Differential responses of soil nirS- and nirK-type denitrifying microbial communities to long-term application of biogas slurry in a paddy soil

Biogas slurry (BS) has been widely used as an organic fertilizer in rice paddy fields in China, with potential impacts on the diversity and activity of many biogeochemically important microbial communities. Among these communities, denitrifiers play a significant role in nitrogen (N) transformation and loss in such ecosystems. Here, we examined the abundance, diversity, and community composition of nirS- and nirK-type denitrifiers in paddy soils after long-term application of BS with different N contents using quantitative PCR and high-throughput sequencing. The experiment included four treatments: 1) mineral fertilizer with 270 kg N ha-1; 2) BS with 270 kg N ha-1; 3) BS with 405 kg N ha-1; and 4) BS with 540 kg N ha-1. Our results showed that BS application increased the soil nitrite reductase (NIR) activity by 5.9-27.5 % compared to mineral fertilizer application. Higher nirS gene abundance was observed in BS fertilized soils with increasing N content and was significantly correlated with NIR activity, but nirK gene abundance did not change with different fertilization intensities. Biogas slurry as a substitute for mineral fertilizer significantly decreased the alpha-diversity of only nirS-type denitrifiers at low application rate, but such negative effect was offset by increasing BS fertilization intensity. The community compositions of both nirS- and nirK-type denitrifiers shifted following changes in fertilizer type or intensity, and were significantly influenced by soil organic carbon (SOC), available phosphorus (AP), zinc (for both groups), and copper (for only nirK-type denitrifiers). The structural equation model indicated that the differences in soil NIR activity were mainly explained by SOC, AP, Zn, and the nirS-type denitrifier communities. Sulfurifustis, Thiobaclillus, Microvirgula and Duganella appeared to be the main genera governing NIR activity in the studied paddy fields. In conclusion, increasing BS application intensity can greatly alter the abundance and structure of the nirS-type denitrifying communities, and to a lesser extent, those of the nirK-type denitrifiers in paddy soils.