Microbial community analysis of nitrifiers producing N2O in the nitrogen-saturated soils by PCR-DGGE method

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Many streams of Kureha Hill, Toyama, Japan, contain high concentration of nitrate. This is believed to be the result of nitrogen saturation, because the nitrogen budget shows the excess leaching of nitrate to the stream water over the nitrogen deposition on the forested watersheds. In the watershed of Hyakumakidani, one of the streams on Kureha Hill, enhanced emission of N_2O by nitrogen saturation has also been observed, even though the soils in the watersheds have been acidified by high-concentration of nitrate. Usually, nitifiers inactivate their nitrification under the pH level less than 5, while the nitrifiers living in the soils of Kureha Hill can still work under the pH of 3.1. The purpose of this study is to identify the species of the nitrifiers which produce N_2O living in the highly-acidified soils of Kureha Hill.The polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method targeting 16S-rDNA combined with DNA-sequencing analysis was applied to identify the nitrifiers. The soils of Kureha Hill, which had been incubated in an incubator for 560days to increase the population of the nitrifiers, were used for the extraction of DNA. 16S-rDNA of the extracted DNA was PCR-amplified with the primers targeting those of nitrifiers. The amplified 16S-rDNA was separated by DGGE method in which different constitution of 16S-rDNA can be separated as a band on a denaturing gradient gel by electrophoresis. As a result, two different bands were seen on the gel which meant that two types of nitrifiers participated in the nitrification in the incubated soils. DNA-sequencing analysis revealed that one of the two types of the nitrifiers showed 98% identity with that of Nitrosospira. sp.