- Email: [email protected]
Electron paramagnetic resonance characteristics of the humic acids from a podzol
Org. Geochem.Vol. 8, No. 6, pp. 427-440, 1985
0146-6380/85 $3.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
Electron paramagnetic resonance characteristics of the humic acids from a podzol M. V. CHESHIRE,B. A. GOODMAN,D. B. McPHAIL and G. P. SPARLING The Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen AB9 2QJ, U.K.
(Received 7 November 1984; accepted 21 May 1985) Abstract--A study has been made of solid and solution electron paramagnetic resonance (EPR) spectra of humic acids from different horizons in a podzolic soil. Hyperfine splitting was observed in the solution spectra of humic acids from all horizons and depended on the strength of alkali and the period of dissolution. The upper organic horizons L, F and O~ contained humic acids with some spectral characteristics in common with lignin. Humic acid from the lower horizons showed different spectra. At least 5 different radical signals were present.
Key words: electron paramagnetic resonance, free radical, humic acid, hyperfine structure, solution spectra, podzol
INTRODUCTION
MATERIALS AND METHODS
The presence of free radicals in humic substances is now firmly established. In the solid state the electron paramagnetic resonance spectra display a single peak with g-values in the range 2.0023-2.0051 (Minderman, 1979). Several authors (e.g. Steelink and Tollin, 1962; Steelink, 1964; Riffaldi and Schnitzer, 1972) have estimated the free radical contents of various humic materials and attempted to relate them to types of humic substance. In solution, humic acids have been reported to exhibit either single peak spectra or hyperfine structure, the latter situation having been equated to humic acids from the more acidic soils (Atherton et al., 1967). However, experimental findings have been variable and some authors have reported failure to find any hyperfine structure where it might have been expected according to the criteria of Atherton et al. In this paper a study has been made on the effect of different concentrations of alkali on the EPR spectra of humic acids from different horizons of a podzolized soil profile. In very acidic soils and peats the decomposition of plant residues is usually slower than in less acid conditions and so plant components such as lignin can persist for long periods. Lignin displays hyperfine structure in EPR spectra (Steelink, 1966) and this study also explores the possible relationship between the free radical of this substance and the humic acid in the different horizons. Many fungi, which produce dark coloured polymeric substances, are particularly resistant to lysis and some accumulation of their residues would be expected in soil (Webley and Jones, 1971). An examination has, therefore, also been made of the dark brown stipes of Marasmius androsaceus which was prevalent at the site.
The soil profile examined occurred under coniferous wood at Blacktop (National Grid reference NJ 866047). The predominant plant species was Scots Pine (Pinus sylvestris L.). The soil was of the Charr series of the Countesswells Association and details of the profile are given in Table 1. Material from each horizon was sampled together with fresh pine needles, yellowing needles and dead needles caught in the branches of the trees. All samples were air-dried. Stipes of the fungus Marasmius androsaceus were collected from the surface litter.
Extraction of litter and soil with alkali Samples of L, F and O horizons of soils were extracted with 0.2 M NaOH (10g soil/100cm 3) for 20 hr. For the mineral-rich horizons, E and Bs, 30 g of sample were used. The mixtures were filtered and humic acid precipitates were obtained by acidification with redistilled 6 M HCI to give solutions 0. l M with respect to HCI. The humic acids were washed with 0.01 M HCI, then water and recovered as solids by freezing, thawing and drying under reduced pressure. The yields are given in Table 1.
Preparation of Mason lignin from fresh pine needles, yellowing pine needles and dead needles (Lai and Sarkanen, 1971)
© The Macaulay Institute for Soil Research, 1984. 427
The samples were continuously extracted with 95% aqueous ethanol and, after drying, with an azeotropic ethanol-toluene mixture using a soxhlet extractor. The residues were treated with 12 M H~SO4 (15 ml per g sample) for 2 h at 21 _+ 2°C before dilution to 0.5 M H2SO4 and hydrolysis at 100°C for 4 hr. The insoluble klason lignin residue was dried under reduced pressure and weighed (Table 2). Samples of the lignin were treated with 0.1 M KOH for 24 hr and the residue extracted with 4 M KOH for 24 hr. In
~vl. \
428
(,ItESHIRF Pi ~1/
'Fable 1. Description of the Charr series soil laorizons, C content and the yield of humic acid Horizon depth (mm) 0 50 50 125 125 250 250 380 380-410 410~460 460 630 630+
Description Dead needles Fresh needle and grass litter Dead needle litler Partially decomposed fibrous litter Black well decomposed organic pH 3.7 Black well decomposed organic some quartz, pH 3.7 Dark grey brown coarse sandy loam Iron pan Yellowish brown sandy loam, pH 4.6 Light yellow brown sandy loam
Table 2. Percentage composition of pine needles and litter 95!',g ethanol soluble
Ethanol toluene soluble
C,,)
('~o)
G3
19,7 122 I 1, I 8,5 2.3 0.2
02 0.6 I. 1 0.9 (1.3 (I.08
33.9 44.3 67.7 62.6" 47.8" 17.3"
Sample Fresh green needles Yellowing needles Dead needles O~ 02 B,
Klason lignin
"Ash free.
each case the soluble matter was separated by filtering on a sintered glass filter. Acidification of the solutions with HC1 precipitated small quantities of dark brown material in each case. These will be referred to as lignin (0.1 M) and lignin (4 M) respectively. Soil from various horizons was also subjected to the same extraction procedure. In the last stage, the residue, which should contain any lignin present, was extracted with 1 M KOH, followed by 4 M KOH.
Extraction of Marasmius androsaceus stipes 25 stipes, about 30 mg, were extracted twice with 1 M KOH (5 cm 3) over a period of 70 hr at 20 + 2°C. Most of the colour was extracted. The orange extracts were acidified with HCI, when part of the material was precipitated. The precipitate was redissolved in 1 M KOH and dialysed against water with no apparent loss of colour. The suspension was evaporated to dryness under reduced pressure to give a yield of 43%.
EPR Spectroscopy All spectra were obtained at room temperature with a Varian E104A X-band spectrometer using 100kHz modulation frequency for 1st derivative spectra and 50 kHz for 2nd harmonic recording. Both modulation amplitude and microwave power were strictly controlled so as to avoid any line broadening or saturation, the settings ranging from 0.050.010 mT for the modulation and from 1-5 mW for the power. For solid samples a known weight of material was added to a quartz tube to occupy ca 1 cm of its length and spin concentrations were
Humic acid ('!~,)
Designation
C%
DN LI t,~, F OE ()~
52.3 49,7 ,0.._''~ 46.6 43.5 8 I
E B B, (
34
47
2.9
1
cj I~ ,~~ 20
32.5 !2
calculated by comparing the spectral area with that of a sample containing a known weight of diphenylpicrylhydrazyl (DPPH). For solution spectra the initial alkaline solutions were fully aerated before being sealed in quartz solution cells for the duration of the experiments. RESULTS
Free radicals in solid humic acid samples The free radical contents of the humic acids from the dead needles and the different soil horizons are shown in Table 3. It can be seen that there is a steady increase in radical content with depth through the predominantly organic horizons of the profile until the mineral horizons, where there is a dramatic decrease in the samples from the E and B~ horizons, respectively.
Free radicals in alkaline solution of the humic acids The alkaline solution spectra of the humic acids showed variations depending on the horizon from which the substance originated and the concentration of the alkali, as well as dissolved oxygen and time related effects.
Spectra obtained shortly after dissolution The 1st derivative spectra obtained within 20 min after dissolution of the humic acids in 0.2 M NaOH are shown in Fig. I. The spectra for the dead needles and litter layer show relatively broad single peaks with little or no evidence of any hyperfine splitting. With the F and O~ horizons the humic acids show increasing evidence of some hyperfine structure superimposed on a broad peak. The 02 horizon Table 3. ESR g value, free radical content and lignin estimated as ~,~ of the humic acid from infrared absorbance Horizon
g value
Free radical cone. (spins g 1 x 10 -16)
Lignin (%)
Dead needles L~ L2 F O~ Oz E BS
2.0037 2.0037 2.0037 2.0037 2.0037 2.0034 2.0035 2.0038
2.7 3.6 8.4 6.8 12.8 I 1.7 0.56 0.29
32 23 29 17 12 0 0 0
Humic acids from a podzol
02
0.1 mT I
I
Fig. 1. EPR spectra of 0.2 M KOH solutions of humic acids.
429
430
M V. ('HESHmE ,'t al.
ff
DN
~jj/" ~J
-.,_,_ IfJ
I1
I"
,/
_f'
J
.j
_i
/
/
_J i /
02
f
~f~
/
d"
.%,
\
f
E
\jr
J
ffP
/ F
/
0.1 mT f
-
-
'C" '/4,/ Fig. 2. EPR spectra of 4 M KOH solutions of humic acids.
~
Humic acids from a podzol shows a distinct 4 peak spectrum which probably arises from 2 inequivalent protons with A~ = 0.1175 and A 2 = 0425 mT, classified as a class I spectrum in the nomenclature devised by Haworth (1971). The spectra obtained within 20 min of dissolution of the humic acids in more concentrated alkali, 4 M KOH (Fig. 2), were different from those in the dilute alkali. The dead needles and the litter horizon humic acids gave spectra which were similar to each other, but complex in character, consisting of more than one component. One species of radical gave a signal corresponding to a quintet, 1:4:6:4:1 with A = 0.106 mT. Other components with much smaller hyperfine splitting, were present and these varied in relative strengths in the humic acids. In the F horizon humic acid the quintet radical signal was much weaker in comparison to other signals and the spectrum was dominated by the central feature of a sharp single peak. The 02 horizon humic acid displayed a broad single peak with some poorly resolved structure. Similar structure was observed in the spectra of the samples from the E and Bs horizons, but with increasingly better resolution with increasing depth. At least two radical species are present in the alkaline solutions of the humic substances from these three horizons, but at different relative strengths in the different horizons. The best-resolved component in the B~ horizon humic acid solution consisted of 4 peaks, probably from 2 inequivalent protons with A~ = 0.102 mT and A 2 = 0.061 mT.
Variation of radical signals with time
(i) Humic acid in 0.2 M alkali The main change observed was that the 4 peak spectra from the humic acid from the lower horizons, were slowly transformed into a second type of 4 peak spectrum with parameters similar to those of the 4-peak component that was seen in 4 M alkali. The rate at which this change occurs depends on the concentration of alkali used. Distinct differences were previously noted for short dissolution periods using a range of alkali concentration 0.1-2 M (Cheshire et al., 1975). (ii) Humic acid in 4 M alkali (a) Humic acids from litter horizons: The changes that occur in material extracted from the L~ horizon and the dead needles are almost identical so only the latter are illustrated in Fig. 3. There is considerable change in detail in the centre of the spectrum over the period of a day, but the quintet feature is unaltered with time. The L2 humic acid spectra also showed the quintet and differed only in showing less developed structure in the minor central components. (b) Humic acid from the F horizon: The signal responsible for the quintet feature present in the litter humic acids was also present at a lower intensity in the F horizon humic acid (Fig. 4a). It shows little change with time over a period of 2 days. On the
431
other hand a sharp central peak more clearly seen in the 2nd derivative spectrum (Fig. 4b) lost about 75-80~o of its intensity during the first 3 hr and had almost disappeared within 21 hr. The relative intensities of other features also vary with time, but, because of the complexity of the spectra, it was not possible to match peaks to separate spectral components, except for a possible doublet with A = 0 . 1 6 m T , which is seen in the 2nd derivative spectra after 21 hr. (c) Humic acid from the O horizons: The initial spectrum obtained with the O~ humic acid possessed a sharp central peak in the 2nd derivative and was almost identical to that obtained with the F humic acid. After several hours this signal decreased in intensity and was replaced by a hyperfine spectrum which was practically identical to that obtained in the later stages with the 02 humic acid. The 2nd derivative signals generated by 02 humic acid in 4 M KOH are illustrated in Fig. 5. There was a steady development of structure with time over a period of 2 days, but no evidence in either 1st (not shown) or 2nd derivative spectra for the presence of the quintet structure seen in the humic acids of higher horizons. The spectra appear to be simpler than those from the upper horizons, but nevertheless contain a number (at least five) of different free radicals whose relative contributions to the spectra vary with time. When a humic acid solution that had developed a spectrum while standing for 2 days in the sealed quartz cell was exposed to air, a broad feature developed immediately and was identical to that obtained with H202 treatment. There was a major change in the hyperfine split components as a result of 02 treatment (Fig. 5) but after H202 treatment all the hyperfine structure was lost. Boiling a freshly-made solution of the 02 horizon humic acid in 4 M KOH gave a similar spectrum to material which had been aged in 4 M KOH over a period of 2 days and exposed to oxygen. (d) Humic acid from the E horizon: These spectra (Fig. 6) show a number of features that display similar behaviour to the components in the 02 horizon humic acid spectra. However, the resolution is somewhat better implying perhaps, that fewer different radicals are contributing to the spectra. Exposure to oxygen of a 6-day aged E horizon humic acid solution gave little evidence of the broad component in the spectrum. The 1st derivative spectra are dominated by a sharp hyperfine split component that is much better resolved than in the 02 horizon humic acid spectra, but the 2nd derivative spectra (Fig. 6) indicate that both samples are dominated at this stage by the same two radical species, present in different relative proportions. (e) Humic acid from Bs horizon: In the early stages the spectra showed some resemblance to that of the E horizon humic acid, but the resolution did not improve with time. Aeration caused a complete loss of signal which was regenerated only very slowly.
432
M
10 min
V CH~SmREet or.
~ ~ " ' Y '
30min
2h
23h
0.1 mT
V
! Fig. 3. Variation with time of the EPR spectrum of dead needles humic acids in 4 M KOH.
Free radicals in lignin extracted from pine needles Lignin (0.1 M). The material derived from green needles dissolved in 4 M KOH gave a spectrum which was similar to that from the yellowing needles except for an additional narrow peak component in the centre of the spectrum. After one day, the spectrum from the yellowing pine needle lignin was very similar
to that of the humic acid from the dead needles and L~ horizons. Lig#n (4 M). When these materials were dissolved in 5 M .~OH the main components in the spectra formed after a short time (Fig, 7) were practically identical to the quintet components in the L and F horizon humic acids. The peaks with an approximate
Humic acids from a podzol
433
O
>
M . V . CHESHIRE Cf ,l/.
434
,\
/ boiled
in 4M
KOH
,
I
Fig. 5. Variation with time of the 2nd derivative EPR spectrum of 02 horizon humic acid in 4 M KOH.
1:4:6:4:1 intensity ratio had a separation of approx. 0.11 mT. Free radicals in 95% ethanol extracts Radicals generated in 5 M KOH were investigated and the spectra were found to bear no resemblance to those obtained with humic acids. The extract from yellowing pine needles (Fig. 8 a, b, c) gave a spectrum which was initially dominated by a 4-peak component from two inequivalent protons (A~ = 0.337 mT, A2 = 0.107 mT). The signal rapidly decreased in strength losing 80~o of its intensity in 1 h, within which time a much sharper 3 peak component from 2 equivalent protons (d =0.079mT) had appeared. This signal grew steadily in intensity over a period of 2 days. Some additional, very much weaker, peaks were also present.
The spectra obtained with the extracts from the O~ and B~ horizons were very complex, but quite different both from each other, from the yellowing pine needle extract spectra, and from the humic acid spectra. The large number and small widths of the component peaks, illustrated in Fig. 8d by the spectra from the Bs horizon humic acid after 3.5 hr, suggest that low molecular weight free radicals are probably responsible, but a detailed analysis of these spectra was not possible. Free radicals in alkali extracts of stipe fiom Marasmius androsaceus A very complex evolved with time (Fig. 9) but these from any of the
set of hyperfine splitting patterns from an initial 5 peak spectrum were found to be quite different spectra from the soil horizons;
Humic acids from a podzol
435
1.5h
7h
24 h
50h
(I/VI Ilffk 0.1 mT
I
I
Fig. 6. Variation with time of the 2nd derivative EPR spectrum of the E horizon humic acid in 4 M KOH.
Further details are given elsewhere (Goodman et al., 1985).
Infrared analysis of the humic acids The infrared spectra of the humic acids showed differences in several regions. There was a gradual decrease in the absorption related to aliphatic hydro-
carbons (2850 and 2920 cm-J) in the humic acids on going from the dead needles down the profile. Similarly an absorbance at 1510 cm -~, which is attributed to an aromatic component, gradually decreases with depth to become no longer visible in the 02 layer. This absorbance is attributed to lignin, and, using a comparable lignin standard, an estimate of the
436
M.V. CHFLSHIREet al.
5rain .y
/
~'i!
•
_//
01 T
,
S
Fig. 7. Variation with time of the EPR spectrum from the 4 M KOH extract of green pine needles lignin in 5 M KOH. lignin has been made in each humic acid. The results are given in Table 3 and selected spectra are shown in Fig. 10. The L2, F and Oi horizon humic acids each had additional absorption bands at 1265 and 1420 cm -t and one at 1540 cm-I, interpreted as peptide structure, was present in B, horizon humic acid with traces in the E horizon material. Some absorption which was attributable to layer silicates was present in the humic acids from the lowest three horizons. DISCUSSION
Previous studies on the hyperfine structure of the EPR spectra of O horizon humic acid from this site have reported a relatively simple 4 peak spectrum in 0.1 M NaOH (Haworth 1971, Cheshire et al., 1975). In more concentrated alkali (2 M) a shift of signal occurred to a second four-peak speet~m. These signals have been tentatively related to the spin polarization of a C - H orbital by the unpaired
electron in a ~ type orbital of a benzene ring and a C-H tr orbital elsewhere (i.e. not on the same ring) (Cheshire et al., 1975). The present results, which extend the concentration of alkali used, give more attention to the effects of time on the changes to the EPR signal and, by examining humic acids from different horizons, show that the spectra are more complicated than was hitherto appreciated. The use of 2nd derivatives in addition to the more usual 1st derivatives of the absorption spectra can be of particular value in resolving small amounts of components with narrow lines. The occurrence of hyperfine splitting in the EPR spectra of humic acids in weakly alkaline solutions appears to depend on the soil having a low pH. Generally, Haworth (1971) found that soils with pH4.2 or less had humic acids that displayed splitting, whereas, when the pH was ~-..ater, they did not. Exceptions could be e x ~ m ~ d by pH changes related to changes in soil use, bearing in mind the great resistance of humic substances to decompo-
Humic acids from a podzol
0,1 mT • 1
~
437
L
Fig. 8. EPR spectra of 95% ethanol extracts of yellowing pine needles after (a) 5 min, (b) I hr, (c) 47 hr and (d) 3.5 hr all in 5 M KOH. sition implied by measurements of mean residence times. Haworth considered that the EPR signal related to condensed aromatic structures whose nature depended on the pH at which their chemical synthesis from phenolic substances had occurred. Other explanations can be put forward. For example, although it is a gross oversimplification to describe soil by a single pH value, acid conditions represented by such a value may distinguish the point at which a predominantly bacterial flora is replaced by a more acidophilic fungal one. Such a change may result in
a different metabolic pathway or other products being present and, subsequently, a change in the nature of the more polymerized organic matter. Fungal tissue itself is frequently dark coloured with humic-like substances. The dark brown stipes of one fungus, Marasmius androsaceus, prevalent in the litter at the site were, therefore, examined and their extracts shown to display an EPR spectrum with hyperfine splitting. This was, however, different from that of the humic acids. Because most of the carbon in soil originates from
438
M
V, ('HESHIRti c/ a t
=c
= o
-5 ,=
,,¢ Ok,
E r~
k~
Humic acids from a podzol I
I
I
I
I
I
1
439 I
I
I
J
I
%T
I
I 30
40
I
I
20
16
I
I 12
I
1 8
1
I 4
~ / 1 O0 crn -1
Fig. 10. Infrared spectra of the humic acids of three soil horizons and the equivalent alkali soluble matter of the dead needles. (a) dead needles; (b) F horizon; (c) O~ horizon; (d) Bs horizon.
vegetation, and recognizable plant remains, e.g. lignin, persist in soils for very long periods, particularly under very acid conditions, radicals present in lignin were also examined by EPR spectroscopy. Alkaline extracts of lignins which gave EPR spectra displaying hyperfine splitting were obtained from the predominant litter-producing plant species at the site. The upper horizons of the podzol examined clearly show the presence of a radical from a lignin component in the humic acid, but this is not apparent in the 02, E and Bs humic acids. The infrared absorption band of the humic acids at 1510 cm -1, characteristic of aromatic structures, showed a similar decrease with depth and indicates that if lignin residues remain in the lowest horizons they do so in a much modified form. The changes in humic acid spin concentration with horizon for the organic horizons fits with the changes in the proportions of lignin and humic acid since it has been observed that there is an increase in the free radical content in the sequence lignin, degraded lignin, fulvic acid, humic acid (Steelink and Tollin, 1962). The lower radical content of the humic acid from the mineral horizons E and Bs may be because O.G. 8/6---D
of qualitative differences, but it could also be partially the effect of a higher content of mineral components which may quench some of the signal. The lignin involved in the present study, being from a gynnosperm, is a dehydrogenation polymer of coniferyl alcohol. The initial formation of the lignin is considered to result from the coupling of radicals formed by the enzymic degradation of the coniferyl alcohol. A schematic constitution of the lignin (Alder et al., 1969) reveals a number of different sites on which radicals could be formed. It is generally believed that the formation of humic substances depends on the prior breakdown of lignin to quinone monomeric units, but there is the possibility that lignin may survive in a partially degraded form (Martin and Haider, 1980). Tollin et al. (1963) appear to have been the first to mention hyperfme splitting in the alkali solution of humic acids. Since then various others have reported similar findings (Haworth and Atherton, 1965; Metcalfe, 1970; Cheshire and Cranwell, 1972; Minderman, 1979). The present results show that the radical species present are a more complex mixture than is implied from previous reports.
M.V. ('HESHIRF ('[ d/
440
Acknowledgements--We would like to thank Dr J. D. Russell and Mr A. R. Fraser for the infrared analyses and Miss Marian Thorn for the elemental analyse~
REFERENCES
Alder E., Larsson S., Lundquist K. and Miksche G. E. (1969) Abstract of the International Wood Chemistry Symposium, Seattle. (Illustrated in Chap. 1, Lignin structure and morphological distribution in plant cell walls by T. Higuchi.) In Lignin Biodegradation: Microbiology, Chemistry and Potential Applications (Edited by Kent T. K., Higuchi T. and Chang H.), Vol. 1, Chap. 1, pp. 1-19. CRC Press 1980. Atherton N. M,, Cranwell P. A., Floyd A. J. and Haworth R. D. (1967) Humic acid--l. ESR spectra of humic acids. Tetrahedron 23, 1653-1667. Cheshire M. V., Goodman B. A. and Mundie C. M. (1975) The composition of soil humus. Welsh soils Discussion Group Report No. 16, pp. 73-90. Cheshire M. V, and Cranwell P. A. 11972) Electron-spin resonance of humic acids from cultivated soils. J. Soil Sci. 23, 424-430. Goodman B. A., Vaughan D., Cheshire M. V. and Sparling G. P. (1985) ESR Investigations of some fungal pigments. Biochem. Soc. Trans. 13, 623-624. Haworth R. D. (1971) The chemical nature of humic acid. Soil Sci. 111, 71-79. Haworth R. D. and Atherton N. M. (1965) Humic acid. Bull. Nat. Inst. Sci, India No. 28, pp. 57-60. gai Y. Z. and Sarkanen K. V. (1971) Isolation and structural studies. In Lignins Occurrence, Formation, Structure
and Reactions (Edited by Sarkanen K. V. and k udwig C. H.), Chap 5, pp, 165-240. Wiley. Martin J. P. and Haider K. (11980) Microbial degradation and stabilization of t4C-labelled lignins, phenols and phenolic polymers in relation to soil humus formation. In l,ignin Biodegradation: Microhioh)gy, Chemi.~trv a,d Powntial Applications (Edited by Kirk T. K., Higuchi T. and Chang H.) Vol. 1. pp. 77 100. CRC Press. Metcalfe J. B. (1970) Some physico-chemical approaches to the study of soil organic matter, Thesis University of Aberdeen. Minderman G. 11979) A tentatitve approach to the molecular structure of humic acids: the spectral evidence for a derivation of humic acids from plant-borne esters, I. The electron paramagnetic resonance (EPR) sectra. Netherlands J. Agric. Sci. 27, 79 91. Riffaldi R. and Schnitzer M. (1972) Electron spin resonance of humic substances. Proc. Soil. SoL Soc. Am. 36, 301 305. Steelink 12'. (1964) Free radical studies of lignm, lignin degradation products and soil humic acids. Geoehim. Cosmochim. A cta 28, 1615-1622. Steelink C. (1966) Stable free radicals in lignin and lignin oxidation products. In Lignin Structure and Reactions. Advances in Chemistry Series No. 59 (Edited by Gould R. F.), Chap. 5, pp. 51-64. American Chemical Society. Steelink C. and Tollin G. (1962) Stable free radicals in soil humic acid. Biochim. Biophys. Acta 59, 25-34. Tollin G., Reid T. and Steelink C. (1963) Structure of humic acid IV. Electron paramagnetic resonance studies. BiochmL Biophys. Acta 66, 444-447. Webley D. M. and Jones D. (197l) Biological transformation of microbial residues in soil. In Soil Biochemistry (Edited by McLaren A. D. and Skujins J.), Vol. 2, Chap. 15, pp. 446485. Marcel Dekker inc.
0146-6380/85 $3.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
Electron paramagnetic resonance characteristics of the humic acids from a podzol M. V. CHESHIRE,B. A. GOODMAN,D. B. McPHAIL and G. P. SPARLING The Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen AB9 2QJ, U.K.
(Received 7 November 1984; accepted 21 May 1985) Abstract--A study has been made of solid and solution electron paramagnetic resonance (EPR) spectra of humic acids from different horizons in a podzolic soil. Hyperfine splitting was observed in the solution spectra of humic acids from all horizons and depended on the strength of alkali and the period of dissolution. The upper organic horizons L, F and O~ contained humic acids with some spectral characteristics in common with lignin. Humic acid from the lower horizons showed different spectra. At least 5 different radical signals were present.
Key words: electron paramagnetic resonance, free radical, humic acid, hyperfine structure, solution spectra, podzol
INTRODUCTION
MATERIALS AND METHODS
The presence of free radicals in humic substances is now firmly established. In the solid state the electron paramagnetic resonance spectra display a single peak with g-values in the range 2.0023-2.0051 (Minderman, 1979). Several authors (e.g. Steelink and Tollin, 1962; Steelink, 1964; Riffaldi and Schnitzer, 1972) have estimated the free radical contents of various humic materials and attempted to relate them to types of humic substance. In solution, humic acids have been reported to exhibit either single peak spectra or hyperfine structure, the latter situation having been equated to humic acids from the more acidic soils (Atherton et al., 1967). However, experimental findings have been variable and some authors have reported failure to find any hyperfine structure where it might have been expected according to the criteria of Atherton et al. In this paper a study has been made on the effect of different concentrations of alkali on the EPR spectra of humic acids from different horizons of a podzolized soil profile. In very acidic soils and peats the decomposition of plant residues is usually slower than in less acid conditions and so plant components such as lignin can persist for long periods. Lignin displays hyperfine structure in EPR spectra (Steelink, 1966) and this study also explores the possible relationship between the free radical of this substance and the humic acid in the different horizons. Many fungi, which produce dark coloured polymeric substances, are particularly resistant to lysis and some accumulation of their residues would be expected in soil (Webley and Jones, 1971). An examination has, therefore, also been made of the dark brown stipes of Marasmius androsaceus which was prevalent at the site.
The soil profile examined occurred under coniferous wood at Blacktop (National Grid reference NJ 866047). The predominant plant species was Scots Pine (Pinus sylvestris L.). The soil was of the Charr series of the Countesswells Association and details of the profile are given in Table 1. Material from each horizon was sampled together with fresh pine needles, yellowing needles and dead needles caught in the branches of the trees. All samples were air-dried. Stipes of the fungus Marasmius androsaceus were collected from the surface litter.
Extraction of litter and soil with alkali Samples of L, F and O horizons of soils were extracted with 0.2 M NaOH (10g soil/100cm 3) for 20 hr. For the mineral-rich horizons, E and Bs, 30 g of sample were used. The mixtures were filtered and humic acid precipitates were obtained by acidification with redistilled 6 M HCI to give solutions 0. l M with respect to HCI. The humic acids were washed with 0.01 M HCI, then water and recovered as solids by freezing, thawing and drying under reduced pressure. The yields are given in Table 1.
Preparation of Mason lignin from fresh pine needles, yellowing pine needles and dead needles (Lai and Sarkanen, 1971)
© The Macaulay Institute for Soil Research, 1984. 427
The samples were continuously extracted with 95% aqueous ethanol and, after drying, with an azeotropic ethanol-toluene mixture using a soxhlet extractor. The residues were treated with 12 M H~SO4 (15 ml per g sample) for 2 h at 21 _+ 2°C before dilution to 0.5 M H2SO4 and hydrolysis at 100°C for 4 hr. The insoluble klason lignin residue was dried under reduced pressure and weighed (Table 2). Samples of the lignin were treated with 0.1 M KOH for 24 hr and the residue extracted with 4 M KOH for 24 hr. In
~vl. \
428
(,ItESHIRF Pi ~1/
'Fable 1. Description of the Charr series soil laorizons, C content and the yield of humic acid Horizon depth (mm) 0 50 50 125 125 250 250 380 380-410 410~460 460 630 630+
Description Dead needles Fresh needle and grass litter Dead needle litler Partially decomposed fibrous litter Black well decomposed organic pH 3.7 Black well decomposed organic some quartz, pH 3.7 Dark grey brown coarse sandy loam Iron pan Yellowish brown sandy loam, pH 4.6 Light yellow brown sandy loam
Table 2. Percentage composition of pine needles and litter 95!',g ethanol soluble
Ethanol toluene soluble
C,,)
('~o)
G3
19,7 122 I 1, I 8,5 2.3 0.2
02 0.6 I. 1 0.9 (1.3 (I.08
33.9 44.3 67.7 62.6" 47.8" 17.3"
Sample Fresh green needles Yellowing needles Dead needles O~ 02 B,
Klason lignin
"Ash free.
each case the soluble matter was separated by filtering on a sintered glass filter. Acidification of the solutions with HC1 precipitated small quantities of dark brown material in each case. These will be referred to as lignin (0.1 M) and lignin (4 M) respectively. Soil from various horizons was also subjected to the same extraction procedure. In the last stage, the residue, which should contain any lignin present, was extracted with 1 M KOH, followed by 4 M KOH.
Extraction of Marasmius androsaceus stipes 25 stipes, about 30 mg, were extracted twice with 1 M KOH (5 cm 3) over a period of 70 hr at 20 + 2°C. Most of the colour was extracted. The orange extracts were acidified with HCI, when part of the material was precipitated. The precipitate was redissolved in 1 M KOH and dialysed against water with no apparent loss of colour. The suspension was evaporated to dryness under reduced pressure to give a yield of 43%.
EPR Spectroscopy All spectra were obtained at room temperature with a Varian E104A X-band spectrometer using 100kHz modulation frequency for 1st derivative spectra and 50 kHz for 2nd harmonic recording. Both modulation amplitude and microwave power were strictly controlled so as to avoid any line broadening or saturation, the settings ranging from 0.050.010 mT for the modulation and from 1-5 mW for the power. For solid samples a known weight of material was added to a quartz tube to occupy ca 1 cm of its length and spin concentrations were
Humic acid ('!~,)
Designation
C%
DN LI t,~, F OE ()~
52.3 49,7 ,0.._''~ 46.6 43.5 8 I
E B B, (
34
47
2.9
1
cj I~ ,~~ 20
32.5 !2
calculated by comparing the spectral area with that of a sample containing a known weight of diphenylpicrylhydrazyl (DPPH). For solution spectra the initial alkaline solutions were fully aerated before being sealed in quartz solution cells for the duration of the experiments. RESULTS
Free radicals in solid humic acid samples The free radical contents of the humic acids from the dead needles and the different soil horizons are shown in Table 3. It can be seen that there is a steady increase in radical content with depth through the predominantly organic horizons of the profile until the mineral horizons, where there is a dramatic decrease in the samples from the E and B~ horizons, respectively.
Free radicals in alkaline solution of the humic acids The alkaline solution spectra of the humic acids showed variations depending on the horizon from which the substance originated and the concentration of the alkali, as well as dissolved oxygen and time related effects.
Spectra obtained shortly after dissolution The 1st derivative spectra obtained within 20 min after dissolution of the humic acids in 0.2 M NaOH are shown in Fig. I. The spectra for the dead needles and litter layer show relatively broad single peaks with little or no evidence of any hyperfine splitting. With the F and O~ horizons the humic acids show increasing evidence of some hyperfine structure superimposed on a broad peak. The 02 horizon Table 3. ESR g value, free radical content and lignin estimated as ~,~ of the humic acid from infrared absorbance Horizon
g value
Free radical cone. (spins g 1 x 10 -16)
Lignin (%)
Dead needles L~ L2 F O~ Oz E BS
2.0037 2.0037 2.0037 2.0037 2.0037 2.0034 2.0035 2.0038
2.7 3.6 8.4 6.8 12.8 I 1.7 0.56 0.29
32 23 29 17 12 0 0 0
Humic acids from a podzol
02
0.1 mT I
I
Fig. 1. EPR spectra of 0.2 M KOH solutions of humic acids.
429
430
M V. ('HESHmE ,'t al.
ff
DN
~jj/" ~J
-.,_,_ IfJ
I1
I"
,/
_f'
J
.j
_i
/
/
_J i /
02
f
~f~
/
d"
.%,
\
f
E
\jr
J
ffP
/ F
/
0.1 mT f
-
-
'C" '/4,/ Fig. 2. EPR spectra of 4 M KOH solutions of humic acids.
~
Humic acids from a podzol shows a distinct 4 peak spectrum which probably arises from 2 inequivalent protons with A~ = 0.1175 and A 2 = 0425 mT, classified as a class I spectrum in the nomenclature devised by Haworth (1971). The spectra obtained within 20 min of dissolution of the humic acids in more concentrated alkali, 4 M KOH (Fig. 2), were different from those in the dilute alkali. The dead needles and the litter horizon humic acids gave spectra which were similar to each other, but complex in character, consisting of more than one component. One species of radical gave a signal corresponding to a quintet, 1:4:6:4:1 with A = 0.106 mT. Other components with much smaller hyperfine splitting, were present and these varied in relative strengths in the humic acids. In the F horizon humic acid the quintet radical signal was much weaker in comparison to other signals and the spectrum was dominated by the central feature of a sharp single peak. The 02 horizon humic acid displayed a broad single peak with some poorly resolved structure. Similar structure was observed in the spectra of the samples from the E and Bs horizons, but with increasingly better resolution with increasing depth. At least two radical species are present in the alkaline solutions of the humic substances from these three horizons, but at different relative strengths in the different horizons. The best-resolved component in the B~ horizon humic acid solution consisted of 4 peaks, probably from 2 inequivalent protons with A~ = 0.102 mT and A 2 = 0.061 mT.
Variation of radical signals with time
(i) Humic acid in 0.2 M alkali The main change observed was that the 4 peak spectra from the humic acid from the lower horizons, were slowly transformed into a second type of 4 peak spectrum with parameters similar to those of the 4-peak component that was seen in 4 M alkali. The rate at which this change occurs depends on the concentration of alkali used. Distinct differences were previously noted for short dissolution periods using a range of alkali concentration 0.1-2 M (Cheshire et al., 1975). (ii) Humic acid in 4 M alkali (a) Humic acids from litter horizons: The changes that occur in material extracted from the L~ horizon and the dead needles are almost identical so only the latter are illustrated in Fig. 3. There is considerable change in detail in the centre of the spectrum over the period of a day, but the quintet feature is unaltered with time. The L2 humic acid spectra also showed the quintet and differed only in showing less developed structure in the minor central components. (b) Humic acid from the F horizon: The signal responsible for the quintet feature present in the litter humic acids was also present at a lower intensity in the F horizon humic acid (Fig. 4a). It shows little change with time over a period of 2 days. On the
431
other hand a sharp central peak more clearly seen in the 2nd derivative spectrum (Fig. 4b) lost about 75-80~o of its intensity during the first 3 hr and had almost disappeared within 21 hr. The relative intensities of other features also vary with time, but, because of the complexity of the spectra, it was not possible to match peaks to separate spectral components, except for a possible doublet with A = 0 . 1 6 m T , which is seen in the 2nd derivative spectra after 21 hr. (c) Humic acid from the O horizons: The initial spectrum obtained with the O~ humic acid possessed a sharp central peak in the 2nd derivative and was almost identical to that obtained with the F humic acid. After several hours this signal decreased in intensity and was replaced by a hyperfine spectrum which was practically identical to that obtained in the later stages with the 02 humic acid. The 2nd derivative signals generated by 02 humic acid in 4 M KOH are illustrated in Fig. 5. There was a steady development of structure with time over a period of 2 days, but no evidence in either 1st (not shown) or 2nd derivative spectra for the presence of the quintet structure seen in the humic acids of higher horizons. The spectra appear to be simpler than those from the upper horizons, but nevertheless contain a number (at least five) of different free radicals whose relative contributions to the spectra vary with time. When a humic acid solution that had developed a spectrum while standing for 2 days in the sealed quartz cell was exposed to air, a broad feature developed immediately and was identical to that obtained with H202 treatment. There was a major change in the hyperfine split components as a result of 02 treatment (Fig. 5) but after H202 treatment all the hyperfine structure was lost. Boiling a freshly-made solution of the 02 horizon humic acid in 4 M KOH gave a similar spectrum to material which had been aged in 4 M KOH over a period of 2 days and exposed to oxygen. (d) Humic acid from the E horizon: These spectra (Fig. 6) show a number of features that display similar behaviour to the components in the 02 horizon humic acid spectra. However, the resolution is somewhat better implying perhaps, that fewer different radicals are contributing to the spectra. Exposure to oxygen of a 6-day aged E horizon humic acid solution gave little evidence of the broad component in the spectrum. The 1st derivative spectra are dominated by a sharp hyperfine split component that is much better resolved than in the 02 horizon humic acid spectra, but the 2nd derivative spectra (Fig. 6) indicate that both samples are dominated at this stage by the same two radical species, present in different relative proportions. (e) Humic acid from Bs horizon: In the early stages the spectra showed some resemblance to that of the E horizon humic acid, but the resolution did not improve with time. Aeration caused a complete loss of signal which was regenerated only very slowly.
432
M
10 min
V CH~SmREet or.
~ ~ " ' Y '
30min
2h
23h
0.1 mT
V
! Fig. 3. Variation with time of the EPR spectrum of dead needles humic acids in 4 M KOH.
Free radicals in lignin extracted from pine needles Lignin (0.1 M). The material derived from green needles dissolved in 4 M KOH gave a spectrum which was similar to that from the yellowing needles except for an additional narrow peak component in the centre of the spectrum. After one day, the spectrum from the yellowing pine needle lignin was very similar
to that of the humic acid from the dead needles and L~ horizons. Lig#n (4 M). When these materials were dissolved in 5 M .~OH the main components in the spectra formed after a short time (Fig, 7) were practically identical to the quintet components in the L and F horizon humic acids. The peaks with an approximate
Humic acids from a podzol
433
O
>
M . V . CHESHIRE Cf ,l/.
434
,\
/ boiled
in 4M
KOH
,
I
Fig. 5. Variation with time of the 2nd derivative EPR spectrum of 02 horizon humic acid in 4 M KOH.
1:4:6:4:1 intensity ratio had a separation of approx. 0.11 mT. Free radicals in 95% ethanol extracts Radicals generated in 5 M KOH were investigated and the spectra were found to bear no resemblance to those obtained with humic acids. The extract from yellowing pine needles (Fig. 8 a, b, c) gave a spectrum which was initially dominated by a 4-peak component from two inequivalent protons (A~ = 0.337 mT, A2 = 0.107 mT). The signal rapidly decreased in strength losing 80~o of its intensity in 1 h, within which time a much sharper 3 peak component from 2 equivalent protons (d =0.079mT) had appeared. This signal grew steadily in intensity over a period of 2 days. Some additional, very much weaker, peaks were also present.
The spectra obtained with the extracts from the O~ and B~ horizons were very complex, but quite different both from each other, from the yellowing pine needle extract spectra, and from the humic acid spectra. The large number and small widths of the component peaks, illustrated in Fig. 8d by the spectra from the Bs horizon humic acid after 3.5 hr, suggest that low molecular weight free radicals are probably responsible, but a detailed analysis of these spectra was not possible. Free radicals in alkali extracts of stipe fiom Marasmius androsaceus A very complex evolved with time (Fig. 9) but these from any of the
set of hyperfine splitting patterns from an initial 5 peak spectrum were found to be quite different spectra from the soil horizons;
Humic acids from a podzol
435
1.5h
7h
24 h
50h
(I/VI Ilffk 0.1 mT
I
I
Fig. 6. Variation with time of the 2nd derivative EPR spectrum of the E horizon humic acid in 4 M KOH.
Further details are given elsewhere (Goodman et al., 1985).
Infrared analysis of the humic acids The infrared spectra of the humic acids showed differences in several regions. There was a gradual decrease in the absorption related to aliphatic hydro-
carbons (2850 and 2920 cm-J) in the humic acids on going from the dead needles down the profile. Similarly an absorbance at 1510 cm -~, which is attributed to an aromatic component, gradually decreases with depth to become no longer visible in the 02 layer. This absorbance is attributed to lignin, and, using a comparable lignin standard, an estimate of the
436
M.V. CHFLSHIREet al.
5rain .y
/
~'i!
•
_//
01 T
,
S
Fig. 7. Variation with time of the EPR spectrum from the 4 M KOH extract of green pine needles lignin in 5 M KOH. lignin has been made in each humic acid. The results are given in Table 3 and selected spectra are shown in Fig. 10. The L2, F and Oi horizon humic acids each had additional absorption bands at 1265 and 1420 cm -t and one at 1540 cm-I, interpreted as peptide structure, was present in B, horizon humic acid with traces in the E horizon material. Some absorption which was attributable to layer silicates was present in the humic acids from the lowest three horizons. DISCUSSION
Previous studies on the hyperfine structure of the EPR spectra of O horizon humic acid from this site have reported a relatively simple 4 peak spectrum in 0.1 M NaOH (Haworth 1971, Cheshire et al., 1975). In more concentrated alkali (2 M) a shift of signal occurred to a second four-peak speet~m. These signals have been tentatively related to the spin polarization of a C - H orbital by the unpaired
electron in a ~ type orbital of a benzene ring and a C-H tr orbital elsewhere (i.e. not on the same ring) (Cheshire et al., 1975). The present results, which extend the concentration of alkali used, give more attention to the effects of time on the changes to the EPR signal and, by examining humic acids from different horizons, show that the spectra are more complicated than was hitherto appreciated. The use of 2nd derivatives in addition to the more usual 1st derivatives of the absorption spectra can be of particular value in resolving small amounts of components with narrow lines. The occurrence of hyperfine splitting in the EPR spectra of humic acids in weakly alkaline solutions appears to depend on the soil having a low pH. Generally, Haworth (1971) found that soils with pH4.2 or less had humic acids that displayed splitting, whereas, when the pH was ~-..ater, they did not. Exceptions could be e x ~ m ~ d by pH changes related to changes in soil use, bearing in mind the great resistance of humic substances to decompo-
Humic acids from a podzol
0,1 mT • 1
~
437
L
Fig. 8. EPR spectra of 95% ethanol extracts of yellowing pine needles after (a) 5 min, (b) I hr, (c) 47 hr and (d) 3.5 hr all in 5 M KOH. sition implied by measurements of mean residence times. Haworth considered that the EPR signal related to condensed aromatic structures whose nature depended on the pH at which their chemical synthesis from phenolic substances had occurred. Other explanations can be put forward. For example, although it is a gross oversimplification to describe soil by a single pH value, acid conditions represented by such a value may distinguish the point at which a predominantly bacterial flora is replaced by a more acidophilic fungal one. Such a change may result in
a different metabolic pathway or other products being present and, subsequently, a change in the nature of the more polymerized organic matter. Fungal tissue itself is frequently dark coloured with humic-like substances. The dark brown stipes of one fungus, Marasmius androsaceus, prevalent in the litter at the site were, therefore, examined and their extracts shown to display an EPR spectrum with hyperfine splitting. This was, however, different from that of the humic acids. Because most of the carbon in soil originates from
438
M
V, ('HESHIRti c/ a t
=c
= o
-5 ,=
,,¢ Ok,
E r~
k~
Humic acids from a podzol I
I
I
I
I
I
1
439 I
I
I
J
I
%T
I
I 30
40
I
I
20
16
I
I 12
I
1 8
1
I 4
~ / 1 O0 crn -1
Fig. 10. Infrared spectra of the humic acids of three soil horizons and the equivalent alkali soluble matter of the dead needles. (a) dead needles; (b) F horizon; (c) O~ horizon; (d) Bs horizon.
vegetation, and recognizable plant remains, e.g. lignin, persist in soils for very long periods, particularly under very acid conditions, radicals present in lignin were also examined by EPR spectroscopy. Alkaline extracts of lignins which gave EPR spectra displaying hyperfine splitting were obtained from the predominant litter-producing plant species at the site. The upper horizons of the podzol examined clearly show the presence of a radical from a lignin component in the humic acid, but this is not apparent in the 02, E and Bs humic acids. The infrared absorption band of the humic acids at 1510 cm -1, characteristic of aromatic structures, showed a similar decrease with depth and indicates that if lignin residues remain in the lowest horizons they do so in a much modified form. The changes in humic acid spin concentration with horizon for the organic horizons fits with the changes in the proportions of lignin and humic acid since it has been observed that there is an increase in the free radical content in the sequence lignin, degraded lignin, fulvic acid, humic acid (Steelink and Tollin, 1962). The lower radical content of the humic acid from the mineral horizons E and Bs may be because O.G. 8/6---D
of qualitative differences, but it could also be partially the effect of a higher content of mineral components which may quench some of the signal. The lignin involved in the present study, being from a gynnosperm, is a dehydrogenation polymer of coniferyl alcohol. The initial formation of the lignin is considered to result from the coupling of radicals formed by the enzymic degradation of the coniferyl alcohol. A schematic constitution of the lignin (Alder et al., 1969) reveals a number of different sites on which radicals could be formed. It is generally believed that the formation of humic substances depends on the prior breakdown of lignin to quinone monomeric units, but there is the possibility that lignin may survive in a partially degraded form (Martin and Haider, 1980). Tollin et al. (1963) appear to have been the first to mention hyperfme splitting in the alkali solution of humic acids. Since then various others have reported similar findings (Haworth and Atherton, 1965; Metcalfe, 1970; Cheshire and Cranwell, 1972; Minderman, 1979). The present results show that the radical species present are a more complex mixture than is implied from previous reports.
M.V. ('HESHIRF ('[ d/
440
Acknowledgements--We would like to thank Dr J. D. Russell and Mr A. R. Fraser for the infrared analyses and Miss Marian Thorn for the elemental analyse~
REFERENCES
Alder E., Larsson S., Lundquist K. and Miksche G. E. (1969) Abstract of the International Wood Chemistry Symposium, Seattle. (Illustrated in Chap. 1, Lignin structure and morphological distribution in plant cell walls by T. Higuchi.) In Lignin Biodegradation: Microbiology, Chemistry and Potential Applications (Edited by Kent T. K., Higuchi T. and Chang H.), Vol. 1, Chap. 1, pp. 1-19. CRC Press 1980. Atherton N. M,, Cranwell P. A., Floyd A. J. and Haworth R. D. (1967) Humic acid--l. ESR spectra of humic acids. Tetrahedron 23, 1653-1667. Cheshire M. V., Goodman B. A. and Mundie C. M. (1975) The composition of soil humus. Welsh soils Discussion Group Report No. 16, pp. 73-90. Cheshire M. V, and Cranwell P. A. 11972) Electron-spin resonance of humic acids from cultivated soils. J. Soil Sci. 23, 424-430. Goodman B. A., Vaughan D., Cheshire M. V. and Sparling G. P. (1985) ESR Investigations of some fungal pigments. Biochem. Soc. Trans. 13, 623-624. Haworth R. D. (1971) The chemical nature of humic acid. Soil Sci. 111, 71-79. Haworth R. D. and Atherton N. M. (1965) Humic acid. Bull. Nat. Inst. Sci, India No. 28, pp. 57-60. gai Y. Z. and Sarkanen K. V. (1971) Isolation and structural studies. In Lignins Occurrence, Formation, Structure
and Reactions (Edited by Sarkanen K. V. and k udwig C. H.), Chap 5, pp, 165-240. Wiley. Martin J. P. and Haider K. (11980) Microbial degradation and stabilization of t4C-labelled lignins, phenols and phenolic polymers in relation to soil humus formation. In l,ignin Biodegradation: Microhioh)gy, Chemi.~trv a,d Powntial Applications (Edited by Kirk T. K., Higuchi T. and Chang H.) Vol. 1. pp. 77 100. CRC Press. Metcalfe J. B. (1970) Some physico-chemical approaches to the study of soil organic matter, Thesis University of Aberdeen. Minderman G. 11979) A tentatitve approach to the molecular structure of humic acids: the spectral evidence for a derivation of humic acids from plant-borne esters, I. The electron paramagnetic resonance (EPR) sectra. Netherlands J. Agric. Sci. 27, 79 91. Riffaldi R. and Schnitzer M. (1972) Electron spin resonance of humic substances. Proc. Soil. SoL Soc. Am. 36, 301 305. Steelink 12'. (1964) Free radical studies of lignm, lignin degradation products and soil humic acids. Geoehim. Cosmochim. A cta 28, 1615-1622. Steelink C. (1966) Stable free radicals in lignin and lignin oxidation products. In Lignin Structure and Reactions. Advances in Chemistry Series No. 59 (Edited by Gould R. F.), Chap. 5, pp. 51-64. American Chemical Society. Steelink C. and Tollin G. (1962) Stable free radicals in soil humic acid. Biochim. Biophys. Acta 59, 25-34. Tollin G., Reid T. and Steelink C. (1963) Structure of humic acid IV. Electron paramagnetic resonance studies. BiochmL Biophys. Acta 66, 444-447. Webley D. M. and Jones D. (197l) Biological transformation of microbial residues in soil. In Soil Biochemistry (Edited by McLaren A. D. and Skujins J.), Vol. 2, Chap. 15, pp. 446485. Marcel Dekker inc.