A STUDY OF THE OPTICAL ROTATION OF LIPIDSEXTRACTED FROM -SOILS, SEDIMENTS,

AND THE ORGUEIL CARBONACEOUS METEORITE*.t

BY BARTHOLOMEW NAGY

DEPARTMENT OF CHEMISTRY, UNIVERSITY OF CALIFORNIA AT SAN DIEGO (LA JOLLA)

Read before the Academy, October 11, 1965, and communicated by H. C. Urey, June 8, 1966

During the past few years a considerable amount of information has been ac-cumulated on the optical rotatory dispersion of biologically significant compounds.Optical rotatory dispersion data has, in several instances, been useful in elucidatingstructural details of molecules, such as those of certain proteins. The optical rota-tions of several lipid and hydrocarbon compounds are also known. On the otherhand, there is very little information available on the optical rotation of lipidswhich occur in soils, marine sediments, and sedimentary rocks. There are someexceptions, such as petroleum and coal. The optical rotations of petroleum dis-tillate fractions and of coal have been studied.'-4 It was found that most petro-leum fractions are dextrorotatory but a few show levorotation.2 The chemicalcomposition of lipids in soils and sediments has also been studied,5-7 and fattyacids, saturated and aromatic hydrocarbons, and so on have been described. Aninvestigation of the optical rotations of soil and sediment lipids may eventually behelpful to elucidate some of the transformation processes that the decayed biologicalmatter is subjected to in the geological habitat. A study of the optical activity ofsoil and sediment lipids may also be of value in explaining the true nature of theoptical activity reported in lipid fractions extracted from carbonaceous meteor-ites.8-"1 The following three topics are discussed in this report: (1) controlexperiments performed in order to evaluate various factors that may cause falseoptical activity readings, (2) optical activity measurements made on soil and sedi-ment, and (3) optical activity measurements on carbonaceous meteorite lipids.

Necessary Precautions for the Measurement of the Optical Rotation of Soil, Sedi-ment, and Meteorite Lipids.-The measurement of the optical rotation of soil,sediment, and meteorite lipids is a most difficult task which can be successfullyaccomplished only if utmost care is exercised. The lipids are usually extractedfrom the sediment samples with a solution of benzene and methanol in a Soxhletapparatus. The extracts are usually yellow to dark brown in color. They maycontain suspended particulate matter and they usually show only a few millidegreesof optical rotation. Investigators working on problems of optical rotatory dis-persion will, of course, realize that such samples could easily give spurious readingsin all commercially available polarimeters unless the necessary precautions aretaken. The major part of this study consisted of control experiments in order todefine the factors that may lead to spurious rotations and to establish precisely theprecautional procedures which one has to follow to avoid artifacts.

In this study, a modified Bendix model 460-C Polarmatic Recording Spectropolar-imeter was used. A series of control experiments were performed to evaluate the in-strument in order to find out if it was indeed suitable for this study. The effects ofthe optical density of the solutions, suspended particulate matter, and the fluores-cence of the solutions were studied in detail, together with such possible sources of

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spurious rotations as the effects of air bubbles in the cells, temperature, strain on cellwindows, and possible inherent, instrumental artifacts. It will be noted below thatsome theoretical studies had been made in the past in an attempt to evaluate sourcesof spurious rotations. It was decided at the beginning of this study to use an experi-mental rather than a theoretical approach to the problem in order to ascertain thatvarious sources of error were detected under the particular experimental conditionsemployed in this laboratory.

1. It is generally known that the optical density of the solutions can be a po-tential source of error. Recently, Resnik and Yamaoka12 conducted an investiga-tion in order to define better the limits of this effect. These authors noted thatspurious rotations can be generated by inorganic, abiological solutions if the opticaldensity is greater than 0.250. It has been confirmed in the present study that falserotations arise when the optical density of the solutions is above a certain limit. Aseries of measurements were made with solutions containing different concentra-tions (and therefore different optical densities) of the synthetic dye isatin (2,3-indolinedione). In addition, the effect of optical density has also been studiedwith a series of naturally occurring, colored lipid samples. As the result of this in-vestigation it was concluded that optical rotation values obtained below 60 per centtransmission (i.e., above 0.223 optical density) must be discarded. Furthermore,in order to obtain entirely reliable optical rotatory dispersion values on soil, sedi-ment, and meteorite lipids, it was decided to use optical rotatory dispersion datameasured above 70 per cent transmission (i.e., below 0.155 optical density) only.Figures 1, 2, 3, and 5 show the results of some of the control experiments.

2. The effect of scattered light from suspended particulate matter has beenknown to be another source of error. The theory of this effect has been describedby Rouy and Carrolll3 and emphasized more recently by Hayatsu.11 In the presentstudy it was observed that various abiological, cloudy-colloidal to murky suspen-sions gave spurious rotations as well as an increase in the recorder noise level (Fig.1). However, nonoptically active suspensions filtered through 0.45-,u pore sizeMillipore filters never showed spurious rotations in this laboratory under the experi-mental conditions employed. Lipid samples in this as well as in previous investiga-tions8' 10 were Millipore-filtered.

FIG. L.-Optical rotatory dispersion+ 4 curves of control samples. Symbols: * =

dark, turbid suspension measured below60% transmission; C = dark, fluorescent

+ \/ - . \ solution measured in the 70-100% trans-mission range. Another control sample isshown in Fig. 5. Note that dark suspen-

at In mr / sions can cause spurious rotations when+ 2 _ ; ' \ their light transmission is lower than 60-

70%. These values, as well as all the othersshown in the following diagrams (except

+1 those specified) were measured in a BendixPolarmatic Recording Spectropolarimeter(modified), model 460-C, on the compressedrange, and they were multiplied by the

o appropriate Verdet constants. All pointsin this diagram and the following diagramsrepresent the average of at least two, but

400 450 500 550 L ,,_inthe great majority three, independentZ4-0V 450 500 550 600 measurements subtracted from similarlymy1 averaged values of the solvent standards.

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3. Another and related source of error can be the presence of air bubbles in thecell. In a series of experiments with water it was found that air bubbles, if suffi-ciently large or abundant, may give rise to spurious rotations. Consequently, itwas ascertained that the cells were always properly scaled and that no evaporationof the solution, leading to the development of air bubbles, was possible.

4. Fluorescent solutions may also cause spurious optical activity. To evaluatethis potential source of error, a rather strongly fluorescent Orgueil fraction wasstudied. This fraction was obtained from the insoluble meteorite organic matterby a process involving ozonolysis. The solution showed rather strong fluorescenceat 480-miA wavelength when excited with 365-my-wavelength radiation in a Beck-man DK-2A fluorescence spectrophotometer. The same solution showed no opticalactivity with the Bendix spectropolarimeter. It appears that the fluorescence of thesample did not interfere with the optical rotation measurement under the experi-mental conditions employed, possibly because of insufficient intensity of the fluo-rescent radiation.

5. It is well known, of course, that optical rotation is affected by temperature.Because optical rotation must be determined by subtracting the measured valuesof the unknown solution from those of the solvent standard, it was ensured thatboth the sample and the solvent standard came to a temperature equilibrium withinthe cell well before measurements were made. Keeping the solutions in the cellwell for a period of 20-30 min prior to the measurements appears to have accom-plished this equilibration effect when 0.1-cm-long cells were used. In addition, thesolvent standard was, at times, measured both before and after the unknown samplewas run. Both the control and the unknown solutions, and the corresponding sol-vent standards, were measured in triplicate.

6. Spurious rotations may also arise from strain that developed within the cellwindows. In a series of experiments, using a demountable 1.0-cm cell, the windowswere subjected to various degrees of strain by tightening the screw caps. Bothempty cells and cells filled with water were examined, and it was assured that thecell always remained in the same position and did not turn around its horizontalaxis. No spurious rotations were observed with the Bendix instrument; however,some were noted with another commercial recording spectropolarimeter where thecells turned around their long axis. The lack of spurious rotation with the Bendixcell may be the result of not applying sufficient strain on the window. Still, toeliminate any possibility of spurious rotation from this source, most experimentswere conducted in a fixed, nondemountable cell, which was glued to a carriage toprevent turning around its horizontal axis, and contained fused-in, nondemount-able windows.

7. To ensure that the Bendix instrument used in this study did not contain stillother, perhaps inherent, sources of error, it was calibrated against both a Carymodel 60 recording spectropolarimeter and a Rudolph nonrecording instrument.Dark, lipid, and petroleum distillate fractions, respectively, were used; the resultsare shown in Figures 2 and 3. The results obtained from the Bendix PolarmaticRecording Spectropolarimeter, under the conditions employed, showed good agree-ment with the values from the other two polarimeters. The most notable disagree-ment was a small measure of discrepancy between the Bendix and Cary polarimetersin the near-ultraviolet range of the spectrum, where the magnitude of the optical

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+ 32

+28

+24

+20

a in rn

+2 - \

-12300 350 400 450 500 550 600

m/1FIG. 2.-Calibration of the Bendix Polarmatic Recording Spectropolarimeter against the

Cary model 60 recording spectropolarimeter. The sample is the nonsaponifiable solvent ex-tract of a soil collected in a pine forest at 7929-ft elevation on Mt. LeConte iv the SierraNevada mountains of California. All measurements were made on Millipore-filtered solu-tions in the 70-100% transmission range as described in the caption of Fig. 1. Symbols:0 = 7.14-mg sample; 0 = approximately 1/3 concentration; An approximately 1/20 con-centration; these measurements were performed with the Bendix polarimeter. The symbolsX and o represent measurements made on the corresponding solutions with the Cary polarim-eter. Note the good agreement between the values obtained from the Bendik and Carypolarimeters in the approximately 1/3 concentrated solution and the less satisfactory agree-ment, particularly in the ultraviolet range, given by the highly dilute solution.

+ 40

+ 30

ainmo+20_--

+10

O 350 400 450 500 550 600 650 700 750

MkLFIG. 3.-Calibration of the Bendix recording spectropolarimeter against a Rudolph photo-

electric polarimeter. Sample: petroleum distillate fraction. Symbols: a = measure-ments made with the Rudolph polarimeter; * = values obtained with the Bendix recordingspectropolarimeter. Vertical lines indicate the range of error; note that the measurementsmade with the Bendix instrument lie within the Rudolph error range.

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rotations was somewhat different in these two instruments (Fig. 2). The Rudolphpolarimeter was one of the three Rudolph instruments that were used originally8 tomeasure the optical rotation of the Orgueil lipids in the visible spectral range.

Consequently, it appears that optical rotatory dispersion measurements of lipidsextracted from soils, sediments, and carbonaceous meteorites may be subject toerror; however, these measurements can be performed accurately and reliably ifscrupulous care is exercised during the analytical process and all necessary precau-tions are taken.

Optical Rotation Measurements of Lipids Extracted from Soils and Sediments.-The optical rotation of the saponifiable and nonsaponifiable fractions of the ben-zene-methanol Soxhlet extracts of ten soils, eight Recent marine sediments, andnine sedimentary rocks has been measured, exercising the experimental precautionsoutlined previously. The extracts have been saponified with KOH in the extractionflask of the Soxhlet apparatus as described previously.8 The saponified compo-nents were extracted with water, acidified with HC1, re-extracted with ether, evap-orated to dryness, and dissolved in CC14. The infrared and ultraviolet spectra ofthese saponifiable fractions were determined in CC14 and methyl alcohol, respec-tively. The samples were also analyzed by thin-layer chromatography. Follow-ing this, the optical rotations of the saponifiable components were measured inCC14. The nonsaponifiable fractions, which formed a separate phase in the Soxhletflask, were evaporated to dryness. Their infrared and ultraviolet spectra and thin-layer chromatograms were determined as those of the saponifiable fractions. Thebenzene solutions were then examined for optical rotation. The chemical composi-tion of the lipid extracts varied; this will be reported separately.The soil and the sediment samples were collected from different geographic areas

and climatic regions. The soil samples included subtropical, semiarid soil samplesfrom California, soils from Connecticut, forest soil samples from Sweden, as well assoil samples collected at 7929-ft, 10,550-ft, and 13,960-ft elevations on Mt. LeContein California. The location of the last sample corresponds to an Arctic climate.The soil samples were collected at approximately 8-10-inch depths, below thehumus layer. The soil samples showed dextrorotation, except one which waslevorotatory in both its saponifiable and nonsaponifiable fractions. This non-saponifiable fraction showed multiple Cotton effects. Specific rotations weredetermined at the green mercury line (X = 5461 A) by considering the entire weightof an extract (which undoubtedly also contains several nonoptically active compo-nents) in the calculations. Specific rotations of soils varied between [a]56 =-8.27° (the nonsaponified soil extract from 7929-ft elevation on Mt. LeConte) to[a]21 = + 14.09° (the saponified soil extract from 10,550-ft elevation). Specificrotations of the other samples were usually +3 to +50. Another soil showed nooptical rotation but this was the sample which was collected on the summit of Mt.LeConte, above the timberline. It consisted of a granite-wash and contained onlytraces of organic matter. Figure 2 shows the observed rotations of the nonsaponi-fiable fraction of a soil sample from a pine forest on Mt. LeConte.Recent marine sediment samples came from both tropical and Arctic regions of

the Pacific Ocean. Only the two cores from the Arctic waters contained sufficientquantities of extractable lipids to show optical activity. One was slightly dextro-rotatory; the other one showed levorotation; the saponified fraction had [a]546

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394 CHEMISTRY: B. NAGY PROC. N. A. S.

= -8.33°. The observed+8 ,data are shown in Figure 4.

Because of the small size ofthe cores, the amount of sam-

-X4 ples was limited.Sedimentary rock samples

42 - *-++ varied between Precambriana in m' ---^--4 and Tertiary in age. Unfortu-

_________ -~-~----~ -,- nately, the rock extracts were

-2ii-Ds>>xJ X-X-X- __X__- dark brown in color and withthe exception of one, their op-

-4 tical density was too high formaking reliable measurements.

300) 350 400 450 500 550- 600 -- - Consequently, they had to bemyI-Lexamined in very dilute solu-

FIG. 4.-Optical rotatory dispersion curves of soil, rock, tions. Lipids from a Pennsyl-and marine sediment samples. Symbols: * = 3.2-mg vanian-age graywacke fromsample of a nonsaponifiable solvent extract of a forest soil,from near Uringe, south of Stockholm, Sweden; A = 1.6- Kansas showed dextrorotation;mg sample of a saponifiable solvent extract of a Pennsyl- it is possible that this was avanian-age sedimentary rock from Kansas; 0 = 1.3-mgsample of a saponifiable extract from a Recent marine sed- petroleum source rock or a rockiment core from the Pacific Ocean, south of Alaska; X = containing residual crude oil1.0-mg sample of the nonsaponifiable fraction of the samemarine sediment sample. All measurements were made (Fig. 4). The saponified frac-on Millipore-filtered solutions in the 70-100% transmis- tion showed [a]"e = +3-33 0

No optical rotations were ob-served in the other sedimentary rock samples; this may have been caused bythe low concentrations that had to be used in order to decrease their optical density.

Optical Rotation Measurements of Lipids in the Orgueil Meteorite.-The Orgueilcarbonaceous meteorite fell in France in 1864; it contains approximately 20 percent water (a part of which has D/H ratios dissimilar to terrestrial waters) and itshows no heat damage below its thin fused crust. This meteorite consists mainly ofclay-type minerals and approximately 7 per cent organic matter. The lipids wereextracted from Orgueil by the identical procedure that was employed with the soilsand sediments. The optical rotation of both the saponifiable and the nonsaponifi-able fractions was determined in a part of the visible spectrum where light trans-mission was higher than 70 per cent (optical density less than 0.15). Extracts fromtwo stones were studied, one from the Mus6e National d'Histoire Naturelle inParis and the other one from the Montauban Museum, Montauban, Tarn etGaronne,France. The weights of the stones extracted were 5.28 gm and 7.37 gm, respec-tively. The lipid extracts were measured in both 1.0-cm and 0.1-cm-long cells; theobserved rotations are shown in Figure 5. All measurements were made by follow-ing the experimental precautions which were outlined previously. It was foundthat the saponifiable fraction was levorotatory, which is in agreement with earlierfindings.8' 10 In this study, the nonsaponifiable fraction was also measured; thisfraction too was levorotatory. The nonsaponifiable fraction of the Paris sampleshowed [a]66 =- 4.65°, and the same fraction of the Montauban sample [a]"6-- 1.250. The specific rotation of the saponifiable lipids had not been calculatedbecause the filtered extracts were estimated to contain at least 50 per cent sulfur.

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The levorotation of the nonsaponi- +

fiable fractions were higher thanthose of the saponifiable lipids from -both the Paris and Montaubanstones. A similar relationship be- a in mo .-O-G5 oO

tween the nonsaponifiable and sa- -2 ,0_.._ponifiable fractions had also been TXnoted in the soil and sediment lipids. -3 (E ,It is interesting that the value of --4

the Orgueil specific rotations is simi- _-1lar to those of petroleum,14 sedi- f-Xments, and soils. 40000 600

Possible Interpretations of the Ori- m/Lgin of Optical Activity in the Orgueil FIG. 5.-Optical rotatory dispersion curves of Milli-Meteorite.-If one observes the ex- pore-filtered Orgueil meteorite extracts and of a con-perimental precautions outlined trolsample. Symbols: A = synthetiedyesolution

with elementary sulfur; 0 = Orgueil saponifiableabove, reliable optical rotation meas solvent extract; o = approximately 2/3 concentra-urements of soil, sediment, and of tion of the same solution; X = Orgueil nonsaponi-'heOr.imeteorite fiable solvent extract, 6.3-mg sample. All measure-

the Orgueil carbonaceous ments were made in the 70-100% transmission range.lipids are possible. The optical ac-tivities appear to be real and not spurious rotations. Hayatsu" suggested thatthe latter may be the cause of the observed optical rotation in Orgueil, and indeedthis could be the case if the necessary experimental precautions had not been takenduring this and the previous studies. Consequently, one may search for the realcause for the presence of optically active lipids in Orgueil. Of course, the first causethat comes to mind is that the Orgueil meteorite became contaminated with terres-trial biological matter after it fell on the soils of southern France or during museumstorage. Biochemicals released from decayed or live organisms might account forthe optically active lipids. It should be noted, however, that free amino acids, oneof such key biochemicals, appear to be absent in Orgueil. Hamilton15 has shownthat the free amino acids in Orgueil can be attributed to contaminations from a singlehuman thumb print.

Stones of the Orgueil meteorite from both Paris and Montauban had beenexamined for the possible presence of a variety of aerobic and anaerobic soil and aircontaminants by Volcani and Bruff.'6 A stone of the Orgueil meteorite, which hadbeen stored in the Paris Museum since 1864, and been kept in this laboratory since1964, was examined for the presence of aerobic and anaerobic air and soil micro-organisms. The media used for the examination are listed in reference 17.Two fragments of this stone were removed aseptically from the container. The

first fragment was gently pulverized in a sterile mortar and divided into three sam-ples. The first was suspended in 0.8 per cent NaCl solution to give 10-20 mg per0.1 ml. The media were inoculated with 0.1 ml of this suspension and incubatedunder the conditions as specified.17 None of the media which were incubatedaerobically showed growth of any microorganisms even after 45 days. Medium 8,incubated anaerobically, showed dense growth after 6 days of incubation. Smallquantities of gas were produced during growth; however, this gas could have re-sulted from the reaction of acidic products of fermentation with the CaCO3 of the

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medium. The other two samples, similarly inoculated into this particular medium,gave similar results. In each case, the samples produced an apparently homoge-nieous culture of a gram-negative, non-spore-forming, nonmotile coccus which wasmost commonly associated in pairs hut also was present as single cells and chains.Several subcultures were made in medium 8; this coccus will not grow aerobically.The second fragment (about 10 mg) of the Paris stone was removed from the

container and dropped directly into medium 8. After 12 days of incubation underanaerobic conditions, a dense growth of nonmotile, non-spore-forming rods of varioussizes and shapes appeared. No gas was formed. Some of the rods were gram-posi-tive; others were gram-negative. No attempt was made to isolate the organismsinto pure culture, although one subculture was made.Two fragments (about 10 mg) of the Orgueil meteorite from the Montauban

Museum were examined only for the presence of anaerobic microorganisms by theuse of medium 8. One fragment produced, after 16 days' incubation, an apparentlyhomogeneous culture of a gram-positive, non-spore-forming coccus, most com-monly associated in clumps. A small amount of gas was formed. After 33 days,the second fragment produced a culture of rods of various sizes and shapes; nomotility or spore-formers were observed. The gram reaction of these bacteria wasnot determined.

In order to obtain evidence that the positive results which are reported abovewere not due to laboratory contamination, sterile saline solution was placed inmedium 8. The bottle was then incubated, as those inoculated with meteoritefragments, for 50 days without evidence of bacterial contamination.

Volcani and Bruffl6 stressed that the results reported above are at best extremelyfragmentary. However, they tentatively concluded that the four fragments of theOrgueil meteorite which were tested contained several different kinds of anaerobicnon-spore-forming bacteria. They found it puzzling that the organisms which arecommon to soil and air and are most resistant to such adverse conditions as desicca-tion, that is, aerobic and anaerobic spore-forming bacteria and fungi, seem to bemissing from at least one of the fragments tested. They considered that perhapsthe anaerobic, non-spore-forming bacteria are merely the "survivors" of a moretypical contaminant population which were picked up by the stones 100 years ago,and that, for some reason, these anaerobic organisms were more resistant to thechemical and physical environment presented by these stones. They also con-sidered two other possibilities, however: first, that these bacteria might be"museum" contaminants, and second, that they might be representatives of organ-isms indigenous to the Orgueil meteorite itself.The Orgueil meteorite contains an abundance of narrow magnesium sulfate veins

as shown by petrographic thin sections. It is an interesting question as to whethersuch biological organisms would grow in films of moisture saturated with magnesiumsulfate under museum conditions. Possibly such an inability would account forthe absence of the common soil and air contaminants.Another puzzling aspect of the Orgueil meteorite lipids is their C13/C12 isotope

ratio. The optically active, saponifiable fraction showed a 6 C11 value of -23.80/oo.Silverman'8 carried out an extensive study of the carbon isotope ratios of petroleum,ancient sedimentary, and Recent biological lipids (Fig. 6). According to thesemeasurements, the Orgueil optically active lipids fall in the ancient sediment range

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VOL. 56, 1966 CHEMISTRY: B. NAGY 397

PLIOCENE

MIOCENE

OLIGOCENE a

EOCENE -i PALEOCENEL

< CRETACEOUS--

JURASSIC __

0en TRIASSIC

j PERM IA N0I PENNSYLVANIAN

MISS ISSIP PIAN

DEVONIAN _

SILURIAN

ORDOVICIAN -

CAM B RIAN

PRECAMBRIAN - I I

-32 -30 -28 -26 -24 -22 -20

8C13FIG. 6.-Carbon isotope ratios of petroleums of different geological age (after Silverman's).

Vertical line illustrates the S C13 value of the optically active saponified Orgueil solventextract. It is possible that certain terrestrial organisms may give S C'3 values similar tothe Orgueil carbon isotope ratio.

rather than that of Recent biological matter. There is, of course, the possibility ofoverlap between the ancient and Recent isotope ratio ranges, and because of thisreason, one cannot rule out entirely the possibility of Recent terrestrial contamina-tions. The C'3/C'2 ratio of the Orgueil carbonate minerals is about +600/ooA2'This ratio is higher than any known terrestrial carbon isotope ratio. This wouldindicate that the carbonate and lipid carbon perhaps originate from different sourcessince no terrestrial process has produced carbonate and carbonaceous material ofsuch markedly different isotopic composition.

Conclusions.-Various sources of spurious rotations have been detected that canaffect the accuracy of optical activity measurements of lipids extracted from soils,sediments, and the Orgueil carbonaceous meteorite. By exercising the necessaryprecautions, however, it was possible to observe low, but real, values of optical rota-tions in soil, sediment, and in the Orgueil meteorite lipids. The results confirmearlier findings that the Orgueil lipids are optically active, but the origin of thiseffect cannot yet be satisfactorily explained. It is possible that the optically activemeteorite lipids are the results of terrestrial contaminations and/or that they areextraterrestrial and indigenous.The author would like to thank Prof. Harold C. Urey for his suggestions and interest in this

study, Prof. B. E. Volcani and Miss B. Bruff for performing the microbiological investigation, andMr. Vincent Modzeleski for his capable technical assistance in the experiments. Theauthor also wishes to acknowledge the assistance of Mr. M. Schwartz of the Bendix Corporation

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in obtaining the Bendix spectropolarimeter, Dr. M. Morrisson of the City of Hope MedicalCenter in Duarte, California, and Mr. R. J. Reynolds of the Applied Physics Corporation inMonrovia, California, for the use of the Cary model 60 spectropolarimeter, and Dr. W. D. Rosen-feld of the Chevron Research Corporation, La Habra, California, who provided the control datafrom the Rudolph polarimeter. The meteorite samples were obtained from Drs. J. Orcel andA. Cavaille.

* This was one of a group of papers read before the Academy by invitation of the Committeeon Arrangements for the Autumn Meeting, held in Seattle, Washington, October 11-13, 1965.

t This investigation was supported by NASA research grant NsG-541.1 Andre, E., and Bloch, A., Ann. Combust. Liquides, 9, 489 (1934).2 Oakwood, T. S., D. S. Shriver, H. H. Fall, W. J. McAleer, and P. R. Wunlz, Indust. Eng.

Chem., 44, 2568 (1952).3Mair, B. J., Geochim. Cosmochim. Acta, 28, 1303 (1964).4Zahn, C., S. H. Langer, B. C. Blaustein, and I. Wender, Nature, 200, 53 (1963).5 Meinschein, W. G., and G. S. Kenny, Anal. Chem., 29, 1153 (1957).6 Smith, P. V., Bull. Am. Assoc. Petrol. Geol., 38, 377 (1954).7Kvenvolden, K. A., Nature, 209, 573 (1966).8 Nagy, B., M. T. J. Murphy, V. E. Modzeleski, G. Rouser, G. Clatus, 1). J. Hennessy, U.

Colombo, and F. Gazzarrini, Nature, 202, 228 (1964).9 Hayatsu, R., Science, 149, 443 (1965).10Nagy, B., Science, 150, 1846 (1965).11 Hayatsu, R., Artifacts in Polarimetry and Optical Activity in Meteorites, preprint, Univ. of

Chicago (1966).12 Resnik, R. A., and K. Yamaoka, Biopolymers, 4, 242 (1966).13 Rouy, A. L., and B. Carroll, Anal. Chem., 33, 594 (1961).14 Hills, I. R., and E. V. Whitehead, Nature, 209, 997 (1966).15 Hamilton, P. B., paper presented at the Carbonaceous Meteorite Symposium iIi La Jolla

(1964).16 Volcani, B. E., and B. Bruff, personal communication (1966).17 The media and incubation temperatures were (1) standard mineral base, pH 7.0, 0.2% Na-

acetate; liquid, aerobic, 230C; (2) 1 + 0.02% NH4Cl, liquid, aerobic, 230C; (3) standard mineralbase, pH 6.5, 0.4% amyl alcohol, 0.02% NH4C1, 2% agar, aerobic, 230C; (4) standard mineralbase, 5% starch, 0.01% yeast extract (Difco), 0.002% NH4C1, 2% agar, aerobic, 230C; (5) 0.5%yeast extract 0.05% MgSO4 7H20, 0.1% K2HPO4, pH 6.8-7.0, aerobic, shaken, 350C; (6) 5 +2% agar; aerobic, 300, 370C; (7) 5 + 0.1% lactose; aerobic, shaken, 350C; (8) 0.5% yeastextract, 0.5% proteose peptone (Difco), 2% glucose, 2% CaCO3; anaerobic in stoppered reagentbottles 300, 350C.The composition of the standard mineral base is solution A, 0.2 M solutions of Na2HPO4 and

KH2PO4 mixed in proportions to give desired pH. Solution B, MgSO4-7H20 20 gm/liter; CaCl21 gm/liter. Solution C, EDTA 0.1 gm/100 ml; ZnSO4 7H20 50 mg/100 ml; MnSO4-H20 50mg/100 ml; CuSO4 10 mg/100 ml; CoSO4 10 mg/100 ml; Na-borate 10 mg/100 ml. Thesestock solutions were mixed in the following proportions and diluted to 1 liter with distilled water.Solution A, 100 ml; solution B, 10 ml; and solution C, 1 ml.

18 Silverman, S., J. Am. Oil Chem. Soc., in press.19 Silverman, S., personal communication; also footnote by Welte, D. H., Bull. Am. Assoc.

Petroleum Geol., 49, 2250 (1965).20 Clayton, R. N., Science, 140, 192 (1963).

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