Name: utilizing soil density fractionation to separate distinct soil carbon pools
Uploaded: 2022-12-16T14:30:00
Description: Watch this Scientific Journal Video about Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools at JoVE.com

For this work, support was provided by National Science Foundation Grants DEB-1257032 to K.L. and DEB-1440409 to the H. J. Andrews Long Term Ecological Research program.

1. Making stock solutions of sodium polytungstate (SPT)
CAUTION: SPT is an irritant and is harmful if swallowed or inhaled. It is toxic to aquatic organisms; avoid its release into the environment.
<ol>
	<li>To make 1 L of SPT solution with a density of 1.85 g/cm3, dissolve 1,051 g of crystalized SPT in approximately 600 mL of deionized distilled (DDI) water. Stir the solution until the SPT has fully dissolved, approximately for 15 min, and then bring the solution...

<ol>
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R., Harden, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sequential+selective+dissolution+method+to+quantify+storage+and+stability+of+organic+carbon+associated+with+Al+and+Fe+hydroxide+phases.">A sequential selective dissolution method to quantify storage and stability of organic carbon associated with Al and Fe hydroxide phases.</a> Geoderma. 312, 24-35 (2018).</li><li>Frosteg&#229;rd, &. #. 1. 9. 7. ;., Tunlid, A., B&#229;&#229;th, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biomass+measured+as+total+lipid+phosphate+in+soils+of+different+organic+content.">Microbial biomass measured as total lipid phosphate in soils of different organic content.</a> Journal of Microbiological Methods. 14 (3), 151-163 (1991).</li><li>Plante, A. F., Conant, R. T., Paul, E. A., Paustian, K., Six, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+hydrolysis+of+easily+dispersed+and+microaggregate-derived+silt-+and+clay-sized+fractions+to+isolate+resistant+soil+organic+matter.">Acid hydrolysis of easily dispersed and microaggregate-derived silt- and clay-sized fractions to isolate resistant soil organic matter.</a> European Journal of Soil Science. 57 (4), 456-467 (2006).</li><li>Eusterhues, K., Rumpel, C., K&#246;gel-Knabner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+soil+organic+matter+isolated+via+oxidative+degradation.">Stabilization of soil organic matter isolated via oxidative degradation.</a> Organic Geochemistry. 36 (11), 1567-1575 (2005).</li><li>Moni, C., Derrien, D., Hatton, P. -. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conceptualizing+soil+organic+matter+into+particulate+and+mineral-associated+forms+to+address+global+change+in+the+21st+century.">Conceptualizing soil organic matter into particulate and mineral-associated forms to address global change in the 21st century.</a> Global Change Biology. 26 (1), 261-273 (2020).</li><li>Sollins, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+C+and+N+stabilization+in+a+forest+soil:+Evidence+from+sequential+density+fractionation.">Organic C and N stabilization in a forest soil: Evidence from sequential density fractionation.</a> Soil Biology and Biochemistry. 38 (11), 3313-3324 (2006).</li><li>Sollins, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+density+fractionation+across+soils+of+contrasting+mineralogy:+evidence+for+both+microbial-+and+mineral-controlled+soil+organic+matter+stabilization.">Sequential density fractionation across soils of contrasting mineralogy: evidence for both microbial- and mineral-controlled soil organic matter stabilization.</a> Biogeochemistry. 96 (1-3), 209-231 (2009).</li><li>Crow, S. E., Swanston, C. W., Lajtha, K., Brooks, J. R., Keirstead, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+fractionation+of+forest+soils:+methodological+questions+and+interpretation+of+incubation+results+and+turnover+time+in+an+ecosystem+context.">Density fractionation of forest soils: methodological questions and interpretation of incubation results and turnover time in an ecosystem context.</a> Biogeochemistry. 85 (1), 69-90 (2007).</li><li>Cotrufo, M. F., Ranalli, M. G., Haddix, M. L., Six, J., Lugato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+carbon+storage+informed+by+particulate+and+mineral-associated+organic+matter.">Soil carbon storage informed by particulate and mineral-associated organic matter.</a> Nature Geoscience. 12 (12), 989-994 (2019).</li><li>Hatton, P. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+litter-derived+N+to+soil+mineral-organic+associations:+Evidence+from+decadal+15N+tracer+experiments.">Transfer of litter-derived N to soil mineral-organic associations: Evidence from decadal 15N tracer experiments.</a> Organic Geochemistry. 42 (12), 1489-1501 (2012).</li><li>Lajtha, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+to+particulate+versus+mineral-associated+soil+carbon+after+50+years+of+litter+manipulation+in+forest+and+prairie+experimental+ecosystems.">Changes to particulate versus mineral-associated soil carbon after 50 years of litter manipulation in forest and prairie experimental ecosystems.</a> Biogeochemistry. 119 (1-3), 341-360 (2014).</li><li>Yeasmin, S., Singh, B., Johnston, C. T., 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=Organic+carbon+characteristics+in+density+fractions+of+soils+with+contrasting+mineralogies.">Organic carbon characteristics in density fractions of soils with contrasting mineralogies.</a> Geochimica et Cosmochimica Acta. 218, 215-236 (2017).</li><li>Wagai, R., Kajiura, M., Asano, M., Hiradate, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+soil+organo-mineral+assemblage+examined+by+sequential+density+fractionation+with+and+without+sonication:+Is+allophanic+soil+different.">Nature of soil organo-mineral assemblage examined by sequential density fractionation with and without sonication: Is allophanic soil different.</a> Geoderma. 241-242, 295-305 (2015).</li><li>Volk, M., Bassin, S., Lehmann, M. F., Johnson, M. G., Andersen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C+isotopic+signature+and+C+concentration+of+soil+density+fractions+illustrate+reduced+C+allocation+to+subalpine+grassland+soil+under+high+atmospheric+N+deposition.">13C isotopic signature and C concentration of soil density fractions illustrate reduced C allocation to subalpine grassland soil under high atmospheric N deposition.</a> Soil Biology and Biochemistry. 125, 178-184 (2018).</li><li>Yonekura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+organic+matter+dynamics+in+density+and+particle-size+fractions+following+destruction+of+tropical+rainforest+and+the+subsequent+establishment+of+Imperata+grassland+in+Indonesian+Borneo+using+stable+carbon+isotopes.">Soil organic matter dynamics in density and particle-size fractions following destruction of tropical rainforest and the subsequent establishment of Imperata grassland in Indonesian Borneo using stable carbon isotopes.</a> Plant and Soil. 372 (1-2), 683-699 (2013).</li><li>Virto, I., Moni, C., Swanston, C., Chenu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnover+of+intra-+and+extra-aggregate+organic+matter+at+the+silt-size+scale.">Turnover of intra- and extra-aggregate organic matter at the silt-size scale.</a> Geoderma. 156 (1-2), 1-10 (2010).</li><li>Poeplau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+a+soil+organic+carbon+fractionation+method+to+derive+RothC+carbon+pools.">Reproducibility of a soil organic carbon fractionation method to derive RothC carbon pools.</a> European Journal of Soil Science. 64 (6), 735-746 (2013).</li><li>Cerli, C., Celi, L., Kalbitz, K., Guggenberger, G., Kaiser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+light+and+heavy+organic+matter+fractions+in+soil+-+Testing+for+proper+density+cut-off+and+dispersion+level.">Separation of light and heavy organic matter fractions in soil - Testing for proper density cut-off and dispersion level.</a> Geoderma. 170, 403-416 (2012).</li><li>Kaiser, K., Guggenberger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+hydrous+aluminium+and+iron+over+density+fractions+depends+on+organic+matter+load+and+ultrasonic+dispersion.">Distribution of hydrous aluminium and iron over density fractions depends on organic matter load and ultrasonic dispersion.</a> Geoderma. 140 (1-2), 140-146 (2007).</li><li>Helbling, E., Pierson, D., Lajtha, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+of+soil+carbon+loss+during+soil+density+fractionation:+Laboratory+loss+or+seasonally+variable+soluble+pools.">Sources of soil carbon loss during soil density fractionation: Laboratory loss or seasonally variable soluble pools.</a> Geoderma. 382, 114776 (2021).</li><li>Nelson, D. W., Sommers, L. E., Sparks, D. L., Page, A. L., Helmke, P. A., Loeppert, R. H. <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. , 539-579 (2015).</li><li>Pierson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mineral+stabilization+of+soil+carbon+is+suppressed+by+live+roots,+outweighing+influences+from+litter+quality+or+quantity.">Mineral stabilization of soil carbon is suppressed by live roots, outweighing influences from litter quality or quantity.</a> Biogeochemistry. 154 (3), 433-449 (2021).</li><li>Kramer, M. 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Throughout the soil density fractionation protocol, there are a few specific procedures that must be monitored closely to help reduce error in the separation and analysis of the soil fractions. A critical step in the soil density fractionation procedure is to repeatedly verify the density of the SPT solution. Moisture in the soil sample will often dilute the SPT solution, thus lowering the density of the SPT. Therefore, the researcher must always ensure that complete separation of the light and heavy solutions has been a...

Soil is the largest store of terrestrial carbon (C), containing upward of 1,500 Pg of C in the top 1 m and almost double that amount in deeper levels globally, thus meaning soil contains more C than plant biomass and the atmosphere combined1. Soil organic matter (SOM) retains water and soil nutrients and is essential for plant productivity and the function of the terrestrial ecosystem. Despite global recognition of the importance of adequate SOM stocks for soil health and agricultural productivity, soil C stocks have been substantially depleted due to unsustainable forest and agricultural management, landscape change, and climate warming2,3. Increased interest in restoring soil health and in using soil C retention as a key player in natural climate solutions has led to efforts to understand the factors that control soil C sequestration and stabilization in diverse environments4,5.
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 (defined here as a material formed by the combination of separate units or items) to highly processed microbial by-products with strong associations with reactive soil minerals6. In cases where it is impractical to identify the full suite of individual compounds in the SOM, investigators often focus on identifying a smaller number of functional pools of C that exist as physical realities and that vary by turnover rates, general chemical composition, and the degree of stabilization with the mineral components of the soil1,7. In order for pools to be critically interpreted and modeled, it is essential that the separated pools be small in number, be directly measurable rather than just theoretical, and exhibit clear differences in composition and reactivity8.
Many different techniques, both chemical and physical, have been employed to isolate meaningful pools of soil C, and these are well summarized by von L&#252;tzow et al.9 and Poeplau et al.10. Chemical extraction techniques aim to isolate specific pools, such as C associated with either poorly crystalline or crystalline Fe and Al11. Organic solvents have been used to extract specific compounds such as lipids12, and either the hydrolysis or oxidation of SOM has been used as a measure of a labile pool of C13,14. However, none of these extraction methods categorize all the pools of C into measurable or modellable fractions. The physical fractionation of soil categorizes all soil C into pools based on size and assumes that the decomposition of plant debris results in fragmentation and increasingly smaller particles. Although size alone cannot separate free plant debris from mineral-associated SOM15, quantifying these two pools is critical for the understanding of soil C stabilization due to common spatial, physical, and biogeochemical differences in formation and turnover16.
The fractionation of soil C based on density is increasingly being used, and it is easy to perform and identifies different pools of C based on the degree of association with different minerals17,18,19; thus, soil density fractionation can help elucidate differing soil C stabilization mechanisms. The primary requirement for soil to be fractionated is the ability to fully disperse the organic and mineral particles. Once dispersed, degraded organic matter that is relatively free of minerals floats in solutions lighter than ~1.85 g/cm3, while minerals typically fall in the range of 2-4.5 g/cm3, although iron oxides may have densities up to 5.3 g/cm3. The light or free particulate fraction tends to have shorter a turnover time (unless there is significant contamination by charcoal) and has been shown to be highly responsive to cultivation and other disturbances. The heavy (&#62;1.85 g/cm3) or mineral-associated fraction often has a longer turnover time due to the resistance to microbially mediated decomposition gained when organic molecules bind with reactive mineral surfaces. However, the heavy fraction may saturate (i.e., reach an upper limit for mineral complexation capacity), while the light fraction can theoretically accumulate almost indefinitely. Thus, understanding the physical distribution of organic matter in pools of mineral-associated versus particulate organic matter helps to elucidate which ecosystems can be managed for efficient carbon sequestration and how different systems will respond to climate change and shifting patterns of anthropogenic disturbance20.
While the use of density fractionation using solutions of sodium polytungstate at different densities has increased greatly in the last decade, the techniques and protocols vary significantly, making the results from different studies and different ecosystems hard to compare. Although a density of 1.85 g/cm3 has been shown to recover the greatest amount of free light fraction with minimal inclusion of mineral-associated organic matter (MAOM)17, many studies have used densities ranging from 1.65-2.0 g/cm3. While most studies have fractionated soils into just two pools (a light fraction and a heavy fraction, hereafter LF and HF), other studies have used multiple densities to further refine the heavy fraction into pools that differ by the minerals that they are associated with, the relative ratio of minerals to organic coating, or the degree of aggregation (e.g., Sollins et al.17, Sollins et al.18, Hatton et al.21, Lajtha et al.22, Yeasmin et al.23, Wagai et al.24, Volk et al.25). In addition, more complex fractionation procedures have been suggested that combine both size and density separation, resulting in a larger number of pools (e.g., Yonekura et al.26, Virto et al.27, Moni et al.15, Poeplau et al.10) but also more room for error, both in the methodology and in relation to the pool size. Further, authors have also used sonication at varied intensities and times in an effort to disperse aggregates and MAOM from mineral surfaces28,29,30.
Here, we describe a robust density fractionation procedure that identifies, first, two unique pools of soil carbon (LF and HF, or POM and MAOM), and we offer both the techniques and the arguments to further separate the HF pool into additional fractions that differ based on their mineralogy, degree of organic coating, or aggregation. The fractions identified here have been shown to differ in terms of their chemical composition, turnover time, degree of microbial processing, and degree of mineral stabilization18,19.
The following procedure separates bulk soil into particulate organic matter (POM) and mineral-associated organic matter (MAOM) by mixing a known quantity of soil in a solution with a specific density. The efficacy of the procedure is measured by the combined recovery of soil mass and carbon relative to the initial soil sample mass and C content. A dense solution is achieved by dissolving sodium polytungstate (SPT) in deionized water. The soil is initially mixed with the dense SPT solution and agitated to thoroughly mix and disperse the soil aggregates. Centrifugation is then used to separate the soil materials that either float (light fraction) or sink (heavy fraction) in the solution. The mixing, isolation, recovery, and washing steps are repeated multiple times to ensure the separation of the light and heavy fractions, along with the removal of SPT from the material. Finally, the soil fractions are dried, weighed, and analyzed for C content. The fractionated material may be used for subsequent procedures and analyses.

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			<td>10847-216</td>
			<td></td>
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			<td>376814</td>
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			<td>Conical rubber gasket for filtering flasks</td>
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			<td>292020001</td>
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			<td>Fisher Scientific</td>
			<td>14-373-25A</td>
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			<td>Glass fiber filter, grade GF/F, 110 mm</td>
			<td>Whatman</td>
			<td>WHA1825110</td>
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			<td>Glass mason jar, 16 oz</td>
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			<td>500 ml beaker or glass weigh dish are also suitable&#160;</td>
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			<td>Polypropylene conical bottle adapter, 250mL</td>
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			<td>369385</td>
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			<td>Porcelain buchner funnel, 90mm</td>
			<td>FisherBrand</td>
			<td>FB966F</td>
			<td></td>
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			<td>Reciprocating shaker, 2-speed</td>
			<td>Eberbach</td>
			<td>E6000.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Sidearm flask, 1000mL</td>
			<td>VWR</td>
			<td>89000-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Polytungstate, crystalline</td>
			<td>Sometu</td>
			<td>SPT-0 or SPT-1, see Discussion for SPT choice</td>
			<td>Shipping via FedEx from Germany</td>
		</tr>
		<tr>
			<td>Swinging bucket centrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>3362020</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 is ideally suited for investigating how soils differ in their particulate and mineral-associated organic matter content. Separating the SOC into these two distinct pools provides an avenue to elucidate the changes in soil C content and stabilization dynamics that may otherwise be unclear when observing trends in bulk soil C content. The further separation of the heavy material (density &#62;1.85 g/cm3) provides additional insight into the changes and trends in soil C stabilization bu...

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Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools

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 ...

This method allows researchers to isolate a small number of functional pools of soil carbon into measurable and modelable fractions as part of soil carbon stabilization research. This technique is simple to perform, allows for greater reproducibility across diverse soil types, and distinguishes different pools of soil carbon based on the degree of association with different minerals. Fractionating soil into light fraction organic matter and heavy fraction mineral components elucidates soil carbon sequestration and stabilization mechanisms for modeling soil carbon turnover rates and mineral organic association pool size.It is easy to lose material during rinsing and transfer, so consistently and carefully accounting for sample mass and carbon content is key to ensuring a minimum of 90%total recovery. Begin by adding 50 grams of the air-dried soil sample sieved to two millimeters into a 250 milliliter conical polypropylene centrifuge tube and record the mass to at least four significant figures. Then add 50 milliliters of the 1.85 gram per cubic centimeter SPT solution to the centrifuge tube and cap the tube tightly.Now, shake the tube vigorously by hand for approximately 60 seconds to break up the non-water-stable aggregates. Next, secure the tightly sealed tube to a platform shaker, and shake for two hours at 40 to 120 rotations per minute. Often, placing the tube on its side aids in soil dispersion by increasing the sloshing force, and reducing the standing height of the soil layer.Periodically remove the tube from the shaker, and shake vigorously by hand to increase the agitation of the denser aggregated material. After removing the tube from the shaker, equalize the centrifuge tube masses across the set of tubes to be centrifuged by carefully adding additional SPT solution wherever required. Ensure to shake vigorously by hand for 30 seconds after adding the SPT solution.Centrifuge the tubes for 10 minutes at 3, 000 g in a swinging bucket centrifuge. After centrifuging, test the density of the supernatant by drawing five milliliters of the same with a pipette and checking the mass by a balance. Adjust the SPT volume if required to achieve the desired density.Shake and centrifuge again if a solution density adjustment was performed. Attach a one liter sidearm flask to a vacuum pump, and place a 110 millimeter glass fiber filter having a 0.7 micron pore size in a porcelain Buchner funnel with an internal diameter of 12 centimeters. Seal the funnel carefully using a conical rubber gasket onto the sidearm flask.Use a vacuum line manifold to run multiple samples simultaneously. Next, attach another one liter sidearm flask to the vacuum pump and place a rubber stopper on it with attached tubing for aspiration having a protruding tube length of about 0.5 meters. Gently aspirate the supernatant, including the suspended material at the top layer, along the sides of the centrifuge tube.Do not touch the pelleted soil surface with the tip of the aspiration tube. If not done carefully, it is easy to aspirate heavy fraction material from the pellet accidentally. To clean the aspiration tube between samples, plunge the tip of the tube very quickly in deionized/distilled, or DDI water, and draw approximately five milliliters of the water through the line by applying the vacuum.Repeat until all the material has been flushed from the vacuum tube. After aspiration, remove the rubber stopper and aspiration tube attachment from the side arm flask, and pour the contents into the top of the Buchner funnel while keeping the vacuum pump switched on. Rinse the flask with DDI water, swirl, and pour the flask contents into the Buchner funnel.Repeat until all residues are removed. Then re-suspend the soil pellet in 50 milliliters of SPT by vigorously shaking the centrifuge tube for 60 seconds by hand to break up the hard pellet at the bottom. Centrifuge the tube for 10 minutes at 3, 000 g.As previously demonstrated, aspirate the supernatant, and collect it in the same flask after filtering through the same Buchner funnel. To wash SPT from the heavy fraction material, add 50 milliliters of DDI water into the centrifuge tube containing the heavy fraction pellet, and shake the tube vigorously by hand for 60 seconds, making sure to break up the hard pellet. Centrifuge the tube for 10 minutes at 3, 000 g.Aspirate the supernatant as described before. Any floating particulates that remain should be added to the funnel with the rest of the light fraction material. Repeat the washing procedure twice.Next, carefully scrape the soil from the centrifuge tube into a clean labeled glass beaker, or jar. Pour enough DDI water into the tube to loosen the remaining soil, and shake the tube before adding the slurry to the glass container. Rinse the tube well using DDI water, and put the washing back into the glass container.Place the glass container in a drying oven set between 40 to 60 degrees Celsius, and dry until a constant dry weight is reached, which typically takes 24 to 72 hours. To ensure the complete removal of the SPT from the light fraction, fill the Buchner funnel containing the light fraction material with DDI water, and filter the contents through glass fiber filters. Once the water has filtered through completely, repeat the washing twice.Remove the funnel from the sidearm flask after turning off the vacuum pump. Now, holding the funnel horizontally over a labeled glass beaker, or jar, gently rinse the particles from the filter using DDI water from a wash bottle. Place the glass container in a drying oven set between 40 to 60 degrees Celsius, and dry until a constant dry weight is reached, which typically takes 24 to 72 hours.To weigh the dry mass of the fractionated materials, take each container and gently scrape all the dried material from it into a plastic weigh boat. Record the mass up to the fourth decimal place before placing the sample into a labeled storage vial, or a bag. Each fraction, as well as the bulk soil, is now ready for analysis.The following figures demonstrate the method effectiveness and insight into soil carbon pools. Here, the recovery of soil organic carbon in different fractions showed distinct effects of the detrital treatments on the light and heavy fractions, especially relative to the effects observed on the bulk content. Additional density fractionation revealed that treatment effects on the mineral-associated organic matter were predominantly confined to the higher density material, but the intermediate fraction show no significant effect, despite greater variability.The carbon-to-nitrogen content of the bulk soil relative to the fractionated pools clearly established the effectiveness of the density fractionation method for separating plant-based particulate material from mineral matter. Pools with densities below 2.20 grams per cubic centimeter responded more to the treatments, as compared to pools with higher densities. The isotopic analysis demonstrated the influence of soil mineralogy on the biogeochemical properties across soil density pools.Further, the analysis of three density pools, as opposed to six, or more, largely captured the isotopic trends. For the light fraction content, oven drying yielded significantly greater carbon loss in the form of dissolved organic carbon, although the amount of loss was insignificant. Secondly, no seasonality was observed in the carbon pool.Diligently verify the SPT solution density throughout to ensure that it remains consistent, and is not diluted by water present in samples. Too low, or too high solution density misrepresents the carbon quantity in samples. Combining 13C, 14C and 15N isotopic analysis and mass spectroscopy provides additional insight into SOC cycling dynamics, while still accounting for site history and soil characteristics.This technique allows researchers to isolate directly measurable pools of carbon differing significantly in turnover time, stabilization mechanism, and chemistry to inform soil carbon modeling more accurately.

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Research

JoVE Journal

Environment

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 ...

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24&#160;hr&#160;after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.

A rainfall simulator was used to apply a consistent rate of uniform rainfall to packed soil boxes in a study of the fate and transport of urea, a nonpoint source environmental contaminant. Under uniform soil and rainfall conditions, antecedent soil moisture content exerted strong control over urea loss in surface runoff.

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

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 ...

Use of Principal Components for Scaling Up Topographic Models to Map Soil Redistribution and Soil Organic Carbon

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it regulates the gravity-driven soil movement induced by runoff and tillage activities. The recent application of Light Detection and Ranging (LiDAR) data holds promise for generating high spatial resolution topographic metrics that can be used to investigate soil property variability. In this study, fifteen topographic metrics derived from LiDAR data were used to investigate topographic impacts on redistribution of soil and spatial distribution of soil organic carbon (SOC). Specifically, we explored the use of topographic principal components (TPCs) for characterizing topography metrics and stepwise principal component regression (SPCR) to develop topography-based soil erosion and SOC models at site and watershed scales. Performance of SPCR models was evaluated against stepwise ordinary least square regression (SOLSR) models. Results showed that SPCR models outperformed SOLSR models in predicting soil redistribution rates and SOC density at different spatial scales. Use of TPCs removes potential collinearity between individual input variables, and dimensionality reduction by principal component analysis (PCA) diminishes the risk of overfitting the prediction models. This study proposes a new approach for modeling soil redistribution across various spatial scales. For one application, access to private lands is often limited, and the need to extrapolate findings from representative study sites to larger settings that include private lands can be important.

Landscape processes are critical components of soil formation and play important roles in determining soil properties and spatial structure in landscapes. We propose a new approach using stepwise principal component regression to predict soil redistribution and soil organic carbon across various spatial scales.

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it ...

Electrostatic Method to Remove Particulate Organic Matter from Soil

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Removing recently deposited and incompletely decomposed plant material from soil samples reduces the influence of temporary seasonal inputs on soil organic carbon measurements. Attraction to an electrostatically charged surface can be used to quickly remove a substantial amount of particulate organic matter.

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of ...