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Relationships between soil fractions (their mass or carbon (C)) and soil organic carbon (SOC) have been used to develop central ideas in SOC research. However, few attempts have been made to quantify the relationship between SOC and all soil fractions, despite the potential for such an effort to address SOC stabilization processes. We identified 41 published studies that used diverse management techniques to cause a change in SOC concentration and disrupted soil into macroaggregates (>250 μm), free microaggregates (53–250 μm) and free silt + clay ( 250 μm, occluded microaggregates, and occluded silt + clay). We used linear hierarchical models to quantify relationships between mass, C concentration and total C of fractions and SOC. Soil mass redistribution toward macroaggregates was associated with SOC accumulation, however total microaggregate mass (free + occluded) did not increase with macroaggregate mass, as would be expected given de novo microaggregate formation within macroaggregates. Instead, high SOC soils exhibited a greater percent of total microaggregates occluded in macroaggregates. Occlusion in macroaggregates was also associated with increased C concentrations of microaggregates (35% higher, SE = 3.2) and silt + clay (30% higher, SE = 3.9) relative to their free counterparts. Taken together, these relationships suggest reduced macroaggregate turnover promotes SOC accumulation via the stabilization of C into occluded fractions. Rates of SOC increase with silt + clay C concentrations failed to increase with mean site-level SOC concentration, indicating of the studied soils (median SOC concentration = 14 g kg−1; max 68), SOC accumulation appears unlikely to be limited by C storage capacity in the silt + clay fraction. For each unit SOC gain, macroaggregates accounted for 83% (95% CI = 74, 91), and occluded microaggregates for 43% (95% CI = 33, 52), consistent relationships that have potential to be used as benchmarks for fraction-based SOC models.

Soil freeze-thaw (FT) and dry-wet (DW) cycles are brief transitory biophysical changes, but these events have important implications in determining the timing and magnitude of N2O emissions and may represent a significant proportion of annual N2O emissions from agricultural systems. It is often assumed that FT and DW cycles influence the processes of N2O production and emission in a similar manner, however, research has yet to systematically identify the similarities and differences in the mechanisms which lead to potentially higher N2O fluxes during FT compared to DW cycles. Herein, we present the first review to do so; in addition, we identify strategic research areas required for improving the understanding of FT and DW processes leading to N2O emissions. There are key differences between the mechanisms that contribute to N2O fluxes during FT and DW cycles, centered on the duration and spatial extent of anaerobiosis, temperature sensitivity of microbial activity, relative gas diffusivity, and soil water dynamics. These differences might increase the risk of N2O emissions during FT cycles relative to soil DW cycles. Current research gaps include (i) the identification of organic substrates made available due to FT and DW cycles, and their contribution to ensuing N2O fluxes, (ii) an understanding of how cryosuction dynamics potentially influence N2O production and emission, (iii) understanding and predicting the air-entry potential of soil as it relates to N2O fluxes, (iv) identifying the relative significance of dissolved N2O in soil water and its solubility changes during FT and DW phases, and (v) determining microbial community and functional changes across soil spatial and temporal scales. Advances in these areas are recommended for improving process descriptions in biogeochemical models in order to more accurately predict N2O emissions from soils prone to FT and DW cycles.

Soil freeze-thaw induces a pulse of nitrous oxide (N2O) emissions fueled by a concomitant increase in available organic carbon (C) and nitrogen (N) substrates. These substrates are hypothesized to originate from the disruption of aggregates and microbial biomass, but experiments designed to falsify these hypotheses have been scarce. We therefore conducted a series of column experiments using intact soil cores of silt loam and loamy sand under different freezing rates and durations, factors previously identified as controlling the magnitude of subsequent N2O emissions. We used a slower freezing rate and shorter freeze duration (6 days) as a Control, to which we compared the same freeze duration but faster freezing rate (Fast, 6 days) and the same freezing rate but longer freeze duration (Long, 21 days). All soils were frozen to ~ -2.5 °C. Upon thaw, only the silt loam emitted N2O; we therefore focused on silt loam C, N, and aggregate dynamics. Control and Fast soils emitted similar amounts of N2O, even though Fast soils maintained aggregate mean weight diameter (MWD). Long soils emitted 1.8 times more N2O than Control, even though Long and Control soils exhibited similar decreases in aggregate MWD. We suggest that because in no instance were differences in N2O emissions between treatments proportional to differences in aggregate disruption, and some soils emitted N2O without any detectable aggregate disruption, that aggregate disruption does not play a significant, universal role in providing substrates for N2O emissions. In contrast, larger N2O emissions due to longer freezing durations were associated with a delayed decrease in microbial biomass. Although we did not assess microbial communities for features other than their biomass, our observations are consistent with longer freezing durations selecting for slower growing microorganisms and freeze-thaw induced N2O emissions being fueled by microbial osmolytes.

Significant reductions in nitrous oxide (N2O) fluxes have been observed with perennial cropping systems compared to annual crops. However, it is unclear if this reduction in N2O flux is linked to soil microbial communities due to plant-specific characteristics stimulating changes in microbial community structure. The response of microbial communities involved in N2O production to liquid dairy manure (LDM) application and tillage in annual and perennial systems has also not been assessed. Our objectives were to contrast changes in the population sizes and community structures of ammonia oxidizer (represented by amoA and crenamoA gene targets) and denitrifier (nirK, nirS, and nosZ gene targets) communities in differently managed annual and perennial fields, and to determine if differences in these microbial communities were related to observed variation in N2O fluxes. Soil was sampled in 2012 and in 2014 in a 4-ha spring-applied LDM grass-legume (perennial) field and two 4-ha corn (annual) fields under fall or spring LDM application. Soil DNA was extracted and used to target N-cycling genes via qPCR (n = 6) and for next-generation sequencing (Illumina Miseq) (n = 3). Significantly higher field-scale N2O fluxes were observed in the annual fields compared to the perennial system; however N2O fluxes increased 10-fold after plough down of the perennial field. Nonmetric multidimensional scaling (NMS) and multi-response permutation procedure (MRPP) indicated statistical differences in N-cycling communities between annual and perennial cropping systems, and in some cases, communities became indistinct in ordination space between annual and perennial fields after ploughing. Indicator species analysis was used to identify sequences clustered into operational taxonomic units (OTUs) most responsible for community shifts related to management. The abundance of N2O-reducing soil microbial communities (nosZ gene copies) and specific nirK, nirS and nosZ OTUs were proposed as important with respect to the production and consumption of N2O in these soils. Our results illustrate that ammonia oxidizer and denitrifier soil bacterial communities are sensitive to agricultural management (annual or perennial crop type, LDM management, and ploughing), highlighting their significance for the development of effective N2O mitigation strategies.

Crop residues are sources of carbon and nitrogen (N) after harvest, releasing inorganic N through mineralization or protecting soil N through immobilization. Inorganic N controls nitrous oxide (N2O) emissions, a potent greenhouse gas (GHG) from agriculture. Hence, crop residues are accounted for as N2O sources in national GHG inventories. For locations where post-harvest N2O emissions occurs due to freeze-thaw, it is not known if crop residues contribute to emissions, and if tillage or residue type impact this contribution. This is of concern since crop residue and freeze-thaw emission factors (EF) may be ‘double-counting’ N2O sources. We conducted an experiment over two non-growing seasons (NGS) in a long-term corn, soybean and winter wheat trial to (i) compare N2O emissions for different crop residues within simple or diverse crop rotations under no-tillage (NT) or conventional tillage (CT); (ii) determine the importance of above- and below-ground residue addition to spring-thaw N2O emissions as affected by rotation and tillage. A15N residue enrichment study was used to directly trace above- and below-ground residue 15N into 15N2O fluxes and derive EF. Higher N2O emissions were observed for CT than NT, regardless of rotation. Soybeans induced higher N2O emissions than corn residue and the same crop residue (e.g. corn or soybean) showed trends of higher N2O in the long-term diverse rotation. In all cases, crop residues contributed minimally to spring-thaw N2O emissions (

Agriculture accounts for a majority of global soil nitrous oxide (N2O) emissions. Increased crop diversity can change soil nitrogen (N), physicochemical properties and alter belowground microbial communities, leading to changes in potential N2O emissions when inorganic N fertilizer is applied. Nitrification and denitrification are two main N cycling pathways under investigation due to their contribution to soil N cycling and production of N2O. The goal of this study was to understand the impacts of 35 years of crop diversification in shaping diversity and the community size and activity of nitrifier and denitrifier bacteria and N2O emissions after N fertilization in corn. In 2017, after fertilizer addition, N2O emissions were continuously measured using automatic chambers from a long-term experiment of simple (corn-corn-soybean-soybean) and diverse (corn-corn-soybean-wheat underseeded with red clover cover crop) four year rotations under second year corn. Soil was collected during peak N2O emissions using high frequency temporal sampling to capture shifts in 16S rRNA, amoA, nirS, nirK, nosZ1 and nosZ2 genes and gene transcripts in soil where urea-ammonium-nitrate (UAN) was applied. An N2O emission event occurred in both rotations following UAN application but was higher in the diverse rotation, which also had persistent higher total and denitrifying (nirK and nosZ2) bacterial abundance. Bacterial amoA significantly increased in both rotations shortly after UAN addition, but gene detection decreased significantly in the simple rotation and remained elevated in the diverse. Transcripts for atypical nosZ2 were consistently detected but higher after UAN addition. Total bacterial diversity did not differ between simple and diverse rotations, however, abundance of microbial pathways leading to soil N2O emissions, ammonia oxidizers and denitrifiers, were elevated in soil with a more diverse cropping history. Best management practices (BMPs) that include crop diversification need to consider microbial communities and greenhouse gas (GHG) production, in order to fully quantify the soil ecosystem services.

In agroecosystems, efficient fertilizer use is key to optimizing productivity and reducing nutrient losses that can be detrimental for the environment, such as nitrous oxide (N2O) emissions. Because microbial communities regulate nitrogen (N) fate in soils, some agrochemicals inhibit specific transformations to reduce N losses. Our study aimed to describe short-term dynamics of N-cycling genes and transcripts and N2O emissions after fertilization with urea-ammonium nitrate (UAN) with or without the addition of nitrification plus urease inhibitors (NUI). The experiment consisted of 4-ha corn plots located in SE Ontario, Canada, where field-scale N2O emissions were monitored continuously using micro-meteorological techniques. Soil samples (0–10 cm) were taken 10 days before (baseline) and 2, 6, 9, 13 and 16 days after fertilization, and immediately flash-frozen. We co-extracted DNA and RNA and, using real-time PCR, quantified genes/transcripts targeting total bacteria (16S rRNA) and key N-cycling groups: ureolytic (ureC), ammonia-oxidizers (bacterial/archaeal amoA), nitrite-reducers (nirK/nirS) and N2O-reducers (clade I/II nosZ). The addition of NUI did not prevent an N2O flux event but reduced its duration and magnitude by more than 50%, and net cumulative N2O emissions for the sampling period by ~68%. NUI effects on N-cycling microorganisms were evident on day 9, as a transient reduction (40–56%) of ammonia-oxidizers and denitrifiers. Changes in transcripts were minor and only detectable on ureC (day 2), nirS and clade II nosZ (day 9). NUI did not interfere with temporal fluctuations in nirK and nirS, but it differentially affected nosZ response to a later rainfall event. Unexpectedly, N2O emissions were negatively associated with the ratio between nitrite-reducers and N2O-reducers. NUI effects on N-cycling microorganisms were minor and transient but resulted in a field-scale reduction in N2O emissions, possibly due to a combination of environmental factors and legacy effects from previous years of treatment.

The abatement of anthropogenic nitrous oxide (N2O) emissions depends on sound management of manure nitrogen (N) and an ability to track and predict manure-derived microbial N2O production in soils. The objective of this study was to investigate the utility of applying a novel combination of micrometeorological, stable isotope, and molecular methods to determine the short-term dynamics of N2O production processes in soil. Nitrous oxide emissions were continuously monitored in two drought-stressed agricultural fields treated with liquid dairy manure applied either in the fall or spring over an N2O emission event triggered by a heavy rainfall. In situ δ15N–N2O and δ18O–N2O measurements were used in conjunction with abundance (DNA) and potential activity (cDNA) measurements of key microbial N cycling gene and transcript (mRNA) targets to evaluate if these two techniques provided similar inferences about N2O soil processes occurring over the emission event. Soil gas was sampled at 4 depths along a profile from 10 to 50 cm below the surface and a multi-layer diffusion model was used to calculate the vertical N2O fluxes, the change in storage, and the net production of N2O in each layer. The rainfall event triggered N2O production in both fields at all depths, but the uppermost soil layer was the main source of N2O emissions throughout. Soil concentrations and surface emissions of N2O increased rapidly reaching a maximum 4 d following rainfall and were accompanied by a significant increase in the abundance of both nitrifier and denitrifier genes and total bacterial and archaeal genes and transcripts. A similar increase in the transcription of functional genes was not detected, but relatively high levels of archaeal ammonia monooxygenase (crenamoA) and bacterial nitrite reductase (nirS) transcripts were present in both fields throughout the study, indicating nitrification and denitrification were occurring concomitantly. High levels of H2O–NOx oxygen exchange at our study site precluded the use of δ18O–N2O values to separate different sources. Prior to the rain, both fields had very small amounts of soil ammonium and the stable nitrogen isotope characterization of N2O in the dry soil implicated denitrification as the main source of N2O rather than nitrifier-denitrification or hydroxylamine oxidation. Following the rain event, the δ15N–N2O values showed a change in production process as nitrifier-denitrification became a more dominant N2O source. By pairing stable isotope measurements with molecular analyses we were able to verify that these two approaches provide similar inferences about soil N2O production processes during an emission event in manure-applied fields. Although significantly higher N2O flux and N2O accumulation in the soil profile were observed for the field receiving manure in the spring, differences in isotope signal and molecular analysis between fields were not observed. To our knowledge this is the first time that isotope and molecular techniques have been used to study processes resulting in N2O production in manure-amended soils in situ. Whereas stable isotopes were useful for directly tracking the pathways of N2O production, molecular analyses revealed the status of the N cycling communities before, during and after the emission event. This information helped explain the observed differences in emissions between fields, and it helped to support our isotope-based conclusions.

Agricultural soils are the main global source of nitrous oxide (N2O) emissions. Nitrous oxide is produced by nitrifying and denitrifying microbes in soils. In cold regions such as Canada, N2O emissions during spring thaw can account for the majority of annual emissions. The objectives of this study were: (i) to quantify gene expression in nitrifying and denitrifying communities by targeting key functional gene transcripts in the nitrogen cycle (amoA, nirS, nosZ), and, (ii) to compare microbial activity to timing of N2O flux in two conventionally tilled corn fields over spring thaw, one field with residues removed (−R), and one with residues returned (+R). In both fields, initial sampling contained populations with amoA and nirS transcripts, and these quantities did not significantly change over the spring thaw sampling period. In contrast, the –R field had a denitrifying population that transcribed significantly less nosZ, and abundance increased significantly over the sampling period, N2O flux over the spring thaw was inversely proportional to nosZ transcription. The combination of anaerobic soil conditions favoring denitrification, nutrient availability, and gene expression in both nitrifiers and denitrifiers present at the onset of the spring thaw provide evidence of de novo denitrification. To the best of our knowledge, ours is the first study to relate the analysis of the quantity and activity of N cycling soil microbes in situ to the timing of a spring thaw N2O emission event.