Production and reduction of nitrous oxide (N2O) by soil denitrifiers influence
atmospheric concentrations of this potent greenhouse gas. Accurate projections of the net
N2O flux have three key uncertainties: (1) short- vs. long-term responses to warming,
(2) interactions among soil horizons, and (3) temperature responses of different steps in the
denitrification pathway. We addressed these uncertainties by sampling soil from a boreal forest
climate transect encompassing a 5.2 ∘C difference in the mean annual temperature and
incubating the soil horizons in isolation and together at three ecologically relevant temperatures
in conditions that promote denitrification. Both short-term exposure to warmer temperatures and
long-term exposure to a warmer climate increased N2O emissions from organic and mineral
soils; an isotopic tracer suggested that an increase in N2O production was more important
than a decline in N2O reduction. Short-term warming promoted the reduction of organic
horizon-derived N2O by mineral soil when these horizons were incubated together. The
abundance of nirS (a precursor gene for N2O production) was not sensitive to
temperature, whereas that of nosZ clade I (a gene for N2O reduction) decreased
with short-term warming in both horizons and was higher from a warmer climate. These results
suggest a decoupling of gene abundance and process rates in these soils that differs across
horizons and timescales. In spite of these variations, our results suggest a consistent, positive
response of denitrifier-mediated net N2O efflux rates to temperature across timescales
in these boreal forests. Our work also highlights the importance of understanding cross-horizon
N2O fluxes for developing a predictive understanding of net N2O efflux from
soils.
Introduction
Nitrous oxide (N2O) is a potent greenhouse gas with ∼300 times the global warming
potential of carbon dioxide on a 100-year timescale and uncertain climate feedback effects (Ciais et
al., 2013; Portmann et al., 2012). Although increases in atmospheric N2O are attributed to
nitrogen (N) fertilizer use (Mosier et al., 1998), emissions from natural systems dominate terrestrial fluxes
(Ciais et al., 2013) and experimental manipulations indicate that warming may enhance these fluxes
(Benoit et al., 2015; Billings and Tiemann, 2014; Kurganova and Lopes de Gerenyu, 2010; Szukics et
al., 2010; Wang et al., 2014). One of the most important biogeochemical pathways of N2O
formation in natural systems is denitrification – the stepwise reduction of NO3- to
N2. In this pathway, soil denitrifiers can both produce and reduce N2O, and the
incomplete reduction of N2O during the final step to N2 can result in
N2O release to the atmosphere (Baggs, 2011; Firestone and Davidson, 1989). Soil
microorganisms play a critical role in climate change (Cavicchioli et al., 2019); however, how sensitive the denitrification pathway is to a warming climate remains unclear.
Translating empirically derived knowledge about soil denitrifiers into climate projections is
difficult due to the dynamic and variable nature of the many interacting steps and their controls
(Butterbach-Bahl et al., 2013). The indirect influences of temperature on strong, proximate
controls of denitrification (i.e., the availability of C, NO3-, or soil
O2) are likely important features governing soil denitrifier response to climate change
(Butterbach-Bahl and Dannenmann, 2011; Wallenstein et al., 2006). Here, we instead address three key
challenges that are associated with the temperature sensitivity of denitrification. First, we do not
know if short-term responses of denitrifying communities to warming (Billings and Tiemann, 2014;
Kurganova and Lopes de Gerenyu, 2010; Szukics et al., 2010; Wang et al., 2014) are maintained across
longer timescales. Therefore, we are uncertain if laboratory studies can provide the empirical data
needed to project longer-term fluxes. Studies of heterotrophic soil CO2 efflux suggest
that enhanced rates of microbial respiration with warming may be dampened over the long-term,
prompted by a combination of microbial acclimation and adaptation (Billings and Ballantyne, 2013;
Bradford, 2013), and it is feasible that denitrifying communities may also exhibit only ephemeral
responses to warming. Such a response is consistent with inconclusive results of multiple in situ
warming experiments, although such studies necessarily reflect both denitrification and other
N2O-producing processes in soils (Bai et al., 2013; Butler et al., 2012; Dijkstra et al.,
2012; McDaniel et al., 2013). Assuming microbial acclimation, denitrifying communities may be more
effective at NO3- reduction and transformation to N2 within their acclimated
climate's typical temperature range. In principle, this could result in relatively lower rates of
N2O loss in that particular temperature regime (i.e., more complete denitrification)
compared with less effective processing by those microbial communities if the mean temperature were to
shift. Although this phenomenon has not been demonstrated for the more complicated soil
denitrification with its multiple enzymatic steps, the so-called “home field advantage” has been
demonstrated in studies exploring rates of other soil microbial processes (Alster et al., 2013;
Wallenstein et al., 2013).
A second knowledge gap limiting our ability to project future soil N2O climate feedbacks
is potential variation with temperature in interactions between microbial production and reduction
of N2O across soil horizons. Implicit in the concept that such cross-horizon interactions
may control net profile N2O efflux is the assumption that soil denitrifiers have different
patterns of production and reduction in different horizons. This may arise because the conditions
that control N2O production or reduction differ between horizons, or it may arise because
the metabolic potentials of the soil microbial community in different horizons are intrinsically
different (Blume et al., 2002; Fierer et al., 2003). Consistent with this idea, Goldberg and Gebauer
(2009) illustrated clear variation in patterns of δ15N of N2O across soil
depth in response to drought, which could have been caused by variations in either N2O
production or reduction (Billings, 2008). Thus, the exchange of substrates between soil horizons can
be an important process dictating whole-soil N2O efflux, and it may contribute to apparent
inconsistencies between warming effects in the laboratory and the field (reviewed in Bai et al.,
2013). Indeed, profile interactions have recently been demonstrated as important drivers of soil
CO2 efflux: temperature responses of whole-soil core respiration can be distinct from the
sum of those observed for horizons incubated in isolation from each other, which is likely due to the exchange of
substrates and microbes among horizons (Podrebarac et al., 2016). Although evidence suggests that
N2O produced in one soil horizon may be reduced in another (Goldberg and Gebauer 2009),
the degree to which this may occur, and why, has not been determined.
A third feature challenging our ability to project soil N2O effluxes in a warmer climate
regime is the potentially different response to warming of distinct steps in the denitrification
pathway (this may be for one or multiple microbes within the community that carry out the enzymatic
steps). For instance, if the activity of nosZ, a gene that codes for an enzyme catalyzing
N2O reduction, experiences a different response to temperature than nirK, a gene
coding for an enzyme catalyzing NO2- reduction (and, thus, N2O production), the
net flux of N2O may either increase or decrease with temperature depending on the
direction and magnitude of both responses. Although gene abundances sometimes exhibit decoupling from
function (Peterson et al. 2012), quantifying any changes in these functional gene abundances with
temperature can help discern the propensity for temperature responses from relevant microbial
communities' structure and, thus, the driving mechanisms for net N2O production
responses. Differential responses of these genes' abundances to short-term temperature manipulation
have been observed in grassland soils (an increase in nosZ with short-term temperature
increases; Billings and Tiemann, 2014), but it is unknown whether these observations are relevant
for soil microbial communities subjected to long-term exposure to distinct temperature regimes.
In this study, we explore the following three issues: short- vs. long-term responses of soil denitrifying
communities' net production of N2O to warming, the exchange of denitrification-derived
N2O among horizons as a driver of the temperature response of net N2O efflux, and
the potentially different responses of the relative abundances of microbial genes linked to
N2O production vs. reduction to temperature. We invoked a space-for-time substitution to
test our long-term warming hypothesis, using a climate transect along which the mean annual temperature
(MAT) varies but dominant vegetation, soil type, and soil moisture are similar. To elucidate both the
short- and long-term temperature responses of soils' denitrifying communities, we incubated soils
that came from different latitudes and climate regimes along this transect (long-term warming) for
60 h at 5, 15, and 25 ∘C (short-term warming) in order to reflect typical current (5
and 15 ∘C) and projected future (25 ∘C) soil
temperatures. Specifically, laboratory incubations of mesic organic and mineral boreal forest soil
horizons were established under conditions that promote denitrification. To understand the potential
for interactions among soil horizons as a driver of the temperature response of net N2O
efflux, we incubated organic and mineral soils both individually and in combination. We measured net
rates of N2O efflux and abundances of representative functional genes linked to the production
and reduction of N2O and estimated N2O reduction using an isotopic tracer.
We expected that short-term warming would enhance net N2O production in these boreal
soils, as in the majority of past incubation studies (Billings and Tiemann, 2014; Kurganova and
Lopes de Gerenyu, 2010; Szukics et al., 2010; Wang et al., 2014). As outlined above, we also tested
the hypothesis that a warmer temperature regime over a longer timescale would show the opposite
effect: a dampened net N2O efflux from the historically warmer soils, where organic N
turnover is faster (Philben et al., 2016) and where denitrifying communities can presumably
function effectively as transformers of NO3- to N2 at warmer temperatures
compared with their more northern counterparts. Here, we define “effective” as a denitrifier community
that is able to transform NO3- to the end product, N2. We also hypothesized
that N2O produced in one horizon would be reduced in the other when incubated together,
resulting in lower net N2O efflux than a simple linear combination of these horizons'
individual efflux rates. Specifically, we anticipated that organic soils, which are relatively rich in
microbial abundance and diversity compared with mineral soils, would reduce mineral-produced
N2O, following dominant diffusion gradients. Finally, we hypothesized that soils that
exhibit higher rates of net N2O production would also exhibit some combination of increased
nir abundance and decreased nos abundance as well as the associated higher ratios of
nir : nos gene abundances, reflecting shifts in microbial genetic potentials
with temperature regime.
Materials and methodStudy site and soil sampling
Soil was collected from three mature forest stands at each of three regions along the Newfoundland
and Labrador Boreal Ecosystem Latitudinal Transect (NL-BELT), Canada (Table 1, Fig. 1; Ziegler et
al., 2017). The NL-BELT spans the north–south extent of the balsam-fir-dominated boreal biome in
Eastern Canada, from southwest Newfoundland to southeast Labrador. This transect has long-term
(century-scale) temperature regime differences but otherwise similar conditions. For instance, the
three study regions along this transect (from south to north), the Grand Codroy, Salmon River, and
Eagle River watersheds (Fig. 1), have similar Orthic Humo-Ferric Podzols (Spodosols; Soil
Classification Working Group, 1998) and vegetation dominated by balsam fir (Abies balsamea). The difference in the MAT and precipitation is 5.2 ∘C and 431 mm, respectively,
between the Grand Codroy (southernmost) and Eagle River (northernmost) climate stations (Environment
and Climate Change Canada, 2020). The soils are mesic, and the regions have an evaporative demand
gradient (Table 1) that considerably reduces the precipitation gradient, making the transect an
excellent proxy for investigating soil temperature responses while mitigating confounding features
of differing soil moisture. Three replicate forest stands were established in each of the three
climate regions, allowing us to assess the influence of long-term differences in the MAT (and the associated
differences in climate) along the transect without concerns about pseudoreplication, which is a rarity in
large-scale space-for-time substitutions (Ziegler et al., 2017).
(a) Map and (b) pictures of the three forests in each region along the
Newfoundland and Labrador Boreal Ecosystem Latitude Transect (NL-BELT) in Canada.
Characteristics of the nine forests in the three study regions along the Newfoundland and Labrador Boreal Ecosystem Latitude Transect (NL-BELT) in Canada.
RegionCoolestIntermediateWarmestForest IDMuddySheppard'sHarry'sHare BayTucka-Catch-A-O'RegansMapleSlugPondRidgePondmoreFeederRidgeHillLatitude53∘33′ N53∘33′ N53∘35′ N51∘15′ N51∘9′ N51∘5′ N47∘53′ N48∘0′ N48∘0′ NLongitude56∘59′ W56∘56′ W56∘53′ W56∘8′ W56∘0′ W56∘12′ W59∘10′ W58∘55′ W58∘54′ WWatershedEagle River Salmon River Grand Codroy Closest weather stationaCartwright (53∘42′ N, 57∘02′ W) Main Brook (51∘11′ N, 56∘01′ W) Doyles (47∘51′ N, 59∘15′ W) Mean annual precipitation (mm)1073.5 1223.9 1504.6 MA PET (mm)b432.9 489.1 608.1 Mean annual temperature (∘C)0.0 2.0 5.2 Organic horizon depth (cm)6.54.66.19.47.46.67.98.84.3Bulk density (organic; gcm-3)0.090.070.100.090.090.120.090.140.10Bulk density (mineral; gcm-3)0.800.720.760.590.591.200.680.680.66Soil pH (organic)5.35.35.44.44.45.74.33.74.6Soil pH (mineral)5.05.05.04.84.85.94.54.74.9
a Climate normal data (1981–2000) were sourced from http://climate.weather.gc.ca/climate_normals/index_e.html (last access: 23 August 2020).
b “MA PET” refers to the mean annual potential evapotranspiration.
Two large (30 cm2) peds of organic (LFH or O horizon) and mineral (B horizon) soil were
collected at each forest stand on a different calendar date but an equivalent ecological date:
22–24 October 2013 in Eagle River, 4–5 November 2013 in Salmon River, and 22–23 November 2013 in Grand Codroy. This pre-freeze, post-growing season period typically exhibits relatively large
and active microbial biomass in northern-latitude organic soils (Buckeridge et al., 2013). The
Ah and Ae horizons were not present at all sites; therefore, they were not included in
the incubation at any site. Each collection was shipped to the University of Kansas (4–5 d
transit in insulated coolers, on ice) and processed immediately. Because regions were processed as
separate experimental blocks we cannot separate the region and block effects. However, we
confounded these factors knowingly, because we believed the ecological date and rapid processing were
more important than minimal differences in laboratory practice between blocks.
Incubation and headspace gas collection
Aboveground vegetation (i.e., moss, herbaceous plants, and tree seedlings) was removed from the peds with
scissors. The two peds of organic and mineral soil from each forest site were pooled within horizon
and mixed by hand, producing an organic and mineral sample for each forest. This process was
repeated nine times – for the three forests in each of the three regions. Subsamples (the fresh mass of the organic sample was
50 g, and the fresh mass of the mineral sample was 40 g) were placed in half-pint (237 mL) Mason jars. To test
the potential for N2O producers and reducers from one horizon to interact with their
counterparts in the other horizon, “combined” samples were also prepared: an open
container of mineral soil (20 g) was placed within a jar next to organic soil
(25 g) such that they had a shared headspace but were not physically mixed. Each sample was
replicated for three temperature incubation scenarios (5, 15, and 25 ∘C), and three
blank jars (no soil) were included for each temperature. To maximize the potential for
denitrification, we promoted anaerobic conditions and substrate diffusion by evacuating headspace
air and replacing it with He, and we adjusted the water-holding capacity to 80 % with a
K15NO3-–N solution (δ15N 3000 ‰) that added 18 and
1.3 µgNg-1 dry weight (dw) soil to the organic and mineral soil samples, respectively
(18× background levels at the time of sampling, although within the annual range of soil
NO3- availability based on unpublished field data). Our approach was distinct from a
potential denitrification assay, which calls for non-limiting C and NO3-
additions to soils (Pell et al., 1996); instead, we intended to promote conditions conducive to
denitrification using natural C pools and as close to natural NO3- concentrations as
was feasible. Therefore, this experiment is not predictive of bulk soil N2O rates and
instead explores controls on N2O rates in soil zones with low O2
concentrations. Such “hot spots” for biogeochemical cycles in soils are well-documented (McClain
and others 2003).
Over 60 h of incubation, we collected headspace gas eight times for determination of the
N2O concentration. The first sample was collected immediately after initiating the
incubations, the second sample was collected at ∼3h, and further samples were then
collected every 10 h. At each collection point, 14 mL of headspace gas was
removed with a needle and gastight syringe and injected into pre-evacuated 12 mL
borosilicate vials with a silicone septum and aluminum crimp (Teledyne Instruments, Inc., CA, USA);
at the second and last collection an additional 14 mL of headspace gas was removed and injected
into pre-evacuated Exetainers (Labco Ltd., High Wycombe, UK) for isotopic analysis of N2O
in the headspace. After each gas sampling, He of an equivalent volume was injected into the
incubation vessels to maintain pressure in the containers. At the end of the incubation all jars
were opened and soils were destructively harvested to quantify soil inorganic N as well as for DNA
extraction.
N2O concentration and isotope analysis
Headspace samples were analyzed for N2O concentration in an auto-injected 5 mL
subsample on a gas chromatograph fitted with an electron capture detector (CP-3800, Varian), and they were
calibrated against a four-point standard curve that encompassed the sample range. Blank-corrected
headspace concentrations were adjusted for the dilution at each sampling with He replacement and were
converted to the rate of net N2O–N production (nggdw-1h-1) by
application of the ideal gas law (PV=nRT), multiplication by the molar mass
of N in N2O, and correction by the dry weight of soil (in grams) in the sample and the change
in time since the previous sample. Then rates of net N2O production were calculated as the
average of the eight sample collections' rates. Net N2O flux changed throughout the course of
the 60 h incubation (Fig. S1 in the Supplement); we focus on the average of these rates to
integrate both production and reduction into an aggregate value across the whole incubation. Samples
for isotope analysis (δ15N of N2O) were submitted to the University of
California, Davis, Stable Isotope Facility, where they were analyzed on a Thermo Finnigan
GasBench + PreCon trace gas concentration system interfaced to a Thermo Scientific Delta V Plus
isotope ratio mass spectrometer (Bremen, Germany). The analysis was conducted with four standards of
0.4–10 ppmN2O in He, with a precision (standard deviation on five replicate
natural abundance standards) of 0.1‰15N.
The change in the percentage of added 15N found in the N2O between incubation
sampling times at 3 and 60 h was used to quantify the gross reduction of N2O
to N2 (Billings and Tiemann 2014). Because our tracer contained far more 15N
than is present naturally, any natural fractionation during N2O reduction was negligible
compared with the isotopic signature of the tracer in the N2O pool, and we can use
15N2O abundance as a means of assessing N2O production vs. reduction. If
15N2O abundance at 60 h is higher than at 3 h, it suggests that the tracer
was continuing to flow into the N2O pool more so than out of it and, thus, that
N2O production outpaced N2O reduction (transformation into N2) at that
time point. In contrast, if 15N2O abundance at 60 h is lower than at
3 h, it suggests that the tracer was flowing out of the N2O pool at a greater pace
than it was flowing into it and, thus, that N2O reduction outpaced N2O production
at that time point. We calculated 15N2O by multiplying the isotopic ratio of the sample
by the concentration of N2O in that sample. We then computed the change in the percentage of the
15N tracer added that was found in headspace N2O across incubation time as follows:
Change in 15N2O(%)=15N2O15NO3–N added×100final-15N2O15NO3–N added×100initial,
where 15N2O represents nanograms of 15N in headspace N2O per gram of dry weight soil,
15NO3-–N represents nanograms of 15N in NO3- per gram of dry weight soil, “final”
refers to the end of the incubation (∼60h), and “initial” refers to the first time
point at which the change in 15N of N2O was assessed (∼3h).
To assess the potential for N2O to be reduced to N2 by denitrifiers in the other
horizon when incubated together, we calculated the combination effect (ng N2O–Ngdw-1h-1) as the difference between observed net N2O fluxes when soil
horizons shared the incubation headspace (observed) and the expected flux determined as the linear,
additive effect of the rate for horizons in separate headspaces
(((organic+mineral)/2)=expected). The combination effect was also
expressed as a percentage of the expected flux:
Combination effect (%)=observed-expectedexpected×100,
where a negative combination effect implies reduction caused by the inclusion of one of the horizons.
Soil nutrient analysis
To observe changes in extractable inorganic N during the incubation, we extracted soil subsamples
prior to and following the incubation (fresh mass of the organic sample was 12 g, and fresh mass of the mineral sample was 10 g) by
shaking for 1 h with 40 mL 0.5 MK2SO4. After shaking all of the
samples were filtered and the extracts were frozen at -20∘C until further analysis. Soil
NO3-–N and NH4+–N in the extracts were analyzed on a Lachat QuikChem 8500
autoanalyzer (Hach Co., Loveland, CO, USA) using the cadmium reduction and phenol red methods,
respectively.
Functional gene abundance
Soil DNA was extracted from approximately 0.25 g fresh weight soil using a MoBio PowerSoil
DNA extraction kit and purified with a MoBio PowerClean DNA clean-up kit (MoBio Laboratories,
Carlsbad, CA, USA; now Qiagen). DNA was quantified with a Qubit 2.0 fluorometer (Invitrogen,
Carlsbad, CA, USA), diluted by a factor of 10, and stored at -20∘C until further
analysis. We assayed several functional gene primers in the denitrification pathway via polymerase chain reaction (PCR)
(nirK, Henry et al., 2006; nirS, Throbäck et al., 2004; norB, Braker
and Tiedje, 2003; nosZ, Rösch et al., 2002; nosZ clade II, Jones et al., 2013;
Table S1 in the Supplement) and selected nirS and nosZ as the most tractable
indicators of N2O production and reduction in these soils using quantitative PCR (qPCR),
based on successful amplification of these genes across all samples. qPCR was accomplished using
ABI StepOnePlus (Applied Biosystems) with Brilliant III Ultra-Fast SYBR® Green QPCR
Master Mix (Agilent/Life Technologies, Carlsbad, CA, USA). Each reaction consisted of
5 µL (∼2ng) genomic DNA, 400 nM of primer, 300 nM of
reference dye, and 1 X Brilliant III in a final volume of 20 µL. The qPCR program
consisted of an initial denaturing temperature of 95 ∘C for 3 min followed
by 40 cycles of denaturing at 95 ∘C for 5 s and a combined annealing and
extension step of 10 s at 60 ∘C for both nirS and nosZ
genes. Melt curves were calculated at the end of each qPCR run to confirm product specificity. Each
qPCR plate contained one primer pair, three negative controls, and a four-point standard curve
(ranging from 300 to 300 000 copies). Standard curves were generated using genomic DNA from a lab
stock of cultured Pseudomonas fluorescens, and gene copy numbers were calculated assuming a
mass of 1.096×10-21g per base pair (Wallenstein and Vilgalys, 2005), one gene
copy per genome, and a genome size of 7.07 Mb (NCBI). All gene abundance data were corrected
by soil ovendry mass based on the dry : fresh mass ratio of an oven-dried subsample collected
post-incubation.
Statistical analysis
We used a three-way ANOVA to assess the influence of the fixed effects of soil horizon, “region”
(historical temperature), “temperature” (short-term, incubation temperature), and their
interactions on inorganic N pools, net N2O flux averaged across the incubation, change in
percent of added 15N tracer found in headspace N2O, the effects of mixing
horizons in the incubation on net N2O flux, and functional gene abundances. For all
analyses, we followed up significant main effects with a Tukey's post hoc analyses and report
adjusted P values. For all variables, we assessed whether they met the assumptions required for
performing these statistical tests, and we log-transformed variables before analysis when required. All
statistical analyses were performed in R (R Core Team, 2014), using the MASS package (Venables and
Ripley, 2003). All significant (α=0.05) results and interactions are reported except for
significant main effects that have the significant interactions of their terms reported instead. Errors
reported are one standard error (±1SE) of the mean.
ResultsChanges in inorganic N pools after the incubation
Temperature altered the pool sizes of NH4+–N differently in each region and
horizon (temperature×region×horizon: P=0.05), increasing relative
to preincubation pool sizes in the organic soils at some of the incubation temperatures (coolest
region, 25 ∘C: P=0.04; intermediate region, 25 ∘C: P=0.02;
warmest region, 15 ∘C: P<0.0001, 25 ∘C: P=0.0001), as shown in Fig. 2a and
b. Mineral soil NH4+–N pool sizes post-incubation did not differ from
preincubation pool sizes.
Soil NH4+–N and NO3-–N pools in the organic (a, c) and
mineral (b, d) soil, preincubation (“Pre-inc.”) and at the end of the incubations at
5, 15, and 25 ∘C of soils from along a boreal forest latitudinal
transect. Preincubation values for nitrate are calculated as ambient concentrations including added
NO3-–N. Note the different y axis values. “MAT” refers to the mean annual temperature. The
“coolest” region is the Eagle River watershed (northern boreal), the “intermediate” region is
the Salmon River watershed (mid-boreal), and the “warmest” region is the Grand Codroy watershed
(southern boreal). See the text for site descriptions. Values are provided as the mean ± 1SE (n=3 forests per latitudinal region).
Temperature also altered the pools sizes of NO3-–N differently for each region
and horizon (temperature×region×horizon: P=0.03), decreasing
relative to preincubation pool sizes in the organic soils at all temperatures in all regions
(coolest, 5 ∘C: P=0.001, 15 ∘C: P=0.0007, 25 ∘C:
P=0.003; intermediate, 5 ∘C: P=0.04, 15 ∘C: P=0.002,
25 ∘C: P=0.008; warmest, 5 ∘C: P<0.0001, 15 ∘C:
P<0.0001, 25 ∘C: P<0.0001). NO3-–N pool sizes also
decreased in the mineral soils at all temperatures in the coolest (5 ∘C: P=0.0005,
15 ∘C: P=0.0008, 25 ∘C: P=0.002) and intermediate
(5 ∘C: P=0.02, 15 ∘C: P=0.002, 25 ∘C:
P=0.0004) regions, although not in the warmest region (Fig. 2c, d). These results imply that
the anaerobic conditions we generated by replacing headspace air with He and maintaining an 80 % water-holding capacity generally supported denitrification and limited nitrification.
N2O net production rates with short- and long-term warming
Net N2O flux was influenced by regions (P=0.002), incubation temperature (P=0.006),
and soil type (P<0.0001) without any significant effect of any interaction among or between these
independent variables. When averaged across all incubation temperatures and the two soil horizons,
the warmest region (3.8±0.8ngN2O–Ng-1h-1) had a
higher rate than the intermediate (1.9±0.6ngN2O–Ng-1h-1, P=0.008) and coolest region (1.2±0.3ngN2O–Ng-1h-1, P=0.003), whereas the intermediate-latitude and
coolest regions' net N2O production did not differ from each other (Fig. 3). Averaged
across all regions and the two soil types, the warmest incubation temperature
(3.4±0.8ngN2O–Ng-1h-1) exhibited a higher net
N2O flux than the lowest temperature (1.1±0.3ngN2O–Ng-1h-1, P=0.003). Averaged across all regions and soil temperatures, the organic
soil (4.9±0.8ngN2O–Ng-1h-1) exhibited a higher rate
than the mineral soil (0.6±0.2ngN2O–Ng-1h-1,
P<0.0001) and the combined incubation (1.3±0.3ngN2O–Ng-1h-1, P<0.0001), which had a higher rate than the mineral soil alone
(P=0.005).
Net N2O flux (“production rate”) averaged for 60 h of incubation at 5,
15, and 25 ∘C from organic soil alone (a), combined organic and mineral
soil (b), and mineral soil alone (c) from three regions along a boreal forest
latitudinal transect. “Combined” refers to incubations with organic and mineral soil in the same
jar, which are physically isolated but share the headspace. “MAT” refers to the mean annual temperature. The
“coolest” region is the Eagle River watershed (northern boreal), the “intermediate” region is
the Salmon River watershed (mid-boreal), and the “warmest” region is the Grand Codroy watershed
(southern boreal). See the text for site descriptions. Values are provided as the mean ± 1SE (n=3 forests per latitudinal region).
We used N2O emission from organic and mineral soil in isolation (Fig. 3a, c) to compute the
expected net N2O flux for the combined soils (Fig. 4a, b). Observed rates of net
N2O production in the headspace surrounding the combined organic and mineral soils (Fig. 3b)
were less than the expected values (Fig. 4a, b) and often exhibited net N2O reduction,
implying inter-profile interactions and differential temperature responses of the two horizons. The
absolute effect of the combined horizons' reduction of N2O differed by incubation
temperature (P=0.002), with higher net reduction in the warmest incubation compared with the
coolest (25 vs. 5 ∘C: P=0.001) and a trend towards more reduction in the
intermediate-latitude region compared with the coolest (P=0.098). In proportional terms, the
effect of combining horizons decreased the combined net N2O flux by up to 175 % of the
expected combined net production rate, and this effect differed by temperature (P=0.009). In
particular, it was more pronounced at 15 ∘C relative to 5 ∘C
(P=0.004). There was no significant interaction between region and temperature on this
combined-horizon rate.
The combination effect of shared headspace surrounding physically separated organic and
mineral horizons on the absolute net N2O flux (a) and as a percentage of the
expected N2O production rate (b), at the end of a 60 h incubation at 5,
15, and 25 ∘C, for soils from three regions along a boreal forest latitudinal
transect. The combination effect (negative denotes reduction) is calculated as the difference between
observed net N2O fluxes when soil horizons shared the incubation headspace (observed)
and the linear, additive effect of rate differences between horizons in separate headspaces
(((organic+mineral)/2)=expected). The percent combination effect
was calculated as ((observed-expected)/expected)×100. The
nonzero values suggest that the shared headspace generated a nonlinear, interactive effect on
net N2O effluxes. “MAT” refers to the mean annual temperature. The “coolest” region is the
Eagle River watershed (northern boreal), the “intermediate” region is the Salmon River watershed
(mid-boreal), and the “warmest” region is the Grand Codroy watershed (southern boreal). See the text
for site descriptions. Values are provided as the mean ± 1SE (n=3 forests per
latitudinal region).
We used the change in 15N in the N2O (t60h-t3h) as a
proxy for estimating how the relative contribution of the production and reduction of N2O
varied among regions, across horizons, and with incubation temperature. Specifically, a negative net
15N abundance in N2O from t60h to t3h would indicate that
consumption outpaced production, given that all the 15NO3- was reduced over this
period. Instead, the change in 15N abundance in N2O across the incubation time was
consistently positive, suggesting that rates of N2O production consistently outpaced rates
of N2O reduction during the 60 h incubation. These values differed by region
(P=0.001), which was a feature driven by the warmest region exhibiting the largest change compared with the
coolest region (P=0.0007), and a similar trend was found between the warmest and intermediate-latitude
regions (P=0.081; Fig. 5). There was no significant effect of incubation temperature or soil type
or any interaction between temperature, region, and soil type on this change in
N2O–15N.
Change in the percentage of added 15N observed in the headspace N2O over the
course of a 60 h incubation at 5, 15, and 25 ∘C
(t60h-t3h) for organic (a), combined organic and mineral
(b), and mineral (c) soils from three regions along a boreal forest latitudinal
transect. “Combined” refers to incubations with organic and mineral soil in the same jar, which are
physically isolated but share the headspace. “MAT” refers to the mean annual temperature. The
“coolest” region is the Eagle River watershed (northern boreal), the “intermediate” region is
the Salmon River watershed (mid-boreal), and the “warmest” region is the Grand Codroy watershed
(southern boreal). See the text for site descriptions. Values are provided as the mean ± 1SE (n=3 forests per latitudinal region).
Functional gene abundance
At the end of the 60 h incubation period, the abundance of one functional gene indicative of
N2O production, nirS, did not vary significantly by incubation temperature or
region but differed strongly by soil horizon (P<0.0001). There was a higher abundance of this
gene in the organic soil (0.73×106±0.04×106g-1) vs. the mineral
soil (0.18×106±0.02×106g-1), as shown in Fig. 6. There was no significant
effect of any interaction among or between the independent variables on nirS
abundance. Functional gene abundance for N2O reduction, nosZ, differed by region
(P=0.0002), incubation temperature (P=0.04), and soil (P<0.0001). It was higher in soils from
the warmest region (8.4×106±1.9×106g-1) relative to the
intermediate-latitude region (4.0×106±0.8×106g-1, P=0.0006) and
the coolest region (4.9×106±1.1×106g-1, P=0.001), at the coolest
(6.7×106±1.6×106g-1) relative to the warmest incubation
temperature (5.2×106±1.7×106g-1, P=0.02), and in organic
(10.55×106±0.95×106g-1) relative to mineral soils
(0.98×106±0.08×106g-1). There was no significant effect of any
interaction among or between the independent variables on nosZ abundance, although there
was a near-significant trend for soil type to alter the regional effect (P=0.052). The resulting
nirS : nosZ ratio ranged from 0.03 to 0.55 and displayed an interaction between
region and soil horizon (P=0.04), driven by lower nirS : nosZ ratios in
organic soil in the warmest relative to intermediate-latitude region (P<0.0001) and the warmest
relative to the coolest region (P=0.003); these effects were not exhibited in the mineral soil.
Functional gene abundances during a 60 h incubation at 5, 15, and
25 ∘C from soil from three boreal forest regions along a latitudinal transect:
nirS in the organic (a) and mineral (b) soil; nosZ in the
organic (c) and mineral (d) soil; and the ratio of
nirS : nosZ in the organic (e) and mineral (f) soil. Note that the
y axis scales differ for each row and between panels (c) and (d). “MAT” refers to the mean
annual temperature. The “coolest” region is the Eagle River watershed (northern boreal), the
“intermediate” region is the Salmon River watershed (mid-boreal), and the “warmest” region is
the Grand Codroy watershed (southern boreal). See the text site descriptions. Values are provided
as the mean ± 1SE (n=3 forests per latitudinal region).
Discussion
By promoting the denitrification pathway we aimed to (1) distinguish short- (via laboratory
manipulations) and long-term (via a natural climate gradient) responses of the denitrification-derived
net N2O flux to temperature, (2) assess the degree to which net N2O fluxes in
these soils are sensitive to interactions between soil horizons, and (3) leverage the abundance of
genes responsible for denitrifier production and reduction of N2O as a means of assessing
differences in these processes' responses to short- and long-term temperature responses. Our first
hypothesis was not supported: although short-term warming enhanced net N2O effluxes from
these soils, soils from a historically warmer environment exhibited a greater net N2O efflux
than those from cooler environments, suggesting a positive response of net N2O fluxes to
both short- and long-term warming (Fig. 3). Indeed, an isotopic proxy for N2O reduction
derived from the use of a stable isotope tracer suggests that enhancement of net N2O
production with long-term warming can be greater than any enhancement in N2O reduction
(Fig. 5). Our second hypothesis was supported in that the combined incubation of mineral and organic
soils exhibited net N2O efflux rates that did not match the linear sum of separate
incubation flux rates. However, we observed the reduction of N2O by mineral soil and not by
organic soil as we predicted. Specifically, net N2O production was tempered by more
mineral soil N2O reduction at warmer incubation temperatures (Figs. 4, 5), indicating
that soil horizon interactions may be critical to rates of net N2O efflux to the
aboveground atmosphere. Finally, our third hypothesis that linked gene abundance to process rates
was only partially supported. NosZ decreased at the warmest incubation temperature
(i.e., lower N2O reduction gene abundance with warming, Fig. 6), consistent with
rates. However, in the organic soils, nosZ was higher under higher historical temperature
(i.e., higher N2O reduction gene abundance with warming, Fig. 6), which was inconsistent with rates
that increase with warming. There was no response to short- or long-term warming in
nirS abundance in either soil horizon nor to long-term warming in nosZ abundance
in the mineral soil. Combined, these data suggest complex microbial responses to short- and
long-term exposure to distinct temperature regimes, which we expand upon below.
Warming-induced enhancement of N2O production exceeds that of N2O reduction
Long-term climate gradients substitute space for time and encompass variation in multiple ecosystem
phenomena driven by centuries of exposure to distinct climate regimes. For instance, we know that
in situ soil N cycling is more rapid (Philben et al., 2016) and likely supports greater
forest productivity in the relatively warm, southernmost boreal forests of this transect (Ziegler
et al., 2017). The net N2O efflux rate data from this set of lab incubations suggest
that, especially in the organic soil horizons, both short-term warming and a long-term warmer
climate enhance net N2O production, which is a result consistent with the stable isotope tracer
data (Fig. 5). These data correspond with the enhanced, short-term warming-induced N2O
fluxes observed in several systems (Billings and Tiemann, 2014; Kurganova and Lopes de Gerenyu,
2010; Szukics et al., 2010; Wang et al., 2014). The apparent lack of long-term, denitrifier
adaptation to rising temperatures (i.e., the continued enhancement of N2O production with
long-term exposure to warmer temperatures that outstrips enhancement of N2O reduction) is
consistent with recent work in soils from these same sites demonstrating no change in the responses
of microbial biomass-specific decay or CO2 efflux rates to warmer temperatures over
decadal timescales (Min et al., 2019). However, results from the current study contrast with our
hypothesis of microbial adaptations to a warmer climate over the long term, which assume that a soil
denitrifying community that is well adapted to its temperature regime is effective at complete
denitrification with relatively little N2O byproduct. Such predictions arise from more
conceptual studies presenting ideas about microbial metabolic responses to warming (Billings and
Ballantyne, 2013; Bradford, 2013) and not collective longer-term warming effects, such as substrate
or microbial community compositional changes, that may further control microbial responses.
The similar difference in net N2O rates between the northern region and southern region
(2.6 ngN2O–Ng-1h-1) and between the coolest and warmest
incubation temperature (2.3 ngN2O–Ng-1h-1, both 68 %
of the average range across treatments) indicates that net rates were enhanced to a similar degree
by both short-term warming of 20 ∘C and a long-term MAT difference of
5 ∘C. Temperature sensitivity (i.e., change per degree Celsius) of net
N2O flux increased at lower latitudes, and the isotopic tracer experiment indicated that
N2O production increases outpaced N2O reduction increases in warmer
regions. Enhanced soil organic matter inputs and nitrogen availability and cycling rates in the
warmer climate forests (Philben et al., 2016; Ziegler et al., 2017) may contribute to greater net
N2O production in the incubations and in situ. In this short-term incubation, the pulse
of NO3- added minimized any differences in NO3- availability for
denitrifiers, likely leaving varying abilities of the soil denitrifier community to respond to warming
as a key difference across the incubated soils. Therefore, the additive, positive result from both
historically warmer soils and warmer incubation temperatures suggests that community-level
denitrifier effectiveness declines (i.e., more incomplete denitrification) in warmer temperatures if
they are from soils with historically warmer temperatures. This pattern contradicts a home field
advantage (Wallenstein et al., 2013) for denitrifiers. More N2O production in warmer
climates may arise from multiple changes that overcome adaptive home field advantages, such as
shifts in the community composition (Delgado-Baquerizo et al., 2016) and an increased number of
inefficient N2O producers, increases in the number of microbial cells and transfer points
involved in the denitrification pathway (i.e., nitrifier-denitrification in a single organism
vs. coupled nitrification–denitrification in distinct organisms (Butterbach-Bahl et al., 2013), or a
changed contribution of alternate, possibly less-efficient electron donors (i.e., co-denitrification;
Spott et al., 2011).
Despite increased net N2O production with higher temperatures, soil horizon interactions
temper the response to warming. Two of our methods supported the potential for mineral soil
N2O reduction: (1) calculated differences in flux values between shared headspace
N2O flux values and the isolated headspace N2O flux values of the two isolated
horizons, and (2) the change in the isotopic enrichment of the shared and isolated headspace
N2O. The first method demonstrated that short-term warming enhanced the degree of
inter-profile interaction that increased N2O reduction during the incubation, whereas
long-term warming did not significantly influence inter-profile N2O dynamics (Fig. 4a, b). The similarities in the net N2O flux between the combined and mineral soil incubations
(Fig. 3b, c), and the fact that both of these incubations have lower flux than the organic soil
alone, indicate that the mineral soil served as a net N2O reducer, especially in response
to short-term temperature increases. A caveat to this soil horizon interaction is that while our
O2-limited experimental environment was necessary to promote denitrification, this design
may have exaggerated total soil reduction processes that occur naturally in anaerobic microsites.
Our second method of detecting horizon interactions driving net N2O efflux used
15N2O headspace differences from the start to the end of the incubation as an indicator
of reduction. We expected an increase in the 15N in the headspace N2O as
15NO3- is reduced, followed by a decline in 15N in the headspace
N2O as the tracer flows into the N2 pool, with balance of these processes over
the 60 h incubation indicating net production or reduction (Billings and Tiemann,
2014). NO3- pools declined and the change in our 15N2O abundance was
positive, suggesting that N2O production still outweighed reduction at the end of the
60 h for both the individual horizons and the combination incubation (Fig. 5a). Large
variation in 15N2O abundance among forest sites led to no significant difference
between soil horizons and did not allow us to confirm the direction of horizon interactions. Horizon
interactions drove net profile N2O fluxes in a field drought manipulation in a Norwegian
spruce forest, during which soils exhibited a net N2O sink via upper mineral soil
reduction of deep mineral soil N2O production (Goldberg and Gebauer, 2009). It remains
unknown if the relatively shallow mineral soils we sampled are analogous reducers of deeper mineral
soil N2O produced in this system or if they could continue to reduce large portions of
organic soil N2O efflux (Fig. 4) in situ. Contrary to our original hypothesis, shallow
mineral soils in situ may be better suited than organic soils to N2O reduction, as mineral
soils experience frequent inputs of leached NO3- and DOC (dissolved organic carbon) from the surface organic
soils and represent a sudden change in the soil structure and porosity towards well-packed fines
and smaller pores. These conditions may promote leachate pooling, anaerobic microsites, and a
microbial community that proves more effective at reduction.
Mineral soil reduction of organic soil-generated N2O becomes most relevant when diffusion
of N2O from the upper soil profile to the atmosphere is restricted, and N2O
produced in those surface layers diffuses downwards according to Fick's Law as has been discussed in
the literature for soil CO2 dynamics (Oh et al., 2005; Richter et al., 2015). Such a
situation is likely to occur in “hot spots” (McClain et al., 2003) such as frozen surface soil
patches during winter. Similarly, “hot moments” may occur in the spring snow melt or in winter,
despite cold temperatures reducing N cycling rates: subnivean N2O production can be an
important contribution to annual N budgets in pastures (reviewed in Uchida and Clough, 2015), and
winter N dynamics also appear to be important in northern temperate forest systems. For example,
winter N2O production equaled ∼30 % of the summer N2O production in a
southeastern Canadian forest (Enanga et al., 2016) and ∼60 % of the annual atmospheric N inputs in a
northeastern US forest (Morse et al., 2015). Mineral soil reduction of winter organic soil-generated
N2O may temper net fluxes and may be an important feature of N cycling in these forests
that likely varies with snowpack dynamics.
Linking biogeochemical process rates to genetic potential
The functional gene associated with N2O reduction that we could quantify in these soils
was sensitive to both short-term and historical temperature, although it was not consistently
associated with process rates. While we did not detect the atypical nosZ clade II in
these soils, other, yet unknown genes that we did not measure may be responsible for N2O
reduction. Beyond this possibility, our results suggest a decoupling of process rates and
denitrifier genetic controls or that the long-term temperature-related increase in genetic
potential for N2O reduction did not translate to rates as effectively as the short-term
temperature-related decrease in genetic potential for N2O reduction.
Consistent with enhanced net N2O production in these soils at warmer incubation
temperatures, the nosZ abundances were reduced after 60 h exposure to
25 ∘C relative to cooler incubations. Although functional gene abundances are
assumed to integrate longer-term changes in the microbial community and, thus, have a reduced dynamism
relative to instantaneous rates (Petersen et al., 2012), our results appear to reflect a capacity of
denitrifiers to respond rapidly to temperature, as indicated in other laboratory incubations that
assayed temperature responses of denitrification functional gene abundances (Billings and Tiemann,
2014; Cui et al., 2016; Keil et al., 2015). However, inconsistent with enhanced net N2O
production in the soils from warmer historical temperatures, we found a reduced
nirS : nosZ ratio in the southern forest soils. A possible explanation for this
apparent decoupling between gene abundances and biogeochemical outcomes may be an interference
between potential and transcription (i.e., better detected with mRNA) or inadequate measurement of
all genes relevant to N2O dynamics in these soils. Although our experimental setup
promoted denitrification, our incubation may also have supported dissimilatory nitrate reduction to
ammonium (DNRA; Schmidt et al., 2011). This pathway is poorly characterized, but it has been detected
in both aerobic and anaerobic environments of many soil types; it may account for a large proportion
of NO3-–N reduction in forest soils (Bengtsson and Bergwall 2000). DNRA
represents a process that can reduce NO3- via a different nitrite reduction enzyme
(nrf) than denitrification (nir) and can result in an accumulation of
NH4–N, as we observed during our incubation. The process also produces and reduces
N2O (Luckmann et al., 2014). The potential existence of this alternate pathway of
NO3- reduction and N2O production and reduction does not negate the observed
N2O efflux nor the nosZ response to short-term and historical temperature shifts;
however, it does imply that a deeper understanding of the complex genetic N cycle is required to
link soil process rates to genetic potential.
Contrasting efficiencies of N2O scavenging is another possible explanation for the
decoupling between gene abundances and biogeochemical fluxes in these soils, as the catalytic
efficiency of enzymes can vary with community structure and resource availability (Tischer et al.,
2015), which are conditions that vary between boreal soil horizons. The observation that mineral soil has the
capacity to reduce a substantial amount of organic soil-derived N2O even as nosZ
abundances are reduced in mineral compared with organic soil, provides a strong indication that
nosZ in mineral soil is more efficient at scavenging N2O from the headspace than
nosZ in the organic horizon. Alternatively, it would be beneficial to increase efforts to
detect the nosZ clade II in boreal forest soil organic and mineral horizons, as this clade
is not detected by the nosZ primer and has a higher N2O consumption capacity than
nosZ in European mineral soils (Jones et al., 2014). Consistent with our combination
samples in the current study, there is increasing evidence that soils can serve as sinks for
atmospheric N2O (Chapuis-Lardy et al., 2007) and, interestingly, that this phenomenon can
be particularly evident when soil water is limited (Goldberg and Gebauer, 2009). Therefore, given
the varying gene abundance and enzyme efficiency with depth implied in this study, a likely fruitful
area of research would be to explore the mineral soil N2O sink capacity and mineral soil
genetic response as moisture availability varies – the occurrence of which is particularly notable during snowmelt periods
and in fall within these boreal soils.
Conclusions
The sensitivity of soil N2O efflux to global change factors such as rising temperature can
be high, as supported by this study, but the mechanisms driving N2O sources and sinks
remain challenging to elucidate. Indeed, variation of net soil denitrifier N2O efflux
within climate region in this study, although less than variation across regions, warrants further
consideration of within-region controls on N2O efflux. The meaningful differences in the
responses to temperature that we observed across regions, however, permitted us to address the three
critical issues framed at the outset of this study; we conclude with three observations and
questions for future research. To improve Earth system models of greenhouse gas emissions we need to
address the importance of varying N2O dynamics with soil depth. Indeed, this research
highlights the potentially different effectiveness of organisms possessing N2O-relevant
functional genes as we move across depth. Is it ubiquitous that organisms possessing nosZ
are more effective at reducing N2O to N2 in subsurface soils? We have taken the
first step towards this characterization, but similar studies should address this question in
diverse ecosystems. Our results also illustrate that both denitrifier-mediated rates of
N2O production and reduction can increase with warming, over both short- and long-term
timescales, in boreal forest soils. In situ variables would undoubtedly alter the
ex situ fluxes observed in this study, but we demonstrate that the net response to warming in these boreal forest soils is dominated by
N2O production when conditions promote
denitrification. Finally, we remain uncertain of the relative importance of the
denitrification pathway in N2O emissions in boreal forest soils (i.e., compared with
nitrification, co-denitrification, DNRA, and others) and suggest similar approaches to explore the
importance of the historic climate regime, shorter-term temperature variation, and interactive responses
among soil horizons in other biochemical pathways of soil N2O emission.
Code and data availability
The data and code for the figures and analysis are publicly available at
https://doi.org/10.5281/zenodo.3934598 (Buckeridge, 2020).
The supplement related to this article is available online at: https://doi.org/10.5194/soil-6-399-2020-supplement.
Author contributions
KMB and SAB designed the experiment, and KAE, SEZ, and SAB conceptualized the site
aims and managed research for the site. KAE conducted the field sampling, and KMB
and KM carried out the lab incubations and analysis. KMB prepared the
paper with contributions from all co-authors.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
We gratefully acknowledge field assistance from Andrea Skinner and
laboratory assistance from Carl Heroneme, Samantha Elledge, Yanjun Chen, and
Mitch Sellers. Research support was provided by an Association for Women
Geoscientists Graduate Research Scholarship (the University of Kansas)
and the Kansas Biological Survey Graduate Summer Research Fund to Kyungjin Min. The
Canadian Forest Service of Natural Resources Canada provided valuable
logistical support.
Financial support
This research has been supported by the National Science Foundation, Division of
Environmental Biology (grant no. NSF-DEB 0950095) and the Natural Sciences and Engineering
Research Council of Canada (grant no. RGPIN#341863).
Review statement
This paper was edited by Steven Sleutel and reviewed by three anonymous referees.
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