LETTERS

PUBLISHED ONLINE: 11 MARCH 2012 | DOI: 10.1038/NCLIMATE1448

Biotic carbon feedbacks in a materially closed

soil–vegetation–atmosphere system
Alexandru Milcu1 *, Martin Lukac1,2 , Jens-Arne Subke3 , Pete Manning1,4 , Andreas Heinemeyer5,6 ,
Dennis Wildman1 , Robert Anderson1 and Phil Ineson5,6

The magnitude and direction of the coupled feedbacks between
the biotic and abiotic components of the terrestrial carbon cycle
is a major source of uncertainty in coupled climate–carbon-
cycle models1–3 . Materially closed, energetically open biological
systems continuously and simultaneously allow the two-way
feedback loop between the biotic and abiotic components
to take place4–7 , but so far have not been used to their
full potential in ecological research, owing to the challenge
of achieving sustainable model systems6,7 . We show that
using materially closed soil–vegetation–atmosphere systems
with pro rata carbon amounts for the main terrestrial carbon
pools enables the establishment of conditions that balance
plant carbon assimilation, and autotrophic and heterotrophic
respiration fluxes over periods suitable to investigate short-
term biotic carbon feedbacks. Using this approach, we tested
an alternative way of assessing the impact of increased CO2
and temperature on biotic carbon feedbacks. The results
show that without nutrient and water limitations, the short-
term biotic responses could potentially buffer a temperature
increase of 2.3 ◦ C without significant positive feedbacks to
atmospheric CO2 . We argue that such closed-system research
represents an important test-bed platform for model validation
and parameterization of plant and soil biotic responses to
environmental changes.
The magnitude and sign of terrestrial carbon feedbacks to
environmental changes constitute a major uncertainty in predicting
future atmospheric CO2 concentrations and temperatures1–3 . Plant
and soil responses to environmental change underpin the terrestrial
climate–carbon feedback1,3 , but despite recent modelling advances,
uncertainty remains large as important aspects of the soil–
vegetation–atmosphere interactions are poorly understood2,8 .
An alternative and complementary, yet underexplored, way to
investigate the impact of biotic feedbacks on the carbon cycle
is to use materially closed, energetically open, physical/analogue
models6,7 . A materially closed approach is well suited and relevant to
study biotic carbon responses to climate change as it continuously
and simultaneously allows for the two-way feedbacks between
the biotic and abiotic components to take place, while excluding
external interference7 . Furthermore, closed-system approaches
permit identification of subtle feedbacks through pool changes over
time that are beyond the resolution of materially open systems and
well suited for mass balance studies9–12 .
Here we established simplified materially closed, but energet-
ically open, soil–vegetation–atmosphere systems (mcSVASs) with
the aim of investigating plant and soil carbon feedback to increased
CO2 and temperature. This approach has received little attention in
ecological research owing to the difficulty of achieving sustainable
model systems that balance carbon assimilation and respiration
fluxes (from autotrophic as well as heterotrophic sources)6,7,13,14 .
Our mcSVASs were set up with scaled-down carbon pools ap-
proximating best estimates of preindustrial global terrestrial carbon
pools in soil, plants and the atmosphere15,16 . Preliminary studies
aimed at exploring the importance of carbon stocks for achieving
systems that balance the autotrophic and heterotrophic carbon
fluxes showed that recreating the pro rata stocks of the terrestrial
carbon cycle is a crucial starting point7 . This was achieved by setting
the initial atmospheric concentration of CO2 to ∼280 ppmv and
establishing pro-rata soil and vegetation carbon pools equivalent to
the preindustrial global atmospheric (560 Gt), vegetation (900 Gt)
and soil (2,011 Gt) carbon levels, thus falling within the upper range
of available global carbon estimates15,16 .
Fifteen mcSVASs were established in custom-built, sealed
polycarbonate containers with a volume of 120 l (Supplementary
Fig. S1). Each mcSVAS was housed in an individual climate-
controlled walk-in growth chamber17 . The design enabled non-
invasive monitoring and control of temperature and atmospheric
CO2 concentrations in the containers, with external control of
photosynthetically active radiation (PAR) following a day–night
regime of 14–10 h (see Methods for details). All units were initially
maintained isothermally at 15 ◦ C ± 0.2 s.e.m. Subsequently, three
scenarios/treatments with five replicated mcSVASs per scenario
were applied to assess plant and soil biotic carbon feedbacks to
increased CO2 and temperature. These were: first, a control scenario
with no CO2 additions and no climate sensitivity (isothermal
15 ◦ C), hereafter referred to as the control scenario (S15 ); second, a
scenario with simulated CO2 additions (15 ppmv CO2 every second
day, equivalent of the Intergovernmental Panel on Climate Change
B1 scenario18 of ∼930 Gt carbon cumulative by the year 2100)
and no climate sensitivity (that is, isothermal at 15 ◦ C), hereafter
referred to as the CO2 -addition scenario (S15CO2 ); and third, a
scenario with the same simulated CO2 additions as in the second
scenario, but with emulated temperature sensitivity to CO2 , here-
after referred to as the emissions-with-feedback scenario (S13CO2 ).
S15CO2 and S13CO2 scenarios started after 12 days of establishment
in which no CO2 additions were made and the temperature was
isothermal 15 ◦ C for all replicates (establishing phase). As the
atmospheric CO2 concentration rose or fell in the S13CO2 scenario,
an internal CO2 –temperature feedback control (±0.2 ◦ C s.e.m.)

1 NERC Centre for Population Biology, Division of Biology, Imperial College London, Silwood Park Campus, Ascot SL5 7PY, UK, 2 School of Agriculture, Policy
and Development, University of Reading, Berkshire RG6 6AR, UK, 3 School Natural Sciences, Institute of Biological and Environmental Sciences, University
of Stirling, Stirling FK9 4LA, UK, 4 University of Newcastle, School of Agriculture, Food and Rural Development, Newcastle upon Tyne NE1 7RU, UK,
5 Stockholm Environment Institute, Environment Department, University of York, York YO10 5DD, UK, 6 NERC National Centre for Earth Observation,

York-Centre, Department of Biology, University of York, York YO10 5DD, UK. *e-mail: a.milcu@imperial.ac.uk.

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© 2012 Macmillan Publishers Limited. All rights reserved.
LETTERS
Table 1 | Carbon mass balance comparing the initial amounts of carbon in the main pools with those found at the end of the
experiment in the isothermal 15 ◦ C (S15 ), isothermal 15 ◦ C + CO2 additions (S15CO2 ) and CO2 additions + feedback 1T2 = 3 (S13CO2 )
scenarios (n = 5).
Time of measurement Initial carbon amounts (mg)
Source/scenario
Air 17.2
CO2 emissions n.a.
Plant 40.0
Soil 71.0
Contamination n.a.
Total 128.2
Unexplained n.a.
Note that the small departure from the target pro rata carbon stocks in the column presenting the initial carbon amounts has been probably introduced with the plant biomass as we can only estimate the
amount of carbon in a live plant; n.a., not applicable.
enabled precise internal temperature regulation. The relationship
between CO2 concentrations and temperature in this scenario was
based on the most likely climate sensitivity of 3 ◦ C (refs 19,20),
defined as the equilibrium response of global mean temperature to
doubling the atmospheric CO2 concentration (1T2 ; ref. 19).
The carbon mass balance analysis carried out at the end of the
experiment indicated that mcSVASs were successful in conserving
the initially introduced carbon (Table 1), that is, no carbon losses
or gains to or from the external environment occurred during
the experiments. The CO2 dynamics confirmed results from initial
trials7 where the net carbon flux to the atmosphere in the S15
scenario stabilized within two weeks of closing containers. This was
indicated by a weekly slope of atmospheric CO2 change that was not
different from zero (Supplementary Table S1, Fig. 1a,b) and atmo-
spheric CO2 concentrations with a strong diurnal pattern, driven by
the presence/absence of light. In the S15CO2 and S13CO2 treatments,
despite an initial increase in the atmospheric CO2 content, the
atmospheric CO2 concentration also stabilized at just below 500
ppmv from the fifth experimental week onwards (Fig. 1a,b, Supple-
mentary Table S1). Furthermore, stabilization of atmospheric CO2
concentrations in the S13CO2 scenario occurred despite a tempera-
ture increase of 2.3 ◦ C, and was not significantly higher than in the
S15CO2 scenario. The increase in temperature in the S13CO2 scenario
led to higher total (plant and soil) dark respiration in the mcSVASs,
but only after seven weeks of CO2 additions and temperature
increase; the temperature sensitivity of dark respiration was ∼2. In
contrast, in the S15CO2 treatment the dark respiration was not differ-
ent from the control (Fig. 1c). The stabilization of atmospheric CO2
concentration in the S15CO2 and S13CO2 scenarios was explained by
the significantly higher plant CO2 uptake relative to the S15 scenario
after five weeks (Fig. 1d; Supplementary Table S2). Furthermore,
CO2 uptake in the S13CO2 scenario was significantly higher than
in the S15CO2 treatment (Fig. 1d; Supplementary Table S2). Despite
higher total dark respiration in the S13CO2 scenario (Fig. 1c), the
enhancement of CO2 uptake through photosynthesis (Fig. 1d; Sup-
plementary Table S2) led to the removal of 62% of the total injected
atmospheric carbon, thus limiting the increase in atmospheric CO2
in the S13CO2 relative to the S15CO2 chambers to only 6%. If upscaled
to the Gt C unit, used as a reference for the establishment of the
pro rata carbon pools in the mcSVASs, the observed sensitivity
to CO2 (that is, the change in carbon uptake per ppmv unit of
atmospheric CO2 increase, estimated from the experimental weeks
5–9) was equivalent to 2.29 Gt C ppmv−1 CO2 . The sensitivity of
the mcSVASs to the imposed temperature increase (that is, the
change in net carbon uptake per ◦ C increase) estimated for the same
period from the difference between the average atmospheric CO2
concentration in the S13CO2 and S15CO2 scenarios, was equivalent to
−34.51 Gt C ◦ C−1 (see Supplementary Table S3 for details).
282
© 2012 Macmillan Publishers Limited. All rights reserved.
NATURE CLIMATE CHANGE DOI: 10.1038/NCLIMATE1448
Carbon amounts (mg) at the end of the experiment for each temperature treatment
S15 S15CO2 S13CO2
18.3 ± 2.0 25.3 ± 3.0 26.0 ± 3.0
n.a. 30 30
62.0 ± 4.0 76.0 ± 7.0 84.1 ± 7.0
50.5 ± 3.0 51.0 ± 1.0 51.0 ± 3.0
0.8 1.0 <0.1
131.5 ± 6.0 152.3 ± 3.0 160.9 ± 4.0
4.9 −5.9 2.7
The capacity of the systems to recover after the cessation of the
emissions was examined over the last 16 days of the experiment,
where the CO2 additions were stopped after 31 CO2 injections, two
months after the first CO2 addition. Halting CO2 additions revealed
the size of the system’s carbon sink in the S15CO2 and S13CO2 scenarios
(Fig. 1b). Interestingly, the recovery of atmospheric CO2 was not to
the levels of the establishing phase and the S13CO2 retained a memory
of the perturbation in gross fluxes (Fig. 1a,b), with significantly
higher respiration rates (Fig. 1c).
Our simple materially closed systems provide several important
insights. First, by using pro rata amounts of carbon for the
main terrestrial pools it is possible to establish materially closed
systems where the heterotrophic and autotrophic carbon fluxes to
and from the atmospheric carbon pool are balanced, making the
investigation of biotic carbon feedbacks feasible. Second, we found
a strong plant-driven negative feedback on atmospheric CO2 —that
is, photosynthesis was stimulated by 33.8% under increased CO2 in
the S15CO2 . This is accompanied by an increase in photosynthetically
active plant biomass (foliage) of 75.2% at the end of the experiment
(Supplementary Table S4). However, in the S13CO2 scenario where
the temperature was adjusted according to the CO2 concentration
by up to 2.3 ◦ C, the photosynthetic rate increased by 64.1% (and the
photosynthetically active plant biomass by 142.1% relative to the
S15 scenario, Supplementary Table S4), preventing a switch of the
non-atmospheric part of mcSVASs from a net carbon sink to carbon
source. This additional increase in carbon uptake in the S13CO2 is
higher than the documented increase of CO2 uptake at increased
CO2 observed in the most responsive plant functional group—
that is, trees21 —indicating an interactive effect of increasing CO2
concentration and temperature. Relative to the S15CO2 treatment
this is an increase of 22.7%, equivalent to ∼9.9% per ◦ C. Although
studies reviewing the interactive effects of increasing CO2 and
temperature found little unequivocal evidence for large differences
in plant growth in response to CO2 at different temperatures, there
is evidence that this might be attributable to numerous confounding
factors such as different acclimation times and different patterns
of growth allocation22 . There remains a strong theoretical and
physiological basis for expecting interactive effects of CO2 and
temperature on photosynthesis23,24 .
The results from our mcSVASs indicate that without nutrient
and water limitations, the short-term biotic responses of plant–soil
systems could potentially buffer a temperature increase of 2.3 ◦ C
without significant positive feedbacks to atmospheric CO2 . This is
because the observed sensitivity of terrestrial carbon uptake to CO2
and temperature was in the higher and lower ranges, respectively,
of CO2 and temperature responses included in the coupled carbon-
cycle models25 . However, we fully recognise that our model systems
are simplistic representations of natural plant–soil–atmosphere
NATURE CLIMATE CHANGE | VOL 2 | APRIL 2012 | www.nature.com/natureclimatechange
NATURE CLIMATE CHANGE DOI: 10.1038/NCLIMATE1448 LETTERS
a 28 b
800
S15 10 S15
26

Slope CO2 change (ppmv)

700 S15CO S15CO
2 8 ∗∗∗ ∗ 2

SΔ3CO SΔ3CO
Atmospheric CO2 (ppmv)
600 2
24 6 2

Cumulative

Temperature (°C)
500 CO2 additions 4
22
2
400
20 0
300 ¬2
18
200 ¬4

Establishment

Week 1¬2

Week 3¬4

Week 5¬6

Week 7¬9

Recovery
100 16

0 14
1 7 14 21 28 35 42 49 56 63 70 77 85 92
Experimental day

c 11 d 0

Net photosynthesis (ppmv CO2 d¬1)

Night-time respiration (ppmv CO2)

S15 ¬5
10 ∗∗
S15CO
2 ∗
9 SΔ3CO ¬10
2

8
¬15
7
¬20 S15
6
S15CO
2
5 ¬25 SΔ3CO
2 ∗ ∗∗ ∗∗
0
Establishment

Week 1¬2

Week 3¬4

Week 5¬6

Week 7¬9

Recovery

Establishment

Week 1¬2

Week 3¬4

Week 5¬6

Week 7¬9

Recovery
Figure 1 | Effects of climate scenarios (S15 , S15CO2 and S13CO2 ) on atmospheric CO2 (ppmv), slopes of CO2 change, respiration and net photosynthesis.
a, Average atmospheric CO2 concentrations and average temperature changes (right axis) for the different periods of the experiment. CO2 additions were
carried out by injecting a constant amount of pure CO2 calculated to result in a 15 ppmv CO2 increase every second day. The cumulative CO2 additions
curve indicates what level the CO2 would have reached purely as a result of additions in systems without biological activity. The temperature in the
mcSVASs that included the CO2 temperature feedback was externally adjusted every second day (between CO2 injections) as a function of the
atmospheric CO2 concentration, assuming a climate sensitivity of 3 ◦ C. b, Slope of CO2 concentration change (expressed as average change in CO2
concentration per day ± s.e.m.) for the different periods of this experiment. The establishment phase represents the first 12 days after sealing the mcSVAS,
whereas the recovery phase represents the last 16 days of the experiment after the cessation of the CO2 additions. c, Night-time respiration (expressed as
amount of ppmv CO2 added during night-time hours ± s.e.m.) for the different periods of the experiment. d, Net photosynthesis (expressed as net change
in ppmv CO2 during daylight hours ± s.e.m.) for the different periods of the experiment. We assume that daytime respiration is of similar magnitude to
night-time respiration. Stars represent significant differences (∗ = P > 0.05, ∗∗ = P < 0.01, ∗∗∗ = P < 0.001) obtained from an analysis contrasting the S15
scenario versus the S15CO2 and S13CO2 scenarios.

systems and several caveats need to be acknowledged, most of which well understood and quantified. However, even after many years
are also common to materially open experimental approaches. The of research into the responses of simple carbon flux components
experimental time may be too short to include potential acclimation to abiotic factors our understanding of these processes remains
responses to increased CO2 and temperature. The length of the incomplete, and may not be fully achievable26 . In contrast, any
experiments did not allow for short-term carbon gains in the unquantified or indeed unknown plant or soil feedback will still
vegetation pool from enhanced growth to be propagated into soil inherently occur in physical analogues if the scale of representation
organic matter pools following the death of plants or plant parts, allows them to take place. To our knowledge, the parameterization
where it would ultimately contribute to heterotrophic respiration. of the CO2 fertilization effect has never been derived from systems
The results therefore address system responses to initial stages where the plant response directly impacts on the ambient CO2
of perturbations, not steady-state conditions following long-term concentration and temperature, which in turn is known to feed
adjustments. The systems were not nitrogen or water limited, back on the photosynthesis rate. Hence, we argue that there
which is important to soil and plant responses to increased CO2 is a gap in understanding plant ecophysiological responses on
and temperature. Furthermore, the model plant species was a the plant physiology scale (leaf and minutes to hours scale)
herbaceous one, whose photosynthetic stimulation by increased versus experiments where the biotic and abiotic components have
CO2 is generally considered to be lower than that of trees21 . sufficient time to feed back on each other. The incorporation
Despite these drawbacks, the mcSVAS approach offers a number of these continuous and simultaneous plant–soil–atmosphere-
of important opportunities. In contrast to computer simulations, temperature feedbacks in our mcSVAS presumably explains the
there is no need to a priori parameterize key processes such as the surprising ability of the vegetation to buffer a temperature increase
temperature sensitivity of soil respiration26 and plant sensitivity to of 2.3 ◦ C without significant positive feedbacks to atmospheric CO2 .
CO2 (ref. 21) and temperature27 . Such complex dynamic feedbacks The results from our materially closed approach indicate the
can be parameterized in computer simulations only once they are urgent need to implement experimental systems that realistically

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© 2012 Macmillan Publishers Limited. All rights reserved.
LETTERS
represent the soil–vegetation–atmosphere feedbacks. Expanding
the size, duration and complexity of mcSVAS by incorporating
elements of global biotic and climatic heterogeneity represents a
major, but achievable, challenge. We argue that materially closed
system approaches have the potential to uncover key properties of
the processes driving global biotic feedbacks, which may ultimately
help to improve our predictions of future Earth system changes
using coupled climate–carbon-cycle models.
Methods
McSVASs. Transparent chambers manufactured from welded polycarbonate
(10 mm wall thickness and 120 l volume, Supplementary Fig. S1) were set up
in the Ecotron facility17 at Silwood Park. Each chamber was connected to a
separate measuring cell mounted under a rotating open-path infrared gas analyser
(OP-2 Open Path CO2 /H2 O Analyser, ADC Bioscientific, Herts, UK), allowing
non-invasive and continuous real-time air CO2 concentration measurements6 .
Temperature, atmospheric pressure, relative humidity, O2 concentration, soil
moisture and PAR were continuously measured and recorded by the TREND 963
data logging supervisor (Trend Control Systems, Horsham, UK). The temperature
was controlled by continuously operating a heater and a cooler, both integrated
within each individual mcSVAS. The moisture content of the soil/sand mixture was
monitored by a soil-moisture probe (Theta Probe, Delta-T Devices, Cambridge,
UK) and homeostatically maintained between 35 and 60% by triggering watering
events of ∼20 ml of water from an internal reservoir of 280 ml each time the soil
moisture dropped below 35% (see ref. 6 for more details).
Leakage estimates. Although the chambers were constructed to full glove box
technology standards, maintaining perfectly materially closed systems over the
long-term duration of the experiments was a major engineering challenge. Internal
and external pressure changes were used to calculate the volume of air that was
exchanged with the outside environment over the course of the experiments
using the combined ideal gas law. Continuous CO2 recordings in- and outside the
boxes, together with information on the volume of exchanged air, allowed for the
calculation of the amount of carbon exchange and net contamination for each
experimental unit28 throughout the experiments (3 × 10−5 ± 1.7 × 10−4 g C s.e.m.).
The net deviation from the target carbon amounts at the end of the experiment was
below 4% (Table 1) and was most likely introduced as plant biomass during the
experimental initiation, as it is intrinsically difficult to introduce precise carbon
amounts as living vegetation on this small scale.
Biological components. We aimed to match preindustrial ratios of soils, terrestrial
vegetation and atmospheric carbon (Table 1). Dry weight arable soil, 2.85 g
(2.13% C, 0.16% N) was used together with 0.528 g fresh weight (14% dry
weight, 38% C dry weight, 1.85% N dry weight) of the C3 plant Pilea glauca
(Urticaceae). After screening a total of eight plant species, P. glauca was selected
as the experimental choice because of its slow growth rate and known suitability
as a durable species in bottle gardens. A carbon-free sand (550 g, <0.001% C,
<0.01% N) was used as additional inert matrix for plant roots. External lighting
was provided by a mixture of halogen and fluorescent tubes with PAR adequate
for this understory plant species (120 µmol m−2 s−1 ). Trial experiments indicated
that a 14–10 h photoperiod was the most suitable light-to-dark ratio to balance
respiration and photosynthesis (as achieved in the S15 scenario).
Statistical analysis. All mcSVASs had independent temperature control and were
consequently treated as true replicates. The R statistical package (version 2.10.1;
ref. 29) was used to carry out repeated measures ANOVA on the effects of three
temperature and CO2 scenarios on the weekly slope of CO2 concentration change
and the weekly CO2 uptake (photosynthesis) and release (night-time respiration)
rates. ANOVAs followed by contrast analysis were carried out for each week
to test if the response of the treatments including increased-temperature and
CO2 –temperature feedbacks differed from the control scenario (isothermal 15 ◦ C).
Received 30 September 2011; accepted 13 February 2012;
published online 11 March 2012
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Acknowledgements
We gratefully acknowledge financial support by the Natural Environment Research
Council. Great support and advice has been offered by G. Mace and the Ecotron
Steering Committee (M. Press, S. Hartley and J. Roy). We thank T. Sloan, M. Saunders
and H. Vallack for technical support, CO2 additions and sample analysis. We also
thank M. J. Crawley for the advice on statistical analysis and A. Fitter for comments
on the manuscript.
Author contributions
A.M. wrote the manuscript. P.I. was responsible for the original concept and acquiring
financial support and with A.M. and M.L. designed the experiments. A.M. and M.L.
carried out the experiments and the statistical analyses. J-A.S. analysed the soil and
plant samples. D.W. and A.M. designed specific pieces of equipment and engineered the
materially closed systems. D.W. implemented the open-path infrared gas analyser and
carried out the TREND programming. R.A. contributed to the TREND programming
and database maintenance. P.M. and A.H. conducted preliminary experiments. All
authors discussed the results and the structure of the paper, commented and revised
the manuscript text.
Additional information
The authors declare no competing financial interests. Supplementary information
accompanies this paper on www.nature.com/natureclimatechange. Reprints and
permissions information is available online at www.nature.com/reprints. Correspondence
and requests for materials should be addressed to A.M.
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 SUPPLEMENTARY INFORMATION
DOI: 10.1038/NCLIMATE1448

Milcu et al. 2012 1/7

Biotic carbon feedbacks in a materially closed soil-vegetation-

atmosphere system

Alexandru Milcu, Martin Lukac, Jens-Arne Subke, Pete Manning, Andreas Heinemeyer,

Dennis Wildman, Robert Anderson & Phil Ineson

- Supplementary information -

This includes:

1) Supplementary Tables S1- S4

2) Supplementary figure S1

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 2/6

1) Supplementary Tables

Supplementary Table S1| T-test derived p-statistics testing whether the slopes of
atmospheric CO2 change are significantly different from zero for all scenarios and
experimental periods. The recovery phase refers to the period where the addition CO2
has been stopped (in S15CO2 and SΔ3CO2 scenarios). Figures in bold are significant at
p<0.05.

Time /Scenarios S15 S15CO2 SΔ3CO2

Establishing week 0.010 0.003 0.002

Week 1-2 0.648 0.001 0.001

Week 3-4 0.408 0.027 0.050

Week 5-6 0.160 0.525 0.210

Week 7-9 0.293 0.585 0.929

Recovery 0.085 0.442 0.283

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Supplementary Table S2 | ANOVA table for the effect of experimental scenarios S15, S15CO2 and SΔ3CO2 on the slope of CO2 change,
night-time respiration (average daily rate of CO2 release) and net photosynthesis (net change in CO2 concentration during daylight)
performed for the individual periods of the experiment and as repeated measures ANOVA (time x treatment) for the two phases of the
experiment (the CO2 additions phase and the recovery phase without CO2 additions). Figures in bold are significant at p<0.05.

Slope CO2
CO2 release (night-time
Time / Source concentration respiration) CO2 uptake (photosynthesis)

Establishment week F2,12 = 0.01, p = 0.909 F2,12 = 0.03 p = 0.962 F2,12 = 0.00, p = 0.930

Week 1-2 F2,12 = 36.41, p < 0.001 F2,12 = 0.41, p =0.669 F2,12 = 0.79, p = 0.474

Week 3-4 F2,12 = 6.23, p = 0.028 F2,12 = 0.76, p = 0.487 F2,12 = 2.13, p = 0.160

Week 5-6 F2,12 = 4.08, p = 0.066 F2,12 = 0.62 p = 0.551 F2,12 = 4.78, p = 0.029

Week 7-9 F2,12 = 2.12, p = 0.183 F2,12 5.34, p = 0.021 F2,12 = 11.19, p = 0.002

Time x Treatment F8,48 = 0.97, p = 0.343 F8,48 = 1.88, p = 0.085 F8,48 = 9.65, p < 0.001

Recovery F1,8 = 2.48, p = 0.153 F2,12 = 14.32, p = 0.002 F2,12 = 15.96, p < 0.003

Time x Treament F2,12 = 5.86, p = 0.032 F2,12 = 2.03, p = 0.174 F2,12 = 0.28, p = 0.754

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Supplementary Table S3 | Temporal variation of the carbon uptake sensitivity to CO2 and temperature in the materially closed Soil-
Vegetation-Atmosphere Systems.

Sensitivity to CO2 concentration Sensitivity to temperature

Time / Sensitivity (Gt C/ppmv CO2) (Gt C/ºC.)

Week 1-2 1.14 -72.80

Week 3-4 1.03 -32.76

Week 5-6 1.80 -34.78

Week 7-9 2.80 -34.25

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Supplementary Table S4 | ANOVA table and planned comparisons output for the effect of scenarios S15, S15CO2 and SΔ3CO2 on plant
root, shoot, leaf and total biomass at the end of the experiment. Ns. represents non-significant differences at p>0.10 whereas the
figures in bold are significant at p<0.05.

ANOVA Planed comparisons (Contrasts)

Source S15 vs. S15CO2 S15 vs. SΔ3CO2 S15CO2 vs. SΔ3CO2

Root biomass Ns. Ns. Ns. Ns.

Shoot biomass F2,12 = 3.77, p = 0.053 Ns. F1,12 = 7.54, p = 0.017 Ns.

Leaf biomass F2,12 = 5.07, p = 0.025 F2,12 = 0.76, p = 0.067 F2,12 = 9.89, p = 0.008 Ns.

Total biomass F2,12 = 2.80, p = 0.099 Ns. F1,12 = 5.59, p = 0.035 Ns.

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2) Supplementary Figures

Supplementary Figure S1

Figure S1| Photograph of one materially closed Soil-Vegetation-Atmosphere

Systems (mcSVAS). (A) Plant (Pilea glauca). (B) Pot containing carbon free sand and

soil. (C) Soil moisture probe. (D) Light (PAR) sensor. (E) Pressure and temperature

sensors. (F) Heater. (G) Chiller. (H) Water reservoir. (I) Peristaltic irrigation pump. (J)

Air pump providing continuous air circulation between mcSVAS and the open-path

infrared gas analyser. (K) Fans maintaining continuous internal air mixing.

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