Description and working steps
We modified the original techniques by 1) substitution of ultrasonication to mechanic soil dispersion in wet paste and 2) light fraction (LF, d< 2.0 g cm-3) into LF> 50 µm and LF< 5 µm by the wet sieving of light fraction through 50 µm sieve. [Original procedure elaborated by Shaimukhametov et al (1984) involved ultrasonic dispersion of soil micro-aggregates].
We substituted ultrasonication to mechanic dispersion in wet paste, since ultrasound even at low power input (40 W) resulted in the losses of new SOC of C4 origin as DOC. Preliminary experiments with our Haplic Chernozem showed the increase of DOC δ13C by 1 ppm.
The separation of light fraction (LF, d< 2.0 g cm-3) into LF> 5 0µm and LF< 50 µm by the wet sieving of light fraction through 50 µm sieve led to isolating 4 main fractions: 2 heavy fractions, Clay and Heavy fraction d > 2.0 g cm-3 and 2 light fractions d < 2.0 g cm-3, Light fraction > 50 µm and Light fraction < 50 µm.
Time required for fractionating ONE soil replicate if one person is working at it – 18 h. The most time-consuming procedure is the separation of the clay fraction.
First step is the size fractionation (clay sampling). The second step is the combination of density fractionation with wet sieving, i.e. partitioning of the rest soil on heavy fraction and two light fractions > 50 µm and < 50 µm.
Step 1: isolation of clay fraction by sedimentation in water
Fraction < 2 µm (clay)
Soil samples were sieved through 1 mm. 3 replicates, 10 g, were used for each of the samples. Soil micro-aggregates were disrupted by 15 min dispersion in wet paste (3 ml deionized water per 10 g soil) were used for the dispersion. Then the paste was transferred to 500 ml sedimentation cylinders through 0.25 mm sieve. The rest coarse sand and coarse POM on the sieves were combined with floatable POM into one fraction of POM and coarse sand; typically, the mass of POM alone in agricultural soils is less than 100 µg. After that, cylinders were filled with deionized water to the volume of 500 ml and shaken up during 1 min. Soil on the sieve (i.e. soil fraction 1 – 0.25 mm) was washed with deionized water and dried at 50-60 °C.
Clay fraction was isolated by exhaustive extraction, with 150 ml samplings every 8 hrs; totally 24-30 extractions were used depending on the clay content. The suspensions were centrifuged during 15 min at 10000 rpm. The 100 ml portion of supernatant was freeze-dried to determine DOC content by CHN analyzer 932 Leco. The clay fraction residue was dried at 50-60 °C.
Step 2: density fractionation
The rest soil after clay extraction (dried at 50-60 °C) was placed into 50 ml plastic centrifugation tubes. Then 10 ml of sodium polytungstate solution (D= 2.0 g cm-3) was added to the tubes, and the suspension was shaken during 10 min at 150 rpm. Then it was centrifuged during 15 min at 3000 rpm, and floating material was filtered through 45 μm membrane filters. Then the treatment with the recovered polytungstate solution was repeated 3 times resulting 4 treatments in total until no material is floating. Finally the light fraction on the filter (LF) and the heavy fraction on the centrifuge tube bottom were washed 3-5 times with 20 ml portions of deionized water. Afterwards, the light fraction was sieved through 50 µm sieve to separate fractions LF >50 µm and LF <50 µm. All the fractions were dried at 50-60 °C.
Shaymukhametov, M.S., Titova, N.A., Travnikova, L.S., Labenets, Y.M., 1984. Use of physical fractionation methods to characterise soil organic matter. Soviet Soil Science 16 (4), 117-128.