Identifying Ammonia-Oxidizing Bacteria Active in Highly Acidified Soils by the PCR-DGGE Method

Abstract

The soils in the watersheds on Kureha Hill, Toyama, Japan have been acidified by high-concentration of nitrate owing to nitrogen-saturation. Generally, nitrifiers inactivate their nitrification at a pH level below 5. However, the nitrifiers present in the soils of Kureha Hill continue to be active at the pH of 3.1. The purpose of this study is to identify the species of ammonia-oxidizing bacteria (AOB) as nitrifiers present 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 these AOB. The DNA was extracted from the soil of Kureha Hill, which was incubated in an incubator for 660 days to increase their AOB population. 16S-rDNA of the extracted DNA was PCR-amplified with the primers targeting that of AOB. The amplified 16S-rDNA was separated by the DGGE method, in which the different constitution of 16S-rDNA can be separated into bands on a denaturing gradient gel by electrophoresis. As a result, two different bands were observed on the gel, which meant that two types of AOB participated in the nitrification in the incubated soil. DNA-sequencing analysis revealed that one of the types of AOB showed 98% identity with that of Nitrosospira. sp. En13.