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Article Effects of Low-Temperature Plasma-Sterilization on Analog Soil Samples Mixed with radiodurans

Janosch Schirmack 1,*, Marcel Fiebrandt 2, Katharina Stapelmann 2 and Dirk Schulze-Makuch 1,3

1 Research Group, Center for Astronomy and Astrophysics, Technical University Berlin (TUB), Berlin 10623, Germany; [email protected] or [email protected] 2 Biomedical Applications of Plasma Technology, Institute for Electrical Engineering and Plasma Technology (AEPT), Ruhr University Bochum (RUB), Bochum 44801, Germany; fi[email protected] (M.F.); [email protected] (K.S.) 3 School of the Environment, Washington State University, Pullman, WA 99164, USA * Correspondence: [email protected]; Tel.: +49-30-3147-9484

Academic Editor: David Deamer Received: 1 March 2016; Accepted: 23 May 2016; Published: 26 May 2016

Abstract: We used Ar plasma-sterilization at a temperature below 80 ˝C to examine its effects on the viability of when intermixed with tested soil. Due to a relatively low temperature, this method is not thought to affect the properties of a soil, particularly its organic component, to a significant degree. The method has previously been shown to work well on parts. The selected for this test was Deinococcus radiodurans R1, which is known for its remarkable resistance to radiation effects. Our results showed a reduction in microbial counts after applying a low temperature plasma, but not to a degree suitable for a sterilization of the soil. Even an increase of the treatment duration from 1.5 to 45 min did not achieve satisfying results, but only resulted in in a mean cell reduction rate of 75% compared to the untreated control samples.

Keywords: plasma sterilization; Deinococcus radiodurans; Mars; regolith

1. Introduction In order to design a life detection mission to Mars, we were searching for an effective sterilization method as a negative control. At the same time the selected method should not affect soil properties, particularly not the organic content of a chosen soil. As a method of choice we tried low temperature Ar-plasma sterilization (below 80 ˝C), because heat sterilization like autoclaving might be too destructive to the organic contents of a soil. In a previous study by Stapelmann et al. [1] it could be shown that plasma sterilization was a suitable method for sterilization of certain parts of a space craft. In that study, stainless steel screws were spiked with spores of the two bacterial strains 168 and SAFR-032. The samples were exposed to increasing time steps of plasma (varying from 100 to 400 W, H2 or H2/O2 gas mixture [20 sccm]). The spore inactivation rates estimated via incubation and colony forming units (CFU) were below the detection level (10´7) when the spores were exposed to the plasma for at least 300 s. The objective in our test series was to quantify survival rates of microorganisms mixed within Martian simulant soil by applying a low-temperature plasma. We used three different Martian regolith analogs and mixed the soil particles with cells of Deinococcus radiodurans, which has been shown to be especially radiation resistant [2,3]. Since the sterilizing effect of the plasma treatment is also based on UV photons [1], the radiation resistant D. radiodurans is a most suitable test organism.

Life 2016, 6, 22; doi:10.3390/life6020022 www.mdpi.com/journal/life Life 2016, 6, 22 2 of 7

2. Materials and Methods

2.1. Incubation Medium D. radiodurans cells were incubated on 2ˆ TGY medium, either on agar plates or in liquid. The 2ˆ TGY had the following composition (per 1000 mL): 10 g Trypticase-Pepton BBLTM (Becton, Dickinson and Company, Sparks, MD, USA), 6 g Bacto Yeast-Extract (Becton, Dickinson and Company) and 2 g D-Glucose-Monohydrate (AppliChem, Darmstadt, Germany). For the plates 15 g of agar (Carl Roth, Karlsruhe, Germany) were added to the mixture. The PBS buffer solution contained (per 1000 mL): 7 g Na2HPO4 ˆ 2H2O, 3 g KH2PO4 and 4 g NaCl (all Sigma-Aldrich, Munich, Germany).

2.2. Growing and Handling of Cells The cells of Deinococcus radiodurans R1T (=ATCC 13939T = DSM 20539T) were incubated on TGY plates for up to four days. A single colony was then transferred to 25 mL of liquid TGY and incubated until the cell density reached 3 ˆ 107 cells¨ mL´1 (counted microscopically with a Thoma chamber of 0.01 mm depth). The cell suspension was centrifuged (3000ˆ g, 15 min), the supernatant was discarded and the pellet was washed with 25 mL of PBS buffer. The centrifugation was repeated and after discarding the supernatant the pellet was resolved in 2.5 mL PBS buffer. The dissolved cells were transferred in PBS aliquots of 0.2 mL to sterile serum bottles with 1.25 g of Mars analog soils. The three Mars analog soils tested were JSC Mars-1A, P-MRA and S-MRA, which are described in detail in [4] (Table1). The bottles were desiccated for 72 h inside a plastic box capped with a lid and filled with the drying agent Köstrolith® (CWK, Chemiewerk Bad Köstritz GmbH, Bad Köstritz, Germany). After the dried samples were filled into 100 mL glass bottles (Figure1a–d). One part was sent to the Ruhr University Bochum for applying plasma sterilization, the other part of the samples was used to estimate the starting cell numbers as a control (counts as described in Section 2.4.).

Table 1. Mineralogical composition of JSC Mars-1A, P-MRA and S-MRA: Composition as weight percent (wt %) of the mixture. Data for JSC Mars-1A were obtained from Morris et al., 2010 [5]; data for P-MRA and S-MRA were obtained from Dr. Jörg Fritz, Museum für Naturkunde Berlin, Germany. (Adapted from Schirmack, et al., 2015 [4]).

Mineral Phase JSC Mars-1A (wt %) P-MRA (wt %) S-MRA (wt %) Plagioclase Feldspar 64 - - Olivine 12 - - Magnetite 11 - - Pyroxene and/or Glass 9 - - Fe2O3 - 5 - Montmorillonite - 45 - Chamosite - 20 - Kaolinite - 5 - Siderite - 5 - Hydromagnesite - 5 - Quartz - 10 3 Gabbro - 3 31 Dunite - 2 16 Hematite 5 - 17 Goethite - - 3 Gypsum - - 30

2.3. Plasma Sterilization Procedure Two separate test runs were conducted with a double inductively coupled plasma system (DICP) operated at a pressure of 10 Pa. The first test consisted of all three regolith mixtures (JSC Mars-1A, S-MRA and P-MRA) and had an exposure time of 1.5 min. An argon-nitrogen mixture (100:5 sccm) Life 2016, 6, 22 22 3 of 7 and P-MRA) and had an exposure time of 1.5 min. An argon-nitrogen mixture (100:5 sccm) was used withwas usedcontinuous with continuous power of power500 W, of as 500 it W, emits as it significantly emits significantly higher higher amounts amounts of radiation of radiation in the in bactericidalthe bactericidal wavelength wavelength region region from from100 nm 100 to nm 400 to nm 400 compared nm compared to an argon- to an argon-oxygen mixture mixture (100:5 sccm)(100:5 [6]. sccm) The [6 absolute]. The absolute values valuespresented presented in [6] are in not [6] areapplicable not applicable to the results to the shown results here, shown due here, to modificationdue to modification of the power of the powercoupling coupling of the plasma of theplasma system. system. Nevertheless, Nevertheless, the significantly the significantly higher emissionshigher emissions of the argon-nitrogen of the argon-nitrogen mixture mixtureare shown. are Ab shown.solute Absolute values of valuesthe radiation of the radiationdose in the dose range in fromthe range 100 nm from to 100400 nm of to the 400 system nm of theconfiguration system configuration used in this used study in were this studymeasured were [7]. measured A possible [7]. radicalA possible that radicalcan interact that can with interact the samples with the is atom samplesic nitrogen. is atomic As nitrogen. atomic nitrogen As atomic is nitrogennot known is notfor etchingknown forof organic etching materials, of organic we materials, assume wethat assume microbial that inactivation microbial inactivation in the argon-nitrogen in the argon-nitrogen mixture is basedmixture on isradiation based on effects. radiation Only effects. JSC Mars-1A Only JSCanalog Mars-1A soil was analog used in soil the was second used trial. in the In secondthis case, trial. an argon-oxygenIn this case, an mixture argon-oxygen (100:5 mixturesccm) was (100:5 used sccm) as was it produces used as it producesreactive oxygen reactive species oxygen speciesdue to dissociationdue to dissociation of molecular of molecular oxygen oxygen in the inplasma. the plasma. Since Sinceradiation-based radiation-based sterilization sterilization is faster is fasterthan sterilizationthan sterilization due to due oxidation, to oxidation, the treatment the treatment time was time increased. was increased. As this Asleads this to leads heating to heatingof the sample of the ˝ abovesample 80 above °C in 80the continuousC in the continuous plasma, we plasma, used a wepulsed used mode, a pulsed switching mode, the switching plasma theon and plasma off at on a frequencyand off at aof frequency 1 kHz and of a duty 1 kHz cycle and of a duty10%. cycleTo acco ofunt 10%. for To this account fact, the for treatment this fact, time the treatment was increased time towas 45 increasedmin, yielding to 45 a plasma min, yielding exposure a plasmatime of 4.5 exposure min. Furthermore, time of 4.5 min.the power Furthermore, was increased the power to 1500 was W, yieldingincreased a tomean 1500 power W, yielding of 150 a W, mean as it power is only of applied 150 W, as10% it isof only the time. applied The 10% plasma of the conditions time. The plasmaare not matchedconditions to areeach not other matched as the tomain each objective other as was the to main observe objective to what was degree to observe the cells to of what D. radiodurans degree the werecells ofaffectedD. radiodurans by radiationwere or affected reactive by species radiation and or not reactive which speciescomponent and notis more which efficient. component As the is samplesmore efficient. were placed As the in samples an ICP weredischarge, placed in antowards ICP discharge, the samples ions are towards only accelerated the samples by are several only tenthaccelerated of eV. byThus, several sputtering tenth of of eV. the Thus, sample sputtering due to of thebombardment sample due can toion be bombardmentneglected as possible can be inactivationneglected as mechanism. possible inactivation mechanism. In parallel, control control tests tests with with only only a a vacuum treatment treatment were were done done for for the the same same time time period. period. These controlscontrols werewere exposed exposed to to the the vacuum vacuum inside inside of of the the plasma plasma chamber chamber for thefor samethe same amount amount of time of timeas the as plasma the plasma samples, samples, but not but treated not treated with with the plasma. the plasma. After After the specific the specific treatment treatment the samples the samples were weresent backsent toback the to TU the Berlin TU Berlin to determine to determine the respective the respective survival survival rates. An rates. additional An additional transport transport control controlsample sample was shipped was shipped to the Ruhrto the University Ruhr University of Bochum of Bochum and sent and backsent back to the to TU the Berlin TU Berlin without without any anyadditional additional treatment, treatment, to estimate to estimate the cell the loss cell during loss during shipping shipping of the samples.of the samples. Details Details of the facilityof the facilityused in used which in thewhich experiments the experiments were conducted were conducted are provided are provided by [6]. by [6].

Figure 1. Desiccated regolith analog samples: (a) 1.25 g regolith mixed with cells of D. radiodurans in Figure 1. Desiccated regolith analog samples: (a) 1.25 g regolith mixed with cells of D. radiodurans in 100 mL glass bottles with wide opening and screw caps; Close ups showing (b) S-MRA; (c) P-MRA; 100 mL glass bottles with wide opening and screw caps; Close ups showing (b) S-MRA; (c) P-MRA; (d) JSC Mars-1A. (d) JSC Mars-1A.

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2.4.2.4. Colony Colony Forming Forming Unit Unit (CFU) (CFU) CountsCounts AfterAfter treatmenttreatment the the desiccated desiccated regolit regolithh samples samples were liquefied were liquefiedwith 5 mL withof PBS 5 buffer. mL ofEach PBS 0.1 buffer.mL 1 2 3 Eachof the 0.1 resolved mL of the sample resolved was sampleplated on was TGY plated agar on plates TGY in agar a serial plates dilution in a serial of 101 dilution, 102, 103 ofand 10 10,4 10 and, 10 4 ˝ andincubated 10 and for incubated two days for at 37 two °C. days The atgrowing 37 C. colonies The growing on the coloniesplates with on the the highest plates dilutions with the were highest ´ dilutionscounted were to estimate counted the to number estimate of theinitial number cells·g of−1 for initial each cells regolith¨ g 1 sample.for each A regolith CFU test sample. with desiccated A CFU test withcell desiccated regolith samples cell regolith before samples shipment before to Bochum shipment serv toed Bochumas a starting served control as a of starting the cell control numbers. of the All cell numbers.CFU counts All CFUwere countsperformed were in performedtriplicate. in triplicate.

3.3. Results Results TheTheD. D. radiodurans radioduranscell cellcounts counts withinwithin all three regolith regolith analogs analogs and and an an exposure exposure time time of of1.51.5 min min toto the the plasma plasma revealed revealed nono significantsignificant reduction reduction in in cell cell numbers, numbers, except except for JSC for JSCMars-1A Mars-1A (Figure (Figure 2). A 2). Acomparison of of the the cells cells count count showed showed significant significant lower lower cell cell numbers numbers before before and and after after the the plasma plasma treatment,treatment, when when compared compared toto thethe transporttransport contro controll but but not not when when compared compared to tothe the vacuum vacuum control. control. ThereThere was was a slighta slight but but significant significant reductionreduction of 57% in cell cell counts counts in in the the JSC JSC Mars-1A Mars-1A analog analog soil. soil. For For the the otherother two two tested tested regolith regolith analogs analogs (P-MRS(P-MRS and S-MRS) S-MRS) there there was was no no reduction reduction in incell cell numbers numbers detectable. detectable.

FigureFigure 2. 2.Results Results ofof thethe firstfirst experimental setup: setup: esti estimatedmated via via plate plate count; count; Only Only for JSC for JSCMars-1A Mars-1A a a significantsignificant reductionreduction inin cell cell numbers numbers could could be observedbe observed before before (Start) (Start) and afterand theafter treatment the treatment (Plasma). Error(Plasma). bars indicate Error bars the indicate standard the deviation, standardn deviation,= 3. The plasma n = 3. The sterilization plasma sterilization parameters parameters for this test for were: Argon-Nitrogenthis test were: Argon-Nitrogen Plasma (100:5) atPlasmap = 10 (100:5) Pa, P =at 500p = 10 W, Pa,T < P 80= 500˝C; W, duration: T < 80 °C;t = duration: 1.5 min. t = 1.5 min.

In the second setup of the experiment, only JSC Mars-1A was tested because a significant In the second setup of the experiment, only JSC Mars-1A was tested because a significant reduction reduction was shown in the first experimental setup. The exposure time in the second experiment was shown in the first experimental setup. The exposure time in the second experiment was increased was increased to 45 min to account for the pulsed mode of the plasma. Furthermore, changing the togas 45 minmixture to account from argon-nitrogen for the pulsed to mode argon-oxygen of the plasma. results Furthermore, in a plasma changingwith many the more gas mixtureof reactive from argon-nitrogenoxygen species, to but argon-oxygen far less radiation results in in the a plasmarange from with 100 many nm moreto 400 ofnm. reactive The change oxygen to species,a radical but faroxygen less radiation species based in the plasma range fromcaused 100 only nm a to slightly 400 nm. increased The change reduction to a radical of cell numbers oxygen species (Figure based3). plasmaThe initial caused microbial only a slightlycount decreased increased from reduction 2.7 ± 0.6 of cell× 106 numbers cells·g−1 to (Figure 6.7 ± 2.33). × The 105 initial cells·g microbial−1 after low-T count 6 ´1 5 ´1 decreasedplasma exposure, from 2.7 which˘ 0.6 ˆequates10 cells to a¨ gmeanto survival 6.7 ˘ 2.3 rateˆ 10of 0.25cells (survival¨ g after rate low-T = N/N plasma0; with N exposure, = cell whichnumber equates after plasma to a mean treatment survival and rateN0 = cell of 0.25 number (survival of the starting rate = N/N control)0; with and thus N = a cell total number reduction after plasmarate of treatment 75%. This andcompares N0 = cellto the number reduction of the of 2.3 starting ± 0.5 × control) 106 to 9.3 and ± 2.3 thus × 10 a5 totalafter exposing reduction JSC rate Mars- of 75%. This1Acompares samples with to the D. reduction radiodurans of 2.3cells˘ 0.5in theˆ 10 first6 to experiment 9.3 ˘ 2.3 ˆ 10only5 after for 1.5 exposing min, resulting JSC Mars-1A in a mean samples withsurvivalD. radiodurans rate of 0.43cells (and in thus the a first reduction experiment rate of only57%). for There 1.5 is min, not resultingmuch of an in additional a mean survival benefit to rate ofchange 0.43 (and the thusplasma a reduction from a radiation rate of based 57%). to There a reactive is not oxygen much based of an approach. additional In benefit both experimental to change the plasmasetups from there a is radiation no significant based difference to a reactive between oxygen the based cell numbers approach. of the In both vacuum experimental control sample setups and there isthe no significantplasma sample, difference although between both values the cell are numbers lower than of the the transport vacuum control. control sample and the plasma sample, although both values are lower than the transport control.

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Figure 3. Results of the second experimental setup: estimated via plate count (here only JSC Mars-1A was used). Cell Cell numbers numbers of of the the plasma plasma sample sample we werere reduced reduced compared compared to the to thestarting starting sample. sample. No Nosignificant significant difference difference in counts in counts between between the theplasma plasma sample sample and and thethe vacuum vacuum control control sample sample could could be beobserved. observed. Error Error bars bars indica indicatete standard standard deviation, deviation, n n== 3. 3. The The plasma plasma sterilization sterilization parameters parameters of this test were:were: Argon-Oxygen Plasma (100:5) at p = 10 Pa, P = 1500 W (1 kHz, 10% duty cycle), T < 80 ˝°C;C; duration: t = 45 min.

4. Discussion Discussion Our experiment to test the effecteffect ofof lowlow temperaturetemperature (<80(<80 ˝°C)C) plasma sterilization on a Martian regolith analoganalog mixedmixed withwith cells cells of ofD. D. radiodurans radioduransrevealed revealed only only a minimuma minimum inactivation inactivation of of the the cells cells in thein the samples. samples. A slightly A slightly larger larger reduction reduction of the of cell the numbers cell numbers was possible, was possible, when argon-oxygen when argon-oxygen plasma wasplasma used, was but used, insufficient but insufficient to use the to applieduse the methodapplied method as a negative as a negative control forcontrol life detection. for life detection. The sterilizing effect of the methodmethod is mainlymainly basedbased onon chemicallychemically reactivereactive species,species, ions, and (V)UV(V)UV photons. Therefore, Therefore, the the observation observation of of a high a high survival survival rate rate of cells of cells of D. of radioduransD. radiodurans is notis that not thatsurprising, surprising, because because this thisstrain in known in known for its for exce its exceptionalptional radiation radiation resistance resistance [3]. Nevertheless, [3]. Nevertheless, the theeffectiveness effectiveness of plasma of plasma for inactivating for inactivating bacterial bacterial spores spores has haspreviously previously been been demonstrated demonstrated for this for thissetup setup [8], where [8], where a log a 6 log reduction 6 reduction of Bacillus of Bacillus atrophaeus atrophaeus was achievedwas achieved within within 60 s treatment 60 s treatment time. With time. Withanother another low-pressure low-pressure low-temperature low-temperature plasma plasma device, device, it was it was shown shown that that it itis is possible possible to to inactivate 1088 spores—contaminated dropwise on stainlessstainless steel screws—of the highly tolerant Bacillus pumilus SAFR-032 within 5 min treatment time [1]. [1]. Neverthe Nevertheless,less, the experiments experiments performed here, reveal only little effecteffect ofof plasmaplasma onon spikedspiked MarsMars regolithregolith analog.analog. In addition to the highhigh radiationradiation resistanceresistance ofof D. radiodurans, thethe regolithregolith particlesparticles mightmight havehave helpedhelped thethe cellscells toto survive the treatment. A thin layer of soilsoil isis capablecapable to to shielding shielding the the cells cells from from harmful harmful radiation, radiation, as couldas could be shownbe shown previously previously [9]. A[9]. thin A layerthin layer of an of artificial an artificial meteorite powder powder helped helped spores spores to survive to survive exposure exposure of space of space conditions conditions in the in BIOPANthe BIOPAN exposure exposure experiment, experiment, whereas whereas unprotected unprotecte sporesd spores were were almost almost completely completely inactivated. inactivated. This isThis in goodis in good agreement agreement of experiments of experiments done ondone spores on spores by [10 by], who [10], showed who showed that even that a even few sporea few layersspore arelayers sufficient are sufficient to effectively to effectively protect protect the remaining the remaining spores spores against against plasma. plasma. Although Although we used we used in our in experimentour experiment only aonly very a thinvery layer thin oflayer soil (<1of soil mm) (<1 and mm)D. radioduransand D. radioduransdoes not formdoes spores,not form it seeminglyspores, it wasseemingly sufficient was to sufficient help the cellsto help to survive the cells the to treatment. survive the Additionally, treatment. Additionally, desiccated cells desiccated of D. radiodurans cells of, asD. wereradiodurans used in, as this were test, used have inshown this test, an have increased shown survival an increased rate under survival various rate stressunder conditionsvarious stress [2]. Theconditions main effect [2]. The reducing main the effect cell reducing numbers oftheD. cell radiodurans numbersseems of D. to radiodurans be related toseems extreme to be dehydration related to ratherextreme than dehydration radiation rather and oxidative than radiation chemical and speciesoxidative based chemical on the species similar based results on the between similar plasma results sterilizationbetween plasma and vacuum sterilization control and (Figure vacuum3). This control is also (Figure consistent 3). This with is earlier also experimentsconsistent with conducted earlier byexperiments [11]. The reasonconducted for the by different[11]. The survival reason for rates the of differentD. radiodurans survivalon rates the different of D. radiodurans regolith analogs on the indifferent the first regolith experimental analogs setup in isthe not first fully experime understood.ntal Asetup possible is not explanation fully understood. could be theA differentpossible grainexplanation size distributions, could be the which different is most grain obvious size distributions, for P-MRS versuswhich JSCis most Mars-1A obvious soil for (Tables P-MRS2 and versus3). JSC Mars-1A soil (Tables 2 and 3). In P-MRS, the grain size component between 50 and 150 µm is

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In P-MRS, the grain size component between 50 and 150 µm is dominant, which might have increased the shielding effect of the regolith against the influence of the plasma.

Table 2. Grain size distribution of P-MRS and S-MRS: distribution as weight percent (wt %) of the mixture. Data were obtained from Jörg Fritz, Museum für Naturkunde Berlin, Germany.

Grain Size [µm] P-MRS S-MRS 1000–550 13% 30% 550–300 11% 26% 300–150 12% 16% 150–50 50% 14% 50–0 14% 14%

Table 3. Grain size distribution of JSC Mars-1A: distribution as weight percent (wt %) of the mixture. Data were obtained and adapted from Wan et al., 2016 [12].

Grain Size [µm] JSC Mars-1A 1000–500 20% 500–250 30% 250–150 20% 150–100 12% 100–50 10% 50–0 8%

Thus, the method of low temperature plasma sterilization as it was used in our experiment was ineffective to reduce the cell numbers of a soil sample sufficiently. It appears this method is better suited for a cleanroom in spacecraft assembly facilities for treatment of surfaces, but based on our limited testing, not for sterilization if microbial cells are intermixed with soil.

Acknowledgments: This work (J.S. and D.S.M.) was financially supported by ERC Advanced Grant HOME (339231) and the German Research Foundation (DFG) Paketantrag (PlasmaDecon PAK 728) to M.F. (AW 7/3-1). The authors like to thank Ralf Möller (DLR—German Aerospace Center, Cologne, Germany) for providing the strain of Deinococcus radiodurans R1 as well as Dirk Wagner (GFZ—German Research Center for Geoscience, Potsdam, Germany) for the permission to use his lab facilities for the cultivation of cells and the analysis of the experimental samples. Author Contributions: J.S. and D.S.M. conceived and designed the experiments; J.S. performed the experiments, M.F. performed the plasma sterilization; M.F. and K.S. provided the plasma system; J.S. and D.S.M. analyzed the data; J.S. wrote the paper with contributions from all others. Conflicts of Interest: The authors declare no conflicts of interest.

Abbreviations The following abbreviations are used in this manuscript: CFU Colony forming units TGY (medium) Trypticase—Glucose—Yeast extract cultivation medium PBS Phosphate-buffered saline JSC Mars-1A Johnson Space Center Mars regolith analog P-MRS Phylosilicatic Mars regolith analog S-MRS Sulfatic Mars regolith analog DICP Double inductively plasma system

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