The overall goal of this sequential fumigation incubation procedure is to estimate labile carbon and the potential carbon turnover rate in soil. This method can answer key things in the soil microbiology field, such as the amount of substrate that soil microbes use to transform nutrients in the soil. One of the main advantages of this technique is that it's relatively simple to use and inexpensive.
Prior to collection, identify differences in soil properties and vegetation within the collection site. Using the appropriate coefficients of variation, determine the number of samples needed and the distance of variability. For heterogeneous soil conditions, designate a grid sample pattern wherein the distance between sample points on each transect is smaller than the distance of variability within an experimental unit.
Clear organic matter from the soil surface at the sample points. Using an auger, collect each soil sample. For each transect of the grid, mix the samples in a single container.
Place each composite sample in a cooler with ice packs immediately after collection. Once field work is completed, sieve the soil samples. Clean the mesh with water after sieving each sample.
For each composite soil sample, place three individual 100 gram samples into beakers. Cover the beakers with laboratory film and pre-incubate the beakers at 25 degrees Celsius for 10 days. Then, for each sample, remove the film and place one gram of soil in a pre-weighed aluminum weigh boat.
Record the combined weight of the moist soil and weigh boat. Dry each sample in the weigh boats at 105 degrees Celsius for 48 hours. Check the weight every eight hours for the first 24 hours and then every six hours until no more weight is lost.
Using the known weights of the weigh boats, determine the weight of the dry soil. Calculate the dry to moist soil weight ratio. To begin the fumigation, place a damp paper towel at the bottom of each of two glass vacuum desiccators with porcelain plates.
Designate one desiccator for fumigation. For each sample, prepare three narrow glass vials, each containing 30 grams of soil. Label each vial in pencil on laboratory tape.
For each set, place two vials in the fumigation desiccator and the third vial in the other desiccator. Place the fumigation desiccator in a fume hood. Cover the bottom of a 100 milliliter beaker with boiling stones.
In the fume hood, pour 50 milliliters of ethanol-free chloroform into the beaker. Place the beaker in the center of the fumigation desiccator and close the desiccator. Connect the fumigation desiccator to a vacuum pump.
Turn on the vacuum pump and allow the chloroform to boil for 30 seconds. Then, turn off and disconnect the pump to allow air into the desiccator. Repeat this step twice more.
Boil the chloroform a fourth time for two minutes. Then, close off the desiccator to maintain the vacuum. Turn off and disconnect the vacuum pump.
Close and seal the other desiccator. Store both desiccators in a dark area at room temperature for 24 hours. Next, place the fumigation desiccator in a fume hood.
Vent the desiccator and remove the paper towel. Place vials with soil into the desiccator, close the desiccator, connect the vacuum pump, and open the vacuum to remove chloroform gas from the soil samples. Run the vacuum for five minutes, and then vent the desiccator.
Perform this step four more times, then remove the soil-filled vials from the desiccator to prepare them for incubation procedures. To prepare incubation containers, label wide-mouth polypropylene bottles for the fumigated and non-fumigated subsamples. Label three more bottles as no soil controls for later titration.
For each labeled bottle, drill a hole in the center of a size 10 rubber stopper and insert a 15 centimeter length glass rod into the hole. Affix a 40 milliliter glass vial to the glass rod with a rubber band. Then, add one gram of soil from the original pre-incubated soil sample to each of its three subsamples.
Place one milliliter of deionized water into one of the bottles labeled for fumigated soil. Place a vial of fumigated soil into the bottle. Pipette one milliliter of two molar sodium hydroxide into the glass vial of the stopper assembly.
Carefully stopper the bottle and seal the bottle with laboratory film. Repeat this process for the remaining soil samples. Prepare the no soil containers in the same way but without soil samples.
Store the incubation chambers in a dark area at room temperature for 10 days. After incubation, carefully remove the vial of sodium hydroxide from an incubation container. Pipette two milliliters of one molar barium chloride into the vial.
Add one drop of phenolphthalein to the vial. Add a magnetic stir bar to the vial, and begin stirring the mixture on a stir plate. With a burette, slowly add 0.1 normal hydrochloric acid until the solution appearance changes from red to colorless.
Record the volume of hydrochloric acid added. Repeat the titration for all of the incubation chambers noting the no soil values separately. Using the titration data and the dry-moist soil weight ratio, calculate the carbon dioxide mineralized during incubation of the soil samples.
From this, calculate the microbial biomass carbon for each set of samples. Perform seven more rounds of fumigation and incubation on the fumigated samples, calculating the mineralized carbon dioxide after each cycle. Using this data, determine the amount of labile organic carbon and the potential turnover time for each fumigated sample by non-linear regression.
The sequential soil fumigation and incubation method identify differences in labile organic carbon, or LOC, and potential carbon turnover rates among different vegetation conditions. Soybean conditions have the highest LOC values, which was associated with biomass deposition during soybean harvests. Loblolly pine plantations had the highest potential carbon turnover rates, which was associated with acidic pine litter being the dominant source of soil organic matter.
The loblolly pine and switchgrass alley cropping system had the broadest array of sampling site conditions and the switchgrass pasture had the greatest variation in soil textures among sampling sites. Both of these conditions exhibited wide ranges of both LOC values and potential carbon turnover rates. Once mastered, this technique takes about 30 minutes per sample over the course of an 80 day period that it takes to run each sample.
To conduct this procedure accurately, some of the things to remember are to make sure the desiccators are sealed tightly, as well as the incubation chambers, and to not breathe too closely to the vials that contain sodium hydroxide. In tandem with this procedure or following this procedure, there's several different enzyme assays that can be conducted in soil to better understand soil microbial function. After watching this video, you should have a good understanding of how to conduct Labile Carbon Analysis using the sequential fumigation incubation procedure.
Don't forget that working with chloroform can be extremely hazardous. You need personal protection equipment and a fume hood to conduct these procedures safely.
