Five Years of Biochar Steers Nitrogen Toward Retention in Deep Paddy Soil

Long-term biochar amendment can quietly rewire the nitrogen economy of alkaline paddy soil, and a new field study suggests the rewiring runs deeper than anyone expected. In a five-year experiment in Ninghe, Tianjin, researchers found that annual applications of rice straw biochar promoted dissimilatory nitrate reduction to ammonium (DNRA) — a pathway that conserves nitrogen in the soil — while suppressing denitrification and anaerobic ammonium oxidation (anammox), the two processes that strip nitrogen away as gas, in the deeper layers of the profile. The work, published open access in Biochar on 8 September 2026, offers some of the most detailed evidence yet that biochar’s effects on soil nitrogen cycling depend dramatically on depth.

The research team, led by Qiannan Yang and Lili Wang with colleagues including Guilong Zhang, Jie Li, Hu Li, and Lukas Van Zwieten, worked in paddy fields on Eutric Cambisols developed from sandy loam deposits, where calcium carbonate from groundwater had accumulated at depth and pushed soil pH above 8. Starting in 2019, they spread rice straw biochar — pyrolyzed at 500 °C for 8 hours under oxygen-limited conditions — on the surface at a modest annual rate of 0.05 tonnes per hectare, incorporating it into the tillage layer with a rotary tiller. After the rice harvest in 2024, they collected soil from four depth intervals: 0–20, 20–40, 40–60, and 60–80 centimeters, in both biochar-amended and control plots.

To disentangle the three competing fates of nitrate, the team used a soil slurry-based 15N tracer pairing technique coupled with membrane inlet mass spectrometry. Fresh soil was homogenized under anaerobic conditions, incubated with water enriched to 100 μM 15N-labeled nitrate, and sampled every six hours over 24 hours. By tracking the production of 29N2, 30N2, and 15NH4+, they could simultaneously quantify potential denitrification, anammox, and DNRA rates — a critical advantage, because measuring only one process risks misjudging total nitrogen flux.

The depth patterns were striking. Across the profile, DNRA rates ranged from 5.03 to 17.90 nanomoles of nitrogen per gram per hour, and the proportion of nitrate flowing through DNRA rose from 25.7 percent near the surface to 91.1 percent at 60–80 centimeters. Denitrification, by contrast, ranged from 1.23 to 37.86 nanomoles per gram per hour, with its share collapsing from 67.9 percent to 7.2 percent with depth. Anammox rates varied from 0.32 to 13.85 nanomoles per gram per hour, its contribution falling from 23.5 percent to just 1.9 percent. In other words, the deeper you dig, the more nitrate is converted back into plant-available ammonium rather than lost as gas.

Biochar’s fingerprint depended on where you looked. In the surface 0–20 centimeter layer, biochar significantly increased the absolute rates of all three processes, and denitrification’s share of nitrate reduction actually rose by 6.2 percent. The authors attribute this to biochar’s porous structure providing habitat for denitrifying bacteria such as Pseudomonas and Micrococcus and anammox bacteria such as Candidatus Brocadia, along with a surge of readily degradable dissolved organic carbon in the root-influenced plow layer. Under those high-carbon conditions, denitrification holds a competitive edge, and it claimed the largest gains.

Below the plow layer, the story inverted. Biochar amendment cut denitrification’s proportional share by 28.8 percent in the 20–40 centimeter layer, 17.8 percent in the 40–60 centimeter layer, and 27.8 percent in the 60–80 centimeter layer, while raising DNRA’s share by 31.4, 22.2, and 37.0 percent respectively. The denitrification-to-total ratio fell by roughly a third in the deeper layers. Anammox proportions declined at every depth, dropping most sharply — by 9.4 percent — in the deepest interval, where rates fell from 10.8 percent to 1.90 percent of nitrate reduction under biochar.

Molecular analysis helped explain the shift. Quantitative PCR detected the napA gene and the denitrification genes nirK, nirS, and nosZ, but the nrfA gene for DNRA and the hzs gene for anammox were below detection, likely due to the high alkaline pH. Overall nitrate-reduction gene abundance plummeted from log10 8.20 to −0.07 copies with depth, with napA falling from 6.06 to 1.05. Biochar had opposing effects by depth: it boosted denitrification gene abundances in the surface layer but significantly reduced all four genes between 20 and 80 centimeters. Sequencing of nirK, nirS, and nosZ communities, deposited in the NCBI Sequence Read Archive, showed distinct community compositions between treatments, with genera such as Thiobacillus and Rhodoferax declining and Pseudomonas and Azoarcus increasing under biochar.

Random forest and structural equation modeling pinpointed the drivers: soil pH, the SOC/NO3− ratio, and Fe2+ concentration emerged as key edaphic controls, acting largely through nosZ and napA gene abundance. The models explained more than 90 percent of the variance in denitrification and DNRA rates, with biochar rate and Fe2+ influencing denitrification indirectly via nosZ (R2 = 0.85) and the denitrification-to-DNRA ratio governed through napA (R2 = 0.95). In surface soil, biochar raised pH from 6.8 to 7.4 and increased SOC/NO3− and Fe2+, stimulating both pathways. In deep layers, however, biochar pushed pH to 8.10–8.12 — past the near-neutral optimum for nosZ enzyme activity, where inhibition sets in above roughly pH 8 to 8.5 — while lowering the SOC/NO3− ratio as labile carbon dwindled.

The fate of the missing nrfA signal is itself revealing. The authors argue that the observed DNRA in deep alkaline soil may be driven substantially by non-biochemical nitrate reduction: under anaerobic conditions, abundant Fe2+ can chemically reduce nitrite directly to ammonium without any nrfA-encoded enzyme. Biochar amendment also increased available phosphorus, which the researchers describe as a protective effect that sustains DNRA-related microbial communities even as dissolved organic carbon declines with depth. Meanwhile, anammox was squeezed from both sides — pH approaching its inhibitory threshold and nitrite scarcity, since suppressed denitrification meant fewer nitrite intermediates and DNRA consumed nitrate toward ammonium instead.

The authors caution that biochar aging — changes in surface area, pore volume, pH, and labile carbon over time — was not resolved in this study and remains a key question for future work. Still, the implications are considerable for the alkaline paddy soils widespread across arid and semi-arid irrigated agriculture. By steering deep-layer nitrate away from gaseous loss pathways and toward ammonium retention, long-term biochar amendment appears to enhance soil nitrogen conservation, potentially supporting plant uptake and yield. The study’s core message is that biochar is not a one-layer intervention: it stimulates nitrogen-transforming activity at the surface while re-partitioning the deep profile toward retention, and any biochar-based nitrogen strategy must account for soil depth and starting pH.

Subject of Research: Effects of long-term biochar amendment on nitrate reduction pathways in deep alkaline paddy soil

Article Title: Long-term biochar amendment increased dissimilatory nitrate reduction to ammonium concomitantly suppressing denitrification and anaerobic ammonium oxidation in deep alkaline paddy soil

Article References: Yang, Q., Zhang, G., Li, J., Li, H., Van Zwieten, L., & Wang, L. (2026). Long-term biochar amendment increased dissimilatory nitrate reduction to ammonium concomitantly suppressing denitrification and anaerobic ammonium oxidation in deep alkaline paddy soil. Biochar, 8(1), Article 136. https://doi.org/10.1007/s42773-026-00645-5

Image Credits: AI Generated

DOI: 10.1007/s42773-026-00645-5

Keywords: biochar, paddy soil, DNRA, denitrification, anammox, nitrogen cycling, 15N tracer, alkaline soil, soil pH, napA, nosZ, nitrogen retention