Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Summary

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.

Abstract

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.

Introduction

Due to its vital roles in carbon (C) and nutrient cycling and its sensitivity to soil change, soil LOC is an important parameter to measure as an indicator of soil organic matter quality. Forests and agroecosystems to a large degree depend on the mineralization of nutrients in soil organic matter as a source of nutrients. Management activities can change the pool size and turnover rate of soil organic C, resulting in changes in nutrient supply¹. Soil organic C consists of two primary fractions of recalcitrant C, which has turnover rates of several thousand years, and LOC, which has turnover rates from a few weeks to a few years^2,3,4. Soil labile C consists of readily decomposable substrates such as microbial biomass C, low-molecular-weight compounds (amino acids, simple carbohydrates) from plant rhizodeposition, and decomposition byproducts and leachates from plant litter^1,4,5. Because soil labile C is readily decomposable, it is highly sensitive to management practices and natural phenomena that disturb or alter soil⁶. Soil labile C serves as the primary energy source for soil microorganisms in the decomposition of organic matter⁷. As such, LOC impacts nutrient cycling to a greater degree than does stable forms of soil organic C⁸. Soil microorganisms are also responsible for the majority of heterotrophic respiration that occurs during decomposition of recalcitrant soil organic matter facilitated by the priming effect of LOC^9,10,11. This respiration plays a substantial role in global C cycles because soil organic C is approximately double that of atmospheric C¹¹.

As a result of its importance in terrestrial ecosystems, several methods have been developed to estimate soil LOC. These methods can be delineated into three general classifications: physical, chemical, and biochemical. Densitometric separation methods are physical methods that consist of separating soil organic C into heavy or light fractions or into coarse and fine particulate organic C^12,13,14,15. Separation methods are relatively easy to perform, but they do not often produce consistent results because these fractions vary with soil type mineral composition, plant material size and density, and soil aggregate consistency^13,15. Separation methods also produce only quantitative information about LOC¹⁵.

Several chemical methods are available for LOC estimation. Aqueous extraction of organic carbon is relatively easy to perform, and the methods often provide easily reproducible results. However, these extractions do not involve the whole spectrum of available substrates for microorganisms¹⁵. Several oxidation methods for chemical fractionation of soil organic C have been developed. Oxidation methods have the advantage of characterizing the quantity and quality of labile organic C, although some methods require work with hazardous chemicals and there is variability among the methods in reproducibility of results¹⁵. The acid hydrolysis extraction method is another type of chemical fractionation procedure that can measure the quantity and quality of LOC, but results of this method do not facilitate interpretation of its biological properties^13,15.

Biochemical methods for interpretation of soil LOC have been developed. Labile organic C can be measured as CO₂ released by microorganisms in respiration assays. These assays provide estimates of true mineralizable organic matter, but typically only the most labile compounds are mineralized during the assays¹⁵. Soil microbial biomass C measured by fumigation-incubation¹⁶ and fumigation-extraction¹⁷ has been used to develop inferences about LOC. However, these procedures provide estimates of C in microbial biomass rather than LOC. Both fumigation procedures include subtraction of values from non-fumigated soil to determine microbial biomass C, but it has been suggested that values obtained without subtraction of non-fumigated soil provide a measure of labile organic fractions of C in addition to microbial biomass¹⁸.

The sequential fumigation-incubation (SFI) procedure¹³ for measuring LOC is a biochemical method adapted from the fumigation-incubation procedure¹⁶ for soil microbial biomass C measurement. The SFI method has some advantages relative to other methods of estimating LOC. A conceptual basis for the method is that LOC is the microbially degradable C that governs microbial growth and that LOC is physically accessible and chemically degradable by soil microorganisms. Under field conditions, microbial growth is typically limited by carbon availability, nutrient availability, available pore space, and/or predation. These factors are nearly eliminated by fumigation, creating unimpeded conditions for microbial growth. No nutrients are removed during the incubation period of the method. Over the course of multiple fumigation and incubation cycles, microbial growth becomes limited by C quantity and quality (lability)¹³. The accumulated CO₂ respired during the incubation cycles is used to extrapolate LOC with a simple negative exponential model^11,13,19. The potential C turnover rate can also be derived from the slope of the exponential model, so the SFI method has the advantage over most other LOC methods of simultaneously estimating the concentrations and potential turnover rate of LOC¹¹. For other methods, information on the potential turnover rates of LOC can only be ascertained if tracers such as ¹⁴C are used¹³. The SFI method is thus a relatively simple and inexpensive technique for obtaining measurements of both LOC and its potential turnover rates.

Protocol

1. Collect Soil to Get Samples Representative of Conditions within the Experimental Area and within Experimental Units²⁰

1. Identify any differences in site properties such as slope and soil properties including texture, bulk density, pH, organic horizon depth, and/or nutrient concentrations. Identify any differences in vegetation type within plots. Use known or published estimates of coefficients of variation for site properties to estimate the number of samples required to attain a pre-specified relative error.
2. Sample soil using an auger or other collection device in a pattern based on site and experimental unit conditions.
   1. For homogeneous conditions, use a random sampling pattern within each experimental unit.
      1. Assign sample points at either completely random locations within the experimental unit or in a zigzag pattern.
      2. Sample soil at each random point or at points assigned in a zigzag pattern. Brush aside organic matter from the surface of mineral soil before using the auger or other collection device to excavate soil sample.
         NOTE: The SFI method was developed using soil horizons from the Oa and below¹³. Further testing is necessary if horizons above the Oa can be tested using the SFI method.
      3. Combine all samples collected within the experimental unit into a single container and physically mix the individual samples within the container to create a composite sample for each experimental unit.
   2. For heterogeneous conditions, which are much more common, use a systematic sampling pattern within each experimental unit.
      1. Sample soils along the transect in the center of each experimental unit such that the distance between sample points within the transect is smaller than the distance needed to represent variability within the experimental units.
      2. Sample soils along multiple transects within each experimental unit that form a grid pattern in relatively large experimental units or experimental units with multiple sources of variability.
      3. Combine all samples collected along each transect into a single container and physically mix the individual samples within the container to create a composite sample for each transect.

2. Prepare Soil for the SFI Assay

1. Place samples in an ice-pack filled cooler immediately after collection in the field.
2. Upon arrival at the facility at which samples are to be stored until analysis, place samples in a refrigerator at 4 °C until sample preparation and SFI procedures are conducted.
3. Sieve soil samples through a 6.4 mm x 6.4 mm mesh sieve. Clean the mesh with water between each sample to prevent contamination between samples.
4. For each sample, measure three 100 g subsamples and place the 100 g subsamples in a 250 ml beaker. Cover each beaker with Parafilm and leave them on a countertop for 10 days at 25 °C.

3. Take Subsamples for Oven-dry Weight Determination

1. At the end of the 10 day pre-incubation of soil samples, remove Parafilm from each sample.
2. Record the weight of an aluminum weigh boat. Take 1 g of soil from all samples and place in weigh boat.
3. Record the weight of the moist soil and weigh boat.
4. Place the weigh boats with soil in an oven at 105 °C. After the samples reach a constant weight, which is typically after 48 hr, record weights of the weigh boats and soil.
5. Subtract weigh boat weight from the weights taken of the moist soil and dry soil within the weigh boat to get moist and dry soil weight. Derive the dry:moist soil ratio by dividing dry soil weight by the moist soil weight.

4. Fumigate Soil Samples

1. Place a damp paper towel in the bottom of at least two (more may be necessary depending on the number of samples) 10.5 L glass vacuum desiccators with porcelain plates.
2. For all samples, weigh 30 g of soil into three separate glass vials. Use vials large enough to hold 40 g of soil and narrow enough to fit within a 40 mm opening if the incubation container design described in section 5 is used.
3. If using labeling tape to identify each 30 g soil subsample, use pencil because fumigation degrades ink.
4. Place two of the three 30 g subsamples for each soil sample into a vacuum desiccator for fumigation and one subsample into a vacuum desiccator that will not conduct fumigation.
5. In a 100 ml beaker, place a layer of boiling stones sufficient to cover the bottom of the beaker.
6. Pour 50 ml of ethanol-free chloroform (CHCl₃) into the 100 ml beaker with a layer of boiling stones. Place the 100 ml beaker with boiling stones and CHCl₃ in the center of a desiccator filled with the 30 g soil subsamples. Conduct this step under a fume hood.
7. Under a fume hood, use a vacuum to boil the CHCl₃ to fumigate two sets of subsamples per soil sample.
   1. Connect the vacuum to the vacuum desiccator with vacuum tubing. Start the vacuum and watch as CHCl₃ begins to boil.
   2. Allow CHCl₃ to boil for 30 sec and disconnect the vacuum tubing from the desiccator to allow air to flow back into the desiccator. This step promotes CHCl₃ gas entry into the soil samples. Repeat twice.
   3. Perform a fourth and final boil of CHCl₃, allowing it to boil for 2 min.
   4. With vacuum still running, close the seal on the vacuum desiccator so that the vacuum within the desiccator is maintained. Turn off the vacuum and disconnect the vacuum tubing from the desiccator.
8. Seal the desiccator containing the non-fumigated samples by placing a lid on the desiccator and sealing the vacuum stopper. Place the desiccators (fumigated and non-fumigated) in a darkened area (such as a cabinet) for 24 hr. Do not repeat the vacuum procedures of subsection 4.7 on the desiccator containing non-fumigated samples.

5. Assemble Containers for Soil Sample Incubation

1. Push a 15 cm length glass rod through a size 10 rubber stopper with a hole drilled in the center. The rod diameter should be sufficient to fit through the hole snugly.
2. Label 0.5 L translucent wide mouth polypropylene bottles with identification that corresponds to the fumigated and non-fumigated subsample identification.

6. Evacuate Chloroform from Desiccators Under a Fume Hood

1. Open the stopper on a vacuum desiccator to allow airflow into the desiccator. Remove the lid from the desiccator, and take the samples and the damp towel out of desiccator.
2. Use a vacuum to evacuate CHCl₃ gas from soil samples.
   1. Place the lid on the desiccator. Connect the desiccator to a vacuum with vacuum tubing.
   2. Turn on the vacuum pump and allow pump to run for five minutes. Disconnect the vacuum tubing from the desiccator to allow airflow into the desiccator.
   3. Repeat step 6.3.2 four times.

7. Move Each Soil Subsample into an Incubation Container (Figure 1) to Conduct a 10 Day Incubation

1. Pipette 1 ml of deionized water into the incubation container. Connect an empty glass vial to the glass rod extending from the size 10 stopper using a rubber band. The open end of the glass vial should face the base of the stopper. The glass vial should be of a size sufficient to hold up to 40 ml of fluid.
2. Place a vial containing the 30 g soil subsample into the incubation container.
3. Add 1 g of non-fumigated soil from the original soil sample to each of its corresponding subsamples (fumigated and non-fumigated) as inoculum.
4. Pipette 1 ml of 2 M NaOH into the glass vial connected to the stopper/glass rod. Push the stopper/glass rod onto the top of the incubation container. Cover the top of the incubation container with Parafilm.
5. Create an incubation container that contains no soil. Assemble three to five no-soil incubation containers.
   NOTE: The acid used to titrate samples of the no-soil container is essential to the determination of CO₂ mineralization during the incubation period, which is described below in subsection 9.3. As such, multiple no-soil containers are created as a safeguard against incorrect handling or titration of a no-soil incubation container that would create an error in CO₂ mineralization calculation for all samples. The acid used to titrate samples from the no-soil containers should be close in values; a highly dissimilar acid value among the no-soil container samples is likely the result of incorrect sample handling or titration.
   1. Follow the procedures of section 5 to assemble incubation containers.
   2. Follow the procedures of 7.1 and 7.4.
6. Place all incubation containers in a darkened storage area at 25 °C. Leave all incubation containers in the storage area for 10 days.

8. Perform Titration on Each Subsample to Quantify CO₂ Produced by Microbial Respiration during the Incubation Period

1. Remove the glass vial containing the 2 M NaOH from the incubation container.
2. Pipette 2 ml of 1 M BaCl₂ into the glass vial containing 2 M NaOH.
3. Add one drop of phenolphthalein (C₂₀H₁₄O₄) from a pipette or medicine dropper into the glass vial containing the mixture of BaCl₂ and NaOH. Place a magnetic stir bar in the glass vial and place the glass vial on a stir plate.
4. With the stir plate activated, slowly add 0.1 N HCl with a burette until the red coloration of the mixture in the glass vial turns clear.
5. Record the amount of HCl required to change the coloration of the mixture in the glass vial.

9. Determine Microbial Biomass C from Data Collected during the First Fumigation-incubation Cycle^16,21,22

1. Determine the dry weight of soil in each subsample by multiplying its moist weight by the dry:moist weight ratio obtained in step 3.8.
2. Determine the average amount of HCl used to titrate the no-soil incubation containers.
3. Calculate CO₂ mineralized during the 10-day incubation using the formula:
   
   where CO₂ = CO₂ mineralized during the 10-day incubation
   NS = Acid used to titrate samples in no-soil incubation container
   S = Acid used to titrate samples that contained soil in the incubation container
   M = molarity of the HCl
   E = 6, the equivalent weight
   W = dry weight of soil contained in the incubation container
4. Calculate microbial biomass C using the formula:
   
   where BioC = microbial biomass C
   F = CO₂ mineralized from soil subsamples that were fumigated
   NF = CO₂ mineralized from soil subsamples that were non-fumigated
   K = fraction of microbial biomass C mineralized to CO₂
   1. Determine the value for K by either direct measurement of ¹⁴C mineralization in preliminary tests with the soil or published values²². A value of 0.45 is commonly used for K for this assay²³.
5. Perform sequential fumigation and incubation cycles by repeating sections 4-8 seven times for the soil subsamples that were fumigated in the first fumigation-incubation cycle.

10. Determine Labile C and Potential C Turnover Rate Using CO₂ Mineralized over the Course of the Eight Fumigation and Incubation Cycles

1. Use the following formula to determine a correction factor for the soil inoculum added to samples after each fumigation:
   
   Where IC = Correction factor for inoculum
   C' = Amount of CO₂ from the non-fumigated subsample during the first 10-day incubation
   r = Weight ratio of inoculum soil to fumigated soil in the first fumigation incubation cycle
   C_t = incubation cycle (1, 2 … 8), such that C_t-1 = 0 when t = 1
2. Use the following formula to estimate the CO₂ released during each incubation for each subsample:
   
   where Ct = CO₂ released during incubation
   NS = Acid used to titrate samples in no-soil incubation container
   S = Acid used to titrate samples that contained soil in the incubation container
   IC = Correction factor for inoculum (determined in step 10.1)
   E = 6, the equivalent weight
   W = dry weight of soil contained in the incubation container
3. Derive labile organic C using non-linear regression.
   1. Organize a spreadsheet that includes for each sample identifiers for the sample, incubation cycle number (1, 2 …8), and CO₂ released during the incubation (derived in step 10.2).
   2. Using software capable of non-linear regression, fit the following model to the dataset:
      
      where Csum = the sum of CO₂ released during the eight incubation cycles
      LOC = soil labile organic C
      k = potential turnover time
      t = incubation cycle (1, 2 …8)
4. Convert potential turnover time from step 10.3.2 into days by multiplying the inverse of k by 10 due to the 10 day incubation cycle.

Results

The SFI method has been used as described in this paper in a series of experiments conducted in the southeastern United States^24,25,26,27. Together, these experiments encompassed a variety of vegetation types, including loblolly pine (Pinus taeda L.), switchgrass (Panicum virgatum L.), cottonwood (Populus deltoides Bartram ex Marsh.), and soybean (Glycine max L. Merr.). The method was sensitive at determining differences in LOC and/or potenti...

Discussion

The SFI method is an effective protocol for detecting differences in soil LOC and potential C turnover rates over a range of management practices (such as fertilization, tillage, vegetation control, and harvest practices) and soil conditions. Soil LOC content and C turnover rate can be used to understand alterations of nutrient cycles. The SFI method also provides measurement of microbial biomass C from the first fumigation-incubation event. The ability to measure soil LOC, C turnover, and microbial biomass C concurrentl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Soil auger sampling kit	JMC	PN039	Several other manufacturers of punch augers are available
Parafilm	Curwood	PM999
Aluminum weighing boats	Fisherbrand	08-732-103
General purpose drying oven	Fisher Scientific	15-103-0511	Many other manufacturers of general purpose laboratory ovens are available
10.5 L vacuum desiccator	Corning	3121-250
Glass scintillation vial	Wheaton	968560
Glass threaded vials, 41 ml	Fisherbrand	03-339-21N
Chloroform, stabilized with amylenes	Sigma-Aldrich	67-66-3
Boiling chips	Fisher Scientific	S25201
Glass rod	Fisherbrand	S63449
Size 10 rubber stopper	Fisherbrand	14-130P	Rubber stoppers can be purchased as solid and drilled in center to install glass rod or bought with a hole to insert glass rod
Wide-mouth PPCO bottle, 0.5 L	ThermoScientific	3121050016
Sodium hydroxide, reagent grade	Sigma-Aldrich	S5881
Barium chloride	Sigma-Aldrich	202738
Phenolphthalein indicator	Fisher Scientific	S25466
Hydrochloric acid solution, 0.1 N	Fisher Scientific	SA54-4