Report Nsec- 118

REPORT NSEC- 118

REPORT

A STUDY ON THE ROLE OF RADIOACTIVITY AND HYDROTHERMAL PROCESSES IN PROTOBIOCHEMSTRY

R. L. Bogner S. L. Hood E. R. White S. Somani

June 1965

Submitted to: Office of Space Science and Applications Bioscience Programs Division National Aeronautics and Space Administration

Contract No. NASW-989

NUCLEAR SCIENCE & ENGINEERING CORPORATION P. 0. Box 10901 Pittsburgh, Pennsylvania 15236 PREFACE

This study was conducted under Contract No. NASW-989 for the Office of Space Sciences, National Aeronautics and Space Administration. Program direction was provided by Dr. R. R. Edwards, Technical Director. The authors wish to acknowledge the technical assistance of Dr. R. C. Koch and Philip Battaglia. The cooperation of Dr. R. Lumb and P. Orlosky at the Western New York Nuclear Research Center in Buffalo, and Mr. Robert Reitz at the Mellon Institute in Pittsburgh is also acknowledged. Grateful appreciation is expressed for the opportunities to discuss aspects of this work with Dr. Carl Bruch, Bioscience Programs, Office of Space Sciences, NASA, Dr. Sidney W. Fox, School of Environmental and Planetary Sciences, University of Miami, Dr . Cyril Ponnamperuma, Exobio- logy Division, NASA, Ames Research Center, Dr. Paul Kuroda, Department of Chemistry, University of Arkansas, and Dr. Truman P. Kohman, Depart- ment of Chemistry, Carnegie Institute of Technology.

ii ABSTRACT

This report describes the initial phases of a study designed to test the novel and unique hypothesis that organic synthesis and chemical evolution in primitive planetary environments may have proceeded in radioactive hydrothermal systems. It was demonstrated that an abundance of biochemically significant organic compounds can be formed from the simplest chemical resources in hot, aqueous environments under the influence of ionizing radiation. Furthermore, it was shown that high-molecular weight peptides and proteinoids can be produced in the same unique model system thus providing mechanisms for the generation of macromolecules, an essential development for the evolutionary progression from chemical simplicity to prebiotic molecular complexity. This experimental verification of the possible geological origin of biochemical substances (chemical abiogenesis) suggests that spontaneous primordial chemical reactions might well have led to the production and proliferation of organic molecules in favorable local environments, e. g. hot springs, early in the history of the planet Earth. These ground-based studies assume an immediate significance for lunar and planetary exploration in view of the fact that propitious geochemical events and conditions on the moon and Mars, similar to those present earlier in the history of the Earth, may provide primitive hydrothermal radioactive systems in which abiogenic formation of extraterrestrial organic compounds proceeds at present.

iii TABLE OF CONTENTS

Page

PREFACE ii

ABSTRACT iii

LIST OF TABLES iv

LIST OF FIGURES V

INTRODUCTION 1

EXPERIMENTAL 3

I. Methods 3

11. Results 8

I DISCUSSION 21

SUMMARY 42

BIBLIOGRAPHY 43

TABLES 48

FIGURES 59 TABLES

Page Table I Experimental conditions 48

Table I1 Composition of reaction mixtures 49

Table I11 Columns for gas chromatography 50-51

Table IV Summary of chemical classes produced from 52 primitive earth compounds

Table V Gas chromatographic data from Experiments 53 6 to 11 I Table VI One-dimensional paper chromatographic data 54 from Experiments 6 to 11

Table VI1 One-dimensional paper chromatography: 55 Numbers of spots from chromium carbide

Table VIII Compounds from irradiation of aqueous methane, 56 Experiment 20

Table IX Compounds from irradiation of aqueous acetylene, 57 Expe riment 2 2

Table X Amino acids from irradiated experiments 58

iv FIGURES

Page Figure 1. Photomicrograph of amber, protein-like 59 spherule s precipitated from an irradiated aqueous amino acid solution, Experiment 12

Figure 2. Autoradiogram of methanol-C- 14 irradiation 60 products, Experiment 10

Figure 3. Autoradiogram of methanol-C- 14 irradiation 61 products after removal of volatile compounds

Figure 4. Autoradiogram of irradiation products from 62 formate-C- 14, Experiment 14

Figure 5. Autoradiogram of irradiation products from 63 formate-C- 14 in the presence of FeCl 3’ Experiment 16

V IN TROD UC TION Investigations of protobiochemistry, "origin of life", are generally concerned with studies of organic synthesis and chemical evolution under simulated primitive planetary environments. Protobiochemistry is under stood to be the process by which organic chemical compounds of the types found in present day biological organisms are formed in the absence of life (abiogenically) by normal chemical processes which occur in a particular environment and using as raw materials simple inorganic and one-carbon organic chemicals which could have existed on the primitive earth. Such investigations involve making assumptions about the nature of conditions on the earth approximately 4 to 5 billion years ago, setting up analogous model systems in the laboratory, and examining these systems for the production of biochemical type compounds. Indeed, evidence to suggest that abiogenic formation of basic biochemicals may have occurred on the primitive earth or in space has been obtained from several studies of a variety of model systems with various energy sources. However, no general scientific agreement has been reached on the probable system and pattern that actuaiiy was responsibie for primordiai synthesis and chemicai evolution. The significance of hydrothermal processes in experimental protobiochemistry has received surprisingly little attention, this in spite of the ubiquitous hydrothermal activity of the primitive earth, the associated energy sources due to the high levels of radioactivity, and the presence of earth's most primitive known organisms in modern hydrothermal waters, From our current knowledge, it seems entirely reasonable to speculate that radioactivity and hydrothermal processes played an important role in the origin of life. Such a model provides for more than an adequate amount of inorganic chemical resources and energy (heat and ionizing radiation) in local regions and in satisfactory sequences of conditions (hydrous and anhydrous) that would be exceedingly favorable to chemical evolution and prebiological development.

- 1- I It would appear reasonable to direct protochemical research to models I of attractive and favorable local environments rather than to concentrate upon I studies which find it necessary to consider equilibrium conditions evenly dis-

I tributed throughout the planet, particularly if it is agreed that the origins of life probably represented low-probability, isolated events. The scope of the present study, then, was to undertake a research program aimed at developing the consistencies of a model, incorporating radioactivity and hydrothermal processes, with other knowledge in regard to protobiochemistry. The initial program was aimed at exploring syntheses under the conditions of the model, beginning with a hot aqueous system in the presence of ionizing radiation and followed by modulation, if necessary, to an anhydrous system in the presence of radioactivity. If the experimental results appeared compatible with the model, further studies were envisioned to pursue more complex syntheses and polymerizations, to analyze extant samples of the model materials, and to investigate the primitive nature of organisms sur- viving in modern systems of the model.

-2- EXPERIMENTAL I. Methods Irradiation Methods. High voltage electron irradiation was achieved with a Van de Graaf accelerator at the Western New York Nuclear Research Center, Buffalo, New York. The machine delivered 5 x 108 rads of 1.5 Mev electrons to samples within a few minutes. The dose rate was limited by the induced heating of the aqueous samples, so the irradiation period was generally set at 45 minutes to minimize temperature rise, The radiation dose was calculated from the beam current absorbed by the sample. The sample consisted of a 20 ml aqueous solution in a 75 mm diameter aluminum dish covered with a thin aluminum foil to reduce evaporation losses. Mixing was provided by a teflon-covered magnetic stirrer, and temperature was controlled to 53-55OC by a thermostated water bath. Temperature was measured by an immersed iron-constantan thermocouple. For the irradiation of methane, a stainless steel capsule was used which contained 2.0 ml water at pH 8.5 with NaOH, and 6.0 cc free gas space. The gas phase used was 1 atmosphere of air plus 47 lbs. /sq. in. of methane. The solubility of methane in the aqueous phase under these conditions was not determined, but is 9 cc per 100 ml at 1 atm. Irradiation was performed at Radiation Applications, Inc. Long Island City, New York. The capsule was irradiated at ambient temperature with cobalt-60 gamma rays at 5 the rate of 1.43 x 10 rads per hour to the aqueous phase. The total absorbed 8 dose in this phase was 10 rads. Under these conditions the radiation ab- 6 sorbed by the gas phase of the system was about 1%, or 10 rads. The gas phase reactions of the methane with the water vapor and water fragments such as H*, OH', etc., therefore probably were much less significant than the aqueous solution reactions. However, the pure hydrocarbon products, representing methane polymerizations, most probably were formed in the gas phase. The irradiated capsule was attached to a gas analysis vacuum line so that there was no possibility of loss of volatile products.

-3- For the irradiation of acetylene, a reaction mixture of 2.0 ml water at pH 8.5 with NaOH and 1 atmosphere of acetylene (air excluded) was irradiated with CO-60 gamma rays as described above for methane. Hydrothermal experiments were run without irradiation as control experiments using portions of the same reaction mixtures as were irradiated. Time, temperature and mixing were the same. Other portions of the reaction mixtures were stored cold as further controls. Samples were stored at 4' or -15OC until analysis. Analytical Methods. The first products of the model systems were expected to be common organic compounds of moderate molecular weights up to a few hundred. Many of these small molecules are quite volatile, indicating a requirement for analytical methods for volatiles. The gas chromatograph was the instrument of choice for this purpose as well as for volatile derivatives of the sugars and amino acids. Columns, programs, and standards suitable for the direct separations of the chemical classes hydrocarbons, (C3 to Clo), alcohols and glycols, carbonyls (aldehydes, ketones), amines, sulfur compounds, acids were established. Derivative methods used included trimethyl- silyl sugars, n-butyl-N-trifluoracetyl amino acids (1)- and methyl esters of organic acids, The high sensitivity flame ionization detection method was used with an F and M Model 720 Gas Chromatograph. Table III presents the column types used for the various chemical classes measured. The second major analytical method used paper chromatography for compounds not detected by the gas methods, including higher molecular weight compounds and polymers. Difficulties were presented by the very large numbers of products present in the samples and the very low concentrations of individual compounds. The low concentrations have made it necessary to spot large quantities of sample, producing overloading of some papers. The number and variety of products produced some interferences in the migration patterns. Introduction of additional steps and modifications to the procedures such as preliminary group separations, desalting, or substituting some ion exchange, electrophoretic or thin-layer chromatographic methods should im- prove the situation. In particular, the sugars and organic acids appeared to distort the paper patterns.

-4- In conjunction with the paper and related methods the use of carbon- 14 labeled substrates provided several advantages. Autoradio- graphy of papers provided greater certainty of locating spots corresponding to all compounds present in even minute amounts. The papers are not harmed and can be used for further chemical tests. The darkening of the film indicates approximate quantities, which can be determined precisely by isotopic counting methods using the separated spot. Several, but not all, of our experiments have included tracers. The labeled substrates have been carbonate, formate and methanol. The carbonate experiment was unsuccessful and should be re- peated. The other two substrates, methanol and formate, both showed large numbers of labeled products, and very few of these were common to both sub- strate s. The paper chromatographic solvents chosen for general use after numerous trials with standards were Solvent C; secondary butanol, tertiary butanol, 2-butanone, water, diethylamine or ammonium hydroxide (8, 8, 16, 9, 0.2 V/V): and Solvent D: n-butanol, acetic acid, water (4, 1, 5 V/V). Four or five other solvents plus several concentration variants were tried and discarded. Spots were located after chromatographic separation by a combination of methods. Autoradiography was discussed above. Ultra- violet fluorescence indicated numerous spots not detected by any other method (therefore, presumably being products of non-carbon- 14 labeled substrates). The chemical nature of these products is as yet undetermined, but is is possible that they may include interesting cyclic nitrogen compounds such as pyrimidines and purines. Numerous chemical tests w-ere used. These include ninhydrin and isatin for amino acids and some peptides and amines, silver nitrate for sugars, bromcresol green for acids, and molybdenum blue for sugar phosphates. Identification of separated and located compounds also required a combination of methods based on chemical properties and comparisons with knowns. These include Rf values of knowns, cochromatography, color tests, absorption spectrophotometry, and derivative formation. Concentrating

-5- the samples was often necessary before spotting the samples onto the paper, A Rinco apparatus was used for vacuum evaporation at room temperature to 40°; volatile constituents were probably removed wholly or in part during this step. Analysis of a heterogenous polypeptide mixture obtained by irradiating a mixture of eighteen amino acids was both difficult and different from those protein methods directed toward single structures. Here an individual molecular structure was expected to be present in extremely small concentration and with very many very closely related structures. Physical separations based on molecular size indicate the presence of polymers of various sizes. Dialysis, precipitation, and ion exchange chromatography were used to give some indication of size distribution, Comparisons of quantities of amino acids in hydrolyzed versus unhydrolyzed samples indicated the presence of peptide bonds, but did not indicate polymer sizes. Chemical tests for peptides, such as the biuret and Folin tests, were unseccessful because of small sample size. The UV absorption spectra obtained were characteristic of peptides, but again were non-specific unless the samples could be fractionated more completely. Samples were also analyzed on a Spinco Amino Acid Analyzer in cooperation with Mellon Institute, Pittsburgh, Pennsylvania. It is clear from the type of results obtained that the analytical program could be extended indefinitely. Additional classes and individual compounds within each class could be sought and the identification of compounds could be verified and made more positive. However, it is equally obvious that the analytical methods employed detected large numbers of products of biological and chemical interest. Experimental Conditions. Twenty-three experiments were performed. Table I indicates the irradiation conditions, composition of the reaction mixtures and other parameters of the various experiments. Detailed composi- tions of each reaction mixture are presented in Table II. Most experiments were arranged in pairs, one was irradiated and the other served as a control hydrothermal experiment. The upper limit of 55OC was imposed by the desire to work at atmospheric pressure and below the boiling point of methanol as well as below the decomposition point of ammonium carbonate.

-6- The pH of 8.5 was chosen as representative of the aqueous systems which would result under postulated primitive earth conditions of an atmosphere containing ammonia. It would probably be desirable to perform additional studies under conditions of both approximate neutrality (pH 7) and mild acidity (pH 2-5) such as would result from solutions of carbon dioxide and acid minerals. The question of whether the primitive conditions included lack of gaseous oxygen was considered to be rather irrelevant to the radioactive hydrothermal model systems, since aqueous systems subjected to the influence of either photochemical or ionizing radiation produce peroxides and other water decomposition products so that oxidation immediately becomes possible. The studies conducted to date have been run in the presence of oxygen. Numerous forms of carbon, including a wide range of oxidation states were compared. The oxidation states of one-carbon compounds ranged from the most oxidized form, carbonate (carbon dioxide), through the progressively more reduced Iorms, carbon monoxide, formate, formaldehyde, and methanol, to the most reduced form, methane. The aqueous systems en- compassed the range of these forms except for methane which has low water solubility and which has been used extensively by others in gas phase (atmas- pheric model) reactions. Methanol may have been the most probable early product from methane, and therefore, was considered a valid subject for the model. The forms of nitrogen and sulfur were also of special interest, but have not been studied in detail to date. The chemical elements which were deemed of importance to include in the reaction mixtures included C, H, 0, N, S, P, C1, and certain cations, The common cations, Na and K, were used, usually excluding the divalent cations such as Ca, Mg, Mn, and heavy metals because of possible complications from insoluble products and catalytic considerations; Fe and Cr were employed in selected experiments.

-7- I' I I. i i a. Results Experiments 1-4 did not yield significant results within a two- week irradiation due to inadequate radiation levels achieved with a 900 mc strontium-90, yttrium-90 sealed source. Table PV summarizes the products by chemical class obtained from various forms of carbon in Experiments 6-19. No organic products were found by gas chromatography of the reaction mixture in Experiment 5 in which carbonate, phosphate, sulfate and ammonia were present. These results indicate the carbonate is an unproductive form of carbon in aqueous systems and may raise a further obstacle to the idea CO was the starting material for that 2

the evolution of organic compounds (21.P In Experiments 6- 11 in which reactions of methanol, cyanide, carbonate, phosphate, sulfide and ammonia were studied, a large number of organic compounds were identified by gas chromatography as shown in Table V. The relative efficiency of the radiation model process was strikingly

demonstrated by the lack Q€ compounds formed in the control hydrothermal Experiments 9 and 11 whereas numerous compounds were formed in the corresponding irradiated Experiments 8 and 10, The control hydrothermal data also indicated the purity of the original reactants, and therefore served in place of

blank analys e 8. Paper chromatographic separations, as already stated, were less satisfactory because of the numbers of compounds, lower sensitivities of chemical detection methods, and mutual interferences in movement. In Table VI previously reported data are gathered for one-dimensional separations. Contrary to the negative results obtained by gas chromatography, spots were found in hydrothermal Experiments 9 and 11. The paper separations may not have been complete enough for full resolution, and spots may consist of several overlapped compounds. Color developments also were complex and overlapped. The silver nitrate reagent, usually used for reducing sugars, may also indicate a number of other compounds so that these spots are listed as unknowns. The UV fluorescent spots have not correlated with any known classes of compounds, including purine and pyrimidine standards,

-8- The sugar phosphate tests using the molybdate reagent for phosphate released by perchlorate hydrolysis were of interest. Yellow and blue spots were produced. In Experiment 10, the single spot was large enough to encompass the standard pasitions of inorganic-P, acetyl-P and carbamyl-P. Acid hydrolysis of portions of each sample results in dis- appearance of the original spots, In Experiments 8 and 10, there was appearance of a new phosphate spot with a high Rf, well beyond that any of the standard phosphates or free sugar8. h Experiments 6 and 7, the initial large spot was replaced after hydrolysis by three well-separated spots, the slowest of which was probably phosphate. The other two may have been carbamyl-P or acetyl-P and an unknown which falls between these two and the high Rf spat of Experiments 8 and 10. Upon standing overnight, the samples of Experiment 6 deveioped a second, blue-colored spot which disappeared on hydrolysis, This perhaps corresponded to the third yellow hudr oly sis compound.

Two-dimensions; separations on paper were not very success- ful because spots tended to be below the threshold of chemical detection. How- ever, when C- 14-labeled methanol was used in Experiment 10, autoradiographs revealed about thirty-two spots.dfter 36-days film exposure of a paper containing the unprocessed irradiated sample from Experiment 10 (Figure 2). Autoradiograms are on 14 x 17 inch x-ray film, exposed 36 days and developed under standard conditions. The paper was larger than the film, so that in the vertical direction the upper third of the paper is not represented. The original autoradiogram shows about twenty- four distinguishable spots, plus about eight more on the part of the paper not shown. It can be seen that all the compounds showed relatively high rates of movement so that none remained near the origin. Overloading of the paper caused elongation (streaking) of many spots. A large mass of material at the center of the upper edge of the figure caused distortion of the migration pattern over most of the paper and made identification of spots by comparison with standard maps almost impossible. This mass of products was considered to be sugars and organic acids from silver nitrate and other chemical tests. Further work with chromatographic

-9- separations of this type could be pursued to provide product identifications. This would involve additional separation steps either on the sample before spotting or upon the individual spots located upon the paper. Elution of individual spots for rechromatography with standards has not yet been undertaken. One spot on this paper alsc showed white ultraviolet fluorescence. When a portion of the sample from Experiment 10 was concentrated by vacuum evaporation prior to spotting, about twelve well-separated spots appeared on the autoradicgram {Figure 3y, one of which was near the methionine-valine map area and another near taurine. The remaining spots showed high Rf's. The difference between the two autoradiograms appeared to be due to losses of volatile constituents during the concentration procedure. These volatiles presumably were detected by the gas chromatograph, and may have been similar to compounds listed in Table V. A single dimensional (Solvent Dj autoradiograph of both original and hydrolyzed sample from Experiment 10 showed at least sixteen distinct separations in each with few changes after hydrolysis. The above data permitted numerous correlations between the

several assumed primitive earth forms of carbon used in this series. There were effects due to the specific form and effects from interactions between forms; with differences in the numbers, types, and quantities of products. Methanol was much more reactive than either cyanide or carbonate. The presence of carbonate resulted in more hydrocarbons, polyhydroxy alcohols and carbohydrates than did cyanide. Cyanide did not change alcohols, oxy compounds, acids, or sulfur compounds relative to methanol alone whereas carbonate reduced the numbers in all classes except acids which were slightly increased. Methanol, cyanide and carbonate together were the least produc- tive mixture, but still permitteq formatton of some thirty-five compounds, compared to none at all for carbonate alone in Experiment 5. Sugars and polyhydroxy alcohols were produced in great quantities; methanol alone being most favorable, closely followed by methanol-carbonate, The general indication is that the most products are formed from methanol, with added cyanide and carbonate reducing the overall reaction efficiency. However, a few more

- 10 - t

hydrocarbons and one additional phosphate hydrolysis product were formed in the presence of carbonate. It should be noted that methanol in the radioactive hydrothermal system represents the most reduced one-carbon form which is readily soluble in water. In Experiments 14- 17, the reactions of dicyandiamide formate and FeCl were investigated. Dicyandiamide, NH2-C(rNH)-NH-CN, is the 3 spontaneously formed dimer of cyanamide which has been proposed as a most probable primitive form of both carbon and nitrogen (3).- It is a probable precursor of such important cyclic nitrogen compounds as pyrimidines and purines. It is relatively reduced, while the other form of carbon used, formate, is more oxidized. By using C-14-labeled formate plus chemical tests, the individual products from the two carbon forme may be distinguished. Details of the composition of reaction mixtures VIP and VU.I are given in Table 11. Ferric chloride was also studied since iron is thought to catalyze many organic reactions such as the synthesis of cyclic nitrogen compounds. At the pH used, 8.5, the iron was present as a flocculent hydroxide precipitate, offering a possible catalytic site. In the irradiated Experiments 14 and 15, Nos. 14 and 16, a clear amber-yellow color was produced. Analysis of the samples by gas chromatography indicated (Table IV) only small number of volatile compounds were produced in contrast to the large number of cornpounds and classes in Experiments 8- 11 using methanol. Nothing was found in the control hydrothermal studies and only traces of three oxygenated compounds in the irradiation studies. The presence of FeCl was ineffective or slightly inhibitory. This indicates that it 3 was not catalytic and may have functioned as a radiation trap to reduce the yield. Another possibility is that products may have been adsorbed on the Fe(0H) 3 precipitate, which was removed by centrifugation before analysis. Formate-C-14 was used in Experiments 14, 15, and 16. The two- dimensional chromatogram s to gethe r with their autoradiogram s yielded much information. After 36 days film exposure, about thirty-two spots of C-14- compounds were seen on the autoradiogram of Experiment 14 (Figure 41, whereas

- 11 - the control hydrothermal Experiment 15 showed about nineteen labeled spots. The majority of the products in Experiment 14 and those in highest quantity

’ showed little mobility from the point of origin. By contrast, the methanol spots in Experiment 10 (Figure 2) demonstrated high mobility as previously mentioned and were, therefore, considered to be different compounds. About six spots coincided fairly closely between methanol, Experiment 10, and formate, Experiment 14. The hydrothermal products in Experiment 15 all corresponded in position to the irradiated ones, but were much less intense. It should be noted that the irradiation levels from the C-14-label in these experiments represented only a tiny fraction of 1% of the electron irradiation dosages used, so that the control hydrothermal products are probably not due to irradiation effects. The inhibitory effect of FeCl can be seen in Figure 5. 3 Ultraviolet examination of paper chromatograms revealed eighteen to twenty-two fluorescent spots in Experiment 14, about six plus traces of three or four more in Experiment 16 (with FeCl ) and none in the 3 control hydrothermal Experiment 15. The fluorescent colors were bright white, yellow, orange, green and blue, and one dark red spot. About six of spots in Experiment 14 corresponded in Rf and shape to the C-14 spots but the remainder were definitely different. This lack of correspondence indicated that most of the spots detected by UV came from dicyandiamide, that dicyandiamide carbon did not interchange with formate carbon, and that only about six compounds were either common to both precursors or were interaction products. In Experiment 16 with FeCl there were four such corresponding 3 spots. The combined radioactive and fluorescent data yielded a total of 48-54 products in irradiated Experiment 14, 31-35 in irradiated Experiment 16 with FeC13, and nineteen in the control hydrothermal Experiment 17. Dicyandia- mide reaction was greatly depressed by FeCl and was absent in the hydro- 3 thermal study, whereas formate reactivity was higher in all three. Several unsuccessful attempts have been made to correlate the UV fluorescence data with ten purine and pyrimidine standards in both the presence and absence of dicyandiamide on the paper. None of the standards had bright fluorescences resembling those observed.

- 12 - t

In Experiments 18 and 19, carbon was introduced in the form of chromium carbide (Cr C ). This compound is quite insoluble and was 32 present as a powder slurry which tended to settle to the bottom of the dish in spite of magnetic stirring. Therefore, its irradiation and reactions were much less than optimal. Sulfur was omitted because of the insolubility of chromium sulfides, Heavy metal and other carbides are probable primitive earth compounds and are present in some meteorites (4).- The heavy metal compounds react somewhat differently from the well-known calcium carbide which hydrolyzes explosively at room temperature to acetylene. Chromium carbide hydrolyzes at about five hundred degrees to form higher-weight hydrocarbons. Other members of the class, especially iron carbide, might be even more interesting, but sources of supply have not been located. During irradiation the pH of the solution of Experiment 18 went from 8.5 to a final value of 2.8. The acid anion has not been identified; simple volatilization of ammonia may have been involved. The gas chromatograph detected no compounds in neither Experiment 18 nor 19. Table VI1 shows some product formation from both irradiation and hydrothermal studies as revealed by paper chromatographic analysis. It has not yet been possible to identify any specific compounds. It is thought that improved experimental conditions, including increased radiation absorption in the solid as well as the use of other metal carbides, will increase the product yield from this interesting class of compounds, In Experiment 20, the irradiation of methane, the major reaction product was a brown, insoluble polymer, similar to the polymers reported by others for gas phase reactions. This polymer has not been analyzed. The fact that the radiation dose to the gas phase was two orders of magnitude less than that to the aqueous phase indicates that the polymer may have formed in the aqueous phase. If so, then it is probably not a simple hydrocarbon but more likely contains oxygen in addition to carbon and hydrogen.

- 13 - I I. 1

I Table VIII presents the compounds identified by gas chromatographic analysis. Since the reaction mixture was simple, and especially since it lacked active forms of nitrogen, the variety and number of products was expected to be smaller than in earlier experiments. Methane, however, represents a highly probable form of primitive earth carbon and furthermore is the most reduced one-carbon com- : pound. The two oxygenated products found deserve special attention, since they usually were not determined by other workers. The production of methanol is most significant since it is an obvious probable radiation product from methane, and methane-containing atmospheres are very frequently postulated in origin of I life studies. Its identification, then, is in line with theoretical expectations, Further, its presence as a primary radiation product justifies the inclusion of I 1 I methanol in second-stage irradiation studies such as those reported in Experi- ments 6-11. Methanol was shown to be highly reactive upon irradiation, leading I to large numbers of more complex products. The small amount, only a trace, found in Experiment 20 is therefore to be expected since the major portion of methanol formed would go on to react further. It is also important to observe (Table VU) that methane reacted to form the carboxyl radical of acetic acid in the absence of nitrogenous or other reactants. The deviation of the nitrogen to oxygen gas ratio from the normal atmospheric value of approximately four to the observed value of 11. 3 deserves an explanation. This high value is an indication of the participation of air oxygen in the radiation induced reaction. The nitrogen is presumed to be unchanged and the oxygen depleted by reaction. A rough calculation indicated that approximately 60% of the oxygen in the irradiation capsule must have reacted; a trace of hydrogen gas was also observed. The two major hydrocarbon products were ethane and propane, both paraffinic hydrocarbons. It is interesting to note that very little olefin was produced. Thus, only a trace of ethylene was detected, while no propylene was detected at all. In addition, only very small traces of C and C olefins were detected. 4 5 These results are consistent with the fact that olefins are very reactive and would be expected to condense further with most reactive species present.

- 14 - In Experiment 22, acetylene was irradiated since acetylene is a product of hydrolysis of certain metallic carbides and carbides are primitive earth compounds, previously discussed. Acetylene is, therefore, a very reactive primary primitive product that might lead to the production of aromatic hydrocarbons. Calculations of absorbed radiation dose from relative electron densities of gas and liquid phases indicated that the gas phase received 0. 34% 5 of the aqueous dose, or 3.4 x 10 rads. Therefore, the gas phase reactions can essentially be ignored, and the system considered to be an aqueous solution of acetylene. The water solubility of acetylene is some eleven times greater than methane, or 100 cc per 100 ml water at 18OC. As in the methane study (Experiment 20), a brown polymer was observed to be the major irradiation product which has not been analyzed. Products identified by gas chromatography are shown in Table IX. Methanol and acetic acid were the only oxygenated products detected in this experiment also. The considerations regarding methanol are of the same great significance as in Experiment 20. However, the acetic acid could be classified as a major product in this run, since the yield was about seven times greater than obtained in Experiment 20. No hydrocarbons were formed as major products. There are two possible explanations for this finding. First of all, the amount of acetylene starting material was less than that used for the methane experiment (No. 20), since acetylene was only added at atmospheric pressure. Secondly, because of the high solubility of acetylene in water, most of the acetylene probably disappeared by a polymerization reaction which took place in the aqueous phase. This is further confirmed by the presence of insoluble brown polymer. Since no unreacted acetylene was detected in this experiment, all of it was apparently used up in the radiati on-induc ed reaction. Amino Acid Production. As mentioned earlier, paper chromatographic methods indicated the presence of many ninhydrin-reactive products in several of the experiments, but the patterns were too complex to permit simple comparison with standard maps and a gas chromatographic method employing n-butyl-N-trifluoracetyl derivatives (l),- as well as a Spinco Amino Acid Analyzer, were utilized to delineate amino acid production.

- 15 - .

Table X lists fourteen of the common amino acids that were produced in one or more of the radioactive hydrothermal experiments. The products included both of the sulfur amino acids, cysteine and methionine. Five common amino acids, arginine, aspartic acid, histidine, phenylalanine and tryptophan, were not found. In the control hydrothermal experiments (non-irradiated), only very faint traces of alanine for Experiment 9, leucine-isoleucine for Experiment 15, and of two unknowns for Experiment 17 were found. The -5 amounts of each were less than 5 x 10 gm per ml original sample. This lack of hydrothermally produced acids both indicates the efficiency of ionizing radiation in promoting reactions and confirms the purity of the original reactants. The identifications of amino acid products, Table X, have elements of uncertainty inherent in the gas and column chromatographic methods. The parameters for identification are elution volume or retention time compared to known compounds. In these methods the retention is rarely unique (peaks may correspond to two or more compounds), data for all possible standards may not be available, and column reproducibility is not absolute. The numbers of small peaks were large and there was much overlapping between adjacent peaks so that although a peak was clearly present, assignment of a specific structure was less precise. Quantitation of amount of compound was based upon peak area multiplied by sensitivity and dilution factors. The numbers from the Amino Acid Analyzer runs are only approximations. The gas chromatographic curves can be read for similar quantitative values if necessary, but have only been reported as indicating major and trace products. A report of Tr indi- -4 cates about 5 x 10 gm per ml original sample. Comparisons between the two methods are given in the columns for Experiments 8 and 14 in Table X. The correlation is good for Experiment 8, and not as good for Experiment 14. However, the large number of unidentified peaks in the Amino Acid Analyzer results indicated the uncertainty to be in compound identification rather than in the presence and absence of peaks. The absolute sensitivities of the two methods for amino acids seems

- 16 - to be nearly the same. We have determined the gas chromatography method -5 limit of sensitivity to be 5 x 10 gm per ml of original sample. This can be reduced by using larger samples. The larger number of peaks, mostly unknown, obtained on the Analyzer probably reflects the sample preparation method. The process of derivative formation for gas chromatographic analysis would eliminate many or most non-amino acid compounds which, however, would be reactive with ninhydrin on the Analyzer. These additional ninhydrin compounds are presumably mostly amines. It would be of interest to study the amines further as a class of protobiochemical compounds. Table X permits comparisons of efficiencies in amino acid production to be made between the simulated primitive reaction mixtures. In terms of the form of carbon, methanol is clearly the most effective reactant both in number of acids and in quantity. The addition of cyanide to methanol, Experiment 8, results in little or no change of major products but a decrease in minor unknown compared to methanol alone, Experiment 10. This is rather surprising; it was expected to furnish both C and N. Carbonate clearly inhibited amino acid formation in Experiment 7 and yielded none at all in Experiment 5. In all of these experiments, ammonia was also present as a source of nitrogen. The dicyandiamide-formate combination, Experiment 14, produced some acids, but less than methanol. With this mixture the addition of FeCl as a possible catalyst, Experiment 16, seems to have decreased the 3 yield of leucine-isoleucine and proline, increased the yield of valine, two unknowns, and possibly glycine and to have increased the total number of gas chromatographic peaks. No comparison of total peaks for the Analyzer data was possible. By performing suitable paper chromatographic studies of the samples from Experiments 14 and 16 it would be possible to determine whether the carbon precursor was dicyandiamide (non-labeled) or formate (C- 14- labeled) Chromium carbide, Experiment 18, was unproductive although further work on this system is indicated.

- 17 - Peptide and Proteinoid Production. In Experiments 12 and 13, eighteen common amino acids in aqueous solution in the proportions of mixture B of Fox and Harada (5)- were studied. The concentrations were such that the solution was almost saturated at 55OC in respect to glutamic and aspartic acids. The irradiated solution, Experiment 12, was a clear amber color with a strong odor as of burned meat, while the hydrothermal solution, Experiment 13, was water-white and odorless. Each solution was dialyzed several days against six changes of distilled water at 4OC until the final dialysate gave a negative ninhydrin test. The dialysate of the indicated Experiment 12 contained part of the original amber color which dialyzed slowly over the first five changes of water. The slowness probably indicated a relatively high molecular weight material. Both sample and dialysate retained their odor and color. During dialysis a brown-purple precipitate slowly formed. The sample from the control hydrothermal Experiment 13 remained colorless and without precipitate. W and visible spectra of the dialyzed samples and dialysates show generalized absorption below 400 mp with traces of peaks or shoulders. These spectra were consistent with spectra of proteins and peptides. The precipitate and dialyzed fractions of Experiment 12 were similar and showed much stronger absorption than the dialysate; both revealed a shoulder at 345 mp and a trace at 300 mp and the precipitate showed another trace at 425 mu. Dialyzed control Experiment 13 had a small concentration of material absorbing below 300 mp with a small peak at 232 mp. and a shoulder at 258 mp, Microscopically, the precipitate of Experiment 12 appeared as small, amber, roughly spherical particles about 0.9 micron in diameter which tended to form loose clumps. Superficially, they resembled the proteinoid particles of Fox, the spherules of Grossenbacher and Knight, and the microspheres of Young (6).- The precipitate formed at pH 5.5 and was soluble in dilute NaOH at pH 8.5. Figure 1 shows a photomicrograph of the

- 18 - precipitate from Experiment 12. This non-dialyzable fraction was interpreted as higher polymers probably representing molecular weights of ten thousand and higher. The precipitate and the soluble non-dialyzable fraction of the sample from irradiated Experiment 12 were redissolved in 0.9% saline at pH 8.5 and fractionated on an 8 x 145 mm Dowex AG 50W-X2 resin column in an attempt to examine the molecular weight distribution. The method is essentially that of Moore and Stein (1,E) wherein elution by sodium acetate separates small peptides containing up to eight or ten acids in the order of increasing weight. It was assumed that dialysis had removed lower peptides, Gradient elution at room temperature was employed starting with 0.2 N sodium acetate, pH 3.2 and increasing to 4N sodium acetate, pH 10.5. The soluble non-dialyzable fraction was eluted with a total volume of two liters and reached a final effluent pH of 5.7. Peptides in the eluted fractions were detected spectrophotometrically at 260 millimicrons (mp). Peptides were eluted continuously in the first 75 fractions, up to about 1400 ml and pH 5.4. The concentration was essentially constant, although there was a slight peak near pH 3.5-3.8 and a gradual decrease between fractions 50 and 75. No peptides were observed in the final 600 ml. The precipitate fraction eluted continuously over the entire range of 1700 ml and final pH about 6.6. The elution was discontinued at this volume although compounds were still being removed. There were slightly elevated concentrations in the first 70 ml and in the 230-300 ml fractions. Concentrations were too low to determine anything but major differences. Slightly higher absorption at 260 mp was observed for fractions 3 and 4, but no other differences were seen. These results were indicative of the presence of a wide range of polymer sizes in both the soluble and precipitate fractions of the sample from Experiment 12 with signs of some small tendencies for preferred structures at one or two weights. In addition, the columns may have retained considerable uneluted material. Fox (9)- has postulated considerable direction or selectivity in anhydrous thermal polymerization of amino acids. Aqueous systems under the influence of ionizing radiation may yield a wider variety of free radicals and other fragments which would lead to a correspondingly

~ i broader range of products. The above data are consistent with this possibility I although further work on fractionation systems is indicated. The several fractions of the sample from Experiment 12 after dialysis were hydrolyzed with HC1 for paper chromatographic study of I changes in ninhydrin-reactive spots. The results have been inconclusive to i date, possibly as a result of incomplete hydrolysis.

- 20 - DISCUSSION The results of this initial one-year study on the role of radioactivity and hydrothermal processes in protobiochemistry demonstrate that an abundance of organic micromolecules as well as complex macromolecules can be produced in hot aqueous solutions under the influence of ionizing radiation and establish the importance of a hydrothermal radioactive model of primitive planetary environments for laboratory or ground-based studies of origins of life. To Oparin (10-13) science owes the origin of credibility to propositions that life on the planet Earth may represent a natural and inevitable consequence of chemical abiogenesis, molecular evolution, macromolecular generation, and prebiotic organization, although recognition must be acknowledged that origins of life may have resulted from intervention of a supernatural force, a matter of theology rather than science, or implantation of life or the prerequisites thereof from extra-terre stria1 sources, accidentally or deliberately via meteorites, comets, or visitations by galactic civilizations. In the absence of information about the metabolic processes, the nutritional requirements, or the ecological limitations of the earliest replicating organisms (life-forms) on Earth, investigations of the origin of life have generally involved studies of chemical abiogenesis or molecular evolution, or the steps by which biochemical compounds as we know them may be formed in prebiological planetary environments by sequential physico-chemical processes. The original classic work of Oparin in 1924 approached the subject of the origin of life largely from a biophilosophical standpoint; since expanded. Blum (14,-- 15) presents a masterful review of modern scientific developments and uses "time's arrowIs, the second law of thermodynamics, to point the way for evolution, both chemical and biological, although his regard for equilibrium may be oversimplifying in a system which has existed for only five billion years in an infinite continuum of time. Other notable discussions related to the origin of life hypotheses have been provided by Haldane (16),- Bernal (17),- Horowitz (18),- Simpson (191,- Urey !s-218, Calvin (22 23), Fox (9 24-26), Pirie (27), Oro (281, Moore and Jackson (291, -7 - -7 - - - - Firsoff Keosian (31). Many points of view, often conflicting, were brought (30),_. -

- 21 - together at a New York Academy of Science Conference in 1956 (321,- at an Inter- national Union of Biochemistry symposium in Moscow in 1957 (33),- and at a conference organized by Florida State University and the National Aeronautics and Space Administration in 1963 at Wakulla Springs, Florida (6).- From a criti- tal review of these and other sources of material, it seems clear that elucidation of the primordial processes which have led to life on earth, and which may have led to replicating biosystems in other parts of our solar system or others, must proceed along two principal lines: (a) the study of chemical processes occurring in non-living systems (gas, liquid, or solid) which are actually obtained from primitive planetary environments, or which simulate primitive planetary systems in great detail, and (b) the study of the most primitive available biological systems in the most primitive planetary ecological conditions capable of sustaining their survival, and perhaps exploring sequentially the development of species capable of thriving under primitive planetary conditions, Abiogenic Organic Synthesis. The first report on the production of biochemically significant organic compounds from presumably primordial or primitive chemical resources was made by Lzb (34)- who synthesized the amino acid, glycine, from a mixture of carbon monoxide, ammonia and water with an electric discharge. However, abiogenic origins of biochemicals were not seriously investigated until Calvin, et a1 (35)- in 1951 reported the reduction of carbon dioxide and water to formaldehyde and formic acid with ionizing radiation (helium ion bombardment in a cyclotron) and Miller (36-38) as early as 1953.synthesixed amino acids by electric discharge in mixtures of methane, ammonia, water, and hydrogen (reducing mixtures of simple gases). Numerous other reports on chemical abiogenesis have appeared in the recent literature (see 6, 9, 13, 28, 31, and 33 for reviews). Surprisingly, little effort has been made in most studies to simulate in detail the actual chemical and physical conditions characteristic of the formative stages of Earth. Experi- ments in aqueous systems at elevated temperatures (36-43) are suggestive of an important role of hydrothermal activity in the evolution of chemical species, as has long been proposed (44).- Ubiquitous hydrothermal activity was certainly a

- 22 - c

prominent feature of the early Earth, and considering that the most primitive known organisms are those found in modern hydrothermal waters, these systems (either actual or synthetic) have received surprisingly little attention. Experi- ments with ionizing radiations (35 45-53) likewise are suggestive that natural -7 - radiation chemistry cannot be discounted as a ---sine qua non of chemical evolution. In addition to providing an abundant source of heat and water for thermal aqueous reactions, the natural hydrothermal and related volcanic systems are associated even in modern times with abnormal concentrations of naturally radioactive materials. Similarly, if one accepts for the sake of argument an abiogenic origin of petroleum (54),- the physical association of petroleum with high-temperature and high-pressure phenomena and its occurrence in intimate mixture with abnormally radioactive waters (55)- suggest means by which organic solvent systems may have participated in important thermal and radiation-induced reactions. The work of Schramm et a1 (56)- has demonstrated the nonenzymatic synthesis of polysaccharides, nucleosides, and nucleic acids in organic polyphosphate- containing systems at elevated temperatures. The existence of such compounds in the scheme of chemical evolution might be difficult to rationalize in terms of aqueous or gaseous (atmospheric) systems. Leaving aside the question of pregeologic formation of simple organic molecules, if one assumes that the principal processes of chemical evolution have occurred since the earth's formation, a number of conditions may be listed which must have been satisfied at some time period during the earth's history -- not necessarily simultaneously, not necessarily on a global scale, but at least in some satisfactory time sequence, and in (perhaps extremely) localized regions of the earth's atmosphere, hydrosphere, and lithosphere. Among these conditions are the following: (a) availability of energy to meet the thermodynamic demands of abiogenic synthesis and chemical evolution; (b) availability of energy -of activation for the evolutionary chemical reactions; characteristically, the activation energy requirement at the

- 23 - 1 molecular (highly localized) level is more stringent than the thermodynamic energy consumption; (c) concentrations of reacting species sufficiently high to meet the demands of reaction kinetics; for example, in the formation of a given peptide link in a mixture of two species, the rate (or probability) of bond formation is proportional to the concentration product of the two, as suggested by biomolecular reaction kinetics, and the formation of large poly- peptides and protein molecules will similarly be a multipowered function of c onc ent ra t ion; (d) inorganic species, e.g., Ca, Fe, P, or inorganic ele- I ments capable of performing functions analogous to those attributed to such elements in modern living systems; (e) water is certainly required at some stages of the evolutionary process; (f) relatively anhydrous conditions, either in solid deposits or in organic systems (or both) are favorable to some of the critical processes in chemical evolution. Each of these conditions has been met at one or more evolutionary stages of Earth, and representative systems of each can in fact be observed on the modern planet, although not necessarily in the proper geographical inter- relationship to support a "continuous creation!' process, whereby all stages of chemical evolution can be observed in any selected current ecological system. The thermal processes accompanying magmatic crystallization appear to offer the most concentrated energy source available to meet the thermodynamic requirements of chemical evolution, and the accompanying hydrothermal processes offer relatively concentrated aqueous solutions of the inorganic species required for the building up of complex bio-organic species, While the role of the atmosphere is limited by its elemental composition, such significant elements as Fe, Ca, and P are abundant in magmatic and hydrothermal deposits (calcareous tufas) provide a medium for anhydrous processes, with or without the participation of hydrocarbon solvents. I The importance of

- 24 - I alternate wet-dry processes in protein and polysaccharide synthesis has been I generally recognized, and is discussed in some detail by Blum (15).- In addition, I the significance of modulation from a hypohydrous to an aqueous system has been

~ discussed and tested by Fox 24 25, 43). (3-3 - - I Radioactivity in the Primitive Planetary Environment. Throughout the I history of Earth, all of the familiar energy sources have been available for several billion years; whether one chooses to believe that solar radiation, electric discharge, cosmic radiation, natural radioactivity, or heat supplied

, supplied the thermodynamic energy of formation of the first self-replicating I molecular systems, it is unlikely that the amounts of energy involved would be I 1 too small to account for the creation of "life" in the periods of time available. In any case, it is clear that all of these sources could have played a role in chemical evolution, both concurrently and sequentially. It is suggested, however, that a quantitative comparison of the magnitude of energy input to the earth from each of these sources is not entirely relevant to the assessment of their relative roles in the processes of chemical evolution. Such comparisons have been made by Miller and Urey (57)- and more recently by Calvin (23),- indicating that natural radioactivity, even in the early earth, could have contributed only a few percent of the total energy available to the outer earth. Since proper combinations of localized energy sources and elemental concentrations are required to explain the observed results of chemical evolution, it seems more fruitful to examine the localized sources of energy in regions of suitable elemental composition. Thus, it is perhaps more important to examine natural radioactivity as a localized source of activation energy for rather improbable reactions than to relegate it to a secondary position because it represents a small fraction of the total energy available on a global scale. Although current levels of natural and artificial radioactivity in man's environments are under extensive investigation, very little is known about the environmental radioactivity on this planet in its primitive stage of development. An attempt can be made to extrapolate the present environmental radio-

- 25 - activity back to a few billion years ago. The results indicate that the radioactivity of the primitive Earth may have been several hundred to several thousand times higher than that of man’s contemporary environment, and perhaps still much higher in localized regions. The satisfactory agreement between the predicted and observed I radiocarbon contents of organic materials of historically known age, as demonstrated by Libby (58),- assures that the cosmic ray radiation intensity has been : approximately the same present, within experimental error, during the as at 4 past 10 years or so. In calculating the cosmic-ray exposure ages of meteorites, it is often assumed that both the cosmic-ray intensity and its spectrum have remained constant in time. This assumption appears justified as long as a very I short geological time is concerned, since a number of cosmic-ray induced radioactivities with half-lives up to of the order of 106 years have been found to

, occur in meteorites in steady-state proportions (59).- Kenna et a1 (60)- have recently reported that the Tc-99/U-238

I ratio in Katanga pitchblende is approximately the same as that calculated from the spontaneous fission half-life of U-238. The T c-99/U-238 ratio in pitchblende is approximately the same as the equilibrium ratio Mo-99/U-238 in depleted uranium, reported earlier by Parker et a1 (61).- These results suggest that the neutron flux in the uranium ore has been approximately the same as at present 6 during the past 10 years or so. From this information, one can be reasonably certain that the cosmic-ray flux on the surface of Earth and the rate of neutron production in rocks must have been approximately the same as at present during the past 6 10 years or so, which is very short indeed compared with the lifetime of Earth, i.e., 4.5~109 years. There are a number of experimental indications, however, that the rate of neutron production in the lithosphere must have been much greater 8 9 10 to 10 years ago than at present. Wetherill and Inghram (62)- studied the abundances of stable xenon isotopes in pitchblende and calculated the degree to

- 26 - *

which the pitchblende deposit was an operating pile. They stated that the deposit was an operating pile. They stated that the deposit was twenty-five percent of the way to becoming a pile, and, if one extrapolates back 2000 million years where the U-235 abundance was six percent instead of 0.7, such a deposit would be closer to being an operating pile. It has long been speculated that at least part of the heavier isotopes of xenon in the earth's atmosphere are fissiogenic, but it was not possible to treat this problem on a quantitative basis until Reynolds (63)- showed that the abundance ratio of the stable xenon isotopes in the Richardton meteorite was different from that in Earth's atmosphere. Kuroda (64)- reported that the difference indicates that at least 10 percent of the atmospheric Xe-136 is fissiogenic. This is much greater than that expected from the U-238 spontaneous fission alone, but can be explained as due to the spontaneous fission of some of the extinct transuranium elements and/or the induced fission of U-235 in the early history of Earth. The contribution from the natural fission processes is estim- 6 ated to be at least 10 times that from the recent artificial nuclear explosions. Kuroda (65) postulated that the extinct transuranium nuclide, 7 Pu-244, played the most important role in the production of the fissiogenic xenon in Earth's atmosphere, and calculated the time interval between nucleo- synthesis and the formation of the planet from the inventory of the fissiogenic xenon in the atmosphere, neglecting the contributions from the U-235 neutron- induced fission and other extinct transuranium elements. Although it is difficult to know which fissionable nuclide was chiefly responsible for the production of the excess;fissiogenic Xe- 136 in the earth atmosphere enables one to calculate a minimum number of fission events which took place in the lithosphere during the entire history of the earth. The 34 number of atoms of fissiogenic Xe-136 in the earth atmosphere is 7. 3x10 (66). Assuming that the fissiogenic Xe-136 was produced in an amount of rock equiva- 24 lent to the mass of the earth's crust, 23.7~10 grams, the fissiogenic Xe-136 9 production per gram of rock is 3.1~10 Xe-136 atoms/gram rock.

- 27 - Further, assuming the fission yield for the mass 136 chain to be 6 percent and the number of fission neutrons produced per fission to be 2. 5, 12 the total number of fission neutrons produced is 1. 3x10 fission neutrons per gram rock. If the spontaneous fission of Pu-244 was chiefly responsible for the production of the fissiogenic Xe-136, the fission event calculated above must have occurred during a period which is comparable to the mean life of Pu-244, 8 or the first 10 years of the earth's history. Thus, an average rate of neutron 8 production during the first 10 years of the earth's history can be calculated as

12 8 1. 3x10 / 1x10 (neutrons/year/gram rock), or 12 1. 3x10 -4 = 4.1~10 (neutrons/ sec/gram rock), (3.16~10~)( lo8)

If, on the other hand, the neutron-induced fission of U-235 was chiefly responsible for the production of the fissiogenic Xe- 136 in the atmosphere, the fission events calculated above must have occurred during a period which is 9 comparable to the mean life of U-235, or the first 10 years of the earth's his- -5 tory. In this case, at least 4.1~10 neutrons per second per gram rock must have been generated from the induced fission of U-235. The rates of neutron production in the early history of Earth calculated above are far greater than the number of neutrons produced per second per gram of contemporary rocks. According to Inghram (671,- this rate -2 -7 is about 5x10 n/gm sec. for a pitchblende and about 10 n/gm sec. in ordinary rock. The above calculation indicates that the neutron density (number of neutrons produced per gram second) in the crustal rock must have been several hundred to several thousand times greater in Earth in its primitive stage of development as compared with the contemporary crustal rocks, and that radioactivity levels in Earth's crust and atmosphere as a consequence of natural fission might have made a substantial contribution to the total environmental radioactivity pattern.

- 28 - I : I The environmental radioactivities of the atmosphere and the hydrosphere of the primitive earth should also be considered. Most accept the view that Earth's atmosphere is of a secondary origin, i. e., the atmosphere was formed by the outgassing of the crust and mantle in the early history of the

I planet, I The following simple relationship holds between the number of atoms of Ar-40 in the atmosphere (Ar-40 ) and the number of atoms of K-40 in A ), as has recently been pointed out by Kuroda and Crouch the crust today (K-40 C (66):-

(AK t X jT BxKtx ) (T-t) (Ar-40)A = (K-40)c e P -e P xK E XKt xP where X and X are the decay constants of K-40 for K-capture and beta decay, K P 9 respectively, T is the age of Earth, 4.5~10 years, and t is its major out- 9 gassing period, which is taken to be a period of 2x10 years following formation. 41 Since (Ar-40)A=9. 85x10 , the average rate of escape of Ar-40 Q from the surface to the atmosphere during the first 2x10' years of Earth's history is 41 9.85~10 - 32 = 5x10 atoms Ar-40/year. 2x 109 On the other hand, Damon and Kulp (68)- have estimated that at 30 the present time the crust is outgassing argon at a maximum rate of 1.5~10 atoms/year. Thus, it would appear that the average rate of outgassing of Ar-40 from the primitive planet was a factor of 300 greater than the current maximum rate of outgassing. This would indicate that the average rate of outgassing of Rn-222 from primitive Earth was also greater by a similar factor than that of today, and hence the radon concentration in the atmosphere of primitive Earth could have been 300 times greater than that of the contemporary planet. Radon concentrations of many of the contemporary thermal waters -8 are known to be as high as to 10 curies/liter (55).- Thermal waters with extremely high radon concentrations are known in various localities of the world.

- 29 - .

-6 In some of the extreme cases, radon concentrations exceeding 10 curies/liter are observed in a number of thermal waters. If it is assumed that the radon concentrations in thermal waters of primitive Earth were on the average a factor of 300 higher than that of today's thermal waters, some of the primitive thermal waters could have contained as much as one millicurie of radon per liter. Similarly, concentrations of radon and other volatile naturally radioactive elements may be assumed to have been higher by two or more orders of magnitude in primitive volcanic gases., Modern observations (55)- have demon- -4 strated local concentrations higher than 10 curies per liter of gas. Primitive concentrations might well have exceeded several millicuries per liter over periods of millions of years in regions of high volcanic and hydrothermal activity. Hydrothermal Radioactive Model Considerations. It is generally presumed that the initial processes leading to the emergence of life required the formation of relatively simple organic molecules such as amino acids, carbohydrates, purines, pyrimidines, etc., which, as they are known today, were the building blocks of proteins, nucleic acids, and polysaccharides, In addition, the availability of high energy phosphorus compounds, specific metal catalysts such as iron porphyrins, etc., were apparently required. It is evident that the initiation of biochemical evolution would depend upon the availability of appropriate chemical starting materials and sources of energy. Such thermodynamically improbable events as origins of life were probably local phenomena. It would follow that they would not necessarily occur to an extent proportionate to an equal distribution of materials and energy sources but rather may have represented outgrowths of effective isolated environments far less encompassing than the entire prebiosphere. An effective community may not have resembled the whole environment, it may have been more or less acid or alkaline, reducing or oxidizing and supplied with different elements from those we recognize as specific or vital in our biosphere. Vanadium, for example, may have served the same function in protobiology that iron does today. Primitive species of tunicates are known that contain large

- 30 - containing sponge that lodged in primitive gill arches of fish (71).- Suggestive evidence of other examples of biochemical complexity exist that may have pre-

we recognize in contemporary life systems. Many original vital activities from the development of early life systems may have been lost and the relative importance of others probably altered. For example, a precarious system dependent upon vanadium in strongly acid solution would have found the more

? common element iron to serve the same function more safely. Thus, investigations of protobiochemistry should not exclude consideration of the factors in local and even perhaps adverse environments that might have led to protobiological systems and should not be restricted to studies of models that necessarily represent the average primitive conditions, at best hypothetical, and rely on only the extant relative biochemical simplicity as guidelines. It would thus seem necessary to search in the inorganic world for the origin of the processes and the materials that couId have led to the organic world and to search in the primitive environments for possible energy sources. It would be equally significant to research the biochemical genealogy of terrestrial life for traces of more primitive processes and materials than are now ordinarily observed on the earth. Previous investigations have utilized model reactions of protobiochemistry utilizing energy sources from electrical discharge, ultraviolet irradiation and ordinary sunlight, heat, and ionizing radiations, as discussed previously. Considerably more attention has been given to the first three than to ionizing radiation as likely energy sources for actual primordial synthesis. When ionizing radiation is employed experimentally, it is commonly because it is a convenient high energy source, and, as stated by Calvin (23),- !'there does not appear to be any marked difference between the products from one type of high-energy radiation and those of another".

- 31 - The failure to attribute a significant vital role to ionizing radiation as an energy source on the primitive earth may be due to several inappropriate I assumptions. The fact that the cosmic-ray radiation has contributed little energy to the earth has precluded serious consideration of ionizing radiation as a factor I in models by several investigations. However, radioactivity in the earth's crust 1 9 1I from K-40, for example, would have contributed appreciable energy 4.5~10 I

I years ago. Synthetic evolution models of the earth's crust, however, have not

~ been pursued. Likewise, it is frequently stated that theories dealing with the

I evolution of life on earth ought of necessity be based upon the availability of ultraviolet radiation as the primary source of energy. This prime role is assigned to the ultraviolet and solar radiation because the calculated sources 2 of energy averaged over the earth (cal/cm /year) show the following approximate relationships (72):- sunlight, 260, 000; ultraviolet light below 2500 AU, 570; ultraviolet light below 2000 AU, 85; ultraviolet light below 1500 AU, 1.6; lightning, 0.9; cosmic rays, 0. 000075; and K-40 in earth!s crust 9 (2.6~10 years ago), 24. Although the solar radiation seems to have been the principal source of energy, photochemical reactions would have taken place principally in the upper atmosphere where reacting materials were scarce and product degradation could be expected. There is no doubt that solar radiation currently supplies the energy for life on earth, but this has probably been true only since photosynthetic mechanisms were developed. 2 If the total ultraviolet light input to the earth is 650 cal/cm / 4 year and the energy is absorbed within a column of air 10 miles (1.6~10 2 3 meters) above 1 cm of earth (1.6~10 liters of air), the average available energy would only be of the order of 0.4 cal/liter/year). As pointed out earlier, radon concentrations of 1 millicurie per liter, and perhaps concentrations many orders of magnitude greater, reflect the rate of radioactivity outgassing from primitive Earth. One Rn disintegration yields an energy of about 20 Mev and 7 15 1 mc (3.7~10 dps or about 10 disintegrations per year) would be roughly 16 equivalent to 2x10 Mev/year, 3.2~10" ergs/year, or 800 cal/liter/year. Thus, it can be seen that the concentration of energy from natural radioactivity at sites of outgassing would exceed the widely distributed levels of solar radiation energy by factors greater than a thousand.

- 32 - It should also be noted, as described above, that local sources of chemicals and energy may have been much more important to rare local origins of life than the distribution of the same factors within the total atmosphere or over the entire surface of the earth. Of course, outgassing proceeded from innumerable sites and certainly more than one energy-favorable source may have existed. Furthermore, hydrothermal waters also presented equivalent concentrations of radioactivity, greater than 1 mc/liter. Hence, the combined radioactivity of hydrothermal systems (gases and liquid) represented relatively high levels of local energy sources suitable for abiogenic chemical s ynthe s e s The primary difference between radiation chemistry and photochemistry is the method of energy absorption, and consequently the primary products of the interaction. In photochemistry, energy is absorbed by a resonance absorption mechanism with the initial products being excited molecules and radicals. In radiation chemistry the interaction with matter is coulombic in nature. This leads to the following primary produced species; ions, electrons, excited molecules and radicals. The ions and molecules thus formed are in various states of excitation. The electrons produced also have a wide range of energies. These species will yield additional ionized and excited ions, molecules, etc. The contribution of excited particles to the overall chemical effects is considerably greater in radiation chemistry than in photochemistry. The high specific absorption (i. e., energy absorbed per unit path length) of ionizing radiation provides local concentrations of excited species far in excess of those obtainable from light absorption. Consequently, the probability of any predictable llfollow-reactionllinvolving two or more excited species would be higher by orders of magnitude if one assumes that the usual laws of polymolecular reaction rates apply. Again, the emphasis is not on equilibrium, but on reaction kinetics (probabilities).

- 33 - It is significant perhaps that the most common primitive organisms now on Earth are frequently found in areas where radioactivity and hydrothermal processes are present, i. e., in hot springs and geysers. It can be safely assumed that no truly primitive organisms have yet been identified on earth. The lower organisms, as determined on the basis of multicellular differentiation, have sustained certain features of an anaerobic era in geologic or protobiologic time, i. e., before the appearance of oxygen in large amounts. The anaerobic photosynthetic bacteria and unicellular algae may be considered to possess metabolic characteristics representative of a preaerobic era. Some of these organisms thrive only in the absence of oxygen but are n0t killed by mild aerobic media. Others are aerobic organisms but continue to thrive under anaerobic conditions. Researches of such examples of border-line organisms may permit detection of contemporary remnants of much earlier biochemical evolution. For example, paleontological evidence suggests that blue- green algae are among the most ancient organisms; calcareous deposits found in pre-Cambrian rocks are attributed to representatives of this group. Today, the blue- greens carry on the same type of photosynthesis as do higher plants. Recent studies of species of greeh and blue-green algae and dino- flagellates have uncovered an intriguing variety of transitions and recombinations between aerobic and anaerobic metabolic patterns. The blue-green algae also appear to be structurally more primitive than the red and green marine algae although they all are capable of performing the complete process of photosynthesis. Definite nuclei and chromosome systems are demonstrable in fungi and in algae but not in the blue-greens. Accordingly, the blue-green algae are placed in the evolutionary scale before the development of genechromosome mechanisms of inheritance. Likewise, it should be noted that green and red photosynthetic bacteria are capable of performing part of the photosynthetic conversion process; they transfer electromagnetic energy from the sun into chemical energy without the evolution of oxygen by using reducing agents other than water in the reduction of carbon, and hence they produce oxidants other than oxygen.

- 34 - Thermophilic algae and bacteria provide exceptions to the rule in biology that the upper temperature limits of life lie near the temperature at which common protein coagulates. Thermophilic algae belong exclusively to the Myxo- phyceae, one of the most primitive classes of algae. Identical species can be found in the hot springs and geysers of Iceland, New Zealand, and the western United States. The waste products formed by the hydrated cadavers of such algae in thermal water is often a viscous unctuous material to which the "healing properties" of the water s are attributed. Thermophilic bacteria, capable of growth at temperatures up to and over 65OC, are aerobic spore-formers of the genus Bacillus and belong mostly to the species Bacillus stearothermophilus and Bacillus coagulans (73-75). Such bacteria may not only be found in hot habitats, but they occur ubiquitously in ordinary top soil (76), even in the Arctic regions (77).- P The nutritional requirements, chemical composition, and biochemical activities of thermophilic bacteria have been investigated, often in cnly a preliminary 1 manner (78-83). It has been demonstrated that cultures grown at 75OC produce a more heat-resistant protein than that formed in a 55OC or a 37OC culture. 1 An apyrase enzyme from a thermophil is heat-stable at 65OC, the optimum temperature for the organism (85).- The amino acid composition of proteins elaborated does not appear to differ markedly from that of proteins formed by mesophils (E),although a high dicarboxylic acid content in thermophils seemed to be characteristic. Thermo- philic sulfate-reducing bacteria can reduce sulfate, sulfite, and thiosulfate to H2S (86).- Lime deposits in thermal springs have been shown to be of thermophilic bacterial origin (87); Si0 -depositing and Fe-storing bacteria also seem to be in- - 2 volved in such formations. The significance of hydrothermal processes in experimental protobiochemistry has received surprisingly little attention, this in spite of the ubiquitous hydrothermal activity of the primitive earth, the associated energy sources due to the high levels of radioactivity, and the presence of earth's most primitive known organisms in modern hydrothermal waters. From current knowledge, it seems entirely reasonable to speculate that radioactivity and hydrothermal

- 35 - processes played an important role in the origin of life. Such a model provides for more than an adequate amount of inorganic chemical resources and energy (heat and ionizing radiation) in local regions and in satisfactory sequences of

I conditions (hydrous and anhydrous) that would be exceedingly favorable to chemical evolution and prebiological development.

Current Status. Laboratory investigations reported herein to test the novel and unique hypothesis that abiogenic organic synthesis and chemical evolution in primitive planetary environments may have proceeded in radioactive hydrothermal systems have established that an abundance of organic micromolecules and complex macromolecules can indeed by produced in hot aqueous solutions under the influence of ionizing radiation. Approximately seventy compounds have been identified and many of the others have been at least partially assigned to chemical classes. Within the chemical classes were found ten hydraearbons, twelve alcohols, six polyhydroxy alcohols, nine aldehydes and ketones, eight fatty acids, thirty-two carbohydrates (sugars), twelve or more amino acids plus I twenty-five or more other compounds such as amines which react with ninhydrin, three sulfur compounds in addition to the two sulfur amino acids, and three sugar phosphates The carbon forms used included the one-carbon oxidation state series starting with methane as the most reduced and extending through methanol to formate and carbonate as oxidized states. In addition, cyanide and dicyandiamide were included as forms already reacted with nitrogen, carbide as another interesting primitive state, and acetylene as a carbide derivative. Results suggest that methanol, dicyandiamide and formate produced the largest numbers of irradiation products and that each tended to produce different products, i. e. that the type of compounds produced is unique with each starting form of carbon. The other indication is, however, that all forms of carbon are reactive to some extent and show differences in degree and in types of product. Comparison of the forms of nitrogen used indicate that dicyandiamide is of much greater interest than cyanide. Ammonia was present in all experiments, so an estimation of its role has not been possible. Sulfur was present as sulfate only in the unproductive early experiments. As sulfide it has yielded several compounds in the methanol series of experiments. - 36 - The laboratory irradiations were. so arranged that both the primary reactants and the reaction products received radiation. This irradiation of products leads to their further reaction, thus simultaneously decreasing their concentration and broadening the total spectrum of final products. Such a situation was quite clear with regard to methanol production in Experiments 20 and 22. It is felt that this situation is realistic in terms of many locales on the primitive earth. It is also possible to visualize geologic situations in which products would have been transported away from the high radiation flux region and would consequently have avoided both further reaction and radiation decomposition. These latter segregation mechanisms would, in the laboratory, certainly permit more precise study of reaction mechanisms and first products. Conditions in the model experiments may be related to the pse- biochemical era through a comparison of the temporal and chemical concentration 8 factors. In the irradiation experiments 5 x 10 rads were delivered in periods of 1 about 40 minutes. In the analysis of radioisotope concentrations in the primitive earth discussed earlier, it was deduced that 4. 5 billion years ago the possible concentrations were far higher than had been previously realized. As an op- timistic maximum it was calculated that radon concentrations were as high as one millicurie per liter in hot springs and other aqueous systems, and volcanic gases and the fall-out areas around volcanoes may have had gas concentrations reaching 0. 1 mc/liter. Using the one millicurie per liter value it can be calculated that even without considering the chain of radon daughter disintegrations but only using the radon itself, doses of the same order of magnitude as those 8 u s e d experimentally, 5 x 10 rads, can be accumulated in only about 20 years. Thus it can be seen that the radiation model system does not require particularly extensive time periods on the geological scale. In terms of radiation flux or 6 intensity this represents a factor of the order of 10 lower radiation rate than the laboratory model used. Such a change in the rate of production of ionizing radiation would result in a much lower density of reactive products such as free radicals, with a consequent corresponding or magnified decrease in the production rate of final products. However, one must consider that periods of tens of millions I of years were available and that product concentrations could build up and accumu- late in selected geographic areas over this entire period. - 37 - -.

All of the compounds produced abiogenically with the possible exception of the hydrocarbons are found in living organisms, and a detailed comparison will show that a large number of the identified compounds are among the most important biochemicals known. For example, the amino acids, several of the sugars such as the pentoses d-ribulose and deoxyribulose, glycerol, ethanol, acetic and other acids are common to almost every known living organism. These types of compounds are important not only of themselves but also as key intermediary metabolites leading to the production and interconver sions of the other compounds of the biochemical system. Thus, the combination of glycerol with fatty acids will produce fats and other lipids. It may be assumed, therefore, that once these compounds are present together with suitable sources af energv and appropriate catalysts that a wide range of additional compounds could readily be formed. It may also be inferred that further analysis of the same systems plus investigation of other related or slightly modified environments and systems would reveal many more compounds of equal interest and significance. Irradiation of amino acids in solution produced high molecular weight peptides and proteinoids, This is a particularly significant observation since most models of abiogenic protein formation have contended that thermodynamic considerations require that peptide bonds be formed in the absence of much water (9).- The synthesis of numerous organic compounds under simulated conditions of the unique hydrothermal radiation model system of a primitive planetary environment has clearly established the validity of employing the model to test the hypothesis of chemical evolution. Additional research on the production of organic compounds in hot, aqueous, irradiated solutians is needed to demonstrate which reactions may be reproduced in the laboratory under the presumed geological conditions of the model. Such ground-based studies are necessary in order to define as far as possible the chemical resources and key compounds that might have played major roles in chemical abiogenesis and molecular evolution. Furthermore, it is necessary to investigate the generation of

- 38 - .

macromolecules such as proteins, nucleic acids, and polysaccharides in order to delineate the geologically plausible conditions compatible with the emergence of the first complex structure prior to the origin of cells and metabolism. Inher e nt limitations cla s sically r e strict experiment s in c he mica1 evolution to earth-bound laboratory investigations. However, evidence exists to suggest that the hydrothermal radioactive model system may be operative --in situ in at least two attainable extraterrestrial locales, the moon and Mars, thus providing a momentous opportunity in the near future for extending these ground- ba sed studies to actual extr ate r r e stria1 studie s . Existing geochemical conditions on the moon and Mars may be similar to those situations postdated in the model for the primitive Earth. Gold has recently proposed (88)- that there is a large amount of water on the moon in the form of a permafrost layer beginning perhaps 300 meters below the surface and extending to about one or two kilometers depth. Below this is

I presumably liquid water and possibly, at even lower depths, steam. This is based on the assumption that there are substantial quantities of radioactive elements in the moon, and hence a substantial selenothermal gradient. In the deeper layers of the moon there would be fairly high temperatures with possible formation of magmas and hydrothermal solutions, which in places would emerge through cracks leading to the surface. Such fumarole-like activity may be the explanation of certain observations of localized transient changes on the moon surface. According to Hapke (89),- volatile substances like water vapor may be continuously diffusing from the interior of the moon, Because of the smaller size of the moon in comparison to the Earth, its rate of heating must have been much slower and chemical differen-, tiation of the lunar material should be much less complete than in the Earth. The moon might thus present situations even now which are similar to those present earlier in the history of the Earth which might have been important in the formation of complex organic compounds critical for the origin of life. If temperatures in the lunar interior are substantially higher than on the surface,

- 39 - radon may be concentrated in the hydrothermal fluids along with compounds such as methane, acetylene, ammonia, hydrogen sulfide and hydrochloric acid, leading to the formation of complex organic compounds through the effect of ionizing radiations, This suggests the desirability of careful examinations and collections of the material issuing from any orifices in the lunar surface, and of deep drilling to determine whether beneath the permafrost layer there are liquid or gaseous reservoirs containing organic compounds, Because Mars, too, is substantially smaller than the Earth, it is likewise probable that the time scale for a chemical differentiation is longer than that on the Earth, and that it may also present opportunities for observing ges- chemical environments pertinent to the origin of life which are no longer present on the Earth. The search for protobiological chemicals on the moon would be facilitated by preliminary surveys of the surface for hot sputs of radioactivity. These would result from emission of small quantities of gases containing radon from the sub-surface hydrothermal systems. Such a survey would be accom- plished by scanning the lunar surface for gamma emissions from an orbiting satellite. This would allow a better than random choice of location sf 2,acding instrumental packages seeking information on selenochemical processes. Such consideration would also apply to Mars, although the presence sf a substantial atmosphere would result in an entirely different distribution of radon and prevent its detection from an orbiting satellite. Abiogenic syntheses conducted during the first year of the program utilized carbon sources such as carbice, cyanide, dicyandiamide, methane, methanol, formate, and carbonate, Future studies would be performed with formaldehyde, thiocyanate, thiocyanide, carbon disulfide, carbamyl phosphate formamide, methylamine, and transition metal carbides. Metal carbides represent important primitive forms of carbon compatible with the hydrothermal radioactive model. Additional significance of the carbides derives from the fact that

I - 40 - Cohenite (Fe C) is an accessory constituent mineral in iron meteorites as well 3 as terrestrial rocks (4).- Properties of carbides vary considerably with the metal involved, For example, the alkali metal carbides hydrolyze spontaneously to give acetylene while transition metal carbides such as chromium carbide only hydrolyze with difficulty and yield higher hydrocarbons. Several members of this latter class of carbides would be examined if they can be obtained, in addition to chromium carbide, which is being used in the first year studies. In contrast to the various studies on the many forms of carbon, little attention has been given by other investigators to comparisons of the primitive forms of nitrogen, sulfur and phosphorus. Several forms of nitrogen and sulfur were included in the first year studies but further investigations of organic compound yield from nitrogen-containing precursors such as cyanide, dicyandiamide, ammonia and thiocyanide should be undertaken. Similarly, model syntheses from sulfur sources such as sulfate, sulfite, sulfide, thiosulfate, and thiocyanate should be pursued, Phosphorus in the forms of meta- or poly- phosphates, pyrophosphates, and carbamyl phosphate should also be included in future model syntheses. The catalytic activity of trace metals such as copper, cobalt, zinc, nickel, etc., would be examined in selected syntheses. The studies of irradiated aqueous amino acid solutions should be broadened to include more complete characterization of peptide and proteinaceous products and to investigate the effects of variations in the initial amino acid composition. The influence of additional reactants such as simple sugars and phosphates on the composition of the products would be followed.

- 41 - SUMMARY An abundance of biochemically significant organic compounds have been formed from the simplest chemical resources in hot, aqueous environments under the influence of ionizing radiation. Furthermore, high-molecular weight peptides and proteinoids have been produced in the same unique model system thus providing mechanisms for the generation of macromolecules, an essential development for the evolutionary progression from chemical simplicity to prebiotic molecular complexity. These experiments establish the importance of a hydrothermal radioactive model system for exobiology investigations of the I primordial chemical reactions that might have led to the production and proliferation of organic molecules in favorable local environments, e. g, hot springs, early in the history of the planet Earth. The new and novel radioactive hydrothermal mode? has been shown io assume an immediate significance in regard to lunar and planetary exploration since propitious geochemical events and conditions on the moon and Mars may reproduce primitive hydrothermal radioactive systems in which abiogenic formation of extraterrestrial organic compounds is proceeding today.

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- 47 - TABLE I

ExDerimental conditions

Irradiations were 5 x 108 rads of 1.5 Mev electrons (Van de Graaf) or gamma rays (CO-60). Zero irradiation indicates a hydrothermal experiment. All temperatures were 55O, solution volumes 20 ml, pH 8.5. Asterisks = C-14 labeled. Reaction mixture details are given in Table II.

Experiment No. Irradiation R eac ti on Mixtur e (aqueous1 - 5 e I. Carbonate, phosphate, sulfate, ammonia - 6 e H. Methanol, cyanide, carbonate, phosphate, sulfide, ammonia. - 7 e 111. Methanol, carbonat e, phosphate, sulfide, ammonia

8 IV. Methanol, cyanide, phosphate, sulfate, 9 ammonia

10% V, Methanol, 4gmethanol-C- 14, phosphate, 11 sulfate, ammonia

12 VI. 18 amino acids 13

14* VIIS Dicyandiamide, formate, *formate-C- 14 15*

-* 16* VIQ. Dicyandiamide, formate, formate-C- 14, 17 FeC13 - 18 e IX. Chromium carbide, phosphate, ammonia 19 0

20 Y X. Methane (gas), NaOH 21 0

22 Y XI. Acetylene (gas), NaOH 23 0

- 48 - .

00 0 0.4 " d

00 0 .. .. *. lno d 00 0 rr)d d NN d

00.. .. z Inno 00 0 0 d NN d z" z 5 5 In co. 0 9 0 0 0 9 m c, ...... a, rn 4 k 5 + 4 9 r;9dd"09 a, N N 3 c, c, m x 5 *2 2 90 0 0 0 9 d Id ...... 1 mrr) d 0 H+ d " rnF4dTprr)" .r( z N N c, PI H 0 W Id a, d W k 0 J .r( w c, 09 0 0 0 9 F9 0 5 0...... 4 d Nrn d d N Ln9d.-("ON E: 0 N B 0 N .d rn c, .r( rn m 5 0 0 P a, E % 090... 0 0 0 0 Id Nrnm d d N U 2 N 0 N Ln k Tp Nd F a, a 0 dd d m d a, d 0

.AE d 4 d a, 0 c, .r( 5 c, Id .rl a, m 0 0 ? z" 0 0 N u zN 4 9 Id c, 0" pc rn !P) i! i3 - 49 - . TABLE III

Columns for gas chromatography

Tubing was copper or stainless steel, 1/8 or 1/4 in. diameter, lengths 18 in. to 12 ft.

Class of compound detected Liquid phase Solid support

Gases: 0, N. H, CO, C02, Molecular sieve 5A 30-60 mesh methane

C -C hydrocarbons 270 diethylhexyl- Silica gel 14 sebacife 30-60 mesh

Diatoport W C - C 15 hydrocarbons 10% Apiezon N 45-60 mesh

' Higher hydrocarbons Silicone rubber

C -C isomeric hydrocarbons 10% dimethyl- Diatoport W 35 sulfolane (1609) 45-60 mesh

C hydrocarbons 3070 dimethyl- Gas pack PA 35-C sulfolane 60-80 mesh

Hydrocarbons 10% UCON 550X Diatoport W 45-60 mesh

Alcohols, amines 20% Carbowax 400 Gas pack F 40-60 mesh

Fatty acids, glycol, glycerol, 10% Carbowax 20 M Diatopor t S C -C5 amines, sulfides, 60-80 mesh 1 mercaptans

C -C fatty acids 1570 Neopentylglycol Firebrick 16 succinate 60-80 mesh

11 2070 11 Gas pack F 40-60 mesh

11 10% 11 Diatoport S 60-80 mesh

Amino acid derivatives Gas pack S 100-120 mesh 1 Amines 570 triethylene - Gas pack F pentaamine and 1570 40-60 mesh tet rahydr oxyethy1 - ethylene diamine (continued) - 50 - TABLE 111 - continued

Class of compound detected Liquid phase Solid support

Alcohols, aldehyde s, ketones 20'70p, P'-oxydipropio- Diatoport S nitrile 60-80 mesh

11 20'70 N, N-bis(2-cyano- Silanized chromo- ethyl) formamide sorb: 60-80 mesh

Sugar derivatives 1% QF- 1, fluorosilicone Gas pack 100-120 mesh

11 1% GE-SE 30 silicone Gas pack 100-120 mesh

11 3% GE-SE 52 silicone Gas pack S 100-120 mesh b Esters 2 0% die thyleneglyeol Gas pack S suc c i na t e 60-80 mesh

11 10% diglycerol Diatoport S 60-80 mesh

- 51 - TABLE TV

Summary of chemical classes produced from primitive earth compounds

Approximate number of compounds; identified t unknown. 0 = not produced, dash = not possible from the reaction mixture.

Methanol Format e Chemical class with or without plus dicyan- Me thane A c etyl ene Cr3C2 cvanide. carbonate diamide

Hydrocarbons 4t2 0 6t4 4tO 0 Alcohols 5t7 o+ 1 1to 1to 0

Polyhydroxy alcohols ' 6tO 0 0 0 0 (glycols, sugar alcohols) Oxy compounds 9tO 1t1 0 0 0 (aldehydes, ketones, oxides)

Fatty acids 5t3 Ot3 1to 1to ot1 ' Carbohydrates 20 t 12 0 0 0 0 Amino acids 10 t 20 8 t 25 - - Ot7 (and unknown amines) Sulfur compounds 3tO (except amino acids) Sugar phosphates 1t2 0 - - - Totals 63 t 46 9 t 30 8t4 6tO Ot8

d Unknowns *gN03 4 8 6 UV fluorescence 3 22 2 visible color 0 4 0 0 0 precipitate 0 1 1 1 - C-14 autoradiography 32 34 Total 39 69 1 1 8 * May duplicate some members of chemical classes.

- 52 - GAS CHROMATOGRAPHIC DATA FROM EXPERIMENTS 6 TO 11

All peaks detected with the flame ionization detector are listed. Identifications are basfd on comparative retention times with standards. Where more than one compound is listed the peak may contain any combination of these. Numbers indicate number of peaks present; Tr equals trace or not fully confirmed. Carbohydrate derivatives may have more than 1 peak per compound.

Exp.6 Exp. 7 Exp. 9 Exp. 10 Exp. 11 Methanol, Methanol, Methanol, Methanol, Methanol Methanol cyanide, carbonate cyanide cyanide :ompound and carbonate =mica1 Class Irradiated Irradiated Irradiated Hydrothermal Irradiated Hydrothermal ydrocarbons: 0 0 c6 aliphatic or olefinic 2t 0 2t 0 c7 - C8 c8- c9 Unknown

.cohols: ethanol 1 1 1 1 isopropanol Tr Tr Tr Tr n:propanol 1 1 sec-butanol Tr Tr isopentanol Tr Tr C6+ alcohols (unknown) 7 7 Unknown 3 4 ilyhydroxy alcohols: 0 0 lycols, sugar alcohols) ethylene glycol 1 1 1 1 propylene glycol TI Tr Tr Tr glycerol 1 1 1 1 exythritol 1 1 1 xylitol, a-xylose 1 1 arabitol, ribitol 1 1 ky compounds: 0 0 ldehydes, ketones, oxides) acetaldehyde 1 1 propionaldehyde 1 1 n-butyraldehyde Tr Tr Tr Tr acetone Tr Tr Tr Tr methyl ethyl ketone ) 1 1 1 1 1 methyl vinyl ketone ) 1 Tr Tr diethyl ketone Tr Tr 1, 2-butylene oxide Tr Tr Tr Tr 2, 3-butylene oxide Tr Tr Tr Tr

-:ids: 0 0 acetic 1 1 1 1 propionic 1 1 1 1 butyric 1 1 1 1 valeric Tr Tr 1 1 isovaleric Tr Tr 1 1 C6 t acids Tr 3-Tr Tr TI

Lrbohydrates: 0 0 180 see polyhydroxy alcohols) threose Tr 1 Tr 1 erythrulose Tr Tr erythrose 1 Tr 1 Cq - Cg desoxy sugars, 4-Tr 5-Tr 4-Tr 6-Tr glycosides, lactones deoxyribose 1 1 digitoxose Tr Tr Tr methyl-p-arabanoside Tr Tr Tr methyl-a-arabanoside Tr Tr Tr r 1bulos e Tr Tr Tr p-arabinose 1 1 rhamnose Tr Tr Tr a rabinolac tone Tr Tr methy1 - a-xylo side Tr D-ribose, p-lyxose, 1-2 1-2 xylonolactone, methyl-p-xyloside D-ribose 7 1 y- ribonolactone Tr fucose Tr Tr a-xylose, (xylitol) Tr Tr kdosan Tr 2-deoxygalactose Tr Tr Unknown 1 7t lfur compounds: 0 0 propyl mercaptan Tr Tr allyl sulfide Tr Tr thiophene Tr Tr - 53 - NI Iln I*

NI lr-

\o m NI NI I1 In N

N mo NI Nm 4

m I1 I+ N m

mI I+ + N N v r.l- a

xQ :ua

- 54 - TABLE VI1

One -dimensional pap e r chromatog r aphy : Numbers of spots from chromium carbide

Chemical class and method of Exp. 18 Exp. 19 mot location Irradiated Hydrothermal

Amino Acids: Ninhydrin (colors) Solvent C 2- 3 2-3 Solvent D 7 4-5

Organic acids, sugars Ninhydrin (white) Solvent C 1-2 1-2 Solvent D 0 0

Unknown &NO3 Solvent C - - Solvent D 4-6 3-5

Unknown UV fluorescence Solvent C - - Solvent D 2 1

- 55 - TABLE .VI11

Compounds from irradiation of aqueous methane, Experiment 20.

M E major product, Tr = minor product

Class and compound Amount

Hvdrocarbons (gas phase)

ethane M propane M n- butane Tr ethylene Tr butene- 2 Tr butadiene Tr C -compounds 4-Tr 5 Gases

Tr H2 O2 N2 = 11.3 N2 '02 Oxy compounds (liquid phase)

methanol Tr acetic acid Tr

- 56 - TABLE IX

Compounds from irradiation of aqueous acetylene, Experiment 22, M = major product, Tr = minor product

Class and compound Amount

Hydrocarbons (gas phase)

methane Tr ethane Tr propane Tr n- butane Tr

Oxy compounds (liquid phase)

methanol Tr acetic acid M

- 57 - 0

.,-la 2 : 43 k 0 b4 c :e m Q) l-4

or(; 4 4

-5 In II m $ k Q) P E m c, k k d &in b Q) cd c F E Q) .r( PI k k Q) 0 Px d Q) -2 a k c kkk kk Q) II c, c b cA br?b bb cd k G F Id k An k cd -4 Q) a E k 0 m 9 k 0 w *m cd rr) 4 m a E .r(u II cd 0 c d m d -z kk k k 4 0 .r( c EF b I3 c, cd u w.r( .r( c, d aQ .,-l k kk k k d b bB B F .r( m Q) .rl c, d .r( h cd Q) c, d k .r( Q) m Q) u d 0 d .r( u 3 k .. k I t s L .r( .r(2

cdcdi, - 58 - i

- 59 - - 60 - .

- 61 - ~~

m ld m d 0 .r(+I 4 d u0 * 4 c, d

.r( k Q) wxa 9- 4 uI I 0) c, ld E k 0 +I E 0 k 4 m +I u =I a 0 k a d 0 .4c, ld % Id k k -4 4 0 E Ki k M 0 m.4 rd ?: 0 c, =I %i

- 62 - 63 -