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Fig. 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).

Fig. 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 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).

Fig. S3. Neighbour-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.

Fig. 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.

Fig. 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.

Fig. 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 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).

Fig. S7. Phylogenetic relationship of representative bacterial 16S rRNA genes clone sequences generated from control rice field soil at day 2 in experiment I. Neighbour-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.

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.

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