We thank Anne Gibson, Katherine Jones, Chris Kolodziejczyk, Alicia Naranjo, Kyle Tiner, and numerous students and temporary technicians for operating the LYCOG facility, conducting sampling, and data processing. L.G.R. acknowledges USDA-NIFA (2010-65615-20632).

1. Collect Soil Monoliths to be used as Weighing Lysimeters
<ol>
	<li>Construct open-ended steel boxes 1 x 1 m square by 1.5 m deep from 8 mm thick steel.</li>
	<li>Press the open-ended boxes vertically into the soil, using hydraulic presses mounted on helical anchors drilled 3 m deep into the soil.</li>
	<li>Excavate the encased monolith using a backhoe or similar equipment.</li>
	<li>Place a fiberglass wick in contact with soil at the base of the monolith. Pass the wick through the steel base into a 10 L reservoir to drain the monolith, and then weld the steel base onto the bottom of the box.</li>
	<li>Kill existing vegetation on the monoliths by applying a non-residual herbicide, such as glyphosate.</li>
</ol>
2. Establish Plant Communities on Soil Monoliths
<ol>
	<li>Plant the monoliths with eight seedlings each of seven species of tallgrass prairie grasses and forbs, for a total density of 56 plants per m2.
	<ol>
		<li>Plant the following Grasses: Bouteloua curtipendula (side-oats grama), Schizachyrium scoparium (little bluestem), Sorghastrum nutans (Indiangrass), Tridens albescens (white tridens)].</li>
		<li>Plant the following Forbs: Salvia azurea (pitcher sage), Solidago canadensis (Canada goldenrod), Desmanthus illinoensis (Illinois bundleflower, a legume).</li>
	</ol>
	</li>
	<li>Plant seedlings in a Latin Square design, re-randomized for each monolith.</li>
	<li>Water the transplants for approximately 2 months following planting. The goal is to minimize water stress during initial establishment. Use any convenient method such as a hand wand or garden sprinkler. The frequency of watering depends on local climate and weather, particularly the occurrence of ambient rainfall.</li>
	<li>Following the initial transplant establishment phase, maintain the transplants under ambient rainfall for as long as necessary while chambers (Section 3) are constructed. Remove unwanted species that emerge in the monoliths during establishment by hand-weeding.</li>
</ol>
3. Chamber Design
<ol>
	<li>Construct two chambers each 1.2 m wide, 1.5 m tall, and 60 m long, divided into ten 5 m long sections. Construct sections from heavy steel of dimensions 5 m x 1.2 m x 1.6 m deep, buried to 1.5 m.
	<ol>
		<li>Install four monoliths in each section, two monoliths each of two of the soil types, in random order. Install each monolith atop a 4,540 kg capacity balance.</li>
		<li>Include Bastsil monoliths in the pairings in even numbered sections.</li>
	</ol>
	</li>
	<li>Join adjacent sections aboveground with a 1 m long x 1 m wide x 0.3 m tall sheet-metal duct to provide a pathway for airflow.
	<ol>
		<li>Supply coolant at 10 &#176;C from a 161.4 kW refrigeration unit to a cooling coil inside each duct.</li>
		<li>Enclose the vegetation with clear greenhouse film (thickness 0.006&#8221;/.15 mm), such as used in other climate manipulation experiments23.</li>
		<li>Fit each cover with a zippered opening backed by a draft flap to allow access to the monoliths for sampling.</li>
		<li>Remove the polyethylene covers at the end of the growing season.</li>
	</ol>
	</li>
</ol>
4. CO 2 and Air Temperature Measurement; Temperature Control
<ol>
	<li>Sample entry and exit CA on both chambers every 2 min through filtered air sample lines located at the entry and exit of superambient and subambient chambers. These data inform CO2 injection and fan speed control.
	<ol>
		<li>Sample CA and water vapor content, and measure air temperature (TA) at the entry and exit of each 5 m section at 20 min intervals.</li>
		<li>Measure all air samples for CO2 and water vapor content in real time using infrared gas analyzers according to manufacturer&#8217;s protocol.</li>
		<li>Measure TA at the entry, midpoint, and exit of each section with shielded fine wire thermocouples.</li>
	</ol>
	</li>
	<li>Regulate the flow of coolant through the cooling coil at the entrance of each section to maintain a consistent mean (mid-section) TA from section to section near the ambient TA.</li>
	<li>Position a quantum sensor to have an unobstructed view of the sky and measure photosynthetic photon flux density according to manufacturer&#8217;s protocol. Light level is an input to the blower control algorithm.</li>
</ol>
5. C A Treatment Application
<ol>
	<li>Daytime
	<ol>
		<li>Mix pure carbon dioxide (CO2) with incoming ambient air to 500 &#181;l L-1 CA, using a mass flow controller in the entrance duct of the superambient leg. See Section 4 for CA measurement details.</li>
		<li>Advect the enriched air through the chambers using blower fans at the entrance to section 1 and in downstream sections.</li>
		<li>Maintain the desired exit CA of 390 &#181;l L-1 (ambient air) by adjusting the blower speed.
		<ol>
			<li>Increase the blower speed if the exit CA is below the set point. This allows less time for plant uptake of CO2, resulting in higher exit CA.</li>
			<li>Decrease the blower speed if exit CA is above the set point.</li>
		</ol>
		</li>
		<li>Use the same approach in the subambient chamber except introduce ambient air and control to achieve exit CA of 250 &#181;l L-1.</li>
	</ol>
	</li>
	<li>Nighttime
	<ol>
		<li>Reverse the direction of air flow.</li>
		<li>Inject CO2 into the daytime exit end of the superambient chamber to achieve 530 &#181;l L-1 CA, and control advection rates to maintain 640 &#181;l L-1 at the nighttime exit (daytime entrance.</li>
		<li>Introduce ambient air at ~ 390 &#181;l L-1 CO2 into the nighttime entrance (daytime exit) of the subambient chamber and control advection rate to maintain 530 &#181;l L-1 at the nighttime exit.</li>
	</ol>
	</li>
</ol>
6. Precipitation Inputs
<ol>
	<li>Apply the mean growing season rainfall amount to each monolith.
	<ol>
		<li>Supply water to each monolith from a domestic water source through a drip irrigation system. Schedule the irrigation events and application amounts to approximate the seasonal rainfall pattern for the experiment location. The exact schedule depends on local climate.</li>
	</ol>
	</li>
	<li>Control application timing with a data logger and measure application volumes with flowmeters.</li>
</ol>
7. Sampling&#160;
<ol>
	<li>Measure vertical profiles of volumetric soil water content (vSWC) weekly during the period of CO2 control, with a neutron attenuation gauge or other appropriate probe.
	<ol>
		<li>Recommended profile increments are 20 cm depth increments to 1 m depth, and one 50 cm increment below a 1 m.</li>
	</ol>
	</li>
	<li>Measure monolith aboveground net primary productivity (ANPP) by harvesting all standing aboveground biomass at the end of the growing season.
	<ol>
		<li>All aboveground biomass is removed each year, consequently standing biomass represents current primary production.</li>
		<li>Sort the sampled biomass by species, dry to constant mass, and weigh.</li>
		<li>Use biomass of individual species to quantify plant species contributions to ANPP.</li>
	</ol>
	</li>
</ol>

<ol>
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E., Nosberger, J., Long, S. P., Norby, R. J., Stitt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+24:+Value:+Perspectives+on+the+Future+of+Free-Air+CO2+Enrichment+Studies.">Ch 24: Value: Perspectives on the Future of Free-Air CO2 Enrichment Studies.</a> Managed Ecosystems and CO2: Case Studies, Processes, and Perspectives. Ecological Studies. 187, 431-449 (2006).</li><li>Mayeux, H. S., Johnson, H. B., Polley, H. W., Dumesnil, M. J., Spanel, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+controlled+environment+chamber+for+growing+plants+across+a+subambient+CO2+gradient.">A controlled environment chamber for growing plants across a subambient CO2 gradient.</a> Funct Ecol. 7 (1), 125-133 (1993).</li><li>Polley, H. W., Johnson, H. B., Mayeux, H. 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The authors have nothing to disclose.

The LYCOG facility achieves its operational goal of maintaining a 250 to 500 &#181;l L-1 continuous gradient of CA concentrations on experimental grassland communities established on three soil types. The change in CA is linear over the prescribed range. Air temperature increased within each section, but was reset by the between-section cooling coils in most sections. As a result, the operational goal of maintaining a consistent mean temperature from section to section was met over most o...

Atmospheric carbon dioxide concentration (CA) has recently increased past 400 &#181;l L-1 from approximately 270 &#181;l L-1 prior to the Industrial Revolution. CA is forecast to reach at least 550 &#181;l L-1 by 21001. This rate of increase surpasses any CA changes observed over the last 500,000 years. The unprecedented rate of change in CA raises the possibility of non-linear or threshold responses of ecosystems to increasing CA. Most ecosystem-scale CA enrichment experiments apply only two treatments, a single level of enriched CA and a control. These experiments have greatly expanded our understanding of the ecosystem impacts of CA enrichment. However, an alternate approach that can reveal the presence of non-linear ecosystem responses to increasing CA is to study ecosystems across a continuous range of subambient to superambient CA. Subambient CA is difficult to maintain in the field, and has most often been studied using growth chambers2. Superambient CA has been studied using growth chambers, open-top chambers, and free-air enrichment techniques3, 4.
CA enrichment occurs across landscapes containing many soil types. Soils properties can strongly affect ecosystem responses to CA enrichment. For example, soil texture determines the retention of water and nutrients in the soil profile5, their availability to plants6, and the amount and quality of organic matter7-9. The availability of soil moisture is a crucial mediator of ecosystem responses to CA enrichment in water limited systems, including most grasslands10. Past field CA enrichment experiments have typically examined only one soil type, and controlled tests of continuously varying CA enrichment over several soil types are lacking. If effects of CA enrichment on ecosystem processes differ with soil type, there is strong reason to expect spatial variation in ecosystem responses to CA enrichment and ensuing changes in climate11, 12.
The Lysimeter Carbon Dioxide Gradient (LYCOG) facility was designed to address questions of spatial variation in non-linear and threshold responses of ecosystems to CA levels ranging from ~ 250 to 500 &#181;l L-1. LYCOG creates the prescribed gradient of CA on perennial grassland plant communities growing on soils representing the broad range of texture, N and C contents, and hydrologic properties of grasslands in the southern portion of the U.S. Central Plains. Specific soils series used in the facility are Houston Black clay (32 monoliths), a Vertisol (Udic Haplustert) typical of lowlands; Austin (32 monoliths), a high carbonate, silty clay Mollisol (Udorthentic Haplustol) typical of uplands; and Bastsil (16 monoliths), an alluvial sandy loam Alfisol (Udic Paleustalf).
The operational principle employed in LYCOG is to harness the photosynthetic capacity of plants to deplete CA from parcels of air moved directionally through the enclosed chambers. The treatment objective is to maintain a constant linear daytime gradient in CA from 500 to 250 &#181;l L-1. To accomplish this, LYCOG consists of two linear chambers, a superambient chamber maintaining the portion of the gradient from 500 to 390 (ambient) &#181;l L-1 CA, and a subambient chamber maintaining the 390 to 250 &#181;l L-1 portion of the gradient. The two chambers are located side by side, oriented on a north-south axis. The CA gradient is maintained during the portion of the year when vegetation photosynthetic capacity is adequate; typically from late April to early November.
The chambers contain sensors and instrumentation needed to regulate the CA gradient, control air temperature (TA) near ambient values, and apply uniform precipitation amounts to all soils. Soils are intact monoliths collected from nearby Blackland prairie installed in hydrologically-isolated weighing lysimeters instrumented to determine all components of the water budget. Water is applied in events of volume and timing that approximate the seasonality of rain events and amounts during an average precipitation year. Thus, LYCOG is capable of evaluating the long-term effects of subambient to superambient CA and soil type on grassland ecosystem function including water and carbon budgets.
LYCOG is the third generation of CA gradient experiments conducted by USDA ARS Grassland Soil and Water Research Laboratory. The first generation was a prototype subambient to ambient gradient that established the viability of the gradient approach13 and advanced our understanding of leaf-level physiological responses of plants to subambient variation in CA14-20. The second generation was a field-scale application of the concept to perennial C4 grassland, with the gradient extended to 200 to 550 &#181;L L-1 21. This field-scale experiment provided the first evidence that grassland productivity increases with CA enrichment may saturate near current ambient concentrations20, in part because nitrogen availability may limit plant productivity at superambient CA22. LYCOG extends this second generation experiment by incorporating replicated soils of varying texture, allowing robust testing for interactive effects of soils on the CA response of grassland communities.

<table>
	<tbody>
		<tr>
			<td>Dataloggers, multiplexers</td>
			<td>Campell Scientific, Logan, UT, USA</td>
			<td>CR-7, CR-10, CR-21X, SDM-A04, SDM-CD16AC, AM25T</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouples: Copper-constantan</td>
			<td>Omega Engineering, Inc., Stamford, CT, USA</td>
			<td>TT-T-40-SLE, TT-T-24-SLE</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum sensor</td>
			<td>Li-Cor Biosciences, Lincoln, NE, USA</td>
			<td>LI-190SB</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2/H2O analyzer</td>
			<td>Li-Cor Biosciences, Lincoln, NE, USA</td>
			<td>LI-7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysimeter scales</td>
			<td>Avery Weigh-Tronix, Houston, TX, USA</td>
			<td>DSL-3636-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Air sampling pump</td>
			<td>Grace Air Components, Houston, TX, USA</td>
			<td>VP 0660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dew-point generator</td>
			<td>Li-Cor Biosciences, Lincoln, NE, USA</td>
			<td>LI-610</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold water chiller</td>
			<td>AEC Application Engineering, Wood Dale, IL, USA</td>
			<td>CCOA-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Chilled water flow control values</td>
			<td>Belimo Air Controls, Danbury, CT, USA</td>
			<td>LRB24-SR</td>
			<td></td>
		</tr>
		<tr>
			<td>Chilled-water cooling coils</td>
			<td>Coil Company, Paoli, PA, USA</td>
			<td>WC12-C14-329-SCA-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide refrigerated liquid</td>
			<td>Temple Welding Supply, Temple, TX, USA</td>
			<td>UN2187</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene film</td>
			<td>AT Plastics, Toronto, ON, Canada</td>
			<td>Dura-film Super Dura 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Blower motor/controller</td>
			<td>Dayton Electric, Lake Forest, IL, USA</td>
			<td>2M168C/4Z829</td>
			<td></td>
		</tr>
		<tr>
			<td>Solenoids</td>
			<td>Industrial Automation, Cornelius, NC, USA</td>
			<td>U8256B046V-12/DC</td>
			<td></td>
		</tr>
		<tr>
			<td>Leachate collection pump</td>
			<td>Gast Manufacturing, Benton Harbor, MI, USA</td>
			<td>0523-V191Q-G588DX</td>
			<td></td>
		</tr>
	</tbody>
</table>

a co2 concentration gradient facility for testing co2 enrichment and soil effects on grassland ecosystem function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

The superambient and subambient portions of the gradient are maintained in separate chambers (Figure 1). However, over seven years of operation (2007 &#8211; 2013), the chambers maintained a linear gradient in CA concentration from 500 to 250 &#181;l L-1 (Figure 2) with only a small discontinuity in CA between the exit of the enriched chambers (Monolith 40) and the entrance of the subambient portion of the gradient (Monolith 41).
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Watch this Scientific Journal Video about A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function at JoVE.com

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

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11.3K Views.  Grassland Soil and Water Research Laboratory. The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 µl L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.