This material is based upon work supported by the National Science Foundation under Grant Number 1215858, by the US Department of Agriculture under Grant Number 2013-68002-20525, and by the US Department of Energy Great Lakes Bioenergy Research Center &#8211; DOE BER Office of Science (DE-FC02-07ER64494) and DOE OBP Office of Energy Efficiency and Renewable Energy (DE-AC05-76RL01830). In-field video and images were recorded at the Wisconsin Integrated Cropping System Trial project of the University of Wisconsin&#8211;Madison. The authors are grateful to Ryan Curtin for skillful videography and editing.

1. Chamber Construction and Anchor Installation
<ol>
	<li>Design and construct chambers &#8211; each consisting of an anchor that is inserted into the soil and a lid that is placed on top of the anchor during flux measurement &#8211; to meet experimental needs.
	<ol>
		<li>In designing chamber shape and size, consider spatial factors such as crop row spacing, fertilizer or manure banding, and plant height. Because protrusion of anchors above the soil surface can contribute to microclimate effects and water ponding, consider having the lids sit as low to the soil surface as possible. Because tradeoffs exist between chamber height and detection sensitivity, design lids to be as short as is feasible for the system under study.</li>
		<li>Build chambers of sturdy, nonreactive material such as stainless steel or PVC, and include a mechanism for sealing the lid onto the anchor. Insulate lids and cover with light-colored or reflective material to prevent heat buildup during measurement. Include a septum to allow sample collection and a vent tube to prevent pressure perturbations during chamber deployment and sample removal. For additional details refer to the Materials table, Parkin and Venterea8, and Clough et al10.</li>
	</ol>
	</li>
	<li>At least 1 day prior to sampling, install chamber anchors in the soil at desired sites. The installation method will depend on chamber design, but in general, apply even pressure across all points so that the anchor does not warp or distort the soil structure. Sink the anchor to a depth of 2.5-13 cm depending on soil type, deployment time, and chamber volume6,11. Leave as little as possible (no more than 5 cm) protruding above the soil surface.</li>
</ol>
2. Calibration and Experimental Design
Note: Prior to beginning the experiment, follow these steps to determine an appropriate sampling time course that will allow data to be fit to an appropriate linear or non-linear flux model (see Parkin et al.12). This will require the use of techniques described in steps 3-5 (Field Sampling, Sample Analysis, and Data Analysis). Optimal timing is a function of both the system under study and the dimensions of chambers being used. Some trial and error may be involved. See Venterea13 for alternate approaches.
<ol>
	<li>Calibration Sampling and Analysis
	<ol>
		<li>Under environmental or management conditions expected to generate relatively high trace gas fluxes, conduct intensive sampling following techniques described in section 3. Using tightly spaced sampling time points, populate a time series of longer duration than would be considered typical. Begin by sampling from several representative chambers at 5-10 evenly spaced time points over the course of an hour.</li>
		<li>Analyze samples by gas chromatography following section 4.</li>
	</ol>
	</li>
	<li>Calibration Interpretation
	<ol>
		<li>For each calibration time series and each gas of interest, plot time-by-concentration.</li>
		<li>Verify that flux rates are on the high end of the expected range. See section 5 for flux calculation. Refer to section 2.3 for troubleshooting tips.</li>
		<li>Inspect graphs for signs of non-linearity, or more specifically, plateauing of gas concentrations over time. 
		Note: The point at which concentration begins to plateau differs by gas type, and is a function of the rate of gas production or consumption within the soil, the concentration of the gas in the chamber headspace, and diffusion between the two zones. It is therefore strongly affected by chamber height, with shorter chambers yielding shorter time before plateau.</li>
		<li>Use the calibration sets to determine optimal chamber deployment time for the experimental system. If linear regression will be used in data analysis (as described here in section 5), select timing that maintains as close to a linear relationship as possible between time and concentration for all gases / systems of interest, while allowing for a minimum of three, preferably four, sampling times within the time series6. For chambers 10-30 cm high used for CO2 and N2O measurements, time series typically range from 20-60 min8,14.</li>
	</ol>
	</li>
	<li>Calibration Troubleshooting
	<ol>
		<li>If there is poor differentiation and/or difficulty discerning linearity or plateau, use tighter calibration time points or longer calibration time series, and check that concentrations are within detection limits. For low rates of flux, a reduction in rate of accumulation may not be observed within the tested timeframe. This should not cause concern.</li>
		<li>If the fluxes are not at the high end of the expected experimental range, repeat calibration, altering treatment or environmental conditions to induce higher flux (by applying fertilizer or irrigation, for example). Alternately, use at least four time points in experimental design, so that if experimental fluxes are significantly higher than those observed during calibration and plateauing does occur, later time points can be excluded while retaining at least three time points for linear regression. Curvilinear regression approaches may also be employed.</li>
	</ol>
	</li>
	<li>Experimental Design
	<ol>
		<li>Based on the optimal timing determined in section 2.2.4, devise an overall sampling scheme that captures all relevant sites, treatments, and/or replicates, and allows personnel to move through chamber sites efficiently. If necessary, divide the chamber sites into several &#8220;rounds&#8221; to be sampled one after the other.
		<ol>
			<li>If measurements are to be taken as representative of a whole day, sample at a time of day when temperatures are moderate relative to daily extremes. In typical temperate cropping systems, the ideal window is mid- to late-morning.</li>
			<li>If samples are to be collected in consecutive rounds, be careful not to introduce a bias by repeatedly sampling the same treatments at the same time of day. Construct rounds out of blocks of replicates rather than treatment-by-treatment.</li>
			<li>Include time for any necessary ancillary measures to be taken either within rounds or before/after, as appropriate. (See section 3.3 for typical ancillary measures.)</li>
			<li>Optionally, include time for collection of ambient air samples for use in non-linear flux models, or as an approximation of starting (time zero, &#8220;T0&#8221;) concentration (not described here).</li>
			<li>Optionally, include time for loading reference gas into vials at the time of sampling to assess possible sample degradation between sampling and analysis. See Parkin and Venterea8 for sample storage considerations.</li>
		</ol>
		</li>
		<li>Determine the frequency of flux measurements that is appropriate for research goals. This may range from a single measurement to daily, weekly, or periodic measurements over the course of months or years. Refer to Rochette et al.14 for a thorough discussion of experimental design considerations.</li>
		<li>If samples are to be collected in cold conditions, plan for inclusion of a warming device such as a hot pack with vials to prevent septa from becoming brittle.</li>
	</ol>
	</li>
</ol>
3. Field Sampling
Note: On each sampling date, follow the sampling scheme established in section 2.4, using the techniques described below. Equipment and sample volume can vary depending on the collection and transfer methods being employed and the amount of sample required for GC analysis8. This protocol utilizes 5.9 ml collection vials and 30 ml syringes, with a flushing method of sample transfer. See Discussion for alternate approaches.
<ol>
	<li>Preparation
	<ol>
		<li>If sampling from multiple chambers per round, prepare a time point reference grid (see Figure 2) to easily track where and when to sample. Alternately, make arrangements to record each time point during sampling.</li>
		<li>Pre-label and arrange collection vials for maximal efficiency and minimal likelihood of confusion during sampling.</li>
		<li>In order to save time during sampling, prepare all materials and equipment beforehand. Include extras of anything that may break or is easily lost (needles, syringes, stopcocks, etc.), and place in a carry tote, bucket, or other container.</li>
		<li>Be prepared to record any delayed time points which can happen due to equipment malfunction or other unforeseen circumstances, and which can be easily corrected during data analysis by adjusting the time associated with a certain sample.</li>
	</ol>
	</li>
	<li>Sample Collection
	<ol>
		<li>Attach and seal the chamber lid to the pre-installed chamber anchor, and start a stopwatch. This is T0.</li>
		<li>Immediately after sealing the lid collect a sample of ambient air from a location adjacent to the chamber, at the approximate height of the chamber top: with an empty 30 ml syringe fitted with a needle and a stopcock in the open position, draw a 30 ml air sample and close the stopcock. This is the T0 sample. Alternatively, take the T0 sample from the chamber6. 
		Note: Tradeoffs exist between the two approaches &#8211; evaluate spatial (distance from site or external microclimate for outside samples) vs. timing (delay between lid closure and sample collection for inside samples) considerations and determine the most appropriate technique for the equipment being used and the system under study.</li>
		<li>With the syringe needle, pierce the septum of a 5.9 ml collection vial that already has another needle poked through near the edge of the septum.</li>
		<li>Open the syringe stopcock and inject approximately 20 ml of the sample into the vial (this causes the previous contents of the vial to be expelled through the extra needle, replaced by sample).</li>
		<li>In a smooth motion, remove the extra needle while continuing to inject as much of the remaining sample (approximately 10 ml) as possible, slightly over-pressurizing the vial to ensure sample integrity and allow analysis of multiple samples if necessary8.</li>
		<li>Close the stopcock and withdraw the syringe needle from the septum. Turn the filled vial upside-down to distinguish from unfilled vials.</li>
		<li>Proceed to the next chamber, repeat steps 3.2.1-3.2.6, sealing the lid on the correct pre-determined T0 time point.</li>
		<li>Continue to repeat steps 3.2.1-3.2.7 until all chambers in the round have been sealed and T0 samples have been collected.</li>
		<li>Return to the first chamber.</li>
		<li>As the time approaches 10 seconds until T1, pierce the septum in the chamber top with the syringe needle.</li>
		<li>Within a 10 second range of T1, withdraw a 30 ml sample of air from inside the chamber and close the stopcock. Remove the syringe needle from the chamber septum.</li>
		<li>Transfer the sample to a collection vial following steps 3.2.3-3.2.6.</li>
		<li>Continue to collect samples following steps 3.2.10-3.2.12, according to the sampling scheme established in section 2.4.</li>
	</ol>
	</li>
	<li>Ancillary Measures
	<ol>
		<li>In order to convert gas concentration to mass, measure the air temperature at the time of sampling. Depending on research goals, record or perform other ancillary measures such as soil temperature and soil moisture content at each location and/or time, daily rainfall, soil bulk density, soil nitrate and ammonium concentrations, etc. Various means exist to obtain these measures &#8211; follow standard protocols.</li>
		<li>Optionally, collect ambient air samples and/or load field standards of known concentrations into vials to assess ambient GHG concentrations and potential storage-vial degradation in the period between sampling and analysis (see sections 2.4.1.4 and 2.4.1.5).</li>
	</ol>
	</li>
</ol>
4. Sample Analysis
<ol>
	<li>Determine the concentration of gases of interest for each sample by gas chromatography, using equipment fitted with an electron capture detector for N2O, an infrared gas analyzer or thermal conductivity detector for CO2, and a flame ionization detector for CH4. 
	Note: It is essential to obtain access to an instrument that is properly configured for GHG analysis and has sufficient run time available. Principles and methods of gas chromatography are described elsewhere5,15,16.</li>
	<li>Convert trace gas concentration from volumetric to mass using the Ideal Gas Law:</li>
</ol>
PV = nRT
Where P = pressure, V = volume, n = moles of gas, R = gas law constant, and T = temperature. Thus:
<img alt="Equation 1" fo:content-width="4in" src="/files/ftp_upload/52110/52110eq1.jpg" width="400" />
5. Data Analysis
<ol>
	<li>For each time series, plot time-by-concentration and evaluate for linearity. Evaluate using goodness of fit or by visual inspection, excluding later time points showing signs of plateau from further analysis. Use a minimum of three time points including T0 for flux calculation (T0, T1, T2 &#8230;). Establish a consistent protocol, and reject any time series that fail to meet that protocol&#8217;s standards for linearity. See Parkin and Venterea8 for a thorough discussion of error, bias, and variance in flux calculation.</li>
	<li>Perform linear regression.</li>
	<li>Use the slope of the regression to calculate flux:</li>
</ol>
F = S &#8226; V &#8226; A-1
Where F = flux, S = slope of the regression, V = chamber volume, and A = chamber area. Thus:
<img alt="Equation 2" fo:content-width="1.3in" src="/files/ftp_upload/52110/52110eq2.jpg" width="130" />
Note: Refer to the Discussion and Parkin et al.12 for non-linear approaches to flux calculation.

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The authors have nothing to disclose.

The static chamber-based approach described here is an efficient method for measurement of GHG flux from soil systems. The relative simplicity of its components makes it especially well suited to conditions or systems in which more infrastructure-intensive methods are infeasible. In order to generate high quality data, however, the static chamber approach must be carried out with strict attention to experimental design6. One notable consideration that must be taken into account is the spatial variability of soil gas fluxes, which can result in high variability among replicate chamber-based measurements. In designing experiments, therefore, it is important to include enough replicates to provide adequate power for statistical analysis. Tradeoffs may exist between the number of treatments which can be studied while maintaining sufficient replication, and a minimum of four replicates per treatment is a general guideline14.
If measured fluxes will be used to estimate daily emissions, diurnal variations in air temperature, soil temperature, and gas emissions must be taken into account. If research goals require measurements to be obtained in mid-morning when temperatures reflect daily averages, the restricted window for sampling may affect the number of chambers that can feasibly be monitored. An additional consideration to be evaluated is the impact that inclusion or exclusion of plant roots and above ground biomass will have on gas fluxes. Chamber placement relative to plant tissue will impact the interpretation of flux data, particularly in the case of CO2 where not only microbial respiration but also root and shoot respiration and photosynthesis must be appropriately balanced. For additional discussion of these factors, see Parkin and Venterea8.
As noted previously, many variations on this methodology exist, including chamber design and sampling volume. One such variation is in the method employed to transfer samples between the syringe and collection vial. The technique described here first flushes the collection vial with sample before filling the vial to positive pressure5. A more commonly used technique is the transfer of samples from syringes to vials that have been pre-evacuated using a vacuum pump, and the use of non-evacuated vials without flushing has also been reported8,17. Another significant point where a range of approaches exists is in data analysis and the selection of the flux model most appropriate to the system under study. In addition to the linear regression method described here, non-linear models may also be employed, particularly when longer deployment times are used. These models include the algorithm developed by Hutchinson and Mosier18 and derivations thereof19,20, the quadratic procedure described by Wagner et al.21, and the non-steady-state diffusive flux estimator described by Livingston et al22. For a thorough discussion of non-linear flux models, refer to Parkin et al.12 and Venterea et al23.
Methods similar to the static chamber approach include the use of flow-through measurement systems with Fourier transfer infrared (FTIR) spectrometry as an alternate to syringe sampling and gas chromatography, as well as automation of chamber closure and sampling through various means. Automated systems enable more frequent measurements with reduced personnel, but also require additional infrastructure investments. Grace et al.24 provide an extensive summary of options and tradeoffs in automated chamber-based N2O measurement.
Characterization of greenhouse gas flux from both managed and natural systems is important to inform process-based models, understand the impacts of management practices and inform mitigation strategies, and to support global accounting and climate change modeling. Thus while individual studies are informative at the local scale, much additional value is derived through contributing to, and drawing from, a global body of knowledge on gas exchange between the landscape and the atmosphere. It is key, therefore, that data be collected and reported in a way that ensures longevity and interoperability with the broader knowledge base. This includes following best practices to ensure data quality, as well as collection of ancillary measures and comprehensive reporting of metadata to allow extension of findings beyond discrete studies. Excellent guidelines for data reporting are available from the GRACEnet project and the GRA25.

Understanding the contributions of both human activities and natural systems to radiative properties of the atmosphere is an area of critical importance as we strive to mitigate anthropogenic contributions to the greenhouse effect. In addition to carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) are also potent GHGs, accounting for an estimated 7% and 19% of global warming, respectively, with the majority of emissions coming from landscape sources1,2. These range from managed systems such as agricultural fields, rice paddies, and landfills, to natural systems such as forest floors, wetlands, and termite mounds. Accurate measurement, supporting well-informed modeling of such landscape-based emissions is critical in order to understand the drivers of climate change as well as to identify mitigation opportunities.
A variety of greenhouse gas measurement strategies exist, each with their own strengths and weaknesses2-5. Mass balance techniques rely on wind-based dispersion of gases and are suited to measurement of flux from small, well-defined sources such as landfills and animal paddocks. Micrometeorological approaches such as eddy covariance are based on real-time direct measurement of vertical gas flux, and can provide direct measurements over large areas. However, homogeneity in source topography is an implicit assumption (in that measurements yield a mean for the area under study), and costly infrastructure can limit deployment possibilities. Finally, chamber-based methods focus on change in gas concentration at the soil surface by sampling from a restricted above ground headspace. They allow measurements to be obtained from small areas and numerous treatments, but are subject to high coefficients of variation due to spatial variation in soil gas flux.
Here we discuss the most prevalent and easily implemented form of chamber-based measurement, utilizing the type of closed chambers without air flow-through commonly referred to as &#8220;static&#8221; or &#8220;non-steady-state non-flow-through&#8221; chambers. In this approach, gas emissions from the soil surface are trapped within a vented chamber, and rates of flux are determined by measuring the change in gas concentration over time within the chamber headspace. The static chamber technique has been widely deployed across both managed and natural landscapes and underpins the bulk of data reporting soil-based flux of greenhouse gases, particularly N2O6,7. It is ideally suited to the study of small experimental plots, diverse sites over variable terrain, or in other situations where multiple distinct locations must be studied without significant infrastructure investments. Typical experimental uses might include the exploration of alternative landscape management practices and their impact on soil-based CO2, N2O, and/or CH4 emissions, examination of landscape-based flux dynamics under artificially induced climate change scenarios such as warming and rainfall exclusion/supplementation, or the descriptive study of natural and agricultural ecosystems and subsystems.
As a critical tool in GHG measurement and flux estimation, the static chamber method has been thoroughly evaluated, and significant efforts have been made towards standardization of techniques and harmonization of data reporting4,6,8,9. Of particular note are the detailed reviews and guidelines produced by the U.S. Department of Agriculture &#8211; Agricultural Research Service&#8217;s Greenhouse gas Reduction through Agricultural Carbon Enhancement network (GRACEnet)8 and by the Global Research Alliance on Agricultural Greenhouse Gases (GRA)9. Such guidelines provide an invaluable resource and platform for coordination, as ultimately the interoperability of data from a myriad of studies is critical for scaling up local findings to global modeling, and for translating research results into viable mitigation strategies.
GRACEnet, GRA, and other reviews also highlight the fact that specific techniques in static chamber-based greenhouse gas flux measurement are extremely diverse, with significant methodological variations possible at nearly every step of the way, including chamber design, temporal and spatial deployment, sampling volumes, sample analysis, and flux calculations. The method described here presents one possible variant, while showcasing best practices and highlighting critical considerations for the generation of high quality, broadly transferrable data. It is intended to provide an accessible overview of this standardized procedure, and a platform from which to explore further nuances and variations described in the literature.

<table border="0" cellpadding="0" cellspacing="0" width="941">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:332px;">5.9 ml soda glass flat bottom 55 x 15.5 mm</td>
			<td style="width:73px;">Labco Limited</td>
			<td style="width:123px;">719W</td>
			<td style="width:413px;">Collection vials</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:332px;">16.5 mm screw caps with pierceable rubber septum</td>
			<td style="width:73px;">Labco Limited</td>
			<td style="width:123px;">VC309</td>
			<td style="width:413px;">Caps for&#160; vials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">90-well plastic vial rack, 17.1 mm well I.D.</td>
			<td style="width:73px;">Wheaton</td>
			<td style="width:123px;">868810</td>
			<td style="width:413px;">Rack for organizing vials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Regular bevel needles 23G x 1&#34;</td>
			<td style="width:73px;">BD</td>
			<td style="width:123px;">305193</td>
			<td style="width:413px;">Needles for sample collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:332px;">Stopcocks with luer connections, 1-way, male slip</td>
			<td style="width:73px;">Cole-Parmer</td>
			<td style="width:123px;">EW-30600-01</td>
			<td style="width:413px;">Stopcocks for syringes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">30 ml syringe, slip tip</td>
			<td style="width:73px;">BD</td>
			<td style="width:123px;">309651</td>
			<td style="width:413px;">Syringes for sample collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Stopwatch or timer</td>
			<td style="width:73px;">Various</td>
			<td style="width:123px;">N/A</td>
			<td style="width:413px;">For timing field sampling</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:332px;">Stainless steel or galvanized utility pans with rim, or fabricated stainless steel or PVC chambers and lids, dimensions as appropriate to experimental system</td>
			<td style="width:73px;">Various</td>
			<td style="width:123px;">N/A</td>
			<td style="width:413px;">Chamber anchor and lid - bottom cut out of anchor, holes for septum and vent tubing bored in lid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:332px;">Gray butyl stoppers 20 mm</td>
			<td style="width:73px;">Wheaton</td>
			<td style="width:123px;">W224100-173</td>
			<td style="width:413px;">Chamber septa for syringe sampling - insert into hole bored in lid top</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:332px;">Tygon tubing 4.0 mm I.D. x 5.6 mm O.D.</td>
			<td style="width:73px;">Sigma-Aldrich</td>
			<td style="width:123px;">Z685623</td>
			<td style="width:413px;">Chamber vent tubing - insert in hole bored in lid side, flush with exterior, approximately 25 cm coiled in lid interior (a 1ml syringe tip may be used as an attachement mechanism)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:332px;">Adhesive foam rubber tape or HDPE O-ring</td>
			<td style="width:73px;">Various</td>
			<td style="width:123px;">N/A</td>
			<td style="width:413px;">Chamber sealing mechanism - fastened to underside of lid rim</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:332px;">Reflective&#160; insulation, 0.3125&#34; thickness</td>
			<td style="width:73px;">Lowe&#39;s</td>
			<td style="width:123px;">409818</td>
			<td style="width:413px;">Insulating and reflective coating - affix to exterior of chamber lid</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:332px;">Large metal binder clips, 2&#34; size with 1&#34; capacity, or manufactured draw latch as appropriate</td>
			<td style="width:73px;">Staples / McMaster</td>
			<td style="width:123px;">831610 (Staples) / 1863A21 (McMaster)</td>
			<td style="width:413px;">Lid attachment mechanism - for clamping lid to anchor during sampling</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:332px;">Gas chromatography equipment fitted with electron capture detector for nitrous oxide, infrared gas analyzer or thermal conductivity detector for carbon dioxide, flame ionization detector for methane</td>
			<td style="width:73px;">Various</td>
			<td style="width:123px;">N/A</td>
			<td style="width:413px;">For sample analysis</td>
		</tr>
	</tbody>
</table>

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.

Prior to beginning a research project with static chambers, it is important to understand the overall workflow, and the organization of in silico, field- and laboratory-based elements (Figure 1). Provided careful experimental design and system calibration (Figure 2), data analysis will generally be relatively straightforward. A rate of flux is determined for each chamber and sampling time by regression of time by concentration using a pre-determined flux model appropriate to the system (Figure 3). However, even following best practices, difficulties may be encountered, and quality control of raw data is critical. For example, failure of a chamber seal or leaky sample vials can result in anomalous concentration values. These are readily identified through visual inspection of time series concentration plots (Figure 4), with CO2 time series often serving as a particularly useful indicator due to the typically more robust and continuous flux of CO2 compared to sometimes negligible, near-detection-limit, or even negative fluxes of N2O or CH4. Once data quality has been confirmed, results may be used to compare gas flux dynamics between treatments or over the course of a season (Figure 5). As can be seen from May and June flux values and error bars, the variation caused by spatial heterogeneity of flux may be significant, and more pronounced under conditions producing high rates of flux. Such variability is not unusual, and underscores the importance of sufficient replication in this technique.
<img alt="Figure 1" src="/files/ftp_upload/52110/52110fig1highres.jpg" width="600" /> 
Figure 1. Workflow overview. Various elements of this protocol will be carried out in the planning stage, in the field, in the laboratory, and in silico. Arrows indicate the sequence of workflow, beginning with chamber design (and construction if necessary), and concluding with data analysis. Multiple boxes/arrows between field sampling and sample analysis represent the possibility of multiple sampling dates over the course of an experiment.
<img alt="Figure 2" src="/files/ftp_upload/52110/52110fig2highres.jpg" width="400" /> 
Figure 2. Sample timing. An example timing scheme for the collection of samples from multiple chambers simultaneously. Chamber numbers are indicated at left and time points at top, with sampling times listed in whole minutes within the grid. In this example, four separate time series of 36 min&#160;each (one for each chamber) are carried out within the space of 46 min, with 12 min&#160;spacing between time points within a series, and 2 min&#160;walking time between chambers. For this hypothetical example, the suitability of 36-min&#160;time series would have been determined by prior calibration. While evenly spaced timing is not necessary, it often simplifies the sampling scheme. Alternately, researchers may individually record each sampling timepoint to determine sampling intervals.
<img alt="Figure 3" src="/files/ftp_upload/52110/52110fig3highres.jpg" width="600" /> 
Figure 3. Flux calculation. A typical static chamber time series, consisting of N2O concentrations measured at four time points over the course of a 36-min&#160;sampling period. The linear regression is displayed, the slope of which yields flux rate.
<img alt="Figure 4" src="/files/ftp_upload/52110/52110fig4highres.jpg" width="600" /> 
Figure 4. Quality control. Paired time series from the same set of samples but different gases are shown in which vial leakage has been identified by visual inspection (red point). A) CO2 concentration over time. B) N2O concentration over time.
<img alt="Figure 5" src="/files/ftp_upload/52110/52110fig5highres.jpg" width="600" /> 
Figure 5. Synthesis results. N2O flux rate from an agricultural field over the course of a single growing season. Flux values represent the mean of six chambers, using four-point time series. Error bars are standard error.

Watch this Scientific Journal Video about Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers at JoVE.com

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

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 ...

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41.0K vistas.  University of Wisconsin-Madison. 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.

Video: Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

Advanced micrometeorological methods have become  increasingly important in soil, crop, and environmental sciences.  For many scientists without formal ...

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A multidimensional gas chromatography method for the analysis of dissolved hydrogen sulfide in liquid crude oil samples is presented. A Deans switch is used to heart-cut light sulfur gases for separation on a secondary column and detection on a sulfur chemiluminescence detector.

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate ...

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the two-spotted spider mite (TSSM), Tetranychus urticae Koch, on greenhouse grown cotton. TSSM is a polyphagous pest which occurs on a variety of field and horticultural crops. It often becomes an early season pest of cotton in damaging proportions as opposed to being a late season innocuous pest in the mid-southern United States. Evaluation of acaricides is important for maintaining the efficacy of and preventing resistance to the currently available arsenal of chemicals and newly developed control agents. Enumeration of spider mites for efficacy evaluations is laborious and time consuming. Therefore, subjective visual damage rating is commonly used to assess density of spider mites. The NDVI (Normalized Difference Vegetation Index) is the most widely used statistic to describe the spectral reflectance characteristics of vegetation canopy to assess plant stress and health consequent to spider mite infestations. Results demonstrated that a multispectral optical sensor is an effective tool in distinguishing varying levels of infestation caused by T. urticae on early season cotton. This remote sensing technique may be used in lieu of a visual rating to evaluate insecticide treatments.

This manuscript describes a multispectral optical sensor that effectively detected damage to early season cotton artificially infested with varying densities of the two-spotted spider mite populations.

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...