Journal of Environmental Management 282 (2021) 111961

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Journal of Environmental Management

journal homepage: http://www.elsevier.com/locate/jenvman

Research article

Nitrogen removal characteristics of a novel heterotrophic nitrification and

aerobic denitrification bacteria, Alcaligenes faecalis strain WT14
Junli Chen a, b, 1, Juan Xu a, c, 1, Shunan Zhang a, *, Feng Liu a, d, Jianwei Peng e, Yingxiang Peng d,
Jinshui Wu a, c
a
Key Laboratory of Agro-ecological Processes in Subtropical Regions, Changsha Research Station for Agricultural & Environmental Monitoring, Institute of Subtropical
Agriculture, Chinese Academy of Sciences, Hunan, 410125, PR China
b
Hunan Jiahe Breeding Intelligence Service Co., Ltd., Hunan, 410199, PR China
c
University of Chinese Academy of Sciences, Beijing, 100049, PR China
d
State Key Laboratory of Heavy Metal Pollution Monitoring for Environmental Protection, Changsha, 410014, PR China
e
College of Resource and Environment, Hunan Agricultural University, Changsha, 410128, PR China

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

Keywords: Alcaligenes faecalis strain WT14 is heterotrophic nitrification and aerobic denitrification bacterium, newly iso­
Alcaligenes faecalis lated from a constructed wetland, and its feasibility in nitrogen removal was investigated. The result showed
Heterotrophic nitrification sodium citrate was more readily utilized by WT14 as a carbon source. The response surface methodology model
High-ammonium wastewater
revealed the highest total nitrogen removal by WT14 occurred at 20.3 ◦ C, 113.5 r⋅min− 1, C/N 10.8, and pH 8.4.
NH+4 -N removal
Under adapted environmental conditions, up to 55.9 mg⋅L− 1⋅h− 1 of ammonium nitrogen (NH+ 4 -N) was removed
Response surface methodology − 1
by WT14, and its NH+ 4 -N tolerance ability reached 2000 mg⋅L . In addition to the reported high NH4 -resistance
+

of Alcaligenes faecalis, WT14 multiplied fast and had strong nitrate or nitrite removal capacity when high strength
nitrate or nitrite was provided as the single nitrogen source; which differed from other Alcaligenes faecalis species.
These results show WT14 is a novel strain of Alcaligenes faecalis and its nitrogen removal pathway will be carried
out in the further study.

1. Introduction
Microbial nitrification/denitrification is one of the most effective
nitrogen removal pathways for wastewater. Generally, the traditional
processes of microbial nitrogen removal include aerobic nitrification
and anaerobic denitrification (Herrero et al., 2015; Zhao et al., 2017).
The nitrification process is dominated by autotrophic nitrifying micro­
organisms. Ammonia oxidizing bacteria convert ammonium to nitrite,
and then nitrite-oxidizing bacteria oxidize nitrite to nitrate (Zhao et al.,
2012). The denitrification process is completed by heterotrophic deni­
trifying microorganisms. They use nitrogen oxides (NO−3 , NO−2 , NO, and
N2O) as electron acceptors to generate energy, and eventually reduce
nitrate into gaseous nitrogen (N2) (Wang et al., 2016). However, because
of the separation of aerobic and anaerobic segments and the long growth
cycle of autotrophic nitrification bacteria, these characteristics eventu­
traditional technology (Zheng et al., 2012). Recently, it has been found
that nitrification and denitrification can be carried out simultaneously in
one system (Zhao et al., 2018), thus overcoming the problems existing in
the traditional process of biological nitrogen removal.
The microorganisms for simultaneous nitrification and denitrifica­
tion are heterotrophic nitrification-aerobic denitrification (HN-AD)
bacteria. Several isolated HN-AD bacteria have been reported, such as
Pannonibacter Phragmitetus (Bai et al., 2019), Paracoccus sp. (Lu et al.,
2019), Acinetobacter sp. (Yao et al., 2013), Enterobacter cloacae (Padhi
et al., 2017), and Klebsiella sp. (Shao et al., 2018). Although these
HN-AD microorganisms can simultaneously perform nitrification and
denitrification, they are mainly used to treat low-strength wastewater
(14–200 mg⋅L− 1) (Bai et al., 2019; Sun et al., 2015). However, Alcali­
genes faecalis, an HN-AD bacterium, is capable of removing high-strength
ammonium nitrogen (NH+ 4 -N). For example, at NH4 -N concentrations of
+

ally lead to a large area, high running cost, and long startup time of 306 and 384 mg⋅L− 1 in wastewater, 99.0 and 100% of NH+ 4 -N, were

* Corresponding author. Changsha Research Station for Agricultural & Environmental Monitoring, Institute of Subtropical Agriculture, Chinese Academy of Sci­
ences, Hunan, 410125, PR China.
E-mail address: zhang-shu-nan@163.com (S. Zhang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.jenvman.2021.111961
Received 25 July 2020; Received in revised form 12 December 2020; Accepted 4 January 2021
Available online 16 January 2021
0301-4797/© 2021 Elsevier Ltd. All rights reserved.
J. Chen et al.
removed by Alcaligenes faecalis strains NR (Zhao et al., 2012) and WY-01
(Wang et al., 2015), respectively. Furthermore, Alcaligenes faecalis C16
and strain No.4 can tolerate and efficiently remove 1200 and 2000
mg⋅L− 1 NH+ 4 -N, respectively (Joo et al., 2006; Liu et al., 2015).
As the HN-AD bacteria, the above Alcaligenes faecalis species can
convert NH+ 4 -N into N2 under aerobic conditions. For Alcaligenes faecalis
No.4, strain NR, and strain WY01, the dominant NH+ 4 -N removal path­
waysare determined using hydroxylamine (NH2OH) (Joo et al., 2005a;
Wang et al., 2015; Zhao et al., 2012). NH+ 4 -N removal by Alcaligenes
faecalis C16 is considered to be the traditional process
(NH+ 4 →NH2OH→NO2 →NO3 , NO3 →NO2 →NO→N2O→N2) (Liu et al.,
− − − −
2015). However, like Alcaligenes faecalis strain NR, WY01, and No.4,
Alcaligenes faecalis C16 cannot grow when nitrate-nitrogen (NO−3 -N) is
utilized as a single nitrogen source (Joo et al., 2005a; Liu et al., 2015;
Zhao et al., 2012), causing the inefficient removal of NO−3 -N in the
treatment of NO−3 -dominated wastewater. Alcaligenes faecalis strain
WT14 (GenBank: MN578054) has recently been isolated from the
sediment of constructed wetlands treating high-strength swine waste­
water (https://www.ncbi.nlm.nih.gov/nuccore/MN578054). It has
been preliminarily confirmed that the bacteria can both thrive with
NH+ 4 -N or NO3 -N as the nitrogen source (Chen et al., 2020a). However,
−
detailed information on the nitrogen removal characteristics of Alcali­
genes faecalis strain WT14 is not yet known.
Therefore, the nitrogen removal characteristic of the novel strain,
WT14, was investigated in this study. The primary objectives were: 1) to
investigate the optimal culture conditions for Alcaligenes faecalis strain
WT14 based on response surface methodology; 2) to identify the fea­
tures of heterotrophic nitrification and aerobic denitrification by WT14;
and 3) to evaluate the potential NH+ 4 -N removal ability of WT14.
2. Materials and methods
2.1. Experimental strain isolation
The sediment sample for strain isolation was collected from a con­
structed wetland vegetated with Myriophyllum aquaticum (28◦ 32′ N,
113◦ 19′ E). The isolation of experimental strains based on the method in
Duan et al. (2015). The sediment sample (fresh weight 10 g) was first
placed into a beaker with 90 mL sterile water. Dilutions in the range of
102‒108 were prepared and inoculated with 100 mL enrichment me­
dium containing 5 g of yeast extract, 5 g of tryptone, 10 g of NaCl in 1 L
of sterile water. The mixtures were incubated at 30 ◦ C and 150 r⋅ min− 1
for 48 h. Following this, the enrichment substances were inculcated in
the solidified heterotrophic nitrification medium at 30 C for 48 h under
◦
aerobic conditions. The heterotrophic nitrification medium consisted of
the following components in 1 L sterile water: 0.472 g of (NH4)2SO4,
4.084 g of C6H5Na3O7, 0.2 g of K2HPO4, 0.05 g of MgSO4⋅7H2O, 0.01 g of
MnSO4⋅4H2O, 0.12 g of NaCl, 0.01g of FeSO4, at a pH of 7.0 (Wang et al.,
2016). To purify the strain, the isolated bacterium was repeatedly
cultivated in the heterotrophic nitrification medium at 30 ◦ C and 150
r⋅min− 1. The isolated bacterium was named strain WT14 for this
following study.
2.2. Identification of microorganism via gene sequence analysis
Amplification of the 16S rDNA gene of colonies was performed at the
species level. Bacterial DNA was amplified via polymerase chain reac­
tion using primers 27f (50-AGAGTTTGATCCTGGCTCAG-30) and 1492r
(50-GGTTACCTTGTTACGACTT-30). The polymerase chain reaction was
carried out under the following conditions: Initial denaturation at 95 ◦ C
for 5 min; 35 cycles of 95 ◦ C for 40 s, 51 ◦ C for 40 s, and 72 ◦ C for 90 s;
final extension at 72 ◦ C for 10 min (Duan et al., 2015). Comparisons of
obtained 16S rDNA sequences with sequences in GenBank were con­
ducted using BLAST. Then, the phylogenetic tree of strain was built
following the adjacency method using MEGA X.
2
Journal of Environmental Management 282 (2021) 111961
2.3. Utilization of carbon substrates
Sodium citrate (C6H5Na3O7), sodium acetate (CH3COONa), glucose
(C6H12O6), sodium succinate (C4H4Na2O4), and sucrose (C12H22O11)
were provided as single carbon sources in the heterotrophic nitrification
medium to assess the utilization of different carbon sources by strain
WT14. When C6H5Na3O7, CH3COONa, C6H12O6, C4H4Na2O4, and
C12H22O11 were added the heterotrophic nitrification medium respec­
tively, the C/N ratio was adjusted to 10 and NH+ 4 -N concentration was
100 mg⋅L− 1. Organisms were cultured at 30 ◦ C and 150 r⋅min− 1 in 250-
mL shaker flasks containing 100 mL medium and 2.5 mL enrichment
culture of strain WT14 for 24 h. 10 mL of medium samples were taken
every 4 h to examine the changes in bacterial growth (OD600) and NH+ 4-
N concentration.
2.4. Optimization of environmental factors by Box-Behnken design
The response surface methodology was used to investigate the im­
pacts of initial temperature, pH, shaking speed, and the ratio of C/N on
the NH+4 -N removal ability of WT14. The shaking speed was set at 30, 90,
and 150 r⋅min− 1 to produce the DO concentrations of 1.31, 4.32, and
6.10 mg⋅L− 1, respectively (Lei et al., 2019; Xia et al., 2020). The het­
erotrophic nitrification medium contained 200 mg⋅L− 1 NH+ 4 -N. K2HPO4
and KH2PO4 were used to adjust the pH and C6H5Na3O7 was used to
adjust the ratio of C/N in heterotrophic nitrification medium. A
four-factor-three-level experiment was defined using the Box-Behnken
design (Table S1). According to the Box-Behnken design, 29 experi­
ments were performed in 250-mL shaker flasks including 100 mL me­
dium and 2.5 mL WT14 enrichment culture for 48 h. In order to subtract
the ammonia volatilized from the 29 experiments, the control treat­
ments without adding bacteria solution were set up simultaneously.
Both experimental and control samples were collected at 0 and 48 h to
detect total nitrogen (TN) change and calculate the TN removal rate.
2.5. Heterotrophic nitrification and aerobic denitrification by WT14 and
its NH+
4 -N removal capability
To clarify nitrogen removal performance by WT14, four nitrogen
sources of NH+ 4 -N, NO3 -N, nitrite nitrogen (NO2 -N), and NH2OH with
− −
concentrations of 100 mg⋅L− 1 were added in heterotrophic nitrification
medium, respectively. When the removal abilities of NO−2 -N, NO−3 -N,
and NH2OH were verified, NH+ 4 -N in heterotrophic nitrification medium
was replaced by NO−2 -N, NO−3 -N, and NH2OH, respectively. Meanwhile,
the ratio of C/N in heterotrophic nitrification medium was adjusted to
10 through the addition of citrate as the carbon source.
To explore the influence of different NH+ 4 -N concentrations on strain
WT14, the initial NH+ 4 -N concentration of the heterotrophic nitrification
medium was adjusted to 100, 400, 700, 1000, 1300, 1600, and 2000
mg⋅L− 1. C6H5Na3O7 was added in the heterotrophic nitrification me­
dium to adjust the C/N ratio to 10. The experimental culture conditions
were the same as described in section 2.3. Precisely 10 mL of medium
was removed after 0, 12, 24, 36, 48, and 60 h to examine the changes in
NH+4 -N, NO2 -N, NO3 -N, and NH2OH concentrations.
− −
2.6. Quality assurance and quality control
Three blank treatments were set for each batch of test. The NH+ 4 -N,
NO−2 -N,NO−3 -N, and NH2OH concentrations in the blank samples were
below the test line. When the four-factor-three-level experiment was
defined using the Box-Behnken design (Table S1), the control treatments
(no inoculation of screened strains) were set simultaneously, and the
recoveries of TN in the control samples were 89.9–99.8% (n = 87),
respectively.
J. Chen et al. Journal of Environmental Management 282 (2021) 111961

2.7. Analytical methods and statistical analysis of Alcaligenes faecalis. The results highlighted the isolated strain WT14
should be identified as the Alcaligenes faecalis specie.
Bacterial growth was monitored at 600 nm (OD600) using a spec­
trophotometer (Eppendorf Bio Photometer® D30, Germany). NH+ 4 -N,
NO−2 -N, and NO−3 -N concentrations were determined through a fully 4 -N removal by strain WT14
3.2. Effect of carbon sources on NH+
automated flow-injection system (AA3, SEAL Analytical, Norderstedt,
Germany). NH+ 4 -N concentration in the medium was monitored using Different carbon sources showed varying effects in influencing the
salicylic acid spectrophotometry, NO−2 -N was analyzed via diazo growth rate and denitrification of WT14. From Fig. 2, low OD600 values
coupling spectrophotometry, and NO−3 -N was determined using a (0.06–0.21) and NHþ 4 -N removal efficiencies (1.3–2.4%) were observed
continuous flow analyzer. NH2OH was analyzed by the method in Frear when C6H12O6 or C12H22O11 was as the sole carbon source. The result
and Burrell (1955). was different from other studies which reported that C6H12O6 can be
Results were represented as the mean ± SD. One-way ANOVA was made available by many heterotrophic nitrification bacteria, such as
used to analyze the data using Origin 8.0 software. To understand the
interaction effect of the four environmental factors on TN removal rate,
response surfaces (3D) and corresponding contours (2D) were plotted by
Design-Expert 11 software (Stat-Ease, Inc., USA) (Liu et al., 2018).
Before fitting the quadratic model, the normal distribution of variables
was checked through a one-sample Kolmogorov–Smirnov test.
The removal efficiency (%) and removal rate (mg⋅L− 1⋅h− 1) were
computed using Equations (1) and (2) (Rout et al., 2017):
Removal efficiency = (Ca − Cb )/Ca × 100 (1)

Removal rate = (Ca − Cb )/(tb − ta ) (2)

Where, Ca and Cb (mg⋅L− 1) are NH+ 4 -N, NO2 -N, and NO3 -N concentra­
− −

tions at time ta and tb, respectively; ta (h) is the initial time of the
experiment, and tb (h) is the end time of the experiment.

3. Results and discussion

3.1. Identification of the study strain

Through enrichment, scribing separation, purification, and

screening, Alcaligenes faecalis strain WT14 was successfully isolated from
the sediment of the three-stage surface flow constructed wetlands,
which were used for swine wastewater treatment (Luo et al., 2018).
Colonies of strain WT14 on the heterotrophic nitrification medium were
pale yellow, irregular in shape, with lobed edges and colony diameters
of 2–3 mm; gram staining was negative, and cells were rod-shaped with
sizes of 0.4–0.5 μm × 1.5–1.6 μm. The 1000 bp 16S rDNA fragment of
WT14 was sequenced. BLAST alignment analysis showed that WT14 had
the highest similarity (99%) with the Alcaligenes faecalis strain JBW4 Fig. 2. The (a) growth characteristic and (b) NH+ 4 -N removal performance of
(Fig. 1). According to the 16S rDNA sequence, a phylogenetic tree was strain WT14 in the presence of different carbon source. Values represent the
constructed, indicating that strain WT14 was clustered in the members mean ± SD (error bars) (n = 3). Values in Figs. 2 and 5 are the same meaning.

Fig. 1. Phylogenetic tree based on 16S rDNA sequence data of strain WT14 and other related strains.

3
J. Chen et al. Journal of Environmental Management 282 (2021) 111961

Halophilic Vibrio diabolicus SF16 (Duan et al., 2015), Alcaligenes faecalis
strain NR (Zhao et al., 2017), and Agrobacterium sp. LAD9 (Chen and Ni,
2012). It is related to the types of heterotrophic nitrifying bacteria,
because different heterotrophic bacteria prefer using different types of
carbon sources (Zhao et al., 2017). C6H5Na3O7, C4H4Na2O4, or
CH3COONa used as carbon source contributed to high OD600 values
(~0.75) and >80% NHþ 4 -N removal efficiencies after the cultivation for
24 h. Similarly, Alcaligenes faecalis strain NR and Vibrio diabolicus SF16
had high NH+ 4 -N removal efficiencies of 91.8% and 92.2% when
C4H4Na2O4 and C6H5Na3O7 were provided as the sole carbon source
(Duan et al., 2015; Zhao et al., 2017; Zheng et al., 2012). C6H5Na3O7
was selected as the optimal carbon source for strain WT14 growth,
achieving the highest removal efficiency of 98% compared to
C4H4Na2O4 and CH3COONa. The reason could be related to the simple
molecular structure of C6H5Na3O7. Citrate ion in C6H5Na3O7 can
directly enter the tricarboxylic acid cycle of bacteria, which is more
conducive to heterotrophic nitrification and aerobic denitrification.
Similar phenomena can be observed in Acinetobacter juniiYB (Ren et al.,
2014), Acinetobacter sp. T1 (Chen et al., 2019), and Acinetobacter sp.
ND7 (Xia et al., 2020), which had an NH+ 4 -N removal efficiency of
92.2–99.0% when C6H5Na3O7 was used as the carbon source.
3.3. Optimal environmental factors for nitrogen removal by strain WT14
In order to assess the effect of temperature, shaking speed, C/N, and
pH on TN removal, a quadratic model reflecting the correlation between
the four environmental factors and TN removal of strain WT14 was
established:
bacteria was mainly regulated by DO (Zhao et al., 2017), pH (Qu et al.,
2015), C/N (Ye et al., 2016), and temperature (Yao et al., 2013).
Furthermore, the predicted results indicated that the optimum condi­
tions for nitrogen removal by strain WT14 were 20.1 ◦ C, 104.4 r⋅min− 1,
C/N 14.7, and pH 7.5, and the TN removal rate was 4.13 mg L− 1⋅h− 1
under these conditions. The TN removal rate in the verification test was
4.11 mg⋅L− 1⋅h− 1 (20 ◦ C, 104 r⋅min− 1, C/N 14.7, and pH 7.5), indicating
that the model fitted the experimental data well. The optimum pH and
shaking speed for strain WT14 were consistent with those of the Han­
seniasporauvarum strain KPL108 (pH 8.2, shaking speed 109.7 r⋅min− 1),
yet the temperature and C/N ratio differed from those of the Hanse­
niaspora uvarum strain KPL108 (Zhang et al., 2018) and Pseudomonas
stutzeri strain ZF31 (Huang et al., 2015), for which the optimum tem­
perature and C/N ratio were 28–28.5 ◦ C and 6.4–6.7 for maximum TN
removal.
The response surface methodology model was used to investigate the
interactive effects of pH, temperature, C/N, and shaking speed on TN
removal (Ye et al., 2016), which was visually marked by response sur­
faces and corresponding contours (Fig. 3). As shown in Table 1, the
interaction effects of temperature and pH on TN removal rate were
significant (p = 0.0006). TN removal rate increased significantly with
neutral pH, while a negative effect between TN removal and the tem­
perature was observed (p < 0.001) (Fig. 3a and b). Compared with pH,
temperature had a more significant effect on TN removal rate (p <
0.0089) (Table 1). Zhang et al. (2018) also found a similar result. TN
removal rate increased significantly with the improvement of C/N and
shaking speed (p = 0.035) (Fig. 3c and d). However, the interactions of
pH and C/N with TN removal were not significant (p = 0.094). A
different result was observed in another study, which reported that TN

Y = 3.56 − 0.319147 ∗ A + 0.85 ∗ B + 0.58 ∗ C − 0.04 ∗ D − 0.41 ∗ AB − 0.40 ∗ AC − 0.80 ∗ AD+

(3)
0.42∗BC + 0.30 ∗ BD + 0.33 ∗ CD − 0.49∗A2 − 0.63∗B2 − 0.61∗C2 − 1.05∗D2

Where Y is TN removal rate (mg⋅L− 1⋅h− 1); A, B, C, and D are the coded
values of temperature, shaking speed, C/N, and pH, respectively.
The quadratic models showed pH, C/N, temperature, and shaking
speed all had a significant effect on TN removal (p < 0.001) (Table 1).
The importance of environmental factors for TN removal rate was as
follows: Shaking speed > C/N > temperature > pH. This result was
similar to other studies which reported that TN removal rate by HN-AD
removal rate of Corynebacterium pollutisoli SPH6 was significantly
affected by the interaction between pH and C/N (Liu et al., 2018). These
results suggested that the optimum environmental conditions varied
with the microbial species.
3.4. Heterotrophic nitrification and aerobic denitrification by WT14

To clarify the nitrogen removal characteristics of WT14, NH2OH,

Table 1
NO−2 -N, NO−3 -N, or NH+ 4 -N was provided for WT14 as sole nitrogen
Analysis of variance (ANOVA) for quadratic models.
sources. When NH2OH was added to the medium alone, strain WT14 did
Source Sum of Squares df Mean F- p-value not grow normally (Supplementary Material) and no decrease in NH2OH
Square value
concentration was observed (Fig. 4a). No utilization of NH2OH by WT14
Model 28.83 14 2.06 15.56 <0.0001** for growth was similar to that of Alcaligenes faecalis strains NR, WY-01,
A-Temperature 1.22 1 1.22 9.236 0.0089** and No.4; however, unlike the inefficient NH2OH removal by WT14,
B-Shaking speed 8.69 1 8.69 65.63
NH2OH can be removed by Alcaligenes faecalis strain WY-01, NR, and
<0.0001**
C–C/N 4.09 1 4.099 30.91 <0.0001**
D-pH 0.02 1 0.021 0.156 0.699 No.4 (Joo et al., 2005a; Wang et al., 2015; Zhao et al., 2012).
AB 0.66 1 0.66 4.99 0.042* When a high concentration (100 mg⋅L− 1) of NO−2 -N was provided,
AC 0.65 1 0.65 4.93 0.043* the rapid growth of WT14 was observed compared to the control
AD 2.57 1 2.57 19.41 0.0006**
(Supplementary Material). The result was different from that of other
BC 0.72 1 0.72 5.43 0.035*
BD 0.36 1 0.36 2.70 0.122 HN-AD bacteria. For example, 0.05 mg⋅L− 1 free nitrous acid (HNO−2 ) in
CD 0.43 1 0.43 3.23 0.094 wastewater causes a remarkable inhibition of the growth of Acineto­
A2 1.56 1 1.56 11.76 0.004** bacter sp. (Weon et al., 2002). Other Alcaligenes faecalis species (strain
B2 2.59 1 2.599 19.6 0.00057** NR, No.4, and WY-01) cannot utilize NO−2 -N for growth (Joo et al.,
C2 2.44 1 2.44 18.40 0.0007**
D2 7.12 1 7.12 53.76 <0.001**
2005a; Wang et al., 2015; Zhao et al., 2012). Generally, the existence of
Residual 1.85 14 0.13 NO−2 -N is toxic to microorganisms, especially at high concentrations
Lack of Fit 1.72 10 0.17 5.20 0.063 (Zhou et al., 2011). However, strain WT14 isolated in this study not only
Pure Error 0.132 4 0.03 proliferated in large numbers but also removed NO−2 -N rapidly even at
Cor Total 30.69 28
NO−2 -N concentrations of up to 100 mg⋅L− 1. Fig. 4b shows that almost all
* and ** represent p values of <0.05 and <0.01, respectively. NO−2 -N was removed within 24 h, faster than NH+ 4 -N and NO3 -N. This
−

4
J. Chen et al. Journal of Environmental Management 282 (2021) 111961

Fig. 3. Response surface (3D) and corresponding contour (2D) plots for TN removal rate by strain WT14: (a, b) TN removal rate as a function of pH and temperature,
(c, d) TN removal rate as a function of C/N and shaking speed, and (e, f) TN removal rate as a function of pH and C/N.

5
J. Chen et al. Journal of Environmental Management 282 (2021) 111961

Fig. 4. Nitrogen concentrations and removal efficiencies by strain WT14 with time:(a) NH2OH,(b) NO−2 -N, (c) NO−3 -N), and (d) NH+
4 -N.

may be due to microbial self-protection through the conversion of

NO−2 -N into NO−3 -N to eliminate the toxicity of NO−2 -N (Chen et al.,
2016).
Similar to NO−2 -N, strain WT14 can either utilize NO−3 -N for growth
or remove it quickly (Fig. 4c). Although previous studies reported that
Alcaligenes faecalis strain NR, No.4, and WY-01 can convert NH+ 4 -N into
gaseous nitrogen, they cannot grow normally or effectively remove
NO−3 -N when it is provided as a nitrogen source (Joo et al., 2005a; Wang
et al., 2015; Zhao et al., 2012). Our results indicated that WT14 might be
a different strain from the previously reported Alcaligenes faecalis spe­
cies. In addition, a previous study showed that a NO−3 -N concentration of
30 mg⋅L− 1 would hinder the growth of denitrifying microorganisms and
therefore inhibit denitrification (Glass et al., 1997). However, at a
NO−3 -N concentration of 100 mg⋅L− 1, the NO−3 -N removal rate of WT14
reached 6.96 mg⋅L− 1⋅h− 1, higher than reported values for other HN-AD
bacteria, such as 1.60 mg⋅L− 1⋅h− 1 for Pseudomonas putida Y-9 (Xu et al.,
2017), 1.46 mg⋅L− 1⋅h− 1 for Marinobacter sp. F6 (Zheng et al., 2012), and
2.69 mg⋅L− 1⋅h− 1 for Bacillus cereus GS-5 (Rout et al., 2017). Like other
HN-AD bacteria, WT14 efficiently removed NH+ 4 -N (Fig. 4d), which is
detailed in section 3.5. These results indicated that strain WT14 was a
highly effective type of HN-AD bacteria.

3.5. The potential NH+

4 -N removal ability of strain WT14

The effect of NH+ 4 -N concentration on the nitrogen removal capacity

was investigated in heterotrophic nitrification medium. The results
indicated that strain WT14 showed efficient NH+ 4 -N removal (Fig. 5a),
− 1 Fig. 5. Nitrogen removal performances of strain WT14 at different initial NH+
4-
and it withstood NH+ 4 -N concentrations of up to 2000 mg⋅L (perhaps
higher), which was higher than other HN-AD bacteria, such as 1200 N concentrations: (a) NH+
4 -N, (b) NO3 -N, and (c) NO2 -N.
− −

mg⋅L− 1 for Alcaligenes faecalis No.4 (Joo et al., 2005b), 800 mg⋅L− 1 for
Alcaligenes faecalis C16 (Liu et al., 2015), and 800 mg⋅L− 1 for Alcaligenes phenomenon might be related to the utilization of carbon sources. Wang
sp. TB (Chen et al., 2016). NH+ 4 -N was removed by almost 100% at a et al. (2016) reported that the carbon source was more likely to be
concentration of 100–400 mg⋅L− 1, more than 70% at 700–1600 mg⋅L− 1 insufficient for microbial utilization under high nitrogen loads than low
NH+ 4 -N, and 2000 mg⋅L
− 1
NH+
4 -N was removed by more than 43%. The nitrogen loads.

6
J. Chen et al. Journal of Environmental Management 282 (2021) 111961

Although NH+ 4 -N removal efficiency decreased with the increasing Chen, J., Gu, S., Hao, H., Chen, J., 2016. Characteristics and metabolic pathway of
Alcaligenes sp. TB for simultaneous heterotrophic nitrification-aerobic
initial NH+4 -N concentration, strain WT14 had a higher NH4 -N removal
+
denitrification. Appl. Microbiol. Biotechnol. 100 (22), 9787–9794.
efficiency compared with other HN-AD bacteria. For example, Pseudo­ Chen, S., He, S., Wu, C., Du, D., 2019. Characteristics of heterotrophic nitrification and
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Junli Chen: Conceptualization, Writing-Original draft preparation. Padhi, S.K., Tripathy, S., Mohanty, S., Maiti, N.K., 2017. Aerobic and heterotrophic
Juan Xu: Methodology, Data curation. Shunan Zhang: Writing- nitrogen removal by Enterobacter cloacae CF-S27 with efficient utilization of
Reviewing and Editing. Feng Liu: Investigation. Jianwei Peng: Super­ hydroxylamine. Bioresour. Technol. 232, 285–296.
Qu, D., Wang, C., Wang, Y., Zhou, R., Ren, H., 2015. Heterotrophic nitrification and
vision. Runlin Xiao: Project administration. Yingxing Peng: Visualiza­
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tion. Jinshui Wu: Funding acquisition. temperatures. RSC Adv. 5 (7), 5149–5157.
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Declaration of competing interest (1–9).
Rout, P.R., Bhunia, P., Dash, R.R., 2017. Simultaneous removal of nitrogen and
The authors declare that they have no known competing financial phosphorous from domestic wastewater using Bacillus cereus GS-5 strain exhibiting
heterotrophic nitrification, aerobic denitrification and denitrifying phosphorous
interests or personal relationships that could have appeared to influence removal. Bioresour. Technol. 244 (Pt 1), 484–495.
the work reported in this paper. Shao, S., Hu, Y., Cheng, C., Cheng, J., Chen, Y., 2018. Simultaneous degradation of
tetracycline and denitrification by a novel bacterium, Klebsiella sp. SQY5.
Chemosphere. 209, 35–43.
Acknowledgments Sun, Y., Li, A., Zhang, X., Ma, F., 2015. Regulation of dissolved oxygen from accumulated
nitrite during the heterotrophic nitrification and aerobic denitrification of
This study was financially supported by the Strategic Priority Pseudomonas stutzeri T13. Appl. Microbiol. Biotechnol. 99 (7), 3243–3248.
Wang, Y., Chen, H., Liu, Y.-X., Ren, R.-P., Lv, Y.-K., 2015. Effect of temperature, salinity,
Research Program of the Chinese Academy of Sciences (XDA23020402 heavy metals, ammonium concentration, pH and dissolved oxygen on ammonium
and XDA23020502). removal by an aerobic nitrifier. RSC Adv. 5 (97), 79988–79996.
Wang, T., Dang, Q., Liu, C., Yan, J., Fan, B., Cha, D., Yin, Y., Zhang, Y., 2016.
Heterotrophic nitrogen removal by a newly-isolated alkalitolerant microorganism,
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Xia, L., Li, X., Fan, W., Wang, J., 2020. Heterotrophic nitrification and aerobic
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