## 4 Discussion  
The results of alternative sucrose supplementation to the raw bulk soil, showing no direct proportion to N\({}_{2}\)O emission (Figure 2), suggested that soil microbial ecosystems in the bulk soil is consisted of diverse community members. Among them, some may show a faster response to sucrose rather than the incomplete denitrifiers. Hence, the soil, that is far from a homogeneous material, probably showed such an alternative response of N\({}_{2}\)O emission to the carbon source added. On the other hand, N\({}_{2}\)O emission-inhibiting effect in the [PERSON]'s mineral solution-based gellan gum soft gel medium was observed only in diquat (**4**) among the herbicides (2-5) and tropolone (**6**) tested at 10 uM (Figure 3). Using gellan gum gel bed for the culture-based N\({}_{2}\)O emission assay, paraquat (**1**) and diquat (**4**), both of which are bipyridylium cation-type Hill reaction inhibitors, showed clear suppression of N\({}_{2}\)O emission by the incomplete denitrifiers of _Pseudomonas_ species [21]. Other chemicals (**2**, **3**, **5**, and **6**) did not show any inhibitory effect on N\({}_{2}\)O emission at 10 uM, indicating that pyridilium cation moiety is necessary for denitrification inhibition. In an autoclaved soil bed, however, two electron-transport inhibiting herbicides, paraquat (**1**) and isouron (**2**) at a final concentration of 50 uM suppressed N\({}_{2}\)O generation from the 2 mM NH\({}_{4}\)NO\({}_{3}\)\({}^{-}\) supplemented model soil bed of fertilized Andisol farmland bulk soil (Figure 4). However, three of the electron-transport inhibiting herbicides (**3**-5), including diquat (**4**), did not show any potent suppressing effect on N\({}_{2}\)O emission. Despite a similar bipyridylium cation structure with **1**, diquat (**4**) did not show any inhibiting activity against N\({}_{2}\)O emission in the soil beds. Thus, N\({}_{2}\)O-producible incomplete denitrification with NO\({}_{3}\)\({}^{-}\) as the substrate was inhibited by **1** and **2** only. These clear differences in the suppressing effects of different Hill reaction inhibitors on the emission of N\({}_{2}\)O from the soil bed culture suggest a specific inhibition of certain oxidoreductase, highly associated with the denitrification process and/or the nitrification process [20]. Conversely, the results shown in Figure 4 probably indicate that not only incomplete denitrifiers but also thermo-tolerant nitrifier is another player in N\({}_{2}\)O emission in the farm soil. In the autoclaved soil, _amoA_-harboring thermo-tolerant archaea may be surviving and emerging as major nitrifiers in the soil bed [32]. In contrast, the inhibitory activity of 100 uM diquat (**4**) on N\({}_{2}\)O quenching by _Chitinophaga_ may indicate that N\({}_{2}\)O production and N\({}_{2}\)O quenching in the soil are separable responses [33]. Oxidoreductase associated with inorganic nitrogen metabolism are often specific to a Hill reaction  
Figure 5: Effect of bipyridylium cation-type Hill reaction-inhibiting herbicides, paraquat, and diquat, on an N\({}_{2}\)O quencher, _Chitinophaga_ strain isolated from Andisol corn farming soil (green arrows). Bars are standard deviation (\(\pm\)SD, \(n\) = 5). inhibiting herbicide. Tropolone (**6**), which inactivates iron-containing heme-dependent cytochrome c or iron-sulfur-cluster containing oxidoreductaseuates showed a relatively high suppression of N\({}_{2}\)O quenching by _Chitinophaga_. Thus, the current data demonstrates the roles of some Hill reaction-inhibiting or other herbicides in the suppression of N\({}_{2}\)O flux from agricultural farming soil [22; 23]. This implication is important because bifunctional herbicides that can effectively prevent the loss of nitrogen by denitrification as well as control the growth of weed can possibly be developed with a molecular design and bio-rational screening systems [9; 10; 34].