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Cumulative response of ecosystem carbon and nitrogen stocks to
chronic CO
2
exposure in a subtropical oak woodland
Bruce A. Hungate
1
, Paul Dijkstra
1
, Zhuoting Wu
1,2
, Benjamin D. Duval
1,3
, Frank P. Day
4
, Dale W. Johnson
5
,
J. Patrick Megonigal
6
, Alisha L. P. Brown
4
and Jay L. Garland
7
1
Department of Biological Sciences and The Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, AZ 86011, USA;
2
US Geological Survey, Flagstaff, AZ 86001,
USA;
3
US Dairy Forage Research Center, USDA-ARS, Madison, WI 53706, USA;
4
Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529, USA;
5
Department of
Natural Resources and Environmental Science, University of Nevada, Reno, NV 89557, USA;
6
Smithsonian Environmental Research Center, Edgewater, MD 21037, USA;
7
Environmental
Protection Agency, Microbiological and Chemical Exposure Assessment Research Division, Cincinnati, OH 45268, USA
Author for correspondence:
Bruce A. Hungate
Tel: +1 928 523 0925
Email: bruce.hungate@nau.edu
Received: 24 January 2013
Accepted: 10 April 2013
New Phytologist (2013)
doi: 10.1111/nph.12333
Key words: carbon cycling, elevated CO
2
,
global change, long-term experiment,
nitrogen cycling, scrub oak, soil carbon,
subtropical woodland.
Summary
Rising atmospheric carbon dioxide (CO
2
) could alter the carbon (C) and nitrogen (N) con-
tent of ecosystems, yet the magnitude of these effects are not well known. We examined C
and N budgets of a subtropical woodland after 11 yr of exposure to elevated CO
2
.
We used open-top chambers to manipulate CO
2
during regrowth after fire, and measured
C, N and tracer
15
N in ecosystem components throughout the experiment.
Elevated CO
2
increased plant C and tended to increase plant N but did not significantly
increase whole-system C or N. Elevated CO
2
increased soil microbial activity and labile soil C,
but more slowly cycling soil C pools tended to decline. Recovery of a long-term
15
N tracer
indicated that CO
2
exposure increased N losses and altered N distribution, with no effect on N
inputs.
Increased plant C accrual was accompanied by higher soil microbial activity and increased C
losses from soil, yielding no statistically detectable effect of elevated CO
2
on net ecosystem C
uptake. These findings challenge the treatment of terrestrial ecosystems responses to elevated
CO
2
in current biogeochemical models, where the effect of elevated CO
2
on ecosystem C
balance is described as enhanced photosynthesis and plant growth with decomposition as a
first-order response.
Introduction
Many experiments have examined the responses of plant produc-
tion and ecosystem carbon (C) balance to rising atmospheric
CO
2
(Reich et al., 2006a; Norby & Zak, 2011). Results from
these feature prominently in assessments of potential feedbacks
between the biosphere and the changing atmosphere (Dolman
et al., 2010). Compared to responses of photosynthesis and plant
growth to elevated CO
2
, the response of soil C is less well under-
stood, because changes in soil C content are difficult to detect
(Smith, 2004). Increased C in soil in response to elevated CO
2
is
sometimes found (Jastrow et al., 2005; Iversen et al., 2008,
2012), although more frequently there is no effect, whether
because of low statistical power or the absence of an important
effect is unclear (Hungate et al., 2009). Ecosystem-scale invento-
ries assessing C balance responses to elevated CO
2
also often
show no effect (Hungate et al., 1997b; Gielen et al., 2005; Gill
et al., 2006; Niklaus & Falloon, 2006; Adair et al., 2009),
although in aggregate some analyses suggest an effect is apparent
(Luo et al., 2006). Thus, global models projecting future C
dynamics of the biosphere have strong support for the effects of
CO
2
on plant growth (Denman et al., 2007), but less empirical
support for assumed effects on total ecosystem C storage. Our
first goal in this work was to construct a complete C inventory
for a subtropical oak woodland after 11 yr of exposure to elevated
CO
2
, to test whether the CO
2
treatment altered total system C
accumulation, and determine how any changes in C accumula-
tion were distributed among plant and soil pools.
Total ecosystem C content is a function of plant growth and
accumulation of plant biomass and detritus and also of C losses
through microbial decomposition. Microbial decomposition is
typically assumed to be a first-order process (Parton et al., 1987),
responding predictably and constantly to changes in substrate
supply, and thus is not expected to respond to elevated CO
2
independently of changes in substrate accumulation (Denman
et al., 2007). Challenging this idea, inputs of C to soil can stimu-
late mineralization of native soil organic matter (Lohnis, 1926;
Broadbent & Norman, 1947; Broadbent & Bartholomew, 1949;
Van Veen et al., 1991), and increased atmospheric CO
2
has been
shown to promote microbial activity (Dieleman et al., 2010) and
even soil C loss (Hoosbeek, 2004; Trueman & Gonzalez-Meler,
2005; Carney et al., 2007; Hagedorn et al., 2008; Paterson et al.,
2008; Taneva & Gonzalez-Meler, 2008; Langley et al., 2009;
Trueman et al., 2009; Drake et al., 2011; Reid et al., 2012).
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This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
Research
Thus, soil processes influence potential C accumulation in
response to increasing atmospheric CO
2
, yet how and to what
extent are not well understood. Our second goal in this work was
to examine changes in soil microbial activity during the 11 yr of
CO
2
enrichment, and to test whether patterns of CO
2
effects on
soil microbial activity might help explain any effects (or lack of
effects) of elevated CO
2
on soil C stocks.
Carbon cycling in ecosystems is linked to cycles of other ele-
ments (Finzi et al., 2011), such as nitrogen (N). Simulations of
land carbon uptake using models with coupled N and C dynam-
ics usually differ, and in many cases differ strongly, from those
ignoring N (e.g. compare Cramer et al., 2001 and Thornton
et al., 2007), because N limits plant growth and C storage
(LeBauer & Treseder, 2008), and because N cycling is sensitive
to environmental change (Galloway et al., 2008). With N cycling
included, simulations project smaller increases in terrestrial C
storage in response to rising CO
2
, because N availability limits
plant growth and its response to elevated CO
2
(Thornton et al.,
2007; McMurtrie et al., 2008; Sokolov et al., 2008; Jain et al.,
2009; Wang & Houlton, 2009; Friedlingstein & Prentice, 2010;
Zaehle et al., 2010).
While model simulations bear out the importance of including
N, these models do not necessarily demonstrate a consistent
pattern of effect. Results differ in magnitude, direction and
mechanism, suggesting that additional data and analyses are
needed to evaluate conditions under which C–N coupling is
important. For example, some simulations project only a modest
limitation of terrestrial C uptake with coupled C–N interactions
in the long term (at equilibrium), but strong effects of C–N
interactions on the dynamics of C cycling and storage after dis-
turbance (Gerber et al., 2010). Although the models generally
agree that including N limitation of plant production reduces the
terrestrial C sink, the magnitude of this effect is highly variable
(Arneth et al., 2010). Experiments also indicate that C–N inter-
actions are critical modulators of the long-term CO
2
fertilization
response, but different experiments provide support for different
mechanisms underlying that modulation. In some cases, C–N
interactions appear to constrain strongly the CO
2
response
(Reich et al., 2006a,b; Norby et al., 2010; Garten et al., 2011),
but in others, plants appear able to access the extra N needed to
support the growth response (Johnson et al., 2006; Drake et al.,
2011). Effects of CO
2
concentration on microbial N transforma-
tions that influence the plant–soil distribution of N are extremely
variable, with negative, positive and neutral effects observed for
the same processes (Dıaz et al., 1993; Zak et al., 1993; Morgan
et al., 1994; Zanetti et al., 1996; Hungate et al., 1997a,c; Johnson
et al., 1997; Hofmockel & Schlesinger, 2007; van Groenigen
et al., 2011, 2012). Furthermore, other concomitant global envi-
ronmental changes will modulate N constraints on C balance
responses to elevated CO
2,
including changes that alter N cycling
directly, such as warming, altered precipitation and atmospheric
N deposition, as well as indirect effects, such as changes in plant
species composition. There is considerable debate as to the mag-
nitude of the impact of such effects on ecosystem C sequestra-
tion, however (Jenkinson et al., 1999; Nadelhoffer et al., 1999;
Arneth et al., 2010). Thus, both model simulations and data can
be invoked to support N cycling constraining, increasing, or hav-
ing little effect on the terrestrial C sink. Our third goal in this
research was to compare C and N inventories in response to 11 yr
of CO
2
exposure in a subtropical woodland, in order to test how
rising CO
2
affects these elements in concert.
One of the challenges in investigating C–N interactions in eco-
system experiments is that the timescale of measurements of N
cycling rates is typically far shorter than the timescale of N
cycling processes that influence ecosystem responses. Elevated
CO
2
can alter multiple processes within the soil N cycle simulta-
neously, with strong temporal dynamics, and with opposing
impacts on plant N availability, making it very difficult to extrap-
olate short-term measurements to long-term effects. Following
an isotope tracer over multiple years can help overcome this chal-
lenge.
15
N tracers reflect short-term effects on N cycling processes
and integrate these into long-term effects on
15
N distribution
among plant and soil components within the system. Because the
15
N is added in labile form, losses of added
15
N will be relatively
larger than losses of total ecosystem N, so can be detected with
greater sensitivity. Our fourth goal in this research was to use a
long-term
15
N tracer to characterize changes in N distribution
and N losses in response to elevated CO
2
.
Here, we report a whole system inventory of the C and N con-
tent of a scrub-oak ecosystem after 11 yr of experimental CO
2
exposure. We also show how soil microbial activity responded to
chronic CO
2
exposure. We also report recovery and distribution
of a
15
N tracer applied early in the experiment, in order to assess
how elevated CO
2
alters the system-level distribution of labile N
over the timescale of a decade.
Materials and Methods
The scrub-oak experiment occurred at the Merritt Island
National Wildlife refuge on the east coast of Florida, USA
(28°38′N, 80°42′W). After controlled burning, 16 open-top
chambers were established over the regrowing vegetation, each
covering 9.42 m
2
ground area, with 8 chambers receiving ambi-
ent air and 8 receiving ambient air +350 ppm V CO
2
(referred
to as the ‘elevated CO
2
’ treatment). A large blower circulated air
through each chamber at a rate of 24–30 m
3
min
1
, replacing
the chamber air volume 1.3–1.6 times min
1
(Dijkstra et al.,
2002). The chambers increased air temperature and vapor
pressure deficit while decreasing light (Dore et al., 2003), micro-
environmental effects that did not significantly alter growth or
species composition (Seiler et al., 2009). The experiment began
in May 1996 and was maintained until June 2007.
In June–July 2007, all aboveground material was harvested
from the chambers (see Seiler et al., 2009), and roots and soils
were collected using multiple cores in each chamber (see Day
et al., 2013). For aboveground biomass, all shoots were cut at the
base of the stem, weighed immediately, and subsampled for the
determination of water content and elemental analysis of leaves
and stems. Ten surface cores (0–10 cm) and five deep cores were
collected from each plot at 10 cm increments; all cores were 7 cm
diameter. Core depth varied among plots from 2 to 3 m due to
differences in the depth to the water table and the spodic (B
h
)
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horizon. For purposes of the element inventory conducted here,
depth increments were combined into 0–10, 10–30, 30–60 and
60–100 cm. Samples were hand-picked to remove large roots,
and subsamples separated into coarse particulate organic matter,
roots and mineral soil. Belowground biomass was also sampled
indirectly using ground-penetrating radar (Stover et al., 2007,
Day et al., 2013). Material on the forest floor was gathered from
1/8th of each plot by hand, collecting until no visibly identifiable
plant fragments remained. Material was dried, sifted to remove
adhering sand, and weighed.
We used a combination of density and biological fractiona-
tions to estimate soil carbon (C) pools of varying turnover
rates. We used incubations to estimate labile and active soil C
pools (and, by difference residual C), using the technique of
Nadelhoffer (1990). We measured CO
2
production from
laboratory incubations, combining short-term incubations of
soils immediately after collection (McKinley et al., 2009) with
541-d incubations conducted in the lab at Northern Arizona
University. We used density fractionations as described previ-
ously (Hungate et al., 2006; Carney et al., 2007), separating light
(<1.5 g cm
3
), medium (1.5–1.8 g cm
3
), heavy (1.8–
2.2 g cm
3
) and residual (>2.2 g cm
3
) organic matter fractions.
Total soil C, N,
15
Nand
13
C were also measured on bulk sam-
ples collected from the cores. Our fractionation analysis focused
on soils from the 0–60 cm depths. For bulk soil analyses where
we measure total C, N and
15
N, we present the data to 1 m to
correspond with the depth of the root biomass inventory.
We measured microbial biomass using the chloroform-fumi-
gation extraction method (Vance et al., 1987) in mineral soil
(0–15 cm) sampled in July 1997; June, July, September and
December 1998; September 1999; and May 2004. Soil subs-
amples (20–25 g at field moisture content) were extracted in
75 ml 0.5 M K
2
SO
4
before and after 24-h fumigation with eth-
anol-free chloroform. The K
2
SO
4
extracts were dehydrated in a
forced-air drying oven at 60°C, the salts ground in a mortar
and pestle, and the resulting powder analyzed for C, N, d
15
N
and d
13
C on a CE 2100 elemental analyzer coupled to a
Thermo DeltaPLUS-XL isotope-ratio mass spectrometer
(http://www.isotope.nau.edu). Microbial biomass was calculated
as the difference in mass (of C, N,
13
Cor
15
N) between fumi-
gated and nonfumigated samples, divided by 0.54 to correct
for extraction efficiency (Vance et al., 1987). For samples col-
lected after the
15
N tracer application (June 1998), we also
measured the
15
N content of mineral soil (0–15 cm depth).
After milling, soil N and
15
N contents were determined as
described above.
The CO
2
added to the elevated-CO
2
treated plots was
depleted in
13
C. We used a two-member mixing model to
determine mineral soil C derived from new photosynthate
(Leavitt et al., 1994; Hungate et al., 1996). Stem tissue pro-
duced in the elevated CO
2
treatment (d
13
C
S,E
) provided an
integrative measure of the d
13
C value of new photosynthate
(average across five sampling dates, -42.6 0.3 &). However,
because mineral soil (d
13
C
M,A
) and stem d
13
C(d
13
C
S,A
)
differed in the ambient C
a
treatment, we calculated the d
13
C
signature of new carbon (d
13
C
new
) as:
d13Cnew ¼d13 CS;Eðd13 CS;Ad13CM;AÞ:Eqn 1
The d
13
C of the mineral soil in the ambient CO
2
treat-
ment was used as the end member for organic matter fixed
before the experiment began. Carbon, N,
15
N, and
13
Cwere
determined for all plant and soil components using coupled
Dumas combustion isotope-ratio mass spectrometry (Carlo-
Erba elemental analyzer and Finnigan Delta-V mass spectrom-
eter) at the Colorado Plateau Stable Isotope Laboratory
(www.isotope.nau.edu).
For testing soil microbial activity, we collected soil and litter
samples in May through July of 2004, after 8 yr of CO
2
treat-
ment. Soil sampling, preparation of microbial inocula, carbon
and nutrient amendments, and incubation conditions are
described in Brown et al. (2009). Carbon substrates included glu-
cose and hot-water extracts of roots and leaf litter collected from
the ambient and elevated CO
2
treatments. Microbial inocula
from litter, rhizosphere and bulk soil communities were also
prepared from the two CO
2
treatments. We used the BD-oxy sys-
tem (BD Oxygen Biosensor System, BD Biosciences, Bedford,
MA, USA (Garland et al., 2003; V€ais€anen et al., 2005; Zabaloy
et al., 2008) to evaluate microbial respiration. The system uses a
fluorophore that fluoresces as O
2
is consumed during the 48 h
incubation. Normalized relative fluorescence was calculated as
relative fluorescence after 48 h normalized by dividing by relative
fluorescence after 1 h. The response to substrate addition was cal-
culated as:
Relative response ¼ðRrRcÞ=Rc100%Eqn 2
(R
c
, normalized relative fluorescence in the absence of resource
addition; R
r
, normalized relative fluorescence with the added
resource. Brown et al. (2009) present data from the ambient CO
2
treatment; here, we expand on this past analysis to evaluate
responses of microbial respiration to elevated CO
2
. We used
ANOVA to test for effects of habitat (rhizosphere, litter or bulk
soil), inoculum source (ambient or elevated CO
2
), substrate
source (ambient or elevated CO
2
), substrate type (litter or root),
N, and P. We used a separate ANOVA to test compare responses
to the addition of glucose vs natural substrates extracted from
roots and litter. Where appropriate, ANOVAs were designed as
split-plots, to account for the nonindependence of inocula col-
lected from individual experimental plots subject to multiple
combinations of resource treatments in the BD-Oxy assay.
We used resampling to infer the effects and estimate the mag-
nitude of the elevated CO
2
treatment on ecosystem C and N
pools and recovery of tracer
15
N. We estimated 5% and 95%
confidence limits for the difference in means between elevated
and ambient CO
2
treatments, using 1000 samples with replace-
ment (n=8 for each treatment).
Results
Elevated CO
2
increased plant biomass, including the mass of C
(g C m
2
) in leaves, stems and coarse roots, and the total mass of
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C in plants (Table 1). The mass of C in fine roots was not signifi-
cantly affected by the elevated CO
2
treatment at the final harvest
(Table 1), although fine roots did exhibit significant increases at
other times during the experiment (Day et al., 2013). On average,
plant C accumulation by the end of the experiment was
71.5 g C m
2
yr
1
higher in elevated compared to ambient CO
2
,
roughly equally distributed aboveground (37.5 g m
2
yr
1
) and
belowground (33.5 g m
2
yr
1
). The C content of the litter layer,
coarse particulate organic matter, total mineral soil C, and the
light and medium density fractions did not significantly respond
to the CO
2
treatment, whereas the heavy density soil C pool sig-
nificantly declined. Elevated CO
2
had no effect on soil C in the
spodic horizon, with no significant effect on total mineral soil C,
or on the light, medium and heavy density fractions (Table 2);
thus, C in the deep soil was also insensitive to the CO
2
treatment.
In general, increased mass of plant C caused by elevated CO
2
did
not translate to increased C storage in other ecosystem reservoirs
(Table 1).
Elevated CO
2
increased the N content of plants above-
ground (Table 3), but the N contents of coarse and fine roots
did not respond to elevated CO
2
, yielding no effect on total
plant N. The N content of most soil fractions was not signifi-
cantly altered by elevated CO
2
, except the medium density
fraction at 30–60 cm, which increased, and the light fraction at
10–30 cm, which declined. Increased C in plant pools with only
small changes in N means higher C to N ratios. Higher C to N
ratios under elevated CO
2
were observed for leaves, coarse roots
and the sum of all plant parts; elevated CO
2
also increased the C
to N ratio of the litter layer (Table 4). Elevated CO
2
did not
increase the C to N ratio of any soil pool; the only soil pool to
respond –the heavy density fraction –actually declined in C to
N ratio. Changes in plant and soil C to N ratios were compensa-
tory, such that elevated CO
2
had no effect on the C to N ratio of
the plant–soil system to 1 m depth.
Elevated CO
2
increased recovery of tracer
15
N in above-
ground plant tissues, but reduced recovery in coarse roots, in the
soil light fraction at 10–30 cm depth, and in the soil residual
fraction at 0–60 cm (Table 5). Together, these changes resulted
in a significant decline in whole-system
15
N recovery under ele-
vated CO
2
. Elevated CO
2
reduced the d
15
N of plant tissue
(weighted average of all plant parts), a dilution of the added
15
N
tracer with unlabeled
15
N. This pattern indicates that elevated
CO
2
increased plant access to N, either through new N inputs
or redistribution from existing ecosystem N reservoirs. But,
because total plant N did not respond to elevated CO
2
, the
increase in inputs of new N to plants were matched by N losses
from plants, such that CO
2
enhanced N turnover through the
plant system. In contrast to plant d
15
N, the d
15
N of soils did
Table 1 Inventory of carbon after 11 yr exposure to increased atmospheric CO
2
in a subtropical oak woodland
a
Carbon (g C m
2
) Ambient Elevated Effect 5% & 95% CLs
Aboveground 624.5 54.6 1043.0 77.5 418.5 (274.8 to 556.9)
Oak leaves 212.2 22.3 318.4 29.6 106.2 (47.6 to 157)
Oak stems 347.1 34.2 621.6 60.8 274.5 (164.8 to 374.2)
Other species 38.3 10.7 63.1 10.2 24.7 (1.7 to 47.4)
Standing dead 26.9 8.4 39.8 13.8 13.0 (9.7 to 39.5)
Litter layer 332 41.2 368.1 42.4 36.1 (57.9 to 127.7)
Roots 2886.7 90.2 3261.3 174.6 374.6 (73.6 to 674.5)
Fine roots 909.4 62.8 803.9 43.3 105.5 (226.8 to 9.7)
Coarse roots 1977.3 102.8 2457.4 177.7 480.1 (168.9 to 790.0)
Plant 3511.2 102.0 4304.3 221.3 793.1 (437.4 to 1172.7)
CPOM (0–100 cm) 1406.5 386.4 1168.5 272.1 238.0 (957 to 354.4)
Soil (0–100 cm) 5513.1 411.5 5025.6 647.4 487.5 (1456.5 to 636.8)
Light, 0–60 cm 2534.7 260.2 2394.4 333.3 140.4 (746.8 to 473.2)
0–10 cm 1530.9 284.8 1415.8 316.8 115.0 (760.1 to 565.5)
10–30 cm 480.2 94.5 331.2 36.5 149.1 (297.9 to 6.7)
30–60 cm 523.7 149.9 647.3 169.2 123.7 (214.9 to 474.5)
Medium, 0–60 cm 1306.3 302 1208.4 177.3 97.9 (633.8 to 380.3)
0–10 cm 660.3 115.3 560.7 108.2 99.6 (346.4 to 158.9)
10–30 cm 370.9 109.2 341.5 55.2 29.4 (222.4 to 147.1)
30–60 cm 275 157.8 306.2 88.6 31.1 (267.6 to 289.2)
Heavy, 0–60 cm 706.3 120.5 396 92.1 310.4 (553.2 to 86.0)
0–10 cm 110.9 27 81.2 19.9 29.7 (81.7 to 22.0)
10–30 cm 148 23.7 83.5 30.6 64.6 (122.9 to 2.0)
30–60 cm 447.4 107.6 231.3 87.2 216.1 (402.7 to 3.3)
Residual, 0–60 cm 965.8 1026.9 1026.9 330.9 61.1 (782.3 to 925.2)
Soil (60–100 cm) 1547.0 129.3 1877.6 359.8 330.7 (274.4 to 925.4)
Total ecosystem 12309.8 582.1 12744.1 444.3 434.4 (723 to 1529.9)
a
Values are means SE of the mean for the Ambient and Elevated CO
2
treatments, the Effect of the CO
2
treatment (E–A), and the bootstrapped 5% and
95% CLs (confidence limits) for the treatment effect. CPOM, coarse particulate organic matter. Soil fractions are density fractions, including light
(<1.5 g cm
3
), medium (1.5–1.8 g cm
3
), heavy (1.8–2.2 g cm
3
) and residual (calculated as total soil total minus the sum of measured density fractions).
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not change with elevated CO
2
, nor was whole-system d
15
N
affected (Table 6).
While elevated CO
2
did not alter total ecosystem C, and
effects on soil C were either nil or negative, several results indicate
that elevated CO
2
increased soil microbial activity. Elevated CO
2
increased C mineralization in laboratory incubations, particularly
for the first 24 h after collection in the field (Fig. 1, and see
McKinley et al., 2009), indicating a larger and more rapidly
cycling labile soil C pool. Elevated CO
2
also increased the pro-
portion of soil organic matter that occurred in the soil microbial
biomass: averaged across seven sample dates from 1997 to 2004,
more soil C, N and
15
N was contained in the soil microbial bio-
mass in the elevated CO
2
treatment (P=0.012 for C, P=0.096
for N, and P=0.049 for
15
N; Fig. 2). When common inocula
were presented with the labile substrates produced by leaves and
roots, substrates produced in the elevated CO
2
treatment were
respired more completely than substrates from the same sources
in the ambient CO
2
treatment (Fig. 3a), indicating that the sub-
strates produced in the high-CO
2
environment were more sus-
ceptible to microbial decay. For the litter and rhizosphere
microbial communities, microbial inocula from the elevated
CO
2
treatment consumed more O
2
than inocula collected from
the ambient CO
2
treatment when presented with a common C
substrate (Fig. 3b). Glucose induced a greater response in bulk
soil inoculum from the ambient treatment (Fig. 3b), which may
reflect CO
2
-depletion of available soil C susceptible to priming
(Brown et al., 2009).
The incorporation of the depleted d
13
C signature into organic
matter pools revealed rates and patterns of flow of ‘new’ C into
the system, where new C is that fixed since CO
2
fumigation
Table 2 Soil carbon (C) in the spodic horizon of the subtropical oak woodland
Ambient Elevated
P-value
Ambient Elevated
P-value%C d
13
C
Total C 0.77 0.10 0.60 0.15 0.383 25.6 0.1 25.1 0.3 0.157
Light 18.3 2.8 11.9 1.2 0.151 25.3 0.1 25.3 0.1 0.943
Medium 14.2 3.8 9.4 2.0 0.288 25.6 0.2 25.3 0.1 0.178
Heavy 13.2 2.3 12.2 1.0 0.710 25.6 0.1 25.2 0.2 0.116
Table 3 Inventory of ecosystem nitrogen
(g N m
2
) after 11 yr exposure to increased
atmospheric CO
2
in a subtropical oak
woodland
a
Ambient Elevated Effect 5% & 95% CLs
Aboveground 8.4 0.8 13.1 0.9 4.7 (3.0 to 6.3)
Oak leaves 4.7 0.6 6.6 0.6 1.9 (0.5 to 3)
Oak stems 3.0 0.3 5.1 0.5 2.2 (1.2 to 3.1)
Other species 0.5 0.1 1.1 0.2 0.5 (0.2 to 0.9)
Standing dead 0.2 0.1 0.3 0.1 0.1 (0.1 to 0.3)
Litter layer 5.7 0.7 6.0 0.9 0.3 (1.4 to 2.2)
Roots 29.3 1.8 27.8 2.5 1.4 (6.4 to 3.1)
Fine roots 8.3 0.8 7.3 0.9 1.0 (2.8 to 0.9)
Coarse roots 21.0 1.2 20.5 2.5 0.4 (4.6 to 4.2)
Plant 37.7 1.8 41.0 2.9 3.3 (1.8 to 8.3)
CPOM (0–100 cm) 20.7 5.7 15.2 3.5 5.4 (15 to 3.0)
Soil (0–100 cm) 159.5 15.0 145.4 17.5 14.2 (44.2 to 15.9)
Light, 0–60 cm 55.9 7.0 54.9 8.9 1.0 (19 to 16.6)
0–10 cm 37.2 7.6 37.6 8.9 0.4 (17.9 to 18.5)
10–30 cm 9.6 1.8 6.5 0.7 3.1 (5.8 to 0.1)
30–60 cm 9.1 2.8 10.8 2.1 1.7 (4.2 to 6.4)
Medium, 0–60 cm 30.7 5.5 30.9 4.1 0.2 (10.9 to 11)
0–10 cm 18.8 3.4 15.4 3.0 3.4 (10.4 to 3.5)
10–30 cm 7.9 2.0 7.0 1.0 0.9 (4.3 to 2.3)
30–60 cm 4.0 1.5 8.5 2.6 4.6 (0.4 to 9.3)
Heavy, 0–60 cm 17.3 2.1 15.6 4.9 1.7 (9.2 to 6.8)
0–10 cm 3.4 0.8 2.4 0.6 1.0 (2.7 to 0.5)
10–30 cm 4.1 0.8 2.5 1.1 1.6 (3.3 to 0.5)
30–60 cm 9.8 1.5 10.7 4.4 0.9 (6 to 8.9)
Residual, 0–60 cm 55.6 44.0 12.3 13.5 43.3 (40 to 17.3)
Soil (60–100 cm) 51.3 3.2 55.3 8.8 4.1 (10.8 to 18.2)
Total ecosystem 274.8 10.9 262.9 13.9 12.0 (38.1 to 18.9)
a
Values are means SE of the mean for the Ambient and Elevated CO
2
treatments, the Effect of the CO
2
treatment (E–A), and the bootstrapped 5% and 95% CLs (confidence limits) for the treatment effect.
CPOM, coarse particulate organic matter. Soil fractions are density fractions, including light (<1.5 g
cm
3
), medium (1.5–1.8 g cm
3
), heavy (1.8–2.2 g cm
3
) and residual (calculated as total soil total minus
the sum of measured density fractions).
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began in May 1996. By 2007, coarse roots contained
740 g C m
2
of new C, 31% of the total C contained in coarse
roots (Fig. 4), yielding a mean C residence time in coarse roots of
35.5 4.2 yr. The total difference in coarse root biomass
between E and A was 480 g C m
2
. This could have been caused
entirely by a stimulation of new root C (probably the most parsi-
monious interpretation), but it is possible that treatments dif-
fered in patterns of use of ‘old’, stored C –an idea which should
not be immediately dismissed, given that these plants use old C
to build new roots (Langley et al., 2002). In the surface soil min-
eral fraction, the percent new C increased linearly (Fig. 5), with
an overall mean residence time of C of 33.6 2.1 yr. In the
spodic horizon, there was no evidence of new C accumulation in
the total mineral soil or in the light, medium, or heavy density
fractions (Table 2). Overall, elevated CO
2
did not significantly
alter the total C content of the system (Table 1), because
increased C in plant reservoirs were compensated by reduced C
from the soil (Fig. 6).
Discussion
In this subtropical oak woodland, 11 yr of exposure to elevated
CO
2
increased plant C by 22%, with a smaller (and not signifi-
cant) effect on plant N of 9%, well within the range of responses
typically observed in plants growing under a wide variety of
experimental conditions (Norby et al., 2005; de Graaff et al.,
2006; Luo et al., 2006). Absolute responses in the mass of C
above- and belowground were similar, consistent with elevated
CO
2
having little impact on the partitioning of biomass above-
and belowground (Tingey et al., 2000), in contrast to the expec-
tation that root growth would increase disproportionately (Stulen
& den Hertog 1993). In our experiment, the relative response
aboveground was actually larger than that belowground, because
most of the biomass in this system is belowground. The mean
residence time of C in coarse roots (revealed by incorporation of
the d
13
C tracer) was sufficiently long that, at the final harvest,
only about one third of the C in coarse roots represented new
growth over the course of this experiment. By contrast, all of the
standing aboveground biomass at the final harvest had accumu-
lated after fire. Thus, repeated cycles of fire disturbance and
recovery might yield a larger cumulative response of new C in
coarse roots.
The increased C content of plants suggests the potential for
elevated CO
2
to enhance ecosystem C uptake. Yet, increased C
contained in plants was not reflected in the C content of soil, nei-
ther in the top meter nor in the deeper spodic horizon. Possibly,
the experiment lacked sufficient power to detect soil C accumula-
tion (Smith, 2004). Alternatively, other mechanisms may have
operated to prevent soil C accumulation in this ecosystem. We
can place boundary conditions on the power problem: integrated
Table 4 Carbon to nitrogen ratios (g : g) in ecosystem components after 11 yr of experimental exposure of a subtropical woodland to increased
atmospheric CO
2a
Ambient Elevated Effect CI
Aboveground 74.7 2.2 79.3 2.1 4.6 (0.3 to 9.1)
Oak leaves 45.8 1.0 48.3 1.0 2.5 (0.2 to 4.9)
Oak stems 120.6 7.9 121.2 7.2 0.6 (13 to 14.5)
Other species 71.9 6.6 60.1 7.4 11.8 (23.7 to 1.3)
Standing dead 108.5 3.8 113.4 4.8 4.9 (4.5 to 13.8)
Litter layer 58.6 1.5 64.2 1.9 5.6 (0.7 to 10.6)
Roots 101.3 7.4 122.0 7.5 20.7 (2.3 to 39.7)
Fine roots 114.0 9.6 116.3 9.8 2.2 (16.8 to 23.6)
Coarse roots 97.0 8.1 128.9 8.1 32.0 (7.1 to 59.3)
Plants 94.6 5.3 106.9 5.6 12.4 (0.8 to 23.4)
CPOM (0–100 cm) 71.1 9.1 76.2 8.7 5.1 (10.6 to 17.9)
Soil (0–100 cm) 36.8 2.2 37.8 2.0 0.9 (3 to 4.6)
Light, 0–60 cm 46.8 3.9 45.0 1.4 1.8 (9.1 to 5.1)
0–10 cm 43.7 6.9 38.8 0.8 4.9 (17.9 to 3.7)
10–30 cm 50.4 2.5 51.9 2.3 1.5 (4.5 to 7.4)
30–60 cm 64.5 9.7 60.8 9.5 3.7 (21.8 to 11.2)
Medium, 0–60 cm 41.2 3.0 39.5 3.0 1.7 (8.2 to 4.1)
0–10 cm 35.4 0.6 37.2 0.7 1.8 (0.3 to 4.4)
10–30 cm 45.6 2.9 48.2 2.9 2.7 (3.4 to 8.8)
30–60 cm 53.2 8.3 44.9 8.6 8.3 (26.8 to 7.7)
Heavy, 0–60 cm 40.2 4.2 29.4 5.1 10.7 (18.5 to 3.5)
0–10 cm 32.8 1.4 33.7 1.4 0.9 (1.6 to 3.6)
10–30 cm 41.2 5.1 38.4 5.1 2.9 (10.8 to 5.3)
30–60 cm 45.5 10.0 27.7 10.6 17.8 (36.6 to 3.4)
Residual, 0–60 cm 30.1 1.4 33.2 1.5 3.1 (0.4 to 6.8)
Soil (60–100 cm) 31.6 9.3 26.0 9.4 5.5 (24.1 to 10.4)
Total ecosystem 45.0 2.1 48.9 1.9 3.9 (0.1 to 8.1)
a
Values are means SE of the mean for the Ambient and Elevated CO
2
treatments, the Effect of the CO
2
treatment (E–A), and the bootstrapped 5% and
95% CLs (confidence limits) for the treatment effect. CPOM, coarse particulate organic matter. Soil fractions are density fractions, including light(<1.5 g
cm
3
), medium (1.5–1.8 g cm
3
), heavy (1.8–2.2 g cm
3
) and residual (calculated as total soil total minus the sum of measured density fractions).
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over the top meter of soil, the mean effect of CO
2
on total soil C
was a decline of 44.3 g C m
2
yr
1
, with the 90% confidence
interval spanning a range of CO
2
effects from more rapid losses
of soil C (132.4 g C m
2
yr
1
) to gain (+57.9 g C m
2
yr
1
).
This range exhibits the power limitations typical when assessing
responses of total soil C to elevated CO
2
(Hungate et al., 2009).
Isolating components of the total soil C reservoir can help over-
come the problem of limited power (e.g. Iversen et al., 2012). In
our case, we found that by year 6 of the experiment, elevated
CO
2
had reduced the C contained in the light density (Carney
et al., 2007) and in the acid-hydrolysable (Langley et al., 2009)
fractions of soil C. These findings are consistent with the
response we observed at the final harvest reported here where ele-
vated CO
2
reduced the heavy density fraction of soil C (Table 1)
and decreased soluble C susceptible to glucose-induced priming
(Fig. 3). The pattern of declining soil C in soil fractions is diffi-
cult to reconcile with the concept of soil C accumulation as a
first-order response to enhanced plant growth.
The second explanation for not finding soil C accumulation in
response to elevated CO
2
is that it does not occur, because
increased C input to soil is compensated by increased C loss.
Elevated CO
2
could enhance export of C through leaching of
dissolved organic matter. But, if elevated CO
2
increased leaching
of C in this experiment, this response had no influence on the C
content or d
13
C composition of the spodic horizon; the absence
of any effect on d
13
C is especially unlikely if leaching was an
important pathway for C loss. These findings indicate that
elevated CO
2
did not substantially alter leaching losses of C from
the system.
In contrast to the absence of any apparent effect on leaching,
there was compelling evidence that elevated CO
2
increased the
rate of C cycling through the soil: elevated CO
2
significantly
increased the size and rate of C flow through the labile soil C
pool (Fig. 1), it enhanced the proportion of soil C (and N, and
15
N) that were cycling through the soil microbial biomass
(Fig. 2), and it increased the decomposability of labile plant sub-
strates and promoted a physiologically more responsive microbial
community (Fig. 3). Elevated CO
2
also increased fungal biomass,
Table 5 Inventory of tracer
15
N (mg excess
15
Nm
2
) after 11 yr exposure to increased
atmospheric CO
2
and 9 yr of integration of
the added
15
N tracer in a subtropical oak
woodland
a
Ambient Elevated Effect 5% & 95% CLs
Aboveground 2.8 0.4 3.7 0.4 0.9 (0.1 to 1.8)
Oak leaves 1.6 0.2 1.8 0.3 0.2 (0.3 to 0.7)
Oak stems 1 0.2 1.6 0.2 0.6 (0.2 to 0.9)
Other species 0.1 0 0.2 0 0.1 (0 to 0.2)
Standing dead 0.1 0 0.1 0 0.0 (0 to 0.1)
Litter layer 2 0.3 2 0.3 0.0 (0.8 to 0.8)
Roots 7.7 1.2 4.5 0.6 3.3 (5.2 to 1.4)
Fine roots 2.1 0.4 1.4 0.2 0.7 (1.5 to 0)
Coarse roots 5.6 1.1 3.1 0.6 2.6 (4.5 to 0.7)
Plant 10.5 1.0 8.2 0.8 2.4 (4.2 to 0.4)
CPOM (0–100 cm) 0.6 0.1 0.7 0.1 0.1 (0.2 to 0.3)
Soil (0–100 cm) 83.7 16.4 59.2 11.4 24.5 (53 to 3.7)
Light, 0–60 cm 28.5 4 29.2 4.8 0.7 (8.9 to 10)
0–10 cm 21.6 4.4 24 5 2.4 (8 to 12.8)
10–30 cm 4 0.7 2.2 0.2 1.8 (2.9 to 0.7)
30–60 cm 2.9 0.9 3.1 0.7 0.1 (1.8 to 1.8)
Medium, 0–60 cm 15.2 2.9 14.9 2.4 0.3 (5.6 to 5.6)
0–10 cm 11.2 2.1 10.1 2.2 1.2 (6 to 3.7)
10–30 cm 2.8 0.7 2.5 0.4 0.3 (1.5 to 0.8)
30–60 cm 1.1 0.4 2.3 0.8 1.2 (0.1 to 2.6)
Heavy, 0–60 cm 5.1 0.8 4.3 1.1 0.8 (2.7 to 1.3)
0–10 cm 1.9 0.5 1.5 0.4 0.3 (1.3 to 0.7)
10–30 cm 1.2 0.2 0.8 0.4 0.4 (1 to 0.3)
30–60 cm 2.1 0.3 2 0.8 0.1 (1.5 to 1.3)
Residual, 0–60 cm 34.8 10.8 14.2 9.5 20.7 (51.7 to 0.8)
Soil (60–100 cm) 5.8 1.1 6.4 1.7 0.6 (2.5 to 4)
Total ecosystem 102.6 15.7 76.4 9.0 26.2 (55.1 to 0.8)
a
Values are means SE of the mean for the Ambient and Elevated CO
2
treatments, the Effect of the CO
2
treatment (E–A), and the bootstrapped 5% and 95% CLs (confidence limits) for the treatment effect.
CPOM, coarse particulate organic matter. Soil fractions are density fractions, including light (<1.5 g
cm
3
), medium (1.5–1.8 g cm
3
), heavy (1.8–2.2 g cm
3
) and residual (calculated as total soil total minus
the sum of measured density fractions).
Table 6 d
15
N signatures (mean SEM) of plant, soil and whole system N
at the final harvest in July 2007
Ambient CO
2
Elevated CO
2
P-value*
Plant 76.3 5.4 55.0 4.2 0.008
Soil 103.7 13.9 85.4 11.6 0.422
System 100.4 11.6 80.8 10.0 0.317
*P-values are for one-way ANOVAs testing the effect of elevated CO
2
.
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as measured by ergosterol (Klamer et al., 2002), by direct mea-
surements of mycorrhizal fungal biomass (Langley et al., 2003),
and by the ratio of fungi to bacteria in the soil microbial biomass,
as indicated by the analysis of phospholipid fatty acid profiles
(Carney et al., 2007). These results indicate that higher microbial
activity was associated with a shift in the composition of the
microbial community.
Increased soil microbial activity may also explain why the
effect of elevated CO
2
on the C : N of plant tissues and the litter
layer was not apparent, and indeed in some cases may even have
been reversed, in soil organic matter. Specifically, elevated CO
2
increased the C : N ratio of individual plant tissues (Table 3) as
commonly observed (Cotrufo et al., 1998; Norby et al., 2001), of
the entire plant biomass, above- and belowground, and of the
litter layer. Yet, this shift was not observed in soil organic matter
after 11 yr of continuous inputs of plant material to the soil
organic matter pool. There are two possibilities for this discrep-
ancy: (1) either the inputs of plant material were too low com-
pared to background soil organic matter to drive a change in soil
organic C : N; or (2) by increasing soil microbial activity and the
processing of C in the soil system, elevated CO
2
caused a com-
pensatory response, tending to reduce soil C : N. Our finding
that elevated CO
2
reduced the total mass of soil N in the medium
density fraction, but increased it in the heavy fraction, is consis-
tent with this second explanation. The medium fraction has a
higher C : N ratio than the heavy fraction, and the medium frac-
tion is thought to cycle into the heavy fraction as the soil organic
matter is processed by microbial activity and interactions with
minerals (Camberdella & Elliott 1992). Thus, the pattern we
observe may indicate increased processing and turnover of soil N,
promoting transfer to pools with lower C : N ratios, and a ten-
dency for CO
2
to decrease soil C : N.
Some previous measurements at this site indicated that ele-
vated CO
2
reduced or had no effect on microbial activity during
the first 18 months of the experiment, with reduced gross N min-
eralization (Hungate et al., 1999) and either reduced or no
impact on microbial biomass N (measured as ninhydrin-reactive
N) and microbial activity (measured as fluorescein diacetate
hydrolysis) in the rhizosphere (Schortemeyer et al., 2000),
although the mechanism(s) for these changes were not apparent.
These early responses were apparently transient, and did not indi-
cate the decadal-scale response of soil microorganisms to elevated
CO
2
. The measurements reported here of microbial biomass, the
size of the labile soil C pool, and the distribution and retention
of
15
N cycling through the system are more representative of the
entire duration of the experiment (e.g. Fig. 2). Results from this
experiment are consistent with the general finding that elevated
CO
2
stimulates soil microbial activity (de Graaff et al., 2006;
Dieleman et al., 2012), and the turnover of soil organic matter
(Marhan et al., 2010; Phillips et al., 2012; Dawes et al., 2013).
Elevated CO
2
can stimulate microbial activity by increasing
soil water content, especially in grasslands (Hungate et al., 1997a;
Morgan et al., 2004), and this response can counterbalance the
increased C inputs from enhanced plant growth at elevated CO
2
,
causing no change in soil C accumulation (Marhan et al., 2010).
In the scrub-oak experiment reported here, elevated CO
2
slightly
0.00
5.00
10.00
15.00
20.00
25.00
30.00 0–10 cm
0.00
2.00
4.00
6.00
8.00
10.00
12.00
10–30 cm
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
1 5 50 500
30–60 cm
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
1 5 50 500
60–100 cm
Soil CO
2
production (µg CO
2
–C g
–1
d
–1
)
Days of soil incubation
(a) (b)
(c) (d)
Fig. 1 CO
2
production during soil incuba-
tions for four soil depths (a, 0–10 cm; b,
10–30 cm; c, 30–60 cm; d, 6–100 cm) in a
subtropical oak woodland exposed to 11 yr
of increased CO
2
. Ambient CO
2
, open
circles; elevated CO
2
, closed circles. Bars
show 2 SEM.
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increased surface soil water content during the first several years
(Hungate et al., 2002), but this effect disappeared with leaf area
development (Li et al., 2007), and elevated CO
2
had no effect on
soil temperature (Hymus et al., 2003). Thus, the changes in
microbial activity and organic matter turnover that we observed
are unlikely to have been driven by differences in temperature,
although increased soil moisture may have played a role early on.
Elevated CO
2
can also increase microbial activity by enhanc-
ing the supply of C substrates to soil microorganisms, a
response consistent with past reports that, in this experiment,
elevated CO
2
stimulated the ‘priming effect’ (Carney et al.,
2007; Langley et al., 2009), the phenomenon where there occurs
‘extra decomposition of native soil organic matter in a soil
receiving an organic amendment’ (Bingeman et al., 1953). In
the experiment described here, the O
2
consumption assay indi-
cates that C derived from the litter and roots is more labile in
the elevated CO
2
treatment (Fig. 3), leading to a larger quantity
of labile organic matter (Fig. 1). The higher rates of microbial
activity observed are consistent with the notion that these new
inputs of labile C to soil increased mineralization of native soil
organic matter (Van Veen et al., 1991; Carney et al., 2007).
This phenomenon has been observed for some time (Lohnis,
1926; Broadbent & Norman, 1947; Broadbent, 1948) and evi-
dence for it has grown: isotope tracer experiments in soil incu-
bations show that substrate additions can more than treble the
decomposition rate of native soil organic matter in the short
term (Cheng & Johnson, 1998; Cheng et al., 2000). Substrate
additions can influence the oxidation of old soil C reservoirs,
for example, in deep soil (Fontaine et al., 2007), and can shape
the response of soil C to elevated CO
2
(Hoosbeek, 2004; Tru-
eman & Gonzalez-Meler, 2005; Carney et al., 2007; Hagedorn
et al., 2008; Paterson et al., 2008; Taneva & Gonzalez-Meler,
2008; Langley et al., 2009; Trueman et al., 2009; Drake et al.,
2011; Reid et al., 2012). Increased oxidation of old soil organic
matter is likely a transient response to a change in the rate of
labile C inputs. In the experiment described here, the reduction
in soil C observed by year 6 (Carney et al., 2007) was
0
500
1000
1500
2000
2500
3000
3500
4000
Litter
extract
Root
extract
Litter
extract
Root
extract
Litter
extract
Root
extract
Bulk soil
inocula
Litter
inocula
Rhizosphere
inocula
*
*
O2 consumption (normalized
relative fluorescence)
0%
40%
80%
120%
160%
200%
Glucose P N
**
Glucose P N
***
Glucose P N
400%
800%
1200%
Relative response of microbial
respiration to resource addition
(a)
(b)
Fig. 3 (a) Total respiration (O
2
consumption, expressed as normalized
relative fluorescence) of microbial inocula from three soil habitats (bulk
soil, litter, rhizosphere) on extracts of litter and roots. Circles, inocula from
ambient CO
2
; squares, from elevated CO
2
. Open symbols, substrates
produced in the ambient CO
2
treatment; closed symbols, substrates
produced in the elevated CO
2
treatment. Significant differences between
substrates produced under ambient and elevated CO
2
conditions (two-
way ANOVAs, effect of substrate origin); *,P<0.050. (b) The relative
responses of microbial respiration to single resource additions (glucose, N,
or P) for microbial inocula from the bulk soil, litter and rhizosphere
communities in the ambient (open circles) and elevated (closed circles)
CO
2
treatments. *Significant differences in resource limitation for
individual comparisons (t-tests) of inocula from the ambient and elevated
CO
2
treatments. For full statistical results, see Supporting Information
Tables S1 and S2. Bars show 2 SEM.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0.00 0.01 0.10 1.00 10.00
Microbial 15N (% of total soil 15N)
0.00%
0.50%
1.00%
1.50%
2.00%
2.50%
3.00%
Microbial N
Microbial N (g N g soil N–1 x 100%)
0.00%
0.10%
0.20%
0.30%
0.40%
0.50%
0.60%
0.70%
Microbial C
Microbial C (g C g soil C–1 x 100%)
Time (years after 15N tracer addition)
(a)
(b)
Fig. 2 (a) Soil microbial biomass nitrogen (N) and carbon (C) from the
ambient (open bars) and elevated (closed bars) CO
2
treated plots.
Microbial C and N (as a proportion of total soil C and N) are shown as
means across seven sample dates spanning 1997 to 2004, years 2–9of
CO
2
exposure. (b) Tracer
15
N in the microbial biomass (as a proportion of
tracer
15
N in total soil) over time after label addition (log scale). Bars
show 2 SEM.
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comparable to that found after 11 yr, suggesting that the sub-
strates susceptible to priming-induced loss had mostly been
degraded during the first 6 yr.
The implications of this response are not limited to C:
increased C input to soil, enhancing microbial activity and turn-
over, can also increasing nutrient availability to plants (Zak et al.,
1993). Observations elsewhere that elevated CO
2
increases
microbial activity in concert with greater plant N acquisition
from soil are also consistent with this interpretation (Drake et al.,
2011), although without direct evidence of increased soil organic
matter turnover, increased root exploration is a simpler explana-
tion. Results presented here call into question the notion that
feedbacks stimulating soil microbial turnover and N availability
necessarily lead to plant N accumulation and increased plant
growth. On the one hand, we did find that elevated CO
2
stimu-
lated plant N uptake and
15
N dilution in plant tissues, likely
driven by increased turnover of soil organic matter mediated by
microorganisms (Figs 1, 3; Johnson et al., 1998, 2001; Finzi
et al., 2007). On the other hand, increased microbial activity
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1994 1996 1998 2000 2002
Year
2004 2006 2008
Coarse root C (g C m–2)
Fig. 4 Coarse root carbon (C) over time in the scrub-oak experiment,
showing ‘old’ (open circles) and ‘new’ (closed squares) carbon for the
elevated CO
2
plots, where new is defined as carrying a
13
C isotopic
signature of the CO
2
added to the elevated CO
2
plots. Modeling % old C
as exponential decay over time yielded a decomposition constant of
0.0325 yr
1
, considerably lower than decomposition assessed by litterbags
(0.22 yr
1
for ambient, 0.29 yr
1
for elevated). Bars show 2 SEM.
–10%
–5%
0%
5%
10%
15%
20%
25%
30%
35%
40%
Oct-95 Jul-98 Apr-01
Month, year
Jan-04 Oct-06 Jul-09
New soil carbon (% of total)
Fig. 5 New carbon in surface mineral soils over time. Bars show 2 SEM.
Fig. 6 Summary of ecosystem carbon (C) and nitrogen (N) inventories in a subtropical woodland after 11 yr of exposure to elevated CO
2
.
New Phytologist (2013) Ó2013 The Authors
New Phytologist Ó2013 New Phytologist Trust
www.newphytologist.com
Research
New
Phytologist
10
likely promoted N losses, accounting for our finding that elevated
CO
2
reduced recovery of added tracer
15
N (Table 4).
In this experiment, spanning more than a decade in a naturally
occurring ecosystem, photosynthesis and aboveground plant
growth exhibited strong responses to chronic exposure to elevated
atmospheric CO
2
(Dijkstra et al., 2002; Seiler et al., 2009), lead-
ing to the increased aboveground C content reported here, as well
as increased C in coarse roots (Day et al., 2013; Fig. 6). The ele-
vated CO
2
treatment did not affect C in fine roots at the final
harvest, although fine roots responded sporadically in this experi-
ment, with particularly strong responses following the initial fire
disturbance and after a hurricane in year 8 (Day et al., 2013). Ele-
vated CO
2
did not increase soil C, and in fact tended to decrease
it, likely a consequence of increased microbial activity. Elevated
CO
2
also increased plant N uptake, possibly driven by higher
microbial activity and increased soil N availability, but these
responses were also associated with reduced recovery of a long-
term
15
N tracer, likely indicating enhanced ecosystem N losses.
Thus, CO
2
altered the C and N cycles in this ecosystem, but not
in ways that promoted large or even detectable increments in
total ecosystem C mass. The effect of elevated CO
2
on soil C
turnover via the ‘priming effect’ was large enough to modulate
net carbon balance. This finding is not unique, and treatment of
this phenomenon in models of soil C cycling is likely warranted
(Heimann & Reichstein, 2008; Chapin et al., 2009). While the
importance of priming is becoming evident, the challenge to
include the phenomenon in models is not trivial: priming is still
poorly quantified and the mechanisms remain inscrutable. Meet-
ing this challenge could improve substantially our understanding
of terrestrial C cycling, replacing, or at least modifying, the stabi-
lizing first-order kinetics of decomposition used in virtually all
current models of the soil C cycle (Luo & Weng, 2011). The
response of soil C to labile substrate inputs suggests a previously
unrecognized sensitivity of what was thought to be a long-term,
stable C sink in the biosphere.
Acknowledgements
This research was supported by the US Department of Energy
(DE-FG-02-95ER61993, and subcontract 95-59, MPOOO02),
and by the National Science Foundation (DEB 9873715,
0092642, and 0445324). The National Aeronautics and Space
Administration at the Kennedy Space Center, the US Fish and
Wildlife Service at Merritt Island National Wildlife Refuge pro-
vided generous support throughout the CO
2
project. Thanks to
Bert Drake for visionary leadership and opportunity. Victoria
Albarracin, Mike Roberts, Mary Hummerick, Jan Bauer and
Lanfang Levine assisted in the laboratory.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Tables S1 & S2 Results from ANOVAs testing responses of soil
microbial respiration to CO
2
treatment, habitat, and substrate
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