Electrochemical Approaches for Chemical and Biological Analysis on Mars Samuel P. Kounaves*[a]

Obtaining in situ chemical data from planetary bodies such as meltwater for a variety of inorganics and chemical parameters. By Mars or Europa can present significant challenges. The one analyzing the chemistry locked in the layers of dust, salt, and ice, analytical technique that has many of the requisite characteristics geologists will be able to determine the recent history of climate, to meet such a challenge is electroanalysis. Described here are water, and atmosphere on Mars and link it to the past. Finally, three electroanalytical devices designed for in situ geochemical and electroanalysis shows its abilities in the detection of possible biological analysis on Mars. The Mars Environmental Compatibility microorganism on Mars or elsewhere in the solar system. To Assessment (MECA) was built and flight qualified for the now identify an unknown microorganism, one that may not even use cancelled NASA Mars 2001 Lander. Part of MECA consisted of four Earth-type biochemistry, requires a detection scheme which makes ™cells∫ containing arrays of electrochemical based sensors for minimal assumptions and looks for the most general features. measuring the ionic species in soil samples. A next-generation Recent work has demonstrated that the use of an array of MECA, the Robotic Chemical Analysis Laboratory (RCAL), uses a electrochemical sensors which monitors the changes in a solution carousel-type system to allow for greater customization of via electrical conductivity, pH, and ion selective electrodes, can be analytical procedures. A second instrument, proposed as part of used to detect minute chemical perturbations caused by the the 2007 CryoScout mission, consists of a flow-through inorganic growth of bacteria and with the correct methodology provide chemical analyzer (MICA). CryoScout is a torpedo-like device unambiguous detection of such life forms. designed for subsurface investigation of the stratigraphic climate record embedded in Mars' north polar cap. As the CryoScout melts KEYWORDS: its way through the ice cap, MICA will collect and analyze the bacteria ¥ electroanalysis ¥ electrochemistry ¥ geochemistry ¥ sensors

During the past 40 years, a variety of scientific instruments have GCMS would not have been able to detected the degradation been usedfor missions to investigatedplanetary bodieswithin products from several million bacterial cells per gram of Martian our solar system. Most of these missions have reliedon remote soil at the ppb level.[6] The life detection and GCMS results form sensing, typically based on optical or radiation detection the basis for the prevalent opinion within the scientific techniques. Mars has been the focus of a large number of such community that it is probably unlikely that any microbial life missions.[1] The first successful flyby of Mars in 1964 returned21 forms were detected on the surface of Mars, and that, in photos with subsequent flyby missions adding more. It was not addition, the chemical and physical conditions are such that it is until 1976, with the success of the two Viking Landers, and in probably unlikely that any organic-basedlife form couldexist on 1997, with the Pathfinder Lander and Rover, that we obtained a the unprotectedsurface. detailed close up view of the surface and its chemical and Unlike Viking, Pathfinder was the first mission to focus on physical properties.[2, 3] Martian geochemistry andmineralogy. Its instruments and The instruments on the Viking I and II Landers included three mobile rover were designed not to directly detect life but to biology experiments, a gas chromatograph/mass spectrometer primarily provide close up optical observation and determine (GCMS), andan X-ray fluorescence spectrometer (XRFS). The the elemental chemical composition of the Martian rocks and results of the biology experiments have been interpretedby a surface material over hundreds of square meters using the alpha majority of the science community as ruling out microbial life on proton X-ray spectrometer (APXS). The Viking XRFS and Path- the surface of Mars.[4] However, there are still some who are finder APXS data have provided a reasonably clear picture of the convincedthe results leave no other conclusion but the elemental composition of the surface material. Even though the presence of life.[5] The big surprise though was that the GCMS detected no organics in the soil samples down to the parts-per- billion (ppb) levels. Many hypotheses have been advanced to [a] Prof. S. P. Kounaves account for the absence of organics andthe possible chemicals Department of Chemistry andreactions that couldaccount for the ambiguous biology Tufts University experiments. These have included reactions involving oxidants Medford, MA 02155 (USA) Fax: (‡1) 617-627-3443 such as hydrogen or superperoxides, smectite clays, and super- http://planetary.chem.tufts.edu oxide radical ions. A recent study has shown that the Viking E-mail:samuel.kounaves(at)tufts.edu

162 ¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1439-4235/03/04/02 $ 20.00+.50/0 CHEMPHYSCHEM 2003, 4, 162 ± 168 Chemical and Biological Analysis on Mars ELECTROCHEMISTRY SPECIAL analyses were from different areas they present a picture of a ppb levels. An array of microfabricatediridiumultramicroelec- rather homogeneous surface composition. The combinedresults trodes was included in the recent MECA instrument for assessing of the Viking andpathfindershow the surface material to be the concentration of such potential toxic metals. The sensor roughly composedof 45% SiO 2, 18% FeO2 ,8%MgO,6%SO3 , measures only millimeters in size andcan easily withstandharsh [7, 8] 7% Al2O3, 1.8% NaO, and0.6% Cl. environments. There are no other devices available which ™in total∫ can fit in a 50 mL volume, consume milliamps of electricity, The Advantages of Electroanalytical Devices andsupply the concentration of metals such as mercury and cadmium at sub-ppb levels. Chemical andbiological analyses are routinely performedon Earth, however, to obtain validin situ analytical datafrom remote harsh environments on planetary bodies, such as Mars or The Mars Environmental Compatibility Europa, presents a truly unique andformidablechallenge. Not Assessment (MECA) only do constraints on such instrumentation include power, size, The MECA instrument was originally designed, built, and flight mass, cost, androbustness, but they it must also be able to qualifiedfor the 2001 Mars Landermission. The mission was survive high g-forces, an eight-month journey through a subsequently cancelled due to the loss of the Mars Polar Lander radiation permeated environment, and possible temperature in 1999. The MECA, anda newer version, the Robotic Chemical variations ranging from ‡60 to À100 8C. Sensors basedon Analysis Laboratory (RCAL), have been proposedfor the 2007 electrochemical transduction schemes have many of the pre- launch opportunity. MECA was designed to evaluate potential requisite properties andcan withstandthe environmental risks geochemical andenvironmental hazardsto which future Mars that will enable them to return significant scientific data under explorers might be exposedandto return datathat wouldhelp such severe constraints. in understanding the geology, geochemistry, paleoclimate, and Because of the history andcomposition of Mars, electro- exobiology of Mars. The MECA instrument package containeda chemical sensors may also be especially well suitedin providing wet chemistry laboratory, an optical andatomic force micro- useful geochemical analyses andbroaderplanetary scientific scope, an electrometer to characterize the electrostatics of the results. The surface of Mars, as described above, appears to soil andits environment, andan array of material patches to contain a large fraction of sulfur andchlorine. These two study the abrasive and adhesive properties of soil grains. elements are most likely in the form of sulfate andchloridesalts. Because of payloadlimitations, the entire MECA package was Basedon what is known about the evolution of the solar system limitedto a mass of 10 kg, a peak power of 15 W, anda volume of andthe evidence returnedby the Mars missions, planetary 35  25  15 cm3. The development of MECA for analyzing the geologists have hypothesizedthat some time in its past Mars surface material in a remote hostile environment poseda unique was coveredby massive oceans andlakes that were eventually set of challenges, especially for remote chemical analysis and desiccated by some planetwide catastrophe or environmental more specifically for electrochemical analysis. change. Geochemical signatures of this wet periodin Mars' history shouldbe preservedin the form of layeredsalt-rich evaporite deposits that would have resulted from such large The Wet Chemistry Laboratory (WCL) Cell bodies of water and also from the geochemical weathering and transport of soluble minerals. Salts wouldalso have been the Containedwithin MECA are four WCLs, each consisting of a byproducts of volcanic gases acting on the Martian soil or thermally insulated, single-use, independent analysis cell, cap- perhaps in areas where microbial activity existed. pedwith a 30 mL pressurizedwater reservoir andactuator The simplest technique for qualitative andquantitative assembly. The actuator assembly consists of a water tank with a analysis of ionic species is the use of potentiometric ion-selective puncture valve, a sample loading drawer, a stirrer, and a solid electrodes (ISEs). These type of sensors possess several desirable reagent pellet dispenser. The sampling ™drawer∫, which holds characteristics, including a very wide dynamic detection range, approximately 1.0 cm3 of sample, receives the soil from the availability for a large variety of chemical species (including Lander's robotic arm and deposits it in the chamber. At the base gases) andproperties, andtheir intrinsic simplicity. Most ISEs can is a spring-loaded flap which retracts and allows the soil to fall be fabricatedin simple, compact, andruggedconfigurations into the cell while a screen prevents particles > 0.5 mm from that will allow them to survive harsh chemical andphysical falling into the receptacle. environments, including the elevated levels of radiation encoun- Figure 1 shows the a single analysis cell with an inside view of teredduringflight. Combinedwith a subsurface sampling the sensor array placement. Each cell was fabricatedfrom an methodologies and sensors for conductivity and redox potential, epoxy resin andmeasured3  3.5 cm2 wide, 3.5 cm deep, and an ISEs can provide not only a picture of the ionic chemical internal volume of approximately 35 mL. The cells were designed composition but of the geological andclimactic history of the to insure a leak rate of < 0.1 cm3 minÀ1, corresponding to a partial planet. pressure of water ten times lower than that of the Mars ambient Another type of electroanalysis which provides unique contribution at 1 cm from the leak, so as to not cause any information is anodic stripping voltammetry (ASV). This precon- frosting on adjacent instruments. Each cell was surrounded by a centration technique allows the analysis of several heavy metal printedcircuit boardcontaining preamplifiers for each sensor. ions, such as copper, lead, cadmium, mercury, and zinc, at sub- Additional information regarding the design, fabrication, cali-

CHEMPHYSCHEM 2003, 4, 162 ± 168 163 S. P. Kounaves

Figure 1. A view of the MECA WCL components showing a) the actuator assembly containing the water reservoir, sample drawer, and WCL, b) the lower cell, and c) a close up of several sensors mounted on the interior of the WCL.

bration, andevaluation of the cells has been previously Figure 2. A photographic and schematic view of a typical ISE sensor element published.[9] used in the MECA. To start a sample analysis, the Lander's robotic arm places soil in a sliding drawer, the drawer is closed, and the chamber and calibratedandtestedfor six months. Even undersuch rigorous cell are resealedandpressurizedto approximately 7 Torr. The conditions all of the ISEs performed well, demonstrating that 8 water reservoir is heatedto about 5 C andthe metal seal on the such sensors are suitable for geochemical measurements in water tank is puncturedto allow water to flow into the cell harsh terrestrial andextraterrestrial environments. A fractional where it mixes with the soil sample. The temperature in the WCL factorial calibration methodsuccessfully describedthe slope, Æ 8 is maintainedat 20 0.5 C during the analysis and monitored intercept, andselectivity coefficients of the ISEs. The results were throughout. Since some chemical reactions may occur instanta- usedto determinethe activities of eight ions in complex samples neously on addition of water to the soil, all the sensors are read of soil leachate simulants andsoil samples. Although some small immediately and then serially and repetitively. The stirring errors were encounteredin the analyses, the overall conclusion homogenizes the solution and drives it towards equilibrium in was that the array successfully predicted the ionic concentra- the shortest possible time. A small reagent pellet can be tions. Simulants andsamples consisting of several salts within a introduced at the end to provide an end-point calibration of the large concentration range were usedto further evaluate the sensors. sensors. The selectivity issues, sensor noise, andincompatibilities that were experiencedare not unlike what might occur if the sensor array hadperformedextraterrestrial chemical analyses. The Ion Sensitive Electrode (ISE) Sensor Array The results showedthat the MECA/RCAL type of electrochemi- cally basedsensor array can accurately andreliably characterize Figure 1 shows the placement of the sensor array within the the type of surface material expectedon Mars. WCL. The array consistedof 26 sensors with the majority being potentiometric, but also included voltammetric, amperometric, and conductivity based devices. The potentiometric devices The CryoScout Mars Inorganic Chemical included ISEs based on polymer membrane and solid pellet Analyzer (MICA) configurations. The ionic species which couldbe analyzed ‡ ‡ ‡ ‡ 2‡ 2‡ ‡ À À À included H ,Li,Na,K,Mg ,Ca ,NH4 ,NO3 , ClO4 ,HCO3 , CryoScout has been defined as a deep subsurface mission to the Ag‡,Cd2‡,ClÀ,BrÀ, andI À. ISEs were also included for dissolved north polar cap of Mars, which will explore the stratigraphic

CO2 andO 2 . Figure 2 shows the configuration for a typical recordof recent climate change in the underlyinglayeredterrain. polymer membrane basedISE. The inner chamber, abutting A ™cryobot∫ thermally driven probe will penetrate the ice to against a Ag/AgCl reference electrode, is filled with a hydrogel examine the borehole wall's stratigraphy andthe chemistry of containing an electrolyte anda fixedconcentration of the the meltwater andentraineddust.Together with surface-station analyte species. The hydrogel is covered by a plasticized PVC observations, these data will provide for a new understanding of membrane into which an analyte specific ionophore is immo- the Martian polar meteorology; present climate andpolar water bilized. exchange; recent polar volatile deposition and erosion; the These PVC membrane basedISE sensors were storedat scale, texture, structure, dust, and volatile content of subsurface À20 8C for over 18 months, repeatedly frozen and thawed, layers; their accumulation rates; the origin of the entraineddust;

164 CHEMPHYSCHEM 2003, 4, 162 ± 168 Chemical and Biological Analysis on Mars ELECTROCHEMISTRY SPECIAL the evolution of the polar cap; andthe role of orbital variations An Electrochemical Microbial Growth Detection in this evolution. These high scientific priority goals, andthis System (LIDA) type of probe andlandingsite, represent important contribution to the Mars andplanetary exploration program. The discovery of any life form elsewhere in the solar system, The ™bubble∫ of meltwater that surrounds the cryobot while it which proved to have an independent history from life on Earth, descends will contain dissolved gases, dust, and soluble species wouldhelp elucidatesome of the most perplexing questions at extracted from the dust. The dust is derived from many sources, the boundaries of biology andchemistry, andcouldreveal much including ancient hydrothermal mineralization, chemical precip- about the origin andevolution of life. It couldreveal what itation in lake beds, floodwaters episodically disgorged from the characteristics are particular to life on Earth andwhich are upper crust, or from moisture-driven mineral differentiation in common everywhere. Is Earth's biology andbiochemistry unique the pedogenic surface. or is it possibly ubiquitous in the Cosmos? The MICA system is an electrochemically basedflow-through The characteristics that microbial life may take on a planet variant of the MECA WCL. Similar to the MECA, the MICA will such as Mars, other than the needfor water, carbon, andenergy, contain a similar array of sensors that will analyze the soluble is a totally open question. In recent years our understanding of components of the Martian dust by measuring a variety of ionic the limits of habitability have undergone a drastic revision. species and properties, including conductivity (0.05 ± Researchers have discovered life distributed throughout the 140 mScmÀ1), pH (0 ± 14 to Æ0.5), cations (Na‡,K‡,Mg2‡,Ca2‡, Earth's crust in a variety of previously unthinkable environments. ‡ À8 À À À Microbial life has been foundin such places as hot springs 200 m andNH 4 to 10 M using ISEs), anions (Cl ,NO3 , HClO4 , and À À8 2‡ 2‡ 2‡ 2‡ below the surface, generating metabolic energy by combining HCO3 to 10 M), metals (Cu ,Cd ,Hg andPb to ppb levels [10] using ASV), reversible andirreversible oxidants in the meltwater hydrogen from the rocks with carbon dioxide; burieddeepin [11] À2 À4 Antarctica's hyperarid, ultracold ice-free valleys; andnear deep (using cyclic voltammetry), dissolved CO2 (10 ±10 M), dis- ocean volcanic vents at temperatures of 1108C andpressures of solvedO 2 (0 ± 14 ppm), oxidation reduction potential (ORP, from [12] ‡1toÀ1 V), andtemperature ( À40 to ‡ 408C). 150 atmospheres. As shown in Figure 3, MICA will normally sample the water Life of course is intimately linkedwith both the geochemical from the meltwater surrounding the cryobot. After flowing and, more importantly, with the electrochemical redox environ- through the sensor array unit the sample is injectedinto a waste ment. Organisms obtain energy by coupling energetically favorable redox pairs with a negative net redox potential. The electroanalytical instruments described above, RCAL and MICA, both have the capability to determine if an environment on Mars has the required™chemical potential∫ to sustain life. However, we have recently discovered the RCAL and MICA sensor arrays also have the ability to detect small perturbations caused by the growth of microbes near or on their surfaces. What is an intractable problem of biofouling on Earth may be the key to detecting extraterrestrial microorganisms.

The Mars Viking Results As mentionedabove, the Mars Viking I and II Landers included Figure 3. Schematic view of the fluidics system consisting of valves, reagent three biology experiments. In the LabeledRelease (LR) experi- containers, and flow-through sensor array. 14 ment, the soil was moistenedwith nutrients (H 2Oand C- labeledorganics) andthen incubated.Microorganisms would consume the nutrients and produce detectable 14C-containing container to prevent contamination of the water ™bubble∫ gases. The Gas Exchange (GEX) experiment partially submerged surrounding the cryobot. The control manifold includes valve- the sample under a simulated Martian atmosphere in a mixture controlled injectors from reservoirs that, in addition to pure of organic andinorganic compounds.Gases such as CO 2 ,O2 , deionized water, may contain calibration solutions or specific CH4 ,H2 , andN 2 produced by organisms were to be detected by reaction reagents. Unlike the MECA wet chemistry cells, these GC. The Pyrolytic Release (PR) experiment incubatedthe soil 14 14 flow-through cells will require no stirring or heating. sample with a CO2/ CO mixture andUV light providedbya It is clear that electrochemically basedsensors have the xenon lamp. No nutrients or water were added. After five days inherent simplicity androbust characteristics that will allow the gases were flushed, the sample heated to 6258C, and them to survive andperform chemical analyses in the type of emittedgases passedthrough a 14C detector. The GCMS, to environments foundin space andon Mars. Even though these everyone's surprise, detected no organics in the soil samples sensors are relatively simple, the data they can return will down to the ppb levels. Later analysis however has shown that provide information that will be vital to both future astronauts the GCMS wouldprobably not have detectedmicrobial cells or andto a variety of planetary scientists. products at the sub-ppb levels.

CHEMPHYSCHEM 2003, 4, 162 ± 168 165 S. P. Kounaves

The Viking experiments though, made several assumptions robust, low-mass, andlow-power devicefor monitoring micro- which contributedto the ambiguity of the results. In the LR bial growth–the Life Detection Array (LIDA). experiment, it was assumedthat microorganisms on Mars would LIDA is basedon several crucial components which ensure a possess biochemical andmetabolic systems similar to Earth definitive conclusion that changes in the growth chambers were organisms andwouldconsume the organic compoundssup- biologically induced. These components include two chambers pliedandproduce 14C-containing gases. The LR results, even containing a differentially monitored pair of sensor arrays with though positive on first analysis, became inconclusive and one chamber for control andthe other for the inoculation, a ambiguous when interpretedalong with the other experiments special sterilization andinoculation procedure,use of the local andthe GCMS data. It appearedthat the Martian soil had soil as a growth medium, and multiple replications. A schematic chemically reacted with the added water and the 14C-labeled of the growth chambers is shown in Figure 4. The chambers are organics to release 14C-labeledgases. The reaction was rapidand identical and each contains sensors for conductivity, oxidation ± ‡ ‡ 2‡ 2‡ À À did not appear biological in origin. The GEX experiment made reduction potential (ORP), pH, Na ,K ,Ca ,Mg ,Cl , andNO 3 . the same assumptions as the LR one. Again, the possibility of More sensors can be added but, as will be seen from our abiotic emission of O2 andother gases madethe results non- preliminary experiments, they may not be needed. Any global definitive. Finally, even though no water or nutrients were added changes due to temperature, pressure, or soil chemistry should in the PR experiment, the same assumptions as the LR experi- affect both chambers identically and thus the differential ment were also made. The possibility of gas exchange with the measurements will remain ™zeroed∫. soil andthe high-temperature heating producednon-definitive results. It is against this backdrop that we must ask ourselves: What assumptions shouldbe made? Where shouldwe look? Andwhat type of instrumentation shouldbe usedthat will enable us to unambiguously characterize the surface chemistry anddetect any microorganisms on Mars? There is no evidence that extraterrestrial life must necessarily be built on terrestrial biochemistry. Thus, we shouldnot assume any commonality except water, carbon chemistry, an energy source, andrepro- duction. The question then arises: How do we detect reproduc- tion? Direct microscopic observation andculturing wouldof course provide definitive evidence, but this is not likely to happen soon. Detection of chemical species such as amino acids, PAHs, lipids, proteins, or DNA/RNA might work, but the major drawback is that many organics can be formed abiotically and there is no basis to assume that DNA/RNA/proteins exactly as we know them are required. Thus, we propose that the best method for detecting microorganisms is to monitor the amplification of the ™chemical disequilibrium∫ that is caused by the reproduction andgrowth of an organism within a transposedportion of its habitat. Figure 4. Schematic diagram of the differential life detection chambers. Both the control and test chambers are identical in every respect until the unsterilized nanogram sample is added to the test chamber. Electrochemical Growth Detection

Bacterial growth in culture media has typically been monitored The experiment starts by delivering into the chambers of an optically, by measuring turbidity, or electrochemically, by con- equal amount of homogenizedsoil sample. The chambers are ductivity, pH, or capacitance.[13, 14] Although never flown, several then filledwith equal amounts of pure sterilizedwater. Once the of these methods have been proposed for detection of water is added, the solutions are then continuously monitored. extraterrestrial microbial life.[15±17] Optical turbidity does not The stirrer andsterilizing heaters are then turnedon andthe appear to be a viable technique because of the problems temperature is increasedto at least 110 8C andmaintainedfor a associatedwith analyzing a particulate soil sample. Modifications predetermined period of time. The temperature is then de- have been proposedto resolve this dilemma, [15] but very little is creasedto just above freezing andthe chambers allowedto gainedandthe final results may still allow ambiguous inter- equilibrate andthe atmosphere above the solution is main- pretation. Even though more reliable, each of the electro- tainedat about 8 Torr CO 2 . chemical techniques by themselves may also be prone to After equilibrium has been ensured, a nanogram quantity or interferences or ambiguous interpretation. However, we pro- surface ™swipe∫ is introduced into the test chamber. Introducing pose that integrating the conductivity, pH, and several ion- such a minute quantity decreases the probability that a chemical selective electrodes as a sensor array and incorporating them reaction with the water or newly solvatedcomponents wouldbe into a multisample micro-laboratory will provide a reliable, responsible for a significant disequilibrium of the bulk solution.

166 CHEMPHYSCHEM 2003, 4, 162 ± 168 Chemical and Biological Analysis on Mars ELECTROCHEMISTRY SPECIAL

The chambers are the monitoredfor the maximum time allowable. Microbial metabolism andexcretion in the test chamber will change the conductivity of the solution and may alter many chemical parameters which can be detected by the various sensors in the array. More importantly, the biofouling of the sensors causedby the metabolic products or by the organisms sensor membranes will result in a signal. Even partial monolayer coverage can effect the transport andcharge properties of ISE membranes. If this experiment was performedin a totally sterile environ- ment, there shouldbe no differencebetween the two monitored chambers. Any differential between the two chambers, that changes as a function of time, must necessarily be the result of the ™substance∫ introduced into the test chamber and whose effects are being ™amplified∫ by some process. If we insure that chemical processes such as catalysis are not responsible, the conclusion can only be a reproducing and growing life form. This substance and/or entity must thus be possess the ability to cause (reproducibly and exponentially) extensive slow changes in the chemistry of the sample even though it was introduced in nanogram quantities. The results in Figure 5 show some of the preliminary data obtainedwith Lactobaccilus casei. Both chambers were sterilized and conductivity was monitored for a day to insure stability and sterility. One chamber was then inoculatedwith a small amount of L. casei. Within about 12 h the conductivity for the inoculated chamber began to show a slow increase. During the next 48 h it Figure 5. Results for monitoring conductivity in the test chamber inoculated with was clear that growth was proceeding in the inoculated L. casei and in the sterile control chamber over a period of 240 h.

Figure 6. Typical changes in the potential of Ca2‡,K‡,Na‡, and ClÀ, ISEs after monitoring a chamber inoculated with B. subtilis for approximately 36 h.

CHEMPHYSCHEM 2003, 4, 162 ± 168 167 S. P. Kounaves chamber. This experiment has been repeatednumerous times [1] Mars: The NASA Mission Reports (Ed: R. Godwin), Apogee Books, and is a good indication that even conductivity by itself is Burlington, ON, 2000. [2] Mars (Eds.: H. H. Kieffer, B. M. Jakosky, C. W. Snyder, M. S. Matthews), sufficient to monitor growth for L. casei. However, for other University of Arizona Press, Tucson, AZ, 1992. bacteria, such as Bacillus subtilis, conductivity was not sufficient [3] M. P. Golombek, R. C. Anderson, J. R. Barnes, J. F. Bell, N. T. Bridges, D. T. to always indicate growth. Britt, J. Bruckner, R. A. Cook, D. Crisp, J. Crisp, T. Economou, W. M. Folkner, Figure 6 shows the results for B. subtilis when monitoredwith R. Greeley, R. M. Haberle, R. B. Hargraves, J. A. Harris, A. F. C. Haldemann, 2‡ ‡ ‡ À K. E. Herkenhoff, S. F. Hviid, R. Jaumann, J. R. Johnson, P. H. Kallemeyn, ISEs for Ca ,K ,Na , andCl . To our surprise, these electrodes H. U. Keller, R. L. Kirk, J. M. Knudsen, S. Larsen, M. Lemmon, M. B. Madsen, indicated drastic changes in ionic concentration. However, J. A. Magalhaes, J. N. Maki, M. C. Malin, R. M. Manning, J. Matijevic, H. Y. seeing that the bacteria couldnot produceor consume such McSween Jr., H. J. Moore, S. L. Murchie, J. R. Murphy, T. J. Parker, R. Rieder, quantities of ionic species that wouldbe neededtocause these T. P. Rivellini, J. T. Schofield, A. Seiff, R. Singer, P. H. Smith, L. A. Soderblom, D. A. Spencer, C. Stoker, R. Sullivan, N. Thomas, S. W. Thurman, M. G. changes, it is clear that the changes in potential are being most Tomasko, R. M. Vaughan, H. Wanke, A. W. Ward, G. R. Wilson, J. Geophys. probably induced by bacterial products adsorbing to the ISE Res. 1999, 104, 8523 ± 8553. membrane surfaces. Whatever the cause of these changes [4] N. H. Horowitz, To Utopia and Back: The Search for Life in the Solar System, (which were not seen in the control), they give an indication that W. H. Freeman, New York, NY, 1986. [5] G. V. Levin, The Viking labeled release experiment and life on Mars in something is most likely growing in the chamber. It is interesting Instruments, Methods and Missions for the Investigation of Extraterrestrial 2‡ to note that after about 500 min the Ca ISE is producing a Microorganisms; Proceedings of the SPIE, vol. 3111 (Ed: R. B. Hoover), SPIE curve typically seen for bacterial growth while at the same time Press, Bellingham, WA, 1997, pp. 146 ± 151. the curves for the ClÀ,K‡, andNa ‡ ISEs are also starting to show [6] D. P. Glavin, M. Shubert, O. Botta, G. Kminek, J. L. Bada, Earth Planet. Sci. Lett. 2001, 185,1±5. changes. These type of results are reproducible and the sterile [7] P. A. Toulmin, A. K. Baird, B. C. Clark, K. Keil, H. J. Rose, R. P. Christian, P. H. chambers showedno such changes. Evans, W. C. Kelliher, J. Geophys. Res. 1977, 82, 4625 ± 4634. These preliminary experiments demonstrated that an array of [8] J. F. Bell, H. Y. McSween, S. L. Murchie, J. R. Johnson, R. Reid, R. V. Morris, electrochemical sensors was capable of detecting bacterial R. C. Anderson, J. L. Bishop, N. T. Bridges, D. T. Britt, J. A. Crisp, T. Economou, A. Ghosh, J. P. Greenwood, H. P. Gunnlaugsson, R. M. Har- growth at reasonably short times. Continuing experiments with graves, S. Hviid, J. M. Knudsen, M. B. Madsen, H. J. Moore, R. Rieder, L. these andother microorganisms will use a larger number of ISE Soderblom, J. Geophys. Res. 2000, 105, 1721 ± 1755. sensors andwill investigate ways to increase sensitivity, decrease [9] S. P. Kounaves, S. R. Lukow, B. P. Comeau, M. H. Hecht, S. M. Grannan- the limits of detection and response time, and increase reliability. Feldman, K. Manatt, S. J. West, X. Wen, M. Frant, T. Gillette, J. Geophys. Res. 2003, in press. Even though some may not be satisfiedthat microbial life has [10] F. H. Chapelle, Nature 2002, 415, 312 ± 315. been discovered until a group of microbiologists lands on Mars, [11] W. C. Mahaney, J. M. Dohm, V. R. Baker, H. E. Newsom, D. Malloch, R. G. V. these type of devices can give a reasonably good indication of Hancock, I. Campbell, D. Sheppard, W. M. Milner, Icarus 2001, 154, 113 ± what may or may not exist. 130. [12] L. J. Rothschild, R. L. Mancinelli, Nature 2001, 409, 1092 ± 1102. [13] M. Lanzanova, G. Mucchetti, E. Neviani, J. Dairy Sci. 1993, 76, 20 ± 28. Funding for most of this work was provided by the National [14] R. E. Madrid, C. J. Felice, M. E. Valentinuzzi, Med. Biol. Eng. Comput. 1999, Aeronautics and Space Administration (NASA). All these projects 37, 789 ± 792. [15] E. L. Merek, V. I. Oyama, Appl. Microbiol. 1968, 16, 724 ± 731. are team efforts and have involved several groups of dedicated [16] M. P. Silverman, E. F. Munoz, Appl. Microbiol. 1974, 28, 960 ± 967. scientists and engineers at the Jet Propulsion Laboratory (Pasade- [17] M. R. Sims, R. E. Cole, W. D. Grant, A. A. Mills, K. Powell, R. W. Ruffles, Simple na, CA), ThermoOrion (Beverly, MA), and Starsys Research (Boulder, techniques for detection of Martian microorganisms in Instruments, CO). A special acknowledgment is made in memory of Kurt Methods and Missions for the Investigation of Extraterrestrial Microorgan- isms; Proceedings of the SPIE, vol. 3111 (Ed: R. B. Hoover), SPIE Press, Lankford at Starsys who was responsible for much of the Bellingham, WA, 1997, 164 ± 174. engineering and fabrication on the MECA and RCAL projects. Received: October 1, 2002 [C 525]

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