Identifying hotspots of N2O formation in the rhizosphere of young maize plants by combining O2 optodes and N2O microsensors

O₂ deficiency is a main prerequisite for denitrification promoting N₂O formation in soils. Increased microbial activity in the rhizosphere of growing plants promotes microbial respiration, which together with root respiration contributes to high O₂ demand and consumption in the rhizosphere creating favorable conditions for denitrification.

To understand the effect of root growth on N₂O formation in the rhizosphere, we developed a novel rhizobox design allowing to monitor soil O₂ concentrations and N₂O fluxes at high spatial and temporal resolution. Rhizoboxes were filled with 2.2 kg of arable soil with silty loam texture and maize (Zea mays L.) was grown for 3-6 weeks. Soil moisture was kept between 70 and 80 % water-filled pore space. The ‘window side’ of the rhizoboxes was equipped with an O₂-sensitive optode, allowing monitoring of O₂ concentrations in the developing rhizosphere and surrounding soil at high spatial and temporal resolution. Root growth was monitored by photographing roots and analyzed using RootPainter software. Surface N₂O fluxes were determined every two to three days using transparent chambers and a LI-COR Trace Gas Analyzer. N₂O concentrations in the soil profile were measured with N₂O microsensors by piercing through the O₂ optode at selected sites in the rhizosphere and bulk soil. On the last day of the experiment, we sampled regions of interest (ROI, 1.6 cm diameter) from the window site of the rhizoboxes and analyzed them for mineral N, total C and N, and the abundance of N cycling genes involved in denitrification and N₂O reduction. Afterwards, the remaining soil was sampled in layers of 5 or 10 cm and analyzed for mineral N and dissolved organic C (DOC).

Soil water content decreased with increasing root length (R²=0.39). Soil O₂ concentrations were positively correlated with root length (R²=0.80), but negatively with soil water content (R²=0.64). Surface N₂O flux rates differed strongly between replicates, yet the overall flux patterns were similar. Root growth and soil moisture were the main controls of N₂O fluxes as confirmed by a linear mixed effect model including an interaction between total root length and soil water content as fixed factors and replicate as random factor.

Analyses of soil sampled at the end of the experiment showed that NO₃^- and DOC content were highest in the uppermost 5 cm of the rhizoboxes and strongly decreased with depth. Similarly, abundance of bacterial 16S rRNA genes, reflecting the overall size of the bacterial community, and genes for denitrification (nirK) and N₂O reduction (nosZII) decreased with increasing sampling depth, which was associated with the lower resource availability (NO₃^-, DOC) in deeper layers.

N₂O concentrations measured with microsensors in soil ranged between 0 and 100 µmol N₂O L^-1 confirming very high heterogeneity of N₂O formation in soils. Highest N₂O concentrations were found in the direct vicinity of roots. Overall, minimum N₂O concentrations were negatively correlated with maximum O₂ concentrations and maximum N₂O concentrations were positively correlated with total N availability indicating that O₂ controlled the onset of denitrification while N availability controlled its magnitude.