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>Fractionation</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-3)</td>
			<td>Allafrance</td>
			<td></td>
			<td>Calibrated at 20 &#176;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 &#181;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>Recycling SPT column</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cation exchange resin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Dowex&#174; Marathon&#8482; 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 &#181;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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14.5K Views.  University of Lausanne. 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.

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 ...

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

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 ...

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 ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Sampling Soils in a Heterogeneous Research Plot

Soils are highly heterogeneous. In general, the number of soil samples required for soil research has always been determined arbitrarily and the associated accuracy is unknown. Here, we present a detailed protocol for efficient and clustered soil sampling in a research plot and, relying on a pilot sampling using this design, for demonstrating soil spatial heterogeneity and informing reasonable sample sizes and associated accuracy for future study. The protocol mainly comprises four steps: sampling design, field collection, soil analysis, and geostatistical analysis. The step-by-step procedure is modified according to former publications. Two examples will be presented to demonstrate contrasting spatial distributions of soil organic carbon (SOC) and soil microbial biomass carbon (MBC) under different management practices. In addition, we present a strategy to determine the sample size requirement (SSR) given a certain level of accuracy based on the plot-level coefficient of variation (CV). The field sampling protocol and the quantitative determination of the sample size will assist researchers in seeking feasible sampling strategies to meet research needs and resources' availability.

The traditional soil-sampling procedure determines the number of soil samples arbitrarily. Here, we provide a simple yet efficient clustered soil-sampling design to demonstrate soil spatial heterogeneity and quantitatively determine the number of soil samples required and the associated sampling accuracy.

Soils are highly heterogeneous. In general, the number of soil samples required for soil research has always been determined arbitrarily and the ...

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...