Denitrification is an important process in the global nitrogen cycle. The genes encoding NirK and NirS (nirK and nirS), which catalyze the reduction of nitrite to nitric oxide, have been used as marker genes to study the ecological behavior of denitrifiers in environments. However, conventional polymerase chain reaction (PCR) primers can only detect a limited range of the phylogenetically diverse nirK and nirS. Thus, we developed new PCR primers covering the diverse nirK and nirS. Clone library and qPCR analysis using the primers showed that nirK and nirS in terrestrial environments are more phylogenetically diverse and 2-6 times more abundant than those revealed with the conventional primers. RNA-and culture-based analyses using a cropland soil also suggested that microorganisms with previously unconsidered nirK or nirS are responsible for denitrification in the soil. PCR techniques still have a greater capacity for the deep analysis of target genes than PCR-independent methods including metagenome analysis, although efforts are needed to minimize the PCR biases. The methodology and the insights obtained here should allow us to achieve a more precise understanding of the ecological behavior of denitrifiers and facilitate more precise estimate of denitrification in environments.
Nitrogen is generally the most limiting nutrient for rice production. In rice paddy soils, various biochemical processes can occur regarding N cycling, including nitrification, denitrification, and nitrogen fixation. Since its discovery in the 1930s, the nitrification-denitrification process has been extensively studied in Japan. It may cause N loss from rice paddy soils, while it can also reduce environmental pollutions such as nitrate leaching and emission of nitrous oxide (N 2 O). In this review article, we first summarize the early and important findings regarding nitrification-denitrification in rice paddy soils, and then update recent findings regarding key players in denitrification and N 2 O reduction. In addition, we also discuss the potential occurrence of other newly found reactions in the N cycle, such as archaeal ammonia oxidization, fungal denitrification, anaerobic methane oxidation coupled with denitrification, and anaerobic ammonium oxidation.
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