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Title ISOPRENOID HYDROCARBONS PRODUCED BY THERMAL ALTERATION OF NOSTOC MUSCORUM AND RHODOPSEUDOMONAS SPHEROIDES
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Author Philp, R.P.
Publication Date 1977-05-01
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ISOPRENOID HYDROCARBONS PRODUCED BY THERMAL ALTERATION OF NOSTOC MUSCORUM AND RHODOPSEUDOMONAS SPHERffiDES
R. P. Philp, S. Brown, and M. Calvin
May 1977
Prepared for the U. S. Energy Research and Development Administration under Contract W -7405 -ENG -48
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., i''- ECE·~ tr ED " .. \V.'! OCT '(~ 0 1977 LIBRARY AND DOCUMENTS SECTION DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor the Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or the Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or the Regents of the University of California. 9 0 7 ISOPRENOID HYDROCAROONS PRODUCED BY THERMAL ALTERATION OF WS'IOC MUS())RJM AND RHOOOPSEUIJCM)NAS SPHEROIDES by 2 R. P. Philp1, S. Brown! and M. calvin ~t. of Olemi.stry, University of california, Berkeley, cal. 94 720 ~rato:ry of Olernical Biodynamics, University of california, Berkeley, cal. 94720 0 0 .} iJ ~i 8 0 ·9 0 8 ABSTRACI' Pure cultures of Nostoc musoorum and Rhodopseudaronas spheroides have been subjected to varying pericxis of thennal alteration. Experinents were cxmducted using both unextracted and extracted cultures in the absence and presence of rrontnorillonite. 'lhe isoprenoid hydrocru:bons produced in these e.xperinents have been examined. 'Ihe major hydrocal:bons produced being phy tane and five isaneric _Fhytenes. It appears that the :relative rate of fonna tion of phytane is greater f:ram the unextracted :residues c:x::IIIpa:red. to the ex tracted :residues, suggesting structural differences in the phycy1 side-chains of the chlorophylls :responsible for the fonnation of these isoprenoid hydro ccu:bons. •. f) 0. ~ i.} u ;!'~~ 8 0 INrroOOcriON In the course of our wo:rk on examining the pJtential of algae and bacteria / as precursors of kerogen (PHILP and CALVIN, 1976), it was necessary to subject pure cultures of certain organisms to varying periOOs of thental alter ation. '!he thennal alteration experiments, perfomEd on cultures of Nostoc ImJSOOrum and RhodopseudcmJnas spheroides, were designed to serve two purposes. First, to see whether or not kerogen fo:r:mi3.tion could be simulated by ther.mal alteration of the organisms. Second, to detennine the rate of for:ma.tion of isoprenoid hydroca.I:bons by the organisms under varying oonditions. c16-c20 It is this latter topic with whidl this paper is ooncerned. '!he results from the first part of the study will be presented elsewhere. '!he origin and net:h.oq of fonnation of pristane and thytane in Crude oil has been the focus of many researdl projects in recent years. '!he unani.nnus conclusion from the studies is that pristane and phytane undoubtedly have their origin from the phytyl side-dlain of dllorophyll. HaNever, the exact ned:lanism of fonnation of these isoprenoid hydrocarbons has been a oonstant souroo of discussion and disagreerrent. A reoont repJrt, by ll Previous to this study, several other workers suggested that certain iso prenoid c:x:npJunds may fonn as a result of lithification or ther.mal alteration of };hytol in a sedim:mt oolumn. 'lhese include dihydrophytol (SEVER and PARKER, 1969), a isoprenoid ketone (6, 10, 14-trinethylpentadecane-2-one; IKAN c18 -2- et al. , 19 73; SIMJNEIT, 19 73) , and c and c . isoprenoid alkanes. In another 19 20 study, MAXWELL et al. (1972, 1973) and rox et al. (1972a) provided stereodlem ical evidence for the foJ:Ination of phytane and intel::nediates fran the phytol ester of chlorophyll at relatively la.v tE!lT{Jeratures. As a J:eSult of this study they (CX>X, et al., 1972b) proposed a schene for the transfonnation of phytol into cl8-c20 isoprenoid alkanes. In the present study, samples of a pure culture of Nostoc musoonnn were heated for two, four, and twelve weeks in .sealed glass tubes at 200°C. 'Ihree different sets of experim:mts were perfo:tmad over each of these tine periods. Firstly, the pure culture was heated alone; seoondly, it was heated in the presence of m::mtnorillonite; finally, the culture was exhaustively extracted with organic solvents and using this J:eSidue, the heating exper.irrents were re peated in the absence of m::>nblorillonite. In a separate set of experinents perfoored for cx::mparative purposes, the photosynthetic bacterium, Rhodopseu dcm::mas spheroides, was heated in the sam: way as above (with and without nont m::>rillonite) except the set of experim:mts using extracted residues was emitted. 'lhe above experiments were not designed to elucidate or propose another nechanism for the source or fonnation of pristane and phytane fran chlorophyll. It is clear that in this set of exper.irrents the source of any isoprenoid hydro carl:x:ms is the algal chlorophyll. Rather, these exper.irrents were designed to see what factors effect the rate of fonnation of the hydrocatbans. Were they preferentially foJ:Ired fran the unbound chlorophyll or the intra-cellular chlo.ro phyll? Was their fonnation catalysed by the presence of a clay-mineral cata lyst, nontm::>rillonite? Could they be fonred "under relatively mild the:anal oon ditions? We roN wish to report the answers to sare of these questions. Our results shew that, in general, increasing arrounts of phytane are fomed, in both of 0 -3- the organisns examined with increasing tine, and the relative arrounts of thy- tenes decrease. fb.rever, in the case of the extracted organisms, the rate of fol11lation of phytane is relatively slower than with the unextracted organisms. z.t:mtnorillonite catalysed the relative rate of fo:rma.tion of thytane in the ex perinents with the Rhodopseudcm:>nas spheroides, but in no experilrent were any Itlytadienes, pristane or pristenes detected. EXPERIMENTAL One-gram sanples of the pure cultures Nostoc rrusoorum and RhOdopseudononas s@eroides (HAN, 1970) were sealed, under nitrogen, in thick-walled glass tubes . . . and subjected to heat treat::mmt at 200°C for vcu:ying periods of tine (two, four and twelve weeks) • '!he experinents were :repeated using the sarre organisms . mixed with m::mtnorillonite in the ratio 1 part organism to 2.5 parts nontnoril- lani~. Finally, the expe.rilrents were :repeated with samples of Nostoc nruscorum whidl had been exhaustively extracted .with toluene/rrethanol (1:1) for fourteen days to renove all _the soluble lipid naterial. At the end of each experilrent, the tubes were opened and the :residues extracted with toluene/rrethanol (1:1) using ultra-sonication. The extracts were fractionated on an alumina· column into heptane, ethyl acetate and rrethanol fractions.· '!he heptane extracts were analysed by gas chrcmatografby ( OC) and spectroiretry (C-OC-MS) • All analytical gas-liquid chranatograthy (OC) was carried out on a ·varian 2700 gas chranatograph. '!he total, nonnal and brandled/cyclic fractions we.i:e c:hranatographed on a 20', x 0~03" i.d. glass column., packed with 3% Dexsil 300 on Gas Olran Q, and ~atu:re prograrared fran 70 to 280°C at 4°/m.in with a helium flew rate of 6 ml/min. ('!he sane OC oonditions were used throughout -4- the study). The various ocrrq;:onents of the mixtures analysed by OC were iden- tified by their retention tines, co-injection of standards, wherever possible, and lCM resolution mass spectra c:btain~ by cx::mbined gas chrorratography-mass spectraretry (GC-MS) • Carbined GC-MS analyses were carried out on a DuPont 492-1 mass spectrateter interfaced with a Varian Aerograph GC r-bdel No. 204 equipped with linear temperature prograrmer. The oolumn used for the GC-MS analyses was also a 20' x 0 .03" i.d. glass capiliary oolurnn packed with 3% Dexsil 300 on Gas 01ran Q. The mass spectral data were acquired and processed using a DuPont 21-094 data system. ('!he insoluble organic residues frcin each set of experinEnts were set aside for characterization by elemental analyses, infra-red spectrosoopy, transmitted and reflected light microscopy and SEM photography. '1be results fran these characterizations will be reported in a separate paper. ) RESULTS AND DISClJSSION Table I sunmarizes the yields of various fractions obtained fran each set of experinents. The gas ciu:ana.tograms cbtained for the hydrocarbon frac- tions fran each set of experiments are shown in Figs. 1-5. In each chranato grarn the peaks which have been labelled with identical nunDe.rs have been shONn by mass spectraretry to arise fran the scure An examination of Table I shaNS that, in general, increasing arrounts of hydrocazbons are prcxluced fran the residues with increasing ti.rre of heating. (Similarly, the arrounts of ethyl acetate and rcethanol fractions also increase with ti.ne of heating.) '!here is not a great deal of difference between the .quantities of hydrocarlxms prcxluced fran the unextracted or the extracted j -~ iJ -5- residres of Nostoc musoorum. Hwe"Ver, in the presence of rrontrrorillonite, the quantities produced are considerably higher. The yields of hydrocarbons from the unextracted culture of Nos toe n'll.iSoonrrn are lo;ver than those from the Hhodopseudorronas spheroides. (The yields of the ethyl acetate and nethanol fractions are also higher fo:t the unextracted Rhodopseudblronas spheroides oom- pared to the Nos toe muscorum.) Prior to discussing the qualitative nature of these results, it is im- portant- to note that the major hydrocarbons obtained from extraction of the pure culture of Nostoc nlliSCbrum were n-c , a mixture of 7.:.. and 8:-nethyl 17136 heptadebane with minor amounts of the n-c H , n-c H and-n-c H ~ hydro 15 32 16 34 18 3 carbons~ It is important to note that no pristane, phytane, phytenes, or phytadienes were detected in the soluble lipid extract, which· agrees with the finding of HAN (1970). From Fig. 1 and Table II it can be seen that with increasing tine of heating; the unextracted Nostoc musconrrn produces rrore phytane and relatively smaller anounts of the phytenes. (No pristane or phytadienes were fonred in any of the experirrents perfonned in this study.) The results from the extract- ed residue of Nostoc musoorum differ in two ways (Fig. 2). First, the n-C 1-f136 and 7-. and 8-methylheptadecanes are absent. Second, the relative rate of for mation of phytane in the first two tine periods appears to be much slo;ver. 'lhere are significant amounts of compounds, tentatively identified by c.:..GC-MS as being rronounsaturated phytenes, present in the products from all three ex- perinents, but it is only after twelve weeks that significant amounts of phy- tane can be detected. In the presence of nontrrorillonite, the relative rate of forrration of phytane from the Nostoc muscorum in the two- and four-week heating periods appears to be reduced (cf. Fig. 3 with Fig. 1). No ready ex- planation can be given for this observation at·this tine. -6- In the two sets of experiments perfonred on the ·Rhodopseudorronas spher oides similar trends were again ooserved. In the two- and four-week experi rrents with the tmextracted residues, phytenes are the najor products (Fig. 4). After bNelve v7eeks phytane is the dc:minant product. In the presence of rront rrorillonite the arrotmt of phytane relative to the phytenes does not appear • to be significant after four weeks, as can be ·seen in Fig. 5, but after twelve weeks the products are again dominated by phytane. Why are there differences between the rates of formation of these hydro carbons from the unextracted and extracted residues of both organisms, espec ially in the initial stages of heating? It would appear that in the unextract ed residues, the phytane and the. phytenes are fonred from the unbound chloro phy11 in . these sarrples. After exhaustiVE:; extraction of the residues, the only sourre of the hydrocarbons is the intracellular chlorophyll. In the unextract ed sample the presence of free phytol could also increase the rate of appear ance of the phytane, but in the extracted samples there is no free phytol pre sent prior to thermal alteration of the residues. Therefore, the rate of phy tane fornation will be slCMTer in the latter case since cleavage of the phytyl side;_chain of chlorophyll has to occur prior to fornation of the isoprenoid hydrocarbons. The n-Crfi and 7- and 8-rrethylheptadecanes would not be expected to be 36 present in the products from the experiments involving the extracted residues. Tnese comp::mnds are rerroved from the residues during the solvent extraction process and are not fonned. by thermal alteration of the residue. · 'Ihe absence of pristane is interesting and could imply that it is not neCEssarily fonned tmder the sarre condi·tions as phytane. HCMTever, if as IKAN et al. (1975) suggest, pristane dres form from phytol via phytenic acid and pristene, it would appear that the rate of fonnation under the tempera- -7- ture cxmditions in this study is insignificant. It is also possible that the pristane found in the study by IKAN et 'al. (1975) was not fonred directly from chlorophyll by the suggested pathway but had an alternative, and as yet nn kno.vn, source. On the other hand, BAYLISS (1968) subjected chlorophyll from • algae and alfalfa to hydrogenqlysis at 800°F in a hydrogen atmosphe:r\~. Under these conditions the ratio of phytane to pristane from the algal chlorophyll was about 3:1. These results, along with our observations, would suggest tha-t the fonnation of phytane via phytol, dihydrophytol and phytene is a relatively mild process but to form pristane, nore severe heating conditions for longer periods of tirre are required. BROJKS et al. (1969) suggested that variations in the pristane:phytane ratio in coals and crude oils may reflect variations in oxidation during- earliest stages of chlorophyll decomposition. '.I.hey proposed that a lON pris- tane:phytane ratio in a crude oil nay be an indication of marine plant origin. '.I.he reason for this is that the fonnation of phytanic acid, the precursor of pristane, is less likely in the nore anaerobic conditions in aquatic environ- nents than it is on land during decay of plant material. Our results support this theory sinre no detectable anonnts of pristane were forrred during our heating e~rirrents which were performed tinder an absence of oxygen. It is noteworthy to rn2ntion that LIJMBACH (1975) has also perfonred sim ilar heating ~irrents on mixed cultures of bacteria both before arid after e.xtraction with chloroform. 'lhese experirrents were perfonred in an attempt to support the theory that the "non-lipid" part of bacterial and algal bodies have contributed to a large extent in· the formation of crude oil. However, the saturate fractions cbtained from Lijrrbach 's work appear to be .dominated by n-alkanes with neither pristane or phytane being fonned by heating of the mixed cultures of bacteria. -8- CDNCLUSION In all five sets of exrerlirents the anount of phytane fanned relative to the phytenes increases with time. In the case of the Nostoc rnuscorum the relative rate of phytane formation was slONer in the experlirents using the • exhaustively extracted residues. 'Ihis observation suggests that there are fundarrental differences between the rrolecular structures of the phyty1 comp::>- . nent in the "free" and intracellular chlorophyll. Alternatively, the presence of free phytol in the unextracted residue could increase the rate of phytane formation. •lhe presence of rrontm:>rillonite appears to enhance the fonnation of the phytenes but not the relative rate of formation of the phytane. •Jhe distribution and types of products formed from both the Nostoc rnus- rorurn and the Rhodopseudom:::mas spheroides exrerlirents were similar, support- ing the theory that they have a CXJmrrDn origin from the chlorophyll noeity. Tne absence of pristane, pristenes and phytadienes from any of these experi- rrents is extrerrely interesting. The only them:y for this observation at the present tine is that the relatively mild temperatures used precluded their formation within the t.iJre limits of this study. In conclusion, therefore, this study has shc:Nm that the relative ra·tes of formation of phytane and phytenes will vary depending on the source of the a.'l.lorophyll responsible for its formation. ACKNOWLEDGMENTS 'lhis work was supported by grants from the National Aeronautics and Space 1-\dm.inistra·tion (NGL 05-003-003), the National Academy of Sciences A. L Day Fund, and in part by the Energy Research and Developrrent Agency. 0 -9- BAYLISS, G. S. (1968) 'Ihe. fonnation of pris·tane, phytane and related isopren oid hydrocarbons by the thermal degradation of chlorophyll. Arter. Chern. Soc. llbstr ~ Papers 155, J-12. BIWKS, J. D. , GOULD, K. and SHI'l'H, J. W. (1969) Isoprenoid hydrocarbons in coal and petroleum. Nature 222, 257-259. ~I CbX, R. E., 1>1AXW£~, J. R., ACKMAN, R. G. and HOOPER, S. N. (1972a) Stereo chemical stuclies of acycli_c isoprenoid compounds - III. The stereochem istry of naturally occurring (marine) 2, 6, 10, 14-tetrarrethylpentade .cane. Can. J. Biocl1em. 50, 1238-1241. COX, 1{. E., MAXWELL, J. R., ACKI\1AN, R. G. and HOOPER, S. N. (1972b) The isola tion of a series of acyclic isoprenoid alcohols from an ancient sediment: approaches to a study of the diagenesis and maturation of phytol~ In: AdvanOJs in Organic Gc"'chemistry, (editors H. R. von Gaertner and H. ~vcimer), pp. 263-276. Pergarron Press. I~, J. (1970) Ch~nica~. studies of terrestrial and extraterrestrial ·life. Ph.D. thesis, U. of California, Berkeley. · TI TI LIJIIJBACH, G. w. M. (1975) On the origin of petroleum. Proc. Ninth ~vorld Petrol. Cong. ~, 357-369. ~1AXWELL, J. R., COX, R. E., AO .MAX\.vEIL, J. R., COX, R. E., EGLIN'IDN, G. and PILLINGER, C. T. (1973) Stereo chemical studies of acyclic isoprenoid. compounds - II; The role of chlorophyll in the derivation of isoprenoid-type acids in a lacustrine seclirrerrt. Geocru.m. Cosrrochim. Acta 37, 297-313. PHILP, R. P. and CALVIN, M. (1976) Possible origin for insoluble organic (kerogen) debris in sediments from insoluble cell-wall materials of algae and bacteria. Nature 262, 134-136. SI:.VER, J. and PARKER, P. L. (1969) Fatty alcohols (norml and isoprenoid) in secliments. Sci~ 164, 1052-1054. "' Table I YIELOO OF SOWBLE PRODUCI'S FlD1 m THERMAL ALTERATION EXPERIMENTS WI'. OF RESIDUE USED WI'S. OF FRACI'IONS FlD1 COLUMN ~HYl HEATING RESIDUE PERIOD ·Heptane Ethyl Acetate ~thanol A B A B A B A B 2 weeks lg 1 g 0.3 2.5 24.3 137.6 178.0 381.5 Pure Culture 4 weeks 1 g 1 g 0.4 1.5 51.5 137.2 190.0 435.8 12 weeks 1 g lg 0.8 20.0 63.6 142.2 182.3 137.0 2 weeks 1g - 0.2 - 34.0 - 301.1 - I ' ...... Extracted 0 Culture2 4 weeks 1g - 0.3 - 96.7 - 373.3 - I 12 weeks 1 g - 0.2 - 147.2 - 355.5 - 1gA 2 weeks 1gB 0.8 1.8 24.5 214.5 35.6 233.7 +2.5 g M-1 +2.5 g M-1 Pure Culture + M:>ntrrorillonite 1 g A 1gB 4 weeks 1.5 3.3 90.5 259.6 165.1 259.4 (M-1) +2.5 g Mv1 +2.5 g .M.'-1 lgA 1gB 12 weeks 13.1 3.0 146.6 76.0 210.5 178.0 +2.5 g Mv1 +2.5 g .MM A Nostoc ItUJSoorum B Rhodopseudaronas s};heroides 1 In rrgs. 2 'Ihis set of experin'ents not attarpted with RhoCbpseudatonas sphel:oid.es • t:_.. t1 0 0 ~.., -11- Table II IDENTI~."'Y OF MAJOR I:IYDROCAHOON ffi'\1PONEN'l'S s· - Molecular Peak No.* Formula Structural Identity 1 n-c aJkene cl7H36 17 2 claH38 7- and 8-IIBthylheptadecane 3 c20H4o Phytene** 4 c20H42 Phytane ' 5 c2oH4o Phytene** 6 c20H4o Phytene** 7 c20H4o Phytene** 8 c20H4o Phytene** * 'Ihese numbers oorrespond to numbers of peaks in Figs. 1-5. ** 1hese five components have been tentatively identified by C-GC-~~ as being IIDnmmsaturated phytenes, although the exact position of the double bond has not been established. -12.., ,, ~· Fig. 1 H;ydrocarlxms produCEd by thermal alteration of non-extracted Nostoc muso::>rum. Identities of ~ts responsible for peaks 1-8 can be found in Table II. GC oonditions as stated in text. 'r 0 o. -13- NON-EXTRACTED NOS TOC MUSCO RUM 2 4 2 WEEKS 7 5 ) 4 w J 4 12 WEEKS I I I (, J I I I 2 • i J 0 20 40 60 80 TIME fdiNS) XBL 7 71-4139 ',''l' -14- J Fig. 2 Hydrocarlxms produced by thermal alteration of extracted Nostoc muscorum. Identities of cxmponents .responsible for peaks l-8 can be found in Table II. ~ a:mdi.tions as stated in text. ,. ; -~~ ,, 0 0 ~~~ v ti 8 0 9 b -15- 8 EXTRACTED 7 6 NOSTOC MUSCORUM 2 WEEKS 8 7 6 w 4 WEEKS 5 a: 0 1- (J w 1- w Cl 12 WEEKS 4 0 20 40 60 . TIME (MIN5) XBL m-4206 -16- Fig. 3 Hydrocarl:x:ms produced by thermal alteration of non-extracted NOstoc nu.iSCbrtml + rront:norillonite. Identities of CClllpOnents :resJ;X>nsilile for peaks 1-8 can be found in Table II. GC a:m ditions as stated in text. '~ ----~ ; ' ¥L • ~~:~ 0 u ~; 'J 8 0 t 9 l 7 -17- NON-EXTRACTED NOS TOC MUSCORUM 8 + MONMORILLONITE 2 2 WEEKS 4 3 Lu -· 12 WEEKS 4 2 0 60 TIMECMIN> xat. nl-4163 -18- Fig. 4 Hydrocarbons praiuced by themal alteration of non-extracted Rhodopsetrlaronas spheroides. Identities of cx:xnponents re sponsible for peaks 1-8 can be found in Table II. GC ron ditions as stated in text. g0 -19- 8 RHODOPS EUD(W~::; SPHEROIDES 6 2 WEEKS w z 4 12 WEEKS 0 20 40 60 Tl ME(MIN) XBL 772-4162 -20- (\ / Fig. 5 Hydroccu:bons produced by thennal alteration of non-extracted Rh.cdopseudcm:mas ·spheroides + mnt:rrorillonite. Identities of a:mponents responsible for peaks 1-8 can be found in Table II. GC oonditions as stated in text. u 9 -21- NON-EXTRACTED RHODOPSEUDOMONAS SFtjf_8QlQE;_S + MONTMORILLONITE (\ 7 2 WEEKS 4 5 4 8 uJ lf) z 0 0.. lf) w 4 WEEKS c:: c:: 5 ·o,_ 3 u ....w w ·o 4 12 WEEKS 0 20 40 60 TIME XBL 772-4183 • u v I •• u J J ,, This report was done with support from the United States Energy Re search and Development Administration. Any conclusions or opinions expressed in this report represent solely those of the author(s) and not necessarily those of The Regents of the University of California, the Lawrence Berkeley Laboratory or the United States Energy Research and Development Administration. . ... TECHNICAL INFORMATION DIVISION LAWRENCE BERKELEY LABORATORY UNIVERSITY OF CALIFORNIA BERKELEY, CALIFORNIA 94720