Short communication
Sequential versus parallel density fractionation of silt-sized organomineral
complexes of tropical soils using metatungstate
C. Shang*, H. Tiessen
Department of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8
Received 1 February 2000; received in revised form 24 May 2000; accepted 18 July 2000
Abstract
A comparison of two density fractionation methods used for studying soil organomineral complexes was carried out on the silt-size fractions of two highly weathered soils from NE Brazil. The dif®culties commonly encountered in isolating organomineral complexes in highly weathered soils were overcome when sodium metatungstate solution in combination with centrifugation on an angular rotor was used. The sequential method caused a greater loss of C (8±10%) than the parallel method (3±5%); however, the sequential method permits direct measurements on C concentration, complexing cations (Fe and Al), and mineralogical composition.q2001 Elsevier Science Ltd. All rights reserved.
Keywords: Density fractionation; Soil organic matter; Tropical soils
Physical fractionation of soil organic matter (SOM) includes mechanical dispersion (e.g. using ultrasonic vibra-tion) to obtain soil size fractions and sedimentation of whole soil or its size fractions in heavy liquids. The information from physical fractionation was considered more relevant to the SOM function in situ (Christensen, 1992) than classical chemical fractionation. The light fraction of SOM, recov-ered by immersing the whole soil in a heavy liquid (e.g. NaI), has a fast turnover and serves as a labile nutrient pool in many agricultural soils (Bremer et al., 1994). Using metatungstate liquids, Cambardella and Elliott (1994) isolated a C- and N-enriched ®ne-silt fraction (2± 20mm) having a density of 2.07±2.22 g cm23from inside macroaggregates of a temperate prairie soil.
There are two contrasting approaches used in the density fractionation of SOM. The sequential separation used by Dalal and Mayer (1986) involves successive extractions with heavy liquids of increasing density. The parallel separation used by Cambardella and Elliott (1992) separates SOM from replicate samples with heavy solutions of differ-ent densities. The mass and C contdiffer-ent of the intermediate fractions are calculated indirectly by taking the difference between two neighbouring (heavier or lighter) fractions
(Cambardella and Elliott, 1992). Sodium metatungstate [Na6(H2W12O40)] was proposed as a new medium for density
gradient centrifugation because of its low toxicity and high density (Plewinsky and Kamps, 1984). Although this inorganic salt has been successfully used for SOM fractio-nation (Baldock et al., 1990; Cambardella and Elliott, 1992; Shang and Tiessen, 1997; Six et al., 1999), the questions regarding C loss and fractionation ef®ciency remain unanswered.
The present study was to compare the two density fractionation schemes on tropical soil separates using sodium metatungstate and to investigate the suitability of this inorganic salt for tropical soil materials.
Two soils (a Ustalf and a Ustox) were collected under native vegetation in Pernambuco State, Brazil. The Al®sol was formed on granitic±gneissic parent material. The clay fraction of the Al®sol contained mainly kaolinite and illite; silts contained quartz, muscovite and K-feldspars as well as iron oxide minerals; sands were dominated by quartz and K-feldspar. The Oxisol was sampled on a plateau of Cretac-eous sediments (Chapada de Araripe). The predominant minerals in this soil were quartz, iron oxides and kaolinite with only traces of 2:1 minerals. The A horizons (0±12 cm for the Al®sol and 0±20 cm for the Oxisol) were used in this study. Samples were air-dried and passed through a 2-mm sieve. The particle-size distribution, determined by H2O2±
metaphosphate dispersion was: 66% sand, 16% silt and 18% clay for the Al®sol, and 79% sand, 2% silt and 19% clay for
Soil Biology & Biochemistry 33 (2001) 259±262
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* Corresponding author. Present address: Department of Chemistry and Biochemistry, South Dakota state University, Brookings, SD 57007, USA. Tel:11-605-688-4262; fax:11-605-688-6364.
C.
Shang,
H.
Tiessen
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Soil
Biology
&
Biochemistry
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(2001)
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Table 1
Comparison of sequential and parallel density separations on the silt-size separates of two highly weathered Brazilian soils (the average of two analyses from combined samples of three replicates)
Density fraction (g cm23) Method Al®sol Oxisol
C conc.a(mg g21) % of fraction mass % of fraction C C conc.a(mg g21) % of fraction mass % of fraction C
,1.8 Parallel ±b ± ± 265 5.2 25
Sequential ± ± ± 265 5.2 27
1.8±2.0 Parallel 258 4.0 34 77 7.0 9.8
Sequential 214 5.1 40 176 5.1 18
2.0±2.2 Parallel 164 4.9 30 329 2.4 15
Sequential 171 3.2 20 106 6.8 14
2.2±2.4 Parallel 36 11 18 84 9.8 16
Sequential 72 3.8 10 92 5.4 9.6
.2.4 Parallel 6.0 81 18 25 76 35
Sequential 9.2 88 30 21 77 32
a Calculated values were given for the C concentrations of intermediate density fractions by parallel method.
the Oxisol. The organic C content of the Al®sol sample was 1.4% and that of the Oxisol 1.1%. More information on the two soils was given by Shang and Tiessen (1998).
Fifty grams of air-dry soil was pre-wetted overnight and ultrasoni®ed in 300 ml of deionized water in a 400-ml beaker at 300 W for 8 min with a 1/2-inch (1.27 cm) probe. Oven-dry weight of soil samples was calculated with known moisture contents. Samples were cooled in an ice bath during sonication. The dispersed sample was passed through a 50-mm sieve to recover the sand, and the silt-size materials (2±50mm) were separated from ,2-mm clay by gravity sedimentation. Comparisons were carried out with silt and clay fractions since isolation of organic matter from sand fraction was straightforward (Shang and Tiessen, 1998).
In the parallel density separation, silt-size materials (0.5 g for the Oxisol and 1 g for the Al®sol) were suspended in 5 ml of metatungstate solutions with densities of 1.8, 2.0, 2.2 and 2.4 g cm23in 15-ml centrifuge tubes. The suspen-sion was stirred with a vortex mixer and then treated ultra-sonically for 1 min at 50 W output using a 1/4-inch (0.64 cm) probe. Material adhering to the probe was washed into the centrifuge tube with 5 ml of heavy solution. The suspension was cooled for one hour at room temperature before centrifuging at 2000g for 10 min using an angular rotor. The supernatant and ¯oating matter were ®ltered through a Millipore membrane (0.10mm pore size) using vacuum suction. The material trapped on the membrane was washed with 5 mM CaCl2, transferred into a pre-weighed
aluminium tray and dried at 558C. The heavy fractions were washed four times with 5 mM CaCl2by centrifugation,
and oven-dried.
In the sequential method, the residual soil (.1.8 g cm23)
from the 1.8-g cm23 separation was washed with dilute CaCl2 solution, oven-dried at 558C and fractionated with
metatungstate solution of 2.0 g cm23 to obtain the 1.8± 2.0 g cm23fraction and so on. Successive density fractions of 2.0±2.2, 2.2±2.4 and.2.4 g cm23were thus recovered using heavy solutions of 2.2 and 2.4 g cm23. The organic matter fractions of three replicates were combined for analysis.
About 50% of total soil C were located in the silt-size fraction, which included silts and silt-size aggregates (Shang and Tiessen, 1998). Density separation by both the parallel and sequential methods produced a similar trend in C concentration for the Al®sol silt (Table 1), i.e. a decrease in C concentration with increasing fraction density. The sequential method gave a relatively lower concentration in the ,2.0 g cm23 fraction but higher concentrations in heavier fractions. The recovery of mass became less comparable between the two methods as the density increased; more mass was recovered in the .2.4 g cm23 fraction by the sequential method (87.9%) than the parallel method (80.5%). The C distribution, as proportion of total fraction C, among density fractions followed the mass distribution.
In the case of Oxisol silt, the parallel method produced a low C concentration for the 1.8±2.0 g cm23fraction and a very high concentration for the 2.0±2.2 g cm23 fraction. The irregularity of C concentration was likely caused by an over-recovery of mass in the ,2.0 g cm23 fraction by the 2.0 g cm23density separation. When subtractions were made, the greater mass of 1.8±2.0 g cm23fraction led to an underestimation of the 2.0±2.2 g cm23 fraction. Conse-quently, the C concentration was lowered by dilution because the material of .2.0 g cm23 density contained
C. Shang, H. Tiessen / Soil Biology & Biochemistry 33 (2001) 259±262 261
less C. Error accumulation in subtraction may also be a factor. Similar amounts of mass for the .2.4 g cm23 frac-tion were recovered by the two methods, but relatively large differences were found for intermediate density fractions. A similar trend was found for C content, expressed as the proportion of total fraction C.
On average 90±92% of C was recovered by the sequential method whereas the recovery of C by the parallel method was 94±96%. The greater C loss with the sequential method is likely related to the greater number of separation steps since carbon loss in general increased with the density of metatungstate solution (Fig. 1). The average C loss was 4% or less when the density of heavy liquids was#2.0 g cm23 (Fig. 1).
Separation of light from heavy materials in metatungstate solutions by gravity sedimentation, used on temperate soils and soil fractions by Cambardella and Elliott (1992, 1994), was also tested and a separation was not achieved due to the coagulation of materials. It seems that a quick separation is necessary to obtain an ef®cient fractionation.
The density fractionation of clay-size materials was not successful probably because of a narrow density range of clay particles (predominant kaolinite) or ¯occulation of clay particles in high ionic strength heavy solutions. The 1.7 g cm23 NaI solution, commonly used to extract light fraction of SOM, failed to recover any signi®cant amount of light fraction (,1.7 g cm23) from the silt-size materials of both soils, and the reason was not clear.
The parallel separation was simple to use and time-saving, but the information for intermediate density frac-tions was derived indirectly by subtracting two neighbour-ing fractions. The sequential separation procedure used by Turchenek and Oades (1979) requires continuous ¯ow centrifugation equipment, which is not commonly available. The sequential separation procedure of Dalal and Mayer (1986) is time-consuming but uses ordinary centrifuges and permits direct measurements such as C concentration, complexing cations (Fe and Al), mineralogical composition and some biochemical properties (Shang and Tiessen,
1998). However, a signi®cant amount of organic matter can be partitioned into liquid phase in this repeated-separa-tion scheme. Using metatungstate solurepeated-separa-tion and centrifuga-tion on an angular rotor, at least for the two highly weathered soils studied, is promising for isolating SOM stabilised in silt-size or larger aggregates.
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