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Biology and Fertility of Soils
Kinetics of short-term carbon mineralization in roots of biofuel crops in soils
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Supplemental Information
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Authors: F. A. Rivas1 • M. A. Tabatabai1 • D. C. Olk2 • M. L. Thompson1
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Corresponding author:
Dr. M.L. Thompson
Department of Agronomy
Iowa State University
Ames, Iowa 50011-1010 U.S.A.
Email: (mlthomps@iastate.edu)
Phone: 515-294- 2415
Fax: 515-294-3163
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Agronomy Department, Iowa State University, Ames, Iowa 50011 USA
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USDA-ARS, National Laboratory for Agriculture and the Environment, Ames, Iowa 50011
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USA
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Supplemental Information
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To determine lignin in the root samples, the acetyl bromide method was adapted from Hatfield et
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al. (1999). Approximately 10-15 mg of root samples were weighed into 30-mL glass tubes with
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screw caps. Then 10 mL of distilled, deionized water were added to each tube. The tubes were
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capped, mixed, and heated at 65oC on a block digester for one hour. The suspensions were
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filtered through a 25-mm diameter glass fiber filter and the root residues were washed
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sequentially with approximately 3-5 mL of water, ethanol, acetone, and diethyl ether to remove
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the water-soluble and semi-water-soluble carbohydrates. After rinsing, the filter papers and their
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root residues were transferred into 50-mL tubes and dried overnight at 70oC. The dried roots
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were then mixed with 2.5 mL of 25% (v/v) acetyl bromide in glacial acetic acid and the samples
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were heated in 50-mL tubes for 2 hours in block digester at 50oC. After cooling, each digestate
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was quantitatively transferred into a 50-mL volumetric flask that contained 10 mL of 2 M NaOH
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and 12 mL of glacial acetic acid and was diluted to volume with glacial acetic acid. The solution
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was shaken and filtered through a 47-mm glass fiber filter to remove colloidal particles that
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could interfere in the spectrophotometric analysis. The absorbance of the filtered sample was
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measured at 280 nm by a UV-Vis spectrophotometer. The results were calculated from
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calibration graphs prepared by using lignin standards (Sigma-Aldrich, St. Louis, Mo)
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Monosaccharides (fucose, arabinose, rhamnose, galactose, glucose, xylose, and mannose)
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were extracted from the root samples by using a procedure adapted from that of Martens and
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Loeffelmann (2002). Here, 20 mg of root materials were weighed into a 15 x 125-mm labeled
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culture tube and soaked in 800 μL of 6 M H2SO4 for 30 minutes. The sample was then diluted
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with 4 mL of deionized water to adjust the concentration of acid to 1 M H2SO4. The diluted
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solution was autoclaved for 30 minutes at 121oC. The samples were cooled, centrifuged, and
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decanted into Falcon tubes. The sample pellet was rinsed twice, each time by adding 1 mL of
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deionized water; the washings were centrifuged and transferred to the originally collected
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supernatant. The pH of the supernatant was then adjusted to pH 5.5 - 6.5 by addition of NaOH
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solution and monitored by a pH meter. The sample was then diluted with deionized water to 10
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mL. A 1-mL aliquot of the sample was further diluted to 10 mL, and 500 μL of the diluted
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sample were pipetted into an auto sampler vial to be analyzed for the monosaccharides noted
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above by ion chromatography. The sum of these sugars was taken as an index of hemicellulose
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(HI) for the root samples. The residues left from the first acid extraction were saved and dried
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overnight at 58 - 60oC. To obtain a cellulose index (CI), the dried residue was soaked for 30
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minutes in 300 𝜇L of concentrated H2SO4. The concentration of the sample solution was adjusted
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to 1.5 M by adding 3.3 mL of deionized water. The samples in the culture tubes were autoclaved
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and glucose was determined by ion chromatography. Monosaccharides comprising both the HI
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and the CI were determined using a Dionex DX-500 Chromatograph (Dionex Corporation,
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Sunnyvale, CA) equipped with a CarboPac PA10 column (2 mm i.d.) and using 5 - 80 mM
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NaOH as the eluent (Martens and Frankenberger, 1990b).
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Water-holding capacity of the soil was determined by saturating a known mass of air-
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dried soil material over a 24-h period, allowing the sample to drain freely in a humid atmosphere,
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and then measuring the wet mass of the sample.
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References
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Hatfield R, Grabber J, Ralph J, Brei K (1999) Using the acetyl bromide assay to determine lignin
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concentrations in herbaceous plants: Some cautionary notes. J Agri Fd Chem 47: 628-
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Martens D A, Loeffelmann KL (2002) Improved accounting of carbohydrate carbon from plants
and soils. Soil Biol Biochem 34:1393-1399