Supplemental material:
Table S1. Composition of bacterial clone library retrieved from control rice field soil
at day 2 in experiment I.
Table S2. Composition of bacterial clone library of rice field soil retrieved from 5
mM nitrate treatment at day 24 (4 days after nitrate amendment) in experiment II,
Nitrate was amended at 20 days after anoxic incubation.
Figure S1. Concentrations of NO3- (A), NO2- (B) and N2O (C) in experiment I; and
concentrations of NO3- (D), NO2- (E) and N2O (F) in experiment II. Nitrate was added
in soil slurries at the beginning (experiment I) and 20 days later after the onset of
anaerobic incubation (experiment II). In experiment II, the ‘day 0’ on the x-axis
corresponds to ‘day 20’ in the entire incubation period. Data are means ± standard
deviations (SD) (n = 3). These data have been published previously (Yuan and Lu,
2009).
Figure S2. Concentrations of NH4+ in control, 5 mM and 10 mM nitrate treatments in
experiment I (A) and experiment II (B). Nitrate was added in soil slurries at the
beginning (experiment I) and 20 days later after the onset of anaerobic incubation
(experiment II). In experiment II, the ‘day 0’ on the x-axis corresponds to ‘day 20’ in
the entire incubation period. Data were means ± SD (n = 3). For the anoxic incubation,
soil slurries were prepared by adding 10 g of soil (d.w.) to 15 ml of anoxic
double-distilled water in 60-ml serum vials. Vials were flushed with N2, capped with
butyl rubber stoppers, and then incubated statically at 25°C. Nitrate was amended at
day 0 or day 20 to produce the final concentration of 5 mM or 10 mM nitrate in soil
1
slurries. The laboratory incubation was carried out following the protocol as described
in (Yuan and Lu, 2009). NH4+ was determined fluorometrically with Infinite F200
microplate reader (TECAN, Switzerland) (Murase et al., 2006).
Figure S3. Neighbor-joining tree based on partial nirK sequences (514 nucleotide
positions). Clones with >95% sequence similarity were considered to be the same
OTU. KD2CK, nirK clones obtained from control rice field soil after 2 days of anoxic
incubation. The number in parentheses represented clone number of each OTU. In
silico T-RF size was showed after the clone name. GenBank accession numbers of
reference sequences were given in square brackets. Bootstrap values (%) were
generated from 1000 replicates, and the values >50% were shown. The sequence of
aniA from Neisseria gonorrhoea served as outgroup.
Figure S4. Composition of nirK (A) and nirS (B) clone libraries retrieved from
control at day 2 in experiment I. Nitrate was added at the beginning of anoxic
incubation of rice field soil.
Figure S5. Composition of nirS clone libraries retrieved from control (A) and 10 mM
nitrate treatment (B) at day 36 (16 days after nitrate amendment) in experiment II.
Nitrate was added at 20 days after anoxic incubation of rice field soil.
Figure S6. T-RFLP profiles of bacterial 16S rRNA genes at day 2 of experiment I (A);
and in control (B), 5 mM (C) and 10 mM (D) nitrate treatments of experiment II.
Nitrate was added in soil slurries at the beginning (experiment I) and 20 days later
after the onset of anaerobic incubation (experiment II). In experiment II, the ‘day 0’
on the x-axis corresponds to ‘day 20’ in the entire incubation period. The graphs show
2
the percentage of individual T-RFs relative to the total integrated fluorescence (mean
– SD, n = 3). The DNA extraction followed the protocols as described in the legend of
Fig. 2. PCR and T-RFLP analysis of bacterial 16S rRNA genes were performed as
described (Rui et al., 2009).
Figure S7. Phylogenetic relationship of representative bacterial 16S rRNA genes
clone sequences generated from control rice field soil at day 2 in experiment I.
Neighbor-joining phylogenetic trees were constructed from the aligned sequences
using ARB software (Ludwig et al., 2004). GenBank accession numbers of reference
sequences were indicated after the clone name; the in silico T-RF size was given in
parentheses.
References:
Yuan, Q., and Lu, Y.H. (2009) Response of methanogenic archaeal community to
nitrate addition in rice field soil. Env Microbiol Rep 1: 362-369.
Murase, J., Noll, M., and Frenzel, P. (2006) Impact of protists on the activity and
structure of the bacterial community in a rice field soil. Appl Environ Microb 72:
5436-5444.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar et al. (2004)
ARB: a software environment for sequence data. Nucleic Acids Res 32:
1363-1371.
Rui, J., Peng, J., and Lu, Y. (2009) Succession of bacterial populations during plant
residue decomposition in rice field soil. Appl Environ Microb 75: 4879-4886.
3
Table S1
4
Table S2
5
Figure S1:
10
14
Control
5 mM nitrate
10 mM nitrate
8
D
10
6
-
NO3 [mM]
-
NO3 [mM]
A
12
4
8
6
4
2
2
0
0
3
6
9
12
0
15
0
5
10
15
20
25
30
35
40
30
35
40
30
35
40
Time [days]
Time [days]
0.3
1.0
0.8
E
0.2
0.6
-
NO2 [mM]
-
NO2 [mM]
B
0.4
0.1
0.2
0.0
0
3
6
9
12
0.0
15
0
5
10
20
25
Time [days]
Time [days]
10
1400
1200
8
C
1000
F
800
N2O [Pa]
N2O [Pa]
15
600
6
4
400
2
200
0
0
3
6
9
12
15
0
0
5
10
15
20
25
Time [days]
Time [days]
6
Figure S2:
7
Figure S3:
8
Figure S4:
9
Figure S5:
10
Figure S6:
A (experiment I)
100
80
Relative abundance [%]
Relative abundance [%]
100
521 bp
509 bp
271 bp
151 bp
146 bp
142 bp
134 bp
60
40
20
0
CK
5 mM
491 bp
474 bp
459 bp
312 bp
200 bp
172 bp
168 bp
162 bp
152 bp
147 bp
146 bp
144 bp
125 bp
121 bp
94 bp
90 bp
80
60
40
20
0
10 mM
B (experiment II, control)
1
4
Treatment
33
48
Time [days]
C (experiment II, 5 mM nitrate amendment)
80
60
40
20
0
27
491 bp 100
474 bp
459 bp
312 bp 80
200 bp
172 bp
168 bp 60
162 bp
152 bp
147 bp 40
146 bp
144 bp
125 bp 20
121 bp
94 bp
90 bp
0
D (experiment II, 10 mM nitrate amendment)
491 bp
474 bp
459 bp
312 bp
200 bp
172 bp
168 bp
162 bp
152 bp
147 bp
146 bp
144 bp
125 bp
121 bp
94 bp
90 bp
Relative abundance [%]
Relative abundance [%]
100
16
1
4
16
27
33
48
1
4
16
27
Time [days]
Time [days]
11
33
48
Figure S7:
12