Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools

Summary

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.

Abstract

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.

Introduction

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 warming^2,3. 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 environments^4,5.

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 soil^1,7. 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 Lützow 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 C^13,14. 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 minerals^17,18,19; 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 (>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 surfaces^28,29,30.

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 stabilization^18,19.

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.

Protocol

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.

1. 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 <1.85 g/cm³ may under-recruit light fraction carbon derived from particulate organic matter^17,18, thus misrepresenting the quantity of carbon in the sample. Thus, an SPT solution density of 1.85 g/cm³ is suggested^8,17 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 <10 %).
2. 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.
3. 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).
   1. 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 <1 ppm and <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.

2. Dissolution of soil in SPT

1. 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.
   1. 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., <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.
   2. Treat soils rich in carbonates to remove inorganic carbonates prior to fractionation³².
2. 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.
3. 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.
4. 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.

3. Performing a coarse soil fractionation

1. 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.
2. 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.
3. Attach a 1 L sidearm flask to a vacuum pump. Place a 110 mm glass fiber filter (0.7 µm 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 °C and rinsed with DDI before use.
4. 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).
5. 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.
   1. 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.
   2. 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.
   3. 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.
6. 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.
7. Repeat step 3.5. Pour the flask contents into the same Buchner funnel as used in step 3.5.2.
8. 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.
9. Repeat step 3.5. Pour the flask contents into the same Buchner funnel as used in step 3.5.2.

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 >2.4 g/cm³ density SPT are shown.

1. Add 50 mL of 2.4 g/cm³ SPT to the centrifuge tube containing the >1.85 g/cm³ soil material from step 3. 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.
2. 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.
3. 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 >2.4 g/cm³.

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.

1. 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.
2. 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.
3. 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 <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.
4. 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.

6. Collection of the heavy fraction material

1. 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.
2. Place the glass container in a drying oven set between 40-60 °C. Dry until a constant dry weight is reached, typically for 24-72 h.

7. Collection of the light fraction material

1. 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.
2. Place the glass container in the drying oven set between 40-60 °C. Dry until a constant dry weight is reached, typically for 24-72 h.

8. Weighing the dry mass of the fractionated material

1. 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.
2. Repeat for all the dried samples.

9. Data collection and analysis for total organic carbon

1. 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.
2. 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 >10%, additional replicate fractionations are recommended.
3. 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 <10 % of the initial SOC in the soil sample.

Results

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 >1.85 g/cm³) provides additional insight into the changes and trends in soil C stabilization bu...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Aspirator/vacuum tubing 1/4 x 1/2"	Kimble	10847-216
Conical polypropylene centrifuge tube, 250mL	Thermo Scientific	376814
Conical rubber gasket for filtering flasks	DWK Life Sciences	292020001
Double flat ended stainless steel spatula/scraper	Fisher Scientific	14-373-25A
Glass fiber filter, grade GF/F, 110 mm	Whatman	WHA1825110
Glass mason jar, 16 oz	Ball Corporation		500 ml beaker or glass weigh dish are also suitable
Polypropylene conical bottle adapter, 250mL	Beckman Coulter	369385
Porcelain buchner funnel, 90mm	FisherBrand	FB966F
Reciprocating shaker, 2-speed	Eberbach	E6000.00
Sidearm flask, 1000mL	VWR	89000-386
Sodium Polytungstate, crystalline	Sometu	SPT-0 or SPT-1, see Discussion for SPT choice	Shipping via FedEx from Germany
Swinging bucket centrifuge	Beckman Coulter	3362020