Bioresource Technology 322 (2021) 124555

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Nitrogen removal by heterotrophic nitrifying and aerobic denitrifying

bacterium Pseudomonas sp. DM02: Removal performance, mechanism and
immobilized application for real aquaculture wastewater treatment
Min Deng a, Xiaoli Zhao b, Yeerken Senbati a, c, Kang Song a, c, *, Xugang He d
a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
b
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China
c
University of Chinese Academy of Sciences, Beijing 100049, China
d
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China

H I G H L I G H T S

• Strain DM02 efficiently removes TAN, nitrate, nitrite under high oxygen environment.
• Diverse organic carbon can be used by strain DM02 for NO−3 -N removal (>95%).
• NO−3 and NO−2 removal efficiency was linearly related to C/N ratio (range 0–10).
• The contribution of N assimilation for N removal increased linearly with C/N ratio.
• Immobilized technology has been used for real aquaculture wastewater treatment.

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

Keywords: A bacterial strain was isolated and identified as Pseudomonas sp. DM02 from an aquaculture system. Strain DM02
Nitrogen removal showed efficient heterotrophic nitrification-aerobic denitrification capability. Total ammonia nitrogen (TAN, 10
Pseudomonas mg/L) could be completely removed by strain DM02 within 12 h under low nutrient condition. Nitrogen mass
Heterotrophic nitrification-aerobic denitrifica­
balance indicated that 70.8% of the initial TAN was translated into gaseous nitrogen and 28.1% was converted
tion
napA
into intracellular nitrogen. Various carbon sources can be used for nitrate removal (>95% within 28 h). The
Immobilized carrier optimal conditions for TAN, nitrate and nitrite removal were pH 7 with carbon/nitrogen (C/N) ratios of 8, 12 and
12, respectively. The napA, nirK, and nosZ functional genes were successful amplified from strain DM02. Both
bioaugmentation and immobilized technology of strain DM02 present ability (>88%) for continuous treatment of
real aquaculture wastewater. This research indicated a great potential for practical application of Pseudomonas
sp. DM02 in aquaculture wastewater treatment.

1. Introduction
Inland freshwater aquaculture produces 64.2% (51.4 million metric
tons) of the world’s farmed food fish in 2016 and the yield has been
raising continuously (FAO, 2018). Nitrogenous compounds are the
major pollutants in aquaculture system and can cause eutrophication of
adjacent water and affect human health (Huang et al., 2017). Protein
rich commercial feed (usually >30%) is the mainly nitrogen source in
aquaculture system. Generally, 1–2 kg of dry feeds are consumed to
produce 1 kg of live food fish (Huang et al., 2020a). However, only about
25% of feed nitrogen can be converted into fish biomass (Hu et al.,
2012). The un-utilized feed nitrogen is excreted to the water as
ammonia, feces, and residual feeds. Meanwhile, organic nitrogen in
feces and residual feeds can also be transformed into ammonia through
microbial metabolism. The un-ionized ammonia (NH3) is toxic to
aquatic animals (Randall and Tsui, 2002) and needs to be effectively and
economically removed from aquaculture system.
Bioremediation is a high-efficient and low-cost technology for

* Corresponding author at: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan
430072, China.
E-mail address: sk@ihb.ac.cn (K. Song).

https://doi.org/10.1016/j.biortech.2020.124555
Received 21 October 2020; Received in revised form 10 December 2020; Accepted 13 December 2020
Available online 16 December 2020
0960-8524/© 2020 Elsevier Ltd. All rights reserved.
M. Deng et al.
wastewater treatment. A series of microorganisms can help remove
nitrogenous compounds from the wastewater treatment plant (WWTP).
Conventionally, total ammonia nitrogen (TAN) can be transformed into
nitrite (NO−2 -N) by ammonia-oxidizing bacteria (AOB), and further
converted to nitrate (NO−3 -N) by nitrite-oxidizing bacteria (NOB). Then,
both NO−2 -N and NO−3 -N can be translated to nitrogen gas and removed
from the system by denitrifying bacteria (Zheng et al., 2018). AOB and
NOB are autotrophic aerobic bacteria while denitrifiers are heterotro­
phic anaerobic bacteria. The distinct difference in physiological and
biochemical properties (i.e. aerobic vs. anaerobic and autotrophic vs.
heterotrophic) increases the complexity of WWTP, the use of land and
operational costs. Recently, heterotrophic nitrification-aerobic denitri­
fication (HN-AD) bacteria have received wide attention because of their
ability to realize nitrification and denitrification simultaneously in one
system (Xia et al., 2020; Yang et al., 2019a, 2019b). Compared with
traditional autotrophic nitrifying and anaerobic denitrifying bacteria,
HN-AD bacteria show many advantages. On one hand, HN-AD bacteria
can reach stationary phase with more than 90% nitrogen removal effi­
ciency in 24 h, but an extended period (weeks) is required to establish a
complete nitrification process (Luo et al., 2020). On the other hand, the
dissolved oxygen (DO) concentration in aquaculture system is usually
higher than 5 mg/L (Wen et al., 2019). But HN-AD bacteria have high
denitrification efficiency and is not limited by oxygen. Recently, most of
reported HN-AD bacteria are applied to treat synthetic wastewater with
high nitrogen concentration (≥50 mg/L) (Lei et al., 2019; Li et al., 2015;
Ma et al., 2015; Sun et al., 2016), which may be unsuitable for treating
the oligotrophic aquaculture wastewater (Huang et al., 2017).
Lately, HN-AD bacteria have been applied in mariculture system in
some researches. Huang et al. (2017) and Liu et al. (2019) have isolated
two different HN-AD strains and assessed their ability for HN-AD. In
simulative aquaculture wastewater treatment system, the effects of
environmental conditions on heterotrophic nitrification have been well
studied (Huang et al., 2017; Liu et al., 2019). However, little is known
about the relationship between environmental factors and aerobic
denitrification performance of HN-AD bacteria for aquaculture waste­
water treatment. Huang et al. (2020a) and Huang et al. (2020b) have
isolated twenty-five HN-AD strains and studied their cooperation in TAN
and NO−2 -N removal in mariculture system. Compared to these studies in
mariculture system, few study on HN-AD bacteria have been reported in
freshwater aquaculture system. Although many HN-AD strains,
including Acinetobacter sp. JR1 (Yang et al., 2019a), Acinetobacter sp.
ND7 (Xia et al., 2020), Pseudomonas putida strain NP5 (Yang et al.,
2019b), and Pseudomonas stutzeri YG-24 (Li et al., 2015), have been
isolated from freshwater environments (such as WWTP and lakes), these
strains have been used for high ammonia wastewater (approximately
100 mg/L) treatment, which is significantly higher than that of aqua­
culture effluent (TAN < 15 mg/L) (Huang et al., 2017). In addition,
microbial immobilized technology can protect the bacteria from being
washed out of the WWTP and has been used widely (Ma et al., 2015).
However, there are few research on aquaculture wastewater treatment
using HN-AD bacterial immobilized carriers.
This study aims to investigate the nitrogen removal performance of
culturable HN-AD bacteria isolated from biofloc of a biofloc-based
recirculating aquaculture system (Deng et al., 2020). Nitrogen mass
balance was used to assess TAN removal potential through assimilation
and HN-AD process. The influences of different environmental factors on
TAN, NO−3 -N and NO−2 -N removal were also detected to improve its
application on aquaculture wastewater treatment. The polymerase chain
reaction (PCR) amplification of functional genes was performed to
identify aerobic denitrification mechanism of isolated HN-AD bacte­
rium. Bioaugmentation and immobilized technology have been con­
ducted to assess the performance and feasibility of continuous real
aquaculture wastewater treatment by HN-AD bacterium.
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Bioresource Technology 322 (2021) 124555
2. Materials and methods
2.1. Culture medium preparation
The low nutrient enrichment medium (LNEM) used for bacteria
enrichment was based on Su et al. (2015) with some modifications. The
LNEM (g/L) contained: 0.1 CH3COONa, 0.02 NH4Cl, 0.02 KH2PO4, 0.01
MgSO4⋅7H2O, 0.013 CaCl2⋅2H2O and 2.0 mL of trace element solution.
The solid LNEM was prepared for bacteria isolation with addition of 2%
agar (w/v). The basic medium (BM) was similar to LNEM except that 0.5
g/L glucose and 0.038 g/L NH4Cl were used as sole organic carbon (OC)
source and nitrogen source, respectively. Based on components of BM,
OC source and nitrogen source were modified for various experiment
requirements. The trace element solution was the same as previous
report (Su et al., 2015). Initial pH value of above mediums was adjusted
to 7.0 and autoclaving was performed at 121 ◦ C for 30 min.
2.2. Isolation and identification
Biofloc sample was collected from a mature biofloc reactor of a
recirculating aquaculture system (Deng et al., 2020) in Wuhan, China
and was utilized to isolate HN-AD bacteria. Fifteen milliliter of biofloc
sample (including water) together with 800 mL autoclaved LNEM me­
dium (pH 7.0) were added into 1000 mL conical flask and incubated on a
rotary shaker (160 rpm) at 30 ◦ C. The supernatant was drained and the
fresh medium was replaced when the TAN removal efficiency was
higher than 90%. This enrichment process was repeated for four times.
The suspension was streaked on LNEM agar plate (Xia et al., 2020) and
twenty-one strains were isolated by purification (Huang et al., 2017).
The nitrogen removal performances of obtained strains were tested
separately using BM with different nitrogen source (TAN or NO−3 -N). A
strain with remarkable nitrogen removal capability was selected for
further assays. Strain DM02, which has both high TAN and NO−3 -N
removal capability, was chosen for further analyses. Strain DM02 added
with 30% glycerol solution was transferred to − 20 ◦ C for long-term
storage.
Qiagen DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) was used
to extract and purify the genomic DNA of strain DM02. The 16S rDNA
was sent to Tianyi Huiyuan biotech company (wuhan, China) for
sequencing (Xia et al., 2020). The homology comparison was carried out
using an online BLAST program (http://www.ncbi.nlm.nih.gov/blast/
Blast.cgi) (Liu et al., 2019). The neighbor-joining method was used to
build a phylogenetic tree (MEGA X, Version 10.1.7) (Xia et al., 2020).
2.3. Fate of TAN utilized by strain DM02
The stored strain DM02 was firstly cultured in Luria-Bertani (LB)
medium (Xia et al., 2020) for 12 h to recover cell activity. Then, the seed
solution was prepared by inoculating 1% (v/v) strain DM02 suspension
into BM (10 mg/L TAN, C/N = 8) for 24 h to reduce nitrogen nutrients
input from seed solution. To investigate the fate of TAN utilized by strain
DM02, 1 mL seed solution was inoculated into three 250-mL flasks with
100-mL BM (10 mg/L TAN, C/N = 8). All the flasks in the present study
were sealed with permeable membranes and cultivated aerobically (160
rpm, 30 ◦ C) unless otherwise stated. Samples were withdrawn periodi­
cally for the measurement of TAN, NO−2 -N, NO−3 -N and total nitrogen
(TN). Cell growth was measured by optical density (OD600) (Xia et al.,
2020). Nitrogen mass balance were calculated to analyze pathway of
TAN utilization. All the determinations were performed in triplicate.
2.4. Effect of environmental factors on HN-AD performance of strain
DM02
The nitrogen removal performance of strain DM02 under different
carbon source, pH and C/N ratio conditions was investigated. Single
factor batch experiment was conducted by using the BM with some
M. Deng et al.
modifications. For carbon source experiment, sucrose, glucose, triso­
dium citrate, sodium succinate, ethanol and sodium acetate were
respectively used as sole OC source, while NO−3 -N (20 mg/L) was used as
sole nitrogen source (C/N = 20). The effect of different pH on TAN
(NH4Cl, 10 mg/L), NO−3 -N (KNO3, 20 mg/L), and NO−2 -N (NaNO2, 5 mg/
L) removal performance was investigated using pH buffer. Na2HPO4 (1/
15 mol/L) and KH2PO4 (1/15 mol/L) was added to the BM to adjust
initial pH to 5, 6, 7, and 8. In addition, 5 M NaOH solution together with
the pH buffer were used to adjust initial pH to 9. For C/N ratio experi­
ments, C/N ratios of 0, 4, 8, 12, 16, and 20 were used for heterotrophic
nitrification (10 mg/L TAN) and C/N ratios of 0, 2, 4, 6, 8, 10, 12, and 14
were used for aerobic denitrification (10 mg/L NO−3 -N and 5 mg/L NO−2 -
N as sole nitrogen source, respectively).
For inoculation, 1% (v/v) strain DM02 LB suspension was cultivated
in 100 mL BM medium using different nitrogen source for 24 h. Then,
these pre-cultured strain DM02 was inoculated (v/v 1%) in 100 mL
sterile medium based on the nitrogen source used in the medium. All
experiments were carried out in triplicate. Samples were collected
periodically to measure the concentrations of TAN, NO−3 -N and NO−2 -N
and the cell density (OD600).
2.5. PCR amplification of denitrification genes
Amplification of denitrification related genes (napA, nirK, nirS, and
nosZ) was conducted using the genomic DNA. All primers and their
amplification conditions were based on previous research (Deng et al.,
2020).
2.6. Semi-continuous treatment
Real aquaculture wastewater from an indoor fish tank in Wuhan,
China (N 30◦ 32′ 48′′ , E 114◦ 21′ 0′′ ) was collected to explore the appli­
cation potential of strain DM02. The aquaculture wastewater was
filtered by 0.45 µm filter and autoclaved at 121 ◦ C before using. The
sodium alginate-kaolin (SAK) carrier was prepared according to Yu et al.
(2020) with some modifications. Nine gram of sodium alginate together
with 2.5 g kaolin powder were dissolved in 150 mL boiling hot water
and then cooled down to room temperature. Ten milliliter of strain
DM02 pre-cultured LB solution was centrifuged (4000 rpm, 5 min) and
washed by sterile water for three times. The cell ball was resuspended by
1 mL sterile water and mixed with the SAK solution. The mixture was
made into gel as reported by Yu et al. (2020) and cutted into 1 cm cubes.
The set-up of three-cycles batch experiments was shown in Table 1.
For bioaugmentation treatment (DM02 and DM02 + OC groups), the
DM02 clone was pre-cultured in 100 mL autoclaved aquaculture
wastewater added with sterile glucose solution (C/N = 10). One percent
(v/v) pre-cultured solution (OD600 = 0.722 ± 0.080) was added to 100
mL aquaculture wastewater and marked as DM02 group. The waste­
water added with pre-cultured strain DM02 solution (1% v/v) and 2 mL
glucose solution (C/N = 10) was marked as DM02 + OC. For immobi­
lized carrier treatment (SAK + OC), 10% (w/v) immobilized SAK car­
riers was added to 100 mL wastewater with 2 mL glucose solution (C/N
Table 1
Batch experiment set-up for immobilized application of strain DM02 treating
real aquaculture wastewater.
Blank DM02 DM02+OC SAK+OC
Real aquaculture wastewater (mL) 100 100 100 100
20 g/L glucose solution (mL) 0 0 2 2
Strain DM02 solution (mL) 0 1 1 0
Cycle times 3 3 3 3
SAK, sodium alginate-kaolin carrier; OC, organic carbon. For DM02 and DM02
+ OC groups, pre-cultured strain DM02 solution was inoculated at the beginning
of cycle 1, and subsequent cycle was inoculated with 1 mL solution from DM02
+ OC treatment.
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Bioresource Technology 322 (2021) 124555
= 10). The wastewater was used as blank group (Table 1). All treatments
were performed in triplicate and shaken at 37 ◦ C and 150 rpm (DO > 5.0
mg/L). All flasks were operated over a cycle of 24 h. At the end of each
cycle, wastewater in flasks was completely replaced by fresh waste­
water, and one liter of inoculum from DM02 + OC treatment was
inoculated into DM02 and DM02 + OC groups for semi-continuous
treatment. The SAK carriers were washed by sterile water for three
times before adding fresh wastewater and glucose solution. The initial
and final concentrations of chemical oxygen demand (COD) and TAN,
NO−2 -N, NO−3 -N, TN were measured.
2.7. Analytical methods
COD, TAN, NO−2 -N, NO−3 -N and TN concentrations were detected
according to standard methods (APHA, 2012). The cell growth rate was
represented by determining OD600 using a UV spectrophotometer. The
dry cell weight (DCW, g/L) was measured according to Duan et al.
(2015). The relationship between OD600 and DCW and between OD600
and intracellular nitrogen were calculated (Feng et al., 2020). Kinetic
analysis was performed with different initial TAN and NO−3 -N concen­
trations, respectively (Jia et al., 2020). The nitrogen removal efficiency
was calculated according to (Huang et al., 2017). One-way ANOVA with
Tukey’s HSD test (P < 0.05) were used for statistical analyses in SPSS
21.0 software.
3. Results and discussion
3.1. Isolation and identification of strain DM02
The colony of strain DM02 on the agar plate was white, circular with
smooth surface (Olympus CKX53, Tokyo, Japan). Strain DM02 was rod
shape in LNEM suspension as shown under the microscope (Olympus
BX53, Tokyo, Japan). The partial 16S rRNA sequence (1399 bp) of strain
DM02 has been submitted to GenBank (MT540002.1). The BLAST result
showed that the sequence of strain DM02 had 100% similarity with the
sequence of Pseudomonas sp. LMB-4 (GenBank accession number:
KR080709.1). A neighbor-joining phylogenetic tree indicated close re­
lationships of DM02 with the members of Pseudomonas (Fig. 1). There­
fore, strain DM02 was classified as Pseudomonas sp. DM02.
3.2. Fate of TAN utilized by strain DM02
The un-ionized ammonia (NH3) is toxic to aquatic animals and hence
should be kept in low concentration in aquaculture system (Randall and
Tsui, 2002). The fate of TAN during its utilization by strain DM02 was
investigated in BM with TAN (10 mg/L) as sole nitrogen source. A C/N
ratio of 8 was conducted, which was similar to that of commercial feed
(Deng et al., 2018). Variations of TAN, NO−2 -N, NO−3 -N, TN and growth
curve (OD600) of strain DM02 were shown in Fig. 2. In initial 3 h, the
average TAN concentration decreased slowly from 9.7 mg/L to 8.7 mg/L
as it was utilized by limited biomass (OD600 ≤ 0.01). Between 3 h and 12
h, the TAN concentration decreased sharply to zero with the OD600
increased of from 0.01 to 0.14. Previous research also found the close
relation between the growth of HN-AD bacteria and TAN removal (Xia
et al., 2020). The maximum cell growth rate and TAN removal rate were
0.03 per hour and 1.28 mg/L/h, respectively, between 6 h and 9 h. The
removal rate was higher than that of Acinetobacter harbinensis HITLi7T
(0.076 mg/L/h) and Bacillus strain N31 (1.0 mg/L/h), which are culti­
vated in heterotrophic nitrification medium with initial TAN of 5.0 mg/L
and 20.0 mg/L, respectively (Huang et al., 2017; Zheng et al., 2018).
Although high TAN removal rates were observed in some strains such as
Acinetobacter sp. ND7 (5.74 mg/L/h) (Xia et al., 2020), Acinetobacter sp.
JR1 (7.75 mg/L/h) (Yang et al., 2019a), Pseudomonas putida strain NP5
(9.96 mg/L/h) (Yang et al., 2019b), the initial TAN concentrations in
these studies were 100 mg/L which is significantly higher than that of
aquaculture effluent (TAN < 15 mg/L) (De Schryver and Verstraete,
M. Deng et al.
Fig. 1. The phylogenetic tree derived from neighbor-joining analysis of partial 16S rRNA gene sequence.
2009; Huang et al., 2017; Yang et al., 2019a).
During the reaction, a small accumulation (<0.1 mg/L) of NO−3 -N
and NO−2 -N was observed, which is consistent with previous researches
on Acinetobacter sp. HA2 (Yao et al., 2013) and Ochrobactrum anthropic
LJ81 (Lei et al., 2019). The inappreciable accumulation of NO−3 -N and
NO−2 -N were probably due to the unbalance of nitrification and deni­
trification. Interestingly, some HN-AD bacteria, such as Acinetobacter
junii YB (Yang et al., 2015), Acinetobacter sp. JR1 (Yang et al., 2019a),
and Cupriavidus sp. S1 (Sun et al., 2016), showed no accumulation of
NO−3 -N and NO−2 -N during TAN removal. Environmental factors may
have significant influence on the balance of HN-AD process. In the
present study, TN was decreased from 13.9 mg/L to 7.1 mg/L, which
was mainly caused by denitrification. Besides, there was a small amount
of TAN regeneration in water from 12 h to 18 h, which is consist with
previous research (Sun et al., 2017). The release of ammonium from the
broken bacteria when nutrients were used up should explain the reason.
The nitrogen mass balance was also calculated to analyze the process
of TAN transformation (Table 2). The result indicated that 70.8% of the
initial nitrogen was translated into gaseous nitrogen through HN-AD,
and 28.1% was transformed into intracellular nitrogen through assimi­
lation. Only 1.1% of the initial TAN was changed into intermediates.
These results suggested that strain DM02 had efficient nitrogen removal
ability and the removal of TAN was primarily caused by the HN-AD
process. This is consistent with Acinetobacter sp. ND7 (Xia et al.,
2020), but inconsistent with Pseudomonas putida strain NP5 (Yang et al.,
2019b), which is mainly due to bacterial assimilation.
3.3. TAN and nitrate removal kinetics by strain DM02
The specific TAN and nitrate removal rates of strain DM02 at
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Bioresource Technology 322 (2021) 124555
different initial TAN and nitrate concentrations are shown in Fig. 2B and
C, respectively. The maximum TAN removal rate (qmax) was 113.8 mgN/
g-DCW/h, which is four times higher than the maximum nitrate removal
rate (27.4 mgN/g-DCW/h). The nitrate removal rate of strain DM02 was
in the same range of other reported denitrifiers (4.6–12.0 mg/L/h) (Sun
et al., 2017, Zhao et al., 2018). The half-saturation coefficient (KS) of
TAN and nitrate were 47.5 mg/L and 11.2 mg/L, respectively. Ruan
et al. (2020a) isolated an aerobic denitrifier, Pseudomonas balearica
RAD-17, from recirculating aquaculture system with a nitrate KS of
180.5 mg/L. Jia et al. (2020) screened an aerobic denitrifying bacte­
rium, Bacillus megaterium Y-4, from electronic waste plant with a nitrate
KS of 29505 mg/L. The low nitrate KS of strain DM02 indicated its ki­
netic affinity to nitrate and favorability for treating low nutrient
wastewater. Moreover, the inhibition coefficient (KI) for TAN and ni­
trate removal were 61.4 mg/L and 190.9 mg/L, respectively. It indicated
that strain DM02 is suitable for treating aquaculture wastewater, in
which TAN and nitrate were usually lower than 10 mg/L and 100 mg/L,
respectively.
3.4. Effect of environmental factors on HN-AD performance of strain
DM02
3.4.1. Carbon sources
OC is energy source for the growth of heterotrophic bacteria and
electron donor for denitrifying bacteria. The cell growth and NO−3 -N
removal performances under different carbon source conditions were
evaluated (Fig. 3). The experiment was performed with 20 mg/L NO−3 -N.
A C/N ratio of 20 was conducted to supply sufficient OC. All OC source
treatments exhibited high NO−3 -N removal capability (>94%) at 28 h.
The results indicated strain DM02 can use a wide range of carbon
M. Deng et al. Bioresource Technology 322 (2021) 124555

Fig. 2. (A) Performance of total ammonia nitrogen (TAN) removal characteristics by strain DM02 in low nutrient basic medium; (B) TAN removal kinetics of
Pseudomonas sp. DM02 fitting by Haldane model; (C) Nitrate nitrogen removal kinetics of Pseudomonas sp. DM02 fitting by Haldane model. Values are means ± SD
(standard deviation) for three replicates.

5
M. Deng et al.
Table 2
Nitrogen mass balance of total ammonia nitrogen removal in BM using TAN as
sole nitrogen source by strain DM02 (unit: mg/L).
TAN NO−2 -N NO−3 -N Intracellular TN Gaseous nitrogen
Initial 9.6 – – 4 13.6 –
Final 0.4 0 0 6.7 7.1 6.8
–: Undetectable. Values are represented as the means ± SD of three replicates.
sources based on the accessibility of local resource. Although the final
NO−3 -N removal efficiencies in sucrose and glucose groups were a little
bit lower than other OC, sucrose and glucose groups showed signifi­
cantly higher NO−3 -N removal efficiency (average 86%) than other
groups (<15.7%) at initial 12 h. These results indicated sucrose and
glucose can be more rapidly utilized by strain DM02 than other carbon
sources.
The cell biomass in sucrose and glucose groups was significantly
higher than the other groups, which indicated high nitrogen assimilation
when using sucrose and glucose as carbon sources (Fig. 3). In addition,
the cell growth was closely correlated with NO−3 -N removal perfor­
mance. NO−2 -N was accumulated at logarithmic phase and undetected at
stationary phase. The maximum NO−2 -N concentrations in sucrose,
glucose, and ethanol groups (average 0.56 mg/L) were significantly
lower than that in trisodium citrate (1.36 mg/L), sodium succinate
(1.73 mg/L) and sodium acetate (2.12 mg/L) groups. NO−2 was toxic to
aquatic animals and should be kept below 1.0 mg/L in aquaculture
system (Huang et al., 2020a). Hence, glucose, sucrose, and ethanol were
suitable carbon sources for strain DM02 for NO−3 -N removal. Ethanol is a
renewable resource and should get more attention in further research. In
the present study, glucose was used as OC for subsequent experiments
because of both good NO−3 -N removal performance and low NO−2 -N
accumulation.
3.4.2. pH
The unbalance of nitrification (produce H+) and denitrification
(consume H+) leads to the variation of pH in wastewater treatment
systems. The pH condition in return has significant influence on
Fig. 3. Effect of different carbon sources on nitrate removal and growth performance of strain DM02.
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Bioresource Technology 322 (2021) 124555
bacterial activity. Here, the effect of different pH value on the removal
performance of TAN, NO−3 -N and NO−2 -N and growth of DM02 were
tested and the results were shown in Fig. 4. A pH range of 5 to 9 was
popular in aquaculture systems and was therefore conducted in the
present study. Previous researches mainly concentrated on the in­
fluences of initial pH on TAN removal performance of HN-AD bacteria
through addition of HCl and NaOH solution (Wang et al., 2016; Yang
et al., 2019a). In the present study, pH buffer (1/15 M Na2HPO4 and 1/
15 M KH2PO4 solution) was added to BM to explore the lasting influence
of pH condition on TAN, NO−3 -N, and NO−2 -N removal by strain DM02.
Interestingly, the maximum TAN removal efficiency at pH 6 was
58.4%, which was significantly lower than pH 5 (83.5%), 7 (90.0%), 8
(89.4%), and 9 (87.6%) at 20 h (Fig. 4A). However, the cell biomass in
pH 6 was 2.0, 1.9, 1.9, and 1.4-folds higher than pH 5, 7, 8, and 9,
respectively. Similarly, pH 6 (87.8%) showed the lowest NO−3 -N removal
efficiency compared with pH 5 (94.3%), 7 (100%), 8 (100%), and 9
(97.6%). Nevertheless, the cell biomass in pH 6 was also higher than pH
5, 7, and 8 (Fig. 4B). The cell biomass in pH 9 was higher than in pH 8
during TAN, nitrite and nitrate removal, which might because of the
addition of 5 M NaOH solution for initial pH adjustion.
The results indicated pH 6 improved TAN and NO−3 -N removal
through nitrogen assimilation, however, it decreased TAN removal
through HN-AD pathway. In total, neutral (pH 7) and alkaline (pH 8 and
pH 9) conditions showed significantly higher TAN and NO−3 -N removal
efficiencies than acidic conditions (p < 0.05) (Fig. 4A, B). Different from
NO−3 -N, strain DM02 was sensitive to pH 5 when using NO−2 -N as sole
nitrogen source for aerobic denitrification (Fig. 4C). Both NO−2 -N
removal efficiency and cell biomass at pH 5 was significantly lower than
pH 6, 7, 8, and 9. Similarly, strain DM02 also showed better cell growth
at pH 6 compared with pH 5, 7, and 8, when using NO−2 -N as nitrogen
source. In addition, the neutral (pH 7) condition showed significantly
higher NO−2 -N removal efficiency than pH 6 and 8 at initial 36 h (P <
0.05). These results showed more details about nitrogen removal under
different pH environment compared with previous researches using only
one sampling point (e.g. sampled at 24 h only) (Wang et al., 2016; Yang
et al., 2019a).
M. Deng et al.
Fig. 4. TAN, nitrate and nitrite removal and growth performance of strain DM02 at different pH (A, B, C) and C/N ratio (D, E, F) conditions.
In total, the results indicated that slightly acidic (pH 6) condition was
better for the nitrogen assimilation of strain DM02, however, it can
significantly decrease HN-AD process when using TAN and NO−3 -N as
sole nitrogen source, respectively. In aquaculture system, the pH was
usually maintained neutral and slightly alkaline because of the aqua­
culture fish, which is suitable for nitrogen removal by strain DM02. The
pH was set to neutral condition in subsequent experiments.
3.4.3. C/N ratio
C/N ratio significantly affected nitrogen removal performance of
HN-AD bacteria (Huang et al., 2013, 2017; Wang et al., 2016). Low C/N
ratio cannot provide sufficient energy and electron donor for cell growth
and denitrification, respectively. On the contrary, high C/N ratio might
result in secondary pollution of organic matter. A negative TAN removal
efficiency was shown without the addition of OC (Fig. 4D). That was
mainly because of the release of ammonium when endogenous organic
matter is used up (Sun et al., 2017). This result also demonstrated that
strain DM02 is a heterotrophic bacterium. Almost all TAN can be
removed when C/N ratio becomes higher than 8, which is consistent
with previous research (Lei et al., 2016). In addition, there was no sig­
nificant accumulation of NO−2 -N (<0.05 mg/L) and NO−3 -N (<0.4 mg/L).
7
Bioresource Technology 322 (2021) 124555
However, the results also indicated that C/N higher than 16 slowed
down the TAN removal rate in initial 12 h. Oversupply of OC also caused
a negative impact on TAN removal by other HN-AD bacteria, such as
Serratia marcescens W5 (Wang et al., 2016) and Acinetobacter sp. ND7
(Xia et al., 2020). In total, C/N = 8 is the optimal condition for TAN
removal for strain DM02, which is lower than Acinetobacter sp. JR1 (C/
N > 16) (Yang et al., 2019a). That is benefit for treating aquaculture
wastewater because of its low organic matter characteristic (Zhao et al.,
2019).
The NO−3 -N and NO−2 -N removal performances were also monitored
under different C/N ratios (Fig. 4E, F). The NO−3 -N and NO−2 -N removal
efficiencies increased gradually from 1.3% and 7.4% to 100% and
99.4%, respectively, when C/N ratio was risen from 0 to 12. There was
no significant accumulation of NO−2 -N (<0.6 mg/L) during NO−3 -N
removal, and the maximal accumulated NO−2 -N concentration was
gradually decreased from 0.50 ± 0.03 mg/L to 0.03 ± 0.01 mg/L when
C/N ratio was increased from 2 to 14.
The relationship between C/N ratio and nitrogen removal efficiency,
especially for NO−3 -N and NO−2 -N, was calculated. The removal effi­
ciencies of NO−3 -N (y = 9.501x − 0.0238, R2 = 0.9992, p < 0.0001) and
NO−2 -N (y = 8.766x + 6.538, R2 = 0.9995, p < 0.0001) was linear
M. Deng et al.
correlated with C/N ratio at 0–10 range. The cell growth of strain DM02
was also positive correlated to initial C/N ratio when TAN (R2 = 0.9862,
p < 0.0001), NO−3 -N (R2 = 0.9986, p < 0.0001), and NO−2 -N (R2 =
0.9959, p < 0.0001) were used as sole nitrogen source, respectively
(Fig. 4). The assimilated nitrogen also gradually increased from 0.0 mg/
L to 5.6, 6.0, and 1.5 mg/L when using TAN, nitrate and nitrite as sole
nitrogen source, respectively. These results demonstrated the contribu­
tion of assimilation for inorganic nitrogen removal by strain DM02
increased with the increasing of initial C/N ratio. High C/N ratio may
lead to the generation of excessive activated sludge when using HN-AD
technology, which should be avoided in wastewater treatment plant (Gu
et al., 2017).
3.5. Nitrogen removal mechanisms of strain DM02
In order to confirm the aerobic denitrification characteristic of strain
DM02, napA (periplasmic nitrate reductase encoding gene), nirK and
nirS (nitrite reductase encoding genes), together with nosZ (nitrous
oxide reductase encoding gene) were amplified by PCR and analyzed
through gel electrophoresis. The napA (152 bp), nirK (473 bp), and nosZ
(276 bp) gene products were amplified from strain DM02 and shown in
Fig. 5. The napA gene is a key functional gene engaged in aerobic
denitrification and is generally regarded as an indicator to identify
aerobic denitrifier (Xia et al., 2020). The periplasmic nitrate reductase,
encoded by napA, is critical for the transformation of NO−3 -N to NO−2 -N in
aerobic environment. Previous studies have also amplified the napA
gene in other Pseudomonas bacteria and confirmed their HN-AD ability
(Carneiro Fidelis Silva et al., 2019; Ruan et al., 2020b; Zhao et al., 2018).
For strain DM02, a Cu-containing enzyme (encoded by nirK), rather than
cytochrome cd1 (encoded by nirS), performed nitrite reduction. The nosZ
gene encodes the last step in denitrification (N2O → N2) and confirms the
complete denitrifying ability of strain DM02. Therefore, the successful
amplification of napA, nirK, and nosZ gene demonstrated that strain
DM02 can conduct aerobic denitrification.
3.6. Bioaugmentation and immobilized application in real aquaculture
wastewater treatment
It is important that strain DM02 must be sustainable and capable of
Fig. 5. The amplification of napA, nirK, nirS, and nosZ gene of Pseudomonas sp. DM02.
8
Bioresource Technology 322 (2021) 124555
continuous treatment of wastewater. Bioaugmentation and microbial
immobilized carriers have been used here to investigate the stability of
strain DM02 in treating real aquaculture wastewater. In the present
study, NO−3 -N (22.7 ± 0.2 mg/L, 96% of total inorganic nitrogen) is the
main nitrogen pollutants in aquaculture wastewater, and was signifi­
cantly removed in all cycles by DM02 + OC and SAK + OC treatment
(>88.2%) (Fig. 6). There was no significantly decrease of NO−3 -N
removal efficiency in DM02 + OC and SAK + OC during different cycles
(p > 0.05). The TN also decreased from 21.4 ± 1.7 mg/L to 6.9 ± 1.3
mg/L in DM02 + OC and SAK + OC treatment, indicating that almost
68% of nitrogen have been removed from the system through gaseous
nitrogen emission. In addition, there was no accumulation of TAN
(average 0.25 ± 0.15 mg/L in effluent) and NO−2 -N (average 0.08 ± 0.23
mg/L in effluent) during NO−3 -N removal. On the contrary, there was a
very low NO−3 -N removal efficiency in DM02 (<2%) and it showed no
significant difference with the blank group. That was mainly because of
the low organic matter concentration (COD = 274.5 ± 0.4 mg/L) in
aquaculture wastewater, which cannot provide enough available
organic carbon for HN-AD bacteria. The COD in DM02 + OC and SAK +
OC significantly decreased from 862 ± 107 mg/L to 197 ± 73 mg/L
during aquaculture wastewater treatment. These results indicated that
strain DM02 can be used for aquaculture wastewater treatment through
both bioaugmentation and immobilized carrier with the addition of
organic carbon source (C/N = 10). Solid carbon source, which can be
used both as carbon source and immobilized carrier for strain DM02
should be employed in subsequent studies.
4. Conclusion
Pseudomonas sp. DM02 was isolated from biofloc of aquaculture
system and exhibited efficient HN-AD capability when using TAN as sole
nitrogen source under low nutrient environment. Strain DM02 can reach
high nitrogen removal efficiency with diverse OC sources and under
both neutral and slightly alkaline environment. Almost 100% of TAN,
NO−3 -N, and NO−2 -N can be removed when increased C/N ratio to 8, 12,
and 12, respectively. The napA, nirK and nosZ genes further proved the
complete aerobic denitrification ability of Strain DM02. Both bio­
augmentation and immobilized carrier of strain DM02 can be used for
real aquaculture wastewater treatment.
M. Deng et al.
Fig. 6. Nitrate removal efficiency in semi-continuous experiment for treating real aquaculture wastewater with bioaugmentation and immobilization of strain DM02.
CRediT authorship contribution statement
Min Deng: Conceptualization, Software, Data curation, Writing -
original draft, Writing - review & editing. Xiaoli Zhao: Supervision.
Yeerken Senbati: Formal analysis. Kang Song: Conceptualization,
Supervision. Xugang He: Supervision, Validation.
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.
Acknowledgments
The research was supported by National Natural Science Foundation
of China (Grant nos. 41877344, 41925031), Study on cleaning mode of
intensive aquaculture in freshwater pond project (Y85Z021301,
Y951040101) from Chinese Academy of Sciences, State Key Laboratory
of Freshwater Ecology and Biotechnology (Grant no. 2019FBZ03).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2020.124555.
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