1

MARS: Dead or Alive?

Gilbert V. Levin

Arizona State University

and

Patricia Ann Straat

NIH (Ret.)

Presented at

Mars Society Convention

League City, TX

August 8, 2014

ABSTRACT

Throughout the thousands of years since we Earthlings first became inquisitive, we have

been taunted by the lugubrious question of whether or not we are alone in the universe. It

was not until 1976 that we developed the technology to launch the first direct search for

extraterrestrial life. That quest was the primary goal of NASA’s Viking Mission to Mars

that landed two spacecraft on the surface of the red planet. At both sites, the Labeled

Release (LR) experiment obtained data that satisfied the pre-mission criteria for the

detection of extant microbial life. Ad hoc experiments performed by the LR on Mars,

new information about the habitability of Mars, the finding of life in extreme

environments on Earth, the similarity among the Viking LR responses and LR responses

from some terrestrial viable soils, and the likelihood that the two planets cross-infect each

other support that conclusion. However, the Viking LR evidence was not generally

accepted initially, and, to this day, while gaining credence, lacks the consensus of the

scientific community. NASA’s current position is that the LR results are, at best,

ambiguous. Inasmuch as the search for life remains the “Holy Grail” of NASA’s

Astrobiology Program, means to resolve this issue are suggested, as is the significance of

what may be achieved.

Primarily, herein is a narrative account of the Viking LR experiment: its thesis,

development, execution, data, the reasons for their lack of acceptance, and relevant post-

Viking findings. The status quo of the LR is presented as of the date of this writing, some

38 years after Viking landed, during which period, remarkably, NASA has not sent

another life detection experiment to Mars. Included in this paper are relevant reported

results, as of the time of this writing, from the Mars Science Laboratory Mission,

“Curiosity.” The authors herein provide the evidence supporting their claim that the LR

did discover living microorganisms on Mars. A method is proposed that can validate the

claim and begin a study of comparative biology.

Background

Since Viking, many new findings have added important information to provide a realistic

background against which to view and evaluate the LR findings. Microorganisms have

been found living in extreme environments rivaling Mars. Cryptoendolithic lichen, B.

2

subtilis and B. pumilus have recently been reported1,2,3

to have survived in naked space

for 1.5 years, the full term of their exposure. Microorganisms have been found4 growing

in perpetual ice at the South Polar Cap. NASA missions have produced data leading to

the conclusion5 that large regions of Mars were habitable in the past. The current diurnal

temperatures6,7,8

over wide areas of Mars rise well above freezing. Perhaps most

importantly for the life issue, liquid water has been measured9 in amounts up to several

percent in surface samples on Mars, amounts well above those in many areas of Earth

heavily populated with microorganisms. In all, it is likely that many forms of terrestrial

life could survive some current environments on Mars. It has also been deduced10

and

supported by laboratory experiments, that Earth and Mars have been seeding each other

with ejecta from meteor and meteorite hits. It has been proposed11,12

that microorganisms

inside such ejecta from one planet could survive to arrive in viable form to infect the

other, as depicted in Figure 1.

FIG. 1. Cross-Infection of Mars and Earth.

In a similar manner, either planet could be infected from other life-bearing sources.

Indeed, in view of the post-Viking information, it could be contended that it might be

very difficult for Mars to be sterile. Thus, this brief background is offered to “level the

playing field” on which to view the possibility of life on Mars and, specifically, on which

to interpret the LR experiment.

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Introduction

In classic terms, the purpose of an experiment is to test a key prediction of a thesis put

forth. If the prediction is supported by the experiment, the thesis is accepted. The thesis

that there might be life on Mars existed for many years before the capability to test it was

developed by NASA. Twenty years of effort were then devoted to preparing carefully

selected experiments. Among these was the LR, originally called the “Radioisotopic

Biochemical Probe for Extraterrestrial Life,” and then “Gulliver,” before being changed

by NASA to the “Labeled Release” experiment when it was accepted as one of the

Viking experiments.

The LR on Mars was a specific experiment designed to test only a narrowly defined

critical aspect of the theory concerning the possibility of life on Mars. The key elements

of that restricted approach were: 1. there is life on Mars, because it is environmentally

sufficiently similar to Earth, 2. as a minimum there is microbial life, because of its

relative simplicity and the need for it to recycle any form of life for continuity, 3. the

microbial life operates on a biochemistry similar to that on Earth, the only sample of life

we have, and 4. the amount of available liquid water required for life is available there

despite the apparent dryness of the planet.

The other Viking life detection experiments were designed to test other specific

hypotheses of possible forms of microbial life on Mars. The Gas Exchange (GEx) life

detection experiment was designed to test for microbial life exposed at first to the

addition of humidity only, and then to immersion in a “chicken soup” of organic nutrients

and supplements. The Pyrolytic Release (PR) experiment was designed to test for

microorganisms that photosynthetically incorporated atmospheric CO2 and/or CO in a

simulated Mars environment without any addition of water. NASA said its selection of

life detection experiments was designed to test different possibilities for life, and that,

were there life on Mars, it would likely respond to only one of the experiments, if any.

Science

The LR thesis was based on the belief that early Mars and Earth possessed similar

environments, each of which were subject to the natural production of Miller-Urey type

organic compounds. These compounds were then available for the genesis of life,

participated in its evolution, and remain to participate in its metabolism today. Therein

lies the presumption of the similarity of the two planetary metabolisms. The LR was

planned to detect life by monitoring for this metabolism over a long period of time, rather

than to seek a “biosignature,” or snapshot, of some particular life-indicating molecule.

Since life is the most complex substance we know, the detection of any biosignature

molecule could suffer by the application of Occam’s Razor. That discriminator of truth

would conclude that the detected substance could more readily have evolved abiotically

than to have required the genesis of life for its production. Most biologists today believe

that the progression of biological compounds had to arise abiotically to be incorporated

into the genesis of life.

The LR married the use of Miller-Urey compounds with a simple and universally used

test for microbial contamination of water or food, and introduced trace radioisotopes to

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enhance the sensitivity of the method. That test13

is still used by health authorities around

the world in determining the presence of E. coli in a sample. The Miller-Urey

compounds were added to the nutrient solution to broaden its appeal to alien microbial

species. Then, all the compounds were tagged with 14

C. This enabled initial detection14

of metabolism in about 30 minutes as opposed to the 48 to 72 hours required by the

Standard Method for the generation of some 1.7 x 109

cells from an inoculum of one in

order to produce a visible bubble of gas. The likely sparse population of cells in the cold

and dry Martian soil, and the precariousness of the landed spacecraft and its vulnerable

communication with Earth made the sensitivity provided by the 14

C highly advantageous.

Final selection of the LR compounds was sodium formate, sodium lactate, glycine,

alanine and calcium glycolate. All are Miller-Urey compounds. In addition, each was

tested with a very broad range of microbial heterotrophs and phototrophs in soils, pure

cultures and mixed cultures. Several thousand tests were made during the development

program. In every case, where life was present, a positive response was obtained. In

some cases, classical methods used for comparison failed to respond while the more

sensitive LR did respond. However, the elimination of the LR response by application of

the control measure showed that the sample had contained life.

Inasmuch as the reason for the preferences of left-handed amino acids and right-handed

carbohydrates by our form of life is unknown, it was originally proposed to send

duplicate LR instruments so both handednesses could be offered separately in case the

Martian life forms exhibited a different handedness from ours. Weight and cost

constraints prohibited this, so both left- and right-handed forms of those LR compounds

that occur in isomeric forms were included in the nutrient. In order to minimize the

possibility of toxicity, the concentrations of these compounds were kept at only 2.5 x 10-4

molar each. For the same reason, each of the 14

C labeled compounds was kept at less

than 12 uCi/ml, with each carbon uniformly labeled with 14

C at 2 uCi/C. Table 1 shows

the makeup of the Viking LR nutrient solution. It was assumed that any living organisms

would be gaining all the supplements needed from the soil, so none was added. Also, no

buffer was used so as not to upset the prevailing pH. Thus, every effort was made to treat

any life present as gently as possible and merely to insinuate a revealing signal into its

normal life process.

TABLE 1. The Viking LR Nutrient Solution

Substrate

Structure and label position (*)

Concentration

µCi ML

-1*

Specific Activity (Ci/Mole)

14C-glycine

14C-DL-alanine

14C-sodium formate

14C-DL-sodium lactate

14C-calcium glycolate

NH3·*CH2·*COOH *CH3·*CH(NH3)·*COOH H*COONa *CH3·*CHOH·*COONa (*CH2OH·*COO)2Ca

2.5 × 10-4

M 5.0 × 10

-4M

2.5 × 10-4

M 5.0 × 10

-4M

2.5 × 10-4

M

4 12 2 12 4

16 48 8 48 16

*Total=34 (6.8 × 10

-7 dpm ml

-1)

5

In early application of the LR, several mg of a suspected sample were placed into 10 ml

of 14

C-labeled nutrient solution. Air was bubbled through the solution and exited through

a tube terminating in an open-ended holder containing a paper pad moistened with

saturated BaOH2 solution. Any CO2 in the gas passing through the pad was trapped by

the BaOH2. Every 15 minutes the pad was replaced with a fresh one. The pads so

collected were then dried and counted for radioactivity. The radioactivity was plotted

cumulatively as a function of time. The resulting curve showed the typical microbial lag

phase before the on-start of the sharp rise attributed to exponential growth. On one field

trip, a single drop of the nutrient solution was placed directly on the ground. The spot

was immediately covered with an inverted planchet that contained a paper absorbent pad

moistened with a saturated solution of BaOH2. Every fifteen minutes the planchet was

replaced with a fresh one containing a newly moistened pad. When the planchets were

dried and counted for radioactivity, it was surprising to see that no lag phase had

occurred. The response was immediate. Figure 2 compares the “wet” and “moist” modes

of performing the LR. From then on, the moist mode was adopted.

Fig. 2. Comparison of “Wet” and “Moist” Modes of LR.

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Not knowing what the optimum moisture content might be for Martian microorganisms,

it was decided to inject a 0.115 ml dose of nutrient solution onto the center of

approximately 0.5 cc of soil sample in the 3.5 cc cylindrical chamber with a 2 cm

diameter. In this way, chromatographic action provided a range of moisture content

starting with liquid at the center and progressing to minimal moisture at the periphery of

the sample. Other environmental conditions chosen for the experiment were a

temperature of 10o C ± 2

o C, Martian atmosphere, a helium overpressure to 85 mb to

assure liquidity in the event the Martian atmosphere were below the triple point,

darkness, and a seven-sol test cycle. The schematic for the LR instrument test cell is

shown in Figure 3. One major advantage of this simple experiment and instrument is

that the signal appears in a gas phase, readily rising out of the liquid phase of the nutrient

solution. Thus, there is no problem in differentiating the signal from the radioactivity of

the mother liquor. This allows for full utilization of the extraordinary sensitivity of the

radioisotope method.

FIG. 3. Schematic of Labeled Release Test Chamber.

While the Standard Method does not require a control, it was felt that a control would be

important for acceptance of the test performed under many unknown conditions on an

alien planet. Initially, the control used was a broad-spectrum antimetabolite, Bard-Parker

Germicide. When a positive response was obtained from a soil sample, a duplicate

sample was treated with Bard-Parker solution and then tested by the LR method. The

germicide was potent enough to significantly reduce the response from viable soils or

cultures. No such reduction would indicate a chemical had been responsible for the

initial positive response. NASA decided the control was a good idea, but asked that it be

changed to the application of heat rather than an antimetabolite, feeling the former was

more universal and more likely to be effective on possible Martian life. NASA proposed

treating the control sample at 160o C for three hours, allowing it to cool and testing it in

the LR instrument. That control method was adopted.

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A paper15

was published making the prediction for the experiment testing the theory for

Martian life as seen in Figure 4. The predicted LR test and control curves were based on

testing of terrestrial soils and cultures as modified by then current knowledge of Martian

environmental conditions.

Fig. 4. Pre-Mission Predicted LR Response from Microbial Life on Mars.

Thus, in classical scientific fashion, the thesis and predicted outcome of an important

experiment were in place.

Instrumentation

The experiment took some 20 years to develop and perform. The first ten years were

spent in developing and verifying the scientific method with the senior author as PI under

contract from NASA to Spherix (originally “Biospherics”). Based on the results, the

method was chosen for inclusion in the Viking Mission from among many submitted to a

NASA-designated national selection committee. The senior author was named

Experimenter when the Viking Mission was created and the LR chosen as a flight

experiment. The co-author then joined as Co-Experimenter. There followed ten more

years of improvement of the method, development of the instrument, testing, and

manufacture of the flight instrument. Over the entire twenty-year development period,

the LR was performed thousands of times, on pure cultures, mixed cultures, heterotrophs,

phototrophs and soils, many of which were provided by NASA from harsh environments

around the world. Field tests were also made in which working models of the instrument

were taken to extreme environments, e.g.: Antarctica, White Mountain above timber line,

Salton Sea flats, Death Valley sands, all of which responded positively. Samples of

naturally sterile soils (Moon, Surtsey, and one Antarctic sample) tested negative, showing

the validity of LR in detecting sterile samples. Not once in all these laboratory and field

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tests did the LR produce a false positive or false negative response when compared to

standard methods by which it was checked. On some occasions, the LR was positive and

the standard method was negative. In these cases, the LR control showed that the sample

had contained living organisms not detected by the standard method. By the time it was

sent to Mars, the LR was completely vetted and merited a high degree of confidence.

The development of the Viking LR instrument was jointly performed under contracts to

Spherix and TRW, Inc. TRW constructed the actual flight hardware. During the final

phase, Dr. Straat lived near the TRW plant at Redondo Beach and worked with the

engineers there to insure accuracy in conveying the science into the hardware. A Test

Standards Module (TSM) of the instrument, duplicating the critical flight parameters and

experimental conditions, was constructed and operated at TRW and later moved to the

NASA Ames Research Center. Shown in Figure 5, this was used to test the various

development stages of the experiment, and to facilitate their incorporation into the flight

instrument.

Fig. 5. Labeled Release Test Standards Module. The Test Standards Module contains

Labeled Release test cells, detectors, nutrient reservoir, valves, and heaters which are

essentially identical to flight components. The instrument was operated by manual

manipulation of the valves and heaters to perform an entire flight sequence. The location

of the flight components is indicated by arrow in the left photograph. These components,

enlarged in the right photograph, are covered during an experiment with a bell jar that

was supplied with a simulated Martian atmosphere. The temperature of the test cell was

regulated to obtain isothermal or diurnal temperature patterns, as desired.

TRW made a final test of the LR experiment on a California (“Aiken”) soil under Mars

experiment conditions in a flight instrument called SN103. This was performed to verify

the fitness of the project, and for comparison with any possible Martian result. Figure 6

presents the results from SN103 on California soil held for three days under Martian

environmental conditions before the sample was inoculated. The results proved prophetic

in magnitude to those obtained on Mars, as seen in Figure 8, although more of the gas

emission occurred later in the active cycle than on Mars.

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Fig. 6. SN103 Results from California (“Aiken”) Soil Under Martian Conditions.

The flight instrument contained four incubation cells of 3.5 cc volume placed in a

carousel that rotated such that each cell could be positioned to receive a soil sample

delivered from the lander distribution box. After sample reception, the LR culture

chamber and the head end of the cell were pressed together with an intervening gold seal

to prevent any gas leakage. The counting chamber contained two solid state beta

detectors. Any gas evolved from the culture rose to the counting chamber through a

narrow 13” tortuous tube which prevented any radioactive dust or aerosol raised by the

injection of the nutrient solution from reaching the counting chamber. The detectors

measured the amount of radioactivity, hence gas, evolved in the culture chamber and

rising into the counting chamber. Measurements were made at an initial frequency of

four minutes for two hours, and then every sixteen minutes for the rest of the test cycle.

The LR hardware contained all necessary plumbing, operated by eight miniaturized

solenoid valves, to manipulate the liquid nutrient and gas components. This included

helium gas for purging the radioactive nutrient of any radioactive gas formed through

self-degradation of the radioisotopes during the long journey from Earth. The culture

chamber also contained a heater and temperature sensors in the top head end for

controlling and recording the temperature during the runs and during heat sterilization.

The LR flight instrument had to fit into about a quarter of a cubic foot. Figure 7 contains

a diagram of the three Viking life detection instruments packaged together.

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Fig. 7. Viking Mission Life Detection and GCMS Instrument Packages.

The Vikings contained one additional instrument that turned out to be of overriding

importance in interpreting the LR results. It was the Molecular Analysis instrument that

consisted of a gas chromatograph-mass spectrometer (GCMS) illustrated in Fig. 7. Its

function was to identify the organic compounds that were expected to have accumulated

on Mars the same as they had on Earth.

The LR on Mars

Vikings 1 and 2 were launched August 20, and September 9, 1975, and landed safely on

Mars July 20 and September 3, 1976, respectively.

Viking 1

On July 30, 1976, sol 10, Viking 1 ran the first LR sample on Martian soil. The sample

was taken from soil that had been scooped up on sol eight by the sampling arm. It

consisted of surface material dredged to a depth of about four cm. The sample had been

stored in the distribution box at approximately 10o

C for the two intervening sols. The

LR response was immediate and strongly positive. After the run was complete, the

critical control phase of the experiment was performed on a duplicate sample of the same

soil. The response was negative. Figure 8 shows these initial Mars LR test and control

results. These results satisfied the pre-mission criteria accepted by NASA for the

detection of life.

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Fig. 8. First Mars LR Test and Control Results.

However, doubts were cast when the experiment was continued past its seven-sol cycle,

after which additional injections of nutrient failed to produce resurgences of gas, but,

instead, caused immediate depletions in the headspace gas, as seen in Figure 9, VL1

cycle 1 extended.

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FIG. 9. Effect of Second Injection of Nutrient on VL1 Cycle 1.

While a resurgence of gas would have been more assertive of biology, the observed effect

could be attributed to death of the microorganisms and reabsorption of CO2 by the

alkaline soil when wetted. As seen in Figure 10, a NASA-bonded test soil, Antarctic 664,

with a pH of 8.1, had acted similarly in the TSM when the LR nutrient dose was repeated.

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FIG. 10. Antarctic Soil 664 LR Response to Second Injection of Nutrient.

In keeping with scientific protocol to verify a positive result, the third run, VL1 cycle 3,

was a duplicate of the first, but with a fresh sample of the soil taken from the same area

as the first. The response was positive, validating the first. Second and third injections

produced the same re-absorption of gas as seen with VL1 cycle 1. The fourth and final

LR run at Viking site 1 was a double injection on a sample from the same area that had

produced the positive response in VL1 cycle 1. However, at the time of the injection for

this VL1 cycle 4 run, the soil had been stored in the distribution box, in the dark, open to

the Martian atmosphere, at temperatures ranging between 10o C and 26

o C for 141 sols.

Two nutrient injections were made, about three hours apart, in order to allow a response

from the first before administering the second. However, each injection resulted in a

negative response. Long-term storage of an active soil under modest environmental

conditions had inactivated an initially active sample. This raised the possibility that,

isolated from their environment, microorganisms had perished during the storage period.

Since the soil had remained active after being held three days (in a previous run) at

approximately 10o C prior to first injection, it seems more difficult to propose a chemical

that survived those conditions for two or three days, but not for the longer period. If the

loss of activity were simply a matter of something evaporating from the soil sample, that

substance would likely have evaporated from the half cc sample during the three days it

was held at approximately 10o C before injection.

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Isolated in a test tube and maintained at a temperature above freezing, a bacterial culture

will lose all vitality over time as short as a month. Hence, cultures are preserved by

desiccating them and holding them at temperatures below the freezing point of water.

The sample held in the Viking distribution box was isolated from its natural environment.

Thus, possibly deprived of nutrients at temperatures above freezing, the organisms might

have died.

All initial Viking site 1 test and control responses are shown in Figure 11.

FIG. 11. All First Injection Cycles of VL1. A fresh sample was used for the active

sequences of cycles 1 and 3 whereas the sample used for active cycle 4 was stored for

approximately 141 Sols at 10-26°C prior to use. For cycle 2, a stored portion of the same

sample used for cycle 1 was heated for 3 hours at 160°C prior to nutrient injection. All

data have been corrected for background counts observed prior to nutrient injection.

Despite duplication of the positive result, the negative control and the biological leaning

of the storage data, doubt remained. It increased greatly when the GCMS failed to detect

any organic matter in the Martian soil or atmosphere.

Viking 2

With the positive LR result duplicated at Viking site 1, the traditional scientific procedure

was followed by attempting to replicate the entire experiment at Viking site 2, some

4,000 miles distant. There, the first sample produced the positive response shown in

Figure 12, essentially duplicating that of VL1 cycle 1.

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FIG. 12. First LR Response at Viking 2 Site.

Since the VL1 LR 160o C control cycle results were not accepted as evidence that

Martian life had been detected , a meeting of the Biology Team was called to discuss

another control. All Members agreed that, if a control sample heated to 50o C for three

hours proved negative, it would be accepted as confirmation for the detection of life. The

engineers, responding to this appeal for an ad hoc experiment, attempted the 50o C

heating, but they reported that the temperature achieved was 51o C. This resulted in a

peculiar response, VL2 cycle 2, consisting of a series of small, sharp spurts of gas over

time, each spurt then being reabsorbed as in the case of repeated injections. Each small

spurt showed kinetics similar to those produced by positive runs upon repeated injections,

but on a much smaller scale.

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FIG. 13. VL2 Cycle 2 LR Response.

It seemed as if numerous attempts had been made to react with the nutrient, but each

attempt quickly succumbed and the wetted alkaline soil then quickly absorbed the gas

that had been emitted. Added together, the pulses totaled only 10% of the amount of gas

evolved in the positive response. The demise of the active agent when heated to only 51o

C for three hours seems more likely attributable to biology than to chemistry. The

possible biological attribution of the pulses adds to this likelihood. The engineers

checked the instrument for a possible intermittent gas leak to account for the loss of gas

after each of the spikes. They reported no problem, and, indeed, subsequent experiments

worked well, supporting that there was no instrument problem. The Team’s agreement

notwithstanding, a biological origin of the positive result was still denied.

It was then contended that the positive responses were attributable to “activation” of the

soil by the intense flux of UV light impacting the surface of Mars. The activated soil, it

was contended, reacted with the nutrients yielding radioactive gas, a false positive for

life. Further discussion with the engineers made it possible to test this theory directly on

Mars. Just at dawn, the sampling arm moved a rock and obtained a sample that the rock

had been shielding from UV light for eons. After two days in the test cell, that sample,

VL2 cycle 3, was tested. It proved strongly positive, nearly as active as the previous

positive samples, dispelling the UV theory.

An attempt was then made to clarify the confusion instilled by the unique, sporadic

response of VL2 cycle 2. A fresh soil sample was heated, aiming for 50o C as before.

This time, however, the temperature attained was only 46o C. The result was very

different from that of the first attempt. The LR VL2 cycle 4 response to this treated

17

sample produced essentially the same kinetics as a positive sample, but with only about

30 percent of its amplitude.

FIG. 14. VL2 Cycle 4 LR Response.

This engendered considerable discussion as to whether its origin was chemical or

biological. It was noted, however, that comparison of the 46o C and 51

o C response

curves is reminiscent of the 37o C v 44

o C responses used to differentiate between E. coli

and the coliform group. Only the E. coli survive at 44o C. It is possible that

microorganisms in the Mars sample heated to 51o

C primarily died, while at least some of

those heated to only 46o

C survived. A chemical agent, displaying such a major change

in sensitivity to a temperature difference of only 5o C is difficult to propose, and none has

been put forth.

VL2 cycle 4 used the last of the LR culture chambers. However, with nutrient solution

still available, an additional run was improvised. This would have to be done in a culture

cell that already contained soil from a previous run. At this point, the Martian winter

approached Viking site 2, and sampling activity was halted for fear of damaging the

sampling arm. Rather than wait for spring, risking possible damage to the spacecraft or

loss of communication, it was decided to re-test the active sample used for the VL2 cycle

3 that had, by then, been held in the sample distribution box for 84 sols at approximately

10o C. As seen in Figure 15, showing all first injection VL2 responses, storage of the

sample used for VL2 cycle 5 had inactivated the soil, producing a negative response.

18

FIG. 15. All First Injection VL2 LR Results.

Moreover, VL2 cycle 5 showed that, although the active agent remained active when held

two sols at 10±2o C (VL2 cycle 3), it succumbed upon long-term storage at approximately

10o C.

Characteristics determined for the active agent discovered in the surface material of Mars

are listed in Table 2. All cycles of VL1 had two injections except cycle 3 that had three.

All cycles of VL2 had two injections except cycle 5 that had only one.

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TABLE 2

Characteristics of Active Agent Detected by Viking LR Experiment

1. Produced positive response when inoculated with nutrient solution, similar in

kinetics and amplitude to responses produced by LR test of a number of terrestrial

viable soils.

2. Inactivated upon heating to 160o C for three hours, similar to terrestrial tests of

active soils.

3. Heating to 51o C for three hours produced sporadic series of small responses,

totaling approximately 10% of a positive response.

4. Heating to 46o C for three hours produced response similar to positive response

but reduced 70% in amplitude.

5. Inactivated upon two months’ isolation in soil distribution box in dark at

approximately 10o C.

6. Activation of soil not caused by UV exposure.

7. Added injection of nutrient solution to positively responding soil caused

approximately 25% of gas already evolved to disappear from detector cell

(probably re-adsorbed into soil), gradually to re-evolve.

Comparison of Martian and Terrestrial LR Responses

The predicted Martian LR response shown in Figure 4 shows a remarkable similarity to

the Martian LR response shown in Figures 11 and 15, thus fulfilling the fundamental

requirement of an experiment for acceptance of the theory behind it. Figure 6, the SN103

result of the California soil held and then tested under Martian conditions also compares

favorably with the Martian results. Figure 16 presents the response from an LR test16

of

scrapping from the interior of an endolithic Antarctic rock. It bears a similarity to the

Martian responses although showing greater gas production later in the cycle than do the

Mars results.

FIG. 16. LR response from Endolithic Antarctic Rock Scrapings. The responses from 0.24 g of material scraped from endolithic microorganism-populated band 10 mm beneath

surface of Antarctic rock, (−−−−) active, and (−−−−) heat-sterilized control. Experiment was

20

performed at room temperature by LR “getter” technique and results are normalized to counting efficiency of

LR flight instrument.

A number of terrestrial viable soils has shown responses quite similar in amplitude and

kinetics to those from Mars. Some are shown in Figure 17 illustrating the range of

responses.

Fig. 17. Viking LR Response Among Terrestrial Viable Soil LR Responses.

As seen, the Viking responses are among those of lower amplitude, including responses

from Barrow and Palmer, Alaska and Antarctic soil 726.

No actual or theoretical non-biological entity meeting the constraints listed in Table 2 has

yet been identified. However, each of these constraints has been shown, as cited herein,

to be met by terrestrial microorganisms of one species or another. This includes the re-

absorption of emitted gas upon second injection of nutrient, death from isolated long-

term storage, and differential susceptibility to small differences in incubation

temperatures.

Challenges to Biological Interpretation

All data from VL1 and VL2 were thus either indicative or supportive of life. However,

the consensus rejected such a conclusion. It was instead contended that the positive

responses were from hydrogen peroxide theorized to be photo-chemically on Mars, or

some oxidant derivative therefrom. Hydrogen peroxide was presumed to rain from the

sky and coat the surface of the planet, where it might be photo-chemically or otherwise

transformed into another strong oxidant. This oxidant was said to be responsible for

destroying any organic matter and, therefore, life, thus explaining the results of the

FIG. 6

Comparison of Terrestrial and Mars LR Active Responses

0.00

10000.00

20000.00

30000.00

40000.00

50000.00

60000.00

70000.00

80000.00

0.00 5.00 10.00 15.00 20.00 25.00

Time (hr)

Cu

mu

lati

ve

Gas

Evo

lve

d (

cp

m)

Barrow, A laska #2

Palmer, A laska

Yuma, Arizona

Needles, California

Sawyers Bar, California

Dunes West o f Yuma, California

Red Rock, Arizona

El Centro , California

Sandy, M aryland

Viking Lander 1 Cycle 1

SN103, A iken, California, M ars Box

Antarctic Endoliths

Antarctic #726

Aiken Soil, Terrestrial Conditions

All data normalized to f light LR

instrument counting eff iciency for

direct comparison

21

GCMS and the LR. This theory was maintained despite the fact that the Viking Magnetic

Properties experiment17

had shown that the surface of Mars was not highly oxidized.

Were the surface material completely oxidized, it would not have been magnetic, and

would not have stuck to the magnets of that experiment. Figure 18 shows that, when the

experiment’s magnets touched the surface, a heavy coat of dust stuck to the magnet.

FIG. 18. Magnetic Properties Experiment Shows Mars Surface Not Completely

Oxidized.

This demonstration that the Martian regolith was not fully oxidized was subsequently

confirmed by Pathfinder as seen in Figure 19 that shows minerals in various states of

oxidation.

22

Fig 19. Pathfinder Confirms Absence of Strong Oxidizing Coating on Mars

NASA reported18

that Curiosity, at its location, similarly showed that the surface of Mars

is not fully oxidized. Further, “A fundamental question for this Mission is whether Mars

could have supported a habitable environment,” said Michael Meyer, lead scientist for

NASA’s Mars Exploration Program. “From what we know now, the answer is yes.”

The report also says, “Scientists identified sulfur, nitrogen, hydrogen, oxygen,

phosphorus and carbon – some of the key chemical ingredients for life – in the powder

Curiosity drilled out of a sedimentary rock near an ancient stream bed in Gale Crater on

the Red Planet last month.” In addition, the reporting scientists discovered a mixture of

oxidized, less-oxidized, and even non-oxidized chemicals capable of supporting an

energy gradient of the sort many microbes on Earth require for survival:

"The range of chemical ingredients we have identified in the sample is impressive, and it

suggests pairings such as sulfates and sulfides that indicate a possible chemical energy

source for micro-organisms," said Paul Mahaffy, a NASA official.

The several demonstrations that Mars was not coated with a strong oxidant did not deter a

series of proposals to that effect from many authors. Over the 38 years since Viking,

some 40 such chemical, physical or otherwise non-biological explanations of the LR data

have been published, even as recently as this year. Each explains away, by experiment or

theory, the LR positive response. Yet, not one of them has reproduced all the stringent

control data. This fact applies also to the finding19,20

of perchlorate on Mars, which the

authors claim explains the failure of the Viking GCMS to detect organics. The texts of

these papers conveniently do not mention the failure to match the control data, but still

claim to have reproduced the LR Mars results. Interestingly, however, no one has

challenged the performance of the LR instruments or the validity of the data they

23

produced, or their ability to find microorganisms in terrestrial soils. Only the

interpretation of the LR data from Mars is disputed.

In addition to the strong oxidant, there were two other primary claims to a non-biological

origin of the LR positive responses. These were the continued acceptance of the lack of

organic matter as reported by the Viking GCMS, and the belief in the absence of life-

essential liquid water in the Martian soil.

Organic Matter

A major difference in sensitivities could explain the seemingly disparate results of the

Viking LR and GCMS. The LR had detected as few as 20 living cells in its test program.

The Experimenter of the Viking GCMS emphasized that the instrument was not a life

detector. He pointed out that the GCMS required the organic matter contained in

millions of bacterial cells to elicit a response. Since he felt that the microbial population,

if any, on Mars would be scarce, the GCMS would rely on the organic content of millions

of dead cells preserved in the environment. Thus, as the author has pointed out, both the

Viking LR and GCMS could have reported correctly, there being enough living cells for

the positive LR response, but too few dead and living cells for the GCMS. In addition, as

cited above, it has been proposed that organics in the Martian soil were oxidized by the

perchlorates when the mixture was heated to 500o

C in the GCMS analysis. Thus, the

rejection of the LR results because of the GCMS’ failure to find organic matter has been

removed.

Liquid Water

The Viking LR experiment was based on the presumption that Martian microorganisms

would operate on an aqueous biochemistry. Thus, liquid water would be essential to their

existence. Viking 2 provided data establishing the presence of liquid water in the surface

regolith. As reported21

, that evidence was in the form of the temperature of the surface

rising with sunlight up to 273 K, and then pausing. This is the unique signature for ice

absorbing heat without increasing its temperature while turning into liquid water. As

cited above, the Pathfinder Mission found that temperatures at the surface of Mars

frequently exceeded freezing, rising into the 20 degrees C. The Odyssey Mission orbiter

found22

hydrogen-containing material within several cm of the surface over wide areas of

the red planet. However, this and a number of additional disclosures of liquid water,

including that incontrovertible data from Curiosity showing water vapor evolving when

the sample was heated to just above freezing, as seen in Figure 20, were ignored along

with their implication for extant life. It is now seen that the Martian surface material

contains from two to several percent liquid water. This is considerably more than the 0.9

percent found in the top sands of Death Valley that supports a thriving microbial ecology

that was readily detected by the LR. Curiosity data have led to the NASA statement that

its principal mission objective has been accomplished: finding that the past environment

of Mars was habitable. However, no mention regarding current habitability was made.

24

FIG. 20. Curiosity Data Show Liquid Water in Mars Surface Material.

The Evidence in Summary

Evidence that the LR detected life on Mars consists of the following elements:

1. Positive responses from soils tested by a universally accepted microbiological

method augmented to improve sensitivity and broaden appeal.

2. The duplication of the Mars VL1 LR test data at Viking 1.

3. Replication of Mars VL1 data at VL2 some 4,000 miles distant.

4. Mars LR responses fall within range of responses from a variety of terrestrial

microbes.

5. Positive Viking LR responses were similar to the Mars simulation test SN103

performed on California soil.

6. Negative response from the 160o control regimen approved by NASA to

confirm the detection of life.

7. Additional, ad hoc low temperature controls rendering a non-biological cause

difficult, especially because of inactivation of the active agent upon

sequestered storage.

This evidence is supported by:

1. Failure of any scientifically sustainable experiment or theory, from the many

tried or proposed over the past 38 years, to provide a non-biological

explanation of the LR data.

2. Odyssey’s finding of water ice or liquid water within several cm of the surface

over wide expanses of Mars.

3. The finding that the temperature of the atmosphere immediately above the

surface of Mars frequently reaches the 20o

C range, sufficient to provide liquid

water from the near-surface ice.

25

4. Acceptance of reasons for the failure of the Viking GCMS to detect organic

matter.

5. Death of terrestrial bacteria upon sequestered storage.

6. The great expansion in knowledge of the terrestrial domain of life into

extreme environments, some of which are as inhospitable as those at Viking 1

and 2 sites.

7. The finding that Mars was habitable in the past, with no explicit reason that it

is not still habitable.

8. Realization of the possible interplanetary contamination by microbes carried

in ejecta expelled by meteorites.

9. The difficulty in conceiving of a sterile Mars in light of the new knowledge of

habitability, extremophiles and the possibility of interplanetary contamination.

The above summation indicates that Carl Sagan’s challenge, “Extraordinary claims

require extraordinary evidence,” as often applied to the Viking LR, has now been met.

The extraordinary claims have become ordinary, and the evidence has become

extraordinary.

Baye’s Rule

Were an objectivist’s view of Baye’s Rule of Inference23

applied to the increasing

knowledge about Mars since Viking, the probability that the LR detected life would be

significantly increased. Assigning weighting factors to each of these new bits of

information for a rigorous application of Baye’s Rule is difficult and would obviously be

inexact. Similarly, assessments would have to be assigned for each of the non-biological

explanations. However, allowing only a very small probability for the existence of life

on Mars at the time of Viking, 1976, the supportive findings since, together with the

difficulties with the non-biological attempts to explain away the LR data, are such as to

instill a significant rise in the Baye’s Rule’s present confidence that the LR detected life.

The fact that nothing antithetical to life on Mars has been discovered by all the post-

Viking research has significant impact on the present probability.

NASA Turning Point

In 2012, following publication24

of a new and independent approach to analysis of the

Viking LR data that indicated it had obtained a biological response, NASA’s Director of

the Mars Exploration Program was quoted25

as saying NASA would now seek direct life

detection experiments, including the use of Curiosity’s hi-resolution camera that can

resolve features as tiny as 12.5 microns to seek possible growths on rocks originally

reported26

with colored patches in Viking images.

Curiosity and Life on Mars

The rover of the Mars Science Laboratory, “Curiosity,” has now been on the surface of

Mars for nearly two years. When NASA announced that the mission carried no life

detection experiment or capability, the senior author laid claim27

to Curiosity’s abilities to

confirm the detection of life by the Viking LR. Specifically, the referenced paper

predicted: 1. that Curiosity would find liquid water in the surface material of Mars,

confirming Viking’s original discovery, and 2. that Curiosity’s liquid extraction method

26

of detecting organic matter would find complex organics in the soil. In addition, the

paper projected that the Hand Lens camera might detect biological features in close-up

examination of the greenish colored spots seen on many rocks in images transmitted by

Curiosity, similar to those seen28

in images of Viking site rocks.

The prediction of finding liquid water has materialized as seen in Figure 20. No results

of the liquid extraction of organic matter have been reported, nor have any close-up

images of the rocks been shown. Repeated requests to NASA for Curiosity data on liquid

water, complex organics and hi-resolution, close-up images of rocks, including a

request29

under the Freedom of Information Act (FOIA), have been answered with

denials that the data exist. It is contended that the liquid extraction method for organics

has not yet been run, that all images taken by Curiosity have been published. It seems

strange that eager scientists would hazard a two-year wait before executing important

experiments. Viking performed its critical experiments as soon as possible for fear of

losing communication with the spacecraft or of some accident or malfunction to systems

exposed to such a hostile environment. Nonetheless, the prediction for Curiosity’s

finding complex organics in support of the Viking LR conclusion remains, as does the

expectation for possible biological evidence in hi-resolution images.

Discussion

This paper attempts to show, in simple narrative fashion, how the Viking LR experiment

was planned and executed in strict conformance with classic scientific principles guiding

exploration and discovery. The hypothesis was formulated that microorganisms existed

on Mars operating under an aqueous biochemistry similar to that on Earth. An

experiment was conceived to test the hypothesis. The experiment was performed. It

produced positive results. The experiment was successfully duplicated. The entire

experiment was then replicated at a site 4,000 miles distant. It was again positive. Its

duplication was also positive. A variety of ad hoc experiments further supported, or were

consistent with, a biological interpretation of the Viking LR data.

The first Viking LR experiment and control satisfied the pre-mission criteria accepted by

NASA and participating scientists for the detection of life. Had it been performed on

Earth, this experiment would readily have been accepted as having detected life. The

remainder of the planned LR experiments and the ad hoc experiments on Mars supported

or were consistent with the conclusion that microbial life had been detected. However,

largely because of the failure of the Viking GCMS to detect any organic matter, the

biological origin of the LR positive signals was not generally accepted. This was despite

the large discrepancy in the sensitivities of the two instruments that could readily explain

the different interpretations of their results. Many other barriers to a biological

explanation have since been raised. A wide variety of non-biological explanations of the

Viking LR results have been proposed, but none has duplicated the test and control data

generated by the LR on Mars. When pressed for reasons for having rejected the

biological interpretation of the Viking LR results, NASA and other scientists have said

that the consensus was against such a conclusion. This ignores the fact that no important

discovery was met with a consensus. If so, it would not have been a discovery. Many

key discoveries have taken scores of years before wide acceptance. The authors believe

27

that such a price has now been paid for acceptance of life on Mars, and ask for

reconsideration in the light of the support that has emerged since Viking.

Beginning with Viking and increasingly over the years since, some scientists

knowledgeable in the field have expressed their opinions on the LR Martian results to the

senior author directly or in public statements. Thinking that other scientists might be

swayed in their opinions by knowing how these experts have evaluated the Viking LR

data, we have prepared a list of those respondents. Depending on what they had said, the

scientists were listed in the category of “Has Detected Life,” or “May Have Detected

Life.” That list was then emailed to those named in it, and permission to include his or

her name was requested. The updated list, with each name newly approved for use in this

paper, is shown in Table 3.

28

TABLE 3. Scientists Stating the Viking LR Detected or May Have Detected Life.

Life on Mars Was Detected by the Viking LR Experiment

NAME INSTITUTION EMAIL

Giorgio Biancardi Siena U., Siena, Italy GBianciardi@yahoo.it

Francisco Carrapico U. Lisbon, Lisbon, Portugal F.Carrapico2fc.ul.pt

Mario Crocco Ministry of Health, Argentine Republic, Buenos Aries, Argentina

Postmaster@neurobiol.cyt.edu.ar

Barry DiGregorio U. Buckingham (UK) Barry.Dig@verizon.net

Richard B. Hoover U. Texas, Athens, (NASA ret.) Entogonia@aol.com

Joop M. Houtkooper Justis-Liebig U., Giessen, DE JoopHoutkooper@gmail.com

Gilbert Levin Arizona State U., LR Experimenter Gilbert.Levin@ASU.Edu

Ron Levin Lockheed-Martin RonLevin@cox.net

Robert Lodder U. Kentucky Lodder@uky.edu

Joseph Miller Am. U. Caribbean, Sch. Med. JMiller2@aucmed.edu

John Newcomb NASA, Viking Manager (ret.) JNewcomb1@cox.net

Elena Pikuta Athens State U., Texas EVPikuta@gmail.com

Dirk Schulze-Makuch Washington State U. DirkSM@wsu.edu

Patricia A. Straat NIH (ret.), LR Co-Experimenter PStraat@comcast.net

Hans Van Dongen Washington State U, Spokane HVD@wsu.edu

Chandra Wickramasinghe

U. Buckingham, UK NCWick@googlemail.com

Life on Mars May Have Been Detected by the Viking LR Experiment

NAME INSTITUTION EMAIL

Ariel Anbar Arizona State U. Ariel.Anbar@asu.edu

Timothy Barker Wheaton College TBarker@wheatonma.edu

Steven Benner U. Florida SBenner@ffame.org

Paul Davies Arizona State U. Paul.Davies@asu.edu

Sergio Fonti U. Salento, Italy Sergio.Fonti@unisalento.it

Robert Hazen Carnegie Institution, DC Hazen@gl.ciw.edu

Bruce Jakosky U. Colorado Bruce.Jakosky@lasp.colorado.edu

Chris McKay NASA Ames Chris.McKay@nasa.gov

Richard Meserve Carnegie Institution, DC RMeserve@ciw.edu

Michael Mumma Goddard Space Flight Center Michael.J.Mumma@nasa.gov

Vincenzo Orofino U. Salento, Italy Vincenzo.Orofino@unisalento.it

John Rummel East Carolina U. (ex NASA) RummelJ@ecu.edu

Andrew Steele Carnegie Institution, DC ASteele@ciw.edu

Carol Stoker NASA Ames Carol.R.Stoker@nasa.gov

Mike Storrie-Lombardi Kinohi Inst., Pasadena, CA Mike@kinohi.org

Henry Sun Desert Research Inst., Reno Henry.Sun@dri.edu

29

Conclusion

A classic, rigorous test has been made of the hypothesis that Mars is inhabited by

microorganisms similar in their biochemistry to terrestrial life. Duplicates and replicates

of the LR experiment to investigate that theory have given strong or supportive evidence

for life on the red planet, with no incompatibilities with life found. The pre-mission

criteria for the detection life have been exceeded. The authors believe the evidence cited

herein establishes the existence of life on Mars. The absence of any tenable non-

biological challenge emphasizes this claim, but is not relied upon, Sherlock Holmes-

like30

, as the basis of this conclusion: “Once you eliminate the impossible, whatever

remains, no matter how improbable, must be the truth.” Further to the point, since

Viking, research into interplanetary microbial contamination has made it extremely

unlikely that Mars could be sterile. All signs now point to another major change in our

ancient anthropocentricity. We are not alone. A way to initiate this historic change

would be for all the evidence, pro and con, on the LR experiment and related issues

bearing on the question of life on Mars to be examined. The evidence would be

submitted to a panel of experts assembled by a major intellectual institution. The panel

should make a formal report on whether or not the evidence proves the existence of

microbial life on Mars. A positive answer would start the needed change in paradigm,

and spur further life detection experimentation. However, even a negative answer would

be very valuable. It would undoubtedly provide information that would influence

NASA’s planetary program, increasing its scientific return and providing significant

economic savings.

Epilogue

In the event the expert panel does not resolve the issue of life on Mars, it is proposed that

the next mission there carry the Chiral LR experiment31

. A schematic of the current

concept for that instrument is shown if Figure 21.

FIG. 21. Schematic of the Chiral LR Instrument.

30

This enhancement of the Viking LR would separately test for the chiral metabolism of

stereoisomer compounds selected as nutrients. All known life forms react only with L-

amino acids and D-carbohydrates. Chemicals cannot distinguish between stereoisomers.

The experiment could be deployed as multiple darts ejected from a landed spacecraft or

from orbit. Each small dart could contain different isomeric compounds for testing.

Duplicate darts could be included for verification and redundancy against the loss of one.

A variety of controls, thermal, chemical and physical, could be incorporated to support or

deny any positive findings. If only one of the isomers of a compound produced an LR

response, and it was confirmed by a control, this would be strong evidence for life. Were

the isomeric preference found to be similar to that on Earth, that would suggest the two

forms of life are related, perhaps by cross-infection, or by seeding from a third source.

However, if the response were to D-amino acids or to L-carbohydrates, it would

constitute strong evidence for an independent genesis of Martian life. Either way, the

discovery would mark the beginning of interplanetary comparative biology. Should Mars

and Earth show independent origins of life, this sample, small as it is, could be viewed as

statistical evidence for life having originated or having been distributed throughout the

cosmos.

Acknowledgements

Thanks are given to Dr. Paul Davies who kindly reviewed this work and made several

important suggestions, including reference to Baye’s Rule.

Dr. Ron Levin is thanked for his scientific support on the issue of liquid water on Mars,

and for his encouragement on the life issue itself, as well as for his preparation of the

PowerPoint figures in this presentation.

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