Maldi-Tof Mass Spectrometry and 16S Rna Approach for The

MALDI-TOF MASS SPECTROMETRY AND 16S RNA APPROACH FOR THE

IDENTIFICATION OF BACTERIA COLLECTED DURING THE PHOENIX

MARS LANDER

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Biological Sciences

Devin L. Lachner

2020 SIGNATURE PAGE

THESIS: MALDI-TOF MASS SPECTROMETRY AND 16S RNA APPROACH FOR THE IDENTIFICATION OF BACTERIA COLLECTED DURING THE PHOENIX MARS LANDER

AUTHOR: Devin L. Lachner

DATE SUBMITTED: Spring 2020

Department of Biological Sciences

Dr. Wei-Jen Lin

Thesis Committee Chair Biological Sciences

Dr. Parag Vaishampayan

Jet Propulsion Laboratory

Dr. Wendy Dixon

Biological Sciences

ACKNOWLEDGEMENTS

I want to thank Dr. Lin who took me into her lab when I was an undergraduate and gave me my first opportunity to perform research. This experience gave me direction for the first time and filled me with excitement for microbiology. Thanks for the countless hours you have invested in me, and for teaching me how to become a better scientist and teacher.

This research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, and was sponsored by the JPL Graduate Student Program and the

National Aeronautics and Space Administration (80NM0018D0004).

A special thanks to Arman Seuylemezian and Parag Vaishampayan, who taught me most of what I learned about lab work at JPL. It was a pleasure learning from you both, thank you for always being there when I needed your help.

Thank you to Dr. Dixon and Dr. Vaishampayan for agreeing to be on my committee and all the work that it has entailed.

A special thank you to my parents who supported me financially through my entire masters and bachelors’ program. Without your help, I never would have accomplished my goals without getting student loans. This has really meant a lot to me, and I can never thank you enough.

A huge thank you to my girlfriend Raven, who has been with me through the entire program supporting me and keeping me motivated.

iii

Thanks to everyone else in the Lin lab for always being available to help. I feel like we all made the best of it together, even during these challenging times. Good luck to all of you!

Finally, a big thanks to NASA and The Biotechnology and Planetary Protection

Group at JPL, without your assistance and funding this research never would have been possible.

ABSTRACT

The spacecraft assembly clean rooms where spacecraft are assembled represent a unique microbiological ecosystem. Stringent bioburden reduction such as controlled temperature, humidity, HEPA filtered air circulation, regular decontamination, and gowning procedures are implemented throughout the assembly and testing operations. The microorganisms that survive these cleaning procedures show a unique microbial diversity and are a complex blend of human and soil associated flora that are often spore-forming.

This taxonomic characterization study is essential for understanding which microorganisms were present on the surface of the Phoenix Mars Lander after microbial reduction measures. Knowing the bacterial contaminants' identities and whether they form spores helps NASA to predict whether the microorganisms pose a forward contamination risk that could affect future planetary protection policy.

A combination of MALDI-TOF MS and 16S rRNA sequencing was used to identify bacterial isolates collected from the Phoenix Mars Lander. There was a total of 637 isolates analyzed using MALDI-TOF MS, approximately 73% (320) of the 438 microorganisms identified to belong to spore-forming genera. For the samples that could not be identified using MALDI-TOF MS method, the 16S rRNA sequencing approach was implemented.

Of the 405 isolates identified with 16S rRNA, there were 312 (77%) isolates that were identified as spore-forming genera. After combining the results from these two methods there were 626 isolates identified, with 460 (74%) of the isolates identified as a spore- forming genus. This bias for spore-forming isolates could be due to many of the samples being heat-shocked at 80◦C for 15 minutes when using the NASA standard assay (NSA) for bioburden detection.

To better understand the relationship between the sample source and the prevalence of spore-forming bacteria, the unique microbiomes of three different locations on the

Phoenix Mars Lander were compared. The three components analyzed were the Phoenix

Mars Lander’s Fairing, MECA (Microscopy, Electrochemistry, and Conductivity

Analyzer), and Robotic Arm. Since some of the sensitive electronics found on the Phoenix

Mars Lander’s MECA and Robotic Arm have different sterilization procedures they could have a different frequency of spore-forming bacteria. A chi-square test of independence was performed to examine the relationship between the sample source and the presence of spore-forming bacteria. The relationship between these variables was found to be significant, and there have been fewer spore-forming isolates identified from the Fairing,

MECA, and Robotic Arm, compared to the other locations. Further understanding of these complex interactions can provide insight for future Planetary Protection missions.

TABLE OF CONTENTS

SIGNATURE PAGE ...... ii

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... v

LIST OF TABLES ...... xi

LIST OF FIGURES ...... xii

1. INTRODUCTION ...... 1

A. A brief introduction of the Jet Propulsion Laboratory ...... 1

B. Phoenix Mars Lander ...... 1

i. History of the Phoenix Mars Lander ...... 1

ii. Objectives of the Phoenix Mars Lander ...... 2

iii. Accomplishments of the Phoenix Mars Lander ...... 3

iv. Construction of the Phoenix Mars Lander ...... 4

C. The Planetary Protection Mission of JPL ...... 6

i. The mission of COSPAR and Planetary Protection ...... 6

ii. The NASA Standard Spore Assay ...... 7

iii. Current JPL Planetary Protection microbial collection ...... 8

iv. Present and future research with GESAM ...... 9

D. Bacterial taxonomic identification using Matrix-Assisted Laser Desorption

Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) ...... 9

i. History of MALDI-TOF MS...... 9

vii

ii. MALDI-TOF MS matrix associated sample preparation ...... 11

iii. MALDI-TOF MS Laser Desorption and Ionization ...... 12

iv. MALDI-TOF MS Time of Flight ...... 13

v. JPL in-house MALDI-TOF MS microbial database ...... 14

vi. Comparison of MALDI-TOF MS proteomic fingerprint to JPL database

...... 15

vii. Advantages of MALDI-TOF MS over 16S rRNA gene sequencing ...... 17

2. OBJECTIVE ...... 19

3. MATERIALS AND METHODS ...... 20

A. Bacterial sample collection from Phoenix Mars Lander ...... 20

B. Revive isolates from JPL Planetary Protection microbial collection ...... 22

C. Rapid and systematic identification using MALDI-TOF MS ...... 22

i. Direct transfer technique for MALDI-TOF MS ...... 23

ii. Direct transfer procedure ...... 23

iii. Tube extraction technique for MALDI-TOF MS...... 24

iv. Tube extraction procedure ...... 25

v. Taxonomic identification using Bruker MALDI Biotyper System ...... 27

D. Zymo DNA extraction of Phoenix Isolates ...... 27

E. Bacterial 16S rRNA gene amplification and sequencing ...... 28

F. Comparison of rRNA gene sequences to the EZBioCloud and the SILVA

LTP type strain databases ...... 30

viii

4. RESULTS ...... 32

A. Retrieval of bacterial isolates from JPL culture collection freezers and the

creation of pure cultures ...... 32

B. Taxonomic identification of Phoenix bacterial isolates using MALDI-TOF

MS ...... 32

i. Assessment of MALDI-TOF MS RTC Results ...... 32

ii. Identification of genera associated with the Phoenix Mars Lander using

MALDI-TOF MS ...... 34

C. Taxonomic identification of Phoenix bacterial isolates using 16S rRNA .... 35

i. Assessment of 16S rRNA gene sequence results ...... 35

D. Taxonomic identification of bacterial isolates by combining MALDI-TOF

MS and 16S rRNA results ...... 37

i. Comparing the isolates that were identified with both methods ...... 37

iii. Frequency of species belonging to the Bacillus genus ...... 41

iv. Frequency of species belonging to the Staphylococcus genus ...... 42

v. Frequency of species belonging to the Sporosarcina genus ...... 44

vi. Frequency of species belonging to the Paenibacillus genus ...... 45

vii. Frequency of species belonging to the Sphingomonas genus ...... 46

viii. Frequency of species belonging to the Agromyces genus ...... 48

ix. Frequency of species belonging to the Microbacterium genus ...... 49

x. The least frequently identified species using MALDI-TOF MS and 16S

rRNA ...... 50

E. Taxonomic identification of bacteria isolated from three distinctive

locations on the Phoenix Mars Lander ...... 53

i. Identification of bacterial isolates from the Fairing ...... 53

ii. Identification of bacterial isolates from the Robotic Arm ...... 55

iii. Identification of bacterial isolates from the MECA ...... 57

iv. Identification of bacterial isolates from the other locations sampled ..... 58

D. Comparing the frequency of spore-forming bacteria isolated from three

different locations on The Phoenix Mars Lander ...... 60

i. Comparison of the bacterial isolates identified using a combination of 16S

rRNA and MALDI-TOF MS ...... 61

5. DISCUSSION ...... 63

A. Comparing the current Phoenix Mars Lander results to previously

published NASA studies ...... 63

B. Comparing the Phoenix Mars Lander and the Mars Science Laboratory . 65

C. Importance of identifying the bacteria isolated from previous missions .... 67

D. Future Planetary Protection Research ...... 68

REFERENCES ...... 70

LIST OF TABLES

Table 1.1 Top 4 taxonomic identification results for isolate PF2-12.4.1...... 18

Table 3.1 Top 5 taxonomic identification results for isolate PF1A 1.2.2...... 30

LIST OF FIGURES

Figure 1.1 Image of Phoenix Mars Lander with Instruments...... 3

Figure 1.2 Final processing of the Phoenix Mars Lander inside the Payload

Hazardous Servicing Facility (PHSF) at the Kennedy Space Center...... 4

Figure 1.3 High Bay 1 cleanroom floor in JPL's Spacecraft Assembly Facility...... 5

Figure 1.4 General schematic for the identification of bacteria by MALDI-TOF MS

(Clark et al., 2013)...... 11

Figure 1.5 Illustration of the analyte mixed with the matrix being struck by a UV laser beam causing the analyte to be desorbed and ionized (Veeravalli et al., 2019).

...... 12

Figure 1.6 Illustration of how mass changes the time of flight of ions and the subsequent impact with the detector...... 14

Figure 3.1 Flowchart of the methods used to acquire the taxonomic identification for each bacterial isolate...... 21

Figure 3.2 Workflow of methods used for MALDI-TOF MS-based bacterial identification...... 23

Figure 3.3 Flow chart of the direct transfer procedure...... 24

Figure 3.4 Flow chart of the tube extraction procedure...... 26

Figure 3.5 Overview of a Polymerase Chain Reaction Cycle...... 29

Figure 4.1 Categorization of MALDI-TOF MS RTC results...... 33

Figure 4.2 Total genus abundance determined with MALDI-TOF MS...... 34

xii

Figure 4.3 Categorization of 16S rRNA results...... 35

Figure 4.4 Frequency of the genera determined with 16S rRNA...... 36

Figure 4.5 Comparison of the isolates that were identified with both 16S rRNA and

MALDI-TOF MS...... 38

Figure 4.6 Genus abundance for the isolates identified using MALDI-TOF MS or

16S rRNA...... 39

Figure 4.7 Genus abundance for the isolates identified using MALDI-TOF MS or

16S rRNA...... 40

Figure 4.8 Species abundance for the Bacillus genus...... 42

Figure 4.9 Species abundance for the Staphylococcus genus...... 44

Figure 4.10 Species abundance for the Sporosarcina genus...... 45

Figure 4.11 Species abundance for the Paenibacillus genus...... 46

Figure 4.12 Species abundance for the Sphingomonas genus...... 48

Figure 4.13 Species abundance for the Agromyces genus...... 49

Figure 4.14 Species abundance for the Microbacterium genus...... 50

Figure 4.15 Species abundance for the genera with less than 10 isolates identified. .52

Figure 4.16 Taxonomic identities for microbial isolates sampled from the Fairing. .55

Figure 4.17 Taxonomic identities of the microbial isolates sampled from the Robotic

Arm...... 56

Figure 4.18 Taxonomic identities determined for microbial isolates sampled from the MECA...... 58

xiii

Figure 4.19 Taxonomic identities determined for microbial isolates sampled from anywhere except for the MECA, Robotic Arm, and Fairing...... 60

Figure 4.20 Number of combined microbial isolates sampled from the Fairing,

MECA, and Robotic Arm...... 62

Figure 5.1 Number of microbial isolates sampled from the Phoenix Mars Lander and the Mars Science Laboratory...... 67

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1. INTRODUCTION

A. A brief introduction of the Jet Propulsion Laboratory

This thesis research took place at the Jet Propulsion Laboratory (JPL) located in La

Cañada Flintridge, California, United States. JPL first started as the Guggenheim

Aeronautical Laboratory on October 31st, 1936, and was based out of the California

Institute of Technology (Caltech) (Corliss, 1971). Ownership of JPL was transferred to

NASA in December 1958; however, JPL has continued to be managed by Caltech. JPL is home to many historic landmarks, including the Space Flight Operations Facility, which has served as the mission control center for NASA’s Jet Propulsion Laboratory since it was built in 1963.

Since being transferred to NASA, JPL’s mission has been to explore space in the pursuit of scientific discoveries that benefit humanity. Since then JPL has been involved with designing, engineering, and operating several manned and unmanned extraterrestrial missions. This includes the historic Ranger and Surveyor missions to the Moon that paved the way for the historic Apollo landing. JPL is significant as a pioneer of interplanetary exploration with the Mariner missions to Venus, Mars, and Mercury. More recently JPL has been involved in numerous solar system exploration missions such as the Phoenix Mars

Lander, JUNO, Mars Pathfinder, Cold Atom Laboratory (CAL), and Mars 2020 Rover

B. Phoenix Mars Lander i. History of the Phoenix Mars Lander

The Phoenix Mars Lander was a lander constructed by JPL and launched by NASA on Aug. 4, 2007. It landed on the Martian surface on May 25, 2008, and was instrumental

1 in NASA’s pursuit of achieving the four science goals of NASA's long-term Mars

Exploration Program. The four goals are to characterize the climate of Mars, determine whether life ever existed on Mars, characterize the geology of Mars, and prepare for human exploration (Greicius, 2015b). Several spacecraft have been involved in the Mars

Exploration Program. For any mission that lands on Mars, NASA’s Planetary Protection engineers continually monitor bioburden on spacecraft and associated surfaces, beginning with the Viking I mission in 1975. This was followed by Mars Pathfinder (1996), Mars

Odyssey (2001), Mars Exploration Rover (2003), Phoenix Mars Lander (2007), Mars

Science Laboratory (2012), Insight (2018), and most recently Mars 2020. Among them,

Phoenix Mars Lander is one of the early missions to study the environment and atmosphere on Mars. ii. Objectives of the Phoenix Mars Lander

The Phoenix Mars Lander mission had two bold objectives to support the goals of

NASA’s long-term Mars Exploration Program. The first was to study the history of water in the Martian arctic, and the second was to search for evidence of a habitable zone while assessing the potential for life in the ice-soil boundary. Phoenix had a planned mission time of three months but due to weather conditions was able to spend five months total studying the Martian ice, soil, and atmosphere. It was able to achieve this with its onboard equipment, which was composed of the Microscopy, Electrochemistry, and Conductivity

Analyzer (MECA), Thermal and Evolved-Gas Analyzer (TEGA), cameras and the Robotic

Arm, which are all shown in Figure 1.1 (Greicius, 2015a).

Figure 1.1 Image of Phoenix Mars Lander with Instruments. This is an artist’s rendition of the Phoenix Mars Lander on the Martian Surface with many of the instruments visualized and labeled (PC: https://www.nasa.gov/mission_pages/phoenix/multimedia/Lt1.html) (NASA, 2013).

iii. Accomplishments of the Phoenix Mars Lander

The Phoenix Mars Lander has been a fundamental tool in helping NASA to achieve the goals of their long-term Mars Exploration Program. By using the portable lab equipment attached to the Lander, NASA was able to confirm the existence of ice water under the Martian surface for the first time. This is a significant discovery because the presence of ice under the surface has long been speculated by scientists, but this was the first time that the ice was able to be detected and analyzed, finally confirming the hypothesis. The presence of water is imperative for life, so this discovery is crucial. By establishing the presence of water on Mars, scientists can use this information to better understand the possible history of life. Since water is also essential for human survival, this discovery allows for future human exploration missions to Mars to be better planned.

3 iv. Construction of the Phoenix Mars Lander

During construction of the Phoenix Mars Lander special precautionary steps were taken to decrease the microbial burden of the spacecraft to protect against the biological contamination of the Martian surface. The many components of the Phoenix Mars Lander were constructed and assembled inside of cleanrooms such as the Payload Hazardous

Servicing Facility (PHSF) at the Kennedy Space Center shown in Figure 1.2. These cleanrooms utilize air filters and sterilization techniques to greatly reduce the number of microorganisms and spores present.

Figure 1.2 Final processing of the Phoenix Mars Lander. The Phoenix Mars Lander was assembled and processed inside the Payload Hazardous Servicing Facility (PHSF) at the Kennedy Space Center (PC: http://www.launchphotography.com/Phoenix_cleanroom.html).

The Phoenix Mars Lander was housed in a 100,000-class cleanroom at Lockheed

Martin Space Systems' facilities near Denver, Colorado. This is the same class as the cleanrooms located at JPL shown in Figure 1.3, (NASA, 2005). Class 100,000 means that for every cubic foot (approximately 28 liters) of air in the room there can be no more than

100,000 particles greater than or equal to one-half of a micron, and fewer than 700 particles that are five microns or bigger.

Figure 1.3 High Bay 1 cleanroom floor in JPL's Spacecraft Assembly Facility. This is one of the primary cleanrooms utilized at JPL and is essential for reducing the microbial burden of any spacecraft that is being constructed (PC: https://www.jpl.nasa.gov/spaceimages/details.php?id=PIA23519).

During construction of a spacecraft, the cleanliness requirements of the cleanroom are dependent on the calculated forward contamination risk associated with the mission, with Landers and Rovers having more stringent requirements than orbital spacecraft. This is because they are potentially going to encounter extraterrestrial surfaces and Martian water which is most likely to be contaminated by physical contact.

C. The Planetary Protection Mission of JPL i. The mission of COSPAR and Planetary Protection

The Committee on Space Research (COSPAR, 2005) was created by the

International Space Committee to promote the sharing of international scientific research in space, with emphasis on the exchange of results, information and opinions, and to provide a forum for all scientists to discuss problems that may affect scientific space research. The COSPAR Planetary Protection Policy is a set of international guidelines practiced by space-faring agencies such as NASA, the European Space Agency, and the

Japanese Aerospace Exploration Agency. This policy was created for two major reasons.

The first reason is to protect the Earth’s biosphere from backward contamination, which is the potential contamination of the Earth and Moon through any mission returning from an extraterrestrial environment. This is essential for controlling potential contaminants from entering the environment and possibly disrupting the ecosystem. The second reason is to prevent forward contamination, which is the unintentional transfer of microorganisms to extraterrestrial environments caused by spacecraft that we launch from Earth. This is necessary to protect extraterrestrial bodies from biological contaminants that might hinder future scientific work.

To minimize forward contamination NASA monitors the total microbial burden of spacecraft as they are assembled. At JPL, the Planetary Protection Group seeks to advance spacecraft cleanliness, sterilization, and validation technologies for NASA's solar system exploration missions. Planetary Protection oversees creating and enforcing new procedures to ensure that spacecraft's meet stringent cleanliness requirements to limit forward contamination.

But even with multiple preventative measures and many forms of sterilization, it is impossible to remove every microbial contaminant, due to the presence of spore formers and other hardy microorganisms. Samples from NASA's JPL, Kennedy Space Center and

Johnson Space Center show the variety of bacteria that make it into the spaceship assembly areas despite the use of cleanrooms to build the craft. Cleanrooms present a unique microbial environment because of the limited nutrients, the variability of surfaces in the cleanroom and on the spaceship, and the multiple methods of sterilization and filtration. A previous study analyzing both live organisms and relic DNA has shown that cleanrooms can have unique microbiomes that include bacteria that can thrive in harsh environments with limited nutrients, extreme temperatures or pH, or high levels of chlorine (La Duc et al., 2007). ii. The NASA Standard Spore Assay

In the 1960s NASA funded research to devise a standard procedure for evaluating the surface contamination of spacecraft surfaces, which eventually became known as the

“NASA Standard Spore Assay”. The NASA Standard Spore Assay is a cultivation dependent assay that selects for spore-forming microorganisms because they present the greatest likelihood of forward contamination. Also, this assay selectively detects heterotrophic, mesophilic, and aerobic spore-forming organisms. These genera of microorganisms are exceptionally hardy and pose the greatest possible risk of forward contamination (Cooper et al., 2011). Since spores are more likely to survive the very harsh conditions of interplanetary travel, they are a bigger concern when looking for novel life signatures.

7 iii. Current JPL Planetary Protection microbial collection

Since the development of the NASA Standard Spore Assay, it has been used to estimate relative surface cleanliness and overall microbial burden, as well as isolate the bacterial cultures. The Biotechnology and Planetary Protection Group at JPL is currently housing a microbial culture collection with a total of 5,494 bacterial samples. These strains have been isolated from spacecraft hardware and the associated surfaces from 8 different

Mars missions, dating back to the first Viking mission in 1975. Among them, there were

824 bacterial isolates collected from the spacecraft and cleanroom surfaces before, during, and after the construction of the Phoenix Mars Lander. The purpose of my research is to identify and characterize these bacterial specimens.

Only a fraction of the isolates collected from the Phoenix Mars Mission has been studied using partial 16S rRNA sequences (Smith et al., 2017). This partial sequencing of less than 1000 bp was insufficient to accurately perform taxonomic identification. No further investigation went into the isolates that were novel species, or into samples that were yielding unsatisfactory 16S rRNA results. Despite the previous work that has been done, greater than 80% of the 824 isolates collected during the construction of the Phoenix

Mars Lander remain unidentified. This presents a large gap in our understanding of which microorganisms have been isolated from the Phoenix Mars Lander. Hopefully, by closing this gap in knowledge we can more accurately document the microorganisms which have been isolated from vehicle assembly rooms and screen for any potential spore-forming bacteria.

8 iv. Present and future research with GESAM

This research has been funded by NASA’s Planetary Protection Research (PPR) program (17-PPR17-0007) to Dr. Vaishampayan of Planetary Protection Group at JPL under the Genome Encyclopedia of Spacecraft Associated Microorganisms (GESAM) project. This project has several goals, the first of which is to perform taxonomic identification of all the bacterial isolates collected from the previous NASA missions.

Using this information, we can determine the most abundant, most recurring, and identify any novel bacterial species. To achieve this, the GESAM project is using a combination of

MALDI-TOF Mass Spectrometry and 16S rRNA. Another goal of the project is to use whole-genome sequencing (WGS) to explore the microorganism's gene contents and establish their functional traits.

Also, NASA can use this information to better understand the microbial communities residing in cleanrooms, allowing them to improve upon their sterilization techniques. This research will greatly assist NASA and JPL’s ability to accurately predict present and future forward contamination risks.

D. Bacterial taxonomic identification using Matrix-Assisted Laser Desorption

Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) i. History of MALDI-TOF MS

Matrix-Assisted Laser Desorption Ionization (MALDI) was pioneered by three different scientists. The first two scientists were Franz Hillencamp and Michael Karas, in

1985 they discovered that the amino acid alanine could be more easily ionized if mixed with tryptophan and irradiated with a 266 nm pulse. This is because of the energy-

9 absorbing properties of tryptophan which assist in ionizing the non-absorbent alanine

(Karas et al., 1987). In 1987, the Nobel laureate Koichi Tanaka found that using a 337 nm nitrogen laser and a mixture of cobalt particles into glycerol, even larger proteins can be ionized (Tanaka et al., 1988). This demonstrated that with the proper combination of matrix and certain laser wavelengths, a large range of proteins could be ionized. The time-of-flight

(TOF) mass spectrometer was first reportedly created and used by A. E. Cameron and D.

F. Eggers Jr with the Y-12 National Security Complex in 1948, which is often paired with the MALDI technique to determine the mass-to-charge ratio of ions (Campana, 1987).

MALDI-TOF MS is becoming increasingly prevalent in clinical laboratories because of its accurate and rapid bacterial identification. The use of this procedure in clinical laboratories has been tested by researchers in several different studies (Bessède et al., 2011; Bizzini et al., 2011). The identification and classification of the microorganisms are possible because of the unique proteomic fingerprint determined using MALDI-TOF

MS, with the complete process demonstrated in Figure 1.4. Past studies have found that

MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (Bessède et al., 2011; Seng et al., 2009). Any discrepancies that were observed were associated with an absence of enough reference organisms within the MALDI-TOF MS database, it was concluded that MALDI-TOF MS can be implemented for routine identification of bacteria in a medical microbiology laboratory

(Carbonnelle et al., 2012; Rahi et al., 2016).

10 ii. MALDI-TOF MS matrix associated sample preparation

To quickly identify the bacterial isolates, each sample that was successfully revived was first identified using MALDI-TOF MS by measuring each protein’s molecular mass.

Bacterial growth was isolated from plated culture media (or concentrated from broth culture by centrifugation in specific cases) and applied directly onto the MALDI test plate.

Samples are then overlaid with α-Cyano-4-hydroxycinnamic acid (HCCA) matrix and dried as shown in Figure 1.4.

Figure 1.4 General schematic for the identification of bacteria by MALDI-TOF MS. After the reagents and biomass from pure cultures are applied to the target it can be placed in the Bruker Biotyper System (Clark et al., 2013).

11 iii. MALDI-TOF MS Laser Desorption and Ionization

Once appropriately processed samples are added to the MALDI plate, overlaid with matrix, and dried, the sample is bombarded by a high energy laser. The analyte is embedded in a matrix compound deposited on a solid surface called a target. After a very brief laser pulse, the irradiated spot is rapidly heated and becomes vibrationally excited (Figure 1.5).

This bombardment results in the sublimation and ionization of both the sample and matrix

(Clark et al., 2013).

Figure 1.5 Illustration of the analyte mixed with the matrix being struck by a UV laser beam causing the analyte to be desorbed and ionized. This diagram shows the simplified mechanism for how proteins are desorped and ionized using MALDI-TOF MS (Veeravalli et al., 2019).

12 iv. MALDI-TOF MS Time of Flight

These generated ions are separated based on their mass-to-charge ratio via a TOF tube, ions with a smaller m/z value (lighter ions) and more highly charged ions move faster through the drift space until they reach the detector (Shimadzu, n.d.). The time of ion flight differs according to the mass-to-charge ratio (m/z) value of the ion, so if all the proteins start their journey at the same time, the lighter ones will arrive earlier at the detector than the heavier ones, which is illustrated in Figure 1.6. This difference in travel time for individual proteins means that each organism has a unique proteomic fingerprint.

➢ Figure 1.6 Illustration of how mass changes the time of flight of ions and the subsequent impact with the detector. If all the proteins start their journey at the same time, those with a lower mass-to-charge ratio will arrive at the detector earlier (PC: https://www.shimadzu.com/an/lifescience/maldi/princpl1.html).

v. JPL in-house MALDI-TOF MS microbial database

The microbiome found on the Phoenix spacecraft and its associated surfaces is

comprised of many spore-forming bacterial species, which could be due to the rigorous 14 sterilization techniques at JPL, and the inherent selective nature of the NASA Standard

Spore Assay. Currently, the Bruker manufacturer-provided MALDI-TOF database is focused on clinical species. Preliminary studies found that this database could successfully yield correct identification for only 8% of organisms, according to 16S rRNA gene-based identification. Due to this technical bottleneck, JPL researchers have been creating a custom database filled with isolates from the JPL microbial culture collection for the last few years (Seuylemezian et al., 2018). vi. Comparison of MALDI-TOF MS proteomic fingerprint to JPL database

The spectral representation of these ions can be generated by MALDI-TOF MS to produce proteomic fingerprints such as the representative mass spectra of different Bacillus species shown in Figure 1.7. These unique mass spectra can be analyzed by the MS software, which can be used to create a unique main spectral profile (MSP). These MSPs were created by taking bacteria that have a strain-level identification and placing biomass onto eight duplicate target spots on the target plate, using MALDI-TOF MS the spectra were obtained in three separate intervals per target spot (Zhang et al., 2015). The isolates can then be classified in real-time by comparing their spectra to the MSPs in the database.

The real-time classification (RTC) profiles generated from a sample can then be systematically compared to the JPL database of reference MSPs and matched to either identical or the most related spectra contained in the database, generating an identification for the bacterial sample (Seuylemezian et al., 2018). If any of the samples could not be identified using that method because no spectra were generated or there was no suitable match in the database, then 16S rRNA was extracted and the bacterial isolates were identified based on the 16S rRNA sequence.

Figure 1.7 Representative MALDI-TOF mass spectra for four different species in the Bacillus genus in the mass range of 2 to 14 kDa. These spectra show the unique proteomic fingerprint that is observed for these four different bacillus species (Lasch et al., 2009). 16 vii. Advantages of MALDI-TOF MS over 16S rRNA gene sequencing

MALDI-TOF MS is not only rapid but relatively inexpensive, with a 90% reduction in reagent costs compared to conventional 16S rRNA gene sequencing (Tran et al., 2015).

MALDI-TOF MS also has many technical advantages over traditional 16S rRNA gene sequencing. When identifying bacteria, MALDI-TOF MS is capable of accurately resolving the taxonomic identity of closely related bacterial species. The equipment is sensitive enough to detect potentially novel species that exhibit >99% sequence similarity with validly described species (Seuylemezian et al., 2018). A major reason for the slow adaptation of this technique for bacterial identification is due to the limited bacterial spectral libraries in its database available for use, and that most existing libraries are specifically for clinical isolates (Vargha et al., 2006).

E. Bacterial taxonomic identification using rRNA gene sequencing and the

EzBioCloud and SILVA 16S rRNA database

RNA sequence databases can use genome sequences from previously identified microorganisms for informative and precise classification and identification of some bacteria. The databases that were used for this research were SILVA and EzBioCloud, which is a quality-controlled database that can provide hierarchal taxonomic identifications using the 16S rRNA gene and genome sequences. Since the current species definition is based on the comparison of genome sequences between different strains in each species, using a genome database with correct taxonomic information is of paramount importance.

This increases the effectiveness when examining prokaryotic diversity, identifying novel

17 species, and for routine identifications of spacecraft associated microorganisms (Yoon et al., 2017).

Since some species share identical or almost identical 16S rRNA sequences when identified based on rRNA they will match closely to several species as shown in Table 1.1 by the isolate PF2-12.4.1 which is a part of the Bacillus subtilis taxonomic group. Because of this, organisms that belong to these groups cannot be differentiated solely by 16S rRNA sequences and are instead placed into taxonomic groups. An example of this is the "Bacillus megaterium group" which is made up of the following species: Bacillus aryabhattai,

Bacillus flexus, Bacillus megaterium, and Bacillus qingshengii. When analyzing a sequence acquired from one of these groups, they cannot be identified correctly based on the 16S rRNA sequence and will instead be identified as the taxonomic group that they belong to (Chun, 2015).

Table 1.1 Top 4 taxonomic identification results for isolate PF2-12.4.1. All 4 matches for the 1426 nucleotide fragment are above the 98.6% cutoff for species-level identification. This is because the rRNA sequence of the top 4 species/subspecies is identical. PF2-12.4.1 Taxonomic Identification % Similarity % Completeness Bacillus subtilis subsp. spizizenii NRRL B- Match #1 23049 99.92987377 100 Match #2 Bacillus tequilensis KCTC 13622 99.92987377 100 Bacillus subtilis subsp. inaquosorum KCTC Match #3 13429 99.92987377 100 Match #4 Bacillus cabrialesii TE3 99.92987377 100

18 2. OBJECTIVE

These microbial isolates are a microbiological ‘time capsule’ which will give us a better understanding of the microorganisms that were present during the construction of the Phoenix Mars Lander. Forward contamination, which is a consequence of space exploration, can potentially cause the confounding of future life-detection experiments on extraterrestrial bodies. However, not all microorganisms have the same potential to cause forward contamination, and understanding which taxa are of greatest abundance and concern due to their functional traits plays a significant role in NASA’s ability to accurately assess and predict Planetary Protection risks.

The objective of this research was to determine if MALDI-TOF MS can be used in combination with 16S rRNA gene sequencing for identifying microorganisms collected from the clean rooms and other locations where spacecraft are assembled. Another goal of this study is to determine whether there is a statistically significant association between the location where the samples were collected, and the spore-forming potential of the bacteria isolated during the construction of the Phoenix Mars Lander. Once pure cultures of these bacteria were isolated each organism was identified using MALDI-TOF MS and/or 16S rRNA sequencing. After identification, two new glycerol stocks were created from every pure culture, and the DNA was extracted, to ensure that there can be future analysis with these microorganisms. Once this was completed the final step was to screen the data for the most recurrent genera and species, and any potential novel isolates, as well as any possible ways to improve the MALDI-TOF MS database at JPL by determining problematic MSPs.

3. MATERIALS AND METHODS

A. Bacterial sample collection from Phoenix Mars Lander

The bacterial samples were collected from the Phoenix Mars Lander flight hardware and any associated surfaces inside the spacecraft-assembly cleanrooms. This was done by using Texwipe TX3211 wipes and Puritan 806-WC cotton swabs. Any samples acquired were then warmed to 80°C and heat-shocked for a total of 15 minutes, then pour plated on TSA plates. They were then incubated at 32°C for 72 hours and sub-cultured to obtain pure cultures (NASA, 2010). These samples were then frozen and stored at -80 °C in JPL’s culture collection freezers, the following procedures take place using these isolates and are outlined in Figure 3.1.

Figure 3.1 Flowchart of the methods used to acquire the taxonomic identification for each bacterial isolate. This flowchart outlines how each isolate was analyzed to determine the taxonomic identification with either MALDI-TOF MS or 16S rRNA sequencing.

B. Revive isolates from JPL Planetary Protection microbial collection

Each bacterial isolate was revived from glycerol stocks stored at -80 °C and incubated on tryptic soy agar (TSA) at 32 °C. The macroscopic morphology of each colony was inspected, and visual purity was confirmed. Upon confirmation of a pure culture, two new glycerol stocks were created with new unique barcodes assigned to that isolate. If any isolates did not show growth within 72 hours of incubation or could not be located at its documented position, then efforts were taken to revive a remaining duplicate stock from the microbial culture collection. All cultures determined to be pure during this step were analyzed using MALDI-TOF MS within 24 hours of showing growth.

C. Rapid and systematic identification using MALDI-TOF MS

All pure cultures obtained were processed using MALDI-TOF MS for a systematic and rapid taxonomic identification of the Phoenix Mars Mission’s bacterial isolates. The procedure for MALDI-TOF MS consists of placing biomass from a pure culture and transferring it to a 96 well stainless-steel target, then the Bruker MALDI Biotyper system generates a unique spectrum that is then compared to several databases (Figure 3.2). Two different techniques can be used for transferring microbial biomass onto the MALDI-TOF

MS target, direct transfer, and tube extraction.

Figure 3.2 Workflow of methods used for MALDI-TOF MS-based bacterial identification. After acquiring pure cultures and using either the direct transfer or tube extraction method to place the biomass onto the target, the Bruker MALDI Biotyper can be used to generate RTCs.

i. Direct transfer technique for MALDI-TOF MS

Direct transfer is the more common technique because it is quicker and uses fewer reagents, however, there are certain microorganisms where the direct transfer technique will not provide results. All isolates that generated no spectra when transferred onto the target using direct transfer were rerun using tube extraction. ii. Direct transfer procedure

When using the direct transfer technique which is shown in Figure 3.3, biomass was removed from isolated colonies using a sterile toothpick and spotted in quadruplicate onto a 96 well MALDI-TOF MS polished steel target. The bacterial cells were then lysed by spotting each well with 1 µL of 70% formic acid. After letting the 70% formic acid dry

23 the next step was to place 1 µL of HCCA matrix (Zymo Research, catalog #R236) onto each of the 96 wells. The HCCA matrix enables highly sensitive MALDI-TOF MS measurements of peptides and proteins ranging in size from 0.7 to 20 kDa (Bruker,

2017). When an isolates spectrum had no peaks that were generated using the direct transfer technique, the organism was retested using tube extraction, and the spectra would replace the no peaks result.

Figure 3.3 Flow chart of the direct transfer procedure. This was the primary method used to place biomass onto the MALDI-TOF MS targets.

iii. Tube extraction technique for MALDI-TOF MS

Tube extraction is not used as frequently as a direct transfer because it is a time- intensive process and requires many reagents in comparison. Most microorganisms that require tube extraction can be determined after incubation due to their unique macroscopic

24 morphology. Any microbial isolates revived that appeared slimy or crusty were run on

MALDI-TOF MS using the tube extraction technique. iv. Tube extraction procedure

When using the tube extraction technique which is shown in Figure 3.4, biomass from pure cultures was first placed into 1 mL of 75% Ethyl Alcohol. The samples were then vortexed for 15 minutes to fully mix the biomass and ethanol. After mixing, the samples were centrifuged for 2 minutes at 15000 RPM to pellet the biomass. After separation, the 1 mL of ethanol was removed and replaced with 100 µL of 70% formic acid, and then vortexed for 15 minutes. Then 100 µL of Acetonitrile was added to the mixture and it was centrifuged for 2 minutes at 15000 RPM. Once completed, 1 µL of the supernatant was placed onto each well of a 96 well MALDI-TOF MS polished steel target.

After letting the supernatant dry the next step is to place 1 µL of HCCA matrix (Zymo

Research, catalog #R236) onto each of the 96 wells.

Figure 3.4 Flow chart of the tube extraction procedure. This method was used for isolates that had a slimy or crusty morphology, and for any of the samples that did not generate spectra when using the direct transfer technique.

26 v. Taxonomic identification using Bruker MALDI Biotyper System

The Bruker MALDI Biotyper System was used for taxonomic identification of the microbial isolates from the Phoenix Mars Mission. By using the RTC software the spectra were obtained from each isolate and compared to the JPL inhouse database of MALDI-

TOF MSPs. These spectra are created using identified bacteria under a reproducible standard operating procedure. Each bacterial isolate was tested in quadruplicate following the procedure outlined in Seuylemezian et al. in 2018. When compared to the MSPs, if the

RTC results in at least 3 identical matches with a log similarity score ≥2.2 they were considered reliably identified at the species level to the database organism. Those isolates producing spectra with no match to the database or a log score of <2.2 were subjected to

16S rRNA gene sequencing-based identification. Each spectrum was analyzed and sorted into groups depending on what taxonomic level the database could identify each isolate.

D. Zymo DNA extraction of Phoenix Isolates

All pure cultures revived from the Phoenix mission were placed into

ZymoBIOMICS MagBead DNA/RNA tubes and sent for DNA Extraction. The

ZymoBIOMICS MagBead DNA/RNA Kit provides a high-throughput, magnetic bead- based purification of both high-quality DNA and total RNA (including small/microRNAs) from the same starting sample. The provided DNA/RNA Shield inactivates infectious agents and is ideal for sample storage at ambient temperatures. The extraction method utilizes magnetic beads for DNA/RNA extraction without the use of phenol and is eluted into ≥50 µl of ZymoBIOMICS DNase/RNase-Free Water. When DNA and RNA are stored in this tube it can then be used for any downstream application including Next-Gen

Sequencing and RT-qPCR (Zymo, n.d.). ZymoBIOMICS extracted the DNA containing the 16S rRNA genes and then sent it to the Planetary Protection Group at JPL for PCR

E. Bacterial 16S rRNA gene amplification and sequencing

Colony PCR was performed by the Planetary Protection Group on a total of 268 bacteria that were isolated from the Phoenix Mars Lander. The DNA containing the 16S rRNA gene was amplified using PCR and two different primers. The two primers that were used for PCR were 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT

TAC CTT GTT ACG ACT T-3′), these primers were used under the following conditions.

First, the mixture was heated for 5 minutes at 95°C before going through 35 thermo- cycles which are shown in Figure 3.5. The 35 thermo-cycles consisted of keeping the sample at 95°C for 50 seconds to denature the DNA and separate it into two single strands.

It was then annealed by being cooled to 55°C for 50 seconds to attach the DNA primers to the 3’ ends of the target sequence. The final step of the thermocycling was elongation or extension, which was done by being placed at 72°C for 55 seconds. Finally, the sample was placed at 72°C for 10 minutes following the procedure outlined by Frank et al. in 2008.

To remove the primers and any excess dNTPs from the amplified PCR products,

10 μl ddH2O, 20 μl of Exonuclease I (New England Biolabs, Ipswich, MA, United States), and 5 μl Antarctic Phosphatase (New England Biolabs, Ipswich, MA, United States), were added to 10 μl amplified PCR product. PCR products and enzyme mix were incubated at

37°C for 45 minutes, then the enzymes were inactivated by placing the mix at 85°C for 15 minutes. The Macrogen Corporation (Rockville, MD, United States) used Sanger sequencing and Lasergene software (DNASTAR, Wisconsin, United States) to produce sequence data.

This sequence data was then processed using CLC Genomics Workbench 20 by

CLC Bio (Cambridge, MA, United States). The forward, internal, and reverse sequences for each isolate were first taken and trimmed to remove any low-quality sequence regions from the downstream analysis. The forward, internal, and reverse sequences were then assembled to obtain near full-length 16S rRNA gene sequences comprised of around 1400 base pairs. The fully assembled sequences were then exported in the form of a FASTA so that the sequences could be compared to a 16S rRNA gene type strain database.

Figure 3.5 Overview of a Polymerase Chain Reaction Cycle. The three steps of the Polymerase Chain Reaction Cycle are denaturing, annealing, and extension. (Cornell, n.d.).

F. Comparison of rRNA gene sequences to the EZBioCloud and the SILVA

LTP type strain databases

To create the EZBioCloud 16S rRNA gene database, the researchers took whole- genome assemblies from the NCBI Assembly Database and screened them for low quality.

As a result, the database is made of 61, 700 species/phylotypes, including 13 ,132 with validly published names, and 62 ,362 whole-genome assemblies that were identified taxonomically at the genus, species and subspecies levels (Yoon et al., 2017). The information from their database was used to provide nucleotide similarity and completeness for each sequence as shown in Table 3.1. EZBioCloud also provides information about genomic properties, such as DNA G+C content, genome size, and the occurrence of a genus or higher taxa in the human microbiome. This bioinformatic tool was used to get bacterial taxonomic identifications using 16S rRNA genome sequences.

The database and search tools are both available at www.ezbiocloud.net/ (Yoon et al.,

2017).

Table 3.1 Top 5 taxonomic identification results for isolate PF1A 1.2.2. PF1A 1.2.2 Taxonomic Identification % Similarity % Completeness Match #1 Bacillus halosaccharovorans E33 99.21540656 97.55766621 Match #2 Bacillus niabensis 4T19 98.51694915 99.72862958 Match #3 Bacillus endolithicus JC267 98.06173726 94.77257298 Match #4 Bacillus weihaiensis Alg07 97.95342272 100 Match #5 Bacillus crassostreae JSM 100118 97.74170783 99.72862958

Sequences were compared to the EZBioCloud database and reconfirmed with the

SILVA LTP type strain database. If a samples sequences had ≥98.6% sequence similarity to a database entry they were identified at the species level. Any sequences with >95% sequence similarity to a database entry were given a genus level identification (Parulekar et al., 2017; Tourlousse et al., 2017). For any sequences that had <95% sequence similarity

30 in its original orientation, the reverse complement of the sequence was compared to the database to determine a taxonomic identification.

4. RESULTS

A. Retrieval of bacterial isolates from JPL culture collection freezers and the creation of pure cultures

There was a total of 884 isolates collected from cleanrooms during and after the construction of the Phoenix Mars Lander. After more than a decade of being analyzed and stored in the Planetary Protection culture collection, some of the isolates were unavailable or had mixed or no growth after incubation. After isolating the pure cultures and reviving all available duplicate frozen samples there were a total of 637 real-time classification

(RTC) profiles generated through MALDI-TOF MS.

B. Taxonomic identification of Phoenix bacterial isolates using MALDI-TOF MS

MALDI-TOF MS captures a larger protein profile than what is represented by the

16S rRNA genes. This allows MALDI-TOF MS to resolve taxonomic differences between bacterial isolates that could not be separated by traditional rRNA gene sequencing

(Seuylemezian et al., 2018). Because of this differential, MALDI-TOF MS was the primary approach used to taxonomically identify the bacterial isolates retrieved from the Phoenix

Mars Lander and its associated cleanrooms. i. Assessment of MALDI-TOF MS RTC Results

The bacterial isolates analyzed by MALDI-TOF MS were classified depending on the results of the RTC software. Of the 637 RTCs created 326 isolates had at least 3 spectra with a log score ≥2.2 and were given a species-level taxonomic identification. A total of

112 isolates are potential novel species/ spectra unavailable and were given a genus level identification because they had at least 3 spectra with a log score ≥2.0. Another 150 RTCs

32 had fewer than 3 spectra with a log score ≥2.0, which do not match to any MSPs at the genus level taxonomic identification and are potentially novel genera. The next group is made up of 41 samples with isolates that were matched to problematic MSPs that had 2 or more spectra matching to different species or genera with a log score of ≥2.2 and ≥2.0, respectively. These problems are indicative of an issue with the MSP database being used to identify these samples, such as a species being mistakenly identified within the MALDI-

TOF MS database. The remaining 8 organisms all had no peaks found for any of the 4 spectra generated, this was a total of only 1% as shown in Figure 4.1.

No Match, 150, 24%

Species ID, 326,

Genus ID, 112, 18%

No Peaks, 8, MSP Issue, 41, 6%

Figure 4.1 Categorization of MALDI-TOF MS RTC results. The 637 sets of four spectra were placed into groups depending on the results of the RTCs created from each isolate.

33 ii. Identification of genera associated with the Phoenix Mars Lander using MALDI-

TOF MS

Of the 326 isolates that had at least three scores above the ≥2.2 log score cutoff for

MALDI-TOF MS, 293 isolates were reconfirmed using the original 16S rRNA gene results or (WGS) Whole Genome Sequence results associated with the MSP. The bacteria identified are mostly comprised of spore-formers (75%), with 172 (59%) of the isolates belonging to the Bacillus genus. (Figure 4.2). The next most recurrent genera are

Staphylococcus with 35 (12%) isolates, Sporosarcina with 29 (10%) isolates, Agromyces with 12 (4%) isolates, and Paenibacillus with 11 (4%) isolates total. The remaining 19 genera make up only 34 (12%) of the total isolates identified from The Phoenix Mars

Lander using MALDI-TOF MS (Figure 4.3).

MALDI-TOF MS Identified Genera 200 180 172 160 140 120 100 80 60 NUMBER OF ISOLATES OF NUMBER 35 40 29 20 12 11 8 5 5 4 3 2 1 1 1 1 1 1 1 0

GENUS

Figure 4.2 Total genus abundance determined with MALDI-TOF MS. The 293 RTCs that had three or more spectra with a log score ≥2.2 were verified by 16S rRNA results and assigned species-level taxonomic identifications to determine the total prevalence of each genus associated with the Phoenix Mars Lander.

C. Taxonomic identification of Phoenix bacterial isolates using 16S rRNA i. Assessment of 16S rRNA gene sequence results

The remaining bacterial isolates were analyzed using 16S rRNA sequencing and were classified depending on the comparative sequence analyses of 16S rRNA genes.

There was a total of 268 sequences confirmed, with 219 isolates that were matched to a database organism above ≥98.6% and given a species-level taxonomic identification. The remaining 49 isolates had a sequence similarity of ≥95% so they were assigned a genus level identification and are potential novel species that will be verified with whole-genome sequencing (Figure 4.3).

Species ID, 219, 82%

Genus ID, 49, 18%

Figure 4.3 Categorization of 16S rRNA results. The 268 isolates were placed into groups depending on the results of the 16S rRNA sequence analysis. Isolates that had a sequence similarity ≥98.6% were assigned a species-level taxonomic identification, and those ≥95% similarity were assigned a genus-level identification.

35 ii. Identification of genera associated with the Phoenix Mars Lander using 16S rRNA genes

Among the 268 bacterial isolates identified by 16S rRNA sequencing, 66% were comprised of spore-formers, with 140 (52%) of the isolates belonging to the Bacillus genus.

(Figure 4.4). The next most recurrent genera are Staphylococcus with 31 (12%) isolates,

Sphingomonas with 14 (5%) isolates, Paenibacillus with 9 (3%) isolates, and Ralstonia with 9 (3%) isolates total. The remaining 20 genera make up only 65 (24%) of the total isolates identified from The Phoenix Mars Lander using 16S rRNA genes (Figure 4.4).

16S rRNA Identified Genera 160 140 140

120

100

NUUMBER OF ISOLATES NUUMBER OF 40 31

20 14 9 9 7 7 6 6 4 4 4 4 4 3 3 2 2 2 2 1 1 1 1 1 0 Dyella Bacillus Leifsonia Ralstonia Variovorax Agromyces Fictibacillus Cupriavidus Brevibacillus Burkholderia Sporosarcina Paenibacillus Lysinibacillus Rhodococcus Sphingomonas Oceanobacillus Psychrobacillus Brevibacterium Staphylococcus Brevundimonas Microbacterium Corynebacterium Novosphingobium Chryseobacterium Methylobacterium GENUS

Figure 4.4 Frequency of the genera determined with 16S rRNA. These 268 isolates had ≥95% sequence similarity and were identified to the genus level using 16S rRNA sequence analysis.

D. Taxonomic identification of bacterial isolates by combining MALDI-TOF MS and 16S rRNA results

To gain a better understanding of the isolates that were sampled from the Phoenix

Mars Lander, the 293 bacteria identified with MALDI-TOF MS, and the 268 bacterial isolates identified with 16S rRNA sequences were combined. When samples were identified by both methods, the species level taxonomic identification determined with

MALDI-TOF MS was used. This was done because 16S rRNA is limited when identifying the species level due to its smaller genomic representation. i. Comparing the isolates that were identified with both methods

There was a total of 218 isolates that were initially identified to the genus level by both MALDI-TOF MS and 16S rRNA. Of these 218 isolates, there were 184 (84.4%) that had the same genus-level taxonomic identification for both methods of identification. For the 87 isolates that were identified to the species level by both methods, a total of 70

(80.5%) matched to the same species or taxonomic group (Figure 4.5). The observable differences between these two methods of identification could have several explanations.

These differences could be caused by a lack of microorganisms within the JPL MSP database or issues caused by outdated or incorrect MSPs within the database. Some of the species-level differences could also be caused by the potential for MALDI-TOF MS to more accurately group microorganisms based on strain-level variations. For isolates that were identified by both methods but to different organisms, they were not added to the list of successful identifications until the conflicting results were corrected.

Comparison of 16S rRNA and MALDI-TOF MS Results

84.4%, 184 15.6%, 34

Genus Identification

80.5%, 70 19.5%, 17

Species Identification

0 50 100 150 200 250 NUMBER OF ISOLATES

Match Don’t Match

Figure 4.5 Comparison of the isolates that were identified with both 16S rRNA and MALDI-TOF MS. The isolates that were identified to the genus level or species level were separated based on whether the two forms of identification matched. ii. Combined identification using MALDI-TOF MS and 16S rRNA To determine the genera associated with the Phoenix Mars Lander the two identification methods were combined to provide a perfect species-level identification for

561 isolates. The bacteria identified were mostly comprised of spore-formers, with 397

(71%) of the organisms identified belonging to a spore-forming genus. The most identified genus was the spore-forming Bacillus genus, with 312 (56%) of the isolates identified

(Figure 4.6). The next most recurrent spore-forming genera were Sporosarcina with 36

(6%) isolates, Paenibacillus with 20 (4%) isolates, Psychrobacillus with 9 (2%) isolates, and Fictibacillus with 9 (2%) isolates. The 3-remaining spore-forming genera were approximately 2% (11) of the total of isolates identified from the Phoenix Mars Lander.

The non-spore-forming genera were very diverse, with isolates belonging to 22 different

38 genera, the most recurrent were Staphylococcus with 66 (12%) isolates, Sphingomonas with 19 (3%) isolates, Agromyces with 16 (3%) isolates, and Microbacterium with 10 (2%) isolates. The remaining 18 non-spore-forming genera make up only 53 (9%) of the total isolates identified from The Phoenix Mars Lander (Figure 4.7).

Other, 82, 15% Microbacterium, 10, 2% Agromyces, 16, 3%

Sphingomonas, 19, 3%

Paenibacillus, 20, 3%

Sporosarcina, 36, 6%

Staphylococcus, 66, 12%

Bacillus, 312, 56%

Figure 4.6 Genus abundance for the isolates identified using MALDI-TOF MS or 16S rRNA. These 561 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

Total Combined Genus Count

Bacillus 312

Staphylococcus 66

Sporosarcina 36

Paenibacillus 20

Sphingomonas 19

Agromyces 16

Microbacterium 10

Ralstonia 9

Psychrobacillus 9

Fictibacillus 9

Methylobacterium 8

Lysinibacillus 6

Cupriavidus 6

Novosphingobium 5

Corynebacterium 5

GENUS Chryseobacterium 4

Oceanobacillus 3

Burkholderia 3

Kocuria 2

Brevibacterium 2

Brevibacillus 2

Variovorax 1

Stenotrophomonas 1

Rhodococcus 1

Pseudarthrobacter 1

Leifsonia 1

Dyella 1

Burkholderia 1

Brevundimonas 1

Acinetobacter 1

0 50 100 150 200 250 300 350 NUMBER OF ISOLATES

Figure 4.7 Genus abundance for the isolates identified using MALDI-TOF MS or 16S rRNA. These 561 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing. 40 iii. Frequency of species belonging to the Bacillus genus

The Bacillus genus is a member of the phylum Firmicutes and is comprised of spore-forming, Gram-positive, rod-shaped bacteria. Because the spores of many Bacillus species are resistant to heat, radiation, disinfectants, and desiccation, they are difficult to eliminate from spacecraft-associated surfaces and are a frequent cause of contamination in cleanrooms (Nicholson et al., 2000). Bacillus was the most prevalent genus identified by

MALDI-TOF MS and 16S rRNA, with 56% of the total isolates being identified as a

Bacillus species. Overall, there were 312 isolates from 37 different species or taxonomic groups represented within the Bacillus genus, there were also 65 (21%) isolates that were not identifiable at the species level due to there being no suitable match within the 16S rRNA database. The top 3 most identified species were B. oceanisediminis with 36 (12%) isolates, B. megaterium with 23 (7%) isolates, and B. licheniformis with 19 (6%) isolates.

The top 3 most identified Bacillus taxonomic groups encompass 17% of the total isolates identified from the Bacillus genus, these include the B. subtilis group with 19 (6%) isolates,

B. pumilus group with 16 (5%) isolates, and B. firmus group with 14 (4%) isolates. The remaining 38% was composed of 31 different Bacillus species and taxonomic groups which are shown in Figure 4.8.

Bacillus Species Count

B. sp. 65 B. oceanisediminis 36 B. megaterium 23 B. subtilis TG 19 B. licheniformis 19 B. herbersteinensis 18 B. pumilus TG 16 B. firmus TG 14 B. flexus 11 B. subtilis subsp. subtilis 9 B. ginsengi 8 B. niabensis 7 B. pumilus 6 B. cereus TG 6 B. muralis TG 5 B. galactosidilyticus 5 B. altitudinis 5 B. encimensis 4 B. amyloliquefaciens TG 4 B. endophyticus TG 3 SPECIES B. endophyticus 3 B. circulans 3 B. axarquiensis 3 B. aerius 3 B. vallismortis 2 B. rhizosphaerae 2 B. niacini 2 B. toyonensis 1 B. tequilensis 1 B. subtilis subsp. inaquosorum 1 B. sonorensis TG 1 B. safensis 1 B. mojavensis TG 1 B. horikoshii 1 B. fumarioli TG 1 B. decisifrondis TG 1 B. clausii 1 B. barcinonensis TG 1 0 10 20 30 40 50 60 70 NUMBER OF ISOLATES

Figure 4.8 Species abundance for the Bacillus genus. These 312 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

iv. Frequency of species belonging to the Staphylococcus genus

The Staphylococcus genus is a member of the phylum Firmicutes and is comprised of non-spore-forming, Gram-positive, sphere-shaped bacteria. Since many of the

Staphylococcus species are a part of the human microbial flora and normally reside on the skin and mucous membranes, this genus could be difficult to eliminate from cleanrooms because of its close association with humans. (Jacquemyn et al., 2013). Staphylococcus was the second most prevalent genus, and the most prevalent non-spore forming genus identified by 16S rRNA, with 12% of the total isolates being identified as a Staphylococcus species. All the Staphylococcus species were identified using MALDI-TOF MS and 16S rRNA sequencing and placed into 12 different taxonomic groups or species. The top 5 most identified species or taxonomic groups within the Staphylococcus genus make up 82% of the isolates belonging to this genus. These include S. warneri with 17 (26%) isolates, the

S. hominis group with 15 (23%) isolates, S. epidermidis group with 11 (17%) isolates, S. saprophyticus subsp. saprophyticus with 7 (11%) isolates, and S. pasteuri with 4 (6%) isolates. The remaining 18% of (12) isolates belong to 7 different taxonomic groups or species (Figure 4.9).

Staphylococcus Species Count

S. warneri 17

S. hominis TG 15

S. epidermidis TG 11

S. saprophyticus subsp. saprophyticus 7

S. pasteuri 4

S. aureus subsp. anaerobius 3

S. equorum TG 2 SPECIESE S. equorum subsp. equorum 2

S. capitis subsp. ureolyticus 2

S. saprophyticus TG 1

S. pettenkoferi 1

S. haemolyticus 1

0 2 4 6 8 10 12 14 16 18 NUMBER OF ISOLATES

Figure 4.9 Species abundance for the Staphylococcus genus. These 66 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

v. Frequency of species belonging to the Sporosarcina genus

The Sporosarcina genus is a member of the phylum Firmicutes and is comprised of spore-forming, Gram-positive, sphere, or rod-shaped bacteria. Since the endospores of some Sporosarcina species have been shown to be resistant to heating and desiccation, they could be difficult to eliminate from spacecraft-associated surfaces and can cause contamination in cleanrooms, though not to the same frequency as the Bacillus genus

(Fahmy et al., 1985). Sporosarcina was the third most prevalent genus, and the second most prevalent spore-forming genus identified by a combination of MALDI-TOF MS and

16S rRNA, with 6% of the total isolates being identified as belonging to the Sporosarcina genus. Overall, there was only 1 species represented within the Sporosarcina genus and there were 7 (19%) isolates that were not identifiable at the species level due to there being 44 no suitable match within the 16S rRNA database. The only identified Sporosarcina species was S. aquimarina with 29 (81%) isolates being identified as this species (Figure 4.10).

S. sp., 7, 19%

S. aquimarina, 29, 81%

Figure 4.10 Species abundance for the Sporosarcina genus. These 36 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

vi. Frequency of species belonging to the Paenibacillus genus

The Paenibacillus genus is a member of the phylum Firmicutes and is comprised of spore-forming, Gram-positive, rod-shaped bacteria. Paenibacillus was originally included in the Bacillus genus but was reclassified as a separate genus in 1993. Like the

Bacillus genus, this genus also produces spores that are resistant to heat, radiation, disinfectants, and desiccation, because of this they are difficult to eliminate from spacecraft-associated surfaces and can cause contamination in cleanrooms (Osman et al.,

2006). Paenibacillus was the fourth most prevalent genus, and the third most prevalent spore-forming genus identified by MALDI-TOF MS and 16S rRNA sequence analysis, with 3% of the total isolates being identified as a Paenibacillus species or taxonomic group.

Overall, there were 5 species and 1 taxonomic group represented within the Paenibacillus genus, there were also 10 (50%) isolates that were not identifiable at the species level due to there being no suitable match within the 16S rRNA database. The identified

Paenibacillus species and taxonomic groups were 69% of the total Paenibacillus genus, the most commonly identified species were P. cucumis with 3 (15%) isolates, P. lactis with

2 (10%) isolates, P. camelliae with 2 (10%) isolates, the P. alvei group with 1 (5%) isolate,

P. campinasensis with 1 (5%) isolates, and P. alvei with 1 (5%) isolate (Figure 4.11).

P. alvei, 1, 5% P. alvei TG, 1, 5%

P. camelliae, 2, 10%

P. campinasensis, 1, 5% P. sp., 10, 50%

P. cucumis, 3, 15%

P. lactis, 2, 10%

Figure 4.11 Species abundance for the Paenibacillus genus. These 20 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

vii. Frequency of species belonging to the Sphingomonas genus

The Sphingomonas genus is a member of the phylum Proteobacteria and is comprised of non-spore-forming, Gram-negative, rod-shaped bacteria. Bacteria in the

Sphingomonas genus are widely distributed in nature and have been shown to survive in environments with low concentrations of nutrients because of their ability to metabolize a

46 wide variety of carbon sources (Nishiyama et al., 1992). This adaptability could explain how this organism can contaminate spacecraft-associated surfaces with limited nutrients.

Sphingomonas was the fifth most prevalent genus, and the second most prevalent non- spore forming genus identified by MALDI-TOF MS and 16S rRNA sequence analysis, with 3% of the total isolates being identified as a Sphingomonas species or taxonomic group. The Phoenix Mars Lander’s bacterial isolates were identified as 1 taxonomic group and 3 species of the Sphingomonas genus, there were also 2 (11%) isolates that were not identifiable at the species level due to there being no suitable match within the 16S rRNA database. The identified Sphingomonas species and taxonomic groups were 89% of the total Sphingomonas genus, with the most commonly identified species and taxonomic groups being S. koreensis with 9 (47%) isolates, S. panaciterrae with 4 (21%) isolates, the

S. trueperi group with 2 (16%) isolates, and S. paucimobilis with 1 isolate (Figure 4.12).

S. trueperi TG, 3, 16%

S. sp., 2, 11% S. koreensis, 9, 47%

S. paucimobilis TG, 1, 5%

S. panaciterrae, 4, 21%

Figure 4.12 Species abundance for the Sphingomonas genus. These 19 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

viii. Frequency of species belonging to the Agromyces genus

The Agromyces genus is a member of the phylum Actinobacteria and is comprised of non-spore-forming, Gram-negative, rod-shaped bacteria. Agromyces is typically found in soil and is a smaller risk for contaminating spacecraft-associated surfaces. Agromyces was the sixth most prevalent genus, and the third most prevalent non-spore forming genus identified by a combination of MALDI-TOF MS and 16S rRNA, with 3% of the total isolates being identified as an Agromyces species or taxonomic group. The only Agromyces species represented within the Agromyces genus was A. soli with 13 (81%) isolates being identified as this species. There were also 2 (13%) isolates in this genus that were identified

48 most closely to the A. mediolanus group, and 1 (6%) isolate that could not be identified at the species level. (Figure 4.13).

A. mediolanus TG, A. sp., 1, 6% 2, 13%

A. soli, 13, 81%

Figure 4.13 Species abundance for the Agromyces genus. These 16 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

ix. Frequency of species belonging to the Microbacterium genus

The Microbacterium genus is a member of the phylum Actinobacteria and is comprised of non-spore-forming, Gram-positive, rod-shaped bacteria. Bacteria in the

Microbacterium genus are widely distributed in various environments and have been isolated from both milk and cheese and could play a role in cheese fermentation.

Microbacterium was the seventh most prevalent genus, and the fourth most prevalent non- spore forming genus identified by MALDI-TOF MS and 16S rRNA sequence analysis, with 2% of the total isolates being identified as a Microbacterium species or taxonomic group. The Phoenix Mars Lander’s bacterial isolates were identified as 1 taxonomic group

49 and 2 species of the Microbacterium genus. The taxonomic group was the M. maritypicum group with 2 (20%) isolates, the identified species were M. ginsengisoli with 5 (50%) isolates, and M. oxydans with 3 (30%) isolates (Figure 4.14).

M. oxydans, 3, 30%

M. ginsengisoli, 5, 50%

M. maritypicum TG, 2, 20%

Figure 4.14 Species abundance for the Microbacterium genus. These 10 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

x. The least frequently identified species using MALDI-TOF MS and 16S rRNA

The remaining 82 isolates were the least frequently identified genera and species that were determined using a combination of MADLI-TOF MS and 16S rRNA and makeup

15% of the total isolates identified. There were 47 (57%) isolates identified as one of 19 different species and 26 (32%) isolates that were identified as one of 9 taxonomic groups, with an additional 9 (11%) isolates from 3 different genera that were not identifiable to the species level due to there being no match in the database. The top 5 most recurrent species or taxonomic groups encompass 44% of the isolates that were identifiable to the species

50 level, these include Psychrobacillus psychrodurans with 9 (12%) isolates, the

Methylobacterium mesophilicum group with 7 (10%) isolates, Ralstonia insidiosa with 6

(8%) isolates, Fictibacillus nanhaiensis with 5 (7%) isolates, and Cupriavidus metallidurans with 5 (7%) isolates. There were 19 (26%) additional isolates that were identified into 8 taxonomic groups, and the remaining 22 isolates were identified as 15 different species and are 30% of the total isolates identified at the species level in this group

(Figure 4.15).

Least Abundant Species Count

Psychrobacillus psychrodurans 9 Methylobacterium mesophilicum TG 7 Ralstonia insidiosa 6 Fictibacillus nanhaiensis 5 Cupriavidus metallidurans 5 Lysinibacillus fusiformis TG 4 Fictibacillus arsenicus TG 4 Corynebacterium afermentans TG 4 Chryseobacterium sp. 4 Ralstonia pickettii 3 Oceanobacillus luteolus 3 Novosphingobium sp. 3 Novosphingobium fluoreni 2 Lysinibacillus sp. 2 Kocuria palustris 2 Burkholderia cepacia TG 2

SPECIES Brevibacterium frigoritolerans TG 2 Brevibacillus limnophilus 2 Variovorax boronicumulans 1 Stenotrophomonas maltophilia 1 Rhodococcus jialingiae 1 Pseudarthrobacter siccitolerans 1 Methylobacterium tardum 1 Leifsonia aquatica TG 1 Dyella terrae 1 Cupriavidus plantarum 1 Corynebacterium mucifaciens 1 Burkholderia contaminans 1 Burkholderia arboris TG 1 Brevundimonas diminuta TG 1 Acinetobacter junii 1

0 1 2 3 4 5 6 7 8 9 10 NUMBER OF ISOLATES

Figure 4.15 Species abundance for the genera with less than 10 isolates identified. These 82 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing.

E. Taxonomic identification of bacteria isolated from three distinctive locations on the Phoenix Mars Lander

A portion of the bacteria isolated from The Phoenix Mars Lander was retrieved from specific locations of the spacecraft. The three locations that these organisms were recorded as being isolated from is the Fairing, the Microscopy, Electrochemistry, and

Conductivity Analyzer (MECA), and the Robotic Arm. The Fairing is a ground support equipment so efforts are made to reduce the microbial burden, but since it is only required for take-off and will never make contact with any extraterrestrial surfaces it is not held to the same level of cleanliness as the actual spacecraft. The MECA and Robotic Arm are examples of subsystems on the Phoenix Mars Lander. These surfaces offer unique microbial environments with each area being composed of different materials and purposes, because of these unique characteristics, they have different sterilization procedures. These differences could cause certain locations to have a higher percentage of spore-formers relative to non-spore-formers. i. Identification of bacterial isolates from the Fairing

The Fairing is a molded structure that forms a protective and aerodynamic nose cone to protect the Phoenix Mars Lander during launch and ascent. The Fairing was the surface that had the highest number of bacterial isolates retrieved during routine swabbing performed by the Planetary Protection group. Since the Fairing is designed to protect the

Phoenix Mars Lander during launch, the materials and components of the Fairing are required to be tolerant of high temperatures. The Fairing’s heat-resistance enabled the components to undergo sterilization procedures where the materials were heated to 110 degrees Celsius or hotter to kill microbial contaminants. This sterilization method is a

53 process that is very effective at killing most microorganisms except spore-formers and thermophiles.

A total of 249 isolates were identified from the Phoenix Mars Lander’s Fairing using MALDI-TOF MS and 16S rRNA. Overall, more than half of the isolates (66%) acquired from the Fairing were related to spore-forming genera. The spore-forming genus that was identified most frequently was Bacillus, with 185 isolates identified as this genus, which is 57% of the total bacteria isolated from the Fairing. The other spore-forming genera identified from the Fairing were Paenibacillus with 8 (3%), Sporosarcina with 7 (3%),

Psychrobacillus with 6 (2%), and Lysinibacillus with 2 isolates.

There were 84 (34%) non-spore-forming isolates identified as 11 different genera, the most prevalent of which were, Staphylococcus with 45 (18%) isolates, Sphingomonas with 10 (4%), Ralstonia with 9 (4%), Methylobacterium with 7 (3%), and

Chryseobacterium with 4 (2%) isolates. The last 9 (4%) isolates were identified as belonging to one of 6 remaining genera (Figure 4.16).

FAIRING Genus Count

Bacillus 142

Staphylococcus 45

Sphingomonas 10

Ralstonia 9

Paenibacillus 8

Sporosarcina 7

Methylobacterium 7

Psychrobacillus 6

GENUS Chryseobacterium 4

Novosphingobium 3

Lysinibacillus 2

Cupriavidus 2

Stenotrophomonas 1

Corynebacterium 1

Acinetobacter 1

Rhodococcus 1

0 20 40 60 80 100 120 140 160 NUMBER OF ISOLATES

Figure 4.16 Taxonomic identities for microbial isolates sampled from the Fairing. These 249 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing. ii. Identification of bacterial isolates from the Robotic Arm

The Robotic Arm (RA) was an instrument designed to dig trenches, scoop up soil and water ice samples, and deliver these samples to the different instruments of the Phoenix

Mars Lander such as the MECA. The RA was sterilized and encased in a protective bio-

55 barrier before final integration on the Phoenix Mars Lander to prevent contamination of the Martian sub-surface with microbes from Earth.

A total of 55 isolates were identified from the Phoenix Mars Lander’s RA using

MALDI-TOF MS and 16S rRNA. Overall, more than half of the isolates (64%) acquired from the RA were related to spore-forming genera. The spore-forming genus that was identified most frequently was Bacillus, with 33 isolates identified as this genus, which is

60% of the total bacteria isolated from the RA. The other spore-forming genera were

Sporosarcina and Fictibacillus which both had 1 isolate.

The most common non-spore-forming genus was Agromyces, the 8 isolates that belong to this genus make up 15% of the entire microbial population sampled from the RA.

The four-remaining spore-forming genera were Sphingomonas with 7 (13%) isolates,

Staphylococcus with 3 (5%), and both Dyella and Burkholderia which had 1 isolate.

(Figure 4.17).

Robotic Arm Genus Count 35 33

10 8 7 NUMBER OF ISOLATES OF NUMBER 5 3 1 1 1 1 0

GENUS

Figure 4.17 Taxonomic identities of the microbial isolates sampled from the Robotic Arm. These 55 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing. 56 iii. Identification of bacterial isolates from the MECA

The Microscopy, Electrochemistry, and Conductivity Analyzer (MECA) was an instrument with multiple probes and wet chemistry labs that could be used to test the

Martian soil. Since the MECA is composed of many advanced electronics, many of the components and electronics associated with the MECA could not be exposed to high temperatures for long periods. Dry heat sterilization is not suited for every Spacecraft

Associated Surface because some components can be damaged, which poses a risk to the long-term reliability of instruments. Also, many instrument sensors cannot be exposed to elevated temperatures without risk of permanent damage, while some instruments and spacecraft structures have critical alignment requirements that limit maximum allowable temperatures. Because of these restrictions, the main sensors and other key electronics of the MECA were sealed and vented through high-efficiency filters to reduce contamination.

A total of 42 isolates were identified from the Phoenix Mars Lander’s MECA using

MALDI-TOF MS. Overall, more than half of the isolates (69%) acquired from the MECA were related to spore-forming genera. The spore-forming genus that was identified most frequently was Bacillus, with 6 isolates identified as this genus, which is 48% of the total bacteria isolated from the MECA. The other spore-forming isolates were matched to the following genera, Oceanobacillus with 3 (7%), Brevibacillus and Psychrobacillus with 2

(5%) isolates each, and Paenibacillus and Fictibacillus which both had 1 (2%) isolate.

The most common non-spore-forming genus was Microbacterium, the isolates that belong to this genus make up 12% of the entire microbial population. The remaining non- spore-forming genera were Agromyces with 3 (7%) isolates, Cupriavidus with 3 isolates, and Staphylococcus and Methylobacterium which both had 1 isolate (Figure 4.18).

MECA Genus Count 25 20 20

10 5

NUMBER OF ISOLATES OF NUMBER 5 3 3 3 2 2 1 1 1 1 0

GENUS

Figure 4.18 Taxonomic identities determined for microbial isolates sampled from the MECA. These 42 taxonomic identities were determined with MALDI-TOF MS or 16S rRNA gene sequencing. iv. Identification of bacterial isolates from the other locations sampled

The other locations sampled are composed of many spacecraft and cleanroom associated surfaces. This group of locations is very diverse with many of these isolates being retrieved directly from the spacecraft, as well as being isolated from the cleanrooms before the arrival of the Phoenix Mars Lander, and after it departed from the cleanrooms.

A total of 215 isolates not sampled from the Faring, MECA, or Robotic Arm were identified from the Phoenix Mars Lander using MALDI-TOF MS and 16S rRNA. Overall, most of the isolates (78%) acquired from these locations were related to spore-forming genera. The spore-forming genus that was identified most frequently was Bacillus with 117 bacteria isolated and identified from these locations. The next most recurrent spore- forming genera were Sporosarcina with 28 (13%) isolates, Paenibacillus with 11 (5%)

58 isolates, Fictibacillus with 7 (3%) isolates, Lysinibacillus with 4 (2%) isolates, and

Psychrobacillus with 1 (<1%) isolate.

The non-spore-forming genera were very diverse, with isolates belonging to 14 different genera, the most recurrent were Staphylococcus with 17 (8%) isolates, Agromyces with 5 (2%) isolates, and Microbacterium with 5 (2%) isolates. The remaining 11 non- spore-forming genera make up only 20 (9%) of the total isolates identified from The

Phoenix Mars Lander’s other locations (Figure 4.19).

Other Locations Genus Count

Bacillus 117

Sporosarcina 28

Staphylococcus 17

Paenibacillus 11

Fictibacillus 7

Agromyces 5

Microbacterium 5

Lysinibacillus 4

Corynebacterium 4

Burkholderia 3

GENUS Kocuria 2

Sphingomonas 2

Brevibacterium 2

Novosphingobium 2

Pseudarthrobacter 1

Psychrobacillus 1

Cupriavidus 1

Brevundimonas 1

Variovorax 1

Leifsonia 1

0 20 40 60 80 100 120 140 NUMBER OF ISOLATES

Figure 4.19 Taxonomic identities determined for microbial isolates sampled from anywhere except for the MECA, Robotic Arm, and Fairing. These 215 taxonomic identities were determined with MALDI- TOF MS or 16S rRNA gene sequencing.

D. Comparing the frequency of spore-forming bacteria isolated from three different locations on The Phoenix Mars Lander

To better understand the relationship between the sample source and the prevalence of spore-forming bacteria, the unique microbiomes of the Phoenix Mars Lander’s Fairing,

MECA, and Robotic Arm were compared. Since some of the sensitive electronics found on the Phoenix Mars Lander’s MECA and Robotic Arm cannot be sterilized using dry heat there could a larger percentage of contamination caused by non-spore-forming genera on these locations, when compared to the Fairing. A chi-square test of independence was performed to determine if there is a relationship between the location that the samples were retrieved and the presence of spore-forming bacteria. i. Comparison of the bacterial isolates identified using a combination of 16S rRNA and MALDI-TOF MS

To gain a better understanding of the isolates that were sampled from the Phoenix

Mars Lander, the bacteria identified with either MALDI-TOF MS or 16S rRNA sequences were combined. After combining these two methods there were a total of 561 isolates that were sampled from the Phoenix Mars Lander. The other locations sampled had the highest percentage (78%) of spore-forming genera. For the MECA, 69% of the isolates were identified as spore-forming microorganisms, while the Faring had 66%, and the Robotic

Arm had the lowest percentage of spore-formers (64%) (Figure 4.20). A chi-square test of independence was performed on these combined results, and the relationship between the location where the samples were collected and their ability to form spores was found to be significant, X2 (3, N = 625) = 59.9, p < .001. These results suggest that the null hypothesis should be rejected and that there is a statistically significant association between the location where the samples were collected, and the number of spore-forming vs. non-spore- forming bacteria identified.

Total Number of Spore-Formers vs. Non- Spore-Formers 180 165 168 160 Spore-Formers 140 Non-Spore-Formers 120

100 84 80

60 47 NUMBER OF ISOLATES OF NUMBER 35 40 29 20 20 13

0 Fairing MECA Robotic Arm Other LOCATION ISOLATES WERE SAMPLED

Figure 4.20 Number of combined microbial isolates sampled from the Fairing, MECA, Robotic Arm, and other locations. These isolates were identified and separated into spore-forming and non-spore-forming genera using a combination of 16S rRNA gene sequencing and MALDI-TOF MS.

5. DISCUSSION

A. Comparing the current Phoenix Mars Lander results to previously published

NASA studies

There have not been any studies that have performed a taxonomic identification on the isolates from the Phoenix Mars Lander, though there have been a few studies that have examined the cleanroom isolates (Ghosh et al., 2010; Vaishampayan et al., 2010). This present study represents a first attempt for the taxonomic identification of the bacteria isolated directly from the spacecraft. There have been multiple studies performed to better understand the isolates identified from other spacecraft and the conditions that existed in the cleanrooms associated with the spacecraft. Previous studies have reported species of the extremely hardy, spore-forming Bacillus genus to comprise >85% of the represented organisms in samples collected from spacecraft assembly facilities (Puleo et al., 1977).

However, these findings were heavily influenced by the NASA standard spore assay and the heat-shock procedures that were used to preferentially detect spores and exclude vegetative cells.

In a previous study by NASA, samples were collected using a cultivation-based method at three different time points, before, during, and after the Phoenix Mars Lander’s presence at the Payload Hazardous Servicing Facility (PHSF), which is the cleanroom where the Phoenix Mars Lander was housed (Ghosh et al., 2010). The study examined extremotolerant bacteria that could potentially survive the conditions that are experienced on the journey to Mars or the surface of the planet. These bacteria were isolated with a series of cultivation-based assays that promoted the growth of spore-formers and other hardy types of bacteria. This study shows there was a reduction in the microbial burden of

63 most bacterial groups, including spore formers, in samples collected during and after the

Phoenix’s arrival at the facility. Analysis of the 262 isolates from the facility demonstrated that there was a shift in predominant cultivable bacterial populations accompanied by a reduction in diversity during and after the departure of the spacecraft from the facility. It is suggested that this shift was a result of increased cleaning when Phoenix was present in the assembly facility and that certain species, such as Acinetobacter johnsonii and

Brevundimonas diminuta, may be better adapted to the environmental conditions that existed during and after the Phoenix Mars Lander was present in the facility. This study also showed that problematic bacteria resistant to multiple extreme conditions, such as

Bacillus pumilus, were able to survive these periods of increased cleaning. These changing environmental conditions in the PHSF could be partially responsible for the various groups of bacteria that were isolated for this study, which included spore formers as well as several non-spore-forming extremophilic species. Previous research studying several of NASA’s cleanrooms has also indicated similar bacterial profiles (La Duc et al., 2007). NASA’s planetary protection concerns about extremophiles that can survive vacuum, UV radiation, and ionizing radiation have been primarily focused on spore-formers, but the results of these studies demonstrate that clean rooms host a variety of non-spore-forming bacteria that can survive extreme conditions which warrant further research into their ability to tolerate other conditions likely to be encountered during space missions.

Another important study performed by NASA compares the samples that were collected from several locations on the cleanroom floor at three different times and analyzed the samples using molecular techniques: before the Phoenix Mars Lander’s arrival, during hardware assembly, and after the spacecraft was removed for launch

(Vaishampayan et al., 2010). For this study, they utilized a combination of clone analysis and a DNA microarray (PhyloChip) approach to avoid the limitations caused by only using a clone library approach. The PhyloChip can distinguish a much larger diversity and will detect less abundant species that are often undetected when using a clone library approach.

This study showed that the bacterial diversity of all major bacterial phyla that were isolated before the Phoenix Mars Lander’s arrival was statistically different from the other time point samples. Due to the stringent cleaning and decontamination protocols that take place during assembly, the bacterial diversity during this time point was dramatically reduced when compared to the other time points. This decrease in community complexity during the assembly process compared to the other periods that the samples were collected, shows the effectiveness of NASA cleaning protocols. However, the persistence of certain Bacillus species throughout the different spacecraft assembly phases emphasizes the need for continued refinement of sterilization technologies and the implementation of safeguards that monitor and inventory microbial contaminants. These results show that just because a microorganism was sampled from the cleanrooms and has been documented in the results of this study, it does not guarantee that the microbe was able to survive future cleaning procedures or that it can pose a serious planetary protection threat.

B. Comparing the Phoenix Mars Lander and the Mars Science Laboratory

As a result of the similar cleaning procedures that take place in the spacecraft cleanrooms, the microbiomes of different spacecraft have the potential to be very similar.

The isolates identified from the Phoenix Mars Lander were a complex blend of human and soil associated flora, with more than half of the organisms belonging to the spore-forming

65 genus Bacillus. As a result of the similar cleaning procedures that take place in the spacecraft cleanrooms, the microbiomes of different spacecraft have the potential to be very similar. The Mars Science Laboratory spacecraft was chosen to be compared to the

Phoenix Mars Lander because of the many similarities between the spacecraft and their missions.

The Mars Science Laboratory had a total of 358 bacterial isolates that were collected and identified after microbial burden reduction measures (Smith et al., 2017).

Most microorganisms (68%) that were identified from this mission belonged to the Bacillus genus, with 78% of the total belonging to a spore former. The second most recurrent genus was the non-spore-forming Staphylococcus which made up 11% of the total isolates. The next most recurring genera isolated from the Mars Science Laboratory were Paenibacillus

(4%), Acinetobacter (3%), and Streptococcus (2%). Much like the Phoenix Mars Lander’s isolates, Bacillus was the most prevalent genus for both missions, though the percentage of Bacillus species and spore-formers was higher in the Mars Science Laboratory. The

Phoenix Mars Lander’s identified isolates were only 57% Bacillus species and 70% spore- formers, which is lower than the 68% and 78% observed on the Mars Science Laboratory

(Figure 5.1). The reason for the increased percentage of non-spore-formers observed in the Phoenix Mars Lander’s results could be due to the large number of environmental samples that were collected from the PHSF.

Total Percentage of Spore-Formers vs. Non- Spore-Formers 100%

90% 22.1%, 79 29.2%, 164 80% 70% 60% Spore-Formers 50% 77.9%, 279 40% 70.8%, 397 Non-Spore-Formers 30% NUMBER OF ISOLATES OF NUMBER 20% 10% 0% Phoenix MSL MISSION ISOLATES WERE SAMPLED

Figure 5.1 Number of microbial isolates sampled from the Phoenix Mars Lander and the Mars Science Laboratory. The isolates from the two missions were identified and separated into spore-forming and non- spore-forming genera using either 16S rRNA gene sequencing or MALDI-TOF MS.

C. Importance of identifying the bacteria isolated from previous missions

Since it has been demonstrated that the isolates sampled from different spacecraft have the potential to be very similar, the most abundant and recurring microbial taxa sampled during other missions pose a serious concern. As a result of this, NASA needs to understand which taxa have been isolated from cleanrooms and their potential to form spores, with this information they can more accurately assess and predict Planetary

Protection risks. This study yields details about the microorganisms that inhabited the surfaces of the Phoenix Mars Lander after microbial reduction measures took place.

Knowing the identities of the bacterial contaminants and whether they form spores helps

NASA to gauge whether the microorganisms pose a forward contamination risk that could affect future planetary protection policy.

D. Future Planetary Protection Research

The continuation of JPL’s Planetary Protection research is an essential part of

NASA’s future missions to construct and launch spacecraft, such as the Mars 2020. Since any future isolates retrieved from the JPL cleanrooms will be run on MALDI-TOF MS, it is necessary for the Planetary Protection Group at JPL to constantly improve and update the MSP database. To improve the JPL MSP database, a list of potentially problematic

MSPs was created using the RTC results for the bacteria isolated from the Phoenix Mars

Lander. This first group of MSPs that was added to this list included the bacterial isolates that received different identifications from MALDI-TOF MS and 16S rRNA. When two or more MSPs belonging to different genera were matched above the 2.0 cutoff to the same isolate, they were both added to the list of problematic MSPs. They were also added to the list when 2 or more MSPs with different species-level identifications were matched >2.2 to an RTC of a single bacterial isolate. The MSPs on this list, as well as the RTCs that were successfully matched to 16S, are currently being analyzed and corrected by the

Planetary Protection group to determine which MSPs are correct, and which ones need to be reclassified.

The duplicate glycerol stocks that were made for each isolate have been frozen and their corresponding barcodes have been saved. If any of the results for these isolates need to be reconfirmed, or have additional testing performed, the frozen samples are available for future testing.

Older taxonomic identifications have the potential to become less accurate over time because the databases are constantly being updated with novel species, and organisms are reclassified over time. This makes it essential to periodically reconfirm the older 16S

68 rRNA identifications, which is accomplished by reblasting the isolated sequences against the current SILVA species database.

Previous studies that tested the survival of different Earth microorganisms in simulated Martian conditions have focused on the bacteria of the Bacillus genus, and other spore-forming microorganisms, this is because spores are a hardy form of terrestrial life with the highest potential to survive Mars-like conditions (Fajardo-Cavazos et al., 2010;

Tauscher et al., 2006). As a result of this, there have been multiple studies to examine spore-forming Bacillus species isolated from cleanroom facilities, and their potential to survive exposure to Mars-like environmental conditions (Nicholson et al., 2002; Setlow,

2006). Though there have been some studies that have used the isolates collected from spacecraft during mission preparations and tested their potential to contaminate Mars

(Smith et al., 2017). NASA’s understanding of the potential planetary protection risk that

Phoenix Mars Lander microorganisms face could be improved if future studies were performed on the isolates. These studies could analyze the non-spore-forming extremophiles and spore-forming isolate's ability to survive Mars-like environmental conditions, this would help the Planetary Protection group to better understand the potential for isolates collected from this mission to contaminate Mars. The future development of appropriate cleaning and sterilization protocols will require the physiological capabilities of cleanroom microorganisms to be understood.

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