# Comparison of PCR Primers for Analyzing Dentirifying Microorganisms in the Hyporheic Zone
## 4 Discussion  
The nitrogen cycle is a microorganism-mediated process that occurs in terrestrial and aquatic ecosystems and is linked between these systems. If nitrogen transported to aquatic ecosystems from terrestrial ecosystems is not cycled, it leads to environmental issues, including eutrophication and an excess of dissolved oxygen [27]. The process of nitrogen removal from wastewater from livestock sheds and chicken houses takes place in aerobic and anaerobic environments. In an aerobic environment, ammonia is oxidized to nitric acid by autotrophic nitrifying microorganisms, and in an anaerobic environment, nitric acid is reduced to nitrogen gas by heterotrophic denitrifying microorganisms [28]. Of the genes identified by the amplification of specific bands from a large number of samples, \(nosZ\) is known to code for nitrous oxide reductase, which reduces nitrous oxide to nitrogen gas; \(nirS\) has been reported to code for nitrite reductase, which reduces nitrogen dioxide to nitric oxide [9]. It has been reported that \(nirK\) is positively correlated [29] and \(nirS\) is negatively correlated [30] with nitrous oxide emission.  
Key denitrifying microbial communities mainly consist of microorganisms that belong to the \(\alpha\), \(\beta\), and \(\gamma\) subclasses of _Proteobacteria_[31], where the key denitrifying microorganism genera are _Agrobacterium_, _Campylobacter_, _Morococcus_, _Digella_, and _Sphingobacterium_[32]. Although denitrification by  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Aqueous** & \(narG\) & \(napA\) & \(nirS\) & \(nirK\) & \(cnorB\) & \(qnorB\) & \(nosZ\) \\
**Sample** & **174** & **152** & **425** & **514** & **389** & **637** & **760** \\ \hline SW*1 & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & \(\bullet\) \\ SW 2 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & & \(\bullet\) \\ SW 3 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & & \(\bullet\) \\ GW*1 & & & & \(\bullet\) & & & & \(\bullet\) \\ GW 2 & \(\bullet\) & & \(\bullet\) & & & & & \(\bullet\) \\ GW 3 & \(\bullet\) & & \(\bullet\) & & & & & \(\bullet\) \\ HW*1 & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\blacktriangle\)* & \(\bullet\) & \(\bullet\) & \(\bullet\) \\ HW 2 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & \(\bullet\) & \(\bullet\) \\ HW 3 & \(\bullet\) & \(\bullet\) & & & & & \(\blacktriangle\) & \\ HW 4 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & \(\bullet\) & \(\bullet\) \\ HW 5 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & \(\bullet\) & \(\bullet\) \\ HW 6 & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & \(\bullet\) & \(\bullet\) \\ HW7 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & \(\bullet\) & \(\bullet\) \\ HW 8 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & \(\bullet\) & \(\bullet\) \\ HW 9 & & & & & & & & \(\bullet\) \\ HW 10 & \(\bullet\) & & & & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 3: PCR primer set validation of denitrification-associated genes using sixteen aqueous environmental samples.  
microorganisms generally occurs under anaerobic conditions, it has been reported to also occur in microaerophilic and aerobic conditions by various bacteria and fungi [33].  
Functional gene-based approaches for studying microorganism communities may be useful for the advancement of ecological theory and predictions regarding how microorganisms respond to environmental change [34]. Although DGGE, temperature gradient gel electrophoresis, and terminal restriction fragment length polymorphism allow for seasonal and regional analysis of a large number of samples, these techniques have drawbacks as only a small number of genes may be cloned and sequence analysis needs to be performed. It has been reported that the use of microarrays (DNA/RNA chips) for the analysis of denitrifying microorganism diversity allows for the analysis of specific microbial communities in nitrogen cycle processes [15].