Methods in Ecology and Evolution 2011, 2, 103–109 doi: 10.1111/j.2041-210X.2010.00058.

Non-intrusive monitoring of atmospheric CO2 in

analogue models of terrestrial carbon cycle
Martin Lukac1*, Alexandru Milcu1, Dennis Wildman1, Rob Anderson1, Tom Sloan1 and
Phil Ineson2
1
NERC Centre for Population Biology, Imperial College London, Silwood Park Campus, Ascot, Berkshire, UK; and
2
NERC Centre for Terrestrial Carbon Dynamics (CTCD), University of York, Biology Department, York, YO10 5YW, UK

Summary
1. Closed ecological systems (CES) are small man-made ecosystems which do not have any material
exchange with the surrounding environment. Recent ecological and technological advances enable
successful establishment and maintenance of CES, making them a suitable tool for detecting and
measuring subtle feedbacks and mechanisms.
2. As a part of an analogue (physical) C cycle modelling experiment, we developed a non-intrusive
methodology to control the internal environment and to monitor atmospheric CO2 concentration
inside 16 replicated CES. Whilst maintaining an air-tight seal of all CES, this approach allowed for
access to the CO2 measuring equipment for periodic re-calibration and repairs.
3. To ensure reliable cross-comparison of CO2 observations between individual CES units and to
minimize the cost of the system, only one CO2 sampling unit was used. An ADC BioScientific OP-2
(open-path) analyser mounted on a swinging arm was passing over a set of 16 measuring cells. Each
cell was connected to an individual CES with air continuously circulating between them.
4. Using this setup, we were able to continuously measure several environmental variables and CO2
concentration within each closed system, allowing us to study minute effects of changing tempera-
ture on C fluxes within each CES. The CES and the measuring cells showed minimal air leakage
during an experimental run lasting, on average, 3 months. The CO2 analyser assembly performed
reliably for over 2 years, however an early iteration of the present design proved to be sensitive to
positioning errors.
5. We indicate how the methodology can be further improved and suggest possible avenues where
future CES based research could be applied.
Key-words: CES, CO2 monitoring, ecological modelling

outside environment can dilute such effects and confound

Introduction
Closed ecological systems (CES), most commonly defined as
biological systems closed to material but open to energy
exchange, are able to self-sustain by continuously cycling all
vital substances (Gitelson, Lisovsky, & MacElroy 2003).
A closed system provides an environment where small effects
of biological activity accumulate over time (Dempster 2008)
and where matter cycling and turnover times are vastly acceler-
ated (Nelson et al. 2003). Normally not detectable in open
systems, subtle mechanisms and feedbacks can be observed
and measured inside a CES, however the element of closure is
of crucial importance. Even a small material exchange with
*Correspondence author. E-mail: m.lukac@imperial.ac.uk
Correspondence site: http://www.respond2articles.com/MEE/
interpretation. Although primarily used in bio-regenerative life
support research (Salisbury, Gitelson, & Lisovsky 1997;
Nelson et al. 2003), CES have already been successfully applied
to ecological research (Taub 1974). Initially used to construct
models of aquatic environments, CES were first used in simple
experiments testing the optimal conditions for long-term
system self-sustainability such as eukaryote ⁄ prokaryote ratios
(Obenhuber & Folsome 1983). Much later, grazer populations
were able to sustain themselves for at least several weeks in
small and simple CES, allowing for observations and manipu-
lations of algae-grazer dynamics and the recording of internal
O2 and pH measurements (Taub 2009). The complexity of
reconstructed and enclosed ecosystems gradually increased,
together with increasing technical competence. Tamponnet &
Savage (1994) still mention technological challenges of

 2010 The Authors. Methods in Ecology and Evolution  2010 British Ecological Society
104 M. Lukac et al.
setting-up and maintaining CES as the main drawback of
their wider deployment in ecological research, while noting
their enormous potential for research applications.
To date, the largest and the most complex attempt at creat-
ing a CES was the Biosphere 2 project (Allen & Nelson 1999).
Housing several humans and internal sections representing
various biomes, this undertaking highlighted the need for clos-
ing the water and nutrient cycles, together with managing the
C cycle (Nelson, Dempster, & Allen 2009).
In order to complement our knowledge of processes and
feedbacks that govern the global C cycle, which is currently vir-
tually unexplored outside computer models, we set out to har-
ness the CES approach and create analogue (physical) models
of terrestrial biosphere. Since studying the rules that govern
the behaviour of materially closed systems has led to many
advances in other fields of science (Jones 1996), we surmised
that making use of the element of closure could offer important
insights into the inner working of the biological C cycle. To
date, there are no experimental observations of C cycling in
terrestrial materially-isolated systems with biotic components
below the scale of the planet. Here, we attempted to model glo-
bal C pools and fluxes, since we currently know very little
about feedbacks and their effects on these C pools and fluxes.
As part of a project aiming to establish physical analogues of
the terrestrial C cycle (SCALE - Sealing Carbon and Life in the
Ecotron) experiment, we created 16 replicate models of terres-
trial biosphere with proportional amounts of C in the atmo-
sphere, plant biomass and soil. For the purpose of clarity and
to distinguish them from the generic concept of CES, our
model systems will be referred to as materially closed chambers
(MCC). This paper outlines the technical and engineering solu-
tions we employed to create airtight MCC and to monitor
internal environmental conditions. The biggest technical chal-
lenge to be overcome was the reduction of the gaseous
exchange between the MCC and exterior, while achieving
accurate and continuous measurements of atmospheric CO2
concentration. There are several limitations associated with
integrating CO2 monitoring devices within each MCC, such as
the inability to perform maintenance or recalibration during
an experiment without contamination. Consequently, we
developed an engineering solution allowing for non-
invasive monitoring of CO2 by one analyser without cross-
contamination between individual MCC. We describe the
technical design and the performance of MCC units and of the
CO2 monitoring apparatus and outline the potential for
further improvement of this technology.
Methodology
MATERIALLY CLOSED CHAMBERS
We utilized the Ecotron (Lawton 1996), a controlled environ-
ment facility at Silwood Park, Imperial College London, to
provide the housing and the supporting computer and control
systems for this experiment. The Ecotron is comprised of 16
individually controlled chambers centrally monitored by a
TREND 963 supervisor (Trend Control Systems Ltd.,
 2010 The Authors. Methods in Ecology and Evolution  2010 British Ecological Society, Methods in Ecology and Evolution, 2, 103–109
Horsham, UK). A single 120 L MCC was installed within each
Ecotron chamber (Fig. S1). Manufactured from transparent
10 mm polycarbonate with welded joints (Glove Box Technol-
ogy Ltd., St Ives, UK) the MCC were designed to be air-tight
and to house independent climate control and monitoring
systems (Fig. S2). Separate heating and chilling systems were
operating continuously to provide accurate temperature
control, while optimal air circulation within each box was
maintained by a set of fans. Each box contained a single free-
draining plant pot filled with soil and sand representing the
biotic part of the system. Rooting medium water content was
continuously monitored by a Theta Probe (Delta-T Devices
Ltd., Cambridge, UK), automatically triggering a 20 mL
watering event (Etatron DS peristaltic pump) from a 280 mL
water reservoir whenever soil moisture decreased below 35%.
Excess water draining from the pot and condensing on the chil-
ler was diverted back into the reservoir.
Each MCC also contained temperature (National Semicon-
ductor LM35, integrated circuits), relative humidity (RH, Hon-
eywell HIH 3610), air pressure (Honeywell ASDX030A24R)
photosynthetically active radiation (PAR, Delta-T PAR quan-
tum sensor) and atmospheric oxygen sensors (City Technology
CiTiceL 70X-V). All electrical and data connections were led
out of the box via 25-way sealed ‘D’ type Bulgin ‘Buccaneer’
and ‘Mini-Buccaneer’ connectors (Elektron Components Ltd.,
UK), clamping to a stainless steel plate held in a perspex hollow
ring and backfilled with epoxy resin to a depth of about 20 mm
to maintain an air-tight seal. Once an experiment was estab-
lished and the access hatch on the side of the MCC was sealed
(Fig. S1), the internal environment was monitored and con-
trolled autonomously from within each MCC. Three
8 mm · ¼ inch BSP (British Standard Pipe) push-fit connec-
tors were installed on one side of each MCC, two for linking
the box to a CO2 measuring cell and one for purging the inter-
nal environment. Continuous circulation of internal atmo-
sphere between the box and the measuring cell (1Æ5 L min)1)
was provided by an aquarium pump installed inside the box.
CO2 MONITORING
A single open-path analyser was utilised to monitor the CO2
levels in the MCC. An infrared ADC BioScientific OP-2 device
(Herold, Thomas, & Foken 2005; Velasco et al. 2009), using 4-
path folded beam was attached to a custom-made welded alu-
minium bracket and mounted vertically on a rotating pillar
(Fig. S3). The beam source and detector were located within a
cylinder, while the main mirror was mounted on a frame sup-
ported rigidly by pillars machined in the instrument body, pro-
viding approximately 200 mm of clear space between the
electronics assembly and the mirror. An adjustment system
was put in place to allow for accurate alignment of the mirror
and source ⁄ detector assembly.
Each MCC was connected to a separate infrared (IR) mea-
suring cell via two 8 mm pneumatic nylon tubes, allowing for
continuous circulation of air between the MCC and the IR
measuring cell. The length of the tubing varied between 4 and
10 m, according to the position of individual MCC relative to

the OP-2. The IR measuring cells were arranged in a circle cen-
tred on the rotating pillar, so that the OP-2 beam source and
analyser assembly would pass over and the mirror under each
IR measurement cell. Several millimetres of clear space were
provided to avoid the OP-2 assembly and the sample cells com-
ing in physical contact. The IR measuring cells were manufac-
tured from solid aluminium and gold-plated to enhance IR
reflectance and to minimize CO2 adhesion to internal surfaces.
Each cell had two sapphire glass windows sealed with an inter-
nal ‘O’ ring, one each at the top and bottom to allow for the
passage of the measurement beam through the internal volume
of the cell. Out of the total of 18, 16 IR measuring cells were
connected to individual MCC, a ‘zero’ cell was connected to
the internal airspace of the OP-2 assembly and the ‘calibration’
cell had connections which were freely accessible from outside
to perform system drift measurements.
Rotation and positioning of the OP-2 arm was provided by
an off-the-shelf antenna rotator motor and gearbox (Protistel
PST641D, Vine Antennas Ltd, Powys), which rotated the
OP-2 pillar with a toothed-belt. Gearing ratio was incorpo-
rated within the system to minimize the angular error of the
OP-2 positioning over each measuring cell. A potentiometer
was fitted to the pillar’s axle to act as the primary position feed-
back mechanism. Initially this was used purely as an indication
of angle, but was later found to be more accurate than the rota-
tor system’s original device and thereafter used as the primary
sensor. The CO2 analyser, all measuring cells, and associated
electronics were enclosed inside a Perspex cabinet. Since ca.
13% of the infrared beam length passed though the open
spaces between the OP-2 device and the sample measuring
cells, we monitored the internal CO2 concentration via the
‘zero’ cell and maintained it as low as possible (approx
5 p.p.m.). To achieve this we used an aquarium pump to draw
air from the bottom of the box and continuously pass it, in ser-
ies, through the ‘zero’ measuring cell, then through a soda lime
cylinder and a Drierite cylinder, before returning it to the inter-
nal space. As a back-up, a pure N2 cylinder was attached to the
box to flush the whole cabinet if the internal concentration of
CO2 exceeded 20 p.p.m.
CONTROL SYSTEM
The sampler system was connected to and continuously inter-
faced with the existing Trend BMS (Trend Control Systems
Ltd., Horsham, UK) which connected to the Ecotron Trend
LAN (Fig. S4). Although off-the-shelf hardware and software
is available to effect such connection, in this instance we elected
to apply a custom-made solution to ensure modifications could
be easily applied. In brief, the Trend controller calculated the
angle to which the OP-2 was to be moved, and signalled this to
the interface box as a DC voltage. An Analogue to Digital
Converter (ADC) interpreted this as a 9-bit number, specifying
a range of 0 (1st cell) to 357 (18th cell). When the system was
ready to move to this angle, a ‘go’ signal was given by the
Trend BMS to the antenna rotator. The output from the posi-
tion feedback potentiometer was fed to the Trend Controller
to indicate the actual position of the OP-2 device. In autono-
 2010 The Authors. Methods in Ecology and Evolution  2010 British Ecological Society, Methods in Ecology and Evolution, 2, 103–109
Closed ecological systems and carbon cycling 105
mous mode, the sampler started at 0 (notionally 1 volt dc posi-
tion feedback) and read the CO2 concentration within the 1st
IR measurement cell. The reading continued for about 65 s to
allow for a running average to be taken and stored, before
moving to the next cell. Once the last (18th cell) was measured
at 238, the system rotated the OP-2 arm back to the starting
position to initiate a new sequence of CO2 observations.
Results and discussion
MCC PERFORMANCE
While we made use of some of environmental control capabili-
ties of the Ecotron, the use of such facility for CES research is
not strictly necessary. The external conditions of each MCC
do not have to be controlled, provided that the internal control
mechanism is sufficiently robust to cancel out variations of out-
side environment. The main goal of our experiment was to
study the C cycle, the design of our MCC was therefore subor-
dinated to the need to have viable models of the terrestrial bio-
sphere (Fig. S1). The internal volume of 120 L was chosen as
the minimum necessary to obtain manageable C pools in the
plant and in the soil, relative to the C content of enclosed
atmosphere. Prior to closure, each MCC was equipped with a
pot containing 2Æ85 g of dry arable soil mixed with C-free sand
and 0Æ528 g of plant (total biomass fresh weight). The MCC
units were sealed and CO2 concentration of internal atmo-
sphere adjusted to pre-industrial level of 280 p.p.m. in order to
obtain atmosphere:plant:soil ratio of 1 : 2 : 4Æ5 (0Æ0136 g C in
the atmosphere, 0Æ0276 g C in the vegetation and 0Æ0606 g C in
the soil), falling within the upper range of available global C
estimates found on Earth (McCarthy and Intergovernmental
Panel on Climate Change. Working Group II 2001). We tested
several plant species for suitability; an ideal plant had to grow
very slowly as not to skew the C ratios, but at the same time it
would be able to survive for prolonged periods inside closed
environment. Pilea glauca (fam. Urticaceae), a tropical semi-
shade tolerant C3 species, was selected for experimentation
since its photosynthetic capacity was able to force a pro-
nounced diurnal signal in internal CO2 concentration (Fig. 1),
Fig. 1. Diurnal oscillation of internal CO2 concentration in selected
materially closed chambers (MCC) in response to 12-hour day and
night cycle. Small errors are due to OP-2 measurement arm misposi-
tioning.
106 M. Lukac et al.
while its biomass did not expand appreciably during experi-
ments lasting 2–3 months. Due to the size of our MCC, we
were able to place only a single plant inside each unit, the size
of which had to be precisely controlled. We attempted to estab-
lish an MCC with a precise ratio of C, whilst having to weigh
fresh plant biomass. The accuracy and the plant survival rate
could be increased if larger MCC were used, allowing for big-
ger plants. On average, plant survival within MCC proved to
be better than expected, with only 3 instances of plant death
during 7 experimental runs involving 15 replicate MCC each.
Towards the end of each experiment lasting up to 3 months, a
slight discolouration of plant leaves could be detected, possibly
caused by nutrient limitation due to very small quantity of soil,
constrained by the global C ratios. The pH (5Æ9 at 20C) of
water in the reservoir was not affected by the length of an
experiment or by the CO2 concentration inside each MCC.
Initially, during an establishment phase lasting approxi-
mately 2 weeks after closure, all MCC were maintained at
15C. Each MCC was equipped with independent temperature
control provided by continuously operating heater and cooler,
allowing for rapid and precise adjustments of internal tempera-
ture. Since both heat exchangers were operating at the same
time, the temperature was controlled by the relative output of
both units. Using this system, we were able to control MCC
temperature with minimal divergence from the set point when
the temperature was kept constant (Fig. 2a) or adjusted daily
(a)
(b)
Fig. 2. Temperature control in materially closed chambers (MCC)
with stable set point of 15C (a) and with set points changed on a daily
basis according to internal CO2 concentration (b).
 2010 The Authors. Methods in Ecology and Evolution  2010 British Ecological Society, Methods in Ecology and Evolution, 2, 103–109
(Fig. 2b). A background temperature of 15C was chosen to
closely resemble the average global temperature (Solomon
et al. 2007) and was kept constant in MCC assigned to the
‘control’ treatment. Some of the climate scenarios we tested
involved daily changes of internal temperature in ‘treatment’
MCC in a feedback loop driven by the CO2 concentration. The
temperature control proved to be very good, as an example,
the standard deviation was 0Æ048C over a 3 day period if tem-
perature was kept constant at 15C. Changing the temperature
set point daily to account for changing CO2 concentration (ca.
0Æ1–0Æ4C per day) introduced a small disturbance to the
MCC, resulting in slightly higher standard deviation of
0Æ115C at 16Æ5C mean temperature. Temperature control
was operated as an autonomous system and was able to
rapidly respond to external influence. Figure 2a shows regular
peaks and troughs deviating from the set point of 15C caused
by the 12-hour on ⁄ off setting of lighting inside each Ecotron
chamber.
Achieving a good level of closure is pivotal to CES research,
without achieving reliable long-term closure we might not be
able to detect subtle feedbacks or accumulations (Dempster
2008). Once connected to an allocated sample IR measurement
cell and sealed, the air-tightness of each MCC was leak-tested
by filling it up with pressurized helium. All leaks detected by
screening with a portable helium detector were fixed and
re-tested. Despite testing, a small rate of contamination (pre-
sumably via gaseous diffusion, as helium detection did not
reveal physical leaks) was unavoidable due to long-term nature
of our experiments and materials used. We were able to calcu-
late long-term contamination of each MCC separately on the
basis of ideal gas law. The internal air volume of each MCC
was known – we submerged a prototype version of all internal
equipment to estimate its volume – and remained unchanged
because of sturdy nature of MCC walls, air tubes and measur-
ing cells. Since we had accurate observations of atmospheric
temperature and pressure inside each MCC, we could calculate
the amount of air lost or gained through undetectable leaks.
The goal of the SCALE experiment was to observe C cycle
inside a closed system, atmospheric CO2 leakage was therefore
of primary interest to us. Expressed as the amount of CO2, we
estimate that 0Æ00011 ± 0Æ00062 g S.E. of CO2 was exchanged
with external atmosphere over the course of a 3-month experi-
ment (Fig. 3), representing 0Æ1% of initial C content of each
MCC. The length of tubing connecting the MCC to the OP-2
ranged from ca 4–10 m according to the position of each
Ecotron chamber relative to the OP-2. We did not detect any
relationship between the length of tubing and the leakage rate.
CO2 OBSERVATION
The tubes connecting each MCC with the OP-2 were wrapped
in Airtec double insulation along their entire length, and a
trace heater cable was enclosed in the insulation. This arrange-
ment avoided water condensation inside the tubes and ensured
that air returning to the MCC had approximately the same
temperature as the unit itself, reducing the strain on the tem-
perature control. An open-path analyser was used to measure

Fig. 3. Mean weekly CO2 exchange between each materially closed
chambers (MCC) and outside environment.
atmospheric CO2 concentration inside each IR measuring cell
(Fig. S3). Although sensitive to changes in atmospheric pres-
sure, temperature and water vapour content (Auble & Meyers
1992), open-path gas analysers have been applied in a large
number of ecosystem C flux studies (e.g. Hirano et al. 2003)
and their output compares well with that of closed-path analy-
sers (Jarvi et al. 2009). An important issue affecting the accu-
racy and comparability of observations from open-path
analysers is the stability of the environmental conditions. We
minimized the effect of external conditions on CO2 readings by
housing the analyser inside a closed cabinet with CO2-free
atmosphere and by insulating the cabinet. We calibrated the
OP-2 analyser prior to each experiment by passing a sequence
of air samples with a range of CO2 concentrations (ca. 50–
1000 p.p.m.) through serially connected IR measurement cells,
while measuring the voltage output of the OP-2 for each mea-
surement cell. A second-order polynomial equation was then
fitted to the data (R2 = 0Æ994 on average across cells), the
parameters of which were applied to the appropriate Trend
controllers to allow voltage-to-CO2 level conversion.
Overall, the OP-2 assembly performed well, with only one
period of technical downtime during more than 2 years of con-
tinuous operation. Due to the number of measuring cells, one
cycle took ca. 30 min to complete, giving us at least hourly
observation of CO2 concentration inside each MCC. The infra-
red beam proved to be very sensitive to the interaction between
the presence of small impurities on sapphire glasses and posi-
tioning accuracy. Prior to the first experiment, inside and out-
side surfaces of both sapphire windows were thoroughly
cleaned to ensure smooth data profiles from all sample cells.
Even a slight mispositioning of the OP-2 resulted in a step
increase or decrease of output voltage, despite the fact that an
average measurement over 65 s was taken for each cell. Once
the OP-2 was settled, resulting CO2 observations allowed us to
observe real-time changes of atmospheric composition both
over diurnal cycle and over the duration of an experiment. As
expected, there was no possibility or evidence for cross-con-
tamination of atmospheres enclosed within individual closed
systems. One key experiment was a CO2 emission simulation
scenario, which involved injecting pure CO2 into ‘treatment’
MCC via a 3-way valve adaptor (Fig. 4). All CO2 injections
 2010 The Authors. Methods in Ecology and Evolution  2010 British Ecological Society, Methods in Ecology and Evolution, 2, 103–109
Closed ecological systems and carbon cycling 107
Fig. 4. Effect of pure CO2 additions on internal CO2 concentration.
A step-increase in treatment materially closed chambers (MCC) (5, 9
and 12 – full lines) is clearly visible immediately after injection, sug-
gesting optimal atmosphere mixing. There is no concurrent increase
in CO2 concentration in control MCC (1, 2 and 4 – dotted lines).
are clearly identifiable and show immediately with the next
pass of the OP-2 over the appropriate measuring cell. Control
MCC, on the other hand, did not exhibit any change of CO2
concentration, confirming a complete separation of MCC.
Limitations and potential improvements
One of the biggest drawbacks, specifically relating to the exper-
iments we were performing, was the size of the MCC. Target-
ing pro rata analogue models of C pools of the terrestrial
biosphere, together with the internal air volume and its CO2
content somewhat limited our ability to accurately reconstruct
the plant-soil subsystem. A larger MCC would have allowed
for bigger, or more, plants and for greater soil mass to provide
nutrition to the plants. It is likely that such a setup would
improve the robustness of the biotic part of our models, while
at the same time giving us a better control over the precision of
C pools installed inside the MCC. Larger MCC, however, need
a mechanism to account for atmospheric pressure differences
between the external and internal environments (Dempster
2008). The ratio between the exterior wall surface area and its
thickness was favourable in the MCC used in our experiment,
allowing us to technically ignore the forces acting on the walls
due to pressure difference. Since a small pressure difference
applied over a large surface area results in significant force, a
larger MCC would need a ‘lung’ to compensate for inter-
nal ⁄ external pressure differentials to prevent wall rupture. In
eliminating pressure difference between the closed system and
its exterior, and such an arrangement would also help to mini-
mize leakage from or into the MCC.
In the extant system, each MCC was linked to a CO2 mea-
suring cell via two lengths of nylon tubing. While the perme-
ability of nylon to CO2 molecules was considered sufficient for
our purpose, where higher precision is required to study very
low concentrations of compounds, the use of welded stainless
steel or PTFE tubing with internal aluminium coating is advis-
able.
We made use of 16 experimental MCC and installed 2 extra
‘technical’ IR measurement cells, with the infra-red CO2
108 M. Lukac et al.
analyser therefore monitoring 18 cells arranged in a large arc.
The size of the measuring cells and the length of the OP-2 arm
between them dictate the number of MCC that can be moni-
tored by one analyser. Replication rate can be increased either
by decreasing the width of the measuring cells or by extending
the OP-arm to cover a larger arc. The latter approach, how-
ever, is only viable if a significant improvement of the position-
ing accuracy over the current design can be achieved. The CO2
readings were sensitive even to small positioning errors;
improved gearing, the use of stepper motors or a linear
arrangement of measuring cells could achieve this improve-
ment.
Open-path analysers are sensitive to changes of some envi-
ronmental variables; atmospheric pressure, temperature and
CO2 concentration all affect the readings of the analyser used
in this research. To minimize these errors, the OP-2 was housed
inside a sealed cabinet with internal CO2 concentration main-
tained as close to zero as possible. However, even small
changes of ambient temperature during initial testing (e.g.
lighting or air conditioning) were found to affect the readings.
We insulated the OP-2 cabinet to minimize these effects by
wrapping it in Airtec double insulation, thus maintaining inter-
nal temperature between 18Æ5 and 19Æ5C. To ensure better
control over internal temperature, a more precise control sys-
tem similar to those employed inside the MCC could be
included in the design of infrared analyser cabinet.
Future applications
We applied CES approach to the study of C fluxes and climatic
feedbacks in models of terrestrial C cycle, following atmo-
spheric CO2 concentration as the main response variable.
There are a multitude of further scientific questions which
could benefit from using unexplored advantages that materi-
ally-closed but energetically open systems may offer. Funda-
mental processes and relationships, such as CO2-temperature
feedbacks or the importance of biological diversity, have
remained virtually unexplored in such systems. This approach
can be used not only for testing the sensitivity of biotic
responses (e.g. Q10s) to future environmental conditions, such
as changes in ambient temperature or atmospheric CO2 con-
centrations and their interactions, but also for recreating and
observing the behaviour of paleoenvironments. The role of
biodiversity in ecosystem stability may be studied by establish-
ing simplified ecosystems with a gradient of diversity and iso-
lating them inside CES, perhaps by using larger MCC which
could accommodate a greater diversity of organisms. The CES
approach also has an unique advantage of being able to estab-
lish systems in which the dynamic interplay between the biotic
and abiotic components offers the opportunity to study the
laws that govern self-regulation and potential Gaian feedbacks
(Lenton & Wilkinson 2003).
Studies of other phenomena in different ecological settings
can be envisaged, perhaps involving non-intrusive monitoring
at other wavelengths or by using alternative analytical equip-
ment. Accumulation of contaminants or nutrient release and
cycling can also be studied in minute detail, e.g. the nitrogen or
 2010 The Authors. Methods in Ecology and Evolution  2010 British Ecological Society, Methods in Ecology and Evolution, 2, 103–109
phosphorus cycling. We have shown that observations of inter-
nal environment can be carried out in real time, even by analyt-
ical equipment which requires maintenance and re-calibration
during the course of an experiment. We further show that
recent technological advances allow us to make a full use of
Closed Ecological Systems as an experimental approach for
studying biological processes and feedback mechanisms.
Acknowledgements
We gratefully acknowledge financial support by the Natural Environment
Research Council, practical and organizational support and advice have been
offered by G. Mace and the Ecotron Steering Committee (M. Press, S. Hartley
and J. Roy). We also thank J. Subke, P. Manning, A. Heynemeyer, C. Denham,
H. Vallack, M.Saunders and all the participants of the workshop on CES orga-
nized by the CPB.
References
Allen, J. & Nelson, M. (1999) Biospherics and Biosphere 2, mission one (1991-
1993). Ecological Engineering, 13, 15–29.
Auble, D.L. & Meyers, T.P. (1992) An open path, fast response infrared-
absorption gas analyzer for H2O and CO2. Boundary-Layer Meteorology,
59, 243–256.
Dempster, W.F. (2008) Tightly closed ecological systems reveal atmospheric
subtleties - experience from Biosphere 2. Advances in Space Research, 42,
1951–1956.
Gitelson, I.I., Lisovsky, G.M. & MacElroy, R.D. (2003) Manmade Closed Eco-
logical Systems. Taylor & Francis, London.
Herold, G.M., Thomas, C. & Foken, T. (2005) The OP-2 Open Path Infrared
Gas Analyser for CO2 and H2O. Universitat Bayeruth, Bayeruth, Germany,
Available at: http://132.180.112.26/mikrometeorologie/ARBERG/ARBER
G27.pdf, accessed May 2010.
Hirano, T., Hirata, R., Fujinuma, Y., Saigusa, N., Yamamoto, S., Harazono,
Y., Takada, M., Inukai, K. & Inoue, G. (2003) CO2 and water vapor
exchange of a larch forest in northern Japan. Tellus Series B-Chemical and
Physical Meteorology, 55, 244–257.
Jarvi, L., Mammarella, I., Eugster, W., Ibrom, A., Siivola, E., Dellwik, E.,
Keronen, P., Burba, G. & Vesala, T. (2009) Comparison of net CO2 fluxes
measured with open- and closed-path infrared gas analyzers in an urban
complex environment. Boreal Environment Research, 14, 499–514.
Jones, T.H. (1996) Biospherics, closed systems and life support. Trends in Ecol-
ogy & Evolution, 11, 448–450.
Lawton, J.H. (1996) The Ecotron facility at Silwood Park: the value of ‘‘big
bottle’’ experiments. Ecology, 77, 665–669.
Lenton, T.M. & Wilkinson, D.M. (2003) Developing the Gaia theory - a
response to the criticisms of Kirchner and Volk. Climatic Change, 58, 1–12.
McCarthy, J.J. & Intergovernmental Panel on Climate Change. Working
Group II. (2001) Climate Change 2001: Impacts, Adaptation, and Vulnerabil-
ity: Contribution of Working Group Ii to the Third Assessment Report of the
Intergovernmental Panel on Climate Change. Cambridge University Press,
Cambridge, UK; New York, USA.
Nelson, M., Dempster, W.F. & Allen, J.P. (2009) The water cycle in closed eco-
logical systems: perspectives from the Biosphere 2 and Laboratory Biosphere
systems. Advances in Space Research, 44, 1404–1412.
Nelson, M., Allen, J., Alling, A., Dempster, W.F. & Silverstone, S. (2003) Earth
applications of closed ecological systems: relevance to the development of
sustainability in our global biosphere. Space Life Sciences: Closed Artificial
Ecosystems and Life Support Systems, 31, 1649–1655.
Obenhuber, D.C. & Folsome, C.E. (1983) Eucaryote procaryote ratio as
an indicator of stability for closed ecological-systems. Biosystems, 16, 291–
296.
Salisbury, F.B., Gitelson, J.I. & Lisovsky, G.M. (1997) Bios-3: Siberian experi-
ments in bioregenerative life support - Attempts to purify air and grow food
for space exploration in a sealed environment began in 1972. BioScience, 47,
575–585.
Solomon, S. & Intergovernmental Panel on Climate Change & Intergovern-
mental Panel on Climate Change. Working Group I. (2007) Climate Change
2007: The Physical Science Basis: Contribution of Working Group I To The
Fourth Assessment Report of the Intergovernmental Panel on Climate Change.
Cambridge University Press, Cambridge, New York, USA.

Tamponnet, C. & Savage, C. (1994) Closed ecological systems. Journal of Bio-
logical Education, 28, 167–174.
Taub, F.B. (1974) Closed ecological systems. Annual Reviews of Ecology and
Systematics, 5, 139–160.
Taub, F.B. (2009) Community metabolism of aquatic Closed Ecological Sys-
tems: effects of nitrogen sources. Advances in Space Research, 44, 949–957.
Velasco, E., Pressley, S., Grivicke, R., Allwine, E., Coons, T., Foster, W.,
Jobson, B.T., Westberg, H., Ramos, R., Hernandez, F., Molina, L.T. &
Lamb, B. (2009) Eddy covariance flux measurements of pollutant gases in
urban Mexico City. Atmospheric Chemistry and Physics, 9, 7325–7342.
Received 25 June 2010; accepted 26 July 2010
Handling Editor: Robert P Freckleton
Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
Figure S1. Side elevation of a materially closed chamber (MCC); (a)
plant, (b) pot containing carbon free sand and soil, (c) soil moisture
probe, (d) light (PAR) sensor, (e) pressure and temperature sensors,
 2010 The Authors. Methods in Ecology and Evolution  2010 British Ecological Society, Methods in Ecology and Evolution, 2, 103–109
Closed ecological systems and carbon cycling 109
(f) heater, (g) cooler, (h) water reservoir, (i) peristaltic irrigation
pump, (j) air pump, (k) air fans.
Figure S2. Schematic diagram of a materially closed chamber (MCC),
showing environment control and monitoring system arrangement.
Figure S3. An open path (OP-2) infrared analyser inside Perspex
cabinet; (a) analyser assembly, (b) rotation cylinder, (c) air fan clean-
ing dust from infrared (IR) measuring cells, (d) IR measuring cells,
(e) nylon tubing connecting IR measuring cells and materially closed
chamber (MCC).
Figure S4. Schematic diagram of open-path (OP-2) and Trend
control arrangement.
As a service to our authors and readers, this journal provides support
ing information supplied by the authors. Such materials may be
re-organized for online delivery, but are not copy-edited or typeset.
Technical support issues arising from supporting information (other
than missing files) should be addressed to the authors.