## 4 Discussion
### Considerations When Estimating Numbers of NR and DN Bacteria by PCR  
Our results show that the simple microtiter plate assay we used is a valuable complement to methods using denitrifier MPN tubes [65] and to PCR-based methods in detecting culturable NR and DN bacteria. With the microtiter plate assay, 54 strains out of 75 were detected as NR or DN bacteria (Table 2), while just ten strains out of the 54 were confirmed to possess the correct functional genes for nitrate reduction or denitrification after sequencing the PCR product, translating the codons into amino acids, and subjecting the translated sequence to BlastP analysis (Supplemental Table S3, Table 3). Therefore, although primers for nitrate reduction and denitrification genes produced PCR products from a number of strains, many of those products could not be confirmed as the expected sequence after analysis, suggesting caution when these primers are used to estimate population sizes of denittifying bacteria [70; 71]. The lack of amplification of NR and DN gene fragments from strains known to be capable of nitrate reduction or denitrification has been previously reported [68; 69; 72; 73], suggesting that reliance on PCR amplification to determine population sizes could lead to underestimates. In addition, reliance on a limited number of genes and primers may cause some important pathways, such as DNRA, to be overlooked [16]. In our study, 44 strains reduced nitrate/nitrite when grown in nitrate broth in the microtiter plate assay, although no PCR products were produced using primers for nitrate/nitrite reductase genes (Supplementary Table S1) in these 44 strains (Supplementary Table S3). Our results suggest that the primers used in this study likely were not perfectly complementary to the nitrate/nitrite reductase genes in these 44 strains. Our results show that all four isolates (e.g., I-1, I-2, I-4, and I-65) that were confirmed to possess one or more of the denitrification genes showed positive results (e.g., DN positive) in the microtiter plate assay (Table 3). Agreement between the detected requisite genes and the predicted phenotype is not always the case. Two strains of _Bosea_ were confirmed to possess gene sequences with high similarity to _tapA_, although they did not reduce nitrate in the microtiter plate assay (Table 3, Supplementary Table S4). However, [PERSON] et al. (2007a) [68] were able to isolate denitrifying _Bosea_ strains after enrichment using denitrifier MPN tubes. With respect to denitrification, even with having more than one gene in the denitrification pathway, microorganisms may still lack the ability to denitrify under certain conditions due to their inability to express the genes they possess because those genes must be transcribed and translated into the denitrification enzymes and these enzymes must be active under the given environmental conditions [11]. Estimating population sizes of \"denitrifying\" bacteria by qPCR of single denitrification genes is complicated by cases in which microbes possess an incomplete denitrification pathway (e.g., the absence of one or more denitrification genes in the denitrification pathway) and, as a result, denitrification stops before the production of nitrogen gas [70; 74; 75; 76]. Conversely, a substantial proportion of denitrifiers may lack _nosZ_[69], which would confound predictions of denitrification rates and N\({}_{2}\)O emissions if population estimates were based on other genes in the denitrification pathway [70]. On the other hand, there is a wide variety of bacteria that possess _nosZ_ and reduce N\({}_{2}\)O yet do not contain any other denitrification pathway genes [74]. As noted by [PERSON] et al. (2018) [77], these atypical or \"Clade II\" _nosZ_ genes accounted for the majority of _nosZ_ sequences found in a metagenome survey of native prairie soil.