Introduction
Life originated on Earth possibly as a physicochemical process; thus, geological environments and their hypothetical characteristics on early Earth are essential for chemical evolution studies (Hazen, Reference Hazen2005). Chemical evolution, as Calvin defined, is the hypothetical period that starts when the Earth was formed and lasts until the first living beings appeared. In this time span, chemical reactions might have occurred, starting from simple compounds and transforming into more complex ones, forming living entities (Calvin, Reference Calvin1955). Those reactions could happen in one or more geologic scenarios, and for prebiotic chemistry studies, hydrothermal systems have been considered plausible sites for the origin of life (Colín-García et al., Reference Colín-García, Heredia, Cordero, Camprubí, Negrón-Mendoza, Ortega-Gutiérrez and Ramos-Bernal2016). A hydrothermal system forms when magma emerges on the surface and causes the heating of surrounding water (Nisbet and Sleep, Reference Nisbet and Sleep2001). A hydrothermal environment consists of two basic components: a heat source and a fluid phase. Heat fluids circulate at different temperatures and pressures, and the system could survive long enough to form anomalous concentrations of metallic minerals (Pirajno, Reference Pirajno2009). Also, they constitute geochemical habitats that harbour microbial communities (Martin et al., Reference Martin, Baross, Kelley and Russell2008). There are several types of hydrothermal systems. For chemical evolution studies, they can be classified in a simplified way on submarine, subaerial and impact-generated systems. Many of the prebiotic chemistry studies that involve hydrothermal systems focus on submarine environments, due to their association with magmas near to mid-ocean ridges, and with the origin of life on Earth (Colín-García et al., Reference Colín-García, Heredia, Cordero, Camprubí, Negrón-Mendoza, Ortega-Gutiérrez and Ramos-Bernal2016). This is due to the possibility that volcanism was more active on early Earth than it is today, and ‘black smoker’ vents could exist in abundance. However, subaerial systems could also exist if plate tectonics were already active. Acasta is a metamorphic heterogeneous complex, comprises rocks as tonalites, granidorites, granites, until ultramafic rocks. This place is an evidence that continental crust formed since 4.2 Ga (Iizuka et al., Reference Iizuka, Horie, Komiya, Maruyama, Hirata, Hidaka and Windley2006). Furthermore, they are a source of organic compounds, and those environments can harbour life without direct photosynthesis (Nisbet and Sleep, Reference Nisbet and Sleep2001; Martin et al., Reference Martin, Baross, Kelley and Russell2008). In most solar system bodies that have a solid surface, impact cratering is the most important process of surface modification (Koeberl, Reference Koeberl2013). Although it is still in debate, it has been proposed that during the Hadean, a heavy bombardment of asteroids and comets occurred on Earth, which means an increment of the impact rate at 4.0–3.8 Gyr (Kring, Reference Kring2002; Gomes, et al., Reference Gomes, Levison, Tsiganis and Morbidelli2005); hence, hydrothermal systems might have generated on primitive Earth as a result of those impacts. The record of lunar craters shows that the impact rate of size and flux decreased drastically after this period. Moreover, its end seems to coincide with the origin of life (Farmer, Reference Farmer2000). Even though impact events can produce environmental disturbances, the associated craters could have certain characteristics that make them adequate geological sites for chemical evolution, specifically those that develop the hydrothermal activity. The role of impacts on the origin of life has not been studied in detail (Cockell, Reference Cockell2006). In oceanic environments, the problem that comes up is about the concentration of reactants because the volume of water is great. Thus, a plausive alternative to this problem can be impact-generated hydrothermal systems (Chatterjee, Reference Chatterjee2016). Those systems can work as a favourable geologic area for chemical evolution for three reasons: They have an energy source (thermal) for carrying out chemical reactions; a localized concentration of molecules can exist, and catalysis could arise; and they are systems that can persist for long periods (from 10 000 years to 2 Myr) (Daubar and Kring, Reference Daubar and Kring2001; Cockell, Reference Cockell2006). Mineral and clay mineral surfaces have been considered as possible as participants in the origin of life, thanks to their physicochemical properties, as they are capable of acting as catalysts, concentrating agents and as assembling molds for prebiotic molecules (Bernal, Reference Bernal1951; Goldschmidt, Reference Goldschmidt1952; Cairns-Smith, Reference Cairns-Smith1966; Hazen, Reference Hazen2012). For this reason, in prebiotic chemistry studies, it is important to take them into account and find the role they represent in this research area (Negrón-Mendoza, Reference Negrón-Mendoza and Seckbach2004; Hazen, Reference Hazen2005; Hazen and Sverjensky, Reference Hazen and Sverjensky2010; Cleaves et al., Reference Cleaves, Michalkova Scott, Hill, Leszczynski, Sahai and Hazen2012).
Along with thermal energy, ionizing radiation could play an important role in chemical evolution because, on the early Earth, the levels of radiation were significantly higher than they are today. Radiation dose from geologic emitters has changed because of the evolution of the continental crust and the relative abundances of radionuclides (Karam and Leslie, Reference Karam and Leslie1999). Also, the intensity of ionizing radiation coming from the exterior of the planet was higher. Chemical changes produced by this type of energy could be the cause of modification of prebiotic molecules that led to the subsequent emergence of life, absorbing energy either directly or indirectly through the presence of other radiolytic products on the medium (Zagórski and Kornacka, Reference Zagórski and Kornacka2012). Production of radiolytic molecules relevant for life has been considered in primordial aqueous environments due to 40K decay (Draganić, Reference Draganić2005).
α-keto acids are compounds that have a carbonyl group adjacent to a carboxylic group. Keto acids are formed as intermediates during metabolic interconversions of sugars, carboxylic acids and amino acids. Those analogous to natural amino acids have great importance in intermediary metabolism. Other metabolic processes in which α-keto acids play an important role are the Krebs cycle and glycolysis. As the precursor to citric acid, Acetyl-CoA is exposed to chemical conversions in which keto acids such as α-ketoglutaric and oxaloacetic acid are involved (Nelson and Cox, Reference Nelson and Cox2012). α-ketoglutaric acid (AKG) is a small molecule (C5H6O5) that possibly played a role in the period of chemical evolution; for this reason, the aim of this work is to study the behaviour of this molecule under radiation and thermic energy conditions, simulating chemical reactions that might have occurred in a primitive environment such as impact-generated hydrothermal system.
Materials and methods
Analytical techniques
For the analysis of the α-ketoglutaric acid, a derivatization method is needed. In this work, ο-phenylenediamine (OPDA) was used as a derivatizing agent, according to Montenegro et al. (Reference Montenegro, Valente, Moreira Gonçalves, Rodrigues and Araújo Barros2011). The chromatographic analysis was carried out on a UHPLC UltiMate 3000 chromatographic system (Thermo Scientific®, USA), with a UV-Vis Dionex UltiMate 3000 VWD (Thermo Scientific®) detector and a Halo® C8 column (50 × 4.6 µm) for the separations. The mobile phase was prepared with 90% ammonium acetate (8 mM, pH 4.5) and 10% methanol at 0.3 ml min−1 a 25°C. A fixed sample volume injection of 20 ml was used. The samples were detected at 340 nm. In addition, gas chromatography was used for the analysis of AKG and radiolytic products. The instrument was a gas chromatograph HP-5890A with a capillary column filled with methyl silicon. The samples were derivatized to their corresponding methyl ester, according to Negrón-Mendoza and Ponnamperuma (Reference Negrón-Mendoza and Ponnamperuma1976).
Materials and glassware
Three different minerals were used for the experiments: SWy-2 Na-montmorillonite (Source of Clay Minerals Repository, Wyoming, USA), pyrite (from Spain) and zeolite (stilbite and heulandite, from Pune, India). AKG was from Sigma Aldrich®. To avoid organic contamination on irradiation experiments, glass material was treated according to radiation chemistry procedures (Draganić, Reference Draganić2005).
pH stability analysis
To learn about the behaviour of AKG in basic pH, stability experiments were carried out. The pH of AKG standard solution was 3, and generally, in impact-generated hydrothermal systems, the reported pH range was slightly alkaline or near neutral (Osinski, Reference Osinski2005; Osinski, et al., Reference Osinski, Lee, Parnell, Spray and Baron2005). Three samples were analysed: The first one was the standard solution without any pH modification. For the second, a pH modification to 6.5 was done using NaOH 0.01 M. For the last sample, the pH was increased to 8. The stability was evaluated immediately after the pH modification, as well as 1, 2 and 3 weeks later.
Sorption studies
Before the assays, mineral samples were treated to remove organic contamination, using a 3% KOH solution (10 ml g−1 mineral), stirring for 30 min, rinsing with distilled water, stirring again for 30 min with a 3% HNO3 solution (10 ml g−1 mineral), rinsing and letting dry.
Sorption assays were performed using 2.5 ml of aqueous standard solution of α-ketoglutaric acid 0.001 M and 50 mg of mineral (mineral-AKG systems). Samples were stirred at different times (1, 2, 4, 6 and 24 h) in centrifuge polyallomer tubes 16 × 76 mm (Beckman Coulter®). Then, the tubes were centrifuged at 26 000 rpm (Beckman Coulter Allegra 64R® centrifuge) for 25 min for the separation of phases. The volume needed for the derivatization was taken from the liquid phase. The sorption percentage was calculated considering the change in the peak area in relation to the standard peak. Sorption studies were done in triplicate.
Sorption studies at different pH
When zeolite or pyrite interacts with the aqueous solution of AKG, the pH of the system is 3.5 (pHi). On the other hand, the montmorillonite-AKG system pH is 5.8. A sorption test with fixed pH (pHf) was conducted to observe if there is a difference in sorption capacity in these systems at 24 h fixed time (Table 1).
Changes on pH of the systems
Radiolysis
Aqueous solution
Aside from the thermal energy effect, ionizing radiation was selected as another energy source on early Earth. Samples of tridistilled aqueous solutions of AKG 1 × 10−3 M were irradiated with γ rays from a 60Co source located at the Gammabeam 651-PT of the Instituto de Ciencias Nucleares, UNAM. Radiation doses were from 5 to 50 kGy, and the dose intensity was 167 Gy min−1. Radiolysis experiments were done in duplicate and with oxygen-free solutions. Additionally, GC-MS analysis was performed for the identification of radiolytic products (carboxylic acids) through their methyl esters.
Mineral-AKG systems
To study the effect of the presence of minerals when AKG is exposed to γ radiation, samples of each mineral and AKG were prepared as in section ‘Materials and glassware’ at a fixed time of contact of 24 h. Once the contact time was reached, samples were irradiated at 5, 30 and 50 kGy, then centrifuged, and the supernatant was collected.
Thermolysis
Thermolysis assays were carried out with oxygen-free 1 × 10−3 M aqueous solution of AKG. Two different systems were used for this purpose. The first one consisted of a heating static system composed of a flask assembled to a condensation column for the recirculation of organic solvents. The organic solvent was contained inside the flask while it was heated to its boiling point at 582 mm Hg. The flask had four orifices to insert tube samples; these tubes did not come in direct contact with the solvent. Three different solvents were used to reach different temperatures: toluene, dimethylformamide and nitrobenzene (100, 135 and 180°C, respectively). The second system was a temperature-controlled (± 2°C) stove (Venticell 22 Eco line MMM Group®). Sealed glass ampules were used as sample containers for this system. The temperature used in the stove was 180°C.
Results
pH stability analysis
The measurements concerning the stability of AKG 10−3 M at different pH ranges indicate that the molecule is stable after 3 weeks of the preparation of the standard solution (pH 3) and the fixed pH solutions (Table 2).
Stability of AKG at different pH values
Sorption assays
All three minerals showed sorption of AKG; pyrite and montmorillonite have more sorption capacity (35%) than zeolite (15%). Poor sorption was observed at 1 h of contact. However, the sorption increases at 6 h and the maximum is reached at 24 h, where sorption capacity seems to remain constant (Fig. 1).
AKG sorption onto different minerals.
Sorption at different pH
Figure 2 shows that sorption capacity slightly increases when pH is higher (pHf from Table 1), especially with zeolite (~5%), these results suggest that if the environment has pH gradients between 3.5 and 6.5, sorption capacity has no major alterations.
Differences in sorption capacity of minerals when pH is modified.
Radiolysis
Aqueous solutions
Decomposition of AKG was followed at different radiation doses. AKG shows to be labile when it is exposed to γ radiation (Fig. 3). A 0.1 M solution was made for a better tracking of decomposition, but the effect of radiation in both irradiated concentrations (0.1 and 1 × 10−3 M) was the same.
Decomposition of AKG by γ radiation (Left: irradiation of 0.1 M solution. Right: irradiation of 1 × 10−3 M solution).
GC-MS analysis showed that the main radiolytic product is succinic acid (Fig. 4). Malonic and glutaric acids were also detected.
Mass spectrum of succinic acid (methyl ester) formed by irradiation of AKG at 30 kGy.
Mineral-AKG systems
The results of the irradiation of mineral-AKG systems demonstrate that all minerals have a protective effect versus radiation, preventing AKG decomposition at 5 and 30 kGy doses. It was observed (Fig. 5) that pyrite has a high protective effect, followed by zeolite and montmorillonite, respectively. At doses of 50 kGy, the systems exhibit total decomposition of AKG.
Comparison of the radiolysis of aqueous AKG and mineral-AKG systems.
Thermolysis
Several experiments were conducted at different times and temperatures for thermolysis of AKG. Table 3 shows the obtained results. Some problems were encountered in managing samples in the static system at temperatures above 100°C; thus, a temperature-controlled stove was used. AKG seems to be stable in high-temperature conditions. Further analysis is needed to track the stability of the compound.
Thermolysis of AKG 1 × 10−3 M at different conditions
Discussion
Considering AKG as a possible precursor for the production of different molecules relevant for chemical evolution and through the study of the stability of this compound, we can suggest clues about its possible availability on early Earth. AKG is stable in alkaline and high-temperature conditions, notwithstanding it is labile under high radiation doses. Minerals such as montmorillonite, pyrite and zeolite can act as concentrators and protectors of α-ketoglutaric acid. Minerals and physicochemical variables for this study were selected based on a review of the literature, shown in Table 4. Preliminary results show that one of the main radiolytic products in aqueous solution is succinic acid, which suggests a dehydroxylation and decarboxylation reactions. Analysis of the radiolytic products of all three mineral-AKG systems is needed to understand the nature of the reactions. It has been reported that in the radiolysis of AKG sorpted onto montmorillonite, the number of radiolytic products decreases, and the main decomposition pathway is decarboxylation, producing succinic acid and CO2 (Negron-Mendoza and Ramos-Bernal, Reference Negron-Mendoza and Ramos-Bernal1998).
Impact-generated hydrothermal system and their main characteristics
The purpose of choosing AKG is because in previous studies (Negrón-Mendoza and Ponnamperuma, Reference Negrón-Mendoza and Ponnamperuma1976; Cruz-Castañeda et al., Reference Cruz-Castañeda, Colín-García and Negrón-Mendoza2014; Negrón-Mendoza and Ramos-Bernal, Reference Negrón-Mendoza and Ramos-Bernal2015; Negrón-Mendoza et al., Reference Negrón-Mendoza, Colín-García and Ramos-Bernal2018), starting from the irradiation of a member molecule of Krebs cycle (i.e. acetic, citric, succinic acids), various compounds (also Krebs cycle members) are produced, although AKG is not found. To get an insight for the lack of its detection, we compare the results of this work with the radiolysis of some carboxylic acids, and from these data, we calculate according to Criquet and Karpel Vel Leitner (Reference Criquet and Karpel Vel Leitner2011, Reference Criquet and Karpel Vel Leitner2012), parameters of degradation versus radiation dose. These results are presented in Fig. 6, where C 0 is the initial concentration of the acid, C is the concentration at dose D in kGy, and k is the dose constant (linearly, the slope is k). It is important to remark that this equation is only descriptive and is not a model of the physicochemical processes of the radiolysis, but it gives a simple approximation about the reactivity towards the degradation of the compounds. These calculations show that citric and isocitric acids are the most stable acids under high radiation doses, while pyruvic and α-ketoglutaric acids (both keto acids) are the most reactive and the decomposition occurs at lower radiation doses with a high k (1.19 × 10−2) compared, for example, with isocitric acid (3 × 10−5). This lability under high radiation conditions may explain why it is not easy to detect AKG in prebiotic experiments. Nevertheless, AKG is converted into products with higher stability (i.e. succinic acid), as seen in Fig. 4. Table 5 shows the k values obtained.
Decomposition of some Krebs cycle carboxylic acids in aqueous solution, acidic pH, exposed to different doses of γ radiation.
Dose constants of some carboxylic acids
Conclusions
Studies about impact-induced hydrothermal systems and their role in prebiotic chemistry are scarce. AKG is capable of existing on environments such as impact-induced hydrothermal systems, which are complex geological systems that present different characteristics, such as pH and temperature gradients, and might also have existed on primitive Earth. AKG is easily degraded under a high radiation field, despite this fact, minerals present in those environments could support the persistence of AKG by concentrating molecules for further reactions and protecting them through inherent and external factors like heat and ionizing radiation, respectively. Other metabolism-relevant carboxylic acids can be produced by AKG decomposition, such as succinic acid.
Acknowledgements
This work was performed at Instituto de Ciencias Nucleares, UNAM. MLRV acknowledge the support from Posgrado en Ciencias de la Tierra, UNAM and the CONACYT fellowship 490940, Dr Karina Cervantes-de la Cruz and Dr María Colín-García for their advice and for the donation of mineral samples. The support of the projects PAPIIT IN110919 and PAPIME PE206918 is also acknowledged. We are grateful to Benjamin Leal, M.Sc., Francisco Flores, Phys., and Claudia Camargo, Chem., for their technical assistance.
