Thermal stability of amino acids in siliceous ooze under alkaline hydrothermal conditions

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Organic Geochemistry Organic Geochemistry 38 (2007) 1897–1909 www.elsevier.com/locate/orggeochem

Thermal stability of amino acids in siliceous ooze under alkaline hydrothermal conditions Kyoko Yamaoka a,*, Hodaka Kawahata a, Lallan P. Gupta b, Miho Ito c, Harue Masuda c a

b

Graduate School of Frontier Sciences and Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan Center for Deep Earth Exploration (CDEX/JAMSTEC), Yokohama Institute for Earth Sciences, 3173-25 Showa-machi, Kanazawa-ku, Yokohama, Kanagawa 236-0001, Japan c Department of Geosciences, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Received 28 December 2006; received in revised form 13 July 2007; accepted 16 July 2007 Available online 21 July 2007

Abstract Hydrothermal systems have been suggested as being favourable environments for the origin and evolution of life on the primitive Earth. To test this hypothesis, it is necessary to investigate under hydrothermal conditions the behaviour of biomolecules such as amino acids (AAs), a major component of organisms. In contrast to submarine hydrothermal systems, hot springs in the Rift Valley in eastern Africa on the thick continental crust often have a high pH (alkaline conditions) as a result of enrichment via Na2CO3 and volatile gases. We reacted siliceous ooze with an aqueous solution of NaCl and Na2CO3 at elevated temperature (100–300 °C) to evaluate the thermal stability of the AAs under alkaline hydrothermal conditions. The AAs in the sediment in peptide form were released to the liquid phase and decomposed through hydrolysis of peptide bonds. Comparison of the results with those from similar experiments using the same sediment sample under neutral conditions reveals that the rates of decomposition of the AAs are significantly inhibited under alkaline conditions. Moreover, AAs remained in both the solid and liquid phases even after heating at 300 °C for 240 h. Our results indicate that AAs are more thermally stable in alkaline solution, indicating that these hydrothermal conditions are more favourable for the evolution of primitive life on the thick continental crust. They also imply that alkaline hydrothermal systems on other planets might be plausible places for extraterrestrial life. Ó 2007 Elsevier Ltd. All rights reserved.

1. Introduction Hydrothermal systems supply thermal energy and chemicals from the Earth’s interior to the surface through circulation of hydrothermal water. * Corresponding author. Tel.: +81 3 5351 6429; fax: +81 3 5351 6438. E-mail address: [email protected] (K. Yamaoka).

The discovery of thermophilic bacteria in hydrothermal vents and high temperature dispersal fluids (reviewed by Deming and Baross, 1993) stimulated biogeochemical interest in such systems. Using the following geochemical assumptions, Baross and Hoffman (1985) and Corliss (1990) hypothesized that hydrothermal systems were a favourable environment for the origin of life on Earth: (1) such systems were more active on the primitive Earth than

0146-6380/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2007.07.003

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on the present day Earth, (2) the thermal energy and reactants for the prebiotic synthesis of organic compounds were readily available, (3) organic compounds were produced easily in the reducing conditions of the hydrothermal systems and (4) the earliest organisms in hydrothermal systems in the deep crust could escape damage by ultraviolet irradiation and meteorite bombardment. In addition, a molecular biological approach has demonstrated that the thermophilic microbial inhabitants of the active seafloor and continental hot springs populate the deepest branches of the universal phylogenetic tree – that is, the hydrothermal ecosystem is the oldest continuously inhabited ecosystem on Earth (Reysenbach and Shock, 2002). Since amino acids (AAs) and the proteins derived therefrom are essential for all living organisms, their behaviour under hydrothermal conditions should be investigated to examine the possibility of the emergence of life in hydrothermal systems. Early experiments showed that AAs rapidly degrade in aqueous solution at 250 °C and suggested that maintaining high concentrations in hydrothermal systems would be difficult (Bernhardt et al., 1984; White, 1984; Miller and Bada, 1988). On the basis of a reinterpretation of data reported by Miller and Bada (1988), Shock (1990) suggested that AAs could approach metastable equilibrium in hydrothermal solution, but Qian et al. (1993), in determining AA decomposition rates, concluded that equilibrium would never be reached under hydrothermal conditions. Experiments in which oxygen fugacity was constrained showed that decomposition rates of some AAs were lower than in non-buffered experiments; nevertheless, metastable or stable equilibrium was not attained (Andersson and Holm, 2000). Recently, Ito et al. (2006) performed experiments with a seafloor sediment and aqueous NaCl under simulated hydrothermal conditions and found that AAs did not survive in hydrothermal water above 250 °C unless they were protected by a solid phase, such as clay minerals. On the other hand, AAs have been synthesized under simulated hydrothermal conditions at high temperature (Marshall, 1994). Several of them and abundant amines were synthesized by reaction of NH4HCO3 solution with C2H2, H2, and O2 at 200–275 °C over 0.2–2 h (Marshall, 1994). AAs and amines were not formed below 150 °C. Islam et al. (2001) heated an aqueous solution containing KCN, HCHO, and NH4HCO3 in a supercritical water flow reactor at 50–400 °C and

found that some non-protein AAs with high carbon number were formed only above 300 °C. Alargov et al. (2002) found that reaction of HCHO and NH3 above 350 °C produced some AAs, but the products rapidly decomposed under the hydrothermal conditions. Thus, high concentrations of AAs cannot be attained unless their rate of formation exceeds their rate of decomposition, or they are removed to a milder environment. Most previous experiments have been conducted under neutral to acidic conditions. The results from these studies would be applicable to modern hydrothermal systems along mid-oceanic ridges; however, the properties of hydrothermal systems depend on their tectonic setting. In contrast to submarine hydrothermal water at a mid-oceanic ridge, hot springs in the Rift Valley in eastern Africa often have a high pH (alkaline conditions) due to enrichment via Na2CO3 and volatile gases. By analogy with such springs, Kempe and Degens (1985) postulated a ‘‘soda ocean’’ model, in which the ancient sea had high alkalinity, high pH, and low Ca and Mg concentrations. Alkaline hydrothermal systems are basically the result of chemical weathering of the continental crust by carbonic acid. The continental crust was likely formed early in the Earth’s history (around 4 billion years ago) in the presence of the ocean (Wilde et al., 2001). Therefore, alkaline hydrothermal systems derived from the continental crust would have existed on the early Earth when primitive life emerged and evolved. The purpose of this study was to assess the thermal stability of AAs under alkaline hydrothermal conditions and to investigate the possibility of continental alkaline hydrothermal systems as sites for the origin and evolution of life. 2. Materials and methods 2.1. Seafloor sediment starting material A siliceous sediment core (44.67 m long) was recovered at 41°33.65 0 N, 141°52.06 0 E in the northwestern North Pacific (water depth 975 m). The bottom part of the core (43.50–44.67 mbsf) was used. The sample was freeze dried and manually ground to a fine powder. X-ray diffractometry (Rigaku Geigerflex RAD-IA) revealed that the constituent minerals were quartz, calcite, plagioclase and clay minerals such as illite, smectite, and chlorite.

K. Yamaoka et al. / Organic Geochemistry 38 (2007) 1897–1909

2.2. Hydrothermal experiment Based on the chemical composition data of alkaline hot springs (Kempe and Degens, 1985), 0.55 M NaCl and 0.2 M Na2CO3 aqueous solution (hereafter called the alkaline solution) was used as the reaction solution for a simple simulation of the alkaline hydrothermal systems on the continents. The solution was prepared in Milli-QÒ water, which did not contain any detectable amount of AAs. The initial pH was 11.5 ± 0.1. The sediment was reacted with the alkaline solution in two different sets of experiments under the same temperature and pressure conditions as used by Ito et al. (2006). In one set (Method I), 5.0 g powdered sediment was placed in a titanium vessel (160 cm3) with 150 ml of the alkaline solution. The vessel was tightly closed after flushing with Ar gas to prevent oxidation of AAs and was heated in a mantle heater at 100, 120, 150 and 200 °C (± 1 °C) for 240 h. While the temperature was kept constant, aliquots of the liquid phase were collected at predetermined time intervals. After pH measurement the aliquots were filtered through a PTFE (polytetrafluoroethylene) membrane filter (AdvantecÒ, 0.2 lm pore size) using a disposable syringe and stored in a sealed glass ampoule under refrigeration ( 20 °C) until analysis. The residual phase (hereafter called the solid phase as water was removed from the residue) was freeze dried, ground to a fine powder (silt-clay size) and kept at room temperature until analysis. In the second set of experiments (Method II), 0.5 g powdered sediment was mixed with 15 ml of the alkaline solution in a Teflon jar (25 cm3), which was tightly closed in a stainless steel vessel, and heated in a muffle oven. The vessel was heated at 230, 250, 280, and 300 °C (± 0.5 °C) for 240 h. After cooling the vessel to room temperature, the liquid and solid phases were processed as described above. To examine the effect of adsorption by clay minerals on the thermal stability of AAs, a small amount of powdered commercially available tablets of Chlorella, a green unicellular freshwater alga (< 1 g aliquots) were dissolved in 15 ml of the alkaline solution and heated with 0.5 g montmorillonite or illite at 250 °C for 120 h (Method II). The AA composition of Chlorella corresponded well with that of phytoplankton reported by Cowie and Hedges (1992). Also, 19 commercially available AAs were dissolved in the alkaline solution without adding sediment and heated at 200 °C for 120 h (Method

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II). The concentration of each AA added was ca. 10 nmol/ml prior to heating. The volume of the gas phase in the vessel at the beginning of all the experiments was 10 ml. 2.3. Analytical procedure To analyze total hydrolysable amino acids (THAAs) in the solid phase, the sediment (ca. 10– 30 mg) was hydrolyzed (3 ml 6 N HCl; 22 h at 110 °C) in a sealed glass ampoule under Ar gas. In the case of the liquid phase, 1 ml solution was hydrolyzed with 1 ml of 11.6 N HCl. Fifty microliters of internal standard solution (norleucine) were also added to each ampoule prior to hydrolysis. After cooling to room temperature, the hydrolyzed sample was filtered through a PTFE membrane filter (0.5 lm pore size) using a disposable syringe. The ampoule was rinsed twice with AA-free MilliQ water, which was also filtered and added to the sample. Then, the sample was dried (40 °C) using a rotary evaporator to remove unreacted HCl. The residue was redissolved in Milli-Q water (1 ml) and 20 ll were derivatized with 20 ll AccQ-Fluor reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbonate, Waters) in a borate buffer solution (60 ll). After derivatization, samples were heated in a heating block (55 °C for 10 min) to convert a minor side product of tyrosine to the major monoderivatized compound. To extract the dissolved free amino acids (DFAAs) from the starting sediment, 1.0 g of sample was mixed with Milli-Q water (5 ml) and agitated in an ultrasonic bath (10 min). After centrifugation (3000 rpm; 5 min), the clear solution was transferred to a pear shaped flask. This procedure was repeated twice, but without ultrasonic agitation. The solution was dried, redissolved in MilliQ water (1 ml), and derivatized following the same procedure as above to measure DFAAs in the liquid phase. All samples were analyzed using high performance liquid chromatography (HPLC) with an ion exchange column (AccQ-Tag Amino Acid Analysis column, Waters, USA). The column was maintained at 37 °C. Injection volume was 5 ll. A standard solution containing 0.1, 1.0, 5.0, and 10.0 pmol/ll of each AA was analyzed in every run of a set of 15 samples. Five pmol/ll standard solution were analyzed after every 7 or 8 samples to check peak area reproducibility. Detection was with a Waters 2475 Multi k Fluorescence Detector. Peak area

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quantification was performed using the Empower Software (Waters, USA). Reproducibility was better than ± 10% for absolute and ± 5% for relative molar concentrations for most of the AAs. The relative concentration of an AA (mol%) was calculated by dividing its mole concentration (nmol/ml or nmol/mg) by the mole concentration of THAA and multiplying by 100. This method does not distinguish aspartic acid from asparagine and glutamic acid from glutamine, because the sample hydrolysis with HCl converts asparagine and glutamine to aspartic acid and glutamic acid, respectively. The detailed analytical procedure has been reported by Gupta et al. (2006). 3. Results 3.1. AAs in starting material The concentrations of individual AAs in the THAAs in the starting sediment is shown in Table 1. THAA concentration was 16.04 nmol/mg. The neutral AAs were the most abundant (58.6 mol%), followed by acidic (19.1 mol%), basic (11.8 mol%), non-protein (5.1 mol%), aromatic (4.7 mol%) and sulfur-containing AAs (0.8 mol%). Glycine (17.3 mol%) and aspartic acid (11.5 mol%) were predominant, followed by alanine, glutamic acid and valine. The total concentration of DFAAs was 1.8 nmol/mg, accounting for 11.4% of the THAAs. 3.2. Changes in THAA concentration in the liquid phase during heating The THAA concentration in the solutions heated at 100 and 120 °C (Method I) increased during the first 96 h, and then remained nearly constant (Fig. 1). In the experiments at 150 and 200 °C, however, the concentration increased rapidly during the initial phase of heating and reached a maximum after 9 h. After reaching a maximum, the concentration in the solution heated at 150 °C decreased after 48 h and then remained almost steady, whereas that in the solution heated at 200 °C gradually decreased over the duration of the reaction. The THAA concentration of the solutions heated for 240 h at 230, 250 and 280 °C (Method II) was 279.7, 228.3 and 45.7 nmol/ml, respectively. It is especially noteworthy that even a solution heated at 300 °C contained 8.0 nmol/ml of THAA. Among the AAs remaining in the liquid phase after heating at 100–300 °C for 240 h, glycine was

Table 1 Concentration (nmol/mg) and relative mole content of AAs in starting sediment Amino acid

Concentration (nmol/mg)

Mol%

Acidic Aspartic acid (ASP) Glutamic acid (GLU) Subtotal

1.84 1.22 3.06

11.5 7.6 19.1

Basic Histidine (HIS) Lysine (LYS) Arginine (ARG) Subtotal

0.26 0.89 0.75 1.90

1.6 5.5 4.7 11.8

Neutral Threonine (THR) Serine (SER) Glycine (GLY) Alanine (ALA) Valine (VAL) Isoleucine (ILE) Leucine (LEU) Proline (PRO) Subtotal

0.99 0.89 2.78 1.37 1.00 0.67 0.87 0.81 9.39

6.2 5.5 17.3 8.6 6.3 4.2 5.4 5.1 58.6

Aromatic Tyrosine (TYR) Phenylalanine (PHE) Subtotal

0.24 0.51 0.76

1.5 3.2 4.7

Sulfur-containing Methionine (MET)

0.12

0.8

Non-protein Ornithine (ORN) b-Alanine (BALA) c-Aminobutyric acid (GABA) Subtotal

0.16 0.35 0.30 0.81

1.0 2.2 1.9 5.1

Total

16.04

100

the most prominent and arginine was not detected at any temperature (Fig. 2). The relative abundance of glycine accounted for over 50 mol% of THAA at 250 °C, followed by alanine (18.8 mol%) and valine (11.9 mol%). Small amounts of 9 AAs (glycine, lysine, b-alanine, aspartic acid, alanine, serine, proline, tyrosine and glutamic acid) were detected in the liquid phase even after heating at 300 °C. 3.3. Changes in AA composition in solid phase after heating for 240 h at 100–300 °C The amounts of THAA in the solid phase after 240 h reaction at 100, 120, 150 and 200 °C (Method I) were 4.1, 2.4, 2.0, and 1.1 nmol/mg, respectively. These amounts decreased to 0.81 and 0.89

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Fig. 1. Change in THAA concentration in liquid phase with time at 100–200 °C.

Fig. 2. Change in relative composition (mol%) of AAs in liquid phase after heating for 240 h at 100–300 °C (abbreviations as in Table 1).

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nmol/mg after reaction at 230 and 250 °C (Method II), respectively. After heating at 280 and 300 °C (Method II), only a small amount of THAA ( 0.2 nmol/mg) remained in the solid phase. The AA composition in the solid phase after reaction at relatively low temperature (100–120 °C) was not significantly different from that of the starting sediment (Fig. 3). The THAA composition changed

considerably after heating at 200 °C. The relative amounts of glycine (43.8 mol%) and proline (26.5 mol%) accounted for ca. 70% of all AAs, followed by alanine (7.3 mol%) and glutamic acid (5.2 mol%). After heating above 200 °C, only neutral AAs and glutamic acid were detected. After heating at 300 °C, the following 6 AAs were detected at concentrations of more than

Fig. 3. Change in relative composition (mol%) of AAs in starting sediment and solid phase after heating for 240 h at 100–300 °C (abbreviations as in Table 1).

K. Yamaoka et al. / Organic Geochemistry 38 (2007) 1897–1909

0.01 nmol/mg in the solid phase: glycine, proline, serine, alanine, glutamic acid and threonine (72.8, 11.3, 4.0, 3.3, 2.4, and 2.3 mol%, respectively). 4. Discussion 4.1. Decomposition of AAs Fig. 4 shows the changes in AA distribution in the THAAs in the solid and liquid phases, relative to that of the starting material, after 240 h of reaction at various temperatures. DFAA/THAA ratio values in the liquid phase are also depicted in Fig. 4. The THAA content in the solid phase decreased with increasing temperature, from 25.7% at 100 °C to 1.4% at 300 °C. The THAA content of the liquid phase also decreased with increasing temperature. Most of the AAs in the starting sediment were present in chemically bound form (i.e., peptides), because DFAA accounted for only 11.4% of the THAA. Thus, the increase in DFAA content after thermal reaction resulted from the hydrolysis of peptides in the liquid phase. Qian et al. (1993) reported that hydrolysis was the dominant reaction for dipeptides in aqueous solution. Although the samples in our study included not only dipeptides but also oligo- and polypeptides,

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we infer from the trends in the distribution of AAs in the DFAA and THAA with temperature that hydrolysis of peptide bonds was the dominant reaction for our samples. The amount of THAA in the liquid phase is determined by the balance between the rate of release from the sediment and the rate of decomposition in the liquid (Ito et al., 2006). As shown in Fig. 1, the rate of release increased with increasing temperature during the first few hours of heating. At relatively low temperatures (100 and 120 °C), both the rate of release and the rate of decomposition were so slow that they seemed to achieve a nearly steady state after 144 h of reaction. At temperatures above 150 °C, the rate of release was much faster than the rate of decomposition during the first 9 h, and most of the AAs in the sediment were probably released during this period. A steady state was gradually attained after about 48 h of heating at 150 °C, whereas the amount of THAA decreased continuously at 200 °C due to an enhanced rate of decomposition. The decrease in the proportion of THAAs in the liquid phase with increasing temperature (Fig. 4) indicates that the rate of AA decomposition exceeded the rate of release at elevated temperatures. The DFAA/ THAA ratio increased with temperature up to

Fig. 4. Change in amount of AAs in solid and liquid phases after 240 h heating at different temperatures when THAA in the starting sediment is assumed to be 100%. Numbers on graph correspond to DFAA/THAA ratio in liquid phase. *Very low concentration of THAA at 300 °C gave low precision data.

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200 °C and then decreased gradually except for 300 °C. The exceptionally high ratio at 300 °C resulted from poor analytical precision owing to the extremely low concentration of AAs, and is not discussed further. The ratio depends on the balance between the rate of hydrolysis of peptide bonds and the rate of decomposition of AAs in the liquid. In other words, it increases when the rate of hydrolysis of peptides exceeds the rate of decomposition of DFAA, and decreases in the opposite case. According to Qian et al. (1993), the rate of hydrolysis of dipeptides in aqueous solution increases exponentially with increasing temperature in the range 100–220 °C. In contrast, in a study of the thermal stability of AAs in aqueous solution, Bernhardt et al. (1984) demonstrated that a number of AAs were stable at temperatures up to 200 °C. The results from these studies corroborate our observations, which suggest that the rate of decomposition of AAs is faster than the rate of hydrolysis of peptides at temperatures above 200 °C. On the basis of thermodynamic calculations, Shock (1992) proposed that elevated temperatures increased the stability of peptide bonds in aqueous solution. Our results suggested that the stability of peptides increased relative to that of the AAs in DFAA at temperatures above 200 °C. However, the continuous decrease in THAA in the liquid phase confirms that decomposition is still the dominant process and that the increase in the stability of peptides hardly contributes to the stability of AAs in the temperature range (100–300 °C) investigated.

4.2. Effect of pH on AAs To discuss the effect of pH on the behaviour of the AAs under hydrothermal conditions, we compared results from this study with those from similar experiments conducted with the same sediment sample and 3% NaCl aqueous solution (Ito et al., unpublished results). Profiles of the change in THAA concentration in the liquid phase after heating at various temperatures for 240 h are shown in Fig. 5. Below 150 °C, the low concentration of THAA under neutral vs. alkaline conditions can be explained by the slow rate of release under neutral conditions. Fig. 6 shows the change in the proportion of THAA remaining in the solid phase after 240 h heating at various temperatures when the percentage of THAA in the starting sediment is assumed to be 100%. The proportion of THAA in the solid phase decreased with increasing temperature under both neutral and alkaline conditions regardless of the type of sediment. However, the proportion of THAA in the solid phase under alkaline conditions was less than half that under neutral conditions at relatively low temperatures (100 and 120 °C). Although the proportion of THAA in the carbonaceous sediment became lower than that under alkaline conditions at temperatures > 200 °C, the proportion of THAA in the siliceous sediment under neutral conditions was still more than double that in the same sediment under alkaline conditions. This difference indicates that the rate of release of peptides depends on the type of sediment.

Fig. 5. Concentration of THAA in liquid phase after heating for 240 h. Data from experiments under neutral conditions are from Ito et al. (unpublished study).

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Fig. 6. Change in amount of AAs in solid phase after 240 h heating at different temperatures when THAA in the starting sediment is assumed to be 100%. Data from experiments under the neutral condition using carbonaceous sediment and siliceous sediment are from Ito et al. (2006) and Ito et al. (unpublished study), respectively.

Nevertheless the rate of release is much faster under alkaline conditions. Adsorption of AAs by marine sediment can be attributed to ion exchange, electrostatic interaction and chemical interaction with surface organic functional groups (Wang and Lee, 1993). In particular, basic (cationic) AAs are strongly adsorbed by the negatively charged faces of clay minerals (Hedges and Hare, 1987). In alkaline solution, AAs are negatively charged and exist as anions rather than as cations or zwitterions. Thus, under alkaline conditions, the similarity in ionic charge prevents adsorption of AAs by clay minerals in the sediment. Furthermore, rapid release of AAs under alkaline conditions can be explained by the release of organic matter that is combined with the AAs. Our results are in agreement with the observations of Wang and Lee (1993), who found that the adsorption of some AAs by marine sediment decreased after humic substances and other organic matter were removed from the sediment by extraction with 0.1 N NaOH. At temperatures > 150 °C, the THAA concentration decreased rapidly under neutral conditions (Fig. 5). In contrast, the concentration under alkaline conditions was approximately three times that under neutral conditions at 200 °C and decreased gradually to a small, but not negligible, concentration at 300 °C (Fig. 5). This result strongly suggests that the rate of decomposition of AAs is signifi-

cantly inhibited in alkaline solution than in neutral solution. The dominant pathway for decomposition of AAs in aqueous solution at high temperature is decarboxylation, producing CO2 and an alkylamines (Li and Brill, 2003). Snider and Wolfenden (2000) reported that the rate of decarboxylation of glycine in strongly acidic or alkaline solutions was approximately one tenth that in neutral solution over a temperature range between 170 and 260 °C. In other words, the anionic and cationic forms of glycine are at least 10 times as stable as the zwitterion. Li et al. (2002) studied the kinetics of the cationic and anionic forms of alanine and demonstrated that the decarboxylation rate of these forms is three times slower than that of the zwitterion at 280–330 °C. Moreover, Li and Brill (2003) observed that the rate of decarboxylation of five neutral AAs (serine, threonine, phenylalanine, proline and methionine) were independent of pH in the range 3.0–8.5, which is the buffered zwitterion region. Fig. 7 shows the concentrations of the individual AAs remaining in solution after reaction for 240 h at 150–250 °C under the alkaline and neutral conditions. Ornithine, b-alanine and c-aminobutyric acid are non-protein AAs, so are not discussed here. The concentrations of the AAs, which belong to the neutral AA group except for serine, were higher in alkaline solution at temperatures > 200 °C. The concentrations of lysine and glutamic acid were also

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Fig. 7. Difference between amount of AA remaining in liquid phase after reaction under alkaline conditions and neutral conditions for 240 h at 150–250 °C ([THAA concentration]alkaline  [THAA concentration]neutral). Abbreviations as in Table 1.

higher under alkaline conditions. Thus, the thermal stability of these AAs is significantly enhanced under alkaline conditions. The observation that the thermal stability of neutral AAs improved mainly under alkaline conditions is consistent with previous studies, which showed that the anionic and cationic forms of neutral AAs were more stable than zwitterions (Snider and Wolfenden, 2000; Li et al., 2002). The effect of pH on the thermal stability of lysine and glutamic acid is more complicated to interpret. Vallentyne (1968) studied the thermal reaction kinetics of some AAs and found that the rate of decomposition of lysine decreased with increasing reaction time. He attributed this decrease to the increase in pH during decomposition. An earlier study of the thermal stability of glutamic acid showed that it rapidly formed a stable cyclic structure (lactam) in aqueous solution (Povoledo and Vallentyne, 1964), so its thermal under alkaline conditions may be explained by the effect of pH on the lactam rather than on glutamic acid itself. 4.3. Implications for the origin of life Our results suggest that alkaline hydrothermal systems would have been more favourable for the origin and evolution of primitive life on the early Earth than neutral hydrothermal systems. However, present day submarine hydrothermal systems are slightly acidic (pH  4), and the thermal stability of AAs in these systems would be low because the rate of decarboxylation of neutral AAs is independent of pH in the range 3.0–8.5 (Li and Brill, 2003). Judging from present day hot spring data, alkaline hydrothermal systems exist only on the

continental crust. The Earth’s continental crust seems to have been formed almost synchronously with the formation of the primordial ocean at least 4.4 billion years ago (Wilde et al., 2001). On the other hand, 13C-depleted graphite in > 3.8 billion year old rocks is currently regarded as putative evidence for the oldest life on Earth (Mojzsis et al., 1996; Rosing, 1999). Since liquid water is a basic requirement for life, primitive life presumably emerged after the formation of the continental crust. Even if the Archaean Ocean was not the ‘‘soda ocean’’ proposed by Kempe and Degens (1985), alkaline hydrothermal systems would have existed on the thick continental crust. The amount of AAs available to form higher order structures under hydrothermal conditions is determined by the balance between their rate of synthesis and their rate of decomposition. Previous studies simulating hydrothermal conditions demonstrated that some AAs are rapidly synthesized at temperatures > 200 °C (Marshall, 1994; Islam et al., 2001; Alargov et al., 2002). However, it is unlikely that such AAs remain in high concentration under neutral to acidic hydrothermal conditions because of their rapid decomposition. In contrast, under alkaline hydrothermal conditions, a higher concentration of AAs could be attained owing to the inhibited rate of decomposition in alkaline media, as demonstrated in our study. Furthermore, some researchers reported that peptide formation is accelerated in alkaline media at 80–150 °C (Zamaraev et al., 1997; Huber and Wa¨chtersha¨user, 1998; Bujda´k and Rode, 1999). These studies support our hypothesis that primitive life evolved in alkaline hydrothermal systems on the continental crust.

K. Yamaoka et al. / Organic Geochemistry 38 (2007) 1897–1909

Sediment plays an important role in preserving AAs and peptides in hydrothermal systems. The Earth’s continental crust consists mainly of granite, in which aluminosilicates dominate (Nesbitt and Young, 1984). Their hydrothermal alteration generates various clay minerals, depending on temperature and the chemical properties of the fluid (e.g., dissolved components, pH). Ito et al. (2006) indicated the importance of clay minerals in protecting AAs against release and decomposition under neutral hydrothermal conditions. The small amount of AAs detected in the solid phase in our experiments at high temperatures also might be preserved inside clay minerals. However, the montmorillonite and illite did not adsorb AAs under alkaline conditions (Table 2). Therefore, clay minerals would probably have been more important as catalysts for prebiotic synthesis than as protectants in alkaline hydrothermal systems. The formation of oligomers catalyzed by clay minerals has been reported (Ferris et al., 1996; Bujda´k and Rode, 1999). Rode et al. (1999) suggested that the Si–O and Al–O functional groups at the mineral surface were responsible for peptide chain elongation and stabilization. In the present study, we found that, in the absence of sediment, the histidine, threonine, serine and pro-

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line in an AA mixture in alkaline solution heated at 200 °C decomposed completely after only 120 h (Table 3), whereas they were still detected in the solution heated at higher temperature in the presence of sediment or clay minerals (Fig. 2 and Table 2). These observations suggest that some AAs could be stabilized in a natural environment by coexisting minerals or other organic matter. Our proposal that AAs in continental alkaline hydrothermal systems would have accumulated in the circulating hydrothermal fluid of early Earth has implications for the exploration for extraterrestrial life. Kempe and Kazmierczak (1997) proposed a model for an alkaline Martian hydrosphere based on the assumption that the early environment on Earth was similar to that on Mars. Most recently, new data from the Mars Express OMEGA revealed the presence of clay minerals in old Martian rocks and soil, suggesting the existence of an alkaline aqueous environment during an ancient era in Martian history (Bibring et al., 2006). Since alkaline hydrothermal systems require reaction of silicate rocks with water and CO2, the planets that possess or have possessed these materials are promising targets for future exploration for evidence of living systems.

Table 2 Concentration (nmol/mg) of AAs in clay minerals (montmorillonite and illite) and concentration (nmol/ml) in the starting solutions, and final concentration in the clay minerals and in the solutions after heating at 250 °C for 120 h (abbreviations as in Table 1) Amino acid

Montmorillonite

Solution

Illite

Solution

Initial (nmol/mg)

After 120 h (nmol/mg)

Initial (nmol/mg)

After 120 h (nmol/mg)

Initial (nmol/mg)

After 120 h (nmol/mg)

ASP GLU HIS LYS ARG THR SER GLY ALA VAL ILE LEU PRO TYR PHE MET ORN BALA GABA

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.01 0.00 0.00 0.00 0.00 0.02 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

8.0 10.0 2.0 8.2 6.3 5.4 3.7 10.4 8.4 5.5 3.6 7.4 5.7 1.7 3.7 0.0 0.0 0.1 0.0

0.0 1.3 0.0 0.0 0.0 0.0 0.2 1.5 0.2 0.4 0.2 0.2 0.4 0.0 0.0 0.0 0.1 0.0 0.0

0.03 0.02 0.00 0.01 0.00 0.04 0.12 0.31 0.04 0.03 0.02 0.02 0.17 0.01 0.00 0.00 0.04 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.00 0.00 0.00 0.07 0.00 0.00 0.00 0.00 0.00 0.00

7.2 8.9 1.5 6.9 5.6 4.6 3.1 9.2 7.3 4.6 3.0 6.1 4.5 1.2 3.1 0.0 0.0 0.0 0.0

0.0 1.6 0.1 0.1 0.0 0.0 0.4 1.9 0.3 0.5 0.2 0.3 0.4 0.0 0.0 0.0 0.1 0.0 0.1

Total

0.43

0.08

90.3

4.5

0.86

0.17

76.6

6.0

Initial (nmol/mg)

After 120 h (nmol/mg)

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K. Yamaoka et al. / Organic Geochemistry 38 (2007) 1897–1909

Table 3 Concentration (nmol/ml) of aqueous AAs in alkaline solution heated without sediment at 200 °C and the proportion of remaining AAs after 120 h of heating Amino acid

After 120 h (nmol/ml)

Remained (%)

ASP GLU HIS LYS ARG THR SER GLY ALA VAL ILE LEU PRO TYR PHE MET ORN BALA GABA Total

0.0 7.0 0.0 2.3 0.0 0.0 0.0 9.9 5.9 5.7 5.0 5.6 0.0 0.1 6.0 0.2 1.9 0.0 6.4 56.0

0 70 0 23 0 0 0 99 59 57 50 56 0 1 60 2 19 0 64 29

Initial concentration of each AA is 10 nmol/ml (abbreviations as in Table 1).

5. Conclusions Under alkaline hydrothermal conditions, AAs existing in sediment in peptide form were rapidly released to the liquid phase. The amount of THAA in the liquid phase decreased with increasing temperature owing to AA decomposition, but a small amount of AAs was still detected after reaction at 300 °C for 240 h. Although the rate of hydrolysis of peptides was faster than the rate of decomposition of AAs in the liquid phase at relatively low temperatures, decomposition of AAs predominated at temperatures above 200 °C. Above 200 °C, the amount of THAA in alkaline solution was greater than that in neutral solution because the stability of neutral AAs is significantly enhanced under alkaline conditions. On the basis of the present day occurrence of alkaline hydrothermal systems on the continents, we propose that AAs synthesized in continental alkaline hydrothermal systems at high temperature (> 200 °C) would have accumulated in the circulating hydrothermal fluid of the early Earth. On the other hand, oligomerization catalyzed by clay minerals would have proceeded in mild environments (< 150 °C). Our results suggest that alkaline hydrothermal systems on the continen-

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