Chemosphere 285 (2021) 131322

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

The effects of Fe(III) and Fe(II) on anammox process and the

Fe–N metabolism
Yao Chen, Fangxu Jia *, Yingjie Liu, Wanrou Yu, Weiwei Cai, Xiaofan Zhang, Haodong He,
Hong Yao
Beijing Key Laboratory of Aqueous Typical Pollutants Control and Water Quality Safeguard, Department of Municipal and Environmental Engineering, School of Civil
Engineering, Beijing Jiaotong University, Beijing, 100044, PR China

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

Handling Editor: Dr A Adalberto Noyola This study aims to compare the effects of different Fe stress on anammox (anaerobic ammonium oxidation)
process, therefore seven identical reactors were operated under different Fe(II)/Fe(III) concentrations. After 38
Keywords: days of operation, the anammox activity was highest (10.49 ± 0.41 mg-TN/(g-VSS⋅h)) under conditions of 5 mg/
Anammox L-Fe(II), while under 30 mg/L-Fe(III) displayed severe inhibition. The results showed that continuous addition of
Ferric
30 mg/L-Fe(III) would damage the composition of EPS (extracellular polymeric substances) and make anammox
Ferous
bacteria more sensitive to environmental stress. While high Fe(II) concentrations could result in precipitates
Fe(II)-dependent nitrate reduction
Nitrogen removal encasing granular sludge, affecting substrate utilization. Moreover, the results of ΔNO-3-N/ΔNH+ 4 -N indicated
that Fe(II)-dependent nitrate reduction was induced in reactors added with Fe(II). OM27_clade and nor­
ank_f__Burkholderiaceae might be candidates for this process according to the correlation of genera and functional
genes (based on the PICRUSt 2 functional prediction). Overall, this research is expected to provide new ideas to
the effects of Fe(II)/Fe(III) on anammox and to the practical application of coupled system based on anammox in
wastewater treatment.

1. Introduction
Anaerobic ammonium oxidation (anammox) is a promising nitrogen
removal processing for treating high ammonia wastewater. Compared to
the conventional nitrification denitrification process, it is more cost-
effective and environmentally friendly as it can reduce oxygen de­
mand and the emission of CO2 and N2O with no organic carbon con­
sumption and lower sludge yields (Op Den Camp et al., 2006; Kuenen,
2008). However, anammox bacteria (AnAOB) are quite sensitive to the
surrounding environment and have a long generation cycle of 11–20
days (Jetten et al., 2009). Therefore, the recovery of anammox activity is
time-consumed once inhibited by interfering substances contained
within wastewater (such as heavy metal, antibiotics, etc. (Pulicharla
et al., 2015; Chen et al., 2016). Consequently, increasing the activity of
anammox and accelerating the recovery of reactors remain important
challenges.
Iron can promote the synthesis of heme c and Fe–S proteins in
AnAOB which are essential for their metabolism (Ferousi et al., 2017).
Besides, AnAOB contain a large number of dense iron particles (van
Niftrik et al., 2008) which may relate to iron respiration. Therefore, the
addition of Fe might be a potential method to enhance the activity of
AnAOB. To date, previous studies showed that the addition of Fe could
stimulate anammox bacteria. For example, the start-up period reduced
by 20 days (Bi et al., 2014) and the specific anammox growth rate
enhanced by 45.8% (Liu and Ni, 2015) with the addition of 5 mg/L-Fe
(II). The activity of AnAOB increased five times with the addition of
3.68 mg/L-Fe(III) (Chen et al., 2014). Moreover, the promotion of
zero-valent iron (Ren et al., 2015, 2016; Zhang et al., 2017), Fe3O4 (Gao
et al., 2014), and Fe electrode (Zhang et al., 2012; Xie et al., 2020) on
anammox have also reported, while reports on the inhibition of anam­
mox by Fe are relatively few. Therefore, concentrations of 5 mg/L
(relatively optimal), 15 mg/L and 30 mg/L (relatively high) were chosen
to investigate the facilitation and suppression of Fe(II)/Fe(III) on
anammox. More importantly, the majority of continuous experiments
have been conducted in a single reactor, in which the Fe concentration
was increased sequentially in a stepwise manner, rather than compared
directly in several parallel (independent) reactors. A sequential experi­
mental scheme makes it difficult to determine the actual effective

* Corresponding author.
E-mail address: jiafx@bjtu.edu.cn (F. Jia).

https://doi.org/10.1016/j.chemosphere.2021.131322
Received 20 April 2021; Received in revised form 14 June 2021; Accepted 20 June 2021
Available online 25 June 2021
0045-6535/© 2021 Elsevier Ltd. All rights reserved.
Y. Chen et al.
concentration of Fe as it can easily precipitate and be absorbed into the
anammox sludge. Therefore, comparative experiments are required to
help understand and reduce the cumulative effects of Fe.
On the other hand, Fe is a potential energy source that can be used as
either the electron donor or acceptor with its different valence states.
The redox reactions of Fe could be carried out by microbes or abiotically.
Moreover, Fe plays a significant role in nitrogen removal and nitrogen
cycling (Melton et al., 2014), such as anaerobic ammonium oxidation
coupled with ferric reduction (Feammox), Nitrate-dependent Fe(II)
oxidation (NDFO), and Fe(II)-dependent dissimilatory nitrate reduction
to ammonium (Fe(II)-dependent DNRA). Feammox coupled with
anammox contributes to nitrogen removal in wetlands and paddy soils
(Yang et al., 2012; Ding et al., 2014, 2020). Meanwhile, Fe
(II)-dependent nitrate reduction (NDFO and DNRA) could reduce ni­
trate with Fe(II) as the electron donor, providing a new pathway for total
nitrogen removal (Han et al., 2020; Li et al., 2020a). Generally, there is a
coupling of anammox, nitrification, DNRA, Feammox, and NDFO in
majority anammox reactors (Shu et al., 2016). However, these Fe–N
metabolism pathways are often neglected, even though they are likely to
be present within reactors due to the continuous addition of Fe. Thus, it
is necessary to take these reactions into consideration when researching
the effects of Fe on anammox. Furthermore, how to establish a coupled
system to achieve high treatment efficiency also requires continued
study and could potentially be beneficial to the engineering application
of anammox.
Consequently, the main objectives of this study were to (1) compare
the facilitation and suppression of different Fe(II)/Fe(III) concentrations
(5, 15 and 30 mg/L) on anammox process; (2) explore the reactions
which might be induced into systems under different Fe stress. There­
fore, seven identical reactors were operated and nitrogen removal per­
formance, EPS, and community structure of reactors were investigated.
Meanwhile, the correlation of genera and genes (results of PICRUSt 2
functional prediction) were also assessed to determine the potential
functional bacteria of reactions related to iron metabolism.
2. Materials and methods
2.1. Experimental set-up and operation strategy
Seven identical UASB, made of polymethyl methacrylate and a
working volume of 0.42 L (diameter of 3.0 cm and height of 60.0 cm),
were utilized for experiments. A thermostatic water-box was equipped
to maintain a fixed temperature of 35 ± 1 ◦ C. The schematic diagram
and reactor designations are shown in Fig. 1. Anammox granular sludge,
refrigerated (4 ◦ C) for over one year, was inoculated into the reactors
with an initial biomass concentration of 8.0–10.0 g-VSS/L. NH+ 4 -N and
Fig. 1. The schematic diagram and the influent conditions (valence and con­
centration of iron) of the reactors.
2
Chemosphere 285 (2021) 131322
NO−2 -N were in form of NH4Cl and NaNO2, respectively. The composi­
tion of synthetic wastewater referred to previous publication (Bi et al.,
2014), but no trace element solution I was added to avoid extra Fe(II)
addition. Each reactor was fed a different Fe(III)/Fe(II) concentration
(sequestrated by EDTA) (Fig. 1). Influent dissolved oxygen (DO) con­
centration was controlled to be under 0.1 mg/L and the pH was adjusted
to 7.0 ± 0.2.
Operation strategies are summarized in Table 1 and were the same
for each reactor. After reaching the stable state, nitrogen loading rate
(NLR) was raised by increasing the influent nitrogen concentration and/
or the flow of influent. Daily water samples were taken to determine the
concentrations of nitrogen compounds. At the end of each phase, the
mixed liquor was withdrawn from the reactors for subsequent batch
tests. Before that, the sludges were washed three times with oxygen-free
water to remove residual NH+ 4 -N and NO2 -N. At which point the specific
−
anammox activity (SAA), EPS contents, SS and VSS of sludges were
determined.
2.2. Determination of SAA
Batch tests were performed in serum flasks (250 mL). Approximately
150 mL mixture was added into each serum flask. The initial concen­
trations of NH+ 4 -N and NO2 -N were 30 and 40 mg/L, respectively, and
−
pH was adjusted to 7.0 ± 0.2. After flushing with 99.99% nitrogen gas,
the serum flasks were sealed with silicon/Teflon gaskets and poly­
propylene caps, and then incubated on an orbital shaker (180 rpm, 35 ±
1 ◦ C) in the dark. Experiments were conducted in triplicate. Water
samples were collected periodically over 8 h by a syringe to determine
the concentrations of NH+ 4 -N, NO2 -N, and NO3 -N.
− −
2.3. EPS extraction and determination
EPS was extracted by the ultrasound method according to Jia et al.
(2017): 40 mL mixed liquors were taken from each reactor and washed
three times using PBS buffer and the supernatant was discarded after
sedimentation. Next the granules were crushed (mortar and pestle) and
resuspended in PBS for sonication (20 kHz and 5.59 W/mL for 1 min);
Finally, the suspension was centrifuged (20 min, 20000 g), and super­
natant were collected after filtering by 0.45 μm polytetrafluoroethylene
filters. The content of proteins, humic acid, and polysaccharides was
measured according to Badireddy et al. (2010). DNA content was
measured by the diphenylamine method (De Mey et al., 2006).
2.4. Illumina MiSeq sequencing
Anammox biomass collected from the seven reactors (triplicate
samples for each reactor) on day 38 was explored by Illumina MiSeq
sequencing to determine the microbial community structure. Samples
were sent to Majorbio Company (Shanghai, China). The universal
primer pair 338 F (5′ -ACTCCTACGGGAGGCAGCAG-3′ ) and 806 R (5′ -
GGACTACHVGGGTWTCTAAT-3′ ) was utilized for amplification and
sequencing of the 16 S rRNA gene V3 ~ V4 region. All data analysis were
finished by I-sanger Cloud Platform (www.i-sanger.com). Sequencing
reads were clustered into operational taxonomic units (OTUs) at an
identity threshold of 0.97. The functional genes were established using
PICRUSt2 function prediction. 31,152 reads were obtained from each
sample after subsampling and the coverage was greater than 99.4%,
indicating that the diversity of the microbial community could be well
Table 1
Experimental conditions of continuous-flow experiments.
Phase NH+
4 -N (mg/L) NO−2 -N (mg/L) HRT (h) Duration time (d)
1 28.55 ± 2.00 37.68 ± 1.73 2.7 1–10
2 54.64 ± 0.98 83.44 ± 3.51 2.7 11–24
3 114.96 ± 3.55 147.78 ± 4.38 1.8 25–38
Y. Chen et al.
represented by the sequences. After cultivation, the 16 S rRNA gene
community diversity of RII-30 was significantly lower than other re­
actors according to the Shannon and Simpson index (Table S1). In
general, the community richness and diversity of RII-5 ~ RII-30 were
lower than that of RIII-5 ~ RIII-30. Additionally, all original sequencing
data have been submitted to the National Center for Biotechnology In­
formation (NCBI) repository with BioProject PRJNA693360.
2.5. Other analytical analysis
Water samples were analyzed immediately after filtrating with a
0.45 μm polyether-sulfone filter. The NH+ 4 -N, NO2 -N, and NO3 -N con­
− −
centrations and the VSS and SS of sludge were determined according to
standard methods (Eaton, 2005). The pH was determined by a WTW
Multi340i pH meter (WTW, Germany). The significance of the results
and linear fittings were performed with OriginPro 2020 (OriginLab,
Northampton, MA, USA) and p < 0.05 represented statistically signifi­
cant value. The Pearson Indices (r) and Significant Correlation (p) of the
correlations between microbial community structure and functional
genes under Fe(II) or Fe(III) stress were calculated by R (v 3.6.3) using
the RStudio (v1.2.5033, using psych, reshape 2 packages) environments.
3. Results
3.1. Effects of Fe(III) and Fe(II) on AnAOB activity
The variation of nitrogen removal performance of the seven reactors
after 38 days of operation is showed in Fig. 2(a ~ c). During the phase 1,
the influent NH+4 -N and NO2 -N concentrations were 28.54 ± 2.00 mg/L
− tion of most reactors were stable at ~0 mg/L after a period of adaption,
and 37.68 ± 1.73 mg/L, respectively. NLR was in the range of 0.61–0.72
kg-N/(m3⋅d). The maximum NH+ 4 -N concentration in effluents of the
seven reactors was at 23.0–25.0 mg/L (NO−2 -N was 21.0–25.8 mg/L) on
day 1 and stabilized at 0 mg/L after 3–4 days. At the end of this period,
3
Chemosphere 285 (2021) 131322
the nitrogen removal rate (NRR) of the seven reactors had stabilized at
0.48–0.54 kg-N/(m3⋅d) and the NH+ 4 -N and NO2 -N removal efficiencies
−
were above 96.0% and 96.3% (not shown in the figure), respectively.
Although the nitrogen removal performances of the seven reactors were
similar, RIII-30 had the highest SAA, at 4.21 ± 0.54 mg-TN/(g-VSS⋅h),
while RII-30 was the lowest (2.35 ± 0.21 mg-TN/(g-VSS⋅h)) (Fig. 2(d)).
During phase 2, the effluent NH+ 4 -N and NO2 -N concentrations dropped
−
below 1 mg/L within 2 days (3 days for RIII-30) even the influent ni­
trogen concentrations were doubled. The maximum NH+ 4 -N concentra­
tion in effluent of RIII-30 was 22.0 mg/L (NO−2 -N was 30.1 mg/L) after
increasing the NLR, while all other reactors were below 10.4 mg/L
(NO−2 -N was below 13.0 mg/L). Together this indicated that inhibition
by high Fe(III) concentration (30 mg/L) emerged gradually. Meanwhile,
the SAA of RIII-30 (4.20 ± 0.37 mg-TN/(g-VSS⋅h)) did not increase as
compared to phase 1 (Fig. 2(d)). The SAA of RII-5, RII-15 and RII-30
(5.45 ± 0.29, 5.50 ± 0.14 and 4.71 ± 0.19 mg-TN/(g-VSS⋅h), respec­
tively) surpassed that of their Fe(III) counterparts, RIII-5, RIII-15 and
RIII-30 (4.30 ± 0.51, 4.11 ± 0.66 and 4.20 ± 0.37 mg-TN/(g-VSS⋅h),
respectively). This showed that the activity of AnAOB in the presence of
Fe(II) was higher than that with Fe(III) after 24 days of cultivation. But
only the SAA of RII-5 and RII-15 were higher than the control (R0, 5.27
± 0.52 mg-TN/(g-VSS⋅h)). Notably, the SAA of RII-30 increased
dramatically during phase 2 as compared to phase 1.
During phase 3, the influent NH+ 4 -N and NO2 -N concentrations were
−
approximately 114.96 ± 3.55 mg/L and 147.78 ± 4.38 mg/L while the
HRT was halved, therefore the NLR was tripled as compared to phase 2.
At this point, the differences among reactors in nitrogen removal per­
formance began to emerge. The effluent NH+ 4 -N and NO2 -N concentra­
−
however, those of RIII-30 remained above 95.0 mg/L and 117.1 mg/L,
respectively, indicating that anammox activity within RIII-30 was
dramatically inhibited. To further elucidate the differences in perfor­
mance of each reactor after increasing the NLR, linear fitting was
Fig. 2. Operation performance of the seven reactors
with the continuous addition of different Fe (Fe(III)/
Fe(II)) concentrations: The variation of (a) ammonia
(b) nitrite concentrations. (c) NLR and NRR
throughout the experiment. The variation of (d) SAA
and (e) VSS/SS ratios at the end of each phase. (f) The
linear fitting of each reactor during the adaptation
period in phase 3. “k” is the slope of the fitting curve,
representing the adaptive rate of reactors. Asterisks
indicate a significant correlation. *P < 0.05, **P <
0.01.
Y. Chen et al. Chemosphere 285 (2021) 131322

performed, and the adaptive rate (slope of the fitting curve, k) of ni­
trogen load impact was calculated during the adaption period (Fig. 2(f)).
The adaptive rate of RII-5 and R0 were the highest (0.98 and 0.86 kg-N/
(m3⋅d2)) followed by that of RII-15 and RII-30 (0.67 and 0.49 kg-N/
(m3⋅d2)), with RIII-5 and RIII-15 having the lowest rates (0.32 and 0.27
kg N/(m3⋅d2)). However, the SAA of RII-15 and RII-30 (8.40 ± 0.13 and
7.88 ± 0.30 mg-TN/(g-VSS⋅h), respectively) were lower than that of
RIII-5 and RIII-15 (8.66 ± 0.30 and 8.52 ± 0.23 mg-TN/(g-VSS⋅h),
respectively). These contrary results were caused by the accidental
washing out of partial sludge in RIII-5 and RIII-15 during phase 3. The
SAA of RII-5 (10.49 ± 0.41 mg-TN/(g-VSS⋅h)) was the highest and the
SAA of all reactors, except that of RIII-30, increased dramatically.
Meanwhile, the VSS/SS ratios of reactors were also calculated and are
showed in Fig. 2(e). The ratios of RII-5, RII-15 and RII-30 were generally
lower than that of others and shows a downward trend (from 0.84 ±
0.03 to 0.63 ± 0.01). In total, the ratios of RII-15, RII-30 and RIII-30
decreased dramatically with the consistent addition of corresponding
Fe(II)/Fe(III) concentrations (from 0.83 ± 0.00 to 0.75 ± 0.01, from
0.82 ± 0.00 to 0.63 ± 0.01 and from 0.87 ± 0.01 to 0.81 ± 0.03,
respectively).

3.2. Difference of stoichiometric ratio in reactors under different Fe stress

To discern which reactions occurred in each reactor, the molar ratios

ΔNO−2 -N/ΔNH+ 4 -N and ΔNO3 -N/ΔNH4 -N during the stable state were
− +

calculated for each phase (Fig. 3). In all three reactors amended with Fe
(III) (RIII-5 ~ RIII-30), the ΔNO−2 -N/ΔNH+ 4 -N ratios were generally
higher than the theoretical value (1.32) of the anammox reaction,
especially for phases 2 (1.32–1.47) and 3 (1.34–2.91) (Fig. 3(a)).
However, there was no obvious correlation between ΔNO−2 -N/ΔNH+ 4 -N
and Fe(III)/Fe(II) concentration. On the contrary, the ΔNO−3 -N/ΔNH+ 4 -N
ratios increased with increasing Fe(III) concentration in each phase
(Fig. 3(b)), from 0.24 ± 0.04 to 0.28 ± 0.06, from 0.27 ± 0.02 to 0.31 ±
0.03 (r = 0.534, P < 0.01) and from 0.24 ± 0.03 to 1.05 ± 0.49 (r = Fig. 3. The (a) ΔNO−2 -N/ΔNH+ 4 -N and (b) ΔNO3 -N/ΔNH4 -N of reactors in
− +

0.718, P < 0.01), respectively. Notably, the ΔNO−2 -N/ΔNH+ 4 -N and
ΔNO−3 -N/ΔNH+ 4 -N ratios of RIII-30 deviated highly from the theoretical
value during phase 3 as its performance was severely damaged. In
general, these results demonstrated that continuous addition of Fe(III)
would induce the excessive transformation of NO−2 -N and NO−3 -N.
phases 1, 2, and 3. The black solid triangle represents the mean value of data.
The black solid pentacle above and below the box represents the maximum and
minimum values of the data, respectively. The upper and lower edges of the box
represent the 25th and 75th percentile of the data. The heatmaps inside the blue
box represent the correlation between ΔNO−2 -N/ΔNH+ 4 -N (in (a)) and ΔNO3 -N/
−

In the reactors which had been amended with Fe(II) (RII-5 ~ RII-30), ΔNH+ 4 -N (in (b)) with Fe(III) concentrations. Meanwhile, the heatmaps inside
the ΔNO−2 -N/ΔNH+ the red box represent the correlation between ΔNO−2 -N/ΔNH+ 4 -N (in (a)) and
4 -N ratios (1.29–1.44) were also generally higher
ΔNO3 -N/ΔNH+
−
4 -N (in (b)) with Fe(II) concentrations. Asterisks indicate a sig­
than the theoretical values, while the ΔNO−3 -N/ΔNH+ 4 -N ratios declined
nificant correlation. *P < 0.05, **P < 0.01. (For interpretation of the references
with increasing Fe(II) concentrations in each phase, from 0.25 ± 0.07 to
to colour in this figure legend, the reader is referred to the Web version of
0.21 ± 0.04 (r = − 0.501, P < 0.05), from 0.28 ± 0.02 to 0.23 ± 0.02 (r this article.)
= − 0.569, P < 0.01) and from 0.27 ± 0.03 to 0.19 ± 0.02 (r = − 0.614, P
< 0.01), respectively. The ΔNO−2 -N/ΔNH+ 4 -N of RII-5 was close to the
theoretical value (0.26) of anammox and the reactors with relatively
high Fe(II) concentrations were lower. In general, the ΔNO−2 -N/ΔNH+ 4 -N
molar ratios of the seven reactors were all higher than the theoretical
value throughout the entire experiment, and the ΔNO−3 -N/ΔNH+ 4 -N ratio
was heavily dependent on the valence and concentration of iron ions. In
total, the results indicated that some reactions other than anammox
were induced by Fe addition and the difference was quite significant
between the reactors to which Fe(III) and Fe(II), respectively, had been
added.

3.3. The variation of EPS contents

EPS plays an essential role in the formation of multilevel porous

structures on the cell surface for self-protection and to promote micro­
bial aggregation (Henriques and Love, 2007). Fe(III)/Fe(II) could either
affect anammox activity directly, or via the EPS. Therefore, the variation
of EPS content for each reactor was analyzed (Fig. 4). Except for reactor
RIII-30, the total EPS contents and PN/PS (proteins/polysaccharide
ratio) ratios of the sludges increased with time, especially in phase 3.
The total EPS content of RIII-30 (268.2 ± 11.9 mg/(g-VSS)) was lower
Fig. 4. The concentrations of EPS contents and the PN/PS ratio in the seven
reactors at the end of each phase.
than that of the other reactors (268.8–300.8 mg/(g-VSS)), and the
PN/PS ratio displayed a similar trend. Notably, the PN/PS ratio of
RIII-30 in phase 3 (0.73 ± 0.04) was lower than that in phase 2 (0.84 ±

4
Y. Chen et al.
0.01), indicating that excess Fe(III) addition had a greater effect on
protein content. The EPS content of reactors with high Fe addition
concentrations (265.51 ± 27.8, 268.4 ± 21.4, 221.18 ± 29.9 and 221.5
± 25.9 mg/(g-VSS) in RIII-15, RIII-30, RII-15, and RII-30, respectively)
were higher than those with low concentrations (204.3 ± 32.5 and
189.4 ± 22.3 mg/(g-VSS) in RIII-5 and RII-5, respectively) in phase 1. It
should also be noted that reactors amended with Fe(III) were higher
than those amended with Fe(II) at the same concentration. However, the
increased rates of RII-5 (5.2 mg/(g-VSS⋅d)) and RIII-5 (3.4
mg/(g-VSS⋅d)) were higher than others (data not shown). Therefore, the
EPS content decreased with increasing Fe(III) (from 300.0 ± 18.2 to
268.9 ± 12.0 mg/(g-VSS)) and Fe(II) (from 335.0 ± 19.7 to 270.2 ± 26.1
mg/(g-VSS)) concentrations in phase 3. Moreover, only the total EPS
contents of RIII-5, RIII-15, and RII-5 were higher than the control, which
was in accordance with the SAA results of anammox.
3.4. Microbial community evolution
A total of 11 abundant phyla (abundance ≥ 0.01%) were detected in
all samples (Fig. S2). The most dominant phylum was Chloroflexi which
accounted for 56.0–69.0% of sequences, followed by Planctomycetes
(11.8–28.6%), Proteobacteria (4.8–12.6%), and Bacteroidetes (2.6–4.8%).
Candidatus Brocadia was the representative genus for Planctomycetes in
this study, given that its abundance variation was highly in accordance
with that of Planctomycetes as a group (r = 0.999, P < 0.01). As shown in
Fig. 5(a), the abundance of Ca. Brocadia in RIII-30 (11.6 ± 4.6%) was
significantly lower than that of other reactors (18.3–28.4%) (P < 0.05).
The relative abundance of Ca. Brocadia roughly decreased with
increasing concentration of Fe(II) or Fe(III), with the highest Ca. Bro­
cadia abundance (28.4 ± 2.6%) observed with 5 mg/L-Fe(II). Addi­
tionally, the abundance of Ca. Brocadia in RIII-5 and RIII-15 (25.6 ± 2.2
and 22.8 ± 5.8%, respectively) were higher than that of the control (R0)
(18.4 ± 1.5%). While the abundance of other two reactors (RII-15 and
RII-30, 19.7 ± 3.4 and 18.2 ± 5.8%, respectively) were similar to that of
R0. These results were supported the SAA results.
As mentioned previously, the addition of Fe(III)/(II) could induce
5
Chemosphere 285 (2021) 131322
other reactions in the systems, therefore it was necessary to analyze
other potential functional bacteria, as well as their correlation with Fe
and N related functional genes (Fig. 5(b)). The significance of difference
among the 21 samples was also analyzed. After 38 days of cultivation
with continuous Fe addition, the community structure of RIII-30 was
significantly different from the others (P < 0.01) as reactor had been
severely destroyed. The abundance of NOB, including Nitrospira and
Candidatus Nitrotoga, was higher in reactors which had been amended
with Fe(III) (P < 0.05). The abundance of denitrifying bacteria,
including Comamonas and Denitratisoma, displayed a similar trend.
Meanwhile, the abundance of Thermomonas and Simplicispira, which
were respectively related to Fe–Mn (Hou et al., 2020) and As(III) (Cui
et al., 2018) oxidation, was higher in the reactors with high Fe(III)/Fe
(II) concentrations (P < 0.01 and P < 0.01, respectively). Members of the
genus Paludibaculum have been reported to be capable of fermentation
and dissimilatory Fe(III) reduction (Kulichevskaya et al., 2014), but its
abundance was reduced as Fe(III)/Fe(II) concentrations increased. The
genus Ignavibacterium, reported as potential functional Feammox bac­
teria (Takao et al., 2010), increased with reactor operation time, how­
ever its proportion was always less than 0.1%. The correlations between
genera and functional genes were quite different within the different Fe
valences circumstances (as shown in Fig. 5 (b)). When Fe(III) was less
than 15 mg/L (RIII-5 ~ RIII-15), Ca. Brocadia was closely and positively
correlated with feoB gene which is speculated to be the only iron
transporter gene in anammox genomes (Ferousi et al., 2017). However,
there was no significant correlation between Ca. Brocadia and feoB
under Fe(II) stress (RII-5 ~ RII-30). These results indicated that the feoB
gene was inactive in Fe(II)-rich environments. NOB, denitrifying bac­
teria, SWB02 and Ignavibacterium showed significant positive correla­
tions with fieF (ferrous-iron efflux pump) and nitrate reduction genes
(nirA, nrfA and/or nirS) only under Fe(III) exposure. OM27_clade and
norank_f__Burkholderiaceae showed significant positive correlation with
nirS gene, but they were positive correlated with feoA, ftnA and/or fur
only under Fe(II) stress.
Fig. 5. Analysis of the microbial community struc­
ture (a) at genus level (“others” represents all classi­
fied taxa that were < 1%) and (b) the correlation
between genera and functional genes under Fe(III) or
Fe(II) stress (bubble matrix). The heatmap shows the
relative abundance of functional bacteria at genus
level. The diameter of bubble represents the value of
correlation coefficient r. Orange and blue bubbles
represent positive and negative correlations, respec­
tively. The RIII-30 was excluded since its community
structure was quite different compared with others
and was severely inhibited. The data with r < 0.5 or p
> 0.05 were filtered out. “△” and “☆” represents the
significance of difference among seven samples
(heatmap) and the significance of the correlation
(bubble). △, P < 0.05; ☆, P < 0.01. (For interpre­
tation of the references to colour in this figure legend,
the reader is referred to the Web version of this
article.)
Y. Chen et al.
4. Discussion
4.1. Effects of Fe(III) and Fe(II) on anammox
Our results showed that the seven reactors displayed similar per­
formance during the initial period (Fig. 1), which might be due to the
high biomass concentrations (8.0–10.0 g-VSS/L) and low substrate
concentrations. The difference emerged only after increasing the NLR.
The activity of anammox could be enhanced slightly by the continuous
addition of relatively low concentrations of Fe(III) (<15 mg/L), but
would be severely inhibited by high concentration (30 mg/L). The ni­
trogen removal of RIII-30 was almost undetectable during phase 3
(Fig. 2) and the relative abundance of AnAOB was greatly reduced
(Fig. 5). This meant the amount of Fe(III) addition could have been
beyond the iron adsorption capacity of sludge and inhibiting the activity
and growth of AnAOB. However, Li et al. (2014) and Yuan and Wang
(2019) concluded that there was no obvious inhibition by excess Fe(III)
(5–65 mg/L and 10–40 mg/L, respectively) during batch tests. They
believed inhibition by high Fe(III) was avoided due to the precipitation
of Fe(III) with OH− produced during the anammox reaction. This pre­
cipitation phenomenon was also observed during the present study, with
the majority of sediments accumulating at the bottom of the reactors.
That was why the VSS/SS ratio of RIII-30 was slightly decreased and
those of RIII-5 and RIII-15 were relatively unchanged. But the total EPS
contents, especially the protein contents, of RIII-30 were obviously
lower than of other reactors in phase 3 (Fig. 4), the same decrease
tendency of protein content was also observed with long-term exposure
to 50 mg/L-Fe(III) according to Zhang et al. (2021). Given these results,
it is reasonable to speculate that the continuous addition of high con­
centration of Fe(III) could damage the composition of EPS (especially
the protein component) and make AnAOB more sensitive to the envi­
ronmental inhibitory factors (such as nitrite), rather than inhibit the
anammox directly. Therefore, the activity of RIII-30 was inhibited
severely when the NLR was tripled in phase 3.
As mentioned above, the reactors with the addition of Fe(II) were
generally superior to that with Fe(III) and the optimal concentration of
Fe(II) is 5 mg/L, which was in agreement with previous studies (Qiao
et al., 2013; Mak et al., 2019). This phenomenon can be explained in two
aspects according to previous studies. Firstly, most of these cellular irons
are divalent and the Fe(II) can be utilized directly while Fe(III) needs to
be reduced to Fe(II) first (Ferousi et al., 2017). Secondly, the presence of
Fe(II) helps maintain an anaerobic environment for anammox. Besides,
The anammox activity of RII-30 was slightly inhibited during phase 1
and promoted during phases 2 and 3 (compared to R0), revealing that
inhibition of 30 mg/L-Fe(II) was recoverable in the prophase cultiva­
tion. The same recoverable inhibition was also observed by Ding et al.
(2021), the inhibition by 56 mg/L-Fe(II) could recover in 24 h without
changing any conditions. At the end of experiment, the SAA and rela­
tively abundance of AnAOB in RII-15 and RII-30 were close to the
control. However, the VSS/SS ratios of RII-15 and RII-30 were greatly
decreased (Fig. 3(b)), especially that of RII-30. Compared to Fe(III), the
deposition rate of Fe(II) was relatively slower such that the precipitate
was more likely to be absorbed into the EPS and even accumulate on the
surface of the granular sludge, which might affect substrate utilization
and mass transfer (Wang et al., 2017). This absorption could be the main
cause of inhibition by Fe(II) on anammox. Therefore, it was reasonable
to presume that the activity of RII-15 and RII-30 might be inhibited
during subsequent reactor operation (>38 d) since the amount of pre­
cipitate accumulating in the sludges would be considerable and possibly
even encapsulate the sludge particles completely. In comparison, Mishra
et al. (2020) found that inhibition of anammox by 30 mg/L-Fe(II) was
unrecoverable, while Li et al. (2020b) found that anammox activity was
not inhibited severely until influent Fe(II) concentration reached 378.6
mg/L. These differences might be due to the different reactor types
(UASB and UBF, respectively), sludge concentrations, operation condi­
tions, etc. Additionally, these two experiments were both conducted in a
6
Chemosphere 285 (2021) 131322
single reactor where the influent Fe(II) concentration was gradually
increased (from 2 to 50 mg/L and from 0 to 378.6 mg/L, respectively),
thus it was hard to determine the true effective Fe(II) concentrations in
the reactors. In total, the mechanisms of influence (especially inhibition)
of Fe(II) and Fe(III) still remain elusive, therefore further studies and
more advanced technologies are needed to ascertain the effect of Fe on
anammox.
4.2. The coupling system induced by Fe(III) and Fe(II)
In this study, the extra consumption of NO−2 -N was observed
throughout the experiment and the ΔNO-3-N/ΔNH+ 4 -N also deviated
from the theoretical value (Fig. 3). It could be presumed that other re­
actions, besides anammox, were present in the reactors. In reactors
amended with Fe(III) (RIII-5, RIII-15, and RIII-30), two genera of NOB
were observed and the abundance of NOB was higher than that with Fe
(II). One possible explanation was that the existence of Fe(II) could
consume the residual DO (Yan et al., 2019) (<0.1 mg/L) within a reactor
to the point that the growth of NOB was inhibited. Feammox, as a newly
discovered reaction, can oxidize ammonia to NO−x -N and N2 using Fe(III)
as the electron acceptor (Park et al., 2009). It may provide nitrite for
anammox without aeration (if well controlled), which can replace par­
tial nitrification and save on energy costs. Therefore, the coupling of
Feammox and anammox in wastewater treatment might have unex­
pected benefits. However, Feammox did not exist, or was not dominant
yet, in these reactors. This could have been partly due to the short in­
cubation time (the cultivation period of Feammox is generally long,
more than 6 mouths), and partly to substrate competition (like NH+ 4 -N)
between Feammox and anammox.
In reactors amended with Fe(II) (RII-5, RII-15, and RII-30), the
ΔNO-3-N/ΔNH+ 4 -N ratio was lower than the theoretical value and
decreased with increasing Fe(II) concentration. This extra consumption
of NO−3 -N has been reported in other studies as well (Bi et al., 2014; Ren
et al., 2015; Shu et al., 2016). However, denitrifying bacteria in these
reactors were no more abundant than in the Fe(III) reactors. Therefore
denitrification might not be the main reason, but rather NDFO or Fe
(II)-dependent DNRA instead. These reactions could utilize Fe(II) as
electron donor to reduce NO−3 -N (produced by anammox process) to
nitrite and ammonia, and further support their central metabolism
(anammox) in a coupled system. Therefore, it could improve nitrogen
removal performance of reactors and even achieve zero nitrogen emis­
sions. Similar coupled systems were created by other researcher groups
through the addition of Fe (Bi et al., 2019, 2020; Li et al., 2020a) or
NO−3 -N (Yang et al., 2020) into the original reactor. Besides, there were a
few candidates, even we could not definitively link any specific func­
tional microorganisms to these metabolisms. As mentioned above (sec­
tion 3.3), the denitrifiers, Thermomonas and Simplicispira, might have the
ability of reduce nitrate in conjunction with Fe(II) oxidation since they
were related to the metal oxidizing in environment. Two genera,
OM27_clade and norank_f__Burkholderiaceae, might also be candidates for
nitrate reduction with Fe(II) oxidation since they showed close, positive
correlation with feoA, ftnA, and/or fur only under Fe(II) stress. Mean­
while the strain Candidatus Brocadia sinica was also reported to be
capable of oxidizing Fe(II) with the reduction of nitrate (Oshiki et al.,
2013).
Overall, this coupled system could be more beneficial for the appli­
cation of anammox process since nitrate is generally present at much
higher concentration than nitrite in wastewater and Fe is a very cheap
and widely available element. Wastewater rich in Fe or nitrate and
sludge produced by Fenton reaction could be utilized as substrates for
the coupled system to turn waste into treasure. Moreover, various iron-
based or iron-modified fillers could be used in anammox system to form
a coupled system. However, the morphology and redox of Fe are
complicated in the reactor environment, and it was hard to determine
the actual effective concentration of Fe. Therefore, the mechanism by
which Fe affects these functional bacteria requires continued study.
Y. Chen et al.
Moreover, the best dosing method and dosing concentration need to be
developed to build a coupled system that does not inhibit anammox
activity.
5. Conclusion
In this work, the effects of three Fe(III) and Fe(II) concentrations (5,
15, and 30 mg/L) on anammox were investigated. It was found that 5
mg/L-Fe(II) was optimal for anammox with the SAA of 10.49 ± 0.41 mg-
TN/(g-VSS⋅h), while the continuous addition of 30 mg/L-Fe(III) would
inhibit AnAOB severely. The results showed that long-term addition of
high Fe(III) concentration damaged the composition of EPS, while that
of high Fe(II) concentration affected the mass-transfer of anammox
granular. Besides, lower ΔNO-3-N/ΔNH+ 4 -N ratios was observed in re­
actors with Fe(II) addition indicated that the process of Fe(II)-dependent
nitrate reduction was induced in reactors. And the genera, OM27_clade
and norank_f__Burkholderiaceae, might be the candidate organisms. This
reaction could reduce the nitrate produced by anammox and improve
the TNRR of reactors.
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.
Acknowledgements
This work was supported by Beijing Outstanding Young Scientist
Project (grant numbers: C19H100010) and National Natural Science
Foundation of China (grant numbers: 51908029).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.chemosphere.2021.131322.
Author contribution
Yao Chen: Conceptualization, Formal analysis, Investigation, Visu­
alization, Writing – original draft; Fangxu Jia: Conceptualization,
Writing – review & editing, Supervision, Resources, Funding acquisition;
Yingjie Liu: Formal analysis, Investigation; Wanrou Yu: Investigation;
Weiwei Cai: Writing – review & editing; Xiaofan Zhang: Validation;
Haodong He: Visualization; Hong Yao: Resources, Funding acquisition,
Writing – review & editing
References
Badireddy, A.R., Chellam, S., Gassman, P.L., Engelhard, M.H., Lea, A.S., Rosso, K.M.,
2010. Role of extracellular polymeric substances in bioflocculation of activated
sludge microorganisms under glucose-controlled conditions. Water Res. 44 (15),
4505–4516.
Bi, Z., Huang, Y., Zhang, W., Song, G., 2020. Impacts of chosen parameters on Fe-
dependent nitrate reduction in anammox consortia: performance and bioactivity.
Water 12 (5), 1379.
Bi, Z., Qiao, S., Zhou, J., Tang, X., Zhang, J., 2014. Fast start-up of Anammox process
with appropriate ferrous iron concentration. Bioresour. Technol. 170, 506–512.
Bi, Z., Zhang, W., Song, G., Huang, Y., 2019. Iron-dependent nitrate reduction by
anammox consortia in continuous-flow reactors: a novel prospective scheme for
autotrophic nitrogen removal. Ence Total Environ. 692.
Chen, H., Chen, Q.Q., Jiang, X.Y., Hu, H.Y., Shi, M.L., Jin, R.C., 2016. Insight into the
short- and long-term effects of Cu(II) on denitrifying biogranules. J. Hazard Mater.
304, 448–456.
Chen, H., Yu, J.J., Jia, X.Y., Jin, R.C., 2014. Enhancement of anammox performance by
Cu(II), Ni(II) and Fe(III) supplementation. Chemosphere 117, 610–616.
Cui, J., Du, J., Tian, H., Chan, T., Jing, C., 2018. Rethinking anaerobic As(III) oxidation
in filters: effect of indigenous nitrate respirers. Chemosphere 196, 223–230.
De Mey, M., Lequeux, G., Maertens, J., De Maeseneire, S., Soetaert, W., Vandamme, E.,
2006. Comparison of DNA and RNA quantification methods suitable for parameter
7
Chemosphere 285 (2021) 131322
estimation in metabolic modeling of microorganisms. Anal. Biochem. 353 (2),
198–203.
Ding, B., Qin, Y., Luo, W., Li, Z., 2020. Spatial and seasonal distributions of Feammox
from ecosystem habitats in the Wanshan region of the Taihu watershed, China.
Chemosphere 239, 124742.
Ding, J., Seow, W., Zhou, J., Zeng, R.J., Zhou, Y.J., 2021. Effects of Fe(II) on anammox
community activity and physiologic response. Front. Environ. Sci. Eng. 15 (1).
Ding, L.-J., An, X.-L., Li, S., Zhang, G.-L., Zhu, Y.-G., 2014. Nitrogen loss through
anaerobic ammonium oxidation coupled to iron reduction from paddy soils in a
chronosequence. Environ. Sci. Techno. 48 (18), 10641–10647.
Eaton, AndrewD., 2005. Standard Methods for the Examination of Water & Wastewater.
American Public Health Association, Washington, DC, USA.
Ferousi, C., Lindhoud, S., Baymann, F., Kartal, B., Jetten, M.S., Reimann, J., 2017. Iron
assimilation and utilization in anaerobic ammonium oxidizing bacteria. Curr. Opin.
Chem. Biol. 37, 129–136.
Gao, F., Zhang, H., Yang, F., Li, H., Zhang, R., 2014. The effects of zero-valent iron (ZVI)
and ferroferric oxide (Fe3O4) on anammox activity and granulation in anaerobic
continuously stirred tank reactors (CSTR). Process Biochem. 49 (11), 1970–1978.
Han, X., Peng, S., Zhang, L., Lu, P., Zhang, D., 2020. The Co-occurrence of DNRA and
Anammox during the anaerobic degradation of benzene under denitrification.
Chemosphere 247, 125968.
Henriques, I.D.S., Love, N.G., 2007. The role of extracellular polymeric substances in the
toxicity response of activated sludge bacteria to chemical toxins. Water Res. 41 (18),
4177–4185.
Hou, D., Zhang, P., Wei, D., Zhang, J., Yan, B., Cao, L., Zhou, Y., Luo, L., 2020.
Simultaneous removal of iron and manganese from acid mine drainage by
acclimated bacteria. J. Hazard Mater. 396, 122631.
Jetten, M.S.M., Niftrik, L.V., Strous, M., Kartal, B., Keltjens, J.T., Op Den Camp, H.J.M.,
2009. Biochemistry and molecular biology of anammox bacteria. Crit. Rev. Biochem.
Mol. Biol. 44 (2–3), 65–84.
Jia, F.X., Yang, Q., Liu, X.H., Li, X.Y., Li, B.K., Zhang, L., Peng, Y.Z., 2017. Stratification
of extracellular polymeric substances (EPS) for aggregated anammox
microorganisms. Environ. Sci. Techno. 51 (6), 3260–3268.
Kuenen, J.G., 2008. Anammox bacteria: from discovery to application. Nat. Rev.
Microbiol. 6 (4), 320–326.
Kulichevskaya, I.S., Suzina, N.E., Rijpstra, W.I.C., Damsté, J.S.S., Dedysh, S.N., 2014.
Paludibaculum fermentans gen. nov., sp. nov., a facultative anaerobe capable of
dissimilatory iron reduction from subdivision 3 of the Acidobacteria. Int. J. Syst.
Evol. Microbiol. 64 (Pt_8), 2857–2864.
Li, X., Huang, Y., Wu, C., Wang, M.-k., Yuan, Y., 2014. Effect of Fe2+ and Fe3+ on the
activity of ANAMMOX. Environ. Sci. 35 (11), 4224–4229.
Li, Z., Peng, Y., Gao, H., 2020a. Enhanced long-term advanced denitrogenation from
nitrate wastewater by anammox consortia: dissimilatory nitrate reduction to
ammonium (DNRA) coupling with anammox in an upflow biofilter reactor equipped
with EDTA-2Na/Fe(II) ratio and pH control. Bioresour. Technol. 305.
Li, Z., Peng, Y., Gao, H., 2020b. A novel strategy for accelerating the recovery of a Fe(II)-
inhibited anammox reactor by intermittent addition of betaine: performance,
kinetics and statistical analysis. Chemosphere 251, 126362.
Liu, Y., Ni, B.-J., 2015. Appropriate Fe (II) addition significantly enhances anaerobic
ammonium oxidation (anammox) activity through improving the bacterial growth
rate. Sci. Rep. 5 (1).
Mak, C.Y., Lin, J.G., Chen, W.H., Ng, C.A., Bashir, M.J.K., 2019. The short- and long-term
inhibitory effects of Fe (II) on anaerobic ammonium oxidizing (anammox) process.
Water Sci. Technol. 79 (10), 1860–1867.
Melton, E.D., Swanner, E.D., Behrens, S., Schmidt, C., Kappler, A., 2014. The interplay of
microbially mediated and abiotic reactions in the biogeochemical Fe cycle. Nat. Rev.
Microbiol. 12 (12), 797–808.
Mishra, P., Burman, I., Sinha, A., 2020. Performance Enhancement and Optimization of
the Anammox Process with the Addition of Iron. Environmental Technology.
Op Den Camp, H.J.M., Kartal, B., Guven, D., Van Niftrik, L.A.M.P., Haaijer, S.C.M., Van
Der Star, W.R.L., Van De Pas-Schoonen, K.T., Cabezas, A., Ying, Z., Schmid, M.C.,
Kuypers, M.M.M., Van De Vossenberg, J., Harhangi, H.R., Picioreanu, C., Van
Loosdrecht, M.C.M., Kuenen, J.G., Strous, M., Jetten, M.S.M., 2006. Global impact
and application of the anaerobic ammonium-oxidizing (anammox) bacteria.
Biochem. Soc. Trans. 34 (1), 174–178.
Oshiki, M., Ishii, S., Yoshida, K., Fujii, N., Ishiguro, M., Satoh, H., Okabe, S., 2013.
Nitrate-dependent ferrous iron oxidation by anaerobic ammonium oxidation
(Anammox) bacteria. Appl. Environ. Microbiol. 79 (13), 4087.
Park, W., Nam, Y.-K., Lee, M.-J., Kim, T.-H., 2009. Anaerobic ammonia-oxidation
coupled with Fe reduction by an anaerobic culture from a piggery wastewater
acclimated to NH4+/Fe3+ Medium. Biotechnol. Bioproc. Eng. 14 (5), 680–685.
Pulicharla, R., Das, R.K., Brar, S.K., Drogui, P., Sarma, S.J., Verma, M., Surampalli, R.Y.,
Valero, J.R., 2015. Toxicity of chlortetracycline and its metal complexes to model
microorganisms in wastewater sludge. Sci. Total Environ. 532, 669–675.
Qiao, S., Bi, Z., Zhou, J., Cheng, Y., Zhang, J., 2013. Long term effects of divalent ferrous
ion on the activity of anammox biomass. Bioresour. Technol. 142, 490–497.
Ren, L.-F., Lv, L., Zhang, J., Gao, B., Ni, S.-Q., Yang, N., Zhou, Q., Liu, X., 2016. Novel
zero-valent iron-assembled reactor for strengthening anammox performance under
low temperature. Appl. Microbiol. Biotechnol. 100, 8711–8720.
Ren, L.-F., Ni, S.-Q., Liu, C., Liang, S., Zhang, B., Kong, Q., Guo, N., 2015. Effect of zero-
valent iron on the start-up performance of anaerobic ammonium oxidation
(anammox) process. Environ. Sci. Pollut. Res. 22, 2925–2934.
Shu, D., He, Y., Yue, H., Yang, S., 2016. Effects of Fe(II) on microbial communities,
nitrogen transformation pathways and iron cycling in the anammox process:
kinetics, quantitative molecular mechanism and metagenomic analysis. RSC Adv. 6
(72), 68005–68016.
Y. Chen et al.
Takao, I., Koji, M., Yoshihito, U., Tatsunori, N., Shigeaki, H., Ken-Ichiro, S., 2010.
Ignavibacterium album gen. nov., sp. nov., a moderately thermophilic anaerobic
bacterium isolated from microbial mats at a terrestrial hot spring and proposal of
Ignavibacteria classis nov., for a novel lineage at the periphery of green sulfur
bacteria. Int. J. Syst. Evol. Microbiol. 60 (6), 1376–1382.
van Niftrik, L., Geerts, W.J.C., van Donselaar, E.G., Humbel, B.M., Yakushevska, A.,
Verkleij, A.J., Jetten, M.S.M., Strous, M., 2008. Combined structural and chemical
analysis of the anammoxosome: a membrane-bounded intracytoplasmic
compartment in anammox bacteria. J. Struct. Biol. 161 (3), 401–410.
Wang, R., Yang, C., Zhang, M., Xu, S.-Y., Dai, C.-L., Liang, L.-Y., Zhao, H.-P., Zheng, P.,
2017. Chemoautotrophic denitrification based on ferrous iron oxidation: reactor
performance and sludge characteristics. Chem. Eng. J. 313, 693–701.
Xie, F., Ma, X., Zhao, B., Cui, Y., Zhang, X., Yue, X., 2020. Promoting the nitrogen
removal of anammox process by Fe-C micro-electrolysis. Bioresour. Technol. 297,
122429.
Yan, Y., Wang, Y., Wang, W., Zhou, S., Wang, J., Guo, J., 2019. Comparison of short-term
dosing ferrous ion and nanoscale zero-valent iron for rapid recovery of anammox
activity from dissolved oxygen inhibition. Water Res. 153, 284–294.
Chemosphere 285 (2021) 131322
Yang, W.H., Weber, K.A., Silver, W.L., 2012. Nitrogen loss from soil through anaerobic
ammonium oxidation coupled to iron reduction. Nat. Geosci. 5 (8), 538–541.
Yang, Y., Xiao, C., Lu, J., Zhang, Y., 2020. Fe(III)/Fe(II) forwarding a new anammox-like
process to remove high-concentration ammonium using nitrate as terminal electron
acceptor. Water Res. 172, 115528.
Yuan, M., Wang, D., 2019. Effect of metal ions on nitrogen removal efficiency in
anammox sludge. Environ. Pollut. Control 41 (5), 515–525.
Zhang, J., Zhang, Y., Li, Y., Zhang, L., Qiao, S., Yang, F., Quan, X., 2012. Enhancement of
nitrogen removal in a novel anammox reactor packed with Fe electrode. Bioresour.
Technol. 114, 102–108.
Zhang, S., Zhang, L., Yao, H., Rong, H., Li, S., 2021. Responses of anammox process to
elevated Fe(III) stress: reactor performance, microbial community and functional
genes. J. Hazard Mater. 414, 125051.
Zhang, Z.-Z., Xu, J.-J., Shi, Z.-J., Bai, Y.-H., Cheng, Y.-F., Hu, H.-Y., Jin, R.-C., 2017.
Unraveling the impact of nanoscale zero-valent iron on the nitrogen removal
performance and microbial community of anammox sludge. Bioresour. Technol.
(243), 883–892.