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Complexity Analysis of the Viking Labeled Release Experiments - Int'l Journal of Aeronautical & Space Science

Complexity Analysis of the Viking Labeled Release Experiments - Int'l Journal of Aeronautical & Space Science

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Reassessment of NASA's Viking experiments for life on Mars based on knowledge and findings since the 1970's. Finds that there is indeed life on the red planet. 'Deep analysis' includes: Signal vs. noise, entropy
vs.negentropy, periodicity vs. aperiodicity, order vs. disorder, etc.

By Giorgio Bianciardi, Joseph D. Miller, Patricia Ann Straat & Gilbert V. Levin
Reassessment of NASA's Viking experiments for life on Mars based on knowledge and findings since the 1970's. Finds that there is indeed life on the red planet. 'Deep analysis' includes: Signal vs. noise, entropy
vs.negentropy, periodicity vs. aperiodicity, order vs. disorder, etc.

By Giorgio Bianciardi, Joseph D. Miller, Patricia Ann Straat & Gilbert V. Levin

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Categories:Types, Research, Science
Published by: aynoneemouse on Apr 13, 2012
Copyright:Attribution Non-commercial


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The Korean Society for Aeronautical & Space SciencesReceived: November 19, 2011 Accepted: February 21, 2012
http://ijass.org pISSN: 2093-274x eISSN: 2093-2480
Technical Paper
Int’l J. of Aeronautical & Space Sci. 13(1), 14–26 (2012)DOI:10.5139/IJASS.2012.13.1.14
Complexity Analysis of the Viking Labeled Release Experiments
Giorgio Bianciardi*
Department of Patologia Umana e Oncologia, Università degli Studi di Siena, Via delle Scotte 6, 53100 Siena, Italy 
Joseph D. Miller**
Department of Cell and Neurobiology, Keck School of Medicine at USC, 1333 San Pablo St./BMT401, Los Angeles, CA 90033,USA jdm@usc.edu 323-442-1629
Patricia Ann Straat***
830 Windy Knoll, Sykesville, Maryland 21784
Gilbert V. Levin****
Beyond Center, College of Liberal Arts and Sciences, Arizona State University, Tempe, AZ 85287 
Te only extraterrestrial lie detection experiments ever conducted were the three which were components o the 1976 VikingMission to Mars. O these, only the Labeled Release experiment obtained a clearly positive response. In this experiment
Cradiolabeled nutrient was added to the Mars soil samples. Active soils exhibited rapid, substantial gas release. Te gas wasprobably CO
and, possibly, other radiocarbon-containing gases. We have applied complexity analysis to the Viking LR data.Measures o mathematical complexity permit deep analysis o data structure along continua including signal vs. noise, entropy  vs.negentropy, periodicity vs. aperiodicity, order vs. disorder etc. We have employed seven complexity variables, all derived romLR data, to show that Viking LR active responses can be distinguished rom controls via cluster analysis and other multivariatetechniques. Furthermore, Martian LR active response data cluster with known biological time series while the control datacluster with purely physical measures. We conclude that the complexity pattern seen in active experiments strongly suggestsbiology while the dierent pattern in the control responses is more likely to be non-biological. Control responses that exhibitrelatively low initial order rapidly devolve into near-random noise, while the active experiments exhibit higher initial order which decays only slowly. Tis suggests a robust biological response. Tese analyses support the interpretation that the VikingLR experiment did detect extant microbial lie on Mars.
Key words:
Astrobiology, extraterrestrial microbiology, Mars, Viking lander labeled release
1. Introduction
Te possibility o extraterrestrial lie has excited the humanimagination or hundreds o years. However, the rst (andonly) dedicated lie detection experiments on another planet were not perormed until the Viking Landers o 1976. Oneexperiment in particular, the Labeled Release (LR) experimento Levin and Straat [1-4] satised a stringent set o prior agreed-upon criteria or the detection o microbial lie on Mars (i.e. asignicant increase in evolved radioactive carbon-containinggas over baseline ater
C radiolabeled nutrient administrationto a Mars soil sample, and abolition o that response by pre-
This is an Open Access article distributed under the terms of the Creative Com-mons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution and reproduc-tion in any medium, provided the original work is properly cited. 
Corresponding author : E-mail : gbianciardi@unisi.it
Retired (NIH)
Giorgio Bianciardi
Complexity Analysis of the Viking Labeled Release Experiments
heating the soil to 160° C). However, controversy has reignedever since over these ndings. Until recently, chemicalinterpretation o the LR results has dominated but discoverieso Martian atmospheric methane [5, 6], sub-surace water iceon Mars [7], drops o liquid water at the Phoenix landing site[8], and the incredible hardiness o terrestrial extremophiles[9] have all led to the re-examination o the possibility o extant Martian microbial lie.In past work [10], we have shown that the “active” (gas-evolving) Viking LR experiments exhibited strong circadianrhythms in radiolabeled gas release. Tese oscillations rapidly grew in amplitude and regularity in the rst two sols (onesol=24.66 hr, a Martian solar day) o the active experiments toreach a near steady state o constant amplitude and period.Perhaps, this reects the synchronization o a population o microbes to the temperature cycle imposed by the Vikinglanders. When tested, heat-treated (control) samples o thesame soil showed a greatly attenuated rhythm, or no rhythm whatsoever. In the two experiments in which the active soilsamples were stored or several months beore administeringthe nutrient solution, rhythmicity was almost completely absent.
2. New Approach
 We now report a new methodological approach to thesedata, complexity analysis. Due to the high order present inbiological systems [11] , time series o biological variables, withtheir short- and long-range correlations, scale-invariance,complex periodic cycles, quasi-periodicities, positive andinverse “memory” and the like, exhibit behaviours thatare dierent rom the complete unpredictability o purerandom physical processes (white noise). Moreover, they are also distinguishable rom the trivially smooth landscapeo a completely predictable deterministic process, otenmaniesting themselves with icker (pink) noise (temporalscale statistical invariance) [12, 13]. We have now ound thata set o complexity measures (appendix#1 or denition)unambiguously distinguishes the active LR experiments, orportions thereo, rom various abiotic controls (p<0.001).Tese measures very strongly suggest, in agreement withterrestrial analyses, that the active LR experiments in alllikelihood detected microbial lie on Mars.
3. LR Results on Mars
Summary o initial analysesIn the thousands o tests that were conducted on a wide variety o terrestrial microorganism-laden soils in 20 years o testing beore and ater the Viking mission, radiolabeled gas,presumably CO
(or possibly CO
plus some other carbon-containing gas such as CH
) was produced by cellularmetabolism, always evolving immediately ater the injectiono the radiolabeled LR nutrient (e.g. Biol 5, see Methods).Heat-treated control soils produced insignicant responses(e.g. Biol 6). Tere was never a alse positive or ambiguousresult in the terrestrial experiments. In the current study,terrestrial LR pilot experiments using bacteria-laden active(Biol 5) and sterilized (Biol 6) soil samples were analyzed,using the same nonlinear approaches that were employedor analysis o the Martian data.On Mars, injected soil samples evolved radioactive gas [3,14] rapidly, subsequently approaching plateaus o 10,000 –15,000 cpm ater several sols (Fig 3, top panel). Tese “actives”(VL1c1, VL1c3, VL2c1, VL2c3), were run at Viking Landersites 1 and 2, with similar results. In contrast, the LR responsein VL1c2, the 160° C control, was very low, essentially nil,thereby, in conjunction with the active experiment results,satisying the pre-mission criteria or lie (see appendix #2or a brie description o the Viking LR results).Martian soil heated or three hours at 51° C produced anerratic succession o declining low-amplitude oscillations,each rising or about a sol, then precipitously alling tobaseline (VL2c2). Soil treated or three hours at 46° Cresponded with typical “active” kinetics, but 70% reducedin amplitude (VL2c4). Further, ormerly “active” soils storedat 10° C or three and ve months, at Lander 2 (VL2c5), andLander 1 (VL1c4), respectively, ailed to respond to thenutrient [15]. A second nutrient injection was made to each “active” soilater seven sols (VL1c1, VL2c1, VL2c3) or 16 sols (VL1c3).Each time, the gas briey spiked, ollowed immediately by a24% mean decrease in the accumulated
C gas. Laboratory simulations [16] showed absorption o CO
by wetted Marsanalog soils (pH 7.2) indicating that the Viking LR gas was,at least in part, CO
. In a terrestrial experiment, upon secondinjection [17] to an Antarctic soil with known bacterialcontent (pH 8.1) a brie spike also occurred, ollowed by adecrease in the accumulated gas. CH
, now known to be acomponent o the Martian atmosphere (6) and a possiblebiological metabolite, is virtually insoluble in aqueousmedia at temperatures and pressures recorded in the VikingLanders. I produced in such experiments, then it must haveremained in the non-reabsorbed
C-labeled gas raction.Tese results indicate that a signicant raction o the
C-labeled gas evolved on Mars was CO
, at least a part o  which (~24%) was reabsorbed on wetting o what was likely an alkaline soil [18], while the unabsorbed raction could
Int’l J. of Aeronautical & Space Sci. 13(1), 14–26 (2012)
have contained CH
. As mentioned in the Introduction, circadian oscillations inthe evolved LR gas developed gradually ater the rst nutrientadministration in the active experiments. Te oscillations were superimposed on the initial rise in cpm, and also on thesubsequent linear rise ollowing the mean 24% reduction incpm ater the second nutrient administration. Te oscillations were relatively stable in amplitude, but phase-delayed by about two hrs compared to the daily oscillation in the landertemperature. Tese oscillations were not slavishly drivenby the diurnal temperature cycle. All these eects are morecharacteristic o a biological rhythm than a purely physicaltemperature-driven process [10]. Our detailed considerationo the possible eects o soil pH, thermal variation in CO
 solubility, and a review o the ground-based and LR controlsound that CO
absorption and release could account orat most about 50% o the oscillatory response. Tus, someraction o the circadian oscillations as well as the evolvedgas remaining in the headspace o the instrument ollowingsecond or third nutrient injection could have been any  water-insoluble carbon-containing gas, such as CH
, whichgas, since Viking, has become o possible biological interestin studies o Mars [19].
4. Materials and Methods
Nine LR experiments (VL1c1, VL1c2, VL1c3, VL1c4, VL2c1, VL2c2, VL2c3, VL2c4, VL2c5) were perormed on Mars soilsamples collected with a robotic arm rom the surace to adepth o about 4 cm. Each sample (0.5 cc) (Levin and Straat,1976a)[2] was injected with 0.115 ml o a solution o ormate,glycine, glycolate, D-lactate, L-lactate, D-alanine andL-alanine, each at 2.5 x 10
M, with each ingredient uniormly labeled with
C. Te soil samples were monitored or theevolution o 
C gas as preliminary evidence o microbiallie. L-lactate and D-alanine were included to detect alienmetabolism that might require amino acids and sugars witha chirality dierent rom ours [20](Levin et al., 1964) (usingopposite chirality enantiomers in separate experiments waslater proposed as a ollow-on lie detection experiment [21](Levin, 1987) ). o conrm a positive response, a second soilsample was heated to sterilize it without destroying possibleinorganic chemical agents, these agents presumably beingar more heat resistant than the biological mechanism thatmight plausibly have produced the positive response. Tus,a negative LR response rom a heat-treated soil conrmedthat the initial response in the active experiments was likely biological, rather than inorganic.Four experiments (VL1c1, VL1c3, VL2c1, VL2c3) wereperormed on untreated soil samples. Another soil sample was heat-treated (“sterilized”) or three hours at 160° C(VL1c2). wo experiments utilized samples that were heat-treated (46°C and 51°C) or three hrs (VL2c4, VL2c2). wosoil samples (VL1c4, VL2c5), ater sub-samples showedactive responses, were stored at 10°C in the dark sampledistribution box or 3 and 5 months, respectively, beorenutrient solution was administered. In all experimentsexcept VL2c5, a second nutrient injection was given at leastour sols ater rst injection, and gas measured as above.Biol 5 and Biol 6 data were obtained rom pre-ight testsconducted in a test instrument that was essentially identicalto the ight instrument. In these tests, LR nutrient was addedto “active” terrestrial soil with a known microbial population(Biol 5) or to soil that had been heated or three hours at160°C (Biol 6). Te results or Biol 5 showed immediate andrapid
C-labeled gas evolution, typical o terrestrial soils with modest microbial populations, whereas Biol 6 results were essentially nil. We employed both positive and negative controls (knownpresence or absence o lie) to urther characterize the LRexperiments. Pre-nutrient administration backgroundradioactivity, a series o internal Viking Lander 1 temperaturemeasurements (1980 data points each taken sequentially every 960 sec), a series o external Mars atmosphere temperaturereadings (1000 data points each taken sequentially every hr) and a terrestrial heat-sterilized sample test (Biol 6)constituted negative controls. A terrestrial bacteria-ladenactive test (Biol 5) and a 23- day series o core temperaturereadings taken every minute rom a rat in constant darknessconstituted positive controls.
Complexity analysis
Nonlinear indices (Relative LZ Complexity or LZ; HurstExponent H; Largest Lyapunov Exponent
; CorrelationDimension CD; Entropy K; BDS Statistic; and Correlationime
; see appendix #1 or denitions) were calculated(Chaos Data Analyzer Pro, J.C: Sprott & G. Rowlands, American Institute o Physics, 1995) as an operationalnumerical method to measure quantitatively the complexity o the LR signal during active and control experiments on the Viking landers and in terrestrial pilot experiments on sterileand bacteria-laden soil samples. Moreover, negative controlsincluded complexity analyses o variations in pre-injectionbackground radioactivity, Mars atmosphere temperature andlander temperature. A positive terrestrial control consistedo a twenty-three day series o rat core temperature measuresthat were taken every minute. Data were analyzed in threedierent ways: 1) all usable data rom a given experiment

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