1 Aerobic denitrification by Pseudomonas stutzeri TR2 has the

28 Pages·2010·0.38 MB·English

AEM Accepts, published online ahead of print on 21 May 2010
Appl. Environ. Microbiol. doi:10.1128/AEM.01983-09
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Aerobic denitrification by Pseudomonas stutzeri TR2 has the potential to reduce nitrous
2 oxide emission from wastewater treatment plants
3
4 Running title; Aerobic denitrification by Pseudomonas stutzeri TR2
D
o
w
5
n
lo
a
6 Morio Miyahara,1 Sang-Wan Kim,1 Shinya Fushinobu,1 Koki Takaki,1 Takeshi d
e
d
7 Yamada,2,** Akira Watanabe,3 Keisuke Miyauchi,2 Ginro Endo,2 Takayoshi Wakagi,1 fr
o
m
8 and Hirofumi Shoun1* h
t
t
p
:
9 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The /
/
a
e
10 University of Tokyo, Bunkyo-ku, Tokyo 113-8657,1 Faculty of Engineering, Tohoku Gakuin m
.
a
s
11 University, Tagajo, Miyagi 985-8537,2 and Ebara Engineering Service Co., Ltd., 11-1 m
.
o
r
g
12 Haneda Asahi-cho, Ohta-ku, Tokyo 144-8610,3 Japan /

o
n

13 A
p
r
14 * Corresponding author. Mailing address: Department of Biotechnology, Graduate School of il 1
2
,

2
15 Agricultural Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
0
1
9
16 Phone and fax: [phone]. E-mail: [email protected] b
y

g
17 **Present address: Department of Ecological Engineering, Toyohashi University of u
e
s
t
18 Technology, Tempaku-cho, Toyohashi 441-8580, Japan.
1
1 ABSTRACT
2 In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 (strain TR2)
3 produces low levels of nitrous oxide (N O) even under aerobic conditions. We compared the
2
4 denitrification activity of strain TR2 with various denitrifiers in an artificial medium that was
D
o
w
5 derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and
n
lo
a
6 produced little N2O under all conditions tested. Its growth rate under denitrifying conditions d
e
d

7 was near comparable to that under aerobic conditions, showing a sharp contrast to lower fr
o
m
8 growth rate of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to h
t
t
p
:
9 toxic nitrite, even utilizing it as good denitrification substrate. When both nitrite and N O /
2 /a
e
m
10 were present, strain TR2 reduced N O in preference to nitrite as the denitrification substrate.
2 .
a
s
m
11 This bacterial strain was readily able to adapt to denitrifying conditions by expressing the
.
o
r
g
12 denitrification genes, cytochrome cd1 nitrite reductase (nirS) and nitrous oxide reductase /
o
n

13 (nosZ). Interestingly, nosZ was constitutively expressed even under non-denitrifying, aerobic A
p
r
14 conditions, in consistent with our finding that strain TR2 preferred N2O to nitrite. These il 1
2
,

2
15 properties of strain TR2 concerning denitrification are in sharp contrast to those of
0
1
9
16 well-characterized denitrifiers. These results demonstrate that some bacterial species, such as b
y

g
17 strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen u
e
s
t
18 respiration. The bacterium was also shown to contain the potential to reduce N O emission
2
19 when applied to sewage disposal fields.
20
2
1 INTRODUCTION
2 Wastewater treatment processes produce one of the major greenhouse effect gases, nitrous
3 oxide (N O) (7, 25, 30). The global warming potential of N O relative to carbon dioxide
2 2
4 (CO ) is 298 for a 100 year time horizon, and its concentration in the atmosphere continues to
2 D
o
w
5 increase by about 0.26% per year (9). Nitrogen removal in wastewater treatment plants is
n
lo
a
6 essentially based on the activity of nitrifying and denitrifying microorganisms, both of which d
e
d

7 are inhabitants of activated sludge. Nitrifying bacteria aerobically oxidize ammonium to fr
o
m
8 nitrite (NO2–) and nitrate (NO3–), which are then reduced anaerobically by denitrifying h
t
t
p
:
9 bacteria to gaseous nitrogen forms such as N O and dinitrogen (N ). It was long been known /
2 2 /a
e
m
10 that N O can be produced during both nitrification and denitrification processes of
2 .
a
s
m
11 wastewater treatment (3, 19, 23), while the cause of the N O emission during the nitrification
2 .
o
r
g
12 process was not clear. We recently showed, however, that the N2O emission during the /
o
n

13 nitrification process depends on the denitrification by ammonia-oxidizing bacteria A
p
r
14 (Nitrosomonas), using the activated sludge grown under the conditions that mimicked a il 1
2
,

2
15 piggery wastewater disposal (18). On the other hand, it is believed that denitrifying bacteria
0
1
9
16 produce N2O as a by-product when anaerobiosis is insufficient during the denitrification b
y

g
17 process, because N O reductase is the enzyme that is mostly sensitive to oxygen (6). Piggery u
2 e
s
t
18 wastewater, in particular, contains a high concentration of ammonia, and N O emission tends
2
19 to take place during the nitrogen removal process (5, 10). Experiments on the removal of
20 ammonia and organic carbon using the aerobic denitrifier Pseudomonas stutzeri SU2 (24)
21 and the heterotrophic nitrifier-aerobic denitrifier Alcaligenes faecalis No. 4 (16, 17) have
3
1 been reported as examples of bioaugmentation in piggery wastewater treatment. Reduction
2 of N O emission from pig manure compost by addition of nitrite-oxidizing bacteria has also
2
3 been reported (11). However, there have been no reports of methods for reducing N O
2
4 emission by bioaugmentation using aerobic denitrifying bacteria.
D
o
w
5 Takaya et al. isolated the aerobic denitrifying bacterium Pseudomonas stutzeri TR2 (strain
n
lo
a
6 TR2) (26). Denitrification activity of strain TR2 was monitored in batch and continuous d
e
d

7 cultures using denitrification and artificial wastewater media, and the strain was found to fr
o
m
8 keep a distinct activity (producing N2 from NO3–) and produce a very low level of N2O at h
t
t
p
9 dissolved oxygen (O ) concentration of 1.25 mg L–1. Therefore, strain TR2 should be useful :/
2 /a
e
m
10 in the future for reducing N O emission from wastewater treatment plants by
2 .
a
s
m
11 bioaugmentation. To investigate the feasibility of using strain TR2 for future application to
.
o
r
g
12 wastewater treatment processes, we examined its denitrification activity, N2O production, /
o
n

13 growth rate, and expression of denitrifying genes in batch cultures using a medium that A
p
r
14 mimics the composition of nitrogen removal wastewater plants. Comparison of the properties il 1
2
,

2
15 of strain TR2 with those of well-characterized denitrifying bacteria revealed characteristics
0
1
9
16 of the strain that favor denitrification, although it can also respire oxygen. b
y

g
17 u
e
s
t
18 MATERIALS AND METHODS
19 Strains. P. stutzeri TR2 and Ralstonia pickettii K50 were isolated in the previous study
20 (26). Paracoccus denitrificans JCM 20620 and Pseudomonas aeruginosa JCM 2412 were
21 obtained from the Japan Collection of Microorganisms. Pseudomonas stutzeri ZoBell ATCC
4
1 14405 was obtained from the American Type Culture Collection.
2
3 Media. Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) was
4 used for seed culture and preculture. In order to mimic the actual conditions of a sewage
D
o
w
5 disposal field, the basal medium used was derived from a field where a swine herd is kept
n
lo
a
6 (Aichi, Japan). Piggery wastewater, comprising urine, washing water, and feces, was first d
e
d

7 digested by anaerobic methane fermentation, and the resulting supernatant after filtration was fr
o
m
8 used as digestion liquid after methane fermentation of piggery wastewater (DLMF) that h
t
t
p
9 contained about 4000 mg L–1 NH +-N. DLMF was further treated in a membrane sequencing :/
4 /a
e
m
10 batch bioreactor (MSBR) to remove nitrogen after supplementation with acetic acid, to give
.
a
s
11 MSBR-treated water (TW). TW still contained 60 mg L–1 NH +-N and 160 mg L–1 total-N m
4 .
o
r
g
12 (pH 8.3). Mixed liquor medium (MLM) was prepared by mixing TW and DLMF in a ratio of /

o
n
13 59:1, followed by supplementation with a trace element solution (1 ml L–1). This MLM was A
p
r
14 used as the basal medium throughout this work. The trace element stock solution consisted of il 1
2
,

2
15 20% MgSO •7H O, 1% FeSO •7H O, 1% FeCl •7H O, 0.2% ZnSO •7H O, 0.4%
4 2 4 2 3 2 4 2 0
1
9
16 CuSO4•5H2O, 0.05% NaMoO4•2H2O, 0.01% MnCl2•4H2O, 0.01% H3BO4, 0.03% Na2SeO3, b
y

g
17 and 1% citric acid. Before cultivation, the media were sterilized by filtration through a u
e
s
t
18 membrane filter (pore size 0.2 µm).
19
20 Seed culture and preculture. The denitrifying bacteria were inoculated from an LB agar
21 plate to 5 mL of LB in a test tube, and the tube was shaken at 150 rpm at 30°C for 24 h (seed
5
1 culture). Two milliliters of seed culture were transferred to 200 mL of LB medium in a
2 500-mL baffled Erlenmeyer flask, and the flask was shaken aerobically at 150 rpm at 30°C
3 for 20 h (preculture). The preculture was collected by centrifugation, washed twice with
4 MLM, suspended with an appropriate volume of MLM to adjust the optical density (OD)
D
o
w
5 (re-suspended preculture), and then transferred to the main cultures.
n
lo
a
6 d
e
d

7 Culture. The growth of various bacterial strains between aerobic and denitrifying fr
o
m
8 conditions was compared by a culture in L-shaped test tube (Monod tube) (Fig. 1). Nine h
t
t
p
:
9 microliters of the re-suspended preculture, whose OD at 660 nm (OD ) was adjusted to 5.0, /
660 /a
e
m
10 was inoculated into 9 mL MLM medium (pH 6.8) in an L-shaped test tube (27 mL volume).
.
a
s
m
11 Thus the initial OD of each culture was 0.005. Sodium acetate (10 mM) and the indicated
660 .
o
r
g
12 amount of 15N-labeled NO3– or NO2– were added to the MLM medium. For aerobic culture, /
o
n

13 the test tube was plugged with an air-permeable foam plug and shaken at 120 rpm on a A
p
r
14 reciprocal shaker at 30°C. For denitrifying culture, the test tube was sealed with a double il 1
2
,

2
15 butyl rubber stopper after purging the headspace with argon gas and replacing its portion
0
1
9
16 (1%) with O2 gas using a gas-tight syringe (Hamilton, Reno, NV). b
y

g
17 For characterizing denitrification by strain TR2 and P. denitrificans under varying u
e
s
t
18 conditions, cultures in test tubes were employed (Figs. 2, 3, and 5). MLM (8.3 mL),
19 supplemented with the indicated amounts (final concentrations) of sodium acetate and
20 15N-labeled NO – or NO – (pH 8.3), was placed in a 50-mL test tube, which was sealed with a
3 2
21 double butyl rubber stopper after purging its headspace with argon gas, with or without
6
1 subsequent replacement of its proportion (3%) with O gas. To examine N O utilization (Fig.
2 2
2 3), 2.1 mL (2.5 mmol L–1 of head space gas; total, 84 µmol) N O gas was also injected.
2
3 Precultures of strain TR2 and P. denitrificans were harvested at late exponential phase (OD
660
4 = 2.5 for strain TR2 and 5.0 for P. denitrificans), re-suspended in MLM, injected using a
D
o
w
5 gas-tight syringe to give the same bacterial concentrations as grown (OD = 2.5 and 5.0,
660 n
lo
a
6 respectively) and the final volume of 16.6 mL, and then cultured. For mixed culture with d
e
d

7 activated sludge grown in MSBR (Fig. 5), a re-suspended preculture of strain TR2 was mixed fr
o
m
8 with the sludge and injected in the same manner as above to give the final concentrations of h
t
t
p
9 microorganisms as: strain TR2, 1 g L–1 in dry weight; sludge, indicated. The test tube for each :/
/
a
e
m
10 culture (Figs. 2, 3, 5) was shaken at 120 rpm at 30°C, and the gas phase was monitored
.
a
s
m
11 periodically using gas chromatography-mass spectrometry (GCMS).
.
o
r
g
12 When periodical extraction of culture broth (sampling) was required to monitor bacterial /

o
n

13 growth and gene expression, culture was performed on a large scale in a 500-mL baffled A
p
r
14 Erlenmeyer flask containing 200-mL MLM medium supplemented with 50 mM sodium il 1
2
,

2
15 acetate (pH 8.3) (Fig. 4). The preculture was grown in the medium in which powder
0
1
9
16 containing the ingredients of LB (tryptone, yeast extract, and NaCl) was dissolved in TW, pH b
y

g
17 7.0. The preculture was inoculated into the flask so that the initial OD was adjusted to 0.25, u
660 e
s
t
18 giving the amount of cells in dry weight of 19 mg (P. denitrificans) and 28 mg (strain TR2),
19 respectively. The flask was plugged with an air-permeable foam plug. Other details are given
20 in the relevant figure legend.
21 BOD of each culture was 540- (Figs. 1 and 5), 3140- (Figs. 2 and 3), and 2620 mg L–1 (Fig.
7
1 4), respectively.
2 Three replicate experiments were performed for all results, however, error bars were
3 indicated only for Fig. 1, because a small difference in time lag makes a large difference in
4 gas evolution. So a typical result was employed for the presentation.
D
o
w
5
n
lo
a
6 Determinations. Gas was monitored periodically by GCMS with a Shimadzu d
e
d

7 GCMS-GP5050 (Shimadzu, Kyoto, Japan) equipped with a CP-PoraPLOT-Q column fr
o
m
8 (Varian, Palo Alto, CA); total flow was 118 mL min-1, voltage 0.9 V, and the temperature of h
t
t
p
9 the oven, injector, and detector was 50°C. Concentrations of NO –, NO –, and NH + ions were :/
2 3 4 /a
e
m
10 measured by ion chromatography with a Metrohm ion chromatograph 861 Advanced
.
a
s
m
11 Compact IC (Metrohm, Zofingen, Switzerland). Bacterial growth was monitored by
.
o
r
g
12 measuring OD660 with a mini photo 518R photometer (TAITEC, Saitama, Japan; Fig. 1) and /
o
n

13 UV/VIS spectrophotometer (Jasco, Tokyo, Japan; Fig. 4). Dissolved oxygen (DO) A
p
r
14 concentration was measured by a DO meter (ABLE, Tokyo, Japan). il 1
2
,

2
15
0
1
9
16 Quantitative real-time PCR analysis. A primer set comprising EUB 338f (5'-ATC CCT b
y

g
17 ACG GGA GGC AGC-3') (2) and EUB 907r (5'-CCG TCA ATT CMT TTR AGT T-3') (29) u
e
s
t
18 was used to amplify bacterial 16S rRNA. A primer set comprising nirS cd3aF and nirS R3cd,
19 and a set comprising nosZ-F and nosZ 1622R were used to amplify the cytochrome cd nitrite
1
20 reductase (nirS) and nitrous oxide reductase (nosZ) genes, respectively (27). For real-time
21 PCR analysis of denitrification genes, 0.5 mL cell culture suspension was transferred to a
8
1 1.5-mL centrifuge tube containing 10 µL of 10 mg mL–1 rifampicin. After centrifugation, the
2 pellet was quick-frozen in liquid nitrogen and stored at –80°C before extraction of RNA.
3 Total RNA was extracted using the TRIzol Reagent kit (Invitrogen, Carlsbad, CA). The
4 sample was treated with DNAse I (Takara, Ohtsu, Japan) and RNase inhibitor (Toyobo,
D
o
w
5 Osaka, Japan), followed by phenol-chloroform extraction and ethanol precipitation. Reverse
n
lo
a
6 transcription (cDNA synthesis) was carried out using the ReverTra Ace -α- kit (Toyobo) and d
e
d

7 random primers. DNA samples to generate the standard curve for real-time PCR analysis fr
o
m
8 were prepared as follows. Genomic DNA was extracted from cells of denitrifying bacteria by h
t
t
p
:
9 phenol-chloroform treatment and amplified using the KAPA2G Fast DNA Polymerase /
/
a
e
m
10 (KAPA Biosystems, Woburn, MA). The PCR product was purified by extracting bands on
.
a
s
11 agarose gel electrophoresis, and a dilution series (50 µL each) was constructed (102 to 108 m
.
o
r
g
12 copies per µL) using EASY Dilution (Takara). Real-time PCR was performed using SYBR /

o
n

13 Premix Ex Taq (Takara) and LightCycler (Roche Diagnostics, Tokyo, Japan), under A
p
r
14 following conditions: initial denaturation, at 95°C for 10 s; followed by 30 cycles each of il 1
2
,

15 which consisted of denaturation for 5 s at 95°C, annealing for 20 s at 50°C (16S rRNA), 58°C 2
0
1
9
16 (nirS), and 60°C (nosZ), respectively, and extension for 15 s at 72°C. b
y

g
17 u
e
s
t
18 RESULTS
19 Comparative analyses of the growth of strain TR2 and other denitrifiers under
20 aerobic and denitrifying conditions. In all experiments of this study, we performed batch
21 cultures of denitrifying bacteria using MLM as the medium that mimics the sewage from a
9
1 piggery. For the growth experiments (Fig. 1), MLM was supplemented with a low
2 concentration of acetate (10 mM) to give the low C/N ratio of 0.85. This mimics the actual
3 sewage disposal field in which carbon sources are always limited. Five denitrifiers were
4 compared with respect to the growth under aerobic and initially microaerobic (denitrifying)
D
o
5 conditions and varying electron acceptors (O , NO –, and NO –) (Fig. 1). Strain TR2 grew the w
2 2 3 n
lo
a
6 most rapidly under all the conditions examined, with shortest lag periods. The other d
e
d

7 denitrifiers than strain TR2 showed little growth or began to grow after a long lag period fr
o
m
8 when incubated under denitrifying conditions. Strain K50 and Ps. aeruginosa did not grow at h
t
t
p
:
9 all during the incubation time of 36 h under the both denitrifying conditions (in the presence /
/
a
e
10 of NO – or NO –; Figs. 1B and 1C). P. denitrificans and Ps. stutzeri Zobell grew well in the m
2 3 .
a
s
11 presence of NO –, however, only after long lag periods (Fig. 1B). They showed a poorer m
3 .
o
r
g
12 growth with a longer lag period in the NO2– medium, respectively (Fig. 1C). By contrast, /
o
n

13 strain TR2 grew well without a lag period even under denitrifying conditions with the same A
p
r
14 rate (0.32 h–1) for the growth in the NO3–- and NO2–-containing media. The growth rate was il 1
2
,

15 near comparable to those for the aerobic growth of the same strain (0.56 h–1) and other 2
0
1
9
16 denitrifiers (Ps. stutzeri Zobell, 0.41; Ps. aeruginosa, 0.39; strain K50, 0.36; P. denitrificans, b
y

g
17 0.32 h–1). Among the other denitrifiers than strain TR2, P. denitrificans exhibited the best u
e
s
t
18 adaptation to the MLM medium. Therefore, we compared strain TR2 with P. denitrificans in
19 the following experiments.
20
21 Strain TR2 produces a low level of N O from nitrite. Time-dependent denitrification of
2
10