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

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

<ol>
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I., 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=Persistence+of+soil+organic+matter+as+an+ecosystem+property.">Persistence of soil organic matter as an ecosystem property.</a> Nature. 478 (7367), 49-56 (2011).</li><li>Billings, S. A., 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+carbon+is+not+just+for+soil+scientists:+Measurement+recommendations+for+diverse+practitioners.">Soil organic carbon is not just for soil scientists: Measurement recommendations for diverse practitioners.</a> Ecological Applications. 31 (3), 02290 (2021).</li><li>von L&#252;tzow, M., 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=SOM+fractionation+methods:+Relevance+to+functional+pools+and+to+stabilization+mechanisms.">SOM fractionation methods: Relevance to functional pools and to stabilization mechanisms.</a> Soil Biology and Biochemistry. 39 (9), 2183-2207 (2007).</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=Isolating+organic+carbon+fractions+with+varying+turnover+rates+in+temperate+agricultural+soils+-+A+comprehensive+method+comparison.">Isolating organic carbon fractions with varying turnover rates in temperate agricultural soils - A comprehensive method comparison.</a> Soil Biology and Biochemistry. 125, 10-26 (2018).</li><li>Heckman, K., Lawrence, C. R., Harden, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sequential+selective+dissolution+method+to+quantify+storage+and+stability+of+organic+carbon+associated+with+Al+and+Fe+hydroxide+phases.">A sequential selective dissolution method to quantify storage and stability of organic carbon associated with Al and Fe hydroxide phases.</a> Geoderma. 312, 24-35 (2018).</li><li>Frosteg&#229;rd, &. #. 1. 9. 7. ;., Tunlid, A., B&#229;&#229;th, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biomass+measured+as+total+lipid+phosphate+in+soils+of+different+organic+content.">Microbial biomass measured as total lipid phosphate in soils of different organic content.</a> Journal of Microbiological Methods. 14 (3), 151-163 (1991).</li><li>Plante, A. F., Conant, R. T., Paul, E. A., Paustian, K., Six, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+hydrolysis+of+easily+dispersed+and+microaggregate-derived+silt-+and+clay-sized+fractions+to+isolate+resistant+soil+organic+matter.">Acid hydrolysis of easily dispersed and microaggregate-derived silt- and clay-sized fractions to isolate resistant soil organic matter.</a> European Journal of Soil Science. 57 (4), 456-467 (2006).</li><li>Eusterhues, K., Rumpel, C., K&#246;gel-Knabner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+soil+organic+matter+isolated+via+oxidative+degradation.">Stabilization of soil organic matter isolated via oxidative degradation.</a> Organic Geochemistry. 36 (11), 1567-1575 (2005).</li><li>Moni, C., Derrien, D., Hatton, P. -. J., Zeller, B., Kleber, 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+fractions+versus+size+separates:+does+physical+fractionation+isolate+functional+soil+compartments.">Density fractions versus size separates: does physical fractionation isolate functional soil compartments.</a> Biogeosciences. 9 (12), 5181-5197 (2012).</li><li>Lavallee, J. M., Soong, J. L., Cotrufo, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conceptualizing+soil+organic+matter+into+particulate+and+mineral-associated+forms+to+address+global+change+in+the+21st+century.">Conceptualizing soil organic matter into particulate and mineral-associated forms to address global change in the 21st century.</a> Global Change Biology. 26 (1), 261-273 (2020).</li><li>Sollins, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+C+and+N+stabilization+in+a+forest+soil:+Evidence+from+sequential+density+fractionation.">Organic C and N stabilization in a forest soil: Evidence from sequential density fractionation.</a> Soil Biology and Biochemistry. 38 (11), 3313-3324 (2006).</li><li>Sollins, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+density+fractionation+across+soils+of+contrasting+mineralogy:+evidence+for+both+microbial-+and+mineral-controlled+soil+organic+matter+stabilization.">Sequential density fractionation across soils of contrasting mineralogy: evidence for both microbial- and mineral-controlled soil organic matter stabilization.</a> Biogeochemistry. 96 (1-3), 209-231 (2009).</li><li>Crow, S. E., Swanston, C. W., Lajtha, K., Brooks, J. R., Keirstead, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+fractionation+of+forest+soils:+methodological+questions+and+interpretation+of+incubation+results+and+turnover+time+in+an+ecosystem+context.">Density fractionation of forest soils: methodological questions and interpretation of incubation results and turnover time in an ecosystem context.</a> Biogeochemistry. 85 (1), 69-90 (2007).</li><li>Cotrufo, M. F., Ranalli, M. G., Haddix, M. L., Six, J., Lugato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+carbon+storage+informed+by+particulate+and+mineral-associated+organic+matter.">Soil carbon storage informed by particulate and mineral-associated organic matter.</a> Nature Geoscience. 12 (12), 989-994 (2019).</li><li>Hatton, P. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+litter-derived+N+to+soil+mineral-organic+associations:+Evidence+from+decadal+15N+tracer+experiments.">Transfer of litter-derived N to soil mineral-organic associations: Evidence from decadal 15N tracer experiments.</a> Organic Geochemistry. 42 (12), 1489-1501 (2012).</li><li>Lajtha, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+to+particulate+versus+mineral-associated+soil+carbon+after+50+years+of+litter+manipulation+in+forest+and+prairie+experimental+ecosystems.">Changes to particulate versus mineral-associated soil carbon after 50 years of litter manipulation in forest and prairie experimental ecosystems.</a> Biogeochemistry. 119 (1-3), 341-360 (2014).</li><li>Yeasmin, S., Singh, B., Johnston, C. T., Sparks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+carbon+characteristics+in+density+fractions+of+soils+with+contrasting+mineralogies.">Organic carbon characteristics in density fractions of soils with contrasting mineralogies.</a> Geochimica et Cosmochimica Acta. 218, 215-236 (2017).</li><li>Wagai, R., Kajiura, M., Asano, M., Hiradate, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+soil+organo-mineral+assemblage+examined+by+sequential+density+fractionation+with+and+without+sonication:+Is+allophanic+soil+different.">Nature of soil organo-mineral assemblage examined by sequential density fractionation with and without sonication: Is allophanic soil different.</a> Geoderma. 241-242, 295-305 (2015).</li><li>Volk, M., Bassin, S., Lehmann, M. F., Johnson, M. G., Andersen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C+isotopic+signature+and+C+concentration+of+soil+density+fractions+illustrate+reduced+C+allocation+to+subalpine+grassland+soil+under+high+atmospheric+N+deposition.">13C isotopic signature and C concentration of soil density fractions illustrate reduced C allocation to subalpine grassland soil under high atmospheric N deposition.</a> Soil Biology and Biochemistry. 125, 178-184 (2018).</li><li>Yonekura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+organic+matter+dynamics+in+density+and+particle-size+fractions+following+destruction+of+tropical+rainforest+and+the+subsequent+establishment+of+Imperata+grassland+in+Indonesian+Borneo+using+stable+carbon+isotopes.">Soil organic matter dynamics in density and particle-size fractions following destruction of tropical rainforest and the subsequent establishment of Imperata grassland in Indonesian Borneo using stable carbon isotopes.</a> Plant and Soil. 372 (1-2), 683-699 (2013).</li><li>Virto, I., Moni, C., Swanston, C., Chenu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnover+of+intra-+and+extra-aggregate+organic+matter+at+the+silt-size+scale.">Turnover of intra- and extra-aggregate organic matter at the silt-size scale.</a> Geoderma. 156 (1-2), 1-10 (2010).</li><li>Poeplau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+a+soil+organic+carbon+fractionation+method+to+derive+RothC+carbon+pools.">Reproducibility of a soil organic carbon fractionation method to derive RothC carbon pools.</a> European Journal of Soil Science. 64 (6), 735-746 (2013).</li><li>Cerli, C., Celi, L., Kalbitz, K., Guggenberger, G., Kaiser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+light+and+heavy+organic+matter+fractions+in+soil+-+Testing+for+proper+density+cut-off+and+dispersion+level.">Separation of light and heavy organic matter fractions in soil - Testing for proper density cut-off and dispersion level.</a> Geoderma. 170, 403-416 (2012).</li><li>Kaiser, K., Guggenberger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+hydrous+aluminium+and+iron+over+density+fractions+depends+on+organic+matter+load+and+ultrasonic+dispersion.">Distribution of hydrous aluminium and iron over density fractions depends on organic matter load and ultrasonic dispersion.</a> Geoderma. 140 (1-2), 140-146 (2007).</li><li>Helbling, E., Pierson, D., Lajtha, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+of+soil+carbon+loss+during+soil+density+fractionation:+Laboratory+loss+or+seasonally+variable+soluble+pools.">Sources of soil carbon loss during soil density fractionation: Laboratory loss or seasonally variable soluble pools.</a> Geoderma. 382, 114776 (2021).</li><li>Nelson, D. W., Sommers, L. E., Sparks, D. L., Page, A. L., Helmke, P. A., Loeppert, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+carbon,+organic+carbon,+and+organic+matter.">Total carbon, organic carbon, and organic matter.</a> Methods of Soil Analysis: Part 3 Chemical Methods. , 539-579 (2015).</li><li>Pierson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mineral+stabilization+of+soil+carbon+is+suppressed+by+live+roots,+outweighing+influences+from+litter+quality+or+quantity.">Mineral stabilization of soil carbon is suppressed by live roots, outweighing influences from litter quality or quantity.</a> Biogeochemistry. 154 (3), 433-449 (2021).</li><li>Kramer, M. 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. 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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">
	<tbody>
		<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...

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Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools

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

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

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

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Research

JoVE Journal

Environment

2.4K Görüntüleme.  Oregon State University. Bu yöntem, araştırmacıların toprak karbon stabilizasyonu araştırmasının bir parçası olarak az sayıda fonksiyonel toprak karbon havuzunu ölçülebilir ve modellenebilir fraksiyonlara ayırmalarını sağlar. Bu tekniğin uygulanması kolaydır, çeşitli toprak tiplerinde daha fazla tekrarlanabilirliğe izin verir ve farklı minerallerle ilişki derecesine bağlı olarak farklı toprak karbon havuzlarını ayırt eder. Toprağın hafif fraksiyonlu organik maddeye ve ağır fraksiy...