Characterisation of fulvic and humic acids from leaves of
Eucalyptus camaldulensis
and from decomposed hay
S.M. Harper
a,*, G.L. Kerven
b, D.G Edwards
b, Z. Ostatek-Boczynski
ca
Queensland Department of Primary Industries, Gatton Research Station, Locked Mail Bag 7 MS 437, Gatton, Qld 4343, Australia b
School of Land and Food, The University of Queensland, Brisbane, Qld 4072, Australia c
Bureau of Sugar Experiment Station, Meiers Rd, Indooroopilly, Qld 4068, Australia
Accepted 2 February 2000
Abstract
Soluble soil organic components including fulvic acid (FA) and humic acid (HA) have the ability to complex cations, such as plant toxic monomeric aluminium (Al). We describe the chemistry of FA and HA extracted from two sources before their use in solution culture experiments aimed at determining their ability to ameliorate Al toxicity. Aqueous extracts containing FA and HA were obtained from aerobically incubated leaves of Eucalyptus camaldulensis (Eucalyptus) and decomposed grass and lucerne hay (Compost). The FA and HA were separated and concentrated by precipitation and ion adsorption using XAD-7 resin, puri®ed and dried. Selected elemental properties, size exclusion chromatograms and solid state13C-NMR spectra obtained for the FA and HA from both sources showed distinct structural dierences. The FA obtained from the extensively decomposed Compost consisted of simpler and smaller components. The younger FA and HA obtained from the freshly prepared extract of Eucalyptus were very similar in their chemical attributes.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Aluminium toxicity;Eucalyptus camaldulensis; Fulvic acid; Humic acid; Monomeric aluminium
1. Introduction
The decomposition and transformation of organic material in soils ultimately results in the formation of fulvic acids (FAs) and humic acids (HAs) (Kononova, 1966). Fulvic and humic acids are important com-ponents in soil and natural water systems, and they are able to strongly complex both plant essential and toxic elements (Schindler et al., 1973; Plankey and Pat-terson, 1987; Lobartini and Orioli, 1988; Suthipradit et al., 1990; Lakshman, 1993). In particular, their role in ameliorating Al toxicity in acid soils is of considerable interest.
Fulvic and humic acids possess similar character-istics in that both acids are composites of smaller and
similar molecular units. The myriad of these smaller units includes components, such as aliphatic and aro-matic groups, oils, amino acids, phenols, phenolic acids, phenolic esters, fatty acids, alkanes, tannins, monosaccharides and polysaccharides (Hayashi and Nagai, 1961; Kunc, 1972; Kononova and Alexandrova, 1973; Almendros and Gonzalez, 1987; Piccolo et al., 1990; Chernikov et al., 1991). Moreover, the assigning of molecular weights to both FA and HA is dicult since neither of these exist as discrete compounds in their own right.
Nutrient solution studies have often been conducted to determine the direct eects of FA and HA on plant growth (Schnitzer and Poapst, 1967; Linehan, 1976; Rauthan and Schnitzer, 1981). The volumes of sol-ution used in these studies were small (R600 ml for extended growth periods), probably because of the lim-ited availability of FA and HA and the diculty involved in their extraction. Commercial preparations
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* Corresponding author. Tel.: +61-7-5466-2222; fax: +61-7-5462-3223.
of these acids are available but expensive, generally of unknown origin, and hence, not likely to be represen-tative of the organic components found in agricultural systems (Malcolm and MacCarthy, 1986).
The quantity recovered from soils is generally low; hence, harsh extractants are often used which can alter the structure of the FA and HA making them less representative of the in-situ acids (Bremner and Lees, 1949). Kononova and Alexandrova (1973) also suggested that the alkali extraction technique used to extract HA from soils has a problem in that it coex-tracts plant tissue components which are not constitu-ents of HA.
We describe a method whereby large quantities of FA and HA (ca. 100 g) were extracted from two sources; a decomposed grass and lucerne hay (Com-post) and from a timber species, Eucalyptus camaldu-lensis. The extracted FA and HA were puri®ed, characterised and compared with each other and held for subsequent solution culture studies.
2. Materials and methods
2.1. Preparation and extraction of Eucalyptus FA and HA
Air dry leaf material (1 kg) of E. camaldulensis
Dehnh. (collected from Gympie State Forestry Research Station, Qld, Australia, latitude 26810'S longitude 152840'E) was aerated continuously in 15 l H2O in each of the ®ve 25 l plastic trays. The trays
were maintained at 308C in a temperature controlled water bath. After 35 days, a bulked (and, hence, unre-plicated) aqueous extract was obtained and succes-sively ®ltered through 800, 400, 200 and 45 mm nylon screens. The ®ltrate was centrifuged at 20,000g rela-tive centrifugal force (RCF) for 20 min to remove micro-particulate and microbial matter. The super-natant was decanted and acidi®ed to pH 1.5±2.0 to precipitate the HA component. This suspension was then centrifuged at 10,400g RCF for 15 min. The HA precipitate was redissolved in 100 mM KOH, acid-i®ed to pH 1.5±2.0, and centrifuged to remove any FA contaminant. The HA was then dialysed against triple deionised H2O for 24 h.
The supernatants containing the non-HA com-ponents, taken from the two precipitations, were passed through a 640 ml capacity glass column (300 mm 52 mm) packed with Amberlite XAD-7 poly-meric resin conditioned to pH 2.0. The solution was pumped at a ¯ow rate of 10 ml minÿ1
using a Master-¯ex pump (model 7015-20). The adsorbed FA, of dark brown colour and pungent or aromatic odour, was eluted with about 800 ml of 0.1 M KOH. The eluted FA was further passed through a column (21 cm3.3
cm) packed with Dowex MSC-1 macroporous cation exchange resin, in the H-form, to remove adsorbed cations.
2.2. Preparation and extraction of Compost FA and HA
Moist well-decomposed Johnson grass (Sorghum halepense L.) and lucerne (Medicago sativa L.) hay (Compost) was collected from a farm in the Lockyer Valley, Qld. Five kilograms of the moist material (about 30% moisture by weight) was added to each of the 10 25 l plastic trays. About 15 l of water was added to each tray and the mixture was aerobically incubated for 3 days to extract the FA and HA. A sol-ution extract was decanted from each tray and sequen-tially ®ltered through an 800 and 200mm nylon screen and bulked giving a single unreplicated source of sol-ution. This turbid solution was left untouched for sev-eral hours and when the micro-particulates had settled, the supernatant was siphoned into a 25 l black poly-thene drum. This solution was then acidi®ed to pH 1.5±2.0 to precipitate the HA. The solution containing FA and a small amount of unsettled HA was passed through a 640 ml capacity glass column (300 mm52 mm) packed with Amberlite XAD-7 polymeric resin, conditioned to pH 2.0. The solution was pumped at a rate of 20±30 ml minÿ1 using a Master¯ex variable speed pump model 7554-20. By means of a ¯oat arrangement, solution was drawn from the surface until about 2.5 cm of the solution remained in the drum. This residual solution was held for further puri-®cation. Thus, the solution passed through the column was, in terms of high molecular weight organic acids, predominantly FA.
The FA (and its small HA contaminant) were eluted from the XAD-7 column with about 800 ml 100 mM KOH. The eluted FA and HA extract was reacidi®ed to pH 1.5±2.0 and centrifuged at 10,400g RCF to remove the precipitated HA contaminant. The FA supernatant was again passed through the XAD-7 col-umn and eluted using 0.1 M KOH. The eluted FA was further passed through a column (21 cm 3.3 cm) packed with Dowex macroporous cation exchange resin, in the hydrogen form, to remove adsorbed cat-ions.
deio-nised water. Exhaustive extractions of FA and HA were made from the initial 10 trays of Compost.
Both the FA and HA extracted from the Eucalyptus and Compost were dried in a vacuum oven at 558C at a pressure of 5.3 kPa. Industrial grade dry N2 was
bled into the oven. The dried FA and HA were stored in sealed plastic bottles at room temperature.
2.3. Elemental analysis
Selected elemental properties were determined for the FA and HA extracted from both sources. The or-ganic acids were solubilised using 1 M KOH, acidi®ed, esparged with He, and element concentrations were then measured using inductively coupled plasma atomic emission spectroscopy (ICPAES).
2.4. Size exclusion chromatography analysis
A 1 ml sample of each dissolved organic acid frac-tion (about 1 mg acid in 1 ml) was passed through a Pharmacia C10/40 column (300 mm10 mm), packed with Fractogel TSK HW-40 (S). The mobile phase consisted of 100 mM NaHCO3 solution with 2%
acetonitrile and a ¯ow rate of 1 ml minÿ1. A Knauer Model 87 variable wavelength detector at 254 nm was used to detect signals and data was processed using Waters Maxima 820.
2.5. Solid-state13C-NMR spectra
Solid-state 13C-NMR spectra of the samples were obtained on a Bruker MSL300 spectrometer operating at 75.482 MHz for13C. A13C ®eld strength of 68 kHz was used, corresponding to p=2 pulse times of 3.7 ms. Samples were spun in zirconium oxide rotors at the magic-angle at a spinning speed of 3 kHz. Spinning side-bands were eliminated using TOSS pulse sequence.
2.6. Statistics
The extraction technique involved the processing of a large volume of liquid (75 1 for the Eucalyptus and 150 1 for the Compost). The processing of such a large volume has eectively averaged variability across the whole bulked sample. The data presented are the measured results from this bulked sample. The equip-ment used to measure eleequip-mental composition is very precise and repeatable results were achieved on the same sample. The ICPAES takes 10 measurements over 10 s giving a mean reading with a standard error. If high standard errors be recorded (>5%), the equip-ment is re-calibrated against the standards.
3. Results and discussion
Selected elemental properties of the Eucalyptus and Compost FA and HA are presented in Table 1. The C content (g kgÿ1
) was higher in the Eucalyptus FA than in the Eucalyptus HA and in the Compost FA com-pared with the Compost HA; values ranged from 610 g kgÿ1 in the Eucalyptus FA to 468 g kgÿ1 in the Compost HA. This is an unusual result as HAs gener-ally contain a higher C content due to the greater pro-portion of aromatic components (Hatcher et al., 1981) and the presence of stronger aromatic peaks in the FAs than in their counterpart HAs from the same source may explain this anomaly. Furthermore, the C content of the Eucalyptus acids, tended to be higher than that of the Compost acids, presumably due to the presence of stronger aromatic peaks and, hence, greater C condensation.
The greater concentration of most elements in the HA than in the FA fractions undoubtedly resulted from the limited ability of dialysis to desorb cations from the HA; the passage of the FA through a cation exchange resin in the hydrogen form was more eec-tive in desorbing cations. The concentration of the inorganic elements was higher in the Compost FA and HA than in the Eucalyptus FA and HA ; this data is consistent with the recorded ash content which was highest in the Compost HA, followed by Compost FA and least in the Eucalyptus FA.
The size exclusion chromatography (SEC) elution
Fig. 1. Elution curves for mm (i) Eucalyptus FA, (ii) Eucalyptus HA, (iii) Compost FA and (iv) Compost HA with Fractogel TSK HW-40 (S) column, 10300 mm. (Mobile phase: 10 mM NaHCO3
curves for the four acids are presented in Fig. 1. The Eucalyptus FA and HA chromatograms (Fig. 1(i) and (ii)) showed two well-de®ned peaks; the larger peak (time 7±8 min) eluted in the void volume and the mol-ecular weight was accordingly greater than 10,000 Da. The smaller peak (elution time 14.75 min for the FA and 12.92 min for the HA) was less than 5000 Da. A third less well-de®ned peak (elution time of 12.00 min) was observed in the Eucalyptus FA chromatogram. The earlier elution of the Eucalyptus HA peak indi-cated that the molecular weight of this fraction was greater than that of the FA fraction. The basic simi-larity in the chromatograms for Eucalyptus FA and HA is probably related to the newly formed nature of these acids.
The SEC elution curves for the Compost FA and HA Fig. 1(iii) and (iv) were quite dierent. The Compost FA chromatogram showed four well-de®ned peaks (elution times > 8.33 min) representing fractions of less than 10,000 Da and a less well-de®ned peak (elution time 8.33 min) representing a fraction greater than 10,000 Da. The Compost HA chromato-gram consisted of only two peaks, one representing a fraction greater than 10,000 Da (elution time 7.42 min) and the other a fraction less than 10,000 Da (elution time 17.06 min). The later eluted fraction was of about the same molecular size as the fourth eluted fraction (16.83 min) in the Compost FA.
The13C-NMR spectra are presented in Fig. 2, and a summary of the major components found in the FA and HA preparations is presented in Table 2. The Eucalyptus FA and HA spectra (Fig. 2(i) and (ii) are very similar; indeed, the spectra for the two acids are almost indistinguishable from each other. In contrast, the spectra for the Compost FA and HA (Fig. 2(iii) and (iv)) show some similarities and some clear
dier-ences. All acids had well-developed carboxyl peaks (at 172 ppm) and hydroxyl peaks (at 55 ppm). The Euca-lyptus FA and HA and Compost HA spectra showed peaks at 70 ppm which correspond to the hydroxyl C of carbohydrate groups; this peak was much smaller in
Table 1
Some chemical properties of the FA and HA extracted from leaves ofEucalyptus camaldulensis(Eucalyptus) and from decomposed grass and lucerne hay (Compost)
Chemical property Organic acid
Eucalyptus FA Eucalyptus HA Compost FA Compost HA C (g kgÿ1
) 610 556 563 468
Ash (g kgÿ1) < 0.1 4.2 6.9 12.6
Ba 2 2 4 2
Caa 0 2 0 48
Pa 57 166 114 404
Mga 2 1 1 16
Cua 3 13 18 88
Fea 13 199 18 750
Mna 1 12 1 2
Zna 1 12 3 20
Sa 47 238 215 1000
Ala 24 135 40 425
a
Units are mg kgÿ1
.
Fig. 2.13C-CP-MASS NMR spectra of (i) Eucalyptus FA, (ii)
the Compost HA spectra. High concentrations of car-boxyl and hydroxyl groups in HA infer the ability to compex cations (Lobartini and Orioli, 1988).
Both the Compost FA and HA had well-de®ned peaks at 147 ppm (corresponding to diphenols) which were absent in the Eucalyptus FA and HA. In con-trast, the Eucalyptus FA and HA spectra showed well-developed peaks at 154 ppm (corresponding to mono-phenols) which were absent in the Compost FA and HA. Further, four peaks detected in the Eucalyptus FA and HA (at 115, 107, 37 and 17 ppm) highlighted a high proportion of aliphatic components which were not observed in the spectra for the Compost FA and HA.
Leaves of Eucalyptusspp. have a high proportion of long chain aliphatic hydrocarbons with aldehyde, alco-hol and ester components, as well as ¯avonoids and terpenoids (Li et al., 1997). In contrast, the constitu-ency of grasses is of short chain aliphatic components (Newman and Tate, 1991), this is con®rmed in the
13
C-NMR spectra. The Compost FA spectrum showed a peak at 42 ppm which was not observed in the spec-tra of the other FA and HA, and represented a further type of unsubstituted aliphatic C (probably alkyl C). The Compost HA had a peak at 30 ppm (probably acetyl aliphatic C) which was not present in the other acids. It is likely that the structural dierences in FA and HA extracted from the two sources were directly related to the inherent structural dierences in the parent plant material from which the FA and HA were derived.
Information from the 13C-NMR spectra supports that from the SEC spectra, where the chromatograms for the Eucalyptus FA and HA were very similar, while those for the Compost FA and HA were quite dierent. Visually, the Compost material from which the FA and HA were extracted was very well decom-posed and bore little resemblance to the parent organic material, whereas the Eucalyptus material was newly decomposed and had not undergone the same amount of transformation. This might explain why the SEC
and 13C-NMR spectra for the Compost FA and HA were markedly dierent from each other, as well as from the Eucalyptus FA and HA. The presence of diphenols in the Compost FA and HA, and not in the Eucalyptus FA and HA, may be related to the fact that the Compost FA and HA were more heavily transformed. Similarly, the presence of monophenols in the Eucalyptus FA and HA, and not in the Com-post FA and HA, may be indicative of the less degraded nature of the Eucalyptus FA and HA. Many more de®ned peaks were evident in the Eucalyptus FA and HA spectra than in the Compost FA and HA spectra which indicates the greater heterogeneity of the Eucalyptus acids, probably due to their lower degree of degradation. The Compost FA, which exhibited fewer components in its 13C-NMR spectrum, was clearly of simpler composition than the Eucalyptus FA and the two HAs. Also, most of the fractions in the Compost FA were of relative molecular mass (RMM) less than 10,000 Da which contrasted to those in the Eucalyptus FA and the other two HAs which were predominantly greater than 10,000 Da.
A 70 ppm peak in the 13C-NMR spectrum for the Compost FA was absent and only a small 70 ppm peak was present in the Compost HA. This contrasted with the large peaks at 70 ppm in both the Eucalyptus FA and HA spectra and suggests that the latter still contained considerable proportions of undecomposed polysaccharide components. Kogel et al. (1988) found pronounced peaks at 72 and 105 ppm in three forest litter extracts with further peaks at 22, 55, 60±90 115, 130, 150 and 175 ppm. This range of spectral peaks is consistent with the range of peaks determined for the Eucalyptus FA and HA and is analogous to constitu-ents of woody materials Kogel et al. (1988).
The Compost FA had broad and strongly developed
13
C-NMR spectral peaks in the range of 15±55 ppm emphasising the highly aliphatic nature of this acid Piccolo et al. (1990); further strong peaks were recorded at 130, 147 and 172 ppm. The prominence of these components of the spectra is consistent with that
Table 2
Components of the FA and HA extracted from leaves ofEucalyptus camaldulensisand from decomposed grass and lucerne hay (Compost) as determined by solid-state13C-NMR spectra
Organic acid Resonance peaks (ppm)a,b
172 154 147 130 115 107 83 70 55 42 37 30 17
Eucalyptus FA
Eucalyptus HA
Compost FA
Compost HA
a
Resonance peak values correspond to the following components: 172; carboxylic C; 154; aromatic Monophenol; 147; aromatic diphenol; 130; alkene C (non-protonated aryl); 115: alkene c; 107; alkene C; 83; alkyne C; 70; hydroxyl C (carbohydrate); 55; hydroxyl C (methoxyl); 42; ali-phatic C (alkyl); 37; aliali-phatic C; 30; aliali-phatic C (acetyl); 17; aliali-phatic.
b
recorded by Newman and Tate (1991) on HAs extracted from a grassland soil. In contrast, the Com-post HA spectrum was less pronounced in the region up to 50 ppm indicating a smaller proportion of ali-phatic components. The two peaks at 70 and 85 ppm in the Compost HA represented cellulose and hemicel-lulose components. The Compost HA spectrum was well developed in the region from 100 to 150 ppm representing aromatic C.
Larger 13C-NMR spectral peaks at 170 ppm (corre-sponding to carboxyl C) were observed in the Compost FA and HA, than those present in the Eucalyptus FA and HA. This is likely to be indicative of oxidation of hydroxyl groups to carboxyl groups over longer aging periods or, alternatively, due to inherent dierences in the parent organic materials. The smaller incidence of carboxyl carbon in the Eucalyptus FA and HA high-lighting the C present in these acids, is of a more con-densed structure. These ®ndings are consistent with those recorded by Kononova and Alexandrova (1973), who found that recently formed HA had a diverse structure and was low in carboxylic groups, but mature HA had a more uniform structure and a higher proportion of carboxylic groups. The presence of greater proportions of carboxylic C in the Compost FA than in the other FA and HA is likely to infer it with a greater capacity to complex cations including toxic Al3+. The importance of carboxyl groups in complexation reactions is well documented (Hue et al., 1986; McColl and Pohlman, 1986; Lobartini and Oriolli, 1988).
Acknowledgements
This study formed part of an Australian Centre for International Agricultural Research Project ``Manage-ment of Acid Soils for Sustainable Food Crop Pro-duction in the Humid Tropics of Asia''. Assistance from The Centre for Magnetic Resonance, The Uni-versity of Queensland, St. Lucia, Qld, Australia is gratefully acknowledged.
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