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 volume to 1 L with DDI. 
	NOTE: Carbon recovery using a solution density &#60;1.85 g/cm3 may under-recruit light fraction carbon derived from particulate organic matter17,18, thus misrepresenting the quantity of carbon in the sample. Thus, an SPT solution density of 1.85 g/cm3 is suggested8,17 in order to be more inclusive of carbon associated with particulate organic matter for a typical soil sample (i.e., most sand, silt, and clay loams with C content &#60;10 %).</li>
	<li>To make 1 L of SPT solution with a density of 2.40 g/cm3, dissolve 1,803 g of solid SPT in approximately 500 mL of DDI water. Stir the solution until the SPT has fully dissolved, and then bring the solution volume to 1 L with DDI. 
	NOTE: Beyond the potential use for soil fractionation, a solution with a density greater than 1.85 g/cm3 is often required for the adjustment of the SPT solution at later steps in the protocol (see step 3.2). If an extra 2.40 g/cm3 solution is leftover, the solution may be diluted to 1.85 g/cm3 with deionized water and used for soil fractionation.</li>
	<li>Prior to use in fractionation, analyze the SPT for C and N content. Perform this analysis by using a solid or liquid elemental analyzer (example methods: ISO 10694:1995, ISO 20236:2018).
	<ol>
		<li>Perform a 1:100 dilution of the solution from step 1.1 for the liquid elemental analyzers to reduce the deterioration of the elemental scrubbers and catalysts. The tolerance for C and N contamination in the SPT solution will depend on the sample and the subsequent uses of the soil fractions. Typically, an SPT solution with a C and N content &#60;1 ppm and &#60;0.1 ppm, respectively, is considered suitable for use, as solutions such as this present minimal capacity for altering the much larger soil C and N pools.</li>
	</ol>
	</li>
</ol>
2. Dissolution of soil in SPT
<ol>
	<li>Add 50 g of soil that is air-dried and sieved to 2 mm to a 250 mL conical polypropylene centrifuge tube. Record the mass to at least four significant figures. Do not use oven-dried soil as this may increase the soluble carbon due to heat-induced cell lysis31. 
	NOTE: Field moist soil may be used31, but further adjustment is required in the later steps to maintain the target density of the SPT solution. Sieving the soil material to 2 mm is recommended to remove large material that may skew the fractionation results, such as rocks and woody debris.
	<ol>
		<li>Adjust the soil mass to ensure an adequate mass of each fraction is recovered to avoid significant error in the quantification. The most common reason for mass adjustment is low POM content (e.g., &#60;2% of the total soil mass). For such soils, provide additional soil mass to accurately quantify the POM recovery. Overall, it is acceptable to adjust the soil mass for each individual sample, since changing the sample mass will not alter the proportion of POM to MAOM. However, it is often useful to use a consistent mass to aid the balancing of the centrifuge.</li>
		<li>Treat soils rich in carbonates to remove inorganic carbonates prior to fractionation32.</li>
	</ol>
	</li>
	<li>Add 50 mL of 1.85 g/cm3 density SPT to the centrifuge tube, and replace the lid tightly. As with the soil amounts, adjust the SPT volume as needed. In POM-rich surface soils (e.g., many temperate forest soils), use a larger ratio of soil to SPT (e.g., 30 g of soil to 60 mL of SPT) to achieve adequate separation of the light and heavy fraction materials.</li>
	<li>Shake the tube vigorously by hand for ~60 s to break up non-water-stable aggregates. The forceful collision of the soil aggregates with the side walls of the centrifuge tube is desired, meaning simply vortexing the solution may be insufficient.</li>
	<li>Secure the tube to a platform shaker. Often, placing the tube on its side aids in soil dispersion by increasing the sloshing force of the solution and reducing the standing height of the soil layer. Take care that the tube is tightly sealed, and shake for 2 h at 40-120 rpm. Periodically remove the tube from the shaker and shake vigorously by hand to increase the agitation of the denser aggregated material.</li>
</ol>
3. Performing a coarse soil fractionation
<ol>
	<li>Remove the tube from the shaker. Equalize the centrifuge tube masses by carefully adding additional SPT solution to reach a consistent mass across the set of tubes to be centrifuged, ensuring to shake vigorously by hand for 30 s after adding the SPT solution. Centrifuge for 10 min at 3,000 x g in a swinging bucket centrifuge.</li>
	<li>Before aspirating the sample, test the density of the supernatant by drawing off 5 mL of the solution with a pipette and checking the mass on a balance. Adjust the SPT density as necessary to achieve the desired density. Shake and centrifuge again if a solution density adjustment was performed.</li>
	<li>Attach a 1 L sidearm flask to a vacuum pump. Place a 110 mm glass fiber filter (0.7 &#181;m pore size) in a 12 cm internal diameter (ID) porcelain Buchner funnel. Seal the funnel carefully using a conical rubber gasket onto the sidearm flask. 
	NOTE: The glass fiber filters should be pre-washed in a drying oven at 150 &#176;C and rinsed with DDI before use.</li>
	<li>Set up one additional 1 L sidearm flask attached to the vacuum. Place a rubber stopper in the top of the flask with a ~0.5 m protruding length of tubing attached for aspiration. 
	NOTE: It may be helpful to attach a plastic tip (such as a 5 mL disposable pipet tip, with the end clipped off at an angle) to the end of the aspiration tubing to improve the control of the suction during aspiration (see Figure 1).</li>
	<li>Gently aspirate the supernatant and suspended material that has settled at the top layer of the solution along the sides of the centrifuge tube, being careful not to touch the tip of the aspiration tube to the pelleted soil surface underneath. 
	NOTE: If any soil pellet material (heavy fraction) is mistakenly aspirated along with the suspended (light fraction) material, the fractionation procedure should be repeated. If unnoticed, such an error will result in a heavier than expected light fraction mass with a lower than expected C content, which may be evident through the data analysis of samples with similar soil properties.
	<ol>
		<li>To clean the aspiration tube between samples, plunge the tip of the tube quickly (e.g., submerge for 0.1 s) in DDI water, and draw ~5 mL of DDI water through the line with the vacuum pump on. Repeat until all the material has been flushed from the vacuum tube.</li>
		<li>Remove the rubber stopper and aspiration tube attachment from the sidearm flask, and pour the contents into the top of the Buchner funnel with the vacuum pump on.</li>
		<li>Rinse the flask with DDI water, swirl, and pour the flask contents into the Buchner funnel. Repeat until all the residue adhered to the sides of the flask is removed.</li>
	</ol>
	</li>
	<li>Add 50 mL of SPT to the centrifuge tube, and shake vigorously by hand for 60 s (or use a shaker table if the soil does not rapidly disperse), making sure to break up the hard pellet at the bottom of the tube so that all the residue is resuspended. Centrifuge for 10 min at 3,000 x g.</li>
	<li>Repeat step 3.5. Pour the flask contents into the same Buchner funnel as used in step 3.5.2.</li>
	<li>Add 50 mL of SPT to the centrifuge tube, and shake vigorously by hand, making sure to break up the hard pellet at the bottom of the tube. Centrifuge for 10 min at 3,000 x g.</li>
	<li>Repeat step 3.5. Pour the flask contents into the same Buchner funnel as used in step 3.5.2.</li>
</ol>
4. Additional density separation(s) using higher-density SPT
NOTE: If performing more than one additional density fraction, the subsequent fractionations must be performed in order of increasing density. Here, steps for isolating using 1.85-2.4 g/cm3 and &#62;2.4 g/cm3 density SPT are shown.
<ol>
	<li>Add 50 mL of 2.4 g/cm3 SPT to the centrifuge tube containing the &#62;1.85 g/cm3 soil material from step 3. Shake vigorously by hand (&#62;60 s), making sure to break up the hard pellet at the bottom of the tube. Centrifuge for 10 min at 3,000 x g.</li>
	<li>Before aspirating the sample, test the density of the supernatant by drawing off 5 mL of the solution with a pipette and checking the mass on a balance. Adjust the SPT density as necessary to achieve the desired density. Shake and centrifuge again if a solution density adjustment was performed.</li>
	<li>Repeat step 3 using a 2.4 g/cm3 SPT solution in place of the 1.85 g/cm3 SPT solution used previously. At the end of step 3, the material isolated in the Buchner funnel will have a density between 1.85-2.4 g/cm3, while the material remaining in the centrifuge tube will have a density &#62;2.4 g/cm3.</li>
</ol>
5. Washing the SPT from the heavy and light fraction samples
NOTE: The following washing steps must be performed for all the fractionated material. If the SPT solution is not completely rinsed from the material, the corresponding fraction weights will be inaccurate.
<ol>
	<li>Add 50 mL of DDI water to the centrifuge tube with the heavy fraction material, and shake vigorously by hand (60 s), making sure to break up the hard pellet at the bottom of the tube. Centrifuge for 10 min at 3,000 x g.</li>
	<li>Aspirate as in step 3.5. At this point, all the light fraction material should have been removed. Dispose of the clear aspirate in a waste bucket instead of adding it to the filter funnel.</li>
	<li>Repeat steps 5.1-5.2 twice. Before finally aspirating the solution in the tube, use a transfer pipette to draw off 25 mL of the supernatant, and check the density by dividing the solution weight by the volume to ensure that the SPT has been adequately removed from the sample. If the density is &#60;1.01 g/mL, proceed to the next step. If the density is 1.01 g/mL or greater, perform additional water washes as above until the density is less than 1.01 g/mL.</li>
	<li>To ensure the complete removal of the SPT from the light fraction, fill each Buchner funnel with DDI water, and filter the contents through glass fiber filters. Once the water has filtered through completely, repeat this twice more. If the soil is high in organic matter, filtration may take up to 48 h.</li>
</ol>
6. Collection of the heavy fraction material
<ol>
	<li>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; replace the cap and shake, and then add the slurry to the glass container. Rinse all the remaining soil from the centrifuge tube, and transfer into the glass container using deionized water.</li>
	<li>Place the glass container in a drying oven set between 40-60 &#176;C. Dry until a constant dry weight is reached, typically for 24-72 h.</li>
</ol>
7. Collection of the light fraction material
<ol>
	<li>Turn off the vacuum pump, and remove the funnel from the sidearm flask. Holding the funnel horizontally over a labeled glass beaker or jar, gently rinse the particles from the filter using a DDI water wash bottle. 
	NOTE: It may be necessary to gently scrape the filter using a spatula and to rinse both sides of the filter to remove all the residue.</li>
	<li>Place the glass container in the drying oven set between 40-60 &#176;C. Dry until a constant dry weight is reached, typically for 24-72 h.</li>
</ol>
8. Weighing the dry mass of the fractionated material
<ol>
	<li>Gently scrape all the dried material from each container into a plastic weigh boat. Record the mass up to the fourth decimal place. Place the sample into a labeled storage vial or bag.</li>
	<li>Repeat for all the dried samples.</li>
</ol>
9. Data collection and analysis for total organic carbon
<ol>
	<li>Follow the analysis procedures in accordance with the instrument to be used for the analysis of the elemental C content (e.g., ISO 10694:1995). 
	NOTE: Grinding the dried fraction material into a fine powder is a common practice to ensure the homogeneity of the fractionated sample before elemental analysis.</li>
	<li>Ensure that the cumulative mass of all the fractions is equal to at least ~90% of the original soil sample mass. If the losses of material are &#62;10%, additional replicate fractionations are recommended.</li>
	<li>Quantify the cumulative recovery of soil organic carbon (SOC) in the fractions. Losses of SOC may not correlate perfectly with mass loss due to the disproportionate loss of fraction material and the loss of dissolved organic carbon. Yet, losses of SOC should also be &#60;10 % of the initial SOC in the soil sample.</li>
</ol>

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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. G., Lajtha, K., Thomas, G., Sollins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contamination+effects+on+soil+density+fractions+from+high+N+or+C+content+sodium+polytungstate.">Contamination effects on soil density fractions from high N or C content sodium polytungstate.</a> Biogeochemistry. 92 (1-2), 177-181 (2009).</li><li>Throop, H. L., Lajtha, K., Kramer, M. <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+and+13C+reveal+changes+in+soil+carbon+following+woody+encroachment+in+a+desert+ecosystem.">Density fractionation and 13C reveal changes in soil carbon following woody encroachment in a desert ecosystem.</a> Biogeochemistry. 112 (1-3), 409-422 (2013).</li><li>Amelung, W., Zech, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimisation+of+organic+matter+disruption+during+particle-size+fractionation+of+grassland+epipedons.">Minimisation of organic matter disruption during particle-size fractionation of grassland epipedons.</a> Geoderma. 92 (1-2), 73-85 (1999).</li><li>Kaiser, M., Asefaw Berhe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+sonication+affect+the+mineral+and+organic+constituents+of+soil+aggregates?-A+review.">How does sonication affect the mineral and organic constituents of soil aggregates?-A review.</a> Journal of Plant Nutrition and Soil Science. 177 (4), 479-495 (2014).</li></ol>

The authors have nothing to disclose.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>Aspirator/vacuum tubing 1/4 x 1/2&#34;</td>
			<td>Kimble</td>
			<td>10847-216</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical polypropylene centrifuge tube, 250mL</td>
			<td>Thermo Scientific</td>
			<td>376814</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical rubber gasket for filtering flasks</td>
			<td>DWK Life Sciences</td>
			<td>292020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Double flat ended stainless steel spatula/scraper</td>
			<td>Fisher Scientific</td>
			<td>14-373-25A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass fiber filter, grade GF/F, 110 mm</td>
			<td>Whatman</td>
			<td>WHA1825110</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass mason jar, 16 oz</td>
			<td>Ball Corporation</td>
			<td></td>
			<td>500 ml beaker or glass weigh dish are also suitable&#160;</td>
		</tr>
		<tr>
			<td>Polypropylene conical bottle adapter, 250mL</td>
			<td>Beckman Coulter</td>
			<td>369385</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcelain buchner funnel, 90mm</td>
			<td>FisherBrand</td>
			<td>FB966F</td>
			<td></td>
		</tr>
		<tr>
			<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...

Watch this Scientific Journal Video about Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools at JoVE.com

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 ...

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2.6K Views.  Oregon State University. 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.

Video: Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools

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 ...

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 ...

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...