THE EXPERIMENTAL INVESTIGATION OF CHEMICAL EVOLUTION

Attempts have been made to simulate and approximate models of primitive Earth conditions for abiogenic synthesis, and successful synthesis of essential biochemical constituents necessary for maintaining life has been partly accomplished.

Urey ([ref.11]) has clearly pointed out the possible role of a reducing atmosphere in the synthesis of prebiological organic molecules. Miller ([ref.12]) synthesized a variety of amino acids in a reducing atmosphere by means of an electrical discharge. A variety of organic compounds have been synthesized by the action of various energy sources upon reducing atmospheres, and several investigators have extended the Urey-Miller-type reactions to synthesize nucleic acid components ([ref.13]), adenosine triphosphate ([ref.14]), and a host of biologically essential organic compounds.

It is likely that in the synthesis of organic moieties, simple and specific molecules were first produced when the planets had a reducing atmosphere. Further complexity or degradation of the organic compounds produced varied, depending on the geochemical changes of the planet's surface, the atmospheric constituents, the degree of interaction between surface and atmosphere, and the rate of the organic synthesis. Oparin ([ref.15]) presented the most detailed mechanisms for the spontaneous generation of the first living organism arising in a sea of organic compounds synthesized in a reducing atmosphere on Earth.

It is generally accepted that, under favorable conditions, life can arise by spontaneous generation. A primary requirement for this initiation is that there be abundant organic compounds concentrated in one or more specific zones. These simple organic molecules would undergo modification to develop a greater structural complexity and specificity, finally giving rise to a "living" organism. Therefore, because of the ease with which organic compounds can be synthesized under reducing conditions, planetary surfaces may contain an abundant source of similar organic matter. However, difficulties arise in postulating steps for further organization or modification of the above synthesized organic matter into a living state. Most of the original organic matter produced in the primary reducing atmospheres of the various planets may have been quite similar. However, major variations between planets, in chemical evolution beyond the prebiotic stage, must have been the rule rather than the exception.

The primary interest in this area of research has been the realization of the possible existence of organic molecules on planetary surfaces and, particularly, Mars. Pertinent synthesis may be either biological or abiological. Research conducted in the simulation of cosmochemical synthesis has used most of the available solar spectrum. Simulation experiments devised to study the effects of these energies on the assumed early atmosphere of the Earth have yielded products that play a dominant role in molecular and biochemical organization of the cell.

Calvin ([ref.16]) irradiated water and carbon dioxide in a cyclotron, obtaining formaldehyde and formic acid. Miller ([ref.17]) found that when methane, ammonia, water, and hydrogen were subjected to a high-frequency electrical discharge, several amino acids were produced along with a variety of other organic compounds.

Corroborating experiments established that the synthesis of amino acids occurred readily. The apparent mechanism for the production of amino acids is as follows: aldehydes and hydrogen cyanide are synthesized in the gas phase by the electrical discharge. These substances react together and also together with ammonia in the water phase of the system to give hydroxy and amino nitriles, which are then hydrolyzed to hydroxy and amino acids. Among the major constituents were aspartic acid, glutamic acid, glycine, a-alanine, and -alanine.

The "Miller-Urey" reaction mixture has been extended and several modifications introduced. OrÓ ([ref.18]) introduced hydrogen cyanide into the system as the primary gas component. Adenine was obtained when OrÓ heated a concentrated solution of hydrogen cyanide in aqueous ammonia for several days at temperatures up to 100°C. Adenine is an essential component of nucleic acids and of several important coenzymes. Guanine and urea were the two other products identified in the hydrogen cyanide reaction. OrÓ further obtained guanine and uracil as products of nonenzymatic reactions by using certain purine intermediates as starting materials.

Ponnamperuma ([ref.19]) also obtained adenine upon irradiation of methane, ammonia, hydrogen, and water, using a high-energy electron beam as the source of energy of irradiation. These results indicate that adenine is very readily synthesized under abiotic conditions. Adenine, among the biologically important purines and pyrimidines, has the greatest resonance energy, thus making its synthesis more likely and imparting greater radiation stability to the molecule.

The formation of adenine and guanine, the purines in RNA and DNA, by a relatively simple abiological process lends further support to the hypothesis that essential biochemical constituents of life may have originated on Earth by a gradual chemical evolution and selection. In this respect, the examination of planetary surfaces—specifically Mars—presents practical implications for current research on the problem of chemical evolution.

When Ponnamperuma et al. ([ref.14]) exposed adenine and ribose to ultraviolet light in the presence of phosphate, adenosine was produced. When the adenine and ribose were similarly exposed in the presence of the ethyl ester of polyphosphoric acid, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) were produced. The abiological formation of ATP was a major stride along the path of chemical evolution, since ATP is the principal free energy source of living organisms.

Oparin ([ref.15]) postulated that a-amino acids could have been formed nonbiologically from hydrocarbons, ammonia, and hydrogen cyanide at a time when the Earth's atmosphere contained these substances in high concentrations. Oparin's hypothesis has received strong experimental support, as evidenced by the work of Miller ([ref.12]). Bernal ([ref.20]) has emphasized the role played by ultraviolet light in the formation of organic compounds at a certain stage of the Earth's evolution.

Generally it has been believed that the first proteins or foreprotein were nonbiologically formed by the polycondensation of preformed free amino acids ([ref.21]). Akabori ([ref.22]) proposed a hypothesis for the origin of the foreprotein and speculated that it must have been produced through reactions consisting of the following three steps.

The first step is the formation of aminoacetonitrile from formaldehyde, ammonia, and hydrogen cyanide.

CH₂O + NH₃ + HCN ————> H₂N—CH₂—CN + H₂O

The second is the polymerization of aminoacetonitrile on a solid surface, probably absorbed on clay, followed by the hydrolysis of the polymer to polyglycine and ammonia.

x H₂N—CH₂—CN ————> (—NH—CH₂—C—)_x

""

NH

"

" + x H₂O

V

(—NH—CH₂—CO—)_x + x NH₃

The third step is the introduction of side chains into polyglycine by the reaction with aldehydes or with unsaturated hydrocarbons. Akabori has demonstrated experimentally the formation of cystinyl and cysteinyl residue in his above-postulated mechanism.

Fox's theory of thermal copolymerization ([ref.23]) suggests that proteins or like molecular units could have been formed in the Earth's crust, under geothermal conditions. The accumulated amino acids were heat polymerized and transported into the primary oceans for further modifications. Fox has obtained polymers consisting of all 18 amino acids usually present in proteins. The polymerization is generally done at 160°C to 200°C, although in the presence of polyphosphoric acid it can be accomplished at temperatures below 100°C. Molecular weights increased from 3600 in a proteinoid made at 160°C to 8600 in one made at 190°C.

Fox showed that when hot saturated solutions of thermal copolymers containing the 18 common amino acids were allowed to cool, large numbers of uniform, relatively firm, and elastic spherules separate. These range from 0.2µ to 60µ in diameter and are quite uniform within each preparation. Various chemical observations suggest the presence of peptide bonds in the structural organization of these proteinoids. Continuing observations of these microspheres have established further characteristics that point to the possibility of their interpretation as a kind of primitive protein macromolecule with self-organizing properties, such that a primitive form of cell, with boundary and other properties, might form.

In laboratory experiments the behavior of gram-negative and gram-positive microspheres in dilute alkali parallels that of gram-negative and gram-positive bacteria ([ref.23]). Furthermore, time-lapse studies indicate that the proteinoid microspheres undergo a septate kind of fission, mimicking cell division as shown in figure 1. Cytochemical studies show that the microsphere's boundary is membranelike in having a primitive selectivity. Electron micrographs of sections of stained microspheres also indicate the presence of a boundary.

Oparin ([ref.15]) states that the type of organization peculiar to life could only result from the evolution of a multimolecular organic system separated from its environment by a distinct boundary but constantly interacting with this environment. In his concept of coacervates as precell models, Oparin ([ref.24]) indicates that present-day protoplasm possesses a number of features similar to coacervate structure. These coacervates could represent the starting point for evolution leading to the origin of life. Moreover, in the course of their evolution the initial systems may gradually become more complex. Oparin also showed ([ref.15]) that mixing solutions of different proteins and other substances of high molecular weight produced these coacervate droplets. These droplets are characterized by the formation of a surface layer with altered structure and mechanical properties, thus providing a somewhat selective barrier in which to house a molecular system capable of replication. However, these coacervates are unstable structurally.

The NASA program has further provided considerable impetus for continuing research with respect to the chemical evolution of life, since its life-detection experiments may encounter prebiological molecules in their search for extraterrestrial life on other planetary surfaces.

In the area of exobiological research, the significant accomplishments to date have been—

1. The reconstruction of some of the pathways which may have led to the origin of life, by means of laboratory simulation of processes yielding prebiological organic molecules

2. The developments in experimental and theoretical biology; specifically, the role of nucleic acid-protein interactions in storage and transmission of information both within living cells and from generation to generation of cells

3. The suspected role of DNA in information storage and the development of new concepts of the coding mechanism in DNA that may lead to a universal biological theory embracing evolutionary, as well as homeostatic, adaptation to environment and learned behavioral systems

With the essential biochemical constituents of life and the mechanism of replication beginning to be understood, the challenge for the synthesis of living matter by abiogenic experimental techniques has become to many scientists the ultimate goal of the scientific era.

NASA has established an exobiology laboratory at Ames Research Center in addition to the sizable support of research at various academic centers of excellence for the continuation of abiogenic synthesis.

Although research on organochemical evolution is in its infancy, the data from relatively few experiments have already created an immense enthusiasm for knowledge of the biochemical pathways of evolution. This kind of research will ultimately elucidate the terrestrial evolution of life and, perhaps, the nature of life on other planetary bodies and the distribution of life in our galaxy.

This program, with its vast demands on the scientific community at large, is coordinated with related endeavors of a number of Federal agencies. It is allied with certain biochemical studies at the National Institutes of Health for the eventual elucidation of the dynamic pathways in cosmochemical synthesis of life's essential biochemical constituents.

METEORITES AND ORGANIC GEOCHEMISTRY

CONCEPTS FOR DETECTION OF EXTRATERRESTRIAL LIFE

It is not possible to present completely convincing evidence for the existence of extraterrestrial life. The problem often reduces to probabilities and to estimates of observational reliability. In almost all cases the evidence is optimistically considered strongly suggestive of—or, at the worst, not inconsistent with—the existence of extraterrestrial life. Alternatively, there is a pessimistic view that the evidence advanced for extraterrestrial life is unconvincing, irrelevant, or has another, nonbiological explanation.

In studies of the laboratory synthesis of life-related compounds and its significance concerning the origin of life, several results seem to suggest that organochemical synthesis is a general process, occurring perhaps on all planets which retain a reducing atmosphere. The temperature ranges must be such that precursors and reaction products are not thermally dissociated. The reaction rates for the synthesis of more complex organic molecules diminish to a negligible value when the temperature range is below 100°C.

Besides the planetary parameter of temperature, an even more fundamental necessity for a living state exists—a liquid solvent system. For terrestrial life forms, water serves this purpose. Water has this and other properties of biological significance because of hydrogen bonding between adjacent molecules in the liquid state.

Ultraviolet radiation could serve as an extraterrestrial energy source for organic synthesis. Research shows that, while an atmosphere is important, living systems can survive a wide range of ambient pressures and are little affected by a wide range of magnetic field strengths.

Oxygen is not a prerequisite for all living systems. While it is sometimes concluded that free oxygen is needed for all but the simplest organisms, less efficient metabolic processes coupled with higher food collection efficiency—or a more sluggish metabolism—would seem to do just as well. Earth is the only planet in the solar system on which molecular oxygen is known to be present in large amounts. Since plant photosynthesis is the primary source of atmospheric oxygen, it seems safe to infer that no other planet has large-scale plant photosynthesis accompanied by the production of oxygen.

The possibility of the existence of extraterrestrial life raises the important question of man's being able to detect it. Research on extraterrestrial life detection is predicated on the ability to develop ways to detect it even when the living systems are based on principles entirely different from those on Earth.

The substitution of various molecules for those of known biological significance to living organisms as we know them has been investigated; the substitution of NH₂ for OH in ammonia-rich environments leads to a diverse, and biologically very promising, chemistry. The hypothesis that silicon may replace carbon does not support the construction of extraterrestrial genetics based on silicon compounds. (Silicon compounds participate in redistribution reactions which tend to maximize the randomness of silicon bonding, and the stable retention of genetic information over long time periods is thus very improbable.)

Evidence relevant to life on Mars has been summarized by Sagan (ch. 1 of [ref.10]):

The Origin of Life

In the past decade, considerable advances have been made in our knowledge of the probable processes leading to the origin of life on Earth. A succession of laboratory experiments has shown that essentially all the organic building blocks of contemporary terrestrial organisms can be synthesized by supplying energy to a mixture of the hydrogen-rich gases of the primitive terrestrial atmosphere. It now seems likely that the laboratory synthesis of a self-replicating molecular system is only a short time away from realization. The syntheses of similar systems in the primitive terrestrial oceans must have occurred—collections of molecules which were so constructed that, by the laws of physics and chemistry, they forced the production of identical copies of themselves out of the building blocks in the surrounding medium. Such a system satisfies many of the criteria for Darwinian natural selection, and the long evolutionary path from molecule to advanced organism can then be understood. Since nothing except very general primitive atmospheric conditions and energy sources are required for such syntheses, it is possible that similar events occurred in the early history of Mars and that life may have come into being on that planet several billions of years ago. Its subsequent evolution, in response to the changing Martian environment, would have produced organisms quite different from those which now inhabit Earth.

Simulation Experiments

Experiments have been performed in which terrestrial micro-organisms have been introduced into simulated Martian environments, with atmospheres composed of nitrogen and carbon dioxide, no oxygen, very little water, a daily temperature variation from +20° to -60°C, and high ultraviolet fluxes. It was found that in every sample of terrestrial soil used there were a few varieties of micro-organisms which easily survived on "Mars." When the local abundance of water was increased, terrestrial micro-organisms were able to grow. Indigenous Martian organisms may be even more efficient in coping with the apparent rigors of their environment. These findings underscore the necessity for sterilizing Mars entry vehicles so as not to perform accidental biological contamination of that planet and obscure the subsequent search for extraterrestrial life.

Direct Searches for Life on Mars

The early evidence for life on Mars—namely, reports of vivid green coloration and the so-called "canals"—are now known to be largely illusory. There are three major areas of contemporary investigation: visual, polarimetric, and spectrographic.

As the Martian polar ice cap recedes each spring, a wave of darkening propagates through the Martian dark areas, sharpening their outlines and increasing their contrast with the surrounding deserts. These changes occur during periods of relatively high humidity and relatively high daytime temperatures. A related dark collar, not due to simple dampening of the soil, follows the edge of the polar cap in its regression. Occasional nonseasonal changes in the form of the Martian dark regions have been observed and sometimes cover vast areas of surface.

Observations of the polarization of sunlight reflected from the Martian dark areas indicate that the small particles covering the dark areas change their size distribution in the spring, while the particles covering the bright areas do not show any analogous changes.

Finally, infrared spectroscopic observations of the Martian dark areas show three spectral features which, to date, seem to be interpretable only in terms of organic matter, the particular molecules giving rise to the absorptions being hydrocarbons and aldehydes. [However, see p. 7 and Rea et al. ([ref.9]).]

Taken together, these observations suggest, but do not conclusively prove, that the Martian dark areas are covered with small organisms composed of familiar types of organic matter, which change their size and darkness in response to the moisture and heat of the Martian spring. We have no evidence either for or against the existence of more advanced life forms. There is much more information which can be garnered from the ground, balloons, Earth satellites, Mars flybys, and Mars orbiters, but the critical tests for life on Mars can only be made from landing vehicles equipped with experimental packages....

Results of Kaplan et al. ([ref.31]) indicate that Mars has no detectable oxygen, but does contain small amounts of water vapor, more abundant carbon dioxide, possibly a large surface flux of solar ultraviolet radiation, and estimated daily temperature variations of 100°C at many latitudes. Studies have shown that terrestrial micro-organisms can survive these extremely harsh environments. Furthermore, a variety of physiological and ecological adaptations might enable the biota to survive the low nighttime temperatures and intracellular ice crystallization.

Less evidence is available to support the possibility of extraterrestrial life on other planets. The Moon has no atmosphere, and extremes of temperature characterize its surface. However, the Moon could have a layer of subsurface permafrost beneath which liquid water might be trapped. The temperatures of these strata might be biologically moderate.

Studies by Davis and Libby ([ref.32]) on the atmosphere of Jupiter support the possibility of the production of organic matter in its atmosphere in a manner analogous to the processes which may have led to the synthesis of organic molecules in the Earth's early history. It is difficult to assess the possibility that life has evolved on Jupiter during the 4- or 5-billion-year period in which the planet has retained a reducing atmosphere.

The question of extraterrestrial life and of the origin of life is interwoven. Discovery of the first and analysis of its nature may very well elucidate the second.

The oldest form of fossil known today is that of a microscopic plant similar in form to common algae found in ponds and lakes. Scientists know that similar organisms flourished in the ancient seas over 2 billion years ago. However, since algae are a relatively complex form of life, life in some simpler form could have originated much earlier. Organic material similar to that found in modern organisms can be detected in these ancient deposits as well as in much older Precambrian rocks.

Although the planets now have differing atmospheres, in their early stages the atmospheres of all the planets may have been essentially the same. The most widely held theory of the origin of the solar system states that the planets were formed from vast clouds of material containing the elements in their cosmic distribution.

It is believed that the synthesis of organic compounds preceding the origin of life on Earth occurred before its atmosphere was transformed from hydrogen and hydrides to oxygen and nitrogen. This theory is supported by laboratory experiments of Calvin ([ref.16]), Miller ([ref.33]), and OrÓ ([ref.34]).

The Earth's present atmosphere consists of nitrogen and oxygen in addition to relatively small amounts of other gases; most of the oxygen is of biological origin. Some of the atmospheric gases, in spite of their low amounts, are crucial for life. The ultraviolet-absorbing ozone in the upper atmosphere and carbon dioxide are examples of such gases.

Significant in the search for extraterrestrial life are the data (e.g., planet's temperature) transmitted by Mariner II, which was launched from Cape Canaveral on August 27, 1962, and flew past Venus on December 14, 1962. Mariner II's measurements showed temperatures on the surface of Venus of the order of 800°F, too hot for life as known on Earth.

The question "Is life limited to this planet?" can be considered on a statistical basis. Although the size of the sample (one planet) is small, the statistical argument for life elsewhere is believed by many to be very strong. While Mars is generally considered the only other likely habitat of life in our solar system, Shapley ([ref.35]) has calculated that more than 100 million stars have planets sufficiently similar in composition and environment to Earth to support life. Of course, yet unknown factors may significantly reduce or even eliminate this probability.

SPACECRAFT STERILIZATION

The search for extraterrestrial life with unmanned space probes requires the total sterilization of the landing capsule and its contents. Scientists agree that terrestrial organisms released on other planets would interfere with exobiological explorations (refs. [ref.36]-[ref.43]). Any flight that infects a planet with terrestrial life will compromise a scientific opportunity of almost unequaled proportions. Studies on microbiological survival in simulated deep-space conditions (low temperature, high ultraviolet flux, and low dose levels of ionizing radiation) indicate that these conditions will not sterilize contaminated spacecraft (refs. [ref.44]-[ref.48]). Furthermore, many terrestrial sporeformers and some vegetative bacteria, especially those with anaerobic growth capabilities, readily survive in simulated Martian environments (refs. [ref.49]-[ref.54]). It has been estimated that a single micro-organism with a replication time of 30 days could, in 8 years of such replication, equal in number the bacterial population of the Earth. This potential could result not only in competition with any Martian life, but in drastic changes in the geochemical and atmospheric characteristics of the planet. To avoid such a disaster, certainly the first, and probably many succeeding landers on Mars, must be sterile—devoid of terrestrial life ([ref.55]). Since the space environment will not in itself kill all life aboard, the lander must leave the Earth in a sterile condition.

The sterility of an object implies the complete absence of life. The presence of life or the lack of sterility may be proven; but the absence of life or sterility cannot be proven, for the one viable organism that negates sterility may remain undetected. Many industrial products which must be guaranteed as sterile cannot be tested for sterility in a nondestructive manner. A similar situation exists in determining the sterility of a spacecraft. Certification of sterility—based on experience with the sterilizing process used, knowledge of the kinetics of the death of micro-organisms, and computation of the probability of a survivor from assays for sterility—is the only accurate approach to defining the sterility of such treated items.

Macroscopic life can be readily detected and kept from or removed from the spacecraft, but the detection and removal of microscopic and submicroscopic life is an extremely difficult task. The destruction of micro-organisms can be achieved by various chemical and physical procedures. Sterilizing agents have been evaluated not only for their ability to kill microbial life on surfaces and sealed inside components, but also for the agents' effects on spacecraft reliability as well (refs. [ref.56]-[ref.59]). Of the available agents, only heat and radiation will penetrate solid materials. Radiation is expensive, hazardous, difficult to control, and apparently damages more materials than does heat. Heat, therefore, has been selected as the primary method of spacecraft sterilization and will be used, except in specific instances where radiation may prove to be less detrimental to the reliability of critical parts ([ref.60]).

The sterilization of spacecraft is a difficult problem if flight reliability is not to be impaired. The development of heat-resistant parts will enable the design and manufacture of a heat-sterilizable spacecraft. Without careful microbiological monitoring of manufacture and assembly procedures, many bacteria could be trapped in parts and subassemblies. To permit sterilization at the lowest temperature-time regimen that will insure kill of all organisms, the microbiological load inside all parts and subassemblies must be held to a minimum.

The role of industrial clean rooms in reducing the biological load on spacecraft is currently being defined. NASA-supported studies indicate that biological contamination in industrial clean rooms for extended time periods is about 1 logarithm less (tenfold reduction), compared with conditions in a well-operated microbiological laboratory ([ref.61]). With the use of clean-room techniques and periodic decontamination by low heat cycles or ethylene oxide treatment, it should be possible to bring a spacecraft to the point of sterilization with about 10⁶ organisms on board ([ref.60]).

The sterilization goal established for Mars landers is a probability of less than 1 in 10000 (10^-4) that a single viable organism will be present on the spacecraft. Laboratory studies of the kinetics of dry-heat kill of resistant organisms show that at 135°C the number of bacterial spores can be reduced 1 logarithm (90 percent) for every 2 hours of exposure (refs. [ref.58] and [ref.62]). The reduction in microbial count needed is the logarithm of the maximum number on the spacecraft (10⁶) plus the logarithm of the reciprocal of the probability of a survivor (10⁴), or a total of 10 logarithms of reduction in microbial count. Thus, with an additional 2 logarithms added as a safety factor, a total of 12 logarithms of reduction in count has been accepted as a safe value which can be achieved by a dry-heat treatment of 135°C for 24 hours. This is the heat cycle that is currently under study and being developed for use in spacecraft sterilization ([ref.60]). However, other heat treatments at temperatures as low as 105°C for periods of 300 hours or longer are under study ([ref.63]).

Based on results to date, it is reasonable to believe that a full complement of heat-sterilizable hardware will be available when needed for planetary exploration. Every effort is being made to improve the state of the art to a point where spacecraft can not only withstand sterilization temperatures, but will be even more reliable than the present state-of-the-art hardware that is not heated.