Investigation of the Growth and Survival of Bacteria from

Louisiana State UniversityLSU Digital Commons

LSU Master's Theses Graduate School

2012

Investigation of the Growth and Survival ofBacteria from Mars Analog Environments WhenExposed to Mars-like ConditionsBharathi VallalarLouisiana State University and Agricultural and Mechanical College

Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses

This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSUMaster's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected].

Recommended CitationVallalar, Bharathi, "Investigation of the Growth and Survival of Bacteria from Mars Analog Environments When Exposed to Mars-likeConditions" (2012). LSU Master's Theses. 3601.https://digitalcommons.lsu.edu/gradschool_theses/3601

https://digitalcommons.lsu.edu?utm_source=digitalcommons.lsu.edu%2Fgradschool_theses%2F3601&utm_medium=PDF&utm_campaign=PDFCoverPages

https://digitalcommons.lsu.edu/gradschool_theses?utm_source=digitalcommons.lsu.edu%2Fgradschool_theses%2F3601&utm_medium=PDF&utm_campaign=PDFCoverPages

https://digitalcommons.lsu.edu/gradschool?utm_source=digitalcommons.lsu.edu%2Fgradschool_theses%2F3601&utm_medium=PDF&utm_campaign=PDFCoverPages

https://digitalcommons.lsu.edu/gradschool_theses?utm_source=digitalcommons.lsu.edu%2Fgradschool_theses%2F3601&utm_medium=PDF&utm_campaign=PDFCoverPages

https://digitalcommons.lsu.edu/gradschool_theses/3601?utm_source=digitalcommons.lsu.edu%2Fgradschool_theses%2F3601&utm_medium=PDF&utm_campaign=PDFCoverPages

mailto:[email protected]

INVESTIGATION OF THE GROWTH AND SURVIVAL OF BACTERIA FROM MARS

ANALOG ENVIRONMENTS WHEN EXPOSED TO MARS-LIKE CONDITIONS

A Thesis

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of Master of Science

in

The Department of Biological Sciences

by Bharathi Vallalar

B.S., Louisiana State University, 2010 December 2012

ii

ACKNOWLEDGEMENTS

My eternal gratitude goes out to Dr. Fred A. Rainey, who graciously gave me a chance at

joining the Master of Science program at LSU. I strongly believe that this opportunity is

singularly responsible for my acceptance into a Ph.D. program, and thus by extension, is

responsible for all successes I will subsequently experience in my career. Working in RaineyLab

has helped me realize where my scientific interests lie, and Dr. Rainey’s firm guidance and high

expectations have aided my personal and academic growth, as well as significantly advancing

my knowledge and skills as a scientific researcher.

I would like to extend my appreciation to my other committee members, Dr. Brent

Christner and Dr. Paul LaRock, for their time and advice.

I must thank Rachael Stebbing for patiently guiding me through laboratory procedures

and experimental techniques like PCR and setting up sequencing reactions. Initiation into the

lab setting would have been much more difficult without her presence to answer my numerous

questions and concerns. I would also like to thank my friends and family whose encouragement

and support greatly aided in my persistence in finishing this thesis.

I also thank Lyle Whyte and Dale Anderson for providing the permafrost samples used in

this study. Finally, I would like to thank NASA (NASA EPSCoR Research Project NNX10AN07A –

MARSLIFE) for making this study possible with their funding, and Priscilla Milligan in the

Department of Biological Sciences for her guidance and advice in making sure I fulfilled the

requirements for my Master of Science degree.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS……………………………………………………………………………………………………..ii

LIST OF TABLES………………………………………………………………………………………………………………….v

LIST OF FIGURES………………………………………………………………………………………………………………..vii

ABSTRACT………………………………………………………………………………………………………………………….viii

CHAPTER I: INTRODUCTION……………………………………………………………………………………………...1

CHAPTER II: GROWTH OF BACTERIAL ISOLATES FROM THE ATACAMA DESERT AT HIGH CONCENTRATIONS OF PERCHLORATE….……………………………………………...5

Introduction…………………………………………………………………………………………………..5 Materials and Methods………………………………………………………………………………….7 Perchlorate-containing culture media…………………………………………….7 Bacterial isolates and type strains……………………………………………….….8 Incubation and growth……………………………………………………………………8 Plating of Atacama soils on perchlorate-containing media……………..8

Survival of Atacama strains in liquid media containing higher concentrations of perchlorate…………………………………………………….…..9 Soil perchlorate determination…………………………………………………….…10

Results and Discussion………………………………………………………………………………..10 Growth of bacterial strains in the presence of perchlorates……………10

Chaotropic effects and water activity of perchlorate-containing media……………………………………………………………………………………………..13 Gram-positive versus Gram-negative growth in the presence of perchlorates………………………………….………………………………………….….…14 Bacterial growth in the presence of MgSO4, KNO3, and NaCl……….…16 Correlation between bacterial growth and perchlorate concentration of Atacama soils…………………………………..……………….….23 Bacterial survival at high perchlorate concentrations in liquid media…………………………………………………………………………………....25 Plating soils on perchlorate-containing media…………………………………26

Conclusion……………………………………………………………………………………….………….28

CHAPTER III: ISOLATION AND CHARACTERIZATION OF IONIZING RADIATION RESISTANT BACTERIA FROM PERMAFROST…….……………………………………………………………….32 Introduction…………………………………………………………………………………………………..32

Materials and Methods……………………………………………………………………………….…34 Irradiation, isolation, and cultivation……………………………………………...34 Bacterial strains and type strains…………………………………………………….35 Phylogenetic analysis………………………………………………………………………35 Morphological, biochemical, and physiological characteristics……….36 Irradiation of isolates and desiccation resistance……………………………37

iv

Results and Discussion…………………………………………………………………………………..38 Cfu/g in permafrost (irradiated and unirradiated)…………………………..38

Isolation and identification of Hymenobacter strains………………………40 Morphological and phenotypic characterization of

Hymenobacter isolates…………………………………………………………………….41 Irradiation of Hymenobacter pure cultures and

desiccation tolerance………………………………………………………………..……..44 Comparison of strains to existing Hymenobacter species…………..…….46

Conclusion……………………………………………………………………………………………………...48 CHAPTER IV: CONCLUSION………………………………………………………………………………………………….50

REFERENCES……………………………………………………………………………………………………………………..…52

APPENDIX A: STRAINS USED IN PERCHLORATE TESTING………………………………………………………61

APPENDIX B: PHOTOMICROGRAPHS OF HYMENOBACTER ISOLATES FROM PERMAFROST............................................................................................................................67

APPENDIX C: BACTERIAL GROWTH IN THE PRESENCE OF PERCHLORATES (TABLES)…………….75

VITA…………………………………………………………………………………………………………………………………..…79

v

LIST OF TABLES

Table 2.1. The highest concentration of perchlorate at which control strains exhibited growth………..……………………………………………………………………….………11

Table 2.2. The highest concentration of perchlorate at which Gram(+) and Gram(-) control strains exhibited growth. NG – no growth observed.……..……….........16

Table 2.3. Growth of Blastococcus strains and Bacillus subtilis in presence of high salt concentrations. NG – no growth observed……..…………………..……………….20

Table 2.4. Growth of control strains in the presence of NaCl, KNO3, and MgSO4………………20

Table 2.5. Growth of all Blastococcus strains in the presence of NaCl, KNO3, and MgSO4……………………………………………………………………………………...21

Table 2.6. Growth of other arid soil strains in the presence of NaCl, KNO3, and MgSO4………………………………………………………………………………………22

Table 2.7. Growth of Deinococcus strains in the presence of NaCl, KNO3, and MgSO4………22

Table 2.8. Chemical analysis of Atacama soil samples showing concentrations of chloride, sulfate, nitrate, and perchlorate……...........................................24

Table 2.9. Highest perchlorate concentration tolerated by certain strains and their survival (+ or -) in liquid media containing 600 and 800 mM Mg-perchlorate (MgCl2O8) and Ca-perchlorate (CaCl2O8) concentrations……………………………………………………………………………………………25

Table 2.10. Cfu/g of soil when plated on 1/10 PCA containing 200 – 600 mM concentrations of Mg-perchlorate (MgCl2O8) and Na-perchlorate (NaClO4) TMTC – too many to count………………………………………………………….27

Table 2.11. Cfu/g of soil when plated on 1/100 PCA containing 200 – 600 mM concentrations of Mg-perchlorate (MgCl2O8) and Na-perchlorate (NaClO4) TMTC – too many to count…….……………………………..…………………….27

vi

Table 3.1. Cfu/g of LH Tundra soil plated on R2A, MA, 1/10 PCA, and 1/100 PCA at 4°C. Radiation dosage and dilution are shown in the left column…………………………………………………………38

Table 3.2. Cfu/g of FSS soil plated on R2A, MA, 1/10 PCA,

and 1/100 PCA at 4°C. Radiation dosage and dilution are

shown in the left column……………………………………………………………………………39

Table 3.3. Cfu/g of LH Tundra soil plated on R2A, MA, 1/10 PCA, and 1/100 PCA at 15° C. Radiation dosage and dilution are shown in the left column……………………………………………………………………..39

Table 3.4. Cfu/g of FSS soil plated on R2A, MA, 1/10 PCA, and 1/100 PCA at 15° C. Radiation dosage and dilution are shown in the left column. TMTC – too many to count.…………………….….40

Table 3.5. Organisms recovered from soils exposed to gamma radiation. Organisms recovered from soils exposed to gamma radiation. The number in parentheses refers to the number of strains belonging to that genus that were isolated from each exposure. Strains were isolated from various media incubated at either 4° or 15° C………………………………………………………………………………………………………41

Table 3.6. Strain data for 15 isolates falling within the radiation of the genus Hymenobacter, and two isolates related to Pontibacter, also isolated from high radiation doses ……………………..………………………………42

Table 3.7. Growth of Hymenobacter isolates after exposure to doses of 1, 3, 9, and 12 kGy gamma radiation while in pure culture………………………...45

Table 3.8. Phenotypic data of Hymenobacter and related BV-irLH and BV-irFSS strains. Nd – not determined; w – weak growth.……………...............47

vii

LIST OF FIGURES

Figure 2.1. Percentage of strains growing at high concentrations

of Na-perchlorate (NaClO4)……………………………………………………………………………………………..11

Figure 2.2. Percentage of strains growing at high concentrations of Mg-perhclorate (Mg(ClO4)2)…………………………………………………………………………..…………..12

Figure 2.3. Percentage of strains growing at high concentrations of Ca-perchlorate (Ca(ClO4)2)………………………………………………………………………………….………12

Figure 2.4. Percentage of strains growing in the presence of 0.5, 1, and 2 M NaCl…………………………………..…………………………………………………………………………..17

Figure 3.1. 16S rRNA gene sequence based phylogeny showing the relationship of permafrost isolates to previously isolated and described Hymenobacter species. Scale bar represents 1 inferred nucleotide substitution per 100 nucleotides. Strains in green represent isolates from permafrost from Canadian arctic and Tibet; dark blue are from glacial ice and snow in Pico de Orizaba; light blue are from basal ice in Victoria Upper Glacier; red are from arid soils from the Atacama, Oman, and Sonoran; orange are from air samples……………………………………………………………………………………………..43

viii

ABSTRACT

The purpose of this study was to test the growth of organisms from terrestrial Mars

analog environments when exposed to Mars-like conditions.

First, the growth of over 75 bacterial strains from the Atacama Desert was tested in the

presence of high concentrations (50 mM to 1 M ) of three different perchlorate salts – NaClO4 ∙

H2O, Mg(ClO4)2 ∙ 6 H2O, and Ca(ClO4)2 ∙ 4 H2O found on the Martian surface. Strains tested

belonged to a variety of genera such as Blastococcus, Microvirga, Geodermatophilus, and

Kocuria. Control strains used for comparison included a number of Gram-positive and Gram-

negative bacteria, many that were isolated from other extreme environments. In general, a

decrease in bacterial growth was observed as perchlorate concentration increased. Atacama

Desert isolates, especially Blastococcus and Kocuria sp., grew at higher perchlorate

concentrations than any of the bacterial control strains. It was noted that Gram-positive

organisms are capable of growing at higher perchlorate concentrations than Gram-negative

organisms. Results from these tests have applications in research involving bacterial

perchlorate tolerance, as well as the search for life on Mars.

Secondly, the diversity of ionizing-radiation resistant bacteria in permafrost soil was

studied by exposing two soil samples from the Canadian High Arctic to various doses of gamma

radiation. Samples were exposed to 0 (unirradiated), 1, 2, 3, and 11.1 kGy doses and

subsequently dilution plated onto a variety of nutrient media including marine agar, R2 agar,

1/100 PCA, and 1/10 PCA. Cfu ranged from 1.5 x 105 to 2.3 x 105 for unirradiated samples and

3.0 x 102 to 1.8 x 103 for samples exposed to 11 kGy. Colonies were selected from irradiated

ix

samples and 16S rRNA gene sequencing was used to identify the isolates as belonging to a

number of genera including Hymenobacter, Pontibacter, Arthrobacter, Salinibacterium, and

Paenibacillus. Phylogenetic analysis showed that nine strains were related to species of the

genus Hymenobacter, and these were chosen for further characterization. This study identifies

radiation resistant, psychrotolerant bacteria from permafrost and demonstrates the use of this

selective technique to isolate them from the total bacterial community.

1

CHAPTER I INTRODUCTION

Research interest in the field of astrobiology, especially concerning possible life on

Mars, has been on the rise in recent years. Mars Exploration Rovers and landers have been

launched by NASA with the goal of detecting life on Mars through geological and geochemical

analysis. The primary reason for the heated search for Martian life is the discovery of signs of

liquid water having been present in Mars’ history, a determination reached by multiple studies

based on data collected from Mars rovers and orbital surveying efforts (Carr 2012; Cull 2010;

Ehlmann et al. 2011; Malin and Carr 1999; McKay 2010). One of the goals of NASA’s Mars

Exploration Program is to determine whether life ever existed on Mars, and studying the

planet’s past water activity is the starting point in making that determination

(http://marsrovers.jpl.nasa.gov). It is apparent that life on Earth originated soon after surface

conditions were conducive to the formation of liquid water (Abramov and Mojzsis 2009), and

thus, tracing water signatures back to an origin on Mars may very well lead to the remnants of

whatever life may have existed or still exists on the planet.

In addition to water signatures, it has been determined that other environmental

conditions of early Mars were similar to that of early Earth, namely warmer temperatures and

dense atmospheric conditions (McKay 2010; Pollack 1987). Therefore, it can be hypothesized

that if life did evolve on Mars under these conditions, it could share an evolutionary similarity

to life on Earth. If so, this could mean that Martian life may be similar in appearance and/or

habitat, metabolism, and mechanisms of survival to organisms capable of tolerating similar

conditions on Earth. Any past or present life on Mars would be subjected to many

http://marsrovers.jpl.nasa.gov/

2

environmentally harsh conditions including low nutrient and water availability, freezing

temperatures, exposure to high levels of radiation, and the presence of high concentrations of

salt compounds in the soil (Clark 1998; Clark and Van Hart 1981; Crisler et al. 2012; Vaniman et

al. 2004). Somewhat comparable conditions can be found in various environments on Earth,

and researching the life forms in these areas is the key to understanding the origin of life on

Earth, which in turn provides clues as to the possibility of life on other planets. The objective of

this study is to investigate the characteristics of bacteria isolated from extreme environments

on Earth, and test their growth and limits of survival when exposed to one or more conditions

currently present on Mars.

Current surface conditions on Mars have been determined to be inhospitable to all

known life forms (McKay 2010). The average surface temperature of -60 °C (McKay 2010) would

certainly be one factor limiting bacterial growth, although studies have found that low-level

microbial metabolic activity occurs in Alaskan tundra soils incubated at -39 °C (Panikov et al.

2005) and in laboratory conditions ranging from -80 to -196 °C (Junge et al. 2006). Extremely

low atmospheric pressure is another obstacle for life on Mars, with a global average pressure of

0.69 kPa (Berry et al. 2010), near the triple point of water; currently, microbes have only been

known to grow at as low as 2.5 kPa (Berry et al. 2006; Schuerger et al. 2006). Perhaps the most

lethal surface condition is the exposure to high levels of UVC radiation (λ = 200-280 nm) that

penetrates through the thin Martian atmosphere (Newcombe et al. 2005; Osman et al. 2008b).

Much of the focus in studying the effects of extreme UV radiation on bacteria has been in

relation to survival of vegetative cells or spore-formers like Bacillus subtilis (Berry et al. 2010;

3

Newcombe et al. 2005; Schuerger et al. 2003; Tauscher et al. 2006) as bacterial growth and

replication in Martian surface conditions is not probable.

Subsurface soil conditions on Mars do not prove to be much more hospitable to life than

the surface, although it has been demonstrated that subsurface soil may largely be protected

from the lethal effects of UV radiation (Osman et al. 2008b). Other inhibitory factors are

present, however, as high concentrations of oxidizing minerals, heavy metals, as well as

chlorine- and sulfur-based salts that are present as chlorides, perchlorates, and sulfates (Berry

et al. 2010; Clark and Van Hart 1981; Crisler et al. 2012; Vaniman et al. 2004). As such, an

increasing number of studies have been investigating the growth and survival of bacteria in the

presence of high salt concentrations found in soil on Mars, and indeed have demonstrated

numerous examples of halotolerant and halophilic bacteria capable of proliferating in these

conditions (Berry et al. 2006; Crisler et al. 2012; Grant 2004; Hallsworth et al. 2007).

The first aspect of this study involved testing the ability of bacterial strains to survive

and grow in the presence of perchlorates. Perchlorate salts have been determined to currently

exist in Martian surface soil (Hecht et al. 2009; Kounaves et al. 2009). Any life forms present in

Martian soil would need to be capable of tolerating high concentrations of perchlorates and

other salts previously mentioned. The test subjects used in these experiments were bacteria

isolated from the extremely arid Atacama Desert, an extensively studied environmental analog

of Mars, also known to contain high concentrations of naturally occurring perchlorate salts.

The second aspect of the study was to determine the presence and identity of ionizing

radiation resistant bacteria in Arctic permafrost. Martian permafrost shares many similarities to

4

permafrost found on Earth (Smith and McKay 2005), and as such, studying organisms from

terrestrial permafrost provides a compelling example of what fossilized life preserved in

Martian permafrost might look like. Radiation resistance could be considered an important

characteristic for any microbial resident of permafrost when taking into consideration the

constant background radiation that such stable permanently frozen soil is exposed to over long

periods of time. Isolates from this study may be considered analogs of hypothetical Martian life,

or at the very least, display characteristics that life on Mars would have needed for survival.

5

CHAPTER II GROWTH OF BACTERIAL ISOLATES FROM THE ATACAMA DESERT AT

HIGH CONCENTRATIONS OF PERCHLORATE

Introduction

Recent data obtained from the Phoenix Mars Lander, which landed in the northern

plains of Mars in 2008, demonstrated the presence of the perchlorate anion distributed

throughout the soil column at the landing site (Hecht et al. 2009; Kounaves et al. 2009).

Perchlorates are highly oxidizing salt compounds that are extremely soluble in water. Calcium,

magnesium, and sodium perchlorate have been identified in Martian soil in concentrations

within the range of 0.4-0.6 wt% (Hecht et al. 2009); geochemical modeling based on data from

the Phoenix lander indicates that magnesium perchlorate may exist in the greatest abundance

(Marion et al. 2010). The existence of perchlorate on Mars is perhaps most significant when

considering the implications that this discovery has on the existence of liquid water on Mars. It

has been suggested that liquid perchlorate brines could be present in Martian conditions, and

may remain liquid for at least up to a few hours (Chevrier et al. 2009). Another study by Cull et

al. (2010) explains that the distribution of perchlorate in the soil on Mars is indicative of the

perchlorate having been solubilized and translocated by thin film liquid water.

Terrestrial perchlorate is primarily synthetic and is utilized in industrial lubricants and

pyrotechnics (Trumpolt et al. 2005); Perchlorates are generally regarded as an environmental

pollutant, especially of groundwater (Coates and Achenbach 2004; Motzer 2001). As such, the

extent of knowledge involving the relationship between bacteria and perchlorate is limited to

the area of bioremediation (Coates and Achenbach 2004). Organisms such as Dechloromonas

6

and Dechlorospirillum are known to anaerobically reduce perchlorate, and bioremediation

methods involving these bacteria have been developed (Coates and Achenbach 2004).

Naturally occurring perchlorate is only found in a few environments on Earth, primarily

in hyper-arid deserts and in the stratosphere (Catling et al. 2010). Deserts in West Texas and

Bolivian playas are known to contain perchlorates in lower concentrations than Mars soils

(Dasgupta et al. 2005; Orris et al. 2003). The only area on Earth determined to contain

perchlorates within the range of concentrations observed on Mars, is the Atacama Desert in

Chile, South America (Catling et al. 2010; Parro et al. 2011). Known as the driest place on Earth

(Hickman 1998), this hyper-arid desert is extensively studied as an environmental analog of

Mars. Low levels of organic matter, limited to no water availability, and oligotrophic conditions

in many areas of the desert result in extremely low numbers of culturable microorganisms

(Bagaley 2006; Connon et al. 2007; Navarro-Gonzales et al. 2003; Warren-Rhodes et al. 2006).

The average concentration of perchlorate in the nitrate ores in the Atacama is determined to be

~0.03 wt% (Michalski et al. 2004), and can reach up to ~0.6 wt% (Ericksen 1981); comparing this

with Martian perchlorate concentrations of 0.4 – 0.6 wt% (Hecht et al. 2009), the significant

similarities between the two environments are apparent. Understanding the source of

perchlorates in the Atacama Desert may provide insight into the origin of perchlorate on Mars.

It is hypothesized that perchlorate in the Atacama forms through a series of gas phase

photochemical reactions in the atmosphere involving chlorine ion oxidation into perchloric acid

(Catling et al. 2010). This same mechanism is suggested to be responsible for the formation of

Martian perchlorate as well (Catling et al. 2010). If the two environments have or had similar

processes leading to the formation of perchlorate, then testing the effects of perchlorate on the

7

growth of bacteria from the Atacama may have possible implications for the search for life on

Mars and for future planetary protection measures.

A recently published study investigated the effect of MgSO4, another salt compound

found in abundance in Martian soil, on the growth of halotolerant bacteria isolated from The

Great Salt Plains of Utah. Results showed that the bacteria were capable of growing at high

concentrations (up to 2 M) of MgSO4 (Crisler et al. 2012), and it was determined that bacterial

halotolerance is linked to tolerance of MgSO4. A comparison of results from this study will

hopefully serve to elucidate important similarities or differences between bacterial growth at

high concentrations of perchlorates and sulfates.

Material and Methods

Perchlorate-containing culture media

R2 agar (Difco) was used for growth of all strains in this study. The perchlorate salt was

dissolved in deionized water, R2A powder was added as per manufacturer instructions, pH was

adjusted to 7.2, volume was adjusted to 500 mL, and autoclaved. Perchlorates used in this

study include sodium perchlorate monohydrate (NaClO4 ∙ H2O), magnesium perchlorate

hexahydrate (Mg(ClO4)2 ∙ 6 H2O) , and calcium perchlorate tetrahydrate (Ca(ClO4)2 ∙ 4 H2O). R2A

with 50, 100, 200, 400, 600, 800, and 1000 mM Na perchlorate, and 50 – 400 mM for Mg- and

Ca-perchlorate were tested due to chaotropic effects on agar above 400 mM. An agar

concentration of 3% w/v was required for solidification of the gel above concentrations of 200

mM for Mg- and Ca-perchlorate. Thus, 3% agar was added to all media used in this study. The

same method was used to make R2A with MgSO4 ∙ 7 H2O, KNO3, and NaCl. R2A containing 0.5,

8

1, and 2 M NaCl was used to test the halotolerance of the Atacama strains. R2A with 0.5 and 1

M KNO3 and 1 M MgSO4 were also tested.

Bacterial isolates and type strains

Type strains, control strains, and Deinococcus strains used in this study were obtained

from DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany. All

Atacama Desert isolates were obtained from previous studies in RaineyLab (Bagaley 2006). For

complete list of strains used in perchlorate testing refer to Appendix A (p.62).

Incubation and growth

A standardized amount of cell material from each strain was suspended in 0.85% w/v

saline and was subsequently spotted onto the perchlorate containing media in 10 µL aliquots. A

control of R2A + 3% agar and no added perchlorate was used for comparison of growth. Plates

were incubated at 28° C for all Blastococcus strains and other Atacama Desert isolates; All other

organisms that were tested on perchlorate were incubated at their respective optimum growth

temperatures, ranging from 10 – 37 °C for Deinococcus strains and 10 – 28 °C for control strains

(see Appendix A on p. 69 for a list of all strains and incubation temperatures). Plates incubated

at ≥ 28° C were incubated for 1 week, while those at 10° C were incubated for 2 weeks; When

checking growth, the control plate was used for comparison and extent of growth was

determined and scored relative to the control plate.

Plating of Atacama soils on perchlorate-containing media

1/10 concentration plate count agar (PCA) and 1/100 concentration PCA containing

either Na- or Mg-perchlorate was prepared. Media was prepared with 200 and 400 mM Mg-

perchlorate and 200, 400, and 600 mM Na-perchlorate. Ten soils were chosen for dilution

9

plating onto the perchlorate-containing agar. Eight soils were from the Atacama Desert,

including AT05-22, AT03-49, AT04-161, AT03-34, AT04-166, AT03-33, AT03-41, and AT03-37. Of

the remaining two soils, SS05-2 was a sample from the Sahara Desert and 33-40Q was from a

desert in Oman, United Arab Emirates. 1 g of soil was mixed in 9 mL of 0.85% w/v saline

solution, and 10 µL aliquots were pipetted onto the perchlorate containing agar plates. Sterile

plastic spreaders were used to evenly distribute the soil solution on the surface of the agar.

Plates incubated at 25, 28, and 37 °C were checked for growth at 1 week, during which time

colony counts were performed. Plates incubated at 10 °C were incubated for 14 days before

counting colonies. For each media, 1/10 PCA and 1/100 PCA, a control set was also used that

did not contain perchlorate.

Survival of Atacama strains in liquid media containing higher concentrations of perchlorate

Five organisms that survived high (≥ 1 M Na-perchlorate and ≥ 200 mM Ca- and Mg-

perchlorate) were tested for growth at higher concentrations of perchlorate dissolved in R2

broth (R2B). Organisms tested include KR-187, AT03-49-2, MCC-63, SS05-2-113, and 33-40Q-

101A. R2B was prepared as per instructions from the manufacturer with concentrations of 600

and 800 mM Mg- and Ca-perchlorate. These concentrations were unable to be tested with solid

media. One mL of perchlorate containing R2B, as well as a control set of R2B without

perchlorate, was pipetted into the wells of a 24 well microwell plate. A standardized amount of

cell material from each of the strains was placed into the well, and plates were incubated at 28°

C for 1 week. 10 µL aliquots from each well were then pipetted onto R2A, spread with a sterile

inoculating loop, and incubated at 28° C for one week. Survival of each strain was determined

based on whether growth was observed on R2A after 1 week incubation.

10

Soil perchlorate determination

Results were obtained from Birgit Hagedorn, UAA, Anchorage, Alaska.

Results and Discussion

Growth of bacterial strains in the presence of perchlorates

Increasing concentration of all perchlorate salts had a negative effect on bacterial

growth and survival. Of all the organisms tested, Blastococcus species grew at the highest

concentrations of perchlorate salts – see Figures 2.1 – 2.3 (reference additional data in

Appendix A, p. 65). These Blastococcus strains were mostly isolates from the Atacama Desert,

two came from the Sahara Desert, and three were previously described strains. Eighty-eight

percent of 49 Blastococcus strains tested grew at concentrations up to 400 mM Na-perchlorate,

whereas only 45% of 33 other isolates from the Atacama grew at concentrations of 400 mM Na-

perchlorate. Three species of Blastococcus, one being the previously described strain

Blastococcus jejuensis, grew at 1 M Na-perchlorate. Some other Atacama strains that survived

at exceptionally high concentrations (≥400 mM Na-perchlorate) include Kocuria sp. (AT04-166-

1), Geodermatophilus sp. (KR-56), and Modestobacter sp. (AT04-159-22). Other bacteria that

were used for comparison with the Atacama isolates generally did not grow at high perchlorate

concentrations. A group of Hymenobacter isolates from permafrost did not grow at the lowest

concentration tested (50 mM) of any perchlorate. Twenty one Deinococcus strains that were

tested did not grow at greater than 200 mM Na-perchlorate. Less than 30% of these grew at

100 mM Mg- and Ca-perchlorate. Of a group of control strains from various taxonomic groups

and different environments (refer to Appendix A for complete list of strains), Bacillus subtilis

grew at concentrations similar to the isolates from the Atacama Desert, up to 600 mM Na-

11

0

20

40

60

80

100

120

50 100 200 400 600 800 1000

% S

trai

ns

Sho

win

g G

row

th

NaClO4 Concentration (mM)

% Blastococcus

% Other Atacama Isolates

% Control Strains

% Deinococcus

Control

Strain Designation Species Name R2A + 3% Agar 400 mM 600 mM 50 mM 100 mM 50 mM 100 mM

DSM 14746T Sphingomonas aerolata ++ - - - - - -

DSM 10604T Pseudomonas syringae +++ - - - - - -

DSM 15428T Paenisporosarcina macmurdoensis ++++ - - +++ +++ - -

DSM 21396T Deinococcus gobiensis ++++ - - - - - -

DSM 14418T Georgenia muralis ++++ +++ - ++++ - ++ -

DSM 21230T Deinococcus aetherius + - - - - - -

DSM 10T Bacillus subtilis ++++ +++ ++ ++++ ++++ +++ +++

DSM 30147T Rhizobium radiobacter ++++ - - ++ - - -

DSM 40313T Streptomyces albus ++++ ++++ - ++++ ++++ ++++ -

DSM 21212T Deinococcus aerius ++++ - - - - - -

DSM 21991T Sporosarcina antarctica ++++ - - - - - -

NaClO4 MgCl2O8 CaCl2O8

perchlorate and up to 100 mM Mg- and Ca-perchlorate. Two other species that grew at

relatively high concentrations were Streptomyces albus and Georgenia muralis (See Table 2.1).

Table 2.1. The highest concentration of perchlorates at which control strains exhibited growth

Figure 2.1. Percentage of strains growing at high concentrations of Na-perchlorate (NaClO4)

12

Figure 2.3. Percentage of strains that growing at high concentrations of Ca-perchlorate (Ca(ClO4)2)

Figure 2.2. Percentage of strains that growing at high concentrations of Mg-perchlorate (Mg(ClO4)2)

13

Chaotropic effects and water activity of perchlorate-containing media

The concentration at which growth occurred for all organisms was dependent on the

type of perchlorate salt. In general, bacterial strains could grow at greater concentrations of Na-

perchlorate than either Mg- or Ca-perchlorate. Additionally, strains grew at higher

concentrations of Mg-perchlorate than Ca-perchlorate. These results likely reflect the varying

degrees of water activity and chaotropicity exhibited by the three different perchlorates.

Chaotropicity refers to the characteristic of disrupting or denaturing molecular structures such

as proteins, water, and other macromocules (Hallsworth et al. 2003). Water activity is defined

as the vapor pressure of air in equilibrium with a solution to that of pure water (Grant 2004).

Simply put, the higher the solute concentration of a given system, the lower the water activity,

and consequently, greater osmotic stress inflicted on organisms living in that system.

It has previously been proposed in a study involving the effects of MgCl2 on bacterial

growth, that chaotropic effects of MgCl2 are solely responsible for inhibiting bacterial growth

rather than water activity (Hallsworth 2007). The specific mechanism of bacterial perchlorate

tolerance was not determined in this study, but it appears that chaotropicity and water activity

are closely related, and thus it is possible that in perchlorate solutions a combination of the two

factors result in inhibition of bacterial growth.

Initially, before addition of an extra 3% agar, 200 mM was the cutoff concentration for

both Mg- and Ca-perchlorate before agar failed to solidify. After an additional 3% agar,

concentrations up to 400 mM solidified. At 3% added agar, a concentration of 1 M Na-

perchlorate could be solidified. Comparing this data with bacterial growth, a correlation is

observed between growth of bacteria and concentration at which agar cannot solidify. Only

http://chemistry.about.com/od/chemistryglossary/g/Chaotropic-Definition.htm

14

28% of Atacama isolates grew at 200 mM Mg-perchlorate, and only 9% grew at 200 mM Ca-

perchlorate. This concentration was initially the concentration at which media could not solidify

without an extra 3% agar. At the new limit for solidification, 400 mM for Mg- and Ca-

perchlorate, there was near zero growth of any isolates. This suggests that inhibition of

bacterial growth is due to chaotropic effects. The organisms that could survive at

concentrations beyond the limit of agar solidification, which include a few strains of

Blastococcus and a Kocuria sp. that survived up to 1 M Na-perchlorate, are worth noting for

future studies. These particular strains may serve as the best models for studying perchlorate

resistance in bacteria.

Gram-positive versus Gram-negative growth in the presence of perchlorates

A comparison between growth of Gram-positive and Gram-negative bacteria used in

this study demonstrates noticeable differences in perchlorate tolerance. Gram-negative

organisms exhibited limited growth at high concentrations of perchlorate relative to Gram-

positive bacteria; organisms surviving some of the highest perchlorate concentrations tested

were all Gram-positive bacteria. Blastococcus strains, for example, are Gram-positive organisms

and consistently showed growth at higher perchlorate concentrations than other bacteria. A

Kocuria strain from the Atacama Desert is another Gram-positive organism that grew at up to 1

M Na-perchlorate, 200 mM Mg-perchlorate, and 100 mM Ca-perchlorate. Of the 11 control

strains, Georgenia muralis, Bacillus subtilis, Paenisporosarcina macmurdoensis, and

Streptomyces albus were the only organisms capable of growing at concentrations ≥400 mM

Na-perchlorate and up to 100 mM Mg-perchlorate; all four of these strains are Gram-positive.

B. subtilis and P. macmurdoensis are known spore-formers which may provide some insight into

15

perchlorate tolerance, but another spore-former, Sporosarcina antarctica, interestingly did not

grow at any concentration of perchlorate tested. A probable explanation for this result is that S.

antarctica was incubated at 10 °C, and water activity is known to decrease at lower

temperatures (Gunde-Cimerman et al. 2003), resulting in a greater inhibitory effect on S.

antarctica than organisms incubated at higher temperatures.

Of the 11 Gram-negative Microvirga strains from both the Atacama and Oman, most

exhibited limited growth on all perchlorate media, i.e. < 400 mM Na-perchlorate and < 100 mM

Mg- and Ca-perchlorate. One exception was a Microvirga strain, 33-40Q-101A, capable of

growth at up to 800 mM Na-perchlorate, 200 mM Mg-perchlorate, and 100 mM Ca-perchlorate;

these results are comparable with the majority of Blastococcus strains. Deinococcus strains,

with cell walls structurally similar to Gram-negative organisms, also exhibited very limited

perchlorate tolerance compared with other tested strains (See Figures 2.1 – 2.3). The majority

of the Gram-negative control strains exhibited no growth at any concentration of perchlorate

tested, and those that did grow could only tolerate concentrations of < 100 mM of any

perchlorate (see Table 2.2). Rhizobium radiobacter was capable of growth at 100 mM Na-

perchlorate and 50 mM Mg-perchlorate; this was the only Gram-negative organism in the

control group that displayed growth in the presence of perchlorate, and it is also known to

produce extracellular polysaccharide (EPS). Therefore, it is possible that production of EPS may

provide some protection against inhibition of growth by perchlorate.

16

Strain Designation Species Name Gram Type NaClO4 MgCl2O8 CaCl2O8

DSM 21991T Sporosarcina antarctica (+) NG NG NG

DSM 40313T Streptomyces albus (+) 400 mM 100 mM 100 mM

DSM 15428T Paenisporosarcina macmurdoensis (+) NG 100 mM NG

DSM 10T Bacillus subtilis (+) 600 mM 100 mM 100 mM

DSM 14418T Georgenia muralis (+) 400 mM 50 mM 50 mM

DSM 21230T Deinococcus aetherius (-) NG NG NG

DSM 14746T Sphingomonas aerolata (-) NG NG NG

DSM 30147T Rhizobium radiobacter (-) 100 mM 50 mM NG

DSM 21396T Deinococcus gobiensis (-) 100 mM NG NG

DSM 21212T Deinococcus aerius (-) NG NG NG

DSM 10604T Pseudomonas syringae (-) NG NG NG

Based on these data, a correlation is observed between higher perchlorate tolerance

and cell wall type. It is possible that a thicker peptidoglycan layer in Gram-positive bacteria

could provide a mechanistic basis for perchlorate tolerance.

Bacterial growth in the presence of MgSO4, KNO3, and NaCl

All strains of bacteria that were tested for growth on perchlorate containing media were

also tested for growth on R2A containing high concentrations of each of three different salts,

MgSO4 ∙ 7H2O, KNO3, and NaCl. The objective of this experiment was to observe any existing

relationships between epsotolerance, halotolerance, and perchlorate tolerance. The

Blastococcus strains from the Atacama Desert grew at the highest concentrations of NaCl,

compared with the other Atacama isolates and other control organisms tested. It is important

to note that whereas 61% of Blastococcus strains grew at 1 M NaCl, less than 4% of the same

strains grew at 1 M Na perchlorate; this result clearly highlights the greater chaotropicity and

lower water activity of perchlorates, which results in greater inhibitory effects on bacterial

Table 2.2. The highest concentration of perchlorate at which Gram(+) and Gram(-) control strains exhibited growth. NG - no growth observed

17

growth. Considering % w/v of the two solutions reveals the significant difference in available

water. Whereas a 1 M NaCl solution is 6% w/v, a 1 M Na-perchlorate solution is 13% w/v; thus,

the greater inhibitory effect of Na-perchlorate versus NaCl may be a result of the greater

amount of solute present at the same molarity, resulting in less available water.

Of the control strains, Pseudomonas syringae, Paenisporosarcina macmurdoensis,

Georgenia muralis, and Streptomyces albus were capable of growth at up to 1 M NaCl, and

Bacillus subtilis exhibited growth at 2 M NaCl (refer to Figure 2.2). Deinococcus strains displayed

extremely limited growth in the presence of NaCl, with only 29% of strains growing at 0.5 M

NaCl and no further growth at higher NaCl concentrations.

Figure 2.4. Percentage of strains growing in the presence of 0.5, 1, and 2 M NaCl

18

Growth in the presence of KNO3 was limited in comparison with perchlorate and MgSO4,

with about 41% of Blastococcus strains growing at 0.5 M KNO3, which then decreased to 8% at

1 M KNO3. Again, other Atacama isolates and Deinococcus strains exhibited lower growth on

KNO3-containing media compared with Blastococcus strains. Two of the control strains, Bacillus

subtilis and Streptomyces albus, grew at 1 M KNO3.

96% of Blastococcus isolates were capable of growing at 1 M MgSO4. This is interesting

when considering that at 400 mM Mg-perchlorate concentration, few Blastococcus exhibited

growth. Again, this result illustrates the lower water activity and greater chaotropic effects of

perchlorate versus MgSO4. Considering % w/v, a 1 M solution of KNO3 is 10% w/v, compared

with a 1 M MgSO4 solution which is 12% w/v. The greater number of organisms observed

growing at 12% w/v MgSO4 versus 10% w/v KNO3 is most likely due to the fact that MgSO4 ∙ 7

H2O was used in this study. Comparing these values with Mg-perchlorate, a 400 mM solution of

Mg-perchlorate is 9% w/v. It is therefore evident that, due to molecular mass, more solute is

present in perchlorate solutions than other salts at similar molarities, thus reducing the water

activity.

Previous studies have found that high sulfate systems have higher water activities than

high chloride systems at identical concentrations (Crisler et al. 2012; Grant 2004; Hallsworth et

al. 2003; Marion et al. 2010); these systems with high chloride concentrations are far less

permissive of bacterial growth than the high sulfate systems. This comparison of water

activities and % w/v calculations between chlorides, sulfates, and perchlorates explains the

results observed in this study with perchlorates. An analysis of bacterial growth at similar

19

concentrations of the salts tested in this study indicates that high-perchlorate solutions exhibit

lower water activities than sulfate or chloride-dominated systems, making high perchlorate

systems more inhibitory to bacterial growth. When taking into account % w/v of these different

salt solutions, it is evident that inhibition of bacterial growth is a result of a greater amount of

solute present in perchlorate solutions. Incidentally, it is also fitting to point out that MgSO4 is

present in far higher concentrations than any perchlorates on Mars, by a ratio of 4:1 (Crisler et

al. 2012). This correlates with the greater inhibitory effects of perchlorate on bacterial growth,

but more importantly, emphasizes the ability of these Blastococcus strains from a Mars analog

environment to grow at high concentrations of multiple salts found in abundance on the

Martian surface.

A relationship is observed between bacteria that display perchlorate tolerance and

tolerance to other salts like MgSO4, KNO3, and NaCl. One example is the Blastococcus strain

AT03-49-2, that grew at up to 2 M NaCl, 1 M KNO3, and 1 M MgSO4 – the highest

concentrations tested in this experiment. This same strain grew at up to 1 M Na perchlorate

and up to 400 mM Mg- and Ca-perchlorate, the only strain able to do so. AT03-34-7, another

Blastococcus strains, grew at 1 M Na-perchlorate, and also grew at all of the previously

mentioned highest concentrations of NaCl, KNO3, and MgSO4. On the other hand, the strain

AT03-40-35 only grew at 0.5 M NaCl, not exhibiting growth at any tested concentrations of

KNO3 and MgSO4. Accordingly, this strain only showed growth at < 100 mM Mg- and Ca-

perchlorate – only 4% of Blastococcus were not capable of growing at 100 mM Mg-perchlorate;

thus, this result clearly highlights the relationship between bacterial growth across various salts.

Essentially, strains that are sensitive to salts like MgSO4, KNO3, and NaCl also exhibit increased

20

Strain Designation Species Name NaClO4 MgCl2O8 CaCl2O8 NaCl KNO3 MgSO4

AT03-49-2 Blastococcus sp. 1 M 400 mM 400 mM 2 M 1 M 1 M

AT03-34-7 Blastococcus sp. 1 M 200 mM 200 mM 2 M 1 M 1 M

AT03-40-35 Blastococcus sp. 400 mM 50 mM 50 mM 0.5 M NG NG

AT03-33-58 Blastococcus sp. 400 mM 50 mM 100 mM 0.5 M NG NG

DSM 10T Bacillus subtilis 600 mM 100 mM 100 mM 2 M 1 M 1 M

Highest Concentration at which Growth Occurred

Strain Designation Species Name R2A + 3% Agar 0.5 M NaCl 1.0 M NaCl 2.0 M NaCl 0.5 M KNO3 1.0 M KNO3 1.0 M MgSO4

DSM 10604T Pseudomonas syringae ++++ ++++ + - + - ++++

DSM 15428T Paenisporosarcina macmurdoensis ++++ ++++ + - - - ++++

DSM 14418T Georgenia muralis ++++ ++++ ++++ - ++ - ++++

DSM 40313T Streptomyces albus ++++ ++++ ++++ - ++++ ++++ ++++

DSM 10T Bacillus subtilis ++++ ++++ ++++ ++++ ++++ ++++ ++++

DSM 21212T Deinococcus aerius ++ - - - - - -

DSM 30147T Rhizobium radiobacter ++++ ++++ - - - - ++++

sensitivity to perchlorates. Higher tolerance to MgSO4, KNO3, and NaCl results in higher

tolerance of perchlorate as well. However, perchlorate compounds have the greatest inhibitory

effects on bacterial growth in comparison to other salt compounds at similar concentrations.

Table 2.4. Growth of control strains in the presence of NaCl, KNO3, and MgSO4

Table 2.3. Growth of Blastococcus strains and Bacillus subtilis in presence of high salt concentrations. NG – no growth observed.

21


AT04-159-25 Blastococcus sp. ++++ ++++ ++++ - - - ++++

AT04-159-34 Blastococcus sp. ++++ ++++ - - ++ - ++++

AT04-159-37 Blastococcus sp. ++++ ++++ - - - - ++++

AT04-159-106 Blastococcus sp. ++++ +++ - - - - ++++


AT03-33-18 Blastococcus sp. ++++ ++++ + - - - ++++

AT03-33-24 Blastococcus sp. ++++ ++++ + - - - +++

AT03-33-58 Blastococcus sp. ++++ +++ - - - - -

AT03-34-7 Blastococcus sp. ++++ ++++ ++++ + ++++ ++++ ++++





AT03-40-15 Blastococcus sp. ++++ ++++ +++ - - - ++++

AT03-40-35 Blastococcus sp. ++++ ++++ - - - - -


AT03-43-14 Blastococcus sp. ++++ ++++ + - + - ++++

AT03-49-2 Blastococcus sp. ++++ ++++ ++++ ++++ ++++ ++++ ++++

AT04-151-2 Blastococcus sp. ++++ ++++ +++ - ++ - ++++

AT04-153-2 Blastococcus sp. ++++ ++++ ++++ - - - +++




AT04-161-28 Blastococcus sp. +++ +++ - - - - ++++

AT04-161-23 Blastococcus sp. ++++ ++++ ++ - - - ++


AT04-161-96 Blastococcus sp. ++++ ++++ - - ++++ - ++++

AT04-164-3 Blastococcus sp. ++++ ++++ +++ - ++++ - ++++




AT04-165-16 Blastococcus sp. ++++ ++++ +++ - + - ++++





AT04-166-109 Blastococcus sp. ++++ ++++ + - - - ++++

AT04-166-169 Blastococcus sp. ++++ ++++ ++++ - ++++ - ++++


SS05-2-14 Blastococcus sp. ++++ ++++ ++++ - ++++ ++ ++++

SS05-2-113 Blastococcus sp. ++++ ++++ - - ++++ - ++++

MCC-59 Blastococcus sp. ++++ + - - - - ++++

KR-187 Blastococcus sp. ++++ ++++ ++++ - - - ++++

KR-206 Blastococcus sp. ++++ ++++ ++++ - +++ - ++++




B. jeju Blastococcus jejuensis ++++ ++++ ++++ - +++ +++ ++++

B. ag Blastococcus aggregatus ++++ ++++ ++ - +++ - ++++

B. sax Blastococcus saxobsidens ++++ ++++ +++ - +++ - ++++

Table 2.5. Growth of all Blastococcus strains in the presence of NaCl, KNO3, and MgSO4

22


AT04-159-22 Modestobacter sp. ++++ ++++ + - ++ - ++++

G. ob. ob Geodermatophilus obscuris obscuris ++++ - - - - - ++++

M. veri Modestobacter versicola ++++ ++++ - - +++ - ++++

ATCC-BAA-817 Balinemonas flocculans ++++ - - - - - -

KACC-12744 Microvirga aerilata ++++ + - - - - -

M. guang Microvirga guangxiensis ++++ + - - - - -

M. aerophila Microvirga aerophila ++++ - - - - - -

10-39Q-44 Microvirga sp. ++++ ++++ - - - - -

KR-477 Microvirga sp. ++++ + - - - - -

KR-450 Microvirga sp. ++++ ++++ - - + - ++++

KR-393 Microvirga sp. ++++ ++++ - - + - ++++

M. subterranea Microvirga subterranea ++++ ++++ - - - - +

AT03-41-2 Frankia sp. ++++ ++++ - - - - ++++

AT03-38-1 Frankia sp. ++++ ++++ + - ++++ ++ ++++

KR-515 α-proteobacterium ++++ - - - - - +

NG05-4-23 Microvirga sp. ++++ - - - - - -

AT04-159-59 Mycobacterium celatum ++++ +++ - - - - -

AT04-166-1 Kocuria sp. ++++ ++++ ++++ + ++++ - ++++

AT03-40-39 Ornithinicoccus hortensis ++++ ++++ +++ - - - -

AT04-159-4 Hymenobacter sp. ++++ - - - - - -

KR-91 Microvirga sp. ++++ - - - - - -

NG05-R6A-48 Microvirga sp. ++++ ++++ - - - - -

NG05-4D6A-29 Microvirga sp. ++++ ++++ +++ - ++++ + ++++

KR-28 Microvirga sp. ++++ - - - - - -

33-40Q-130 Microvirga sp. ++++ - - - - - -


DSM 15974T Deinococcus saxicola ++ - - - - - -

DSM 12027T Deinococcus radiopugnans ++++ ++++ - - +++ - ++++

DSM 20539T Deinococcus radiodurans ++++ ++++ - - + - ++++

DSM 3963T Deinococcus grandis ++++ - - - - - +

DSM 17005T Deinococcus yunweiensis ++++ - - - - - -

KCTC-12553T Deinococcus caenii ++++ - - - - - -

KCTC-12552T Deinococcus aquaticus ++++ - - - - - ++++

LMG 22133T Deinococcus hopiensis ++++ - - - - - -

LB-34T Deinococcus maricopensis ++++ - - - - - -

CCUG 51391T Deinococcus ficus ++++ + - - - - +++

DSM 15307T Deinococcus indicus ++++ ++ - - - - ++++

NG-371 Deinococcus sp. ++++ - - - - - -

KR-35 Deinococcus apachensis ++ - - - - - -

KR-40T Deinococcus hohokamensis ++++ + - - - - -

KR-200T Deinococcus peraridilitoris ++++ - - - - - -

KR-235T Deinococcus pimensis ++ - - - - - -

DSM 17424T Deinococcus mumbaiensis ++++ - - - - - +

DSM 11300T Deinococcus geothermalis ++++ - - - - - -

PO-04-20-132T Deinococcus radiomollis ++++ - - - - - -

Table 2.6. Growth of other arid soil strains in the presence of NaCl, KNO3, and MgSO4

Table 2.7. Growth of Deinococcus strains in the presence of NaCl, KNO3, and MgSO4

23

Correlation between bacterial growth and perchlorate concentration of Atacama soils

Perchlorate concentrations were determined for a number of Atacama soil samples

from which many of the isolates tested in this study originated (Table 2.8). A possible

correlation was observed between the perchlorate concentration at specific sites in the

Atacama Desert and the organisms isolated from those sites. The perchlorate concentration at

AT04-166 was measured at 0.27 µg/g, and at AT04-153 the perchlorate concentration was

measured at 0.1 µg/g. Comparison of growth data for Blastococcus strains isolated from both

sites reveals that whereas 100% of strains from AT04-166 survived at 400 mM Na-perchlorate,

only 50% of the strains from AT04-153 survived that concentration. Looking at Mg-perchlorate

results, 60% of Blastococcus strains. from AT04-166 survived up to 200 mM Mg-perchlorate,

and there were no survivors from site AT04-153 at 200 mM Mg-perchlorate. However, growth

data for Ca-perchlorate did not show any correlating results as concentrations greater than 100

mM were lethal to all Blastococcus strains, and concentrations below 100 mM allowed for

similar, incomparable growth of isolates from different areas. It is interesting to note, however,

that Blastococcus isolate AT03-34-7 grew at Na-perchlorate concentrations exceeding 800 mM

and was isolated from site AT03-34, at which the perchlorate concentration was recorded at

0.42 µg/g, double that of soil from site AT04-166. Another strain growing at > 800 mM Na-

perchlorate, Kocuria strain. designated AT04-166-1, was isolated from AT04-166 containing

0.27 µg/g perchlorate. Although these results point towards some correlation between soil

perchlorate concentration and perchlorate resistance of organisms from that soil, more data is

needed in order for definitive conclusions to be made. Bacterial resistance to and growth in the

24

Chloride Sulfate Nitrate Perchlorate

site mg/g soil mg/g soil mg/g soil ug/g soil

AT03-34 0.20 7.27 0.19 0.42

AT03-37 0.15 7.21 0.21 0.31

AT03-41 0.13 6.95 0.14 0.20

AT03-44 <LOD1 7.28 0.18 0.37

AT03-45 <LOD 7.18 0.08 0.20

AT03-49 0.17 7.50 0.13 0.29

AT04-152 0.18 8.12 0.16 0.36

AT04-153 0.12 2.07 0.06 0.10

AT04-161 0.15 7.38 0.15 0.24

AT04-166 0.11 7.06 0.08 0.27

presence of high concentrations of perchlorates has not previously been studied, and it is

possible that the mechanism is a complex one.

Considering that Atacama Desert isolates grew at overall higher concentrations of

perchlorate than bacteria from other sources and taxonomic groups further supports the

probable relationship between perchlorate tolerance and the perchlorate concentration in the

soil from which strains were originally isolated. It would be reasonable to assume that higher

perchlorate levels in the environment led to an adaptive perchlorate tolerance in these

Blastococcus strains as well as the other isolates from the Atacama Desert. Further

experimentation and collection of more data could solidify these initial observations.

Table 2.8. Chemical analysis of Atacama soil samples showing concentrations of chloride, sulfate, nitrate, and perchlorate

1Level of detection

25

Strain Designation Soil Origin Species Name NaClO4 MgCl2O8 CaCl2O8 MgCl2O8 CaCl2O8

AT03-49-2 Atacama Blastococcus sp. 1000 400 400 (+) 600, (+) 800 (-) 600, (-) 800

33-40Q-101A Oman Microvirga sp. 1000 200 200 (+) 600, (+) 800 (+) 600, (-) 800

SS05-2-113 Sahara Blastococcus sp. 400 200 400 (-) 600, (-) 800 (-) 600, (-) 800

MCC-63 Atacama Blastococcus sp. 400 100 100 (-) 600, (-) 800 (-) 600, (-) 800

Highest concentration at which growth occurred (mM) Survival at higher concentrations (mM)

Bacterial survival at high perchlorate concentrations in liquid media

Survival of four isolates that grew at high concentrations of perchlorate in R2A was

examined by incubating them in liquid R2 broth containing 600 mM and 800 mM Mg- and Ca-

perchlorate (see Table 2.9). It is interesting to note that one of the strains surviving 600 and 800

mM Mg- and Ca-perchlorate is a Gram-negative bacterium of the genus Microvirga, a group

that otherwise consistently exhibited limited growth on perchlorate media. It is also worth

pointing out that the Blastococcus sp. from the Sahara was not capable of survival at 600 or 800

mM concentrations of either perchlorate, but a Blastococcus strains from the Atacama survived

both concentrations of Mg-perchlorate. Another interesting observation is that the

Blastococcus and Microvirga strains surviving 600 and 800 mM Mg-perchlorate did not survive

similar concentrations of Ca-perchlorate; the Microvirga strain did survive 600 mM Ca-

perchlorate, however. This trend of lower tolerance to Ca-perchlorate is prominent in general

growth results (refer to Figures 2.1 – 2.3), and is most likely due to lower water activity in Ca-

perchlorate solutions.

Table 2.9. Highest perchlorate concentration tolerated by certain strains and their survival (+ or -) in liquid media containing 600 and 800 mM Mg-perchlorate (MgCl2O8) and Ca-perchlorate (CaCl2O8) concentrations

26

Plating soils on perchlorate-containing media

Eight soils from the Atacama Desert, one from the Sahara Desert, and one from a desert

in Oman were plated on perchlorate-containing 1/10 PCA and 1/100 PCA. After one week

incubation, plates exhibited a diversity of microorganisms growing at lower perchlorate

concentrations (200 mM Na- and Mg-perchlorate), and diversity and Cfu/g decreased at higher

concentrations (400, 600 mM Na-perchlorate and 400 mM Mg-perchlorate). On 1/10 PCA,

there were no observable colonies at either 200 or 400 mM Mg-perchlorate for any soil, except

AT05-22. Another sample, AT03-37, yielded no colonies on any media, including the control

1/10 PCA; it is worth mentioning that this result is consistent with previous data of cfu/g

calculated for this soil on 1/10 PCA (Bagaley 2006). In most cases, the cfu/g of the control plates

that did not contain perchlorate was higher than cfu/g on media containing perchlorate.

Exceptions were noted, however, such as AT03-34 in which the same number of colonies were

recovered from 1/10 PCA control and 1/10 PCA + 200 mM Na-perchlorate. Other soils like

AT03-49 (on 1/10 PCA) and AT03-33 (on 1/100 PCA) yielded more colonies on perchlorate-

containing media than the control plates (see Tables 2.10 – 2.11). In general, cfu/g was also

greater on 1/10 PCA versus 1/100 PCA, except in the case of AT03-33, AT05-22, and 33-40Q

which all yielded equal or greater cfu/g on 1/100 PCA containing 200 mM Mg-perchlorate than

1/10 PCA containing 200 mM Mg-perchlorate.

Data from this study suggest that recovery of organisms from soils with higher

perchlorate concentrations decreased in comparison with soils containing less perchlorate due

to the inhibitory effects on growth observed as perchlorate concentration is increased; it is also

important to note that the perchlorate concentration of the media would be present in

27

Perchlorate Concentration Control

Soil Origin in Soil (ug/g) 1/10 PCA 200 mM 400 mM 200 mM 400 mM 600 mM

AT03-34 Atacama 0.42 1 x 102 0 0 1 x 102 0 0

SS05-2 Sahara 0.32 TMTC 0 0 2.2 x 103 1 x 102 0

AT03-37 Atacama 0.31 0 0 0 0 0 0

AT03-49 Atacama 0.29 1 x 102 0 0 4 x 102 0 0

AT04-166 Atacama 0.27 TMTC 0 0 2 x 103 4.7 x 103 1 x 102

AT04-161 Atacama 0.24 3.8 x 104 0 0 2 x 102 0 0

AT03-41 Atacama 0.20 2 x 102 0 0 0 0 0

AT03-33 Atacama N/A 9.3 x 103 0 0 0 0 0

AT05-22 Atacama N/A TMTC 6 x 102 0 TMTC 5.1 x 104 4 x 102

33-40Q Oman N/A TMTC 0 0 TMTC 1.1 x 103 1 x 102

1/10 PCA + MgCl2O8 1/10 PCA + NaClO4

Perchlorate Concentration Control

Soil Origin in Soil (ug/g) 1/100 PCA 200 mM 400 mM 200 mM 400 mM 600 mM

AT03-34 Atacama 0.42 0 0 0 0 0 0

SS05-2 Sahara 0.32 TMTC 0 0 8 x 103 1.5 x 103 0

AT03-37 Atacama 0.31 0 0 0 0 0 0

AT03-49 Atacama 0.29 0 0 0 0 0 0

AT04-166 Atacama 0.27 0 0 0 0 0 0

AT04-161 Atacama 0.24 2.7 x 103 0 0 0 0 0

AT03-41 Atacama 0.20 0 0 0 0 0 0

AT03-33 Atacama N/A 0 1 x 102 0 0 0 0

AT05-22 Atacama N/A TMTC 6 x 102 0 TMTC 5.4 x 103 2 x 102

33-40Q Oman N/A TMTC 1.3 x 103 0 8.5 x 103 2.6 x 103 0

1/100 PCA + MgCl2O8 1/100 PCA + NaClO4

addition to whatever perchlorate was contained in the soil. However, considering the

perchlorate concentration measured for each soil, there was no clear correlation between cfu/g

from soils with higher perchlorate concentration and those with less perchlorate concentration.

One possible example may be demonstrated by AT03-34 plated on 1/10 PCA at 200 mM Na-

Table 2.10. Cfu/g of soil when plated on 1/10 PCA containing 200 – 600 mM concentrations of Mg-perchlorate (MgCl2O8) and Na-perchlorate (NaClO4). TMTC – too many to count

Table 2.11. Cfu/g of soil when plated on 1/100 PCA containing 200 – 600 mM concentrations of Mg-perchlorate (MgCl2O8) and Na-perchlorate (NaClO4). TMTC – too many to count

28

perchlorate, which had a cfu/g of 1 x 102. This soil contained the highest perchlorate

concentration, 0.42 ug/g, of all soils plated on perchlorate-containing media. At 200 mM Na-

perchlorate, most of the other soils yielded higher cfu/g than AT03-34. Alternatively, it could be

hypothesized that if soil containing a higher perchlorate concentration contained a greater

number of organisms adapted to tolerate these conditions, a difference in cfu’s may not then

be noticed. Further study and collection of more data could clarify whether soils with higher

perchlorate concentrations contain a lower cfu/g than soils with lower perchlorate

concentrations, or if they contain a greater number of perchlorate-tolerating microorganisms.

Conclusion

Ultimately, bacterial growth is inhibited by high concentrations of perchlorate. Bacteria

from the Atacama Desert, especially members of the Blastococcus genus, grew at higher

concentrations of perchlorate than organisms from other environments. As the Atacama Desert

is one of few locations that contain naturally occurring perchlorate, the higher tolerance of

these isolates is likely attributable to an adaptive tolerance mechanism developed for survival.

Correlating data for higher perchlorate at isolation site and higher strain resistance is observed

to some extent, although gathering additional data would further validate these findings.

Another correlation is seen between bacterial growth at high perchlorate concentrations and

growth at high concentrations of other salts such as MgSO4, KNO3, and NaCl. It may be that

bacteria capable of tolerating high concentrations of one salt compound are equipped with

mechanisms to tolerate others as well. Interestingly, Gram-positive bacteria generally showed

greater tolerance to perchlorates than Gram-negative bacteria. Further study with a focus on

the differences between perchlorate tolerance of Gram-positive versus Gram-negative bacteria

29

could clarify whether this cell wall structural characteristic is involved in tolerance to

perchlorate. It would also be worth investigating whether or not there is a relationship between

production of extracellular polysaccharide and resistance to inhibition of growth by

perchlorates.

The large difference between the numbers of bacteria capable of surviving the same

high concentration of NaCl versus NaClO4 indicates that the mechanism for halotolerance and

perchlorate tolerance may be dissimilar, or that the mechanism for halotolerance is not

efficient enough to counter the more severe negative effects of perchlorates. Furthermore, the

larger inhibitory effect observed in bacterial growth in the presence of perchlorates versus

other salts suggests that chaotropicity and low water activity may play significant roles in

cellular disruption. Pure water has a water activity value (aw) of 1, and most bacteria are

inhibited by aw values below 0.9; but examples of haloarchaea have been demonstrated that

can grow at aw of 0.75 - 0.8 (Brewer 1999; Grant 2004). An example of a natural formation with

extremely low water activity is Don Juan Pond in Antarctica. Don Juan Pond is a body of water

saturated with CaCl2 and has a aw calculated at 0.45 (Grant 2004, Siegel 1979). Interestingly, no

signs of life have currently been determined to exist in Don Juan Pond (Grant 2004, Siegel

1979). In fact, current data indicates that 0.61 may be the lowest aw at which life, in the form of

fungi, can proliferate or survive (Berry et al. 2010; Brewer 1999; Grant 2004). It is important to

point out that NASA scientists have predicted that the water activity of Martian surface soil is

likely less than 0.1, significantly below the currently determined minimum for microbial growth

(Beaty et al. 2006; Berry et al. 2010). This prediction further supports the hypothesis that while

life may not be found in surface soils, that it may be preserved deep within the regolith where

30

water may be more prevalent, or within previously mentioned liquid salt brines. Increased

research is necessary into the limits of bacterial growth in low water activity environments.

Based on the results of bacterial growth in the presence of perchlorates, it can be assumed that

water activity of perchlorate solutions may fall within a range that is lower than other salt

solutions at equivalent molarities. These results also contribute to further studies involving life

at low water activity.

Specific organisms show survival at extremely high concentrations of Mg- and Ca-

perchlorate in liquid media. Testing a greater number of bacteria for survival in these conditions

may reveal more information about the types of organisms capable of surviving high

concentrations of perchlorate. Plating soils on perchlorate-containing media yielded varying

levels of cfu/g depending on the media (1/10 or 1/100 PCA), perchlorate salt (Mg or Na), and

perchlorate concentration. Generally, fewer cfu’s were recovered on 1/100 PCA versus 1/10

PCA. Fewer cfu’s/g were also obtained on Mg-perchlorate containing plates versus Na-

perchlorate containing plates, and cfu/g decreased as perchlorate concentration increased.

Repeating the experiment with more soils and a wider range of concentrations may reveal

trends that were not apparent in this study, and what effects the perchlorate concentration in

the soil has on cfu/g of the soil.

Future directions for this work could be to further characterize Atacama strains and

investigate the mechanism of perchlorate resistance. It would also be interesting to test

whether there are organisms in Atacama soil that are capable of metabolizing, or reducing, the

perchlorate as particular bacterial anaerobes are known to do. The Blastococcus isolates in this

31

study could be used as model organisms for studying the mechanisms of perchlorate resistance

in bacteria. Implications of such study could range from genetic engineering of microbes for use

in bioremediation, to updating planetary protection measures, or searching for life on Mars.

32

CHAPTER III ISOLATION AND CHARACTERIZATION OF IONIZING RADIATION RESISTANT BACTERIA FROM

PERMAFROST

Introduction

Since the early 1900’s, it has been known that viable microorganisms reside in

permafrost (Isachenko 1912; Omelyansky 1911). However, due to the lack of adequate

methods and technology for the better part of the century, microbiological analysis of

permafrost biodiversity was often impossible or considered invalid. It is only recently that

drilling core samples in permanently frozen regions has been modified to be aseptic and the

microbial diversity can truly be studied. Studies in Siberian permafrost have shown that

microbes can remain viable for millions of years, likely preserved in a dormant state at subzero

temperatures (Gilichinsky et al. 1992). Numerous studies have investigated cell counts which

can range from 102 – 108 cfu/g, depending on whether it is Antarctic or Siberian permafrost,

Siberian permafrost having much higher cell counts (Gilichinsky et al. 2007; Vorobyova et al

1997). Some bacterial residents that have been identified in both Antarctic and Siberian

permafrost samples include Arthrobacter, Bacillus, and Enterobacteriaceae (Blanco et al. 2012;

Dobrovolskaya et al. 1996; Gilichinsky et al. 2007, 2008; Mannisto and Haggblom 2006; Nelson

and Parkinson 1978; Parinkina 1989).

Microbial inhabitants of permafrost must overcome various extreme conditions, ranging

from below-zero temperature to constant bombardment by background radiation. Elements

such as U, Th, and K emit a constant low-level radiation that amounts to about 0.2 rad/yr (Smith

and McKay 2005), depending on the sediments present. Although it is not continuously present

at lethal levels, the total radiation given off over an extended period of time can accumulate to

33

high doses that would be lethal to many microorganisms. Bacteria that have been preserved in

frozen soil for millions of years could be exposed to hundreds of thousands of rads, and

therefore must have some level of radiation resistance to remain viable and repair their DNA. It

has also been discovered that bacteria within frozen soil at temperatures between -20 to -25 °C

have a much higher resistance to radiation than if they were exposed in a thawed state

(Gilichinsky et al. 2008), so it may be that the frozen soil itself acts to protect bacteria from the

effects of radiation. It has also been proposed that the low rate of metabolic activity at low

temperatures may result in less damage to cellular components by ionizing radiation

(Gilichinsky et al. 2008).

A great deal of interest currently lies in exploring permafrost in the northern arctic

regions of Mars. Martian permafrost is analogous in many ways to permafrost found on Earth

(Hoover 2003), and studying the life in permafrost here may provide insights into possible life

on Mars. One major difference between the permafrost on both planets is that on Mars it is

said to have been frozen for anywhere from 3 – 4 billion years (Smith and McKay 2005), far

longer than that which is found on Earth. Considering the radioactivity previously discussed, the

radiation exposure over this period of time would be extremely high. For this reason, it is

believed that while living organisms may not be found, the fossils of any past Martian life would

be well-preserved in such soil. On the other hand, it has previously been suggested and proven

that DNA damage repair is capable of occurring at below zero temperatures (Amato et al. 2010;

Carpenter et al. 2000; Gilichinsky et al. 2008; Gilichinsky et al. 1995; Price 2000; Price and

Sowers 2004; Rivkina et al. 2000, 2004), so it is not wholly implausible that organisms could

survive if they harbor the capacity to repair damaged DNA at a faster rate than damage is

34

occurring. Continued research in permafrost bacteria here on Earth is necessary, and could

answer many important questions concerning the origin of life and possible life on Mars. In this

study, ionizing radiation resistant bacteria belonging to the genus Hymenobacter are isolated

from permafrost and characterized in order to better understand microbial life in this extreme

environment.

Species of the Hymenobacter genus has been consistently isolated from or detected in

extreme environments, including glacial ice, arctic soil, and permafrost (Aislabie et al. 2006a, b;

Costello et al. 2009; Dai et al. 2009; Hirsch et al. 1998; Klassen and Foght 2008; Saul et al. 2005;

Yergeau et al. 2007; Zhang et al. 2006, 2007, 2008a, 2009). The species of this genus have been

shown to survive and grow at subzero temperatures, but one species has also been isolated

from irradiated food (Collins et al. 2000), thereby placing them in the polyextremophile

category. This is confirmed by other studies in which species of Hymenobacter were isolated

from various other extreme environments including sterilized clean rooms and aerosols

(Buczolits et al. 2002, 2006; Osman et al. 2008a; Polymenakou et al. 2008; Venkateswaran et al.

2003), as well as temperate desert soil (Fredrickson et al. 2008; Rainey et al. 2005). The

Hymenobacter isolates in this study are unique in that they are capable of tolerating subzero

temperatures and surviving high doses of gamma radiation.

Materials and Methods Irradiation, isolation, and cultivation

Two samples of permafrost were obtained from the Canadian High Arctic, denoted LH

Tundra (LH) and Exp Fiord Surface Soil (FSS). One gram aliquots of each soil were placed into

microcentrifuge tubes, put on ice, and exposed to gamma radiation using a JL Sheppard model

35

484 60Co irradiator. An unirradiated sample from each of the two soils was also used for

comparison. Four tubes of each sample were placed in the irradiator so that each one may be

removed after receiving the necessary dosage. Tubes of soil were removed after receiving

exposures of 1, 2, 3, and 11.1 kGy. The soils were subsequently serially diluted in 0.85% wt/vol

saline solution. 100 µL aliquots from each dilution were pipetted and spread onto the surface of

nutrient media including R2 agar (R2A), marine agar (MA), 1/10 concentration plate count agar

(1/10 PCA), and 1/100 concentration plate count agar (1/100 PCA). Plates were incubated at 4°

and 15° C for up to 30 days with cfu counts performed at 14 days and 30 days, after which time

colonies were selected from all plates for isolation and identification. Selected colonies were

purified and stored in the appropriate medium containing 15% (vol/vol) glycerol at -80° C.

Bacterial strains and type strains

Strains used for taxonomic comparison in this study included Hymenobacter aerophilus

LMG 19657T, Hymenobacter algoricola LMG 26018T, Hymenobacter antarcticus LMG 26017T,

Hymenobacter elongatus LMG 26016T, Hymenobacter fastidiosus LMG 26015T, Hymenobacter

glaciei LMG 26019T, Hymenobacter norwichensis LMG 21876T, Hymenobacter psychrophilus

LMG 25548T, and Hymenobacter roseosalivarius DSM 21212T. Strains were obtained from LMG,

Ghent, Belgium and Deutsche Sammlung von Mikroorganismen und Zellkulturen,

Braunschweig, Germany. All Hymenobacter strains, including reference strains were incubated

at 10 °C.

Phylogenetic analysis

DNA extraction was performed using the MoBio UltraClean Microbial DNA Isolation Kit;

procedure was performed according to instructions from the manufacturer. PCR amplification

36

of the 16S rRNA gene was performed according to Rainey et al. (1996). UltraClean PCR Clean-

Up Kit (MoBio) was used to purify PCR reactions; the procedure was performed according to

instructions from the manufacturer (MoBio). Sequencing of the purified PCR products were

performed using primers as described by Rainey et al. (1996). The identities of the 16S rRNA

gene sequences were analyzed using the BLAST (blastn) facility at the National Center for

Biotechnology Information website (www.ncbi.nlm.nih.gov/BLAST/) and EzTaxon

(www.eztaxon.org). In concordance with the results obtained, the sequences were analyzed

against representative reference sequences of members of the lineage to which they were

assigned using MEGA 4.0 editor (Tamura et al. 2007). Jukes and Cantor method (Jukes and

Cantor 1969) was used to calculate evolutionary distances. Phylogenetic dendrograms were

generated and bootstrap analyses were performed using various algorithms contained in MEGA

4.0.

Morphological, biochemical, and physiological characteristics

Colony morphologies were determined after growth on R2A incubated at 4° or 15° C.

Cell morphology was determined using phase-contrast microscopy and differential interference

contrast microscopy. Cells were suspended in 0.85% saline and 10 µL aliquots were placed onto

glass slides and covered with a coverslip, after which time photomicrographs were taken. The

temperature range for growth of the strains was determined by plating the strains on R2 agar

and incubating them at 4, 10, 15, 25, and 37° C. Halotolerance was examined using R2 agar

supplemented with NaCl to achieve concentrations of 1, 2, 5, 7, and 10% (w/v). Strains were

suspended in 0.85% saline and 10 µL aliquots were spotted onto the plates that were then

incubated at 15° C. A control plate of R2 agar without added NaCl was also incubated

http://www.ncbi.nlm.nih.gov/BLAST/

37

simultaneously. Growth at different pH values was tested by adjusting R2 agar with either

NaOH or HCl to achieve pH values of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; the plating procedure was

performed as described for halotolerance testing. Catalase activity and cytochrome oxidase

activity were determined according to standard methods (Hurst et al. 2007).

Irradiation of isolates and desiccation resistance

Pure cultures of the strains were exposed to radiation in order to determine radiation

resistance of the isolates after they were cultured from the environmental sample. Cells were

taken from the growth media and were transferred to microcentrifuge tubes containing 1 mL of

0.85% saline. Tubes were then irradiated as described for as the soils, and were exposed to

doses of 1, 3, 9, and 12 kGy. The strains were re-plated immediately onto R2 agar

simultaneously with a control of each strain that was not exposed to radiation, and all plates

were incubated at 15°C for up to 28 days. Growth of irradiated strains was compared to growth

on the control plates to determine relative growth strength that was signified with one to three

(+) signs, or scored with (-) for no growth.

Desiccation resistance was examined by pipetting 10 µL aliquots of a cell suspension

into microwell tissue culture plates, allowing spots to dry, and placing the plates in a

desiccation chamber with relative humidity controlled at 5%. CaSO4 (Dri-Rite) was placed in the

chamber and the whole chamber was incubated at 4° C. Cell material was rehydrated and

plated onto R2 agar at 7, 14, 28, 42 days and at 3 and 6 months. Plates were incubated at 15° C,

and survival of cells was determined by growth observed after 14 days.

38

R2A MA 1/10 PCA 1/100 PCA

0 kGy 1.5 x 105 8.1 x 105 8.6 x 103 2.0 x 103

1 kGy 1.27 x 105 3.3 x 105 7.3 x 104 2.0 x 103

2 kGy 1.6 x 103 1.0 x 101 7.5 x 102 2.0 x 101

3 kGy 2.1 x 103 3.0 x 101 1.2 x 103 1.0 x 101

11.1 kGy 1.8 x 102 0 0 1.0 x 101

LH Tundra (4 °C)

Results and Discussion CFU/g in permafrost (irradiated and unirradiated)

For the unirradiated control soils, colonies were observed on all media and at both

temperatures for dilutions ranging from 10-1 through 10-4 (see Tables 3.1 – 3.4). In general,

more colonies were present on all media incubated at 15° C versus media incubated at 4° C. A

greater number of CFU/g was generally observed on R2A and 1/10 PCA than other media, with

generally fewer colonies growing on MA for both irradiated and unirradiated samples (~102 –

104 less colonies on MA). Irradiated soil produced significantly less CFU/g than unirradiated soil

and observable colonies decreased in number as dosage increased. For the LH sample at 4°C on

R2A, CFU/g dropped from 1.3 x 105 at 1 kGy to 1.8 x 103 for 11.1 kGy. For the FSS sample with

the same media and temperature, CFU/g decreased from 4.4 x 103 to 3 x 102 when the dose

increased from 1 to 11.1 kGy. The CFU/g from the FSS sample was generally much less than

what was observed from the LH sample under all conditions and radiation exposures.

Table 3.1. Cfu/g calculated for LH Tundra soil plated on R2A, MA, 1/10 PCA, and 1/100 PCA at 4°C. Radiation dosage and dilution are shown in the left column

39


0 kGy 1.5 x 105 1.0 x 103 8.9 x 104 9.2 x 104

1 kGy 4.4 x 102 8.0 x 101 3.7 x 102 7.1 x 102

2 kGy 3.0 x 102 1.0 x 101 6.0 x 101 9.0 x 101

3 kGy 3.3 x 102 2.0 x 101 2.1 x 102 0

11.1 kGy 3.0 x 102 0 0 1.0 x 101

Fiord Surface Soil (4 °C)


0 kGy 2.3 x 105 3.4 x 105 2.5 x 105 2.0 x 105

1 kGy 1.3 x 105 4.6 x 104 1.0 x 105 1.5 x 104

2 kGy 2.9 x 104 7 x 102 2.2 x 104 1.81 x 104

3 kGy 1.6 x 104 0 2.9 x 104 0

11.1 kGy 2.9 x 103 0 2.9 x 103 0

LH Tundra (15 °C)

Table 3.2. Cfu/g calculated for FSS soil plated on R2A, MA, 1/10 PCA, and 1/100 PCA at 4°C. Radiation dosage and dilution are shown in the left column

Table 3.3. Cfu/g calculated for LH Tundra soil plated on R2A, MA, 1/10 PCA, and 1/100 PCA at 15° C. Radiation dosage and dilution are shown in the left column

40


0 kGy 1.9 x 105 1.9 x 105 2.2 x 105 TMTC

1 kGy 5.6 x 103 4.0 x 102 2.8 x 103 1.4 x 104

2 kGy 1.0 x 103 2.0 x 103 5.0 x 103 1.0 x 104

3 kGy 5.0 x 102 0 1.2 x 103 1.0 x 102

11.1 kGy 1.0 x 102 0 1.0 x 102 1.0 x 102

Fiord Surface Soil (15 °C)

Isolation and identification of Hymenobacter strains

Forty-two colonies from radiation exposed soils were selected for isolation and

identification. Isolates were identified as belonging to a variety of genera including

Herbaspirillum, Pontibacter, Arthrobacter, Salinibacterium, Paenibacillus, Planococcus, and

Hymenobacter (see Table 3.5). Sixteen of the strains falling into the radiation of the genus

Hymenobacter were chosen for further characterization (See Table 3.6). The strains were given

designations depending on the soil that it came from, LH or FSS. Phylogenetic relationships to

existing members of the Hymenobacter genus can be seen in Figure 3.1. Analysis of

phylogenetic data and sequence similarity to existing species suggests that these strains

represent novel species within this genus.

Table 3.4. Table 7C. Cfu/g calculated for FSS soil plated on R2A, MA, 1/10 PCA, and 1/100 PCA at 15° C. Radiation dosage and dilution are shown in the left column. TMTC – too many to count

41

Exposure (kGy) Identity of Organisms Isolated

Arthrobacter (10)

Planomicrobium (1)

Streptomyces (6)

Paenibacillus (1)

Salinibacterium (1)

Planococcus (1)

Hymenobacter (1)

Arthrobacter (1)

Hymenobacter (1)

Arthrobacter (1)

Pontibacter (2)

Herbaspirillum (1)

Hymenobacter (6)

Paenibacillus (1)

Hymenobacter (7)

Plantibacter (1)

0

1

2

3

11.1

Morphological and phenotypic characterization of Hymenobacter isolates

Cells are observed as short rods, approximately 2 – 4 um in length (refer to

photomicrographs in Appendix B). Strains irLH24 and irLH25 show signs of bleb formation,

though this is not present in any other Hymenobacter strain. Colonies have smooth margins and

are circular, shiny, and convex with bright red or pink pigmentation. Average colony size at

optimum temperature is approximately 5.0 mm. Growth occurs between 4° and 25° C but does

not occur at 37° C. A slight change in pigment was noted with increased incubation

temperature, with colors shifting from shades of pink and red to salmon or beige at 25° C.

Growth was not observed in liquid media. Optimum growth temperature is between the 10° to

15° C range. Colonies were generally gelatinous in consistency or had hardened exteriors,

clumping together and they do not disperse in liquid.

Table 3.5. Organisms recovered from soils exposed to gamma radiation. The number in parentheses refers to the number of strains belonging to that genus that were isolated from each exposure. Strains were isolated from various media incubated at either 4° or 15° C

42

Stra

inD

ose

(kG

y)M

edia

Des

crip

tion

Isol

atio

n Te

mp

(°C)

Iden

tity

Sequ

ence

Mat

ch (%

)

BV-i

rLH

243

MA

pale

pin

k-be

ige,

cir

cula

r, s

hiny

, con

vex

15Po

ntib

acte

r92

.375

BV-i

rLH

253

MA

brig

ht re

d-or

ange

, cir

cuar

, shi

ny, c

onve

x15

Pont

ibac

ter

93.1

03

BV-i

rLH

3411

.1R2

Abr

ight

ora

nge-

red,

cir

cula

r, s

hiny

, con

vex

4H

ymen

obac

ter

95.6

81

BV-i

rLH

3511

.1R2

Alig

ht p

ink,

cir

cula

r, s

hiny

, con

vex

4H

ymen

obac

ter

95.0

21

BV-i

rLH

3611

.1R2

Alig

ht p

ink,

cir

cula

r, s

hiny

, con

vex

4H

ymen

obac

ter

95.2

08

BV-i

rFSS

2111

.11/

10 P

CAda

rk re

d-pi

nk, c

ircu

lar,

shi

ny, c

onve

x15

Hym

enob

acte

r95

.816

BV-i

rFSS

253

1/10

PCA

pink

, tra

nslu

cent

, cir

cula

r, fl

at15

Hym

enob

acte

r89

.353

BV-i

rFSS

272

1/10

PCA

dark

pin

k-re

d, c

ircu

lar,

shi

ny, c

onve

x15

Hym

enob

acte

r93

.125

BV-i

rFSS

301

R2A

beig

e-or

ange

, cir

cula

r, s

hiny

, con

vex;

jelly

-lik

e co

nsis

tenc

y15

Hym

enob

acte

r94

.363

BV-i

rFSS

3111

.11/

100

PCA

pink

-vio

let,

flat

, cir

cula

r, s

hiny

4H

ymen

obac

ter

94.5

72

BV-i

rFSS

3211

.1R2

Alig

ht p

ink,

cir

cula

r, s

hiny

, con

vex

4H

ymen

obac

ter

91.8

44

BV-i

rFSS

3311

.11/

10 P

CAda

rk re

d, c

ircu

lar,

shi

ny, c

onve

x4

Hym

enob

acte

r90

.909

BV-i

rFSS

353

R2A

pink

-ora

nge,

cir

cula

r, s

hiny

, con

vex

4H

ymen

obac

ter

89.6

19

BV-i

rFSS

363

R2A

light

pin

k, c

ircu

lar,

shi

ny, s

light

ly e

leva

ted

4H

ymen

obac

ter

88.3

33

BV-i

rFSS

373

1/10

PCA

light

pin

k-re

d, fl

at, c

ircu

lar,

shi

ny, c

onve

x4

Hym

enob

acte

r90

.976

BV-i

rFSS

383

1/10

PCA

dark

pur

ple-

red,

cir

cula

r, s

hiny

, con

vex

4H

ymen

obac

ter

89.0

87

Tab

le 3

.6. S

trai

n d

ata

for

15

iso

late

s fa

llin

g w

ith

in t

he

rad

iati

on

of

the

gen

us

Hym

eno

ba

cter

, an

d t

wo

iso

late

s re

late

d t

o

Po

nti

ba

cter

, als

o is

ola

ted

fro

m h

igh

rad

iati

on

do

ses

43

Figure 3.1. 16S rRNA gene sequence based phylogeny showing the relationship of permafrost isolates to previously isolated and described Hymenobacter species. Scale bar represents 1 inferred nucleotide substitution per 100 nucleotides. Strains in green represent isolates from permafrost from Canadian Arctic (this study) and Tibet; dark blue are from glacial ice and snow in Pico de Orizaba; light blue are from basal ice in Victoria Upper Glacier; red are from arid soils from the Atacama, Oman, and Sonoran; orange are from air samples.

44

Halosensitivity was exhibited by all strains as growth was only noted at concentrations <

0.5% NaCl. Two strains, that were close relatives of Pontibacter spp., were capable of surviving

≤ 1% NaCl. These results are consistent with those observed for other Hymenobacter species

previously described (Klassen and Foght 2011). This lack of halotolerance may also be extended

to the general microbial community of the permafrost samples as cfu/g prior to irradiation was

lower on marine agar than any other media - Difco Marine Agar contains about 2% NaCl by

volume. The pH range permitting growth was in the range of 6 – 11 with > 50% of strains

growing at pH 11. All strains grew optimally at pH values between 7 and 8. There were many

strains exhibiting identical growth levels for a range of pH values, i.e. 7 – 11, 6 – 11, or 6 – 8.

The optimum pH range for growth differed slightly for specific strains.

Irradiation of Hymenobacter pure cultures and desiccation tolerance

After exposure to radiation while in pure culture, all strains demonstrated growth up to

3 kGy with one strain surviving 12 kGy exposure (see Table 3.7). This is in support of the idea

that the soil matrix provides some level of shielding to microbial cells since initially, at least 50%

of Hymenobacter were isolated from samples exposed to 11.1 kGy; this observation is

supported by previously published data (Osman et al. 2008b). It is important to point out that

all irradiation in this study was done at 4° C on ice, and samples were not in a frozen state. It

has been observed by Gilichinksy et al. (2008) that bacteria frozen in soil can withstand far

greater doses of radiation than if they were exposed in a thawed state. Therefore, these

bacteria that already survive 3 kGy while suspended in liquid may actually be capable of

surviving greater doses of radiation when in their natural habitat, frozen in permafrost. It is

45

Strain Designation 1 3 9 12

BV-irFSS27 (+++) (+++) (-) (-)

BV-irFSS21 (+++) (+++) (++) (++)

BV-irFSS25 (+++) (+++) (-) (-)

BV-irFSS35 (+++) (+++) (-) (-)

BV-irFSS32 (+++) (++) (-) (-)

BV-irFSS30 (+++) (+++) (-) (-)

BV-irFSS31 (+++) (++) (-) (-)

BV-irFSS33 (+++) (+++) (-) (-)

BV-irFSS37 (+++) (+++) (-) (-)

BV-irFSS38 (+++) (+++) (-) (-)

BV-irFSS36 (+++) (+++) (-) (-)

BV-irLH34 (+++) (+++) (-) (-)

BV-irLH36 (+++) (+++) (-) (-)

Radiation Dosage (kGy)

possible that these strains represent bacteria capable of surviving for millions of years in the

presence of background radiation.

A high level of desiccation resistance was also demonstrated by many of the strains. The

majority survived 7 days of desiccation, 50% survived up to 14 days, and 25% of strains

continued to show signs of viability after 3 months in the desiccator. Individual results for

growth are sporadic and inconsistent; for example, no viable cells were observed for some

strains at 14 days but strong growth was observed again at 42 days. This is likely a procedural

problem involving the inability to collect all viable cells from the microwell plate. Regardless of

previous results for each strain, positive growth data at any time during desiccation was

considered survival of that strain up to that point. Osmotic desiccation of cells is said to occur at

subzero temperatures as the lower vapor pressure of surrounding ice forces water out of cells

Table 3.7. Growth of Hymenobacter isolates after exposure to doses of 1, 3, 9, and 12 kGy gamma radiation while in pure culture

46

(Gilichinsky et al. 2008), so it is likely that the observed desiccation resistance is attributable to

environmental adaptation. The exhibited radiation resistance may also be a product of

desiccation resistance, a relationship proposed by Mattimore and Battista (1996), and

demonstrated at the ecological level by Rainey et al. (2005).

Comparison of strains to existing Hymenobacter species

Hymenobacter strains isolated from permafrost in this study were compared with

existing members of the genus. Phenotypic characteristics of previously described species were

obtained from the respective publications. Overall phenotypic characteristics of the proposed

novel isolates fall within range of characteristics previously described for Hymenobacter

species. It is interesting to note that four strains with comparatively limited pH ranges for

growth (BV-irFSS33, BV-irFSS36, BV-irFSS37, and BV-irFSS38) are closely related to H. perfusus,

H. flocculans, and H. metalli (Figure 3.1), which also display very similar limited range of growth,

suggesting this trend has a phylogenetic basis. These organisms grow at pH values of 6 – 8 or 6

– 9 as opposed to 5 – 11 or 6 – 11 as exhibited by many of the other strains. This indicates that

these organisms were isolated from sites where the pH may remain relatively constant.

Organisms with a wider range of growth at different pH values may have been inhabiting sites

with high fluctuation in pH. Table 3.8 shows phenotypic characteristics of permafrost isolates

from this study and those of previously described Hymenobacter species. It is expected that

completion of metabolic profiling, fatty acid analysis, and determination of major lipids and

menaquinones will provide data supporting the description of these strains as novel species in

the genus Hymenobacter.

47

Hymenobacter strain

Gram Type

Catalase Test

Oxidase Test

Growth Temperature

(°C)

Relative Growth

Rate

Max % NaCl

Growth pH

range Tolerated

H. roseosalivariusa - + + 10, 18(w) slow < 0.5 6 - 12

H. actinosclerusb - + + 4 - 28 fast 2 5 - 12

H. aerophilusc - + + 4 - 28 fast 2 5 -12

H. riguid - - + 4 - 28 fast 1 5 - 12

H. norwichensise - + + 4 - 28 fast 1 5 - 12

H. chitinovoranse - + + 4 - 28 fast 1(w) 5 - 12

H. gelipurpurascense - + + 4 - 28 fast 1 5 - 12

H. ocellatuse - + + 10 - 28 fast 1 5 - 12

H. xinjiangensisf - + + 4 - 28 fast 1 5 - 11

H. psychrotoleransg - + + 4 - 28 fast 1(w) 5 - 11

H. solih - + + 4 - 28 fast 0.5 5 - 11

H. desertii - + + 4 - 40 nd < 2 5 - 11

H. daecheongensisj - + + 4 - 30 fast < 1 5 - 10

H. psychrophilusk - + + 1 - 20 nd 1 nd

H. yonginensisl - + + 10 -30 nd 3 7 - 8

H. perfususm - + - 4 - 30 nd nd 6 - 9

H. flocculansm - + - 4 - 30 nd nd 6 - 9

H. metallim - + - 4 - 30 nd nd 6 - 9

H. algoricolan - - + 4 - 18 slow < 0.5 6 - 11

H. antarcticusn - - + 4 - 18 slow < 0.5 6 - 11

H. fastidiosusn - - + 4 - 18 slow < 0.5 7 - 10

H. elongatusn - - + 4 - 18 slow < 0.5 6 - 11

H. glacein - + + 4 - 28 slow < 0.5 5 - 11

BV-irLH34 - + - 4 - 28 slow < 0.5 7 - 11

BV-irLH35 - + + 4 - 28 slow 1(w) 7 - 11

BV-irLH36 - + - 4 - 28 slow < 1 6 - 11

BV-irFSS21 - + + 4 - 28 slow < 1 6 - 11

Table 3.8. Phenotypic data of Hymenobacter and related BV-irLH and BV-irFSS strains.

Nd – not determined; w – weak growth

48

(Table 3.8 cont’d)

Hymenobacter strain

Gram Type

Catalase Test

Oxidase Test

Growth Temperature

(°C)

Relative Growth

Rate

Max % NaCl

Growth pH

range Tolerated

BV-irFSS25 - + - 4 - 28 slow < 1 5 - 11

BV-irFSS27 - + - 4 - 28 slow < 1 6 - 11

BV-irFSS30 - + (w) - 4 - 28 slow 1(w) 6 - 8

BV-irFSS31 - + - 4 - 28 slow < 1 6 - 11

BV-irFSS32 - + - 4 - 28 slow < 1 6 - 9

BV-irFSS33 - + - 4 - 28 slow < 1 6 - 9

BV-irFSS35 - + - 4 - 28 slow 1(w) 6 - 9

BV-irFSS36 - + - 4 - 28 slow < 1 6 - 8

BV-irFSS37 - + - 4 - 28 slow 1(w) 6 - 9

BV-irFSS38 - + - 4 - 28 slow < 1 6 - 8 Phenotypic data of previously described Hymenobacter species was obtained from the following: a

Hirsch et al. 1999 b

Collins et al. 2000 c Buczolits et al. 2002

d Baik et al. 2006

e Buczolits et al. 2006

f Zhang et al. 2007

g Zhang et al. 2008

h Kim et al. 2008

I Zhang et al. 2009

j Xu et al. 2009

k Zhang et al. 2011

l Joung et al. 2011

m Chung et al. 2011

n Klassen and Foght 2011

Conclusion Selective isolation of bacteria from permafrost exposed to gamma radiation yielded

highly radiation resistant, psychrotolerant members of the genus Hymenobacter. Phylogenetic

analysis using the 16S rRNA gene sequence, as well as phenotypic comparison with existing

members of this genus, suggests that these organisms represent novel species level taxa within

this genus. The most noted characteristic of these strains is their desiccation and radiation

resistance, two important characteristics to consider when contemplating life in Martian

49

permafrost. Data obtained from Martian meteorites indicate that the background radioactivity

of Martian soil may be similar to what has been measured in Siberian permafrost (Smith and

McKay 2005, Stoker et al. 2003); and since life has been identified and cultured within

permafrost on this planet, this can be extended to the search for life on Mars. Although the

permafrost on Mars, being billions of years old, would have emitted enough radiation for

complete sterilization of all life forms presently known to science, past life would have endured

it for perhaps millions of years as it did here on Earth. There are also the organisms currently

being studied with extremely efficient DNA repair mechanisms that can counteract DNA

damage by radiation at low temperatures. A recent study demonstrates macromolecular

synthesis in Psychrobacter cryhohalolentis and Psychrobacter arcticus occurred at sufficient

levels at -15° C to counteract DNA damage from ionizing radiation (Amato et al. 2010). Thus,

these Hymenobacter represent analogs of hypothetical Martian life, and could provide insight

into the DNA-repair mechanisms necessary for survival under Martian conditions

Further research in this area could study the specific mechanism of radiation resistance

or DNA damage repair that is occurring in these strains. Further analysis of the microbial

biodiversity of permafrost could also be carried out to gain better understanding as to what

role these Hymenobacter play in the overall microbial community, since they are clearly

prominent enough to be found in permafrost from multiple locations, as well as other distinct

extreme environments. If in fact these microbes have been preserved for millions of years in

permafrost, it would be yet another supporting factor in the theory for life in Martian

permafrost.

50

CHAPTER IV CONCLUSION

The focus of this study was to examine the characteristics of bacteria from extreme

environments and observe their growth response under certain Mars-like conditions. Bacterial

growth in the presence of perchlorate salts was pushed to extreme levels, with some strains of

the genus Blastococcus isolated from the Atacama Desert tolerating perchlorate concentrations

in excess of 1 M Na-perchlorate and 400 mM Ca- and Mg-perchlorate. The overall greater

tolerance of Atacama isolates demands the increased study of microbes from this location to

achieve better understanding of interactions between perchlorate salts and bacteria. This

perchlorate experiment adds to existing literature concerning the effects of other salts like

MgSO4 on bacterial growth, and as such, interesting relationships have been identified between

tolerances of various salts. The concept of water activity and chaotropicity have both been

mentioned in relation to the inhibition of bacterial growth in concentrated salt solutions, but

the literature concerning these effects is vague and limited (Crisler et al. 2012; Hallsworth et al.

2007; Grant 2004). Studies involving water activity are primarily focused on food preservation,

as water activity is a determinant of estimating shelf life of foods (Katz 1981; Labuza 1980; Roos

YH 1993; Slade 1991). Therefore, water activity in relation to high salt environments and

bacterial habitability is in dire need of increased study.

Permafrost, an environment characterized by extremes in temperature and exposure to

large doses of radiation over time, is habitat to a broad range of microorganisms. Using high

doses of gamma radiation, a segment of that biodiversity was isolated and identified using

culture-based techniques. Bacteria belonging to the genus Hymenobacter were prominent

among isolates from all radiation exposures, many being identified in samples exposed to 11.1

51

kGy. After re-exposure to gamma radiation in pure culture, the strains survived a 3 kGy dose,

and one survived up to 12 kGy. This radiation resistance may be useful for surviving in

permafrost for thousands of years while exposed to constant background radiation. These

Hymenobacter display characteristics that would be expected of life in Martian permafrost, and

may be further studied to gain insight into radiation damage repair at low temperatures.

Rapid advances in technology are constantly improving the efficiency and rate at which

data is gathered from Mars. Although the landing of space crafts on Mars has been happening

since the 1970’s, the most successful missions have only occurred in the past 10 years. The

latest rover, Curiosity, is scheduled to land in August 2012 and is equipped with an on-board

laboratory with the primary objective of searching for evidence of organic material in rocks and

soil that could potentially be of biological origin (http://msl-scicorner.jpl.nasa.gov/ScienceGoals/).

Finding such evidence will be a world-changing discovery with implications ranging across all

fields of study, from philosophy to biology. The place to begin the study of life, however, is here

on Earth where it is known to exist. Research, like what has been conveyed here, is invaluable

in understanding the origin and dynamics of life in different environments. This work can then

be applied in the search for life on other planets. With continued research in the field of

astrobiology along with the aforementioned advances in aerospace technology, a scientific

breakthrough is imminent. If there is life on Mars, we will find it.

http://msl-scicorner.jpl.nasa.gov/ScienceGoals/

52

REFERENCES

Abramov O and Mojzsis SJ (2009) Microbial habitability of the terrestrial biosphere during the late heavy bombardment. Nature, 459: 419–422

Achenbach L A, Bruce RA, Michaelidou U and Coates JD (2001) Dechloromonas agitata gen. nov., sp.

nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position. International Journal of Systematic and Evolutionary Microbiology, 51: 527– 533

Ader M, Chaudhuri S, Coates JD and 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(30): 605-613

Aislabie JM, Broady PA and Saul DJ (2006a) Culturable aerobic heterotrophic bacteria from high altitude,

high latitude soil of La Gorce Mountains (86_300S, 147_W), Antarctica. Antarctic Science, 18: 313–321

Aislabie JM, Chhour K-L, Saul DJ, Miyauchi S, Ayton J, Paetzold RF and Balks MR (2006b) Dominant

bacteria in soils of Marble Point and Wright Valley, Victoria Land, Antarctica. Soil Biology and Biochemistry, 38: 3041–3056

Amato P, Doyle SM, Battista JR and Christner BC (2010) Implications of subzero metabolic activity on

long-term microbial survival in terrestrial and extraterrestrial permafrost. Astrobiology, 10(8): 789-798

Baik KS, Seong CN, Moon EY, Park Y, Yi H and Chun J (2006) Hymenobacter rigui sp. nov., isolated form

wetland freshwater. International Journal of Systematic and Evolutionary Microbiology, 56: 2189-2192

Bagaley DR (2006) Uncovering Bacterial Diversity On and Below the Surface of a Hyper-Arid

Environment, The Atacama Desert, Chile. MS Thesis. Louisiana State University, Baton Rouge, 2006. Print.

Beaty DW, Buxbaum KL, Meyer MA, Barlow NG, Boynton WV, Clark BC, Deming JW, Doran PT, Edgett KS,

Hancock SL, Head JW, Hecht MH, Hipkin V, Kieft TL, Mancinelli RL, McDonald V, McKay CP, Mellon MT, Newsom H, Ori GG, Paige DA, Schuerger AC, Sogin ML, Spry JA, Steele A, Tanaka KL and Voytek MA (2006) Findings of the Special Regions Science Analysis Group. Astrobiology, 6: 677-732

Berry BJ, Jenkins DG and Schuerger AC (2010) Effects of Simulated Mars Conditions on the Survival and

Growth of Escherichia coli and Serratia liquefaciens. Applied and Environmental Microbiology, 76(8): 2377-2386

Blanco Y, Prieto-Ballesteros O, Gomez MJ, Moreno-paz M, Garcia-Villadangos M, Rodriguez-Manfredi JA,

Cruz-Gil P, Sanchez-Roman M, Rivas LA and Parro V (2012) Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica). Environmental Microbiology, [Epub ahead of print] DOI: 10.1111/j.1462-2920.2012.02767.x

53

Brewer MS (1999) Traditional preservatives – sodium chloride. Encyclopedia of Food Microbiology, vol.

3. ed Robinson RK, Blatt CA, Patel PD, pp 1723-1728. London: Academic. Buczolits S, Denner EBM, Vybiral D, Wieser M, Kampfer P and Busse HJ (2002) Classification of three

airborne bacteria and proposal of Hymenobacter aerophilus sp. nov. International Journal of Systematic and Evolutionary Microbiology, 52:445–456

Buczolits S, Denner EBM, Kampfer P and Busse H-J (2006) Proposal of Hymenobacter norwichensis sp.

nov., classification of ‘Taxeobacter ocellatus’, ‘Taxeobacter gelipurpurascens’ and ‘Taxeobacter chitinivorans’ as Hymenobacter ocellatus sp. nov., Hymenobacter gelipurpurascens sp. nov. and Hymenobacter chitinivorans sp. nov., respectively, and emended description of the genus Hymenobacter Hirsch et al. 1999. International Journal of Systematic and Evolutionary Microbiology , 56:2071–2078

Carpenter EJ, Lin SJ and Capone DG (2000) Bacterial activity in South Pole snow. Applied and

Environmental Microbiology, 66:4514–4517 Carr MH (2012) The Fluvial History of Mars. Philosophical Transactions. Series A, Mathematical, Physical,

and Engineering Sciences, 370(1966):2193-215 Catling DC, Clair MW, Zahnle KJ, Quinn RC, Clark BC, Hecht MH and Kounaves S (2009) Atmospheric

origins of perchlorate on Mars and in the Atacama. Journal of Geophysical Research, 115: 1-15 Chevrier VF, Hanley J and Altheide TS (2009) Stability of perchlorate hydrates and their liquid

solutions at the Phoenix landing site, Mars. Geophysical Research Letters. 36:L10202 Chung AP, Lopes A, Nobre MF and Morais PV (2010) Hymenobacter perfusus sp. nov., Hymenobacter

flocculans sp. nov., and Hymenobacter metallic sp. nov. three new species isolated from an uranium mine waste water treatment system. Systematic and Applied Microbiology, 33(8):436-443

Clark BC and Van Hart DC (1981) The salts of Mars. Icarus, 45:370-378 Clark BC (1998) Surviving the limits to life at the surface of Mars. Journal of Geophysical Research,

103:28545-28556 Coates JD and Achenbach LA (2004) Microbial perchlorate reduction: rocket-fueled metabolism. Nature

Reviews Microbiology, 2(7): 569-80 Collins, MD, Hutson RA, Gran IR and Patterson MF (2000) Phylogenetic characterization of a novel

radiation-resistant bacterium from irradiated pork: description of Hymenobacter actinosclerus sp. nov. International Journal of Systematic and Evolutionary Microbiology, 50: 731-734

Connon SA, Lester E, Shafaat HS, Obenhuber DC and Ponce A (2007) Bacterial diversity in the hyperarid

Atacama Desert soils. Journal of Geophysical Research, 112: G04S17

54

Costello EK, Halloy SRP, Reed SC, Sowell P and Schmidt SK (2009) Fumarole-supported islands of biodiversity within a hyperarid, high-elevation landscape on Socompa Volcano, Puna de Atacama, Andes. Applied and Environmental Microbiology, 75: 735-747

Crisler JD, Newville TM, Chen F, clark BC and Schneegurt MA (2012) Bacterial growth at the high

concentrations of magnesium sulfate found in Martian soils. Astrobiology, 12(2): 98-106 Cull SC, Arvidson RE, Catalano JG, Ming DW, Morris RV, Mellon MT and Lemmon M (2010) Concentrated

perchlorate at the Mars Phoenix landing site: Evidence for thin film liquid water on Mars. Geophysical Research Letters, 37:L22203

Dai J, Wang Y, Zhang L, Tang Y, Luo X, An H and Fang C (2009) Hymenobacter tibetensis sp. nov., a UV-

resistant bacterium isolated from Qinghai-Tibet plateau. Systematic and Applied Microbiology, 32: 543–548

Dasgupta PK, Martinelango PK, Jackson WA, Anderson TA, Tian K, Tock RW and Rajagopalan S (2005) The

origin of naturally occurring perchlorate: the role of atmospheric processes. Environmental Science and Technology, 39(6): 1569-1575

Davis WL and McKay CP (1996) Origins of life: A comparison of theories and application to Mars. Origins

of Life and Evolution of Biospheres, 26: 61–73 Dobrovolskaya TG, Lysak LV and Zvyagintsev DG (1996) Soils and microbial diversity. Eurasian Soil

Science, 29: 630-634 Ehlmann BL, Mustard JF, Murchie SL, Bibring JP, Meunier A, Fraeman AA and Langevin Y (2011)

Subsurface water and clay mineral formation during the early history of Mars. Nature, 479(7371): 53-60

Ericksen GE (1981) Geology and origin of Chilean Nitrate Deposits. United States Geological Survey

Professional Paper, 1188 Fredrickson JK, Li S-mW, Gaidamakova EK, Matrosova VY, Zhai M, Sulloway HM, Scholten JC, Brown MG,

Balkwill DL and Daly MJ (2008) Protein oxidation: key to bacterial desiccation resistance? International Society for Microbial Ecology Journal, 2: 393-403

Gilichinsky DA, Vorobyova EA, Erokhina LG, Fyordorov-Dayvdov DG and Chaikovskaya NR (1992) Long-

term preservation of microbial ecosystems in permafrost. Advances in Space Research, 12(4): 255–263

Gilichinsky DA and Wagener S (1995) Microbial life in permafrost (A historical review). Permafrost

Periglacial Processes, 5: 243–250 Gilichinsky DA, Wilson GS, Friedmann EI, McKay CP, Sletten RS, Rivkina EM, Vishnivetskaya TA, Erokhina

LG, Ivanushkina NE, Kochkina GA, Shcherbakova VA, Soina VS, Spirina EV, Vorobyova EA, Fyodorov-Davydov, DG, Hallet B, Ozerskaya SM, Sorokovikov VA, Laurinavichyus KS, Shatilovich

55

AV, Chanton JP, Ostroumov VE and Tiedje JM (2007) Microbial populations in Antarctic permafrost: biodiversity, state, age, and implication for astrobiology. Astrobiology, 7(2): 275-311

Gilichinsky D, Vishnivetskaya T, Petrova M, Spirina E, Mamykin V and Rivkina E. (2008) “Bacteria in

Permafrost”. Psychrophiles: From Biodiversity to Biotechnology. Margesin R, Schinner F, Marx JC, Gerday, C. Heidelberg: Springer-Verlag Berlin Heidelberg, 2008. pp. 83-99

Grant WD (2004) Life at low water activity. Philosphical Transactions of the Royal Society London, 359:

1249-1267 Gunde-Cimerman N, Sonjak S, Zalar P, Frisvad JC, Diderichsen B, Plemenitas A (2003) Extremophilic fungi

in arctic ice: a relationship between adaptation to low temperature and water activity. Physics and Chemistry of the Earth, Parts A/B/C, 28(28-32): 1273-1278

Hallsworth JE, Heim S, Timmis KN (2003) Chaotropic solutes cause water stress in Pseudomonas putida.

Environmental Microbiology, 5(12): 1270-1280 Hallsworth JE, Yakimov MM, Golyshin PN, Gillion JL, D’Auria G, de Lima Alves F, La Cono V, Genovese M,

McKew BA, Hayes SL, Harris G, Giuliano L, Timmis KN and McGenity TJ (2007) Limits of life in MgCl2-containing environments: chaotropicity defines the window. Environmental Microbiology, 9: 801-813

Hecht MH, Kounaves SP, Quinn RC, West SJ, Young SMM, Ming DW, Catling DC, Clark BC, WV Boynton,

Hoffman, J, DeFlores LP, Gospodinova K, Kapit J and Smith PH (2009) Detection of Perchlorate and the Soluble Chemistry of Martian Soil at the Phoenix Lander Site. Science, 325(5936): 64-67

Hickman J (1998) New from the End of the Earth: a portrait of Chile. St. Martin’s Press, Scholarly and

Reference Division, New York, NY. pp. 2-3 Hirsch P, Ludwig W, Hethke C, Sittig M, Hoffmann B and Gallikowski CA (1998) Hymenobacter

roseosalivarius gen. nov., sp. nov. from continental Antarctic soils and sandstone: bacteria of the Cytophaga/Flavobacterium/Bacteroides line of phylogenetic descent. Systematic and Applied Microbiology 21: 374–383

Hoover RB and Rozanov AY (2003) Microfossils, Biominerals and Chemical Biomarkers in Meteorites.

Perspectives in Astrobiology. Proceedings of the NATO Advanced Study Institute on Perspectives in Astrobiology, 4939: 10-255

Hurst CJ, Crawford RL, Garland JL, Lipson DA, Mills AL, Stetzenbach LD (2007) Manual of environmental

microbiology, 3rd edn. ASM Press, Herndon, VI Isachenko B (1912) Some data on permafrost bacteria. Izvestiya Sankt-Peterburgskogo Botanicheskogo

Sada, vol 12, N 5–6:140 (in Russian) Jukes TH and Cantor CR (1969) Evolution of protein molecules. Mammalian Protein Metabolism, p. 21-

132. In H. N. Munro (ed.), Mammalian protein metabolism. Academic Press, New York

56

Junge K, Eicken H, Swanson BD and Deming JW (2006) Bacterial incorporation of leucine into protein down to -20° C with evidence for potential activity in sub-eutectic saline ice formations. Cryobiology, 52(3): 417-429

Joung Y, Cho S-H, Kim H, Kim SB and Joh K (2011) Hymenobacter yonginensis sp. nov., isolated from a

mestrophic artificial lake. International Journal of Systematic and Evolutionary Microbiology, 61(7): 1511-1514

Katz EE and Labuza TP (1981) Effect of Water Activity on the Sensory Crispness and Mechanical

Deformation of Snack Food Products. Journal of Food Science, 46(2):403-409 Kim K, Im W and Lee S (2008) Hymenobacter soli sp. nov., isolated from grass soil. International Journal

of Systematic and Evolutionary Microbiology, 58: 941-945 Klassen JL and Foght JM (2008) Differences in carotenoid composition among Hymenobacter and related

strains support a tree-like model of carotenoid evolution. Applied and Environmental Microbiology ,74: 2016–2022

Klassen JL and Foght JM (2011) Characterization of Hymenobacter isolates from Victoria Upper Glacier,

Antarctica reveals five new species and substantial non-vertical evolution within this genus. Extremophiles, 15: 45-57

Kounaves SP, Hecht MH, West SJ, Morookian JM, Young SM, Quinn R, Grunthaner P, Wen X, Weilert M,

Cable C, Fisher A, Gospodinova K, Kapit J, Stroble S, Hsu P, Clark B, Ming D and Smith P (2009) The MECA Wet Chemistry Laboratory on the 2007 Phoenix Mars Scout Lander. Journal of Geophysical Research, 114: E00A19

Labuza TP (1980) The effect of water activity on reaction kinetics of food deterioration. Food

Technology, 34(4): 36-41 Malin MC and Carr MH (1999) Groundwater formation on Martian valleys. Nature, 397: 589–591 Mannisto M and Haggblom M (2006) Characterization of psychrotolerant heterotrophic

bacteria from Finnish Lapland. Systematic and Applied Microbiology, 29: 229-243 Marion GM et al (2010) Modeling aqueous perchlorate chemistries with applications to Mars.

Icarus, 207(2): 675 - 685 Mars Exploration Rovers - NASA Jet Propulsion Laboratory: California Institute of Technology webpage.

[Online.] Available from http://marsrovers.jpl.nasa.gov. [cited 7 May 2012] Mars Science Laboratory - NASA Jet Propulsion Laboratory: California Institute of Technology webpage.

[Online.] Available from http://msl-scicorner.jpl.nasa.gov/ScienceGoals/. [cited 13 June 2012]

Mattimore V and Battista JR (1996) Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. Journal of Bacteriology, 178(3): 633-637

http://marsrovers.jpl.nasa.gov/

http://msl-scicorner.jpl.nasa.gov/ScienceGoals/

57

McKay CP and Stoker CR (1990) Could the early environment of Mars have supported the development

of life? Earth Space, 2(5): 10-2 McKay CP (2010) An Origin of Life on Mars. Cold Spring Harbor Perspectives in Biology, 2: a003509 Michalski G, Bohlke JK and Thiemens M (2004) Long term atmospheric deposition as the source of

nitrate and other salts in the Atacama desert, Chile: New evidence from mass-independent oxygen isotopic compositions. Geochimica et Cosmochimica Acta, 68: 4023– 4038

Motzer WE (2001) Perchlorate: problems, detection, and solutions. Environmental Forensics, 2: 301-311 Navarro-González R, Rainey FA, Molina P, Bagaley DR, Hollen BJ, de la Rosa J, Small AM, Quinn

RC, Grunthaner FJ, Cáceres L, Gomez-Silva B and McKay CP (2003) Mars-like soils in the Atacama Desert, Chile, and the dry limit of microbial life. Science, 302(5647): 18-21

Nelson LM and Parkinson D (1978) Growth characteristics of three bacterial isolates from an arctic soil. Canadian Journal of Microbiology, 24: 909-914

Newcombe, DA, Schuerger AC, Benardini JN, Dickinson D, Tanner R and Venkateswaran K (2005) Survival of spacecraft associated microbes under simulated Martian UV irradiation. Applied and Environmental Microbiology, 71: 8147-8156

Omelyansky V (1911) Bacteriological investigation of Sanga mammoth and nearby soil. Arkhiv biologicheskikh nauk, 16(4): 335–340 (in Russian)

Orris GJ, Harvey GJ, Tsui DT and Eldrige JE (2003) Preliminary analyses for perchlorate in selected natural

materials and their derivative products. United States Geological Survey, Open File Rep, 03-314 Osman S, La Duc MT, Dekas A, Newcombe D and Venkateswaran K (2008a) Microbial burden and

diversity of commercial airline cabin air during short and long durations of travel. International Society for Microbial Ecology Journal, 2: 482–497

Osman S, Peeters Z, Myron TLD, Mancinelli R, Ehrenfreund P and Venkateswaran K (2008b) Effect of

shadowing on Survival of Bacteria under conditions Simulating the Martian Atmosphere and UV Radiation. Applied and Environmental Microbiology, 74(4): 959-970

Panikov NS, Flanagan PW, Oechel WC, Mastepanov MA and Christensen TR (2005) Microbial activity in

soils frozen to below -39 °C. Soil Biology and Biochemistry, 38(4): 785-794

Parinkina OM (1989) Microflora of tundra soils: ecological geographical features and productivity. Nauka, Leningrad (in Russian)

Parro V, de Diego-Castilla G, Moreno-Paz M, Blanco Y, Cruz-Gil P, Rodríguez-Manfredi JA, Fernández-Remolar D, Gómez F, Gómez MJ, Rivas LA, Demergasso C, Echeverría A, Urtuvia VN, Ruiz-Bermejo M, García-Villadangos M, Postigo M, Sánchez-Román M, Chong-Díaz G and Gómez-Elvira J (2011) A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars. Astrobiology, 11(10): 969-96

http://www.ncbi.nlm.nih.gov/pubmed?term=Navarro-Gonz%C3%A1lez%20R%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Rainey%20FA%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Molina%20P%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Bagaley%20DR%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Hollen%20BJ%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=de%20la%20Rosa%20J%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Small%20AM%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Quinn%20RC%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Quinn%20RC%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Grunthaner%20FJ%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=C%C3%A1ceres%20L%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Gomez-Silva%20B%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=McKay%20CP%5BAuthor%5D&cauthor=true&cauthor_uid=14605363

http://www.ncbi.nlm.nih.gov/pubmed?term=Parro%20V%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=de%20Diego-Castilla%20G%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Moreno-Paz%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Blanco%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Cruz-Gil%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Rodr%C3%ADguez-Manfredi%20JA%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Fern%C3%A1ndez-Remolar%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Fern%C3%A1ndez-Remolar%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=G%C3%B3mez%20F%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=G%C3%B3mez%20MJ%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Rivas%20LA%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Demergasso%20C%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Echeverr%C3%ADa%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Urtuvia%20VN%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Ruiz-Bermejo%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Ruiz-Bermejo%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Garc%C3%ADa-Villadangos%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Postigo%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=S%C3%A1nchez-Rom%C3%A1n%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=Chong-D%C3%ADaz%20G%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=G%C3%B3mez-Elvira%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

http://www.ncbi.nlm.nih.gov/pubmed?term=G%C3%B3mez-Elvira%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22149750

58

Pollack JB, Kasting JF, Richardson SM and Poliakoff K (1987) The case for a wet, warm climate on early Mars. Icarus, 71: 203-224

Polymenakou PN, Mandalakis M, Stephanou EG and Tselepides A (2008) Particle size distribution of

airborne microorganisms and pathogens during an intense African dust event in the Eastern Mediterranean. Environmental Health Perspectives, 116: 292–296

Price P (2000) A habitat for psychrophiles in deep Antarctic ice. Proceedings of the National Academy of

Sciences USA, 97: 1247–1251 Price PB and Sowers T (2004) Temperature dependence of metabolic rates for microbial growth,

maintenance, and survival. Proceedings of the National Academy of Sciences USA, 101: 4631–4636

Rainey FA, Ray K, Ferreira M, Gatz BZ, Nobre MF, Bagaley D, Rash BA, Park M-J, Earl AM, Shank NC, Small

AM, Henk MC, Battista JR, Kampfer P and da Costa MS (2005) Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample. Applied and Environmental Microbiology, 71: 5225-5235

Rainey FA, Ward-Rainey N, Kroppenstedt RM and Stackebrandt E (1996) The genus Nocardiopsis

represents a phylogenetically coherent taxon and a distinct actinoymycete lineage: proposal of Nocardiopsaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology, 46(4): 1088-1092

Rivkina EM, Friedmann EI, McKay CP and Gilichinsky DA (2000) Metabolic activity of permafrost bacteria

below the freezing point. Applied and Environmental Microbiology, 66: 3230–3233 Rivkina E, Laurinavichius K, McGrath J, Tiedje J, Shcherbakova V and Gilichinsky D (2004) Microbial life in

permafrost. Advances in Space Research, 33: 1215–1221 Roos YH (1993) Water Activity and Physical State Effects on Amorphous Food Stability. Journal of Food

Processing and Preservation, 16(6): 433-447 Siegel BZ, McMurty G, Siegel SM, Chen J and LaRock P (1979) Life in the calcium chloride environment of

Don Juan Pond, Antarctica. Nature, 280: 828-829 Saul DJ, Aislabie JM, Brown CE, Harris L and Foght JM (2005) Hydrocarbon contamination changes the

bacterial diversity of soil from around Scott Base, Antarctica. Federation of European Microbiological Sciences Microbiology Ecology, 53: 141–155

Schuerger, AC, Berry BJ and Nicholson WL (2006) Interactive effects of low pressure, low temperature,

and CO2 atmospheres inhibit the growth of Bacillus spp. under simulated Martian conditions. Icarus, 185: 143-152

Slade L, Levine H and Reid DS (1991) Beyond water activity: Recent advances based on an alternative

approach to the assessment of food quality and safety. Critical Reviews in Food Science and Nutrition, 30(2-3): 115-360

59

Smith HD and McKay CP (2005) Drilling in ancient permafrost on Mars for evidence of a second genesis

of life. Planetary and Space Science, 53: 1302–1308 Stoker CR, Gooding JL, Roush T, Banin A, Burt D, Clark BC, Flynn G and Gwynne O (1993) The physical

and chemical properties and resource potential of Martian surface soils. In: Resources of Near-Earth Space. Edited by J Lewis, MS Matthews, and ML Guerrieri. University of Arizona Press, Tucson, AZ. pp. 659-707

Tamura K, Dudley J, Nei M and Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24: 1596-1599

Tauscher C, Schuerger AC and Nicholson WL (2006) Survival and germinability of Bacillus subtilis spores

exposed to simulated Mars solar radiation: implications for planetary protection and lithopanspermia. Astrobiology, 6: 592-605

Trumpolt CW, Crain M, Cullison GD, Flanagan SJ, Siegel L and Lathrop S (2005) Perchlorate: Source, uses

and occurrences in the environment. Remediation Journal, 16: 65–89 Vaniman DT, Bish DL, Chipera SJ, Fialips CL, Carey JW and Feldman WC (2004) Magnesium sulphate salts

and the history of water on Mars. Nature, 431(7009): 663-665 Venkateswaran K, Hattori N, La Duc MT and Kern R (2003) ATP as a biomarker of viable microorganisms

in clean-room facilities. Journal of Microbiological Methods, 52: 367–377 Vorobyova E, Soina V, Gorlenko M, Minkovskaya N, Zalinova N, Mamukelashvili A, Gilichinsky D, Rivkina

E and Vishnivetskaya T (1997) The deep cold biosphere: facts and hypothesis. FEMS Microbiological Reviews, 20: 277-290

Warren-Rhodes KA, Rhodes KL, Pointing SP, Ewing SA, Lacap DC, Gomez-Silva B, Amundson R, Friedmann

EI and McKay CP (2006) Hypolithic cyanobacteria, dry limit of photosynthesis, and microbial ecology in the hyperarid Atacama Desert. Microbial Ecology, 52(3): 389-398

Xu J, Liu Q, Yu H, Jin F, Lee S and Im W (2009) Hymenobacter daecheongensis sp. nov., isolated from

stream sediment. International Journal of Systematic and Evolutionary Microbiology, 59: 1183-1187

Yergeau E, Newsham KK, Pearce DA and Kowalchuk GA (2007) Patterns of bacterial diversity across a

range of Antarctic terrestrial habitats. Environmental Microbiology, 9: 2670–2682 Zhang S, Hou S, Ma X, Qin D and Chen T (2006) Culturable bacteria in Himalayan ice in response to

atmospheric circulation. Biogeosciences Discussion, 3: 765–778 Zhang Q, Liu C, Tang Y, Zhou G, Shen P, Fang C and Yokota A (2007) Hymenobacter xinjiangensis sp. nov.,

a radiation-resistant bacterium isolated from the desert of Xinjiang, China. International Journal of Systematic and Evolutionary Microbiology, 57: 1752–1756

60

Zhang G, Niu F, Busse H-J, Ma X, Liu W, Dong M, Feng H, An L and Cheng G (2008a) Hymenobacter psychrotolerans sp. nov., isolated from the Qinghai-Tibet Plateau permafrost region. International Journal of Systematic and Evolutionary Microbiology, 58: 1215–1220

Zhang X, Ma X, Wang N and Yao T (2008b) New subgroup of Bacteroidetes and diverse microorganisms

in Tibetan plateau glacial ice provide a biological record of environmental conditions. FEMS Microbiology Ecology, 67: 21–29

Zhang L, Dai J, Tang Y, Luo X, Wang Y, An H, Fang C and Zhang C (2009) Hymenobacter deserti sp. nov.,

isolated from the desert of Xinjiang, China. International Journal of Systematic and Evolutionary Microbiology, 59: 77-82

Zhang D, Busse H, Liu H, Zhou Y, Schinner F and Margesin R (2011) Hymenobacter psychrophilus sp. nov.,

a psychrophilic bacterium isolated from soil. International Journal of Systematic and Evolutionary Microbiology, 61: 859-863

61

APPENDIX A STRAINS USED IN PERHCLORATE TESTING

Table of Blastococcus Strains – All Blastococcus strains were incubated at 28 °C.

Strain Designation

Species Designation

Isolation Media

Sample Site Location GPS Coordinates

West South

AT04-159-25 Blastococcus sp. 1/10 PCA AT04-159

Atacama Desert NA NA





AT04-159-106 Blastococcus sp. NA AT04-159




AT03-33-18 Blastococcus sp. 1/100 PCA AT03-33 Atacama Desert

69° W 51.840'

24° S 4.112′

AT03-33-24 Blastococcus sp. MA AT03-33 Atacama Desert

69° W 51.840'

24° S 4.112′

AT03-33-58 Blastococcus sp. NA AT03-33 Atacama Desert

69° W 51.840'

24° S 4.112′


69° W 51.806'

24° S 4.103′


69° W 51.806'

24° S 4.103′


69° W 51.854'

24° S 3.836′


69° W 51.891'

24° S 3.734′


69° W 51.891'

24° S 3.734′

AT03-40-15 Blastococcus sp. PCA AT03-40 Atacama Desert

69° W 52.451'

24° S 3.637′


69° W 52.451'

24° S 3.637′


69° W 53.091'

24° S 3.666′


69° W 54.637'

24° S 3.445′


69° W 52.632'

24° S 4.545′

AT04-151-2 Blastococcus sp. MA AT04-151

Atacama Desert

69° W 51.858'

24° S 3.720′

62

Strain Designation

Species Designation

Isolation Media


West South


Atacama Desert

69° W 51.423′

24° S 3.585′


Atacama Desert

69° W 51.423′

24° S 3.585′


Atacama Desert

69° W 51.423′

24° S 3.585′


Atacama Desert

69° W 51.423′

24° S 3.585′


Atacama Desert

69° W 51.423′

24° S 3.585′


Atacama Desert

69° W 54.065'

24° S 4.440′


Atacama Desert

69° W 54.065'

24° S 4.440′


Atacama Desert

69° W 54.065'

24° S 4.440′


Atacama Desert

69° W 54.065'

24° S 4.440′


Atacama Desert

69° W 52.560'

24° S 3.660′


Atacama Desert

69° W 52.560'

24° S 3.660′


Atacama Desert

69° W 52.560'

24° S 3.660′


Atacama Desert

69° W 52.680'

24° S 4.140′


Atacama Desert

69° W 52.680'

24° S 4.140′


Atacama Desert

69° W 52.680'

24° S 4.140′


Atacama Desert

69° W 52.260'

24° S 4.200′


Atacama Desert

69° W 52.260'

24° S 4.200′


Atacama Desert

69° W 52.260'

24° S 4.200′


Atacama Desert

69° W 52.260'

24° S 4.200′


Atacama Desert

69° W 52.260'

24° S 4.200′


Atacama Desert

69° W 52.260'

24° S 4.200′

SS05-2-14 Blastococcus sp. 1/10 PCA SS05-2 Sahara Desert NA NA

(Table of Blastococcus Strains cont’d)

63

Strain Designation

Species Designation

Isolation Media


West South

SS05-2-113 Blastococcus sp. 1/10 PCA SS05-2 Sahara Desert NA NA

MCC-59 Blastococcus sp. R2A

AT01-19 Atacama Desert NA NA

MCC-63 Blastococcus sp. R2A


KR-187 Blastococcus sp. RM AT97-3B


KR-206 Blastococcus sp. PCA AT97-3B






KR-214 Blastococcus sp. NA AT97-3B


B. jeju Blastococcus

jejuensis SC-SW agar NA Beach - Jeju,

Korea NA NA

B. ag Blastococcus aggregatus

Bunt & Rovira NA

Mediterranean Basin NA NA

B. sax Blastococcus saxobsidens

Bunt & Rovira NA

Mediterranean Basin NA NA

(Table of Blastococcus Strains)

cont’d

64

Table of Atacama Isolates – All Atacama isolates were incubated at 28 °C

Strain Designation Species Name

Isolation Media


West South

AT04-159-22 Modestobacter sp. 1/10 PCA


AT03-40-39 Ornithinicoccus

hortensis 1/100 PCA

AT03-40 Atacama Desert

69° W 52.451'

24° S 3.637′

G. ob. ob Geodermatophilus obscuris obscuris BHI Agar NA Soils - US NA NA

M. veri Modestobacter

versicolor BG11-PGY NA Colorado Plateau NA NA

ATCC-BAA-817

Balneimonas flocculans NA NA Japan – hot spring NA NA

KACC-12744 Microvirga aerilata NA NA Air Sample, China NA NA

M. guang Microvirga

guangxiensis GYM agar NA Guangxi Province,

China NA NA

M. aerophila Microvirga aerophila NA NA Air Sample, China NA NA

10-39Q-44 Microvirga sp. 1/10 PCA 10-39Q Oman, UAE NA NA

KR-477 Microvirga sp. RM N97-2D Mojave Desert, US NA NA

KR-450 Microvirga sp. RM N97-2C Mojave Desert, US NA NA

KR-393 Microvirga sp. NA N97-2C Mojave Desert, US NA NA

M. subterranea

Microvirga subterranea YE NA

Great Artesian Basin, Australia NA NA

AT03-41-2 Frankia sp. Strain

AVN17S PCA AT03-

41 Atacama Desert 69° W

53.091' 24° S

3.666′

AT03-38-1 Frankia sp. PCA AT03-


52.088′ 24° S

3.645′

KR-515 α-proteobacterium 1/10 PCA N97-2H Mojave Desert, US NA NA

NG05-4-23 Microvirga sp. NA NG05-4 Negev, Israel NA NA

AT04-159-59

Mycobacterium celatum 1/10 PCA


AT04-166-1 Kocuria sp. MA AT04-166 Atacama Desert

69° W 52.260'

24° S 4.200′

AT04-159-4 Hymenobacter sp. 1/10 PCA AT04-159 Atacama Desert NA NA

33-40Q-101A Microvirga sp. NA 33-40Q Oman, UAE NA NA

NG05-R6A-48 Microvirga sp. NA

NG05-R6A Negev, Israel NA NA

NG05-4D6A-29 Microvirga sp. NA

NG05-4D6A Negev, Israel NA NA

65


Isolation Media


West South

KR-56 Geodermatophilus sp. RM S97-3J Sonoran Desert NA NA

KR-28 Microvirga sp. RM S97-3F Sonoran Desert NA NA



AT03-33-7 Brevibacillus PCA AT03-

33 Atacama Desert NA NA

AT04-162-12 Micrococcus luteus PCA


69° W 53.465'

24° S 4.200′

AT03-34-3 Williamsia muralis PCA AT03-


51.806' 24° S

4.103′

AT03-33-2 Modestobacter multiseptatus 1/10 PCA


69° W 51.840'

24° S 4.112′

AT03-33-23 Promicromonospora

enterophila MA AT03-


51.840' 24° S

4.112′

AT04-166-15 Kocuria erythromyxa MA


69° W 52.260'

24° S 4.200′

AT04-161-18 Cellulomonas cartae MA


69° W 54.065'

24° S 4.440′

Table of Atacama Isolates cont’d

66

Table of Control Strains


Incubation Temp °C Isolate Origin

DSM 14746T Sphingomonas aerolata 25 airborne dust

DSM 10604T Pseudomonas syringae 25 lilac plant

DSM 15428T Paenisporosarcina macmurdoensis 25 landfill site

DSM 21396T Deinococcus gobiensis 28 Gobi Desert soil

DSM 14418T Georgenia muralis 28 medieval wall painting

DSM 21230T Deinococcus aetherius 28 stratosphere

DSM 10T Bacillus subtilis 28 desert soil

DSM 30147T Rhizobium radiobacter 28 rhizosphere

DSM 40313T Streptomyces albus 28 soil

DSM 21212T Deinococcus aerius 28 high atmosphere

DSM 21991T Sporosarcina antarctica 10 Antarctic soil

Strain Designation Species Name Incubation Temp °C Isolate Origin

DSM 11300T Deinococcus geothermalis 37 Hot springs

DSM 17424T Deinococcus mumbaiensis 37 TGYE Agar

DSM 20539T Deinococcus radiodurans 28 Irradiated meat

CCUG 51391T Deinococcus ficus 28 rhizosphere

LB-34T Deinococcus maricopensis 28 Sonoran Desert

DSM 17005T Deinococcus yunweiensis 28 YE-ME agar

DSM 3963T Deinococcus grandis 28 elephant feces

KCTC-12553T Deinococcus caenii 28 activated sludge

KCTC-12552T Deinococcus aquaticus 28 fresh water

LMG 22133T Deinococcus hopiensis 28 Sonoran Desert

KR-35 Deinococcus apachensis 28 Sonoran Desert

NG-371 Deinococcus sp. 28 Negev Desert

DSM 12027T Deinococcus radiopugnans 28 NA

KR-200T Deinococcus peraridilitoris 28 arid soil - Chile

KR-40T Deinococcus hohokamensis 28 Sonoran Desert

DSM 12807T Deinococcus frigens 10 Antarctic soil

DSM 12784T Deinococcus marmoris 10 Antarctic marble

DSM 15974T Deinococcus saxicola 10 Antarctic sandstone

PO-04-20-132T Deinococcus radiomollis 10 Alpine soil

DSM 15307T Deinococcus indicus 28 Indian aquifer

KR-235T Deinococcus pimensis 28 Sonoran Desert

Table of Deinococcus strains

67

APPENDIX B PHOTOMICROGRAPHS OF HYMENOBACTER ISOLATES FROM PERMAFROST

irFSS21

irFSS25

68

irFSS27

BV-irFSS30

69

BV-irFSS32

BV-irFSS31

70

BV-irFSS33

BV-irFSS35

71

BV-irFSS36

BV-irFSS37

72

BV-irFSS38

BV-irLH24 (A)

73

BV-irLH24 (B)

BV-irLH25

74

BV-irLH34

75

Co

ntr

ol

Str

ain

De

sig

na

tio

nS

pe

cie

s N

am

eR

2A

+ 3

% A

ga

r5

0 m

M1

00

mM

20

0 m

M4

00

mM

60

0 m

M8

00

mM

1 M

50

mM

10

0 m

M2

00

mM

40

0 m

M5

0 m

M1

00

mM

20

0 m

M4

00

mM

AT

04

-15

9-2

5B

last

oco

ccu

s a

gg

reg

atu

s(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(++

++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

04

-15

9-3

4B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(-

)(+

++

+)

(++

++

)(-

)(-

)(+

++

+)

(-)

(-)

(-)

AT

04

-15

9-3

7B

last

oco

ccu

s a

gg

reg

atu

s(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(-

)(+

++

+)

(++

)(-

)(-

)(+

++

+)

(-)

(-)

(-)

AT

04

-15

9-1

06

Bla

sto

cocc

us

ag

gre

ga

tus

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

)(-

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

(++

++

)(+

+)

(-)

(-)

AT

04

-15

9-1

13

Bla

sto

cocc

us

ag

gre

ga

tus

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(-)

(++

+)

(++

+)

(-)

(-)

(++

++

)(+

+)

(-)

(-)

AT

03

-33

-18

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

)(-

)(-

)(-

)(+

++

)(+

++

+)

(++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

03

-33

-24

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

+)

(-)

(-)

(-)

(++

+)

(++

++

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

AT

03

-33

-58

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

)(-

)(-

)(-

)(+

++

+)

(+)

(-)

(-)

(++

++

)(+

++

)(-

)(-

)

AT

03

-34

-7B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

+)

(++

)(+

)(+

++

+)

(++

++

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

AT

03

-34

-9B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(-)

(++

)(-

)(-

)(-

)(+

+)

(++

)(-

)(-

)(+

++

+)

(-)

(-)

(-)

AT

03

-36

-18

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(+

++

)(+

++

+)

(++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

03

-37

-6B

last

oco

ccu

s a

gg

reg

atu

s(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

)(-

)(-

)(-

)(+

++

+)

(++

++

)(-

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

03

-37

-10

Bla

sto

cocc

us

ag

gre

ga

tus

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

03

-40

-15

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

)(-

)(-

)(-

)(+

++

)(+

++

+)

(-)

(-)

(++

++

)(+

+)

(-)

(-)

AT

03

-40

-35

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

+)

(+)

(-)

(-)

(-)

(+)

(-)

(-)

(-)

(++

+)

(-)

(-)

(-)

AT

03

-41

-4B

last

oco

ccu

s a

gg

reg

atu

s(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

)(-

)(-

)(-

)(+

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

)(-

)(-

)

AT

03

-43

-14

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(+)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

03

-49

-2B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(+)

(++

)(+

+)

(++

++

)(+

++

+)

(+)

(+)

(++

++

)(+

++

+)

(+)

(+)

AT

04

-15

1-2

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

+)

(-)

(-)

(-)

(++

+)

(++

++

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

AT

04

-15

3-2

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(+

+)

(++

++

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

AT

04

-15

3-4

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-15

3-1

5B

last

oco

ccu

s a

gg

reg

atu

s(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(-

)(+

++

+)

(++

++

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

AT

04

-15

3-2

4B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

)(-

)(-

)(-

)(+

++

+)

(++

++

)(-

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

04

-15

3-4

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

)(-

)(-

)(-

)(+

++

+)

(++

++

)(+

++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

04

-16

1-2

8B

last

oco

ccu

s a

gg

reg

atu

s(+

++

+)

(++

++

)(+

+)

(+)

(-)

(-)

(-)

(-)

(++

)(+

)(-

)(-

)(+

+)

(+)

(-)

(-)

AT

04

-16

1-2

3B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

+)

(++

+)

(-)

(-)

(-)

(++

)(+

++

)(-

)(-

)(+

++

)(+

++

)(-

)(-

)

AT

04

-16

1-1

00

Bla

sto

cocc

us

ag

gre

ga

tus

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

+)

(-)

(-)

AT

04

-16

1-9

6B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

4-3

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(+

++

+)

(++

++

)(+

++

+)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

4-2

3B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

4-2

7B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

+)

(++

++

)(+

++

+)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

5-1

2B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

5-1

6B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

5-2

7B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

6-2

3B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(++

+)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

6-2

5B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

AT

04

-16

6-6

4B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(++

++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

04

-16

6-1

09

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(+)

(-)

(-)

(-)

(++

++

)(+

++

)(-

)(-

)(+

++

)(-

)(-

)(-

)

AT

04

-16

6-1

69

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(+

++

+)

(++

++

)(+

++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

AT

04

-16

6-1

59

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

)(-

)(-

)

SS

05

-2-1

4B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(++

+)

(-)

(++

++

)(+

++

+)

(-)

(-)

SS

05

-2-1

13

Bla

sto

cocc

us

sp.

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(+)

(+)

MC

C-5

9B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(-

)(-

)(-

)(-

)(+

++

+)

(++

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

MC

C-6

3B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

)(-

)(-

)(-

)(+

++

+)

(++

+)

(-)

(-)

(++

++

)(+

++

)(-

)(-

)

KR

-18

7B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

)(-

)(-

)

KR

-20

6B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

)(-

)(-

)

KR

-20

7B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

KR

-20

9B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

KR

-21

4B

last

oco

ccu

s sp

.(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

(++

++

)(+

++

+)

(-)

(-)

B.

jeju

Bla

sto

cocc

us

jeju

en

sis

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(+)

(+)

(++

++

)(+

++

+)

(++

++

)(-

)(+

++

+)

(++

++

)(+

++

)(-

)

B.

ag

Bla

sto

cocc

us

ag

gre

ga

tus

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(+)

(-)

(++

++

)(+

++

+)

(++

)(-

)(+

++

+)

(++

++

)(-

)(-

)

B.

sax

Bla

sto

cocc

us

sax

ob

sid

en

s(+

++

+)

(++

++

)(+

++

+)

(++

++

)(+

++

+)

(-)

(-)

(-)

(++

++

)(+

++

+)

(+)

(-)

(++

++

)(+

++

+)

(-)

(-)

Na

ClO

4M

gC

l 2O

8C

aC

l 2O

8

AP

PEN

DIX

C

BA

CTE

RIA

L G

RO

WTH

IN T

HE

PR

ESEN

CE

OF

PER

CH

LOR

ATE

S (T

AB

LES)

Gro

wth

of

Bla

sto

cocc

us

iso

late

s in

per

chlo

rate

-co

nta

inin

g m

edia

76

Co

ntr

ol

Stra

in D

esi

gnat

ion

Spe

cie

s N

ame

R2A

+ 3

% A

gar

50 m

M10

0 m

M20

0 m

M40

0 m

M60

0 m

M80

0 m

M1

M50

mM

100

mM

200

mM

400

mM

50 m

M10

0 m

M20

0 m

M40

0 m

M

G. o

b. o

bG

eod

erm

ato

ph

ilus

ob

scu

ris

ob

scu

ris

(+++

+)(+

+++)

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(+++

+)(+

+++)

(-)

(-)

(+++

)(+

++)

(+)

(-)

M. v

eri

Mo

des

tob

act

er

(+++

+)(+

+++)

(+++

+)(+

+++)

(+++

+)(-

)(-

)(-

)(+

+++)

(+++

+)(-

)(-

)(+

+++)

(-)

(-)

(-)

ATC

C-B

AA

-817

Ba

lnei

mo

na

s(+

+++)

(+++

+)(+

)(-

)(-

)(-

)(-

)(-

)(+

+)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

KA

CC

-127

44M

icro

virg

a a

erila

ta(+

+++)

(+++

+)(+

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

M. g

uan

gM

icro

virg

a g

ua

ng

xien

sis

(+++

+)(+

+++)

(+++

+)(-

)(-

)(-

)(-

)(-

)(+

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

M. a

ero

ph

ila

Mic

rovi

rga

aer

op

hila

(+++

+)(+

+++)

(+)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

10-3

9Q-4

4M

icro

virg

a s

p.

(+++

+)(+

+++)

(++)

(+)

(+)

(-)

(-)

(-)

(+)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

KR

-477

Mic

rovi

rga

sp

.(+

+++)

(+++

+)(+

+)(-

)(-

)(-

)(-

)(-

)(+

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

KR

-450

Mic

rovi

rga

sp

.(+

+++)

(+++

+)(+

+++)

(+)

(+)

(-)

(-)

(-)

(++)

(-)

(-)

(-)

(+)

(-)

(-)

(-)

KR

-393

Mic

rovi

rga

sp

.(+

+++)

(+++

+)(+

+++)

(+++

)(+

)(-

)(-

)(-

)(+

+)(-

)(-

)(-

)(+

)(-

)(-

)(-

)

M. s

ub

terr

ane

aM

icro

virg

a s

ub

terr

an

ea(+

+++)

(+++

+)(+

+)(+

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

AT0

3-41

-2Fr

an

kia

sp

. St

rain

AV

N17

S(+

+++)

(+++

+)(+

+++)

(+++

+)(+

++)

(-)

(-)

(-)

(+++

+)(+

+++)

(-)

(-)

(+++

+)(+

++)

(-)

(-)

AT0

3-38

-1Fr

an

kia

sp

.(+

+++)

(+++

+)(+

+++)

(+++

+)(+

+++)

(-)

(-)

(-)

(+++

+)(+

+++)

(-)

(-)

(+++

+)(+

++)

(-)

(-)

KR

-515

α-p

rote

ob

acte

riu

m(+

+++)

(+++

+)(+

+)(-

)(+

++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

NG

05-4

-23

Mic

rovi

rga

sp

.(+

+++)

(+++

+)(+

++)

(++)

(+)

(-)

(-)

(-)

(+)

(-)

(-)

(-)

(+)

(-)

(-)

(-)

AT0

4-15

9-59

Myc

ob

act

eriu

m c

ela

tum

(+++

+)(+

+++)

(+++

+)(+

+++)

(+)

(-)

(-)

(-)

(+++

+)(+

+++)

(+++

+)(-

)(+

+++)

(+++

+)(-

)(-

)

AT0

4-16

6-1

Ko

curi

a s

p.

(+++

+)(+

+++)

(+++

+)(+

+++)

(+++

+)(+

)(+

)(+

+++)

(+++

+)(+

+++)

(+++

+)(-

)(+

+++)

(+++

+)(+

)(-

)

AT0

4-15

9-4

Hym

eno

ba

cter

sp

.(+

+++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

33-4

0Q-1

01A

Mic

rovi

rga

sp

.(+

+++)

(+++

+)(+

+++)

(+++

+)(+

+++)

(++)

(+)

(+)

(+++

+)(+

+++)

(+++

+)(-

)(+

+++)

(+++

+)(+

)(-

)

NG

05-R

6A-4

8M

icro

virg

a s

p.

(+++

+)(+

+++)

(++)

(-)

(-)

(-)

(-)

(-)

(++)

(-)

(-)

(-)

(++)

(-)

(-)

(-)

NG

05-4

D6A

-29

Mic

rovi

rga

sp

.(+

+++)

(+++

+)(+

+++)

(+++

+)(+

+++)

(+)

(+)

(-)

(+++

+)(+

+++)

(++)

(-)

(+++

+)(+

++)

(-)

(-)

KR

-56

Geo

der

ma

top

hilu

s(+

+++)

(+++

+)(+

+++)

(+++

+)(+

++)

(++)

(+)

(-)

(+++

+)(+

+++)

(+++

)(-

)(+

+++)

(+++

+)(+

)(+

)

KR

-28

Mic

rovi

rga

sp

.(+

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(+

)(+

)(-

)(-

)

33-4

0Q-7

5M

icro

virg

a s

p.

(+++

+)(+

+++)

(+++

+)(+

+++)

(+++

)(+

)(-

)(-

)(+

+++)

(+++

+)(+

)(-

)(+

)(+

)(-

)(-

)

33-4

0Q-1

30M

icro

virg

a s

p.

(+++

+)(+

+++)

(+++

+)(+

+)(+

)(+

)(-

)(-

)(+

)(-

)(-

)(-

)(+

)(+

)(-

)(-

)

AT0

3-33

-7B

revi

ba

cillu

s++

++(+

+++)

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(-)

(-)

AT0

4-16

2-12

Mic

roco

ccu

s lu

teu

s++

++(+

+++)

(+++

+)(+

+)(-

)(-

)(-

)(-

)(+

+++)

(+++

+)(-

)(-

)(-

)(-

)(-

)(-

)

AT0

3-34

-3W

illia

msi

a m

ura

lis++

++(+

+++)

(+++

+)(+

)(-

)(-

)(-

)(-

)(+

+++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

AT0

3-33

-2M

od

esto

ba

cter

mu

ltis

epta

tus

++++

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(-)

(+++

+)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

AT0

3-33

-23

Pro

mic

rom

on

osp

ora

en

tero

ph

ila++

++(+

+)(+

+++)

(-)

(-)

(-)

(-)

(-)

(+++

+)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

AT0

4-16

6-15

Ko

curi

a e

ryth

rom

yxa

++++

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(-)

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(-)

(-)

AT0

4-16

1-18

Cel

lulo

mo

na

s ca

rta

e++

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

NaC

lO4

MgC

l 2O

8C

aCl 2

O8

B

acte

rial

Gro

wth

in t

he

Pre

sen

ce o

f P

erch

lora

tes

con

t’d

Gro

wth

of

Ata

cam

a is

ola

tes

in p

erch

lora

te-c

on

tain

ing

med

ia

77

Co

ntr

ol

Stra

in D

esi

gnat

ion

Spe

cie

s N

ame

R2A

+ 3

% A

gar

50 m

M10

0 m

M20

0 m

M40

0 m

M60

0 m

M80

0 m

M1

M50

mM

100

mM

200

mM

400

mM

50 m

M10

0 m

M20

0 m

M40

0 m

M

DSM

113

00T

Dei

no

cocc

us

geo

ther

ma

lis(+

+++)

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(-)

(+++

+)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

DSM

174

24T

Dei

no

cocc

us

mu

mb

aie

nsi

s(+

+++)

(+++

+)(+

+++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

DSM

205

39T

Dei

no

cocc

us

rad

iod

ura

ns

(+++

+)(+

+++)

(+++

+)(+

+)(-

)(-

)(-

)(-

)(+

++)

(++)

(-)

(-)

(++)

(+)

(-)

(-)

CC

UG

513

91T

Dei

no

cocc

us

ficu

s(+

+++)

(+++

+)(+

+++)

(++)

(-)

(-)

(-)

(-)

(+++

+)(+

+)(-

)(-

)(+

+)(+

)(-

)(-

)

LB-3

4TD

ein

oco

ccu

s m

ari

cop

ensi

s(+

+++)

(+++

+)(+

++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

DSM

170

05T

Dei

no

cocc

us

yun

wei

ensi

s(+

+++)

(+++

+)(+

+)(+

+)(-

)(-

)(-

)(-

)(+

+)(+

+)(-

)(-

)(+

+)(+

)(-

)(-

)

DSM

396

3TD

ein

oco

ccu

s g

ran

dis

(+++

+)(+

+++)

(+++

+)(+

)(-

)(-

)(-

)(-

)(+

+++)

(++)

(-)

(-)

(+)

(+)

(-)

(-)

KC

TC-1

2553

TD

ein

oco

ccu

s ca

enii

(+++

+)(+

+++)

(+++

+)(+

)(-

)(-

)(-

)(-

)(+

++)

(++)

(-)

(-)

(+)

(+)

(-)

(-)

KC

TC-1

2552

TD

ein

oco

ccu

s a

qu

ati

cus

(+++

+)(+

+++)

(+++

+)(+

)(-

)(-

)(-

)(-

)(+

++)

(++)

(-)

(-)

(+)

(+)

(-)

(-)

LMG

221

33T

Dei

no

cocc

us

ho

pie

nsi

s(+

+++)

(+++

+)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

KR

-35

Dei

no

cocc

us

ap

ach

ensi

s(+

+)(+

++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

NG

-371

Dei

no

cocc

us

sp.

(+++

+)(+

+++)

(+)

(+)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

DSM

120

27T

Dei

no

cocc

us

rad

iop

ug

na

ns

(+++

+)(+

+++)

(+++

+)(+

)(-

)(-

)(-

)(-

)(+

+++)

(-)

(-)

(-)

(+++

)(-

)(-

)(-

)

KR

-200

TD

ein

oco

ccu

s p

era

rid

ilito

ris

(+++

+)(+

+++)

(-)

(+)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(+)

(-)

(-)

(-)

KR

-40T

Dei

no

cocc

us

ho

ho

kam

ensi

s(+

+++)

(+++

+)(+

++)

(+)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(+++

)(-

)(-

)(-

)

DSM

128

07T

Dei

no

cocc

us

frig

ens

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

DSM

127

84T

Dei

no

cocc

us

ma

rmo

ris

(+++

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

DSM

159

74T

Dei

no

cocc

us

saxi

cola

(+++

+)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)(-

)

PO

-04-

20-1

32T

Dei

no

cocc

us

rad

iom

olli

s(+

+++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

DSM

153

07T

Dei

no

cocc

us

ind

icu

s(+

+++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

KR

-235

TD

ein

oco

ccu

s p

imen

sis

(++)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

NaC

lO4

MgC

l 2O

8C

aCl 2

O8

B

acte

rial

Gro

wth

in t

he

Pre

sen

ce o

f P

erc

hlo

rate

s co

nt’

d

Gro

wth

of

Dei

no

cocc

us

stra

ins

on

per

chlo

rate

-co

nta

inin

g m

edia

78

Bac

teri

al G

row

th in

th

e P

rese

nce

of

Pe

rch

lora

tes

con

t’d

Gro

wth

of

con

tro

l str

ain

s o

n p

erch

lora

te-c

on

tain

ing

med

ia

Co

ntr

ol

Stra

in D

esi

gnat

ion

Spe

cie

s N

ame

R2A

+ 3

% A

gar

50 m

M10

0 m

M20

0 m

M40

0 m

M60

0 m

M80

0 m

M1

M50

mM

100

mM

200

mM

400

mM

50 m

M10

0 m

M20

0 m

M40

0 m

M

DSM

147

46T

Sph

ing

om

on

as

aer

ola

ta(+

+)-

--

--

--

--

--

--

--

DSM

106

04T

Pse

ud

om

on

as

syri

ng

ae

(+++

)-

--

--

--

--

--

--

--

DSM

154

28T

Pa

enis

po

rosa

rcin

a m

acm

urd

oen

sis

(+++

+)-

--

--

--

(+++

)(+

++)

--

--

--

DSM

213

96T

Dei

no

cocc

us

go

bie

nsi

s(+

+++)

(+++

+)(+

+++)

--

--

--

--

--

--

-

DSM

144

18T

Geo

rgen

ia m

ura

lis(+

+++)

(+++

+)(+

+++)

(+++

)(+

++)

--

-(+

+++)

--

-(+

+)-

--

DSM

212

30T

Dei

no

cocc

us

aet

her

ius

(+)

--

--

--

--

--

--

--

-

DSM

10T

Ba

cillu

s su

bti

lis(+

+++)

(+++

+)(+

+++)

(+++

)(+

++)

(++)

--

(+++

+)(+

+++)

--

(+++

)(+

++)

--

DSM

301

47T

Rh

izo

biu

m r

ad

iob

act

er(+

+++)

(+++

+)(+

+++)

--

--

-(+

+)-

--

--

--

DSM

403

13T

Stre

pto

myc

es a

lbu

s(+

+++)

(+++

+)(+

+++)

(+++

+)(+

+++)

--

-(+

+++)

(+++

+)-

-(+

+++)

--

-

DSM

212

12T

Dei

no

cocc

us

aer

ius

(+++

+)-

--

--

--

--

--

--

-

DSM

219

91T

Spo

rosa

rcin

a a

nta

rcti

ca(+

+++)

--

--

--

--

--

--

--

-

CaC

l 2O

8N

aClO

4M

gCl 2

O8

79

Vita

Bharathi Vallalar was born in Chidambaram, India on February 23rd, 1988. He was raised in

Lafayette, LA and graduated from Ovey Comeaux High School. He then moved to Baton Rouge, LA to

pursue a degree in Biological Sciences at Louisiana State University. Upon completion of the Methods

for Microbial Diversity course taught by Frederick A. Rainey, Bharathi gained interest in microbiological

research. He therefore changed his major to microbiology and began working in RaineyLab as a student

worker in his third year of undergraduate studies. Upon completion of his B.S. in Microbiology, Bharathi

worked in RaineyLab as a contingent employee for one semester before Dr. Rainey presented him with

an opportunity to complete a Master of Science Degree. Hence, Bharathi began his graduate career at

LSU in the summer of 2011, and completed his research and this thesis by the summer of 2012. Upon

completion of his defense, he will have completed all requirements for the degree of Master of Science

in Biological Sciences, and will collect his diploma in December of 2012. Bharathi has been accepted into

a Ph.D. program in Earth and Environmental Sciences at the University of Illinois in Chicago, where he

will continue research in the field of geomicrobiology.

Documents

Investigation of the Growth and Survival of Bacteria from