- Email: [email protected]
Early stage incorporation of biolipids into kerogen in a lacustrine sediment: Evidence from alkaline potassium permanganate oxidation of sedimentary lipids and humic matter
Oral. t;cochem. Vol. 4. No. 34. pp. 179 184, 1983 Printed in Great Britain
0146-638083,030179-06503.001) Pergamon Press l.td
Early stage incorporation of biolipids into kerogen in a lacustrine sediment: Evidence from alkaline potassium permanganate oxidation of sedimentary lipids and humic matter RYOSHI ISHIWATARIand TSUTOMUMACHIHARA Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Fukazawa. Setagaya-ku, Tokyo 158, Japan Lipids,fnlvic acid, humic acid and kerogen were isolated from a lacustrine sediment in which the organic matter is probably derived predominantly from phytoplankton (Lake Haruna, JapanL An alkaline KMnO4 oxidation study of the organic matter showed that distributions of polymethylene chain lengths in the lipids, humic acid and kerogen fractions are almost the same. The polymethylcne chains in the sediment are dominant in the kerogen, lipids and humic acid, their relative abundance estimated by the oxidation being: kerogen (42'~, of the total amounts of polymethylene chains estimated) > lipids (38%) > humic acid (19°.o)> fulvic acid (l~o), It was concluded that algal lipids ma,, have been incorporated into the kerogen and humic acid fractions after the death of the algae and during, after, their deposition. Abstract
INTRODUCTION MANY investigators consider that polymethylene chains [(CH2)n, n _> 4] in algal kerogen originate from polymethylene chain-containing compounds present in algae (Abelson, 1963; Philp and Calvin, 1976: Larter and Douglas, 1980). In supporting the above idea, we claimed in a previous paper that lipids in phytoplanktons could make a greater contribution to polymethylene chains in kerogens which are mainly derived from algal lipid components than the cell-wall material (lshiwatari and Machihara, 1982). However, the time and pathways of incorporation of these lipids into kerogen are not well known. On the basis of the laboratory experiment Latter and Douglas (1980) proposed a mechanism that the lipids react with melanoidins (polymers of carbohydrates and amino acids) changing into kerogen during diagenesis. In addition to insoluble organic matter (kerogen), alkali-soluble organic matter (fulvic and humic acids) represent an important part of the organic matter in recent sediments. Nissenbaum and Kaplan (1972) claimed from carbon isotope study a possible pathway of polymerization of fulvic acid into kerogen through humic acid. However, as far as polymethylene chains in kerogen are concerned, the mechanism of their formation is considered to be different from that claimed by Nissenbaum and Kaplan (1972). One of the ways to elucidate the mechanism of incorporation of lipids into algal kerogen is to study the behavior of polymethylene chain-containing materials during sedimentation and diagenesis. In the present paper we describe the isolation of four organic fractions (lipids, fulvic acid, humic and kerogenl from a surface lacustrine sediment, the examination of the polymcthylene chains by alkaline KMnO4 oxidation and discuss the genetic relations among these frac-
tions as well as the incorporation of lipids into kerogen. EXPERIMENTAL
Sediment sample The sediment sample was taken from the deepest part of Lake Haruna, which is a representative mesotrophic freshwater lake in Japan (altitude 1084 m, area 1.23km 2, maximum depth 13.0m, volume 0.01 km3). The main source of organic matter in the sediment is considered to be phytoplankton populations (Ishiwatari et al., 1980).
Extraction and separation of lipid& fidric acid, humic acid and kerogen The solvent-extractable lipids were obtained by 17 successive extractions from the freeze-dried sediment sample with benzene/methanol (6:4) by using a homogenizer (10,000rpm: 15-30min). The lipids were concentrated under vacuum to dryness and used for the oxidation study. The non-extracted material was subsequently extracted with 0.5 N NaOH solution on mechanical shaking at room temperature to obtain alkali-soluble humic matter (fulvic and humic acids). The material was then centrifuged and the solid again treated as described: this procedure was repeated until the supernatant became almost colorless. The combined supernatant was acidified with 6 N HCI to pH 1 and the humic acid was obtained by centrifugation. The humic acid was purified by recycling between acid and basic solutions and was finally precipitated from an acid solution and separated by centrifugation (10,000 rpm). The acid solution containing fulvic acids was demineralized by dialysis against double-distilled water and recovered by freeze-drying. This procedure caused the loss of part of the fulvic 179
IN()
RYOSHI ISHIWA'IARIand TSUTOMU MACHIHARA
acid (low molecular weight compounds such as amino acids and carbohydrates), which passed through the membrane. The residue after the alkali extraction was digested with 46~},iHF/6 N HC1 (1 : 1) at 6(>70:C with mechanical shaking and the kerogen was purified by refluxing with 6 N HC1 to remove inorganic material and extracting with benzene/methanol (6:4) by sonication ( × 31, washed with redistilled water and freezedried.
Ovidatio~l study A sample (10 20mg) of lipids, fulvic acid, humic acid or kerogen was mixed with 10 ml of 20% KMnO4 in 1% KOH aqueous solution and allowed to react at 60 C for 1 hr with mechanical shaking. At the end of the reaction, the solution was acidified with cone, HeSO~ to p H I and then the excess permanganate and MnO2 were reduced by addition of powdered NaHSO3. The oxidation products were then extracted from the reaction mixture with ethyl acetate (10ml × 3), washed with distilled water (5ml x 1) and evaporated to dryness under reduced pressure. After silylation with Silyl-8 (Pierce Chemical Co.), the organic products were analyzed by gas chromatography (GC) or gas chromatography-mass spectrometry (GC/MS). Carbon dioxide in the reaction mixture was analyzed as follows: 100/A of the solution was taken out from the reaction mixture and injected into a 10 ml glass ampoule with silicon stopper containing 3 ml of 0.5", SnC12 in 0.INH2SO,, solution to reduce the excess permanganate and MnO2. CO_, generated was determined by a method similar to that of Menzel and Vaccaro (19641. K2CO 3 solution was used as a standard for CO: analysis. The error in the COz analysis was within 5{3~,.
detector. GC separations were performed using a glass column (2 m × 3 mm i.d.l packed with 1.0% OV-1 on Chromosorb W-AWDMCS (8(>100 mesh) and carrier gas (Nx) flow rate of 40ml/min. The column temperature was programmed from 90 280C at 6 C/rain. The degradation products on the gas chromatograms were quantified by measuring their peak areas with a Shimadzu Chromatopack C-RIA digital integrator. The degradation products were identified with a Shimadzu-LKB 9000 gas chromatograph mass spectrometer. The temperature and column conditions were the same as used for GC analyses. Helium gas was used as a carrier at the flow rate of 30 ml/min. The mass spectrometer was operated at an electron energy of 70 eV, accelerator voltage of 3.5 kV, and an ion source temperature of 310C. The molecular separator was maintained at 290 C.
GC and (;(-'/MS analyses GC analyses were carried out on a Shimadzu 4BM or 6AM gas chromatograph with a flame ionization
RESULTS
Table 1 gives the chemical data for the organic fractions. Since fulvic acid was obtained after dialysis for removing inorganic salts, a considerable amount of lower molecular weight compounds may have lost during dialysis, leading us to underestimate the amount of this fraction. According to a previous study (Ishiwatari, 1970), the amount of fulvic acid should be approximately 40%o more than that of humic acid for this lake sediment.
KMn04 degradation products As shown in Table 2, all fractions produced a large amount of CO2 on the KMnO4 oxidation. The amount of CO2 evolved from kerogen or humic acid is higher than that from lipids or fulvic acid. Since CO2 is easily produced by cleavage of carbon-oxygen bonds, this fact suggests that kerogen and humic acid contain these bonds more than lipids and fulvic acid. The lower degree of CO2 production for fulvic acid
Table l. Chemical data for the organic flactions Fraction
Concentration
Relative
in dry sediment concentration mg/g % Lipids
Elemental Composition(on ash free basis) Ash %
C%
H%
N%
0%
H/C N/C (atomic ratio)
15.7
12.3
15.4
57.1 7.77 i.i0 34.0
1.62
0.017
Fulvic Acid
4.7
3.7
14.0
48.7 6.34 7.39 37.6
1.55
0.130
llumic Acid
24.3
19.1
15.3
51.4 5.71 5.08 37.8
1.32
0.085
Kerogen
31.9
25.1
12.8
61.9 4.72 1.73 31.6
0.91
0.024
50.7
39.8
.
.
.
.
.
.
.
.
127.3
100.0
.
.
.
.
.
.
.
.
Others* Total Organic Matter**
*Organic matter lost during extraction and purification of organic fractions, whose amount was calculated by difference between total organic matter and isolated organic fractions (lipids + fulvic acid + humic acid/. ** Calculated by assuming that total organic matter contains 56"0 carbon.
Incorporation of biolipids into kerogen Table 2. Carbon balance on KMnO,, oxidation Co of the initial carbon) CO 2
Non-oxidized residue
Water-soluble product*
Lipid
14
1
85
Kerogen
25
I0
66
Humic acid
22
1
77
Fulvic acid
16
0
84
* Soluble in pH I solution. may be due to the preferential loss of carbohydrates and amino acids from the fraction during purification by dialysis. Figure 1 shows gas chromatograms of degradation products from four fractions (lipids, fulvic acid, humic acid and kerogen). Table 3 gives quantitative results of degradation products from four fractions. The major degradation products are normal C4 C~2 ~,e)dicarboxylic acids for lipids, humic acid and kerogen. being essentially similar to those for kerogens from
l 81
other young sediments (Machihara and Ishiwatari, 198l). Lipids. Major degradation products of lipids are C2 and n-C4 C~2 zw)-dicarboxylic acids, n-Cs, ~1-C0 and n-C14-C2~, monocarboxylic acids and C1~- isoprenoidal ketone (6,10,14-trimethyl pentadecan-2-onel. n-Cl4 C20 monocarboxylic acids may ha,,e been derived by h~drolysis of the corresponding esters and oxidation of the corresponding alcohols. The C1 s isoprenoidal ketone is known to be formed by oxidation of phytol (Simoneit and Burlingame, 1974: Philp and Calvin. 1976). H-C8. n-C0 monocarboxylic acids and n-C,,-Co :c(,)-dicarboxylic acids are produced in a good yield by oxidation of unsaturated fatty acids such as A'~ Ca
Table 3. Organic compounds produced by oxidation ofcach fraction Type of Product
Yield of oxidation product (mg/g) Lipids
Fulvic acid
Humic acid
Kerogen
0.4 0.3 0.2
1.3 0.3 0.2
Monocarboxylic acid C8
C9 CI0 ~iI ~12 '/_'13 c14 C~l5 ~16 C17 c£i8 ~19 L~20 ~21 c22 c~23 c~24 C25 u26 Total
4.1
1.7 1.0 0.i 0.3 0.2 3.4 0.9 7.2 i.i 2.6 0.2 0.8 0.6 1.6 0.6 2.8 0.3 1.0 30.5
0.0
0.0 0.0 0.0 0.0 0.0 0.01 0.0 0.0~ 0.0 0.02 0.0 0.0 0.0 0.0 0.0 0.0
0.0
0.0
0.1 0.2 0.5 0.i I.I 0.0 0.4 0.1 0.2 0.i 0.2 0.i 0.2
0.0
0.0
0.0 0.1
0.0 4.2
0.2 0.3 0.4 0.2 1.5 0.2 0.7 0.i 0.4 0.2 0.3 0.2 0.3 0.I 0.1 7.0
6.0 0.5 2.9 1.2 0.6 0.6 1.4 0.3 0.2 0.0 0.0 0.0
1.7 0.2 3.3 2.5 2.5 2.9 3.9 2.4 1.3 0.8 0.4 0.2
a, ~-Dicarbox~lic acid C2 C3 C4 C5 C6 C7 C8 C9 CI0 ~ll ~12 c13 c14 Total
1.4 2.5 6.1 5.9 6.2 6.0 8.1 6.7 3.3 2.3 I.i 0.6 0.4
0.0
0.I
0.4 1.0 4.9 4.7 4.6 5.0 5.5 4.7 2.3 1.4 0.9 0.5 0.2
50.6
13.7
22.2
36. i
Cls-ZSoprenoid ketone 3.0
0.0
0.0
0.2
Benzenecarboxylic acid Monocarboxylic Dicarboxylic Tricarboxylic
0.i 0.2 0.0
2.8 0.3 0.3
2.6 0.4 1.0
0.3 0.4 0.7
Total
0.3
3.4
4.0
1.4
182
RYOSHI ISHIWATARIand TSUTOMUMACHIHARA
(A) L i p i d
8
i
4 5
2
6
7
J
8~ 9'
9
Kerogen. Molecular distribution of degradation products of kerogen resembles that of lipids. However, the relative abundance of aliphatic (n-C~,~-C2~,) monocarboxylic acids and C~8-isoprenoidal ketone for kerogen is lower than that for lipids, indicating that precursors of these acids (monocarboxylic acids and phytol) is less abundant in the former than in the latter.
16'
c~ ~0
ill
24'
II
18'
8
4 5 6 7 I 9
22'
26'
(B) Kerogen DISCUSSION
Polymethylene chains in}bur fractions
i, f,y _
~4
(C) Humic acid
8
zJ @
(D) Fulvic acid
56 7
o
io
20
30
rnin. Fig. i. Gas chromatograms of KMnO4 degradation products (TMS esters) of four fractions from Lake Haruna sediment. The carbon numbers of the dicarboxylic acids are indicated by the arabic numbers and mono-carboxylic acids are indicated by the primed arabic numbers. @ etc. indicate benzenecarboxylic acids; ~ indicates C~8 isoprenoid ketone (6,10,14-trimethylpentadecan-2-one).The gas chromatographic conditions are given in the text. acid, J~°C4 and ~l-Cs ct,to-dicarboxylic acids and benzoic acid are much higher than those of other compounds. Oxalic acid may have been predominantly derived by oxidation of carbohydrates. Benzoic acid may have been derived from phenylalanine and/or its derivatives. A portion of n-C4 :~,co-dicarboxylic acid may have originated from lysine. Consequently, the above result indicates that carbohydrates, phenylalanine and other amino acids may be still abundant in this fraction, even though a considerable amount of these compounds may have lost during purification. Humic acid. Molecular distribution of degradation products of humic acid resembles that of kerogen rather than lipids. But benzoic acid, oxalic acid, n-C4, n-Cs z~,~o-dicarboxylicacids and benzenetricarboxylic acid are more abundant for humic acid than for kerogcn. This fact indicates that carbohydrates and amino acids in humic acid are more abundant than in kerogen.
As described already, the lipids, humic acid and kerogen appear to be similar in terms of distribution patterns of ~,og-dicarboxylic acids. The similarity of molecular distribution of aliphatic acids (0c~o-dicarboxylic acids and monocarboxylic acids) for four fractions was checked by plotting the yields of these compounds from fulvic acid, humic acid or kerogen against those from lipids (Fig. 2). As shown in Fig. 2, a fairly good straight line can be drawn for ~,(o-dicarboxylic acids (excluding C2 and C~ acids) from kerogen and humic acid versus those from lipids (r = 0.97-0.99). A good correlation in the molecular distribution of monocarboxylic acids was also observed between lipids and kerogen or humic acid (r = 0.85-0.92). The above facts clearly indicate a common origin for the polymethylenc chains in lipids, kerogens and humic acids. We calculated the amounts of polymethylene chains in the organic fractions in the sediment using the quantitated amounts of four fractions (column 1 in Table 4) and the amounts of aliphatic acids (C4 C~,~~,~o-dicarboxylic acids and Cs C2~, monocarboxylic acids) produced by oxidation of each fraction. In this calculation we assumed that the yield of aliphatic acids from polymethylene chains by KMnO,, oxidation is the same for four organic fractions. The organic fraction which was lost during isolation of
t, 6
A
Lipids-kerogen
o o
.2
~
%/°LipidS~cid
2
~5 2
4
6
8
10
Diocid for lipids ( m g / g l Fig. 2. Relation between yields of ~,~-dicarboxylic acids from lipids and those from kerogen, humic acid or fulvic acid. © kerogen; • humic acid: A fulvic acid.
Incorporation of biolipids into kerogen Table 4. Distribution of polymethylene chains in Lake Haruna sediment Fraction
Concentration
of p o l y m e t h y l e n e
chains in the s e d i m e n t Obtained C4-C14 diacids Lipids Kerogen Ilumic acid
as
O b t a i n e d as C8-C26 monoacid
Relative
(~g/g)
abundance
(%)
C8-C26 monoacid
Totals
Totals C4-C14 diacid
731
477
1208
30.9
59.4
38.1
1109
224
1333
46.8
27.9
42,@
494
102
596
20.9
12.7
18,~
F u l v i c acid-
34
1
35
1.5
0.i
1.1
Others*
.
100.1
100.~
Totals
2368
.
.
.
.
804
.
. 3172
.
.
. i00.i
.
.
* Lost during extraction and purification of organic fractions (the same as in Table I I. amount of algal lipids are still present in the sediment, as shown by chlorophyll pigments which arc a representative lipid component in algae and are present in the sediment (approx 0.4'~; of the total organic matter in the sediment: lshiwatari et al.. 1980l. Furthermore the ratio of fatty acids to chlorophyll pigments for the sediment (approx 3) appears to be the same for living algae IDarley, 1977). Therefore, it seems reasonable to consider that algal lipids contribute considerably to the polymethylene chains in kerogen and humic acid. According to Nissenbaum and Kaplan I19721. ,513C values of fulvic acids are 0.5-3.4",,, heavier than those of humic acids with some exceptions. Stuermer et al, (1978) reported that ,513C values of humic acids are 0.4 3.7",,,, heavier than those of kerogens. We also have obtained the following 613C values for the orImplication on the oriqin o( kerogen and humic acid ganic matter from a marine (Tanner Basin. off CaliThe above results (high abundance of polymethy- fornia) sediment: -19.60;,, (fulvic acidl. -21.7".o lene chains in kerogen and humic acid, and similarity (humic acid), -22.4"i;,, (kerogen) and -24.3",., (lipid) of their distribution pattern with that for the sedimen- (Ishiwatari et al., 1977 and unpublished data). Based tary lipids) may indicate the incorporation of a con- on the carbon isotopic evidence, Nissenbaum and siderable portion of algal lipids into kerogen and Kaplan (1972) proposed a hypothesis that humic acid humic acid after the death of algae during and/or formation and transformation in marine sediments proceed by the following pathway: (1) degraded celluafter their deposition. A hypothesis that unsaturated fatty acids (C~, and lar material-+(2) water-soluble complex containing CI s acids are generally dominant) in living algae pol- amino acids and carbohydrates --+ 13) fulvic ymerize into insoluble form (kerogen and humic acid) acids--+(4) humic acids--+(5) kerogen. They conafter their death (Abelson, 1963) appears to be unable sidered that the continuous polymerization of humic to explain the striking similarity of the polymethylene materials involves loss of fractions enriched in ~3C chain distribution in lipids, humic acid and kerogen, such as oxygenated groups. The reaction pathway proposed by Nissenbaum and Kaplan {1972) may be If the polymerization of C~, and Ct8 unsaturated fittty acids took place and these compounds were operative for the formation of non-lipid-derived part eliminated selectively from lipid fraction, distribution {not involving polymethylene chains) of kerogen and pattern of oxidation products (:z,u)-dicarboxylic acids) humic acid. The abundant presence of polymethylene for humic acid and kerogen should be different from chains in kerogen and humic acid cannot be that for the sedimentary lipids. The unsaturated fatty explained by the polymerization of fulvic acids, as acids and their polymerization products are expected shown in the preceding section. However. the results in this paper indicate that to produce a relatively large percentage of Cs and/or C,~ ~ao-dicarboxylic acids on the KMnO,~ oxidation another interpretation is possible for the isotopic evidence of humic materials cited above. The ditlkrence (Machihara and Ishiwatari, unpublished result). A mechanism that polymethylene chains in kerogen in 3~3C values can be explained by tbe different and bumic acid come predominantly from cell-wall degree of incorporation of algal lipids whose carbon materials in living algae is not likely to occur. A fair is isotopically lighter than the other organic ffaclions
four organic fractions was excluded for the calculation because the fraction is presumably composed predominantly of low molecular weight oxygenated compounds (e.g. carbohydrates). The result of calculation (Table 4) clearly indicates that polymethylene chains in the sediment are predominant in the kerogen, lipids and humic acids, their relative abundance estimated by the oxidation being: kerogen (4T?i, of the total amounts of polymethylene chains in the sediment) > lipids (38'30) > humic acid (19",) > fulvic acid (I!~,,). The abundant presence of polymethylcnc chains in organic solvent-insoluble fraction has not been observed for living algae, where the most (73 941!;l of the polymethylene chains are present in a lipid fraction (Ishiwatari and Machihara, 1982).
184
RYOSHI ISHIWATARIand TSUTOMU MACHIHARA
to the humic material fractions. As shown in Table 3, the degree of contribution of lipids as represented by polymethylene chains to the organic fractions decreases in the order of: lipids > kerogen > humic acid > fulvic acid. This order is the same as that of ~9 3C value (light to heavy) of these fractions. Here we make a rough estimate of the degree of contribution of lipids to kerogen and humic acid in Lake H a r u n a sediment, assuming that all polymethylene chains (C4 Cx4) are derived from sedimentary lipids whose one gram produces 47 mg C4 C~4 :<{odicarboxylic acids (Table 3). Surprisingly, the degree of contribution of lipids was calculated to be 0.74 for kcrogen and 0.43 for humic acid, respectively. We have no evidence, at present, for verifying the correctness of these values. However, the carbon isotope data for the organic matter from Tanner Basin sediment cited above seem to support the above estimate. The degree of contribution of lipids to kerogen and humic acid in this sediment was estimated using their ~51-~C values on the assumption that (1) kerogen and humic acid are produced from lipids and non-lipid materials and (2) ~93C of kerogen and humic acid can be expressed as the sum of ,~lac of lipids (-24.31~J and non-lipid materials whose ~5~~C are represented by that of fulvic acid (-19.6'!i,,,). The calculation showed that the degree of contribution of lipids is 0.60 for kerogen and 0.45 for humic acid, respectively. These results of calculation suggest that lipids play a very important role in the formation of kerogen and humic acid in recent sediments.
CONCLUSIONS 1. The relative adundance of polymethylene chains which is estimated by the oxidative degradation are similar a m o n g lipids, humic acid and kerogen from Lake H a r u n a sediment where organic matter is thought to originate mainly from phytoplanktons. 2. The estimated a m o u n t of polymethylene chains in kerogen is higher than those in other fractions (lipids, humic acid and fulvic acid). 3. It is concluded from the above results that a considerable portion of algal lipids incorporates into kerogen (and humic acid) in the early stage of deposition of dead algae.
Acknowledgements This study was supported in part by a grant from the Ministry of Education, Science and Culture, Japan (grant No. 564700341. We wish to thank Dr P. R. Pmt, P for reviewing the manuscript.
REFERENCES
Abelson, P. H., 1963, Organic geochemistry and the formation of petroleum: Proe. 6th World Pet. Con gr.. Frankfurt, Sec. i, p. 397 407. Darley, W. M., 1977. Biochemical composition, in Werner, D., ed., The Biology of Diatoms, Botanical monographs, Vol. 13, Blackwell, p. 198 223. Ishiwatari, R., 1970, Structural characteristics of recent lake sediments, in Hobson, G. D., and Speers. G. C., eds, Advances in Organic Geochemistry 1966, Pergamon Press, p. 285 311. lshiwatari, R., and Machihara, T., 1982, Algal lipids as a possible contributor to the polymethylene chains in kerogen: Geochim. Cosmoehim. Aeta, v. 46, p. 1459 1464. lshiwatari, R., lshiwatari, M., Rohrback, B. G., and Kaplan, I. R., 1977, Thermal alteration experiments on organic matter from Recent marine sediments in relation to petroleum genesis: Geochim. Cosmoehim. Aetu, v. 41, p. 815 828. lshiwatari, R., Ogura, K., and Horie, S., 1980, Organic geochemistry of a lacustrine sediment {Lake Haruna. Japanl: Chem. Geol.. v. 29, p. 261 280, Latter, S. R.. and Douglas, A. G., 1980, Melanoidins-kerogen precursors and geochemical lipid sinks: a study using pyrolysis gas chromatography {PGC): Geoehim. Cosmoehim. Acta, v. 44, p. 2087 2095. Machihara. T., and Ishiwatari. R., 1981. Characteristics of insoluble organic matter in lake sediments as revealed by alkaline potassmm permanganate oxidation: l'er, Int. l'erein. Limnol., v. 21, p. 244 247. Menzel, D, W.. and Vaccaro, R. F., 1964, The measurement of dissolved organic and particulate carbon in seawater: Limnol. Oeeanogr., v. 9, p. 138 142. Nissenbaum. A., and Kaplan, 1. R., 1972, Chemical and isotopic evidence for the in ,situ origin of marine humic substances: Limnol. Oeeanogr., v. 17, p. 570 582. Philp, R. P., and Calvin, M., 1976, Possible origin for insoluble organic (kerogen) degris of algae and bacteria: Nature, v. 262, p. 134~136. Simoneit, B. R., and Burlingame, A. L., 1974. Ketones derived from the oxidative degradation of Green River Formation oil shale kerogen, in Tissot, B., and Bienner, F., eds, Advances in Organic Geochemistry 1973, Editions Technip, p. 191 202. Stuermer, D. H., Peters. K. E.. and Kaplan. I. R., 1978, Source indicators of humic substances and protokerogen. Stable isotope ratios, elemental compositions and electron spin resonance spectra: Geochim. Cosmochim. Acta, v. 42, p. 989 997.
0146-638083,030179-06503.001) Pergamon Press l.td
Early stage incorporation of biolipids into kerogen in a lacustrine sediment: Evidence from alkaline potassium permanganate oxidation of sedimentary lipids and humic matter RYOSHI ISHIWATARIand TSUTOMUMACHIHARA Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Fukazawa. Setagaya-ku, Tokyo 158, Japan Lipids,fnlvic acid, humic acid and kerogen were isolated from a lacustrine sediment in which the organic matter is probably derived predominantly from phytoplankton (Lake Haruna, JapanL An alkaline KMnO4 oxidation study of the organic matter showed that distributions of polymethylene chain lengths in the lipids, humic acid and kerogen fractions are almost the same. The polymethylcne chains in the sediment are dominant in the kerogen, lipids and humic acid, their relative abundance estimated by the oxidation being: kerogen (42'~, of the total amounts of polymethylene chains estimated) > lipids (38%) > humic acid (19°.o)> fulvic acid (l~o), It was concluded that algal lipids ma,, have been incorporated into the kerogen and humic acid fractions after the death of the algae and during, after, their deposition. Abstract
INTRODUCTION MANY investigators consider that polymethylene chains [(CH2)n, n _> 4] in algal kerogen originate from polymethylene chain-containing compounds present in algae (Abelson, 1963; Philp and Calvin, 1976: Larter and Douglas, 1980). In supporting the above idea, we claimed in a previous paper that lipids in phytoplanktons could make a greater contribution to polymethylene chains in kerogens which are mainly derived from algal lipid components than the cell-wall material (lshiwatari and Machihara, 1982). However, the time and pathways of incorporation of these lipids into kerogen are not well known. On the basis of the laboratory experiment Latter and Douglas (1980) proposed a mechanism that the lipids react with melanoidins (polymers of carbohydrates and amino acids) changing into kerogen during diagenesis. In addition to insoluble organic matter (kerogen), alkali-soluble organic matter (fulvic and humic acids) represent an important part of the organic matter in recent sediments. Nissenbaum and Kaplan (1972) claimed from carbon isotope study a possible pathway of polymerization of fulvic acid into kerogen through humic acid. However, as far as polymethylene chains in kerogen are concerned, the mechanism of their formation is considered to be different from that claimed by Nissenbaum and Kaplan (1972). One of the ways to elucidate the mechanism of incorporation of lipids into algal kerogen is to study the behavior of polymethylene chain-containing materials during sedimentation and diagenesis. In the present paper we describe the isolation of four organic fractions (lipids, fulvic acid, humic and kerogenl from a surface lacustrine sediment, the examination of the polymcthylene chains by alkaline KMnO4 oxidation and discuss the genetic relations among these frac-
tions as well as the incorporation of lipids into kerogen. EXPERIMENTAL
Sediment sample The sediment sample was taken from the deepest part of Lake Haruna, which is a representative mesotrophic freshwater lake in Japan (altitude 1084 m, area 1.23km 2, maximum depth 13.0m, volume 0.01 km3). The main source of organic matter in the sediment is considered to be phytoplankton populations (Ishiwatari et al., 1980).
Extraction and separation of lipid& fidric acid, humic acid and kerogen The solvent-extractable lipids were obtained by 17 successive extractions from the freeze-dried sediment sample with benzene/methanol (6:4) by using a homogenizer (10,000rpm: 15-30min). The lipids were concentrated under vacuum to dryness and used for the oxidation study. The non-extracted material was subsequently extracted with 0.5 N NaOH solution on mechanical shaking at room temperature to obtain alkali-soluble humic matter (fulvic and humic acids). The material was then centrifuged and the solid again treated as described: this procedure was repeated until the supernatant became almost colorless. The combined supernatant was acidified with 6 N HCI to pH 1 and the humic acid was obtained by centrifugation. The humic acid was purified by recycling between acid and basic solutions and was finally precipitated from an acid solution and separated by centrifugation (10,000 rpm). The acid solution containing fulvic acids was demineralized by dialysis against double-distilled water and recovered by freeze-drying. This procedure caused the loss of part of the fulvic 179
IN()
RYOSHI ISHIWA'IARIand TSUTOMU MACHIHARA
acid (low molecular weight compounds such as amino acids and carbohydrates), which passed through the membrane. The residue after the alkali extraction was digested with 46~},iHF/6 N HC1 (1 : 1) at 6(>70:C with mechanical shaking and the kerogen was purified by refluxing with 6 N HC1 to remove inorganic material and extracting with benzene/methanol (6:4) by sonication ( × 31, washed with redistilled water and freezedried.
Ovidatio~l study A sample (10 20mg) of lipids, fulvic acid, humic acid or kerogen was mixed with 10 ml of 20% KMnO4 in 1% KOH aqueous solution and allowed to react at 60 C for 1 hr with mechanical shaking. At the end of the reaction, the solution was acidified with cone, HeSO~ to p H I and then the excess permanganate and MnO2 were reduced by addition of powdered NaHSO3. The oxidation products were then extracted from the reaction mixture with ethyl acetate (10ml × 3), washed with distilled water (5ml x 1) and evaporated to dryness under reduced pressure. After silylation with Silyl-8 (Pierce Chemical Co.), the organic products were analyzed by gas chromatography (GC) or gas chromatography-mass spectrometry (GC/MS). Carbon dioxide in the reaction mixture was analyzed as follows: 100/A of the solution was taken out from the reaction mixture and injected into a 10 ml glass ampoule with silicon stopper containing 3 ml of 0.5", SnC12 in 0.INH2SO,, solution to reduce the excess permanganate and MnO2. CO_, generated was determined by a method similar to that of Menzel and Vaccaro (19641. K2CO 3 solution was used as a standard for CO: analysis. The error in the COz analysis was within 5{3~,.
detector. GC separations were performed using a glass column (2 m × 3 mm i.d.l packed with 1.0% OV-1 on Chromosorb W-AWDMCS (8(>100 mesh) and carrier gas (Nx) flow rate of 40ml/min. The column temperature was programmed from 90 280C at 6 C/rain. The degradation products on the gas chromatograms were quantified by measuring their peak areas with a Shimadzu Chromatopack C-RIA digital integrator. The degradation products were identified with a Shimadzu-LKB 9000 gas chromatograph mass spectrometer. The temperature and column conditions were the same as used for GC analyses. Helium gas was used as a carrier at the flow rate of 30 ml/min. The mass spectrometer was operated at an electron energy of 70 eV, accelerator voltage of 3.5 kV, and an ion source temperature of 310C. The molecular separator was maintained at 290 C.
GC and (;(-'/MS analyses GC analyses were carried out on a Shimadzu 4BM or 6AM gas chromatograph with a flame ionization
RESULTS
Table 1 gives the chemical data for the organic fractions. Since fulvic acid was obtained after dialysis for removing inorganic salts, a considerable amount of lower molecular weight compounds may have lost during dialysis, leading us to underestimate the amount of this fraction. According to a previous study (Ishiwatari, 1970), the amount of fulvic acid should be approximately 40%o more than that of humic acid for this lake sediment.
KMn04 degradation products As shown in Table 2, all fractions produced a large amount of CO2 on the KMnO4 oxidation. The amount of CO2 evolved from kerogen or humic acid is higher than that from lipids or fulvic acid. Since CO2 is easily produced by cleavage of carbon-oxygen bonds, this fact suggests that kerogen and humic acid contain these bonds more than lipids and fulvic acid. The lower degree of CO2 production for fulvic acid
Table l. Chemical data for the organic flactions Fraction
Concentration
Relative
in dry sediment concentration mg/g % Lipids
Elemental Composition(on ash free basis) Ash %
C%
H%
N%
0%
H/C N/C (atomic ratio)
15.7
12.3
15.4
57.1 7.77 i.i0 34.0
1.62
0.017
Fulvic Acid
4.7
3.7
14.0
48.7 6.34 7.39 37.6
1.55
0.130
llumic Acid
24.3
19.1
15.3
51.4 5.71 5.08 37.8
1.32
0.085
Kerogen
31.9
25.1
12.8
61.9 4.72 1.73 31.6
0.91
0.024
50.7
39.8
.
.
.
.
.
.
.
.
127.3
100.0
.
.
.
.
.
.
.
.
Others* Total Organic Matter**
*Organic matter lost during extraction and purification of organic fractions, whose amount was calculated by difference between total organic matter and isolated organic fractions (lipids + fulvic acid + humic acid/. ** Calculated by assuming that total organic matter contains 56"0 carbon.
Incorporation of biolipids into kerogen Table 2. Carbon balance on KMnO,, oxidation Co of the initial carbon) CO 2
Non-oxidized residue
Water-soluble product*
Lipid
14
1
85
Kerogen
25
I0
66
Humic acid
22
1
77
Fulvic acid
16
0
84
* Soluble in pH I solution. may be due to the preferential loss of carbohydrates and amino acids from the fraction during purification by dialysis. Figure 1 shows gas chromatograms of degradation products from four fractions (lipids, fulvic acid, humic acid and kerogen). Table 3 gives quantitative results of degradation products from four fractions. The major degradation products are normal C4 C~2 ~,e)dicarboxylic acids for lipids, humic acid and kerogen. being essentially similar to those for kerogens from
l 81
other young sediments (Machihara and Ishiwatari, 198l). Lipids. Major degradation products of lipids are C2 and n-C4 C~2 zw)-dicarboxylic acids, n-Cs, ~1-C0 and n-C14-C2~, monocarboxylic acids and C1~- isoprenoidal ketone (6,10,14-trimethyl pentadecan-2-onel. n-Cl4 C20 monocarboxylic acids may ha,,e been derived by h~drolysis of the corresponding esters and oxidation of the corresponding alcohols. The C1 s isoprenoidal ketone is known to be formed by oxidation of phytol (Simoneit and Burlingame, 1974: Philp and Calvin. 1976). H-C8. n-C0 monocarboxylic acids and n-C,,-Co :c(,)-dicarboxylic acids are produced in a good yield by oxidation of unsaturated fatty acids such as A'~ Ca
Table 3. Organic compounds produced by oxidation ofcach fraction Type of Product
Yield of oxidation product (mg/g) Lipids
Fulvic acid
Humic acid
Kerogen
0.4 0.3 0.2
1.3 0.3 0.2
Monocarboxylic acid C8
C9 CI0 ~iI ~12 '/_'13 c14 C~l5 ~16 C17 c£i8 ~19 L~20 ~21 c22 c~23 c~24 C25 u26 Total
4.1
1.7 1.0 0.i 0.3 0.2 3.4 0.9 7.2 i.i 2.6 0.2 0.8 0.6 1.6 0.6 2.8 0.3 1.0 30.5
0.0
0.0 0.0 0.0 0.0 0.0 0.01 0.0 0.0~ 0.0 0.02 0.0 0.0 0.0 0.0 0.0 0.0
0.0
0.0
0.1 0.2 0.5 0.i I.I 0.0 0.4 0.1 0.2 0.i 0.2 0.i 0.2
0.0
0.0
0.0 0.1
0.0 4.2
0.2 0.3 0.4 0.2 1.5 0.2 0.7 0.i 0.4 0.2 0.3 0.2 0.3 0.I 0.1 7.0
6.0 0.5 2.9 1.2 0.6 0.6 1.4 0.3 0.2 0.0 0.0 0.0
1.7 0.2 3.3 2.5 2.5 2.9 3.9 2.4 1.3 0.8 0.4 0.2
a, ~-Dicarbox~lic acid C2 C3 C4 C5 C6 C7 C8 C9 CI0 ~ll ~12 c13 c14 Total
1.4 2.5 6.1 5.9 6.2 6.0 8.1 6.7 3.3 2.3 I.i 0.6 0.4
0.0
0.I
0.4 1.0 4.9 4.7 4.6 5.0 5.5 4.7 2.3 1.4 0.9 0.5 0.2
50.6
13.7
22.2
36. i
Cls-ZSoprenoid ketone 3.0
0.0
0.0
0.2
Benzenecarboxylic acid Monocarboxylic Dicarboxylic Tricarboxylic
0.i 0.2 0.0
2.8 0.3 0.3
2.6 0.4 1.0
0.3 0.4 0.7
Total
0.3
3.4
4.0
1.4
182
RYOSHI ISHIWATARIand TSUTOMUMACHIHARA
(A) L i p i d
8
i
4 5
2
6
7
J
8~ 9'
9
Kerogen. Molecular distribution of degradation products of kerogen resembles that of lipids. However, the relative abundance of aliphatic (n-C~,~-C2~,) monocarboxylic acids and C~8-isoprenoidal ketone for kerogen is lower than that for lipids, indicating that precursors of these acids (monocarboxylic acids and phytol) is less abundant in the former than in the latter.
16'
c~ ~0
ill
24'
II
18'
8
4 5 6 7 I 9
22'
26'
(B) Kerogen DISCUSSION
Polymethylene chains in}bur fractions
i, f,y _
~4
(C) Humic acid
8
zJ @
(D) Fulvic acid
56 7
o
io
20
30
rnin. Fig. i. Gas chromatograms of KMnO4 degradation products (TMS esters) of four fractions from Lake Haruna sediment. The carbon numbers of the dicarboxylic acids are indicated by the arabic numbers and mono-carboxylic acids are indicated by the primed arabic numbers. @ etc. indicate benzenecarboxylic acids; ~ indicates C~8 isoprenoid ketone (6,10,14-trimethylpentadecan-2-one).The gas chromatographic conditions are given in the text. acid, J~°C4 and ~l-Cs ct,to-dicarboxylic acids and benzoic acid are much higher than those of other compounds. Oxalic acid may have been predominantly derived by oxidation of carbohydrates. Benzoic acid may have been derived from phenylalanine and/or its derivatives. A portion of n-C4 :~,co-dicarboxylic acid may have originated from lysine. Consequently, the above result indicates that carbohydrates, phenylalanine and other amino acids may be still abundant in this fraction, even though a considerable amount of these compounds may have lost during purification. Humic acid. Molecular distribution of degradation products of humic acid resembles that of kerogen rather than lipids. But benzoic acid, oxalic acid, n-C4, n-Cs z~,~o-dicarboxylicacids and benzenetricarboxylic acid are more abundant for humic acid than for kerogcn. This fact indicates that carbohydrates and amino acids in humic acid are more abundant than in kerogen.
As described already, the lipids, humic acid and kerogen appear to be similar in terms of distribution patterns of ~,og-dicarboxylic acids. The similarity of molecular distribution of aliphatic acids (0c~o-dicarboxylic acids and monocarboxylic acids) for four fractions was checked by plotting the yields of these compounds from fulvic acid, humic acid or kerogen against those from lipids (Fig. 2). As shown in Fig. 2, a fairly good straight line can be drawn for ~,(o-dicarboxylic acids (excluding C2 and C~ acids) from kerogen and humic acid versus those from lipids (r = 0.97-0.99). A good correlation in the molecular distribution of monocarboxylic acids was also observed between lipids and kerogen or humic acid (r = 0.85-0.92). The above facts clearly indicate a common origin for the polymethylenc chains in lipids, kerogens and humic acids. We calculated the amounts of polymethylene chains in the organic fractions in the sediment using the quantitated amounts of four fractions (column 1 in Table 4) and the amounts of aliphatic acids (C4 C~,~~,~o-dicarboxylic acids and Cs C2~, monocarboxylic acids) produced by oxidation of each fraction. In this calculation we assumed that the yield of aliphatic acids from polymethylene chains by KMnO,, oxidation is the same for four organic fractions. The organic fraction which was lost during isolation of
t, 6
A
Lipids-kerogen
o o
.2
~
%/°LipidS~cid
2
~5 2
4
6
8
10
Diocid for lipids ( m g / g l Fig. 2. Relation between yields of ~,~-dicarboxylic acids from lipids and those from kerogen, humic acid or fulvic acid. © kerogen; • humic acid: A fulvic acid.
Incorporation of biolipids into kerogen Table 4. Distribution of polymethylene chains in Lake Haruna sediment Fraction
Concentration
of p o l y m e t h y l e n e
chains in the s e d i m e n t Obtained C4-C14 diacids Lipids Kerogen Ilumic acid
as
O b t a i n e d as C8-C26 monoacid
Relative
(~g/g)
abundance
(%)
C8-C26 monoacid
Totals
Totals C4-C14 diacid
731
477
1208
30.9
59.4
38.1
1109
224
1333
46.8
27.9
42,@
494
102
596
20.9
12.7
18,~
F u l v i c acid-
34
1
35
1.5
0.i
1.1
Others*
.
100.1
100.~
Totals
2368
.
.
.
.
804
.
. 3172
.
.
. i00.i
.
.
* Lost during extraction and purification of organic fractions (the same as in Table I I. amount of algal lipids are still present in the sediment, as shown by chlorophyll pigments which arc a representative lipid component in algae and are present in the sediment (approx 0.4'~; of the total organic matter in the sediment: lshiwatari et al.. 1980l. Furthermore the ratio of fatty acids to chlorophyll pigments for the sediment (approx 3) appears to be the same for living algae IDarley, 1977). Therefore, it seems reasonable to consider that algal lipids contribute considerably to the polymethylene chains in kerogen and humic acid. According to Nissenbaum and Kaplan I19721. ,513C values of fulvic acids are 0.5-3.4",,, heavier than those of humic acids with some exceptions. Stuermer et al, (1978) reported that ,513C values of humic acids are 0.4 3.7",,,, heavier than those of kerogens. We also have obtained the following 613C values for the orImplication on the oriqin o( kerogen and humic acid ganic matter from a marine (Tanner Basin. off CaliThe above results (high abundance of polymethy- fornia) sediment: -19.60;,, (fulvic acidl. -21.7".o lene chains in kerogen and humic acid, and similarity (humic acid), -22.4"i;,, (kerogen) and -24.3",., (lipid) of their distribution pattern with that for the sedimen- (Ishiwatari et al., 1977 and unpublished data). Based tary lipids) may indicate the incorporation of a con- on the carbon isotopic evidence, Nissenbaum and siderable portion of algal lipids into kerogen and Kaplan (1972) proposed a hypothesis that humic acid humic acid after the death of algae during and/or formation and transformation in marine sediments proceed by the following pathway: (1) degraded celluafter their deposition. A hypothesis that unsaturated fatty acids (C~, and lar material-+(2) water-soluble complex containing CI s acids are generally dominant) in living algae pol- amino acids and carbohydrates --+ 13) fulvic ymerize into insoluble form (kerogen and humic acid) acids--+(4) humic acids--+(5) kerogen. They conafter their death (Abelson, 1963) appears to be unable sidered that the continuous polymerization of humic to explain the striking similarity of the polymethylene materials involves loss of fractions enriched in ~3C chain distribution in lipids, humic acid and kerogen, such as oxygenated groups. The reaction pathway proposed by Nissenbaum and Kaplan {1972) may be If the polymerization of C~, and Ct8 unsaturated fittty acids took place and these compounds were operative for the formation of non-lipid-derived part eliminated selectively from lipid fraction, distribution {not involving polymethylene chains) of kerogen and pattern of oxidation products (:z,u)-dicarboxylic acids) humic acid. The abundant presence of polymethylene for humic acid and kerogen should be different from chains in kerogen and humic acid cannot be that for the sedimentary lipids. The unsaturated fatty explained by the polymerization of fulvic acids, as acids and their polymerization products are expected shown in the preceding section. However. the results in this paper indicate that to produce a relatively large percentage of Cs and/or C,~ ~ao-dicarboxylic acids on the KMnO,~ oxidation another interpretation is possible for the isotopic evidence of humic materials cited above. The ditlkrence (Machihara and Ishiwatari, unpublished result). A mechanism that polymethylene chains in kerogen in 3~3C values can be explained by tbe different and bumic acid come predominantly from cell-wall degree of incorporation of algal lipids whose carbon materials in living algae is not likely to occur. A fair is isotopically lighter than the other organic ffaclions
four organic fractions was excluded for the calculation because the fraction is presumably composed predominantly of low molecular weight oxygenated compounds (e.g. carbohydrates). The result of calculation (Table 4) clearly indicates that polymethylene chains in the sediment are predominant in the kerogen, lipids and humic acids, their relative abundance estimated by the oxidation being: kerogen (4T?i, of the total amounts of polymethylene chains in the sediment) > lipids (38'30) > humic acid (19",) > fulvic acid (I!~,,). The abundant presence of polymethylcnc chains in organic solvent-insoluble fraction has not been observed for living algae, where the most (73 941!;l of the polymethylene chains are present in a lipid fraction (Ishiwatari and Machihara, 1982).
184
RYOSHI ISHIWATARIand TSUTOMU MACHIHARA
to the humic material fractions. As shown in Table 3, the degree of contribution of lipids as represented by polymethylene chains to the organic fractions decreases in the order of: lipids > kerogen > humic acid > fulvic acid. This order is the same as that of ~9 3C value (light to heavy) of these fractions. Here we make a rough estimate of the degree of contribution of lipids to kerogen and humic acid in Lake H a r u n a sediment, assuming that all polymethylene chains (C4 Cx4) are derived from sedimentary lipids whose one gram produces 47 mg C4 C~4 :<{odicarboxylic acids (Table 3). Surprisingly, the degree of contribution of lipids was calculated to be 0.74 for kcrogen and 0.43 for humic acid, respectively. We have no evidence, at present, for verifying the correctness of these values. However, the carbon isotope data for the organic matter from Tanner Basin sediment cited above seem to support the above estimate. The degree of contribution of lipids to kerogen and humic acid in this sediment was estimated using their ~51-~C values on the assumption that (1) kerogen and humic acid are produced from lipids and non-lipid materials and (2) ~93C of kerogen and humic acid can be expressed as the sum of ,~lac of lipids (-24.31~J and non-lipid materials whose ~5~~C are represented by that of fulvic acid (-19.6'!i,,,). The calculation showed that the degree of contribution of lipids is 0.60 for kerogen and 0.45 for humic acid, respectively. These results of calculation suggest that lipids play a very important role in the formation of kerogen and humic acid in recent sediments.
CONCLUSIONS 1. The relative adundance of polymethylene chains which is estimated by the oxidative degradation are similar a m o n g lipids, humic acid and kerogen from Lake H a r u n a sediment where organic matter is thought to originate mainly from phytoplanktons. 2. The estimated a m o u n t of polymethylene chains in kerogen is higher than those in other fractions (lipids, humic acid and fulvic acid). 3. It is concluded from the above results that a considerable portion of algal lipids incorporates into kerogen (and humic acid) in the early stage of deposition of dead algae.
Acknowledgements This study was supported in part by a grant from the Ministry of Education, Science and Culture, Japan (grant No. 564700341. We wish to thank Dr P. R. Pmt, P for reviewing the manuscript.
REFERENCES
Abelson, P. H., 1963, Organic geochemistry and the formation of petroleum: Proe. 6th World Pet. Con gr.. Frankfurt, Sec. i, p. 397 407. Darley, W. M., 1977. Biochemical composition, in Werner, D., ed., The Biology of Diatoms, Botanical monographs, Vol. 13, Blackwell, p. 198 223. Ishiwatari, R., 1970, Structural characteristics of recent lake sediments, in Hobson, G. D., and Speers. G. C., eds, Advances in Organic Geochemistry 1966, Pergamon Press, p. 285 311. lshiwatari, R., and Machihara, T., 1982, Algal lipids as a possible contributor to the polymethylene chains in kerogen: Geochim. Cosmoehim. Aeta, v. 46, p. 1459 1464. lshiwatari, R., lshiwatari, M., Rohrback, B. G., and Kaplan, I. R., 1977, Thermal alteration experiments on organic matter from Recent marine sediments in relation to petroleum genesis: Geochim. Cosmoehim. Aetu, v. 41, p. 815 828. lshiwatari, R., Ogura, K., and Horie, S., 1980, Organic geochemistry of a lacustrine sediment {Lake Haruna. Japanl: Chem. Geol.. v. 29, p. 261 280, Latter, S. R.. and Douglas, A. G., 1980, Melanoidins-kerogen precursors and geochemical lipid sinks: a study using pyrolysis gas chromatography {PGC): Geoehim. Cosmoehim. Acta, v. 44, p. 2087 2095. Machihara. T., and Ishiwatari. R., 1981. Characteristics of insoluble organic matter in lake sediments as revealed by alkaline potassmm permanganate oxidation: l'er, Int. l'erein. Limnol., v. 21, p. 244 247. Menzel, D, W.. and Vaccaro, R. F., 1964, The measurement of dissolved organic and particulate carbon in seawater: Limnol. Oeeanogr., v. 9, p. 138 142. Nissenbaum. A., and Kaplan, 1. R., 1972, Chemical and isotopic evidence for the in ,situ origin of marine humic substances: Limnol. Oeeanogr., v. 17, p. 570 582. Philp, R. P., and Calvin, M., 1976, Possible origin for insoluble organic (kerogen) degris of algae and bacteria: Nature, v. 262, p. 134~136. Simoneit, B. R., and Burlingame, A. L., 1974. Ketones derived from the oxidative degradation of Green River Formation oil shale kerogen, in Tissot, B., and Bienner, F., eds, Advances in Organic Geochemistry 1973, Editions Technip, p. 191 202. Stuermer, D. H., Peters. K. E.. and Kaplan. I. R., 1978, Source indicators of humic substances and protokerogen. Stable isotope ratios, elemental compositions and electron spin resonance spectra: Geochim. Cosmochim. Acta, v. 42, p. 989 997.