Nitrate reduction in the reed rhizosphere of a riparian zone: From functional genes to activity and contribution.

The increased nitrogen (N) fertilizer usage caused substantial nitrate (NO3-) leaching into groundwater and eutrophication in downstream aquatic systems. Riparian zones positioned as the link interfaces of terrestrial and aquatic environments are effective in NO3- removal. However, the microbial mechanisms regulating NO3- reduction in riparian zones are still unclear. In this study, four microbial NO3- reduction processes were explored in fine-scale riparian soil horizons by isotopic tracing technique, qPCR of functional gene, high-throughput amplicon sequencing, and phylogenetic molecular ecological network analysis. Interestingly, anaerobic ammonium oxidation (anammox) contributed to NO3- removal of up to 48.2% only in waterward sediments but not in landward soil. Denitrification was still the most significant contributor to NO3- reduction (32.0-91.8%) and N-losses (51.7-100%). Additionally, dissimilatory nitrate reduction to ammonium (DNRA) played a key role in NO3- reduction (4.4-67.5%) and was even comparable to denitrification. Community structure analysis of denitrifying, anammox, and DNRA bacterial communities targeting the related functional gene showed that spatial heterogeneity played a greater role than both temporal and soil type (rhizosphere and non-rhizosphere soil) variability in microbial community structuring. Denitrification and DNRA communities were diverse, and their activities did not depend on gene abundance but were significantly related to organic matter, suggesting that gene abundance alone was insufficient in assessing their activity in riparian zones. Based on networks, DNRA plays a keystone role among the microbial NO3- reducers. As the last line of defense in the interception of terrestrial NO3-, these findings contribute to our understanding of NO3- removal mechanisms in riparian zones, and could potentially be exploited to reduce the diffusion of NO3- pollution.