Utilizing Soil Density Fractionation to Separate Distinct Soil Carbon Pools

This method allows researchers to isolate a small number of functional pools of soil carbon into measurable and modelable fractions as part of soil carbon stabilization research. This technique is simple to perform, allows for greater reproducibility across diverse soil types, and distinguishes different pools of soil carbon based on the degree of association with different minerals. Fractionating soil into light fraction organic matter and heavy fraction mineral components elucidates soil carbon sequestration and stabilization mechanisms for modeling soil carbon turnover rates and mineral organic association pool size.

It is easy to lose material during rinsing and transfer, so consistently and carefully accounting for sample mass and carbon content is key to ensuring a minimum of 90%total recovery. Begin by adding 50 grams of the air-dried soil sample sieved to two millimeters into a 250 milliliter conical polypropylene centrifuge tube and record the mass to at least four significant figures. Then add 50 milliliters of the 1.85 gram per cubic centimeter SPT solution to the centrifuge tube and cap the tube tightly.

Now, shake the tube vigorously by hand for approximately 60 seconds to break up the non-water-stable aggregates. Next, secure the tightly sealed tube to a platform shaker, and shake for two hours at 40 to 120 rotations per minute. Often, placing the tube on its side aids in soil dispersion by increasing the sloshing force, and reducing the standing height of the soil layer.

Periodically remove the tube from the shaker, and shake vigorously by hand to increase the agitation of the denser aggregated material. After removing the tube from the shaker, equalize the centrifuge tube masses across the set of tubes to be centrifuged by carefully adding additional SPT solution wherever required. Ensure to shake vigorously by hand for 30 seconds after adding the SPT solution.

Centrifuge the tubes for 10 minutes at 3, 000 g in a swinging bucket centrifuge. After centrifuging, test the density of the supernatant by drawing five milliliters of the same with a pipette and checking the mass by a balance. Adjust the SPT volume if required to achieve the desired density.

Shake and centrifuge again if a solution density adjustment was performed. Attach a one liter sidearm flask to a vacuum pump, and place a 110 millimeter glass fiber filter having a 0.7 micron pore size in a porcelain Buchner funnel with an internal diameter of 12 centimeters. Seal the funnel carefully using a conical rubber gasket onto the sidearm flask.

Use a vacuum line manifold to run multiple samples simultaneously. Next, attach another one liter sidearm flask to the vacuum pump and place a rubber stopper on it with attached tubing for aspiration having a protruding tube length of about 0.5 meters. Gently aspirate the supernatant, including the suspended material at the top layer, along the sides of the centrifuge tube.

Do not touch the pelleted soil surface with the tip of the aspiration tube. If not done carefully, it is easy to aspirate heavy fraction material from the pellet accidentally. To clean the aspiration tube between samples, plunge the tip of the tube very quickly in deionized/distilled, or DDI water, and draw approximately five milliliters of the water through the line by applying the vacuum.

Repeat until all the material has been flushed from the vacuum tube. After aspiration, remove the rubber stopper and aspiration tube attachment from the side arm flask, and pour the contents into the top of the Buchner funnel while keeping the vacuum pump switched on. Rinse the flask with DDI water, swirl, and pour the flask contents into the Buchner funnel.

Repeat until all residues are removed. Then re-suspend the soil pellet in 50 milliliters of SPT by vigorously shaking the centrifuge tube for 60 seconds by hand to break up the hard pellet at the bottom. Centrifuge the tube for 10 minutes at 3, 000 g.

As previously demonstrated, aspirate the supernatant, and collect it in the same flask after filtering through the same Buchner funnel. To wash SPT from the heavy fraction material, add 50 milliliters of DDI water into the centrifuge tube containing the heavy fraction pellet, and shake the tube vigorously by hand for 60 seconds, making sure to break up the hard pellet. Centrifuge the tube for 10 minutes at 3, 000 g.

Aspirate the supernatant as described before. Any floating particulates that remain should be added to the funnel with the rest of the light fraction material. Repeat the washing procedure twice.

Next, carefully scrape the soil from the centrifuge tube into a clean labeled glass beaker, or jar. Pour enough DDI water into the tube to loosen the remaining soil, and shake the tube before adding the slurry to the glass container. Rinse the tube well using DDI water, and put the washing back into the glass container.

Place the glass container in a drying oven set between 40 to 60 degrees Celsius, and dry until a constant dry weight is reached, which typically takes 24 to 72 hours. To ensure the complete removal of the SPT from the light fraction, fill the Buchner funnel containing the light fraction material with DDI water, and filter the contents through glass fiber filters. Once the water has filtered through completely, repeat the washing twice.

Remove the funnel from the sidearm flask after turning off the vacuum pump. Now, holding the funnel horizontally over a labeled glass beaker, or jar, gently rinse the particles from the filter using DDI water from a wash bottle. Place the glass container in a drying oven set between 40 to 60 degrees Celsius, and dry until a constant dry weight is reached, which typically takes 24 to 72 hours.

To weigh the dry mass of the fractionated materials, take each container and gently scrape all the dried material from it into a plastic weigh boat. Record the mass up to the fourth decimal place before placing the sample into a labeled storage vial, or a bag. Each fraction, as well as the bulk soil, is now ready for analysis.

The following figures demonstrate the method effectiveness and insight into soil carbon pools. Here, the recovery of soil organic carbon in different fractions showed distinct effects of the detrital treatments on the light and heavy fractions, especially relative to the effects observed on the bulk content. Additional density fractionation revealed that treatment effects on the mineral-associated organic matter were predominantly confined to the higher density material, but the intermediate fraction show no significant effect, despite greater variability.

The carbon-to-nitrogen content of the bulk soil relative to the fractionated pools clearly established the effectiveness of the density fractionation method for separating plant-based particulate material from mineral matter. Pools with densities below 2.20 grams per cubic centimeter responded more to the treatments, as compared to pools with higher densities. The isotopic analysis demonstrated the influence of soil mineralogy on the biogeochemical properties across soil density pools.

Further, the analysis of three density pools, as opposed to six, or more, largely captured the isotopic trends. For the light fraction content, oven drying yielded significantly greater carbon loss in the form of dissolved organic carbon, although the amount of loss was insignificant. Secondly, no seasonality was observed in the carbon pool.

Diligently verify the SPT solution density throughout to ensure that it remains consistent, and is not diluted by water present in samples. Too low, or too high solution density misrepresents the carbon quantity in samples. Combining 13C, 14C and 15N isotopic analysis and mass spectroscopy provides additional insight into SOC cycling dynamics, while still accounting for site history and soil characteristics.

This technique allows researchers to isolate directly measurable pools of carbon differing significantly in turnover time, stabilization mechanism, and chemistry to inform soil carbon modeling more accurately.