Microbiology (2015), 161, 829–837
DOI 10.1099/mic.0.000047
Heterotrophic nitrification and aerobic
denitrification at low nutrient conditions by a newly
isolated bacterium, Acinetobacter sp. SYF26
Jun-feng Su,1,2 Kai Zhang,1 Ting-lin Huang,1 Gang Wen,1 Lin Guo1
and Shao-fei Yang1
Correspondence
Jun-feng Su
[email protected]
Received 23 January 2015
Accepted 27 January 2015
1
School of Environmental and Municipal Engineering, Xi’an University of Architecture and
Technology, Xi’an 710055, PR China
2
State Environmental Protection Key Laboratory of Microorganism Application and Risk Control,
Tsinghua University, Beijing 100084, PR China
A new strain, named SYF26, was isolated from the Hei He oligotrophic drinking-water reservoir in
China. Based on its phenotypic and phylogenetic characteristics, the isolate was identified as a
species of genus Acinetobacter. Strain SYF26 was able to grow at low NH4+-N concentrations
(5.46 mg l”1), and the nitrification rate was 0.064 mg NH4+-N l”1 h”1. Low accumulation of
nitrate and nitrite was observed throughout the ammonium removal experiment. Strain SYF26
reduced NO3”-N or NO2”-N. Nitrite reductase and periplasmic nitrate reductase were detectable.
The putative nitrogen removal process carried out by the strain SYF26 is as follows:
NH4+ANH2OHANO2”ANO3”, then NO3”ANO2”AN2. Response surface methodology
analysis demonstrated that the maximum removal of ammonium occurred under the following
conditions: NH4+-N concentration of 22.05 mg l”1, C/N ratio of 4.31, initial pH of 7.78 and
temperature of 29.73 6C, where initial pH and temperature had the largest influence on
ammonium removal.
INTRODUCTION
Over the last few years, a tremendously high amount of
artificial nitrogen fertilizer has been used for achieving high
crop yields to meet the rapidly growing human population
(Galloway et al., 2008; Tilman et al., 2002). Accordingly, a
substantial amount of nitrate and ammonium has entered
into water bodies such as lakes, reservoirs and seas (Zhou
et al., 2007) via tributary rivers; hence, an increasing
number of aquatic ecosystems in China have suffered
hyper-eutrophication and serious algal blooms in recent
years (Guo et al., 2013). Therefore, many researches have
focused on the purification of polluted source water, and
bioremediation technology has received wide attention
because of its low maintenance cost and effective pollutant
removal performance (Jechalke et al., 2010; Perelo, 2010).
However, the conventional system for ammonium removal
consists of two steps: nitrification by autotrophs under
aerobic conditions, and denitrification by heterotrophs
under anaerobic conditions. This type of system is difficult
to operate due to the low rate of nitrification, and the
Abbreviation: RSM, response surface methodology.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
sequence of Acinetobacter strain SYF26 is LC010332.
000047 G 2015 The Authors
complexity of separating nitrification and denitrification
reactors.
Heterotrophic nitrifying–aerobic denitrifying micro-organisms capable of nitrification and denitrification simultaneously under aerobic conditions have drawn increasing
attention (Chen & Ni, 2011; Khardenavis et al., 2007;
Zhu et al., 2012). To date, certain groups of heterotrophic
nitrification–aerobic denitrification micro-organisms, such
as Paracoccus denitrificans (formerly known as Thiosphaera
pantotropha), Alcaligenes faecalis, Pseudomonas stutzeri,
Microvirgula aerodenitrificans and Bacillus have been
isolated from soils and wastewater treatment systems
(Kim et al., 2008; Yang et al., 2011; Joo et al., 2006).
These micro-organisms, due to their high growth rate and
ability to convert ammonia nitrogen to nitrogenous gas
aerobically, have a great number of advantages as applied
for nitrogen removal: (i) procedural simplicity, where
nitrification and denitrification can take place simultaneously; (ii) less acclimation problems; (iii) lesser buffer
quantity needed because alkalinity generated during
denitrification can partly compensate for the alkalinity
consumption in nitrification (Gupta, 1997; Zhao et al.,
2010a; Yao et al., 2013). However, they may meet
acclimation problems at low levels of carbon source, which
could limit the denitrification process. Therefore, a study on
ammonium as a denitrifying substrate was urgent and
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829
J. Su and others
important not only in theoretical research but also in
applied research to better understand the mechanism of
heterotrophic nitrification–aerobic denitrification and the
control of ammonium pollutions at low nutrient conditions.
The objective of this study was to isolate a novel autochthonous heterotrophic nitrification–aerobic denitrification bacterium, Acinetobacter sp. SYF26, from the Hei He
oligotrophic reservoir, which is one of the main drinkingwater resources for citizens of Xi’an, China. Strain SYF26 was
characterized based on its heterotrophic nitrifying–aerobic
denitrifying performance using ammonia, nitrite and nitrate
as substrates under low nutrient conditions. Factors affecting
the performance of Acinetobacter sp. SYF26 were comprehensively evaluated based on response surface methodology
(RSM) analysis. The purpose of the study was to determine
the bacterium’s ability to remove ammonium at low nutrient
conditions, which might also contribute to elucidation of the
general mechanisms of heterotrophic nitrification–aerobic
denitrification.
METHODS
Isolation of heterotrophic nitrifying bacteria. Sediments were
obtained from the Hei He reservoir. A total of 50 g sediment was
suspended in a low-nutrient medium (LNM) to obtain a homogeneous
suspension. The LNM used in this study comprised the following
reagents (l21): CH3COONa, 0.1 g; NH4Cl, 0.02 g; KH2PO4, 0.02 g;
MgSO4.7H2O, 0.01 g; CaCl2, 0.01 g; and trace element solution, 2 ml.
The final pH of the medium was adjusted to 7.0. The components of
trace element solution (g l21) were as follows: EDTA, 5; ZnSO4, 0.3;
MnCl2.4H2O, 0.5; FeSO4.7H20, 0.5; CuSO4.5H2O, 0.2; CoCl2.6H2O,
0.3. Sterilized glass beads were added and culture flasks (500 ml) were
sealed with sterile culture vessel breathable sealing membranes,
following which the flasks were shaken in a rotary shaker at 30 uC
and 120 r.p.m. for 7 days. After cultivation, bacterial suspensions
were inoculated into fresh medium (10 % inoculum). Subculture was
repeated twice, and the nitrogen conversion rate in the upper
supernatant was determined. After 10-fold serial dilution, 0.5 ml each
bacterial suspension was spread on an LNM agar plate (basal medium
with the addition of 1.5 % agar) and incubated at 30 uC. Bacterial
colonies with different apparent characteristics were spread with a
platinum loop on 1.5 % LNM agar plates. Purified isolates were
obtained via repeated streaking on fresh agar plates. To detect the
nitrifying performance, isolates were cultivated in the LNM with
NH4Cl as the sole nitrogen source. A heterotrophic nitrification–
aerobic denitrification strain SYF26 with high nitrogen removal
efficiency was obtained. Two nitrogen compounds, potassium nitrate
and sodium nitrite, were used instead of ammonium chloride in the
LNM to elucidate the denitrification process of the isolate.
Bacterial identification and denitrification gene amplification.
The 16S rRNA gene of the isolate SYF26 was PCR amplified using
bacterial universal primers F27 (59-AGAGTTTGATCMTGGCTCAG39) and R1492 (59-TTGGYTACCTTGTTACGACT-39), under the
following conditions: 5 min at 94 uC; 30 cycles of 1 min at 94 uC,
1 min at 53 uC, 1.5 min at 72 uC; and a final step of 10 min at 72 uC.
PCR products were run and visualized by electrophoresis in a 1 %
agarose gel and ethidium bromide staining. The amplified products
were purified and sequenced by TaKaRa Biotechnology. Finally,
sequences were compared to other relevant micro-organisms in
GenBank/EMBL/DDBJ by BLAST and were themselves submitted to
the databases.
830
The napA gene encoding periplasmic nitrate reductase, which reduces
nitrate to nitrite under aerobic conditions, and the nirK or nirS gene
encoding nitrite reductase, which reduces nitrite to nitric oxide, were
amplified for confirming aerobic denitrification. Primers NAP1/NAP2
were used for napA amplification using the conditions described by
Kong et al. (2006). Primers nirK1F/nirK5R and nirS1F/nirS6R were
used for nirK and nirS amplification, respectively, which was conducted
as described elsewhere (Braker et al., 2000). All PCR products were
detected and sequenced as the 16S rRNA amplicon.
Assessment of ammonium oxidation with batch experiments.
The isolated bacterium SYF26 was inoculated into the LNM and
cultured at 30 uC for 3–4 days. Two hundred millilitres LNM was
placed in a 500 ml shaking flask, and a 10 % preculture of the isolate
was inoculated, followed by cultivation at 30 uC with shaking at
120 r.p.m. for 112 h. During incubation, the cultures were sampled
periodically to determine the optical density at 600 nm, and the levels
of NH4+-N, NO22-N and NO32-N.
Assessment of nitrite and nitrate removal. Two nitrogen
compounds, nitrite and nitrate, were used instead of ammonium in
the LNM to elucidate the denitrification process of the isolate. NO22N and NO32-N were adjusted to 4.65 mg l21 and 2.50 mg l21,
respectively. A 15 ml cell suspension was inoculated into triplicate
250 ml shaking flasks with 150 ml medium and was incubated
aerobically at 30 uC with shaking at 120 r.p.m. for about 100 h for
nitrate and 112 h for nitrite.
Box–Behnken design for optimizing the environmental factors.
RSM was used to investigate the effects of NH4+-N concentration,
C/N ratio, initial pH and temperature on the activity of heterotrophic
nitrification–aerobic denitrification by the strain SYF26. Ten millilitres enriched culture was transferred to 100 ml liquid medium in
150 ml flasks. The levels of four independent variables were defined
according to the Box–Behnken design, and 28 experiments were
required for the procedure (Table 1). A genuine replicate of the whole
matrix was performed to estimate the experimental error. The
statistical and graphical analyses were performed using the MINITAB
program (version 16; Minitab).
Statistical analysis. The ammonium removal rate was calculated
using the formula (C02Cn)/h. C0 is the initial concentration of
NH4+-N (NO22-N or NO32-N). Cn is the final concentration of
NH4+-N (NO22-N or NO32-N) at hour n, and h is the time of SYF26
treatment.
RESULTS AND DISCUSSION
Isolation and identification of strain SYF26
A heterotrophic nitrification–aerobic denitrification bacterium, SYF26, was isolated from the Hei He reservoir.
The strain SYF26 is a Gram-negative, non-motile, rodshaped bacterium with a size of 0.5–0.8 mm60.9–1.3 mm.
Approximately 1450 bp of 16S rRNA sequences were
obtained via PCR and sequencing. A phylogenetic tree
was reconstructed based on the 16S rRNA gene sequence
of the isolate and some other phylogenetically related
strains (Fig. 1). The results indicated that strain SYF26 was
most closely related to Acinetobacter sp. PHEA-2 (similarity
100 %). Therefore, strain SYF26 is proposed to be an
Acinetobacter species.
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Microbiology 161
Heterotrophic nitrification at low nutrient conditions
Table 1. The Box–Behnken experimental design along with the corresponding responses
Run
NH4+-N concentration (mg l”1) (X1)
C/N ratio (X2)
Initial pH (X3)
Temperature (6C) (X4)
Response (ADR %)
5.23
15.70
26.17
15.70
15.70
15.70
15.70
15.70
15.70
15.70
26.17
15.70
26.17
15.70
5.23
26.17
15.70
15.70
5.23
15.70
5.23
26.17
15.70
5.23
26.17
5.23
15.70
15.70
4.50
6.00
3.00
3.00
4.50
4.50
6.00
4.50
4.50
3.00
4.50
6.00
6.00
4.50
4.50
4.50
4.50
3.00
6.00
3.00
3.00
4.50
4.50
4.50
4.50
4.50
6.00
4.50
7.00
7.00
7.00
8.00
7.00
6.00
7.00
8.00
7.00
7.00
7.00
6.00
7.00
7.00
6.00
6.00
6.00
7.00
7.00
6.00
7.00
7.00
8.00
7.00
8.00
8.00
8.00
7.00
33.00
27.00
30.00
30.00
30.00
27.00
33.00
27.00
30.00
33.00
27.00
30.00
30.00
30.00
30.00
30.00
33.00
27.00
30.00
30.00
30.00
33.00
33.00
27.00
30.00
30.00
30.00
30.00
76.00
68.20
80.33
88.73
90.60
60.10
79.89
89.53
92.78
62.95
69.52
74.15
83.27
93.14
85.72
81.97
81.22
80.04
90.40
78.65
75.33
91.42
73.87
85.86
97.38
90.00
80.14
90.77
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
ADR, Ammonium degradation rate.
87 Acinetobacter calcoaceticus strain CIP 81.8 (NR 114922)
Acinetobacter calcoaceticus strain LMG 1046 (NR 114921)
100
Acinetobacter calcoaceticus strain JCM 6842 (NR 113343)
0.01
100
Acinetobacter calcoaceticus strain ATCC 23055 (NR 114958)
Acinetobacter calcoaceticus strain PHEA-2 (NR 102826)
100
100
SYF26
Acinetobacter baumannii strain ATCC 17978 (NR 074737)
83
Acinetobacter sp. strain ADP1 (NR 074752)
Moraxella catarrhalis strain BBH18 (NR 102953)
100
Psychrobacter sp. strain PRwf-1 (NR 074709)
100
100
Marinobacter sp. strain BSs20148 (NR 074735)
Pseudomonas putida strain F1 (NR 074739)
Pseudomonas putida strain KT2440 (NR 074596)
Pseudomonas aeruginosa strain PAO1 (NR 074828)
100
Pseudomonas stutzeri A1501 (NR 074829)
55
70
Pseudomonas mendocina strain ymp (NR 074727)
Fig. 1. The phylogenetic tree derived from neighbour-joining analysis of partial 16S rRNA sequences.
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831
J. Su and others
Ammonium removal by strain SYF26
The ability of heterotrophic organisms to oxidize ammonium has generally been linked to aerobic denitrification.
Therefore, the utilization of ammonium by the isolate was
investigated using the LNM that contained ammonium as
the sole nitrogen source. The growth of strain SYF26 was
significantly different in LNM compared with that in
nitrite and nitrate media. The OD600 value of strain SYF26
increased significantly in the first 16 h without a lag phase
(Fig. 2). However, the OD600 value decreased after 16 h,
followed by a slow second stage of growth between 16 and
24 h. Growth was recorded under low nutrient conditions,
demonstrating that strain SYF26 is able to tolerate low
nutrient conditions.
Strain SYF26 was able to grow at low NH4+-N concentrations (5.46 mg l21). Although the growth rate of strain
SYF26 was low, under low nutrient conditions, it could
oxidize 5.09 mg NH4+-N l21 after 80 h, and the
nitrification rate was approximately 0.064 mg NH4+N l21 h21; its ability to remove ammonium had not been
reported at low NH4+-N concentrations. Low accumulation of nitrate and nitrite was observed during the entire
ammonium removal experiment, and nitrite concentration
reached a maximum value of 0.014 mg l21 at 96 h. The
production of NO32-N and NO22-N was consistent with
that of known heterotrophic nitrification processes where
ammoniumAhydroxylamineAnitriteAnitrate (Taylor et al.,
2009). It has been reported that the nitrification rate of
P. stutzeri strain T1 was approximately 0.60 mg NH4+N l21 h21 (Guo et al., 2013), which is slightly higher than
that of the strain SYF26 (0.064 mg NH4+-N l21 h21).
However, the initial ammonium concentration of the
previous experiment was 101 mg l21, which is much higher
than that used in the present study. In this study, the
bacterial growth might have been restricted at low nutrient
conditions, which resulted in a low ammonium removal
rate. Previous studies have also shown that heterotrophic
nitrifying activity was affected by nitrogen and carbon
components (Brierley & Wood, 2001; Joo et al., 2005; Kim
et al., 2005).
Utilization of nitrite and nitrate by strain SYF26
under aerobic conditions
To clarify the denitrification by the strain SYF26, two
nitrification intermediates, potassium nitrate and sodium
nitrite, were used as nitrogen sources in the medium.
Changes in the levels of various components in the LNM
during the period of nitrogen removal under aerobic
conditions are shown in Fig. 3. When the initial NO22-N
and NO32-N concentrations were 4.65 and 2.50 mg l21,
the removal efficiency of nitrite and nitrate reached a peak
value of 95.70 and 95.60 %, respectively. A lag period was
observed with both compounds at the very beginning,
which probably resulted from the conversion from nitrite
or nitrate to NH4+-N for bacterial assimilation. The
growth of the strain reached stationary phase after 32 h.
The NO32-N concentration decreased slightly in the first
8 h. From 8 to 48 h, the decrease of NO32-N concentration became substantial. NO32-N concentration did not
decrease to an apparent extent after 48 h. Nitrite slightly
accumulated with the decline of nitrate and then decreased
to a final concentration of 0.02 mg l21 (Fig. 3a). The
same trend was observed for the NO22-N concentration.
NO22-N concentration decreased from 4.65 to 0.20 mg l21,
and the denitrification rate of strain SYF26 was approximately 0.04 mg NO22-N l21 h21 (Fig. 3b).
Richardson et al. (1998) indicated that the heterotrophic
nitrification process and aerobic denitrification process are
5.6
4.8
–
NO2-N
0.040
–
OD600
NO3-N
4.4
0.035
4.0
3.6
0.030
3.2
2.8
0.025
2.4
OD600
Concentration (mg l–1)
0.045
+
NH4-N
5.2
2.0
0.020
1.6
1.2
0.015
0.8
0.4
0.010
0
0
8
16
24
32
40
48
56
64
Time (h)
72
80
88
96
104
112
Fig. 2. Growth of strain SYF26 and ammonium removal at low nutrient conditions.
832
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Microbiology 161
Heterotrophic nitrification at low nutrient conditions
NH+
4 -N
OD600
0.024
0.020
0.016
0.012
8 16 24 32 40 48 56 64 72 80 88 96
OD600
0.028
–
4.5
0.036
Concentration (mg l–1)
NO2-N
0.032
0
5.0
–
NO3-N
4.0
–
NO2-N
NO3-N
NH+
4 -N
OD600
0.040
0.036
3.5
3.0
0.030
OD600
(b)
0.040
–
Concentration (mg l–1)
(a) 2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
2.5
0.025
2.0
1.5
0.020
1.0
0.5
0
0
0.015
8 16 24 32 40 48 56 64 72 80 88 96 104112
Time (h)
Time (h)
Fig. 3. Aerobic utilization of nitrate (a) and nitrite (b) by strain SYF26.
coupled via nitrite or hydroxylamine. Ammonium removal
by heterotrophic micro-organisms has usually been reported
to oxidize NH4+-N to NO22-N or NO32-N and to
simultaneously convert NO22-N or NO32-N to N2O and/
or N2 (Chen et al., 2012; Khardenavis et al., 2007; Robertson
et al., 1988). In comparison, for strain SYF26, conversion of
nitrate was observed during the process of ammonium
removal, and both nitrite and nitrate could be denitrified
efficiently under aerobic conditions. Furthermore, the napA
and nirS genes, which are responsible for the aerobic
reduction of nitrate and nitrite, respectively, were successfully amplified. However, the nirK gene failed to be
amplified in strain SYF26 in this study. Based on these
results, the ammonium removal pathway for strain SYF26
was proposed to be via nitrite, that is NH4+A
NH2OHANO22ANO32, then NO32ANO22AN2 (Chen
et al., 2012; Zhang et al., 2013; Robertson et al., 1988).
Box–Behnken design for optimizing the
environmental factors
An RSM with a Box–Behnken design was used to analyse
the interactive effects of important variables that significantly affect the degradation of ammonium by Acinetobacter
sp. SYF26, including NH4+-N concentration, C/N ratio,
initial pH and temperature. The design matrix and
response of the dependent variable are shown in Table 1.
On the basis of the parameter estimate, the following
quadratic polynomial equation using the coded factors was
given to explain the dependence of ammonium degradation rate on different factors.
YSYF26591.82+0.049X1+0.83X2+4.82X3+1.01X4
23.03X1X2+2.78X1X3+7.94X1X421.02X2X3
+7.19X2X429.19X3X420.20X1228.35X22
23.42X32211.28X42
where YSYF26 is the predicted response of degradation,
and X1, X2, X3, X4 are the coded values of NH4+-N
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concentration, C/N ratio, initial pH and temperature,
respectively.
The t values and the corresponding P values, along with the
regression coefficients, are given in Table 2. Both the linear
and quadratic coefficients of initial pH were the most
significant of all factors; its linear effect was more pronounced
(P,0.0007) than the quadratic effect (P,0.0451). Quadratic
coefficients of C/N ratio (P,0.0001) and temperature
(P,0.0001) also had a considerable influence on the specific
degradation rate. The interaction effect of initial pH and
temperature (P,0.0003) exerted a more pronounced positive
Table 2. The least-squares fit and the parameter estimates
Coefficient of correlation (R2)50.9227; coefficient of determination
(adj R2)50.8394; coefficient of variation54.62 %.
Term
Regression coefficient
t value
91.8221
0.0491
0.8342
4.8198
1.0078
20.2048
28.3453
23.4223
211.2809
23.0306
2.7844
7.9386
21.0212
7.1942
29.1927
48.549
0.045
0.764
4.414
0.923
20.133
25.404
22.216
27.305
21.602
1.472
4.197
20.540
3.804
24.860
Constant
X1
X2
X3
X4
X1X1
X2X2
X3X3
X4X4
X1X2
X1X3
X1X4
X2X3
X2X4
X3X4
P value
,0.0001***
0.9648
0.4585
0.0007***
0.3729
0.8965
0.0001***
0.0451*
,0.0001***
0.1331
0.1648
0.0010**
0.5984
0.0022**
0.0003***
*Significant, 0.01,P value ,0.05.
**Very significant, 0.001,P-value ,0.01.
***Vitally significant, P value ,0.001.
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J. Su and others
increased with increasing temperature from 27.00 uC to
30.08 uC, and then gradually decreased above a temperature of 30.08 uC. At the same time, the ammonium
degradation rate increased with increasing C/N from 3.00
to 4.63, and a further increase in C/N (above 4.63) did not
result in further improvement of the ammonium degradation rate. In the scope of operational costs, the optimum
temperature and C/N ratio were determined to be 30.08 uC
and 4.63, respectively, for achieving the optimum ammonium degradation rate with the maximal value of 91.89 %.
Temperature is an external factor that is well known to play
a significant role in bacterial growth. Increased temperatures result in decreased growth and sometimes cessation
of bacterial growth. The optimal temperature of 29.73 uC
was in accordance with the data reported in the literature,
such as for Providencia rettgeri YL (Taylor et al., 2009) and
Bacillus methylotrophicus strain L7 (Zhang et al., 2012).
influence on ammonium removal than the quadratic effect
of initial pH (P,0.0451). The mutual interactions between
the NH4+-N concentration and temperature (P,0.0010),
and between C/N ratio and temperature (P,0.0022) exerted
a pronounced positive influence on ammonium removal.
Other coefficients were insignificant. These results suggested
that, among the test variables, initial pH and temperature
produced the largest effects on the specific heterotrophic
nitrification–aerobic denitrification rate at low nutrient
conditions.
In view of the main and interactive effects of the four
factors evaluated, the optimal conditions for ammonium
removal were determined to be an NH4+-N concentration
of 22.05 mg l21, a C/N ratio of 4.31, an initial pH of 7.78
and a temperature of 29.73 uC by ridge analysis using the
SAS program (version 9.1.3; SAS). The maximum ammonium degradation rate that can be achieved, according to
the model prediction under the optimal conditions, was
94.98 %. Validation experiments were then conducted in
triplicate at this optimal condition. A degradation value of
about 94.98±0.50 % was obtained in the validation tests,
indicating that the model fit well with the experimental
data.
The response surfaces shown in Fig. 5 demonstrate that a
maximum ammonium removal rate of 90.00 % could be
obtained at an initial pH range of 6.68–8.00 and a C/N
ratio range of 3.56–5.49. The optimization values for these
factors were found to be 7.78 for initial pH and 4.53 for
C/N ratio, with the maximum ammonium degradation
rate of 93.52 %. These results vastly differ from those of
previous studies. C/N515 was determined to be the
optimal condition for Bacillus sp. LY (Zhao et al.,
2010b). The nitrate, ammonium and total nitrogen were
all completely consumed at C/N ratios of 8.00 and 10.00,
but the consumption of both nitrate and ammonium was
minimal at a C/N ratio of 4.00, which was mainly due to
exhaustion of the carbon source (Guo et al., 2013).
However, Zhang et al. (2013) reported that the ammonium
removal rate was the highest at a C/N ratio of 10.00. In
particular, at a C/N of 2.00, the ammonium removal rate
was 91.80 % of that at a C/N of 10.00. The NH4+-N
removal percentage was not significantly different among
3D response surface graphs were plotted to evaluate the
interaction of temperature and C/N ratio, initial pH and
C/N ratio, initial pH and NH4+-N concentration, and the
optimization conditions of ammonium degradation rate.
From the response surface curves and contours, it was
easy and convenient to understand the interaction effects
between two independent variables and to locate the
optimum levels.
Fig. 4 shows the response surface and contours of the
ammonium degradation rate as a function of temperature
and C/N ratio as independent variables. The semi-spherical
response surface of ammonium degradation rate gradually
ADR (%)
33.00
80
31.50
90
D: temperature
ADR (%)
100
80
70
60
33.00
6.00
31.80
5.40
30.60
4.80
29.40
4.20
B: C/N ratio
3.60
D: temperature 28.20
27.00 3.00
30.00
4
85
90
28.50
80
80
27.00
3.00
3.60
4.20
4.80
5.40
6.00
B: C/N ratio
Fig. 4. Response surface of ammonium degradation rate (ADR) by strain SYF26 at low nutrient conditions as a function of
temperature and C/N ratio.
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Microbiology 161
Heterotrophic nitrification at low nutrient conditions
ADR (%)
8.00
100
7.50
C: initial pH
ADR (%)
90
80
70
60
8.00
5.40
7.50
7.00
C: initial pH
6.50
6.00 3.00
6.00
4.80
4.20
B: C/N ratio
3.60
7.00
4
90
6.50
80
85
80
75
6.00
3.00
3.60
4.20
4.80
5.40
6.00
B: C/N ratio
Fig. 5. Response surface of ammonium degradation rate (ADR) by strain SYF26 at low nutrient conditions as a function of initial
pH and C/N ratio.
C/N ratios of 2.00 to 20.00 for Bacillus methylotrophicus
strain L7, with the highest removal percentage of 58.00 %
observed at a C/N ratio of 6.00 (Zhang et al., 2012). At
low nutrient conditions, the C/N ratio did not play an
important role in the process of heterotrophic nitrification of strain SYF26. Taking cost effectiveness into consideration, a C/N ratio of 3.50 was used in subsequent
experiments.
As shown in Fig. 6, strain SYF26 exhibited good
denitrifying ability between pH 6.57 and 8.00 at any
NH4+-N concentration, with an NH4+-N removal percentage of 90 %. It is noteworthy that at the condition of
high NH4+-N concentration (22.63–26.17 mg l21) and pH
(7.64–8.00), the ammonium removal rate could even reach
up to 95.00 %. According to Xu et al. (2010), a Salmonella
aerobic denitrifying bacterium showed the highest denitrification efficiency at pH 7.00, whereas under acidic (pH
,5.50) or alkaline (pH .9.00) conditions, it lost its
denitrification ability. Zhang et al. (2012) reported that
Bacillus methylotrophicus strain L7 showed efficient nitrification ability at an initial pH of 7.00–8.00. Furthermore, a
slightly alkaline environment is conducive to heterotrophic
nitrification, because more free ammonia (NH3) is contained in the medium according to the theory that the
substrate used by ammonia monooxygenase is NH3 and not
NH4+ (Mével & Prieur, 2000).
Conclusions
In this study, a new strain, named SYF26, was isolated from the
Hei He oligotrophic drinking-water reservoir, and identified
ADR (%)
8.00
95
100
7.50
C: initial pH
ADR (%)
95
90
85
7.00
4
90
80
6.50
8.00
7.50
7.00
C: initial pH
6.50
17.79
13.61
26.17
21.98
9.47
6.00 5.23 A: ammonium concentration
85
6.00
5.23
9.42
13.61
17.79
21.98
A: ammonium concentration
26.17
Fig. 6. Response surface of ammonium degradation rate (ADR) by strain SYF26 at low nutrient conditions as a function of initial
pH and NH4+-N concentration.
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835
J. Su and others
as a species of genus Acinetobacter. Strain SYF26 was able to
grow at low NH4+-N concentrations (5.46 mg l21), and the
nitrification rate was 0.064 mg NH4+-N l21 h21. Strain
SYF26 reduced NO32-N or NO22-N. Nitrite reductase and
periplasmic nitrate reductase were detectable. The putative
nitrogen removal process carried out by the strain SYF26
is as follows: NH4+ANH2OHANO22ANO32, then
NO32ANO22AN2. RSM analysis demonstrated that maximum removal of ammonium occurred under the following
conditions: NH4+-N concentration of 22.05 mg l21, C/N
ratio of 4.31, initial pH of 7.78 and temperature of 29.73 uC,
where initial pH and temperature had the largest influence
on the ammonium removal.
Joo, H. S., Hirai, M. & Shoda, M. (2006). Piggery wastewater treatment
using Alcaligenes faecalis strain no. 4 with heterotrophic nitrification
and aerobic denitrification. Water Res 40, 3029–3036.
Khardenavis, A. A., Kapley, A. & Purohit, H. J. (2007). Simultaneous
nitrification and denitrification by diverse Diaphorobacter sp. Appl
Microbiol Biotechnol 77, 403–409.
Kim, J. K., Park, K. J., Cho, K. S., Nam, S. W., Park, T. J. & Bajpai, R.
(2005). Aerobic nitrification-denitrification by heterotrophic Bacillus
strains. Bioresour Technol 96, 1897–1906.
Kim, M., Jeong, S. Y., Yoon, S. J., Cho, S. J., Kim, Y. H., Kim, M. J., Ryu,
E. Y. & Lee, S. J. (2008). Aerobic denitrification of Pseudomonas
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ACKNOWLEDGEMENTS
This work was partly supported by the National Key Technology
Research and Development Program of the Ministry of Science
and Technology of China (2012BAC04B02), State Environmental
Protection Key Laboratory of Microorganism Application and Risk
Control (MARC2012D001) and the Key Laboratory of the Education
Department of Shan Xi Province (12JS051).
Mével, G. & Prieur, D. (2000). Heterotrophic nitrification by a thermo-
philic Bacillus species as influenced by different culture conditions. Can
J Microbiol 46, 465–473.
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pollutants in aquatic sediments. J Hazard Mater 177, 81–89.
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Edited by: A. Ball
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