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. 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…

Representative 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

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