Definition of Life

Preliminary Steps
  Geological Survey
  Sample Collection

Present Life
  Spectroscopic Analysis
  Organic Analysis
  Biology Experiments

Past Life
  Thin Section
  Isotope Analysis


Experimental Design

Past Life: Isotope Analysis

Motivation for Isotope Analysis

When we look for fossils on Mars, we do not necessarily expect to find recent fossils, or fossils in unaltered rocks.  Instead, evidence of fossils may be present in heavily metamorphosed and weathered impact crater ejecta, or in rock strata strongly heated and altered by nearby magma flows.  Thus, we need a way to find signs of fossil life in rocks that have been poorly preserved.

The solution to this problem is to go beyond the traditional methods of fossil hunting, to go beyond microscopes and visible fossils and instead look for chemical evidence of life.


Isotope ratio analysis:  Chemical elements exist in multiple forms with different masses, called isotopes.  This includes the elements of life, such as carbon, nitrogen, oxygen, and sulfur.  Different isotopes are denoted by mass number, such as 12C and 13C.  While isotopes of the same element have the same chemical properties, their masses can affect how they are used in chemical reactions.  In particular, the metabolic reactions of life which process the above elements preferentially use lighter isotopes.  In the cases of carbon and sulfur, the relative amounts of the various isotopes are preserved in fossils and surrounding abiotic rocks.  These relative amounts of isotopes can be measured in the form of isotope ratios, and the ratios of interest are 12C/13C and 32S/35S.  Fossils will have higher values of 12C/13C compared to the surrounding non-biological rocks.  Sulfide minerals, created in non-volcanic settings by microorganisms, will have higher values of 32S/35S than sulfate minerals, which formed the raw material used by microbes to form sulfides.  These differences remain even after rocks have been so heavily altered so that no recognizable fossils remain, and only raw organic material remains of the fossils.  These isotope ratios can be measured by mass spectroscopy for various materials in rocks to infer the presence or absence of life.  In addition, different kinds of metabolic reactions produce different 12C/13C and 32S/35S ratios, so these isotope ratios can indicate the types of metabolic reactions used by past life.  The main problem with this method is the need to measure the ratios very accurately, and to account for any non-biological reactions that can produce similar isotope ratios.  Fortunately, these reactions are known, and so their influence can be inferred from the type of rock being sampled.

Molecular fossil analysis:  Even when fossils have been rendered completely unrecognizable due to heavy alteration of their host rocks, and those rocks have been subjected to high heat and pressure for millions of years, some complex organic material will remain.  The molecules present in this leftover tar-like material are called molecular fossils, and can indicate the source of the organic material.  Certain classes of molecules are produced by the decomposition of different kinds of living things, so the specific molecules present can indicate whether the organic material came from fossils or other processes, and even what kind of fossil was originally present.  The molecules present can be determined by simple mass spectroscopy.  The primary drawback to this method is that the kinds of complex organic molecules generated by non-biological processes are not known to a great level of detail, and so results that seem to infer life could actually be caused by non-biological processes.



Since the isotope ratios produced by biological and non-biological processes are known in greater detail than the molecular fossils generated by such processes, we will perform isotope ratio analysis to look for chemical evidence of past life.

Principles of operation

According to William Schopf (1999), a leading expert in the identification of ancient fossils, metabolism leaves isotopic signatures in four different materials: organic carbon (kerogen), carbonates, sulfides, and sulfates.  Metabolic enzymes preferentially use lighter isotopes of carbon and sulfur, leaving heavier isotopes in the non-biological environment.  The metabolic products are preserved as sulfides and kerogen, while the leftovers are preserved in carbonates and sulfates.  The isotopic situation is simple in carbonates and kerogen: the kerogen should have a lower value of 12C/13C, while the carbonates should have a higher value.  The chemistry of sulfur is more complex, since its minerals can be either feedstock for metabolism or metabolic waste, depending on the process. Schopf (1999) described the results of research on with sulfate-reducing bacteria, which would lead to higher 32S/35S in sulfate minerals than sulfides; however, sulfide-oxidizing bacteria are also possible, and would reverse the sulfide vs. sulfate trend.  Regardless, however, the two phases should show different 32S/35S ratios.  The determination of these ratios will be done in the laboratory module.  Separating the kerogen, carbonates, sulfides, and sulfates will be accomplished by combusting the kerogen to CO2 and the sulfides to SO2 at relatively low temperatures, until no kerogen or sulfides remain.  The gases produced will be analyzed by the GC/MS to determine their 12C/13C and 32S/35S ratios.  Then, part of the remaining sample, which now only contains carbon and sulfur in sulfate and carbonate minerals, is directly introduced to and ionized in the mass spectrometer, yielding 12C/13C and 32S/35S isotope ratios for the highly stable carbonates and sulfates.






Power Required

rock grinder


~50 kg

~250 W

GC/MS (see spectroscopy)




analytical balance


~2 kg

~10 W

sterile Al weighing boats (1000)


~5 kg


multi-purpose combustion chamber


~40 kg

~500 W

oxygen gas




miscellaneous lab tools


~2 kg




~50 kg


distilled water


~50 kg




  • Check the field notes to see if the sample contains carbon and/or sulfur.  This determination will be made by field use of an alpha-proton x-ray spectrometer, which is discussed in the spectroscopy section of this report..  If it contains neither, bypass this experiment, since there will be no isotopes of carbon or sulfur present to measure.
  • Use a rock saw to cut off a chunk of rock with a mass of roughly ten grams.  Use the balance to record the exact mass of the chunk obtained.
  • Place a clean , massed weighing boat under the output of the rock grinder, handling it only with tongs.
  • Handling the sample only with tongs, clean it with acetone and dry it in air with slight heat in the combustion chamber, until the GC/MS no longer detects acetone.  If it detected other organic molecules, repeat the rinse and dry process until only acetone can be detected.
  • Still handling the sample only with tongs, place it into the rock grinder.  Grind it to a fine powder, and collect it in the weighing boat.  Record the mass of the powder obtained.
  • Handling the boat only with tongs, place it on the heating unit in the combustion chamber.  Seal the chamber, and flush it with pure oxygen.
  • Heat the sample to approximately 300 °C, under a constant supply of oxygen.  Record the 12C/13C and 32S/35S ratios in the combustion gases.
  • When no additional CO2 or SO2 is generated, turn off the heater.  When the sample has cooled to room temperature, replace the oxygen with air.
  • Using tongs, remove the weighing boat from the combustion chamber.  Use a scoopula to remove a small amount of the sample and introduce it directly into the mass spectrometer’s ionization chamber.  The sample will be directly ionized by an electron beam, and the isotope ratios of the ions produced will be measured.  Record the 12C/13C and 32S/35S ratios.
  • Dispose of the remaining rock powder, and wash the rock grinder with water, followed by acetone.
  • If the results indicate different isotope ratios between the combustion phase and the direct ionization phase, store the remaining part of the sample for Earth return.  If the isotopic differences match a known pattern, earmark the sample for special interest and obtain nearby samples for confirmation.

Data Interpretation

As discussed under principles of operation, the influences of life can be seen in the differences between the 12C/13C and 32S/35S ratios in kerogen, sulfides, carbonates, and sulfates.  Living material and its waste products tend to concentrate lighter isotopes, leaving relatively more heavy isotopes in the environment.  In fossil systems, the kerogen is the remnant of the living material, and the sulfides are its waste products.  Thus, any differences between the 12C/13C and 32S/35S ratios in these materials and the ratios in the non-biological carbonates and sulfates could signify the possible presence of life.  If Martian life uses the same metabolic reactions as Earth life, then these two isotope ratios should be higher in the kerogen and sulfides.  However, other reactions are possible, so other differences in isotope ratios could also signify life.  The final analysis will depend on other factors, including detailed knowledge of the composition and formation conditions of the rock, which will be determined on Earth.


Use of this experiment

Field analysis of samples should indicate the presence of carbon and/or sulfur.  If either is present, the isotopic measurements are performed.


Time for experiment
From 1 hour to 5 or 6 hours, depending on carbon and S2- content.



Cradle of Life: the Discovery of Earth’s Earliest Fossils.  Schopf, J. William.  1999.  Princeton, NJ: Princeton U. Press.


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