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14C-HP-GPC of alkaline extract (Fig. 5) showed that the catechol residues were not homogeneously present within the humic substances and small molecules (200-300 Da in Fig. 5B) disappeared during the incubation (Fig. 5A).

Rong Ji1, Marko Bertmer2, Philippe Corvini1, and Andreas Schäffer1

1 Institut für Umweltforschung (Bio V) – Umweltbiologie und –chemodynamik2 Lehrstuhl für Makromolekulare Chemie RWTH Aachen, D-52056 Aachen, Germany. Rong.Ji@bio5.rwth-aachen.de

Mineralization and Transformation of Catechol in Soil after a Long-Term Incubation

Introduction

Naturally occurring monomeric phenols are ubiqui-tous in soil1-2, regarded as precursors of humic sub-stances3-4 and play important roles in the global car-bon cycle. In soil, they are subject to biotic and abio-tic degradation and to a certain extent immobilized in soil organic and inorganic matrices5-6. However, the immobilization mechanisms and the chemical structures of the stabilized residues are still unclear. Using 13C and 14C-labelled catechol as model com-pound, we studied the mineralization and transfor-mation of phenolic compounds in soil.

References

1. Gallet C, Keller C (1999) Phenolic composition of soil solutions: comparative study of lysimeter and centrifuge waters. Soil Biol. Biochem. 31: 1151– 1160.

2. Siqueira JO, Nair MG, Hammerschmidt R, Safer GR (1991) Significance of phenolic compounds in plant-soil-microbial systems. Crit. Rev. Food Sci. 10: 63-121.

3. Haider K, Martin JP, Filip Z (1975) Humus biochemistry. In: Paul EA, McLaren AD (Eds.) Soil Biochemistry. Marcel Dekker, New York, pp. 198-244.

4. Stevenson FJ (1994) Humus chemistry: genesis, composition, reactions, 2nd edition. John Wiley & Sons, New York.

5. Huang PM (2000) Abiotic catalysis. In: Handbook of soil science (Sumner ME, Editor-in-chief), CRC Press, Boca Raton, B303-B332.

6. Martin JP, Haider K (1980) Microbial degradation and stabilization of carbon-14-labeled lignins, phenols, and phenolic polymers in relation to soil humus formation. In: Kirk TK, Higuchi T, Chang H-M (Eds.) Lignin Biodegradation: Microbiol., Chem., Potential Appl., [Proc. Int. Semin.], Meeting date 1978. CRC, Boca Raton. 1: 77-100.

7. Ji R, Schäffer A (2002) Synthesis of 13C- and 14C-labelled catechol. J. Labelled Compd. Radiopharm. 45: 551-558.

14C-HP-GPC of alkaline extract

Mineralization

Umweltbiologie und -chemodynamik - UBC

Soil was loamy sand, Haplic Phaeozem, pH 6.5; from the Versuchsfeld Ewiger Roggenbau-Mais NPK, Halle, Germany. Labelled catechol was synthesized in our laboratory.7 Incubation was performed in flow-through-systems in the dark at 20°C for 2 years (Fig. 1).

Figure 1 Experimental set-up for the incubation of catechol in soil. The incubation flask (100 ml) contained soil (50 g, < 2 mm, 40% of MWC) spiked with 13C- and 14C-labeled catechol (100 µg g1 dry soil) and the headspace was refreshed continuously by air saturated with H2O at a rate of 6 ml min-1.

Experimental setup

AirAirAir Pump

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NaOHH2O

ttt eeA

24 1082.31011.5 7.232.86

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HA of soil incubated with 13C-catechol for 2 years

13C-Catechol in CDCl3

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Mineralization of catechol in soil (Fig. 2) followed the equation

The modeling indicates that catechol in the soil under the experimental conditions was transformed into two groups of soil carbon with different degradability, one (23.7%) is relatively labile, another (86.2%) is relatively recalcitrant, with half-lives of 18 days and 3.7 years, respectively. Figure 2 Mineralization of catechol

in soil (n = 2)

Here, At: percentage of total substrate remained in soil after time t (year).

Distribution of residual carbon in soil

After incubation, the soil was sequentially extracted with H2O, isopropanol, CH2Cl2, and 0.1 M NaOH to ob-tain water-soluble, hydrophilic, and hydrophobic residu-al, as well as humic substances (Fig. 3). The alkaline extract was fractionated into fulvic and humic acids. The humin was silylated to separate physico-chemically bound residues from chemically bound residues (Fig. 4).

Figure 3 Distribution of the residual carbon of catechol in fractions of soil organic matter after 2 years of incubation at 20°C, compared to control soil, which was spiked with catechol but not incubated. (n = 2)

• Mineralization of catechol followed a two-component-model consisting of one labile and one recalcitrant component.

• After 2 years of incubation, the residues of catechol were mainly located in the fractions of humic and fulvic acids and humin.

• The residues in humin were nearly quantitatively stabilized by chemical bondings.

• Alkali-extractable residues of catechol were bound to humic substances with relative low molecular weights (200 – 11k Dalton).

• The residues of catechol in humic acids were main-ly transformed to carboxyl or carbonyl groups.

SummaryFigure 4 Relative amounts of radiolabel of fulvic acids in alkaline extract (A) and of physico-chemical-ly bound radiolabel in humin (B) in soils either incubated for 2 years or without incubation. (n = 2)

13C-NMR of humic acids

Figure 6 Solid-state 13C-CP-MAS-NMR spectra of HA of soil spiked with 13C-catechol and incubated for 2 years, in comparison with that without incubation and HA of the original soil. Spectrum of pure 13C-catechol was obtained with liquid-state NMR.

A

B

13C-NMR analyses showed that the ring carbons of catechol were mainly transformed into carboxyl or carbonyl groups (160-200 ppm) in humic acids (HA) after 2 years of incubation in soil.

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During the incubation the amount of water-soluble residues decreased strongly, whereas the residues bound to humic substances (NaOH extract) increased (Fig. 3). Amounts of residues bound to the humin decreased also during the incubation (Fig. 3), which is in agreement with the decrease of physico-chemically bound residues in humin (Fig. 4).

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Figure 5 Molecular size distribution of catechol residues (14C) within the humic substances (UV) of the soil spiked with 13C-14C-catechol. (A) incubated for 2 years; (B) without incubation. (n = 2)

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