The Use of Ascorbate as an Oxidation Inhibitor in Prebiotic Amino Acid Synthesis: A Cautionary Note

It is generally thought that the terrestrial atmosphere at the time of the origin of life was CO₂-rich and that organic compounds such as amino acids would not have been efficiently formed abiotically under such conditions. It has been pointed out, however, that the previously reported low yields of amino acids may have been partially due to oxidation by nitrite/nitrate during acid hydrolysis. Specifically, the yield of amino acids was found to have increased significantly (by a factor of several hundred) after acid hydrolysis with ascorbic acid as an oxidation inhibitor. However, it has not been shown that CO₂ was the carbon source for the formation of the amino acids detected after acid hydrolysis with ascorbic acid. We therefore reinvestigated the prebiotic synthesis of amino acids in a CO₂-rich atmosphere using an isotope labeling experiment. Herein, we report that ascorbic acid does not behave as an appropriate oxidation inhibitor, because it contributes amino acid contaminants as a consequence of its reactions with the nitrogen containing species and formic acid produced during the spark discharge experiment. Thus, amino acids are not efficiently formed from a CO₂-rich atmosphere under the conditions studied.     

Formation of amino acids from models of Titan and more oxidized atmospheres

Protein and non-protein amino acids were synthesized following hydrolysis of products obtained by high frequency discharge techniques applied to model atmospheres consisting of N₂ as a nitrogen source together with CH₄ and/or CO₂ as a carbon source. Highest yields were obtained in the absence of CO₂ and from mixtures rich in CH₄. Amino acids would indeed be expected on the frozen surface of Titan with its CH₄–N₂ atmosphere.Paper presented at the 6th College Park Colloquium, October 1981.     

The atmosphere of the primitive Earth and the prebiotic synthesis of amino acids

The atmosphere of the Earth at the time of its formation is now generally believed to have been reducing, an idea proposed by Oparin and extensively discussed by Urey. This atmosphere would have contained CH₄, N₂ with traces of NH₃, water and hydrogen. Only traces of NH₃ would have been present because of its solubility in water. UV light and electric discharges were the major sources of energy for amino acid synthesis, with electric discharges being the most efficient, although most other sources of energy also give amino acids.The first prebiotic electric discharge synthesis of amino acids showed that surprisingly high yields of amino acids were synthesized. Eleven amino acids were identified, four of which occur in proteins. Hydroxy acids, simple aliphatic acids and urea were also identified. These experiments have been repeated recently, and 33 amino acids were identified, ten of which occur in proteins, including all of the hydrophobic amino acids.Methionine can be synthesized by electric discharges if H₂S or CH₃SH is added to the reduced gases. The prebiotic synthesis of phenylalanine, tyrosine and trytophan involves pyrolysis reactions combined with plausible solution reactions.Eighteen amino acids have been identified in the Murchison meteorite, a type II carbonaceous chondrite, of which six occur in proteins. All of the amino acids found in the Murchison meteorite have been found among the electric discharge products. Furthermore, the ratios of amino acids in the meteorite show a close correspondence to the ratios from the electric discharge synthesis, indicating that the amino acids on the parent body of the carbonaceous chondrites were synthesized by electric discharges or by an analogous process.     

Organic syntheses from CH4−N2 atmospheres: Implications for Titan

Numerous experiments have already been performed, simulating the evolution of gaseous mixtures containing CH₄ when submitted to energy flux. From their results, it appears that a variety of organic compounds, including unsaturated hydrocarbons and nitriles such as HCN, can be synthesized into noticeable amounts from CH₄–N₂ mixtures. In particular, systematic studies of the influence of the composition of the mixture on the nature and amount of synthesized compounds show that organic volatile nitriles, and particularly cyanoacetylene and cyanogen, are formed only in media rich in nitrogen. Those nitriles have been identified very recently in the atmosphere of Titan, and thus, data from such laboratory experiments may provide important indirect information on the organic chemistry occuring at the periphery of this satellite of Saturn. However, during these experiments, there is a continuous formation and accumulation of molecular hydrogen, which does not occur in the atmosphere of Titan, because of H₂ escape. In order to reassess the data already available from this type of laboratory studies, experiments on CH₄–N₂ atmospheres, with and without H2 escape, have been recently performed. The influence of this parameter on the chemical evolution of the atmosphere and on the nature and relative quantities of organic compounds has been studied.After reviewing these experiments, implications of the obtained results on the organic chemistry at the periphery of Titan are discussed.Paper presented at the 6th College Park Colloquium, October 1981.     

Biomass fast pyrolysis in a fluidized bed reactor under N2, CO2, CO, CH4 and H2 atmospheres

Biomass fast pyrolysis is one of the most promising technologies for biomass utilization. In order to increase its economic potential, pyrolysis gas is usually recycled to serve as carrier gas. In this study, biomass fast pyrolysis was carried out in a fluidized bed reactor using various main pyrolysis gas components, namely N₂, CO₂, CO, CH₄ and H₂, as carrier gases. The atmosphere effects on product yields and oil fraction compositions were investigated. Results show that CO atmosphere gave the lowest liquid yield (49.6%) compared to highest 58.7% obtained with CH₄. CO and H₂ atmospheres converted more oxygen into CO₂ and H₂O, respectively. GC/MS analysis of the liquid products shows that CO and CO₂ atmospheres produced less methoxy-containing compounds and more monofunctional phenols. The higher heating value of the obtained bio-oil under N₂ atmosphere is only 17.8 MJ/kg, while that under CO and H₂ atmospheres increased to 23.7 and 24.4 MJ/kg, respectively.     

Far UV irradiation of model prebiotic atmospheres

UV light has been the most important energy source on the primitive Earth. However, very few experiments have been performed to test directly the possible role of this energy source on the chemical evolution of the primitive atmosphere, mainly on account of experimental difficulties. Experiments are generally performed with other excitations, mainly electric discharge, and it is frequently assumed that UV irradiation would give similar results.As theoretical considerations make this assumption questionable, direct experimental controls have been undertaken: Model primitive atmospheres have been submitted to 147 nm UV light and the gaseous phase has been analysed. Preliminary qualitative results concerning CH₄–NH₃ atmospheres are reported.Irradiation of pure CH₄ gives rise to the synthesis of a large number of hydrocarbons, mainly saturated hydrocarbons but including also unsaturated ones as, C₂H₂, C₂H₄, C₃H₆, C₃H₄. These insaturated hydrocarbons are synthetized at a very low rate when ammonia is present in the medium.Irradiations of CH₄–NH₃ mixtures give rise, in addition to hydrocarbons, to important amounts of HCN (about 0.1%) and to lesser amounts of CH₃CN and C₂H₅CN. No unsaturated nitriles such as acrylonitrile and cyanoacetylene have been detected. Search for amines is in progress.These results evidence that UV irradiation may contribute largely to synthesis of HCN in CH₄–NH₃ atmospheres and, consequently to the synthesis of many biochemical compounds that can be derivated from HCN. However, synthesis of other compounds, such as pyrimidines, which can derivate from other nitriles, such as cyanoacetylene, cannot be initiated only by UV light, contrary to electric discharges. In addition, if electric discharges are very efficient for synthesis of nitriles in CH₄–N₂ atmospheres, there is not yet evidence that UV light is able to do so.Presented at the 2nd ISSOL Meeting and the 5th ICOL in Kyoto, 5–10 April, 1977.     

Study on the chemical evolution of low molecular weight compounds in a highly oxidized atmosphere using electric discharges

The molecular basis for the chemical evolution of low molecular compounds was studied using electric discharges on a higly oxidized atmosphere comprised of CO₂, N₂ and H₂O. In the gas phase, O₂ and CO were formed by the decomposition of CO₂ and their yields were enhanced by the addition of N₂ to the gas mixture. It was demonstrated that H₂O suppressed the reduction of CO₂ while H₂O also had a role in producing organic compounds such as formic acid and formaldehyde. Infrared analysis of the water soluble products and the inner surface of the reaction vessel indicated the production of compounds more complex than formic acid and formaldehyde. These compounds contained the chemical bonds which were identified to be OH, CO, CN and/or CC.     

Geochemical constraints on the origin of organic compounds in hydrothermal systems

It is proposed that abiotic synthesis of organic compounds occurs in metastable states. These states are permitted by kinetic barriers which inhibit the approach to stable equilibrium in the C-H-O-N system. Evidence for metastable equilibrium among organic compounds in sedimentary basins is reviewed, and further evidence is elucidated from hydrous pyrolysis experiments reported in the literature. This analysis shows that at hydrothermal conditions, organic compounds are formed or destroyed primarily through oxidation/reduction reactions, and that the role of temperature is to lower the kinetic barriers to these reactions. These lines of evidence allow the development of a scenario in which abiotic synthesis can occur at hydrothermal conditions through the reduction of CO₂ and N₂. This scenario can be tested quantitatively with distribution of species calculations as functions of temperature, pressure, hydrogen fugacity (fH₂) and initial composition. One example of such a test is given for an early, sudden outgassing of the Earth, in which CO₂, H₂O, and N₂ are transported from the mantle to the atmosphere by hydrothermal solutions. Activities of metastable aqueous organic species which form as a consequence of this process are evaluated at conditions appropriate for seafloor hydrothermal systems, and are found to maximize at about 200 °C and between the oxidation states set by two mineral assemblages common in the oceanic crust.     

Abiogenic synthesis of peptide-like substances induced by UV-irradiation and heat]

Dry mixtures of glycine and succinic acid as well as of glycine, phenylalanine and succinic acid were exposed to UV-irradiation and heating up to 170 degrees in atmospheres of O2 and N2. Under these conditions, synthesis of substances with peptide bonds was observed. Irradiation or heating of the first mixture results in synthesis of a substance which is water soluble and capable of dialysis, exhibiting therefore low degree of polymerization. This substance contains COOH-groups, but lacks free NH2-groups. The latter reveal themselves during hydrolysis which brings about the appearance of the initial compounds. In the second mixture, which contains an aromatic amino acid, several products are synthesized some of them being presented by the insoluble and non-dialysed precipitate (polymer). Among soluble products, peptide-like substances (without free NH2-groups) and dipeptides were found. UV-irradiation causes photolysis of phenylalanine which is mixed with succinic acid; as the result, tyrosine, alanine and asparagic acid are found among the products synthesized. These amino acids are not formed during heat-induced synthesis. Abiogenic synthesis takes place more readily in the presence of O2 than in a N2 atmosphere. Irradiation of aqueous solution of the product synthesized resulted in the photolysis of the latter. The results obtained indicate that during abiogenic period, synthesis of peptides and peptide-like substances from dry mixtures of amino acids and organic acids induced by UV-irradiation and heat might take place on the litosphere.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.     

Formation of prebiochemical compounds in models of the primitive Earth's atmosphere

In order to understand the formation of organic compounds in the primitive atmosphere, the first steps of evolution in models of the primitive atmosphere were investigated. Mixtures containing C−H−N elements were subjected to a low pressure silent electric discharge for several seconds, and the resulting effluents were analysed mainly by gas chromatography, infrared spectrometry and chemical analysis. The formation of hydrocarbons (i.e. ethylene, acetylene, methylacetylene) and of nitrogen containing compounds (i.e. hydrogen cyanide, cyanogen, saturated nitriles, acrylonitrile, cyanoacetylene) is reported. The influence of the initial mixture composition on the amount of compounds formed was systematically studied. The nature of the nitrogen source (N₂ or NH₃) in the primitive atmosphere has a great influence on the amount and on the very nature of the synthesized products. It is shown that important precursors such as cyanogen and cyanoacetylene are formed only in very rich N₂ mediums. There results show the important role played by the nature of the primitive atmosphere in the determination of the chemical evolution pathways.     

Photocatalytic synthesis of organic compounds from CO and water: Involvement of surfaces in the formation and stabilization of products

Summary ¹⁴C-Formic acid and other¹⁴C-organic compounds are formed on surface materials when mixtures of¹⁴CO,¹²CO₂ or N₂ and water vapor are irradiated with ultraviolet light (UV) of > 250 nm. The rate of organic formation is roughly proportional to the quantity of substratum irradiated. The available evidence suggests that¹⁴CO adsorbed to or in contact with the substratum is excited by the long wavelength UV and reacts with adsorbed H₂O or surface hydroxyl groups yielding the organic products. Photodestruction of the¹⁴C-organics yields¹⁴CO₂ and¹⁴CO. A steady state is attained when organic products reach a concentration such that the rate of photodestruction is equal to the rate of synthesis. The product accumulation is greater and the photodestruction is slower when N₂ is used as diluent gas.Differences in the rates of synthesis, rates of photodestruction and amounts of product accumulation are observed with different silica and alumina substrata. The substrata with large surface areas are most effective for synthesis while maximum photoprotection of organics is afforded by substrata containing high concentrations of surface hydroxyl groups.The observation of the synthesis on a variety of substrata using realistic simulations of atmospheres and solar energies strengthens previous proposals that this process may occur on Mars and may have been important on the primitive Earth.     

The possibility of organic molecule formation in the Venus atmosphere

Based on the detection of ammonia in the Venus atmosphere, and the suggested presence of hydrogen chloride, a structure for the Venus atmosphere was suggested as having 3 cloud layers, consisting of ammonium chloride (30 to 50 km above the ground), a mixture of ammonium bicarbonate and ammonium carbamate (NH₂COOHN₄) from 50–60 km, and water ice crystals above this. There is a strong possibility of electrical discharge in the atmosphere as a result of thermal convective turbulence, which in the case of the slightly reducing atmosphere outlined above could lead to organic compound formation. The hypothesis was tested experimentally by passing a 60 KV spark from platinum electrodes through a gas mixture of composition: N₂(0.2%), NH₃ (2%), Water (5%), O₂ (0.6%), CO₂ (remainder), for 8 hr. The products were analysed by mass spectrometry and amino acid analysis by ion exchange. Methane and formaldehyde were identified by MS, and glycine and alanine by the amino acid analyzer. The presence of organic compounds in the Venus atmosphere is therefore a strong possibility.     

Anaerobic stimulation of root exudates and disease of peas

Summary The relationships between root exudation, root disease and anaerobic root stresses were investigated. Sand culture and mist chamber studies demonstrated that low O₂ and high CO₂ reduced plant growth and increased the exudation of ethanol, amino acids, and sugars by pea roots. The relative loss of ethanol by roots was much greater in treatments with atmospheres of N₂ containing 30% CO₂ than in treatments of air containing 30% CO₂ or N₂. Ethanol was not detected in the nutrient solution of aerated plant roots. Atmospheres of N₂ plus 30% CO₂ caused 500% greater mycelial growth ofFusarium solani f. sp.pisi and 400% more disease of inoculated pea roots. Relative losses of four amino acids and four sugars were much greater in atmospheres of N₂ plus 30% CO₂ than in N₂ or air.     

Prebiotic synthesis in atmospheres containing CH4, CO,and CO2

The prebiotic synthesis of organic compounds using a spark discharge on various simulated primitive earth atmospheres at 25 degrees C has been studied. Methane mixtures contained H2 + CH4 + H2O + N2 + NH3 with H2/CH4 molar ratios from 0 to 4 and pNH3 = 0.1 torr. A similar set of experiments without added NH3 was performed. The yields of amino acids (1.2 to 4.7% based on the carbon) are approximately independent of the H2/CH4 ratio and whether NH3 was present, and a wide variety of amino acids are obtained. Mixtures of H2 + CO + H2O + N2 and H2 + CO2 + H2O + N2, with and without added NH3, all gave about 2% yields of amino acids at H2/CO and H2/CO2 ratios of 2 to 4. For a H2/CO2 ratio of 0, the yield of amino acids is extremely low (10(-3)%). Glycine is almost the only amino acid produced from CO and CO2 model atmospheres. These results show that the maximum yield is about the same for the three carbon sources at high H2/carbon ratios, but that CH4 is superior at low H2/carbon ratios. In addition, CH4 gives a much greater variety of amino acids than either CO or CO2. If it is assumed that an abundance of amino acids more complex than glycine was required for the origin of life, then these results indicate the requirement for CH4 in the primitive atmosphere.     

Energy yields for hydrogen cyanide and formaldehyde syntheses: The hcn and amino acid concentrations in the primitive ocean

Prebiotic electric discharge and ultraviolet light experiments are usually reported in terms of carbon yields and involve a large input of energy to maximize yields. Experiments using lower energy inputs are more realistic prebiotic models and give energy yields which can be used to estimate the relative importance of the different energy sources on the primitive earth. Simulated prebiotic atmospheres containing either CH₄, CO or CO₂ with N₂, H₂O and variable amounts of H₂ were subjected to the spark from a high frequency Tesla coil. The energy yields for the synthesis of HCN and H₂CO were estimated. CH₄ mixtures give the highest yields of HCN while H₂CO is most efficiently produced with the CO mixtures. These results are a model for atmospheric corona discharges, which are more abundant than lightning and different in character. Preliminary experiments using artificial lightning are also reported. The energy yields from these experiments combined with the corona discharge available on the earth, allows a yearly production rate to be estimated. These are compared with other experiments and model calculations. From these production rates of HCN (e.g. 100 nmoles cm⁻² yr⁻¹) and the experimental hydrolysis rates, the steady state concentration in the primitive ocean can be calculated (e.g., 4 × 10⁻⁶ M at pH 8 and 0°). A steady state amino acid concentration of 3 × 10⁻⁴ M is estimated from the HCN production rate and the rate of decomposition of the amino acids by passage through the submarine vents.     

Comparative physiology of field-grown tomatoes during ripening on the plant or retarded ripening in controlled atmospheres

Tomato fruits of six cultivars were harvested at three different stages of maturity or were harvested when mature-green and then stored in a modified gas atmosphere (2·5-4% O₂; 4% CO₂) for 2 months. The fresh and stored fruits were analysed for their contents of sugars, organic acids and free amino acids, while proteins were separated by discontinuous electrophoresis on polyacrylamide gels. In general, the low molecular weight components decreased during storage. A comparison of mature-green fruits before and after storage showed that although total protein was not decreased, a different electrophoretic pattern was obtained following controlled atmosphere (CA) storage. Thus, although controlled atmosphere storage repressed the loss of chlorophyll and synthesis of lycopene, carotenoids and xanthophylls, the biochemical parameters measured showed a controlled change towards the conditions exemplified by ripe fruits. This was not so marked in some cultivars as it was in others.     

Photochemical synthesis of biomolecules under anoxic conditions

We report the long-wavelength UV anoxic photosynthesis of uracil, various sugars (including deoxyribose and glycoaldehyde), amino acids, and other organic photoproducts. These reactions occur in mixtures of water, calcium carbonate, formaldehyde and hydrazine. Our data demonstrate that under several sets of conditions biomolecules can be formed in variety and abundance from reduced compounds (formaldehyde and hydrazine) derived from anoxic dinitrogen/carbon dioxide environments. The formaldehyde concentrations were varied from 10 mM to 0.005 mM, and the hydrazine concentrations were varied from 1 mM to 0.01 mM. The highest of these reactant concentrations were 500 and 6 times greater than those reported for earlier experiments upon the synthesis of these precursors from CO₂ or N₂, while the lowest of reactant concentrations employed here were 0.5 (formaldehyde) and 0.006 (hydrazine). Product yields were greatest when the hydrazine/formaldehyde ratio was 1, and when the reactant concentrations were low. These data suggest that organic products can be formed in variety from those amounts of formaldehyde and hydrazine precursors which are themselves formed under anoxic UV photochemical conditions. Hence these various reactions would seem to have prebiotic relevance. The UV 254 nm photon flux employed was 100 times higher than unattenuated solar flux. Durations of UV exposure were 24 hrs and 72 hrs. No experiments have been addressed to the possibility of UV flux dependency.     

Carbon dioxide effects on ethanol production, pyruvate decarboxylase, and alcohol dehydrogenase activities in anaerobic sweet potato roots

The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O₂ and N₂ gases disappeared quickly and were replaced by CO₂. There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO₂ environment, followed by submergence and a N₂ environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO₂-treated and 100% N₂-treated roots. CO₂ atmospheres also resulted in higher pyruvate decarboxylase activity than did N₂ atmospheres. Concentrations of CO₂ were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO₂ concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase.     

Origin of organic compounds on the primitive earth and in meteorites

The role and relative contributions of different forms of energy to the synthesis of amino acids and other organic compounds on the primitive earth, in the parent bodies or carbonaceous chondrites, and in the solar nebula are examined. A single source of energy or a single process would not account for all the organic compounds synthesized in the solar system. Electric discharges appear to produce amino acids more efficiently than other sources of energy and the composition of the synthesized amino acids is qualitatively similar to those found in the Murchison meteorite. Ultraviolet light is also likely to have played a major role in prebiotic synthesis. Although the energy in the sun's spectrum that can be absorbed by the major constituents of the primitive atmosphere is not large, reactive trace components such as H2S and formaldehyde absorb at longer wavelengths where greater amounts of energy are available and produce amino acids by reactions involving hot hydrogen atoms. The thermal reaction of CO + H2 + NH3 on Fischer-Tropsch catalysts generates intermediates that lead to amino acids and other organic compounds that have been found in meteorites. However, this synthesis appears to be less efficient than electric discharges and to require a special set of reaction conditions. It should be emphasized that after the reactive organic intermediates are generated by the above processes, the subsequent reactions which produce the more complete biochemical compounds are low temperature homogenous reactions occurring in an aqueous environment.