Is There Life on Mars? Bacteria from Mars Analogue Sites from Barren Highland Habitat Types in Iceland

viðskipta- og raunvísindasvið

Háskólinn á Akureyri Viðskipta- og Raunvísindasvið

Námskeið LOK 1126 og LOK 1226

Heiti verkefnis Is there life on Mars? Bacteria from Mars analogue sites from barren highland habitat types in Iceland

Verktími Janúar 2017 – Apríl 2018

Nemandi Hjördís Ólafsdóttir

Leiðbeinendur Oddur Vilhelmsson og Margrét Auður Sigurbjörnsdóttir

Upplag Rafrænt auk þriggja prentaðra eintaka

Blaðsíðufjöldi 24

Fjöldi viðauka Engin

Fylgigögn Engin

Opið verkefni Útgáfu– og notkunarréttur Verkefnið má ekki fjölfalda, hvorki í heild sinni né að hluta nema með leyfi höfundar

Yfirlýsingar

Ég lýsi því yfir að ég ein er höfundur þessa verkefnis og er það afrakstur minna eigin rannsókna

Hjördís Ólafsdóttir

Það staðfestist að verkefni þetta fullnægir að mínum dómi kröfum til prófs í námskeiðunum LOK1126 og LOK1226

Oddur Vilhelmsson

Margrét Auður Sigurbjörnsdóttir

ii Formáli

Verkefni þetta er gildir til B.S gráðu í Heilbrigðislíftækni frá Háskólanum á Akureyri er ritað á formi vísindagreinar með leyfi frá leiðbeinanda og umsjónarmanni lokaverkefni við Auðlindadeild. Verkefnið er með tilliti til þessa skrifað á ensku.

iii Abstract

For centuries scientists and other space enthusiasts have wondered if there is a possibility of life on other planets in our solar system, as well as in others far away. The last decades space institutions have focus on Earths nearest neighbor, Mars and the possibility of life to be found there. Many research mission have been performed to look for potential life in Mars‘s atmosphere and surface. The latest mission is now searching for life underneath the surface with the help of special rover and research equipment. Before going to Mars though, this equipment had to be tested. For that, Icelandic highland was chosen due to its desert-like environment and cold weather. In the year of 2016 numerous samples were taken throughout the highlands of Iceland, they were isolated and identified and their microbial-life analyzed and if there is any change these types of bacteria would be able to survive in the harsh environment, such as on Mars.

Keywords; Astrobiology, microbial ecology, ESA; HABIT, bacteria, PCR

iv Þakkarorð

Ég vil byrja á því að þakka leiðbeinendunum mínum, Oddi Vilhelmssyni og Auði Sigurbjörnsdóttur fyrir samstarfið og alla þá aðstoð sem þau hafa veitt mér í gegnum þessi skrif sem og námið við Háskólann á Akureyri síðustu ár. Einnig vil ég þakka Guðný Völu Þorsteinsdóttur fyrir andlegan stuðning þegar á þurfti á ferðum mínum norður til Akureyrar. Samnemdur mínir fá einnig þakkir fyrir að vera ávallt til staðar undanfarin misseri og þá sérstaklega Anna Lilja Benidiktsdóttir og Elfa Björk Rúnarsdóttir sem hafa staðið sterkar mér við hlið undanfarin ár. Að lokum vil ég þakka fjölskyldu minni og vinkvennahópnum mínum fyrir allan þann stuðning sem þau hafa sýnt mér við gerð verkefnisins sem ásamt allir þeirri hvatningu sem þau hafa gefið mér í gegnum námið í heild sinni.

Hjördís Ólafsdóttir Stóra Núpi, 9 apríl 2018

v Útdráttur

Það hefur ávallt vakið áhuga vísindamanna að rannsaka möguleikana á því hvort líf geti þrifist á öðrum plánetum í sólkerfi okkar. Undanfarin ár hafa augu þeirra beinst að nágranna okkar, plánetunni Mars og möguleikum hennar á þar geti þrifist nokkurskonar líf. Margir rannsóknarleiðangrar hafa verið farnir með könnunarför með þau verkefni að kanna lofthjúp og yfirborð Mars. Næstu leiðangar beinast að því að kanna það sem finnst undir yfirborði og til þess þarf að notast við sérhæfðan búnað. Áður en hægt er að senda slíkan búnað á fjarlæga plánetu er nauðsynlegt að kanna þol hans og virkni, til þess var leitast til Íslands, en umhverfi landsins minnir margt á þá auðn sem finnst á Mars sem og lágt hitastig og þungt veðurfar. Yfir sumarið og haustið 2016 var farið í leiðangra um hálendi Íslands, valdir voru staðir sem þóttu líkja helst til yfirborð Mars. Jarðvegssýni voru tekin og erfðaefni bakteríanna einangruð og tegundagreind. Efnaskipti og örverusamfélag þeirra skoðað sem og lífsmöguleikar þeirra á öðrum plánetum á borð við Mars kannaðir ásamt því hvaða umhverfisþættir það eru sem gætu takmarkað örverulíf við slíkar aðstæður.

Lykilorð; Geimlíffræði, örveruvistfræði, ESA, HABIT, bakteríur, PCR

vi Efnisyfirlit

1. Introduction ...... 1

2. Methods and materials ...... 4

2.1 Sampling ...... 4

2.2 Identification ...... 5

3. Results ...... 7

4. Discussion ...... 9

5. References ...... 11

vii Myndaskrá

Figure 1 - Sampling location in the highlands of Northern Iceland...... 4

Töfluskrá

Table 1 - Sampling locations and GPS coordinates...... 5 Table 2 - Identity of the isolates using 16S rDNA sequencing...... 8

viii Skilgreiningar

HABIT: Habitability, Brine Irradiation and Temperature

ESA: European Space Agency

NASA: The National Aeronautics and Space Administration

TGO: Trace Gas Orbiter

LaRa: Lander Radioscience experiment

PCR: Polymerase chain reaction

Roscosmos: The Roscosmos State Corporation for Space Activiti

ix 1. Introduction

Ever since the 1600s, astronomers and other space enthusiasts have wondered the possibility of life on other planets (Angel, Woolf, & Powell, 1996). Since the discovery of Earth's nearest neighbor, the red planet Mars, ideas have come up for the possibility of life to be found there (Davies, Levine, & Schild, 2010). However, last centuries researches have shown that Mars is relatively small in size which has led to a thin atmosphere and therefore it is impossible for the planet to sustain water (Catling, 2013). There are though evidence showing that Mars had surface water in past history and it is speculated that life may have flourished there in early life. However, today (Lunine, 2005), Mars is a very cold planet, with the average surface temperature of -56°C compared to 15°C on Earth, during the day the temperature on the surface is around -40°C and at night it can go as far down as -90°C. The greenhouse effects are only at 7°C compared to 33°C on Earth (Catling, 2013; Weiss, Yung, & Nealson, 2000). The atmosphere on Earth consists of a nitrogen-oxygen mixture, which allows life to sustain there, however Mars‘s atmosphere contains mostly carbon dioxide with an admixture of nitrogen. Due to this type of atmosphere the possibility for life to sustain on Mars is very unlikely (Lunine, 2005). On Mars, only particles of oxygen can be found which means that the planet is anoxic (Nixon, Cousins, & Cockell, 2013). Due to Mars's thin atmosphere the planet is also very vulnerable to UV radiation, it lacks ozone layer (Léveillé & Datta, 2010; Weiss et al., 2000). The surface of Mars is coated by highly oxidizing material, dominated by hydrogen peroxide. An environment like this will degrade any form of organic materials that might be there. This hostile environment makes Mars essentially frozen (Catling, 2013; Ellery et al., 2002). During the 19th and 20th century a lot has been learned about the early history of Mars and it is speculated that Mars may have been quite similar to Earth billions of years ago, with wet and warmer surface and even the possibility of microbial life forms (Nealson, 1997). Nevertheless, how the water on Mars disappeared is yet not known, although it has been suggested that it may have been lost into space by the impacts of asteroidal or cometary bodies and therefore the water vaporized from the planet. Solar wind may also have been the cause for the disappearance of water (Lunine, 2005). The possibility for life to be found, if it ever existed or still exists is most likely to be found deep under the surface of the planet (Ellery et al., 2002).

Numerous research mission have been performed since the mid-20th century by space agencies in the US, the Russians and European agencies. Many of them have been successful but a lot of them have failed. Their goal has been to search for the possibility that life had and could sustain on Mars. By the late 70s, a team from NASA space center successfully sent the first ever lander to the red planet in a mission called The Viking Invasion, which involved two orbiters and two landers. First, their initial job was mainly to survey the planet for a possible landing site and later it would inspect the planet’s surface as well its atmosphere. The landers’ jobs were to survey the surface itself. They would take soil samples and inspect the winds and atmosphere on the surface. The landers then did numerous experiments to study previous and possible life on the planet (Davies et al., 2010). The Viking did however not detect any organic matter in the Martian soil, neither at the surface nor at the depth of 10 cm (Parnell et al., 2007). Even though the tests indicated bacterial life, the results from the analysis were insufficient and therefore the results were insignificant due to contamination on the road from Earth to Mars. (Gibson, Mckay, Thomas-keprta, & Wentworth, 2001; Whitfield, 2004). Several other research probes were sent on route to Mars in the late 20th century with the intention to photograph and do surface scans. Most of these mission have given scientist great amount of information about Mars (Davies et al., 2010). The next vast step in exploring the red planet is an ongoing project set up by the European Space Agency (ESA) and the Russia‘s space agency (Roscosmos). The two agencies have partnered up for two missions, The ExoMars programme, the former to study the atmosphere of Mars and deliver a lander where they will be using a Trace Gas Orbiter (TGO) to search for evidence of methane along with other gases in the atmosphere of Mars as well as investigate the surface (J. Vago et al., 2013). The latter mission is more substantial, as they plan to send a research rover, equipped with two instruments, the HABIT and LaRa (ESA Exploration, 2016) to the surface of the planet. The aim is to search for traces of past life, as well as profiling the surface of Mars and performing geological and geophysical analysis (Patel, Slade, & Clemmet, 2010; Rull, Maurice, Diaz, Tato, & Pacros, 2011; J. Vago et al., 2013). LaRa‘s task is to reveal details of the internal structure of Mars whereas HABIT‘s task is to investigate the atmosphere for water and other elements (ESA Exploration, 2016) The rover has the capability to drill vertically under the surface of Mars and into surface rocks to collect samples that are well preserved and therefore free from any radiation damage and surface oxidation, and analyze them as well while based on Mars. At each sampling site the

2 rover will do geological measurements and search for any form of bio-signatures (J. Vago et al., 2013). During the recent years, substantial investigation has been on the diversity of microorganism in cold, desert-like environments, such as glacier and arctic ecosystems. Results have shown that there is a greater diversity than expected, with phyla such as Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria and Chloroflexi being the most abundant in separate researches that have previously been made from polar desert soil that can be found in Svalbard and in the Himalayan regions. (Männistö, Kurhela, Tiirola, & Häggblom, 2013; Schütte et al., 2010; Yadav, Yadav, Verma, Sachan, & Saxena, 2017). Similar results have been found in analyzing on microbiota in the Icelandic highlands, where Proteobacteria and Actinobacteria are common in the snow and ice-caps (Lutz, Anesio, Edwards, & Benning, 2015). Before shipping the HABIT complex to Mars it needed to be strain tested, Iceland was chosen for this task since its similarity to the surface of Mars, cold conditions and desert-like environment. Due to Iceland‘s environmental diversity the microbiota is as well very diverse and could give an idea of what types of microbial life there is on Martian surface. The HABIT complex was tested and samples collected for microbial testing to search for the possibility of extraterrestrial life.

The objective of this study is to identify strains of bacteria isolated from barren highland habitat types in Iceland, their characteristics will be determined and the possibility if they would be able to thrive in an environment such as on Mars will be discussed in light of their tolerance to desiccation and irradiation, as judged from available literature on their close relatives.

2. Methods and materials 2.1 Sampling Samples were collected at various sites in the highland of North Iceland, including Lofthellir, Nýjidalur, Öskjuvatn and Fjórðungsvatn (Figure 1) under diverse weather conditions during the summer of 2016. Environmental temperature was measured on site and GPS coordinates are listed in Table 1.

Figure 1 - Sampling location in the highlands of Northern Iceland. The arrows in the figure show the sampling sites at Lofthellir, Nýidalur, Krafla, Öskjuvatn and Fjórðungsvatn. Samples were collected during summer 2016 and consisted of various types of regolith sand, gravel and water samples from hot springs. Samples were collected aseptically with sterile implements into sterile Falcon Tubes and stored on ice until processed in the lab.

The samples consisted of various types of regolith sand, gravel and water samples from near hot springs. The samples were collected aseptically with sterile implements into sterile Falcon tubes (Becton-Dickinson, Franklin Lakes, NJ, USA) and stored on ice until processed in the lab. The samples were diluted in tenfold dilutions in Butterfields Buffer (Potassium dihydrogen phosphate, distilled water, pH 7.2 ± 0.2 at 25°C) and spread-plated in duplicate onto Nutrient Agar (Difco; NA), Nutrient Agar with 10% NaCl (NA + NaCl), NB + 10% NaCl, R2A (both aerobic and anaerobic), R2A + 10% NaCl, 9K (both aerobic and anaerobic). The plates were incubated at 15°C and growth monitored for up to 3 weeks. Based on colony morphology and other growth characteristics, representatives of each morphotype were aseptically picked, streak onto fresh medium identical to the original plate and incubated using the same incubation

4 conditions as the original plates. Stocks of purified isolates were prepared by suspending a loopful of colony growth in 30% glycerol and stored at -80°C.

Table 1 - Sampling locations and GPS coordinates; altitude along with Air and sample temperatures and notes taken by Matin-Torres et al. July, 2016 as well as sampling and notes information from Lofthellir taken by Anett Blischke and Snæbjörn Rolf Blischke, November 2016.

SAMPLE LOCATION GPS COORDINATES; AIRTEMP TSAMPLE NOTES ALTITUDE (°C) (°C) AL1 Öskjuvatn 65.0442 –16.7252; 1065m 12.1 15,8

AL2 Víti við 65.0471, -16.7249; 1121m 2.0 56.3 Near hot spring Öskju IO2 Víti við 65.7149, -16.7507; 598m 61.5 73.3 Kröflu L1 Víti við 65.7182, -16.7544 11.9 11.5 At snow melt on crater slope Kröflu

NC Nýjidalur 64.7823, -18.0038; 884m 8.5 15.6 Basalt sand. Soggy due to snow melt

FL Fjórðungs- 64.8692, -180663; 762m 9.4 13.0 Blustery rain. Sample at lake vatn shore

H16 Lofthellir 16.7220, -65.551 -1

2.2 Identification

2.2.1 16S rRNA gene sequencing A total of 110 isolates were selected for 16S rRNA gene sequencing. From the freezer stock of each isolate, 1 µl of sample was suspended in 25 µl of lysis buffer ( 1% Triton x-100, 20 mM Tris, 2 mM EDTA, pH 8,0) and the suspension heated to 95°C for 10 min and then cooled to 4°C. As a template in a standard PCR reaction, 1 µl of the lysis-suspension was used. The primer pair used were 8F 5'- AGT TTG ATC CTG GCT CAG - 3' and 1522R 5'- AAG GAG GTG ATC CAG CCG CA -3' at a final concentration of 0.2 µM in a total volume of 25 µL of

PCR mixture, containing 0.15 µl of Taq polymerase (New England Biolabs, Ipswich, MA, USA). The PCR reaction was as followed: 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 sec, 50° for 30 sec and 63°C for 90 sec. The final extension was 68°C for seven minutes. Amplicons were visualized on a 1.3% agarose gel using a SYBR safe dye. The electrophoresis was run at 115 volts for 25 minutes and the fragments detected under UV light and documented.

2.2.2 PCR clean up and sequencing Amplicons were cleaned up for sequencing using a mixture of Exonuclease I and Antarctic phosphatase (0,25 µl of Exo, 0,20 µl of Sap and 1,55 µl of MQ water).For each sample, 2.5 µl of EXO-SAP was used. The cleanup reaction occurred at 37°C for 30 minutes and at 95°C for 5 minutes before the final temperature of 4°C. Partial 16S rRNA sequencing was performed on Applied Biosystems 3130XL DNA analyzer (Applied Biosystems, Foster City, USA) at Macrogen Europe, Amsterdam, the Netherlands, using the sequencing primers 519F (5'-CAG CAG CCG CGG TAA TAC-'3) and 926R (5'-CCG TCA ATT CCT TTG AGT TT-'3). After sequencing the results were trimmed and aligned by using 4Peaks (Nucleobytes) and finally aligned against the ref_seq database at EzTaxon (EZbioCloud).

3. Results

A total of 110 isolates were selected for 16S rRNA gene sequencing, the isolates were selected due to their sampling locations and environmental position in the Icelandic highlands. Of the 110 isolates, the gene sequencing was successful for 39 isolates. Those 39 isolates were sequenced, thereof 21 were successfully identified and are listed in Table 2. The similarity varied from 98.88 - 100%. All of the isolates belonged to three phyla as can be seen as well in Table 2. The majority (12) of the identified bacteria belong to the phylum of Proteobacteria, with Massilia eurypsychrophila (99% similarity) being the most abundant, it is known for being able to survive in extreme cold environments (Shen et al., 2015). Five other Proteobacteria strains were identified, Sphingomonas faeni (100%), Polaromonas cryoconiti (99,7%), Pseudomonas moorei (99%), Caulobacter henricii (100%) and brevundimonas variabilis (98%). The less majority (8) belonged to the phylum Actinobacteria with Arthobacter being the most common and showing a great similarity to the species A. ginsengisoli (99%). An individual strain was identified as Bacteroidetes with high similarity to F. swingsii (99%). The majority (12) of the bacteria were isolated from the cave at Lofthelli, the remaining results came from three locations, at Nýjidalur (3), Fjórðungsvatn (3) and Öskjuvatn (3). At Fjórðungsdalur there were exclusively Actinobacter identified and at Nýjidalur and Öskjuvatn there were two types of Proteobacter and one type of Actinobacter identified at each location.

Table 2 - Identity of the isolates using 16S rDNA sequencing. STRAIN # EZTAXON HIT % SIMILARITY PHYLUM

FJÓRÐUNGSVATN EWFLSR1A Arthrobacter ginsengisoli 99.36 Actinobacteria EWFLSR2 Arthrobacter ginsengisoli 99.24 Actinobacteria EWFLSR5A Arthrobacter ginsengisoli 99.33 Actinobacteria ÖSKJUVATN EWAL 1SN1 Microccus yunnanensis 100.0 Actinobacteria EWAL 1SN3 Polaromonas cryoconiti 99.67 Proteobacteria EWAL 1SR1 Pseudomonas moorei 99.24 Proteobacteria NÝJIDALUR EWNCSN 6 Arthrobacter ginsengisoli 99.85 Actinobacteria EWNCSR 2 Caulobacter henricii 100.0 Proteobacteria EWNCSR 11 Caulobacter henricii 100.0 Proteobacteria LOFTHELLIR H16 0502 Sphingomonas faeni 100.0 Proteobacteria H16 0503 Sphingomonas faeni 100.0 Proteobacteria H16 0504 Massilia eurypsychrophila 99.68 Proteobacteria H16 0506 Massilia eurypsychrophila 99.63 Proteobacteria H16 0302 Flavobacterium swingsii 99.66 Bacteroidetes H16 0306 Massilia eurypsychrophila 99.43 Proteobacteria H16 0307 Massilia eurypsychrophila 99.46 Proteobacteria H16 0308 Arthrobacter ginsengisoli 100.0 Actinobacteria H16 0603 Brevundimonas variabilis 98.88 Proteobacteria H16 0604 Arthrobacter humicola 100.0 Actinobacteria H16 0606 Arthrobacter humicola 100.0 Actinobacteria H16 0610 Sphingomonas faeni 100.0 Proteobacteria

4. Discussion

During the year 2016, diverse strains of bacteria were isolated from various sites in the highlands of northern Iceland. The purpose was to consider the likelihood of microbes being able to thrive in harsh environments on different planet, such as Mars. Iceland was chosen for this research due to its cold environmental diversity. Sampling locations varied between sites but the focus was on areas with desert– like environments and cold climate. A total of 110 strains were isolated, thereof 21 were identified belonging to three genera. Due to time restrain, it was not possible to repeat the identification for more and exact results. The bacteria strains were identified by 16S rRNA gene sequencing, showing that the phylum of Proteobacteria, which was isolated from three different locations in Iceland, being in the most abundant (57% of total identified samples, with 67% coming from the same location at Lofthellir). Two other phyla were identified, Actinobacteria (which accounted for 38% of the total identified samples) and Bacteriodetes (5% of the samples). Most Proteobacteria are Gram-negavite, using flagella to move around, they are also chemolithoautotrophs, meaning that they use carbon dioxide from the environment to survive (Gupta, 2000; Yamanaka, 2008). The most abundant Proteobacteria was identified as Massilia eurypsychrophila (19% of total identified samples and 34% of the Proteobacteria phylum). M. eurypsychrophila is described as a gram-negative, rod-shaped bacteria with polar flagella, its color is light-pink to white and grows at temperatures from 0-25°C, with the optimum temperature at 10°C. M.

eurypsychrophila is positive for nitrate reduction but negative for H2S production (Shen et al., 2015). Other Proteobacteria identified were such as Caulobacter henricii, which is more likely to be found in a aqueous environment (Abraham et al., 1999) and Sphingomonas faeni a small, gram-negative bacteria with the optimum growth temperature from 4-28°C (Busse et al., 2003). Three strains of Actinobacteria were identified, Arthrobacter ginsengisoli, Micrococcus yunnanensis and Arthrobacter humincola. Arthobacter species can be found widespread in soil, ice caves, air and plant tissue (Chung, Park, Park, Ahn, & Chung, 2010). These three strains of Actinobacter, M. yunnanensis, A. ginsengisoli and A. humincola are all gram-positive bacteria, their growth temperature is quite broad, from 4-45°C, their NaCl tolerance is fairly similar, from 0-15% (Chung et al., 2010; Siddiqi et al., 2014; Zhao et al., 2009). Only one strain of Bacteroidetes was identified belonging to Flavobacterium swingsii. Bacteroidetes are gram- negatives and can be found in all types of environments such as in soil, sea water and sediments (Slots & Genco, 1984).

F. swingsii has a similar growth temperature as the Actinobacter and Proteobacter strains, from 3 -26°C. Just as M. eurypsychrophila, F. swingsii is able to reduce nitrate (Ali et al., 2009). F. swingsii grows with 0% NaCl, however it does not at 1% NaCl, M. eurypsychrophila is similar although it does grow at 1% NaCl (Ali et al., 2009; Shen et al., 2015). For life to be found in any environment on any planet it is critical to have water, as it is needed for biological reactions and metabolites. However, many microorganisms do not need water to sustain life, they can live in vacuum for years, without any organic matters or oxygen (Yamagishi et al., 2010). Free energy is also necessary and can be obtained through metabolic

pathways such as chemosynthesis in microorganisms. Compounds such as H2, H2S and CH4 can

be produced with the help of CO2 for instance and therefore produce free energy. These factors could be found somewhere in the Martian environment, therefore, life might be sustained there. There is also evidence of water being on Mars and if it is NaCl rich there might be residue of halophilic organisms (Chapelle et al., 2002; J. L. Vago et al., 2017). Other factors that have influence on life on Mars is UV radiation; with high radiation it is hard for life to sustain on the surface, therefore, sub-surface life is more likely to be found (Yamagishi et al., 2010). Yet, recently there have been studies that show some irradiation tolerance with some phylum of hydrated Actinobacteria, with Arthobacter being one of the genera along with its relative Microbacterium schleiferi. Without being exposed irradiation, Arthobacter seems to have lesser tolerance against desiccation and does not have a great chance of surviving for long period of time while M. schleiferi is more likely to have tolerance against desiccation. As desiccated strains, both Arthobacter and M. schleiferi show quite high resistance to irradiation (Osman et al., 2008). On Mars, Proteobacteria is the most likely phylum to be found under the surface since it uses carbon dioxide from the environment for survival as they are chemolithoheterotrophs (Chapelle et al., 2002), as well has having shown relatively high tolerance against irradiation (Chanal et al., 2006). Overall, the results from this study might not give the precise conclusion of whether or not any form of life on Mars can be found. It does however give an idea of what might be expected. It would be interesting to re-identify the samples as well as study more of them for a wider understanding and a better overview of these microorganism that have here been identified.

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