## 3 Results
### PCR Method Selection for Each Gene
Among the three PCR methods tested for _nifR_ gene amplification, method #3 showed no amplification for the sample nucleic acid, and in method #1, only one band of a specific size was formed. However, method #2 showed sufficient amplification of 40% of the samples (10/25), with a detection sensitivity of \(10^{0}\) (Supplementary Figure S8). Among the three PCR primer sets for _nirB_, a specific band did not appear from any of the 25 surface soil total DNA samples (Supplementary Figure S9). Among the six PCR methods expected to amplify the _nirK_ gene, one or two bacterial DNA samples showed amplification by PCR methods #1, #2, #3, and #6, and 6-16 specific bands were detected from the 25 samples by all the PCR methods. Among these, we selected methods #2 and #3, which showed relatively good sample reaction results (Supplementary Figure S10A) and amplified approximately 64% of the samples (16/25), and #3, which showed > 40% sample coverage (Supplementary Figures S10A,B). Among the six PCR methods for _nirS_ amplification, methods #1, #3, and #6 showed no amplification from the bacteria and sampled nucleic acids, and methods #4 and #5 had relatively small amplicon sizes (244 and 257 bp), with many multi-bands (Supplementary Figure S11). By contrast, method #2 produced an amplicon size of 425 nt, and specific amplifications were found from three bacteria and nine samples (Supplementary Figure S11A), with an average detection sensitivity of \(10^{-1}\)-\(10^{-2}\) (average, \(-1.33\)) (Supplementary Figure S11B). Among seven _nosZ_ PCR methods, method #7 did not react with the bacteria and sampled nucleic acids, and methods #1-3 were amplified from 4-6 bacterial DNA samples. PCR methods #1-6 showed a response to 10-12 of 25 samples (Supplementary Figure S12A). However, method #2 formed similar-sized multi-bands, method #3 showed weak amplification with multi-bands, and method #4 showed few sample reactions and multi-bands. Methods #1, #5, and #6 showed detection sensitivity levels of \(10^{-2}\), \(10^{0}\), and \(10^{0}\), respectively. Method #1 produced an amplicon of 267 bp; this was smaller than those of methods #5 and #6, which amplified 300 and 279 bp, respectively. However, method #1 amplified four bacterial DNA and showed excellent detection sensitivity (\(10^{-3}\)-\(10^{-4}\) in bacteria) and sample reaction (Supplementary Figure S12B). Among the five PCR methods for _nrfA_ gene amplification, methods #1-4 showed no amplification in the total DNA samples. Method #5 produced an amplicon size of 236 bp but formed a band of a specific size in approximately 48% of the samples (12/25), with a detection sensitivity of approximately \(10^{-1}\) (Supplementary Figure S13). Among the three PCR methods to amplify two _nxxrA_ genes and one _nxxB_ gene, responses to six and ten samples were observed for the _nxxrA_ PCR methods #1 and #2, respectively, with a detection sensitivity of \(10^{-1}\). According to the detection sensitivity results, we selected method #1, which also had wide sample coverage. In contrast, no reaction was observed among the methods expected to amplify _nxxB_ (Supplementary Figure S14).