We start our series of Primordial Scoops covering the Viking life detection experiments with the experiments that were designed to search for Martian photosynthesis or, in a modern interpretation, Martian autotrophy. Autotrophy is the ability of an ecosystem to get carbon for organic molecules directly from an atmosphere, such as those containing CO₂.

This is arguably the most important of the Viking life detection experiments, as it detects the chemical processes that might sit at the bottom of a food chain in a near-surface Martian ecosystem. The experiment was designed by Jerry Hubbard, Norm Horowitz, George Hobby, and their team (Hubbard et al., 1970). Because it seeks “carbon assimilation” by a “pyrolytic release” analysis, we will refer to it as the CAPyR experiment.

The Setup. A summary

• The CAPyR experiment looked for the movement of radioactive ¹⁴C-label in two ¹⁴C-labeled gases (¹⁴CO₂ and ¹⁴CO) from the gas phase into samples of scooped soil placed in a container. Radioactivity that

(i) remained in the soil after non-absorbed gases were flushed out at 120 °C, and

(ii) later released when the soil was heated at 635 °C, and then

(iii) trapped on Chromosorb-P,

was inferred to be associated with small organic fragments created by “pyrolysis” of large, non-volatile ¹⁴C-labeled organic molecules. To be labeled, they must have been made by Martian autotrophic microbes as part of their growth and metabolism. Chromosorb-P traps any organic molecules larger than methane, and not trap CO₂.

• The trapped ¹⁴C-labeled organic fragments were then oxidized to ¹⁴CO₂ on the Chromosorb-P, where they were released into a radiation detector as¹⁴CO₂. This was counted as “Peak 2”.

• When this workflow was tested on soils from Earth locales where life is sparse due to severe resource limitations (e.g. Atacama Desert soils), the experiment could detect a few hundred cell equivalents of carbon-fixing autotrophs after exposure over a few days.

• Threatening to under-estimate the soil’s carbon fixing capability, the team noted that some of the label in the bio-organics emerged at 635 °C from the soil as ¹⁴CO₂, whose oxygen atom came from components of the biomass. This ¹⁴CO₂ would pass through the Chromosorb-P and directly enter the radiation detector. Its radioactivity was counted as “Peak 1”. This radioactivity, if it came via this process, should also be added to the carbon fixing budget of the soil sample.

• As a further way to underestimate the amount of label fixed in the soil, the CAPyR team showed that the Atacama soils contained oxidants (nitrates > perchorates). At 635 °C, these oxidants burned some of the ¹⁴C-labeled fixed organics to give ¹⁴CO₂. This caused “the pyrolysis [to be] converted to a partial combustion” (Hubbard et al., 1970). The resulting ¹⁴CO₂ would also pass through the Chromosorb-P and directly enter the radiation detector, adding to the “Peak 1” counts. This radioactivity, if it came via this process, should also be added to the carbon fixing budget of the soil sample.

• However, it was apparently not recognized by the CAPyR team that Martian soils might contain enough nitrate oxidant to combust all of the fixed organics. This would give a false negative, if Peak 1 radioactivity were to be ignored as ¹⁴C-label arising via Martian autotrophy.

• As a threat of a false positive, the team found that Terran alkaline soils absorbed ¹⁴CO₂ as bicarbonates or carbonates without reduction, that is, without biology. This threat was (perhaps) mitigated by that workflow that flushed ¹⁴CO₂ absorbed inorganically at 120 °C. However, if this abiologically absorbed label was not released at 120 °C, but was released at 635 flC, it would end up in Peak 1, making some of the Peak 1 signal not assignable to Martian autotrophy.

• This created an inherent ambiguity in quantitating the amount of ¹⁴CO₂ fixed in the soils. If the ¹⁴C-label in both Peak 1 and Peak 2 came from label in ¹⁴C-labeled fixed organics, then Peak 1 and Peak 2 should be counted together to avoid underestimating the amount of carbon fixation in the soil sample. However, only the label in Peak 2 was reliably assigned to fixed organics. To the extent that Peak 1 radioactivity came from abiologically absorbed carbonates, it could not be so assigned.

For those who like to peak at the end of the book to get the conclusion to the story without reading the details, when the CAPyR team interpreted the data collected by this experiment on Mars, they considered the amount of radioactivity in Peak 2 as the only radioactivity arising from ¹⁴C-labeled biologically fixed organics in the Viking soil samples. They observed in Peak 2 an amount of ¹⁴C that corresponded to ~1000 Earth-like cells. They interpreted this as evidence for a Martian autotrophic biosphere. This is comparable to the amounts of cell in Earth soils that are severely starved for resources.

However, had they remembered nitrate, or anticipated another oxidant (perchlorate), and included Peak 1 label as radioactivity also coming from ¹⁴C-labeled biologically fixed organics in the Viking soil samples, but this by nitrate combustion, then the amount of carbon fixation implied by the Viking data corresponded to 100 fold more carbon fixation, ~100,000 Earth-typical cells per gram of Mars soil.

The Viking experiment was developed on Earth with Terran soils

This experiment is my favorite in its concept. It did nothing unnatural to the Martian soil. It looked for the most fundamental process required by a living ecosystem, the uptake of carbon as a resource.

The experiment has been variously called the “carbon assimilation experiment”, the “pyrolytic release experiment,” and the “carbon fixation experiment”. Here, we will refer to it as the CAPyR experiment. Calling it simply the “pyrolytic release” (PR) experiment confuses the goal of the experiment (to detect carbon fixation) with a step in the process workflow.

The CAPyR experiment was designed to seek Martian microbial photosynthesis. More generally, it sought out Martian autotrophy, the ability of life at the bottom of a Martian food chain to get carbon directly from carbon dioxide (or monoxide) from the Martian atmosphere.

The CAPyR experiment assumed that life requires organic molecules. For the novice, “organic molecules” contain carbon atoms that are bonded to other carbon atoms or to hydrogen atoms (molecules with C–C and/or C–H bonds). This is not a controversial assumption, then or today.

Relatively few carbon-containing species are not organic. Carbon dioxide (CO₂) and carbon monoxide (CO) are two of these. Both are available to the near-surface soil of Mars. Approximately 95% of the Martian atmosphere is CO₂; approximately 0.07% is CO. Because the carbon atoms in these molecules are attached only to oxygen atoms, we call these “oxidized carbon atoms”.

Converting these oxidized carbon species into organic carbon involves the formation of C-H and C-C bonds. This process is often called “reduction”. Thus, methane (CH₄) is the most reduced organic carbon atom possible, as all of the four bonds that carbon atoms are allowed to make are bonds to hydrogen atoms. Methane has no bonds to oxygen atoms, nitrogen atoms, sulfur atoms, or any of the other atoms that chemists call ‘heteroatoms’ because they are neither carbon nor hydrogen.

Carbon atoms as such cannot be said to be a limiting resource on the near-surface of Mars. Any microorganism living on Martian surface can get the carbon atoms in indefinite amounts out of the air, if it can fix carbon from CO₂ or CO.

Rather, the scarce resource in making organic molecules on the near Martian surface is hydrogen (and, if one wants to be technical, electrons that might go with them). The atmosphere contains only ~0.01% water vapor, H₂O.* Discussion of the availability of water on the Martian near surface is left to another blog post.

Autotrophy is at the bottom of the food chain in such a biosphere

Autotrophy (from the Greek, meaning “self-feeding”) is the process by which organic carbon is made from inorganic carbon. If the atmosphere is the only source of carbon available to near-surface Martian life, a food chain to support organic life must begin with the reduction of CO₂ and/or CO to give compounds with C-H and C-C bonds.

Thus, one-carbon reduced organics are HCOOH (formic acid), HCHO (formaldehyde), CH₃OH (methanol), and CH₄ (the aforementioned methane), in order of increasing reduction.

Of course, life also requires organic molecules with more than one carbon atom. A biosphere must have metabolic cycles that fix CO₂ and/or CO to give organic species that contain many carbon atoms and many C-C bonds, sometimes called “higher organics”. For example, on Mars, the Curiosity rover recently identified long chain hydrocarbons possibly derived from fatty acids, which have the general structure H₃C-CH₂-{CH₂}_n-CH₂-COOH, where n is an integer (Pavlov et al., 2026). You yourself have these fatty acids. It remains an open question as to whether the long-chain alkanes detected by Curiosity are remnants of fatty acids generated by ancient Martian life.

Nevertheless, one can look for active life in an environment by looking for carbon fixation, the conversion of CO₂ and/or CO, both gases, to give organic molecules that are solids in the Martian soil. You need not know the precise structure of those organic molecules to observe this.

The CAPyR experiment sought movement of gaseous radioactive carbon-14 into the soil

The conversion of a gas into a solid by autotrophic microbes is key to the CAPyR experiment design. The structures of the organic molecules in Martian life are not known. You do know, however, that to be part of a microbe living in a soil, those higher organic species cannot be gases, at least not at the temperature where the organism lives. Thus, carbon fixation can be identified simply by observing the movement of carbon from the atmosphere, in gaseous compounds, into the soil, as non-gaseous compounds.

To follow the movement of carbon from the gas to a solid, the CAPyR experiment exploited the fact that carbon has a radioactive isotope, carbon-14. From a chemical perspective, ¹⁴C behaves (almost exactly) in chemical reactions the same as the majority carbon isotope, ¹²C, which is not radioactive. Every 5730 years, half of the radioactive ¹⁴C atoms decay, spitting out a high energy electron (a beta particle) that can be detected, or “counted”.

The CAPyR experiment planned to send to Mars a mixture of radioactively labeled ¹⁴CO₂ and ¹⁴CO, in approximately the same ratio as these gases are found in the atmosphere of Mars. It planned to expose Martian soil to these gases, for five days, under light. An observation of ¹⁴C ending up in the soil was to be taken as a sign of active Martian photosynthesis or, more generally, Martian autotrophy.

Now for some numbers. In a “mole” of carbon-14 (referring not to the burrowing animal, but to a collection of ~6 x 10²³ atoms, about 14 grams of the stuff), in one minute, ~1.4 x 10¹⁴ carbon-14 atoms decay, each ejecting a beta particle that can be detected by a radiation detector. That is, 14 grams of ¹⁴CO₂ spits out about 140 trillion beta particles per minute. Or, if you prefer, 1.4 x 10¹⁴ beta particles per minute.

This scales. That is, if you have only 14 milligrams of ¹⁴C, you see only 140 billion beta particles per minute.  If you have only 14 micrograms of ¹⁴C, you see only 140 million beta particles per minute. Or, if you prefer, 1.4 x 10⁸ beta particles per minute.

Since 140 beta particles per minute is easily countable above background, such experiments can detect very little ¹⁴C. Thus, 140 electron beta particle “counts” would come from 14 picograms (14 x 10^-12 grams), or 1 picomole (10^-12 mole), or about 6 x 10¹¹ atoms of ¹⁴C. Call it 10¹² atoms of ¹⁴C. The ¹⁴CO₂ was diluted a bit by Martian atmospheric ¹²CO₂, but we will leave this for a later discussion.

Thus, to return a life-positive result, the CAPyR experiment was sensitive enough to easily see the conversion of about 10¹² atoms of ¹⁴C presented as gaseous ¹⁴CO₂/¹⁴CO to non-gaseous ¹⁴C. Subject to caveats discussed below, this would represent carbon fixed into organic molecules in the Martian soil by life living there.

The CAPyR experiment had two attributes. First, carbon fixation must occur in any environment where CO₂/CO is the sole source of organic carbon. There is no way around it, unless we are prepared to propose life that contains no carbon atoms at all, or an unknown source of organic carbon supports a Martian biosphere without autotrophy.

Further, the CAPyR experiment did nothing unnatural to the Martian soil, including nothing that would obviously kill Martian life living there. Martian soil is naturally and continuously exposed to these gases. The CAPyR experiment did not add liquid water to the soil. Liquid water is rare at the Viking sites.

Nor did the experiment make any assumptions about the organic molecules used by Martian life, or what Martians liked to eat. Or what they might find poisonous. These advantages were all noted by the CAPyR team itself in its papers in 1970 and in many papers thereafter.

The false negative problem

But this does not mean that the CAPyR experiment would be totally unambiguous. As they developed the experiment, the designers needed to worry about false positive and false negative results.

In general, a false negative statement is that “life exists in the Martian soil, but we did not detect it.” At one level, this is simply a sensitivity question, something to do with what chemists call a “limit of detection”. Even though we are using radioactivity as a probe, and even though radioactive measurements are the most sensitive that we have available, the life in a Martian soil sample might be too sparse to fix 10¹² carbon atoms.

We could, of course, say that if the amount of Martian life is so little, we are not interested in it. But at least we should make a fair guess to how many cells the CAPyR experiment might detect.

This is easy enough if we are willing to use Earth bacteria as a benchmark. For example, an E. coli cell has ~10¹⁰ carbon atoms  If these atoms were all ¹⁴C, a single bacterial cell would generate ~1.4 high energy electrons per minute. A photosynthetic blue-green algal cell (called cyanobacteria in the trade) has similar amounts of carbon.

This is barely above background, but adequate if you count electrons for 100 minutes. So this “back of the envelope” calculation says that if the Martian soil contained one E. coli cell that divided over the course of its exposure to ¹⁴CO₂ to give a second cell whose carbon came entirely from that gas, one might be able to detect it if one looked for high energy electrons for 100 minutes. Ten cells would be better. One hundred cells for sure.**

This can be compared to the number of cells in soils on Earth that are resource-starved. For example, soils from the high Atacama Desert in Chile contain 100-10000 cells per gram of soil. A gram of glacial ice from the Antarctic plateau glacier contains 10-1000 cells (Christner et al., 2001). Farmland soil, by comparison, contains billions of cells per gram.

Of course, these calculations assume 100% efficient counting of the fixed ¹⁴C. For example, inefficient movement of the ¹⁴C-labeled higher organics in Martian microbes into a spot where its radioactivity could be detected would be a source of false negatives.

Seeing the fixed ¹⁴C directly in soil samples is difficult

The next step of the CAPyR architecture required that one detect the fixed non-volatile radioactive ¹⁴C-labeled carbon in organic molecules in the soil. This is conceivable, and we will propose experiments to do so that exploit the Spacek IMPRESS ride share penetrator architecture in a later Scoop in this set.

However, the beta particles coming from ¹⁴C are not strong enough to penetrate through much soil. The soil absorbs them. Thus, even if one put the soil on top of a Geiger counter, the efficiency of counting of the ¹⁴C decay particles from organics in that soil would be low.

Therefore, the original CAPyR paper developed a workflow that sought to get the fixed non-gas ¹⁴C back out of the soil … as a gas.  Here, the idea was to heat the soil to a temperature high enough to fragment the nonvolatile ¹⁴C-labeled “higher” organics into volatile ¹⁴C-labeled organic fragments. Think of the small organics that you can smell when you heat popcorn up enough to burn it.

What temperature was hot enough to do this “pyrolysis”? Based on experience with Terran organics, the CAPyR team decided to do pyrolysis at temperatures greater than 600 °C. The Viking experiment on the Martian surface eventually used 635 °C as the pyrolysis temperature (Fig. 1).

Figure 1. It is difficult to detect ¹⁴C radioactivity in soil; the soil absorbs many of the beta particles emerging from the decaying ¹⁴C-atoms. Thus, the CAPyR experiment used this workflow to fragment by heat (635 °C, “pyrolysis”) the ¹⁴C-organics made by putative autotrophic Martian microbes in the soil. This would give smaller, more volatile, ¹⁴C-labeled radioactive organic fragments (analogous to the organic fragments that give burnt popcorn its smell). These would come off the soil and be absorbed on Chromosorb P^®, which absorbs ¹⁴C-labeled organic molecules larger than methane, but not ¹⁴CO₂.

This “pyrolysis”, when applied to large biological non-volatile organics, converts them into smaller volatile organics, molecules that still contain reduced carbon, but that evaporate at 635 °C. Think of the organics that come off of over-heated popcorn that give a burnt popcorn its distinctive smell. The CAPyR experiment would detect radioactivity in these, after it fed radioactivity to the growing Martian “corn” for five days.

As we will see in a moment, pyrolysis has complications. If oxygen atoms are around, some of the ¹⁴C-labeled higher organics will end up in ¹⁴CO₂, not as smaller organic fragments.

This will certainly happen when the oxygen atoms that are present in the form of atmospheric O₂. In other words, heating organics in Earth’s atmosphere results in combustion. Try heating popcorn to 635 °C in your kitchen; you will set the popcorn on fire. The ¹⁴CO₂ coming from the burning popcorn (or Martian organics) must not be confused with the ¹⁴CO₂ that we used to feed the corn (or the Martians) in the first place. And that ¹⁴CO₂ will not stick to Chromosorb-P.

The CAPyR team was aware of this, and developed its workflow accordingly. In Earth-based development of the experiment, atmospheric O₂ was excluded. Then, the vapors coming off of the heated Martian soil were passed through something Chromosorb P^® that had been coated with copper oxide (CuO).

So what is Chromosorb P^®? It is a form of diatomaceous earth. This is silica material that is the fossilized skeletons left over from small organisms called diatoms. And its value in the workflow? It absorbs small organic fragments, anything larger than methane. It does not absorb CO₂ (Klein, 1974).

This lets you understand the workflow. The biosignature is the conversion of gaseous radioactivity in the form of ¹⁴CO₂ into non-volatile ¹⁴C-labeled higher organics in the Martian soil. If seen, this will be interpreted to be a sign of Martian photosynthesis, or, more generally, Martian autotrophy.

But the designers could not figure out how to count the radioactivity in the soil directly. Therefore, they decided to get the fixed radioactivity out of the soil by heating it to 635 °C. At that temperature, many organic molecules fragment (pyrolyze) to give small organic fragments that would, at this high temperature, vaporize. Any fragment bigger than methane would be absorbed on to Chromosorb P^®.

Of course, some of the ¹⁴C-labeled higher organics would end up after pyrolysis as volatile ¹⁴CO₂, from oxygen already in the soil). This label, which also should be counted as a biosignature, would pass through the Chromosorb P^® directly into the detector. This ¹⁴CO₂ would be counted as “Peak 1”.

So far so good. But you may have noticed that this workflow so far only trades one problem for another. One cannot count the “higher” non-volatile organics directly in the soil, because the soil would get in the way. But one also cannot count radioactivity the volatile organic fragments in the Chromosorb P^®, because the Chromosorb P^® material would get in the way.

To solve this problem, copper oxide (CuO) was put into the Chromosorb P^®. In the CAPyR workflow, the ¹⁴C-labeled organic fragments were trapped at low temperature in the Chromosorb P^®. At this low temperature, the CuO was inert. Then, the trap would be heated up to 640 °C. At these high temperatures, the CuO would oxidize any trapped ¹⁴C-labeled organic fragments to give ¹⁴CO₂. In this reaction, CuO provides the oxygen atoms..

This newly generated ¹⁴CO₂ would not continue to absorb to the Chromosorb P^®. Instead, it would emerge as a free gas that, at last, could make it to a detector where its beta particles could be counted. The counts in this fraction were called “Peak 2”.

This workflow was put together in an Earth-based experiment, which the CAPyR team published in 1970 in preparation to sell it for the Viking mission (Hubbard et al., 1970). During the Earth-based instrument development, the ¹⁴C electrons were counted with 57% efficiency. The counting period lasted for 100 minutes, to allow a 2-sigma error of <7%. The background was about 14 counts per minute.

Using this device on Earth, CAPyR team was able to detect carbon fixation in a wide variety of soils. These included soils from the Atacama Desert, where the biosphere is as sparse as it gets anywhere on Earth. A few hundred cells were well within its limits of detection

Warning signs of potential false negatives were seen

As the CAPyR team tested the soils from the Atacama Desert, they observed nitrate (NO₃^–) in their samples. It was ~13% by weight. As any schoolchild pyromaniac knows, nitrate burns organics to give CO₂. This is how classical gunpowder works.

The CAPyR team noticed this process as they pyrolyzed organics out the Atacama soils containing nitrate. They remarked that “the pyrolysis was converted to a partial combustion” by the nitrate.

In simpler words, if the soil contained nitrate, even without atmospheric O₂, some of the ¹⁴C fixed into organics would be converted not to organic fragments that could be trapped on Chromosorb P^® and later counted as Peak 2. Rather, nitrate would burn them to give ¹⁴ CO₂, which passed through the Chromosorb P^® trap and counted as Peak 1.

In a passage that foreshadowed the actual results when the CAPyR experiment was run on Mars, the CAPyR team dismissed this by writing: “Nonetheless, even in this poor example [from the Atacama Desert], a significant yield of ¹⁴C was detected in the trapped organic fraction.”

“Poor example”? What they meant was that the Atacama soil contained not enough nitrate oxidant to burn all of the organics that were present in the soil. The left over organics continued to generate burnt-popcorn-smell fragments that were absorbed on Chromosorb P^®.

And here was the first consequential mistake. The team did not remark that if the Martian soil contained more nitrate than the Atacama soils, in particular, nitrate in excess of the higher fixed ¹⁴C-labeled organics, then all of the ¹⁴C-label fixed by Martian life might be lost through the Chromosorb P^® trap as ¹⁴CO₂. A false negative. The fixed label would all appear in Peak 1; none would appear in Peak 2.

In any case, they evidently took no steps to mitigate this possible problem.

Replace “nitrate in Atacama soils” by “perchlorate in Martian soils”, and you are half way towards understanding the results from the CAPyR experiments on the Viking 1976 landers. As we shall see in a later Scoop, Klaus Biemann, who ran the Viking gas chromatograph-mass spectrometer (GC-MS), actually did consider the possibility that nitrate was present in the Martian soils when he analyzed his Viking data. However, he dismissed this possibility in a way that was not wholly logical.

But we are getting ahead of ourselves. The CAPyR experiment was prepared to detect ¹⁴CO₂ coming from fixed organics by a workflow that involved pyrolysis of the organics to give organic vapors, trapping the organic vapors in a material that did not trap carbon dioxide, oxidizing them to ¹⁴CO₂, and counting the ¹⁴CO₂ that emerged from the Chromosorb P^® (Peak 2).  In a pre-flight paper, pyrolytically generated ¹⁴CO₂ in Peak 1 was recognized as being a biosignature as well (Horowitz et al., 1972). Thus, it was counted as Peak 1, both on Earth and on Mars. However, when the experiment was actually run on Mars, the CAPyR team was confused about this.

Warning signs of potential false positives also were seen

The CAPyR team also considered false positives. Prime among these was the possibility that the ¹⁴CO₂ would react with alkaline soils to make bicarbonate and carbonate salts. If this label were released only when the soil was heated to 635°C, it would also contribute to Peak 1. The workflow lacked a strategy to distinguish ¹⁴CO₂ that comes fr0m:

(i) non-biologically absorbed as carbonates that are not flushed out at 120 °C, but released at higher temperatures, versus

(ii) ¹⁴CO₂ from biologically generated organic materials pyrolyzed at 635°C, where oxygen came from the soil.

On Earth as the CAPyR experiment was being developed, the team saw substantial amounts of ¹⁴CO₂ absorbed on to soils that had been sterilized to kill biology. Sterilized Atacama soil (which the CAPyR team numbered Sample 264) absorbed large amounts of CO₂ without biology. This radioactivity was released by pyrolysis, not trapped on Chromosorb P^®, and ended up in Peak 1. There is an ambiguity in the CAPyR interpretation. One does not know how much of the Peak 1 radioactivity should be added to the total organic fixed by soil autotrophs, after Peak 1 material that survived in the soil as non-biologically absorbed carbohydrates.

And this was perhaps the second strategic mistake made by the CAPyR team. It is not clear in their literature whether they thought that bicarbonate/carbonate ¹⁴CO₂ would be released in the 120 °C flush. As we will see, a series of studies could have clarified this issue pre-flight.

Rather, the CAPyR team asked instead whether a small amount of this Peak 1 ¹⁴CO₂ would be caught on the Chromosorb P^®, to be later released in the last step of the workflow into Peak 2. If it were, it would be confused with the ¹⁴CO₂ arising from ¹⁴C-labeled organic fragments that had been oxidized by copper oxide.

The CAPyR team did considered another possible false positive, however. Very few non-biological processes convert gaseous CO₂ into higher organics that would be pyrolyzed to give organic fragments. However, non-biological processes are known that convert gaseous ¹⁴CO₂ into higher organics that would be pyrolyzed to give back ¹⁴CO₂. The CAPyR team considered one, the reaction of phenol (hydroxybenzene) with CO₂ to give salicylic acid. Heating salicyclic acid regenerated ¹⁴CO₂, which was not caught by the Chromosorb P^®, and contribute to Peak 1. In this interpretation, non-trapped pyrolysis radioactivity should not be added to the inventory of biologically fixed organics.

An astute reader can see the incomplete logical that was not resolved in the design: What do we do with radioactivity in Peak 1? Radioactivity in Peak 1 could come from fixed ¹⁴C-labeled organics that were converted at 635 °C to ¹⁴CO₂ by nitrate in the soil, or with other oxygen-containing species in the soil (biological) inventory. This part of Peak 1 should be counted as a biosignature.

However, radioactivity in Peak 1 could come from ¹⁴CO₂ that was absorbed as bicarbonates, carbonates, or organics that released ¹⁴CO₂ back when heated. This part of Peak 1 should not be counted as a biosignature.

Only one feature saves the experiment from this logic. In any case, radioactivity in Peak 2 should be counted as a biosignature.

A final possibility for a false positive was reported by the CAPyR team in 1971 (Hubbard et al., 1971). The Martian surface, like Earth’s, is bathed in sunlight. Because Mars is ~1.52 AU from the Sun on average, it receives ~43% of Earth’s solar input. However, the oxygen atmosphere on Earth creates ozone in the upper layers of the atmosphere. This filters out the harshest ultraviolet light. Unfortunately, on Mars, that harsh ultraviolet light comes all the way down to the surface.

In a series of experiments run on Earth, the CAPyR team discovered that ¹⁴CO, when it interacts with this high energy ultraviolet light, gets fixed to organics without any biology at all. Fearing that this would be mistaken on Mars as photosynthetic carbon fixation, a false positive, the mission did not use Solar light in these experiments. Rather, Viking brought along its own light source that did not present hard ultraviolet light to the soil. This was hoped to be able to avoid this particular false positive.

The CAPyR experiment flew without mitigating these false positive and falses negative possibilities

At the end of its Earth-based development, the CAPyR instrument was commissioned to fly. Its workflow was impeccably natural. Other than the scooping itself, and perhaps the illumination by an artificial lamp, the experiment did nothing to kill indigenous Martians. Not by changing their atmosphere, not by changing the carbon compounds that they had access to, not by poisoning them, and not by putting water on them.

However, the experiment flew with two poorly managed problems.

As the false negative problem, if the Martian soil contained nitrate, some (or all) of the ¹⁴C-labeled organics fixed into higher organics by autotrophic Martians would be burned to give ¹⁴CO₂ upon heating to 635 °C (but not at 120 °C). This ¹⁴CO₂ would pass through the Chromosorb P and end up as Peak 1. If nitrate were present in excess over the fixed ¹⁴C-biological higher organics, all of the fixed ¹⁴C would end up as Peak 1. And as we have seen, there was no logic to distinguish Peak 1 radioactivity from ¹⁴CO₂ generated from bio-organics in the soil, versus radioactivity from ¹⁴CO₂ arising from non-biological absorption as bicarbonates, as carbonates, or reaction with very nucleophilic organics (e.g. phenols) present in the soil.

Now, hindsight is 20-20. However, the CAPyR team actually saw the oxidation problem in their Atacama soils, which contained nitrate. They were (un)fortunate that the nitrate combustion was not complete; in the soils that they had tested on Earth, the nitrate was not present in sufficient amounts to convert all of the fixed ¹⁴C into ¹⁴CO₂. Thus, the team saw only partial combustion, and concluded that this left behind enough fixed organics to still give an affirmative life detection result. They evidently did not consider the possibility that the Mars soils might contain enough nitrate to complete the combustion.

Reading ahead, as it turned out, the Martian Viking soils contained perchlorate in large amounts. Perchlorate in large amounts also combusts higher organics at 635 °C. This process will be detailed in the next scoop.

None of this would be a problem is we simply counted radioactivity in Peak 1 as a biosignature. However, this would encounter the false positive problem. If the Martian soils were alkaline, then they would non-reductively absorb ¹⁴CO₂ as carbonate or bicarbonate minerals. This ¹⁴CO₂ might be released at 635 °C, become “Peak 1”, and be misinterpreted as fixed carbon that had been converted to ¹⁴CO₂ using oxygen atoms from the soil.

The CAPyR literature does not answer clearly whether the CAPyR team understood this problem. It is possible that the CAPyR team thought that by heating the soil to 120°C prior to heating to 635° C and flushing out what came off the soil, they would be able to release the non-reductively absorbed ¹⁴CO₂. However, I have not been able to find a spot where the team says this explicitly.

As we shall see, the Viking version of the CAPyR experiment at two sites on Mars saw some ¹⁴CO₂ fixed that led to burnt popcorn fragments absorbed on Chromosorb P. This ended up as Peak 2. This Peak 2 amount of ¹⁴CO₂ corresponded to ~1000 E. coli cells.

However, the CAPyR experiment saw 100 times more Peak 1 ¹⁴CO₂ emerging directly from the soil when heated at 635 °C, even though the soil had been flushed at 120°C. This could mean that the soil had fixed enough ¹⁴CO₂ for 100,000 cells, with perchlorate destroying the burnt popcorn smell so that this label ended up in Peak 1, indicating 100,000 cell-equivalents of biosynthetic carbon fixation.

Or Peak 1 ¹⁴CO₂ could represent material absorbed as a carbonate/bicarbonate stable at 120 °C but not at 635°C, or absorbed non-biologically as salicylic acid. In either case, the CAPyR experiment biological fixation corresponding only to 1000 Earth-equivalent cells.

Yes, either way, extant autotrophic Martian life was detected by the CAPyR experiment, under the terms of its experimental design. But the difference between 1000 Earth-equivalent cells and 100,000 Earth-equivalent cells is important.

This is the lead to the next blog post. As we shall see, by the time that the CAPyR team got to Mars, they had largely forgotten the possibilities for the false positive and false negative results that they had recognized in their Earth-based experiments. And “perchlorate” evidently never occurred to them.

The article was posted after minor revisions by Jan Spacek on 2/18/2026.
Spacek’s notes:
*The Martian atmosphere holds 10-20 ppmv of H₂ while Earth atmosphere holds only ~0.5 ppmv of H₂, making the Martian hydrogen gas a viable alternative source of hydrogen atoms for carbon fixation via trace gas metabolism, in process similar to one known from Earth’s polar soils – even after we account for the pressure differences between the atmospheres. In absolute values, Mars’ atmosphere holds 3-6x less H₂ than Earth’s atmosphere. If perchlorates are allowed to be used as electron acceptors, water used for metabolism can be generated from H₂ oxidation.
**Hubbard et al (1970) states that the method “easily detects” 100-1000 algae in 0.25 cc of soil after 3-24 hours. However, simple observation of the bulk metabolic rates in a sample cannot distinguish whether Martians are sparse and active metabolizers, or abundant and lazy metabolizers. The results would be the same for sample that contain just one super-active cell that duplicated fully or 1000 “lazy” cells each gaining only 0.1% of new carbon mass using the labeled carbon. Using an analogy from slowly metabolizing life in energy sparse environments on Earth, one might expect more chilled life style.

References

Christner, B. C., Mosley-Thompson, E., Thompson, L. G., & Reeve, J. N. (2001). Isolation of bacteria and 16S rDNAs from Lake Vostok accretion ice. Environmental Microbiology, 3(9), 570–577.  https://doi.org/10.1046/j.1462-2920.2001.00226.x

Hubbard, J. S., Hobby, G. L., Horowitz, N. H., Geiger, P. J., & Morelli, F. A. (1970). Measurement of ¹⁴CO₂ assimilation in soils: an experiment for the biological exploration of Mars. Applied Microbiology, 19(1), 32-38.

Hubbard, J. S., Hardy, J. P., & Horowitz, N. H. (1971). Photocatalytic production of organic compounds from CO and H₂O in a simulated martian atmosphere. Proceedings of the National Academy of Sciences, 68(3), 574-578.

Klein, H. P. (1974). Automated life-detection experiments for the Viking mission to Mars. Origins of life, 5(3), 431-441.

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Pavlov, A. A., Freissinet, C., Glavin, D. P., House, C. H., Stern, J. C., McAdam, A. C., … & Gomez, F. (2026). Does the Measured Abundance Suggest a Biological Origin for the Ancient Alkanes Preserved in a Martian Mudstone?. Astrobiology, doi:10.1177/15311074261417879.

Spacek, J., Benner, S.A. (2022) Agnostic Life Finder (ALF) for large-scale screening of Martian life during in situ refueling. Astrobiology 22, 1255-1263. doi.org/10.1089/ast.2021.0070