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 achieved following centrifugation. If the fractions do not adequately separate, more of the SPT solution should be added, or the mass of the soil should be reduced. Sandy soils separate quickly, while finely textured soils, such as Oxisols in particular, may remain cloudy for a long time during centrifugation due to a high suspended load of fine particles. When the solutions appear cloudy after centrifugation, either the centrifugation time or speed should be increased. As an alternative, an estimate of the C loss from the suspended sediment may be determined by analyzing the C content of the aspirated solution.
Determining the quality of sodium polytungstate (SPT) to purchase depends on the analyses that will be performed after fractionation. Kramer et al.34 found that commercial SPT may be enriched in high 15N ammonium and, thus, may significantly alter the 15N signature of soil fractions. Thus, a high-purity grade SPT (e.g., SPT-0) should be used to ensure minimal C and N contamination for studies in which the isotopic signatures of soils will be measured. However, SPT with a purity grade that is one step lower (e.g., SPT-1) often has minimal N and C enrichment and is less expensive, thus providing a more economical option when isotopes will not be measured.
To avoid significant error, care must be taken to remove all the light fraction from the solution, which often sticks to the side walls of the centrifuge tubes during aspiration and to the funnel during the subsequent filtration steps. Excess light fraction remaining in the heavy fraction material will result in a low estimate of the light fraction C while simultaneously over-estimating the C content of the heavy fraction. A close examination of the final data may help to identify such errors when fractionating a series of samples with similar soil properties.
The loss of dissolved organic carbon (DOC) to solution is typically small, usually &#60;5% of the total soil C, and cannot be avoided (Figure 7)31. However, the losses of DOC can be far greater when fractionating soils with high soluble C pools, such as those found in some desert environments35. In such instances, the water-extractable DOC pool should also be quantified. Typically, errors stemming from soil mass loss, especially of the light fraction, are far greater than the errors caused by DOC loss.
The adsorption of polytungstate to the soil sample is a chemical possibility, and the extent to which such chemical exchanges occur is currently unknown. Further, the binding affinity of polytungstate is expected to vary across soils with different chemical properties. Currently, the correlation between soil mass loss and SOC loss at the end of the procedure provides a straightforward and logistically feasible form of assurance that any mass gains from polytungstate exchange are negligible for the quantification of the SOC in the fractionated material. If the cumulative mass of the soil fraction is greater than the initial sample mass, or if the mass losses are starkly less than the SOC losses, then the samples may absorb the polytungstate. Additional wash steps should first be performed to attempt to resolve such an issue. If the absorption of polytungstate in the fractionated material persists, additional elemental analysis may be required to verify and correct for an increase in the tungsten content of the fractionated material. Notably, such issues involving the inability to rinse out the polytungstate from the soil material are uncommon.
Although density fractionation ideally separates out the free particulate matter (POM) from the mineral-associated organic matter (MAOM), the presence of water-stable aggregates complicates the interpretation of the density fractionation results. Silt and clay may form strong associations and bind with organic matter, and soil biota, roots, and fungal hyphae can form macroaggregates that help protect organic compounds from microbial decomposition. This aggregate-protected organic matter, often referred to as occluded organic matter, is not MAOM but will be recovered in the heavy fraction (i.e., &#62;1.85 g/cm3). The inclusion of occluded POM is likely to be most impactful on the results and interpretation of the intermediate density fractions. While fractions at a density over ~2.40 g/cm3 are likely to contain organic-poor minerals devoid of occluded organic material and material with a density less than 1.85 g/cm3 is assumed to be mineral-free organic matter, intermediate fractions can be mixes of organic-rich heavy minerals, aggregates, and organic-poor light minerals. To date, no common consensus or pervasive method has emerged for the interpretation of differences in C found in the intermediate fraction material. When reporting such information, we suggest acknowledging the potential influence of occluded organic matter and mineralogy on the results.
Various chemical and physical techniques have been employed to disperse aggregates to facilitate the release of occluded POM, with ultrasonic energy representing the most commonly used method. Unfortunately, there is no one sonication energy level that can cause complete dispersion across all aggregates, as aggregate strength and binding mechanisms vary widely over both soil types and aggregate size classes36. Amelung and Zech36 found that microaggregates (20-250 &#181;m) required more ultrasonic energy to disperse than larger macroaggregates but also found that particulate organic matter was disrupted at these higher energies. Further, sequential sonication with increasing intensity continues to yield free occluded organic matter37, again suggesting that there is no single pool of occluded organic matter and that, at higher sonication levels, much of the separated light fraction material could be a colloidal artifact. Kaiser and Guggenberger30 also demonstrated the potential for sonication to alter the density distribution of the mixtures of the light fraction organic matter with different minerals. While incorporating ultrasonic dispersion techniques during or after soil density fractionation provides unique opportunities to disperse and isolate SOM pools, these studies warrant the consideration of the dispersion efficacy and the destruction of the POM and mineral structures.
The most prevalent alternative method for separating soil C into easily measurable pools is size fractionation. Size fractionation is quick and low-cost relative to density fractionation and may provide similar insights into SOM dynamics given the correlation between clay content and MAOM. Indeed, Poeplau et al.10 found no significant difference in SOM turnover rates for C pools separated by size and density fractionation across three different soils. However, particulate organic matter (POM) with a size equivalent to or smaller than clay is common, meaning size fractionation methods alone are not capable of accurately separating POM from MAOM. The incorporation of POM in fine size fractions can, thus, lead to errors in the elemental and organic chemical analysis in certain soils with a significant amount of fine particulate material15. If a need exists to quantify the C content of mineral material at a specific particle size (e.g., sand, silt, clay), the two methods may be combined by performing a single density fractionation followed by the size fractionation of the heavy fraction material.

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><tbody><tr><td>Aspirator/vacuum tubing 1/4 x 1/2&quot;</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&amp;nbsp;</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&amp;nbsp;</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 but increases the complexity of the procedure and the associated interpretation and is associated with additional costs. Nonetheless, the fractionation of the soil into three or more density pools may elucidate complex trends and chemical differences in soil C pools. As with any soil fractionation procedure, the separation of these soil C pools is imperfect, and the potential influence of such errors and the assumptions of the method should be recognized when reporting the results. Finally, practitioners should be aware of the variety of soil fractionation methods that exist and their unique strengths and weaknesses (see reviews and comparisons provided by von L&#252;tzow et al.9 and Poeplau et al.10). Many of these soil fractionation methods are not mutually exclusive and may be appropriately combined to improve or validate analyses of soil C dynamics.
Choosing the number of density fractions to be used is the critical first step before beginning the fractionation procedure. While multiple pools can always be mathematically combined to produce a single light and a single heavy fraction to compare results to other studies, fractionating the heavy fraction into more than one pool adds significant time and expense. Pierson et al.33 quantified the change in SOC across three density pools following a long-term detrital manipulation study. When combining the two heavier pools (Figure 2), the effects of the detrital treatments were distinct between the light (&#60;1.85 g/cm3) and heavy (&#62;1.85 g/cm3) fractions, especially relative to the effects observed from the bulk SOC content. By performing the additional density fractionation at 2.40 g/cm3 (Figure 3), it could be further determined that the treatment effects on MAOM were predominantly confined to the higher-density material (&#62;2.4 g/cm3). Finally, the reported C:N content of the bulk soil relative to the density fractionated pools (Figure 4) presents a clear demonstration of the effectiveness of the density fractionation method for separating plant-based particulate material from mineral matter with relatively low C:N content.
After 50 years of detrital manipulations, Lajtha et al.22 used six sequential density fractionations to closely examine the detrital influences on SOC stabilization and destabilization (Figure 5). By isolating seven SOC pools, the authors were able to observe a greater nuance in the POM and MAOM response following litter addition and removal treatments. Pools with densities &#60;2.20 g/cm3 were more responsive to the treatment, in agreement with the long-standing expectation of a positive relationship between litter input and SOC accumulation. However, the response of SOC pools with densities &#62;2.20 g/cm3 was less pronounced, and specifically for the litter removal treatment, an opposite, negative relationship was identified between the inputs and the SOC (i.e., the inputs were reduced, but the SOC content of the fraction increased). By performing subsequent analyses of 14C, the contributions and losses of more recent C inputs for each pool were ascertained, providing mechanistic insight into the detrital control of the formation and destabilization pathways for MAOM.
Combining isotopic analysis with sequential density fractionation provides additional avenues to investigate the intricacies of SOM dynamics. Yet, investigators should take care to consider the influence of mineralogy on the density fractionation results. Unique mineral structure and reactivity cause inherent differences in particle density distributions between minerals. As an example, Sollins et al.18 examined the isotopic and chemical characteristics of multiple density pools in four forested soils with disparate parent materials and morphologies (Figure 6). The contrasting mineralogy between the four soils contributed heavily to the observed differences in 13C, 14C, and 15N for each density fraction. Without the consideration of mineralogy, such results may be misinterpreted with respect to SOC formation and stabilization dynamics. Finally, returning to the practicality of performing additional density separations, little additional information was gained by Sollins et al.18 from the analysis of six density pools as opposed to only three (Figure 6).
Helbling et al.31 determined the effect of seasonality on the light fraction content of forested soils, as well as the effect of soil drying treatment on the loss of C to the soluble pool (Figure 7). Two significant results emerged from this work. First, while oven-drying the soil yielded significantly greater dissolved organic C loss to the SPT solution, the amount of C lost was insignificant. Secondly, there did not appear to be any seasonality to the light fraction C pool, meaning that the soil sample collection timing did not influence the fractionation results. However, the results may be expected to differ across soils and environments given the differences in POM stocks and decomposition rates.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64759/64759fig01.jpg" /> 
Figure 1: Light fraction aspiration apparatus. Schematic of the vacuum apparatus for the aspiration of the light fraction. *Parafilm may be used to secure and seal the junction between the pipet tip and the vacuum tube. **Cutting the pipet tip at an angle may be useful to increase the size of the tip opening, as well as for applying close suction to the side walls of the centrifuge tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64759/64759fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64759/64759fig02.jpg" /> 
Figure 2: Comparison of the carbon content for bulk, light, and heavy fraction soil. Soil was collected from the Detrital Input and Removal Treatment (DIRT) plots in the H.J. Andrews Experimental Forest after 20 years of treatment (n = 3). The mineral-associated C for the root removal treatments (NR, NI) was significantly increased, contrasting with the observed effects on bulk C content. Further, the fractionated results show that the increase in bulk C from the double wood (DW) treatment was derived from an increase in the light fraction C. The error bars represent the standard error. This figure has been modified from Pierson et al.33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64759/64759fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64759/64759fig03.jpg" /> 
Figure 3: Comparison of the intermediate and heavy fraction pools. Soil was collected from the Detrital Input and Removal Treatment (DIRT) plots in the H.J. Andrews Experimental Forest after 20 years of treatment (n = 3). The results demonstrate the findings from the isolation of an intermediate fraction (1.85-2.40 g/cm3) and a heavier fraction (&#62;2.40 g/cm3). The intermediate fraction C content showed greater variability, and no treatment effects were significant. The error bars represent the standard error. This figure has been modified from Pierson et al.33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64759/64759fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64759/64759fig04.jpg" /> 
Figure 4: Carbon to nitrogen ratios for bulk, light, and heavy fraction soil. Soil was collected from the Detrital Input and Removal Treatment (DIRT) plots in the H.J. Andrews Experimental Forest after 20 years of treatment (n = 3). Given the high C:N content of plant tissue relative to soil minerals, the observed difference in C:N content between the light and heavy fractions clearly demonstrates the capability of soil density fractionation to separate particulate organic matter from bulk soil. The error bars represent the standard error. This figure has been modified from Pierson et al.33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64759/64759fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64759/64759fig05.jpg" /> 
Figure 5: Sequential density fractionation of soil following 50 years of detrital manipulations (n = 4). Separating the soil into seven density fractions provided insight into the nature of the C loading on soil minerals. The double litter treatment, which increased soil C, led to the C loading of mineral material in the 2.00-2.40 g/cm3 fraction, as shown by the change in the fraction C concentrations relative to the control. The losses of soil C from the no litter treatment were greatest in the soil fractions with densities between 1.85-2.20 g/cm3. The error bars represent the standard error. This figure has been modified from Lajtha et al.22. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64759/64759fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64759/64759fig06.jpg" /> 
Figure 6: Effects of mineralogy on soil density pools. Soils with unique morphology were collected from four forested sites. The isotopic analysis demonstrates how soil mineralogy may influence biogeochemical properties across soil density pools. Further, in this instance, the analysis of three density pools, as opposed to six or more, largely captured the trends within and between the different isotopic signatures. This figure has been modified from Sollins et al.18. The original data and graphs are shown in the column with more than six pools; the data were recalculated and displayed to demonstrate the results for only three pools. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64759/64759fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64759/64759fig07.jpg" /> 
Figure 7: Sample collection and preparation effects on soil density fractionation. Helbling et al.31 found that oven-dried soil often yields greater dissolved organic carbon relative to air-dried, field moist, and leached soil. Across sample collection seasons, the proportion of light to heavy fraction mass was not significantly different. This figure has been modified from Helbling et al.31. The error bars represent the standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64759/64759fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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Nanyang Technological University

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2.8K Views.  Oregon State University. Deze methode stelt onderzoekers in staat om een klein aantal functionele pools van bodemkoolstof te isoleren in meetbare en modeleerbare fracties als onderdeel van onderzoek naar koolstofstabilisatie in de bodem. Deze techniek is eenvoudig uit te voeren, zorgt voor een grotere reproduceerbaarheid over verschillende bodemtypen en onderscheidt verschillende pools van bodemkoolstof op basis van de mate van associatie met verschillende mineralen. Het fractioneren van de bodem in lichte fractie or...

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

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

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 ...

Methods of Soil Resampling to Monitor Changes in the Chemical Concentrations of Forest Soils

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental changes. Repeated sampling to monitor these changes in forest soils is a relatively new practice that is not well documented in the literature and has only recently been broadly embraced by the scientific community. The objective of this protocol is therefore to synthesize the latest information on methods of soil resampling in a format that can be used to design and implement a soil monitoring program. Successful monitoring of forest soils requires that a study unit be defined within an area of forested land that can be characterized with replicate sampling locations. A resampling interval of 5 years is recommended, but if monitoring is done to evaluate a specific environmental driver, the rate of change expected in that driver should be taken into consideration. Here, we show that the sampling of the profile can be done by horizon where boundaries can be clearly identified and horizons are sufficiently thick to remove soil without contamination from horizons above or below. Otherwise, sampling can be done by depth interval. Archiving of sample for future reanalysis is a key step in avoiding analytical bias and providing the opportunity for additional analyses as new questions arise.

Repeated soil sampling has recently been shown to be an effective way to monitor forest soil change over years and decades. To support its use, a protocol is presented that synthesizes the latest information on soil resampling methods to aid in the design and implementation of successful soil monitoring programs.

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental ...

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 ...

Improving Infrared Spectroscopy Characterization of Soil Organic Matter with Spectral Subtractions

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic bonds that constitute the organic component of soils. However, the relatively low organic matter content of soils (commonly &#60; 5% by mass) and absorbance overlap of mineral and organic functional groups in the mid-infrared (MIR) region (4,000-400 cm-1) engenders substantial interference by dominant mineral absorbances, challenging or even preventing interpretation of spectra for SOM characterization. Spectral subtractions, a post-hoc mathematical treatment of spectra, can reduce mineral interference and enhance resolution of spectral regions corresponding to organic functional groups by mathematically removing mineral absorbances. This requires a mineral-enriched reference spectrum, which can be empirically obtained for a given soil sample by removing SOM. The mineral-enriched reference spectrum is subtracted from the original (untreated) spectrum of the soil sample to produce a spectrum representing SOM absorbances. Common SOM removal methods include high-temperature combustion (&#39;ashing&#39;) and chemical oxidation. Selection of the SOM removal method carries two considerations: (1) the amount of SOM removed, and (2) absorbance artifacts in the mineral reference spectrum and thus the resulting subtraction spectrum. These potential issues can, and should, be identified and quantified in order to avoid fallacious or biased interpretations of spectra for organic functional group composition of SOM. Following SOM removal, the resulting mineral-enriched sample is used to collect a mineral reference spectrum. Several strategies exist to perform subtractions depending on the experimental goals and sample characteristics, most notably the determination of the subtraction factor. The resulting subtraction spectrum requires careful interpretation based on the aforementioned methodology. For many soil and other environmental samples containing substantial mineral components, subtractions have strong potential to improve FTIR spectroscopic characterization of organic matter composition.

SOM underlies many soil functions and processes, but its characterization by FTIR spectroscopy is often challenged by mineral interferences. The described method can increase the utility of SOM analysis by FTIR spectroscopy by subtracting mineral interferences in soil spectra using empirically obtained mineral reference spectra.

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic ...

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 ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...

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 ...

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

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

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

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

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools

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