when the concentration of ammonia was higher [19,20,23–26]. Adaptation to long-term energy stress is believed to be a crucial factor that distinguishes AOA from AOB [27]. Different specific affinities for substrate between AOA and AOB may explain their different growth patterns under low or high ammonia concentra- tions. N. maritimus exhibited a high affinity for ammonia and was able to grow and convert ammonia at an extremely low ammonia concentration (0.14 mg/L), while the ammonia-oxidizing activity was completely inhibited when the ammonia concentration reached 28 mg/L. The affinity of N. maritimus for ammonia can be more than 1,000-fold greater than that of N. europaea [28]. The affinity for ammonia of N. viennensis fell in between N. maritimus and N. europaea, and their growth was totally inhibited at ammonia concentration of 280 mg/L [7]. Other than ammonia concentra- tion, other environmental traits such as pH [29], oxygen concentration [18] and organic carbon [30] could affect the abundance and diversity of ammonia-oxidizing microorganisms and consequently lead to niche separation of AOA and AOB. Paddy soils are an important component of agricultural ecosystems in Asia, and these soils represent one of the major cultivated soil types in China. China has a long history of rice production, and the total area of the paddy soils in the country is 46 M ha [31]. Thus, research on nitrification in paddy soils is of great significance for improving our understanding of nitrogen fluxes in agricultural and soil ecosystems. The abundance and community structure of ammonia-oxidizing microorganisms in paddy soils have been described [32], but the mechanism underlying the response of these microorganisms to ammonia stress is unclear. Therefore, the main objective of this study was to verify the significant role that ammonia concentrations play in the population shifts of AOA and AOB in paddy soils. Materials and Methods Ethics Statement No specific permits were required for the described field studies. Simulated Soil Column and Sampling The flooded paddy soil used in this study was collected from paddy land (30u26’N, 120u19’E) in Zhejiang Province, China. The upper 50 cm of paddy soil was collected and filled into the soil column. The soil column was divided into three layers due to the definition and classification of soil layer in pedology and the setting of sampling ports, the surface layer (2–5 cm), the mid layer (17– 20 cm) and the deep layer (47–50 cm), to investigate the differences in ammonia-oxidizing microorganisms at different depths. The simulated soil column (Fig. 1) was made of plexiglass and had a height of 70.0 cm and a diameter of 12.5 cm.It had a working volume of 2.5 L and a total volume of 3.0 L. Spherical fillings placed at the bottom of the column acted as a filter to avoid loss of biomass. Paddy soil in agricultural ecosystem were usually irrigated with water from river or rainfall, so natural river water were used in this study to simulate the in situ environment of paddy soil. Therefore, the experimental medium was prepared with natural river water (NH4+-N, 0.5360.1 mg/L; NO2-N, 0.160.03 mg/L; NO3-N, 3.7260.5 mg/L, filtrated by filter) adding ammonia, with final ammonia concentration of 42 mg/ L. medium was introduced at the top of the column by spraying (5L per day), and then permeated to the deeper layer. Due to the consumption of microorganisms and adsorption by the soil, an ammonia concentration gradient formed in the soil column from the surface layer to the deep layer (surface layer, 143 mg NH4+-N g21 dry soil; mid layer, 61.9 mg NH4+-N g21 dry soil; deep layer, Effect of Ammonia on Ammonia-oxidizing Microbes 39.9 mg NH4+-N g21 dry soil). The soil column was placed in thermostatic chamber and the temperature was 3061uC. From the top layer to the deep layer, pH of the soil were 7.2460.12, 7.4160.1, 7.0960.15 and the humidity were 38.761%, 36.560.8% and 34.461.5%, respectively. The oxygen concen- tration in the top soil of the column was 2.48 mg/L. Soil sampling was performed every six months, constituting the first stage, the second stage, the third stage and the last stage of the cultivation process. For each layer, triplicate samples were collected in each sampling port shown in Figure1 (from the point close to the sampling connection to the point off the sampling connection) using miniature soil sampler and mixed together. The samples were used to measure ammonium, nitrite and nitrate and a small subsample was stored at 280uC for DNA extraction. Potential Nitrification Rates The potential nitrification rates in the soil layers were determined via batch tests using 150 mL conical flasks containing 80 mL of liquid. The medium for determining the rates contained 3.5 mg/L NH4+-N. Medium with sterilized soil was incubated as a control. Tests were performed in triplicate for 8 hours at 30uC in the dark, and the concentrations of NH4+-N, NO2–N and NO3–N were periodically monitored per 1.5 hours during incubation. Potential nitrification rates were characterized on the basis of the consumption of NH4+-N. In consideration of some ammonia- oxidizing microorganisms which could accumulate substrates intracellularly [33], the results of potential nitrification rates we obtained may be to some extent higher than the real situation. DNA Extraction and PCR Amplification DNA was extracted using the Power Soil DNA Kit (Mo Bio Laboratories, California, USA) following the manufacturer’s instructions. The extracted DNA was examined in a 1.0% agarose gel by electrophoresis. Figure 1. The simulated paddy soil column used in this study. (1) inlet pail, (2) peristaltic pump, (3) timer, (4) main body of soil column, (5) sprayer, (6) beaumontage, (7) outlet pipe, (8) sample connection. doi:10.1371/journal.pone.0044122.g001 PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e44122