THE DISTRIBUTION OF THE STABLE CARBON ... - Science Direct

Geoderma, 11(1974) 137--145 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE DISTRIBUTION OF THE STABLE CARBON ISOTOPE (13C/1~C) IN FRACTIONS OF SOIL ORGANIC MATTER*

A. NISSENBAUM and K.M. SCHALLINGER

Isotope Department, Weizmann Institute of Science, Rehovot (Israel) Institute for Soils and Water, Agricultural Research Organization, Bet Dagan (Israel) (Accepted for publication October 25, ].973)

ABSTRACT Nissenbaum, A. and Schallinger, K.M., 1974. The distribution of the stable carbon isotope (~3C/12C) in fractions of soil organic matter. Geoderma, 11: 137--145. The carbon-isotopic composition of fulvic and humic acid from the A horizons of eight soil types, developed under a wide variety of climatological conditions, was measured. The fulvic acid is always enriched in ~3C as compared with the humic acid from the same soil by a rather constant factor of 0.9%0. The fulvic acids are isotopically closer to the plant source of the organic matter and thus represent an intermediate stage in the formation of humic substances. Depth sections of peat soil showed that carbon isotopes can be used to evaluate the dynamic nature of the fulvic-acid fraction. With depth, a transfer of carbon groups from polysaccharides to fulvic acid is seen. Based on isotopic evidence it is shown that in addition to formation of ~humus, part of the fulvic acid is condensed with depth to a stable humic fraction -- humin.

INTRODUCTION

Measurements of the distribution of the stable carbon isotopes have been used extensively in geochemistry to evaluate the sources of organic matter in recent and ancient sediments. In addition, the fractionation of carbon isotopes can be used to obtain some insights on the post-depositional diagenetic changes which occur in sedimentary organic matter (e.g., Brown et al., 1972). Concise summaries of the biogeochemistry of the carbon isotopes is given by Degens (1969) and Epstein (1969). As compared with the large amount of data on the distribution of carbon isotopes in organic matter from recent sediments, little use has been made of this technique as applied to soil organic matter, with the exception of measurements related to radiocarbon dating of soil organic matter (Tate, 1972). Yet, on general principles, such studies may contribute towards understanding some of the stages of formation and development of soil organic matter. For * Contribution from the A.R.O., Volcani Center, Bet Dagan, Israel. 1973 Series, no. 157-E.

138

example, Degens et al. (1968) have shown that in marine plankton different biochemical fractions such as lipids, carbohydrates, proteins, etc., have different 13C/12C ratios. The lipids, for example, always contain less 13C than carbohydrates. Although no similar comprehensive study was made of the higher plants, we would expect to find similar variations. For example, the pigments of Italian Stone Pine needles have been shown to be depleted in '3C compared with the total carbon as expected from their biosynthetic pathway, which is that of lipids (Nissenbaum et al., 1972). The fractionation of 13C/nC in plankton biochemicals has been used as a m e t h o d to trace the source of sedimentary organic matter. Thus, the close similarity between the carbon-isotopic composition of the lipid fraction of plankton and that of petroleum, lead to the hypothesis that oil is derived from the lipid fraction of cellular material. It is quite conceivable that similar tracing techniques could be applied in studying soil organic matter. Recently, a chemical and isotopic study of marine and nonmarine humic substances was made by Nissenbaum and Kaplan (1972). It was shown that it is possible to differentiate between marine and nonmarine humates on the basis of carbon-isotopic ratios. Whereas marine humates had values of ~ ~3C= - - 2 0 % 0 (the same as in marine plankton of the subtropical zone), the composition of soil humic substances depended on the isotopic composition of the plant source (ca.--25% o for plants employing the Calvin photosynthetic cycle and ca. --10 to --17 °/oo for plants utilizing the Hatch--Slack cycle). They also showed that in marine sediments the fulvic acid is isotopically closer to the parent planktonic cellular material. The depletion of 13C in the humic acids was ascribed to diagenetic loss of '3C-enriched carbon, during the transformation of fulvic acid to humic acid. The purpose of the present investigation was to check whether similar regularity applies to humic and fulvic acids from soils with a limited geographical range, but from a wide variety of soil types. In addition, we studied in more detail the carbon-isotopic composition of fractions of peat soil, rich in organic matter, as a possible tool for elucidating some of the diagenetic transformations occurring in organic m a t t e r with time. M A T E R I A L S AND METHODS

Eight major soil types were chosen from prevailing Israeli soils (Ravikovitch, 1960). The soil types and sampling locations are given in Table I. The data given in this study refer to surface samples (horizon A). In addition, Table II gives isotopic data on samples taken from a 200-cm profile of peat soil from the area of the dried Hula Swamp (Schallinger, 1968). Further details on the exact sampling locations, climatic conditions, etc., are given by Schallinger (1971). The soil samples were air-dried and the nondecomposed plant material removed by CC14 flotation (Roulet et al., 1963). The flow diagram, showing the techniques used in fractionating the soil organic m a t t e r is given in Fig. 1.

Kfar Hassidim Umm-E1Zinat Wadi A r r a

AshdotYa'akov Massznia

Moledet Gilat dried H u l a swamp

B r o w n alluvial soil (vertisol)

T e r r a rossa

Mountain rendzina

Valley zendzina

Alluvial ( b r o w n ) soil

B r o w n basaltic soil

Loessial (arid) soil

Peat

HA FA

HA FA

HA FA

HA FA

HA FA

HA FA

HA FA

HA FA

Fraction*

54.1 42.5

54.1 35.1

48.6 40.3

46.9 39.0

45.1 40.0

42.0 38.5

45.5 38.9

50.2 38.1

%C

4.7 6.4

8.0 5.6

6.5 6.9

6.6 6.4

5.1 6.3

5.6 6.1

5.8 6.9

5.8 6.7

% H

3.1 3.2

5.1 3.5

5.2 3.6

5.5 3.2

4.8 4.6

4.6 4.3

4.2 3.3

4.9 4.6

%N

39.6 47.9

32.8 55.8

39.7 49.2

41.0 51.4

45.0 49.1

47.8 51.1

44.5 50.9

39.1 50.6

% O

613C vs. P D B s t a n d a r d ; c h e m i c a l c o m p o s i t i o n o n ash-free basis, o x y g e n b y difference. * H A = H u m i c A c i d ; F A = Fulvic Acid. ** A6 ~3C = ~ 13C H u m i c Acid--~ ~3C Fulvic Acid.

Location

Soil t y p e

C h e m i c a l c o m p o s i t i o n a n d ~ ~3C o f h u m i c a n d fulvic acids pairs f r o m s o m e Israeli soils

TABLE I

--18.0 --17.4

--25.7 --24.8

--28.4 --27.8

--28.0 --26.8

--28.9 --28.1

--26.6 --25.6

--29.4 --28.6

--27.0 --26.1

~13C (°/®)

--0.6

--0.9

--0.6

--1.2

--0.8

--1.0

--0.8

--0.9

A~3C** (°/00)

O~ ~D

--17.6 --17.7 --17.6 --17.4 --17.7

(%o),

(°I~)

--17.6 --17.8 --17.8 --17.6 --17.9

Humic acid

Total fraction

" H u m i c Acid" fraction

--18.4 --18.5 --18.4 --18.9 --18.1

(°Ioo)

Hymatomelanic acid

Results given against the PDB standard.

0-- 25 25-- 50 50--100 100--150 150--200

(cm)

Depth

--17.1 --17.9 --17.4 --17.7 --17.9

(%o)

Total fraction

--17.0 --17.6 --17.6 --18.1 --18.6

(%o)

Fulvic acid

"Fulvic A c i d " fraction

--18.5 --18.7 --17.8 --17.7 --17.4

Polysaccharides (%)

Changes in carbon-isotopic c o m p o s i t i o n of fractions of peat organic m a t t e r with d e p t h

TABLE II

--17.8 --17.4 --17.4 --17.6

(°Ioo)

~-humus

141

NaOH-SOLUBLE MATERIAL ocidified with HCI to pH=l PRECIPITATE

1

SOLUTION

1

1

"HUMIC ACID,, reflux withethonol

"FULVIC ACID,, //~acetone . / ~recipitot,on

PRECIPITATE SOLUTION I

I..... [POLYSACCHARIDES] pR odjusfed to 4.8 HYMATOMELANIC [ [ ACD ] PRECIPITATE[ SOLUTION

I HUMICACIDI .Es,oo j

1

'

[FULVICACID

_.oMos] [ t .Es,oo]

Fig.1. Scheme of fractionation of NaOH-soluble organic matter used in this study.

In the text, the humic acid fraction is to be distinguished from the humic acid residue fraction, which is the fraction obtained after removal of the hymatomelanic acid fraction from the humic acid fraction. The later fraction includes all the base-soluble, acid-insoluble material. The same applies to the fulvic acid residue fraction which includes the fraction remaining after the removal of ~-humus and polysaccharides from the total fulvic acid fraction. The separated fractions were dried either b y lyophilization or in a vacuum desiccator (Schallinger, 1971). For carbon-isotope measurements, the samples were burned in a vacuum line at 900°C under sub-atmospheric oxygen flow. The evolved CO2 was purified by distillation through dry ice--alcohol traps. The isotopic measurements were made on an Atlas M-86, dual inlet, dual collector isotope-ratio mass spectrometer. The isotopic data are given relative to the Chicago-PDB standard b y the following relationship: ~13C =

(~3C/~2C sample _ ~ standard 1/ × 1,000

All samples were run in duplicate or triplicate. The standard error is +0.15°/oo . RESULTS

In Table I the results of the chemical analysis and isotopic data on eight humic and fulvic acids samples are given. In all samples the humic acid fraction

142

is consistently depleted in 13C as compared with the corresponding fulvic acid. The depletion ranges between 0 . 6 % 0 and 1 . 2 % o and averages at about 0.9%0 difference. Similar relationships, although with somewhat larger isotopic differences (up to 3.8%0) were found by Nissenbaum and Kaplan (1972) for humic acids of marine origin. In the case of marine samples it has been shown that the carbon-isotope value of the fulvic acid corresponds to that of the plankton, its suggested precursor material. The isotopic difference between the humic acids and the fulvic acids was assumed to occur through diagenetic reactions. Inspecting the data reported here, a similar relationship is found. The fulvic acids are indeed closer to the values found or assumed for the precursor plant material. Although data on the ~sC/12C ratio in humic--fulvic acid pairs from other parts of the world are lacking, the samples analyzed for this study were collected from a fair variety of climates, thus indicating that this difference is of general distribution and is n o t necessarily limited to processes occurring in a particular environment. A different picture emerges where the depth distribution of the carbon isotopes is studied (Table II). In the surface sample, the expected isotopic difference is observed, even if it is only 0.50/00 . With depth the isotopic difference becomes much smaller or disappears. Since the humic acid shows little isotopic variation with depth, the masking of the isotopic difference is due to changes occurring in the fulvic acid fraction. Within the total fulvic acid fraction, the only changes occur in the fulvic acid residue and polysaccharide fractions. The ~humus fraction stays isotopically constant. The changes in the fulvic acid and polysaccharide fractions show inverse relationship. With depth the fulvic acid becomes more enriched in 13C b y a b o u t 1.6%o, whereas the polysaccharides become depleted in 12C by 1.1°/oo . From Table III we can see that in addition to the depth effect on the isotopic composition, a change in the concentration of those fractions occurs as well. The fulvic acid and polysaccharides decrease with depth at a similar rate. For both fractions the decrease of the b o t t o m is by a b o u t 50%. On the other hand, the concentration of the ~-humus fraction shows a marked increase with depth in its relative concentration of the total fulvic acid fraction from 2 % of the top to 6 1 % of the bottom, T A B L E III Changes in c o n c e n t r a t i o n o f f r a c t i o n s o f p e a t organic m a t t e r w i t h d e p t h (in % o f f r a c t i o n ) Depth

"Humic acid" fraction

"Fulvic acid" fraction

H u m i c acid

Hymatomelanic

Fulvic acid

Polysaccharide

~-humus

96.5 95.2 83.0 95.8 90.7

3.5 4.8 17.0 4.2 9.3

74.4 64.1 29.0 29.6 28.6

23.8 22.2 13.4 14.6 10.6

2.0 13.7 56.9 55.8 60.8

(cm) (%)

0-- 25 2 5 - - 50 50--100 100-150 150-200

(%)

(%)

(%)

(%)

143 although this increase is n o t accompanied by an isotopic change. The total humic acid fraction seems to show much greater stability. No significant changes in concentration or isotopic composition were n o t e d either for the humic acid or hymatomelanic acid fractions. DISCUSSION The data given in this study indicate that the fractions of organic soil matter associated with "fulvic acid" according to the c o m m o n scheme of soil organic matter fractionation are in a dynamic state in the soil. Fulvic acid usually has been considered to occupy one of t w o positions in the sequence of transformation of soil organic matter. I t w a s considered either to be a degradation p r o d u c t of soil humic substances or, alternatively, to be a low-molecular-weight precursor in the process of polymerization of degraded plant cellular material to humic acids. The similarity between the carbon-isotope composition of plants and that of the fulvic acid, whereas humic acid is usually depleted in 13C as compared with plants, indicates that the second hypothesis, namely that fulvic acid is an intermediate in the humification process, is much more tenable. Similar conclusions were reached by Nissenbaum and Kaplan (1972 } for humic substances from the marine environment. The rather constant difference in the isotopic composition (0.95+ 0.3%) between the humic and fulvic acids indicates that the fulvic acid to humic acid transformation is n o t gradual, b u t involves the loss of carbon c o m p o u n d s of fixed isotopic composition. Martin et al. (1963) have shown that with the molecular-weight increase from fulvic to humic acid, the percentage of carbon increases and that of oxygen decreases. This was attributed to loss of carboxyl groups. Schnitzer and Desjardins (1965) have indeed shown a decrease of carboxyl functional groups in Podzol B from 9.1 mequiv./g in fulvic acid to L 5 mequiv./g in humic acid. The analytical data given in Table I also show thgtrhumic acids are depleted in oxygen when compared with the fulvic acids. This loss of carboxyl groups can be correlated with the increase in '2C in the humic acid. Abelson and Hoering (1961) have shown that the carboxyl moieties of amino acids formed b y algae are highly enriched in 13C as compared with the amino acid carbon skeleton. Thus, a loss of 13C-enriched carboxyl assumed to originate, at least partially, from the carboxyl moieties of the amino acid c o m p o n e n t s of fulvic acid, w o u l d lead to the formation of ~2Cenriched humic acid. This process, however, seems to occur quite rapidly, and the isotopic difference is characteristic of processes occurring in the upper soil layers. With depth, as can be seen from data for the peat in Table II, this difference disappears, probably due to loss of all the labile amino acid carboxyl moieties of the fulvic acid. Preliminary results indicare that similar processes occur in other soil types as well. It has to be emphasized, though, that so far data on intramolecular distribution of carbon isotopes is available for amino acids only. Thus, we cannot evaluate the possible contribution of carboxyl moieties from other molecular species. The isotopic

144 composition o f the humic and hym at om e l a ni c acids remains rather constant with depth. The percentage of this fraction of the total organic m a t t e r increases only slightly with d e p t h and the percentage of h ym at om el ani c acid of the total humic acid fraction also remains rather constant with depth. This indicates clearly the relative stability of this fraction. The isotopic value for the hymatomelanic acid is quite different from that of the humic acid by being depleted in 13C. The reason for this is not clearly known. We can only suggest t hat probably some o f the lipid material, which is depleted in 13C (Degens et al., 1968), is associated with this fraction. As co mp ar ed to the relatively m o r e stable humic acid fraction, the fulvic acid fraction is mu ch m o r e dynamic. The isotopic value for the "fulvic acid" fraction shows a variation of less than 1% o (Table III), but the fulvic acid and polysaccharides show differences of up to 1 . 8 % o . The inverse isotopic relationship between the fulvic acid and the polysaccharides seems to indicate the transfer of some 13C-depleted m o i e t y from the polysaccharides into the fulvic acid fraction. The nature o f this carbon is unknow n. This transfer of carbon, however, is n o t the only reaction occurring. Because bot h the polysaccharides and fulvic acid decrease in c onc e nt r at i on with depth, the fulvic acid m ust be transformed in to a n o t h e r t ype of c om pound. The/~humus, although it increases its c o n cen tr atio n by a factor of 30, does n o t show m u c h isotopic change. It is therefore suggested that part of the fulvic acid is n o t transformed into humic acid, but into a stable fraction o f the organic matter, probabl y into humin, the insoluble or difficultly soluble fraction of organic m a t t e r in soils. The fulvic acid, therefore, plays the very i m p o r t a n t role of an active intermediate in the polymerization and condensation sequence of soil organic m a t t e r transformation. Carbon-isotope measurements have been shown t o be a valuable natural tracer for the study of the dynamic system of the humification process. ACKNOWLEDGEMENTS We would like to thank Mrs. Ziva Fellous-Hochman and Mrs. Ida Mouravinski of the Weizmann Institute of Science, and Mr. E. Elia of the Agricultural Research Organization f or their skillful technical help. REFERENCES Abelson, P.H. and Hoering, T.C., 1961. Carbon isotope fractionation in formation of amino acids by photosynthetic organisms. Proc. Natl. Acad. Sci., 47: 623--632. Brown, F.S., Baedecker, M.J., Nissenbaum, A. and Kaplan, I.R., 1972. Early diagenesis in a reducing fjord, Saanich Inlet, British Columbia, III. Changes in organic constituents of sediments. Geochim. Cosmochim. Acta, 36: 1185--1203. Degens, E.T., 1969. Biogeochemistry of stable carbon isotopes. In: G. Eglinton and M.T.J. Murphy (Editors), Organic Geochemistry -- Methods and Results. Springer Verlag, Berlin, 828 pp. Degens, E.T., Guillard, R.R.L., Sackett, W.M. and Hellebust, J.A., 1968. Metabolic fractionation of carbon isotopes in marine plankton, 1. Deep Sea Res., 15 : 1--9.

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Epstein, S., 1969. Distribution of carbon isotopes and their biochemical and geochemical significance. In: R.E. Forster, I.T. Edsall, A.B. Otis and F.J.W. Roughton (Editors), CO2 : Chemical, Biochemical and Physiological Aspects. NASA, Washington, D.C., 289 pp. Martin, F., Dubach, P., Mehta, N.C. and Deuel, H., 1963. Bestimmung der Funktionellen Gruppen von Humin-stoffen. Z. Pflanzenern~ihr., I)iing., Bodenkd., 103 : 27--29. Nissenbaum A. and Kaplan, I.R., 1972. Chemical and isotopic evidence for the in situ origin of marine humic substances. Limnol. Oceanogr., 17(4) : 570--582. Nissenbaum, A., Baedecker, M.J. and Kaplan, I.R., 1972. Organic geochemistry of Dead Sea sediments. Geochim. Cosmochim. Acta, 36: 709--722. Ravikovitch, S., 1960. Soils of Israel, Classification of the Soils of Israel. Hebrew University, Jerusalem, Israel, 89 pp. Roulet, N., Dubach, P., Mehta, N.C., Muller-Vemoos, M. and Deuel, H., 1963. Verteilung der organischen Substanz und der Kohlenhydrate bei der Gewinnung wurzelfreien Bodenmaterials durch Schlemensiebung. Z. Pflanzenerniihr., IRing., Bodenkd., 101: 201--214. Schallinger, K.M., 1968. Peat soils in Israel. Proc. Int. Peat Congr., 3rd, Quebec, Que., Canada, 17~ Schallinger, K.M., 1971. The Organic Matter in Soils of Israel -- Nature and Functions of the Polysaccharides. Thesis, Hebrew University, Jerusalem, Israel, 124 pp. (in Hebrew). Schnitzer, M. and Desjardins, J.O., 1965. Molecular and equivalent weights of the organic m a t t e r of podzol. Soil Sci. Soc. Am. Proc., 26: 362--365. Tate, K.H., 1972. Radiocarbon dating in studies of soil organic matter--vegetation relationship. In: Proc. Int. Conf. Radiocarbon Dating, 8th, 2: E27--E39.