Stable isotope probing reveals compositional and functional shifts in active denitrifying communities along the soil profile in an intensive agricultural area

Abstract

Denitrifying microbial communities in the vadose zone play an essential role in eliminating the nitrate leached from agricultural practices. This nitrate could otherwise contaminate groundwater and threaten public health. Here, we utilized stable isotope probing combined with amplicon sequencing and functional gene quantification to inspect the composition and function of heterotrophic denitrifying microorganisms along a 9-m soil profile in an intensive agricultural area. Dramatic differences in the composition of the active denitrifiers were uncovered between the surface soil and deep layers of the vadose zone. The main denitrifying bacterial taxa identified from 13C-DNA fractions were Pseudomonadaceae (Pseudomonas), Rhodocyclaceae (Azoarcus), and Burkholderiaceae in the surface soil (0–0.2 m), and were Pseudomonadaceae (Pseudomonas), Burkholderiaceae, Bacillaceae (Bacillus), and Paenibacillaceae (Ammoniphilus) in the deep layers (0.5–9.0 m). Analysis of the functional genes (nirS, nirK, and nosZ) in isotope-labeled DNA revealed an upward nos/nir ratio with increasing soil depth, which may account for the higher nitrous oxide emission potential in the surface soil, as compared to the deeper sand-rich, low organic carbon layers. This study improves our understanding of active denitrifying microbes in the vadose zone and helps in developing techniques to reduce nitrate pollution in groundwater.