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Org. Geochem. Vol. 29, No. 57, pp. 13451354, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0146-6380(98)00093-X 0146-6380/98 $ - see front matter

Assessing microbial lipid contributions during laboratory degradations of fats and oils and pure triacylglycerols absorbed in ceramic potsherds
STEPHANIE N. DUDD, MARTINE REGERT and RICHARD P. EVERSHED*
Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, U.K. AbstractIn order to better interpret the origins of degraded fats and plant oils, experiments have been performed in the laboratory to simulate the decay of such commodities during burial. The progress of decay of various acyl lipids (milk and olive oil) and pure compounds (triolein and tristearin) has been monitored for up to 126 days. A general pattern emerged of a rapid decrease in triacylglycerol abundance with a concomitant increase in free fatty acids as a result of ester hydrolysis. The hydrolysis was assumed to be mediated largely by micro-organisms since this loss of triacylglycerol was accompanied by an overall consumption of lipid in all experiments. The appearance of microbial lipids occurred in the latter stages of degradation; these included short and long straight-chain, and branched-chain fatty acids ranging between C14 and C20. Ergosterol, a characteristic fungal marker, was also seen in the degradation experiment involving milk. The extent of incorporation of bacterial and fungal markers in decay experiments was minor (<2%) even in the case of highly degraded lipids (>90% consumed), thus fully legitimising the use of absorbed lipids in archaeological investigations. # 1998 Elsevier Science Ltd. All rights reserved Key wordsfatty acids, triacylglycerols, milk, olive oil, biodegradation, bacterial markers, ceramics, potsherds, archaeology

INTRODUCTION

Although lipids have been recognised as surviving absorbed within the fabric of archaeological pottery vessels, e.g. cooking and storage jars, for more than twenty years (Condamin et al., 1976; Condamin and Formenti, 1978; Rottla nder, 1990; Heron and Evershed, 1993, and references therein), the extent of the information that can be recovered from such residues is only just beginning to be fully realised. Advances are being made by exploiting very specic chemical properties that allow the origins of dierent residues to be reliably distinguished. The most commonly occurring residues absorbed in archaeological ceramics derive from animal fats and in many cases lipid residues are present in appreciable amounts (>1000 mg g1 of sherd). The structures and distributions of acyl lipids and especially the stable carbon isotope ratios (d13 C values) of the fatty acid components are proving to be useful in assigning particular animal origins (Evershed et al., 1997a,b). Even in its degraded form, the analysis of the compositional characteristics of fats enables distinctions to be made between subcutaneous fats of ruminant and non-ruminant animals. For example, sherds of Neolithic pottery (Peterborough Ware (ca 5000 BP) and Grooved Ware (ca 4500 BP)) from the Walton Basin in mid Wales, were found to
*To whom correspondence should be addressed. Fax: +44-117-9251295; E-mail: r.p.evershed@bristol.ac.uk

contain substantial amounts of well-preserved animal fat residues, comprising abundant intact triacylglycerols ranging from C44 to C54 together with di- and monoacylglycerols and free fatty acids (Evershed et al., 1997a). Assignments of the original source of the lipid components were based upon a combination of criteria, including the relative proportions of lipid components (including variations in the distributions of triacylglycerols), and the d13 C values of the C16:0 and C18:0 fatty acids. It is inevitable during burial that bacterial action will alter the original distribution of the lipid components derived from vessel use. It is well known that high molecular weight compounds forming storage or structural materials are readily transformed into assimilable components by fungal and bacterial enzymes (Killops and Killops, 1993). Since bacteria adapt and thrive readily where a suitable substrate is available (together with other essential nutrients), there is clearly a need to assess the eect of bacterial contributions to the lipid components derived from the original archaeological commodities. Since we assume that degradation is partly bacterially mediated we would expect lipid extracts of potsherds and indeed other archeological artefacts to contain cellular components of the micro-organisms, or structurally altered products, in addition to the lipid substrates initially present. The most widely recognised markers of bacterial action are the branched-chain fatty acids of the iso- and anteiso- series which occur in many bacteria as the

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major acyl constituents of membrane lipids (Lo sel, 1988; Schweizer, 1989). The contributions of bacterial lipids to archaeological pottery are dicult to assess since commonly occurring ruminant animal fats contain an appreciable abundance of bacterial lipids which derive from the function of the rumen. Such contributions are probably best assessed through laboratory experiments designed to simulate the decay of lipids in archaeological pottery. Since many of the microbiologically mediated changes probably occur quite soon after the vessels have been discarded, it is reasonable to assume that such experiments can provide a reasonable analogue to the natural situation. In this paper we present the results of experiments designed to simulate the decay of lipids in ceramic vessels under oxic conditions. The laboratory decay of milk and olive oil has been studied in view of the archaeological importance of these two commodities. The results obtained complement previous work involving the decay of ruminant adipose fat (Evershed and Charters, 1995; Charters, 1996). The laboratory decay of pure triacylglycerols, namely tristearin and triolein absorbed in potsherds rather than complex natural mixtures, provides a convenient means of assessing possible contributions from microorganisms active in the degradation of the absorbed lipid.
EXPERIMENTAL

7140). The absorption of lipid was facilitated by sonication for 2 20 min. One sherd from each treatment was kept for analysis at Day 0. The sherds were dried to constant weight at room temperature before burial in asks (Duran; 250 ml) of ``mushroom compost'' (Magnolia Brand, mushroom humix manure). The asks were plugged with extracted cotton wool in order to allow diusion of air. Sherds dosed with lipid were incubated in clean, compost-free asks as controls. The potsherds were then incubated at 308C for the fat/oil and pure triacylglycerol standards respectively and removed at intervals for analysis by our established protocol for the extraction of organic residues (Charters et al., 1993; see below). Details of the decay experiments are given in Table 1. Extraction procedure Approximately 2 g samples were taken and their surfaces cleaned using a modelling drill to remove adhering compost. The samples were then ground to a ne powder, accurately weighed and a known amount of internal standard (n-tetratriacontane) added. The lipids were extracted with a mixture of chloroform and methanol (2:1, v/v). Following separation from the ground potsherd, solvent was evaporated under a gentle stream of nitrogen to obtain the total lipid extract (TLE). Preparation of trimethylsilyl derivatives Portions of the total lipid extract were derivatised using N,O-bis(trimethylsilyl)triuoroacetamide (BSTFA; 20 ml; 708C; 30 min) and analysed by gas chromatography (GC) and GC/mass spectrometry (GC/MS). Isolation of the acid and neutral fractions An aliquot of the TLE was submitted to an acid/ neutral separation using an NH2 aminopropyl Bond Elute column (Varian). The neutral fraction was collected in a clean round bottomed ask following elution with 12 ml of DCM/isopropanol solution (2:1, v/v). The acid fraction was eluted with 12 ml

The potsherds used in these experiments come from replica pottery vessels, wheel-thrown and made from a mixture of Pot Clay (1137; Keuper Marl, Staordshire) and sand (3:1, v/v) to provide a porous fabric. Dosing of sherds and decay experiments Sherds (approx. 2 g) were prepared by dosing in solutions of milk (White Goat), olive oil (pure virgin; 25 mg ml1 solution in dichloromethane (DCM)), tristearin (10 mg ml1 in DCM; Sigma T5016) and triolein (10 mg ml1 in DCM; Sigma T-

Table 1. Summary of laboratory decay experiments carried out using animal fat/plant oils and pure triacylglycerols to investigate the degradation of lipids absorbed in archaeological pottery Substrate Olive oil Milk (White Goat) Pure tristearin Pure triolein Olive oil Milk (White Goat)
a b

Experimental conditions oxicb oxic oxic oxic oxic oxic

Internal standarda (mg) Animal fat/plant oil 80 80

Incubation temperature (8C) 30 30 30 30 30 30

Sampling intervals (days) 0, 10, 20, 67, 95 0, 5, 10, 15, 25 0, 6, 14, 32, 57, 84, 126 0, 7, 14, 28, 57, 97, 126 15 15

Pure standard compounds 50 50 Controlsc 80 30

Added per 2 g of powdered sherd. Air allowed to diuse into asks. c Sherds incubated in a clean ask without compost.

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Fig. 1. Partial high temperature-GC (HT-GC) proles of the total lipid extracts of milk fat residues (a) at Day 0 and (b) after 25 days of laboratory decay. Peak identities are: 12, 14, 16 and 18 correspond to n-alkanoic acids containing 12, 14, 16 and 18 carbon atoms, respectively; 16:1 and 18:1 correspond to monounsaturated fatty acids with 16 and 18 carbon atoms respectively; Br15 and Br17 are co-eluting iso- and anteiso-branched alkanoic acids containing 15 and 17 carbon atoms respectively; 16M and 18M refer to monoacylglycerols containing acyl moieties with 16 and 18 acyl carbon atoms, respectively; Ch and Er refer to cholesterol and ergosterol, respectively; IS refers to the internal standard, ntetratriacontane; 26, 28, 3054 etc. refer to triacylglycerols with a total of 26, 28, 3054 etc. acyl carbon atoms; * plasticiser contamination.

of 5% acetic acid solution in ether. Following rotary evaporation to remove excess solvent, the acid fraction was derivatised using BSTFA and diluted in hexane before GC and GC/MS analysis to enable a more detailed investigation of the minor free fatty acid components. Saponication of intact triacylglycerols Methanolic sodium hydroxide (2 ml; 5% w/v) was added to an aliquot of the total lipid extract and heated at 708C for 1 h in order to release the fatty acids. Following acidication to pH 3, lipids were extracted into hexane and the solvent reduced under nitrogen. The fatty acids were derivatised with BSTFA prior to analysis by GC and GC/MS. Analysis of the distribution of fatty acid components comprising the triacylglycerols enabled the composition at Day 0 to be determined. Gas chromatography (GC) The GC analyses were performed on a Hewlett Packard 5890 gas chromatograph, coupled to an Opus V PC using HP Chemstation software, which provided instrument control, data acquisition and post-run data processing facilities. Samples were introduced by on-column injection into a 15 m 0.32 mm i.d. fused silica capillary column, coated with DB1 stationary phase (immobilised dimethyl polysiloxane, 0.1 mm lm thickness; J&W

Scientic). The temperature program consisted of a 2 min isothermal hold at 508C followed by a ramp from 50 to 3508C at 108C min1. The temperature was then held at 3508C for 10 min. Hydrogen was used as carrier gas (head pressure 10 psi). Flame ionisation detection was used to monitor the column euent. Gas chromatography/mass spectrometry (GC/MS) GC/MS analyses were performed using a Finnigan 4500 quadrupole mass spectrometer directly coupled to a Carlo Erba 5160 Mega series gas chromatograph with on-column injection. Operating conditions were as follows: ion source, 1708C; emission current, 400 mA and electron energy, 70 eV. The GC/MS interface was maintained at a temperature of 3508C. Spectra were recorded over the range m/z 50850 every 1.5 s. Data was acquired and processed using an INCOS data system. The GC operating conditions were the same as those described above except that helium was used as carrier gas.

RESULTS

Milk fat In its undegraded form milk fat consists predominantly of triacylglycerols ranging from C26 to C54

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Table 2. Absolute concentrations of lipid components (mg g1 of powdered sherd) present during laboratory decay of milk under oxic conditions Free fatty acids Day 0 5 10 15 25
a

TAGsa C18:1 2.2 19.2 4.2 1.1 0.7 C18:0 0.0 8.2 2.0 0.6 0.4 314.9 19.6 5.1 5.8 5.5

Total lipid

C12:0 0.0 0.7 0.0 0.0 0.0

C14:0 0.0 4.6 0.3 0.2 0.2

C16:0 2.4 19.2 4.3 1.5 0.6

391.5 71.5 15.8 9.2 7.3

TAGS = triacylglycerols.

acyl carbon atoms containing fatty acids between C4 and C20 (Breckenridge and Kuksis, 1969; Smith et al., 1968; Evershed, 1995). After only 10 days of the laboratory decay of milk fat under oxic conditions >95% reduction in the concentration of intact triacylglycerols present absorbed in the potsherds at Day 0 had occurred. The major decay products of the hydrolysis of the triacylglycerols were free fatty acids in the range C8C20, with the C14:0, C16:0 and C18:0 components predominating (Fig. 1). Mono- and diacylglycerols were also produced in relatively minor quantities. Hydrolysis of triacylglycerols appears to proceed rapidly once the rst fatty acid has been cleaved from the glycerol backbone since at no time did monoacylglycerols and diacylglycerols accumulate to any appreciable

extent. The absolute quantitative data for this experiment is given in Table 2. As a result of dosing, 392 mg g1 of lipid had been absorbed by the potsherd (Day 0). The total amount of lipid present decreased throughout the experiment with the total triacylglycerol concentration decreasing from 315 mg g1 to only 20 mg g1 between Day 0 and Day 5 with a slower but gradual decrease thereafter. Figure 2a shows a plot of the changing relative abundances of free fatty acids and intact triacylglycerols during the milk degradation experiment. There was no signicant change in the relative proportions of the major fatty acids (C16:0 and C18:0) during the course of the decay. The proportion of intact triacylglycerols initially decreased with a corresponding increase in the abundance of

Fig. 2. Relative abundances (%) of the lipid components of (a) milk fat and (b) olive oil during laboratory decay experiments.

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Fig. 3. Partial HT-GC proles of olive oil residues (a) at Day 0, (b) after 20 days and (c) after 95 days of laboratory decay. Peak identities are the same as in Fig. 1. Peaks in the expanded region of the chromatogram are: 1, 18:1 1-monoacylglycerol; 2, 18:0 2-monoacylglycerol; 3, sitosterol; 4, internal standard (n-tetratriacontane); 5, 16:0, 18:1 diacylglycerol; 6, 16:0, 18:0 diacylglycerol; 7, 18:1, 18:1 diacylglycerol, and 8, 18:0, 18:1 diacylglycerol.

free acids. After 10 days there was a decrease in the rate of depletion of triacylglycerols relative to the free fatty acids (Fig. 2a). Signicantly during decay, alteration of the distribution of intact triacylglycerols occurred due to the early liberation of shorter-chain fatty acyl moieties. By Day 25 the lower molecular weight triacylglycerols (C26C44) which originally comprised 66% of total triacylglycerols had been reduced to only 34%. Their longer chain counterparts (C46C54), which originally comprised 34% had increased to 67% of the total triacylglycerols. After 25 days of

incubation (Fig. 1a), the shorter-chain free fatty acids characteristic of milk fats (C4C12) were largely undetectable and only a very small proportion of the original lipid remained, approximating to 7 mg g1. A signicant abundance of branched-chain (iso- and anteiso-; I and II, Appendix) and oddchain carbon number components was apparent in the degraded residue from Day 5 onwards. However, their appearance was to be expected due to their natural occurrence in ruminant milk. In contrast, ergosterol ((22E)-ergosta-5,7,22-trien-3bol; III, Appendix) present in the degraded milk resi-

Table 3. Absolute concentrations of lipid components (mg g1 of powdered sherd) present during laboratory decay of olive oil under oxic conditions Free fatty acids Day 0 10 20 67 95
a b

C16:0 0 21.4 75.7 31.3 24.6

C18:1 12.7 164.0 530.9 223.9 175.3

C18:0 0 14.3 35.8 17.4 12.6

MAGsa 9.8 8.9 28.7 4.2 1.4

DAGsb 74.8 105.8 93.1 9.4 3.8

TAGsc 3696.7 2214.6 607.6 67.4 32.6

Total lipid 3795.1 2544.6 1405.7 366.9 262.0

MAGs = monoacylglycerols. DAGs = diacylglycerols. c TAGs = triacylglycerols.

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chain fatty acids not present in the oil at the start of the experiment (Fig. 4). The relative abundance of these compounds increased during the course of the experiment. However, even after 95 days of decay they were present in only very minor amounts, constituting <2% of the total free fatty acids present. The appearance of C14:0, C15:0 and C20:0 components occurred together with smaller amounts of the branched-chain iso- and anteisoC15:0 and C17:0 fatty acids known to be of bacterial origin. The components eluting between 12 and 14 min in the gas chromatogram in Fig. 4 are as yet unidentied, but are possibly oxidation products. Pure triacylglycerols After 7 days of incubation under oxic conditions hydrolysis of triolein had been initiated to produce diacyglycerols, monoacyglycerols and free oleic acid (Fig. 5a). At rst the rate of accumulation of mono- and diacylglycerols by hydrolysis of triacylglycerols appeared to be more rapid than their rate of degradation by micro-organisms. This pattern of decay continued through to Day 57 (Table 4). Between 57 and 97 days, the relative proportion of triolein remained constant (57.8 and 57.4%), whereas a decrease in the relative proportion of the diacylglycerols was observed while the monoacylglycerols and free oleic acid continued to increase in relative abundance. The same general trends occurred during the oxic decay of tristearin absorbed in sherds as for triolein, although the rate of degradation in the case of the former was slower. Diacylglycerols were produced after 6 days with monoacylglycerols and stearic acid appearing between Day 14 and Day 32 (Table 5). After 57 days, additional minor components were detected in both experiments, as shown by the partial gas chromatograms displayed in Fig. 5b. These minor components were identied as saturated, unsaturated and branched-chain fatty acids, including C14:0, branched-chain C15:0 (iso- and anteiso-), C15:0, C16:0, branched-chain C17:0 (iso- and anteiso-), C17:0, C18:0 and C18:1; these minor components were dominated by the aliphatic C14:0 and C16:0 fatty acids.
DISCUSSION

Fig. 4. Partial HT-GC proles of free fatty acids from (a) saponied olive oil at Day 0 and (b) the acid fraction of olive oil after 95 days of laboratory decay. Peak identities are the same as in Fig. 1. * refer to unknown compounds, possibly oxidation products.

dues from Day 5, does not occur naturally in milk and must be derived from the yeast and fungi involved in the degradation process (Goad and Akihisa, 1997, and references therein). Olive oil Intact pure virgin olive oil is composed predominantly of triacylglycerols bearing 50, 52 and 54 acyl carbon atoms, with the C18:1 the most abundant fatty acyl moiety. The major free fatty acids liberated during decay include C16:0, C18:1 and C18:0. The relative proportions of free fatty acids present did not alter signicantly during the course of the experiment (Fig. 3 and Table 3). Initially >3700 mg g1 of lipid was present in the sherd (Day 0), 90% more than was absorbed by the potsherds than in the milk experiment. The greater absorption of olive oil may have been facilitated by the solvent with which it was mixed, since in the milk experiment sherds were dosed in pure milk. A 60% decrease in the concentration of total absorbed lipid was observed by Day 15, with a >90% decrease of total lipid occurring by Day 95 (Fig. 3c and Table 3). Close inspection of the lipid proles revealed the appearance of straight- and branched-

It can be argued that milk fat is dicult to detect in the lipid residues recovered from archaeological pottery vessels primarily due to the fact that the degraded lipid prole of milk becomes indistinguishable from the degraded prole of ruminant adipose fat (Evershed and Charters, 1995; Charters, 1996) through diagenetic alteration. As seen in the simulated degradation of milk under oxic conditions, there was rapid loss of the shorter chain fatty acids (<C14) once cleaved from the parent triacylglycerol. In addition, the rate of decay was faster in the triacylglycerols containing <44 carbon

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Fig. 5. Partial HT-GC proles of (a) triolein after 97 days of oxic decay and (b) tristearin after 126 days of oxic decay in the laboratory. Regions of the chromatograms are expanded in order to show the distributions of minor components present. Peak identities are the same as in Fig. 1, with the addition of 1-MAG and 2-MAG which refer to monoacylglycerols with the fatty acyl moieties esteried at the 1- and the 2-positions respectively; 1,2-DAG and 1,3-DAG refer to diacylglycerols with fatty acyl moieties esteried at the 1,2- and 1,3-positions, respectively; * plasticiser contamination.

Table 4. Compositional data from the laboratory decay of pure triolein (as percentage composition). The data given for mono-, di- and triacylglycerols represent the sum of each class of compound Duration of the experiment Day Day Day Day Day Day Day 0 7 14 28 57 97 126 Free C18:1 (%) nd 2.4 6.7 11.5 9.6 22.8 25.3 Monoacylglycerols (%) nd 0.3 nd 3.6 3.8 2.5 7.5 Diacylglycerols (%) nd 6.0 7.2 22.3 26.1 16.8 23.2 Triacylglycerols (%) 99.2 90.6 83.9 61.0 57.8 57.4 39.8

nd = not detected.

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Table 5. Compositional data from the laboratory decay of pure tristearin (as percentage composition). The data given for mono-, di- and triacylglycerols represent the sum of each class of compound Duration of the experiment Day Day Day Day Day Day Day 0 6 14 32 57 84 126 Free C18:0 (%) nd nd nd 6.2 6.5 17.0 32.0 Monoacylglycerols (%) nd nd nd 0.6 1.6 6.2 6.8 Diacylglycerols (%) 0.3 2.2 9.0 6.5 14.1 34.0 31.7 Triacylglycerols (%) 98.9 95.8 90.0 86.7 77.0 41.0 24.5

nd = not detected.

atoms, resulting in a prole not dissimilar to degraded adipose fat. Thus, it has been conrmed experimentally that the preferential loss of the shorter chain free fatty acids would contribute to our failure to distinguish degraded archaeological milk fats from degraded adipose fats in archaeological pottery. The similarity of the composition of degraded adipose fat and degraded milk fat indicates that caution is required in our interpretations, and previous assignments of animal adipose fats may need to be reconsidered since some of them may actually represent degraded dairy fats. Since milk fat naturally contains such a complex mixture of lipid components, including branchedchain and odd carbon number fatty acids, resulting from the action of bacteria in the rumen (Christie, 1978), comparison of the decay products of pure triacylglycerols and a compositionally less complex plant oil (olive oil) has allowed us to begin to assess the likely contributions of microbial lipids to the degraded acyl lipid proles commonly seen in archaeological pottery. During the laboratory decay of pure triacylglycerols absorbed in sherds, both tristearin and triolein were seen to undergo partial hydrolysis to free fatty acids (stearic and oleic acids, respectively), monoacylglycerols and diacylglycerols, presumably as a result of a combination of microbiological and abiological hydrolysis. The relative contribution of both processes involved is dicult to evaluate, although previous experiments (not involving potsherds) have shown a high rate of hydrolysis of tristearin during the rst week of incubation due to the action of lipases (Hita et al., 1996). The absence of bacterial markers in the early stages of the experiments may suggest that chemical hydrolysis was responsible for initial degradation, however, the possibility cannot be ruled out that these markers are present in low abundance, but are swamped by the high concentrations of added lipid still present at the beginning of the experiment. Thus, bacterial markers might only be detectable after the major proportion of the utilizable lipid has been consumed and dead bacterial biomass is added to the organic matter in the sherds. Based on the absence of marker compounds there was no evidence for microbial activity in the sherds at the beginning of the experiments but the identication of minor components after 57 days of incu-

bation clearly indicates that microbes had been active in the potsherds and were likely responsible for the majority of compositional changes seen in the added lipids. The possibility of migration of lipids from the compost to the sherds (Heron et al., 1991) is ruled out from the results of control experiments involving the incubation of sherds without added lipid; no signicant lipid extract was obtained from any such experiments. The branched chain fatty acids containing 15 and 17 carbon atoms are well-known as bacterial markers (Gillan and Hogg, 1984; Goossens et al., 1986; Marty et al., 1996) produced by G+ bacteria (Paul and Clark, 1996), and monounsaturated acids C16:1 and C18:1 are also present in high concentration in bacteria (Marty et al., 1996) compared with higher organisms. Current work is focusing on more detailed analysis of the positional isomers of the monounsaturated C16 and C18 components, and their stable isotope (d13 C values) composition, which will provide further evidence for the bacterial origin of such compounds. The processes of b-oxidation and reduction have been suggested as being involved in the formation of palmitic acid from oleic acid formed after hydrolysis of acylglycerols (den Dooren de Jong, 1961). A more likely origin, however, is microbial lipids since the halting of b-oxidation after one cycle only is unlikely to lead to an accumulation of C14:0 and would only occur if the system was nutrient limited. Myristic acid is known to occur as a minor component in some bacteria (Goossens et al., 1986), and a review of fungal fatty acids by Lo sel (1988) has shown that myristic acid is one of the principal fatty acids of certain bacteria. The appearance of fatty acids not present in the olive oil at Day 0 are attributed to the activity of microorganisms, with similar characteristics to the minor components seen in the pure tristearin and triolein experiments. However, relative to the amount of lipid present at Day 0, the abundance of these components was minor. Moreover, the original character of the lipid prole was clearly recognisable throughout the decay experiment, despite changes in the relative proportions of the mono-, di-, triacylglycerols and free fatty acids. The rate of decay of olive oil proceeded more slowly than the decay of milk and is attributed to the nutrients

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contained in the milk which would have encouraged microbial activity. It is likely that in archaeological lipid residues the processes of decay would already have been initialised prior to burial due to normal vessel use, e.g. by oxidative, hydrolytic and thermal decomposition processes (Charters et al., 1995; Charters, 1996); and indeed, alteration of the original lipid character prior to burial is evidenced by the presence of mid-chain ketones in numerous vessels where animal fats have been heated to temperatures in excess of 3008C (Evershed et al., 1995; Raven et al., 1997). Moreover, studies of ethnographic vessels that have never been buried showed patterns of hydrolytic change in acyl lipids analogous to those seen in buried potsherds (Charters, 1996; Evershed et al., 1997b). The decay experiments described herein show how similar patterns of degradation occur for acyl lipids of widely varying origin and that, for the most part, the degraded lipid residues can be readily related to the intact fats and oils from which they derive. Signicantly, microbial lipids, although present, constitute only a very minor contribution to the overall lipid distribution of the absorbed residues.

for providing mass spectrometry facilities (GR3/2951, GR3/3758 and F60G6/36/01).

REFERENCES

CONCLUSIONS

The laboratory decay of organic residues absorbed in ceramic vessels allows us to bridge the gap in our knowledge between rapid short-term decay occurring in microbiologically active soils and sediments, and those slower transformations of organic matter occurring on archaeological timescales (102103 yr). Overall, the results of these degradation experiments indicate: (1) That available lipid will be readily and rapidly degraded, by a combination of oxidative, enzymatic and hydrolytic processes. (2) A signicant proportion of lipid comprising plant oil (and adipose fats) appears to have been aorded sucient protection such that it reects the original lipid prole. However, milk fats are rapidly altered in such a way as to aord fatty acid distributions reminiscent of adipose fats. (3) Although bacterial lipids are detectable following decay these represent a minor, and insignicant, contribution to the overall lipid prole. These results validate the use of acyl lipids preserved in ancient ceramics as a source of archaeological information.

Acknowledgements We would like to thank Prof. John Parkes, Ian Mather and Stephanie Charters for valuable discussions and the Fyssen Foundation and NERC for funding (GR3/10153). The NERC are also acknowledged

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