1
Supplementary materials
2
Table S1 The key functional gene markers for soil N2O production and nitrogen cycling
Name
The encoding enzyme
Functioning
Primers
Heterotrophic denitrifiers
napA
The periplasmic nitrate
Reduction of NO3- to v66 TAYTTYYTNHSNAARATHATGTAYGG
reductase
NO2v67 DATNGGRTGCATYTCNGCCATRTT
narG
The membrane-bound nitrate
Reduction of NO3 to narG-F TCGCCSATYCCGGCSATGTC
reductase
NO2narG-R GAGTTGTACCAGTCRGCSGAYTCSG
nirK
The periplasmic copperReduction of NO2 to nirK876 ATYGGCGGVCAYGGCGA
containing nitrite reductase
NO
nirK1040 GCCTCGATCAGRTTRTGGTT
nirS
The heme-containing
Reduction of NO2 to nirSCs3aFm AACGYSAAGGARACSGG
cytochrome cd1 nitrite
NO
nirSR3cdm GASTTCGGRTGSGTCTTSAYGAA
reductase
cnorB
The cytochrome bc-containing Reduction of NO to
norB2 GACAARHWVTAYTGGTGGT
nitric oxide reductase (cNOR) N2O
norB6 TGCAKSARRCCCCABACBCC
nosZ
The multi-copper nitrous oxide Reduction of N2O to nosZ2F CGCRACGGCAASAAGGTSMSSGT
reductase
N2
nosZ2R CAKRTGCAKSGCRTGGCAGAA
AOB
amoA
The ammonia monooxygenase Oxidation of NH3 to amoA1F GGGGTTTCTACTGGTGGT
subunit A
NO2amoA2R CCCCTCKGSAAAGCCTTCTTC
hao
The hydroxylamine
Oxidation of NH2OH haoF1 TGCGTGGARTGYCAC
oxidoreductase
to NO
haoR3 AGRTARGAKYSGGCAAA
cnorB
The cytochrome bc-containing Reduction of NO to
norB1 CGNGARTTYCTSGARCARCC
nitric oxide reductase (cNOR) N2O
norB3 CCYTCVACCCAGASATGCAC
nirK
The copper-containing nitrite
Reduction of NO2 to Cunir3 CGTCTA(C/T)CA(C/T)TCCGC(A/C/G)CC
reductase
NO
Cunir4 GCCTCGATCAG(A/G)TT(A/G)TGG
norYS
The nitric oxide reductase
Reduction of NO to
norY_1143F GGTACCKGGTCATTTKCATTTCT
(sNOR)
N2O
norY_1210R AGTAGGCRAAKCCGATGAGCAT
qnorB
The quinol-dependent nitric
oxide reductase (qNOR)
Reduction of nitric
oxide to N2O
qnorB2F GGNCAYCARGGNTAYGA
qnorB5R ACCCANAGRTGNACNACCCACCA
References
Abed et al.,
2013
Bru et al., 2007
Hallin et al.,
2009
Hallin et al.,
2009
Casciotti and
Ward 2005
Hallin et al.,
2009
Rotthauwe et
al., 1997
Schmid et al.,
2008
Casciotti and
Ward 2005
Casciotti and
Ward 2001
Ishii et al.,
2014
Braker and
Tiedje 2003
NOB
nxrB
AOA
amoA
nirK
DNRA
narG
napA
nrfA
nirB
The nitrite oxidoreductase
Oxidation of NO2- to
NO3-
nxrB169f TACATGTGGTGGAACA
nxrB638r CGGTTCTGGTCRATCA
Pester et al.,
2013
The ammonia monooxygenase
subunit A
The copper-containing nitrite
reductase
Oxidation of NH3 to
NO2Reduction of NO2- to
NO
CrenamoA23f ATGGTCTGGCTWAGACG
CrenamoA616r GCCATCCATCTGTATGTCCA
AnirKa-58F ACBYTATTCGGAAGYACATACACA
AnirKa-579R GYMATTCCGTACATKCCGGA
Tourna et al.,
2008
Lund et al.,
2012
The membrane-bound nitrate
reductase
The periplasmic nitrate
reductase
The cytochrome c nitrite
reductase
The cytoplasmic nitrite
reductase
NO formation from
NO3NO formation from
NO3Reduction of NO2- to
NO or NH3
Reduction of NO2- to
NO or NH3
narG1960f TAYGTSGGSCARGARAA narG2650r
TTYTCRTACCABGTBGC
napA_F CAAGCATATCCGCAGGAAGT
napA_R CTGGACAATGGGATTCAACC
nrfA-F2 ACCTGCTGGAACTGCAARA
nrfA-R5 GTGGAAGCCCACGCTGTT
nir1
GTATTTACAGGAAGCTTATGGGAGGGAATAACGG
nirB-int CACGCAGCATATCTCCTGCTTGACGG
Philippot et al.,
2002
Hanson et al.,
2013
Giacomucci et
al., 2012
Neubauer et
al., 1999
Synthesis of
hydrazine from NO
and NH3
Synthesis of N2H4
from NO and NH3
Oxidation of N2H4 to
N2
Oxidation of N2H4 to
N2
Reduction of NO2- to
NO
Reduction of NO2- to
NO
hzsA_1597F WTYGGKTATCARTATGTAG
hzsA_1857R AAABGGYGAATCATARTGGC
Harhangi et al.,
2012
hzsB_196F ARGGHTGGGGHAGYTGGAAG
hzsB_742R GTYCCHACRTCATGVGTCTG
Not available
Wang et al.,
2012
Li and Gu 2011
hzoF1 TGTGCATGGTCAATTGAAAG
hzoR1 CAACCTCTTCWGCAGGTGCATG
AnnirS379F TCTATCGTTGCATCGCATTT
AnnirS821R GGATGGGTCTTGATAAACA
Not available
Li and Gu 2011
Anommox
hzsA
The hydrazine synthase
hzsB
The hydrazine synthase
hdh
The hydrazine dehydrogenase
hzo
The hydrazine oxidoreductase
nirS
The cd1 nitrite reductase
nirK
The copper-containing nitrite
reductase
Li and Gu 2011
Hira et al.,
2012
3
Table S2 Reported parameters relevant to N2O production for enriched or pure cultures of ammonia-oxidizing microorganisms and denitrfiers
Strains
MY1
MY2
MY3
JG1
AR
CS
Isolation
sources
Taxonomy
pH
Total N2O
production
rates (amol
N2O cell-1 h-1)
N2O yields
(N2O/NO2-)
(%)
δ15NbulkN2O (‰)
δ18ON2O
(‰)
16S
rRNA
gene
References
23.87
-13.53
30.91
21.76
-16.96
29.26
HQ33111
6
JX427546
-16.49
33.56
JX427547
Jung et al.,
2013
Jung et al.,
2013
Jung et al.,
2013
19.62
-15.32
21.59
JF748724
29.83
-12.91
36.11
FJ971112
13.1
-35.54
22.44
JX427545
Site preference (‰)
Via ammonia
oxidation
Via
denitrification
AOA
Nitrosopulimus
AOA
Nitrosopulimus
AOA
Nitrososphaera
6.5
NAa
0.11
6.5
NA
0.11
7.0
NA
0.08
AOA
Nitrososphaera
AOA
Nitrosopulimus
AOA
Nitrosotalea
AOA
Nitrosophaera
AOA
Nitrosopulimus
6.5
NA
0.82
7.5
NA
0.09
5.0
NA
NA
7.5
4.2~4.6
0.07~0.09
NA
NA
NA
FR773157
7.5
NA
0.03~0.05
NA
NA
NA
DQ08509
7
Jung et al.,
2013
Jung et al.,
2013
Jung et al.,
2013
Stieglmeier
et al., 2014
Stieglmeier
et al., 2014
7.8
NA
0.016~0.018
NA
NA
NA
7.8
NA
0.016~0.09
8.7
34
7.0~
7.2
7.5~
7.7
NA
NA
NA
NA
Soil
AOA
Nitrosopulimus
AOA
Nitrosopulimus
AOA
Nitrosopulimus
AOB
NA
NA
14.9
-0.8
-34.0~28.6
8.8~26.
5
HQ33810
9
HQ33810
8
DQ08509
7
BX32185
6
Santoro et
al., 2011
Santoro et
al., 2011
Loscher et
al., 2012
Sutka et al.,
2004
Soil
AOB
7.5~
7.7
NA
NA
33.5
NA
-0.3
38.8
BX32185
6
Sutka et al.,
2006
Soil
AOB
7.5~
7.7
NA
NA
32.5
0.1
-0.3
32.5
L35509
Sutka et al.,
2006
Marine
AOB Nitrosomonas
europaea/
Nitrosococcus
NA
NA
36.3
-10.7
-54.9~15.2
-5~40
NA
Frame and
Casciotti
2010
Agricultural
soil
Agricultural
soil
Tarcontaminate
d soil
Agricultural
soil
Marine
sediment
Acid mine
Nitrosophaera
viennensis
Nitrosopumilus
maritimus
Grassland
soil
Marine
CN75
Marine
CN25
Marine
Nitrosopumilus
maritimus SCM1
Nitrosomonas
europaea ATCC
19718
Nitrosomonas
europaea ATCC
19718
Nitrosospira
multiformis ATCC
25196
Nitrosomonas marina
C-113
Marine
25.09
30.8
34
mobilis’ lineage
AOB
Nitrosospira
Nitrosospira
multiformis ATCC
25196
Nitrosospira tenuis
NV12
Nitrosomonas
europaea ATCC
19718
Soil
7.5~
8.0
7.6
0.08
NA
NA
NA
L35509
Shaw et al.,
2006
7.5~
8.0
7.5~
8.0
2.0
0.06
NA
NA
NA
M96405
58.0
0.45
NA
NA
NA
BX32185
6
Shaw et al.,
2006
Shaw et al.,
2006
Nitrosospira sp.
Strain 40KI
Nitrosospira sp.
Strain NpAV
Nitrosomonas
europaea ATCC
25978
Clay loam
soil
Soil
7.5~
8.0
7.5~
8.0
7.5~
8.0
4.6
NA
NA
NA
NA
X84656
3.9
0.29
NA
NA
NA
Y10127
Nitrosospira briensis
strain 128
Nitrosospira sp.
Strain En13
Nitrosomonas sp.
Soil
AOB Nitrosomonas
europaea/
Nitrosococcus
mobilis’ lineage
AOB Nitrosospira
15.5
0.19
NA
NA
NA
NB07098
2
7.5~
8.0
7.5~
8.0
NA
4.2
0.03
NA
NA
NA
L35505
Craibstone
soil
NA
AOB
Nitrosospira
AOB
5.7
0.70
NA
NA
NA
46
NA
NA
NA
NA
AY85607
9
NA
Nitrosomonas
europaea strain 28
Soil
7.4
229
0.05-1.95
NA
NA
NA
NA
7.5
NA
0.33
-19.88
26.58
BX32185
6
Jung et al.,
2013
Soil
AOB Nitrosomonas
europaea/
Nitrosococcus
mobilis’ lineage
AOB Nitrosomonas
europaea/Nitrosoco
ccus mobilis’
lineage
AOB
Nitrosomonas
europaea ATCC
19718
Soil
Nitrosospira
multiformis ATCC
25196T
Nitrosococcus oceani
strain NS58
Nitrosomonas
europaea ATCC
19718
7.58.0
NA
0.09~0.27
NA
NA
NC_0076
14
Stieglmeier
et al., 2014
Marine
AOB Nitrosococcus
7.8
NA
NA
34.7~36.3
NA
-17.9~-5.8
Soil
AOB Nitrosomonas
europaea/
Nitrosococcus
mobilis’ lineage
Methane oxidizer
7.5
NA
NA
35.9
NA
-15.0
37.8~45
.1
41.8
BX32185
6
Yamazaki et
al., 2014
Yamazaki et
al., 2014
Methylococcus
capsulatus Bath
NA
6.9~
7.1
NA
NA
30.8
NA
0.8
57.3
Soil
Soil
Soil
AOB
Nitrosospira
AOB Nitrosomonas
europaea/
Nitrosococcus
mobilis’ lineage
AOB
AOB
28.94
NA
NA
Shaw et al.,
2006
Shaw et al.,
2006
Shaw et al.,
2006
Shaw et al.,
2006
Shaw et al.,
2006
Goreau et
al., 1980
Remde and
Conrad
1990
Sutka et al.,
2004
Methylosinus
trichosporium ATCC
49234
Pseudomonas
eureofaciens ATCC
13985
Pseudomonas
chlororaphis ATCC
43928
Paracoccus
denitrificans ATCC
35512
Pseudomonas
fluorescens ATCC
13525
Paracoccus
denitrificans ATCC
17741
Fusarium oxysporum
ATCC 12580
Cylindrocarpon
tonkinense ATCC
42055
4
a
NA
Methane oxidizer
6.9~
7.1
NA
NA
35.6
NA
3.4
39.7
NA
Sutka et al.,
2006
NA
Bacterial denitrifier
NA
NA
NA
NA
-0.5
36.7
NA
NA
Sutka et al.,
2006
NA
Bacterial denitrifier
NA
NA
NA
NA
-0.5
12.7
NA
NA
Sutka et al.,
2006
NA
Bacterial denitrifier
7.8
NA
NA
NA
-5.9
15.6
22.4
NA
Yamazaki et
al., 2014
NA
Bacterial denitrifier
7.0~
7.2
NA
NA
NA
23.3
-37.2~14.9
15.7~55
.9
NA
Toyoda et
al., 2005
NA
Bacterial denitrifier
7.0~
7.2
NA
NA
NA
-5.1
-20.0~-7.9
27.9~46
.5
NA
Toyoda et
al., 2005
NA
Fungal denitrifier
NA
NA
NA
NA
37.1
-20.0~-9.3
NA
NA
Fungal denitrifier
NA
NA
NA
NA
36.9
-19.6~-2.6
36.3~38
.6
22.8~40
.0
Sutka et al.,
2008
Sutka et al.,
2008
NA, not available
NA
5
Table S3 Reported isotopic signatures for soil-emitted N2O fluxes from various environments
Site
Amazon tropical forest soils
Tropical agricultural soils
Subtropical agricultural soil
Temperate grassland soil
Temperate grassland soil
Tropical forest soil (nitrification)
Tropical forest soil (denitrification)
Unflooded temperate marsh soil
Flooded temperate marsh soil
Temperate arable soil, Haplic Luvisol
(nitrification)
Temperate arable soil, Gleyic Podzol
(nitrification)
Temperate arable soil, Calcaric Cambisol
(nitrification)
Temperate arable soil, (denitrification)
6
pH
NA
NA
NA
NA
NA
NA
NA
NA
NA
6.1
Site preference (‰) δ15Nbulk-N2O (‰)
9.7
-18.0
1.5
-34.3
9.0
-41.5
6.7
-3.4
2.2
-6.1
-16.8
-91.6
9.4
-39.9
14.8
-5.6
41.8
-12.6
24.3
-52.7
δ18O-N2O (‰)
12.1
26.3
23.3
42.0
26
9.6
27.2
30.6
27.2
25
References
Park et al., 2011
Park et al., 2011
Perez et al., 2001
Yamulki et al., 2001
Bol et al., 2003
Perez et al., 2006
Perez et al., 2006
Bol et al., 2004
Bol et al., 2004
Well et al., 2008
5.6
16
-22.4
20.9
Well et al., 2008
8.0
18.4
-57.5
15.3
Well et al., 2008
6.1
15.3
-34.8
48.4
Well et al., 2006
7
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Bol R, Toyoda S, Yamulki S, et al. Dual isotope and isotopomer ratios of N2O emitted from a
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Giacomucci L, Purdy KJ, Zanardini E, et al. A new non-degenerate primer pair for the
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