The development of this method was supported by the Fond d&#39;Investissement (FINV) of the Faculty of Geosciences at the University of Lausanne. We acknowledge the Uganda National Council for Science and Technology and Uganda Wildlife Authority for granting us permission to collect research samples. The authors further wish to thank Prof. Thierry Adatte for CHN and XRD analyses. We are grateful to Prof. Erika Marin-Spiotta for providing initial training in classical density fractionation. We also thank laboratory manager Laetitia Monbaron for her assistance in securing supplies and equipment.

The authors have nothing to disclose.

The success of CSDF experiments hinges on the selection of appropriate parameters for the method, so that fractions of relatively homogeneous composition may be isolated. Key considerations in the selection of fractionation parameters are discussed below.
The fLF represents organic matter for which interaction with minerals is minimal. Extraction of this fraction is delicate, since the mixing of soil with the dense solution may already break-up some macroaggregates. There are, however, indications that the organic matter present in macroaggregates may be more similar to the fLF stricto sensu than to the oLF released by high-energy sonication18. Some authors have even proposed a low-energy sonication step to isolate the pool of free and weakly mineral-interacting organic matter, termed &#39;intra-aggregate particulate organic matter&#39;, iPOM54.
For the release of occluded organic matter, different techniques exist to disrupt soil aggregates. The most widespread are sonication, agitation with glass beads and the use of chemical dispersants33,62,63. Sonication was chosen here because the output energy can be finely controlled and is believed to distribute more or less uniformly in the sample. By precluding the need to use chemical dispersants, sonication may be considered as relatively preservative towards organo-mineral complexes22, 33. The dispersion step, however, remains one of the most delicate operations. On the one hand, a weak dispersion will leave the aggregates intact and may lead to an over-estimate of hF SOC; on the other hand, a highly vigorous dispersion step could cause re-distribution of SOC across the fractions by partial destruction of organo-mineral complexes. Weak organic-sand associations may be particularly vulnerable to this process. Since occlusion within aggregates and surface sorption are processes occurring along a continuum2, no perfect solution exists. Therefore, the energy level of sonication needs to be adjusted thoughtfully according to the soil properties. Kaiser and Berhe64 have published a very helpful review that proposes a strategy to minimize artifacts caused by ultrasound when dispersing soils.
Reported sonication energies range from 60 to 5,000 J&#183;mL-1. Several research groups have reported that 100 J&#183;mL-1 could be sufficient to destroy macroaggregates and effectively disperse sandy soils, while 500 J&#183;mL-1 would destroy large microaggregates and provide a reasonable dispersion of reactive soils63,65,66,67,68. In physical fractionation schemes, complete dispersion of silt and clay-sized aggregates may not be necessary, since the protection mechanism is likely to become indistinguishable from sorptive stabilization in these size ranges. A reasonable objective of dispersion prior to size or density fractionation may be to disrupt macro- (&#62; 250 &#181;m) and large micro- (&#62; 53 &#181;m) aggregates. Energies of 100 J&#183;mL-1 (sandy soils) to 200 J&#183;mL-1 (loamy soils) may be appropriate choices. An energy of 200 J&#183;mL-1 may already extract a portion of microbial metabolites (supposedly mineral-associated)69, thus the use of higher sonication energies should be subject to caution. However, mineralogically reactive soils with cemented aggregates could require up to 500 J&#183;mL-1 to disperse. It is essential that the dispersion energy be adjusted to match each soil type as well as study objectives. Finally, it is important to remember that even after supposedly complete ultrasonic dispersion, clay-sized microaggregates are likely to persist70.
A difficulty with harmonizing physical fractionation techniques resides in the heterogeneity found in soils, in particular in their mineral composition. The choice of dense solutions should be made on the basis of known or inferred soil mineralogy, with the ultimate goal to isolate fractions which are as homogeneous as possible.
In the article, the dense solution used was SPT - pH 371, 72. The low pH minimizes losses of soluble organic compounds. However, density fractionation may be performed with different dense solutions. Historically, organic liquids were used (tetrabromoethane, tetrachloromethane), but were gradually abandoned at the profit of inorganic salts (sodium iodide, SPT) because of the toxicity of halogenated hydrocarbons and the inherent contamination of soil organics. Nowadays, SPT is the preferred solution because its density can be adjusted between 1.0 to 3.1 g&#183;cm-3, it can be recycled and has a low toxicity (unless ingested)22, 50. Main manufacturers offer a range of SPT grades differing in the level of carbon and nitrogen contamination. For density fractionation of soils, the purest grade is recommended, particularly if the fractions are to be analyzed for isotopic composition.
A solution of density 1.6 g&#183;cm-3 has classically been used to separate light organic from mineral-associated fractions - see for example Golchin et al.21. While some authors have suggested that a density of 1 g&#183;cm-3 (water) could be sufficient to extract most of the light fraction73, 74, others have proposed higher density cut-offs such as 1.62 or 1.65 g&#183;cm-3 based on the idea that some organic components could show densities up to 1.60 g&#183;cm-3&#160;33,75,76. Densities as high as 1.85 g&#183;cm-3 have even been employed50. When selecting a density to separate light from heavy fractions, it should be noted that no perfect solution exists. Indeed, lower densities risk attributing some &#39;light&#39; organics to the heavy fractions, while higher densities risk including some minerals into the light fractions. This last effect can be detected when observing the carbon content of the light fractions, with a % SOC lower than 40-45% indicating some degree of mineral contamination.
For heavy fractions, preliminary analysis such as XRD can provide insight into the mineralogy of the bulk sample60 and help define density cut-offs capable of distinguishing between the main mineral components of a soil, keeping in mind that high organic loadings will lower the density of a mineral compared to its theoretical value. Similarly, for particle-size separation, a textural analysis77,78 can help set appropriate limits. Particle-size separation is a particularly attractive addition to simple density fractionation whenever sequential density fractionation is difficult. This is the case for instance for soils containing large amounts of oxyhydroxides and low-activity clays, which result in sample dispersion and prevent clear separations in heavy liquids. A particle-size separation step is also indicated to separate minerals of similar densities but different sizes (e.g., quartz and illite).
Free calcium ions will react with SPT to form insoluble Ca metatungstate. The procedure is thus inappropriate for alkaline soils containing large amounts of poorly crystalline, pedogenic carbonates. Small amounts of low-reactivity carbonates do not interfere with the fractionation as long as the samples are not left in contact with SPT for too long. Ca metatungstate precipitates will lead to an over-estimate of fraction masses. If LFs are run on an elemental analyzer for C concentration, the problem will be discovered but the fractionation will be compromised.
In addition to these technical difficulties, the fundamental limitation of CSDF (or of any physical fractionation scheme) stems from the fact that reactive minerals in soils rarely occur as discrete separates, but instead as coatings and cements. The occurrence of highly sorptive but very thin coatings on otherwise unreactive minerals (such as quartz) can lead to a biased view of organo-mineral associations. Caution is thus required when interpreting results, particularly for soils whose reactivity is dominated by poorly crystalline and oxide phases. Further characterization of fractions can help alleviate such ambiguities. Nevertheless, detailed physical fractionation methods such as CSDF have an unmatched ability to gain insight into the composition of naturally-occurring organo-mineral complexes. Such insight is expected to yield new understanding of the biogeochemistry of the largest pool of organic matter in soils, the mineral-associated one.

Soil is a complex system which contains elements of geological and biological origin. The study of their inter-relation is a cornerstone of our understanding of ecosystem function1. In particular, organo-mineral interactions are thought to play a key role in soil organic matter (SOM) dynamics2. Unravelling SOM dynamics is presently a very active research area for several reasons. A soil with high SOM stocks will tend to show good intrinsic fertility and may also constitute an environmentally valuable carbon sequestration opportunity3,4.
Organic matter in soil is highly heterogeneous, with some components turning over in the space within a few hours while others may persist for thousands of years5. The determinants of this heterogeneity remain a controversial topic, but association with the mineral matrix is thought to be particularly important6,7, especially for subsoil horizons8. As a result, mineral phases known to closely associate with organic components are receiving increasing interest9,10,11.
Soils contain a wide range of minerals with qualitatively and quantitatively varying sorptive potential towards SOM. Minerals with large specific surface areas and/or highly reactive surfaces have been shown to have a high sorption capacity for organic compounds4,12. In soils, secondary minerals such as high-activity phyllosilicates (e.g., smectites), iron oxyhydroxides and poorly crystalline aluminosilicates have all been shown to engage significantly in the sorptive preservation of some organic compounds13,14,15,16,17. Separating soil into fractions differing in mineralogy could thus help isolate organic matter pools with relative functional homogeneity.
The aim of this paper is to present a methodology to isolate organo-mineral complexes according to composition, which then facilitates the study of their properties. The method combines size and density fractionation to physically separate bulk soil into a sequence of fractions of different composition. Combined size and density fractionation (CSDF) integrates two effective physical fractionation approaches (particle size separation and density separation). The combination of these two approaches brings improved resolution to our understanding of organo-mineral associations in soil.
There are many different approaches (chemical, physical and / or biochemical) that can be used to specify fractions in a bulk soil sample18,19. Simple density fractionation is a physical separation which has been widely used by soil scientists to study SOM dynamics (see for instance Grunwald et al., 2017 and references therein)20. In its classical form, simple density fractionation separates materials lighter than a given cutoff (generally 1.6 to 1.85 g&#183;cm-3) - the light fraction (LF) from heavier materials - the heavy fraction (hF). The LF is sometimes further split into free light fraction (fLF) and occluded light fraction (oLF)21.
In many soils, the largest SOM pool is found in the hF22. SOM in the hF is generally thought to be more stable than that in the LF23, yet it has been shown to retain a high compositional and probably, functional heterogeneity18. This points to the need to further separate the hF into more homogeneous subfractions, with the view of isolating pools of SOM with distinct biogeochemical properties (such as residence time or functionality). Sequential density fractionation, as described by Sollins et al. (2009)24, has indeed proved to be a successful method; yet a separation done solely on the basis of density runs the risk of overlooking differences arising from variation in grain size and thus specific surface area. For instance, kaolinite has approximately the same density as quartz but may be separated on the basis of its size mode (Table 1). CSDF includes consideration of grain size and improves the resolution of the fractionation.
SOM fractionation based on physical, chemical or biochemical properties has a long history. Physical methods such as CSDF are based on physical attributes of soil components, such as size (of particles or aggregates) or density. Chemical methods include selective extractions of specific compounds or classes of compounds, as well as chemical oxidation. Biochemical methods rely on microbial oxidation under various experimental conditions. Chemical and biochemical methods are based on different principles and have different objectives compared to physical methods but are nevertheless briefly reviewed below.
The alkaline extraction (with sodium hydroxide for example) ranks among the earliest methods used to chemically isolate the organic component of soils6. Examples of more modern, chemical methods for SOM fractionation include i) alkaline extraction with Na-pyrophosphate aimed at isolating SOM bound to minerals; ii) acid hydrolysis (HCl) aimed at quantifying old, persistent SOM; and iii) selective oxidation of SOM with chemical agents aimed at attacking free or labile SOM2. While these methods can be useful to gain insight into functionally different organic matter pool, they suffer from several limitations. First, the extractions can be imperfect or incomplete. For example, the classical alkaline method extracts only 50-70% of soil organic carbon (SOC)6. Second, fractionation products may not be representative of the SOM found in situ and may be difficult to categorize5. Third, these chemical methods only offer limited insight into the organo-mineral interaction since many of them do not preserve the original association between organics and minerals.
Biochemical extraction including incubations experiments are used mainly to study labile and reactive SOM (see Strosser32 for a review of biochemical methods). Incubation experiments can be thought of as a measure of biochemical oxygen demand and is intuitively well-suited to the determination of bioavailable organic substrates. However, the need for long incubation times in conditions that differ from the field (temperature, humidity, physical disturbance, absence of new inputs) makes the extrapolation to in-situ SOM dynamics delicate.
Compared to chemical or biochemical methods which are believed to be either transformative or destructive, physical fractionation techniques can be considered as more preservative22 (with the important exception of soluble organic compounds, which are lost during the procedure). At their best, physical soil fractions can be thought of as a &#39;snapshot&#39; of solid-phase soil components as present in the field and could thus relate more directly to SOM dynamics in situ33. Moreover, the non-destructive nature of the technique means that the fractions can be subsequently characterized using a variety of analyses or further fractionated according to chemical or biochemical methods.
Physical fractionation of soils is not a recent idea. Scientific literature about physical separation techniques dates back to the mid-20 century. Applications of density fractionation were reported as early as 196534, 35. During the same period and in the following decades, publications about the dynamics of SOM and its interaction with minerals were already becoming widespread amongst soil scientists36,37,38,39.
Separation based on density, aggregate size or particle size are the most common physical separation methods used currently. One of the main challenges of physical separation is the isolation of homogenous functional SOM pools, as defined by turn-over rate, size or other indicator of function. Combining separation methods or criteria, as in CSDF, may help bring functional resolution to soil fractions; indeed, these methods seem to be used more and more in combination18,40,41,42,43. By combining sequential density separation, able to yield fractions with different organic matter content and mineralogical composition, with size separation, which accounts for differences attributable to specific surface area, CSDF holds the promise of yielding insight into the diversity and function of organo-mineral associations in soil.
CSDF aims to physically fractionate bulk soil samples into fractions of relative mineralogical and textural homogeneity. The density and particle size cut-offs, as well as the dispersion energy used here have been selected based on our soil type, but these parameters may be adapted depending on the samples to be fractioned and the purpose of the study. In this example, we have chosen to use one dispersion step, two density and one size cut-offs, resulting in the separation of the bulk soil into 6 fractions (Table 2). Figure 1 gives a conceptual overview of the method. The materials being fractioned here are tropical soils, but the method can be applied to any soil type as well as sediments. CSDF is generally used as a preparatory step before further analyses, even though the distribution of materials among fractions can be very informative in and of itself. When applied to soils, CSDF yields fractions differing in (1) mineral composition (mineralogy and texture) and (2) SOM concentration and composition.

<table><tbody><tr><td>&lt;strong&gt;Fractionation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium polytungstate</td><td>Sometu</td><td></td><td>SPT 0 (low C and N) is recommended. Lower grade polytungstate may contaminate samples.</td></tr><tr><td>Hydrometers (1-1.5, 1.5-2, 2-2.5, 2.5-3 g.cm&lt;sup&gt;-3&lt;/sup&gt;)</td><td>Allafrance</td><td></td><td>Calibrated at 20 &amp;deg;C, e.g. 3050FG250/20-qp</td></tr><tr><td>Vortex mixer</td><td>Fisher</td><td></td><td>Fixed speed standard vortex mixer, e.g. 02-215-410</td></tr><tr><td>Sonifier</td><td>VWR</td><td></td><td>Qsonica LLC - Q500 system with standard probe 4220</td></tr><tr><td>Sonifier stand</td><td>VWR</td><td></td><td>Large clamp stand</td></tr><tr><td>Sonifier enclosure</td><td>VWR</td><td></td><td>Soundproof cabinet (optional)</td></tr><tr><td>Swinging-bucket centrifuge</td><td>Beckman</td><td></td><td>Able to achieve speeds of 4000 g or more, fitted with rotor accommodating 50 mL Falcon tubes</td></tr><tr><td>High-speed centrifuge with fixed angle rotor</td><td>Beckman</td><td></td><td>Able to achieve speeds of 7500 g or more, fitted with rotor accommodating 250 mL bottles</td></tr><tr><td>50 mL centrifuge Falcon tubes</td><td>Corning</td><td></td><td>e.g. 352070</td></tr><tr><td>250 mL centrifuge bottles</td><td>Beckman</td><td></td><td>Polycarbonate bottles (e.g. 352070) are recommended because they are clearer than other plastics.</td></tr><tr><td>Vaccum filtration units</td><td>Semadeni</td><td></td><td>Polusulfone reusable units, e.g. 3029</td></tr><tr><td>Polypropylene hose</td><td>Semadeni</td><td></td><td>To connect the filtration unit to vaccuum source</td></tr><tr><td>Ultrafiltration disks, 0.45 &amp;micro;m pore size</td><td>Millipore</td><td></td><td>e.g. HAWP04700</td></tr><tr><td>Dessicator cabinet</td><td>Fisher scientific</td><td></td><td>3 shelves, e.g. 305317-0120</td></tr><tr><td>Drierite absorbent indicating</td><td>Millipore</td><td></td><td>Blue drierite, e.g. 10276750</td></tr><tr><td>Scintillation vials</td><td>Fisher scientific</td><td></td><td>HDPE - separated cap 20mL, e.g. 12341599</td></tr><tr><td>150 mL aluminium boats (smooth sides)</td><td>Fisher scientific</td><td></td><td>Any model.</td></tr><tr><td>Laboratory oven</td><td>Fisher scientific</td><td></td><td>Any model.</td></tr><tr><td>&lt;strong&gt;Recycling SPT column&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cation exchange resin</td><td>Sigma-Aldrich</td><td></td><td>Dowex&amp;reg; Marathon&amp;trade; C sodium form, strongly acidic, 20-50 mesh</td></tr><tr><td>Activated charcoal</td><td>Sigma-Aldrich</td><td></td><td>Darco S-51, 4-12 mesh</td></tr><tr><td>Glass wool</td><td>Fisher scientific</td><td></td><td>Pyrex</td></tr><tr><td>Filter paper, 2.5 &amp;micro;m pore size</td><td>Sigma-Aldrich</td><td></td><td>Whatman grade 42, e.g. WHA1442150</td></tr><tr><td>Hydrogen peroxide</td><td>Sigma-Aldrich</td><td></td><td>Reagent grade.</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td></td><td>Reagent grade.</td></tr><tr><td>Polycarbonate 1000mL graduated cylinder</td><td>Semadeni</td><td></td><td>Any model.</td></tr><tr><td>Stand and clamp</td><td>Sigma-Aldrich</td><td></td><td>Size L - 2-prong</td></tr><tr><td>Polypropylene hose</td><td>Semadeni</td><td></td><td>Any model.</td></tr><tr><td>Polypropylene hose clamp</td><td>Semadeni</td><td></td><td>Any model.</td></tr><tr><td>Polypropylene funnels</td><td>Semadeni</td><td></td><td>Any model.</td></tr><tr><td>Polypropylene bottle (1L, 2L)</td><td>Semadeni</td><td></td><td>Any model.</td></tr><tr><td>Heating plate</td><td>Fisher scientific</td><td></td><td>Any model.</td></tr></tbody></table>

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).

Watch this Scientific Journal Video about Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions at JoVE.com

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

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 ...

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14.7K Views.  University of Lausanne. Deze methode is een interessante benadering om organo-mineralen interacties te bestuderen omdat het de organische stof van de bodem verfraait volgens zijn associatie met verschillende mineralen. Het is een fysiek fractioneringsproces, wat betekent dat het de chemie van de breuken niet verandert. Als gevolg hiervan vertelt de analyse van de fractie ons iets over de natuurlijke samenstelling van organo-minerale complexen.Deze methode is echt nuttig als we proberen om meer te leren over h...

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

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

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 ...

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 ...

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

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 ...

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 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 ...

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

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