Origins of humus variation

Org. Geochem. Vol. 9, No. 5, pp. 225-231, 1986 Printed in Great Britain

0146-6380/86 $3.00 + 0.00 Pergamon Journals Ltd

Origins of humus variation M. A. WILSON l, K. M. GOH 2, P. J. COLLIN 1 and L. G. GREENFIELD 3 ICSIRO Division of Fossil Fuels, P.O. Box 136, North Ryde, NSW 2113, Australia 2Department of Soil Science, Lincoln College, Canterbury, New Zealand 3Department of Botany, University of Canterbury, Christchurch, New Zealand (Received 4 June 1985; accepted 27 November 1985)

Abstract--Humus samples derived from peat, moss or ornithogenic sources in the Antarctic continent have been examined by ~3C nuclear magnetic resonance techniques. The ~3C spectra show that the humus has undergone very little chemical modification. A fulvic acid fraction from a moss derived soil is almost entirely ~t- and r-glucose. The results also show that lignin is not a necessary precursor for aromatic components in soils. It is suggested that the aromaticity and carbohydrate content of certain soils is governed by the low temperature which influences the degree of humification. Key words: humus, ~3C NMR, Antarctic, humic acid, fulvic acid, carbohydrates, peat, moss INTRODUCTION Solid state and solution high resolution nuclear magnetic resonance ( N M R ) spectroscopic techniques are established methods for the analysis of the types and forms of organic carbon in soils and extracts from soils. N M R techniques have clearly demonstrated the variability in structure of the organic matter in soils and solvent soluble fractions of soils. Values obtained for aromaticities (fa = aromatic carbon/total carbon) have ranged from 10 to 74% (Hatcher et al., 1981a, b; Skjemstad et al., 1983). Likewise, large variations in carbohydrate content have been reported (Hatcher et al., 1981a, b; Wilson 1981; Wilson et al., 1983c). Nevertheless, little work has been done to establish reasons for these variations. Humic acids with low aromatic content appear to be derived from humus in regions where rainfall is high (Wilson et al., 1981a, b) or in marine systems where lignin content is low (Hatcher et al., 1980, 1981a, b; Wilson et al., 1983a). Little is known of why large amounts of carbohydrates are quite stable in soils. In this paper, we investigate the chemical composition of a number of humic materials derived from terrestrial sources from the Antarctic continent with a negligible input of lignin from vegetation. Because of the cold climate in the Antarctic continent the rate of humification of organic matter in these samples is expected to be slow. The purpose of this work is to elucidate factors responsible for variations in soil aromaticity and carbohydrate concentration.

EXPERIMENTAL

Samples were taken from the Antarctic continent and are derived from an ornithogenic soil, or from an Antarctic moss soil or peat (0-3 cm depth). Details of samples are given in Table 1. When humic extracts were prepared, samples were

finely ground and extracted with 0.1 m o l d m 3 NaOH/0.1 mol dm -3 Na4P207 solutions. They were then acidified to separate the humic and fulvic acid fractions. The humic acid fraction was purified by the rapid method of (Goh, 1970), and the fuivic acid fraction by ion exchange (Goh, 1970). 1H-solution N M R Experiments were performed on a Jeol FX90Q spectrometer operating at 90 MHz. A spectral width of 1500 Hz was used with an acquisition time of 2.73 sec and a pulse delay of 4 sec. Data were collected in 8 K data points and Fourier transformed with line broadening of 0.12 Hz. Approximately 200 scans were collected. The protons from water in the sample were irradiated with optimum r.f. power (Wilson et al., 1983b). An external lithium lock was used and chemical shifts were measured with respect to an internal capillary tube of tetramethylsilane. 13C-solution N M R Solution 13C-NMR spectra were obtained on a Jeol FX90Q instrument operating at 22.5 MHz. A spectral width of 10,000 Hz was used. Data were collected in 8 K points with inverse gated broad band decoupling. Up to 166,904 scans were collected. Line broadening was varied from 5 to 100 Hz. Chemical shifts were

Table 1. Details of samples used Sample Parent material Guano deposits collected from several loOrnithogenic soil cations in Antarctica (i.e. Cape Adare, Cape Hallet, Cape Bird, Cape Crozier and Inexpressible Island) Antarctic moss Under dead mosses consisting of comminuted (whole) and visible organic matter of Bryum antareticum and Bryum argenteum mosses Polytrichum moss from'Signy Island, British Antarctic peat Antarctic Peninsula 225

226

M.A. WILSONet al.

1.9/* 0"92 1-37 3'76, \ 2.70 / ~'~ rvl

measured with respect to an internal capillary of tetramethylsilane. z3C-solid state N M R

Most spectra were determined on a Bruker CXP100 using cross polarization and magic angle spinning. Experiments using conventional and dipolar dephasing techniques were employed (Wilson et al., 1983c). A 7/~sec 90 ° pulse was used and a contact time of 1 msec and a recycle time of 1 sec to 0.7 sec were normally employed. Proton T~p measurements through carbon were made by varying the contact time between 1 and 9 msec. They were found to be similar to those reported for other humic materials (Wilson et al., 1983c). Two delay periods about a 180 ° refocussing pulse were employed in dipolar dephasing experiments (Wilson et al., 1983c). Some dipolar dephasing experiments were performed at 75.5 MHz on a Bruker CXP300 under similar conditions. RESULTS AND DISCUSSION Solid state ~3C, and solution ]3C and 'H spectra are shown in Figs 1-5. Both solution and solid state ]ac spectra were obtained for the humic acid from the ornithogenic soil (Figs 1 and 3) and the solution ~H spectrum was also determined (Fig. 2). The solid state ~3C spectra of the peat, the humic acid from the soil under moss and the humic acid from the peat were also obtained (Fig. 4) but the ~3C solid state spectra of the whole soil under moss or the ornithogenic soil were not obtained because signal to noise was poor due to low carbon content and/or paramagnetics. The fulvic acids were extremely hygroscopic and 13C spectra could only be obtained by solution N M R (Fig. 5).

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Chemicalshift (ppm) Fig. 2. ~H NMR spectrum of humic acid from ornithogenic soil. The water peak has been irradiated. Five major regions of resonance can be recognised in ]3C-spectra of humic substances or soils (Vila et al., 1976; Wilson, 1981; Bayer et al., 1984). These are: 0-50 ppm (alkyl carbon), 50-108 ppm (carbohydrate, alcohol, ether carbon and the or-carbon and/3-carbon of amino acids), 108-160 ppm (aromatic carbon) and 160-200ppm (carboxylic, ester, amide carbon, 200-220 ppm (ketonic or aldehyde carbon). When spectra are detailed, further subdivision is possible. Full details and discussion of assignments can be found elsewhere (Wilson, 1981; Skjemstad et al., 1983; Hatcher et al., 1980, 1981a, b; Wilson et al., 1983a, c). Ornithogenic samples

Figure 1 shows solid state and solution N M R spectra for the ornithogenic humic acid. Considerably more detail is present in the solution spectrum. The relative signal intensities from aromatic and

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Chemicalshift (ppm) Fig. 1. 22.6 MHz ~3C NMR spectra of humic acid from ornithogenic soil. (a) solid state CP/MAS spectrum; (b) solution spectrum.

/,0 $0 Fig. 3. 75.7 MHz ]3C CP/MAS dipolar dephased spectra of ornithogenic humic acid. The dipolar dephasing time Tad is shown alongside the spectra.

227

Origins of humus variation

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182"4~ 208.6.~j Chemica|shift(ppm) Fig. 4. ~3CCP/MAS spectra of(a) Antarctic peat; (b) humic acid from Antarctic peat; (c) 40/~sec dipolar dephased spectrum of humic acid from Antarctic peat; (d) humic acid from soil under moss.

carboxyl resonances are, however, similar in both spectra. The fraction of signal from aromatic carbon (f,) is 19 ___2% by solid state N M R and 16 + 2% by solution NMR. In the latter case the observed f, was independent of pulse delay over the range 0.2-5.0 sec. Resonances from aromatic carbon in the solution spectrum lie mainly at 131 ppm or below. In principle, olefins could contribute to this region of the spectrum. However, in the case of terminal olefins (i.e. 1-alkene compounds) discrete resonances should be observed at about 114 and 140 ppm (Collin et al., 1983) and these are not present. Likewise, proton resonances from olefins in long chain alkenes are observed at < 6 ppm in JH spectra (Collin et al., 1983; Wilson et al., 1985; Jackman and Sternhell, 1969) and these are not present in Fig. 2. We cannot exclude the possibility of aryl conjugated olefins, but the results

clearly suggest that olefinic material is not a major contribution to the structure of this humic material. The absence of significant resonances in the aromatic region of the solution NMR spectrum at > 131 ppm could in principle be an experimental artifact, because non-protonated carbons which normally resonate in this region have long spin lattice relaxation times (Tj's) and hence could be saturated under the experimental conditions used in this work. However, resonances are also weak in the spectrum obtained by solid state techniques where relaxation is rapid. Hence oxygenated aromatic substances such as phenols are not significant contributors to the extract. Carboxyl carbon is present in significant quantities. The carboxyl content was estimated as 16 ___2% by solid state NMR and 12 + 3% by solution NMR. At

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least three resonances are observed in the solution spectrum at 173.6, 178.6 and 182.4ppm. The former is due to carboxyl groups close to electron withdrawing groups, probably other carboxyl groups. The other resonances are more typical of carboxylate anions terminating alkyl chains. Aliphatic resonances are prominent at 22.4 ppm in solution and at 24.5 ppm in the solid state 13C spectra. These resonances are probably from methine units ~cfrom the end of alkyl chains and hence aliphatic chains are mainly branched. This conclusion is confirmed by the significant contribution of methyl groups to the 13C spectrum at 18 ppm and the ~H spectrum at 0.92 ppm, and the relatively small resonance from polymethylene (CH2) . carbons at 29 ppm. The resonance at 52.6 ppm could arise from methoxy carbon or from the protonated C-2 carbons of amino acids. In the solid state these structures can be differentiated by dipolar dephasing experiments (Wilson et al., 1983c) because methyl resonances are

present in spectra obtained at dipolar dephasing times of 40 ttsec but protonated C-2 amino acid carbons are absent. Unfortunately the 52.6 ppm resonance is not well resolved in the solid state spectrum in Fig. I. Hence dipolar dephasing experiments were performed at higher field (75.5 MHz). Typical spectra are shown in Fig. 3. It is clear that the 52.6 ppm resonance is not present in the spectrum prepared with a dipolar dephasing time of 40/~sec. Hence methoxy carbon is not present in significant concentration in this sample, and there is probably some amino acid carbon. Carbohydrates such as those in cellulose give 13C N M R spectra with resonances between 70 and 80 ppm (Earl and Vander Hart, 1980; Atalla et al., 1980; Dudley et al., 1983). Although there is some absorption at 70 ppm the ornithogenic humic acid contains little carbohydrate. The spectrum of the fulvic acid from the ornithogenic soil (Fig. 5c) has a major resonance at

Origins of humus variation

229

Table 2. Chemicalshifts of a- and fl-glucose ppm from tetramethylsilane Carbon number C1 C2 C~ C4 Cs C6 0~-Glucose 93.0 72.5 73.8 70.6 72.3 61.8 fl-Glucose 96,8 75.2 76.7 70.6 76.7 61.8

161-162 ppm. This probably arises from oxalate ion (Stothers, 1972 or polyhydroxy phenols). Other resonances from carbohydrate at 61.6, 70.3, 71.6, 92.3, 97.0ppm are present and also a further carboxyl resonance (174ppm). There is also a broad unresolved underlying resonance from ~ 15-150 ppm.

Peat samples The Antarctic peat sample appears to be almost entirely carbohydrate. Resonances are present at 99.8ppm from C-1 dioxygenated carbons and at around 78.8 ppm and 63 ppm from C2-C6 hydroxylated carbons (Fig. 4a). Likewise, the spectrum of the fulvic acid fraction (Fig. 5b) shows resonances from carbohydrate carbon. The humic acid spectrum (Fig. 4b) also contains carbohydrate resonances at 71 ppm. However, the spectrum is more complex than that of the whole peat. Resonances at 56 ppm are present which, like the ornithogenic humic acid, could arise from amino or methoxy carbon. It is clear however, that the resonance is still present in the dipolar dephased spectrum (Fig. 4c) and hence arises from methoxy carbon and not amino acid carbon. Methoxy carbon in peat is normally attached to aromatics possibly because it is lignin derived. A noteworthy feature of the spectrum (Fig. 4b) is that the methoxy resonance is larger than that of the resonance from oxygenated aromatic carbon (157 ppm). Thus if all the oxygenated aromatic carbon is attached to methyl groups (i.e. is methoxy implying no phenoxy content) all the methoxy content cannot be accounted for. Bearing in mind that there will be a contribution from underlying resonances, the results suggest that some methoxy carbon is present. The aliphatic resonance in the humic acid fraction is centred around 29 ppm which suggests some long straight chain aliphatic resonances are present. It is also noteworthy that, as expected, some of this resonance can be dephased out together with the protonated aromatic resonances at l l 6 p p m and carbohydrate resonances at 71 ppm (Fig. 4c).

Moss samples The spectrum of the fulvic acid fraction from the moss soil is unique in that it contains discrete narrow resonances from individual carbons (Fig. 5a). Resonances from two types of dioxygenated carbons at 97.2 and 93.2 ppm are observed together with at least 5 hydroxylated CH resonances between and including resonances at 77.1 and 71.2ppm. End chain (CH2OH) carbons are also observed at 62.2 ppm. The sum of the signal intensities of the resonances from O.G

95

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the dioxygenated carbons is equal to the signal intensity of the resonance from the end chain carbons. This suggests that two end chain carbons are resonating at 62.2 ppm, and single carbons at 97.2 and 93.2 ppm. Analysis of the signal intensities of the remaining resonances indicates that relatively, eight carbons are present. Thus the spectrum suggests that the fulvic acid consists of two carbohydrates of 6-carbon units and is consistent with a mixture of and fl glucose (Table 2). The Cl resonance of /3 glucose has been reported as 96.8 ppm and the resonance of ~ glucose at 93.0ppm (Stothers, 1972). Likewise the C6 carbons both resonate at 61.8 ppm (Stothers, 1972). Bearing in mind that small chemical shift differences in strong alkali are expected from those recorded in aqueous solution, the other resonances in the 77.1-71.2 ppm region are also consistent with glucose being present (Table 2). Moreover, the resonances at 97.2 and 93.2 ppm are present in intensities of 1.6 : 1 which suggests 62% of the carbohydrate is present as the/3-isomer. This is in excellent agreement with the value obtained by mutarotation studies (Finar, 1959). Hence it is certain that the fulvic acid fraction is a mixture of glucose isomers derived by hydrolysis of cellulose. The humic acid from the moss sample contains aromatic carbon (Fig. 4d) but appears to be substantially different in structure than the other humic acids. There also appears to be a different carboxyl composition since there is clear evidence of ketone/aldehyde carbon present ( ~ 208.6 ppm).

Soil Jorming .factors Our results demonstrate why large variations in the chemical structure of organic matter in soil can occur. Clearly, since aromatic components are present in soils without a lignin input, lignin is not a necessary precursor for aromatic components in soil. Although lignin may increase the aromatic content of soils, other materials may also contribute aromatic material and alter aromaticity. The results also demonstrate that under low humification rates, soils contain considerable quantities of cellulose. Indeed, it has been shown that a fulvic acid fraction can consist almost entirely of ~and fl-glucose. Thus, it would appear that under the extremely cold conditions in Antarctica, the transformation of fresh organic residues to humus is slow and incomplete as temperature variation is an important environmental factor affecting humus formation (Alexander, 1965; Dormaar, 1975; Duchaufour, 1976; Stout et al., 1981). Most of the cellulose is likely to

230

M.A. WILSONet al.

have originated from the residual plant components which have resisted decomposition although it could also be derived from direct microbial syntheses and transformations (Cheshire, 1977). The low aromaticity may be due to the incompletely transformed humus and the absence of lignin in the vegetation. Hurst and Burges (1967) suggest that a range of humic materials may exist from "flavonoid type" to "lignohumate" depending on the lignin content of source material. Polyhydroxy flavonoid units (e.g. phloroglucinol, resorcinol, 2:4 dihydroxytoluene, 3:5 dihydroxybenzoic acid) have been identified in Antarctic moss humus, which contained no units believed to be lignin derived such as vanillic acid, syringic acid, p-hydroxybenzoic acid, guaiacylpropionic acid and syringylpropionic acid (Hurst and Burges, 1967). It is likely that the absence of these aromatic units of lignin in the Antarctic humus accounts for its low aromaticity. There is significant evidence that microbiological activity may affect aromaticity. A podzol studied by Goh (1970) and Wilson et al. (1981a, b), formed under extremely wet conditions which retard the breakdown and mineralisation of newly formed organic matter, has a low aromaticity (fa=0.21). Moreover, the N M R spectra of the podzol show strong resonances from carbohydrate carbon at 73.8 and 103.6ppm (Wilson et al., 1981b) and nominal molecular weight studies suggest that there is a predominance of large ( > 200,000) molecular weight species (Goh and Williams, 1979; Goh et al., 1981). The low aromaticity and high carbohydrate content may be due to low rates of activity of microorganisms. In addition, the presence of excessive moisture, due largely to low evaporation, favours reducing processes. The importance of permafrost in the formation of humus in tundra soils has been stressed by Kononova (1966). On the other hand, high temperature and abundant precipitation in tropical soils and krasnozems, provide conditions for intensive microbiological activity leading to intensive mineralization of organic matter to simple compounds and its final product of CO2. Humic substances in these soils would be expected to be of low aromaticity as reported by Skjemsted et al. (1983). Tnus low aromaticity can be caused by low humus synthesis as in tundra and Antarctic soils or by high humus decomposition rates as in krasnozems and tropical soils. In addition to microbiological activity, the distribution and nature of humic substances are also determined by the vegetation and physico-chemical processes in soils (Kononova, 1966) or lakes. For example, the aromaticity of freshwater lake organic matter derived from a surrounding catchment area is high (J~ = 0.40) even though the catchment vegetation has a low lignin content (Wilson et al., 1981c). This may be explained by physico-chemical processes in lakes which require detailed investigations. Barron et al. (1980) have shown that the aromaticity of the "B" horizon of a podzol is greater than that of the

" A " horizon. These results contrast with those obtained by Walkden (1966) who noted a gradual change from a lignohumate in upper horizons to predominantly a flavonoid type humate in Bh horizons. Many fulvic acid fractions have very low aromaticities (Farmer and Pisaniello, 1985) and high carbohydrate contents (Tan and Clark, 1969) and would be expected to be the more soluble fractions leached from soils. It is possible therefore, that leaching can affect the aromaticity of soils, but the effectiveness of rainfall on organic matter structure will depend on the degree of prior chemical transformations and type of organic input. Although this paper demonstrates that climate and vegetation affect aromaticity, a number of other unknown factors also are important. One explanation that should not be discounted at this stage is that "safe zones" in soils exist in which organic matter is protected from microbiological decomposition, either through clay interaction or some physical phenomena. CONCLUSIONS Examination of the J3C N M R spectra of humus samples derived from various sources in the Antarctic continent shows that: (1) Due to slow rates of decomposition of Antarctic peat, its fulvic acid and the fulvic acid from a moss derived soil are almost entirely carbohydrate. It has been demonstrated that the moss fulvic acid is almost entirely a mixture of ~ and fl glucose in proportions expected from hydrolysis of cellulose. These results clearly demonstrate that slow organic matter turnover rates are important in increasing the carbohydrate content of soils. (2) Humic acids from ornithogenic soil and from Antarctic moss soil or peat contain aromatic carbon. This demonstrates that lignin input is not a prerequisite for aromatic components in soil. (3) Methoxy carbon was found to be a significant contributor to the 13C N M R spectrum of an Antarctic peat sample. Moreover, some of the methoxy carbon could be attached to aliphatic carbon, rather than lignin derived aromatic carbon as normally found. Methoxy carbon is not a significant contributor to the humic acids from moss of ornithogenic soils. Acknowledgements--We thank J. Skjemstad for useful dis-

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