Sensitivity and adaptability of methanogens to perchlorates: Implications for life on Mars

Author’s Accepted Manuscript Sensitivity and Adaptability of Methanogens to Perchlorates: Implications for Life on Mars Timothy A. Kral, Timothy H. Goodhart, Joshua D. Harpool, Christopher E. Hearnsberger, Graham L. McCracken, Stanley W. McSpadden www.elsevier.com

PII: DOI: Reference:

S0032-0633(15)00363-3 http://dx.doi.org/10.1016/j.pss.2015.11.014 PSS4102

To appear in: Planetary and Space Science Received date: 31 August 2014 Revised date: 5 November 2015 Accepted date: 26 November 2015 Cite this article as: Timothy A. Kral, Timothy H. Goodhart, Joshua D. Harpool, Christopher E. Hearnsberger, Graham L. McCracken and Stanley W. McSpadden, Sensitivity and Adaptability of Methanogens to Perchlorates: Implications for Life on Mars, Planetary and Space Science, http://dx.doi.org/10.1016/j.pss.2015.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Sensitivity and Adaptability of Methanogens to Perchlorates: Implications for Life on Mars

Timothy A. Kralab, Timothy H. Goodharta, Joshua D. Harpoola, Christopher E. Hearnsbergera, Graham L. McCrackena, and Stanley W. McSpaddena. a

Dept. of Biological Sciences and bArkansas Center for Space and Planetary Sciences, SCEN-

601, University of Arkansas, Fayetteville, AR 72701, USA

Corresponding Author: Timothy A. Kral, Dept. of Biological Sciences, SCEN-601, University of Arkansas, Fayetteville, AR 72701, phone 479-575-6338, FAX 479-575-4010, e-mail [email protected]

Key Words: Methanogens, Mars, Perchlorate

Abstract: In 2008, the Mars Phoenix Lander discovered perchlorate at its landing site, and in 2012, the Curiosity Rover confirmed the presence of perchlorate on Mars. The research reported here was designed to determine if certain methanogens could grow in the presence of three different perchlorate salt solutions. The methanogens tested were Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum and Methanococcus maripaludis. Media were prepared containing 0, 0.5%, 1.0%, 2%, 5% and 10% wt/vol magnesium perchlorate,

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sodium perchlorate, or calcium perchlorate. Organisms were inoculated into their respective media followed by incubation at each organism’s growth temperature. Methane production, commonly used to measure methanogen growth, was measured by gas chromatography of headspace gas samples. Methane concentrations varied with species and perchlorate salt tested. However, all four methanogens produced substantial levels of methane in the presence of up to 1.0% perchlorate, but not higher. The standard procedure for growing methanogens typically includes sodium sulfide, a reducing agent, to reduce residual molecular oxygen. However, the sodium sulfide may have been reducing the perchlorate, thus allowing for growth of the methanogens. To investigate this possibility, experiments were conducted where stainless steel nails were used instead of sodium sulfide as the reducing agent. Prior to the addition of perchlorate and inoculation, the nails were removed from the liquid medium. Just as in the prior experiments, the methanogens produced methane at comparable levels to those seen with sodium sulfide as the reductant, indicating that sodium sulfide did not reduce the perchlorate to any significant extent. Additionally, cells metabolizing in 1% perchlorate were transferred to 2%, cells metabolizing in 2% were transferred to 5%, and finally cells metabolizing in 5% were transferred to 10%. All four species produced methane at 2% and 5%, but not 10% indicating some success in adapting cells to concentrations higher than 1%. The results reported here indicate that the presence of perchlorate on Mars does not rule out the possible existence of methanogens.

Introduction: On May 25, 2008, the Phoenix spacecraft landed in the northern plains of Mars with its science mission focusing on obtaining the ground truth for the 2002 Odyssey discovery of massive ice deposits under the surface regolith (Smith et al., 2008; Smith et al., 2009; Hecht et

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al., 2009). Onboard the spacecraft was a Wet Chemistry Laboratory (WCL) which measured solution concentrations of Ca2+, Mg2+, K+, NH4+, Na+, H+, Cl-, Br-, and I- using ion-selective electrodes (ISEs). One of the ISEs, a Hofmeister anion ISE was originally intended to measure nitrate, but was repurposed to detect perchlorate. This ISE detected 0.4 to 0.6% ClO4- by mass leached from each soil sample tested, and in fact, most of the soluble chlorine analyzed was in the form of perchlorate (Hecht et al., 2009). In recently refined analyses of WCL data, results suggest that the parent perchlorate molecules were approximately 60% calcium perchlorate and 40% magnesium perchlorate (Kounaves et al., 2014b). The Viking lander’s data were also reexamined and suggest ≤0.1% perchlorate at landing sites 1 and 2 (Navarro-Gonzalez et al., 2010). In 2014(a), Kounaves et al. detected the presence of perchlorate at a concentration of 0.6 +/- 0.1 ppm in the sawdust portion of martian meteorite EETA79001. Its quantity and location appeared to rule out terrestrial contamination. In addition, the Sample Analysis at Mars instrument onboard the Mars Science Laboratory rover (Curiosity) concluded that the best candidate for oxychlorine compounds detected during pyrolysis of martian samples is hydrated calcium perchlorate (Glavin et al., 2013). On Earth, perchlorate is often synthesized for use as a jet propellant, and is used in airbag inflators, fireworks and matches (Nozawa-Inoue et al., 2005; Logan, 2001; Motzer, 2001), and has become a concern in drinking water due to high concentrations in ground and surface waters (Betts, 1999; Logan et al., 2001; Urbansky, 1998; Urbansky, 2000). But in nature it is rarely seen except for very dry places like the Atacama Desert in Chile (Ader et al., 2008; Marion et al., 2010). On Mars, it is not known what the concentration may be in the deep sub-surface environment, however, it has been proposed that high concentrations could conceivably accumulate under the surface (Ader et al., 2008). It has also been suggested that perchlorate salts

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detected at Gale crater by the Curiosity rover may lower the freezing temperature of water forming stable hydrated compounds and liquid solutions in the uppermost subsurface (MartinTorres et al., 2015), and may allow for the formation of the observed recurring slope lineae (Kossacki and Markiewicz, 2014). With respect to future human exploration of Mars, perchlorate could be a source of oxygen and at the same time might be chemically hazardous to astronauts (Davila et al., 2013). The discovery of perchlorate in the soil on the surface of Mars could have an impact on the possibility of microbial life on Mars. Perchlorate salts such as magnesium, calcium and sodium with low eutectic temperatures could lower the freezing point of water substantially (Marion et al., 2009), thus possibly allowing for the existence of life forms such as microbes that can anaerobically reduce perchlorate (Dudley et al., 2007). A number of bacterial species including Dechloromonas, Dechlorosoma (Nozawa-Inoue et al., 2005; Bruce et al., 1999; Coates et al., 1999; Logan et al., 2001; Miller and Logan, 2000; Zang et al., 2002) and Azospirillum (Waller et al., 2004) have been shown to carry out reduction of perchlorate using the following pathway: ClO4- (perchlorate)  ClO3- (chlorate)  ClO2- (chlorite)  Cl- (chloride) + O2 (Rikken et al., 1996). Another microorganism that might exist under martian conditions is the methanogen, an organism in the domain Archaea, that metabolizes molecular hydrogen and carbon dioxide and produces methane. Methanogens have been studied as models for life in the subsurface of Mars for over 20 years (Chastain and Kral, 2012; Chastain and Kral, 2010(a); Chastain and Kral, 2010(b); Kendrick and Kral, 2006; Kral et al., 1998; Kral et al., 2004; Kral et al. 2011; Kral and Altheide, 2013; McAllister and Kral, 2006; Moran et al., 2005; Ormond and Kral, 2006; Ulrich 4

et al., 2010; Kral et al., 2014; Sinha and Kral, 2015). They are ideal candidates because they are anaerobic, non-photosynthetic, and most do not require any organic nutrients. The research reported here examines the ability of certain methanogens to metabolize in the presence of perchlorates, their ability to adapt to higher concentrations of perchlorate, and their survival following exposure to relatively high concentrations of perchlorate for variable lengths of time. Recently, it was reported that permafrost methanogens were more resistant to sodium and magnesium perchlorate than non-permafrost methanogens (Shcherbakova et al., 2015). In those studies, the highest concentration of perchlorate tested was 10 mM, substantially lower than the levels reported here, and the organisms studied were different than those discussed in the research reported here.

2. Materials and Methods 2.1. Organisms and Growth Media Methanothermobacter wolfeii (OCM 36), Methanosarcina barkeri (OCM 38), Methanobacterium formicicum (OCM 55) and Methanococcus maripaludis (OCM 151), were obtained from the Oregon Collection of Methanogens, Portland State University, OR. These four species were chosen because they can metabolize with H2 as an energy source, CO2 as carbon source (Staley, 1989), water and a few inorganic nutrients that are commonly found on Earth and most likely found on Mars. They were grown in their respective growth media, MM, MS, MSF, and MSH, as described by Kral and Altheide (2013). MM (a minimal medium) contains the following per liter: 4.0 g NaOH, 1.0 g NH4Cl, 1.0 g MgCl2*6H2O, 0.4 g CaCl2*2H2O, 0.4 g K2HPO4*3H2O, 1.0 mg resazurin, 5.0 mg Na2-EDTA*2H2O, 1.5 mg CoCl2*6H2O, 1.0 mg MnCl2*4H2O, 1.0 mg FeSO4*7H2O, 1.0 mg ZnCl2, 0.4 mg AlCl3*6H2O, 0.3 mg Na2WO4*2H2O, 0.2 mg CuCl2*2H2O, 0.2 mg NiSO4*6H2O, 0.1 mg H2SeO3, 0.1 mg

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H3BO3 and 0.1 mg NaMoO4*2H2O. MS medium is MM medium with the addition of yeast extract (2.0 g/L), trypticase peptone (2.0 g/L) and sodium 2-mercaptoethanesulfonate (0.5 g/L). MSF medium is MS medium with sodium formate (0.25 g/L) added. Finally, MSH medium is MS medium with the addition of NaCl (29.5 g/L), MgCl2*6H2O (1.7 g/L) and KCl (0.5 g/L), and is used to grow a number of halophilic methanogens. All of the media were saturated with CO2 gas prior to distribution into tubes. Prior to inoculation, 0.15 mL of a sterile 2.5% sodium sulfide solution was added per 10 mL of media to remove any residual O2. 2.2. Preparation of media with perchlorate salts. For media containing perchlorate salts (magnesium perchlorate [Mg(ClO4)2*6H2O], Alfa Aesar, stock #11635, Ward Hill, MA 01835; sodium perchlorate [NaClO4*H2O], SX0694-1, EMD Chemicals Inc., Gibbstown, NJ 08027; calcium perchlorate [Ca(ClO4)2], Alfa Aesar, Stock #11655, Ward Hill, MA 01835), appropriate amounts of the salts were weighed out and added to flasks of growth media to achieve final perchlorate salt concentrations of 0.5%, 1.0%, 2.0%, 5.0% and 10% wt/vol. These media were de-oxygenated in a Coy Anaerobic Chamber (Coy Laboratory Products Inc., Grass Lake Charter Township, MI) and distributed into anaerobic culture tubes (9 or 9.5 mL per tube, depending on further additions in the individual experiments). The tubes were sealed with butyl rubber stoppers, removed from the chamber, crimped and autoclaved at 121 oC at 15 psi for 30 minutes. Control tubes contained growth media without perchlorate salts added. 2.3. Preparation of media with perchlorate salts using membrane filter-sterilized salt solutions. In a separate preliminary experiment, growth media were prepared as described by Kral and Altheide (2013) without added perchlorate salts. Concentrated solutions of the perchlorate

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salts were membrane filter-sterilized (Nuclepore Filter, pore size 0.4 uM, SN110607, Nuclepore Corporation, Pleasanton, CA 94566). Small aliquots (0.5 mL) of the concentrated solutions were added to autoclaved growth media (9 mL) to achieve final concentrations of 0.5% and 1.0% wt/vol.

2.4. Measurement of perchlorate ion concentration and pH following preparation of the media. A perchlorate ion selective electrode (item #EW-27504-24, Cole Parmer, Vernon Hills, IL) was utilized to measure the perchlorate in autoclaved and non-autoclaved MM and MSH media. This electrode is designed to measure perchlorate within the concentration range of 0.7 to 98,000 ppm in the pH range of 2.5 to 11 with no interference from other ions. The electrode was used in conjunction with a Thermo Orion pH/ISE meter (model 720 A+, Thermo Fisher Scientific Inc, Waltham, MA). Media were prepared in quadruplicate as described in section 2.2 containing magnesium perchlorate (1%, 2%, 5% wt/vol), calcium perchlorate (1%, 5% wt/vol), or sodium perchlorate (1% wt/vol). Sodium sulfide (as described) was also added before measurements were taken. Standards included these same concentrations of magnesium, calcium, and sodium perchlorate dissolved in de-ionized water. These same experimental media were also subjected to pH measurements using a Fisher Scientific Accumet Basic pH Meter (Denver Instrument Co., Bohemia, NY).

2.5. Inoculation of media containing perchlorate salts, incubation and methane measurements.

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Exponential-phase cells (approximately 0.1 optical density at 600 nm, Spectronic 20D+, Spectronic Instruments) of the methanogens in standard growth media were added (0.5 mL) to the appropriate culture tubes. All tubes were pressurized with 200 kPa of H2 and then incubated at growth temperatures for the respective methanogens (55 oC for M. wolfeii, 37 oC for M. barkeri and M. formicicum, 25 oC for M. maripaludis). At regular time intervals (typically 3 to 7 days), headspace gas samples were removed with 3-cc sterile syringes and injected into a Varian Micro-GC with a thermal conductivity detector, model CP-4900 (Palo Alto, Ca) for methane analysis. Scotty Analyzed Gases (0.5%, 1.01%, 2.5%, 5.70%, 50.0% CH4; Supelco, Bellefonte, PA) were used to calibrate the gas chromatograph. This experiment was performed in triplicate.

2.6. Use of stainless steel nails as reductants. Cultures tubes containing MM, MSF and MSH media containing 0% and 1.0% wt/vol magnesium perchlorate were prepared as described in Section 2.2. Sterile sodium sulfide (2.5%) was added to each of these tubes as described. In a second set of culture tubes, the same media were prepared without perchlorate salts and without sodium sulfide. A 4.45 cm, 1.75 g stainless steel nail (GripRite Fas’ners, PrimeSource Building Products, Inc., Irving, TX 75038) was added to each of these tubes prior to sealing, crimping and autoclaving. Stainless steel is a steel alloy (iron and carbon) with a minimum of 10.5% chromium (http://web.archive.org/web/20060924043735/http://www.nickelinstitute.org/index.cfm/ci_id/11 021.htm; retrieved 20 October 2015). Following sterilization, the nail-containing tubes were allowed to sit for three to four days at room temperature until the MM medium changed color from blue to clear, indicating that residual O2 had been reduced. Once this had occurred, a round 1.9 cm, 7 g magnet (Tree House Studio, Oklahoma City, OK 73179, magnet strength rating of 6) 8

was used to move the nail up the side of each culture tube until the nail was no longer in contact with the medium. The magnet was then secured to the tube using electrical tape, maintaining the nail at a position outside of the medium (Figure 1). A 20% wt/vol stock solution of magnesium perchlorate in de-ionized water was deoxygenated by bubbling with argon gas for 10 minutes, followed by sealing and autoclaving. A 0.5 mL aliquot of the stock solution was added to half of the nail-containing tubes (9.0 mL) once the O2 had been reduced. The remaining nail-containing tubes served as controls (no perchlorate). There were four tubes for each type of medium. M. wolfeii, M. formicicum and M. maripaludis were grown in standard methanogenic growth media to exponential phase. Cells of each species were washed according to Kral et al. (2014). Cells suspended in bicarbonate buffer without sodium sulfide were inoculated (0.5 mL) into the prepared tubes containing either sodium sulfide or stainless steel nails. The tubes were pressurized with H2 followed by incubation and methane measurements as described above. 2.7. Attempts to adapt methanogens to higher concentrations of magnesium perchlorate. Methanogenic cells were inoculated into magnesium perchlorate-containing media at concentrations of 0% and 1% wt/vol. The 1% culture tube that had the highest methane concentration for each organism following four weeks of incubation was used to inoculate tubes containing 0%, 1% and 2% wt/vol magnesium perchlorate. The 2% culture tube that had the highest methane concentration for each organism was used to inoculate tubes containing 0%, 2% and 5% wt/vol magnesium perchlorate. Finally, the 5% culture tube that had the highest methane concentration for each organism was used to inoculate tubes containing 0%, 5% and 10% wt/vol magnesium perchlorate. In all cases, there were three tubes for each concentration of each

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medium. Tubes were pressurized with H2 and incubated, followed by methane measurements as described above. 2.8. Exposure of methanogens to relatively high concentrations of perchlorate salts. Stock solutions of perchlorate salts were prepared by dissolving magnesium perchlorate, sodium perchlorate or calcium perchlorate in de-ionized water to achieve final concentrations of 5% wt/vol and 25% wt/vol for each. These solutions were autoclaved at 121 oC and 15 psi for 30 minutes. M. wolfeii and M. barkeri cells were washed in sterile 50 mL polycarbonate centrifuge tubes according to McAllister and Kral (2006). The cell pellets were suspended in sterile bicarbonate buffer followed by transfer to sterile 1.5 mL microcentrifuge tubes. These tubes were centrifuged (15,000 rpm) for 10 minutes in a Fisher micro-centrifuge (Model 235A, Fisher Scientific, Pittsburgh, PA). The buffer supernatants were poured off leaving the cell pellets. Stock perchlorate solutions were added (1 mL) to each pellet. Controls consisted of tubes with added sterile buffer. These tubes were then placed into the Coy Anaerobic Chamber for one hour, 24 hours or 72 hours. There were three tubes for each organism in each concentration of each salt solution. Following the designated exposure time, each tube was centrifuged again (15,000 rpm) for 10 minutes followed by removal of the perchlorate solutions. Each pellet was suspended again in sterile buffer and washed as described. The pellets were washed a final time with the appropriate growth media, followed by suspension in the appropriate growth media and transfer to culture tubes of the same medium. Incubation and methane measurements were as previously described.

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3. Results In order to determine if interactions with media components resulted in a reduction in perchlorate ion concentration compared to the same starting concentrations in deionized water, a perchlorate ion selective electrode was used to measure perchlorate in two types of methanogenic growth media, MM and MSH. MM is a minimal medium with the least number of components while MSH has the most in these experiments. The initial perchlorate salt concentrations chosen were those where methane production occurred (up to 5%). In MM medium, there appeared to be very little if any reduction in any perchlorate concentration tested (Figure 2). However, in MSH medium, there was significant reduction of perchlorate at all concentrations tested (roughly 20% [e.g. 5% magnesium perchlorate] to 50% [e.g. 1% calcium perchlorate] reduction in concentration). There appeared to be greater reduction in concentration with calcium perchlorate compared to magnesium or sodium perchlorates. Autoclaving appeared to have no noticeable effect on perchlorate concentrations. This was also confirmed by comparing membrane-filtered perchlorate stock solutions to autoclaved perchloratesupplemented media (data not shown). The pH ranged from 6.6 to 6.9 for MM medium and 6.5 to 6.8 for MSH medium (data not shown). The pH for MM medium without perchlorate was 6.8 while that of MSH medium without perchlorate was 6.6. Table 1 shows the highest headspace methane percentages for M. wolfeii, M. barkeri, M. formicicum and M. maripaludis growing in the presence of magnesium perchlorate (0.5% and 1.0% wt/vol), sodium perchlorate (0.5% and 1.0% wt/vol) and calcium perchlorate (1.0% wt/vol) as well as controls without perchlorate. In many cases, the presence of perchlorate resulted in less headspace methane than in control tubes, and the amount was further reduced as the perchlorate concentration increased. In some cases (e.g. M. formicicum in the presence of 0.5% 11

or 1.0% sodium perchlorate), there was no significant difference between the perchlorate tubes and the control tubes. Results seen in Figure 3 demonstrate methane production in media using sodium sulfide compared to stainless steel nails as reductants in the presence of 0% or 1% wt/vol magnesium perchlorate. In all cases, the three methanogens tested produced substantial methane using stainless steel nails, similar to those with sodium sulfide. For M. maripaludis, the amount of methane produced with the stainless steel nail and 1% magnesium perchlorate present appears to be about half of that produced in the presence of sodium sulfide, however, the error bars do overlap, so the difference may not be significant. Results from experiments designed to adapt the four methanogenic species to higher concentrations of magnesium perchlorate are seen in Figure 4. For M. wolfeii, M. formicicum and M. maripaludis, headspace methane concentrations (12.6%, 24.5% and 22.0%, respectively) were substantial for cells that were transferred from 1% to 2% wt/vol magnesium perchlorate. For M. barkeri, it was much less (1.9%), but significant (increasing with time), nonetheless. For cells transferred from 2% to 5% wt/vol magnesium perchlorate, all species demonstrated methane production, albeit at much lower levels. Table 2 shows survival of M. wolfeii and M. barkeri in standard methanogen growth media following variable exposure (one hour, 24 hours and 72 hours) to two relatively high concentrations (5% and 25% wt/vol) of perchlorate salt solutions (magnesium, sodium and calcium). Highest headspace methane concentration is presented for each tube in a manner previously done by Kral and Altheide (2013) for methanogen survival following exposure to low pressure and desiccation. For M. wolfeii, at least one culture out of three survived all of the

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conditions tested except 25% sodium perchlorate for 72 hours and 25% calcium perchlorate for 24 and 72 hours. For M. barkeri, all cultures exposed to any level of perchlorate showed reduced methane levels compared to controls with virtually no survival at 25% perchlorate for 24 and 72 hours exposure. 4. Discussion The four methanogens used in these studies can metabolize (defined here as methanogenesis) fairly well at concentrations of up to 1% wt/vol of three different perchlorate salt solutions (Table 1). None of the methanogens showed methane production at starting concentrations greater than 1% sodium, magnesium or calcium perchlorate. In general, inhibition by perchlorate increased as concentration increased. Prior to performing the majority of experiments, there was a concern that the heat from autoclaving might diminish the activity of the perchlorate. Thus, a few experiments were conducted where membrane filter-sterilized perchlorate solutions were added to the media to eliminate the heat factor. The results for autoclaved media vs. filtered perchlorate media were virtually indistinguishable. There was also concern that components found in the media might interact with the perchlorate ion, reducing its concentration in the media. Potentially, there are a number of chemical reactions that might have occurred due to the compositions of the four different media and the increased temperature during autoclaving. All four media contained ferrous sulfate and most media in these experiments also contained sodium sulfide. Ferrous sulfide is known as a reducing agent for culturing of anaerobes (Brock and Od’ea, 1977). The important question is whether the perchlorate ion concentration was affected. Indeed, this was the case for MSH

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medium, but not MM medium, therefore the presence of ferrous cation and sulfide anion apparently did not reduce perchlorate concentration to any measureable extent. In some cases, the reduced perchlorate concentration in MSH medium was approximately 50%. MSH medium is MM medium plus yeast extract, trypticase peptone, sodium 2-mercaptoethanesulfonate, and relatively high concentrations of sodium chloride, magnesium chloride and potassium chloride. The final salt concentration in MSH medium is approximately 4%, ideal for growth of many halophilic methanogens. Two of the other media, MS and MSF, also contain yeast extract, trypticase peptone and sodium 2-mercaptoethanesulfonate. With respect to solubility, perchlorate salts in general are highly soluble in an aqueous solution with the exception of potassium perchlorate (1.5 g in 100 mL H2O at 25 oC; http://hazard.com/msds/mf/baker/baker/files/p5983.htm; retrieved 27 October 2015), which has the lowest solubility of any alkali metal perchlorate (https://en.wikipedia.org/wiki/Perchlorate; retrieved 20 October 2015). There are two known sources of potassium in MSH medium, K2HPO4*3H20 and KCl. The combined potassium ion concentration is 0.011 M, which coincidentally, is the same as the highest solubility concentration of potassium perchlorate in H2O at 25 oC. The organism, M. maripaludis, which was grown in MSH medium, was incubated at 25 oC. Therefore, the added potassium should not have combined with perchlorate to any significant degree and should be ruled out as a possible factor in reducing the perchlorate ion concentration. All of the MS media also contain sodium 2-mercaptoethanesulfonate, also known as coenzyme M, which acts as a C1 donor in methanogenesis (Balch and Wolfe, 1979; Taylor and Wolfe, 1974; Thauer, 1998). The sodium salt of 2-mercaptoethanesulfonate is known as Mesna and is used as a chemotherapeutic adjuvant where it detoxifies urotoxic metabolites by

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reaction of its sulfhydryl group with ,-unsaturated carbonyl containing compounds (Thurston, 2007). Interactions of 2-mercaptoethanesulfonate with perchlorate are unknown. MSH, as well as MS and MSF media, are undefined or complex media, ones where the amino acid sources, yeast extract and trypticase peptone, contain a variety of compounds of unknown composition (Atlas, 1993). There was no visible precipitate formation in any of the sodium or magnesium perchlorate-containing tubes. However, in tubes containing calcium perchlorate, there was a precipitate at all concentrations tested in both media. This precipitate consisted of the relatively insoluble calcium phosphate (0.02 g/100 mL H2O at 25 oC; https://www.fishersci.com/shop/msdsproxy?productName=C12912&productDescription=CAL+ PHOS+DIB+USP%2FFCC+HYDR+12KG&catNo=C12912&vendorId=VN00033897&storeId=10652; retrieved 27 October 2015). Phosphate is a normal component of all of the media used in these experiments. In addition to acting as a nutrient source, phosphate is also used to help stabilize pH. Because of this important role, all MM and MSH media that were subjected to perchlorate ion measurement were also subjected to pH measurement. For both media, the range was 0.3 pH units, and each range included the pH of its respected medium without perchlorate addition. Thus, the removal of phosphate ion from solution in calcium perchlorate-supplemented media did not appear to be a significant factor in any of the results observed. All media also contained resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide), a blue dye that is irreversibly reduced to the pink dye resorufin during the preparation of the media. Resazurin is a standard component found in methanogenic growth media as an O2 indicator (Boone et al., 1989). Once the media were allowed to sit in the anaerobic chamber overnight, the resorufin was further reduced to colorless dihydroresorufin. The reduction to dihydroresorufin is reversible in the presence of O2, thus its role as an O2 indicator (O’Brien et

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al., 2000; Guerin et al., 2001; Gonzalez-Pinzon et al., 2012). Since MM medium contains resazurin, and there was virtually no reduction in perchlorate concentration in MM medium, resazurin apparently was not a contributing factor in the reduction of the perchlorate concentration in the MSH medium. What is causing the reduced perchlorate concentration in MSH medium is unknown. The fact that the concentration reduction was greater at lower starting perchlorate concentrations might indicate that the components that were causing the reduction had been exhausted at the higher perchlorate levels. The bottom line is there was reduction in perchlorate concentration in MSH. Perchlorate concentrations mentioned throughout this manuscript are the concentrations of perchlorate salts added to media (wt/vol). Resulting perchlorate concentrations in MM medium, only, would appear to accurately correspond to the added perchlorate salt concentrations. Another concern dealt with the possibility that perchlorate might oxidize the highly reduced methane molecule. The observation in Table 1 that, in some cases, the methane levels in control tubes and 0.5% and 1% perchlorate-containing tubes were the same would seem to indicate that any oxidation of the methane by perchlorate was negligible. A final concern had to do with the addition of sodium sulfide to each medium tube to reduce residual O2. Would this reducing agent neutralize the perchlorate? In a separate experiment, stainless steel nails were used instead of sodium sulfide. In each tube, the nail remained in the liquid medium until the medium became clear, indicating reduction of residual O2. This reduction typically took three to four days at room temperature. Then the nail was removed from the medium using a magnet (Figure 1), thus preventing interaction with the perchlorate that was added after its removal. There was no visible oxidation on any of the nails following removal from the media. The importance of removing the nail from the medium 16

cannot be overstated. Hydrogen gas, which is added to our culture tubes as an energy source, has been shown to reduce perchlorate when in the presence of some metallic catalysts, however iron and chromium were very poor catalysts (Wang et al., 2008). Hydrogen gas by itself does not lead to any measureable reduction of perchlorate. Even though the reaction is thermodynamically favorable (Gibbs free energy of -289 kJ), the reaction is kinetically slow due to its large activation energy (Wang et al., 2008). Also, microbial reduction of perchlorate in the presence of zero-valent iron has been demonstrated. Son et al., (2006) observed complete removal of 65 mg/L of perchlorate in batch reactors in eight days by iron-supported mixed cultures. Metallic (zero-valent) iron was chosen as a reducing agent because it is insoluble in water and easy to remove from solution. The chromium reacts with O2 and water to form a protective oxide film known as a passive layer on the surface of the stainless steel (http://www.eurofer.org/Eurofer%20Stainless/About%20Stainless%20Steel.fhtml; retrieved 20 October 2015). The chromium in the steel continues to react with O2 and water in the environment to strengthen the oxide layer. The half reaction Fe  Fe2+ + 2e- has an oxidation potential of +0.47 volts. The half reaction Cr  Cr3+ + 3e- has an even greater oxidation potential of +0.74 volts (Quagliano and Vallarino, 1969). So the chromium probably played a more significant role in the reduction of O2 than the iron. Nonetheless, the combination of iron and chromium accomplished the task of reducing the residual O2 and allowed for the removal of the reducing agent(s) before addition of perchlorate. Any iron or chromium ions released from the nail would not have combined with the added perchlorate because ferrous perchlorate, ferric perchlorate, and chromium (III) perchlorate, like most perchlorate salts, are highly soluble, as discussed previously. Because there was no significant difference in methane production in 1%

17

magnesium perchlorate when using sodium sulfide or stainless steel as a reductant, it appears that sodium sulfide was not reducing the perchlorate to any significant level. When it had been determined that these methanogens could produce substantial methane in the presence of up to 1% perchlorate, but not higher (Table 1), attempts were made to adapt them to higher levels of magnesium perchlorate. In Figure 3, it can be seen that these attempts were successful up to 5%. No methane production was observed at 10%. The genetic basis for the adaptation to higher levels of perchlorate was not investigated nor was the physiological mechanism for the increased “resistance/tolerance”. In experiments summarized in Table 2, the emphasis switched to survival of M. wolfeii and M. barkeri following exposure to relatively high concentrations of perchlorate salt solutions for variable lengths of time. Cells were exposed to 5% or 25% wt/vol perchlorate in water in an anaerobic chamber for one, 24 or 72 hours. Immediately following exposure, the cells were washed and transferred into growth media. In general, there were M. wolfeii cultures that survived both concentrations for one and 24 hours (except calcium at 25%), and even a few that survived 72 hour exposures. Most M. wolfeii cultures that survived produced methane levels comparable to the controls. M. barkeri, on the other hand, did not fare as well. In all exposures to perchlorate, the highest methane levels were lower than the controls, and fewer survived at 25% perchlorate for 24 or 72 hours. Salts have a major impact on the biosphere, influencing water availability and other biological systems which can cause major cellular stress (Hallsworth et al., 2007). One of those effects is chaotropicity. Chaotropic compounds, such as perchlorate salts, weaken the hydrogen bonds between water and macromolecules (Shcherbakova et al., 2015), and elicit specific stress

18

responses in microbial cells (Hallsworth et al., 2003; Bhaganna et al., 2010; Cray et al, 2013). Other chaotropic salts such as MgCl2 and CaCl2 induce water stress in microbial cells at sublethal concentrations (Hallsworth et al., 2003; Hallsworth et al., 2007; Cray et al., 2013) and at sufficient concentrations are lethal (Hallsworth et al., 2007; Bodaker et al., 2010; Cray et al, 2013). Hallsworth et al. (2007) state that the chaotropicity of MgCl2, rather than reduction in water activity, is the factor that inhibits cells. This chaotropic nature of perchlorates might explain the inhibition exhibited by methanogens in this study. Because of the high salt content of the martian regolith, tolerance to salt would seem to be a requirement for survival on Mars (Shcherbakova et al., 2015). Overall, the results reported here would seem to indicate that methanogens might be able to survive and metabolize below the surface of Mars in the presence of perchlorate. If the perchlorate is in the dry state, as discovered by the Phoenix Lander (Hecht, 2009), it would not be relevant since methanogens, like all known life forms, must have water to metabolize. How long methanogens can survive in the dry state at the low martian pressures has been addressed previously by Kral and Altheide (2013). The research reported here deals with aqueous perchlorate. If the aqueous concentration is as high as 5%, methanogens might be able to metabolize in its presence. If it is much higher (e.g 25%), survival would most likely depend on length of exposure. As stated previously (Kral and Altheide, 2013; Kral et al., 2014), these experiments were not intended to mimic actual martian conditions, but rather, to study metabolism and survival of methanogens exposed to perchlorates under ideal growth conditions.

5. Conclusions

19

M. wolfeii, M. barkeri, M. formicicum and M. maripaludis were able to produce substantial methane in the presence of up to 1% magnesium, sodium or calcium perchlorate solutions. In the case of MSH medium, and presumably, MS and MSF media, perchlorate levels were diminished up to approximately 50%, most likely due to unknown interactions between media components and the perchlorate. In general, increasing perchlorate resulted in decreasing methane production. Stainless steel nails can be used as a removable reducing agent in the media. By comparing stainless steel to sodium sulfide, the standard reducing agent in methanogenic media, it appeared that sodium sulfide did not measurably diminish the effects of perchlorate. All four of the methanogenic species were shown to be able to adapt to magnesium perchlorate concentrations as high as 5%, but not 10%. Further research would be necessary to propose a mechanism for the increased resistance/tolerance. M. wolfeii and M. barkeri were able to survive exposure to 5% and 25% perchlorate for variable lengths of time with M. wolfeii surviving the 25% concentration for greater lengths of time than did M. barkeri. The results reported here indicate that the presence of perchlorate on Mars does not rule out the possible existence of methanogens on Mars.

Acknowledgements The research was supported by a grant from the NASA Astrobiology: Exobiology and Evolutionary Biology Program, #NNX12AD90G.

20

References

Atlas, R.M., 1993, Handbook of Microbiological Media, CRC Press, Inc., Boca Raton, FL.

Ader, M., Chaudhuri, S., Coates, J.D., Coleman, M., 2008. Microbial perchlorate reduction: a precise laboratory determination of the chlorine isotope fractionation and its possible biochemical basis. Earth and Planetary Science Letters 269, 604-612.

Balch, W.E., Wolfe, R.S., 1979. Specificity and biological distribution of coenzyme M (2mercaptoethanesulfonic acid). J. Bacteriol. 137, 256-263.

Betts, K.S., 1999. A wide variety of bugs can break down perchlorate. Environ. Sci. Technol. 33, 515.

Bhaganna, P., Volkers,, R.J.M., Bell, A.N.W., Kluge, K., Timson, D.J., McGrath, J.W., Ruijssenaars, H.J., Hallsworth, J.E., 2010. Hydrophobic substances induce water stress in microbial cells. Microb. Biotechnol. 3, 701-716.

21

Bodaker, I., Sharon, I., Suzuki, M.T., Feingersch, R., Shmoish, M., Andreishcheva, E., Sogin, M.L., Rosenberg, M., Maguire, M.E., Belkin, S., Oren, A., Beja, O., 2010. Comparative community genomics in the Dead Sea: an increasingly extreme environment. ISME J. 4, 399-407.

Boone, D.R., Johnson, R.L., Liu, Y., 1989. Diffusion of the interspecies electron carriers H2 and formate in methanogenic ecosystems and its implications in the measurement of Km for H2 or formate uptake. Appl. Environ. Microbio. 55, 1735-1741.

Brock, T.D., Od’ea, K., 1977. Amorphous ferrous sulfide as a reducing agent for culture of anaerobes. Appl. Environ. Microbiol. 33, 254-256.

Bruce, R.A., Achenbach, L.A., Coates, J.D., 1999. Reduction of (per)chlorate by a novel organism isolated from paper mill waste. Environ. Microbiol. 1, 319-329.

Chastain, B.K., Kral, T.A., 2010a. Zero-valent iron on Mars: an alternative energy source for methanogens. Icarus 208, 198–201.

Chastain, B.K., Kral, T.A., 2010b. Approaching Mars-like geochemical conditions in the laboratory: Omission of artificial buffers and reductants in a study of biogenic methane production on a smectite clay. Astrobiology 10, 889-897.

22

Chastain, B.K., Kral, T.A., 2012. Methane production by methanogens in the presence of Mars regolith analogs. (abstract 271). in American Society for Microbiology General Meeting, San Francisco.

Coates, J.D., Michaelidou, U., Bruce, R.A., O’Connor, S.M., Crespi, J.N., Achenbach, L.A., 1999. Ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria. Appl. Environ. Microbiol. 65, 5234-5241.

Cray, J.A., Russell, J.T., Timson, D.J., Singhal, R.S., Hallsworth, J.E., 2013. A universal measure of chaotropicity and kosmotropicity. Environ. Microbiol. 15, 287-296.

Davila, A.F., Willson, D., Coates, J.D., McKay, C.P., 2013. Perchlorate on Mars: a chemical hazard and a resource for humans. Int. J. Astrobiol. 12, 321-325.

Dudley, M., Salamone, A., Nerenberg, R., 2008. Kinetics of chlorate-accumulating perchloratereducing bacterium. Water Research 42, 2403-2410.

23

Glavin, D.P., Freissinet, C., Miller, K.E., Eigenbrode, J.L., Brunner, A.E., Buch, A., Sutter, B., Archer Jr., P.D., Atreya, S.K., Brinckerhoff, W.B., Cabane, Michael, Coll, P., Conrad, P.G., Coscia, D., Dworkin, J.P., Franz, H.B., Grotzinger, J.P., Leshin, L.A., Martin, M.G., McKay, C., Ming, D.W., Navarro-Gonzalez, R., Pavlov, A., Steele, A., Summons, R.E., Szopa, C., Teinturier, S., Mahaffy, P.R., 2013. Evidence for perchlorates and the origin of chlorinated hydrocarbons detected by SAM at the Rocknest Aeolian deposit in Gale Crater. J. Geophys. Res. (Planets) 118, 1955-1973.

Gonzalez-Pinzon, R., Haggerty, R., Myrold, D.D., 2012. Measuring aerobic respiration in stream ecosystems using the resazurin-resorufin system. J. Geophys. Res. 117, doi:10.1029/2012JG001965, 2012.

Guerin, T., Mondido, M., McClenn, B., Peasley, B., 2001. Application of resazurin for estimating abundance of contaminant-degrading microorganisms. Lett. Appl. Microbiol. 32, 340345.

Hallsworth, J.E., Heim, S., Timmis, K.N., 2003. Chaotropic sloutes cause water stress in Pseudomonas putida. Environ. Microbiol. 5, 1270-1280.

Hallsworth, J.E., Yakimov, M.M., Golyshin, P.N., Gillion, J.L.M., D’Auria,, G., Alves, F.D.L., Cono, V.L., Genovese, M., McKew, B.A., Hayes, S.L., Harris, G., Giuliano, L., Timmis, K.N., 24

Mcgenity, T.J. 2007. Limits of life in MgCl2-containing environments: chaotropicity defines the window. Environ. Microbiol. 9, 801-813.

Hecht, M.H., Kounaves, S.P., Quinn, R.C., West, S.J., Young, S.M.M., Ming, D.W., Catling, D.C., Clark, B.C., Boynton, W.V., Hoffman, J., DeFlores, L.P., Gospodinova, K., Kapit, J., Smith, P.H., 2009. Detection of perchlorate and the soluble chemistry of martian soil at the Phoenix Lander site. Science 325, 64-67.

Kendrick, M.G., Kral, T.A., 2006. Survival of methanogens during desiccation: implications for life on Mars. Astrobiology 6, 546–551.

Kossacki, K.J., Markiewicz, W.J., 2014. Seasonal flows on dark martian slopes, thermal conditions for liquescence of salts. Icarus 233, 126-130.

Kounaves, S.P., Carrier, B.L., O’Neil, G.D., Stroble, S.T., Claire, M.W., 2014(a). Evidence of martian perchlorate, chlorate, and nitrate in Mars meteorite EETA79001: Implications for oxidants and organics. Icarus 229, 206-213.

Kounaves, S.P., Chaniotakis, N.A., Chevier, V.F., Carrier, B.L., Folds, K.E., Hansen, V.M., McElhoney, K.M., O’Neil, G.D., Weber, A.W., 2014(b). Identification of the perchlorate parent

25

salts at the Phoenix Mars landing site and possible implications. Icarus 232, 226-231.

Kral, T.A., Altheide, S.T., 2013. Methanogen survival following exposure to desiccation, low pressure and martian regolith analogs. Planet. Space Sci. 89, 167-171.

Kral, T.A., Altheide, T.S., Lueders, A.E., Schuerger, A.C., 2011. Low pressure and desiccation effects on methanogens: Implications for life on Mars. Planet. Space Sci. 59, 264-270.

Kral, T.A., Bekkum, C.R., McKay, C.P., 2004. Growth of methanogens on a Mars soil simulant. Origins Life Evol. Biosphere 34, 615-626.

Kral, T.A., Birch, W., Lavender, L.E., Bryant, B.T., 2014. Potential Use of Highly Insoluble Carbonates as Carbon Sources by Methanogens in the Subsurface of Mars. Planet. Space Sci. 101, 181-185.

Kral, T.A., Brink, K.M., Miller, S.M., McKay, C.P., 1998. Hydrogen consumption by methanogens on the early Earth. Origins Life Evol. Biosphere 28, 311-319.

26

Logan, B.E., 2001. Assessing the outlook for perchlorate mediation. Environ. Sci. Technol. 35, 482-487.

Logan, B.E., Zhang, H.S., Mulvaney, P., Milner, M.G., Head, I.M., Unz, R.F., 2001. Kinetics of perchlorate- and chlorate-respiring bacteria. Appl. Environ. Microbiol. 67, 2499-2506.

Marion, G.M., Catling, D.C., Zahnle, K.J., Claire, M.W., 2010. Modeling aqueous perchlorate chemistries with applications to Mars. Icarus 207, 675-685.

Martin-Torres, F.J., Zorzano, M., Valentin-Serrano, P., Harri, A., Genzer, M., Kemppinen, O., Rivera-Valentin, E.G., Jun, I., Wray, J., Madsen, M.B., Goetz, W., McEwen, A.S., Hardgrove, C., Renno, N., Chevrier, V.F., Mishna, M., Navarro-Gonzalez, R., Martinez-Frias, J., Conrad, P., McConnochie, T., Cockell, C., Berger, G., Vasavada, A.R., Sumner, D., Vaniman, D., 2015. Transient liquid water and water activity at Gale crater on Mars. Nature Geoscience 8, 357-361.

McAllister, S.A., Kral, T.A., 2006. Methane production by methanogens following an aerobic washing procedure: simplifying methods for manipulation. Astrobiology 6, 819–823.

Miller, J.P., Logan, B.E., 2000. Sustained perchlorate degradation in an autotrophic, gas-phase, packed-bed bioreactor. Environ. Sci. Technol. 34, 3018-3022.

27

Moran, M., Miller, J.D., Kral, T., Scott, D., 2005. Desert methane: Implications for life detection on Mars. Icarus 178, 277-280.

Motzer, W.E., 2001. Perchlorate: problems, detection and solutions. Environ. Forensics 2, 301311.

Navarro-Gonzalez, R., Vargas, E., de la Rosa, J., Raga,, A.C., McKay, C.P., 2010. Reanalysis of the Viking results suggests perchlorate and organics at midlatitudes on Mars. J. Geophys. Res. (Planets) 115, DOI: 10.1029/2010JE003599.

Nozawa-Inoue, M., Scow, K.M., Rolston, D.E., 2005. Reduction of perchlorate and nitrate by microbial communities in vadose soil. Appl. Environ. Microbiol. 71, 3928-3934.

O.Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 54215426.

Ormond, D.R., Kral, T.A., 2006. Washing methanogenic cells with the liquid fraction from a

28

Mars soil simulant and water mixture. J Microbiol Methods 67, 603–605.

Quagliano, J.V, Vallarino, L.M., 1969. Chemistry, 3rd Ed, Prentice-Hall, Inc., Englewood Cliffs, NJ.

Rikken, G.B., Kroon, A.G.M., Van Ginkel, C.G., 1996. Transformation of (per)chlorate into chloride by a newly isolated bacterium: reduction and dismutation. Appl. Microbiol. Biotechnol. 45, 420-426.

Shcherbakova, V, Oshurkova, V, Yoshimura, Y. 2015. The effects of perchlorates on the permafrost methanogens: Implications for autotrophic life on Mars. Microorganisms. Doi:10.3390/microorganisms3030518.

Sinha N., Kral, T.A., 2015. Stable Carbon Isotope Fractionation by Methanogens Growing on Different Mars Regolith Analogs. Planet. Space Science. 112, 35-41.

Smith, P.H., Tamppari, L., Arvidson, R.R., Bass, D., Blaney, D., Boynton, W., Carswell, A., Catling, D., Clark, B., Duck, T., DeJong, E., Fisher, D., Goetz, W., Gunnlaugsson, P., Hecht, M., Hipkin, V., Hoffman, J., Hviid, S., Keller, H., Kounaves, S., Lange, C.F., Lemmon, M., Madsen, M., Malin, M., Markiewicz, W., Marshall, J., McKay, C., Mellon, M., Michelangeli, D., Ming, 29

D., Morris, R., Renno, N., Pike, W.T., Staufer, U., Stoker, C., Taylor, P., Whiteway, J., Young, S., Zent, A., 2008. Introduction to special section on the Phoenix Mission: Landing site characterization experiments, mission overviews, and expected science. J. Geophys. Res. 113, doi:10.1029/2008JE003083.

Smith, P.H., Tamppari, L., Arvidson, R.R., Bass, D., Blaney, D., Boynton, W., Carswell, A., Catling, D., Clark, B., Duck, T., DeJong, E., Fisher, D., Goetz, W., Gunnlaugsson, P., Hecht, M., Hipkin, V., Hoffman, J., Hviid, S., Keller, H., Kounaves, S., Lange, C.F., Lemmon, M., Madsen, M., Markiewicz, W., Marshall, J., McKay, C., Mellon, M., Ming, D., Morris, R., Pike, W.T., Renno, N., Staufer, U., Stoker, Taylor, P., C., Whiteway, J., Young, S., Zent, A., 2009. H2O at the Phoenix landing site. Science 325, 58-61

Son, A., Lee, J., Chiu, P.C., Kim, B.J., Cha, D.K., 2006. Microbial reduction of perchlorate with zero-valent iron. Water Res. 40, 2027-2032.

Staley, J.T., 1989. Bergey’s Manual of Systematic Bacteriology, Vol. 3, Williams and Wilkins, Baltimore.

Taylor, C.D., Wolfe, R.S., 1974. Structure and methylation of coenzyme M (HSCH2CH2SO3). J. Biol Chem. 249, 4879-4885.

30

Thauer, R.K., 1998. Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiology 144, 2377-2406.

Thurston, D.E., 2007. Chemistry and Pharmacology of Anticancer Drugs. CRC Press/ Taylor & Francis, Boca Raton. Pp. 53-54.

Ulrich, R., Kral, T., Chevrier, V., Pilgrim, R., Roe, L., 2010. Dynamic temperature fields under the Mars landing sites and implications for supporting microbial life. Astrobiology. 10, 643-650.

Urbansky, E.T., 1998. Perchlorate chemistry: implications for analysis and remediation. Bioremediat. J. 2, 81-85.

Urbansky, E.T., ed, 2000. Perchlorate in the environment. Kluwer Academic/Plenum Publishers, New York, NY.

Waller, A.S., Cox, E.E., Edwards, E.A., 2004. Perchlorate-reducing microorganisms isolated from contaminated sites. Environ. Microbiol. 6, 517-527.

31

Wang, D.M., Shah, S.I., Chen, J.G., Huang, C.P., 2008. Catalytic reduction of perchlorate by H2 gas in dilute aqueous solutions. Separation and Purification Tech. 60, 14-21.

Zang, H.S., Bruns, M.A., Logan, B.E., 2002. Perchlorate reduction by a novel chemolithoautotrophic, hydrogen-oxidizing bacterium. Environ. Microbiol. 4, 570-576.

Figure Captions

Figure 1. Two anaerobic culture tubes containing MM medium. In the left tube, the medium has been reduced by sodium sulfide. In the right tube, the medium has been reduced by a stainless steel nail that has been removed from the medium by a magnet following reduction.

32

Figure 2. Perchlorate ion concentration in MM and MSH media measured using a perchlorate selective electrode. Media contained different concentrations (wt/vol) of magnesium perchlorate, calcium perchlorate or sodium perchlorate. Half of the media tubes were autoclaved and half were not. This experiment was performed in quadruplicate.

Figure 3. Highest headspace methane concentration following the growth of Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum and Methanococcus maripaludis in the presence of 0% or 1% (wt/vol) magnesium perchlorate following reduction of residual O2 in the media using sodium sulfide or stainless steel nails. This experiment was performed in quadruplicate.

Figure 4. Highest headspace methane concentration following the growth of Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum and Methanococcus maripaludis that were adapted to metabolize in the presence of 2% and 5% (wt/vol) magnesium perchlorate-containing media. This experiment was performed in triplicate.

33

34

6

5

Perchlorate (%)

4

3

2

1

0 Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Mg Ca Ca Ca Ca Ca Ca Ca Ca Na Na Na Na 5

5

5

5

2

2

2

2

1

1

1

1

5

5

5

5

1

1

1

1

1

1

1

1

MM MM MSHMSH MM MM MSHMSH MM MM MSHMSH MM MM MSHMSH MM MM MSHMSH MM MM MSHMSH +

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

+

-

Cation, Perchlorate Salt Concentration (%), Medium and Autoclaved or Not

35

36

Highlights:

1. Some methanogens can produce methane in the presence of a 1% perchlorate solution. 2. Some of these methanogens were able to adapt to 5% perchlorate. 3. Two species survived in 25% perchlorate solution for short periods of time.

Table 1. Methane production by four species of methanogens in perchlorate salt solutions.

Perchlorate Initial Salt Perchlorate Salt Concentration Concentration in (%) MM Medium (M)a None N/A N/A Magnesium

0.5

0.030

Magnesium

1.0

0.060

Sodium

0.5

0.036

Sodium

1.0

0.071

Calcium

1.0

0.084

Organismsb M. w.

M. b.

M. f.

M. m.

41.9 (2.1)c 33.2 (7.0) 32.1 (4.5) 34.5 (5.5) 33.4 (7.1) 25.1 (3.2)

22.8 (8.3) 20.5 (8.5) 9.4 (7.0) 10.8 (3.1) 4.8 (0.9) 19.4 (3.1)

45.1 (1.9) 39.8 (2.3) 38.5 (3.4) 44.5 (3.5) 45.0 (5.1) 20.2 (2.5)

43.8 (8.7) 21.3 (3.4) 12.0 (3.5) 38.5 (1.5) 40.5 (1.9) 40.0 (1.8)

aPerchlorate

measured in MM Medium, used to grow Methanothermobacter wolfeii, corresponded well to the initial salt concentration. Perchlorate measured in MSH medium, used to grow Methanococcus maripaludis, was diminished in concentration (20% - 50%) compared to the initial salt concentration. M. w. = Methanobacterium wolfeii; M. b. = Methanosarcina barkeri; M. f. = Methanobacterium formicicum; M. m. = Methanococcus maripaludis. b

Highest headspace methane percentages and standard deviations were rounded to the nearest tenth of a percent. c

37

Table 2. Methane production by Methanothermobacter wolfeii and Methanosarcina barkeri in standard methanogen growth media following limited exposure to relatively high concentrations of perchlorate salt solutions.

Perchlorate Concentration Perchlorate (%) Concentration in Solution (M) None N/A N/A None N/A N/A None N/A N/A Magnesium 5 0.3 Magnesium 5 0.3 Magnesium 5 0.3 Magnesium 25 1.5 Magnesium 25 1.5 Magnesium 25 1.5 Sodium 5 0.36 Sodium 5 0.36 Sodium 5 0.36 Sodium 25 1.78 Sodium 25 1.78 Sodium 25 1.78 Calcium 5 0.42 Calcium 5 0.42 Calcium 5 0.42 Calcium 25 2.1 Calcium 25 2.1 Calcium 25 2.1

Exposure Time (hr.) 1 24 72 1 24 72 1 24 72 1 24 72 1 24 72 1 24 72 1 24 72

Organisms M. wolfeii M. barkeri 4/4/4a 4/4/4 4/4/4 4/4/3 4/4/0 4/4/4 4/4/4 4/1/1 4/0/0 4/4/0 4/4/0 4/4/0 4/0/0 4/0/0 0/0/0 4/4/2 4/0/0 4/4/0 4/0/0 0/0/0 0/0/0

3/3/3 3/3/3 3/3/3 2/2/2 1/1/1 1/1/1 1/1/0 0/0/0 0/0/0 1/1/1 1/1/1 1/1/1 1/1/1 1/0/0 0/0/0 1/1/1 1/1/1 1/1/0 1/1/1 0/0/0 0/0/0

Highest headspace methane measurements in anaerobic culture tubes containing standard methanogen growth media after 21 weeks of incubation at growth temperatures. Experiments were performed in triplicate. 4 = > 25%; 3 = 15 – 24.9%; 2 = 5 – 14.9%; 1 = 0.5 – 4.9%; 0 = < 0.5%; tubes where methane measurements were less than 0.5% and not increasing with time were classified as containing non-surviving organisms. a

38