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>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Working+Group+I+to+the+Fifth+Assessment+Report+of+the+Intergovernmental+Panel+on+Climate+Change.">Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change.</a> Climate Change 2013: The Physical Science Basis. , 1535 (2013).</li><li>Gerhart, L. M., Ward, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+responses+to+low+CO2+of+the+past.">Plant responses to low CO2 of the past.</a> New Phytol. 188 (3), 674-695 (2010).</li><li>Kimball, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+comparisons+among+free-air+CO2+enrichment,+open-top+chamber,+and+sunlit+controlled-environment+chamber+methods+of+CO2+exposure.">Cost comparisons among free-air CO2 enrichment, open-top chamber, and sunlit controlled-environment chamber methods of CO2 exposure.</a> Crit. Rev. Plant Sci. 11 (2-3), 265-270 (1992).</li><li>Hendrey, G. R., Lewin, K. F., Nagy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+Air+Carbon+Dioxide+Enrichment:+DevelopmentProgress,+Results.">Free Air Carbon Dioxide Enrichment: DevelopmentProgress, Results.</a> Vegetatio. 104/105 (1), 16-31 (1993).</li><li>Weng, E., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+hydrological+properties+regulate+grassland+ecosystem+responses+to+multifactor+global+change:+A+modeling+analysis.">Soil hydrological properties regulate grassland ecosystem responses to multifactor global change: A modeling analysis.</a> J. Geophys. Res. 113 (G3), G03003 (2008).</li><li>Brady, N. C., Weil, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nature and Properties of Soils. , 960 (2002).</li><li>Jenkinson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+decomposition+of+plant+material+in+soil.+V.+The+effects+of+plant+cover+and+soil+type+opn+the+logg+of+carbon+from+14C+labelled+ryegrass+decomposing+under+field+conditions.">Studies on the decomposition of plant material in soil. V. The effects of plant cover and soil type opn the logg of carbon from 14C labelled ryegrass decomposing under field conditions.</a> J. Soil Sci. 28 (3), 424-434 (1977).</li><li>Hassink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+plant+residues+in+soils+differing+in+unsaturated+protective+capacity.">Preservation of plant residues in soils differing in unsaturated protective capacity.</a> Soil Sci. Soc. Am. J. 60 (2), 487-491 (1996).</li><li>Oades, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retention+of+organic+matter+in+soils.">The retention of organic matter in soils.</a> Biogeochemistry. 5 (1), 35-70 (1988).</li><li>Knapp, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+more+extreme+precipitation+regimes+for+terrestrial+ecosystems.">Consequences of more extreme precipitation regimes for terrestrial ecosystems.</a> BioScience. 58 (9), 811-821 (2008).</li><li>Ainsworth, E. A., Long, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+have+we+learned+from+15+years+of+free-air+CO2+enrichment+(FACE)?+A+meta-analytic+review+of+the+responses+of+photosynthesis,+canopy+properties+and+plant+production+to+rising+CO2.">What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2.</a> New Phytol. 165 (2), 351-372 (2005).</li><li>Rogers, A., Ainsworth, E. A., Kammann, C. F. A. C. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+dioxide+and+water+fluxes+of+C3+annuals+and+C4+perennials+at+subambient+CO2+concentrations.">Carbon dioxide and water fluxes of C3 annuals and C4 perennials at subambient CO2 concentrations.</a> Funct Ecol. 6 (6), 693-703 (1992).</li><li>Polley, H. W., Johnson, H. B., Mayeux, H. S., Malone, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+growth+of+wheat+across+a+subambient+carbon+dioxide+gradient.">Physiology and growth of wheat across a subambient carbon dioxide gradient.</a> Ann. Bot. 71 (4), 347-356 (1993).</li><li>Polley, H. W., Johnson, H. B., Marino, B. D., Mayeux, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+C3+plant+water-use+efficiency+and+biomass+over+glacial+to+present+CO2+concentrations.">Increase in C3 plant water-use efficiency and biomass over glacial to present CO2 concentrations.</a> Nature. 361 (6407), 61-64 (1993).</li><li>Polley, H. W., Johnson, H. B., Mayeux, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+CO2:+comparative+responses+of+the+C4+grass+Schizachyrium.+and+grassland+invader+Prosopis.">Increasing CO2: comparative responses of the C4 grass Schizachyrium. and grassland invader Prosopis.</a> Ecology. 75 (4), 976-988 (1994).</li><li>Polley, H. W., Johnson, H. B., Mayeux, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+and+water+requirements+of+C3+plants+grown+at+glacial+to+present+carbon+dioxide+concentrations.">Nitrogen and water requirements of C3 plants grown at glacial to present carbon dioxide concentrations.</a> Funct. Ecol. 9 (1), 86-96 (1995).</li><li>Polley, H. W., Johnson, H. B., Mayeux, H. S., Brown, D. A., White, J. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+and+plant+water+use+efficiency+of+C4+species+grown+at+glacial+to+elevated+CO2+concentrations.">Leaf and plant water use efficiency of C4 species grown at glacial to elevated CO2 concentrations.</a> Int. J. Plant Sci. 157 (2), 164-170 (2012).</li><li>Polley, H. W., Johnson, H. B., Derner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+CO2+from+subambient+to+superambient+concentrations+alters+species+composition+and+increases+above-ground+biomass+in+a+C3/C4+grassland.">Increasing CO2 from subambient to superambient concentrations alters species composition and increases above-ground biomass in a C3/C4 grassland.</a> New Phytol. 160 (2), 319-327 (2003).</li><li>Johnson, H. B., Polley, H. W., Whitis, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongated+chambers+for+field+studies+across+atmospheric+CO2+gradients.">Elongated chambers for field studies across atmospheric CO2 gradients.</a> Funct. Ecol. 14 (3), 388-396 (2000).</li><li>Gill, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+grassland+responses+to+past+and+future+atmospheric+CO2.">Nonlinear grassland responses to past and future atmospheric CO2.</a> Nature. 417 (6886), 279-282 (2002).</li><li>Fay, P. A., Carlisle, J. D., Knapp, A. K., Blair, J. M., Collins, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Productivity+responses+to+altered+rainfall+patterns+in+a+C4-dominated+grassland.">Productivity responses to altered rainfall patterns in a C4-dominated grassland.</a> Oecologia. 137 (2), 245-251 (2003).</li><li>Miglietta, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+performance+of+the+miniface+(free+air+CO2+enrichment)+system+on+bog+ecosystems+in+northern+and+central+Europe.">Spatial and temporal performance of the miniface (free air CO2 enrichment) system on bog ecosystems in northern and central Europe.</a> Environmental Monitoring and Assessment. 66 (2), 107-127 (2001).</li></ol>

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).
<p class="jove_conten...

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 ...

a-co2-concentration-gradient-facility-for-testing-co2-enrichment-soil

Research

JoVE Journal

Environment

11.4K 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.

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

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The system comprises a high-pressure rheometer which is connected to a circulation loop. The rheometer has a rotational flow-through measurement cell with two alternative geometries: coaxial cylinder and double gap. The circulation loop contains a mixer, to bring the crude oil sample into equilibrium with CO2, and a gear pump that transports the mixture from the mixer to the rheometer and recycles it back to the mixer. The CO2 and crude oil are brought to equilibrium by stirring and circulation and the rheology of the saturated mixture is measured by the rheometer. The system is used to measure the rheological properties of Zuata crude oil (and its toluene dilution) in equilibrium with CO2 at elevated pressures up to 220 bar and a temperature of 50 &#176;C. The results show that CO2 addition changes the oil rheology significantly, initially reducing the viscosity as the CO2 pressure is increased and then increasing the viscosity above a threshold pressure. The non-Newtonian response of the crude is also seen to change with the addition of CO2.

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. Rather than discharging treated wastewater to a stream, and thereby directly impacting the stream quality, the effluent is applied to forested and cropped land managed by the University. Concerns related to reductions in soil hydraulic conductivity occur when considering wastewater reuse. The methodology described in this manuscript, matching soil sample size with the size of the laboratory-based hydraulic conductivity measurement apparatus, provides the benefits of a relatively rapid collection of samples with the benefits of controlled laboratory boundary conditions. The results suggest that there may have been some impact of wastewater reuse on the soil&#39;s ability to transmit water at deeper depths in the depressional areas of the site. Most of the reductions in the soil hydraulic conductivity in the depressions appear to be related to the depth from which the sample was collected, and by inference, associated with the soil structural and textural differences.

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of different gall types and prevents the development of normal shoots. Repeated and uncontrolled attacks cause, besides the production of galls and the attendant gall-related reduction in leaf area, progressive corruption of the branch architecture, including the death of branch parts, and an increase in dormant bud activation. Thus far, there have been few attempts to quantify branch architecture damage. Further, the different methods for assessing infestation degree (MAID) that have been developed focus only on the galls' presence and abundance.
Using the leaf area to sapwood area relationship as a green biomass indicator, we developed in a previous study a damage composite index (DCI) that takes into account the most important branch architectural features, allowing for realistic damage assessment during the entire epidemic process. 
The aim of this study is to present this novel method and highlight differences in the damage description with respect to other broadly used indices. Results show how the DCI depicts branch damage better, especially during the epidemic peak, compared to MAID, which tend to underestimate it. We conclude by suggesting how to properly evaluate the overall impact of the pest by means of our composite damage index, the infestation degree using classic methods, and crown transparency evaluations.

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

It is common practice to assess the damage caused by Dryocosmus kuriphilus by considering the abundance of galls alone rather than by also taking related branch corruption into consideration. We propose a composite damage index that takes into account the most important branch features, thus enabling more realistic damage assessment.

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of ...

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and increasing the recovery of coalbed methane. A visualized and constant-volume gas-solid coupling system is introduced here to investigate the influence of CO2 sorption on the physical and mechanical properties of coal. Being able to keep a constant volume and monitor the sample using a camera, this system offers the potential to improve instrument accuracy and analyze fracture evolution with a fractal geometry method. This paper provides all steps to perform a uniaxial compression experiment with a briquette sample in different CO2 pressures with the gas-solid coupling test system. A briquette, cold-pressed by raw coal and sodium humate cement, is loaded in high-pressure CO2, and its surface is monitored in real-time using a camera. However, the similarity between the briquette and the raw coal still needs improvement, and a flammable gas such as methane (CH4) cannot be injected for the test. The results show that CO2 sorption leads to peak strength and elastic modulus reduction of the briquette, and the fracture evolution of the briquette in a failure state indicates fractal characteristics. The strength, elastic modulus, and fractal dimension are all correlated with CO2 pressure but not with a linear correlation. The visualized and constant-volume gas-solid coupling test system can serve as a platform for experimental research about rock mechanics considering the multifield coupling effect.

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal&#8217;s physical and mechanical properties induced by CO2 adsorption.

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and ...