Composition of humic acids with respect to the degree of humification in cultivated soils with and without manure application as assessed by fractional precipitation

Abstract

A fractional precipitation method with 0.01 mol L⁻¹ NaOH–ethanol solutions was used to assess the temporal change in the composition of humic acids (HAs) in upland field soils with respect to the degree of humification as expressed by the degree of darkening. A Typic Hapludult (Togo) and Pachic Melanudand (Kuriyagawa) with and without the application of cattle manure were examined. Humic acids were fractionated into five to eight fractions, and a fraction precipitated at a lower ethanol concentration tended to have a larger degree of humification in each soil. The slight increase in the degree of humification of Togo HAs in both the manured (40 Mg ha⁻¹ year⁻¹) and non-manured plots after 10 years was exhibited as a shift in the dominant fraction. The distribution pattern in the fractionation of Kuriyagawa HAs was similar between the initial soil and the manured (80 Mg ha⁻¹ year⁻¹) and non-manured plot soils after 19 years. However, the degree of humification of the dominant fraction that was precipitated at the lowest ethanol concentration (200 mL L⁻¹) was smaller in the manured plot soil than in the initial and non-manured plot soils. In another Kuriyagawa soil with manure applied at 160 Mg ha⁻¹ year⁻¹ for 19 years, the amounts of three HA fractions with smaller degrees of humification were larger than those in the other Kuriyagawa soils, and the dominant fraction was observed at a higher ethanol concentration (400 mL L⁻¹). Thus, fractional precipitation was effective in assessing changes in the degree of humification of HAs induced by agricultural practices, including manure application.

Key words:

• fractionation
• humic acids
• humification
• manure
• soil organic matter

INTRODUCTION

An increase in the total carbon (T-C) content in soil as a result of the application of manure (Persson and Kirchmann 1994; Schjønning et al. 1994) accompanies an increase in the content of humic acids (HAs), a major humic substance fraction (Aoyama and Kumakura 2001; Yang et al. 2004). The composition or structure of HAs that is related to their degradability and functions may also be altered by manure application. Dorado et al. (2003) observed brighter-coloured HAs under manure amendment compared with those in non-manured treatments. In contrast, no significant differences in the chemical characteristics of HAs between a Chernozemic soil with and without manure application, notwithstanding the increase in the amount of HAs in the former plot, were reported by Campbell et al. (1986). Insignificant temporal variations in the elemental composition and the infrared (IR) and ¹³C nuclear magnetic resonance (NMR) spectra of HAs in a yellow soil (Typic Hapludult), irrespective of manure application, were also observed by Watanabe et al. (2007), whereas the degree of humification of HAs that was evaluated by their degree of darkening slightly increased with time. In an ando soil (Pachic Melanudand), the effect of manure on the degree of humification of HAs became more marked with an increasing rate of application (Aoyama and Kumakura 2001), but variation in the composition of the functional groups was insignificant at a practical application level (Watanabe et al. 2007).

The degree of humification seems to be a very sensitive parameter that reflects the effect of cultivation or manure application on HAs. However, changes in the degree of humification may be diluted by the humification of manure or crop residues occurring in parallel to the degradation of indigenous HAs, and may become more prominent by fractionating HAs. Various fractionation techniques, such as size-exclusion chromatography (Kawahigashi et al. 1995), adsorption chromatography (Yonebayashi and Hattori 1990), reversed-phase chromatography (Woelki et al. 1997) and electrophoresis (Saiz-Jimenez et al. 2006), have been applied to HAs. Among them, a classical precipitation method using alkali–ethanol solutions (Kumada and Kawamura 1968; Kumada 1987) may be most suitable to separate HAs into fractions with different degrees of humification. In the present study, we assessed temporal change in the composition of HAs in upland field soils using the fractional precipitation method. We expected that variation in the composition of HAs with respect to the degree of humification would be reflected in the distribution pattern, for example, an increase in the amount of one or more fractions as a result of an accumulation of manure-derived HAs with small degrees of humification.

MATERIALS AND METHODS

Humic acid samples

Topsoil (Typic Hapludults) collected from two upland field plots, TCF and TCM, at the Nagoya University Farm, Togo, Aichi, Japan, in 1996 and topsoil collected just before the plots were initiated in 1987 were used. TCF was treated with chemical fertilizer consisting of urea, fused magnesium superphosphate and KCl at rates of 370 kg N ha⁻¹ year⁻¹, 420 kg P₂O₅ ha⁻¹ year⁻¹ and 370 kg K₂O ha⁻¹ year⁻¹, and TCM was treated with 40 Mg ha⁻¹ year⁻¹ cattle manure and chemical fertilizer at the same level as that for TCF. Details of the plots, including the type of crops, soil chemical properties and properties of the manure, are described in Katayama et al. (1995, 1998) and Watanabe et al. (2007).

Topsoil (Pachic Melanudands) collected from three plots, KCF, K80M and K160M, at the National Agricultural Research Center for the Tohoku Region 19 years after the plots were initiated as well as the initial topsoil were also used. These plots were grown with potato (first year) and corn (after second year), and (NH₄)₂SO₄, superphosphate and K₂SO₄ were applied at rates of 20–90 kg ha⁻¹ year⁻¹ N, 80–120 kg ha⁻¹ year⁻¹ P₂O₅ and 80–90 kg ha⁻¹ year⁻¹ K₂O. For K80M and K160M, an additional 80 and 160 Mg ha⁻¹ year⁻¹ of cattle manure were applied, respectively. Details of these plots are shown in Sugihara et al. (1979).

Humic acids were prepared by Watanabe et al. (2007) according to the NAGOYA method (Kuwatsuka et al. 1992).

Fractional precipitation in 0.01 mol L⁻¹ NaOH–ethanol solutions

The HAs (48 mg) were dissolved in 12 mL of 0.01 mol L⁻¹ NaOH containing 10 g L⁻¹ of NaCl and 3 mL of ethanol was dropped into the solution while stirring to make a mixing ratio of 200 mL L⁻¹. The suspension was left at 4°C overnight and then the supernatant was separated by centrifugation. The precipitate (designated as 20P) was washed with a small volume of 0.01 mol L⁻¹ NaOH containing 10 g L⁻¹ of NaOH–ethanol (8:2) solution, and the washing separated by centrifugation was combined with the supernatant. Ethanol was added to the supernatant to make a mixing ratio of 300 mL L⁻¹ and the precipitate (30P) was collected using the method outlined for 20P. Thereafter, similar procedures were repeated to obtain five fractions that precipitate at ethanol concentrations of 400, 500, 600, 700 and 800 mL L⁻¹ (40P–80P) and a fraction soluble at an ethanol concentration of 800 mL L⁻¹ (80S). Appropriate fractionation conditions, including the concentration of HAs, temperature and duration after mixing with ethanol, and an addition of NaCl, including the concentration, were examined in preliminary experiments (data not shown).

Fractions 20P–80P were dissolved in 0.01 mol L⁻¹ NaOH, neutralized with 0.1 mol L⁻¹ KH₂PO₄ and dried-up using a rotary evaporator. To remove ethanol completely, each fraction was suspended in pure water and dried-up repeatedly. The ethanol in 80S was also removed in the same manner after the pH was regulated at 7 with 0.1 mol L⁻¹ KH₂PO₄.

Fractionation of each sample was conducted in duplicate.

Determinations of organic C concentration and the degree of humification

After the removal of ethanol, each fraction was dissolved in 0.1 mol L⁻¹ NaOH and the absorbances at 600 and 400 nm were measured with a spectrophotometer (UV-2200; Shimadzu, Kyoto, Japan) immediately. A portion of each fraction was diluted with a fivefold volume of 0.067 mol L⁻¹ KH₂PO₄ and the organic C concentration was determined using a dissolved organic C analyzer (TOC-500; Shimadzu) after bubbling with N₂. The degree of humification was evaluated with two variables, A ₆₀₀/C and log(A ₄₀₀/A ₆₀₀), where C, A ₆₀₀ and A ₄₀₀ are the organic C concentration (mg mL⁻¹) and absorbances at 600 and 400 nm, respectively (Ikeya and Watanabe 2003). These values could not be obtained for some fractions with a small yield.

RESULTS AND DISCUSSION

The content and the degree of humification of unfractionated HAs in each soil sample (Watanabe et al. 2007) are presented in Table 1. In brief, in comparison with the initial soils, the HA content tended to be greater in TCM and K160M, smaller in KCF, and similar in TCF and K80M. The degree of humification of HAs slightly increased in TCF, but the faint increase in TCM was difficult to confirm. The degree of humification of HAs barely varied in KCF, whereas it decreased slightly in K80M and distinctly in K160M.

Table 1 Humic acid content in the soil and the degree of humification of humic acids†

Soil	Plot	Humic acid content (mg C g^−1)	A 600/C ^‡	log(A 400/A 600)^§
Togo	Initial	1.66	0.97	1.00
	TCF^¶	1.62	2.13	0.814
	TCM^††	4.30	1.55	0.971
Kuriyagawa	Initial	25.2	9.52	0.533
	KCF^¶	21.1	9.58	0.534
	K80M^††	25.1	8.93	0.540
	K160M^††	34.2	5.98	0.609
^†From Watanabe et al. (2007).
^‡Absorbance at 600 nm in 0.1 mol L^−1 NaOH per mg C mL^−1.
^§Logarithmic value of the ratio of absorbance at 400 nm to that at 600 nm in 0.1 mol L^−1 NaOH.
^¶Plot with chemical fertilizer.
^††Plot with manure at 40 (TCM), 80 (K80M) or 160 (K160M) Mg ha^−1 year^−1 plus chemical fertilizer.

Figure 1 shows the yield, log(A ₄₀₀/A ₆₀₀) and A ₆₀₀/C of the fractions of Togo HAs. Recovery in the fractionation ranged from 92 to 98% on a C basis. Yields of 20P to 40P were negligible. Although the other five fractions belonged to Type Rp in Kumada's classification (Ikeya and Watanabe 2003), a fraction precipitated at a lower ethanol concentration had a lower log (A ₄₀₀/A ₆₀₀) and a higher A ₆₀₀/C values, that is, a larger degree of humification. Notwithstanding the similar HA content in the soil, the distribution pattern in the fractionation of TCF HAs differed from that of the initial Togo HAs. The increase in the degree of humification of TCF HAs after 10 years was shown as a shift in the dominant fraction from 70P to 60P because the degree of humification of the five fractions was similar between the two HAs. In TCM, the amounts of all the fractions increased after 10 years (Fig. 1c), but the distribution pattern was similar to that of the TCF HAs. The larger accumulation of 60P than the other fractions indicated an increase in the degree of humification of HAs in TCM.

Figure 2 shows the yield, log(A ₄₀₀/A ₆₀₀) and A ₆₀₀/C of the fractions of Kuriyagawa HAs. The recovery ranged from 91 to 98% on a C basis. The arrangement of log(A ₄₀₀/A ₆₀₀) and A ₆₀₀/C along with a variation in ethanol concentration was observed, although the degree of humification of each fraction was not necessarily equal to that from the Togo HAs. Although fractions 80P and 80S belonged to Type Rp, 60P and 70P were classified into Type B. Fractions 20P to 50P belonged to Type A. The yields of eight fractions and their degree of humification were similar between the initial soil and the KCF soil after 19 years. The K80 M HAs after 19 years also showed a similar distribution pattern in the fractionation. However, the A ₆₀₀/C value of 20P that accounted for 49–63% of the total HAs was smaller in the K80 M soil (10.9) than in the initial and KCF soils (12.3). The difference in the A ₆₀₀/C of 20P between the initial Kuriyagawa or KCF soil HAs and the K80M soil HAs (1.4) was more probable than the difference in A ₆₀₀/C between the unfractionated HAs (0.59–0.65; Table 1).

Figure 1  Yield (□), A400/A600 (○) and A600/C (•) of fractions obtained from Togo humic acids by fractional precipitation in 0.01 mol L−1 NaOH–ethanol solutions. (a) Initial soil before the plots were initialised, (b) chemical fertilizer plot after 10 years and (c) plot with cattle manure at 40 Mg ha−1 year−1 plus chemical fertilizer after 10 years. Bars indicate ranges of duplicated data. A400/A600 and A600/C were not determined for 20P–40P.

Figure 2  Yield (□), A400/A600 (○) and A600/C (•) of fractions obtained from Kuriyagawa humic acids by fractional precipitation in 0.01 mol L−1 NaOH–ethanol solutions. (a) Initial soil before the plots were initialized, (b) chemical fertilizer plot after 19 years; (c) and (d) plots with cattle manure at 80 or 160 Mg ha−1 year−1 plus chemical fertilizer after 19 years. Bars indicate ranges of duplicated data. In (d) A400/A600 and A600/C were not determined for 20P.

The fractionation pattern of K160M HAs differed markedly from that of the other Kuriyagawa HAs. In addition to the greater amounts of 70P, 80P and 80S, the dominant fraction shifted from 20P to 40P. These observations were compatible with a smaller degree of humification of the unfractionated K160M HAs (Table 1). The increases in the amounts of 70P, 80P and 80S were probably because of the accumulation of manure-derived HAs having small degrees of humification, which have been suggested from the chemical characteristics of K160M HAs (Watanabe et al. 2007). It is difficult to believe that almost all the HAs comprising 20P had been degraded during the 19-year period. Hence, most of them were probably not precipitated as 20P but as 40P because of the interaction with manure-derived materials. Watanabe et al. (2007) suggested a preferential degradation of the larger HAs among the manure-derived HAs in K80M, whereas their accumulations were observed in K160M. Interaction of such large molecules with highly humified indigenous HAs might have changed their solubility. An elucidation of their relationship, that is, bound covalently, interacted physically in soil or only co-precipitated during the fractionation procedure, may give us helpful information for evaluating the effect of manure application on the dynamics of HAs in agricultural soils.

Conclusion

The fractional precipitation method with 0.01 mol L⁻¹ NaOH–ethanol solutions was useful for expressing the change in the composition of HAs with respect to the degree of humification resulting from agricultural practices, including manure application.

ACKNOWLEDGMENTS

The authors are grateful to Dr M. Saito, National Institute for Agro-Environmental Sciences, for supplying them with the Kuriyagawa soil samples. We also thank Professors M. Kimura and A. Katayama, Nagoya University, for their valuable suggestions.

Notes

Present address: †School of Veterinary Medicine, Kitazato University, Towada, Aomori 034-8628, Japan.