Investigating denitrification from two Missouri claypan soils
Authors:   , Johnson Frank E., II 
Published: December 2023

Denitrification in agricultural soils is responsible for a majority of anthropogenic nitrous oxide (N2O) production, and N2O is a major greenhouse gas with a global warming potential [approximately] 300 times that of carbon dioxide. The objectives of this research were to: 1) compare multiple RNA-based sequencing methods for quantifying denitrification genes in soil; 2) relate denitrification gene abundance in soil to actual and potential denitrification rates in claypan soils; 3) measure actual and potential soil denitrification rates from claypan soils and understand how landscape position influences denitrification; and 4) upscale the estimates of denitrification to the field scale to understand its importance to the N budget in row crop fields. The research sites consisted of two claypan soil fields in Central Missouri. Several sets of soil cores were collected in triplicate in two landscape transects across both fields and N2 and N2O production were measured using a gas flow soil core incubation system. In addition, soil denitrification potential, under non-limiting conditions, was determined on 90 m-grid samples collected from the fields. Potential and actual denitrification rates were not significantly different between fields, but potential denitrification rates were greater by almost two-fold in the toeslope position (p [less than] 0.10) compared to the backslope and summit positions across, fields. In one field, actual denitrification rates were greater in the summit landscape position while rates were greater in the backslope position in the other field. Actual denitrification in these fields predominantly resulted in N2 emissions and N2O accounted for a minor portion of the total flux. Although the high smectitic clay content of upland soils provides environmental conditions suitable for high N2O flux rates, these results suggested that denitrification rates are higher in the toe-slope position due to accumulation of soil C from long-term sediment deposition. Therefore, long-term erosion patterns rather than current or recent crop management systems controlled observed spatial patterns of denitrification on these claypan fields. One set of cores was analyzed for extractable soil RNA, and nosZ gene abundance using three methods: real-time quantitative polymerase chain reaction (RT-qPCR); droplet digital polymerase chain reaction (ddPCR); and nanostring sequencing techniques at two depths (0-15 cm and 15-30 cm). There were significant differences in soil RNA quantities between the two depths, with an average of 54.51 mg RNA kg soil-1 at 0-15 cm and 14.20 mg kg-1 at 15-30 cm. The low soil RNA concentrations in the subsoil prevented quantification of the nosZ gene abundance, and suggested low overall microbial activity below 15 cm depth in these claypan soils. Abundance of nosZ in the surface soil showed that ddPCR resulted in significantly greater gene copy estimates than those of RT-qPCR and nanostring sequencing (p [less than] 0.10). There were no statistical differences between nosZ abundance when comparing RT-qPCR and nanostring sequencing. Variability of nosZ abundance was very minimal in both RT-qPCR and nanostring technologies. Landscape variability of the gene copy estimates in these two fields were not similar to the actual denitrification measurement pattern. These results suggest more research should be conducted to establish the molecular sequencing technique best suited to measure genes involved in denitrification from soil samples or that gene prevalence may not effectively predict denitrification, and on these two Missouri claypan soils, most of the biological community and activity are in the top 15 cm of the soil profile. Actual denitrification, along with other parameters for soil volumetric water content, and soil temperature were used to model and upscale estimates for denitrification at the field scale. Soil O2, temperature, and volumetric water content (VWC) were measured at a depth of 10 cm depth at three landscape positions within each field and were used to establish a relationship between VWC and soil O2 content. It was assumed conditions for denitrification were a soil O2 content [less than or equal to] 5 [percent] and a soil temperature [greater than or equal to] 15oC, and flux estimates were corrected using a Q10 value of 2. For each field, daily and annual denitrification estimates were calculated for years grown under corn or wheat, due to N fertilizer application. Daily total denitrification (N2O+N2) estimates ranged from 0.39 kg N ha-1d-1 to 0.87 kg N ha-1d-1. The highest annual denitrification estimates were for Field 1 in 2016, in which 9.26 kg N ha-1 were estimated. Denitrification accounted for up to 7.6 [percent] of total applied N. There are many facets to denitrification. This study highlights the complex relationship between denitrification and other soil characteristics. Denitrification was more strongly related to differences in soils across landscape position rather than crop management, and served as a major N-loss pathway in both fields. These results will aid in our understanding of denitrification and demonstrates the need for denitrification mitigation strategies.