The authors are indebted to Barry G. Dorman, Robert A. Icenogle, Juan Rodriguez, Morris G. Welch, and Marlin Siegford for technical assistance in experimental measurements, and to Jim Clark and Dexter LaGrand for assistance with computer simulations. We thank XIA LLC for allowing the use of their electronics and detectors in this project. This work was supported by NIFA ALA Research Contract No ALA061-4-15014 "Precision geospatial mapping of soil carbon content for agricultural productivity and lifecycle management".

1. Construction of the INS system
<ol>
	<li>Use the general INS system geometry shown in Figure 1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56270/56270fig1.jpg" /> 
Figure 1. INS System Geometry. <a href="//cloudfront.jove.com/files/ftp_upload/56270/56270fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Use the INS system design shown in Figure 2.10</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56270/56270fig2.jpg" /> 
Figure 2. Overview of the INS System. 
A) first block contains neutron generator, neutron detector, and power system; B) second block contains three NaI (Tl) detectors; C) third block contains equipment for system operation; D) general view of the first block showing individual components; and E) close up view of the gamma detectors.10 <a href="//cloudfront.jove.com/files/ftp_upload/56270/56270fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Use three blocks in the INS system (see Appendix).
	<ol>
		<li>For the first block (A), use a neutron generator (NG) and power system (Figure 2A and 2D). Pulsed neutron output of this generator will be 107 - 108 n/s with neutron energy of 14 MeV. The power system will consist of four batteries (12 V, 105 Ah), a DC-AC Inverter, and a charger. This block will also contain iron (10 cm x 20 cm x 30 cm) and boric acid (5 cm x 20 cm x 30 cm) shielding to protect the gamma detector from neutron irradiation. 
		NOTE: A neutron detector is also included in this block for checking that the NG is functioning properly.</li>
		<li>For the second block (B), use gamma-ray measurement equipment (Figure 2B and 2E). This block will contain three 12.7 cm x 12.7 cm x 15.2 cm scintillation NaI(Tl) detectors with corresponding electronics. The exterior size of the detectors with electronics will measure 15.2 cm x 15.2 cm x 46 cm.</li>
		<li>For the third block (C), use a laptop computer that controls the neutron generator (with DNC software), detectors, and data acquisition system (Figure 2C).</li>
	</ol>
	</li>
</ol>
2. Caution and Personal Requirements
<ol>
	<li>Have each user of the INS system pass radiological training.</li>
	<li>Ensure that each person operating the NG carries a radiation monitoring badge. During measurements, the restricted area boundary (&#62;20 &#181;Sv/h) around the NG will have the radiation symbol with the words &#34;CAUTION, RADIATION AREA.&#34; All edges of the restricted area will be no less than 4 m from the NG.</li>
	<li>In an emergency, immediately push the &#34;Emergency Interrupt&#34; button on the NG, remove the key from the NG, and unplug the NG from the power source.</li>
</ol>
3. Preparation of the INS system for Measurement
<ol>
	<li>Check the power system. The power level indicator on the charger will be green, or more than 3 red lamps must illuminate. If not, connect the charger to a power outlet and wait until batteries become fully charged (green lamp will illuminate) or until an acceptable power level is reached (&#8805;3 red lamps will illuminate).</li>
	<li>Turn on the inverter (green lamp illuminates) and laptop.</li>
	<li>Run the data acquisition program on the laptop to operate the gamma detectors and check the required parameters for each detector. The values of these parameters will be defined and recorded previously at INS system testing.
	<ol>
		<li>Place a Cs-137 control source (any type) within 5 - 15 cm from the detectors.</li>
		<li>Start spectra acquisition for 1 - 3 min; check the centroids of the 662 keV Cs-137 peak for all detectors. They must be at the same channel. If not, use the Energy Coefficient Scale of thedata acquisition program by changing the value to adjust the 662 keV peak centroids.</li>
	</ol>
	</li>
	<li>Turn on the NG by using the special key. The indicator lamp on the NG will illuminate green and yellow.</li>
</ol>
4. Calibration of the INS System
<ol>
	<li>Prepare 4 pits sized 1.5 m x 1.5 m x 0.6 m with homogeneous sand-carbon mixtures (Figure 3). Carbon contents is 0, 2.5, 5 and 10 w%. 
	NOTE: A concrete mixer is used to make synthetic soil composed of construction sand and coconut shell (100% carbon content, average granular diameter &#60; 0.5 mm). Homogeneity of these mixtures is determined visually.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56270/56270fig3.jpg" /> 
Figure 3. View of Pit with Sand and Pit with 10 Cw% Sand-carbon Mixture. <a href="//cloudfront.jove.com/files/ftp_upload/56270/56270fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Take measurements over the pits using the following steps.
	<ol>
		<li>Position the INS system over the pit manually or by towing with a suitable vehicle. Position the INS system such that the projection of the neutron source is centered on the pit.</li>
		<li>Run the DNC software on the laptop that operates the NG generator. In the Faults column on the right side of the DNC program screen, all lamps will illuminate green; if not, click the Clear button. Insert the following parameters: for Pulse Parameters - frequency 5 kHz, duty cycle 25%, delay 0 &#181;s, extension 2 &#181;s; for Beam - high voltage 50 kV, beam current 50 &#181;A (note that these parameters can be different depending on the particular INS system setup and task).
		<ol>
			<li>Activate the switch on the DNC program screen and wait for the NG to enter the working regime where the High voltage and Beam current will come to stable values corresponding to the entered values; Reservoir current also will come to a stable value.</li>
		</ol>
		</li>
		<li>Run the data acquisition software on the laptop to operate the gamma detectors. Start spectra acquisition by running the data acquisition program for 1 h. The two spectra acquisition processes (INS &#38; TNC and TNC) will appear on the screen.</li>
		<li>After 1 h, stop the spectra acquisition and save spectra to the hard drive (File | Save MCA Data | choose the folder and enter the file name. 
		NOTE: There will be two saved spectra (TNC and INS) with filename extensions .mca and _gated.mca, respectively).</li>
		<li>Select second detector (click the arrow in the top left corner) and save the spectra for this detector. Do the same for third detector.</li>
		<li>Click File | Exit to close the software.</li>
		<li>Turn off the DNC software by turning off the switch on the DNC program screen.</li>
		<li>Repeat steps 4.2.1 - 4.2.7 for the other pits.</li>
		<li>Turn off the NG by using the special key. The indicator lamp on the NG will dim.</li>
	</ol>
	</li>
	<li>Determine the INS system background spectra by elevating the whole INS system to a distance greater than 4 m above the ground surface and away from any large objects, and repeat data acquisition steps 4.2.2 - 4.2.9.</li>
	<li>Data processing
	<ol>
		<li>Use a spreadsheet program to open data files saved in step 4.2.4. Find values for output and input count rates (OCR and ICR) and real time (RT) in rows 28, 27, and 30, respectively.</li>
		<li>Calculate the life time (LT) for INS &#38; TNC and TNC spectra for all measurements as 
		LTi = OCRi/ICRi&#183;RTi (1), 
		where OCRi and ICRi are the output and input count rates for the i-th measurement and RTi is real time of the i-th measurement.</li>
		<li>Calculate the gamma spectra in counts per second (cps) by dividing the spectra (rows 33-2080 in the spreadsheet) by the corresponding LT.</li>
		<li>Calculate the net INS spectra from the corresponding measurements for each pit as 
		Net INS Spectrum = (INS&#38;TNC - TNC)Pit - (INS&#38;TNC - TNC)Bkg (2)</li>
		<li>Find the gamma peaks 1.78 MeV (28Si) and 4.44 MeV (12C) in the Net INS spectrum for each pit, and calculate the peak areas (4.44 MeV C peak area, 1.78 MeV Si peak area) using IGOR software.
		<ol>
			<li>Open the software by double clicking the icon. Insert first Net INS Spectrum into the Table.</li>
			<li>Click Windows | New Graph | From Target | &#34;FileName&#34; | Do It. The Spectrum appears in the Graph Window. Click Graph | Show Info. The windows with A and B markers appears under the graph window.</li>
			<li>Place mouse pointer on sign A, push the left mouse button, and drag the cursor to the spectrum on left side of the 1.78 MeV peak. Place mouse pointer on the sign B, push the left mouse button, and drag the cursor to the spectrum on right side of the 1.78 MeV peak.</li>
			<li>Click Analysis | Multi-peak Fit | Start New Multi-peak Fit | From Target | Continue. In the pop-up window marked Use Graph Cursor | Baseline Linear| Auto-locate Peaks Now | Do it | Peak Results. The area of the peak appears in the pop-up window.</li>
			<li>Repeat the same operations for 4.44 MeV peak.</li>
			<li>Repeat all previous operations with the remaining Net INS Spectra.</li>
		</ol>
		</li>
		<li>Find the Net carbon peak areas for each pit by the equation 
		Net C peak areai = 4.44 MeV C peak areai- 0.058 &#183; 1.78 MeV Si peak areai (3)</li>
		<li>Build the calibration line for the INS system as a direct proportional dependence of the Net carbon peak area vs. carbon concentration expressed in weight percent.
		<ol>
			<li>Open new Table in IGOR software: click Window | New Table. Enter pit carbon concentration values in the first column, and the corresponding Net C peak area in the second column.</li>
			<li>Plot the Net C peak area vs pit carbon concentration: click Windows | New Graph. Choose Net C peak area as YWave, and carbon concentrations as XWave. Click Do it. The points appear on the Graph.</li>
			<li>Build the calibration line: Click Analysis | Curve Fitting | Function - line | From Target | Do it. The calibration line and the calibration coefficient (k) will appear in the window.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Conducting Field Soil Measurements in Static Mode
<ol>
	<li>Prepare the INS system for measurement according to step 3.</li>
	<li>Place the system over the site requiring soil carbon content analysis manually or by towing using suitable vehicle. Position the INS system such that the projection of the neutron source is centered over the site being measured.</li>
	<li>Implement actions following steps 4.2.2 - 4.2.9 and 4.4.1 - 4.4.6 for determining Net C peak areas for the study sites.</li>
	<li>Calculate the carbon concentration in weight % using the calibration coefficient as 
	<img alt="Equation 1" src="/files/ftp_upload/56270/56270eq1.jpg" /></li>
</ol>
6. Conducting Field Soil Measurements in the Scanning Mode
<ol>
	<li>Estimate the path that the INS system will travel over the field while accounting for travel speed (&#8804; 5 km/h), field size, INS system footprint (radius ~1 m), and measurement time (1 h) such that the moving trajectory eventually covers the whole field area. For convenience, place flags at turn points along the field perimeter.</li>
	<li>Prepare the INS system for measurement according to step 3.</li>
	<li>Implement actions following steps 4.2.2 - 4.2.3.</li>
	<li>Follow the predetermined travel path for 1 h.</li>
	<li>Implement actions following steps 4.2.4 - 4.2.9 and 4.4.1 - 4.4.6 for determining Net C peak areas for the studied field.</li>
	<li>Calculate the carbon concentration in weight % using the calibration coefficient by equation 4.</li>
</ol>

<ol>
	<li>Potter, K. N., Daniel, J. A., Altom, W., Torbert, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stocking+rate+effect+on+soil+carbon+and+nitrogen+in+degraded+soils.">Stocking rate effect on soil carbon and nitrogen in degraded soils.</a> J. Soil Water Conserv. 56, 233-236 (2001).</li><li>Torbert, H. A., Prior, S. A., Runion, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+return+to+cultivation+on+carbon+(C)+sequestration.">Impact of the return to cultivation on carbon (C) sequestration.</a> J. Soil Water Conserv. 59 (1), 1-8 (2004).</li><li>Stolbovoy, V., Montanarella, L., Filippi, N., Jones, A., Gallego, J., Grassi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soil sampling protocol to certify the changes of organic carbon stock in mineral soil of the European Union. Version 2. , (2007).</li><li>Smith, K. E., Watts, D. B., Way, T. R., Torbert, H. A., Prior, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+tillage+and+fertilizer+application+method+on+gas+emissions+(CO2,+CH4,+N2O)+in+a+corn+cropping+system.">Impact of tillage and fertilizer application method on gas emissions (CO2, CH4, N2O) in a corn cropping system.</a> Pedosphere. 22 (5), 604-615 (2012).</li><li>Seybold, C. A., Mausbach, M. J., Karlen, D. L., Rogers, H. H., Lal, R., Kimble, J., Stewart, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+soil+quality.">Quantification of soil quality.</a> Soil processes and the carbon cycle. , 387-404 (1997).</li><li>Nelson, D. W., Sommers, L. E., Sparks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+carbon,+organic+carbon,+and+organic+matter.">Total carbon, organic carbon, and organic matter.</a> Methods of Soil Analysis., Part 3, Chemical Methods. , 961-1010 (1996).</li><li>Wielopolski, L., Carayannis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+methodology+for+non-destructive+multi-elemental+analysis+of+large+volumes+of+soil.">Nuclear methodology for non-destructive multi-elemental analysis of large volumes of soil.</a> Planet Earth: Global Warming Challenges and Opportunities for Policy and Practice. , (2011).</li><li>Wielopolski, L., Yanai, R. D., Levine , C. R., Mitra, S., Vadeboncoeur, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+non-destructive+carbon+analysis+of+forest+soils+using+neutron-induced+gamma-ray+spectroscopy.">Rapid, non-destructive carbon analysis of forest soils using neutron-induced gamma-ray spectroscopy.</a> Forest Ecol. Manag. 260, 1132-1137 (2010).</li><li>Mitra, S., Wielopolski, L., Tan, H., Fallu-Labruyere, A., Hennig, W., Warburton, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+measurement+of+individual+gamma-ray+spectra+during+and+between+fast+neutron+pulses.">Concurrent measurement of individual gamma-ray spectra during and between fast neutron pulses.</a> Nucl. Sci. 54 (1), 192-196 (2007).</li><li>Yakubova, G., Wielopolski, L., Kavetskiy, A., Torbert, H. A., Prior, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+testing+a+mobile+inelastic+neutron+scattering+system+to+measure+soil+carbon.">Field testing a mobile inelastic neutron scattering system to measure soil carbon.</a> Soil Sci. 179, 529-535 (2014).</li><li>Yakubova, G., Kavetskiy, A., Prior, S. A., Torbert, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+the+inelastic+neutron+scattering+soil+carbon+method.">Benchmarking the inelastic neutron scattering soil carbon method.</a> Vadose Zone J. 15 (2), (2016).</li><li>Knoll, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Radiation Detection and Measurement. , (2000).</li><li>Mitra, S., Dioszegi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexploded+Ordnance+identification+-+A+gamma-ray+spectral+analysis+method+for+Carbon,+Nitrogen+and+Oxygen+signals+following+tagged+neutron+interrogation.">Unexploded Ordnance identification - A gamma-ray spectral analysis method for Carbon, Nitrogen and Oxygen signals following tagged neutron interrogation.</a> Nucl. Instrum. Meth. A. 693, 16-22 (2012).</li></ol>

The authors have nothing to disclose.

Building on the foundation established by previous researchers, the NSDL staff addressed questions critical to the practical and successful use of this technology in real world field settings. Initially, NSDL researchers demonstrated the necessity to account for the INS system background signal when determining net carbon peak areas.11 Another effort showed that the net carbon peak area characterizes the average carbon weight percent in the upper 10 cm soil layer (regardless of carbon depth distri...

Knowledge of soil carbon content is required for optimization of soil productivity and profitability, understanding the impact of agricultural land use practices on soil resources, and evaluating strategies for carbon sequestration1,2,3,4. Soil carbon is a universal indicator of soil quality5. Several methods have been developed for soil carbon measurements. Dry combustion (DC) has been the most widely used method for years6; this method is based on field sample collection and laboratory processing and measurement that is destructive, labor intensive, and time consuming. Two newer methods are laser-induced breakdown spectroscopy, and near and mid infrared spectroscopy7. These methods are also destructive and only analyze the very near-surface soil layer (0.1 - 1 cm soil depth). In addition, these methods only yield point measurements of carbon content for small sample volumes (~60 cm3 for DC method, and 0.01-10 cm3 for infrared spectroscopy methods). Such point measurements make it difficult to extrapolate results to field or landscape scales. Since these methods are destructive, recurring measurements are also impossible.
Previous researchers at Brookhaven National Laboratory suggested applying neutron technology for soil carbon analysis (INS method)7,8,9. This initial effort developed the theory and practice of using neutron gamma analysis for soil carbon measurement. Starting in 2013, this effort was continued at the USDA-ARS National Soil Dynamics Laboratory (NSDL). The expansion of this technological application over the last 10 years is due to two main factors: the availability of relatively inexpensive commercial neutron generators, gamma detectors, and corresponding electronics with software; and state of the art neutron-nuclei interaction reference databases. This method has several advantages over others. An INS system, placed on a platform, could be maneuvered over any type of field that requires measurement. This non-destructive in-situ method can analyze large soils volumes (~300 kg) that can be interpolated to a whole agricultural field using just a few measurements. This INS system is also capable of operating in a scanning mode that determines the average carbon content of an area based on scanning over a predetermine grid of the field or landscape.

<table><tbody><tr><td>Neutron Generator</td><td>Thermo Fisher Scientific, Colorado Springs, CO&lt;br/&gt; DNC software</td><td>MP320</td><td></td></tr><tr><td>Gamma-detector:</td><td></td><td>na</td><td></td></tr><tr><td>- NaI(Tl) crystal</td><td>Scionix USA, Orlando, FL</td><td></td><td></td></tr><tr><td>- Electronics</td><td>XIA LLC, Hayward, CA</td><td></td><td></td></tr><tr><td>- Software</td><td>ProSpect</td><td></td><td></td></tr><tr><td>Battery</td><td>Fullriver Battery USA, Camarillo, CA</td><td>DC105-12</td><td></td></tr><tr><td>Invertor</td><td>Nova Electric, Bergenfield, NJ</td><td>CGL 600W-series</td><td></td></tr><tr><td>Charger</td><td>PRO Charging Systems, LLC, LaVergne, TN</td><td>PS4</td><td></td></tr><tr><td>Block of Iron</td><td>Any</td><td>na</td><td></td></tr><tr><td>Boric Acid</td><td>Any</td><td>na</td><td></td></tr><tr><td>Laptop</td><td>Any</td><td>na</td><td></td></tr><tr><td>mu-metal</td><td>Magnetic Shield Corp., Bensenville, IL</td><td>&amp;nbsp;MU010-12</td><td></td></tr><tr><td>Construction sand</td><td>Any</td><td>na</td><td></td></tr><tr><td>Coconut shell</td><td>General Carbon Corp., Patterson, NJ</td><td>GC 8 X 30S</td><td></td></tr><tr><td>Reference Cs-137 source</td><td>Any</td><td>na</td><td></td></tr></tbody></table>

measurements of soil carbon by neutron-gamma analysis in static and scanning modes

The herein described application of the inelastic neutron scattering (INS) method for soil carbon analysis is based on the registration and analysis of gamma rays created when neutrons interact with soil elements. The main parts of the INS system are a pulsed neutron generator, NaI(Tl) gamma detectors, split electronics to separate gamma spectra due to INS and thermo-neutron capture (TNC) processes, and software for gamma spectra acquisition and data processing. This method has several advantages over other methods in that it is a non-destructive in situ method that measures the average carbon content in large soil volumes, is negligibly impacted by local sharp changes in soil carbon, and can be used in stationary or scanning modes. The result of the INS method is the carbon content from a site with a footprint of ~2.5 - 3 m2 in the stationary regime, or the average carbon content of the traversed area in the scanning regime. The measurement range of the current INS system is &#62;1.5 carbon weight % (standard deviation &#177; 0.3 w%) in the upper 10 cm soil layer for a 1 hmeasurement.

Soil INS &#38; TNC and TNC gamma spectra
A general view of the measured soil gamma spectra is shown in Figure 4. The spectra consist of a set of peaks on a continuous background. The main peaks of interest have centroids at 4.44 MeV and 1.78 MeV in the INS &#38; TNC spectra. The second peak can be attributed to silicon nuclei contained in soil, and the first peak is an overlapping peak from carbon and silicon nucle...

Watch this Scientific Journal Video about Measurements of Soil Carbon by Neutron-Gamma Analysis in Static and Scanning Modes at JoVE.com

Measurements of Soil Carbon by Neutron-Gamma Analysis in Static and Scanning Modes

Here, we present the protocol for in situ measurement of soil carbon using the neutron-gamma technique for single point measurements (static mode) or field averages (scanning mode). We also describe system construction and elaborate data treatment procedures.

The herein described application of the inelastic neutron scattering (INS) method for soil carbon analysis is based on the registration and analysis of ...

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Research

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Engineering

7.3K Views.  Agricultural Research Service, United States Department of Agriculture. Hier presenteren we het protocol voor in situ meting van de bodemkoolstof met behulp van het neutron-gamma techniek voor aanspreekpunt metingen (statische modus) of veld gemiddelden (scanmodus). Ook beschrijven we de bouw van het systeem en gegevens behandeling procedures uit te werken.

Video: Measurements of Soil Carbon by Neutron-Gamma Analysis in Static and Scanning Modes

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 ...

Environment

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 ...

Using Neutron Spin Echo Resolved Grazing Incidence Scattering to Investigate Organic Solar Cell Materials

The spin echo resolved grazing incidence scattering (SERGIS) technique has been used to probe the length-scales associated with irregularly shaped crystallites. Neutrons are passed through two well defined regions of magnetic field; one before and one after the sample. The two magnetic field regions have opposite polarity and are tuned such that neutrons travelling through both regions, without being perturbed, will undergo the same number of precessions in opposing directions. In this case the neutron precession in the second arm is said to &#34;echo&#34; the first, and the original polarization of the beam is preserved. If the neutron interacts with a sample and scatters elastically the path through the second arm is not the same as the first and the original polarization is not recovered. Depolarization of the neutron beam is a highly sensitive probe at very small angles (&#60;50&#160;&#956;rad) but still allows a high intensity, divergent beam to be used. The decrease in polarization of the beam reflected from the sample as compared to that from the reference sample can be directly related to structure within the sample.
In comparison to scattering observed in neutron reflection measurements the SERGIS signals are often weak and are unlikely to be observed if the in-plane structures within the sample under investigation are dilute, disordered, small in size and polydisperse or the neutron scattering contrast is low. Therefore, good results will most likely be obtained using the SERGIS technique if the sample being measured consist of thin films on a flat substrate and contain scattering features that contains a high density of moderately sized features (30 nm to 5 &#181;m) which scatter neutrons strongly or the features are arranged on a lattice. An advantage of the SERGIS technique is that it can probe structures in the plane of the sample.

Progress has been made in utilizing spin echo resolved grazing incidence scattering (SERGIS) as a neutron scattering technique to probe the length-scales in irregular samples. Crystallites of [6,6]-phenyl-C61-butyric acid methyl ester have been probed using the SERGIS technique and the results confirmed by optical and atomic force microscopy.

The spin echo resolved grazing incidence scattering (SERGIS) technique has been used to probe the length-scales associated with irregularly shaped ...

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

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

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

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

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

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 ...

Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools

Soil organic matter (SOM) is a complicated mixture of different compounds that span the range from free, partially degraded plant components to more microbially altered compounds held in the soil aggregates to highly processed microbial by-products with strong associations with reactive soil minerals. Soil scientists have struggled to find ways to separate soil into fractions that are easily measurable and useful for soil carbon (C) modeling. Fractionating soil based on density is increasingly being used, and it is easy to perform and yields C pools based on the degree of association between the SOM and different minerals; thus, soil density fractionation can help to characterize the SOM and identify SOM stabilization mechanisms. However, the reported soil density fractionation protocols vary significantly, making the results from different studies and ecosystems hard to compare. Here, we describe a robust density fractionation procedure that separates particulate and mineral-associated organic matter and explain the benefits and drawbacks of separating soil into two, three, or more density fractions. Such fractions often differ in their chemical and mineral composition, turnover time, and degree of microbial processing, as well as the degree of mineral stabilization.

Soil density fractionation separates soil organic matter into distinct pools with differing stabilization mechanisms, chemistries, and turnover times. Sodium polytungstate solutions with specific densities allow the separation of free particulate organic matter and mineral-associated organic matter, resulting in organic matter fractions suitable for describing the soil response to management and climate change.

Soil organic matter (SOM) is a complicated mixture of different compounds that span the range from free, partially degraded plant components to more ...

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

Biology

Lead Analysis of Soil Using Atomic Absorption Spectroscopy

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Lead occurs naturally in soil, in levels ranging from 10-50 ppm. However, with the widespread use of lead in paint and gasoline in addition to contamination by industry, urban soils often have concentrations of lead significantly greater than background levels &#8211; up to 10,000 ppm in some places. Ongoing problems arise from the fact that lead does not biodegrade, and instead remains in the soil.
Serious health risks are associated with lead poisoning, where children are particularly at risk. Millions of children in the U.S. are exposed to soil containing lead. This exposure can cause developmental and behavioral problems in children. These problems include learning disabilities, inattention, delayed growth, and brain damage. The Environmental Protection Agency has set a standard for lead in soil at 400 ppm for play areas and 1,200 ppm for non-play areas.
Lead is also of concern in soil, when it&#8217;s used for gardening. Plants take up lead from the soil. Therefore, vegetables or herbs grown in contaminated soil can lead to lead poisoning. In addition, contaminated soil particles can be breathed in while gardening or brought into the house on clothing and footwear. It is recommended that soils with lead levels greater than 400 ppm should not be used for gardening. It is further recommended that soil with lead levels between 100 and 400 ppm not be used for leafy vegetables or herbs, because lead can be stored in the leaves. On a similar note, root vegetables should not be grown in this soil, because lead can also accumulate in plant roots.

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Lead occurs naturally in soil, in levels ranging from 10-50 ppm. However, ...

Measurements of Soil Carbon by Neutron-Gamma Analysis in Static and Scanning Modes

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