Introduction

It is now generally thought that life emerged via chemical evolution on the Earth (e.g., Oparin 1952). According to this model, the building blocks of life such as amino acids would have been available from abiotic sources. Since the Miller–Urey experiment (Miller 1953), various types of prebiotic amino acid syntheses have been studied (e.g., Schlesinger and Miller 1983a; Kobayashi et al. 1990). These experiments revealed that the efficiency of the formation of organic compounds, including amino acids, was strongly dependent on the redox state of the gas mixture used, suggesting that the redox state of the early atmosphere would have also played a significant role in the synthesis of organic compounds.

Carbon isotope composition analysis of the kerogen found in ancient sedimentary rocks from Isua in West Greenland indicated that life may have already emerged as early as 3.8 billion years ago (Mojzsis et al. 1996; Rosing 1999). Although the composition of the early Earth atmosphere around this time remains unknown, volcanic gases are thought to have been a major contributor (e.g., Trail et al. 2011). The chemical composition of volcanic gases is largely dependent on the redox state of the upper mantle, and it has been reported that this state was close to that of the present mantle that is approximated by the fayalite-magnetite-quartz (FMQ) buffer (Delano 2001; Trail et al. 2011). Melts with oxygen fugacity close to the FMQ buffer predominantly yield H2O and CO2 (e.g., Zolotov and Shock 2000). Thus, it is now widely believed that the major C-bearing species in the early Earth atmosphere was CO2 (herein referred to as CO2-rich). It is also important to consider the effects of asteroid impacts on the composition of the early Earth atmosphere. Based on a photochemical model and chemical equilibrium calculations, it has been proposed that the early Earth was likely to have had a reducing atmosphere when the accretion phase was almost complete (Kasting 1990; Hashimoto et al. 2007; Schaefer and Fegley 2010). In addition, it has been reported that the escape of hydrogen from the early Earth atmosphere may have been much slower than previously thought (Tian et al. 2005). Thus, the atmosphere of the early Earth may have contained a significant amount of reduced gases such as H2, CH4, and CO.

Lightning is thought to have been one of the major energy sources for prebiotic synthesis on the early Earth (e.g., Chyba and Sagan 1991). This type of energy produces high temperature (>104 K) gases (e.g., Uman 1964). Furthermore, under such high temperature conditions, the chemical composition of a gas phase is thought to be rapidly reaching equilibrium (McKay and Borucki 1997). As gas phases expand and cool, their chemical composition can change. At low temperatures, the gas composition is frozen at the freeze-out temperature because the rates of the chemical reactions become too slow to maintain the equilibrium. For lightning and cometary impacts, the freeze-out temperatures are thought to be in the range of 2,000–3,000 K (Chameides and Walker 1981; McKay and Borucki 1997).

Hydrogen cyanide, formaldehyde, and ammonia have been reported to be important precursors of abiotic organic molecules (e.g., Schlesinger and Miller 1983b). The efficient synthesis of these molecules would therefore be required for the prebiotic synthesis of compounds such as amino acids. In a CO2-rich gas mixture, the chemical equilibrium composition at high temperatures (2000–3000 K) is rich in CO and NO (Chameides and Walker 1981). An experimental study has also shown that neutral atmospheres are favorable for the synthesis of CO and NO (Summers and Khare 2007). Taken together, these results suggest that prebiotic synthesis of amino acids, at least, would not have proceeded efficiently in a CO2-rich atmosphere (Schlesinger and Miller 1983b; Stribling and Miller 1987).

It has been suggested that the previously reported low yields of amino acids resulting from CO2-N2 gas mixtures using a spark discharge may be attributed in part to the oxidation of amino acids or their precursors during acid hydrolysis by the nitrate/nitrite also produced during the spark discharge experiment (Cleaves et al. 2008). More specifically, it has been reported that the yield of amino acids was greatly increased (by a factor of several hundred) when ascorbic acid was added as an oxidation inhibitor to the spark discharge products prior to the acid hydrolysis step. Cleaves et al. (2008) also reported a moderate increase in the yields of amino acids when other oxidation inhibitors were used, such as pyrites (by a factor of 10 relative to the control) and FeSO4 (by a factor of 2 relative to the control). Although the oxidation of free amino acids by nitrate/nitrite during acid hydrolysis has already been reported (Robertson et al. 1987), to the best of our knowledge, there have been no reports in the literature investigating what the carbon source of the amino acids detected after acid hydrolysis with ascorbic acid actually was. With this in mind, the decision was taken to reinvestigate this reaction. Spark discharge experiments were initially performed in a gas mixture of (1) CO2–N2–H2O or (2) 13C-labeled CO2–N2–H2O. The spark discharge products in the aqueous phase were then acid hydrolyzed with (1) ascorbic acid or (2) 13C-labeled ascorbic acid (13C6H8O6) and the amino acids in the hydrolysates were subsequently analyzed by gas chromatography/mass spectrometry following their derivatization.

Experimental

Chemicals

Nitrogen (purity >99.99 %) was purchased from Suzuki Shokan Co., Japan. A (1:1) mixture of carbon dioxide and nitrogen (purity >99.995 % CO2, >99.99995 % N2) was purchased from Taiyo Nippon Sanso Co., Japan. 13C-labeled carbon dioxide (purity >99 % 13C) was obtained from ICON Co., USA. L-ascorbic acid (purity >99.6 %) was purchased from Wako Pure Chemical Co., Japan. 13C-labeled L-ascorbic acid (purity >99 % 13C) was purchased from Omicron Biochemicals, Inc., USA. Deionized water was further purified using a Milli-Q Labo SystemTM. All other chemicals were analytical grade. Prior to the commencement of any experiments, all the glass apparatus was heated in an oven at 500 °C to eliminate any possible sources of contamination.

Instruments

The high performance liquid chromatography (HPLC) system used for amino acid analysis consisted of two Shimadzu LC-10A pumps, a Shimpak ISC-07/S1504, 4 mm i.d. × 150 mm cation exchange column, a Shimadzu RF-535 fluorescence detector, and an o-phthalaldehyde (OPA)/N-acetyl-L-cysteine (NAC)/borate buffer post-column derivatization system. This analytical system was the same as that used by Kurihara et al. (2012). Capillary electrophoresis (CE) (Otsuka CAPI-3300) was used for the analysis of amines, nitric acid, and carboxylic acids. A gas chromatograph/mass spectrometer (GC/MS) (JEOL JMS-600 with Agilent Technologies J&W DB-5 ms 0.25 mm i.d. × 30 m column or Varian Chirasil-L-Val 0.25 mm i.d. × 25 m column) was used for amino acid identification. The temperature profile used for GC/MS analysis was as follows: initial temperature 70 °C for 5 min, increased to 130 °C (4 °C/min), increased to 190 °C (8 °C/min) and held at 190 °C for 20 min.

Spark Discharge Experiment

Figure 1 shows the apparatus used for the spark discharge experiment. A gas mixture (1:1) (800 mbar) of CO2 (or 13C-labeled CO2) and N2 was introduced into the glass apparatus (1.6 L) with deionized water (40 mL). Prior to the gas introduction, air dissolved in the water in the glass apparatus was degassed by freezing, evacuating the headspace, and thawing three times. Spark discharges generated by a BD-50 Tesla coil were fired through two tungsten electrodes for 24 h at room temperature. Water in the glass apparatus was maintained at approximately 293 K throughout the experiment.

Fig. 1
figure 1

Schematic representation of the apparatus for the spark discharge experiment

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Analytical Procedures

The aqueous phase containing the products was hydrolyzed with 6 M HCl in a sealed test tube at 110 °C for 24 h both the presence and in the absence of ascorbic acid (or 13C-labeled ascorbic acid). Following acid hydrolysis, the HCl was evaporated using centrifugal drying equipment, and the amino acids present in the resulting residue were determined by ion-exchange HPLC followed by post-column derivatization with OPA and NAC using the method by reported by Kurihara et al. (2012). In the GC/MS analysis, glycine and alanine in the products were fractionated using reversed-phase HPLC, and further analyzed following derivatization with isopropanol and trifluoroacetic anhydride (Waldhier et al. 2010). The nitric acid, carboxylic acids, and amines in the solution were analyzed by CE using the method reported by Soga et al. (2000).

Results and Discussion

Analysis of the Spark Discharge Products in the Aqueous Phase

The pH of the aqueous phase upon completion of the experiment was 0.94. Ammonia, nitric acid, and formic acid were the major products detected in the spark discharge experiment (see Table 1). Larger molecules, including acetic acid, propionic acid and aminoacetonitrile, which is a glycine precursor in the Strecker reaction, were not detected.

Table 1 Yield of nitric acid, ammonia, and formic acid (mol)
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Based on a theoretical model and experimental studies, the production rate of hydrogen cyanide has been reported to decrease with a decreasing C/O ratio (Chameides and Walker 1981; Schlesinger and Miller 1983b; Stribling and Miller 1987). At low C/O ratios (≤ 1), CO and NO are mainly produced by shock heating (e.g., Mancinelli and McKay 1988). The occurrence of different reaction pathways following the production of CO and NO has been reported and discussed in detail in several studies (Mancinelli and McKay 1988; Summers and Khare 2007; Vantrump and Miller 1973). Based on the result of these studies, it is plausible to suggest that some of the CO may react with OH radicals produced from water vapor to form CO2. Furthermore, CO may also react with H radicals produced from water vapor to form HCO or dissolve in the aqueous phase and undergo hydration to form formic acid. NO may dissolve in the aqueous phase as HNO as a consequence of the reaction between NO and HCO. NO− dissociated from HNO in the aqueous phase may react with NO or itself to form NxO −x (x = 2 or 3). These species could then rapidly decay into NO −x and N2O.

Our experimental results were effectively consistent with the reaction models outlined above. Under the CO2-rich conditions, it was expected that the reduced species such as hydrogen cyanide and ammonia would only be formed in minor quantities, because their formation is thermodynamically unfavorable (e.g., Chameides and Walker 1981). In contrast to our expectation, however, the amount of ammonia formed during the experiments was found to be comparable to that of the nitric acid. This result was consistent with the data reported by Cleaves et al. (2008), who attributed the observation to a redox disproportionation event, although it is also possible that the ammonia may have been formed by the partial reduction of N2 by H radicals produced from the water vapor.

Amino Acid Analysis of the Spark Discharge Products

Ascorbic acid was added to the spark discharge products prior to the acid hydrolysis in an attempt to confirm its anti-oxidation effect. After hydrolysis in the absence of ascorbic acid, no amino acids were detected, whereas after hydrolysis in the presence of ascorbic acid, the yield of amino acids increased significantly. This result was consistent with the data reported by Cleaves et al. (2008). The composition of the detected amino acids, however, depended on the amount of ascorbic acid that had been added to the system. Amino acids with carbon number ≥3, including aspartic acid, alanine, β-alanine and β-aminobutyric acid, increased in proportion as the amount of ascorbic acid added to the system increased. The HPLC chromatograms of the detected amino acids are shown in Fig. 2. Furthermore, a mixture of formic acid, nitric acid, and ammonia was hydrolyzed under acidic conditions with ascorbic acid to confirm whether the amino acids had been formed as a consequence of the reaction between the spark discharge products and ascorbic acid. The HPLC chromatograms of the detected amino acids are shown in Fig. 2. The results revealed that the major products of this reaction were glycine, alanine, α-aminobutyric acid, β-alanine and γ-aminobutyric acid.

Fig. 2
figure 2

a-c HPLC chromatograms of the amino acids detected following the acid hydrolysis of the spark discharge products a in the absence of ascorbic acid, b in the presence of ascorbic acid (the amounts of ascorbic acid and nitric acid were equivalent), and c in the presence of ascorbic acid (that amount of ascorbic acid was five times greater than that of the nitric acid). d-f HPLC chromatograms of the amino acids detected following the acid hydrolysis d a mixture of formic acid, nitric acid, and ammonia, e ascorbic acid, f a mixture of formic acid, nitric acid, ammonia, and ascorbic acid. 1: DL-Aspartic acid; 2: DL-Glutamic acid; 3: Glycine; 4: DL-Alanine; 5: DL-α-aminobutyric acid; 6: DL-β-aminobutyric acid; 7: β-Alanine; 8: DL-γ-aminobutyric acid

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To investigate and identify the carbon source of these amino acid products, the spark discharge experiment was repeated using 13C-labeled CO2. The mass spectrum of the alanine detected in this experiment is shown in Fig. 3b. If the amino acids were formed from CO2, their mass number should be shifted relative to their 12C-amino acid counterparts. Interestingly, however, no changes in the mass spectra were observed and the mass spectrum of glycine also provided similar results to that of alanine, indicating that the majority of the amino acids detected were likely not formed from the labeled CO2. The spark discharge products from a 12CO2–N2–H2O mixture were also hydrolyzed under acidic conditions with 13C-labeled ascorbic acid. The mass spectrum of the alanine detected in this experiment is shown in Fig. 3c. This analysis revealed changes in the mass profile and clearly indicated that the alanine was being formed from the ascorbic acid that reacted with the spark discharge products during the acid hydrolysis. These results therefore suggested that the majority of the amino acids were formed from ascorbic acid during the acid hydrolysis step.

Fig. 3
figure 3

Mass spectra of a standard 12C- DL-alanine derivative, b DL-alanine derivative following acid hydrolysis of a spark discharge sample of a 13CO2–N2–H2O gas mixture with ascorbic acid c DL-alanine derivative following acid hydrolysis of a spark discharge sample of a 12CO2–N2–H2O gas mixture with 13C-labeled ascorbic acid. The structural formulas indicate the structure and corresponding fragments of alanine esterified with 2-propanol and acylated with trifluoroacetic anhydride. *C indicates the positions that could have been replaced by 13C

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Importance of the pH and HCN Concentrations in Prebiotic Chemistry

The Strecker and HCN polymerization reactions are well-known as important mechanisms for the formation of amino acid precursors (e.g., Miller 1955; Ferris et al. 1978), and these reactions require high concentrations of starting materials. The efficient formation of HCN is therefore required for the synthesis of amino acid precursors. As mentioned above, however, HCN is not formed efficiently in CO2-rich atmospheres. For example, it has been previously reported that the efficiency of HCN formation decreases significantly when the H2/CO2 ratio is less than 1 (Schlesinger and Miller 1983b).

For HCN polymerization, the decomposition and polymerization rates were reported to be balanced when the concentration of HCN was 0.01–0.1 M, the pH was 8–9, and the temperature was in the range of 0–60 °C (Sanchez et al. 1967). At low concentrations, the HCN is hydrolyzed into ammonia and formic acid. Miyakawa et al. (2002a, b) proposed that eutectic freezing could be required to provide sufficiently high enough concentrations of HCN to facilitate polymerization, because HCN cannot be concentrated by water evaporation as a consequence of its volatility. Miyakawa et al. (2002b) also showed that adenine could be synthesized from dilute NH4CN solutions (> 0.001 M) via eutectic condensation. With regard to the influence of the pH of the solution, Cleaves et al. (2008) pointed out that a low pH could inhibit HCN reactions by reducing the nucleophilicity of cyanide anion. They also demonstrated that modest yields of amino acids (by a factor of 10) could be achieved by buffering the pH during the reaction. The formation of amino acid precursors from HCN reactions may therefore represent an inefficient process in a neutral atmosphere in the absence of pH buffering and HCN condensation mechanisms.

Conclusions

Ascorbic acid contributes to the formation of amino acid contaminants during hydrolysis reactions with the nitrogen species produced in the spark discharge, and is therefore not an appropriate oxidation inhibitor for investigating the synthesis of amino acids from oxidizing gas mixtures. The cause of the oxidation by nitrite/nitrate may be attributable to the formation of nitrosonium ions from nitrate/nitrite under the strongly acidic conditions. Thus, the use of alkaline conditions should be employed to avoid the potential for oxidation by the nitrate and nitrite species (e.g., Neish et al. 2010).

The ability to effectively estimate which organic compounds could have been present on the early Earth is an important for our understanding of chemical evolution. Under CO2-rich conditions, through lightning-induced reactions, carbon may have been mainly fixed as formic acid, whereas nitrogen may have been fixed predominantly as nitric acid and ammonia in the early oceans. Given that these species do not form amino acid precursors and that the main carbon species in the primitive Earth atmosphere may have been CO2, it is likely that exogenous sources such as interstellar dust particles may have been the dominant prebiotic sources of amino acids (Chyba and Sagan 1992; Greenberg et al. 1994), barring other possible efficient sources such as hydrothermal synthesis.