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Biological diagenesis: dicarboxylic acids in recent sediments
eeochimica etCosmochimics Act&1976. Vol.39, pp. 129 to 136.
Pergamon Press.
Printed inNorthern Ireland
Biological diagenesis: dicarboxylic acids in recent sediments R. B. JOHNS and 0. M. ONDER Department of Organic Chemistry, University of Melbourne, Parkville, 3062, Australia (Received12 Fdmmy
1974; acceptedin rev&d form 2 July 1974)
Abstract-A series of even carbon numbered a, o-dicarboxylic acids ranging from C,, to Cso has been identified in recent sediments from various environments and sampling localities. A lacustrine sediment did not show detectable quantities of diacids. Consideration of quantitative relationships involving the diacids leads us to propose a dual origin for these diacids: deposition by mangroves is their main source in mangal areas while in situ production by sedimentary organisms is the only important source of diacids in a non-mangal marine environment. A fresh water lagoon shows an intermediate situation between these extremes. INTRODUCTION
THE CONCEPT of geochemical diagenesis involving heat and pressure is inadequate to explain the very earliest chemical changes of organic substrates which occur in recently deposited sediments. Allowing for hydrolytic and simple oxidative types of chemical reactions which will occur in the uppermost sedimentary layers with time, it is now generally accepted that early structural transform&ions are largely brought about through the agency of the indigenous micro-organisms (FARRINQTON and Qma~, 1973; OPPENHEIMER, 1960; RHEALIet al., 1971b; JOHNSON and CALDER, 1973) and these changes are conveniently classified as biological diagenesis. The processes of biological diagenesis are incompletely understood and the complexity of interacting environmental variables is often a serious obstacle to the extrapolation of experimental results and the consequent formulation of theories of diagenesis (FARRINGTONand QUINN, 1973). One difficulty is the question of the extent of diagenesis of the originally deposited organic substances; this is expected not only to depend on the sedimentary environment but also on the nature of the substrate itself (BREGER,1966; RHEAD et al., 1971a, b). For example: esterified materials apparently undergo rapid and extensive hydrolysis, unsaturated substances diminish but n-alkanes have often been assumed to reflect the composition of n-alkanes in the source materials (~GRTINet al., 1963; MATHEWSet al., 1972). Within this rationale the search for reliable biological and environmental markers has been actively pursued (AIZENSHTAT,1973; ECGINTON et al., 1966; EGLINTONet al., 1968a; LEO and PARKER,1966; ~IAXWEIXet al., 1973). This paper discusses long chain a, o-dicarboxylic acids, which we have isolated from & number of recent sediments, in relation to the concept of marker substances of use in paleoecological studies. Long chain tc, co-diacids have been reported in some ancient sediments (DOIJGL~L~ et al., 1966; HAUG et al., 1967; SIMONEITand BURLINCUME, 1973), and in a recent lacustrine sediment (EGILINTON et al., 1968a), but there are no previous reports of diacids from recent marine and estuarine samples, nor of unsaturated diacids from a geological source. There is a paucity of reports of diacids, free or esterified in micro-organisms or algae although certain bacteria have been reported to produce 129 8
130
R. B.
JOHNS
and 0. X.
ONDER
and accumulate diacids (KESTERand FOSTER,1963; VAX DERLIRDEX and THIJSSE, 1965). However, esterified diacids are present in the surface layers of many plants (CROTEAUand FAOERSON,1972; LAMBERTON, 1961). The above-mentioned types of organisms taken as a group are the likely source of the major fraction of recent sedimentary organic substances such as are discussed in this paper. The diacid content of the Esthwaite lacustrine sediment (EGLINTON et aE.,1968a) was considered to be a mixture of deposited and diagenetically formed d&ids, but within the context of the present paper, there appears to be no valid reasons to assume that nonbiological chemical reactions are important diagenetic contributors to the diacids under discussion. i%XPLES The following recent sediments, which will be referred to by the symbols in brackets, were selected from several geographical regions: 1. The tidal mud flats on the banks of the Norman River estuary at Karumba, North Queensland. The west bank where deposition is occurring contrasts with the east bank where evidence of erosion suggests that this sediment is older than that from the west bank. (Kl): West bank core; depth, 21-43 cm. No consistent or gross changes with depth have been found in this core. (KZ): Surface sample collected from the same area as (Kl) a year later. (KME): East bank core; depth, O-145 cm. 2. The tidal mud flats of Pt. Franklin Harbour, SE. Victoria. (PFS): Surface sample (O-145 em.) about 5.76 m horizontally displaced from the low water mark. (PF2T); Core (14+-29 cm) collected about 5-76 m horizontally displaced from (PFS) and from low water mark. Regions (1) and (2) were within the tidal zone and were well colonised by mangroves including Awicennia matins. No other vegetation was visible. The time-textured silt from site 2 ranged from brown at the surface through grey to black in the depth range of U-9.6 cm. Sulphides were abundant. Currents which move the silt along pass through extensive mangrove stands before reaching the sampling sites. 3. Tidal flats at Corner Inlet, S.E. Victoria. This is a mangrove-free area in the inter-tidal zone with eel grass (Zoatera spp) growing in patches as the only vegetation. Collections were at low tide. (CB): A layer of soft brown material, probably mainly planktonia remains and at most a few millimetres thick. (CBI): A black layer 4.5-5-5 cm thick directly underlying (CB), and rich in sulphides. (COS): Collected at a depth 6-14-5 em about 86 m seaward from the above sites. Eel grass was growing throughout and shallow water oovered the site. No brown surface layer was noted, the mud being consistently grey-black to a depth of 21.5-28.5 cm and much less compacted than (Cbl). 4. (ML): Leaves and twigs from the mangrove species dvicennia mu&a at Corner Inlet (ML). A sample from Pt. Franklin gave similar analytical results. 5. Lake Bullenmerri. A fresh water lake situated in an extinct volcanic crater in Western Victoria. (LBm): A black nakron mud sample taken at a water depth of about 14.5 cm from the lake’s northern shore. 6. Woodside Lagoon. A shallow fresh water body, which drains surrounding farm land and is located just behind the dunes of the Ninety-Mile Beach at Woodside, E. Victoria. A variety of grasses grow around the perimeter, and some duckweed and other unidentified water p1ant.s grow in the water. The sediments have a dark brown colour, similar to some agricultural S&k3 and are fairly well compacted. The sedimentation rate is very slow.
Biological diagenesie: dicarboxylic acids in recent sediments
131
(WL): O-19 cm section of core taken from the centre of the lagoon at a water depth of 43 cm. (WLC): 29-43 cm section of a core taken from a site 86 cm from (WL). 7. (ISJ): A surface sediment sample from a saline (ISJ) on the Isle of San Jose, Baja California. The saline is replenished with sea water every few weeks. METHODS
Samples were stored at 4%. After freeze-drying, the samples were crushed using a glass mortar and pestle and sieved through 0.048 cm wire mesh to remove any sizeable plant debris. Reagents used were of AR grades, or further purified by distillation. The prepared sample (200 g) was twice refluxed with potassium hydroxide (60 g) in methanol (500 ml) for 6 hr, and &er clarification of the combined supernatants by centrifugation, water (400 ml) was added and the liquid extracted five times with n-heptane portions (200 ml) to remove non-acidic components. After acidification to pH 2 the aqueous phase was again extracted five times with 200 ml portions of heptane to obtain the acid fraction. Following rotary evaporation to dryness, the acid fraction was ester&d using boron trifluoride in methanol (&fErCAI.FE and SCHMITZ, 1961). A solution of the methyl esters in methanol or diethyl ether, was chromatographed on activated silica gel GF, thin layer plates (20 x 20 x 0.1 cm or 20 x 6 x 0.025 cm) using two different solvent systems: (a) Elution with diethyl ether-heptane (5:95, v/v). The monoesters (RF = 04-0.6) were sepsxated, the rest of the plate wss extracted with methanol and the solvent reduced to a small volume for rechromatography. (b) Elution with diethyl ether-heptane (2 : 1. v/v). This gave diesters (RF = 0*7-O-8), a- and /?-monohydroxyesters (R, = 04-0.6) and w-hydroxyesters (RF = O-3&-0.4). The fraction R, < O-3 contained methyl 10, 16dihydroxyhexadecanoate and other compounds. Aliquots from each TLC fraction were run on a PE-900 gas chromatograph using a 14.4 m SCOT column, (o.d. 0.048 cm; SE 30 phase) and temperature programming from 90’ to 245% at 2’/min. Peak areas for individual components were quantitatively determined by measuring heights and widths at half height. Components in a fraction were identified by a combination of coinjection and CC-MS using a PE 270 B gas chromatograph-mass spectrometer at 70 eV. Hydrogenation in methanol over Adam’s catalyst at room temperature and pressure was used to check the identity of presumed unsaturated compounds. Hydroxylated compounds were analysed after trimethylsilylation with bis(trimethylsily1) acetamide, or after oxidation of the saturated compounds with acid dichromate (SCHMLD and B~I, 1971). Compounds were identified by comparison of their maza spectra with those of authentic standardsor withpublished spectra(EGLINTON et al., 1968b; RYEU~E and STENHAGEN, 1960). The identity of hydroxyesters was confirmed by mass spectra of their oxidation products referred to above. In addition, aliquota of 10, 16-dihydroxyhexadecanoic acid were tosylated, or selectively halogenated at either hydroxyl group using standard methods (Vogel 1956), followed by lithium aluminium hydride reduction, before mass spectrometric investigation in order to confirm its structure. RESULTS
AND DISCUSSION
Only one sediment (LBm) was found not to contain ao-diacids, this exception being from a lacustrine environment of low salinity. Nowhere were diacids of chain length outside the range C,&& positively identif?ed, nor were any odd numbered diacids observed. However, one unsaturated ao-diacid C,,:, was identified and in a concentration comparable to that for the saturated C,,-diacid. The occurrence of the G,:, aw-diacid bears strongly on the probable source of the diacids (see Section A below). Table 1 shows a wide range in total diacid content of the various samples. Figure 1A illustrates a degree of parallelism between di- and mono-acid distributions often observed, and Fig. 1B illustrates deviations in this parallelism. Suggested origins for the sedimentary diacids are discussed in the sections below.
132
R.
B.
JOHNSand 0. M. ONDER
Table 1. Diacid and monoacid content and ratios of samples. Relative abundance of dicarboxylic acids Content(ppm)8
Sample*
XL Kl K?ti ii KME
PFS PF2T \VLti
WLdl
CB Cbi COS LBm ISJ
Dioarboxylic acid
Co diacid:C,monoecid
Diaoide
Monoacida
16:O
18: 1
18:O
1400 40
11,000 300
40 20 40 40
300 200 300 300
1 1 1 1
1.9 0.9 0.8 0.9
0.2 0.3 0.2 0.3
: 0.07 0.03 0.08
$ 0.1 0.04 0.1
1
60 50 <1 l-5 1-5 $
300 300 800 300 300 300
1 1 1 1 1 1
0.6 0.7 0.7 0.8 0.8 0.5 11 0.3 0.4
0.4 0.6 0.6 0.5 0.6 0.4 0.6 0.3 0.6
0.3 II 0.3 0.4 0.5 0.5 0.3 0.2 0.6
2’3 . 0.4 0.4 0.4 0.5 0.3 0.2 1.4
1
1.7
1.3
0.7
II
1
20:o
22:o
24:0
16:O
:
0.2 0.3 0.4 0.3
0.1 0.2 0.2 0.1 0.3
II
ratios
18:O
20:o
22:o
24:0
0.2 1.2 1.2 0.8
0.3 0.2 0.4 0.3
$ 0.5 0.2 Or
: 0.5 0.2 0.1
: 0.2 0.1 0.1
0.2 0.4 0.4 0.6 0.6 0.5
0.2 0.6 1.2 0.9 0.9 2.5
0.4 0.4 0.6 1.5 1.2 0.8
0.6 0.5 2.3 I.4 1.0
II 0.9 0.7 6.0 4.0 4.6
II 1.5 0.4 >6 >6 6.4
II 0.07 1.1
0.06 0.02 0.07
0.05 /I 0.08
0.1 0.1 0.2
0.2 0.3 0.5
1.1 0.3 2.6
Ii >3
II
0.2
0.6
0.6
0.9
0.03 0.02 0.02
18:l
* Sample identifioetioa is given in the text. t Sample WM divided into two portions end extracted separately. Aliquot (i) in both oaeee wee not freeze-dried. t Below the level of deteotion. $ Diaoide are approximated to the nearest ten ppm, and the mcnc aoide to the nearest hundred ppm. All aamplee were dr:ed except for (ML). jj No reliable data available.
A. 17laragaZsedinzenh (from Pt. Frc~nkh and Karumbe, Kl, K2, KME, PFS, PFZT) The stands of mangroves near the sampling sites makes it reasonable that they should be contributors to the sedimentary organic chemicals. This expectation is realized by the detection of signScant amounts of 10,16-dihydroxyhexadecanoic acid in the sediment (JOHNSand ONDER, unpublished results). This hydroxyacid, as well as being the major acidic constituent of the mangrove sample investigated, is abundant in higher plants and can be used as a marker to show plant contribution to a sediment (BAKER and HOLLOWAY, 1970; EGLINTON et al., 1968a). The lack of other likely plant contributors nearby makes it reasonable to suppose that the substantial diacid content of the mangal sediments is at least in part contributed by mangroves, which also have a substantial diacid content. Table 1 shows that the total diacid content relative to the total monoacid content of all the mangal samples, lies within a relatively narrow range, and is comparable to the value for mangrove leaves. Inspection of Table 1, however, will show that the molecular distribution of diacids shows a number of differences between mangrove leaves (ML) and the mangal sediments. A higher proportion of diacids containing more than 18 carbon atoms are present in sediments than in the mangrove samples, hence it is necessary to propose at least one other contributory source for the sedimentary diacids. Post-depositional diacid formation has previously been suggested in a lacustrine environment (EGLINTON et al., 1968a), and it seems likely that in our marine sediments also, such an effect is operative. A number of possible pathways of microbial diacid production have been described (KESTER and FOSTER, 1963 ; VAN DEB LINDEN and THIJSSE, 1965). Another difference between mangrove leaves and sediments is the lesser concentration of sedimentary octadecenedioic acid, relative to the saturated diacids, an observation analogous to the reduction of sedimentary unsaturated monoacid content with increasing age of the sediment. Concerning the removal of
Biological diagenesis: dicarboxylic acids in recent sediments
133
0
Corbon no.
Fig. 1. Ratios of d&ids to monoacids in representative samples. (A) Abundance of monoacids (A) and d&ids (D) in a sample (WL) from Woodside Lagoon. The concentrations are adjusted relative to C,,,, monoacid which is taken aa equnl to unity. (B) Ratios of d&id to monoacid concentrationsin represent&ive wplee. The ratios are adjusted relative to the C,, acids which is taken as unity. ML = Mangrove leaves; WL = Woodside Lagoon; COS = Corner Inlet (S.E. Vio.); PFZT = Port Franklin (S.E. Vie.) harbour mud flat core (14-29 cm deep); Norman River estuary at Karumba, Nth. Qld.-K2 = surface sample from the west bank of recently deposited sediment; KME = surface sample of older sediment from the east bank. Detailed descriptions of the sites are given in the text.
sedimentary diacids, studies have shown that under some conditions diacids may be relatively poor microbial substrates (HESTER and FOSTER,1963; VANDERLINDEN and THIJSSE, 1965; TRUST and MILLIS, 1970), and if this is the situation here, the consequently greater rate of removal of C,,:, monoacid relative to the CI.+I diacid, could result in the observed maxima of the plotted ratios at 18: 1 in Fig. 1B (cf. Table 2), at least in mangal sediments. Sample KME is exceptional among the mange1 sediments, because of its lower acid content (Table 1) and lack of a maximum in the di:mono acid ratio at 18: 1 (Fig. lB), which probably reflects its greater age over the west bank samples.
134
R. B. JOIMSand 0. M. ONDER Table 2. Ratios of some monocarboxylic
CB Cbl cos
0.5 1.3 1.7
acids
0.2 0.3 0.3
C lsbr includes both iso- and anteiaocarboxylic acids.
B. Woodside Lagoon samples (WL, WLC) The substantial diacid content of these samples may suggest at first, some plant contribution, but it Ls notable that no 10, 16dihydroxyhexadecanoic acid was detected (JOHNSand ONDER,unpublished results). Table 1 shows a relatively large proportion of high carbon number diacids, and with Fig. 1B show that the diacid: monoacid relationship is in contrast to that of the mangal samples. ,Assuming that the higher carbon number diacids result from in situ production, then it would seem from the above considerations that the d&genetic production of diacids makes a much greater contribution here. This is reasonable in view of the more compacted nature of, and slower sedimentation rete in Woodside Lagoon. However, the environment is also favourable to the input of soil bacteria which are known to form diacids (KUSUNOSEet al., 1964). C. Corner inlet saqdes
(COS, CB, Cbl)
These samples have the lowest diacid content of all except the lacustrine sediment examined. The other quantitative relationships (Tables 1, Fig. 1B) concerning these samples also set them apart from the mangal areas and of particular note is the high abundance of C,, and C,, diacids in (COS). No likely plant contributors to the sedimentary diacids can be suggested, and eel grass, whilst plentiful, does not contain detectable quantities of diacids, hence it is a reasonable conclusion that the major proportion if not all of the sedimentary diacids in this tidal flat environment must arise from in situ production by micro-organisms. The surf&e layer sample (CB) has a very low diacid concentration, and the proportion of branched C,, monoacids is also quite low (Table 2). The presence of the latter has been used as an indicator of bacterial lipids (LEO and PBRRER, 1966). It may be inferred from the low branched Cl5 monoacid content, that microbial processes e.g. diacid production, have not been extensive here, contrasting with the other less recently deposited samples (Cbl) and (COS) w 1lere the d&id content is higher in keeping with an in situ production which parallels the increased content of the branched C,, monoacids. These data point to a biological diagenetic origin for the diacids in these samples. Whilst there is a quantitative relationship between the mono- and di-acids as revealed in Fig. 1B and Table 1 this does not necessarily, however, require a biosynthetic relationship. Sample (ISJ) shows similarities (Table 1) to the Corner Inlet samples in the lack of humic-like material in the sediment, and especially where microbial activity is implicated. Although input of organic material can be presumed to be much higher than for the Corner Inlet environment, the microbial activity on the other hand
Biological diagenesis: dicarboxylic acids in recent sediments
136
e.g. of halophiles, can also be presumed to be greater for this sub-tropical environment. These views find support in the presence of phytanic and pristanic acids in the extracts, and the relatively higher proportions of Cl8 and C,, diacids identified, these observations being consistent with microbial oxidation processes. The molecular distribution of diacids from mangal sediments shows similarities to the diacid distribution in mangrove leaves. Indeed, mangal sediments provide a good example, in their diacid content, of a reflection in the orgsnic extractables of contributing source materials. In other sediments the distributions differ with a lower proportion of C,,:, diacid and a larger proportion of higher carbon number diacids, which latter distribution seems consistent with a biological dirtgenetic origin, involving monoacid precursors. Deviations from parsllelism of di- and monoacid distributions (Fig. 1B) appears consistent with an inter-relationship in the origin(s) of sedimentary diacids. The data and conclusions reported here provide a further perspective for assessing the strength of the concept of biological markers in ancient sediments, which is dependent upon an understanding of pathways of biological diagenesis. This information can only be inferred from the presently limited data on contemporary environments. This paper, whilst pointing to the caution necessary in the interpretation of data from ancient sediments, does show that sediments can reflect organic input, and suggests that diacids may prove of value as indicators of microbial action at the time of deposition. Both conclusions could have value in paleoecologicrd studies. Acknowledgementa-The authors thank the Australian Research Grants Committee and the NuiBeld Foundation for financial support. R. B. Johns acknowledges the support of U.S. Public Health Service grant ES-00603, whilst on Leave at MBRD, Scripps Institution of Oceanography.
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Z. (1953) Perylene and its geochemical significance. Geochim. Coamockim. Acta
87, 559-567.
BAXER E. A. and HOLLOWAYP. J. (1970) The constituent acids of angiosperm cutins. Phytochemistry 9, 1557-1562. BREGERI. A. (1966) Geochemistry of lipids. Trans. Amer. Oil Chem. Sot. 48, 197-202. CROTEAUR. and FAGERSO~I. S. (1972) The constituent cutin acids of cranbury cuticle. Phytochemistry 11,353-363. DOUGLASA. G., DOCRAGHI-Z~EH K., EGLINTONG., MAXWELLJ. R. and RAMSAYJ.N. (1966) Fatty acids in sediments including the Green River Shale (Eocene) and Scottish torbanite (Carboniferous). Advunces in O,rganicGeochemistry, pp. 315-334. Pergsmon Press. EGLINTOXG., H-E>D. H., and DOU~AG~I-ZA~EHK. (1968a) Gas chromatography-mass spectrometric studies of long chain hydroxyacids. Tetrahedron 24, 5929-5941. EGLIXTON G., HTXSE~ D. H. and MCCORMICKA. (1968b) Gas chromatography-mass spectrometric studies of long-chain hydroxy acids. Org. Mass Sptrom. 1,693-611. E~LINTONG., SCOTTP. M., BELSKPT., BURLING~ A. L., RICHTFXW., and CUMIN M. (1966) Occurrence of isoprenoid alkanes in a Precambrian sediment. AcZvancea in Organic Geochemidry, pp. 41-54. Pergamon Press. F~LRRINGTOX J. W. and &KXXXJ.G. (1973) Biogeochemistry of fatty acids in Recent sediments from Narraganset.tBay, Rhode Island. Geochim. Cosmochim. Acta 8’7, 269-268. HAVG P., SCHNOESH. K. and BCRLINGAXEA. L. (1967) Isoprenoid and dicarboxylic acids isolated from Colorado Green River shale (Eocene). Science 158,772-773.
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JOHNSONR. W. and Carson J. A. (1973) Early d&genesis of fatty acid and hydrocarbons in a salt marsh environment. Geochim. Coemochim. Acta 37, 1943-1955. Joxxs R. B. and ONDEE 0. M. Unpublished results. KESTERA. S. and FOSTERJ. W. (1963) Diterminal oxidation of long-chain olkanes by bacteria. J. Bacterial. 86,869-889. KUSUNOSEE., KUSTJNOSE M. and COON M. J. (1964) Enzymatic u-oxidation of fatty acids. J. Biol. Chem. $89, 1374-1380. LAMB~L~TON J. A. (1961) The diabasic acids of Japan wax. Au&. J. Chem. 14,323-324. LEO R. F. and PARKERP. L. (1966) Branched-chain fatty acids in sediments. Science 152, 649-650. VAN DERLINDENA. C. and THIJSSEG. J. E. (1966) The mechanisms of microbial oxidations of petroleum hydrocarbons. Adv. Enzymol. fl,469-546. -TIN R. L., WINTERSJ. C. and WILLIAMSJ. A. (1963) Distributions of n-pars&ns in crude oils and their implications to origin of petroleum. Nature 198, 110-113. MATHEWSR. T., I~UAL X. P., JACXSONK. S. and JOHNSR. B. (1972) Hydrocarbons and fatty acids in the Evergreen Shale, Surat Basin, Queensland, Australia. Geochim. Coemochim. Acta 38, 886-896. MUWELL J. R., Cox R. E., EGLINTONG., PILLINGERC. T., ACEMANR. G. and HOOPERS. N. (1973) Stereochemical studies of acyclic isoprenoid compounds. Geochim. Cosmochim. Acta 87, 297-313. METCALFEL. D. and SCHMITZA. A. (1961) The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33, 363-364. OPPENHEIMER C. H. (1960) Bacterial activity in sediments of shallow marine bays. Geochim. Coemoch~m. Acta 19,244-260. RHEAD M. M. EGLINTONG., DRAFIFAN G. H. and ENGLANDP. J. (1971a) Conversion of oleic acid to saturated fatty acids in Severn Estuary sediments. Nature 232, 327-330. REED M. M., EGLINTONG. and ENGLANDP. J. (1971b) Products of the short-term diagenesis of oleio acid in an estucsrinesediment. Advaracee in Organic Geochemistry, pp. 323-333. Pergamon Press. RYEAGE R. and STENHAQENE. (1960) Mass spectrometry in lipid research. J. Lipid Res. 1, 361-390. SCHYID H. H. 0. end BANDI P. C. (1971) Naturally occurring long-chain /3-hydroxyketones. J. Lipid Res. 12, 198-202. SIMONEITB. R. and BURLINGA~EA. L. (1973) Carboxylic acids derived from Tasmanian tasmanite by extractions and kerogen oxidation& Geochim. Cosmochim.Acta 37, 595-610. TRUST T. J. and MILLISN. F. (1970) The isolation and characterization of alkane-oxidising organisms and the effect of growth substrate on isocitric lyase. J. Gen. _Uic~obioE.61,245-254. VOGELA. I. (1966) Practical Organic Chemistry, 3rd Edition, pp. 320-321. Longmans, Green & co.
Pergamon Press.
Printed inNorthern Ireland
Biological diagenesis: dicarboxylic acids in recent sediments R. B. JOHNS and 0. M. ONDER Department of Organic Chemistry, University of Melbourne, Parkville, 3062, Australia (Received12 Fdmmy
1974; acceptedin rev&d form 2 July 1974)
Abstract-A series of even carbon numbered a, o-dicarboxylic acids ranging from C,, to Cso has been identified in recent sediments from various environments and sampling localities. A lacustrine sediment did not show detectable quantities of diacids. Consideration of quantitative relationships involving the diacids leads us to propose a dual origin for these diacids: deposition by mangroves is their main source in mangal areas while in situ production by sedimentary organisms is the only important source of diacids in a non-mangal marine environment. A fresh water lagoon shows an intermediate situation between these extremes. INTRODUCTION
THE CONCEPT of geochemical diagenesis involving heat and pressure is inadequate to explain the very earliest chemical changes of organic substrates which occur in recently deposited sediments. Allowing for hydrolytic and simple oxidative types of chemical reactions which will occur in the uppermost sedimentary layers with time, it is now generally accepted that early structural transform&ions are largely brought about through the agency of the indigenous micro-organisms (FARRINQTON and Qma~, 1973; OPPENHEIMER, 1960; RHEALIet al., 1971b; JOHNSON and CALDER, 1973) and these changes are conveniently classified as biological diagenesis. The processes of biological diagenesis are incompletely understood and the complexity of interacting environmental variables is often a serious obstacle to the extrapolation of experimental results and the consequent formulation of theories of diagenesis (FARRINGTONand QUINN, 1973). One difficulty is the question of the extent of diagenesis of the originally deposited organic substances; this is expected not only to depend on the sedimentary environment but also on the nature of the substrate itself (BREGER,1966; RHEAD et al., 1971a, b). For example: esterified materials apparently undergo rapid and extensive hydrolysis, unsaturated substances diminish but n-alkanes have often been assumed to reflect the composition of n-alkanes in the source materials (~GRTINet al., 1963; MATHEWSet al., 1972). Within this rationale the search for reliable biological and environmental markers has been actively pursued (AIZENSHTAT,1973; ECGINTON et al., 1966; EGLINTONet al., 1968a; LEO and PARKER,1966; ~IAXWEIXet al., 1973). This paper discusses long chain a, o-dicarboxylic acids, which we have isolated from & number of recent sediments, in relation to the concept of marker substances of use in paleoecological studies. Long chain tc, co-diacids have been reported in some ancient sediments (DOIJGL~L~ et al., 1966; HAUG et al., 1967; SIMONEITand BURLINCUME, 1973), and in a recent lacustrine sediment (EGILINTON et al., 1968a), but there are no previous reports of diacids from recent marine and estuarine samples, nor of unsaturated diacids from a geological source. There is a paucity of reports of diacids, free or esterified in micro-organisms or algae although certain bacteria have been reported to produce 129 8
130
R. B.
JOHNS
and 0. X.
ONDER
and accumulate diacids (KESTERand FOSTER,1963; VAX DERLIRDEX and THIJSSE, 1965). However, esterified diacids are present in the surface layers of many plants (CROTEAUand FAOERSON,1972; LAMBERTON, 1961). The above-mentioned types of organisms taken as a group are the likely source of the major fraction of recent sedimentary organic substances such as are discussed in this paper. The diacid content of the Esthwaite lacustrine sediment (EGLINTON et aE.,1968a) was considered to be a mixture of deposited and diagenetically formed d&ids, but within the context of the present paper, there appears to be no valid reasons to assume that nonbiological chemical reactions are important diagenetic contributors to the diacids under discussion. i%XPLES The following recent sediments, which will be referred to by the symbols in brackets, were selected from several geographical regions: 1. The tidal mud flats on the banks of the Norman River estuary at Karumba, North Queensland. The west bank where deposition is occurring contrasts with the east bank where evidence of erosion suggests that this sediment is older than that from the west bank. (Kl): West bank core; depth, 21-43 cm. No consistent or gross changes with depth have been found in this core. (KZ): Surface sample collected from the same area as (Kl) a year later. (KME): East bank core; depth, O-145 cm. 2. The tidal mud flats of Pt. Franklin Harbour, SE. Victoria. (PFS): Surface sample (O-145 em.) about 5.76 m horizontally displaced from the low water mark. (PF2T); Core (14+-29 cm) collected about 5-76 m horizontally displaced from (PFS) and from low water mark. Regions (1) and (2) were within the tidal zone and were well colonised by mangroves including Awicennia matins. No other vegetation was visible. The time-textured silt from site 2 ranged from brown at the surface through grey to black in the depth range of U-9.6 cm. Sulphides were abundant. Currents which move the silt along pass through extensive mangrove stands before reaching the sampling sites. 3. Tidal flats at Corner Inlet, S.E. Victoria. This is a mangrove-free area in the inter-tidal zone with eel grass (Zoatera spp) growing in patches as the only vegetation. Collections were at low tide. (CB): A layer of soft brown material, probably mainly planktonia remains and at most a few millimetres thick. (CBI): A black layer 4.5-5-5 cm thick directly underlying (CB), and rich in sulphides. (COS): Collected at a depth 6-14-5 em about 86 m seaward from the above sites. Eel grass was growing throughout and shallow water oovered the site. No brown surface layer was noted, the mud being consistently grey-black to a depth of 21.5-28.5 cm and much less compacted than (Cbl). 4. (ML): Leaves and twigs from the mangrove species dvicennia mu&a at Corner Inlet (ML). A sample from Pt. Franklin gave similar analytical results. 5. Lake Bullenmerri. A fresh water lake situated in an extinct volcanic crater in Western Victoria. (LBm): A black nakron mud sample taken at a water depth of about 14.5 cm from the lake’s northern shore. 6. Woodside Lagoon. A shallow fresh water body, which drains surrounding farm land and is located just behind the dunes of the Ninety-Mile Beach at Woodside, E. Victoria. A variety of grasses grow around the perimeter, and some duckweed and other unidentified water p1ant.s grow in the water. The sediments have a dark brown colour, similar to some agricultural S&k3 and are fairly well compacted. The sedimentation rate is very slow.
Biological diagenesie: dicarboxylic acids in recent sediments
131
(WL): O-19 cm section of core taken from the centre of the lagoon at a water depth of 43 cm. (WLC): 29-43 cm section of a core taken from a site 86 cm from (WL). 7. (ISJ): A surface sediment sample from a saline (ISJ) on the Isle of San Jose, Baja California. The saline is replenished with sea water every few weeks. METHODS
Samples were stored at 4%. After freeze-drying, the samples were crushed using a glass mortar and pestle and sieved through 0.048 cm wire mesh to remove any sizeable plant debris. Reagents used were of AR grades, or further purified by distillation. The prepared sample (200 g) was twice refluxed with potassium hydroxide (60 g) in methanol (500 ml) for 6 hr, and &er clarification of the combined supernatants by centrifugation, water (400 ml) was added and the liquid extracted five times with n-heptane portions (200 ml) to remove non-acidic components. After acidification to pH 2 the aqueous phase was again extracted five times with 200 ml portions of heptane to obtain the acid fraction. Following rotary evaporation to dryness, the acid fraction was ester&d using boron trifluoride in methanol (&fErCAI.FE and SCHMITZ, 1961). A solution of the methyl esters in methanol or diethyl ether, was chromatographed on activated silica gel GF, thin layer plates (20 x 20 x 0.1 cm or 20 x 6 x 0.025 cm) using two different solvent systems: (a) Elution with diethyl ether-heptane (5:95, v/v). The monoesters (RF = 04-0.6) were sepsxated, the rest of the plate wss extracted with methanol and the solvent reduced to a small volume for rechromatography. (b) Elution with diethyl ether-heptane (2 : 1. v/v). This gave diesters (RF = 0*7-O-8), a- and /?-monohydroxyesters (R, = 04-0.6) and w-hydroxyesters (RF = O-3&-0.4). The fraction R, < O-3 contained methyl 10, 16dihydroxyhexadecanoate and other compounds. Aliquots from each TLC fraction were run on a PE-900 gas chromatograph using a 14.4 m SCOT column, (o.d. 0.048 cm; SE 30 phase) and temperature programming from 90’ to 245% at 2’/min. Peak areas for individual components were quantitatively determined by measuring heights and widths at half height. Components in a fraction were identified by a combination of coinjection and CC-MS using a PE 270 B gas chromatograph-mass spectrometer at 70 eV. Hydrogenation in methanol over Adam’s catalyst at room temperature and pressure was used to check the identity of presumed unsaturated compounds. Hydroxylated compounds were analysed after trimethylsilylation with bis(trimethylsily1) acetamide, or after oxidation of the saturated compounds with acid dichromate (SCHMLD and B~I, 1971). Compounds were identified by comparison of their maza spectra with those of authentic standardsor withpublished spectra(EGLINTON et al., 1968b; RYEU~E and STENHAGEN, 1960). The identity of hydroxyesters was confirmed by mass spectra of their oxidation products referred to above. In addition, aliquota of 10, 16-dihydroxyhexadecanoic acid were tosylated, or selectively halogenated at either hydroxyl group using standard methods (Vogel 1956), followed by lithium aluminium hydride reduction, before mass spectrometric investigation in order to confirm its structure. RESULTS
AND DISCUSSION
Only one sediment (LBm) was found not to contain ao-diacids, this exception being from a lacustrine environment of low salinity. Nowhere were diacids of chain length outside the range C,&& positively identif?ed, nor were any odd numbered diacids observed. However, one unsaturated ao-diacid C,,:, was identified and in a concentration comparable to that for the saturated C,,-diacid. The occurrence of the G,:, aw-diacid bears strongly on the probable source of the diacids (see Section A below). Table 1 shows a wide range in total diacid content of the various samples. Figure 1A illustrates a degree of parallelism between di- and mono-acid distributions often observed, and Fig. 1B illustrates deviations in this parallelism. Suggested origins for the sedimentary diacids are discussed in the sections below.
132
R.
B.
JOHNSand 0. M. ONDER
Table 1. Diacid and monoacid content and ratios of samples. Relative abundance of dicarboxylic acids Content(ppm)8
Sample*
XL Kl K?ti ii KME
PFS PF2T \VLti
WLdl
CB Cbi COS LBm ISJ
Dioarboxylic acid
Co diacid:C,monoecid
Diaoide
Monoacida
16:O
18: 1
18:O
1400 40
11,000 300
40 20 40 40
300 200 300 300
1 1 1 1
1.9 0.9 0.8 0.9
0.2 0.3 0.2 0.3
: 0.07 0.03 0.08
$ 0.1 0.04 0.1
1
60 50 <1 l-5 1-5 $
300 300 800 300 300 300
1 1 1 1 1 1
0.6 0.7 0.7 0.8 0.8 0.5 11 0.3 0.4
0.4 0.6 0.6 0.5 0.6 0.4 0.6 0.3 0.6
0.3 II 0.3 0.4 0.5 0.5 0.3 0.2 0.6
2’3 . 0.4 0.4 0.4 0.5 0.3 0.2 1.4
1
1.7
1.3
0.7
II
1
20:o
22:o
24:0
16:O
:
0.2 0.3 0.4 0.3
0.1 0.2 0.2 0.1 0.3
II
ratios
18:O
20:o
22:o
24:0
0.2 1.2 1.2 0.8
0.3 0.2 0.4 0.3
$ 0.5 0.2 Or
: 0.5 0.2 0.1
: 0.2 0.1 0.1
0.2 0.4 0.4 0.6 0.6 0.5
0.2 0.6 1.2 0.9 0.9 2.5
0.4 0.4 0.6 1.5 1.2 0.8
0.6 0.5 2.3 I.4 1.0
II 0.9 0.7 6.0 4.0 4.6
II 1.5 0.4 >6 >6 6.4
II 0.07 1.1
0.06 0.02 0.07
0.05 /I 0.08
0.1 0.1 0.2
0.2 0.3 0.5
1.1 0.3 2.6
Ii >3
II
0.2
0.6
0.6
0.9
0.03 0.02 0.02
18:l
* Sample identifioetioa is given in the text. t Sample WM divided into two portions end extracted separately. Aliquot (i) in both oaeee wee not freeze-dried. t Below the level of deteotion. $ Diaoide are approximated to the nearest ten ppm, and the mcnc aoide to the nearest hundred ppm. All aamplee were dr:ed except for (ML). jj No reliable data available.
A. 17laragaZsedinzenh (from Pt. Frc~nkh and Karumbe, Kl, K2, KME, PFS, PFZT) The stands of mangroves near the sampling sites makes it reasonable that they should be contributors to the sedimentary organic chemicals. This expectation is realized by the detection of signScant amounts of 10,16-dihydroxyhexadecanoic acid in the sediment (JOHNSand ONDER, unpublished results). This hydroxyacid, as well as being the major acidic constituent of the mangrove sample investigated, is abundant in higher plants and can be used as a marker to show plant contribution to a sediment (BAKER and HOLLOWAY, 1970; EGLINTON et al., 1968a). The lack of other likely plant contributors nearby makes it reasonable to suppose that the substantial diacid content of the mangal sediments is at least in part contributed by mangroves, which also have a substantial diacid content. Table 1 shows that the total diacid content relative to the total monoacid content of all the mangal samples, lies within a relatively narrow range, and is comparable to the value for mangrove leaves. Inspection of Table 1, however, will show that the molecular distribution of diacids shows a number of differences between mangrove leaves (ML) and the mangal sediments. A higher proportion of diacids containing more than 18 carbon atoms are present in sediments than in the mangrove samples, hence it is necessary to propose at least one other contributory source for the sedimentary diacids. Post-depositional diacid formation has previously been suggested in a lacustrine environment (EGLINTON et al., 1968a), and it seems likely that in our marine sediments also, such an effect is operative. A number of possible pathways of microbial diacid production have been described (KESTER and FOSTER, 1963 ; VAN DEB LINDEN and THIJSSE, 1965). Another difference between mangrove leaves and sediments is the lesser concentration of sedimentary octadecenedioic acid, relative to the saturated diacids, an observation analogous to the reduction of sedimentary unsaturated monoacid content with increasing age of the sediment. Concerning the removal of
Biological diagenesis: dicarboxylic acids in recent sediments
133
0
Corbon no.
Fig. 1. Ratios of d&ids to monoacids in representative samples. (A) Abundance of monoacids (A) and d&ids (D) in a sample (WL) from Woodside Lagoon. The concentrations are adjusted relative to C,,,, monoacid which is taken aa equnl to unity. (B) Ratios of d&id to monoacid concentrationsin represent&ive wplee. The ratios are adjusted relative to the C,, acids which is taken as unity. ML = Mangrove leaves; WL = Woodside Lagoon; COS = Corner Inlet (S.E. Vio.); PFZT = Port Franklin (S.E. Vie.) harbour mud flat core (14-29 cm deep); Norman River estuary at Karumba, Nth. Qld.-K2 = surface sample from the west bank of recently deposited sediment; KME = surface sample of older sediment from the east bank. Detailed descriptions of the sites are given in the text.
sedimentary diacids, studies have shown that under some conditions diacids may be relatively poor microbial substrates (HESTER and FOSTER,1963; VANDERLINDEN and THIJSSE, 1965; TRUST and MILLIS, 1970), and if this is the situation here, the consequently greater rate of removal of C,,:, monoacid relative to the CI.+I diacid, could result in the observed maxima of the plotted ratios at 18: 1 in Fig. 1B (cf. Table 2), at least in mangal sediments. Sample KME is exceptional among the mange1 sediments, because of its lower acid content (Table 1) and lack of a maximum in the di:mono acid ratio at 18: 1 (Fig. lB), which probably reflects its greater age over the west bank samples.
134
R. B. JOIMSand 0. M. ONDER Table 2. Ratios of some monocarboxylic
CB Cbl cos
0.5 1.3 1.7
acids
0.2 0.3 0.3
C lsbr includes both iso- and anteiaocarboxylic acids.
B. Woodside Lagoon samples (WL, WLC) The substantial diacid content of these samples may suggest at first, some plant contribution, but it Ls notable that no 10, 16dihydroxyhexadecanoic acid was detected (JOHNSand ONDER,unpublished results). Table 1 shows a relatively large proportion of high carbon number diacids, and with Fig. 1B show that the diacid: monoacid relationship is in contrast to that of the mangal samples. ,Assuming that the higher carbon number diacids result from in situ production, then it would seem from the above considerations that the d&genetic production of diacids makes a much greater contribution here. This is reasonable in view of the more compacted nature of, and slower sedimentation rete in Woodside Lagoon. However, the environment is also favourable to the input of soil bacteria which are known to form diacids (KUSUNOSEet al., 1964). C. Corner inlet saqdes
(COS, CB, Cbl)
These samples have the lowest diacid content of all except the lacustrine sediment examined. The other quantitative relationships (Tables 1, Fig. 1B) concerning these samples also set them apart from the mangal areas and of particular note is the high abundance of C,, and C,, diacids in (COS). No likely plant contributors to the sedimentary diacids can be suggested, and eel grass, whilst plentiful, does not contain detectable quantities of diacids, hence it is a reasonable conclusion that the major proportion if not all of the sedimentary diacids in this tidal flat environment must arise from in situ production by micro-organisms. The surf&e layer sample (CB) has a very low diacid concentration, and the proportion of branched C,, monoacids is also quite low (Table 2). The presence of the latter has been used as an indicator of bacterial lipids (LEO and PBRRER, 1966). It may be inferred from the low branched Cl5 monoacid content, that microbial processes e.g. diacid production, have not been extensive here, contrasting with the other less recently deposited samples (Cbl) and (COS) w 1lere the d&id content is higher in keeping with an in situ production which parallels the increased content of the branched C,, monoacids. These data point to a biological diagenetic origin for the diacids in these samples. Whilst there is a quantitative relationship between the mono- and di-acids as revealed in Fig. 1B and Table 1 this does not necessarily, however, require a biosynthetic relationship. Sample (ISJ) shows similarities (Table 1) to the Corner Inlet samples in the lack of humic-like material in the sediment, and especially where microbial activity is implicated. Although input of organic material can be presumed to be much higher than for the Corner Inlet environment, the microbial activity on the other hand
Biological diagenesis: dicarboxylic acids in recent sediments
136
e.g. of halophiles, can also be presumed to be greater for this sub-tropical environment. These views find support in the presence of phytanic and pristanic acids in the extracts, and the relatively higher proportions of Cl8 and C,, diacids identified, these observations being consistent with microbial oxidation processes. The molecular distribution of diacids from mangal sediments shows similarities to the diacid distribution in mangrove leaves. Indeed, mangal sediments provide a good example, in their diacid content, of a reflection in the orgsnic extractables of contributing source materials. In other sediments the distributions differ with a lower proportion of C,,:, diacid and a larger proportion of higher carbon number diacids, which latter distribution seems consistent with a biological dirtgenetic origin, involving monoacid precursors. Deviations from parsllelism of di- and monoacid distributions (Fig. 1B) appears consistent with an inter-relationship in the origin(s) of sedimentary diacids. The data and conclusions reported here provide a further perspective for assessing the strength of the concept of biological markers in ancient sediments, which is dependent upon an understanding of pathways of biological diagenesis. This information can only be inferred from the presently limited data on contemporary environments. This paper, whilst pointing to the caution necessary in the interpretation of data from ancient sediments, does show that sediments can reflect organic input, and suggests that diacids may prove of value as indicators of microbial action at the time of deposition. Both conclusions could have value in paleoecologicrd studies. Acknowledgementa-The authors thank the Australian Research Grants Committee and the NuiBeld Foundation for financial support. R. B. Johns acknowledges the support of U.S. Public Health Service grant ES-00603, whilst on Leave at MBRD, Scripps Institution of Oceanography.
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