Aerosol and Air Quality Research, 13: 1126–1132, 2013
Copyright © Taiwan Association for Aerosol Research
ISSN: 1680-8584 print / 2071-1409 online
doi: 10.4209/aaqr.2012.07.0199
Isolation of an Aerobic Denitrifying Bacterial Strain from a Biofilter for Removal
of Nitrogen Oxide
Zhidao Wu1,2, Shaobin Huang1,2,3,4*, Yunlong Yang1,2, Fuqian Xu1,2, Yongqing Zhang1,3*,
Ran Jiang3,5
1
College of Environmental Science and Engineering, South China University of Technology, Guangzhou, China
Guangdong Provincial Key Laboratory of Atmospheric Environment and Pollution Control, South China University of
Technology, Guangzhou, China
3
The Key Laboratory of Environmental Protection and Eco-Remediation of Guangdong Regular Higher Education
Institutions, Guangzhou, China
4
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China
5
Pearl River Water Resources Institute, Pearl River Water Resources Commission, Guangzhou, China
2
ABSTRACT
A strain of bacteria CP1 with high nitrogen removal efficiency was newly isolated from the biofilm of a biofilter for
removal of NOx from flue gas. The isolate was identified as Pseudomonas aeruginosa based on its physiological and
biochemical characteristics and the results of 16S rRNA gene homology analysis. The new isolate had a high denitrifying
ability, removing 98.49% of the nitrate in a 24-h period under aerobic conditions, with no nitrite accumulation. With
regard to the nitrogen balance, the percentage of nitrogen lost in the flask culture was estimated to be 32.3%, which was
presumed to be converted to nitrogen gas. An analysis of its denitrification activity showed that the optimal C/N and
temperature were 12 and 30°C–40°C, respectively. By using glucose, sodium citrate and succinate, CP1 removed nitrate
with high denitrification efficiency. The change in DO did not influence the effects of denitrification when it varied from 0
to 7.2 mg/L. The results show that CP1 could be a good candidate for the process of aerobic denitrification.
Keywords: Aerobic denitrification; Pseudomonas aeruginosa; Nitrogen removal; Isolation and identification.
INTRODUCTION
Nitrogen oxides (NOx) generally comprises of N2O, NO,
NO2, N2O5, N2O3, and NO5. However, this term often
refers specifically to nitric oxide (NO) and nitrogen dioxide
(NO2), both of which are hazardous air pollutants that lead
to the formation of acid rains, airborne particulates, and
increase ground level ozone (Barman and Philip, 2006; Han
et al., 2011). Emissions of NOx in China have undergone
severe increase over the past decade, thus an effective
technology is needed to decrease its concentration in the
air (Chang et al., 2012). However, the traditional methods
used to control NOx emissions from power plant, such as
selective catalytic and non-catalytic reduction, require high
temperatures or the use of catalysts, resulting in high
installation and maintenance costs, and possibly cause
secondary pollution (Flanagan et al., 2002; Jin et al., 2005).
*
Corresponding author. Tel.: 86-20-39380587
E-mail address: chshuang@scut.edu.cn;
zhangyq@scut.edu.cn
In comparison, the biological method by which nitrogen
oxides are converted to nitrogen gas is a promising alternative
(Hsieh et al., 2011). Among these methods, a biotrickling
filter (BF), usually comprising inert packing material which
can provide sufficient surface area for gas/liquid mass transfer
and biofilm growth, is a good example and has been applied
to the flue gas treatment (Jiang et al., 2009a, b).
Denitrification was considered to be an anaerobic process
by previous investigators as the enzyme system was inhibited
under certain oxygen concentrations (Apel and Turick, 1993;
Ferguson, 1994). However, with the increasing number of
reports on denitrifying bacteria, aerobic denitrification
attracted a lot of attention for its easier operation and
higher denitrification rate than anaerobic denitrification.
There are recent reports of aerobic denitrifying species
isolated from canals, ponds, soils, and activated sludge that
can simultaneously utilize oxygen and nitrate as electron
acceptors. These include Paracoccus (Lukow and Diekmann,
1997), Pseudomonas (Kesser et al., 2003), Bacillus (Kim et
al., 2005), Alcaligenes (Robertson and Kuenen, 1983), and
etc.
We had developed a BF system where a mature biofilm
grew in the filter for 18 months with the property to
Wu et al., Aerosol and Air Quality Research, 13: 1126–1132, 2013
remove NOx from desulfurized flue gas at the temperature
of about 40°C to 50°C in a coal-fired power plant. The flue
gas contains 5–10 percent of O2. NOx removal efficiency
reached more than 85% in the best condition when the flow
of the flue gas was approximately 10000 m3/h. In biotrickling
filter system, NOx in the vapor phase are absorbed by the
biofilm and part of NOx can be oxidized into nitrate by both
nitrification and chemical oxidation under aerobic conditions
(Wang et al., 2006; Reddy et al., 2012). Thus, toxic nitrogen
oxides, nitrate and nitrite are reduced to inert nitrogen gas by
denitrifying bacteria in liquid phase. To achieve treatment
system that has a consistently high efficiency, we should
first understand the characteristics of microorganisms resided
in the BF system. Therefore, we isolated one of the dominant
aerobic denitrifier from the BF system and investigated its
denitrification activity under different environment factors
to find an effective kind of aerobic denitrification bacteria
and to provide useful information for operating aerobic
denitrification process in the biofilter system engineering
application.
MATERIALS AND METHODS
Media
To screen the aerobic denitrifiers, we used the modified
seed medium (SM) and bromothylmol blue (BTB) medium.
The SM consisted of (g/L): peptone 8.6, NaCl 6.4, KNO3
1.0, sodium succinate 1.5 at pH 7.0. The formula of BTB
medium included (g/L): KNO3 5.0, Na2HPO4·7H2O 7.9,
KH2PO4 1.5, MgSO4·7H2O 0.1, disodium succinate 15.0,
agar 10.0, BTB (1% in ethanol) 1 mL/L, trace element
solution 2 mL/L at pH 7.0. To test denitrification activity,
we used a modified denitrification medium (DM) containing
the following components (g/L): KNO3 1.0, Na2HPO4·7H2O
7.9, KH2PO4 1.5, MgSO4·7H2O 0.1, disodium succinate 9.4,
trace element solution 2 mL/L at pH 7.0. The trace element
solutions for the bacterial growth consisted of the following
components (g/L): EDTA 50.0, ZnSO4 2.2, CaCl2 5.5,
MnCl2·4H2O 5.06, FeSO4·7H2O 5.0, (NH4)6Mo7O2·4H2O
1.1, CuSO4·5H2O 1.57, CoCl2·6H2O 1.61 at pH 7.0.
Screening of Aerobic Denitrifier
Aerobic denitrifying organisms were enriched from the
biofilm of an on-site BF for removal of NOx from the flue
gas at the Ruiming coal-fired power plant (Guangzhou,
China). The biofilm samples were put into 100 mL of the
SM in 250-mL flasks with eight layers of gauze and
incubated at 30°C on a rotary shaker at 160 rpm for 1d. A
10% (volume ratio) of bacteria suspension was added to
freshly autoclaved SM and incubated for 1d under the
same conditions. This procedure was repeated three times.
After doubling dilution, the resulting bacterial suspension
were streaked onto BTB medium agar plates and incubated
at 30°C for 1–3 days. This method was based on the
increase in the pH of the medium due to consumption of
NO3– by denitrification (Takaya et al., 2003). Blue colonies
were selected from the BTB agar plates, transferred to DM
and incubated on a rotary shaker. The bacteria suspension
with high nitrogen removal rate was streaked onto BTB
1127
again. The rescreening was repeated three times. One of the
main strain was isolated from the biofilm, which was named
CP1.
Identification of the Isolated Bacterium
Standard physiological and biochemical characteristics
were examined according to previously described methods
(MacFaddin, 1984). For pure cultures, 1 mL of log phase
cultures was centrifuged for 1 min at 12,000 rpm. The pallet
was used for DNA extractions with a bacterial genomic
DNA isolation kit (Bioteke Corporation, Beijing, China).
The genes encoding 16S rRNA were amplified by using the
total DNA (0.1 μg) as the template. PCR was performed in
a Mastercycler gradient (Eppendorf 5331, Germany) using
the following primers: 16SF (5′-AGAGTTTGATCATGGC
TCAG-3′) and 16SR (5′-GGTACCTTGTTACGACTT-3′).
Genes were amplified by 30 cycles of denaturation at 94°C
for 1 min, annealing at 56°C for 1 min, and extension at
72°C for 2 min, followed by final extension at 72°C for
7min. The PCR products were sequenced by Invitrogen
Company (Shanghai, China). The 16S rRNA sequences
were examined in blastn (NCBI, USA) for similarities.
Aerobic Denitrification by the Isolated Bacterium
Capacity of Isolate for Aerobic Denitrification
After precultured in SM at 30°C on a rotary shaker at
160 rpm for 12 h, the isolated bacterium (1% v/v) was
inoculated to 100 mL of DM and incubated under the same
condition for 24 h. Samples were taken from the flasks
every three hours for measurement of optical density (OD),
nitrate, nitrite, pH and total nitrogen (TN). All tests were
performed in triplicate.
Effect of Various Conditions
The same amount of isolates was cultivated in flasks, and
1% of those mixed cells were used as an inoculum. The
inoculum was inoculated to modified DM (varying C/N
ratios, carbon sources and dissolved oxygen (DO)) and
incubated under different temperature for 24 h. Disodium
succinate and potassium nitrate were used as the carbon
and nitrogen sources, respectively. The C/N ratios in the
medium were adjusted to 1, 3, 6, 9, 12 and 15 by changing
the amount of the carbon source and by maintaining a
constant nitrogen concentration at 138.5 mg/L. To investigate
the electron donor specificity of CP1, glucose, citrate,
sucrose, acetate, and succinate were added to the DM as
carbon sources, respectively. The concentration of DO and
temperature were adjusted by the rotary shaker. After 24 h
of cultivation under different environment factors, the
concentrations of nitrate, nitrite and OD in the DM were
analyzed. All of the experiments were performed in triplicate.
Analytical Methods
The optical density was determined by spectrophotometry
(TU1810; Pgeneral, China) at a wavelength of 600 nm.
The estimations concentration of NO3–-N and TN were
determined based on standard procedures as described in
Standard Methods (APHA, 1992). Meanwhile, NO2–-N
was determined by colorimetry at a wavelength of 540 nm
Wu et al., Aerosol and Air Quality Research, 13: 1126–1132, 2013
1128
(Murray, 1994). DO and pH were measured with a
multifunctional water quality monitor (Mnlti 340i; Germany
WTW).
Definitions and Efficiency Reporting
The efficiency of denitrification was expressed as
denitrification efficiency (DE) and accumulation rate of nitrite
(NA), defined in Eqs. (1) and (2), as a function of the initial
inorganic-nitrogen (Ci0) and last inorganic-nitrogen (Ci1), the
initial nitrite-nitrogen (Cn0) and last nitrite-nitrogen (Cn1).
 C 
DE  1  i1   100%
 Ci 0 
(1)
 C  Cn 0
NA   n1
 Ci 0
(2)

  100%

RESULTS AND DISCUSSION
Isolation and Identification of the Aerobic Denitrifying
Bacterium CP1
Eight strains were isolated from the first screening and
they formed blue colonies and/or halos on the BTB agar
plates due to an increase of pH. After rescreening, the nitrateremoval rate was examined in DM under aerobic conditions.
One strain, which we designated CP1, had a 97.5% nitrate
removal rate and was selected for further characterization.
It stained gram negative and showed catalase and oxidase
activities (Table 1). Strain CP1 was grown on succinate agar
plates in opaque, round, smooth, sandy beige, wet-looking
colonies. A BLAST search of available data in the DDBJ/
EMBL/GenBank database showed a high similarity (99.0%)
with Pseudomonas aeruginosa strain N002 (Fig. 1). Thus, we
designated this bacterium Pseudomonas aeruginosa strain
CP1.
Aerobic Denitrification by the Isolated Bacterium
Capacity of Isolate for Aerobic Denitrification
The time for nitrate, nitrite, TN reduction and the growth
of strain CP1 are shown in Figs. 2(a) and 2(b).The first six
hours were the lag phase. When cell growth reached the
exponential phase, the cell concentration increased quickly
from 0.099 to 1.133 OD600 (optical density at 600 nm), and
the nitrate and TN concentration decreased rapidly. There
was no significant change during the stationary phase. After
24h, the nitrate had decreased to 2.06 mg/L from the initial
136.87 mg/L with no nitrite accumulation (dentrification
efficiency 98.49%). But the concentration of nitrite increased
at first and then decreased during the whole denitrification
process. Nitrite peaked at 87.8 mg/L after 12 h of cultivation
and was exhausted at 18 h, probably because the activity of
nitrite reductase did not enhance until the concentration of
nitrite accumulated to a certain amount. As shown in Fig.
2(b), the pH increased from 7.13 to 9.23 and TN decreased
from 137.76 to 93.25 mg/L after 24 h of cultivation. From
the nitrogen balance, the percentage of nitrogen lost in the
flask culture was estimated to be 32.3%, which was presumed
Table 1. Taxonomical characteristics of the thermophilic
aerobic denitrifying CP1.
Test
Result
Morphological test
Colony morphology
Round
Margin
Irregular
Elevation
Applanation
Surface
Smooth
Density
Opaque
Pigment
Sandy beige
Gram’s reaction
–
Shape
Rod
Size
Short
Physiological test
Growth temperature 4°C
–
Growth temperature 41°C
+
Growth under anaerobic condition
+
Biochemical test
Oxidation/fermentation (O/F)
O
Catalase test
+
Oxidase test
+
H2S production
–
Voges Proskauer test
–
Gelatin liquefaction
+
Nitrate reduction
+
D-Glucose fermentation
+
Fructose fermentation
+
Lysine decarboxylase
–
ο-Nitrophenyl-β-D-galactopyranoside
–
Amylolysis test
–
Pyocyanin test
+
Methyl red test
–
Arginine dihydrolase
+
Citrate utilization
+
(+) positive reaction; (–) negative reaction.
to be converted to nitrogen gas. These results demonstrate
that CP1 is an aerobic denitrifying bacterium.
Effect of C/N Ratios on Cell Growth and Denitrification
The precultured isolated bacterium (1% v/v) was
inoculated into the DM with different C/N ratios and
incubated at 30°C for 24 h under aerobic conditions. The
effect of C/N on the denitrification efficiency is shown in
Fig. 3. At a C/N ratio of 1, the value of OD600 was extremely
low and the denitrification efficiency was only 5.68%.
Removal efficiency and cell growth both increased as more
carbon was added to the medium. While the C/N ratio was
increased to 12, the denitrification efficiency reached to
97.91% and there was no significant change even if the
C/N ratio continued to grow. However, the value of OD600
was still on the increase while the C/N ratio increased from
12 to 15, probably as a result of the fact that the electron
flow provided energy for cell growth by utilizing oxygen
as electron acceptors while the nitrogen sources were
exhausted. When the C/N ratio was less than 12, the strain
growed at an insufficient carbon concentration, and therefore
the electron flow was too low to provide enough energy for
Wu et al., Aerosol and Air Quality Research, 13: 1126–1132, 2013
cell growth. Because carbon was essential for cell growth
and nitrate reduction processes, the optimal quantity of
carbon was a key parameter in the denitrification process
1129
(Patureau et al., 2000). Therefore, it was important to
optimize the C/N ratio for each denitrifier. In this study,
the optimal C/N ratio of CP1 was 12.
c/mg·L-1
(a)
140
1.4
120
1.2
100
1
80
0.8
nitrate
nitrogen
nitrite
nitrogen
OD
60
40
0.6
0.4
20
0.2
0
0
0
3
6
9
12
t/h
15
18
21
24
140
9.5
130
9
120
8.5
pH
c/mg·L
-1
(b)
OD600
Fig. 1. Phylogenetic tree based on a comparison of the 16 S rRNA gene sequence. The phylogenetic tree was generated
using the neighbor-joining method. Bootstrap values, expressed as percentages of 1000 replications, are given at branching
points. Bar shows two nucleotide substitutions per 1000 nucleotides.
TN
110
8
pH
100
7.5
90
7
0
3
6
9
12
15
18
21
24
t/h
Fig. 2. Temporal variation of nitrite, nitrate and TN concentration, the value of pH and OD600 for aerobic denitrification of
strain CP1.
Wu et al., Aerosol and Air Quality Research, 13: 1126–1132, 2013
1130
denitrification
efficiency
accumulation rate
of nitrite
OD
100.00%
1.2
60.00%
0.9
40.00%
0.6
20.00%
0.3
OD 600
Rate
80.00%
1.5
0.00%
0
1
3
6
C/N
9
12
15
Fig. 3. The effect of C/N ratios on cell growth and denitrification by strain CP1.
Electron Donor Specificity
The denitrification electron donor specificity of CP1 was
investigated in DM containing the same C/N ratio (12) and
initial nitrate concentration (138.5 mg/L) under aerobic
conditions. The use of different carbon sources as electron
donors affected the denitrification activity of this bacterium
(Table 2). Of the carbon sources tested, CP1 removed
nitrate by use of from glucose, citrate, succinate with high
denitrification efficiency and no nitrite accumulated.
However, 23.22% denitrification efficiency and almost
halved accumulation of nitrite indicated that acetate was
not a suitable carbon source for CP1. There was hardly any
removal efficiency when sucrose was used as carbon source.
Effect of Temperature on Cell Growth and Denitrification
To characterize the effect of varying temperature on CP1
for cell activity and aerobic denitrification, flask cultures
were incubated at 20°C, 25°C, 30°C, 37°C, 40°C, 45°C for
24 h under aerobic conditions, respectively. The growth and
denitrification efficiency profiles of denitrifier were plotted
in Fig. 4. At a temperature of 20°C, the cell activity was
inhibited. Therefore, the value of OD600 and denitrification
efficiency had only reached 0.391 and 21.79%, respectively,
in addition of sizeable proportion of nitrite accumulation.
The rise of temperature improved both the growth of strain
CP1 and the removal of nitrogen significantly. When the
temperature was increased to 30°C, the denitrification
efficiency and OD600 quickly increased to a maximum of
97.54% and 1.214, respectively, and nitrite was detected
only at extremely low concentrations. When temperature
was maintained in a range of 30°C to 40°C, there was no
significant change on cell activity. It indicated that the
strain CP1 grew well and maintained a high level of
denitrification efficiency while temperature varied from
30°C to 40°C. Under the condition of 45°C, the OD600 was
slightly lower and the nitrate was almost completely reduced.
However, the rate of nitrite accumulation increased rapidly
to 85.68%. These phenomena could be explained in that
the activity of nitrite reductase was inhibited and thus nitrite
was accumulated at this temperature.
Effect of Dissolved Oxygen
The effect of DO on denitrification efficiency from 138.5
mg/L NO3–-N during a 24-h growth period at 30°C was
investigated (Table 3). Studies have shown that the level in
DO concentration in any solution is strongly affected by
variations in shaking speed (McDaniel et al., 1969; Ho and
Chou, 2000; Joo et al., 2005; Qi et al., 2006). With
increased shaking speeds there was a corresponding increase
in DO concentration. In this study, the adjustments of DO
concentration in cultures were made by changing the
shaking speed levels. In one group of experiments, the flask
was evacuated and pure nitrogen was fully pressurized into
the flask before autoclaving. The mouth of flask was sealed
with a rubber plug and DO concentration was maintained at
essentially zero. The results revealed that the denitrification
efficiencies were not affected by DO concentration and the
strain maintained denitrification efficiency above 95% when
the DO concentration varied from 2.1 to 7.2 mg/L. For the
denitrification efficiency, there was not much difference
between anoxic condition and aerobic condition. The
bacterium performed simultaneous O2 and nitrate respiration.
Most studies considered that there exists a strong
relationship between DO concentration and denitrification
efficiency (Taylor et al., 2009). However, part of existing
research (Su et al., 2001) had the distinct conclusion that
the existence of oxygen did not inhibite the activity of
nitrate reductase and nitrite reductase. The results indicated
that the aerobic denitrifier CP1 was with strong adaptability
in the application for practical engineering in field of NOx
remove from flue gas and denitrification for wastewater
under high oxygen conditions.
CONCLUSION
To explore the characteristic of microorganisms used in
the BF system for removal of NOx from flue gas in a coalfired power plant, a dominant aerobic denitrifier of
Pseudomonas aeruginosa CP1 was isolated from biofilm
in the BF system. The newly isolated strain CP1 had a high
denitrifying ability, removing 98.49% of the nitrate in a
24-h period in an aerobic environment, with no nitrite
accumulation. From the nitrogen balance, the percentage of
nitrogen lost in the flask culture was estimated to be 32.3%,
which was presumed to be converted to nitrogen gas. The
removal ability was not inhibited by high oxygen conditions
Wu et al., Aerosol and Air Quality Research, 13: 1126–1132, 2013
1131
Table 2. The denitrification activity by CP1 using different carbon sources.
Carbon source
Sucrose
Acetate
Glucose
Citrate
Succinate
Denitrification efficiency (%)
0.17 ± 0.06
23.22 ± 2.34
87.34 ± 3.39
92.31 ± 4.16
95.82 ± 3.27
Accumulation rate of nitrite (%)
0.66 ± 0.09
9.23 ± 0.14
0.09 ± 0.01
0
0
Table 3. The effect of DO on denitrification activity by CP1.
DO (mg/L)
0
2.1
3.3
5.1
7.2
Denitrification efficiency (%)
96.90 ± 1.93
95.12 ± 2.21
95.84 ± 1.79
97.50 ± 2.04
96.92 ± 1.76
Accumulation rate of nitrite (%)
0.03 ± 0.02
0.04 ± 0.01
0.13 ± 0.02
0.13 ± 0.01
0.10 ± 0.01
100.00%
1.4
1.2
80.00%
40.00%
20.00%
0.8
0.6
OD600
Rate
1
denitrification
efficiency
accumulation
rate of nitrite
OD
60.00%
0.4
0.2
0.00%
0
20
25
30
37
40
45
T/℃
Fig. 4. The effect of temperature on cell growth and denitrification by strain CP1.
and occured best under conditions of C/N ratio 12, 30–40°C
using glucose, citrate, succinate as carbon source. These
results can be useful information for operating aerobic
denitrification process in the BF system engineering
application. The CP1 was isolated from BF system for
treating NOx and hence, should be suitable for the removal
of NOx by means of immobilized cells. The strain was
shown to have an excellent denitrifying capability under
aerobic conditions. Thus, this strain may also be an attractive
candidate for application in high concentration of nitrate
and nitrite containing wastewater treatment.
ACKNOWLEDGMENTS
This research was financially supported by National
Natural Science Foundations of China (Grants No.
20777019), Guangdong Provincial Department of Science
and Technology Department (Grants No. 2011B090400284
and 2011B010100029).
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Received for review, July 30, 2012
Accepted, November 15, 2012