# Comparison of PCR Primers for Analyzing Dentirifying Microorganisms in the Hyporheic Zone
## 2 Materials and Methods  
### Study Area and Sampling  
The study area is situated in the Haean basin located in the northeast part of Yanggu County, Kangwon province, northeastern South Korea, along the demilitarized zone (DMZ) between South and North Korea (Figure 2a) [23,24]. Geologically, the \"punchbowl\" shaped basin is composed of Precambrian gneiss at the higher elevations, with a Jurassic biotite granite intrusion that was subsequently eroded in the central portion of the catchment. The basin consists of highly weathered biotite granite at the basin's bottom, surrounded by metamorphic rocks forming mountain ridges. [24].  
Figure 1: Genes related to denitrification in the nitrogen cycle.  
The average annual precipitation in the Haean basin is 1520 mm, with approximately 70% of the rainfall occurring during the monsoon period in June and July [23; 24].  
Figure 2: A map showing the sampling locations. (**a**) Location of study area and sampling points (**b**) Installed piezometers (**c**) Points of monitoring wells and piezometers  
The entire basin has an area of 64 km\({}^{2}\) and is a major agricultural area. Rice paddies cover 11.4% of the arable land, whereas the four most important dryland crops (radish, bean, potato, and cabbage) account for approximately 30% of the cropped area [25]. The use of total fertilizer for crops was 181-259 kg\(\cdot\)N\(\cdot\)ha\({}^{-1}\).yr\({}^{-1}\) in 2010, 248 kg\(\cdot\)N\(\cdot\)ha\({}^{-1}\).yr\({}^{-1}\) in 2011, and 279 kg\(\cdot\)N\(\cdot\)ha\({}^{-1}\).yr\({}^{-1}\) in 2012 [24]. Tributaries draining this area flow into Lake Soyang and ultimately the Han River, providing a major drinking water source to the metropolitan city of Seoul [24]. Groundwater exists throughout the top of the weathered rock and the bottom of the fractured bedrock. The aquifer thickness of this study area was 10 m and the water table ranged from 1.1-8.0 m below the surface in the dry season and from 0.8-5.5 m below the surface in the wet season [24]. The Darcian velocity of the groundwater was estimated to be \(2.1\times 10^{-5}\)-3.0 \(\times 10^{-5}\) cm\(\cdot\)s\({}^{-1}\). The experimental investigation focused on the stream Mandae, a tributary of Soyang. The study site was approximately 25 m long and 15 m wide. The streambed was highly permeable. The investigated stream's reach was characterized by longitudinal sequences of pools and riffles (Figure 2a). The stream was well connected to the aquifer. Therefore, the stream was chosen based on its representativeness regarding characteristic hydro-geomorphic streambed conditions, such as groundwater upwelling and stream water downwelling (Figure 2b). The calculated hyporheic zone depths of the site using heat transfer analysis ranged from 9.2 to 15 cm [22,23]. Moreover, ten hyporheic water samples, three stream water samples, and three groundwater samples were collected in December 2018. At the transect (Figure 2b,c), a total of 10 piezometers were inserted at a 0.1 m depth [23] beneath the streambed adjacent to each seepage meter installation point at regular intervals (Figure 2c). Before water sampling took place, hyporheic water extraction was performed using a peristaltic pump (Precise peristaltic pump BT100-2J, Loggger precision pump Co., Ltd., West Yorkshire, UK) until a specific volume had been extracted and the physicochemical parameters had stabilized (Figure 2c). Water samples were collected in acid-washed, 2000 mL polypropylene bottles for microbial analysis after filtration through 0.20 \(\upmu\)m membrane filters.