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Intracrystalline lipids within sulfates from the Haughton Impact Structure—Implications for survival of lipids on Mars
Icarus 187 (2007) 422–429 www.elsevier.com/locate/icarus
Intracrystalline lipids within sulfates from the Haughton Impact Structure—Implications for survival of lipids on Mars Stephen A. Bowden ∗ , John Parnell Department of Geology and Petroleum Geology, University of Aberdeen, Aberdeen AB24 3UE, United Kingdom Received 22 March 2006; revised 5 July 2006 Available online 5 December 2006
Abstract Lipids can be present within gypsum as intracrystalline inclusions if they become incorporated within the mineral as is it precipitates. The lipids that comprise these inclusions are protected against alteration or destruction by an external oxidising chemical environment because a protective mineral matrix surrounds them. Sulfate minerals are abundant on the surface of Mars and were present in the samples that were analysed by the Viking landers. The quantities of secondary intracrystalline fossil-lipids that are present in samples of gypsum and gypsum-rich soils from the Haughton Impact Structure, Devon Island, Canadian High Arctic are sufficient to suggest that if a similar concentration of fossil lipids was present in the sulfate-rich samples analysed by the Viking Landers then they could have been detected. Possible reasons why a secondary fossil-lipid signature was not detected include a poor rate of conversion during pyrolysis, exposure of intracrystalline lipids during periods of weathering to oxidative martian diagenesis, a low level of biological productivity or an absence of a source for lipids on the surface of Mars. Polycyclic aromatic hydrocarbons of meteoritic origin, and terpane biomarkers such as hopanes and steranes, are not present in the Haughton gypsum in sufficient quantities to have been readily detected. © 2006 Elsevier Inc. All rights reserved. Keywords: Astrobiology; Exobiology; Mars; Mineralogy
1. Introduction The presence of sulfates on the surface of Mars was indicated by a strong Mg–S correlation in the fluorescence data obtained by the Viking landers (Clark et al., 1976). Sulfates were subsequently identified as being resent in the soil at the Pathfinder landing site at a concentration of ∼10% MgSO4 (Wänke et al., 2001). Data from the Opportunity rover indicates that sulfates of probable evaporite origin are present in Eagle crater, Meridiani Planum where the sulfates constitute ∼40% by weight of the bedrock and could have a formed by direct precipitation from water or during early burial (Squyres et al., 2004). Thermal emission spectrometer data suggest that the major sulfate phases are magnesium and calcium sulfates (Christensen et al., 2004). Similar deposits occur in evaporitic environments on Earth which support life. Therefore the * Corresponding author. Fax: +44 (0) 1224 272785.
E-mail address: [email protected] (S.A. Bowden). 0019-1035/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.icarus.2006.10.013
presence of evaporitic sulfate deposits could indicate that the surface of Mars was also potentially habitable to similar organisms during the periods of sulfate deposition (McKay, 1996). Laboratory experiments suggest that in the highly oxidising conditions of the martian regolith, small organic compounds would not survive for long periods of time (although more recalcitrant larger molecular weight humic acids similar to kerogen might be more resistant to oxidation), and the absence of this pool of organic matter could explain the negative results obtained by the Py–GC–MS (pyrolysis–gas chromatography– mass spectrometry) instruments deployed on the Viking Landers (Stoker and Bullock, 1997; McDonald et al., 1998). Furthermore, even if compounds are not completely destroyed, oxidising conditions may convert large organic molecules to smaller organic molecules that would form organic salts that would not be amenable to detection by Py–GC–MS (Benner et al., 2000). Nevertheless, it has been proposed that even in the highly oxidising conditions of the martian surface, there is potential for a small quantity of organic molecules to be preserved within mineral phases as intracrystalline inclusions (Parnell et
Intracrystalline lipids—Survival of chemical fossils on Mars
al., 2002). Although the oxidation of organic matter by sulfate mineral phases may be thermodynamically possible, the preservation of organic matter within sulfate minerals over geological periods of time is kinetically reasonable at low temperatures and low values of pH (Sumner, 2004). This is because the abiotic reduction of sulfate (SO2− 4 ) to hydrogen sulfide H2 S is slow at temperatures less than 200 ◦ C (Ohmoto and Lasaga, 1982). However, other oxidised sulphur species are formed during the oxidation of sulfate to sulfide, so it is possible that these compounds have oxidising potential (even if weak) that may break down organic matter over geological time scales. Therefore there is some uncertainty over the stability of organic matter in sulfates over geological time. Within the types of samples that have been analysed on Earth, intracrystalline, organic compounds within fluid inclusions have been found to be highly resistant to chemical degradation and oxidation. This is best illustrated by the methods some workers have used to remove adhering organic matter from samples of carbonate and quartz sandstone prior to petroleum-bearing fluid inclusions being extracted and analysed: After extraction samples have been treated with hydrogen peroxide (George et al., 1997) to break down noninclusion organic compounds in a method similar to that used (McDonald et al., 1998) to simulate the oxidative effects of the martian regolith. Intracrystalline biomarkers included within the mineral phases as fluid inclusions are not oxidised by hydrogen peroxide as they are effectively contained within a sealed environment, and they can be extracted and analysed after further crushing. In theory, sulfates should also be capable of preserving lipid and fossil-lipid biomarkers as intracrystalline inclusions in the same way that oil is preserved as fluid inclusions in sandstones (George et al., 1997) and lipids can be incorporated within carbonate (Xie et al., 1998; Stern et al., 2001; Ingalls et al., 2003). Lipids within these minerals may be autochthonous, i.e. indigenous to the environment of deposition and therefore constitute a primary biomarker record or they may constitute a secondary signature derived from biological activity outside of the environment of deposition (i.e. allochthonous). However, uncertainty remains about the quantity of organic compounds and fossil lipids that could be preserved within sulfate minerals as intracrystalline as opposed to nonintracrystalline material. On the surface of Mars, where it is likely that non-intracrystalline organic compounds are destroyed in the highly oxidising conditions of the martian surface or converted to non-GC amenable organic salts (Benner et al., 2000), the size of a potential pool of intracrystalline organic matter is very important. Biological productivity will control the quantities of primary organic material that is initially preserved, but later geological processes and the nature of the host mineral substrate and its ability to sequester biomolecules or their biomarkers will control the quantities of allochthonous and secondary organic material that may survive over long periods of time as intracrystalline inclusions. To assess the viability of analysis of intracrystalline lipid biomarkers within mineral phases we have analysed gypsum-
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rich materials that are present in the Recent/Quaternary aged (<1.6 Ma) soil and Eocene aged (impact dated to 39 Ma) impact-related, hydrothermal deposits in the Haughton Impact Structure, Devon Island, Canadian High Arctic. Devon Island is a pertinent sampling locality because of its dry, cold arctic environment (75◦ , 22 N and 89◦ , 41 W ), the oxidising conditions present in surface soils and associated low levels of biological productivity (Cockell et al., 2001). We have quantified the lipids that were amenable to recovery by simple solvent extraction as well as the intracrystalline lipids that can only be recovered by the complete dissolution of the mineral matrix (the intracrystalline lipids that would be expected to be resistant to oxidation and destruction). 2. Sample collection and setting 2.1. Geology The Haughton Impact Structure was formed about 39 Ma (Sherlock et al., 2005) in a ∼1750-m-thick series of Lower Paleozoic sedimentary rocks dominated by a sulfate-bearing carbonate facies overlying a Precambrian crystalline basement. The crater is filled with carbonate-rich impact melt rocks (Osinski and Spray, 2001), which contain clasts of basement, indicating an excavation depth of 2 + km. Limited Tertiary lacustrine sediments lie upon the melt rocks. The rocks within the central part of the crater include hydrothermal mineral veins of quartz and calcite (Osinski et al., 2001). Country rocks around the crater are predominantly brown dolomites with a sucrosic, porous texture, and are generally unveined. The surface deposits analysed for this study comprise a crystal of gypsum formed during a late-stage of the impact-related hydrothermal system (Osinski et al., 2001), a sample of hydrothermal gypsum colonised by modern cyanobacteria (Parnell et al., 2004) and a pale gray coarse-grained alluvial soil consisting of fragments of dolomite, calcite, gypsum and crystalline basement (fragments of basement are mostly banded gneiss rich in quartz). 2.2. Ecology The alluvial terraces of Devon Island, Arctic Canada have a vegetation cover ranging from 2 to 11%, although micro-oases exist that have vegetation coverage between 2 and 98% and bacterial counts that are an order of magnitude greater than outside of the micro-oases (Cockell et al., 2001). Endolithic colonisation by Chroococcidiopsis, Aphanothece, unicellular chlorophytes and Gloeocapsa sp. of both impact-shocked and unshocked rocks occurs across the island (Cockell et al., 2002). One unique endolithic habitat exploited by cyanobacteria within the impact crater is outcrops of high purity gypsum formed during the late stages of impact-induced lowtemperature hydrothermal activity (Parnell et al., 2004). 3. Materials and methods Two samples of hydrothermal gypsum (CaSO4 ·2H2 O), one colonised by Gloeocapsa and another non-colonised sample
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were cleaned prior to analysis. Cleaning comprised brief immersion in 20% HCl (hydrochloric acid), immersion in MeOH (methanol) followed by immersion in DCM (dichloromethane). The samples were then rinsed with MeOH and the cleaning process repeated at the end of which the samples were rinsed with DCM and allowed to dry. Samples of gypsum were crushed in a pestle and mortar and dissolved in 20% HCl prior to extraction. Liquid–liquid extraction of the dissolved gypsum samples was performed in calcined-glass vials with excess HCl added to lower pH and inhibit the formation of carboxylic salts to enable the partitioning of lipids to an organic extracting solvent phase. A five-stage liquid–liquid extraction was then performed on the dissolved samples using 4:1 v/v DCM:MeOH and the extracts combined. Powder X-ray diffraction confirmed the presence of dolomite, calcite, gypsum and quartz mineral phases in the soil sample and microscopic analysis identified secondary sulfate and calcite mineral phase overgrowths. The powdered soil sample was soxhlet extracted for 48 h with 93:7 v/v DCM:MeOH and the extract recovered. Two further soxhlet extractions were performed each lasting 24 h and using 93:7 v/v DCM:MeOH as the extracting solvent. The final extract had no visible colour and the extracted soil was treated with hydrogen peroxide to oxidise any residual organic material prior to being split. One sample split was treated with 20% HCl to dissolve the most soluble evaporite mineral phases (calcite and gypsum); the other split was not treated with 20% HCl but used as a control for later stages. Powder X-ray diffraction revealed that the dominant calcite and gypsum peaks were reduced by 50 and 40% subsequent to the treatment with HCl. Following treatment a sample split treated with HCl and its control sample (the control sample was not treated with HCl) were extracted using a five stage sonic extraction. The starting composition of the extracting solvent was 4:1 v/v DCM:MeOH but later extraction stages contained progressively less MeOH so that the final sonic extraction was performed with DCM. Sonication was used to agitate the samples for brief periods of time (not exceeding 15min duration). All of the products recovered by extraction were separated by silica gel adsorption chromatography using a short Pasteur pipette packed with coarse-grained silica into hydrocarbon and polar fractions by elution with 3:1 v/v hexane:DCM and 2:1 v/v DCM:MeOH, respectively. The polar fraction was silyated by heating in the presence of excess BSTFA (N ,Obis(trimethylsilyl)trifluoroacetamide) to convert alkanols into their silyated ethers and alkenoic acids into their silyated esters. GC–MS analysis was performed on a HP (Hewlett Packard) 5890 series 2 GC fitted with an SGE BP-X5 column (fused silica; 0.5-µm film thickness; 30 m × 0.32 mm ID) and connected to a HP 5970 mass detector (ionisation energy 70 eV; SIM mode). The oven-heating program was 50 to 175 ◦ C at 4 ◦ C/min and then from 175 to 300 ◦ C at 4 ◦ C/min finally holding at 290 ◦ C for 20 min. Both full scan and SIM data were collected, and compound identification was based on elution order and examination of the spectra for diagnostic ions. Quantification was performed relative to an internal standard
Table 1 Yields of n-alkanoic acid and n-alkanol lipids Extract compositiona Hydrocarbons (%)
Polar (%)
Polar lipidsb µg/g sulfate
Colonised gypsum
<1
>99
14
Non-colonised gypsum
<5
>95
7
Soil Non-intracrystalline
17
83
2c
Intracrystalline Calcite Sulfate Control
<0.1 – – –
>99.9 – – –
– 9d 6a 0.09
a b c d
Extract composition based on column chromatography. Yields of polar lipids based on quantification by GC–MS. Soxhlet extracts combined from three stages. Yields of lipids were proportioned to the calcite and sulfate mineral phases according to the changes in dominant (XRD) 2θ -peak heights observed with acid treatment and assuming an equal proportion of organic mineral within each mineral phase.
(silyated ester of nonadecanoic acid) added prior to GC–MS analysis. 4. Results The yields obtained for the different samples are shown in Table 1. 4.1. Polar lipids The soil sample and the sample of gypsum colonised by cyanobacteria contain n-alkanoic and n-alkenoic fatty acids that range in carbon number from C14 to C26 . In these samples the most prominent straight chain fatty acids are the mono- and unsaturated-C16 and C18 homologues and the most prominent methyl branched iso- and anteiso-alkenoic acids are the C15 and C17 homologues (Fig. 1). A sample of the hydrothermal gypsum with no visible colonisation was also analysed and yielded C16 and C14 n-alkanoic acids and monosaturated C16 n-alkanoic acids. The most abundant compound was C17 n-alkanol. 4.2. Hydrocarbon products Hydrocarbons did not constitute a significant proportion of the extracts of the samples of gypsum or intracrystalline organic matter (less than 5% of extract weight). n-Alkanes were present in all extracts (Fig. 2). The n-alkanes do not show a strong oddover-even (or even-over-odd) predominance. The biosynthesised hopane diploptene (C30 17β,21α(H)hop-22(29)-ene) and several of the geohopanoids (predominantly C29 and C30 hopanes with the 17α,21β(H) and 17β, 21α(H) configurations, products of the diagenetic conversion of biohopanoids) were detected in the hydrocarbon fractions of the colonised gypsum and soil sample (Fig. 3). Diplopterol and other C35 biohopanoids were not detected within the polar fractions.
Intracrystalline lipids—Survival of chemical fossils on Mars
Fig. 1. GC–MS traces of the summed m/z 75 + 117 ion chromatograms of the polar fractions derivatized with BSTFA. Silyated derivatives of: 14:0 = tetradecanoic acid; i15:0 = 13-methyltetradecanoic acid; a15:0 = 12-methyltetradecanoic acid; 15:1 = pentadecenoic acid (location of double bond unspecified); 15:0 = pentadecanoic acid; 15OH = pentadecanol; i16:0 = 14-methylpentadecanoic acid; a16:0 = 13-methylpentadecanoic acid; 16:1 = hexadecenoic acid (location of double bond unspecified); 16:0 = hexadecanoic acid; i17:0 = 15-methylhexadecanoic acid; a17:0 = 14-methylhexadecanoic acid; 17:1 = heptadecenoic acid (location of double bond unspecified); 17:0 = heptadecanoic acid; 17OH = heptadecanol; 18:1 = octadecenoic acid (location of double bond unspecified); 18:0 = octadecanoic acid; ∗ = plasticizer contamination thought to originate during sample storage.
4.3. Intracrystalline sulfate-bound lipids Following treatment with hydrogen peroxide, the intracrystalline lipids within the sulfate and calcite mineral phases were recovered by treating the sample with 20% HCl. Treatment with acid is most likely to have preferentially dissolved secondary mineral overgrowths that formed around cores of bedrock clasts. The lipids extracted from the acid-treated sample contain low proportions of the C15 and C17 n-alkanols. The C15 and C17 iso- and anteiso-fatty acids are also relatively more abundant in the lipids recovered by dissolution of the calcite and sulfate mineral phases than the geolipids recovered from the bulk soil extract (Fig. 1). The hydrocarbon fraction of intracrystalline lipids also bears a greater similarity to the hydrocarbon
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Fig. 2. GC–MS trace of the m/z 85 ion of the hydrocarbon fractions. nC17 = heptadecane, etc.; br = methyl branched alkanes and alkyl-cyclohexanes. Note that the early part of the chromatogram pertaining to heptadecane has also been included as insert with an exaggerated vertical scale.
fraction extracted from the hydrothermal gypsum than from the soil; e.g. the peak heights for the C29 αβ and C30 αβ hopanes are equal and C27 Tm (C27 17α(H)-22,29,30 trisnorhopane) hopane is present (Fig. 3). 5. Discussion 5.1. Polar lipids The C16 and C18 fatty acids extracted from the cyanobacteria-colonised gypsum and sample of soil correspond to the main phospholipid-constituents of the cell membranes of both prokaryotes and eukaryotes. However, an origin from a microbial source is most likely for lipids extracted from the sample of soil as well as for the sample of colonised gypsum as, except for micro-oases that exhibit species- and spatially-limited vegetation cover (Cockell et al., 2001), higher plants are not significant in the arctic polar desert of Devon Island. Endolithic colonisation of shocked gneiss by Chroococcidiopsis spp. has been reported by Cockell et al. (2002), who also observed communities of Chroococcidiopsis, Aphanothece and Gloeocapsa (cyanobacteria) as well as chlorophytes (green algae) on the undersides and other surfaces of many rocks, whereas Gloeocapsa and Nostoc commune (also cyanobacteria) are the domi-
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Fig. 3. GC–MS trace of the m/z 191 ion of the hydrocarbon fractions. Tm = C27 17α(H)-22,29,30 trisnorhopane; C29 αβ = C29 17α, 21β(H) hopane; C29 βα = C29 17β, 21α(H) hopane; C31 αβ S = C31 17α, 21β(H) (22S) hopane; C31 αβ R = C31 17α, 21β(H) (22R) hopane; Dip = diploptene (C30 17β,21β(H)-hop-22(29)-ene); ∗ = unidentified contaminant.
nant chasmoendoliths present within the microbe bearing gypsum (Parnell et al., 2004). Fungi (eukaryotes) have also been cultured from alluivial material collected from Devon Island (Cockell et al., 2001). The lipid fingerprints of the extracts of the colonisedgypsum and the intracrystalline lipids extracted from the soil are characteristic of predominantly prokaryote sources. This can be inferred from the significant proportions of branched C15 and C17 methyl branched fatty acids which are generally present in higher proportions in bacteria (Volkman et al., 1980; Harwood and Russell, 1984), and the absence of polyunsaturated fatty acids [polyunsaturated fatty acids (C18 and greater) are generally present in chlorophytes but not abundant in prokaryotes (Volkman et al., 1980, 1998)]. The saturated C16 and C18 n-alkanoic acids are also commonly held to be present in all fungi and are also present in many heterotrophic bacteria (Harwood and Russell, 1984), so it is possible that some of the non-intracrystalline fatty acids present in the soil may have a fungal origin or derive from bac-
teria that are not phototrophic (e.g. other than cyanobacteria). The distribution of the lipids extracted from the sample of non-colonised hydrothermal gypsum is quite different to that seen in the other samples. The mechanism by which the modern cyanobacteria colonise the gypsum deposits is relatively well constrained and is dependant on the opening of mineral cleavage planes open during weathering. Gloeocapsa, a cyanobacterium prevalent in the sample area, penetrates along opened cleavage planes and seems most capable to exploit this unusual ecological niche (Parnell et al., 2004). As the non-colonised sample of gypsum analysed during this study contained no visible colonisation, and appeared not to have been weathered, it is unlikely that the sample has been colonised by cyanobacteria during recent times. Therefore, the lipids extracted from the gypsum are not likely to derive from modern chasmoendolithic bacteria and it is reasonable to explore the possibility that the origin of any lipids is related to the impact-induced hydrothermal system that precipitated the gypsum (Osinski et al., 2001). The gypsum is interpreted to have precipitated from a low temperature hydrothermal system that persisted for some time after the event that created the Haughton Impact Structure (Osinski et al., 2001). A similar distribution of lipids (to those shown in Fig. 1) with prominent C17 n-alkanol and low proportions of C18 fatty acids (order of prominence C17 n-alkanol > C16 n-alkanoic acid) has been reported in microbial mats dominated by Chloroflexus in low-temperature hot springs (<65 ◦ C) in Yellowstone Park (van der Meer et al., 2000, 2003). In other instances the distribution of alkanoic acids relative to alkanols in microbial mats living in hydrothermal springs, in which Chloroflexus and other non-cyanobacteria photosynthetic bacteria are prominent, is not always dominated by C17 n-alkanol, although one particular alcohol dominates in most instances, e.g. C18 n-alkanol, C17 n-alkanol or phytol (Shiea et al., 1991). Methyl branched fatty acids are also less common constituents of the cultures of the green and purple bacteria found in these hydrothermal spring communities (Shiea et al., 1991). Thus the distribution of the lipids extracted from the non-colonised gypsum is consistent with, although not necessarily conclusive proof for, an origin from a microbial community dominated by non-cyanobacterial photosynthetic prokaryotes that existed in an ancient hydrothermal system. As microbial mat structures are not present within the transparent gypsum, it is likely that the lipids are allochthonous/secondary and were transported by hydrothermal fluids and incorporated within the gypsum as it precipitated. The incorporation of stearic acid within magnesium sulfate as intracrystalline lipids has been replicated within the laboratory (Bowden et al., 2005), so this is certainly possible. 5.2. Hydrocarbon products None of the sample extracts contain an n-alkane distribution with a strong odd over even predominance even though odd carbon numbers (greater than C25 ) generally dominate the n-alkanes extracted from algae and plants. The lack of an odd over even preference in n-alkanes, while unusual in extracts of
Intracrystalline lipids—Survival of chemical fossils on Mars
living organisms, has been observed in the extracts of cultures of Chlorobium sp. and the purple bacteria Chromatium tepidum (Shiea et al., 1991). However, fossil fuels that have been heated during burial also typically have n-alkanes with a distribution in which odd carbon numbers do not significantly predominant over even carbon numbers. As the dolomitic bedrock within the sample locality often contains small quantities of fossil fuel, which also have no odd over even predominance (Parnell et al., 2004), an origin from this source can not be ruled out, particularly for the non-intracrystalline fraction of the soil extract. Given the pervasive colonisation of the sample locality by cyanobacteria, diploptene is most likely to have originated from a modern cyanobacterial (rather than a plant) source. However, the most prominent hopane in the non-colonised gypsum was the geohopanoid C30 17α,21β(H) hopane, a compound that results from the diagenetic conversion of biologically produced hopanoids (Seifert and Moldowan, 1980). The prominence of this geohopanoid (with the 17α,21β(H) configuration) relative to any biohopanoids (with the 17β,21α(H) configuration) further indicates that many of the compounds within the sample of non-colonised gypsum may pertain to syn-hydrothermal ecological communities, although the dominance of this single hopane is unusual. 6. Yields and quantitative considerations with respect to the Viking missions The lipid yields of 7 ppm for the sample of non-colonised gypsum and 6 ppm for the secondary mineral phase for the intracrystalline lipids released by acidification of the soil sample are of similar orders of magnitude to the quantities of intracrystalline lipids detected in carbonate sediment (Ingalls et al., 2003, 2004). Orders of magnitude more lipid material have been detected in stalactites in caves (Xie et al., 1998) and in secondary aragonite present within cavities in Zechstein carbonates (Peckmann et al., 1999). The latter two examples may have included primary contributions from microbes that lived within the samples (as was the case for the Gloeocapsa colonised gypsum). If modern microbial life is growing on a sample then there is clearly no upward limit on the concentration of autochthonous lipids that might be present. However there may be a limit for the total amount of intracrystalline fossil lipids of allochthonous/secondary origin, dependant on transport mechanisms and the ability of the host mineral to sequester lipids. There are few studies of fossil lipids within sulfates (e.g. Grice et al., 1998), and to the authors knowledge no studies of secondary intracrystalline lipids within sulfates (e.g. instances where the lipids did not originate from biological productivity within the sample or in the environment where the sulfate phase formed). A value of 7 ppm was recorded during this study, but it is probably appropriate to moderate this number by taking into account the data derived from studies of carbonates (Xie et al., 1998; Peckmann et al., 1999): therefore at least 5–10 ppm of secondary lipids may typically be preserved by incorporation as intracrystalline organic matter in calcite or sulfate mineral phases. These intracrystalline lipids could survive oxidation within the
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martian regolith and would not be expected to be destroyed (Stoker and Bullock, 1997; McDonald et al., 1998) or converted to small organic molecules that in turn form non-GC amenable organic salts (Benner et al., 2000). If the sulfates analysed in this study are analogous to the sulfates present in the martian regolith, is there a sufficient quantity of intracrystalline lipids for the Viking Py–GC–MS to have detected? A conservative estimate for the quantities of lipids that could be present would assume that only allochthonous secondary intercrystalline lipids would be present e.g. the surface of Mars does not appear to possess a surface biosphere that produces large quantities of lipids at the present time, therefore no recent biological input is likely. A concentration for intracrystalline lipids of 5 to 10 ppm for a sulfate mineral phase and a sulfate composition of 10% for the martian regolith (Toulim et al., 1977; Wänke et al., 2001) would equate to 500 to 1000 ppb of intracrystalline lipids in secondary sulfate mineral phases. Some of the samples analysed by the Viking landers were heated to 500 ◦ C which is sufficient to thermally decompose any magnesium and calcium sulfates within the regolith, thus releasing any intracrystalline lipids that may have been present. However, pyrolysis of organic matter is relatively inefficient and the conversion rate to GC-amenable volatiles was assumed to have been 10% (Biemann et al., 1977), thus further reducing any yield to at least 50 to 100 ppb. This concentration would still have been within the detection limits of the Viking Py–GC–MS (Biemann et al., 1977) for both aliphatic hydrocarbons and aromatic hydrocarbons, potential products from the pyrolysis of fatty acids. The intracrystalline n-alkanes and other hydrocarbons present in sulfates from Devon Island examined in this study are present at very low concentrations (an equivalent yield of less than 0.5 ppb for intracrystalline organic matter within the sulfates in the martian regolith) and would probably not have been readily detectable by the Py–GC–MS deployed during the Viking missions. The negative results obtained by the Py–GC–MS deployed on the Viking landers, with respect to the detection of potential biomarker nitrogen-containing compounds derived from amino acids, can be explained by the creation of non-volatile nonGC amenable pyrolysis products in an oxidising environment (Benner et al., 2000) and the venting of water and carbon dioxide by the effluent divider during the elution of volatile amines (Glavin et al., 2001). However, in addition to the technical and analytical considerations regarding the nature of the sample matrix, for intracrystalline lipids there are many other factors that may explain why the Viking landers did not detect the pyrolysis products of the intracrystalline lipids that could have been present. (i) If very low concentrations of lipids are present in the parent solution then low concentrations of intracrystalline lipids will be present in minerals precipitating from that solution. Consequently there is no lower limit to the amount of lipids that might be present, but there may be an upper limit at about the 10 ppm level for lipids in secondary mineral phases. This maybe for a number of reasons, but the incorporation of carboxylic acids within mineral phases has been
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suggested to be linked to the creation of ionic bonds between carboxylic acid groups and mineral surfaces (Fenter and Struchio, 1999). The importance of this mechanism may explain why compounds with polar groups have been observed to be incorporated in greater quantities than apolar compounds (Bowden et al., 2005). Therefore, factors limiting the bonding of organic acids to mineral surfaces (such as the surface area of particles in solution) may limit the quantities of organic acids that become incorporated as intracrystalline inclusions. As alkanoic acids are not as a significant component of the cell membranes of archaea as they are of prokaryotes and eukaryotes, a similar type of biological productivity that did not produce alkanoic acids could also reduce the amount of intracrystalline lipids that can be preserved in sulfates. (ii) The concentration of intracrystalline lipids has been shown to decrease with diagenesis, which may arise from partial destruction of the mineral matrix and the exposure of any included organic matter (Ingalls et al., 2003, 2004). Thus martian diagenesis and surface processes should reduce the concentration of intracrystalline lipids, particularly the ablation of sulfate grains during transport and reworking of the martian regolith by aeolian processes prior to the reconstituting of sulfates as a secondary cement phase (Thomas et al., 1999). Intracrystalline lipids exposed to the surface environment in this way would be expected to proceed along the diagenetic scheme proposed by Benner et al. (2000). Thus a low concentration of lipids in the parent solution from which a sulfate mineral phase precipitated and a heavy degree of reworking by surface processes would all reduce the potential yield of volatile hydrocarbons from intracrystalline lipids to a level that may not have been detectable by the Py–GC–MS carried by the Viking landers. 7. Conclusions A significant proportion of the lipids preserved within the samples of sulfate deposits from the Haughton Impact Structure are present as intracrystalline inclusions and are resistant to chemical oxidation. Some lipids derive from a recent phase of endolithic colonisation by cyanobacteria (Parnell et al., 2004); whilst some intracrystalline lipids are interpreted to be the product of an extinct hydrothermal system (Osinski and Spray, 2001), dating to a period of time soon after the impact. If the sulfate minerals that are present in the Haughton Impact Structure are used as an analogue for Mars, then the quantities of intracrystalline n-alkanoic acids and n-alkanols of secondary origin that could have potentially been present should have been detectable by the Py–GC–MS instrument deployed on the Viking Landers. However this assumes that the conversion of lipids to GC-amenable compounds was 10% efficient (Biemann et al., 1977). If conversion of organic matter to GC-amenable volatiles by pyrolysis was less than 0.1%, intracrystalline lipids might not have been detected. Intracrystalline hydrocarbons (e.g. PAH and hopanes) are not present
within the secondary sulfates at concentrations above the detection limits of the Py–GC–MS deployed on the Viking landers. Therefore both intracrystalline PAH that could derive from meteoritic input (Basile et al., 1984) and intracrystalline ancient biomarkers similar to the oldest (2.7 Ga) reported molecular biomarkers on Earth (Brocks et al., 1998) would probably have not been detected by the Viking Py–GC–MS. Acknowledgments This work was carried out under the auspices of the NASA Haughton–Mars Project. We are grateful to the Polar Continental Shelf Project (Natural Resources Canada), the Nunavut Research Institute, the Communities of Grise Fiord and Resolute Bay for their support, and to the many participants of the NASA Haughton–Mars Project. This work was supported by EPSRC Grant GR/S47267/01. References Basile, B.P., Middleditch, B.S., Oro, J., 1984. Polycyclic aromatic hydrocarbons in the Murchison meteorite. Org. Geochem. 5, 211–216. Benner, S.A., Devine, K.G., Matveeva, L.N., Powell, D.H., 2000. The missing organic molecules on Mars. Proc. Natl. Acad. Sci. USA 97, 2425–2430. Biemann, K., Oro, J., Toulmin III, P., Orgel, L.E., Nier, A.O., Anderson, D.M., Simmonds, P.G., Flory, D., Diaz, A.V., Rushneck, D.R., Biller, J.E., Lafleur, A.L., 1977. The search for organic substances and inorganic volatile compounds in the surface of Mars. J. Geophys. Res. 82, 4641–4658. Bowden, S.A., Cooper, J.M., Parnell, J., 2005. The extraction of organic compounds from sulfate minerals for astrobiological exploration. Lunar Planet. Sci. XXXVI. Abstract 1325. Brocks, J.J., Logan, G.A., Buick, R., Summons, R.E., 1998. Archean molecular fossils and the early rise of eukaryotes. Science 285, 1033–1036. Christensen, P.R., Wyatt, M.B., Glotch, T.D., Rogers, A.D., Anwar, S., Arvidson, R.E., Bandfield, J.L., Blaney, D.L., Budney, C., Calvin, W.M., Fallacaro, Fergason, R.K., Gorelick, N., Graff, T.G., Hamilton, V.E., Hayes, A.G., Johnson, J.R., Knudson, A.T., McSween Jr., H.Y., Mehall, G.L., Mehall, L.K., Moersch, J.E., Morris, R.V., Smith, M.D., Squyres, S.W., Ruff, S.W., Wolff, M.J., 2004. Mineralogy at Meridiani Planum from the Mini-TES experiment on the Opportunity Rover. Science 306, 1733–1739. Clark, B.C., Baird, A.K., Rose, H.J., Toulmin, P., Keil, K., Castro, A.J., Kelliher, W.C., Rowe, C.D., Evans, P.H., 1976. Inorganic analyses of martian surface samples at Viking landing sites. Science 194, 1283–1288. Cockell, C.S., Lee, P., Schuerger, A.C., Hidalgo, L., Jones, J.A., Stokes, M.D., 2001. Microbiology and vegetation of micro-oases and polar desert, Haughton impact crater, Devon Island, Nunavut, Canada. Arctic Antarctic Alpine Res. 33, 306–318. Cockell, C.S., Lee, P., Osinski, G., Horneck, G., Broady, P., 2002. Impactinduced microbial endolithic habitats. Meteorit. Planet. Sci. 37, 1287–1298. Fenter, P., Struchio, N.C., 1999. Structure and growth of sterae monolayers on calcite: First results of an in situ X-ray reflectivity study. Geochim. Cosmochim. Acta 63, 3145–3152. George, S.C., Krieger, F.W., Eadington, P.J., Quezada, R.A., Greenwood, P.F., Eisenberg, L.I., Hamilton, P.J., Wilson, M.A., 1997. Geochemical comparison of oil-bearing fluid inclusions and produced oil from the Toro sandstone, Papua New Guinea. Org. Geochem. 26, 155–173. Glavin, D.P., Shcubert, M., Botta, O., Kminek, G., Bada, J.L., 2001. Detecting pyrolysis products from bacteria on Mars. Earth Planet. Sci. Lett. 185, 1–5. Grice, K., Shouten, S., Nissenbaum, N., Charrach, J., Sinninghe Damsté, J.S., 1998. Unusual distribution of monomethylalkanes in Botryococcus brauniirich samples: Origin and significance. Geochim. Cosmochim. Acta 28, 349– 359. Harwood, J.L., Russell, N.J., 1984. Lipids in Plants and Microbes. Allen & Uwin, London, pp. 35–59.
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Ingalls, A.E., Lee, C., Druffel, E.R.M., 2003. Preservation of organic matter in mound-forming coral skeletons. Geochim. Cosmochim. Acta 67, 2827– 2841. Ingalls, A.E., Aller, R.C., Lee, C., Wakeham, S.G., 2004. Organic matter diagenesis in shallow water carbonate sediments. Geochim. Cosmochim. Acta 68, 4363–4379. McDonald, G.D., de Vanssay, E., Buckley, J.R., 1998. Oxidation of organic macromolecules by hydrogen peroxide: Implications for stability of biomarkers on Mars. Icarus 132, 170–175. McKay, C.P., 1996. Origins of life: A comparison of theories and application to Mars. Origins Life Evol. Biosphere 27, 263–289. Ohmoto, H., Lasaga, A.C., 1982. Kinetics of reactions between aqueous sulfates and sulfides in hydrothermal systems. Geochim. Cosmochim. Acta 46, 1727–1745. Osinski, G.R., Spray, J.G., 2001. Impact-generated carbonate melts: Evidence from the Haughton structure, Canada. Earth Planet. Sci. Lett. 215, 357–370. Osinski, G.R., Spray, J.G., Lee, P., 2001. Impact-induced hydrothermal activity within the Haughton Impact Structure, Arctic Canada: Generation of a transient, warm, wet oasis. Meteorit. Planet. Sci. 36, 731–745. Parnell, J., Mazzini, A., Honghan, C., 2002. Fluid inclusion studies of chemosynthetic carbonates: Strategy for seeking life on Mars. Astrobiology 2, 43–57. Parnell, J., Lee, P., Cockell, C.S., Osinski, G.R., 2004. Microbial colonisation in impact-generated hydrothermal sulfate deposits, Haughton Impact Structure, and implications for sulfates on Mars. Int. J. Astrobiol. 3, 247–256. Peckmann, J., Paul, J., Thiel, V., 1999. Bacterially mediated formation of diagenetic aragonite and native sulphur in Zechstein carbonates (Upper Permian, Central Germany). Sediment. Geol. 126, 205–222. Seifert, W.K., Moldowan, J.M., 1980. The effect of thermal stress on sourcerock quality as measured by hopane stereochemistry. Phys. Chem. Earth 12, 229–237. Sherlock, S.C., Kelley, S.P., Parnell, J., Green, P., Lee, P., Osinsk, G.R., Cockell, C.S., 2005. Re-evaluating the age of the Haughton impact event. Meteorit. Planet. Sci. 40, 1777–1787. Shiea, J., Brassell, S.C., Ward, D.M., 1991. Comparative analysis of extractable lipids in hot spring microbial mats and their component photosynthetic bacteria. Org. Geochem. 17, 309–319. Squyres, S.W., Grotzinger, J.P., Arvidson, R.E., Bell III, J.F., Calvin, W., Christensen, P.R., Clark, B.C., Crisp, J.A., Farrand, W.H., Herkenhoff, K.E.,
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Johnson, J.R., Klingelhöfer, G., Knoll, A.H., McLennan, S.M., McSween Jr., H.Y., Morris, R.V., Rice Jr., J.W., Rieder, R., Soderblom, L.A., 2004. In situ evidence for an ancient aqueous environment at Meridiani Planum, Mars. Science 306, 1709–1714. Stern, B., Abbott, G.D., Collins, M.J., Armstrong, H.A., 2001. Development and comparison of different methods for the extraction of biomineral associated lipids. Ancient Biomol. 2, 321–334. Stoker, C.R., Bullock, M.A., 1997. Organic degradation under simulated martian conditions. J. Geophys. Res. 102 (E5), 10881–10888. Sumner, D.Y., 2004. Poor preservation potential of organics in Meridiani Planum hematite-bearing sedimentary rocks. J. Geophys. Res. Planets 109 (E12), doi:10.1029/2004JE002321. Thomas, P.C., Malin, M.C., Carr, M.H., Danielson, G.E., Davies, M.E., Hartmann, W.K., Ingersoll, A.P., James, P.B., McEwen, A.S., Soderblom, L.A., Veverka, J., 1999. Bright dunes on Mars. Nature 397, 592–594. Toulim, P., Baird, A.K., Clark, B.C., Keil, K., Rose, H.J., Christian, R.P., Evans, H.P., Kelliher, W.C., 1977. Geochemical and mineralogical interpretation of the Viking inorganic chemical results. J. Geophys. Res. 82, 4625–4634. van der Meer, M.T.J., Shouten, S., de Leeuw, J.W., Ward, D.M., 2000. Autotrophy of green non-sulphur bacteria in hot spring microbial mats: Biological explanations for isotopically heavy organic carbon in the geological record. Environ. Microbiol. 2, 428–435. van der Meer, M.T.J., Shouten, S., Sinninghe Damsté, J.S., de Leeuw, J.W., Ward, D.M., 2003. Compound-specific isotopic fractionation patterns suggest different carbon metabolisms among Chloroflexus-like bacteria in hotspring microbial mats. Appl. Environ. Microbiol. 69, 6000–6006. Volkman, J.K., Johns, R.B., Gilian, F.T., Perry, G.J., 1980. Microbial lipids of an intertidal sediment. I. Fatty acids and hydrocarbons. Geochim. Cosmochim. Acta 44, 1133–1143. Volkman, J.K., Barrett, S.M., Blackburn, S.I., Mansour, M.P., Sikes, E.L., Gelin, F., 1998. Microalgal biomarkers: A review of recent research developments. Org. Geochem. 29, 1163–1179. Wänke, H., Brückner, G., Rieder, R., Ryabchikov, I., 2001. Chemical composition of rocks and soils at the Pathfinder site. Space Sci. Rev. 96, 317– 330. Xie, S., Yi, Y., Huang, J., Hu, C., Cai, Y., Collins, M., Baker, A., 1998. Lipid distribution in a subtropical southern China stalagmite as a record of soil ecosystem response to paleoclimate change. Quat. Res. 60, 340–347.
Intracrystalline lipids within sulfates from the Haughton Impact Structure—Implications for survival of lipids on Mars Stephen A. Bowden ∗ , John Parnell Department of Geology and Petroleum Geology, University of Aberdeen, Aberdeen AB24 3UE, United Kingdom Received 22 March 2006; revised 5 July 2006 Available online 5 December 2006
Abstract Lipids can be present within gypsum as intracrystalline inclusions if they become incorporated within the mineral as is it precipitates. The lipids that comprise these inclusions are protected against alteration or destruction by an external oxidising chemical environment because a protective mineral matrix surrounds them. Sulfate minerals are abundant on the surface of Mars and were present in the samples that were analysed by the Viking landers. The quantities of secondary intracrystalline fossil-lipids that are present in samples of gypsum and gypsum-rich soils from the Haughton Impact Structure, Devon Island, Canadian High Arctic are sufficient to suggest that if a similar concentration of fossil lipids was present in the sulfate-rich samples analysed by the Viking Landers then they could have been detected. Possible reasons why a secondary fossil-lipid signature was not detected include a poor rate of conversion during pyrolysis, exposure of intracrystalline lipids during periods of weathering to oxidative martian diagenesis, a low level of biological productivity or an absence of a source for lipids on the surface of Mars. Polycyclic aromatic hydrocarbons of meteoritic origin, and terpane biomarkers such as hopanes and steranes, are not present in the Haughton gypsum in sufficient quantities to have been readily detected. © 2006 Elsevier Inc. All rights reserved. Keywords: Astrobiology; Exobiology; Mars; Mineralogy
1. Introduction The presence of sulfates on the surface of Mars was indicated by a strong Mg–S correlation in the fluorescence data obtained by the Viking landers (Clark et al., 1976). Sulfates were subsequently identified as being resent in the soil at the Pathfinder landing site at a concentration of ∼10% MgSO4 (Wänke et al., 2001). Data from the Opportunity rover indicates that sulfates of probable evaporite origin are present in Eagle crater, Meridiani Planum where the sulfates constitute ∼40% by weight of the bedrock and could have a formed by direct precipitation from water or during early burial (Squyres et al., 2004). Thermal emission spectrometer data suggest that the major sulfate phases are magnesium and calcium sulfates (Christensen et al., 2004). Similar deposits occur in evaporitic environments on Earth which support life. Therefore the * Corresponding author. Fax: +44 (0) 1224 272785.
E-mail address: [email protected] (S.A. Bowden). 0019-1035/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.icarus.2006.10.013
presence of evaporitic sulfate deposits could indicate that the surface of Mars was also potentially habitable to similar organisms during the periods of sulfate deposition (McKay, 1996). Laboratory experiments suggest that in the highly oxidising conditions of the martian regolith, small organic compounds would not survive for long periods of time (although more recalcitrant larger molecular weight humic acids similar to kerogen might be more resistant to oxidation), and the absence of this pool of organic matter could explain the negative results obtained by the Py–GC–MS (pyrolysis–gas chromatography– mass spectrometry) instruments deployed on the Viking Landers (Stoker and Bullock, 1997; McDonald et al., 1998). Furthermore, even if compounds are not completely destroyed, oxidising conditions may convert large organic molecules to smaller organic molecules that would form organic salts that would not be amenable to detection by Py–GC–MS (Benner et al., 2000). Nevertheless, it has been proposed that even in the highly oxidising conditions of the martian surface, there is potential for a small quantity of organic molecules to be preserved within mineral phases as intracrystalline inclusions (Parnell et
Intracrystalline lipids—Survival of chemical fossils on Mars
al., 2002). Although the oxidation of organic matter by sulfate mineral phases may be thermodynamically possible, the preservation of organic matter within sulfate minerals over geological periods of time is kinetically reasonable at low temperatures and low values of pH (Sumner, 2004). This is because the abiotic reduction of sulfate (SO2− 4 ) to hydrogen sulfide H2 S is slow at temperatures less than 200 ◦ C (Ohmoto and Lasaga, 1982). However, other oxidised sulphur species are formed during the oxidation of sulfate to sulfide, so it is possible that these compounds have oxidising potential (even if weak) that may break down organic matter over geological time scales. Therefore there is some uncertainty over the stability of organic matter in sulfates over geological time. Within the types of samples that have been analysed on Earth, intracrystalline, organic compounds within fluid inclusions have been found to be highly resistant to chemical degradation and oxidation. This is best illustrated by the methods some workers have used to remove adhering organic matter from samples of carbonate and quartz sandstone prior to petroleum-bearing fluid inclusions being extracted and analysed: After extraction samples have been treated with hydrogen peroxide (George et al., 1997) to break down noninclusion organic compounds in a method similar to that used (McDonald et al., 1998) to simulate the oxidative effects of the martian regolith. Intracrystalline biomarkers included within the mineral phases as fluid inclusions are not oxidised by hydrogen peroxide as they are effectively contained within a sealed environment, and they can be extracted and analysed after further crushing. In theory, sulfates should also be capable of preserving lipid and fossil-lipid biomarkers as intracrystalline inclusions in the same way that oil is preserved as fluid inclusions in sandstones (George et al., 1997) and lipids can be incorporated within carbonate (Xie et al., 1998; Stern et al., 2001; Ingalls et al., 2003). Lipids within these minerals may be autochthonous, i.e. indigenous to the environment of deposition and therefore constitute a primary biomarker record or they may constitute a secondary signature derived from biological activity outside of the environment of deposition (i.e. allochthonous). However, uncertainty remains about the quantity of organic compounds and fossil lipids that could be preserved within sulfate minerals as intracrystalline as opposed to nonintracrystalline material. On the surface of Mars, where it is likely that non-intracrystalline organic compounds are destroyed in the highly oxidising conditions of the martian surface or converted to non-GC amenable organic salts (Benner et al., 2000), the size of a potential pool of intracrystalline organic matter is very important. Biological productivity will control the quantities of primary organic material that is initially preserved, but later geological processes and the nature of the host mineral substrate and its ability to sequester biomolecules or their biomarkers will control the quantities of allochthonous and secondary organic material that may survive over long periods of time as intracrystalline inclusions. To assess the viability of analysis of intracrystalline lipid biomarkers within mineral phases we have analysed gypsum-
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rich materials that are present in the Recent/Quaternary aged (<1.6 Ma) soil and Eocene aged (impact dated to 39 Ma) impact-related, hydrothermal deposits in the Haughton Impact Structure, Devon Island, Canadian High Arctic. Devon Island is a pertinent sampling locality because of its dry, cold arctic environment (75◦ , 22 N and 89◦ , 41 W ), the oxidising conditions present in surface soils and associated low levels of biological productivity (Cockell et al., 2001). We have quantified the lipids that were amenable to recovery by simple solvent extraction as well as the intracrystalline lipids that can only be recovered by the complete dissolution of the mineral matrix (the intracrystalline lipids that would be expected to be resistant to oxidation and destruction). 2. Sample collection and setting 2.1. Geology The Haughton Impact Structure was formed about 39 Ma (Sherlock et al., 2005) in a ∼1750-m-thick series of Lower Paleozoic sedimentary rocks dominated by a sulfate-bearing carbonate facies overlying a Precambrian crystalline basement. The crater is filled with carbonate-rich impact melt rocks (Osinski and Spray, 2001), which contain clasts of basement, indicating an excavation depth of 2 + km. Limited Tertiary lacustrine sediments lie upon the melt rocks. The rocks within the central part of the crater include hydrothermal mineral veins of quartz and calcite (Osinski et al., 2001). Country rocks around the crater are predominantly brown dolomites with a sucrosic, porous texture, and are generally unveined. The surface deposits analysed for this study comprise a crystal of gypsum formed during a late-stage of the impact-related hydrothermal system (Osinski et al., 2001), a sample of hydrothermal gypsum colonised by modern cyanobacteria (Parnell et al., 2004) and a pale gray coarse-grained alluvial soil consisting of fragments of dolomite, calcite, gypsum and crystalline basement (fragments of basement are mostly banded gneiss rich in quartz). 2.2. Ecology The alluvial terraces of Devon Island, Arctic Canada have a vegetation cover ranging from 2 to 11%, although micro-oases exist that have vegetation coverage between 2 and 98% and bacterial counts that are an order of magnitude greater than outside of the micro-oases (Cockell et al., 2001). Endolithic colonisation by Chroococcidiopsis, Aphanothece, unicellular chlorophytes and Gloeocapsa sp. of both impact-shocked and unshocked rocks occurs across the island (Cockell et al., 2002). One unique endolithic habitat exploited by cyanobacteria within the impact crater is outcrops of high purity gypsum formed during the late stages of impact-induced lowtemperature hydrothermal activity (Parnell et al., 2004). 3. Materials and methods Two samples of hydrothermal gypsum (CaSO4 ·2H2 O), one colonised by Gloeocapsa and another non-colonised sample
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were cleaned prior to analysis. Cleaning comprised brief immersion in 20% HCl (hydrochloric acid), immersion in MeOH (methanol) followed by immersion in DCM (dichloromethane). The samples were then rinsed with MeOH and the cleaning process repeated at the end of which the samples were rinsed with DCM and allowed to dry. Samples of gypsum were crushed in a pestle and mortar and dissolved in 20% HCl prior to extraction. Liquid–liquid extraction of the dissolved gypsum samples was performed in calcined-glass vials with excess HCl added to lower pH and inhibit the formation of carboxylic salts to enable the partitioning of lipids to an organic extracting solvent phase. A five-stage liquid–liquid extraction was then performed on the dissolved samples using 4:1 v/v DCM:MeOH and the extracts combined. Powder X-ray diffraction confirmed the presence of dolomite, calcite, gypsum and quartz mineral phases in the soil sample and microscopic analysis identified secondary sulfate and calcite mineral phase overgrowths. The powdered soil sample was soxhlet extracted for 48 h with 93:7 v/v DCM:MeOH and the extract recovered. Two further soxhlet extractions were performed each lasting 24 h and using 93:7 v/v DCM:MeOH as the extracting solvent. The final extract had no visible colour and the extracted soil was treated with hydrogen peroxide to oxidise any residual organic material prior to being split. One sample split was treated with 20% HCl to dissolve the most soluble evaporite mineral phases (calcite and gypsum); the other split was not treated with 20% HCl but used as a control for later stages. Powder X-ray diffraction revealed that the dominant calcite and gypsum peaks were reduced by 50 and 40% subsequent to the treatment with HCl. Following treatment a sample split treated with HCl and its control sample (the control sample was not treated with HCl) were extracted using a five stage sonic extraction. The starting composition of the extracting solvent was 4:1 v/v DCM:MeOH but later extraction stages contained progressively less MeOH so that the final sonic extraction was performed with DCM. Sonication was used to agitate the samples for brief periods of time (not exceeding 15min duration). All of the products recovered by extraction were separated by silica gel adsorption chromatography using a short Pasteur pipette packed with coarse-grained silica into hydrocarbon and polar fractions by elution with 3:1 v/v hexane:DCM and 2:1 v/v DCM:MeOH, respectively. The polar fraction was silyated by heating in the presence of excess BSTFA (N ,Obis(trimethylsilyl)trifluoroacetamide) to convert alkanols into their silyated ethers and alkenoic acids into their silyated esters. GC–MS analysis was performed on a HP (Hewlett Packard) 5890 series 2 GC fitted with an SGE BP-X5 column (fused silica; 0.5-µm film thickness; 30 m × 0.32 mm ID) and connected to a HP 5970 mass detector (ionisation energy 70 eV; SIM mode). The oven-heating program was 50 to 175 ◦ C at 4 ◦ C/min and then from 175 to 300 ◦ C at 4 ◦ C/min finally holding at 290 ◦ C for 20 min. Both full scan and SIM data were collected, and compound identification was based on elution order and examination of the spectra for diagnostic ions. Quantification was performed relative to an internal standard
Table 1 Yields of n-alkanoic acid and n-alkanol lipids Extract compositiona Hydrocarbons (%)
Polar (%)
Polar lipidsb µg/g sulfate
Colonised gypsum
<1
>99
14
Non-colonised gypsum
<5
>95
7
Soil Non-intracrystalline
17
83
2c
Intracrystalline Calcite Sulfate Control
<0.1 – – –
>99.9 – – –
– 9d 6a 0.09
a b c d
Extract composition based on column chromatography. Yields of polar lipids based on quantification by GC–MS. Soxhlet extracts combined from three stages. Yields of lipids were proportioned to the calcite and sulfate mineral phases according to the changes in dominant (XRD) 2θ -peak heights observed with acid treatment and assuming an equal proportion of organic mineral within each mineral phase.
(silyated ester of nonadecanoic acid) added prior to GC–MS analysis. 4. Results The yields obtained for the different samples are shown in Table 1. 4.1. Polar lipids The soil sample and the sample of gypsum colonised by cyanobacteria contain n-alkanoic and n-alkenoic fatty acids that range in carbon number from C14 to C26 . In these samples the most prominent straight chain fatty acids are the mono- and unsaturated-C16 and C18 homologues and the most prominent methyl branched iso- and anteiso-alkenoic acids are the C15 and C17 homologues (Fig. 1). A sample of the hydrothermal gypsum with no visible colonisation was also analysed and yielded C16 and C14 n-alkanoic acids and monosaturated C16 n-alkanoic acids. The most abundant compound was C17 n-alkanol. 4.2. Hydrocarbon products Hydrocarbons did not constitute a significant proportion of the extracts of the samples of gypsum or intracrystalline organic matter (less than 5% of extract weight). n-Alkanes were present in all extracts (Fig. 2). The n-alkanes do not show a strong oddover-even (or even-over-odd) predominance. The biosynthesised hopane diploptene (C30 17β,21α(H)hop-22(29)-ene) and several of the geohopanoids (predominantly C29 and C30 hopanes with the 17α,21β(H) and 17β, 21α(H) configurations, products of the diagenetic conversion of biohopanoids) were detected in the hydrocarbon fractions of the colonised gypsum and soil sample (Fig. 3). Diplopterol and other C35 biohopanoids were not detected within the polar fractions.
Intracrystalline lipids—Survival of chemical fossils on Mars
Fig. 1. GC–MS traces of the summed m/z 75 + 117 ion chromatograms of the polar fractions derivatized with BSTFA. Silyated derivatives of: 14:0 = tetradecanoic acid; i15:0 = 13-methyltetradecanoic acid; a15:0 = 12-methyltetradecanoic acid; 15:1 = pentadecenoic acid (location of double bond unspecified); 15:0 = pentadecanoic acid; 15OH = pentadecanol; i16:0 = 14-methylpentadecanoic acid; a16:0 = 13-methylpentadecanoic acid; 16:1 = hexadecenoic acid (location of double bond unspecified); 16:0 = hexadecanoic acid; i17:0 = 15-methylhexadecanoic acid; a17:0 = 14-methylhexadecanoic acid; 17:1 = heptadecenoic acid (location of double bond unspecified); 17:0 = heptadecanoic acid; 17OH = heptadecanol; 18:1 = octadecenoic acid (location of double bond unspecified); 18:0 = octadecanoic acid; ∗ = plasticizer contamination thought to originate during sample storage.
4.3. Intracrystalline sulfate-bound lipids Following treatment with hydrogen peroxide, the intracrystalline lipids within the sulfate and calcite mineral phases were recovered by treating the sample with 20% HCl. Treatment with acid is most likely to have preferentially dissolved secondary mineral overgrowths that formed around cores of bedrock clasts. The lipids extracted from the acid-treated sample contain low proportions of the C15 and C17 n-alkanols. The C15 and C17 iso- and anteiso-fatty acids are also relatively more abundant in the lipids recovered by dissolution of the calcite and sulfate mineral phases than the geolipids recovered from the bulk soil extract (Fig. 1). The hydrocarbon fraction of intracrystalline lipids also bears a greater similarity to the hydrocarbon
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Fig. 2. GC–MS trace of the m/z 85 ion of the hydrocarbon fractions. nC17 = heptadecane, etc.; br = methyl branched alkanes and alkyl-cyclohexanes. Note that the early part of the chromatogram pertaining to heptadecane has also been included as insert with an exaggerated vertical scale.
fraction extracted from the hydrothermal gypsum than from the soil; e.g. the peak heights for the C29 αβ and C30 αβ hopanes are equal and C27 Tm (C27 17α(H)-22,29,30 trisnorhopane) hopane is present (Fig. 3). 5. Discussion 5.1. Polar lipids The C16 and C18 fatty acids extracted from the cyanobacteria-colonised gypsum and sample of soil correspond to the main phospholipid-constituents of the cell membranes of both prokaryotes and eukaryotes. However, an origin from a microbial source is most likely for lipids extracted from the sample of soil as well as for the sample of colonised gypsum as, except for micro-oases that exhibit species- and spatially-limited vegetation cover (Cockell et al., 2001), higher plants are not significant in the arctic polar desert of Devon Island. Endolithic colonisation of shocked gneiss by Chroococcidiopsis spp. has been reported by Cockell et al. (2002), who also observed communities of Chroococcidiopsis, Aphanothece and Gloeocapsa (cyanobacteria) as well as chlorophytes (green algae) on the undersides and other surfaces of many rocks, whereas Gloeocapsa and Nostoc commune (also cyanobacteria) are the domi-
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Fig. 3. GC–MS trace of the m/z 191 ion of the hydrocarbon fractions. Tm = C27 17α(H)-22,29,30 trisnorhopane; C29 αβ = C29 17α, 21β(H) hopane; C29 βα = C29 17β, 21α(H) hopane; C31 αβ S = C31 17α, 21β(H) (22S) hopane; C31 αβ R = C31 17α, 21β(H) (22R) hopane; Dip = diploptene (C30 17β,21β(H)-hop-22(29)-ene); ∗ = unidentified contaminant.
nant chasmoendoliths present within the microbe bearing gypsum (Parnell et al., 2004). Fungi (eukaryotes) have also been cultured from alluivial material collected from Devon Island (Cockell et al., 2001). The lipid fingerprints of the extracts of the colonisedgypsum and the intracrystalline lipids extracted from the soil are characteristic of predominantly prokaryote sources. This can be inferred from the significant proportions of branched C15 and C17 methyl branched fatty acids which are generally present in higher proportions in bacteria (Volkman et al., 1980; Harwood and Russell, 1984), and the absence of polyunsaturated fatty acids [polyunsaturated fatty acids (C18 and greater) are generally present in chlorophytes but not abundant in prokaryotes (Volkman et al., 1980, 1998)]. The saturated C16 and C18 n-alkanoic acids are also commonly held to be present in all fungi and are also present in many heterotrophic bacteria (Harwood and Russell, 1984), so it is possible that some of the non-intracrystalline fatty acids present in the soil may have a fungal origin or derive from bac-
teria that are not phototrophic (e.g. other than cyanobacteria). The distribution of the lipids extracted from the sample of non-colonised hydrothermal gypsum is quite different to that seen in the other samples. The mechanism by which the modern cyanobacteria colonise the gypsum deposits is relatively well constrained and is dependant on the opening of mineral cleavage planes open during weathering. Gloeocapsa, a cyanobacterium prevalent in the sample area, penetrates along opened cleavage planes and seems most capable to exploit this unusual ecological niche (Parnell et al., 2004). As the non-colonised sample of gypsum analysed during this study contained no visible colonisation, and appeared not to have been weathered, it is unlikely that the sample has been colonised by cyanobacteria during recent times. Therefore, the lipids extracted from the gypsum are not likely to derive from modern chasmoendolithic bacteria and it is reasonable to explore the possibility that the origin of any lipids is related to the impact-induced hydrothermal system that precipitated the gypsum (Osinski et al., 2001). The gypsum is interpreted to have precipitated from a low temperature hydrothermal system that persisted for some time after the event that created the Haughton Impact Structure (Osinski et al., 2001). A similar distribution of lipids (to those shown in Fig. 1) with prominent C17 n-alkanol and low proportions of C18 fatty acids (order of prominence C17 n-alkanol > C16 n-alkanoic acid) has been reported in microbial mats dominated by Chloroflexus in low-temperature hot springs (<65 ◦ C) in Yellowstone Park (van der Meer et al., 2000, 2003). In other instances the distribution of alkanoic acids relative to alkanols in microbial mats living in hydrothermal springs, in which Chloroflexus and other non-cyanobacteria photosynthetic bacteria are prominent, is not always dominated by C17 n-alkanol, although one particular alcohol dominates in most instances, e.g. C18 n-alkanol, C17 n-alkanol or phytol (Shiea et al., 1991). Methyl branched fatty acids are also less common constituents of the cultures of the green and purple bacteria found in these hydrothermal spring communities (Shiea et al., 1991). Thus the distribution of the lipids extracted from the non-colonised gypsum is consistent with, although not necessarily conclusive proof for, an origin from a microbial community dominated by non-cyanobacterial photosynthetic prokaryotes that existed in an ancient hydrothermal system. As microbial mat structures are not present within the transparent gypsum, it is likely that the lipids are allochthonous/secondary and were transported by hydrothermal fluids and incorporated within the gypsum as it precipitated. The incorporation of stearic acid within magnesium sulfate as intracrystalline lipids has been replicated within the laboratory (Bowden et al., 2005), so this is certainly possible. 5.2. Hydrocarbon products None of the sample extracts contain an n-alkane distribution with a strong odd over even predominance even though odd carbon numbers (greater than C25 ) generally dominate the n-alkanes extracted from algae and plants. The lack of an odd over even preference in n-alkanes, while unusual in extracts of
Intracrystalline lipids—Survival of chemical fossils on Mars
living organisms, has been observed in the extracts of cultures of Chlorobium sp. and the purple bacteria Chromatium tepidum (Shiea et al., 1991). However, fossil fuels that have been heated during burial also typically have n-alkanes with a distribution in which odd carbon numbers do not significantly predominant over even carbon numbers. As the dolomitic bedrock within the sample locality often contains small quantities of fossil fuel, which also have no odd over even predominance (Parnell et al., 2004), an origin from this source can not be ruled out, particularly for the non-intracrystalline fraction of the soil extract. Given the pervasive colonisation of the sample locality by cyanobacteria, diploptene is most likely to have originated from a modern cyanobacterial (rather than a plant) source. However, the most prominent hopane in the non-colonised gypsum was the geohopanoid C30 17α,21β(H) hopane, a compound that results from the diagenetic conversion of biologically produced hopanoids (Seifert and Moldowan, 1980). The prominence of this geohopanoid (with the 17α,21β(H) configuration) relative to any biohopanoids (with the 17β,21α(H) configuration) further indicates that many of the compounds within the sample of non-colonised gypsum may pertain to syn-hydrothermal ecological communities, although the dominance of this single hopane is unusual. 6. Yields and quantitative considerations with respect to the Viking missions The lipid yields of 7 ppm for the sample of non-colonised gypsum and 6 ppm for the secondary mineral phase for the intracrystalline lipids released by acidification of the soil sample are of similar orders of magnitude to the quantities of intracrystalline lipids detected in carbonate sediment (Ingalls et al., 2003, 2004). Orders of magnitude more lipid material have been detected in stalactites in caves (Xie et al., 1998) and in secondary aragonite present within cavities in Zechstein carbonates (Peckmann et al., 1999). The latter two examples may have included primary contributions from microbes that lived within the samples (as was the case for the Gloeocapsa colonised gypsum). If modern microbial life is growing on a sample then there is clearly no upward limit on the concentration of autochthonous lipids that might be present. However there may be a limit for the total amount of intracrystalline fossil lipids of allochthonous/secondary origin, dependant on transport mechanisms and the ability of the host mineral to sequester lipids. There are few studies of fossil lipids within sulfates (e.g. Grice et al., 1998), and to the authors knowledge no studies of secondary intracrystalline lipids within sulfates (e.g. instances where the lipids did not originate from biological productivity within the sample or in the environment where the sulfate phase formed). A value of 7 ppm was recorded during this study, but it is probably appropriate to moderate this number by taking into account the data derived from studies of carbonates (Xie et al., 1998; Peckmann et al., 1999): therefore at least 5–10 ppm of secondary lipids may typically be preserved by incorporation as intracrystalline organic matter in calcite or sulfate mineral phases. These intracrystalline lipids could survive oxidation within the
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martian regolith and would not be expected to be destroyed (Stoker and Bullock, 1997; McDonald et al., 1998) or converted to small organic molecules that in turn form non-GC amenable organic salts (Benner et al., 2000). If the sulfates analysed in this study are analogous to the sulfates present in the martian regolith, is there a sufficient quantity of intracrystalline lipids for the Viking Py–GC–MS to have detected? A conservative estimate for the quantities of lipids that could be present would assume that only allochthonous secondary intercrystalline lipids would be present e.g. the surface of Mars does not appear to possess a surface biosphere that produces large quantities of lipids at the present time, therefore no recent biological input is likely. A concentration for intracrystalline lipids of 5 to 10 ppm for a sulfate mineral phase and a sulfate composition of 10% for the martian regolith (Toulim et al., 1977; Wänke et al., 2001) would equate to 500 to 1000 ppb of intracrystalline lipids in secondary sulfate mineral phases. Some of the samples analysed by the Viking landers were heated to 500 ◦ C which is sufficient to thermally decompose any magnesium and calcium sulfates within the regolith, thus releasing any intracrystalline lipids that may have been present. However, pyrolysis of organic matter is relatively inefficient and the conversion rate to GC-amenable volatiles was assumed to have been 10% (Biemann et al., 1977), thus further reducing any yield to at least 50 to 100 ppb. This concentration would still have been within the detection limits of the Viking Py–GC–MS (Biemann et al., 1977) for both aliphatic hydrocarbons and aromatic hydrocarbons, potential products from the pyrolysis of fatty acids. The intracrystalline n-alkanes and other hydrocarbons present in sulfates from Devon Island examined in this study are present at very low concentrations (an equivalent yield of less than 0.5 ppb for intracrystalline organic matter within the sulfates in the martian regolith) and would probably not have been readily detectable by the Py–GC–MS deployed during the Viking missions. The negative results obtained by the Py–GC–MS deployed on the Viking landers, with respect to the detection of potential biomarker nitrogen-containing compounds derived from amino acids, can be explained by the creation of non-volatile nonGC amenable pyrolysis products in an oxidising environment (Benner et al., 2000) and the venting of water and carbon dioxide by the effluent divider during the elution of volatile amines (Glavin et al., 2001). However, in addition to the technical and analytical considerations regarding the nature of the sample matrix, for intracrystalline lipids there are many other factors that may explain why the Viking landers did not detect the pyrolysis products of the intracrystalline lipids that could have been present. (i) If very low concentrations of lipids are present in the parent solution then low concentrations of intracrystalline lipids will be present in minerals precipitating from that solution. Consequently there is no lower limit to the amount of lipids that might be present, but there may be an upper limit at about the 10 ppm level for lipids in secondary mineral phases. This maybe for a number of reasons, but the incorporation of carboxylic acids within mineral phases has been
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suggested to be linked to the creation of ionic bonds between carboxylic acid groups and mineral surfaces (Fenter and Struchio, 1999). The importance of this mechanism may explain why compounds with polar groups have been observed to be incorporated in greater quantities than apolar compounds (Bowden et al., 2005). Therefore, factors limiting the bonding of organic acids to mineral surfaces (such as the surface area of particles in solution) may limit the quantities of organic acids that become incorporated as intracrystalline inclusions. As alkanoic acids are not as a significant component of the cell membranes of archaea as they are of prokaryotes and eukaryotes, a similar type of biological productivity that did not produce alkanoic acids could also reduce the amount of intracrystalline lipids that can be preserved in sulfates. (ii) The concentration of intracrystalline lipids has been shown to decrease with diagenesis, which may arise from partial destruction of the mineral matrix and the exposure of any included organic matter (Ingalls et al., 2003, 2004). Thus martian diagenesis and surface processes should reduce the concentration of intracrystalline lipids, particularly the ablation of sulfate grains during transport and reworking of the martian regolith by aeolian processes prior to the reconstituting of sulfates as a secondary cement phase (Thomas et al., 1999). Intracrystalline lipids exposed to the surface environment in this way would be expected to proceed along the diagenetic scheme proposed by Benner et al. (2000). Thus a low concentration of lipids in the parent solution from which a sulfate mineral phase precipitated and a heavy degree of reworking by surface processes would all reduce the potential yield of volatile hydrocarbons from intracrystalline lipids to a level that may not have been detectable by the Py–GC–MS carried by the Viking landers. 7. Conclusions A significant proportion of the lipids preserved within the samples of sulfate deposits from the Haughton Impact Structure are present as intracrystalline inclusions and are resistant to chemical oxidation. Some lipids derive from a recent phase of endolithic colonisation by cyanobacteria (Parnell et al., 2004); whilst some intracrystalline lipids are interpreted to be the product of an extinct hydrothermal system (Osinski and Spray, 2001), dating to a period of time soon after the impact. If the sulfate minerals that are present in the Haughton Impact Structure are used as an analogue for Mars, then the quantities of intracrystalline n-alkanoic acids and n-alkanols of secondary origin that could have potentially been present should have been detectable by the Py–GC–MS instrument deployed on the Viking Landers. However this assumes that the conversion of lipids to GC-amenable compounds was 10% efficient (Biemann et al., 1977). If conversion of organic matter to GC-amenable volatiles by pyrolysis was less than 0.1%, intracrystalline lipids might not have been detected. Intracrystalline hydrocarbons (e.g. PAH and hopanes) are not present
within the secondary sulfates at concentrations above the detection limits of the Py–GC–MS deployed on the Viking landers. Therefore both intracrystalline PAH that could derive from meteoritic input (Basile et al., 1984) and intracrystalline ancient biomarkers similar to the oldest (2.7 Ga) reported molecular biomarkers on Earth (Brocks et al., 1998) would probably have not been detected by the Viking Py–GC–MS. Acknowledgments This work was carried out under the auspices of the NASA Haughton–Mars Project. We are grateful to the Polar Continental Shelf Project (Natural Resources Canada), the Nunavut Research Institute, the Communities of Grise Fiord and Resolute Bay for their support, and to the many participants of the NASA Haughton–Mars Project. This work was supported by EPSRC Grant GR/S47267/01. References Basile, B.P., Middleditch, B.S., Oro, J., 1984. Polycyclic aromatic hydrocarbons in the Murchison meteorite. Org. Geochem. 5, 211–216. Benner, S.A., Devine, K.G., Matveeva, L.N., Powell, D.H., 2000. The missing organic molecules on Mars. Proc. Natl. Acad. Sci. USA 97, 2425–2430. Biemann, K., Oro, J., Toulmin III, P., Orgel, L.E., Nier, A.O., Anderson, D.M., Simmonds, P.G., Flory, D., Diaz, A.V., Rushneck, D.R., Biller, J.E., Lafleur, A.L., 1977. The search for organic substances and inorganic volatile compounds in the surface of Mars. J. Geophys. Res. 82, 4641–4658. Bowden, S.A., Cooper, J.M., Parnell, J., 2005. The extraction of organic compounds from sulfate minerals for astrobiological exploration. Lunar Planet. Sci. XXXVI. Abstract 1325. Brocks, J.J., Logan, G.A., Buick, R., Summons, R.E., 1998. Archean molecular fossils and the early rise of eukaryotes. Science 285, 1033–1036. Christensen, P.R., Wyatt, M.B., Glotch, T.D., Rogers, A.D., Anwar, S., Arvidson, R.E., Bandfield, J.L., Blaney, D.L., Budney, C., Calvin, W.M., Fallacaro, Fergason, R.K., Gorelick, N., Graff, T.G., Hamilton, V.E., Hayes, A.G., Johnson, J.R., Knudson, A.T., McSween Jr., H.Y., Mehall, G.L., Mehall, L.K., Moersch, J.E., Morris, R.V., Smith, M.D., Squyres, S.W., Ruff, S.W., Wolff, M.J., 2004. Mineralogy at Meridiani Planum from the Mini-TES experiment on the Opportunity Rover. Science 306, 1733–1739. Clark, B.C., Baird, A.K., Rose, H.J., Toulmin, P., Keil, K., Castro, A.J., Kelliher, W.C., Rowe, C.D., Evans, P.H., 1976. Inorganic analyses of martian surface samples at Viking landing sites. Science 194, 1283–1288. Cockell, C.S., Lee, P., Schuerger, A.C., Hidalgo, L., Jones, J.A., Stokes, M.D., 2001. Microbiology and vegetation of micro-oases and polar desert, Haughton impact crater, Devon Island, Nunavut, Canada. Arctic Antarctic Alpine Res. 33, 306–318. Cockell, C.S., Lee, P., Osinski, G., Horneck, G., Broady, P., 2002. Impactinduced microbial endolithic habitats. Meteorit. Planet. Sci. 37, 1287–1298. Fenter, P., Struchio, N.C., 1999. Structure and growth of sterae monolayers on calcite: First results of an in situ X-ray reflectivity study. Geochim. Cosmochim. Acta 63, 3145–3152. George, S.C., Krieger, F.W., Eadington, P.J., Quezada, R.A., Greenwood, P.F., Eisenberg, L.I., Hamilton, P.J., Wilson, M.A., 1997. Geochemical comparison of oil-bearing fluid inclusions and produced oil from the Toro sandstone, Papua New Guinea. Org. Geochem. 26, 155–173. Glavin, D.P., Shcubert, M., Botta, O., Kminek, G., Bada, J.L., 2001. Detecting pyrolysis products from bacteria on Mars. Earth Planet. Sci. Lett. 185, 1–5. Grice, K., Shouten, S., Nissenbaum, N., Charrach, J., Sinninghe Damsté, J.S., 1998. Unusual distribution of monomethylalkanes in Botryococcus brauniirich samples: Origin and significance. Geochim. Cosmochim. Acta 28, 349– 359. Harwood, J.L., Russell, N.J., 1984. Lipids in Plants and Microbes. Allen & Uwin, London, pp. 35–59.
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