Imprint of denitrifying bacteria on the global
terrestrial biosphere
Benjamin Z. Houlton1 and Edith Bai
Department of Land, Air, and Water Resources, University of California, Davis, CA 95616
denitrification 兩 isotope 兩 nitrogen 兩 ecosystem 兩 microbe
W
e present a simple nitrogen (N) isotope model to investigate the partitioning of gaseous ( fgas) vs. leaching
( fleaching) pathways of N removal from the nonagricultural (i.e.,
unmanaged) terrestrial biosphere. N⬘s isotopes, 14N and 15N, are
stable but display a high degree of variation in biogenic materials
owing to isotope fractionation, particularly kinetic fractionation,
whereby organisms’ enzymes more rapidly transfer the light
isotope from substrates to products. 15N is rare (⬇0.37%), and
small deviations in its concentration relative to 14N have been
widely used in the examination of N cycling on land and in the
sea (1). In the case of the N isotope composition of the terrestrial
biosphere, we consider a steady-state approximation to examine
the partitioning of global N losses:
fgas ⫽
␦ 15NTB ⫺ ␦ 15NI ⫹ ␧ L
␧L ⫺ ␧G
[1]
in which ␦15N ⫽ ((15N/14Nsample/15N/14Nstd ⫺ 1) ⫻ 1,000) of the
terrestrial biosphere (TB) or atmospheric inputs (I); ␧ is the
isotope effect associated with leaching (L) or gaseous (G) N
removal; and 1 ⫽ fgas⫹ fleaching. We define ␧ as the ratio of the
rate constants of the heavy and light isotopes (i.e., ␧ ⫽ (15k/
14k ⫺1) ⫻ 1,000) at steady state, whereby instantaneous isotope
effects are equivalent to equilibrium ones. Here, we constrain
global N losses by: (i) estimating the ␦15N of natural plants
and soils worldwide; (ii) compiling data on the ␦15N of N inputs,
and potential fractionations via leaching vs. gaseous N losses;
and (iii) parameterizing Eq. 1 to quantify the N lost via microbial
denitrification.
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0912111106
Results and Discussion.
Across natural terrestrial environments, 15N/14N of bulk soils
(integrated to 50-cm depth) and foliage vary systematically with
climate (2–4); hence, large-scale (i.e., regions, biomes) terrestrial
15N/14N ratios can be estimated from precipitation and temperature fields (3). Although this model can introduce errors at
smaller scales, it reasonably captures the shift in plant and soil
␦15N across biomes, pointing to its robustness at the global scale
(Fig. S1 and Tables S1 and S2). Taking advantage of this model
(3) and eliminating those areas of land that are managed by
humans (Methods and SI Text), we estimate that the areaintegrated ␦15N of natural soil and vegetation leaves are equal to
5.4‰ (⫾2.2) and 0.43‰ (⫾2.5), respectively (Fig. 1A). Plants
are known to have a lower 15N/14N than soils owing to various
mechanisms (5, 6), consistent with the offset in Fig. 1. Although
plants are not expected to alter the 15N/14N of bulk soils under
quasiequilibrium conditions (7), we account for any such N
recycling effects by combining components of the biosphere into
a single vector. Because roots, shoots, and leaves are similar in
their N isotope compositions (8) (Table S3), the 15N/14N of leaves
also reflects that of total vegetation. Using estimates (9) of the
N stored in vegetation and soils worldwide, we show that the
mass- and area-weighted ␦15N of the unmanaged terrestrial
biosphere is equal to 5.3‰ (Fig. 1 A).
The 15N/14N of the natural terrestrial biosphere is elevated
compared with external N inputs (Fig. 1 A). Biological N2 fixation,
the biochemical conversion of N2 to ammonia, imparts a minor
isotope effect; its ␦15N ranges from 0 to ⫺2‰ (10). The other
major N input to natural systems occurs via atmospheric deposition,
originating from natural and from fossil fuel sources, producing
mainly NOx that rains down as nitrate on land (11). We show in Fig.
1B that the ␦15N of bulk nitrate deposition observed for various
latitudes (Table S4), altitudes and biomes (from deserts to tropical
rainforests; n ⫽ 187) approach a median of ⫺1.3‰, with most
values clustered between ⫺3 and 1. Although ammonium and
dissolved organic N compounds can also contribute deposition,
their 15N/14N ratios either overlap with or are somewhat 15Ndepleted with respect to that of nitrate in bulk precipitation (7, 12,
13). We therefore infer that there must be a pathway of N loss that
preferentially removes 14N from the land, acting to elevate the
terrestrial biosphere’s 15N/14N over the long term.
Two mechanisms could underlie terrestrial 15N enrichment. First,
leaching of N to waterways could remove low 15N/14N compounds
from soil (␧L) (Eq. 1), thereby elevating the 15N/14N of remaining
ecosystem pools (1). In particular, nitrifying bacteria can produce
low 15N/14N nitrate (␧ ⬇ ⫺15 to ⫺35‰) (1); if 15N-depleted nitrate
were to escape ecosystems via streams, it would increase the 15N/14N
Author contributions: B.Z.H. designed research; B.Z.H. and E.B. performed research; B.Z.H.
and E.B. analyzed data; and B.Z.H. and E.B. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
To whom correspondence should be addressed at: Department of Land, Air, and Water
Resources, University of California, One Shields Avenue, Davis, CA 95616. Email:
bzhoulton@ucdavis.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0912111106/DCSupplemental.
PNAS 兩 December 22, 2009 兩 vol. 106 兩 no. 51 兩 21713–21716
ECOLOGY
Loss of nitrogen (N) from land limits the uptake and storage of
atmospheric CO2 by the biosphere, influencing Earth’s climate
system and myriads of the global ecological functions and services
on which humans rely. Nitrogen can be lost in both dissolved and
gaseous phases; however, the partitioning of these vectors remains controversial. Particularly uncertain is whether the bacterial
conversion of plant available N to gaseous forms (denitrification)
plays a major role in structuring global N supplies in the nonagrarian centers of Earth. Here, we use the isotope composition of N
(15N/14N) to constrain the transfer of this nutrient from the land to
the water and atmosphere. We report that the integrated 15N/14N
of the natural terrestrial biosphere is elevated with respect to that
of atmospheric N inputs. This cannot be explained by preferential
loss of 14N to waterways; rather, it reflects a history of low 15N/14N
gaseous N emissions to the atmosphere owing to denitrifying
bacteria in the soil. Parameterizing a simple model with global N
isotope data, we estimate that soil denitrification (including N2)
accounts for ⬇1/3 of the total N lost from the unmanaged terrestrial biosphere. Applying this fraction to estimates of N inputs, N2O
and NOx fluxes, we calculate that ⬇28 Tg of N are lost annually via
N2 efflux from the natural soil. These results place isotopic constraints on the widely held belief that denitrifying bacteria account
for a significant fraction of the missing N in the global N cycle.
ENVIRONMENTAL
SCIENCES
Communicated by William H. Schlesinger, Cary Institute of Ecosystem Studies, Millbrook, NY, October 21, 2009 (received for review June 6, 2009)
Fig. 2. 15N/14N of N leaching losses to small streams across the terrestrial
biosphere. These data were compiled from the primary literature (see SI Text).
The small black symbols are individual observations. The large black symbols
reflect site means for the ␦15N of stream nitrate; the gray symbols reflect the
means for the ␦15N of total dissolved N in small streams. The regression line is
drawn through the site means.
Fig. 1. 15N/14N of soil, vegetation and inputs to the terrestrial biosphere. (A)
␦15N of soil and vegetation were estimated from multiple regression models
(3) and integrated at the global scale in the absence of human-managed
environments (See Methods and SI Text). The ␦15N of the terrestrial biosphere
was estimated as follows: (␦15Nsoil ⫻ Nsoil ⫹ ␦15Nveg ⫻ Nveg)/(Nsoil ⫹ Nveg), where
N is total N (i.e., 14N⫹15N). The solid lines delineate the ␦15N range for
biological dinitrogen fixation (10); the dashed line is the median of bulk
nitrate deposition from B. Errors for soil and vegetation reflect the SE of the
regression models, which were propagated to the terrestrial biosphere (i.e.,
2
2
(SEsoil
⫹ SEveg
)1/2). (B) 15N/14N of bulk nitrate deposition from different locations around the globe, including sites spanning temperate to tropical biomes,
and low to high altitudes (see SI Text).
of remaining terrestrial matter. Alternatively, by discriminating
against 15N, gaseous N losses (␧G) (Eq. 1) could impact the
terrestrial 15N/14N system in manner similar to that of leaching (7).
However, nitrate leaching does not seem to significantly discriminate against ecosystem 15N (Fig. 2). Across major biomes and
latitudes, including arctic tundra, temperate and boreal forest, and
tropical lowland and montane forest (Table S5), there is good
agreement between the ␦15N of nitrate in small drainage streams
and the ␦15N of soil particulate matter (Fig. 2). Together the slope
(0.98) and intercept (⫺0.85) of this significant (r2 ⫽ 0.82; n ⫽ 63;
P ⬍ 0.001) linear relationship point to ␧L ⬍ ⫺1‰ that is systematically conserved across these diverse biomes. Missing from Fig. 2
are arid sites with high average soil ␦15N (⬇4–15‰) (14, 15).
Although leaching is limited in such environments (i.e., evaporation ⬎ precipitation), observations of high nitrate-␦15N in biologically active soils (8–15‰) (14) and in water that percolates from
soils to ground water (10–13‰) (16) suggest a similarly small
leaching effect in natural arid lands. Finally, although other forms
21714 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0912111106
of dissolved N might also influence soil ␦15N, neither is there
evidence for major 15N-discriminations against soil N isotopes by
total N leaching losses (gray circles) (Fig. 2). Thus the available
evidence points to a small leaching term; based on the intercept in
Fig. 2, it seems that leaching could at most raise the ␦15N of the
terrestrial biosphere by ⬇1‰ above N inputs (see Fig. 1 A)
The only remaining mechanism involves isotope fractionation
via gaseous pathways, particularly bacterial ones. Ammonia
volatilization is not likely to affect the integrated 15N/14N of the
terrestrial system; the short atmospheric lifespan of ammonia
means that this N form mainly recycles within and among
terrestrial regions (17). Regional-scale anomalies in ␦15N, which
contain important information beyond the global mean, may be
influenced by processes such as volatilization; but our integrated
estimate would be minimally affected by this process. Although
emission and transport of ammonia from the terrestrial biosphere to the ocean has increased in response to anthropogenic
activities (18), particularly N fertilizer applications, ammonia
emissions from natural soils are relatively small (i.e., 2.4 ⫺ 10 Tg
N/yr) (19, 20). By contrast, bacterial denitrifiers produce substantial quantities of long-lived gaseous compounds (N2, N2O)
which mix evenly in the atmosphere before returning to land.
Denitrification can also produce NOx, especially in dry environments (21), some of which rapidly washes out of the atmosphere
as deposition (17, 22). Nevertheless, this term is small compared
with the combined losses of NOx, N2O, and (especially) N2 (22).
Proximally, whether originating from denitrification or nitrifier-denitrification, bacterial pathways of gaseous N removal
would be expected to elevate the 15N/14N of nitrate relative to
any upstream isotope effects (23, 24). Indeed, the average
isotope effect of denitrification on nitrate is substantial in both
pure culture (⫺20 ⫾1.0‰) and in natural soil communities
(⫺16 ⫾1.6‰) (Table S6). The slightly lower (P ⬍ 0.1; two-tailed
t test) value for soil might be due to heterogeneities in nitrate
consumption, which reduces expression of the isotope effect
relative to homogenous (culture) conditions (7, 25). Perhaps
more important, the isotope effect of denitrification seems to
control entire ecosystems: the near ⬇1:1 relationship in Fig. 2
can only be explained by fractionating losses via denitrification
under steady state conditions (7). The similarities between
nitrate ␦15N and soil ␦15N indicates that the rather high isotope
Houlton and Bai
Table 1. Global isotopic constraints on N2 efflux from natural
terrestrial soil (Tg N/yr)
Inputs
Gaseous N emissions
Fixation ⫹ deposition
134.64
130
N2O
12.0†
6.1‡
8.0§
NOx
5.0†
9.7‡
7.4§
N2*
N2/N2O
26.6
27.9
28.3
2.2
4.6
3.5
effect of nitrification is not fully expressed (i.e., closed system)
and that any low 15N/14N nitrate produced during nitrification
passes through zones of denitrification in the soil before leaching
(7). In sum, the elevated 15N/14N of the terrestrial biosphere (Fig.
1 A) points to substantial 14N removal via denitrifying bacteria—
consistent with how this bacterial group elevates the 15N/14N of
the global nitrate reservoir in the oceanic water column (23, 26).
Parameterizing Eq. 1 with the mid-point of inputs (⫺1‰) and
the observed isotope effect of leaching (␧L ⫽ ⫺0.8‰; Fig. 2) and
denitrifying bacteria in pure culture (␧G ⫽ ⫺20‰; Table S6) we
estimate that denitrification accounts for a substantial fraction
(mean ⫽ 28%; maximum sensitivity range ⫽ 26% to 31% given
by changing the mean ␦15NTB and ␦15NI by ⫾10%) of fixed N loss
from the unmanaged terrestrial biosphere (square; Fig. 3). This
estimate is probably a lower bound: any microzones of complete
nitrate consumption in soil would not elevate the 15N/14N of
remaining pools (7). Using the mean isotope effect observed for
native denitrifier communities in various soils (⫺16‰) to help
account for such isotopic underexpression (circle; Fig. 3) increases the contribution of denitrification to ⬇36% (maximum
sensitivity range ⫽ 32% to 40% given by changing the mean
␦15NTB and ␦15NI by ⫾10%) Additional information on denitrification’s isotope effect will help to constrain our estimate
further (Fig. 4 and Table S6). Other uncertainties in our model
could arise from our steady-state assumption, because N may be
accumulating in terrestrial systems (17). For instance, given
modern rates of terrestrial C accumulation (⬇2 Pg C/yr) (27), a
C/N ratio that partitions this C into soil (15/1) and plant materials
(200/1) equally (28), and assuming that all of the additional C
results from new N inputs, we estimate that ⬇18 Tg N/yr is
accumulating on land. This value drops to ⬇10 Tg N/yr if all of
the C is assumed to be stored in plant biomass, consistent with
other estimates (e.g., 9 Tg N/yr) (29). Even integrated over 100
years, however, such rates of N accumulation would only have
altered total terrestrial N by ⬍0.1% (9). Thus, our results, which
Houlton and Bai
Methods
The ␦15N of the terrestrial soil and plant matter were derived from global
climate regressions, integrated across the terrestrial surface in the absence of
human-managed areas of land (see also SI Text). The integrated soil (or plant)
␦15N was obtained by applying the zonal tools algorithm in ArcView GIS
Spatial Analyst to a raster estimate of the unmanaged soil (or plant) ␦15N
surfaces. The global unmanaged surface was based on the biome classification
PNAS 兩 December 22, 2009 兩 vol. 106 兩 no. 51 兩 21715
ECOLOGY
Fig. 3. Steady-state solutions of the fraction of N lost via denitrification at
the global scale. Isoclines are based on Eq. 1. The midpoint of inputs is based
on our compilation in Fig. 2 and estimates of the ␦15N of biological dinitrogen
fixation. The isotope effect of leaching (␧L) is based on the calculated intercept
in Fig. 2. The isotope effect of denitrification (␧G) is shown for culture studies
(square) and native soil communities (circle) (see SI Text).
are based on total terrestrial N, mainly reflect N losses via
denitrification vs. leaching pathways.
We conclude that the elevated 15N/14N of the natural (i.e.,
unmanaged) terrestrial biosphere bears the long-term imprint of
microbial denitrification (including NOx, N2O, and N2), providing a quantitative tracer of gaseous N emissions from the soil.
The 15N/14N of the biosphere integrates many regional patterns
thereby reflecting the relative strengths of leaching vs. denitrification pathways as a whole. Arid environments, for example,
are among the most 15N enriched of the terrestrial biomes; yet
denitrification rates are likely low in arid soils compared to other
global ecosystems (30). Rather, our analysis suggests that gaseous N efflux is comparatively high relative to leaching in sites
with high ␦15N, not necessarily large in absolute flux, consistent
with global N cycle models (30, 31). Future work on regional
patterns will help in identifying the dominant source regions of
N release from the land to the water and atmosphere.
For decades, N2 has been prominently purported in the
missing N of the global N cycle (9, 17, 22, 29, 32, 33). Using
current estimates of global N inputs (mean ⫽ 132 Tg N/yr; Table
1) to natural ecosystems and subtracting fluxes of NOx and N2O
from natural soils, we place isotopic constraints on N2 efflux
from the unmanaged terrestrial biosphere (see Table 1). Specifically, we estimate that ⬇28 Tg of N are lost annually via
denitrification to N2 in the terrestrial soil beneath natural
vegetation, with an N2/N2O ratio ranging from 2.2 to 4.6 at the
global scale. This implies that unmeasured N2 losses are indeed
substantial. The range in N2/N2O ratios is driven (largely) by
variations in global estimates of NOx and N2O (Table 1); the
lower end of the range is approximately twice as high as the mean
of point measures for upland ecosystems (i.e., N2/N2O ⫽ 1),
although within the very wide range of the estimates reported
(⬇0–32) (29). Moreover, the total denitrification flux (44 Tg
N/yr) we estimate for the unmanaged terrestrial biosphere
agrees reasonably well with estimates for natural soils as based
on numerical simulation models (e.g., 58 Tg N/yr) (31). Like other
approaches, our isotope-based calculation relies on knowledge of N
inputs, which suffer from their own uncertainties; the inputs we
used here fall on the lower end of the spectrum for natural N2
fixation (100–290 Tg N/yr) (34). Combining fluxes with isotopebased models seems like a promising way forward in addressing
denitrification and N2 losses quantitatively (e.g., ref. 35), an approach that may be amenable to agricultural sites as well.
ENVIRONMENTAL
SCIENCES
Inputs to natural systems from Gruber and Galloway (130 Tg N) (22) and van
Drecht et al. (134.64 Tg N/yr) (30).
*This study, where denitrification ⫽ 33% of total N losses; and N2 ⫽ average
N inputs (i.e., 132 Tg N) ⫻ 0.33 ⫺ (N2O ⫹ NOx).
†Bowden (36).
‡Potter et al. (37).
§Galloway et al. (17), preindustrial (unmanaged) soil.
soil communities were collated from the primary literature. Details of our
literature syntheses can be found in the SI Text.
scheme of VUB and VITO, derived from a full year cycle (1998 –1999) of 10 daily
composites of SPOT-VEGETATION (www.geosuccess.net/Geosuccess). Areas
classified as croplands, urban and built-up, and cropland and natural vegetation mosaic were considered as human-managed.
Data on the ␦15N of N leaching losses to small streams and atmospheric N
inputs, and the isotope effect of denitrification in pure culture and in native
ACKNOWLEDGMENTS. We thank Bryant A. Browne for influential discussions,
and Audrey Niboyet for comments. This work was supported by the Andrew
W. Mellon Foundation.
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Houlton and Bai