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STABLE CARBON ISOTOPE FRACTIONATION IN THE SEARCH FOR LIFE ON EARLY MARS L. J. Rothschild and D. DesMarais NASA—Ames Research Center, MS 239—12, Moffert Field, CA 94035, U.S.A.

ABSTRACT 3C to 12C in organic relative to inorganic deposits, are useful Isotopic measurements and, more specifically, ratios of ‘ in reconstructing past biological activity on Earth. Organic matter has a lower ratio of ‘3C to ‘2C due largely to the preferential fixation of ‘SC over the heavier isotope by the major carbon-fixation enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase, although other factors (e.g., availability of source carbon, fixation by other carboxylating enzymes and diagenesis of organic material) also contribute to fractionation. Would carbon isotope discrepancies between inorganic and organic carbon indicate past biological activity on Mars? In order to answer this question, we analyse what is known about terrestrial biologic and abiologic carbon fixation and its preservation in the fossil record, and suggest what the isotope discrimination during possible biologic and abiologic carbon fixation on Mars might have been like. Primarily because isotopic signatures of abiotically fixed carbon overlap with those ofbiotic fixation, but also because heterotrophy does not significantly alter the isotopic signature of ingested carbon, fractionation alone would not be definitive evidence for life. However, a narrow range of fractionation, including no fractionation, would suggest biotic processes. Never-the-less, isotopic ratios in organic deposits on Mars would be extremely useful in analysing prebiotic, if not biotic, carbon transformations on Mars. INTRODUCTION The Viking life detection experiments of the mid 1970’s were unable to detect extant life on Mars. Yet, the possibility of extinct life persists. Unfortunately, it is not obvious how to search for traces of a (as yet) hypothetical extinct life form. What is needed is one or more chemical or morphological signatures that we can both detect and unequivocally assign to an indigenous Martian biota. One possible approach is to look for traces of biological carbon fixation by identifying differences in the stable carbon isotope compositions between organic and inorganic deposits in rocks. The two largest carbon reservoirs in the Earth’s crust, marine carbonate and organic matter in sedimentary rocks, have 6’3C values typically in the range of -2 to +2 and -20 to -35 permil (°/~), respectively. This isotopic contrast has been observed in relatively well-preserved rocks of all ages. It is interpreted to be indicative of biological carbon fixation on Earth primarily because, in the modern biosphere, the photosynthetic carbon-fixation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39) preferentially fixes ~2CO 3C0 over 2 ‘ 2 by an amount similar to that seen in the rock record /1,2/. Here we ask the question: Would carbon isotope discrepancies between inorganic and organic carbon be a definitive signature of extinct life on Mars? A positive answer depends upon three assumptions. First, if there was life on Mars, it fractionated carbon isotopes. Second, we could detect this fractionation. Third, there are no other (i.e., abiotic) explanations for the observed fractionation pattern. In this paper we will discuss all three assumptions in an attempt to estimate the utility of carbon isotope fractionation for Mars exobiology. IF LIFE AROSE ON MARS, HOW DID IT FRACTIONATE CARBON? Carbon isotope discrimination is frequently associated with biotic carboxylation reactions (the conversion of inorganic to organic carbon.) Here we assume thatwhere an early Martian biota evolved this ability to fix carbon. The rationale 3C values, *

Isotope abundances are reported as 6’ 5~3~

(~/~ ) = [~sc/s~c

~

~

1000

The standard used is carbonate, usually the Peedee belemnite (PDB) standard which has a 53C0 12C0 3C value of the PDB is defined as 0 pertoil (C ~ .) 3 to 2 ratio of 0.0112372. Thus, the 5 ‘

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behind this assumption is as follows. Martian life would have been based on carbon because on early Mars (and early Earth) carbon dioxide was abundant (e.g., /3,4,5/), and CO 2 is known to form organic compounds under conditions thought to have been present on early Mars and early Earth (e.g., /6/). Furthermore, we know that life can be based on organic carbon because all known life forms are. After life originated, organic carbon soon may have become scarce. An organism with the ability to fix its own carbon dioxide would have had a competitive advantage. Witness the success of carbon-fixing organisms on a similar planet, the Earth. Assuming that an early Martian biota could fix carbon, would this process have entailed isotope fractionation? Again we argue by analogy with the terrestrial biota by reviewing the factors that influence terrestrial biological carbon fractionation and its preservation in the fossil record in order to determine which aspects would have applied to a Martian biota. Factors that Affect Carbon Isotope Fractionation on Earth On Earth, the carbon isotope ratio of organi: carbon in autotrophs is affected by the isotopic ratio of the source carbon, the availability of CO2 and its uptake rate, and the enzymatic pathway used for fixation and physical factors that affect discrimination during fixation. The preservation of the isotopic signature depends on tissue-specific signatures and diagenesis. Isotopic ratio of source carbon. The theory behind isotopic studies is that there has been a fractionation of the source carbon. Thus, the isotopic composition of the sampled compound only makes sense relative to the isotopic composition of the source carbon. If the isotopic ratio of the source carbon for fixation is significantly different from that of the standard used for comparison, the apparent fractionation will not reflect the actual fractionation that has occurred. This problem can occur because locally different pools of inorganic carbon are possible. For example, in greenhouses and the bottoms of dense forests, CO2 may be more ‘SC depleted, and therefore leaves from these 3C values, it is imperative coeval inorganic carbon, suchspecimens as carbonates, be analysed as the standard. areas are several permilthat more negative than comparable from elsewhere /7/. Thus, in determining 5’ Availability of CO 2 . Isotope discrimination is correlated with the availability of CO3 relative to the turnover rate of the carboxylating enzyme. When CO2 is abundant, maximum fractionation is expressed, and when CO2 is limiting, discrimination is repressed /8,9/’. For example, in slow-moving or stagnant water there may be a local depletion of CO2 which leads to decreased discrimination /10,11/. In laboratory experiments with the protist Chiamydomonas reinhardtii, a decrease in the concentration of CO2 in the culture medium from 3,300 ~l 1’ CO2 to 200 pl l’ CO2 long enough to allow CO2 -concentrating mechanisms to be activated caused a decrease in the isotopic composition of the cell material from 20-29 5/a, to 4 a/s, /12/. This precipitous drop in discrimination was the result of maintaining a high fixation rate relative to the amount of available substrate. In the past, Earth may have had a partial pressure of CO2 in the range of from a few tenths of a bar /4/ to 10 bar /3/. Today the pressure of atmospheric CO2 is 0.3 mbar, yet many photosynthetic organisms still express a strong biological isotope discrimination. Like Earth, Mars is thought to have had a high pressure of CO2 in the past (—.0.75-5 bar /5/) to account for the presence of liquid water, even with a lower solar luminosity. Even today the pressure of Martian atmospheric CO2 is high (-.6 mbar) relative to today’s Earth. This suggests that during Mars history there has been plenty of CO2 available to express discrimination with terrestrial biological fixation systems. However, CO2 could still have become limiting if the saturation values for the carboxylating enzymes were proportionally higher or if the CO2 was inhibited from reaching the enzyme as can happen in modern microbial mats. There is no way to know this, but given that (1) there is currently isotopic fractionation associated with terrestrial biological carbon fixation, (2) carboxylating systems that might have evolved on Mars were similar to terrestrial systems, and (3) the present terrestrial atmospheric pressure of CO2 is 0.3 mbar and that the equivalent figure for early Mars was —0.75-5 bar, that is, 2500-16,667 times higher, then the carboxylation rate of an early 3C Martian carboxylating enzyme would have had to have been over 2500-16,667 times higher than current terrestrial rates in to order for CO2 to have relative inorganic carbon, thislimited might carboxylation. suggest that COIn any case, if Martian organic carbon was depleted in ‘ 2 was not limiting. Conversely, if no fractionation is found on Mars, it is possible that there was biologic carbon fixation, but that CO2 was limiting so isotopic discrimination did not occur. Enzymatic pathway and physical factors that affect carboxylation. The greatest affect on carbon isotope fractionation is associated with the enzymatic pathway used in carbon fixation. Part of this fractionation is due to the intrinsic fractionation properties of the carbon-fixation enzyme(s) involved. In the majority of biological carbon-fixation on Earth, the carboxylating enzyme is RuBPCase. The isotopic discrimination of RuBPCase is between -20 to -40 5/a, /13,14/. Many organisms, including the C3 plants, algae, cyanobacteria and purple photosynthetic bacteria use RuBPCase as their only or principal carboxylating enzyme for primary production. In the C4 plants and anaerobic and some 2C is used preferentially, the source carbon becomes enriched in ‘3C. If *

the goes an to completion, of the Thisreaction is assuming open system.theInisotopic a closedsignature system, as the ‘fixed carbon wiU be the same as that of the source carbon.

Carbon Isotope Fractionation

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facultatively anaerobic bacteria, inorganic carbon is fixed first by the enzyme phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31.) The fixed carbon is released intracellularly where it is re-fixed by RuBPCase. The fractionation associated with PEPCase is only -2 ‘/~, /8/, possibly because bicarbonate rather than CO 2 is fixed Csee discussion under pH.) Owing to other factors, such as diffusion, the overall fractionation associated with C3 plants and C4 plants is —-28 and —-14, respectively /8/. The CAM plants and possibly some protists such as the diatom Cyiindrotheca /15/ use PEPCase under some environmental conditions. Consequently, fractionation patterns vary depending on whether PEPCase is utilized or not. For example, during the day CAM plants mimic C3 plants, and during dark fixation they mimic C4 plants by using PEPCase. Other biological carbon-fixation reactions are known. These include those used in primary production, such as the reductive carboxylic acid cycle used by some photosynthetic bacteria, and carbon-fixation steps found in intermediary metabolism. Carboxylating enzymes associated with these pathways include acetyl CoA carboxylase (EC 6.4.1.2) and carbamoylphosphate synthetase (EC 6.3.4.16), an enzyme essential in pyrimidine cases, 3C = -6 to biosynthesis. -40 (see /1/). In In some the case of isotope the enzymes fractionation used in intermediary values for these metabolism, enzymes fractionation are known, and hasrange not been fromstudied. S’ While the carbon-fixation reaction steps found in intermediary metabolism do nor, always lead to the net synthesis of organic carbon, their fractionation properties would be of interest because they provide data on the range of fractionation values associated with the biological carboxylation. There are environmental factors which may alter the isotopic discrimination displayed by a particular pathway (rev. in /8,16/.) Most of these effects are small relative to overall discrimination. For example, light levels affect the photosynthetic pathway used by CAM plants, and thus overall fractionation (see above.) In addition, C3 plant fractionation may vary as much as 7 °/~in response to light intensities /8/, but as light intensities vary, there may be no net effect in isotopic composition. In plankton, decreased fractionation has been correlated with an increase in temperature /16,8/. This may be the result of differences in CO 2 availability at different temperatures or different fixation pathways 3C (rev, in /17/. of 02inhas been inshown to affect fractionation in C3 Likewise, plants where a 0.25 results from The a 1%level increase oxygen the range of 4-21 % oxygen /16/. in timothy ~grassdecrease a 1-2 °/~~ in difference 5’ in isotope content was caused by changes in nutritional status /8/. Salinity affects isotope discrimination in some organisms but not in others /8/. For example, in the halophytic plants Salicornia europaea and Puccin cilia nuttalliana, higher salt concentrations causes a decrease in isotope discrimination /18/, while in other organisms it does not. DesMarais et al. /19/ have shown that the S’3C values of a microbial mat that lives in a range of salinities decrease slightly with increasing salinities. Perhaps this is because at higher salinities there is a lower photosynthetic rate, so the available dissolved inorganic carbon increases relative to the photosynthetic demand /19/. In Nicotiana tabacum, increasing the amount of deuterium increases S’3C values by 6 °/,~when going from —0 to 60% deuterium (a 4000-fold increase), and similar responses have been found with two Chloreila species /20,8/. The interpretation is that this was a result ofmany changes in the cell. It is possible that the D:H ratio on Marscould have increased four-fold over time as the lighter isotope was lost preferentially /21,22/. Thus, the small changes in carbon isotope fractionation associated with increasing concentrations of deuterium suggest that the loss of deuterium on Mars would not have significantly altered 5’3C values. A more important environmental impacter on fractionation may be pH. This parameter affects the species ofinorganic carbon available for fixation. Some organisms use CO 3C value of2+1 but°/~s taketoup-2HCO’ °/~~ and andthe dehydrate 5’3C ofitCO with the enzyme carbonic anhydrase. As seawater HCO~has a 5’ 3C value2 ofis the 7 o/~~ organic to -10 carbon ~ /7/, a difference in carbon species algae, assimilated wouldgrown be expected to affect the S’ produced. In studies of unicellular the species at low pH, and thus having CO 3CO 2 passing throught the cell membrane, was 7 0/~~ more depleted in ‘ 2 than in algae where HCO~is taken up /9/. While this study was small, it suggests that the species of inorganic carbon transported across the cell membrane does affect fractionation. Some marine algae may use HCO’. If an enzyme uses HC0~,as does PEPCase, a small discrimination may result because (a) HCO~in equilibrium with CO2 is several permil heavier under ambient temperatures, and (b) HCO~ has greater mass than CO2 so the kinetic isotope effect is smaller3C /16,7/. /1/. Unfortunately, Consistent withit the is difficult latter possibility to assess fully are SD values the importance that are of pH approximately on 513C values five without times more additional negativedata. thanSeveral 5’ of the carboxylating enzymes of intermediary metabolism (e.g., pyruvate carboxylase and acetyl CoA carboxylase) use HCO’ as a substrate. It would be very interesting to measure S’3C values from these and other carboxylases in vitro to determine whether hydration of CO 2 has a major effect on 3Cisotope values produced discrimination. by purified It should RuBPCase be noted /14/. in passing that pH variations from 7 to 9 do not affect significantly S’ In summary, there are several major factors that affect isotope fractionation. These are the availability of source carbon, the enzyme system is used in carboxylation, and possibly whether the carboxylating enzyme uses CO 2 or HCO~as a substrate. Preservation of the isotopic signature. After biological carbon fixation, the organic carbon may be remineralized and released during respiration. The few studies that have been done on this suggest that little isotope fractionation

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is associated with this step /8,23/. Alternatively, after fixation the carbon may be present in a variety of different compounds with different isotopic compositiols /24,25/. The form of fixed carbon may have a major impact on the 3C relative to polysaccharides eventual isotopic signature of a deposit. For example, lignin is relatively depleted in ‘ such as hemicellulose and cellulose, yet the latter are decomposed preferentially during the early stages of diagenesis. This means that for plants with lignin, the isotopic signature found in the fossil record might show a 5’3C value lower that that of the original plant (e.g., 4 °/~,lower for Spartina detritus /26/.) This sort of problem is also relevant for microbes because lipids in, for example, Escherichia coli can be 2.7±0.90/00 depleted in ‘3C relative to source glucose /23/. There may or may not be shifts in the isotopic composition of organic matter during diagenesis. Low temperature (100-150°C)diagenetic reactions such as racemization do not cause a significant shift in the stable isotopic composition of amino acids /27/. However, there may be a 2-3 0/,~ enrichment of ‘3C in mature kerogens’, mostly because volatiles, which are enriched in 12C, are preferentially removed /28/. Isotopic shifts occur because there is preferential breakage of the ‘2C-’2C bond under heat and because isotopically light gases and hydrocarbons which are formed are preferentially lost /28/. In summary, the range of 5’3C values found in sedimentary carbon reflects the range of values found in the extant organisms discussed above. In general, the 5’3C values range from -10 to -40, with a distinct narrowing of the range to -25±5during the last 600 my /1/. At high temperatures there can be a reequilibration of isotopes between coexisting sedimentary carbonate and organic carbon, starting with low-grade metamorphism (300-450°C)and increasing with metamorphic temperature. Equilibration can be approached in the high-temperature range ( 650°C/16/. This sort of process can shift S’3C values from -25 0/,, to -10 0/,~ or less. Schidlowskj /2/ invokes high temperature equilibration to explain why Isua metasediments (-.3.8 x l0~yr) have lower S’3C values than the rest of the carbonates (3.5 Gyr to recent) and why these organic deposits show less negative 5’3C values than subsequent deposits. Thus, there can be a significant alteration in the isotopic composition of organic carbon during metamorphosis. On Earth, metamorphic activity is estimated by determining the ratio of hydrogen to carbon in a sample because hydrogen tends to be stripped from organic carbon during metamorphosis /29/. Anyway, this may not be a problem on Mars because there has been much less reworking of the Martian lithosphere /30/. Because carbonates should be used as a standard to determine isotope discrimination in organic sediments, it is important to know whether coeval carbonates reflect the isotopic ratio of the source carbon at the time of carbon fixation. Fortunately, the isotopic composition of carbonate rocks is the same as that of the parent muds ±10/,, /1/. A small isotopic shift of +1 °/~,to +3 0/00 is associated with equilibrium fractionation between HCO~and C0~. However, diagenetic carbonates (carbonates formed from CO 2 released during burial diagenesis) will show isotopic ratios reflecting source organic carbon. The following carbon isotopic signatures relative to PDB have been associated with these diagenetic events: sulfate reduction and thermally-induced decarboxylation 2C (-15 —.~75 too/~, -30 ), 0/,, the simultaneously /16/. Because released the CH4 CO produced by methanogens during fermentation is isotopically light (5’ 2 must therefore be somewhat heavy, perhaps +15 °/,, /31/. COULD WE DETECT FRACTIONATION? To determine isotopic discrepancies, inorganic and organic carbon deposits must be found. So far, neither has been found on Mars. It is possible that there is a carbonate signature in the Mariner 9 IRIS data /32/. Kahn also argues that the formation of carbonate rock deposits is the most reasonable reservoir for unaccounted-for CO2 that must have been outgassed from Mars ifMars had an early wet, warm period. More recently, McKay and Nedell /33/ have suggested that there was no carbonate signal from the Mariner 6/7 infrared spectrometer data because the spectral footprints obtained do not lie over layered deposits which are possibly aquatic sediments. As these deposits are the most likely locations for carbonate deposits, the lack of a carbonate signal is inconclusive. In addition, a thin eolian mantle could cover underlying carbonates, thus preventing their detection /33/. The success of this approach also rests on locating organic deposits on Mars. The Viking data do not indicate the presence of organic carbon in the top 10 cm of soil /34/. It is possible that there are organic deposits in locations not yet sampled, such as in ancient stream beds or other formerly aquatic areas. Alternatively, there could be organic carbon buried deeper than Viking sampled. It is also possible, however, that all the organic carbon that has been on Mars has been degraded by various mechanisms (e.g., /35,36,37/). ARE THERE OTHER POSSIBLE EXPLANATIONS FOR FRACTIONATION? Clearly the discovery of carbon isotope discrepancies on Mars would provide definitive evidence for the existence of life only if there are no other reasonable explanations for the data. In addition to biological synthesis of organic carbon, abiotic synthesis can occur under conditions simulating the Martian atmosphere /38/ and must have occurred prior to the evolution of life on Earth. Would deposits that resulted from abiotically-synthesized organic carbon also be isotopically light? *

Kerogens are the highly polymerized, acid-insoluble result of diagenesis.


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If an organic synthesis occurs in either an open system or one in which the formation of product is low relative to 2C species reacts just 2% faster the 13C~substrate, the product will have a c5’3C the substrate available, and if the ‘ value of-20 °/°°relative to the source carbon. In a series of experiments, Chat,g et al. /6/ demonstrated that such an isotope fractionation, which would be in the range of a biological synthesis, can occur. For example, early in a sparking reaction with CH 3C values of the products ranged from -10 to -25 0/0, relative to the starting methane. The 4 as products the substrate, produced the S’included acetylene, as well as other unsaturated and saturated hydrocarbons, with the more negative 5’3C values correlated with the longer chain length products. Similarly, sparking experiments with different combinations of CH 4, N2, NH3 and H2O as reactants showed discriminations 3C was in amino in the range acids, followed of -10bytoacid-insoluble -15 0/,,~ . In material, experiments lightwith hydrocarbons CH4, NH3 and andcarbon H2O, the oxides. greatest The explanation depletion in proposed ‘ was that more complex molecules require more steps in synthesis, and each step entails a fractionation. Thus, more complex molecules should have more negative S’3C values. Most importantly for this discussion, Chang et al. /6/ showed that in spark experiments using CO 2 for3Ca values carbonof source at least and-20 with °/,° anrelative excess to of H2, the starting (1) organic material. carbonFor is prodeced, example, and (2) this organic can have hydrocarbons S’ acetylene, one of the carbon most abundant produced, was depleted —.23 0/,0 in ‘3C relative to the starting inorganic carbon. In the few experiments done on post-fixational changes in the isotopic signature of these abiotically-produced organics, little change in signature has been found. When different products from HCN polymerization experiments and the electric discharge experiments were heated, the 5’3C values were within 1 0/,, of initial value regardless of time or temperature during heating /6/. Thus, isotopic fractionation that occurred during synthesis of these compounds was preserved. Because of the results of the sparking experiments, as well as similar experiments using ultraviolet photolysis as an energy source, it is clear that isotopic bias per se cannot distinguish between bioticallyand abiotically-synthesized organic carbon. There is another possible scenario to explain the presence of isotopic discrepancies. Many studies have shown that the overall stable carbon isotope composition of animals (i.e., heterotrophs) closely reflects the isotopic composition ofdietary carbon (rev, in /39/). Thus, if life arose on Mars but was heterotrophic, or at least the preserved isotopic signal was derived from heterotrophs that consumed abiotically-fixed carbon, the isotopic signature found on Mars would be that of the dietary (i.e., abiotically-fixed) carbon. This suggests that one could not necessarily discriminate between life vs. no life on the basis of the presence or absence of an isotopic difference between organic and inorganic carbon, but, at best, between biotic and abiotic fixation (see below). An isotopic difference interpreted as ‘abiotic fixation’ would not preclude the possibility of heterotrophic life. AN ISOTOPIC SIGNATURE OF LIFE? Because both biological and nonbiological chemical reactions can be isotopically selective, the mere presence of isotopically different organic and inorganic carbon reservoirs on Mars cannot constitute unequivocal evidence of life. The carbon isotopic record in ancient Earth sediments is compelling evidence of biological activity because not only is the preserved organic carbon depleted in ‘3C relative to coeval carbonates, it is typically depleted by -25 to -40 0/,,, Had this organic matter been synthesized abiotically, it would display a much wider range of isotopic compositions because of the large variety of reaction pathways and environments associated with abiotic syntheses. In contrast, few reactions for organic synthesis from CO 2 have survived the evolutionary process. Most prominent among these is the Calvin cycle with its CO2 -fixing enzyme, RuBPCase. The characteristic -35 to -40 °/~~ depletion observed in rock organic matter matches remarkably closely the values expected for Calvin cycle enzymes which assimilated CO2 having an isotopic composition similar to that predicted from the carbonate isotope data. Furthermore, this CO2 uptake occurred under conditions where the carbon isotope selectivity of RuBPCase could be expressed to almost its maximum possible extent. Such would be possible for organisms living in aquatic environments having access to an essentially unlimited reservoir of CO3 . Thus, the carbon isotopic record in rocks helps to establish not only the presence of ancient life on Earth, but also possibly the enzymes and environments associated with the biota. We cannot assume that if life existed on Mars it would have utilized enzymes having a discrimination similar to that expressed by the Calvin cycle. But conceivably, Martian life, as ancient life on Earth, became established with only a small number of the countless possible reactions for carboxylation. Perhaps this narrow field of react~nns, either the result of a limited range of evolutionary innovations or selection, created a relatively narrow range of isotopic compositions in the preserved organic matter. If this had been so, the chemical order created by Martian life would have left an isotopic order on the organic matter that it produced, as has life on Earth. Of course, it is also possible that there was biotic carbon fixation, or abiotic carbon fixation with or without the evolution of heterotropic organisms. These latter possibilities would all have left a range of isotopic values. CONCLUSION In conclusion, although we feel that the presence of a narrow isotopic range in organic deposits on Mars probably would be indicative of biological carbon frac’ionation, and thus life, this information would have to be combined with other independent data suggesting an extinct life form to obtain a definitive answer. Conversely, while a broad

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isotopic range would suggest that no biological carbon fixation occurred, this would not provide definitive evidence to rule out the possibility that there was once life on Mars. In any case, we do recommend an analysis of the carbon isotopic ratios in any discovered organic deposits relative to coeval carbonates as a way to analyse ancient carbon fixation mechanisms on Mars. Because there has been less thermal activity and reworking of the Martian surface, any such discoveries may provide clues to ans:ient carbon-fixation mechanisms on Earth that have long since been obscured by metamorphic activity. ACKNOWLEDGEMENTS This work was performed while the senior author was supported by a National Research Council postdoctoral fellowship. REFERENCES

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