Chloroflexi. These phyla were
commonly found in anammox bioreactors in the previous
studies (Zhang et al., 2019b). Proteobacteria have been reported
to play an important role in the nitrogen cycle (Chen et al.,
2019; Lu et al., 2014). With sCOD addition, Proteobacteria as
the most dominant phylum, significantly increased from ap-
proximately 25.9% to 51.9%. In contrast, the Planctomycetes, a
subset of anammox bacteria, decreased from 10.2% to 4.7%,
which demonstrated that the Planctomycetes were sensitive to
 journal of environmental sciences 124 (2023) 146–155
Fig. 4 – Microbial community structure and relative in the sludge at (a) the phylum levels, (b) at the genus levels, (c)
Percentage of the dominant anammox bacteria at the genus levels, Genera occurring at < 1% abundance in the samples
were defined as “Others”.
COD addition. This phenomenon was also discovered in other
studies (He et al., 2018; Zhang et al., 2020). The percentages
of Chloroflexi, related to some heterotrophic microorganisms
(Shu et al., 2015), remained stable. This may confirm a signifi-
cant role of those bacteria in COD removal.
Based on the analysis at the genus level (Fig. 4b), two
species of the functional microorganisms were detected. Can-
didatus Kuenenia and Candidatus Brocadia of phylum Proteobac-
teria were predominant anammox bacteria. The relative abun-
dances of Candidatus Kuenenia and Candidatus Brocadia de-
creased respectively to 1.3% and 0.4% in the presence of
sCOD. The relatively low percentage of autotrophic anammox
bacteria was mainly caused by their low growth rate. How-
ever, despite the low abundance of anammox bacteria, the
high abundance of anammox genes (Fig. 3a) demonstrated
that anammox bacteria still acted as the functional bacteria
in the system. Although Candidatus Brocadia was reported to
be more competitive in the acetate metabolism (Feng et al.,
2019), in this system Candidatus Kuenenia became dominant
instead of Candidatus Brocadia with sCOD addition (Fig. 4c).
Du et al. (2017) discovered that Candidatus Kuenenia grew better
than Candidatus Brocadia at a low substrate concentration due
to the lower half-saturation constant (Ks) of Candidatus Kuene-
nia than Candidatus Brocadia under limited NO2 − -N conditions.
In this study, limited NO2 − -N was observed, which could fur-
ther confirm that Candidatus Kuenenia had a better advantage
in adapting to such conditions than Candidatus Brocadia.
For denitrifiers, Calorithrix (15.6%) and Denitratisoma (7.1%)
occupied a large portion in the control sample. Calorithrix
can utilize proteinaceous and carbohydrate substrates to
generate acetate, propionate, and lactate for their growth
(Kompantseva et al., 2017). Additionally, Denitratisoma has
been detected in a novel denitrifying anaerobic ammonium
oxidation (DEAMOX) process to participate in the denitrifica-
tion (Du et al., 2017). With sCOD addition, Thauera became
dominant and accounted for 33.1%, while Denitratisoma and
151
Calorithrix decreased to 4.2% and 7.7%, respectively. This in-
dicated that sodium acetate as organic carbon had an ob-
vious preference for Thauera. Thauera has been reported to
play an important role in PDN with stable NO2 − -N accumu-
lation, containing much more NO3 − -N reductase (Nar) than
NO2 − -N reductase (Nir) (Ma et al., 2017). The phenomenon
that high abundance of genes for NO2 − -N reduction than
that of NO3 − -N reduction is shown in Fig. 3a. Nir carried
by Candidatus Kuenenia (Wang et al., 2016b) and Denitratisoma
(Zhan et al., 2020) might contribute to the high abundance
of NO2 − -N reduction, thereby being responsible for NO2 − -N
reduction. Qiao et al. (2022) also discovered a similar multi-
species cooperation for biological nitrogen removal in a bio-
electrochemical system coupled an anammox reactor. Anaer-
obic digestion bacteria, such as Thauera, Proteobacteria and
Thiobacillus, reduced NO3 − -N to NO2 − -N, and the generated
NO2 − -N and NH4 + -N were removed by anammox bacteria
(mainly Candidatus Kuenenia).
A low sCOD/N ratio allow to successfully enhance nitro-
gen removal in the anammox reactor by developing stable
PDN/anammox and denitrification processes, in which NO3 − -
N produced by anammox was converted back to NO2 − -N.
Anammox process played a predominant role in nitrogen re-
moval despite the low abundance of anammox bacteria. The
introduction of organic matter could play an important role in
regulating the dominant genera. Thauera and Candidatus Kue-
nenia were responsible for PDN/anammox, while Denitratisoma
mediated denitrification.
2.4. Effect of influent COD/N and carbon composition on
nitrogen removal performance and N2 O production
Low sCOD concentrations were beneficial for the anammox
systems with enhanced denitrification. However, pCOD was
also a potential carbon source for denitrification in spite of
a relatively low denitrification rate. In this study, batch tests
152 journal of environmental sciences 124 (2023) 146–155

were conducted to investigate the effect of the influent COD/N

ratio (i.e., 0.5, 1, and 2) and sCOD% (i.e., 100%, 50%, 30%, and
15%) (sodium acetate as sCOD and starch as pCOD) on ni-
trogen removal. NO2 − -N was almost removed by anammox
without the sCOD addition (Appendix ATable S3), which could
be hypothesized that further utilization of NO2 − -N via anam-
mox occurred from NO3 − -N by PDN. Fig. 5 shows the TN re-
moval efficiency and contribution of PDN/anammox and den-
itrification to nitrogen removal at the different COD/N ratios
with decreasing sCOD%. At the COD/N ratios of 0.5 and 1,
the nitrogen removal efficiency of sCOD% = 50% was not sig-
nificantly different (p>0.05) compared with the control group
(sCOD%=100%).
With the increasing percentage of pCOD, statistical anal-
ysis showed significant differences (p<0.05) in the TN re-
moval efficiency between low sCOD% (30% and 15%) and
Fig. 5 – Total nitrogen removal efficiency and contribution of
high sCOD% (100%). The contribution of denitrification grad-
PDN/anammox and denitrification to nitrogen removal in
ually decreased with the decreasing sCOD% removal, while
different treatments. Asterisks represent p < 0.05
the PDN/anammox still played the key role in nitrogen re-
compared with the control of the batch experiment.
moval. pCOD has a more complex metabolic pathway of
carbon sources which results in a lower denitrification rate
(Elefsiniotis and Li, 2006). Liu et al. (2016) reported that the
driver for denitrification. It could be hypothesized that sCOD
transformation of pCOD to sCOD reached the maximum after
was first consumed by PDN for anammox and then utilized
8 hours and then could then be easily utilized. In this study,
by denitrification. However, the high percentage of pCOD and
taking the short HRT (4 h) into account, sCOD was the main
its low retention time resulted in an insufficient amount of

Fig. 6 – Model predictions vs. measured data during validation in the batch experiments (scatters: measured data; lines:
model predictions; black (solid squares, solid line): NH4 + -N; blue (solid triangles, dashed line): NO2 − -N; red (solid circles,
dashed dotted line): NO3 − -N; pink (empty squares, short dashed line): N2 O.
 journal of environmental sciences 124 (2023) 146–155
sCOD for denitrification, and thus caused a decreasing con-
tribution of denitrification. Although it was reported that car-
bon storage and reutilization by pCOD could ensure efficient
PDN performance, prolonging the pCOD retention time to 520
min might be one of the key factors for denitrification process
(Shi et al., 2021).
N2 O is mostly produced as an intermediate of incomplete
heterotrophic denitrification, especially in the presence of
NO2 − -N. Based on the effect of pCOD on PDN, the dynamics of
N2 O emission were investigated by an optimized ASM1 model.
The predicted N2 O, shown in Fig. 6, corresponded well with
the measured N2 O under the four operational conditions. As
shown in Fig. 6a-b, with the same amount of sCOD, more pCOD
caused the accumulation of N2 O in the liquid phase (from
0.025 to 0.10 mg N/L), accompanying with the increase in the
peak value of N2 O in the liquid phase (from 0.18 to 0.25 mg
N/L). Okabe et al. (2011) reported that the active N2 O produc-
tion zone was located in the inner part of anammox granules,
and N2 O production occurred mainly by heterotrophic denitri-
fication. Therefore, it could be hypothesized that the increas-
ing N2 O production was due to the diffusion limitation of the
pCOD for denitrifiers, which affected N2 O reduction (higher
Ks = 40 mg COD/L) more than NO2 − -N reduction (lower Ks = 20
mg COD/L) (Hiatt and Grady, 2008).
Additionally, the peak value of N2 O decreased dramatically
from 0.15 to 0.02 mg N/L at the COD/N ratio increased from 0.5
to 2, respectively, under the content of sCOD% of 50% (Fig. 6c-
d). The contribution of denitrification accounted for 2.6% and
12.3% at COD/N ratios of 0.5 and 2, respectively (Fig 5). It could
be hypothesized that the condition of low COD/N conditions
could not provide a sufficient amount of carbon source for
complete denitrification, which resulted in a high N2 O pro-
duction from incomplete denitrification. Zhang et al. (2016) re-
ported that N2 O accumulation at low COD/N ratios was mainly
caused by qnorB/nirK as these genes were directly involved in
NO consumption (qnorB) and production (nirK). Further stud-
ies on interactive metabolism of microbial community are
still required to fully understand the main mechanism for au-
totrophic nitrogen removal.
Although PDN and anammox processes were simultane-
ously achieved in the presence of sCOD in previous research
(Zhan et al, 2020), the pCOD also played a crucial role in ni-
trogen removal. In this study, high percentage of pCOD (15
and 30% of sCOD%) was explicitly proved to suppress the ni-
trogen removal efficiency when treating low COD/N wastew-
ater owing to the weakened denitrification process. Moreover,
incomplete denitrification led to a high N2 O production. In
practice, a complex pretreatment, including chemically en-
hanced primary treatment, need to be applied in WWTPs
to capture pCOD (Han et al., 2016; Li et al., 2018). Moreover,
Li et al. (2020b) also indicated that the residual pCOD af-
ter different organic capture pathways was a critical prob-
lem for B-stage in an A/B process when treating municipal
sewage via anammox. This study provides a new insight for
a cost-effective and efficient wastewater pretreatment strat-
egy, in which controlling the sCOD content in the range of 50-
100% can effectively avoid negative effects of pCOD. Moreover,
the hydrolysis of pCOD was a limiting step for pCOD utiliza-
tion by denitrifying bacteria (Liu et al., 2016), which depended
on the reactor HRT. Therefore, the HRT should be subject
153
to further optimization when treating wastewater containing
pCOD.
3. Conclusion
PDN/anammox and denitrification were simultaneously
maintained at a low sCOD/N ratio of 0.5, resulting in a high
TN removal efficiency of 85%. The PDN/anammox process
played the key role in nitrogen removal. Both qPCR and
microbial community analysis further indicated that Thauera,
Denitratisoma and Candidatus Kuenenia were the active species
for both PDN/anammox and denitrification processes. The
high activity of the functional genes (napA, nirS, nosZ and
hzo) (increased by 161%-247%) related to anammox and den-
itrification was the key for improving the nitrogen removal
performance. With the addition of a mixture of sCOD and
pCOD, a high percentage of pCOD not only inhibited the
nitrogen removal efficiency by 8%-17%, but also caused a high
N2 O production (the peak value up to 0.25mg N/L).
Declaration of Competing Interest
The authors declare that they have no known competing fi-
nancial interests or personal relationships that could have ap-
peared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Science Foundation
of China (Nos. 51978487 and 51678424), National Key Research
and Development Program of China (No. 2019YFC1906402) and
the National Science Centre (No. 2017/26/D/ST8/00967).
Appendix A Supplementary data
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jes.2021.11.007.
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