Name: a co2 concentration gradient facility for testing co2 enrichment and soil effects on grassland ecosystem function
Uploaded: 2015-11-21T13:00:00.000+00:00
Duration: 10 min 19 s
Description: 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

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>

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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 of the gradient. Temperature and CA control are easily maintained during the spring and early summer when soil moisture is relatively high and plants are at their highest photosynthetic capacity.
Critical Steps in the Protocol
Control of blower speed is the most critical aspect of maintaining the prescribed CO2 gradient. Control is based on a combination of feedback and feed-forward techniques to match air flow to the vegetation carbon uptake. The feedback technique adjusts fan speed based on the difference between measured and target exit CO2 concentration. Feed-forward control anticipates changes in photosynthetic rate and rapidly (5 sec response time) adjusts fan speed, based on changes in photosynthetically active radiation measured with the quantum sensor. Feed-forward control considerably improves control over that achieved by feedback control alone. The maximum airflow rate through the chambers is on the order of 1 m sec-1, or about 3.6 km hr-1, which is on the low-end of wind speeds these plants see in the field. Thus, varying fan speed is unlikely to affect plant responses.
Another critical aspect of maintaining the CO2 gradient is the presence of adequate photosynthetic capacity. The steeper the gradient, the greater the canopy photosynthetic capacity required to draw down the CO2 concentration. Species or communities with more leaf area, higher photosynthetic rates, or longer chamber length all increase the CO2 draw-down that can be achieved. Care should also be taken the monolith volume and depth is chosen to provide a realistic rooting volume for the established plant communities. The species used here have rooting depths of 1 &#8211; 1.5 m, but other species may be shallower or deeper, and monolith volume should be adjusted accordingly. The final critical aspect is the importance of reliably supplying and controlling the flow of chilled water to the cooling coils between each section, in order to match chamber temperatures to outside diurnal and seasonal variation in outside ambient temperature.
Modifications to the Technique
The first year of operation revealed that the prairie vegetation was marginally capable of adequate CO2 drawdown. This was remedied by converting a total of 20 monoliths chosen from the Houston and Austin soil series to monocultures of switchgrass, Panicum virgatum. Switchgrass is a highly productive native tallgrass, and is well-watered throughout the growing season, which insures adequate C uptake capacity along the gradient even during the hot summer months. The first year also revealed greater than anticipated aerodynamic resistance in the chambers, which degraded flow rates in downstream chambers, leading to overheating. This issue was remedied by installation of additional downstream blower fans to boost the flow rates. We recommend installing new polyethylene covers each growing season to maintain maximum light transmittance.
Limitations of the Technique
The system poses certain operational issues that create both opportunities and limitations on the research questions the facility can support. Control of the gradient becomes more difficult from mid-summer through the end of the growing season, because higher summer temperatures lower soil moisture, increasing plant water stress and lowering photosynthetic capacity. This in turn requires slower air flow rates to achieve the CA draw-down needed to meet the target CA concentrations, which in turn further raises temperatures. This dynamic illustrates the limited ability of this system for studies of drought interactions with CO2 concentration. Temperature increases within each 5 meter section are unavoidable because of the linear flow design of the experiment. Long-wave energy accumulates within each chamber until air passes through the cooling coil and entering the next chamber. Within-section temperature increases are of similar magnitude to some of the higher estimates for future temperature increases expected with some climate change scenarios. Thus, the within-section temperature variation represents an opportunity to analyze grassland responses to interactions between CA and warming. Finally, the dimensions of the chamber limit the vegetation to a maximum height of about one meter, and the monolith area limits the vegetation to herbaceous species with smaller basal areas. The use of tree species, for example to study woody encroachment into grassland, would be impractical beyond the seedling stage.
Significance compared to Other Techniques
LYCOG is considerably more economical to operate compared to techniques such as FACE and OTC. LYCOG uses approximately 3,700 L per month of CO2, which is greater than the CO2 use in MiniFACE systems24, but much less than the CO2 consumption of FACE and OTC approaches3, 12. The major expense of maintaining the experiment comes from temperature control, which costs approximately $30,000 per year, comparable to estimates of the CO2 expense for open top chamber CA enrichment costs but still much less than that of the CO2 expense of Free Air CO2 Enrichment systems3. The economic advantages come in addition to the unique capability of supporting studies at subambient CO2 and along a continuous CO2 gradient.
Current and Future Applications 
Current research is examining ecosystem responses other than ANPP, including soil CO2 efflux, and evapotranspiration, which will expand our understanding of soil-specific variation in CA effects on grassland carbon and water cycling. Future possibilities for research include combining temperature and CO2 treatments, for example by operating both chambers as superambient but maintaining one chamber at a warmer temperature differential with respect to ambient. Current vegetation can easily be replaced with other species or communities to study how variation in community structure influences CO2 effects on ecosystem function. Other ecologically important atmospheric constituents such as methane or ozone could be added to test for interactions with CO2.

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).
Air temperature and vapor pressure deficit remained constant from section to section in both the superambient and subambient chambers, except in section 10 of the superambient chamber, and sections 19 and 20 of the subambient chamber, where air temperature averaged ~ 3 &#176;C warmer than other sections (Figure 2). However there can be pronounced temperature increases of 5 &#8211; 7 &#176;C within each section, and corresponding increases in vapor pressure deficit.
Averaged over the 2007 &#8211; 2013 growing seasons, vSWC varied linearly along the CA gradient on two of the three soils (Figure 3). vSWC in the top 20 cm of the soil profile increased by 3.1% per 100 &#181;l L-1 increase in CA on the sandy loam (Bastsil series) soil (R2 = 0.34, p = 0.01), and by 1.7% per 100 &#181;l L-1 CA on the clay soil (Houston series). However there was no change in 0-20 vSWC in the silty clay (Austin series) soil (p = 0.13).
Plant productivity also varied linearly with by CA, and the magnitude of the CA response differed among soils. ANPP (Figure 4A) of monoliths with Blackland prairie plant communities had the smallest response to CA on the clay soil, increasing by 59 g m-2 per 100 &#181;l L-1 increase in CA (R2 = 0.22, p = 0.02). The ANPP response to CA enrichment was intermediate on the silty clay soil, increasing by 76 g m-2 per 100 &#181;l L-1 of CO2 (R2 = 0.22, p = 0.02), and greatest on the sandy loam soil, where ANPP gained 131 g m-2 per 100 &#181;l L-1 of CO2 (R2 = 0.55, p &#60; 0.001).
These soil-specific responses of ANPP to CA corresponded closely to the soil-specific responses of a mesic C4 tallgrass, Sorghastrum nutans, the most abundant grass species in the experimental plant communities. Aboveground biomass of S. nutans increased most strongly with increased CA on the sandy loam soil, gaining over 200 g m-2 for every 100 &#181;l L-1 increase in CA (R2 = 0.40, p = 0.005). In contrast, S. nutans gained only 100 g m-1 per 100 &#181;l L-1 increase in CA on the silty clay soil (R2 = 0.50, p &#60; 0.0001), while S. nutans responded marginally to CA on the clay soil (R2 = 0.12, p = 0.07; Figure 4B).
The soil-specific increase in ANPP with CA enrichment occurred despite decreases in productivity on the two more responsive soils by the xeric C4 mid-grass Bouteloua curtipendula (Figure 4C). B. curtipendula was the second most abundant species in the experimental communities. On the silty-clay soil, B. curtipendula was the dominant grass at subambient CA concentrations but decreased most strongly with CA enrichment on the silty clay soil (69 g m-2, per 100 &#181;l L-1 increase in CA; R2 = 0.36, p &#60; 0.008), decreased less strongly on the sandy loam soil (44 g m-2, per 100 &#181;l L-1 increase in CA; R2 = 0.36, p = 0.008), and did not vary with CA enrichment on the clay soil (p = 0.46).
<img alt="Figure 1" src="/files/ftp_upload/53151/53151fig1.jpg" /> 
Figure 1.&#160;Arrangement of chambers and soils. The two linear sequences of chambers containing grassland vegetation growing on intact soil monoliths (photo), and schematic of the distribution of the three soil types along the CO2 gradient. Plot numbers 1 - 40 are located along the 500 &#8211; 380 &#181;l L-1 portion of the gradient, and numbers 41 - 80 on the 380 &#8211; 250 &#181;l L-1 portion. Photo: Philip Fay.
<img alt="Figure 2" src="/files/ftp_upload/53151/53151fig2.jpg" /> 
Figure 2.&#160;Microclimate along the CA gradient. Daytime growing season carbon dioxide (CO2) concentration, air temperature, and vapor pressure deficit for the 80 monoliths in the enriched and subambient chambers. Values are measured at the air entry and exit of each section, and estimated from linear interpolation for other positions. Data points represent means for the 2007 through 2013 growing seasons. Error bars omitted for clarity; mean standard errors were 3.5 for CO2, 0.82 for air temperature, and 0.18 for vapor pressure deficit.
<img alt="Figure 3" src="/files/ftp_upload/53151/53151fig3.jpg" /> 
Figure 3.&#160;Soil moisture on each soil type along the CO2 gradient. Growing season volumetric soil water contents (vSWC) for 0 &#8211; 20 cm in the soil profile for each soil type, plotted by position along the CO2 concentration gradient. Linear regressions are plotted for soils with significant relationships of vSWC to CO2 concentration. Data points represent means of 2007 through 2013 growing seasons. Error bars omitted for clarity; mean standard errors on the three soils ranged from 0.74 to 0.99.
<img alt="Figure 4" src="/files/ftp_upload/53151/53151fig4.jpg" /> 
Figure 4.&#160;Plant productivity on each soil type along the CO2 gradient. (A) Mean aboveground net primary productivity (ANPP), the sum of the current-year biomass of all species in the 60 monoliths with Blackland Prairie plant communities; and the current year biomass of (B) the mesic C4 tallgrass, Sorghastrum nutans, and (C) the xeric C4 midgrass Bouteloua curtipendula plotted by position along the CO2 concentration gradient. Linear regressions are plotted for soils with significant relationships of ANPP or species biomass to CO2 concentration. Data points represent means of 2007 through 2013 growing seasons. Error bars omitted for clarity; mean standard errors on the three soils ranged from 34.9 to 42.5 for ANPP, 21.8 to 34.4 for S. nutans, and 7.4 - 24.8 for B. curtipendula.

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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Nanyang Technological University

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11.4K Views.  Grassland Soil and Water Research Laboratory.  De Lysimeteronderzoek Carbon Dioxide Gradient Facility creëert een 250-500 pi L -1 lineaire kooldioxide gradiënt in temperatuur-gecontroleerde kamers huisvesting graslanden plantengemeenschappen op klei, lemige klei en zandgrond monolieten. De faciliteit wordt gebruikt om te bepalen hoe het verleden en de toekomst van kooldioxide niveaus invloed grasland koolstofcyclus. 

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

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer&#39;s particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays,&#160;but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

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 ...

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

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 ...

Measurements of CO2 Fluxes at Non-Ideal Eddy Covariance Sites

This protocol is an example of utilizing the eddy covariance (EC) technique to investigate spatially and temporally averaged net CO2 fluxes (net ecosystem production, NEP), in non-typical ecosystems, on a currently reforested windthrow area in Poland. After a tornado event, a relatively narrow &#8220;corridor&#8221; was created within surviving forest stands, which complicates such kind of experiments. The application of other measuring techniques, such as the chamber method, is even more difficult under these circumstances, because especially at the beginning, fallen trees and in general great heterogeneity of the site provide a challenging platform to perform flux measurements and then to properly upscale obtained results. In comparison with standard EC measurements carried out in untouched forests, the case of windthrow areas requires special consideration when it comes to the site location and data analysis in order to ensure their representativeness. Therefore, here we present a protocol of real-time, continuous CO2 flux measurements at a dynamically changing, non-ideal EC site, which includes (1) site location and instrumentation setup, (2) flux computation, (3) rigorous data filtering and quality control, and (4) gap filling and net fluxes partitioning into CO2 respiration and absorption. The main advantage of the described methodology is that it provides a detailed description of the experimental setup and measurement performance from scratch, which can be applied to other spatially limited ecosystems. It can also be viewed as a list of recommendations on how to deal with unconventional site operation, providing a description for non-specialists. Obtained quality-checked, gap filled, half-hour values of net CO2, as well as absorption and respiration fluxes, can be finally aggregated into daily, monthly, seasonal or annual totals.

Measurements of CO2 Fluxes at Non-Ideal Eddy Covariance Sites

The presented protocol uses the eddy covariance method at non-typical locations, applicable to all types of short-canopy ecosystems with limited area, on a currently reforested windthrow site in Poland. Details of measuring site setup rules, flux calculations and quality control, and final result analysis, are described.

This protocol is an example of utilizing the eddy covariance (EC) technique to investigate spatially and temporally averaged net CO2 fluxes (net ecosystem ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Monitoring Pedogenic Inorganic Carbon Accumulation Due to Weathering of Amended Silicate Minerals in Agricultural Soils.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in agricultural soils. To this end, the core soil samples taken at different depth (including 0-15 cm, 15-30 cm, and 30-60 cm profiles) are collected from an agriculture field, the topsoil of which has already been enriched with an alkaline earth metal silicate containing mineral (such as wollastonite). After transporting to the laboratory, the soil samples are air-dried and sieved. Then, the inorganic carbon content of the samples is determined by a volumetric method called calcimetry. The representative results presented herein showed five folded increments of inorganic carbon content in the soils amended with the Ca-silicate compared to control soils. This compositional change was accompanied by more than 1 unit of pH increase in the amended soils, implying high dissolution of the silicate. Mineralogical and morphological analyses, as well as elemental composition, further corroborate the increase in the inorganic carbon content of silicate-amended soils. The sampling and analysis methods presented in this study can be adopted by researchers and professionals looking to trace pedogenic inorganic carbon changes in soils and subsoils, including those amended with other suitable silicate rocks such as basalt and olivine. These methods can also be exploited as tools for verifying soil inorganic carbon sequestration by private and governmental entities to certify and award carbon credits.

The verification method described here is adaptable for monitoring pedogenic inorganic carbon sequestration in various agricultural soils amended with alkaline earth metal silicate-containing rocks, such as wollastonite, basalt, and olivine. This type of validation is essential for carbon credit programs, which can benefit farmers that sequester carbon in their fields.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in ...

Modeling Methanotroph Niches – The Gradient Syringe Model Ecosystem

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists' understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs' natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.

Author Spotlight: Designing Simple and Inexpensive Techniques to Grow Methane-Oxidizing Bacteria in the Laboratory

A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

JenaTron - An Experimental Approach to Study the Effects of Plant History and Soil History on Grassland Ecosystem Functioning

The global loss of biodiversity has motivated many studies that experimentally vary plant species richness and examine the consequences for ecosystem functioning. Such experiments generally show a positive relationship between above- and below-ground biodiversity and the functioning of terrestrial ecosystems. Moreover, this relationship tends to strengthen over time, seen as enhanced functioning of diverse plant communities and reduced functioning of low-diversity plant communities. Differences in multitrophic community assembly and biotic interactions in high- versus low-diversity plant communities are hypothesized to affect plant performance by altering consumer community structure and function and driving plastic or micro-evolutionary responses of plant species in the plant communities. To resolve this complex interplay of community history, we separated these effects into plant and soil history. Plant history refers to all plant-level responses to past abiotic and biotic selection pressures experienced in their communities, while soil history relates to all abiotic and biotic soil properties developed as a legacy of plant-soil interactions under variable plant diversity. We set up a biodiversity experiment in an Ecotron, a terrestrial mesocosm facility that allows controlling environmental conditions above- and below-ground, to test whether the strengthening biodiversity-ecosystem functioning relationship is due to soil history, plant history, or a combination of both. We established a plant diversity gradient consisting of 1, 2, 3, and 6 grassland plant species and factorially nested with soil history and plant history treatments for each level of plant species richness. Representative results demonstrate the successful establishment of target treatments in the Ecotron experiment, observing the effects of plant and soil history on initial plant development and final plant growth. Additionally, we provide a case study for data analysis of individual response variables. We outline research objectives and methods to comprehensively assess the multifunctional responses to the experimental treatments necessary to ultimately address the overarching hypothesis.

Here we present a study established in the iDiv Ecotron, terrestrial mesocosm chambers with controlled environmental conditions above- and below-ground, to study the independent and interactive effects of plant history and soil history on ecosystem functioning of grassland communities along a plant diversity gradient.

The global loss of biodiversity has motivated many studies that experimentally vary plant species richness and examine the consequences for ecosystem ...

Enhancing CO2 Sequestration: A Soil Biota Approach to Boost Mineral Weathering

Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies on accelerating the natural process of mineral weathering in soils by manipulating the abiotic variables that govern this process, in particular mineral grain size and exposure to acids dissolved in water. EW mainly aims at reducing atmospheric CO2 concentrations by enhancing inorganic carbon sequestration. Until now, knowledge of EW has been mainly gained through experiments that focused on the abiotic variables known for stimulating mineral weathering, thereby neglecting the potential influence of biotic components. While bacteria, fungi, and earthworms are known to increase mineral weathering rates, the use of soil organisms in the context of EW remains underexplored. 
 This protocol describes the design and construction of an experimental setup developed to enhance mineral weathering rates through soil organisms while concurrently controlling abiotic conditions. The setup is designed to maximize weathering rates while maintaining soil organisms' activity. It consists of a large number of columns filled with rock powder and organic material, located in a climate chamber and with water applied via a downflow irrigation system. Columns are placed above a fridge containing jerrycans to collect the leachate. Representative results demonstrate that this setup is suitable to ensure the activity of soil organisms and quantify their effect on inorganic carbon sequestration. Challenges remain in minimizing leachate losses, ensuring homogeneous ventilation through the climate chamber, and avoiding flooding of the columns. With this setup, an innovative and promising approach is proposed to enhance mineral weathering rates through the activity of soil biota and disentangle the effect of biotic and abiotic factors as drivers of EW.

Author Spotlight: Unraveling the Role of Earthworms in Enhancing Mineral Weathering for CO2 Removal 

Here we present the construction and operation of an experimental setup to enhance mineral weathering through the activity of soil organisms while concurrently manipulating abiotic variables known to stimulate weathering. Representative results from the functioning of the setup and sample analyses are discussed together with points for improvement.

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies ...

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

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