Soil samples were collected from the upper 10 cm soil layer of a rice paddy field at Niigata Agricultural Research Institute (37°20′N, 138°45′E), Japan, on April 7 (before the cultivation period) in 2009. Ten soil samples were randomly collected from the field, which were combined together and sieved through a 2-mm mesh, and stored at 4°C until use. The soil type is gley soil, and its physicochemical properties are as follows: pH (H2O): 5.6; total carbon: 14.0 g kg-1 dry soil; and total nitrogen: 1.3 g kg-1 dry soil.
Soil microcosm and isolation
To perform functional single-cell isolation of denitrifying bacteria, a denitrification-inductive soil microcosm was set up according to the method of [Tago et al. (2011]), with some modifications. In brief, 1.0 g of the soil sample was placed in a 10 ml glass serum vial (Nichiden-Rika Glass, Kobe, Japan), which was submerged in sterile MilliQ water (3 ml) for 1 week at 30°C (pre-incubation). A 2.5 ml layer of clear water was removed, and 0.05 mg-C succinate (1.4 μmol) and 0.01 mg-N nitrate (2.1 μmol) were added as an electron donor and acceptor for denitrification, respectively. Nalidixic acid (final concentration, 20 μg g−1 dry soil), pilomidic acid (10 μg g−1 dry soil), and pipemidic acid (20 μg g-1 dry soil) were also added to the soil as bacterial cell division inhibitors. The vial was sealed with a butyl rubber stopper and the headspace air was replaced with Ar-C2H2 (90:10) gas. The prepared soil microcosm was incubated at 30°C for 16 h to elongate bacterial cell ready to grow by succinate assimilation under denitrification-inductive conditions ([Tago et al. 2011]).
After live staining with 5-carboxyfluorescein diacetate-acetoxymethyl ester (CFDA-AM), single elongated cells were individually captured using a micromanipulator under a fluorescent microscope (Functional single-cell isolation, [Ashida et al. 2010]), and transferred into separate 10 ml vials containing 100-fold diluted nutrient broth medium supplemented with 0.3 mM sodium nitrate and 4.4 mM sodium succinate (DNB-NS) liquid medium. After anaerobic cultivation at 30°C for 2 weeks, each cell suspension was streaked onto DNB-NS agar medium and incubated anaerobically at 30°C for 2 weeks to obtain well-isolated single colonies ([Tago et al. 2011]). Denitrifying bacteria was isolated from the incubated soil using the standard agar plate dilution (APD) method. An aliquot (100 Âµl) of the diluted soil suspension was spread onto DNB-NS agar, and incubated anaerobically at 30Â°C for 2 weeks to obtain single colonies.
Measurement of potential activity of denitrification
The potential activities of denitrification, proportion (%) of nitrate reduced to N2O in two weeks, of denitrifying bacteria obtained using the FSC and APD methods were determined with the acetylene-block method ([Tiedje 1994]), as described previously ([Tago et al. 2011]). The vials containing DNB-NS medium were inoculated with each strain, and the head-space air was replaced with Ar-C2H2 (90:10) gas. After incubation at 30°C for 2 weeks, a portion (0.5 ml) of the head-space gas was analyzed for N2O by gas chromatography ([Saito et al. 2008]). The quantity of water-dissolved N2O gas was calculated as the Bunsen absorption coefficient ([Tiedje 1994]). Strains reducing more than 20% of the added nitrate to N2O were considered to be denitrifying bacteria in this study ([Tiedje 1994]; [Tago et al. 2011]). A two-sided, unpaired Student’s t-test was performed to statistically analyze the denitrifying activity.
DNA extraction and PCR amplification
DNA was extracted from single colonies as described previously ([Ashida et al. 2010]), and used for PCR amplification. The bacterial 16S rRNA gene was amplified using the m-27 F (5′-AGRGTTTGATYMTGGCTCAG-3′) primer and the m-1492R (5′-GGYTACCTTGTTACGACTT-3′) primer pair ([Tyson et al. 2004]), as described previously ([Tago et al. 2011]).
Amplified ribosomal DNA restriction analysis (ARDRA)-based profiling
The PCR amplicons of 16S rRNA gene were purified with a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and condensed to a final volume of 20 μl in sterilized-MilliQ water. Aliquots (3 μl) of the DNA samples were individually digested with HaeIII (Toyobo, Tokyo, Japan) and RsaI (Takara Bio), according to the manufacturer’s instructions. To determine the precise length of generated DNA fragments, the digested DNA samples were subjected to MetaPhor Agarose (Takara Bio) gel (3%) electrophoresis. Grouping of the denitrifying isolates was performed based on the ARDRA gel profiles.
Sequencing and phylogenetic analysis
The 16S rRNA gene PCR amplicons derived from one to three representative strains from each ARDRA group, i.e., a total of 47 strains, were purified and directly sequenced as described previously ([Ashida et al. 2010]). DNA sequencing reactions were performed using a BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), according to the manufacturer’s protocol, and analyzed using a PE Applied Biosystems Automated DNA Sequencer (model 3130xl). Based on the gene sequences, taxonomic assignments of the strains at the genus level were performed using RDP Naïve Bayesian classifier, with an 80% bootstrap cutoff ([Cole et al. 2009]). Multiple alignments were performed using CLUSTAL W (ver. 1.83) ([Thompson et al. 1994]). Phylogenetic trees were constructed using the neighbor-joining method with Kimura-2 parameters and 500 bootstrap replicates, in the MEGA4 program ([Tamura et al. 2007]).
Accession numbers of nucleotide sequences
The sequence data of the bacterial 16S rRNA gene produced in this study have been deposited in the DDBJ database under accession numbers AB696837 to AB696882.