Viking lander biological experiments

The two Viking landers each carried four types of biological experiments to the surface of Mars in 1976. These were the first Mars landers to carry out experiments to look for biosignatures of microbial life on Mars. The landers used a robotic arm to put soil samples into sealed test containers on the craft. The two landers were identical, so the same tests were carried out at two places on Mars' surface, Viking 1 near the equator and Viking 2 further north.

The experiments
Four experiments are presented here in the order in which they were carried out by the two Viking landers. The biology team leader for the Viking program was Harold P. Klein (NASA Ames).

Gas chromatograph — mass spectrometer
A gas chromatograph — mass spectrometer (GCMS) is a device that separates vapor components chemically via a gas chromatograph and then feeds the result into a mass spectrometer, which measures the molecular weight of each chemical. As a result, it can separate, identify, and quantify a large number of different chemicals. The GCMS (PI: Klaus Biemann, MIT) was used to analyze the components of untreated Martian soil, and particularly those components that are released as the soil is heated to different temperatures. It could measure molecules present at a level of a few parts per billion.

The GCMS measured no significant amount of organic molecules in the Martian soil. In fact, Martian soils were found to contain less carbon than lifeless lunar soils returned by the Apollo program. This result was difficult to explain if Martian bacterial metabolism was responsible for the positive results seen by the Labeled Release experiment (see below). A 2011 astrobiology textbook notes that this was the decisive factor due to which "For most of the Viking scientists, the final conclusion was that the Viking missions failed to detect life in the Martian soil."

Experiments conducted in 2008 by the Phoenix lander discovered the presence of perchlorate in Martian soil. The 2011 astrobiology textbook discusses the importance of this finding with respect to the results obtained by Viking as "while perchlorate is too poor an oxidizer to reproduce the LR results (under the conditions of that experiment perchlorate does not oxidize organics), it does oxidize, and thus destroy, organics at the higher temperatures used in the Viking GCMS experiment. NASA astrobiologist Chris McKay has estimated, in fact, that if Phoenix-like levels of perchlorates were present in the Viking samples, the organic content of the Martian soil could have been as high as 0.1% and still would have produced the (false) negative result that the GCMS returned. Thus, while conventional wisdom regarding the Viking biology experiments still points to "no evidence of life", recent years have seen at least a small shift toward "inconclusive evidence"."

According to a 2010 NASA press release: "The only organic chemicals identified when the Viking landers heated samples of Martian soil were chloromethane and dichloromethane -- chlorine compounds interpreted at the time as likely contaminants from cleaning fluids." According to a paper authored by a team led by Rafael Navarro-González of the National Autonomous University of Mexico, "those chemicals are exactly what [their] new study found when a little perchlorate -- the surprise finding from Phoenix -- was added to desert soil from Chile containing organics and analyzed in the manner of the Viking tests." However, the 2010 NASA press release also noted that: "One reason the chlorinated organics found by Viking were interpreted as contaminants from Earth was that the ratio of two isotopes of chlorine in them matched the three-to-one ratio for those isotopes on Earth. The ratio for them on Mars has not been clearly determined yet. If it is found to be much different than Earth's, that would support the 1970s interpretation." Biemann has written a commentary critical of the Navarro-González and McKay paper, to which the latter have replied; the exchange was published in December 2011.

Gas exchange
The gas exchange (GEX) experiment (PI: Vance Oyama, NASA Ames) looked for gases given off by an incubated soil sample by first replacing the Martian atmosphere with the inert gas Helium. It applied a liquid complex of organic and inorganic nutrients and supplements to a soil sample, first with just nutrients added, then with water added too. Periodically, the instrument sampled the atmosphere of the incubation chamber and used a gas chromatograph to measure the concentrations of several gases, including oxygen, CO2, nitrogen, hydrogen, and methane. The scientists hypothesized that metabolizing organisms would either consume or release at least one of the gases being measured. The result was negative.

Labeled release
The labeled release (LR) experiment (PI: Gilbert Levin, Biospherics Inc.) gave the most promise for exobiologists. In the LR experiment, a sample of Martian soil was inoculated with a drop of very dilute aqueous nutrient solution. The nutrients (7 molecules that were Miller-Urey products) were tagged with radioactive 14C. The air above the soil was monitored for the evolution of radioactive 14CO2 gas as evidence that microorganisms in the soil had metabolized one or more of the nutrients. Such a result was to be followed with the control part of the experiment as described for the PR below. The result was quite a surprise, considering the negative results of the first two tests, with a steady stream of radioactive gases being given off by the soil immediately following the first injection. The experiment was done by both Viking probes, the first using a sample from the surface exposed to sunlight and the second probe taking the sample from underneath a rock; both initial injections came back positive. Subsequent injections a week later did not, however, elicit the same reaction, and according to a 1976 paper by Levin and Patricia Ann Straat the results were inconclusive. In 1997, Levin, Straat and Barry DiGregorio co-authored a book on the issue, titled Mars: The Living Planet.

A CNN article from 2000 noted that "Though most of his peers concluded otherwise, Levin still holds that the robot tests he coordinated on the 1976 Viking lander indicated the presence of living organisms on Mars." A 2006 astrobiology textbook noted that "With unsterilized Terrestrial samples, though, the addition of more nutrients after the initial incubation would then produce still more radioactive gas as the dormant bacteria sprang into action to consume the new dose of food. This was not true of the Martian soil; on Mars, the second and third nutrient injections did not produce any further release of labeled gas." The 2011 edition of the same textbook noted that "Albet Yen of the Jet Propulsion Laboratory has shown that, under extremely cold and dry conditions and in a carbon dioxide atmosphere, ultraviolet light (remember: Mars lacks an ozone layer, so the surface is bathed in ultraviolet) can cause carbon dioxide to react with soils to produce various oxidizers, including highly reactive superoxides (salts containing O2&minus;) When mixed with small organic molecules, superoxidizers readily oxidize them to carbon dioxide, which may account for the LR result. Superoxide chemistry can also account for the puzzling results seen when more nutrients were added to the soil in the LR experiment; because life multiplies, the amount of gas should have increased when a second or third batch of nutrients was added, but if the effect was due to a chemical being consumed in the first reaction, no new gas would be expected. Lastly, many superoxides are relatively unstable and are destroyed at elevated temperatures, also accounting for the "sterilization" seen in the LR experiment."

In a 2002 paper published by Joseph Miller, he speculates that recorded delays in the system's chemical reactions point to biological activity similar to the circadian rhythm previously observed in terrestrial cyanobacteria.

On 12 April 2012, an international team including Levin and Straat published a peer reviewed paper suggesting the detection of "extant microbial life on Mars", based on mathematical speculation through cluster analysis of the Labeled Release experiments of the 1976 Viking Mission.

Pyrolytic release
The pyrolytic release (PR) experiment (PI: Norman Horowitz, Caltech) consisted of the use of light, water, and a carbon-containing atmosphere of carbon monoxide (CO) and carbon dioxide (CO2), simulating that on Mars. The carbon-bearing gases were made with carbon-14 (14C), a heavy, radioactive isotope of carbon. If there were photosynthetic organisms present, it was believed that they would incorporate some of the carbon as biomass through the process of carbon fixation, just as plants and cyanobacteria on earth do. After several days of incubation, the experiment removed the gases, baked the remaining soil at 650 °C (1200 °F), and collected the products in a device which counted radioactivity. If any of the 14C had been converted to biomass, it would be vaporized during heating and the radioactivity counter would detect it as evidence for life. Should a positive response be obtained, a duplicate sample of the same soil would be heated to "sterilize" it. It would then be tested as a control and should it still show activity similar to the first response, that was evidence that the activity was chemical in nature. However, a nil, or greatly diminished response, was evidence for biology. This same control was to be used for any of the three life detection experiments that showed a positive initial result.

Scientific conclusions
Organic compounds seem to be common, for example, on asteroids, meteorites, comets and the icy bodies orbiting the Sun, so detecting no trace of any organic compound on the surface of Mars came as a surprise. The GC-MS was definitely working, because the controls were effective and it was able to detect traces of the cleaning solvents that had been used to sterilize it prior to launch. At the time, the total absence of organic material on the surface made the results of the biology experiments moot, since metabolism involving organic compounds were what those experiments were designed to detect. However, the general scientific community surmise that the Viking's biological tests remain inconclusive. Most researchers surmise that the results of the Viking biology experiments can be explained by purely chemical processes that do not require the presence of life, and the GC-MS results rule out life.

Despite the positive result from the Labeled Release experiment, a general assessment is that the results seen in the four experiments are best explained by oxidative chemical reactions with the Martian soil. One of the current conclusions is that the Martian soil, being continuously exposed to UV light from the Sun (Mars has no protective ozone layer), has built up a thin layer of a very strong oxidant. A sufficiently strong oxidizing molecule would react with the added water to produce oxygen and hydrogen, and with the nutrients to produce carbon dioxide (CO2).

On August 2008, the Phoenix lander detected perchlorate, a strong oxidizer when heated above 200 °C. This was initially thought to be the cause of a false positive LR result. However, results of experiments published in December 2010 propose that organic compounds "could have been present" in the soil analyzed by both Viking 1 and 2, since NASA's Phoenix lander in 2008 detected perchlorate, which can break down organic compounds. The study's authors found that perchlorate can destroy organics when heated and produce chloromethane and dichloromethane as byproduct, the identical chlorine compounds discovered by both Viking landers when they performed the same tests on Mars. Because perchlorate would have broken down any Martian organics, the question of whether or not Viking found organic compounds is still wide open, as alternative chemical and biological interpretations are possible.

In 2013, astrobiologist Richard Quinn at the Ames Center conducted experiments in which perchlorates irradiated with gamma rays seemed to reproduce the findings of the labeled-release experiment. He concluded that neither hydrogen peroxide nor superoxide is required to explain the results of the Viking biology experiments.

Did the experiments detect biology or reactive chemistry?
The Viking landers (operating on Mars from 1976-1982), are the only spacecraft so far to search directly for life on Mars. They landed in the equatorial regions of Mars. With our modern understanding of Mars, this would be a surprising location to find life, as the soil there is thought to be completely ice free to a depth of at least hundred meters, and possibly for a kilometer or more. It is not totally impossible though, as some scientists have suggested ways that life could exist even in such arid conditions, using the night time humidity of the atmosphere, and possibly in some way utilizing the frosts that form frequently in the mornings in equatorial regions.

The Viking results were intriguing, and inconclusive.

There has been much debate since then between a small number of scientists who think that the Viking missions did detect life, and the majority of scientists who think that it did not.

The Viking lander had three main biological experiments, but only one of these experiments produced positive results.


 * The Gas Chromatograph/Mass Spectrometer searched for organics, and found no trace of them.
 * The Gas Exchange experiment searched for any gases that evolved from a sample of the Mars soil left in a nutrient solution in simulated martian atmosphere for twelve days. This experiment did detect gases, but so did the control, which repeated the experiment with a sample heated to sterilize it of any possible life. This suggests a chemical explanation.
 * The labeled release experiment used nutrients tagged with 14C. It then monitored the air above the experiment for radioactive 14CO2 gas as evidence that the nutrients had been taken up by micro-organisms. This experiment produced positive results. Also, in this case, the control experiments came out negative. Normally this would suggest a biological explanation. For this experiment the microbes don't have to grow, reproduce. They just have to metabolize the organics and produce the 14CO2 gas in the process.

The conclusion at the time, for most scientists, was that the Labeled release experiment had to have some non biological explanation involving the unusual chemistry on Mars. One idea put forward by Albert Yen of JPL was that first carbon dioxide could react with the soil to produce superoxides in the cold dry conditions with UV radiation, which could then react with the small organics of the LR experiment to produce carbon dioxide. The other two experiments seemed to rule out any possibility of a biological explanation.

Some of the LR data remained hard to explain as chemistry and the experimenter's Principle Investigator Gilbert Levin maintained from the beginning that his experiment probably detected life. Here are some of the things that any theory has to explain, in addition to the non detection of organics by the other instruments:


 * The LR response produced a lot of carbon dioxide rapidly, which some criticized as "too much too soon" for the levels of life expected there.
 * A second injection of nutrient actually lead to a 20% decrease in the previously evolved 14CO2
 * A sample maintained at 10 °C in darkness for two months at one site and three months at another had no response to the nutrient
 * A sample heated to 46 °C produced 60% less gas
 * A sample heated to 51 °C became erratic and produced 90% less gas

His comments on how this could be explained biologically are that first, the amount of 14CO2 released is comparable to a sample from Antarctica and less than is usually released in tests on Earth. The second injection seems to have just wetted the sample and lead to absorption of 14CO2 and his conclusion is that the life died during the experiment, which is not too surprising given that most microbes even on Earth can't be cultivated in the laboratory. The difference in effect between 46 °C and 51 °C he considers to be strongly suggestive of life and hard to explain chemically for such a small change. The results for the samples kept in darkness he also considers to be hard to explain without biology.

Most other scientists at the time continued to regard the experiments as inconclusive.

Before the discovery of the oxidizer perchlorate on Mars in 2008, Joop Houtkooper and Dirk Schulze Makuch made another proposal in 2007 to explain the results, that life on Mars may be using a mixture of water and biogenically created hydrogen peroxide as its internal solvent. This possible form of life might explain some puzzling aspects of the Viking experiments. On cooling, the eutectic of 61.2% (by weight) mix of water and hydrogen peroxide has a freezing point of −56.5 °C, and also tends to super-cool rather than crystallize. It is also hygroscopic, an advantage in a water-scarce environment. . It would prefer regions with lower temperatures, and would avoid liquid water. Conditions at the poles would be optimal, but it could also survive in the equatorial regions visited by Viking

Upon heating, Houtkooper and Schulze Makuch's putative organisms might have auto-oxidized catastrophically, leaving the detected gases and very little solid residue. The release of oxygen in dry conditions could be the result of one of its metabolic pathways, decomposition of hydrogen peroxide to water and oxygen, and in wet conditions due to decomposition through hyperhydration. Organic synthesis occurred under dry and not under wet conditions, which again coudl be explained by this form of light. Some of the other data could be the result of microbes coping and failing to cope in the extremely unmartian conditions of high temperatures combined with saturation of water vapour.

Houtkooper and Schulze Makuch reasoned at the time that no known Earth organisms can reproduce the Viking results, however no known Earth chemistry can either, and any explanation must be based on the differing geochemistry as well as the potential organisms.

In 2006, Mario Crocco proposed creation of a new nomenclatural rank Gillevinia straata with this potential biochemistry based on the experimental results. Normally the organism's DNA has a major role in identification and naming. There is precedent for naming a new species for teaching purposes, raising visibility and encouraging further research. However this classification is seen by other biologists as premature, as it could lend inappropriate credibility to life detection, and the response could also be due to a mix of species.

Work since then has suggested a possible re-evaluation of those results.

First, some have suggested that the gas chromatograph may not have been sensitive enough to detect the organics. Though other scientists have suggested that they could have detected low levels of organics....

Then in 2002, Joseph Miller, a specialist in circadian rhythms thought he spotted these in the Viking data. He was able to get hold of the original Viking raw data (using printouts kept by Levin's co-researcher Pat Straat) and on re-analysis this seemed to confirm his conclusions.


 * The data, though it follows temperature changes, is smoother than you'd expect from a purely chemical reaction response.
 * It is also delayed by 2 hours. From analysis of the experiment he concluded that though a 20-minute delay could be explained using variability in CO2 solubility, 2 hours seems too much of a delay to explain that way.
 * There are signs of a change of rhythm after the second nutrient injection.
 * In an accidental experiment, one of the samples was kept for two months in cold and darkness before it was used. This showed no daily cycle. This is quite hard to explain on basis of chemistry.

Another paper published in 2012 uses cluster analysis cluster analysis and suggested once more that they may have detected biological activity.

There is progress in the chemical explanations too. In 1999, experiments with hydrogen peroxide-saturated titanium dioxide produced similar results.

On the other hand, a paper published in 2013 by Quinn has refined the chemical explanations suggested for the labeled release observations, using radiation damaged perchlorates. By simulating the radiation environment on Mars, he was able to duplicate radioactive 14CO2 emission from the sample.

In short, the findings are intriguing but there is no consensus yet on whether the correct interpretation is biological or chemical. Most scientists still favour the chemical explanation, though a few scientists have recently shown renewed interest in a possible biological explanation.

Critiques
James Lovelock argued that the Viking mission would have done better to examine the Martian atmosphere than look at the soil. He theorised that all life tends to expel waste gases into the atmosphere, and as such it would be possible to theorise the existence of life on a planet by detecting an atmosphere that was not in chemical equilibrium. He concluded that there was enough information about Mars' atmosphere at that time to discount the possibility of life there. Since then, methane has been discovered in Mars' atmosphere at 10ppb, thus reopening this debate. Although in 2013 the Curiosity rover failed to detect methane at its location in levels exceeding 1.3ppb. later in 2013 and in 2014, measurements by Curiosity did detect methane, suggesting a time-variable source. The planned ExoMars Trace Gas Orbiter, launched on March 2016, will implement this approach and will focus on detection, characterization of spatial and temporal variation, and localization of sources for a broad suite of atmospheric trace gases on Mars and help determine if their formation is of biological or geological origin. The Mars Orbiter Mission is also attempting —since late 2014— to detect and map methane on Mars' atmosphere. A press commentary argued that, if there was life at the Viking lander sites, it may have been killed by the exhaust from the landing rockets. That is not a problem for missions which land via an airbag-protected capsule, slowed by parachutes and retrorockets, and dropped from a height that allows rocket exhaust to avoid the surface. Mars Pathfinder's Sojourner rover and the Mars Exploration Rovers each used this landing technique successfully. The Phoenix Scout lander descended to the surface with retro-rockets, however, their fuel was hydrazine, and the end products of the plume (water, nitrogen, and ammonia) were not found to have affected the soils at the landing site.

Future missions
The question of life on Mars will probably not be resolved entirely until future missions to Mars either conclusively demonstrate the presence of life on the planet, identify the chemical(s) responsible for the Viking results, or both. The Mars Science Laboratory mission landed the Curiosity rover on August 6, 2012, and its goals include investigation of the Martian climate, geology, and whether Mars could have ever supported life, including investigation of the role of water and planetary habitability. Astrobiology research on Mars will continue with the ExoMars Trace Gas Orbiter in 2016, ExoMars rover on 2018, and the Mars 2020 rover in 2020.

In 2008, the Thermal and Evolved Gas Analyzer was operated at Mars, which could chemically analyze 8 samples.

The Urey instrument was a funded study for sensitive organic compound detector, but has not been sent to Mars but was considered for ExoMars program of the 2000s

Proposed missions
The Biological Oxidant and Life Detection (BOLD) is a proposed Mars mission that would follow up the Viking soil tests by using several small impact landers. Another proposal is the Phoenix lander-based Icebreaker Life.