Root vertical spatial stress drives targeted enrichment of rhizospheric aerobic denitrifying microorganisms for enhanced nitrogen removal in constructed wetlands.

Aerobic denitrification (AD) can overcome the limitations of traditional denitrification in constructed wetlands (CWs) caused by heterogeneous dissolved oxygen distribution and carbon source scarcity, yet effectively enriching functional aerobic denitrifying microorganisms (ADMs) to enhance nitrogen removal efficiency remains a challenge. In this study, the microbial community structure and nitrogen removal pathway were regulated in small-scale CWs by root vertical spatial stress and different plants species. Reducing substrate depth from 60 cm to 10 cm significantly increased the average root number (1.55-fold), radial oxygen loss (ROL, 1.33-fold), and root activity (1.27-fold). Concurrently, the ADMs community in the rhizosphere of shallow constructed wetlands (SCWs) were significantly enriched, raising AD rates from 0.77 to 1.26 to 0.91-1.41 μmol N/(g h) and NO3--N removal contribution from 33 to 35 % to 42-44 %. The average total nitrogen removal efficiency improved from 71.25 % to 74.96 % in CCWs to 93.91 %-98.44 % in SCWs. Furthermore, plant species and growth stages influenced the structure and function of microbial communities. Higher ROL and root activity promoted ADMs enrichment, while root quantity and weight affected denitrification gene abundance. Ketones (3-Methyl-4-Heptanone) and esters (isobutyl butyrate, isobutyl isobutyrate, butyl butyrate) in root exudates facilitated the enrichment of Exiguobacterium and Pseudonocardia. Therefore, high ROL conditions facilitated the colonization of aerobic microorganisms, and these key exudates drove the targeted enrichment of specific ADMs in the rhizosphere. This study provides a novel insight into the enrichment strategy of ADMs, thereby enhancing nitrogen removal in CWs.