Differential responses of nirK- and nirS-carrying bacteria to denitrifying conditions in the anoxic rice field soil.

Denitrification occurs actively in rice field soils. In the present study, the responses of nirK and nirS denitrifier communities to nitrate addition in the anoxic rice soil were determined through molecular analyses of nitrite reductase genes nirK and nirS and 16S rRNA genes. Denitrification occurred rapidly when nitrate was added at the beginning of anoxic incubation (experiment I). The structure of nirK-type denitrifiers did not change; but their abundance as determined by quantitative (real-time) PCR increased in nitrate treatments compared with control. Both the structure and abundance of nirS denitrifiers remained unaffected in experiment I. The rate of denitrification was slowed down when nitrate was added 20 days after the onset of anoxic incubation (experiment II). The structure and abundance of nirK-type denitrifier community did not respond to nitrate addition; but the nirS community changed substantially in this experiment. The copy number of nirS genes increased by an order of magnitude in the treatments of 5 mM and 10 mM nitrate compared with control. The terminal restriction fragment length polymorphism (T-RFLP) analysis of nirS genes revealed that the 100 bp T-RF substantially increased in the nitrate treatments. Cloning and sequence analysis indicated that this T-RF had similarity of up to 90% with Herbaspirillum sp. T-RFLP profiles of the bacterial 16S rRNA genes also showed that Herbaspirillum sp. increased after nitrate amendments. Collectively, the nirK-type denitrifiers were probably active at the beginning of anaerobic incubation, while the nirS denitrifiers, especially those related with Herbaspirillum sp. probably were more active when anaerobic condition was fully developed.