Recognition of the sources of isoprenoid alkanes in recent environments

Recognition of the sources of isoprenoid alkanes in recent environments Recognition of the sources of isoprenoid alkanes in recent environments Recognition of the sources of isoprenoid alkanes in recent environments Recognition of the sources of isoprenoid alkanes in recent environments Recognition of the sources of isoprenoid alkanes in recent environments Recognition of the sources of isoprenoid alkanes in recent environments Recognition of the sources of isoprenoid alkanes in recent environments

Recognition of the Sources of Isoprenoid Alkanes in Recent Environments M M QUIRK*, R L PATIENCE*, J R MAXWELL* and R E WHEATLEY** * Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol BS8 ITS, England **Department of Microbiology, Macaulay Institute for Soil Research, Aberdeen AB9 Qj, Scotland ABSTRACT Alkanes are ubiquitous components of contemporary aquatic and t e r r e s t r i a l environments and derive from natural and/or p o l l u t a n t sources In many cases, a detailed consideration of the structure and stereochemistry of individual components is necessary to distinguish the sources of these components High resolution gas chromatography and computerised gas chromatography-mass spectrometry allow determination of the s t r u c t u r e s , including stereochemistry, of certain w i d e l y - d i s t r i b u t e d alkanes in recent sediments Three examples of such compounds have been chosen to i l l u s t r a t e the p r i n c i p l e s by which these s t r u c t u r a l determinations permit assignment to p a r t i c u l a r sources: ( i ) pristane (2,6,10,14-tetramethylpentadecane) from a lacustrine sediment is shown to derive from a mixed source ( b i o l o g i c a l and p o l l u t a n t ) ; ( i i ) 17ßH-hopane in the same sediment appears to derive from reduction of hop-22(29)-ene, an abundant alkene of Microcystis aeruginosa, present in the lake; ( i i i ) 17a(H)homohopane in a sphagnum peat arises mainly as a r e s u l t of bacterial decay of Sphagnum cuspidatum Keywords: p r i s t a n e , 173(H)-hopane, 17a(H)-homohopane, Microcystis aeruginosa, Sphagnum cuspidatum, peat, sediments INTRODUCTION Alkanes occur widely in recent sediments as complex mixtures readily amenable to analysis by c a p i l l a r y gas chromatography (GLC) and computerised gas chromatography-mass spectrometry (C-GC-MS) ( e g Giger and Schaffner, 1978; Thompson and E g l i n t o n , 1978) These compounds can derive from a number of sources, which include a contribution from l i v i n g organisms ( e i t h e r d i r e c t l y , or i n d i r e c t l y by way of a precursor compound converted to the alkane in the sediment) Alkanes may derive from a v a r i e t y of p o l l u t a n t sources In some cases the gross structure of an i n d i v i d u a l alkane, or the d i s t r i b u t i o n of a series of alkanes, r e f l e c t s a p a r t i c u l a r i n p u t For example, the presence of 7- and 8-methylheptadecanes is c h a r a c t e r i s t i c of a d i r e c t contribution from 23 24 M M Quirk et al blue-green algae ( e g Han, McCarthy and Calvin, 1968; E g l i n t o n , Maxwell and P h i l p , 1974) Since steroidal alkanes (steranes) ( e g Mülheim and Ryback, 1975) are geological maturation products of s t e r o l s , t h e i r presence in contemporary environments is good evidence of a f o s s i l fuel-derived input (Cardoso, 1976; Brassell and others, 1978) The origins of certain other alkanes which are ubiquitous in recent sediments can only be determined from elucidation of t h e i r stereochemistry Three examples have been chosen to i l l u s t r a t e t h i s approach: 2,6,10,14-tetramethylpentadecane (pristane) ( I ) , 17ß(H)-hopane ( I I ) and 17ot(H)-homohopane ( I I I , R=CH3) Pristane occurs widely in marine organisms, and in Zooplankton and a higher marine organism comprises solely the 6(R),10(S) isomer ( l a ) (Cox and others, 1972; Patience, Rowland and Maxwell, 1979) In mature geological samples, including petroleum, the alkane comprises a mixture of la : lb + Ic in the r a t i o 1:1 (Patience, Rowland and Maxwell, 1979) Thus, determination of the configuration of pristane in a recent sediment should allow d i s t i n c t i o n between a biological and a p o l l u t a n t source ^χ-s ^ t S v L x * - ^ ^ s s v Compound I I also occurs widely in | | | ] T22 R recent sediments ( e g Brooks and I I I 1^ J I others, 1977), but possible sources y // s> ( ^^^ ^ \^\^^ are more d i f f i c u l t to assign I t : j I i n does not occur in petroleum, so i s X^^As^^X unlikely to derive from a p o l l u t a n t y^^ source; there is only one report of i t s occurrence in an a c i d o p h i l i c bacterium (De Rosa and others, 1973) Petroleum triterpanes are mainly of the 17a(H)-hopane type and are characterised (> C3-1) by the presence of ca 1:1 mixtures of C-22 diastereoisomers (e.g Ensminger and others, 1974J7 The occurrence of such a d i s t r i b u t i o n in recent sediments has been taken as evidence for petroleum-derived p o l l u t i o n (Dastillung and Albrecht, 1976) However, in many recent sediments the C31 member ( I I I ) occurs as an unequal mixture, with the l a t e r - e l u t i n g isomer (GLC) more abundant; this points to an additional ( b i o l o g i c a l ) o r i g i n for t h i s isomer although i t has never been found in organisms (Brooks and others, 1977) In the present study the o r i g i n of each of these compounds in a p a r t i c u l a r sediment has been determined by application of gas chromatography and computerised gas chromatography-mass spectrometry techniques: ( i ) pristane in a lacustrine sediment, ( i i ) 17$(H)-hopane in the same sediment, ( i i i ) 17a(H)-homohopane in a sphagnum peat Recognition of Sources of Isoprenoid Alkanes EXPERIMENTAL Sample Collection Rostherne sediment Cores were collected using a Gilson mud sampler, and stored frozen before sectioning and analysis (Gaskell, 1974) Microcystis aeruginosa A sample of the alga was collected from the surface of the lake An a l i q u o t was kept f o r analyses A second portion was suspended (18 wk) j u s t below the lake surface (0.5m) in a conical flask with the neck packed with s t e r i l e glass wool; subsequently, i t was allowed to stand (53 wk) on the lake bed (30m) The laboratory culture was grown by Mr.B Capel at Porton Microbiological Research Establishment Growth occurred (7d) at ambient in an aerated polycarbonate vessel (40£) containing a mineral salts solution with aqueous garden s o i l e x t r a c t The c e l l s were harvested by centrifugation Sphagnum cuspidatum A fresh sample was collected from the Lyne of Skene peat bog Part was kept for analysis w h i l s t a second portion was allowed to decay (ca 13 mnth) in a culture room at 30OC under aerobic conditions in the dark In a d d i t i o n , a sample of the moss base was collected f o r analysis Extraction and Separation Samples were extracted using e i t h e r a Dawes soniprobe (sediment samples; i PrOH/hexane, : ) , a Soxhlet apparatus (algal samples; (CH3)2C0 followed by CH2Cl2/MeOH, : ) , a d i r e c t r e f l u x (moss base and decayed moss, O^CWhexane; l i v i n g moss, (CH3J2CO followed by C^C^/MeOH, : ) In each case, the ' n e u t r a l ' f r a c t i o n of the t o t a l organic e x t r a c t was separated from the ' a c i d ' f r a c t i o n by shaking with aqueous (or methanolic) KOH (4 10% w/v; ca_ 50ml) followed by extraction with hexane or Ch^C^ The neutral f r a c t i o n was separated by t h i n - l a y e r chromatography (TLC) on SiO? (CH2CI2 developer) to y i e l d a hydrocarbon band (Rf * ) Urea adduction (3X) separated the hydrocarbons i n t o an adduct and non-adduct (branched and c y c l i c ) components; the l a t t e r was f u r t h e r fractionated by TLC (10% AgN03/SiÛ2; hexane developer) to y i e l d the branched and c y c l i c alkanes (Rf * ) Gas Chromatography Diastereoisomers of pristane were separated using a modified Perkin Elmer F-17 gas Chromatograph (flame i o n i s a t i o n d e t e c t o r ) This was f i t t e d with a DEGS glass c a p i l l a r y column (100m) using helium (30 psig) as c a r r i e r gas The oven temperature was programmed from 40°C to 80°C at 2°/min Gas Chromatography-Mass Spectrometry A l l samples were analysed using a Finnigan 9610 gas Chromatograph coupled d i r e c t l y to a Finnigan 4000 mass spectrometer The Chromatograph was f i t t e d with an 0V-1 glass c a p i l l a r y column (20m) using helium (ca 10 psig) c a r r i e r gas Temperature programme conditions were 50°C to 260°IT~at 6°/min Typical mass spectrometer conditions were: ion source temperature 200°C, electron energy 70 eV, filament current 430 yA Data were collected (ca 1.5 sec per scan) on a DEC PDP 8/e (32K core) laboratory computer Mass Tragmentograms and mass spectra were p l o t t e d using a Calcomp 565 p r i n t e r 25 M M Quirk et 26 al RESULTS AND DISCUSSION Rostherne Mere, Cheshire (U.K.) is a small (0.5 km ), eutrophic lake with permanent oxygen depletion at the deepest part (c_a 30m) The dominant algal species is the blue-green Microcystis aeruginosa, which is p a r t i c u l a r l y abundant as intense blooms during the summer months I t is probably the major contributor of organic matter to the bottom sediment (Belcher and Storey, 1968; Reynolds and Rogers, 1976) Skene Moss (0.5 km2) is situated near the Lyne of Skene, Aberdeenshire (U.K.) and is an o l i g o t r o p h i c , raised moss The upper 2.5m of peat i s composed mainly of Sphagnum cuspidatum remains; the peat has been used as a fuel so the present surface dates from the sub-Boreal period (2000-3000 yr B.P.) except for the upper 1cm deposited w i t h i n the l a s t 100 years (Wheatley, Greaves and Inkson, 1976) A l l the hydrocarbons were i d e n t i f i e d from comparison of mass spectra with standards, except for those in the moss which were assigned by mass fragmentography (m/e 191, 205) and retention data Pristane in Rostherne Sediment Pristane was analysed by GLC on diethyleneglycol succinate (DEGS) The d i a stereoisomer separation f o r three sediment sections (0-7cm, 7-18cm, 18-30cm), a sample of the alga collected from the lake surface, and a standard (1:1 mixture of the 6(R),10(R) and 6(R),10(S) isomers, lb,a) is shown in F i g l The Fig GLC traces of pristane (100m DEGS): i ) 1:1 mixture 6(R),10(R) and 6(R),10(S) isomers; i i ) - i v ) from d i f f e r e n t depths of Rostherne Mere sediment; v) from M.aeruginosa in Rostherne r e l a t i v e contribution from the 6(R),10(S) isomer ( l a ) varied among the samples and was at a maximum in the alga Since pristane of b i o l o g i c a l o r i g i n should comprise solely the 6(R),10(S) isomer (Cox and others, 1972; Patience, Rowland and Maxwell, 1979) each sample therefore contains a contribution from a p o l l u t a n t source A p l o t of the proportion of pollutant-derived pristane f o r each sample shows a r e l a t i v e decrease for t h i s component with increasing depth of sediment, as would be expected ( F i g ) The alga, although containing mainly the phytol-derived 6(R),10(S) isomer ( l a ) , s t i l l has a s i g n i f i c a n t cont r i b u t i o n from a contamination o r i g i n The most l i k e l y source of the p o l l u t i o n is petroleum-derived hydrocarbon material entering the lake via a small sewage Recognition of Sources of Isoprenoid Alkanes effluent and/or run-off from a nearby trunk road o C3|ocß x PRISTA 7o POLLUTANT -DERIVED Fig Plot of percent contribution of pollutant-derived pristane and 17a(H)-homohopane in Rostherne alga and d i f f e r e n t sediment depths 17ß(H)-Hopane in Rostherne Sediment Pentacyclic triterpanes of the hopane type show an abundant ion at m/e 191 in t h e i r mass spectra and can be recognised readily in complex mixtures by mass fragmentography, using t h i s i o n F i g 3D shows the m/e 191 fragmentogram of the alkane f r a c t i o n from a sediment core (0-20cm) The extended (> C31) 17a(H)-hopanes ( I I I , R=C2H5,n-C3H7,n-C4Hg,n-C5H]-|) occur as ca_ 1:1 doublets for the two C-22 diastereoisomers ( I I I ) The d i s t r i b u t i o n is t h a t t y p i c a l l y observed in mature geological samples and indicates that these components arise from a petroleum-derived source (Dastillung and Albrecht, 1976) The C30 alkane, 173(H)-hopane is also a s i g n i f i c a n t component ( F i g 3D) but cannot derive from a p o l l u t a n t source (see above) Formally, i t can only arise from a contemporary b i o l o g i c a l source or from an erosion of an immature ancient sediment where the 173(H)-hopanes have not been replaced by the more stable 17a(H) series t y p i c a l of mature samples ( e g Ensminger and others, 1974; Van Dorsselaer, Albrecht and Ourisson, 1977) In Rostherne Mere, the geology of the area is such that the l a t t e r p o s s i b i l i t y is not l i k e l y and a b i o l o g i c a l o r i g i n is more f e a s i b l e Mass fragmentography reveals, however, the absence of 173(H)-hopane in the cultured sample of M.aeruginosa ( F i g 3A) The two most abundant hopane derivatives recognised (2 and 390 ppm dry weight respect i v e l y ) were hop-13(18)-ene ( I V ; Fig 3A) in the alkane f r a c t i o n (hindered double bond) and hop-22(29)-ene (V) in the alkenes In the sample of the alga from the lake surface and the sample suspended in the lake p r i o r to lowering 27 M M Quirk et 28 al to the lake bed, the concentrations (ppm dry weight) were: hop-13(18)-ene, and respectively (Fig 3B,C); hop-22(29)-ene, 820 and 450; 17B(H)-hopane, 0.3 and (Fig 3B,C) This is strong circumstantial evidence that the hopane originates from reduction of the abundant hop-22(29)-ene present The culture sample, although not axenic, had no detectable iso- or anteiso-acids (Ci and Ci7) of bacterial origin, whereas the lake and TäFe bed samples had both isoand anteiso-acids present (70 and 1290 ppm t o t a l , respectively) (Quirk anci~ Maxwell, unpublished results) I t is possible, therefore, that the hopane arises from bacterial reduction of the alkene This may represent a general pathway for the origin of the widespread 17ß(H)-hopane in recent sediments ΗΟΡ-13(1β)-ΕΝΕ (X) ^Ju^J^J B D C ôòHOPANE C AA~JLJ'H^y^-A^ Fig ^-ôòHOPANES^ C 32 C 33 C34 35 O Mass fragmentograms (m/e 191) of branched/cyclic alkanes from: A Culture of M aeruginosa; B Rostherne surface alga; C Rostherne alga (lake bed experiment); D Rostherne sediment (0-20cm) Of the two C-22 diastereoisomers of l7a(H)-homohopane ( I I I , R=CH3) in the sediment (Fig 3D), the second isomer (by GLC) is in much higher relative abundance, unlike the C32 - C35 members of pollutant origin alone The second isomer i s , therefore, mainly of biological origin, and the pollutant contribution (both isomers in ca 1:1 ratio) decreases (Fig 2) with increasing depth of sediment (cf pristane) 17a(H)-Homohopane in Lyne of Skene Peat More detailed information about the origin of the second eluting isomer in the sedimentary environment was obtained by examination of the pentacyclic t r i t e r panes in this peat and in a major contributor of organic matter, Sphagnum Recognition of Sources of Isoprenoid Alkanes cuspidatum GC-MS analysis of the alkanes from various depths of a peat core showed the major t r i t e r p a n e by f a r to be 17a(H)-homohopane ( I I I , R=CH3) (Quirk and Maxwell, unpublished r e s u l t s ) A s i m i l a r s i t u a t i o n has been observed i n another peat (Gaskell, 1974) and in a l i g n i t e (Van Dorsselaer, Albrecht and Connan, 1977) and i t is possible that t h i s remarkable predominance in the branched and c y c l i c alkanes may be c h a r a c t e r i s t i c of certain peat types and t h e i r ancient counterparts The branched and c y c l i c alkanes of the l i v i n g moss showed only a trace amount of 17a(H)-homohopane, which was only revealed by mass fragmentography of m/e 191 ( F i g 4A) and 205 (not shown) The alkane was present as a ca 1:1 r a t i o of the two C-22 isomers, i n d i c a t i n g a p o l l u tant o r i g i n In "tiïe moss base ( c o l l e c t e d immediately below the l i v i n g moss, age ca yr) a dramatic increase in the r e l a t i v e abundance of the second e l u t i n g isomer was observed ( F i g 4B) This s i t u a t i o n was paralleled in the branched and c y c l i c alkane f r a c t i o n from the moss sample allowed to decay under dark, aerobic conditions ( F i g 4C) The o r i g i n of the abundant second isomer of 17a(H)-homohopane ( I I I , R=CH3) in the peat, t h e r e f o r e , appears to be associated with b a c t e r i a l decay I t is not known i f i t arises from a l t e r a t i o n of a precursor in the moss or i f i t is solely a bacterial product The l a t t e r appears more l i k e l y since extended (> C30) hopane derivatives have only been found in bacteria and blue-green algae ( e g Rohmer and Ourisson, 1976) Fig Mass fragmentograms (m/e 191) of branched/cyclic alkanes from S cuspidatum: A Living moss; B Moss base; C Decayed moss (laboratory) ^^^jjl ^KJVA^JU^ 29 M M Quirk et 30 al ACKNOWLEDGEMENTS We thank Mrs A.P Gowar and Mr A Turrington f o r technical assistance Two of us (MMQ and RLP) thank the Science Research Council and the Natural Environment Research Council (NERC) respectively, for Research Studentships Acknowledgement is made to the Donors of The Petroleum Research Fund, administered by the American Chemical Society, f o r p a r t i a l support We are also grateful to NERC f o r f i n a n c i a l support (GR3/2951) REFERENCES Belcher, J.H and J.E Storey (1968) The phytoplankton of Rostherne and Mere meres, Cheshire N a t u r a l i s t ( H u l l ) , 905, 57-61 B r a s s e l l , S.C., G E g l i n t o n , J.R Maxwell and RTPT PhiIp (1978) Natural backgroundof alkanes in the aquatic environment In Hutzinger, L.H van Lelyveld and B.C.J Zoetman (Eds.), Aquatic P o l l u t a n t s , Transformat i o n and Biological E f f e c t s , J_» 69-86 Pergamon Press, U.K Brooks, P.W., G E g l i n t o n , S.J Gaskell, D.J McHugh, J.R Maxwell and R.P Philp (1977) Li pi ds of recent sediments, Part I I Branched and c y c l i c alkanes and alkanoic acids of some temperate lacustrine and sub-tropical l a g o o n a l / t i d a l - f l a t sediments Chem.Geol., 20, 189-204 Cardoso, J.C (1976) The organic geochemistry o f T q u a t i c sediments Ph.D Thesis University of B r i s t o l , B r i s t o l , U.K Cox, R.E., J.R Maxwell, R.G Ackman and S.N Hooper (1972) Stereochemical studies of acyclic isoprenoid compounds I I I The stereochemistry of naturally-occurring (marine) 2,6,10,14-tetramethylpentadecane Can.J Biochem., 50, 1238-1241 D a s t i l l u n g , M and P Albrecht (1976) Molecular t e s t for o i l p o l l u t i o n in surface sediments Marine Pol ut.Bui 11., _7, 13-15 De Rosa, M., A Gambacorta, L Minale and J Bu'Lock (1973) Isoprenoids of Bacillus Acidocaldarius Photochemistry, 12, 1117-1123 Eglinton, G., J.R Maxwell and R.P Philp (197477 Organic geochemistry of sediments from contemporary aquatic environments In B Tissot and F Bienner (Eds.), Advances in Organic Geochemistry 1973, 941-961 Editions Technip, France Ensminger, A., A Van Dorsselaer, C Spyckerelle, P Albrecht and G Ourisson (1974) Pentacyclic triterpenes of the hopane type as ubiquitous geochemical markers: o r i g i n and s i g n i f i c a n c e In B Tissot and F Bienner (Eds.), Advances in Organic Geochemistry 1973, 245-260 Editions Technip, France Gaskell, S.J (1974) The environmental geochemistry of s t e r o l s Ph.D Thesis University of B r i s t o l , B r i s t o l , U.K Giger, W and C Schaffner (1978) Determination of polycyclic aromatic hydrocarbons in the environment by glass c a p i l l a r y gas chromatography Analyt Chem., 5.0, 243-249 Han, J , E.D McCarthy and M Calvin (1968) Hydrocarbon constituents of the blue-green algae Nostoc muscorum, Anacystjs nidulans, Phorinidium 1uridum and Chlorogloea f r i t s c h i ' T XỴïïem.Soc (C), 2785-2791 Mülheim, L.J and G Ryback (197F]7 Stereochemistry of some steranes from geological sources Nature, 256, 301-302 Patience, R.L., S.J Rowland and J.R Maxwell (1979) The e f f e c t of maturat i o n on the configuration of pristane in sediments and petroleum Geochim Cosmochim Acta, 43, In press Reynolds, C.S and D.A Rogers (1976) Seasonal variations in the v e r t i c a l d i s t r i b u t i o n and buoyancy of Microcystis aeruginosa Kutz emend Elenkin in Recognition of Sources of Isoprenoid Alkanes Rostherne Mere, England Hydrobiologia, 48, 17-23 Rohmer, M and G Ourisson (197FJ~! Structure ïïês Bacteriohopanetetrols d' Acetobacter xylinum Tetrahedron L e t t e r s , 40, 3633-3636 Thompson, S and G Eglinton (1978) Composition and sources of pollutant hydrocarbons in the Severn Estuary Marine P o l l u t B u l l , 9, 133-136 Van Dorsselaer, A , P Albrecht and J Connan (1977) Changes Tn composition of polycyclic alkanes by thermal maturation In R Campos and J Goni (Eds.), Advances in Organic Geochemistry 1975, 53-59 ENADIMSA Van Dorsselaer, A , P Albrecht and G Ourisson (1977) I d e n t i f i c a t i o n of novel 17a(H)-hopanes in shales, coals, l i g n i t e s , sediments and petroleum Bull.Soc.Chim.France, ( - , P t ) , 165-170 Wheatley, R.E., M.P Greaves and R.H.E Inkson (1976) The aerobic bacterial f l o r a of a raised bog Soil Biol.Biochem., 8, 453-460 31 ... information about the origin of the second eluting isomer in the sedimentary environment was obtained by examination of the pentacyclic t r i t e r panes in this peat and in a major contributor of organic... V ; Fig 3A) in the alkane f r a c t i o n (hindered double bond) and hop-22(29)-ene (V) in the alkenes In the sample of the alga from the lake surface and the sample suspended in the lake p r... presence in contemporary environments is good evidence of a f o s s i l fuel-derived input (Cardoso, 1976; Brassell and others, 1978) The origins of certain other alkanes which are ubiquitous in recent