Accepted Manuscript

Ammonium removal characteristics of an acid-resistant bacterium Acinetobact-

er sp. JR1 from pharmaceutical wastewater capable of heterotrophic nitrifica-
tion-aerobic denitrification

Jing-Rui Yang, Ying Wang, Hu Chen, Yong-Kang Lyu

PII: S0960-8524(18)31484-6
DOI: https://doi.org/10.1016/j.biortech.2018.10.052
Reference: BITE 20616

To appear in: Bioresource Technology

Received Date: 16 August 2018

Revised Date: 19 October 2018
Accepted Date: 20 October 2018

Please cite this article as: Yang, J-R., Wang, Y., Chen, H., Lyu, Y-K., Ammonium removal characteristics of an
acid-resistant bacterium Acinetobacter sp. JR1 from pharmaceutical wastewater capable of heterotrophic
nitrification-aerobic denitrification, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.
2018.10.052

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Ammonium removal characteristics of an acid-resistant bacterium Acinetobacter
sp. JR1 from pharmaceutical wastewater capable of heterotrophic nitrification-
aerobic denitrification
Jing-Rui Yang a, 1, Ying Wang a, 1, Hu Chen b, Yong-Kang Lyu a, *
a Key Laboratory of Coal Science and Technology, Ministry of Education and Shanxi Province, Taiyuan
University of Technology, Taiyuan 030024, Shanxi, China
b College of Environmental Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, Shanxi,
China
* Corresponding author. E-mail address: lykang@tyut.edu.cn (Y.-K. Lyu).
1 These authors contributed to the work equally and should be regarded as co-first authors.

Abstract
A new acid-resistant bacterium Acinetobacter sp. JR1 was isolated, and its
feasibility in nitrogen removal was investigated under acidic condition. Results show
that JR1 indicated excellent ammonium and nitrate removal abilities with no
accumulation of intermediates, and the maximum ammonium and nitrate removal
efficiencies were 98.5% and 91.1%, respectively. Further experiments demonstrated
that JR1 preferred to use ammonium with ammonium and nitrate as the mixed N-
sources. For JR1, ammonium was assimilated directly as nutrients into cells and also
converted into N2 through heterotrophic nitrification-aerobic denitrification. Under
acidic condition, JR1 performed comparable nitrogen removal abilities to other strains
under neutral or weak alkaline environment, and the efficient removal of ammonium
occurred at pH 4.5–10, C/N 12–24, 20–40 °C, DO ≥ 4.72 mg/L, 0–1.5% of salinity,
10 mg/L Zn2+ or 20 mg/L Mn2+. All these make JR1 a promising candidate for
treating acidic wastewater containing nitrogen.
Keywords: Acinetobacter sp., Acid-resistant bacterium, Heterotrophic nitrification,
Aerobic denitrification, Nitrogen removal pathway
1. Introduction
Nitrogen is an important contaminant in water bodies, mainly from urban
domestic sewage, industrial wastewater and fertilizers (Gao et al., 2014; Zhou et al.,
2007). Excessive nitrogen can cause deterioration of water environment quality,
resulting in eutrophication of water body, and a serious threat to human production
and life and ecological balance (Ahn, 2006; Shi et al., 2013), so reducing nitrogen
levels from the discharges is necessary. Biological methods for nitrogen removal have
drawn more attentions for their easy implementation, high-efficiency, and cost
benefit. Traditional biological methods include the nitrification of autotrophic bacteria
under aerobic conditions and the denitrification of heterotrophic bacteria under
anaerobic conditions. Obviously, the requirements for oxygen and organic substrates
in traditional process are completely different, undoubtedly making the operation
complex and increasing the investment cost.
In 1980s, Paracoccus denitrificans with aerobic denitrification ability was first
discovered (Robertson & Kuenen, 1984). After that, a number of heterotrophic
nitrification-aerobic denitrification bacteria have been isolated and intensively studied
as potential microorganisms in biological nitrogen removal, such as Alcaligenes
faecalis strain WY-01 (Wang et al., 2016b), Cupriavidus sp. S1 (Sun et al., 2016),
Enterobacter cloacae CF-S27 (Padhi et al., 2017), Bacillus methylotrophicus strain
L7 (Zhang et al., 2012), Acinetobacter harbinensis HITLi7T (Zheng et al., 2018), and
Serratia marcescens W5 (Wang et al., 2016a). These bacteria exhibit much higher
growth rate than autotrophs and provide a potential way to combine time and space
into an integrated one, apparently more cost-effective and manageable than traditional
biological methods.
However, pH is a key factor affecting nitrogen removal process. As far as we
know, most of the reported heterotrophic nitrification-aerobic denitrification bacteria
prefer neutral or alkaline conditions, which may not be suitable for treating acid
wastewater stemming from mining, metallurgical and pharmaceutical industries. It
has also been reported that nitrification and denitrification are strongly inhibited at pH
below 6 (Ren et al., 2014; Wang et al., 2016a; Zhang et al., 2012). In this case, the
implementation of biological process would be very challenging under the acidic
condition, and adding alkaline to neutralize the acid would increase the operation cost.
Recently, some acid-resistant microorganisms have been reported for nitrogen
removal (Chen et al., 2014; Ma et al., 2015), but there is still a lack of detailed
researches on nitrogen removal under acidic condition and the resistant abilities to
other environmental impact, which would provide specific guidance for practical
applications in acid wastewater treatment. Thus, it is important to isolate and evaluate
a new microbe that exhibits efficient nitrogen removal capability under acidic
condition.
As a candidate for heterotrophic nitrification and aerobic denitrification, strain
JR1 was newly isolated from acidic pharmaceutical wastewater by our lab and
identified as Acinetobacter sp.. And it is amazing to find that strain JR1 showed high
nitrogen removal efficiency in acid synthetic wastewater. However, few studies have
focused on the heterotrophic bacteria that can effectively remove nitrogen under acid
condition. In this paper, the heterotrophic nitrification and aerobic denitrification
performance under acid condition was investigated systematically, and then the
performance of simultaneous nitrification and denitrification in mixed nitrogen
sources was studied under acid condition. Nitrogen balance, gas detection, enzyme
assay and gene amplification experiments were carried out in order to fully
understand the possible nitrogen removal pathway by strain JR1. Finally, single-factor
experiments were conducted to study the influence of pH, carbon source, C/N ratio,
incubation temperature, salinity, dissolved oxygen (DO), initial ammonium
concentration and heavy metal on the ammonium removal under acid condition.
2. Materials and methods
2.1. Media
The bacteria enrichment medium (EM) consisted of following components (per
liter): 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, pH = 7.5.
The basal medium (BM) used for bacteria isolation and single-factor experiments
contained (per liter): 0.472 g of (NH4)2SO4, 3.316 g of fumaric acid, 0.75 g of
K2HPO4·3H2O, 0.25 g of NaH2PO4·2H2O, 0.12 g of NaCl, 0.01 g of MnSO4·H2O,
0.05 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, pH = 4.5.
The denitrification medium (DM) used for nitrate reduction studies contained (per
liter): 0.722 g of KNO3, 3.316 g of fumaric acid, 0.75 g of K2HPO4·3H2O, 0.25 g of
NaH2PO4·2H2O, 0.12 g of NaCl, 0.01 g of MnSO4·H2O, 0.05 g of MgSO4·7H2O, 0.01
g of FeSO4·7H2O, pH = 4.5.
The simultaneous nitrification and denitrification mixed media (SNDM) contained
(per liter): 0.236 g of (NH4)2SO4, 0.361 g of KNO3. Except for different nitrogen
sources, other components were the same as the BM medium, pH = 4.5. All the media
were autoclaved for sterility at 121 °C for 20 min before use. Solid media were
prepared from above media with the addition of 2% agar. All chemicals were of
analytical grade and used without further purification.
2.2. Enrichment and isolation of bacteria
Raw water was collected from a pharmaceutical factory located in Qingxu County
(Shanxi, China), and 10 mL of water sample was added to a 250 mL Erlenmeyer flask
containing 90 mL of sterile EM media. The flask was sealed with sterile breathable
films and then incubated in a rotary shaker at 30 °C and 120 rpm. After 2 days
cultivation, 10 mL of cell suspension was transferred to 90 mL of fresh EM media and
then incubated for another 2 days under the same culture conditions. Thereafter, 1 mL
of the bacterial suspension was transferred to a 250 mL Erlenmeyer flask containing
100 mL of sterile BM media for incubating heterotrophic nitrifying bacteria (This
process was repeated three times). Finally, the enriched bacterial culture was
streaking on BM agar plates using the gradient dilution method and then incubated at
30 °C in an isothermal incubator until visible colonies were formed. Five single
strains with different shapes and colors were selected and cultivated in liquid BM
media at 30 °C and 120 rpm for 48 h to test their aerobic ammonium removal ability
under acidic condition. By repeatedly streaking on BM agar plates and testing aerobic
nitrification ability at least three times, a purified strain named JR1 with the highest
ammonium removal efficiency was obtained from the five strains for further study.
The strain JR1 was suspended in 25% glycerol solution at −80 °C for long-term
storage.
2.3. Identification of strain JR1
A gram staining experiment was carried out to observe the morphology and size
of the strain under optical microscope (OPTEC BK 5000). The partial 16S rRNA gene
of the isolate was amplified by polymerase chain reaction (PCR) using bacterial
universal primers 27F (5'-AGAGTTTGATCATGGCTCAG-3') and 1492R (5'-
TACGGTTACCTTGTTACGACTT-3') and sequenced by TaKaRa Biotechnology
(Dalian, China) Co., Ltd. The 16S rRNA gene sequences were compared with those
of other microorganisms by BLAST (http://www.ncbi.nlm.-
nih.gov/BLAST/Blast.cgi). A phylogenetic tree was constructed in MEGA 5.1
program using the neighbor-joining (NJ) method with 1000 bootstrap replicates and
the maximum composite likelihood model.
2.4. Assessment of nitrogen removal performance under the acidic condition
The BM medium was used to evaluate the nitrification performance with an initial
ammonium nitrogen (NH +4 –N) concentration of 100 mg/L, and the DM medium was
used to investigate the aerobic denitrification ability with 100 mg/L of initial nitrate
nitrogen (NO 3- –N) as the sole nitrogen source. The above experiments were all
conducted in triplicate with the inoculation size of 1% (v/v) in 250 mL Erlenmeyer
flasks containing 100 mL of sterile media, and non-seeded sample experiments were
also conducted as controls. The flasks were cultivated at 30 °C with a shaking speed
of 120 rpm. Samples were taken from the bottles periodically to determine OD600 and
pH, and then centrifuged at 10,000 rpm for 10 min to obtain supernatant for the
determination of NH +4 –N, NH2OH–N, NO 3- –N, NO 2- –N, total nitrogen (TN) and
COD.
2.5. Single-factor influencing the ammonium removal performance of the strain JR1
under the acidic condition
Single-factor experiments were designed to study the heterotrophic nitrification
characteristics of strain JR1 under different culture conditions, including pH, carbon
source, C/N ratio, incubation temperature, salinity, dissolved oxygen (DO), initial
ammonium concentration and heavy metal. The initial pH was adjusted to 4, 4.5, 6, 7,
8, 9, 10 and 11 through addition of 5 mol/L HCl or 5 mol/L NaOH. In the carbon
source experiments, sodium acetate, sodium succinate, sodium pyruvate, sodium
citrate, fumaric acid and sodium ethoxide were used as the sole carbon source in the
BM. In the C/N ratio experiments, the initial C/N ratio was adjusted to 4, 8, 12, 16, 20
and 24 with a fixed ammonium nitrogen concentration of 100 mg/L. To determine the
effects of incubation temperature, DO and salinity on ammonium removal, the
incubation temperature was set at 10, 20, 30, 37 and 40 °C, the shaking speed was
adjusted to 0 (DO ≈ 2.4 mg/L), 40 (DO ≈ 2.98 mg/L), 80 (DO ≈ 3.23 mg/L), 120 (DO
≈ 4.72 mg/L), 160 (DO ≈ 5.64 mg/L) and 200 rpm (DO ≈ 6.34 mg/L), and the salinity
was set at 0, 5, 10, 15, 20, 25 and 30 g/L NaCl in the BM. To further explore the
ammonium nitrogen removal potential of strain JR1, the initial ammonium
concentrations were adjusted to approximately 100, 300, 500, 800 and 1000 mg/L
with a fixed C/N ratio of 16. Finally, the effects of heavy metals on ammonium
removal were also discussed with Cu2+, Zn2+, Ni2+ and Mn2+, which were adjusted to
0, 1, 5, 10 and 20 mg/L. All of above single-factor experiments were conducted in
triplicate with the inoculation size of 1% (v/v) in 250 mL Erlenmeyer flasks
containing 100 mL of sterile media, and non-seeded samples were also conducted as
controls. Unless the single-factor was adjusted according to experimental design, the
media were cultured at C/N 16, pH 4.5, 30 °C and 120 rpm with a constant NH +4 –N
concentration of 100 mg/L. Samples were taken from the bottles periodically to
determine OD600 and pH, and then centrifuged at 10,000 rpm for 10 min to obtain
supernatant for the determination of NH +4 –N, NH2OH–N, NO 3- –N, NO 2- –N, TN
and COD.
2.6. Enzyme assay
 After cultivating in BM media at 30 °C for 6 h, strain JR1 was harvested by
centrifugation at 4 °C and 10,000 rpm for 20 min, and then resuspended in 20 mM
phosphate buffer (PBS, pH = 7.4). Thereafter, cell-free extracts were obtained by
centrifugation at 4 °C and 10,000 rpm for 20 min after being lysed using
ultrasonication treatment. Hydroxylamine oxidoreductase (HAO), nitrate reductase
(NR) and nitrite reductase (NIR) have been reported as the three key enzymes in the
nitrogen biodegradation process (Huang et al., 2015; Ji et al., 2014), and their
activities were analyzed according to our previous methods (Wang et al., 2015).
Protein concentration in the cell-free extract was determined by the Bradford Protein
Assay Kit (Sangon, Shanghai, China). One unit of enzyme activity (U) is defined as
the amount of enzyme that catalyzes the conversion of 1 µmol of substrate per minute.
The specific activity (U/mg) is defined as the concentration of enzyme units divided
by the total amount of protein in mg. All tests were implemented in triplicate.
2.7. PCR amplification of nitrate and nitrite reductase genes
DNA was extracted from the bacterial suspension of strain JR1 using a bacterial
genomic DNA extraction kit (TIANGEN, Beijing, China) following the
manufacture’s instruction. The genomic DNA was used as the template for nitrate
reductase gene (napA) and the nitrite reductase gene (nirS) amplification. Primers
used for napA amplification were napA-F (5'-AAGTGCTGTACGGGTCATGG-3')
and napA-R (5'-CGCTGTTTTCTATGGGCGTG-3'). Primers used for nirS
amplification were nirS-F (5'-GCATCACCAATCGATGCCAC-3') and nirS-R (5'-
GTATGGATTTTGCCGACGCC-3'). The PCR amplification were conducted as
follows: Pre-denaturation at 94 °C for 5 min; followed by 32 cycles of denaturation at
94 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 50 s; and final
extension at 72 °C for 10 min. The PCR products was electrophoresed on 1.2%
agarose gel to determine the target DNA fragments, which were sequenced by Sangon
biotechnology Co., Ltd. (Shanghai, China). The resulting amplified gene sequences
were analyzed by the BLAST tool of NCBI database (http: //www.ncbi.nlm.
nih.gov/BLAST/Blast.cgi).
2.8. Gas detection and nitrogen balance experiment
One milliliter of bacterial suspension of strain JR1 was incubated into 250 mL
glass serum bottle containing 100 mL of BM medium. The bottle was then fully
aerated with high-purity oxygen gas and then hermetically cultivated at 30 °C and 120
rpm for 24 h; meanwhile, the system without inoculation was used as a control. Gas
samples (400 µL) from the headspace were detected at 0 h and 24 h using a gas-tight
syringe to detect N2 and O2 by gas chromatography (GC) equipped with a thermal
conductivity detector. Meanwhile, samples were taken from the bottles at 0 h and 24 h
to determine OD600 and pH, and then centrifuged at 10,000 rpm for 10 min to obtain
supernatant for the determination of NH +4 –N, NH2OH–N, NO 3- –N, NO 2- –N, TN
and COD. The media without inoculum were used as control.
2.9. Analytical methods and calculations
The growth of the strain was assayed by measuring the wavelength of 600 nm
(OD600) using a spectrophotometer (SP-752, Spectrum Shanghai, China). NH +4 –N,
- -
NO 3 –N, NO 2 –N and TN were detected according to standard methods (APHA,
1998). NH +4 –N was determined by the method of Nessler's reagent photometry at a
wavelength of 420 nm, NO 2- –N was measured by N-(1-naphthalene)-diaminoethane
spectrophotometer method at 540 nm, and NO 3- –N was measured by phenol
disulfonic acid method at 410 nm. TN and intracellular nitrogen were detected by the
alkaline persulfate oxidation with a UV spectrophotometric method. Hydroxylamine
was analyzed by indirect spectrophotometry (Frear & Burrell, 1955). Dissolved
oxygen (DO) and pH were measured with a DO meter (HI98193, HANNA, Italy) and
a pH meter (PB-10, Sartorius, Germany), respectively. COD was measured by
potassium dichromate method with a COD analyzer (DR 1010, HACH, USA).
The nitrogen and COD degradation efficiencies were calculated by the formula
(C0 − Ct) / C0 × 100% and their removal rates by the formula (C0 − Ct) / t, where C0
is the initial concentration of COD or nitrogen (NH +4 –N, NH2OH–N, NO 3- –N, NO 2-
–N, TN) and Ct represents their concentrations at a given time, t is the time of strain
JR1 treatment. Intracellular nitrogen content was calculated by subtracting the TN of
inoculated medium following centrifugation (8000 rpm for 10 min) from the TN of
non-centrifuged medium (Rout et al., 2017). Data in this experiment were analyzed by
one-way ANOVA with Turkey's HSD test (p < 0.05) using SPSS Statistics 17.0
software and figures were made by OriginPro 8 software. Each experiment had three
repetitions, and the results are presented as means ± SD (standard deviation of
means).
3. Results and discussions
3.1. Isolation and identification of strain JR1
The strain named JR1 with the highest ability for ammonium removal was
selected for further study under acidic condition. It was a Gram-negative, non-motile,
rod-shaped bacterium and its colony on the agar plate was rounded, light yellow,
salient with smooth surface and a regular edge.
Almost the entire 16S rRNA gene of strain JR1 was obtained by PCR
amplification and its DNA sequences (1451 bp) were deposited in GenBank database
under the accession number of MG188323. The BLAST results indicate that strain
JR1 was closely related to the members of genus Acinetobacter, showing the highest
similarity of 99%. A neighbor-joining phylogenetic tree was constructed on the basis
of 16S rRNA gene sequence of strain JR1 and some other strains with heterotrophic
nitrogen removal capability (Fig. 1). The result further reveals that strain JR1
obviously belonged to the genus Acinetobacter. Therefore, strain JR1 is proposed to
be an Acinetobacter species.
(Fig. 1)
3.2. Heterotrophic nitrification and aerobic denitrification performance of strain JR1
under the acidic condition
The ability of heterotrophic nitrification by strain JR1 was investigated using
ammonium as the sole nitrogen source under the initial pH of 4.5. As depicted in Fig.
2a, strain JR1 directly entered an exponential phase at the initial pH of 4.5 as the
OD600 increased from 0.013 to 1.15 in the first 18 h, indicating that strain JR1 grew
well under acidic condition. Meanwhile, ammonium with an initial concentration of
103 mg/L declined rapidly to 15.5 mg/L at 18 h with the maximum ammonium
removal rate of 7.75 mg/L/h between 6 and 12 h. After that, ammonium continued to
drop and eventually reached zero around at 24 h as the OD600 value reached the
maximum of 1.26, but a slight increase of ammonium occurred after 24 h of
incubation, which might be due to the release of ammonium from the dead bacterial
cell caused by insufficient organic compound (Chen et al., 2014; Lei et al., 2016). All
these results indicate that ammonium removal was closely related to the growth of
strain JR1. During the whole ammonium removal process, no accumulation of nitrate
and nitrite was detected. This result is consistent with Acinetobacter junii YB (Yang
et al., 2015) and Cupriavidus sp. S1 (Sun et al., 2016), but quite different from
Acinetobacter sp. HA2, in which the concentration of nitrate increased to a maximum
and then declined (Yao et al., 2013). Moreover, the removal profiles of TN and COD
were very analogous to that of ammonium nitrogen (Fig. 2a), and the maximum
removal efficiencies of NH +4 –N, TN and COD were 98.5%, 97.9% and 93.5%,
respectively, indicating a simultaneous removal of nitrogen and organic carbon.
As we all know, most of heterotrophic nitrifying-aerobic denitrifying bacteria
prefer to grow in neutral or alkaline conditions, and their optimal pH range is 6–9 (He
et al., 2016; Wang et al., 2016b; Yang et al., 2018). Zhang et al. (2012) reported that
acidic (pH 5–6) or alkaline (pH 9–10) condition is harmful to the growth of strain L7,
and the ammonium removal efficiency in 108 h does not surpass 20.0% under this
acidic (pH 5–6) or alkaline (pH 9–10) condition. However, strain JR1 in this study
grew fast in the initial pH of 4.5 with a maximum ammonium removal efficiency of
98.5% (Fig. 2a), confirming that strain JR1 could grow and remove ammonium well
in the acidic condition. Moreover, the average ammonium removal rate within 24 h
was 4.22 mg/L/h, which is comparable to those obtained by some heterotrophic
nitrifying-aerobic denitrifying bacteria under neutral or alkaline conditions of pH 7–9,
such as Acinetobacter junii YB (4.14 mg/L/h) (Yang et al., 2015) and Klebsiella
pneumoniae CF-S9 (4.3 mg/L/h) (Padhi et al., 2013). As a result, strain JR1
performed good heterotrophic nitrification ability under acidic culture condition,
indicating its potential for treating acid wastewater.
Obviously, the formation of intermediate products hydroxylamine, nitrate and
nitrite was insignificant, probably because these products could be denitrified
instantly by strain JR1 during the ammonium removal process. Thus, N2 production
was tested in a closed system using gas chromatography to evaluate the aerobic
denitrifying performance of JR1, and the result indicates that strain JR1 converted
ammonium into N2 ultimately when ammonium was used as the sole N-source at the
initial culture pH of 4.5, further suggesting the aerobic denitrification ability of strain
JR1 under acidic condition. Meanwhile, the nitrogen balance under acidic condition
indicates that 68.9% and 19.4% of initial NH +4 –N (10.2 mg) were converted into
intracellular nitrogen and N2, respectively, while there were low accumulations of
hydroxylamine (0.00310 mg), nitrate (0.0632 mg) and nitrite (0.0110 mg).
Approximately 9.80% of initial NH +4 –N was lost during this ammonium removal
process, which might be due to the other removal form of gaseous nitrogen or may be
attributed to the measurement errors caused by different analytical methods (Rout et
al., 2017). From above results, strain JR1 had the ability of heterotrophic nitrification
and aerobic denitrification under acidic condition.
 In general, the step that nitrate is reduced to nitrite via nitrate reductase is often
considered to be the first step for denitrification. Thus, nitrate was used as the sole
nitrogen source under aerobic conditions at the initial pH of 4.5 to further confirm the
aerobic denitrification capacity of JR1. As shown in Fig. 2b, strain JR1 directly
entered logarithmic phase with no lag phase, and the OD600 value reached a maximum
of 1.14 at 24 h. Meanwhile, nitrate decreased almost linearly in 24 h with a maximum
nitrate removal rate of 4.12 mg/L/h, and 91.1% of nitrate was finally removed by
strain JR1. All these further indicate that nitrate removal was closely related with the
growth of strain JR1. Moreover, the removal trends of TN and COD were basically in
agreement with that of nitrate, and pH increased from the initial of 4.5 to 9.3 in the
whole nitrate removal process. Almost no accumulation of nitrite was observed in the
process of nitrate degradation, but a small amount of NH +4 –N was accumulated when
strain JR1 entered decline phase, probably owing to the release of ammonium from
the dead bacteria cell caused by insufficient organic compound (Chen et al., 2014; Lei
et al., 2016). As the final product of nitrification process, nitrate could be utilized as
the sole nitrogen source by strain JR1, further demonstrating the aerobic
denitrification of strain JR1 under acidic condition.
(Fig. 2)
3.3. Simultaneous nitrification and denitrification performance of strain JR1 under
the acidic condition
Ammonium (53.7 mg/L) and nitrate (51.6 mg/L) were used as mixed nitrogen
sources (SNDM) to further investigate the simultaneous nitrification and
denitrification ability of strain JR1 under acid condition, and the results are shown in
Fig. 3. The OD600 indicated a significant increase and reached a peak value of
approximately 1.2 at 24 h. This growth trend was in accordance with the conditions
when ammonium and nitrate were separately used as the sole N-source. After 24 h of
incubation, OD600 decreased obviously, indicating that strain JR1 entered decline
growth phase.
For nitrogen removal, ammonium decreased rapidly and was removed in 12 h
with the maximum ammonium removal rate of 6.68 mg/L/h, while nitrate
concentration decreased by only about 5 mg in the first culture time of 12 h. When
ammonium was almost completely degraded by JR1, nitrate began to decrease from
45.9 mg/L at 12 h to 16.4 mg/L at 48 h with the maximum nitrate removal efficiency
of 68.3%. Moreover, the maximum nitrate removal rate was 2.82 mg/L/h, much lower
than above maximum ammonium removal rate (6.68 mg/L/h) in the mixed N-sources,
further demonstrating that ammonium was removed faster than nitrate. All these
results indicates that strain JR1 preferred to remove ammonium in the mixed N-
source, which might be attributed to the higher enzyme activity of ammonium
oxidization than that of nitrate reduction. It could also be concluded that the pattern of
TN removal was in line with the sum of ammonium nitrogen and nitrate nitrogen as
there was no accumulation of nitrite and hydroxylamine during the whole
biodegradation process. The maximum TN removal efficiency was 76.3% at 24 h and
the corresponding maximum removal rate was 7.33 mg/L/h. After 24 h, TN
concentration kept unchanged, probably because the decrease in nitrate was equal to
the increase in ammonium.
(Fig. 3)
3.4. Aerobic ammonium removal pathway of strain JR1
To investigate the aerobic pathway of strain JR1 involved in this ammonium
removal process, the activities of three key enzymes HAO, NR and NIR were
evaluated under aerobic conditions. The results show that all the enzymes were
detected in cell-free extracts, and the specific activities of HAO, NR and NIR were
0.093, 0.034 and 0.057 U/mg proteins, respectively. HAO has been reported to be
responsible for the oxidation of hydroxylamine to nitrite (Otte et al., 1999). It is also
well known that NR and NIR are the key enzymes involved in the denitrifying
process, and the presence of NR and NIR further demonstrated the aerobic
denitrification ability of strain JR1.
To further confirm the aerobic denitrification pathway, the PCR amplifications of
the two denitrifying genes (napA and nirS) were carried out. As shown in Fig. 4a,
napA and nirS were amplified with the fragment length of 951 bp and 791 bp,
respectively. The napA and nirS genes of strain JR1 showed high similarities to those
of other Acinetobacter strains (Fig. 4b-c), which were consistent with the 16S rRNA
gene phylogeny, demonstrating that these two genes had co-evolved with the
chromosomal genes in Acinetobacter species. The napA gene, which belongs to the
nap gene cluster, encodes the large catalytic subunit of periplasmic nitrate resuctase
(NAP) (Ji et al., 2015). Obviously, this periplasmic NAP in strain JR1 is independent
of nitrate transport via cytoplasmic membrane, and thus is essential for the conversion
of nitrate under aerobic conditions (Bell et al., 1990). Moreover, recent studies
indicated that nirS is more likely involved in aerobic denitrification because it has
been amplified from many aerobic denitrifying bacteria, such as Bacillus cereus GS-5
(Rout et al., 2017), Pseudomonas stutzeri YG-24 (Li et al., 2015), Acinetobacter junii
YB (Yang et al., 2015), and Pseudomonas stutzeri strain XL-2 (Zhao et al., 2018), and
it encodes the homodimer cytochrome cd1 nitrite reductase (cd1-NIR). As we all
know, periplasmic NR and cd1-type NIR have been reported to be responsible for the
aerobic conversion of nitrate to nitrite and nitrite to nitrogenous gas, respectively
(Richardson & Watmough, 1999). Therefore, the two genes napA and nirS may be
involved in the aerobic denitrification process of strain JR1.
As mentioned above, strain JR1 could convert ammonium into N2 under aerobic
conditions and utilize nitrate as the sole nitrogen source. Thus, ammonium removal by
strain JR1 was achieved through heterotrophic nitrification coupled with aerobic
denitrification. This plausible nitrogen removal pathway of strain JR1 is largely
consistent with that of some previously reported strains (Huang et al., 2013; Ren et
al., 2014; Silva et al., 2018; Zhao et al., 2018), but different from the degradation
pathway of A. calcoaceticusi (Zhao et al., 2012) and A. faecalis (Wang et al., 2016b).
In these strains, neither NR nor NIR activity is detected under aerobic conditions, and
HAO is responsible for the transformation of hydroxylamine to gaseous nitrogen.
Moreover, 68.9% of initial NH +4 –N in nitrogen balance test was converted into
intracellular nitrogen, indicating ammonium was mainly utilized as nitrogen source
for cell synthesis through assimilation, which is in accordance with many
heterotrophic nitrification-aerobic denitrification strains (Huang et al., 2015; Shi et al.,
2013; Zhao et al., 2012). As a result, two different metabolic pathways are proposed
for ammonium removal in strain JR1 under acidic condition. One is that ammonium is
mainly utilized for cell synthesis through assimilation, the other is that ammonium is
converted into N2 through heterotrophic nitrification coupled with aerobic
denitrification.
(Fig. 4)
3.5. Effects of different factors on heterotrophic nitrification performance under acid
condition
3.5.1. Initial pH
The effect of pH on ammonium degradation was investigated and the results are
shown in Fig. 5a. Growth was observed in the entire pH range from 4 to 11 and the
maximum OD600 value was 1.27 at pH 4.5. At pH 6–10, strain JR1 grew well and the
OD600 value reached about 1.0 after 24 h of culture. However, the activity of strain
JR1 was affected seriously at pH 4 and 11 with OD600 values only of 0.042 and 0.180,
respectively. After 24 h cultivation, above 96.0% of total ammonium was removed by
strain JR1, demonstrating its amazing NH +4 –N removal abilities from pH 4.5 to 10.
After subtracting the additional ammonium loss caused by the increase in pH value,
the highest NH +4 –N removal rates at pH 4.5, 6, 7, 8, 9 and 10 were 7.75, 5.42, 7.41,
6.44, 6.36 and 6.12 mg/L/h, respectively. TN showed a similar removal trend with
NH +4 –N, and the maximum TN removal efficiencies at pH 4.5, 6, 7, 8, 9 and 10 were
97.9%, 98.0%, 96.9%, 96.0%, 96.7% and 96.9%, respectively. In contrast, acidic (pH
5–6) or alkaline (pH 9–10) condition is harmful to the growth of strain L7 (Zhang et
al., 2012). A thermophilic strain Bacillus MS 30 exhibits excellent nitrification ability
at pH 7.5–8, while the best growth is achieved at pH 6.0–6.5 (Mével & Prieur, 2000).
Strain W5 exerts high ammonium removal abilities at both neutral and alkaline
conditions with a highest NH +4 –N removal efficiency of 91.0% at pH 9 (Wang et al.,
2016a).
As reported above, most of heterotrophic nitrification-aerobic denitrification
bacteria prefer neutral or weak alkaline environment, and the optimum pH range is 6–
9 (Chen et al., 2014; Guo et al., 2013a; Zhang et al., 2012). Few strains have been
reported to grow at pH 4.5 (Guo et al., 2013b; Wang et al., 2016b), probably because
such acidic condition could affect bacterial activity (Jagadevan et al., 2012).
However, strain JR1 grew well in the pH range from 4.5 to 10, and thus degraded
ammonium in both acidic and alkaline condition. These findings indicate that the
strain JR1 could tolerate a very wide pH range from 4.5 to 10, achieving more than
90.0% of ammonium removal efficiency after incubation for 48 h (date not shown in
Fig. 5a).
3.5.2 Carbon source
Carbon source serves as both energy and electron source for heterotrophic
bacteria, and is an important factor influencing NH +4 –N removal and cell growth.
Effects of different carbon sources on the growth and degradation of NH +4 –N, TN
and COD were investigated at the initial pH of 4.5. As shown in Fig. 5b, strain JR1
did not utilize sodium acetate and sodium ethoxide as the sole carbon source for
growth. Whereas it separately used sodium succinate, sodium pyruvate, fumaric acid
and sodium citrate as the sole carbon sources to degrade NH +4 –N under acidic
condition, and more than 80.0% of ammonium was removed by strain JR1, indicating
that strain JR1 had good nitrification ability under these four carbon sources.
After incubation for 24 h, when fumaric acid was used as the sole carbon source,
the highest degradation efficiencies of NH +4 –N, TN and COD among the carbon
sources tested (p < 0.05), were obtained as 98.5%, 97.9% and 87.3%, respectively.
Meanwhile, the highest OD600 value was reached 1.26 at 24 h. As a result, fumaric
acid was used as the optimal carbon source for subsequent experiments.
3.5.3 C/N ratio
The effect of C/N ratio on NH +4 –N removal was investigated under acidic
condition (Fig. 5c). As C/N ratio increased from 4 to 12, cell density (OD600), NH +4 –
N and TN removal efficiency increased from 0.580, 35.4% and 35.0% to 1.13, 83.1%
and 82.7% in 48 h, respectively, but above 90.0% of COD was removed by strain JR1
at C/N ratio of 4–12. Obviously, this low degradation efficiency of NH +4 –N and TN
was due to the exhaustion of carbon source, which would impair both microbial
growth and electron donors for nitrogen removal (Guo et al., 2013a; Lin et al., 2010;
Yang et al., 2011; Zheng et al., 2012). When C/N ratio was beyond 16, there was no
significant difference in the degradation of NH +4 –N and TN (P > 0.05), and above
96.0% of NH +4 –N and TN was removed. However, both cell density and COD
removal declined at C/N ratio ≥ 16. This result indicates that the exhaustion of
nitrogen source might result in low degradation of COD as the supply of carbon was
higher than its demand. By taking the removal of NH +4 –N, TN and COD into
consideration, C/N ratio of 16 might be the optimal condition for nitrogen removal.
3.5.4 Dissolved oxygen
As is well known, DO acts as the main control parameter in the nitrogen removal
process (Hocaoglu et al., 2011). The DO in medium is controlled by adjusting shaking
speed, and the effect of DO on ammonium removal was studied at the initial pH of
4.5. As shown in Fig. 5d, ammonium removal increased significantly from 15.1% at 0
rpm (DO ≈ 2.40 mg/L) to 98.9% at 120 rpm (DO ≈ 4.72 mg/L). While there was no
apparent variance in ammonium removal when the shaking speed was above 120 rpm
(DO ≈ 5.64 mg/L) (P > 0.05). Appropriately increasing shaking speeds significantly
promoted the cell growth and ammonium removal (P < 0.05) owing to the increase of
the mass transfer coefficient of oxygen. Besides, properly increasing rotation speed
could promote the contact strength and mixing degree between bacteria and
ammonium, which could stimulate the growth of stain JR1 and enhance the
degradation rate of ammonium. It was also found that the OD600 decreased gradually
when the shaking speed exceeded 120 rpm, probably owing to negative effects of
vigorous shaking on bacterial growth. Therefore, the shaking speed was fixed at 120
rpm for further study.
(Fig. 5)
3.5.5 Effects of temperature, salinity, ammonium concentration and heavy metal on
heterotrophic nitrification performance under the acidic condition
The ammonium removal characteristics of JR1 at different temperatures were
investigated at the initial pH of 4.5, and the results are shown in Fig. 6a. The
variations of NH +4 –N and OD600 were marginal at 10 °C, and the maximum removal
efficiencies of ammonium were all above 80.0% within 20–40 °C, achieving the best
removal efficiency of 97.9% at 30 °C for 24 h. Moreover, the trend of cell growth was
consistent with that of ammonium removal efficiency, and the maximum OD600 value
was 1.27 at 30 °C. Therefore, the optimum incubation temperature for JR1 was 30 °C.
This might be due to the increase of the enzyme activity of heterotrophic nitrification
and the concentration of free ammonia, the substrate of ammonia monooxygenase
(AMO), at high temperatures (20–33 °C) (Kim et al., 2006). At present, researches on
heterotrophic nitrification-aerobic denitrification are mainly carried out under
mesophilic conditions (30–37 °C) (Liu et al., 2016; Ma et al., 2015; Rout et al., 2017),
while the resistance of JR1 against temperature is comparable to the bacteria reported
before (Ren et al., 2014; Wang et al., 2016a), allowing its application in a wide
temperature range.
Salinity is another parameter that affects the nitrogen removal ability of nitrifying
bacteria. The effects of salinity on the ammonium removal of strain JR1 were
evaluated under acidic condition. As shown in Fig. 6b, in the culture time of 48 h, JR1
could degrade ammonium when the salinity was less than 20 g/L, and more than
96.0% of NH +4 –N was removed by strain JR1 as the salinity increased from 0 to 15
g/L. However, strain JR1 hardly removed NH +4 –N when the salinity increased up to
25 and 30 g/L, probably because too high salinity may cause cell plasmolysis and
activity loss of microorganism (Uygur & Kargı, 2004). The results here reveal that
JR1 could be identified as a halotolerant microbe (Ventosa et al., 1998). Wang et al.
(2016a) reported that above 90% of ammonium is removed by strain W5 in 0–25
mg/L salinity under neutral condition (pH = 7). A halotolerant microbe isolated by
Zhang et al. (2012) can remove more than 58.7% of ammonium in 0–30 g/L NaCl
under the initial pH of 7. Another halotolerant strain HN-02 also shows strong
ammonium removal ability at a salinity of 20 g/L at pH = 7 (Chen et al., 2014).
Obviously, the salinity tolerance of strain JR1 under acidic condition is comparable to
that of these halotolerant bacteria under neutral or slightly alkaline conditions. Thus,
the acid-resistant strain JR1 could grow in 0–20 g/L salinity under acidic condition,
which greatly expands its application scope.
Fig. 6c depicts the effect of initial ammonium concentrations on ammonium
degradation under acidic condition. The maximum removal efficiencies of NH +4 –N
under low (100 mg/L), intermediate (300 and 500 mg/L) and high (800 and 1000
mg/L) ammonium loads were 98.5%, 61.8%, 49.0%, 41.0% and 28.0%. In contrast,
Yang et al. (2011) reported that the maximum NH +4 –N removal efficiencies of
Bacillus subtilis A1 under low (105.58 mg/L), intermediate (257.23 mg/L) and high
(536.21 and 1014.17 mg/L) ammonium concentrations are 24.9%, 13.1%, 4.10% and
2.30% after culturing for 288 h, which is much lower than those of strain JR1. The
removal efficiency declined with the increase of initial NH +4 –N concentration, which
might be due to the inhibition of highly free ammonia caused by increased pH (Kim et
al., 2006). As a result, JR1 could tolerate high concentration of ammonium nitrogen
under acidic condition, suggesting its potential in application for treatment of acidic
wastewater with high concentration of ammonium nitrogen.
Effects of heavy metals (Cu2+, Zn2+, Ni2+ and Mn2+) on the heterotrophic
nitrification performance of JR1 were investigated under acidic condition. Fig. 6d
shows that both Cu2+ and Ni2+ had stronger inhibitory effects than Zn2+ and Mn2+.
When both Cu2+ and Ni2+ reached 5 mg/L in the medium, the ammonium nitrogen
removal efficiencies were only 8.80% and 4.89%, respectively, and the ammonium
removal nearly stopped as the concentration of Cu2+ and Ni2+ continued to increase up
to 10.0 mg/L. However, 90.5% of ammonium was removed by strain JR1 at 20.0
mg/L of Mn2+, which was nearly the same as the control group. This is probably due
to the fact that Mn2+ not only acted as a kind of microelement to promote the growth
of bacteria, but also played a crucial role in enzyme composition and activation. At
the concentration of Zn2+ ≤ 10 mg/L, more than 90.0% of ammonium was removed by
strain JR1, while the ammonium removal was inhibited partly at 20.0 mg/L Zn2+ with
the ammonium removal efficiency of only 50.0%. The above results demonstrate that
both Cu2+ and Ni2+ were the most toxic among the four metals, whereas Mn2+ was the
least toxic metal. Kim et al. (2005) found that 0.5 mM Cu2+ and Zn2+ (31.77 mg/L
Cu2+ and 32.70 mg/L Zn2+) do not affect the growth and ammonium removal of
Bacillus sp. PK15. However, Chen et al. (2014) found that Aeromonas sp. HN-02 is
extremely sensitive to Cu2+ (0.5 mg/L Cu2+). Some studies also showed that 1.0 mg/L
Cu2+, Ni2+ or Zn2+ is sufficient to reduce nitrification (Pettet, 1955). All these indicate
that metal ions have different effects on different bacteria. Appropriate dosage of
heavy metals would not affect the growth of microorganism, but there is no doubt that
excessive amounts of heavy metals are harmful to the growth of microorganisms.
(Fig. 6)
4. Conclusions
Acinetobacter sp. JR1 was newly isolated from pharmaceutical wastewater, and
tested for the performance and pathway of nitrogen removal under acidic condition.
Strain JR1 could use ammonium and nitrate efficiently with no intermediate
accumulation. Further studied indicated that ammonium was assimilated directly by
strain JR1 for cell synthesis and also converted into N2 through heterotrophic
nitrification coupled with aerobic denitrification. From single-factor tests, strain JR1
exhibited high ammonium removal efficiency (above 80%) under different culture
conditions (pH 4.5–10, C/N ratio 12–24, 20–40 °C, 0–1.5% salinity). Therefore, JR1
shows great potential for treating acidic wastewater containing nitrogen.
Acknowledgements
This study was financially supported by the Program for the National Natural
Science Foundation of China (Grant No. 51778397), National Key Research and
Development Program of China (Grant No. 2016YFB0600502), International
Cooperation Projects of Shanxi Province (Grant No. 201603D421040), and the Key
research and Development Program of Shanxi Province (Grant No. 201603D321010).
We are thankful to Professor Xu-Guang Liu (Taiyuan University of Technology,
Shanxi) for English language editing during the preparation of this article. The
authors also thank the reviewers for their valuable suggestions and comments on the
manuscript.
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Fig. 1. Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences of
strain JR1 and some other related bacteria.
 Fig. 2. The growth and nitrogen removal characteristics of strain JR1 under acidic
condition. (a) ammonium; (b) nitrate. Values are means ± SD (error bars) for three
replicates.

Fig. 3. Simultaneous nitrification and denitrification ability of strain JR1 under acidic
condition. Values are means ± SD (error bars) for three replicates.

Fig. 4. (a) The amplification of napA and nirS genes of Acinetobacter sp. JR1.
Marker: DL 5000 DNA Marker (TaKaRa, Japan). Phylogenetic trees of Acinetobacter
 sp. JR1 based on napA (b) and nirS (c) gene sequences show the phylogenetic
relationship of JR1 with other strains. Bootstrap values of 1000 replications are
indicated at the interior branches.

Fig. 5. Effects of pH (a), carbon source (b), C/N ratio (c) and shaking speed (d)
on nitrification ability of strain JR1 under acidic condition in 24 h. Values are
means ± SD (error bars) for three replicates.
Fig. 6. Effects of temperature (a), salinity (b), initial ammonium concentration (c) and
heavy metal (d) on ammonium removal abilities of JR1 under acidic condition.
Values are means ± SD (error bars) for three replicates.

Highlights
 A newly acid-resistant strain JR1 was isolated from pharmaceutical wastewater.
 A high nitrogen removal could be achieved by JR1 under the acidic condition.
 Nitrate began to be utilized as substitute N-source after exhausting of ammonium.
 Ammonium was utilized through assimilation along with heterotrophic
nitrification.