LABORATORY 9

SOIL ORGANIC MATTER AND EXTRACTION OF HUMUS

IObjectives

Gain familiarity with a method for quantifying soil organic matter. Extract humus and demonstrate cation exchange and flocculation / dispersion behavior.

IIIntroduction

ASoil Organic Matter

Plants continuously produce tremendous quantities of organic matter that would otherwise accumulate near the soil surface if it were not decomposed by soil organisms. Soil organisms depend on the continuous supply of plant and animal residues as a source of energy and essential nutrients. This biological activity which continuously produces and destroys organic matter, recycles nutrients and produces humus is a major characteristic that distinguishes soils from geologic deposits.

This laboratory introduces a method commonly used to determine the total concentration of organic matter in soil. In this method organic C is oxidized by Cr2O72- and the reaction is aided by heat generated when H2SO4 is added to the soil-dichromate mixture. Since a known mass of Cr2O72- is added, the mass of C present (which is converted to CO2) is calculated by difference with remaining dichromate. Unreacted Cr2O72- is titrated with FeSO4$ 7H2O using o-phenanthroline as the indicator.

Although this is a fast and convenient method, not all soil C is oxidized. But based on work with many different soils, 75 % of the C present is oxidized. Therefore, organic C can be fairly well estimated from measured C.

BExtraction of Humus

Soil organic matter is a very general term that includes everything organic Bliving biomass, partially decomposed organic residues and largely amorphous and colloidal substances no longer identifiable as tissue. The latter category of organic substances is referred to as humus.

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Humus is formed by microbial decomposition and synthesis reactions. It includes humic and non-humic substances. The non-humic substances are identifiable biomolecules such as proteins, hemicelluloses, cellulose, fats, waxes and lignins. Humic substances include fluvic acid, humic acid and humin. Fulvic acid has the lowest molecular weight and least resistance to decomposition by microorganisms. Humin has the highest molecular weight and greatest resistance to decomposition.

Humus is very dark in color. It has a large number of negative charges per unit mass, thus, very high CEC. The large molecules of humus bind to clay particles and greatly increase aggregate formation and stability. Physical as well as chemical soil properties of soils are greatly improved by humus.

This laboratory also introduces an abbreviated method for the extraction and fractionation of humus. The alkaline extraction procedure allows separation of fulvic acid, humic acid and humin. Whereas fulvic and humic acids are extracted by base, humin is not. Fulvic acid is separated from humic acid by subsequent precipitation of humic acid with acid.

The organic soil sample is pretreated with HCl. You will qualitatively test the acid wash for the presence of Ca2+, thereby demonstrating cation exchange. You will also induce flocculation and dispersion.

If cation exchange sites of humus are primarily occupied by Ca+2 and Mg+2, humus particles remain flocculated and bind soil particles together. But if exchange sites are largely saturated with Na+, the soil pH is high and humic colloids are dispersed. Under these conditions humus loses its stabilizing influence, and may be leached from the soil as individual particles or rise to the surface of the soil as water moves upward during evaporation. It is deposited there, resulting in what is called a "black alkali" soil.

IIIProcedure

ADetermination of Soil Organic Matter

Wear safety glasses, gloves and lab coat or apron. You are working with a mixture of a strong oxidizer and strong acid.

  1. Weigh duplicate samples (1.00 to 4.00 g, depending on whether expected organic C is high or low, such as in sandy soils).
  1. Transfer to a 250 mL Erlenmeyer flask.

Your lab instructor will do steps 3 through 6 for you.

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  1. Add exactly 10.0 mL of 1.0 N K2Cr2O7 from a repipette.

A soil-free blank must also be run. Follow steps beginning with 3 using a third Erlenmeyer flask but without any soil in the flask.

  1. While gently swirling flask, add 20 mL of concentrated H2SO4 from a repipette. Swirl for 1 min.
  1. Set hot flask (120 C) aside for 30 min.
  1. Add 100 mL of distilled water from a graduated cylinder and swirl.
  1. Gravity filter contents through Whatman No. 2 filter paper into a 250 mL Erlenmeyer flask.
  1. Add 4 drops of o-phenanthroline indicator to each flask.
  1. Titrate with 0.5 N FeSO4$ 7H2O. Record mL required to nearest 0.1 mL. During titration the color change is from blue-green to dark reddish brown. The solution becomes olive green, dark green, then blue-green before the end point. Color change at the end point occurs abruptly in one drop.
  1. Transfer contents of flasks to designated waste container.
  1. Calculations.

Determine the normality of the ferrous sulfate solution from the mLs required to titrate the blank, then calculate the percent organic C in each sample from

% OC = [(meq K2Cr2O7 - meq FeSO4$ 7H2O) x 0.003 x 1.33 x 100 %] / g soil

meq = mL x N

Normality of FeSO4$ 7H2O is calculated from the blank titration, since the meq of K2Cr2O7 is known

N FeSO4$ 7H2O = meq K2Cr2O7 / mL FeSO4$ 7H2O

The factor 0.003 is g of C per meq of C oxidized. The factor 1.33 adjusts for the 75 % efficiency of the method in oxidizing organic C. Use g of oven-dry soil.

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BExtraction, Flocculation, Dispersion and Cation Exchange Behavior of Humus

Extraction

  1. Weigh 1 g of an organic soil and transfer to a funnel lined with filter paper. Set a test tube under the funnel.
  1. Slowly add 3 mL of HCl to the organic soil. Pour leachate through the soil.
  1. Wash the soil with three 5 mL increments of distilled H2O.
  1. Collect about 1 cm of leachate in the first test tube, then move the funnel to a second test tube. Save leachate in the first tube for Test for Exchanged Ca2+ (below).
  1. Once the distilled H2O has drained from the soil, add 5 mL of 1:5 NH4OH.
  1. When the leachate begins to show a dark brown color, move the funnel to a third test tube and collect the leachate. Discard leachate in the second tube.
  1. Wash soil with distilled H2O until there are 5 cm of dark brown leachate in the third test tube.
  1. Answer questions # 1 below.

Flocculation

  1. Pour 1/2 of the leachate from the third test tube into another tube.
  1. Dilute both samples of leachate with water to a coffee brown color.
  1. Add a spatula tip full of Ca(OH)2 powder to one tube, shake well, and let stand.
  1. Put a short piece of litmus paper in the other tube and add dilute HCl drop by drop until the leachate is acidic (litmus turns red).
  1. Let stand and observe the results. Use this tube in the next section.
  1. Answer questions # 2.

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Dispersion

  1. Add dilute NaOH dropwise to the acidified leachate from section Flocculation of Humus (above) until it is alkaline.
  1. Shake and let stand.
  1. Answer question # 3.

Test for Exchanged Ca+2

  1. Add 1:2 NH4OH slowly, with constant shaking to the first test tube containing the HCl leachate until the leachate is alkaline. A light brown precipitate of iron and aluminum hydroxides will develop. Avoid an excess of NH4OH.
  1. Filter the contents of the test tube into a clear test tube.
  1. Add 5 drops of ammonium oxalate to the filtrate. Watch for a white precipitate to occur.
  1. Answer question # 4.

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IVWorksheet

ADetermination of Soil Organic Matter

Table 1.

Sample / Mass Soil (g) / mL Ferrous Sulfate / Milliequivalents / % Organic C
Blank
Rep #1
Rep #2
Average

Calculations

BExtraction, Flocculation, Dispersion and Cation Exchange Behavior of Humus

1.What was the predominant cation on the soil CEC after step 3 of the extraction procedure? After step 7?

What caused the dark brown color in the leachate in step 6?

2.Did flocculation of the humus occur? If so, in which tube or tubes?

Describe the difference in appearance of Ca-saturated and H-saturated humus.

3.Describe the appearance of humus after the NaOH was added.

4.What was the white precipitate that formed?

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