Bollettino della Società Paleontologica Italiana, 52 (1), 2013, 63-70. Modena

ISSN 0375-7633 doi:10.4435/BSPI.2013.11

Considering the relations between carbonates and microbiological activities on Earth (e.g., Burne and Moore, 1987; Folk & Chafetz, 2000; Riding, 2011; Guido et al., 2007a, 2011, 2012a, 2013; Riding, 2011), the identification of these deposits on Mars could be extremely useful in identifying extinct Martian life and reconstructing the planet’s paleoclimate (Ehlmann et al., 2008).

Fourier-Transform Infrared (FT-IR) spectroscopy has been used to characterise kerogens through measurement of the energy absorbed by the molecules in transition to different vibrational states. Because each functional group tends to absorb infrared radiation in a specific wavelength range, it is possible to detect and discriminate different chemical compounds (Mastandrea et al., 2011; Guido et al., 2012b). An extensive investigation of different species of recent bacterial cells with FT-IR spectroscopy was undertaken by Naumann and co-workers (Naumann et al., 1991; Helm et al., 1991a). These studies showed that FT-IR absorption spectra contain specific fingerprints of microbial cells and can make microbial characterisation possible down to subspecies level when multivariate statistical methods are employed to analyze the spectra (Helm et al., 1991b).

In kerogen, infrared analysis does not target particular molecules but instead detects classes of molecules. Each organic group (i.e., aliphatic C-H bonds) is present in many compounds with a particular wavelength and this

INTRODUCTION

The search for evidence of water and organic compounds, including those of possible biological origin, is one of the major goals of the Mars exploration programs of the National Aeronautics and Space Administration (NASA) and the European Space Agency (ESA).

Biosignatures in early terrestrial rocks are highly relevant in the search for traces of life on Mars because it has been maintained that conditions on early Mars were similar to those of early Earth. If life originated on early Earth via organic compounds, it is reasonable to infer that similar kinds of organic matter were also formed on primitive Mars. These materials might have been preserved on Mars to the present (Aubrey et al., 2006; Westall, 2008), but the presence of organic compounds on Mars remains uncertain (Glavin et al., 2001; Aubrey et al., 2006). Opportunities to analyze samples from Mars have been provided by meteorites ejected from its surface and delivered to Earth. The bulk of the organic carbon in meteorites consists of insoluble macromolecular material of complex structure (Botta & Bada, 2002; Martins et al., 2006, 2007, 2008; Martins, 2011), generally referred to “kerogen-like” material (Peters et al., 2005). Studies of insoluble organic compounds in chondrites have been performed similarly to those in coal, oil shale and petroleum (Robl & Davis, 1993; Murae,1994).

Biotic vs abiotic carbonates: characterisation of the fossil organic matter with Fourier-Transform Infrared (FT-IR) Spectroscopy

Adriano Guido, Adelaide Mastandrea & Franco russo

Dipartimento di Biologia, Ecologia e Scienze della Terra, Università della Calabria, Via P. Bucci, Cubo 15b, I-87036, Rende (CS) Italy. (aguido@unical.it, a.mast@unical.it, f.russo@unical.it)

KEYWORDS: carbonates, organic matter, FT-IR spectroscopy, Calcare di Base, northern Calabria (Italy).

ABSTRACT - The investigated carbonates, belonging to the Messinian “Calcare di Base” Formation, crop out in northern Calabria. The organic matter preserved in these problematic carbonates has been characterised through Fourier-Transform Infrared (FT-IR) spectroscopy. This methodology allowed the origin and thermal maturity of organic matter in these sediments to be recognized. The discrimination between biotic and abiotic material requires the support of other techniques, such as optical and Scanning Electron Microscopy and Gas Chromatography-Mass Spectrometry spectroscopy. Carbonates, which are strictly connected with biological activity on the Earth and have been discovered also in the Nili Fossae region of Mars, could provide evidence of ancient life on the Red Planet. In the next space missions to Mars, this procedure is not applicable in situ for the analytical composite method required, therefore it is necessary to return suitable Martian rocks to Earth.

RIASSUNTO - [Caratterizzazione della materia organica fossile tramite Spettroscopia ad Infrarossi (FT-IR) per discriminare la biogenicità nei carbonati] - I processi biotici, che sulla Terra inducono la precipitazione di minerali carbonatici con caratteristiche tessiture microbialitiche, possono essere riconosciuti anche nei carbonati marziani grazie alle loro micromorfologie. Considerando le strette relazioni tra i carbonati e le attività biologiche sulla Terra, la recente identificazione di sedimenti simili anche su Marte apre nuovi orizzonti per l’individuazione di tracce di vita marziana.

Un nuovo approccio per la caratterizzazione dei lipidi preservati in carbonati microbialitici per mezzo della Spettroscopia ad Infrarossi in Trasformata di Fourier (FT-IR) ha permesso il riconoscimento di tracce biotiche altrimenti non individuabili. Tale metodologia, testata su carbonati tardo miocenici della formazione del “Calcare di Base” affioranti nel bacino di Rossano in Calabria settentrionale, è risultata valida sia per la caratterizzazione biogeochimica di tali sedimenti sia per la valutazione dell’evoluzione termica dei composti organici. E’ stato quindi dimostrato che questi sedimenti carbonatici derivano da processi metabolici microbici. La Spettroscopia ad Infrarossi rappresenta un efficace metodo di indagine per l’identificazione di tracce di vita nella storia geologica della Terra e di Marte, metodo che può essere applicato a tutti i sedimenti dai quali è possibile estrarre resti di materia organica.

Bollettino della Società Paleontologica Italiana, 52 (1), 201364

PREVIOUS DATA ON MICROFACIES AND ORGANIC MATTER OF THE “CALCARE DI BASE” FORMATION

The studied samples, belonging to the Messinian “Calcare di Base” (CdB) formation, have been collected near the Cropalati village, Rossano Basin (Northern Calabria) (Guido et al., 2007a, b). In this area the CdB consists of two massive fine-grained calcareous beds interlayered with decimetric laminated marls (Fig. 1). We collected three samples for each calcareous bed, numbered CB1-CB2-CB3 and CB4-CB5-CB6, from bottom to top respectively (Fig.1). Three thin sections were observed for each sample, in order to put in evidence their microfacies. Guido et al. (2007a, b) demonstrated that CdB carbonates, from Cropalati, preserve their original aragonitic mineralogy and consist of small crystals (micrite) organized in peloids, which form clots with antigravity fabric (Fig. 2). The bright fluorescence (Fig. 2a) indicates that this peloidal micrite is rich in organic matter, palynomorphs and lipid biomarkers (molecular fossils) that were analyzed by Rock-Eval pyrolysis, palynofacies and molecular-fossil recognition. The Rock-Eval Tmax value of ~400 °C, and the presence of unsaturated fatty acids and ββ-hopanoids, indicate that the carbonates suffered very low thermal stress and that the organic matter is very immature (Fig. 3). The transitional composition of kerogens between Type II and Type III, suggests a mixture of marine and terrigenous organic matter (OM) (Fig. 3). These different OM sources are supported by biomarker analyses (for further information see Guido et al., 2007a). The n-alkane distribution indicated the presence of three main biological signatures: algal (nC18-nC20 compounds),

provides a high sensitivity for mixtures (Borrego et al., 1995; Anderson et al., 2005). The major classes of biomolecules that are readily distinguished by FT-IR, include proteins, lipids, carbohydrates, and nucleic acids.

A broad variety of microorganisms, whether phototrophic or not, are involved in the formation of carbonate (Castanier et al., 1999; Perry et al., 2007; Dupraz et al., 2009). Their direct characterisation in ancient sedimentary systems is limited by the extremely low fossilisation potential of most microorganisms, especially bacteria (Thiel et al., 1997). Many studies have shown that organic geochemical techniques can be used to identify the original presence of microorganisms (Tissot & Welte, 1984; Thiel et al., 1997; Heindel et al., 2010, 2012; Guido et al., 2013).

In this paper we present a new approach to detect lipids with Fourier-Transform Infrared (FT-IR) spectroscopy and to characterise Mars return samples. The main goal of this study was to assess FT-IR spectroscopy as a tool for geochemical characterisation of carbonates with regard to their organic-matter content and thermal evolution. This approach was tested on carbonates of the Late Miocene “Calcare di Base” (CdB) formation in Calabria (Guido et al., 2007a, b). The origin of these deposits has long been uncertain due to the absence of any evident fossils. In particular it was unclear whether they formed abiotically in hypersaline environments, or biotically in normal marine settings. Guido et al. (2007a) demonstrated through the study of the micromorphologies of the mineral matrix and biogeochemical characterisation of the remains of organic matter that these deposits precipitated biotically via microbially induced mineralization.

Fig. 1 - “Calcare di Base” carbonate beds cropping out near the Cropalati village (Rossano Basin, Northern Calabria) with the location of the studied samples. Refer to Guido et al. (2007a, b) for the geologic and stratigraphic settings.

65A. Guido et alii - Biotic vs abiotic carbonate discrimination with FT-IR Spectroscopy

Samples were centrifuged after each treatment and the supernatant was collected. To concentrate the organic compounds, the solution was evaporated and the dry residue was re-suspended in a few millilitres of dichloromethane/methanol. Several drops of this extract were placed onto the crystal of the ATR apparatus and dried under a nitrogen flow.

Considering that the absorption bands of kerogen overlap in some regions of the spectrum, we used Fourier self-deconvolution (Kauppinen et al., 1981a, b) to determine the content of functional groups semiquantitatively (Starsinic et al., 1984; Solomon & Carangelo, 1988; Kister et al., 1990; Landais & Rochdi, 1990; Sobkowiak & Painter, 1992; Ruau et al., 1997).

terrestrial (nC27-nC29), and bacterial (nC26-nC28, with no odd-even carbon-number predominance) (Tissot & Welte, 1984; Baranger & Disnar, 1988; Baranger et al., 1989; Thiel et al., 1997; Guido et al., 2007a). The autochthonous acidic fraction (<C22) and the level of unsaturated compounds validated the bacterial signature, which was further supported by the presence of hopanes and branched n-alkane. These data point to a marine depositional setting, modified by episodic freshwater inputs, in which carbonate precipitation was induced by bacterial ammonification of amino acids in aerobic conditions (Guido et al., 2007a; Mastandrea et al., 2010, 2011).

F O U R I E R T R A N S F O R M - I N F R A R E D SPECTROSCOPY METHODOLOGY

Fourier-Transform Infrared techniques were performed on six samples with three different methodologies: standard petrographic thin section (6 x 4.5 cm), powder, and organic matter extracted from the mineral matrix. The spectroscopic analyses were made in the mid infrared area (4000-400 cm-1) by accumulating 256 scans for analysis. A Perkin Elmer Spectrum 100 spectrophotometer with a Universal ATR (Attenuated Total Reflectance) was employed in the following arrangement: a K-Br beamsplitter and a LiTaO3 detector. In this configuration, the resolution was 4 cm-1. Spectral bands were assigned taking into account the literature (Solomon & Carangelo, 1988; Sobkowiak & Painter, 1992; Vescogni et al., 2011).

To avoid mineral disturbance, we extracted organic compounds from the carbonate matrix by acidic separation. Small fragments of samples, visibly free of alteration, were hand ground in an agate mortar and three grams of powder were ultrasonically treated three times with a mixture of dichloromethane/methanol (1:1) to extract the organic compounds. The choice of bulk sediments without any kind of alteration is essential to prevent contamination of the original organic remains with organics occurring in diagenetic fluids or interstitial waters.

Fig. 2 - Photomicrographs of clotted peloidal microfacies: a) transmitted light; b) epifluorescence UV. The bright fluorescence put in evidence the high content and distribution of the organic matter in the clotted peloidal microfabric. Note that both pictures were acquired in the same area of the sample CB2.

Fig. 3 - Classification of kerogens on a pseudo-van Krevelen diagram (HI-Tmax). The diagram shows the position of “Calcare di Base” organic matter which falls in the immature region (modified from Mastandrea et al., 2011).

Bollettino della Società Paleontologica Italiana, 52 (1), 201366

vibration of organic matter appears between 1300 and 1100 cm-1.

INTERPRETATION AND DISCUSSION

Orofino et al. (2007, 2009, 2010) and Blanco et al. (2011) studied the infrared (IR) spectral modifications induced by thermal processing in various calcium carbonate fossils, to discriminate skeletal carbonates from their abiotic counterparts. These authors, analyzing the mineral fraction of the carbonate, did not consider the diagenetic processes that could have altered the original biominerals.

In contrast, organic matter tends to be more stable during diagenesis because the lipids are not water soluble (Peters et al., 2005). The main factors that promote diagenetic modification in lipid biomarkers are biological activities and, during deep burial, increased pressure and temperature. During diagenesis and catagenesis, the first two alteration levels (Fig. 3), lipid biomarkers of former organisms are still preserved. The original molecular matter is completely lost during metagenesis that typically occurs at burial depths greater than 3 km and temperatures higher than 150°C (Peters et al., 2005). The capacity to analyze organic matter remains that have been extracted from any kind of mineral matrix permits application of this methodology to a wide spectrum of materials collected on Earth or Martian surfaces.

The results of measurements of carbonate thin sections and powders show strong bands in the region near 1400

RESULTS

The following absorption bands of organic compounds were recorded: stretching of aromatic C-H groups in the region from 3100 to 3000 cm-1, stretching of aliphatic C-H groups in the region from 3000 to 2800 cm-1, oxygenated groups and aromatic/alkenic region from 1550 to 1750 cm-1, bending modes of CH2 and CH3 groups at 1450 cm-1, absorption bands of CH3 groups at 1375 cm-1, and aromatic out-of-plane C-H deformation region from 700 to 900 cm-1 (Fig. 4).

Aragonite shows strong bands, due to the asymmetric C-O stretching modes, at 1445, 1081, 853, 713, or 697 cm-1. IR spectra from bulk sediment also show bands at 640 and 607 cm-1 (Fig. 4a). These are characteristic of the skeletal vibrations of sulphate minerals probably related to celestine (SrSO4).

Fourier self-deconvolution resolved the region around 700 cm-1, discriminating a peak at 720 cm-1 due to the skeletal vibration of more than four methylene groups [γ(CH2)4]. The aliphatic C-H stretching region (3000-2800 cm-1) was resolved into three spectral bands at 2950 (asymmetrical CH3 stretching), 2920 (asymmetrical CH2 stretching), and 2850 cm-1 (symmetrical CH2 stretching) (Fig. 5). The CH2/CH3 ratio was calculated by using the asymmetrical stretching of these bands (2920/2950 cm-

1) following Lin & Ritz (1993). Fourier deconvolution revealed methyl (δCH3; 1370 cm-1), and methyl-methylene [δ(CH2 + CH3); 1460 cm-1] groups (Fig. 6).

The spectra display the band assigned to carbonyl or carboxyl groups (νC=O; 1740 cm-1), or both. The νC-O

Fig. 4 - Comparison between IR spectra obtained from the extracted organic fraction (a) and powdered dry sediment (b).

67A. Guido et alii - Biotic vs abiotic carbonate discrimination with FT-IR Spectroscopy

continental) are distinguishable by their different H/C and O/C ratios (Peters et al., 2005).

To test the consistency of FT-IR analyses with the intent to determine kerogen origin, we compared the carbonyl/aliphatic (νC=O/νCHali) ratio with the Rock-Eval data (Hydrogen Index and Oxygen Index). The carbonyl groups, mainly composed of C=O bonds, can be correlated to the oxygen index, while the aliphatic groups, formed mainly of C-H bonds, can be correlated to the hydrogen index.

In the studied samples, the νC=O/νCHali ratio increase upward within each carbonate layer (from 0.19 to 0.33 in the basal layer and from 0.21 to 0.23 in the upper layer). This trend can be correlated to the increase of terrestrial input in the sedimentary environment, that increase the vibration intensity of carboxyl groups (νC=O). The νC=O/νCHali ratio shows the same trend of hydrogen and oxygen index that Guido et al. (2007a) attributed to periodic increases in the deposition of continental organic matter. The authors demonstrated a transitional composition between Type II and Type III kerogen revealing a mixture of marine and continental OM whit an increase of the oxygen index OI

cm-1 due to the asymmetric C-O stretching modes and weak bending mode frequencies in the regions near 785 cm-1 and 700 cm-1 (Anderson et al., 2005). These bands, when the organic matter content is very low as in some ancient carbonates, overlie and mask those of organic compounds, which prevent the FT-IR methodology detecting and characterising organic matter when applied directly to powdered sediments and thin sections (Fig. 4b). In the herein analysed CdB samples, the concentration of organic matter ranges from 0.06% to 0.19% and is almost homogeneously dispersed in the mineral matrix. These two characteristics make organic compounds unrecognizable in FT-IR spectroscopy because it is very difficult to distinguish the signal from the background (Fig. 4b). To overcome this problem and to increase the sensitivity of the IR analysis it was necessary to extract and concentrate the organic compounds.

The ratio of carbonyl to aliphatic groups (νC=O/νCHali) permits distinction between marine and continental inputs, and it is possible to obtain an indication of the kerogen origin by infrared spectrum analyses. This is reasonable, taking into account that kerogens (lacustrine, marine and

Fig. 5 - Infrared spectra of extracted organic matter. Note the uniformity of the molecular compounds indicating the homogeneity of the carbonate precipitation processes.

Bollettino della Società Paleontologica Italiana, 52 (1), 201368

samples are characterised by the absence of peaks in the three aromatic regions: the aromatic C-H stretching region (3000-3100 cm-1), the aromatic ring stretching (~1600 cm-1), and aromatic C-H out-of-plane deformation bands (700-900 cm-1) (Fig. 6a). Our spectra fit very well with the data of Lis et al. (2005) that relate the spectral distribution of the organic compounds with the lowest thermal maturity (Fig. 6). Organics have not yet been detected on the Martian surface (ten Kate, 2010). The major components of carbonaceous matter in carbonaceous chondrites are poorly characterised macromolecular complexes that resemble terrestrial kerogen (Hayatsu et al., 1983). Although a number of investigators have applied a variety of analytical methods to characterise these molecules in carbonaceous chondrites the structure of the carbonaceous matter remains poorly understood.

Murae (1994) carried out a comparative study of the chemical structures detectable by IR spectroscopy

values upward within each carbonate layer. These new data confirm the reliability of the proposed carbonyl/aliphatic ratio and therefore the FT-IR analyses.

The ratio of chain lengths (CH2/CH3) and the degree of branching [ν(CH2)4/νCHali] allow evaluation of the organic matter thermal maturity (Borrego et al., 1995; Lis et al., 2005). In the CdB samples, the CH2/CH3 ratio was determined with 2800-3000 cm-1 aliphatic-stretching region by Fourier deconvolution (Lin & Ritz, 1993). The value of this ratio ranges from 2.41 to 2.51, confirming the immaturity of the organic compounds, which was already indicated by biomarker data and the maximum hydrocarbon-generation temperature (Tmax).

The comparison between the FT-IR spectra of the studied samples and data from Lis et al. (2005), regarding the thermal maturity of type-II kerogens from Devonian black shales, corroborates the immaturity of the organic compounds preserved in the CdB (Fig. 6). The studied

Fig. 6 - a) Functional groups of the organic compounds extracted from the mineral matrix of sample CB1; b) data from Lis et al. (2005) regarding the thermal maturity of type-II kerogens from Devonian black shales.

69A. Guido et alii - Biotic vs abiotic carbonate discrimination with FT-IR Spectroscopy

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in carbonaceous chondrites of different classes and in terrestrial coals of different types. All the spectra obtained from carbonaceous chondrites closely resemble each other, which suggests that the main types of carbonaceous matter are similar in chemical structure. The IR spectra of several coals reveal that the structure of mature terrestrial kerogens is similar to that of organics in carbonaceous chondrites. Murae (1994) maintained that the similarity between terrestrial and extraterrestrial kerogens is close when similar thermal conditions prevailed.

In our view, the structural complexity of terrestrial kerogens originates from biological molecule syntheses. In contrast, subsequent geological maturation processes simplify the structure of kerogens, which finally ends with graphite.

The detection of thermal maturity and geochemical composition of the organic compounds furnishes a new tool to characterise Mars return samples, to compare the data with those reported for chondrites, and to evaluate the preservation possibilities of biomarkers.

CONCLUSIONS

We utilized IR spectroscopy to characterise fossil organic matter preserved in a problematic terrestrial carbonate, the CdB. This study shows the efficacy of FT-IR for the detection and classification of organics even when they are present in very low amounts and dispersed in mineral matrices. In this case, due to interference with the carbonate functional groups, the spectra obtained from powdered carbonates and thin sections, did not yield useful information regarding the organic molecules. Nonetheless, the utility of the FT-IR method is demonstrated by the analyses performed on organic matter extracted from the carbonate. Therefore, in the geochemical characterisation of the main kerogen types, the FT-IR can be considered a complementary technique to Rock-Eval pyrolysis and Gas Chromatography-Mass Spectrometry. Furthermore, FT-IR analysis makes possible to recognize the presence, origin, and thermal maturity of the organic matter preserved in carbonate sediments. The proposed methodology does not appear to be applicable in situ, considering the required multiple analytical approaches, nonetheless it does offer a useful tool for the discrimination between biotic and abiotic material of Mars sample return missions.

ACKNOWLEDGMENTS

We are grateful to Armando Blanco, Katrin Heindel and an anonymous reviewer for their suggestions that improved the quality of the manuscript. We thank Robert Riding who kindly checked the English of the text. This research has been funded by Regional Operative Program (ROP) Calabria ESF 2007/2013 - IV Axis Human Capital - Operative Objective M2-Action d.5 (Post-Doctoral Fellowship-Adriano Guido).

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