Energy & Fuels 19948, 481-486
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Occurrence of Bound 3&Carboxysteroids in Geological Samples Assem 0. Barakat' Department of Chemistry, Faculty of Science, Alexandria University, P.O.Box 426, Alexandria 21321, Egypt
Jurgen Rullkotter' Institute of Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky Universitat, Oldenburg, P.O.Box 2503,D-26111 Oldenburg, Germany Received August 23, 1993. Revised Manuscript Received December 27, 1 9 9 P
Three main series of bound steroidal acids have been tentatively identified, in low concentrations, in the hydrolysates of kerogens and the extractable polar fractions of sediments from the Nardlinger Ries (southern Germany), and in a kerogen sample from the Monterey Formation (California). The results provide further evidence of the widespread occurrence of 3-carboxysteroids and indicate that they are chemically bonded to the complex network of kerogen through ester linkages. This suggests that these compounds could represent a yet unrecognized family of compounds occurring in the biosphere.
Introduction Fossil steroids occur in almost all ancient sediments and crude oils. These compounds retain structural and isotopic characteristics inherited from precursor lipids, and because of their characteristic carbon skeletons are extremely useful for geochemical purposes, in particular for providing a record of thermal history and for correlating oils with source r0cks.l They may also give information on paleoenvironmental conditions and on the precursor organisms that contributed part of their biomass to the fossil organic matter.24 A particular class of steroid components, i.e., those possessing a methyl group at C-4, have been the subject of recent interest, in view of the potential of certain of them as markers for dinoflagellates.kS More recently, there has been a number of reports on the occurrence of fossil steroids with unprecedented carbon substituent patterns on ring A. These novel sedimentary steroids include 2- and 3-alkylsteranes,10-12 2- and 3-methyltriaromatic steroid hydroThe recognition carbons,l3 and 3/3-~arboxysteranes.~~J~ of such an unusual structural modification of the sterane
* Abstract published in Adoance ACS Abstracts, February 1, 1994. (1) Mackenzie,A. S.;Braasell,S. C.; Eglinton, G.; Maxwell, J. R. Science
1982,217, 491-504. (2) Huang, W. Y.; Meinschein,W. G. Geochim.Cosmochim. Acta 1979, 43.739-745. '(3) Moldowan, J. M.; Seifert, W. K.; Gallegos, E. J. Bull. Am. Assoc. Pet. Geol. 198& 69, 1225-1268. (4) Braasell, S. C.; Eglinton, G.; Mo, F. J. Org. Geochem. 1986, 10, 927-45. (5) Robinson, N.; Eglinton, G.; Cranwell, P. A. Nature 1984,308,43941. (6) Wolff, G. A.; Lamb, N. A.; Maxwell, J. R. Ceochim. Cosmochim. Acta 1986,50, 335-342. ~
(7)Summons, R. E.; Volkman, J. K.; Boreham, C. J. Ceochim. Cosmochim. Acta 1987,51, 3075-82. (8) Summons, R. E.; Thomas, J.; Maxwell, J. R.; Boreham, C. J. Geochrm. Cosmochim. Acta 1992,56, 2437-2444. (9) Goodwin, N. S.; Mann, A. L.; Patience, R. L. Org. Geochem. 1988, 12,495-506.
(IO) Summons, R. E.; Capon, R. J. Geochim. Cosmochim. Acta 1988, 52, 2733-36.
(11) Summons, R. E; Capon, R. J. Geochim. Cosmochim. Acta 1992, 55, 2391-2395.
skeleton is of geochemical,biochemical, and paleobiological interest.11J6 Despite the structural assignments previously achieved, the origin of these compounds and aspects of their biogeochemistry are still unknown. Evidence is reported here from capillary column gas chromatography-mass spectrometry (GC-MS)data for the presence of steroidal acids in the hydrolysates of kerogens and extractable polar fractions of sulfur-rich lacustrine sediments from the Nordlinger Ries (southern Germany), and in a kerogen sample from the Monterey Formation (California), all of Miocene age.
Experimental Section Lacustrine sediments in the Nerdlinger Ries, a former impact crater in southern Germany formed during the Miocene, were deposited under slightly saline conditions and comprise several black shale layers rich in immature organic matter and ~ u l f u r . ~ e The five samples used in this study were taken at various depths from the most bitumen-rich layers in two boreholes (NR-10and NR-30) of BEB Erdgas und ErdSl GmbH. The Miocene Monterey Formation sample was supplied by the Union Sciences and TechnologyDivision, Brea, CA. This sample, rich in carbon and sulfur, was taken at 1350 m depth from a well in the Santa Maria Basin. The ground sediments were exhaustively extracted with dichloromethane in a Soxhlet apparatus. The total lipids were separated into several compound classes by medium pressure liquid chromatography (MPLC)." The neutral polar fractions were saponifiedwith KOH/CHaOH, and bound carboxylicacids (12) Dahl, J.; Moldowan,J. M.; McCaffrey,M. A; Lipton,P. A.Nature, 1992,355, 154-157. (13) Lichtfouse, E.; Riolo, J.;Albrecht. P. Tetrahedron Lett. 1990.31. . . 3937-40. (14) Dany, F.; Riolo, J.; Trendel, J. M.; Albrecht, P. J. Chem. SOC., Chem. Commun. 1990,1228-30. (15) Schaeffer, P.; Fache-Dany, F.; Trendel, J. M.; Albrecht, P. Org.
Geochem., in press. (16) RullkBtter,J.;Littke,R.;Schaefer,R.G. InGeochemistryofSulfur in Fossil Fuels; Om W. L., White C. M., Eds.; ACS Sympdum Seriee 249; American Chemical Society: Washington, DC, 1890; pp 149-169. (17)Radke, M.; Willach, H.; Welte, D. H. A w l . Chem. 1980,52,406-
411.
0887-0624/94/2508-0481$04.50/0
0 1994 American Chemical Society
Barakat and Rullkirtter
482 Energy & Fuels, Vol. 8, No. 2, 1994 A’
NR-10 ( 1 5 1 . 5
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Kerogen-bound acids
Kerogen-bound acids
17794048
501760 b
5
108
.-
-
.+---A
m _ -
LA,
miz 275
29491 2
0
1500
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Figure 2. Mass chromatograms (ml2 207, 221, 275, and 289) and total ion current (TIC)obtained by GC-MS analysis of the carboxylic acid fraction generated by saponification of a the kerogen sample of a Nerdlinger Ries sediment from 151.5m depth of well NR-10. Labeled peaks are identified in Table 1. A
K A’ B L M
B’ L’ N C’
0 P
compound structure 3fl-carboxy-5fl(H)-cholestane(?) I,R-H 3@-carboxy-4a-methyl-5a(~-cholestane 11, R = H 3@-carboxy-5a(H)-cholestane I,R=H 3@-carboxy-24-methyl-5@(H)-cholestane(?) I, R = CHs 3fl-carboxy-23,24-dimethyl-5a(H)-cholest- I11 22-ene 3~-carboxymethyl-5a(H)-cholestane IV,R=H 3fl-carboxy-24-methyl-5a(H)-cholestane I, R = CHs 111 isomer of L [5@(H)?I 3@-carboxy-4a,24-dimethyl-5a(H)-cholestane 11, R = CHa 3@-carboxy-24-ethyl-5a(H)-cholestane I, R C & 5 3@-carboxymethyl-24-methyl-5a(H)-cholestane IV, R = CHs 3@-carboxy-24-ethyl-4a-methyl-5a(Zf)11, R CzHs
cholestane
Q 3fl-carboxymethyl-24-ethyl-5~(H)-cholestane IV,R = CzH6 a
See text for structures.
were separatedon a column of silica gel impregnatedwith KOH. The procedure for isolation of the kerogen concentrates,kerogen saponification, carboxylic acid isolation,and derivatization,and the instrumental conditione used for gas chromatography (GC) and computerized gas chromatography-mass spectrometry (CGC-MS),were described earlier.’*
Results and Discussion GC-MS analyses revealed the presence of three series of steroidal acids in the esterified kerogen-boundcarboxylic acid fractions. Figure 1shows part of the reconstructed ion current (TIC) chromatogram of the carboxylic acid fraction obtained by saponification of the kerogen of a Ndrdlinger Ries sediment from 151.5m depth of well NR10. The assignments of the peaks labeled in Figure 1are given in Table 1. Recognition of each series was made on the basis of their mass spectral fragmentation patterns. Analogous to that of the corresponding alkanes, this fragmentation is governed by ions corresponding to M+ and M+ - 15, and by fragments at mlz 275 or 289 (base peak; cleavage in ring D through C-13(17) and C-14(15)), mlz 207 or 221 (cleavage through C-9(11) and C-8(14)), and mlz 290 or 304 (C-13(17) and C-15(16) cleavage). Confirmation of the structural assignments was obtained (18) Barakat, A. 0.; Rullketter, J. Fuel, in press.
by comparison of mass spectra with reference spectra of natural and synthesized model compounds and from retention time data reported in the literature.14J5Jg Extensive use was also made of mass fragmentography to aid compound recognition (see, for example, Figure 2). The mass spectra of several members of these series are shown in Figure 3. Assignment of the position of the methyl group in ring A at C-4 is based on the reported occurrence of this compound series in evaporitic sediments from the Mulhouse basin.16 According to characterization by GC-MS data, the compounds with key fragments a t mlz 207 and 275 consist of a pseudohomologous series of 38-carboxysteranes with 28-30 carbon atoms (including the carboxylgroup) as pairs of epimers most likely at C-5 detected for the two lower carbon number species. Corresponding to this, cm-c31 4a-methyl-38-carboxy-5a(H)-steranes together with c29-c3138-carboxymethyl-5a(H)-ster~esrepresent two pseudohomologous series with key fragments at mlz 221 and 289. The 38-carboxysterane and 4a-methyl-38carboxy-5a(H)-sterane series were observed in the hydrolysates of the kerogen samples from the three Niirdlinger Ries sediments from well NR-10. The 38carboxymethyl-5a(H)-sterane series were observed in the hydrolysate of Nardlinger Ries kerogen sample from well was found NR-10 (151.5 m), and only one component (M) in the correspondingfractions from the sediments obtained at 170.5 and 250.0 m depth from the same well. The two isomeric 38-carboxycholestanes (A and A’) are always the most prominent members with the 5a(H) epimer being consistently more abundant than the 58(H) epimer. The site of epimerization at C-5 is derived from the retention time difference in analogy to that for 5D(H)- and 5a(H)steranes and the fact that epimerization at (2-20 to an extent indicated by the AIA’ ratio is very unlikly in the Nordlinger Ries sediments considering the low maturity of the organic matter.l6 Epimers at C-24 would virtually have identical GC retention times as shown by Dany et Epimers A and A’ do not show, however, the characteristic intensity difference of the AB-ring fragment (19)Trifilieff,S. Thhe de Doctorat, Universit.4 Louis Paeteur, Strasbourg, 1987.
*-'
36-Carboxysteroids in Geological Samples
,
Energy & Fuels, Vol. 8, No. 2,1994 483
275 HJCOOC
io' i
A
430
415 I
100
200
300
d
500
400
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200
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.
I.,. 100
200
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300 m/z
/
vvc
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100
-
100
200
400
300 m/z
500
m/r
415430
500
100
200
do. '
300
400
500
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Figure 3. Mass spectra and proposed structures of selected carboxysteroids labeled in Figure 1.
HOOC
HOOC
HOOC
HOOC H
I
H
I
CH3~
at mlz 207 and mlz 209 analogous to that at mlz 149 and mlz 151 in the corresponding 5a(H)-and 5@(H)-steranes. This may, however, be due to the presence of the functional group at C-3 which may have an influence on the fragmentation pattern. Both compounds are the only two steroidal acids bound to the kerogens of the Monterey sediment and the Nordlinger Ries sediment from well NR30 (222.9 m). Besides the three main series, two monounsaturated steroidal acids (L and L' in Figure 1) having virtually identical mass spectra were also found in the Ndrdlinger Ries samples, although in relatively low concentrations. The mass spectrum of component L is shown in Figure 4. It is characterized by a molecular ion at mlz 456, a base peak at mlz 69, and main fragment ions at mlz 315,317, 344, and 413 (M+- 43). Based on this, compounds L and L' are tentatively identified as isomeric methyl esters of 3~-carboxy-23,24-dimethylcholest-22-ene (possibly the 5a(H)and 5/3(H) epimers). The intense ion at mlz 69 and
111
IV
the fragment ions at mlz 315/317 and 344 indicate a A22 double bond analogous to the mass spectra of 23,24dimethyl A22-steroids. Main fragmentation for these steroids occurs by cleavage of the side chain at C-17/C-20 and C-20/C-22, respectively.20 Other than in the kerogen hydrolysates, only 38carboxysteranes (CZS-C~S) and 38-carboxy-4a-methylsteranes (Cw-Cd were detected in the acid fractionsbound to the neutral polar components of two of the Nordlinger Ries bitumens [samples from well NR-10 (151.5 and 170.5 m), see,for example, Figure 51. Contraryto kerogen-bound 3-carboxysteroids, polar-bound acids were always dominated by the 4-methylsteroid acids (N and P). Considering the fact that there is a strong dominance of cholest-4- and -5-enes (Cn)in the extractablehydrocarbonsand a complex mixture of C~TCWsulfur-bearing steroid hydrocarbons in the Nordlinger Ries bitumens,lsthis indicatesthat there (20)Wyllie, S.G.;Djerassi, C. Org. Chem. 1968.33, 305-13.
484 Energy & Fuels, Vol. 8, No. 2, 1994
Barakat and Rullkotter
315
\
100
200
344
413
456
I
300
400
mlr
Figure 4. Mass spectrum and proposed structure of peak L in Figure 1. Polar-bound acids
100
1210.
100
]
I"
TIC
3800
4000
P 4200
238592.
4400
Scan number
Figure 5. Mass chromatograms (mlz 275 and 289) and total ion current (TIC) obtained by GC-MS analysis of the carboxylic acid fraction generated by saponificationof the polar fraction of a NBrdlinger Ries sediment from 151.5 m depth of well NR-10. Labeled peaks are identified in Table 1.
are several modes of selective preservation of steroids in the Sediments studied which may be of different paleoenvironmental significance. There are remarkable differences in the occurrence of the steroid carboxylic acids in the kerogens and polar fractions of the sediments investigated in this study (Figure 6). 38-Carboxysteroids have never been observed in living organisms. They were found in the extractable lipids of immature phosphatic sediments from Timahdit (Morroco)21and in a sediment from the Monterey formation (A. De Lemos Scofield, unpublished results). 3PCarboxysteroids (including the C30 and C3l 4-methyl derivatives; N and P in Table 1) were also obtained by oxidation of petroleum asphaltenes with ruthenium tetroxide. On the basis of the selectivity of the ruthenium tetroxide oxidation, it was suggested that the carboxylic function was generated by oxidation of an aromatic entity attached to a sterane skeleton at position 3, although the release of carboxysteranes by hydrolytic cleavage from a polycondensed substrate was not ex~1uded.l~Recently, two Bj3-carboxy-24-ethyl-5a(H)-steranes(24R and 24s) were isolated from an immature carbonate sediment; in the latter case, an origin by cleavagefrom a macromolecular framework was considered unlikely because carboxysteranes were not found when the polar fraction of the extract or the insoluble residue (kerogen) were subjected to ruthenium tetroxide 0xidation.1~ (21) Meunier-Christmann, C. These de Doctorat, Universit4 Louis Pasteur, Strasbourg, 1988.
It is now generally accepted that geochemical fossils may be trapped in the kerogen network, or alternatively bound to kerogen by chemical bonds and later released from kerogen with increase in depth and temperature.22 Specific chemical degradation is particularly useful in structural analysis of kerogen because it provides details on the nature of linkages by which the released molecules are attached to the polycondensed matrix (see ref 23 for an overview). The fact that 38-carboxysteroids are released from the kerogen matrix by mild alkaline hydrolysis indicates that they are chemically bonded to the complex network of kerogen through an ester linkage. This provides circumstantial evidence to suggest that the anomalous steroidal acids reported here may have originated from precursor organisms which have contributed part of their macromolecular biomass to the kerogen. In this way, the S@-carboxysteraneswere preserved from chemical or microbial degradation during diagenesis. This is corroborated by the occurrence of the tentatively (comidentified 3~-carboxy-23,24-dimethylcholest-22-enes ponents L and L' in Table 1) which indicates that even carbon-carbon double-bond functionalized members of the carboxysterane series survived because they were attached to the kerogen matrix. 38-Carboxysteranes may represent still unrecognized constituents of marine and fresh-water algae. Chemical reactions during diagenesis or increasing thermal stress may result in the release of these molecules from the macromolecular fractions of the organic matter in sediments. They may then be further reduced to the corresponding 3P-methylsteranes which have been found in sediments and petroleum.1° This would be analogous to hopanoid triterpenes, which are abundant components in sediments and petroleum and have also been identified (as carboxylic acids) in kerogen oxidation and kerogen Their ubiquitous occurrence subsequently led to the recognition of a C35bacteriohopanetetrol precursor that could be isolated from various microorganismsz6and was more recently identified in kerogen degradation products.27 The occurrence of 4-methylcarboxysteroids in the Nordlinger Ries sediments indicates that dinoflagellates may be among the source organisms for the organic matter of these sediments. This is in contrast to the absence of 4-methylsteranes or -sterenes in the bitumen fractions of the same sediments and of morphologically recognizable dinoflagellates in the microscopically observed maceral assemblages.lG Similar discrepancies have been observed before, however. For example, in the Messel oil shale (Eocene, Germany) 4-methylsterols are among the most abundant extractable organic substituents,28 whereas maceral analysis shows small algal bodies not related to dinoflagellatesz9but identified as green algae by electron microscopy.30 Apparently, the cell wall components of dinoflagellates are less resistant to early diagenesis than those of other planktonic organisms. Also, in the case of ~
~
~~~
(22) Tissot, B. P.;Welte, D. H. Petroleum Formation and Occurrence, 2nd ed.; Springer: Heidelberg, 1984; 699 pp. (23) Rullkbtter, J.; Michaelis, W. Org. Geochem. 1990, 16, 829-52. (24) Barakat, A. 0.; Yen, T. F. Org. Geochem. 1990,15, 299-311. (25) Barakat, A. 0.Energy Fuels, in press. (26) Ourisson, G.; Albrecht, P.; Rohmer, M. Pure Appl. Chem. 1979, 51, 709-729. (27) Mycke, B.; Narjes, F.; Michaelis, W. Nature 1987,326, 179-181. (28) Robinson, N.; Eglinton, G.; Cranwell, P. A.; Zeng, Y. B. Chem. Geol. 1989, 76, 153-173. (29) Rullkbtter, J.; Littke, R.; Hagedorn-Gbtz,I; Jankowski, B. Cour. Foursch.-Inst. Senckenberg 1988,107, 37-51. (30) Goth, K.; de Leeuw, J. W.; Pfittmann, W.;Tegelaar, E. W. Nature 1988,336, 759-761.
Energy &Fuels, Vol. 8, No. 2, 1994 485
3g-Carboxysteroids in Geological Samples ppm of TOC
NR-10 (151.6 m) Kerogen-bound acids
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Scan n u m b e r Scan number Figure 6. Abundance (in ppm of TOC) of 3@-carboxysteroidsin the hydrolysates of kerogens and extractable polar fractions of Ndrdlinger Ries sediments and in a kerogen sample from Monterey Formation.
the Nordlinger Ries sediments, 4-methylcarboxysteroidpossibly being part of biomacromolecules in the precursor organisms-were selectively preserved in the kerogen, while free 4-methylsterols eventually present in the same
organisms did not survive sedimentation and early diagenesis. This is somewhat similar to the selective preservation of biogenic organic compounds in fossil sulfurbridged organic macromolecule^.^^
486 Energy &Fuels, Vol. 8, No. 2,1994
Conclusions Steroid carboxylic acids were found in the hydrolysates of kerogens and polar bitumen fractions of sediments from the Nerdlinger Ries (southern Germany)and the Monterey Formation (California). They occur as series of 38carboxy-, 3@-carboxy-4-methyl-,and 3/3-carbomethoxysteranes in varying relative abundance8 in the samples studied. The observation of these compounds provides evidence of a more widespread occurrence of 38-carboxysteroids than previously known. The mode of occurrence shows that they are preferentially bound to the macromolecular structures of kerogens and polar lipids by ester functions. The presence of 4-methylcarboxysteroidsand side-chain unsaturated components not represented by related components in the extractable bitumens is another example of selective preservation of biogenic lipids in geological samples. Release of 3@-carboxysteroidsand (31)SinningheDamst4,J,S.;Rijpetra, W.I.C.;Kock-vanDalen,A.C.; de Leeuw, J. W.;Schenck, P.A. Ceochim. Cosmochim. Acto 1989,53, 1343-1355.
Barakat and Rullkctter subsequent reduction of the carboxyl group may be one of the sources of 3-alkylsteranes detected in sediments containing more mature organic matter and in petroleums.
Acknowledgment. We are grateful to BEB Erdel und Erdgas GmbH (Hannover) for providing the Niirdlinger Ries samples and permission for publishing the results. We are also grateful to J. Curiale of Union Sciences and Technology Division in California for providing the Monterey shale sample. Liquid and gas chromatographies were supervised by Dr. M. Radke and Dr. R. G. Schaefer (KFA Jiilich, Germany), respectively. Technical assistance by U. Disko, A. Fischer, R. Harms, B. Kammer, F. J. Keller, and S. de Waal is gratefullyacknowledged.A.O.B. is grateful to the Alexander von Humboldt Foundation for a fellowship and for donation of a GUMS data processing unit to Alexandria University. This work was begun a t the Institute of Petroleum and Organic Geochemistry a t KFA Jiilich, FRG, and was completed at the Department of Chemistry, Alexandria University, Egypt.
