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Habitability of Saturn's moon Titan for acetylenotrophy: laboratory culturing and energetics as tools for the search for life

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Habitability of Saturn's moon Titan for acetylenotrophy: laboratory culturing and energetics as tools for the search for life

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Content Copyright 2024 Maya Danielle Yanez
HABITABILITY OF SATURN’S MOON TITAN FOR
ACETYLENOTROPHY:
LABORATORY CULTURING AND ENERGETICS AS TOOLS
FOR THE SEARCH FOR LIFE
by
Maya Danielle Yanez
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GEOLOGICAL SCIENCES)
August 2024

ii
DEDICATION
This dissertation is dedicated to Dr. Jan Amend.
He gave me the opportunity to achieve great things and explore my scientific curiosities on my terms;
I never got to thank him for that.
As the first doctorate in my family, I dedicate this to them—Yanez and Edgett alike,
¡Si se puede!

iii
ACKNOWLEDGEMENTS
Science is never done in isolation; bridging fields and working on an interdisciplinary project is only
possible with the help of collaborators. Firstly, thank you to my committee for seeing me through
qualifying exams, several sets of unprecedented circumstances, and for believing in me even when I
was uncertain: Morgan Cable & Doug LaRowe have been my rocks so I did not get washed away by
the torrent of graduate school; Frank Corsetti for being my favorite professor to TA for, and for
asking some of the best questions about what life is or is not; Steven Finkel for coming in at the end
and saving the day; I’m so grateful for all the advocacy on my behalf from this committee! Advice
given by every one of you made a difference. To my collaborators around the country--thank you all
for the science help, mama bear-ness, and encouragement: Denise Akob, Shaun Baesman, Mike
Malaska, and D’Arcy Meyer-Dombard.
To the Amend Lab: We didn’t know we were going to be the last of Jan Amend’s students, but I’m
so glad we knew each other. I could not imagine graduate school without you all as my companions
on this journey. Thank you to Jayme Fehyl-Buska for helping me learn what it means to be a
microbiologist and how to stand up for myself in my PhD. The largest thanks to Heidi Aronson for
guiding me on becoming a microbiologist—I grew and learned from the lectures and advice you
gave me ☺ I would be less of a scientist and person without the support, tears, and good food we
shared. Thank you to Didi Bojanova for video game discussions and long dog pets, and for giving me
my first chance to work on a project in the lab!
I think the lab would have exploded, been even more of mess, and definitely had more broken parts
without the oversight of Pratixa Savalia—thank you for filling in always and for going the extra mile
to keep us running. To Roman Barco, thanks for being the easiest officemate and for always stopping
and spending time to talk about a weird thing some microbe is doing. To Kilian, Caleb, and Juli—I
am so excited to see what your projects become and the great work you’ll achieve, thank you for
listening to my longwinded stories and unsolicited advice! I can’t thank you all enough for bolstering
each other in what were several unprecedented events; I was so glad to be one small role in teaching
you all a bit about lab work. I am grateful to have been a mentor to and received assistance from
three exceptional students: Karli Rodrigues, Itzel Vazquez-Salazar, and Julia Bustos. You all are
future leaders with remarkable passion and resolve to accomplish what you want—¡Si se puede!
Thank you and a gigantic hug to my friends in Earth Sciences: Katie, Ryley, Brianna, and Tori—
friends make every day easier and you all always lent an ear for complaints or tears. We all deserve
to be here and I cannot wait to see how we change the world! Jess Stellmann—our joint passion for
social justice and holding those with power accountable made us friends and I appreciate having a
partner in crime, that will also play nerdy games with me! Thank you for absorbing so many tears as
I reached the end.
Lastly, I want to thank Lee, my partner of 12 years. Those who have interacted with me know
that I am loud, opinionated, and strong-willed. Yet, Lee manages to get a word in edgewise. He
remains at my side throughout graduate school and all of that life has thrown at us. I could never
have done this without his support. His laughter, grand smile, and sturdiness have fostered a
belief in myself that I lost at moments along this journey. He was ready with a distraction when
needed and always a stubborn belief in me as I pushed through the end. Thank you for many
hugs, words of encouragement, unwavering support, and frequent reminders that I am capable of
far more than I give myself credit for.

iv
TABLE OF CONTENTS
Dedication...................................................................................................................................... ii
Acknowledgements....................................................................................................................... iii
List of Tables .................................................................................................................................vi
List of Figures...............................................................................................................................vii
Abstract..........................................................................................................................................ix
Chapter 1: Introduction & Background
1.1 Astrobiology & Habitability ..............................................................................1
1.2 Titan..................................................................................................................3
1.3 Acetylenotrophy ...............................................................................................5
Figure .....................................................................................................................8
Chapter 2: Bioenergetics and Environmental Applications..............................................................9
2.1 Gibbs Free Energy & Predicting Novel Metabolisms ......................................9
2.2. Acetylenotrophy & Methanogenesis: A Quick Comparison ..........................11
Figure ...................................................................................................................17
Chapter 3: Acetylenotrophy on Titan .............................................................................................18
3.1 Introduction & Background ............................................................................18
3.1.1 Subsurface Ocean..............................................................................20
3.1.2 Surficial Impact Melt Ponds................................................................21
3.2 Methods..........................................................................................................23
3.3 Results ............................................................................................................28
3.4 Discussion.......................................................................................................30
3.5 Outlook ...........................................................................................................33
Figures & Tables..................................................................................................35
Chapter 4: The Intersection of Scientific Research & Education......................................................48
4.1 Philosophy on Scientists’ Role in Education...................................................48
4.2 Astrobiology: Bringing the Search for Aliens to the Elementary Classroom...50
Figures & Tables..................................................................................................58
Chapter 5: Conclusion
Holistic Interdisciplinary Habitability ..........................................................................65
Bibliography .................................................................................................................................67

v
Appendix A...................................................................................................................................80
A.1 Additional Growth Curves.............................................................................80
A.2 Acetate Data...................................................................................................81
A.3 Astrobiology Lesson Plans............................................................................82

vi
LIST OF TABLES
Table 3.1: A summary of the conditions and Gibbs energies calculated for different
environments and those replicated in the lab. All other concentrations were held at 10µm........35
Table 3.2: A list of the components of DMSZ Media 293 used for the marine strain
GhAcy1 and used for marine-like experiments............................................................................36
Table 3.3: A list of the components of DMSZ Media 298 used for the freshwater strain
WoAcy1 and used for freshwater experiments ............................................................................37
Table 3.4: A list of the components of DMSZ Trace element solution SL-10 used in all
laboratory experiments to provide various trace metals...............................................................38
Table 3.5: A list of the energy costs per cell used to predict cell densities shown in
Figure 3.8. The first three rows were calculated from experiments. The final one is taken
from the reference ........................................................................................................................39
Table 4.1: Recreated here from the text of Lesson Two; this table highlights the true
names of the instruments used by spacecraft, a more simplified name, and a brief simple
description of what the instrument does. Imaging Science Subsystem is the name used on
the Cassini mission.......................................................................................................................58
Table 4.2: Grade level specific NGSS alignment from The Next Generation Science
Standards (NGSS, 2013) .........................................................................................................59-60

vii
LIST OF FIGURES
Figure 1.1: Schematic describing the potential biological cycling on Titan’s surface and
subsurface. Beginning with atmospheric production of a molecule of interest, how it might
travel to the ocean and be altered, and then returned back to the surface as a biosignature
able to be detected by spacecraft. This graphic is from the NASA Astrobiology Institute
funded Habitability of Hydrocarbon Worlds: Titan and Beyond grant
https://astrobiology.nasa.gov/nai/teams/can-8/jpl-titan/ ................................................................8
Figure 2.1: Filled contour plots of the Gibbs energies, -∆Gr
, for hydrogenotrophic
methanogenesis (a, c) and acetylenotrophy (b, d) as a function of temperature and substrate
concentration (H2 for panels a and c and C2H2 for panels b and d). Energy yields are shown
in kJ/mol C. The top two panels represent Titan subsurface ocean pressure, 250 MPa, and
neutral pH and the bottom two represent Enceladus’ ocean pressure, 10 MPa, and pH = 8.5.
The starred locations are representing the most likely substrate concentration for each ocean
world considered, at a temperature of 1C and assumed water activity = 1.0. Contour lines
for panels a and c represent 25 kJ differences and those for panels b and d represent 3 kJ
increments ....................................................................................................................................17
Figure 3.1: An image of Syntrophotalea acetylenica marine strain GhAcy1. Scalebar
represents 5µm. Cells are stained using SYBR-Green I nucleic acid stain and viewed using
fluorescence microscopy. The picture contrast is increased to better visualize the cells................40
Figure 3.2: Gibbs energy of A) acetoin and B) acetylene fermentation, Reactions 7 and 2,
as a function of temperature and substrate concentration. The white stars refer to the
conditions prevailing in the laboratory experiments noted in Section 3.2 as optimal. Acetoin
thermodynamic properties were extrapolated from other known data and calculated using
equations of state..........................................................................................................................41
Figure 3.3: Growth curves for the marine and fresh strains of S. acetylenica at 20C and
optimal conditions as described by the DSMZ. These represent the mean of three cultures.......42
Figure 3.4: Growth rates as a function of temperature for the marine and fresh strains of S.
acetylenica at pH = 7.5, [NaCl] = 20 g/L for the marine strain, and [NaCl] = 1 g/L for the
fresh strain, optimal conditions as described by the DSMZ ........................................................43
Figure 3.5: Growth rates as a function of sodium chloride (salinity) concentrations for the
marine and fresh strains of S. acetylenica at 30C and pH = 7.5, optimal conditions as
described by the DSMZ................................................................................................................44
Figure 3.6: Growth curve of the marine strain of S. acetylenica coupled with measurements
of the acetylene in the headspace. This experiment was performed at 20C, pH = 7.5, and
instead of acetoin, had 1mL of gaseous acetylene added to the headspace prior to
inoculation....................................................................................................................................45
Figure 3.7: Consumption of acetylene with time as measured via gas chromatography. The

viii
blue line represents a control containing no cells; whereas the red, green, and orange lines
represent the culture in triplicate. Acetylene can be seen to be completely consumed in the
cultures and not in the control......................................................................................................46
Figure 3.8: Predicted cell densities for acetylenotrophs across three environments: An
impact melt pond in Selk Crater on Titan (green), Titan’s subsurface ocean (blue), and
Enceladus’ subsurface ocean (purple). Four energy costs were used for these predictions;
1mL-C2H2 (leftmost) represent the average energy cost per cell of the marine and fresh
experiments where 1mL of acetylene was added. Marine 2mL-C2H2 and Fresh 2mL-C2H2
represent the same but where 2mL of acetylene was added. The right most energy cost
represents the minimum energy cost to produce a cell as modelled for E. coli published in
Ortega-Arzola, et al. 2024 ............................................................................................................47
Figure 4.1: Images from the Fall 2022 WonderKids Program. A Highlights of post-test
results from the 3-5 grade group, noting specific facts covered in the lessons, indicating
comprehension. B Pre-test results from the K-2 group: Aryana and Zelda (top B) and
Allison (bottom B) represent a lack of comprehension. Zhanel’s response (middle B)
represents comprehension as it is related to astrobiology. Students explain their drawings to
aid in these determinations. C Post-test results from the K-2 group. Victoria and Giovanna’s
answers feature either Ms. Maya (the expert for the week) or themselves doing science,
which we consider to demonstrate comprehension. Allison’s inclusion of aliens is also
comprehension of astrobiology. Photo Credit: M. D. Yanez .......................................................61
Figure 4.2: A summary figure representing Yanez’s research ....................................................62
Figure 4.3: Depictions of each lesson are shown. 1 is an image of the final product of Lesson
1, an edible Saturn model. 2 is the answer key to Lesson 2 showing all instruments fitting in
the cargo fairing. 3 is from the teacher professional development workshop where teachers
are actively playing the microbe energy game in Lesson 3. 4 is an image of the final
“bacteria soup” bottle from Lesson 4, also from the teacher professional development
workshop. Photo Credit: 1, 2, M. D. Yanez and 3, 4, Kathrin Rising..........................................63
Figure 4.4: Energy yields reproduced from Yanez’ research. These energy yields (color bar)
are a function of how much food is available for each diet (y-axis) and the temperature of the
environment (x-axis). The left plot represents pink-eaters or methanogens and the right plot
represents blue-eaters or acetylenotrophs (see Figure 2). Values used in Lesson 3 are
denoted by 1-4. Bottom boxes 1-5 represent the pink and blue marbles in each environment
of Lesson 3....................................................................................................................................64

ix
Abstract
The search for life beyond Earth has been closely tied to the presence of liquid water.
Often, astrobiologists focus on three key criteria for life: liquid water, nutrient availability,
and metabolic energy sources. The putative energy sources are defined by whether the
reactants for known metabolisms on Earth are found in abundance on those other worlds, in
environments collocated with available nutrients and liquid water. Our observations of
Saturn’s moon Titan describe a world comprised of chemicals and bedrock layers so
different from Earth, yet still likely environments to meet the aforementioned criteria. Titan
is comprised of a surface shrouded by a thick atmosphere with stable liquids (albeit not
liquid water) in lakes and seas; fluvial and aeolian erosion that are altering a changing
landscape; and a deep subsurface liquid water ocean bounded by shells of thick water ice.
Utilizing methods that have been proven to work on Earth for exploring environments in
search of novel microbes or unique metabolisms, could prove fruitful for those
extraterrestrial environments with limited access and information, such as the subsurface
ocean on Titan. In the coming years, missions like NASA’s New Frontiers mission
Dragonfly will arrive on Titan’s surface and complete measurements aimed at identifying
any possible life found therein, and in turn provide a more detailed view of surface
environments. However, Dragonfly’s instrument capabilities and possible measurements
will be constrained by the type of life expected to be found on Titan. As environmental
microbiology research has bloomed in recent decades, there is an ever-growing list of
possible microbial metabolisms. On Earth, we predict undiscovered metabolisms given the
energy yields of novel catabolic reactions and search for environments where those energy
yields are possible.
Our search for life elsewhere can only be informed by our understanding of life’s origins

x
and evolutions on Earth. One plausible source of metabolic energy is acetylene (C2H2), a
simple organic compound that is the second most abundant photochemical product in Titan’s
atmosphere. Acetylenotrophy, or the microbial fermentation of acetylene, is utilized by
microbes on Earth in a number of environments. Here I provide an energetics analysis of
acetylenotrophy for Titan including predicted cell densities based on laboratory culturing of
Syntrophotalea acetylenica, the type strain of acetylenotrophs. In addition, I advocate for the
translation of these research projects into lesson plans for elementary students to foster
excitement and engagement in the field of astrobiology. Overall, I describe the potential
habitability of Titan for acetylenotrophs as a small contribution to the knowledge needed to
guide future missions aiming to search for life on ocean worlds.

1
Chapter One: Introduction and Background
1.1 Astrobiology and Habitability
Astrobiology is the study of understanding life’s origin(s) and evolution on Earth as well
as the search for life beyond Earth. In particular, our knowledge of Earth and terrestrial life
have rightfully influenced our exploration of life beyond Earth. Single-celled microbes have
existed for nearly 3 billion years on Earth (Allwood et al., 2006)—multicellular life has
existed for at least 600 million years (Mills et al., 2022; Ros-Rocher et al., 2021). Microbial
life is a far more likely candidate to exist in extraterrestrial environments: microbes are
ubiquitous and found wherever we find life and throughout the history of the Earth, life has
been mainly microbial.
By studying microbes living under a diverse array of environments on Earth, we can
anticipate the type of life possibly inhabiting other worlds. However, best practices include
anticipating life as we know it (as found on Earth) and life as we do not know it (unlike life
on Earth). Too narrow of a definition for life could prove ineffectual at discovering and
identifying life elsewhere. Yet strictly defined requirements with multiple lines of evidence
would be the only metrics for life that could convince scientists. Therefore it behooves us to
consider novel and unique Earth life that may stretch our understanding and in turn, expand
the references used to contextualize our observations in the search for life.
In the decades since humanity’s first landing on the Moon, NASA has sent hundreds of
missions into space. Robotic spacecraft have landed on only four planetary bodies in the
entire Solar System: Earth’s moon, Mars, Venus (by the Soviet Space Program), and
Saturn’s moon, Titan (by the joint NASA-ESA mission, Cassini-Huygens). Two missions
have visited asteroids as well to return samples back to Earth, Hayabusa (JAXA) and

2
OSIRIS-Rex (NASA). The Viking Landers to Mars in the 1970s and the New Frontiers
Dragonfly mission to Titan are some of the only missions whose science goals expressly
include searching and discovering evidence of life. However the search for life has been a
critical goal of NASA’s science initiatives since the 1970s. Most missions are orbital
spacecraft that exist to “flyby” the objects of interest, often flying kilometers above the
surface and only dipping into the atmospheres. Yet only missions that interact with the
planetary surface (e.g. landers and rovers) can provide data at a high enough resolution to
attempt to determine the presence or absence of life. Few if any universally accepted
biosignatures can be viewed at the global macroscales that orbital missions provide. But the
cost of landers and rovers make them a rare and infrequent mission type. Such limitations
combined with the likelihood that any life to be found would be microscopic have set a high
bar for the search for life.
Habitability on Earth is often discretely defined for a particular organism or known
community (e.g. ecosystems, biomes, consortia). Yet as we explore other potentially
habitable worlds, current efforts focus on broad definitions with no specific organism in
mind in order to account for life as we do not know it. To remain agnostic, specific
metabolisms/diets are considered because those reactions would remain the same while the
energy associated with them may be different in extraterrestrial environments. Calculating
the energetics of these metabolisms under the conditions of different planetary bodies is
possible even with the low resolution data available from flyby missions.
To most astrobiologists, a habitable world is one that may have the potential to harbor
life: there is likely the presence of liquid water, available nutrients, and an energy source.
Saturn’s moon Titan is one of these worlds. I propose that we may be able to narrowly

3
define which environments on these bodies are more likely to be habitable, or support extant
life, by focusing on the environmental conditions that could prove energy-yielding for
specific metabolisms. Furthermore, using laboratory cultures to derive estimates for cell
densities within these energy-yielding (hospitable) conditions provides detection limits for
future instrumentation on astrobiology-focused missions.
1.2 Titan
Astrobiologists consider several ocean worlds, planets or moons that sustain large liquid
water oceans, as astrobiology targets given their likelihood of being habitable. Throughout
this thesis, I focus on Saturn’s moon Titan. Titan is about 40% the volume of Earth, but its
atmosphere is 50% thicker at the surface than Earth’s (Zebker et al., 2009). This thick
atmosphere is comprised of several haze layers with the dominant species being nitrogen
(N2) and methane (CH4). Photochemistry alters nitrogen and methane to form many other
organic molecules; this is fueled by high energy radiation from Saturn’s magnetosphere
(Vuitton et al., 2019). The organic products are then deposited on the surface of Titan (Singh
et al., 2016), a rich geologically active landscape. Beneath the organic regolith lies a thick
water-ice shell encasing a global subsurface ocean (Lopes et al., 2020; Malaska et al., 2020).
Titan’s rocky core is also covered by a shell of water-ice, leaving Titan’s subsurface ocean
wedged between shells water-ice, sequestered from the surface environment in modern times
(Journaux et al., 2020; Vance et al., 2018). There are little to no environments on Earth that
can act as analogues for Titan’s water-ice surface/bedrock, although ice-capped lakes may
have the closest resemblance to Titan’s subsurface ocean (e.g. Lake Vostok, Antarctica).
Furthermore, the complex organic sludge that is Titan’s surface regolith also has no
analogue on Earth. As such, any geophysical phenomena (such as the cause of labyrinth

4
terrain, cryovolcanism, etc.) that could exchange material between the surface and
subsurface are relatively unconstrained (MacKenzie et al., 2021a; Malaska et al., 2020).
It does seem, however that, Titan has a complex and active surface environment. The
Cassini mission orbited Saturn for ~13 years and made 115 flybys of Titan; these
observations revealed a landscape of diverse terrains, as well as evidence of aeolian and
fluvial erosion actively altering the surface. There are dunes, lakes and seas of liquid
methane/ethane, labyrinth terrain, hummocky mountains, and plains (Lopes et al., 2020).
Some surface features could possibly be evidence of cryovolcanism sourced from the waterice bedrock (Marusiak et al., 2022; Mousis and Schmitt, 2008; Sohl et al., 2014). A window
to Titan’s surface was carved by the descent of the Huygens lander in 2005: this probe
recorded images and atmospheric data while it approached the surface of Titan after
deployment from the Cassini orbiter. It relayed data post-landing for an additional hour on
the surface. Even with a brief visit from a lander, knowledge gained about Titan led to more
questions. The Huygens probe observed rounded rocks that were rather Earth-like as they
were altered due to liquid flowing across the surface.
Lakes and seas are present on Titan’s surface composed of liquid methane/ethane (Hayes,
2016). These liquid bodies and the thick atmosphere fuel a methane cycle akin to Earth’s
water cycle: methane rains down from clouds and is evaporated from the surface to form
clouds (Hayes et al., 2018). While these surficial liquids create a dynamic landscape that is
Earth-like, Titan’s subsurface ocean is the exciting candidate for a habitable environment. At
18 times the volume of Earth’s oceans and putative water-rock interactions at its origin
providing ample nutrients, it is an enticing target for life beyond Earth (MacKenzie et al.,
2021a). However, this ocean remains inaccessible to current spacecraft. Throughout this

5
thesis, I consider two possible aqueous environments on Titan where liquid water and
acetylene ought to be co-located: freshwater impact melt ponds on the surface, and a salty
subsurface ocean that is sequestered from the surface. See Figure 1.1 for a schematic crosssection of Titan and potential biosignature cycling between surface and subsurface.
The most abundant solid photochemical product on Titan is acetylene (C2H2) formed via
Reaction 1, photolysis of ethylene, the final reaction along a chain of radical chemistry.
Acetylene is formed at ~200km and ~800km above Titan’s surface, above and below the
haze layer found at ~600km. Once enough molecules are formed they will coalesce into a
solid particle and be deposited on the surface (Singh et al., 2016; Vuitton et al., 2019).
C4H2 + ℎ𝜈 → C2H2 + C2
(1)
The rate of acetylene deposition is 14 mm/Myr (Vuitton et al., 2019); this equates to 1500
moles deposited per second onto Titan’s surface globally. With such an abundance of
acetylene, perhaps there are lifeforms that can take advantage of it.
1.3 Acetylenotrophy
Acetylene (C2H2) is the simplest alkyne and is most commonly used on Earth as the fuel
for welding torches. Most of the acetylene produced on Earth comes from anthropogenic
sources such as the burning of various fuels and debris. Despite its low atmospheric
concentration on Earth today, 0.04 parts per billion (Oremland and Voytek, 2008), acetylene
is a highly energy-yielding food source for those bacteria capable of consuming it. While
these bacteria are found in many different environments across the globe, acetylenotrophy is
relatively understudied (Akob et al., 2018). Acetylenotrophy includes the microbial
fermentation of acetylene:
2C2H2 + 3H2O → CH3CH2OH + CH3COO
− + H
+ (2)

6
Acetylene fermentation is catalyzed with the acetylene hydratase (AH) enzyme to
produce acetaldehyde. Two molecules of acetaldehyde are formed where one is oxidized to
ethanol and the other is reduced to acetate (Akob et al., 2018; Miller et al., 2013; Schink,
1985). The AH enzyme is the only known enzyme capable of using acetylene as a substrate
(Abt, 2001). Syntrophotalea acetylenica, the type strain of acetylenotrophs, can consume
some of the ethanol to form additional acetate and hydrogen gas (H2) (Miller et al., 2013;
Seitz et al., 1990). Of the seven identified species that appear to consume acetylene in
laboratory cultures, there are sixteen different strains, with five of those being anaerobic and
demonstrated to consume acetylene via Reaction 2. The aerobic strains produced the
intermediary acetaldehyde, but were not observed producing ethanol or acetate (Akob et al.,
2018).
Acetylenotrophy is a known syntrophic metabolism: the metabolic products are substrates
for other nearby microorganisms, allowing acetylenotrophs to fuel a microbial community
(Akob et al., 2018; Mao et al., 2017; Oremland and Voytek, 2008; Seitz et al., 1990).
Furthermore, acetylene is often inhibitory for microbes, as it can preferentially bind to the
metal cofactors of enzymes needed for methanogenesis, sulfate reductions, nitrogen fixation,
organo-halide reduction, and others (Mao et al., 2017). On early Earth as life arose,
acetylene was possibly a million times more concentrated than today (Oremland and Voytek,
2008). Yet methanogenesis and sulfate reduction were some of the first (if not the first)
metabolisms to arise on Earth. The presence of acetylenotrophs in an environment can
reduce the concentration of acetylene, provide metabolic products that are useful substrates
for methanogens and sulfate reducers, and establish microbial communities as life originates
and develops. It is possible that acetylenotrophs cleared the way for life’s colonization of the

7
soils on early Earth (Culbertson et al., 1988; Oremland, 1989; Oremland and Voytek, 2008).
Its potential role in the life’s origins on Earth and the abundance of acetylene found on Titan
make acetylenotrophy an exciting candidate for astrobiology studies. I present the
environmental energetics of acetylenotroph, laboratory culturing of known acetylenotrophs,
and the application of those results to the search for life on worlds like Titan.

8
Figure
Figure 1.1: Schematic describing the potential biological cycling on Titan’s surface and subsurface.
Beginning with atmospheric production of a molecule of interest, how it might travel to the ocean and be
altered, and then returned back to the surface as a biosignature able to be detected by spacecraft. This
graphic is from the NASA Astrobiology Institute funded Habitability of Hydrocarbon Worlds: Titan and
Beyond grant https://astrobiology.nasa.gov/nai/teams/can-8/jpl-titan/

9
Chapter Two: Bioenergetics and Environmental Applications
Microbes can be difficult to pinpoint in an environment and to study in the lab. One
mechanism to explore their potential is to focus on their metabolism, specifically catabolic
reactions, and perform thermodynamic modeling of that metabolism. Given the temperature,
pressure, ionic strength, concentrations of reaction components, and activity coefficients one
can determine if a reaction will be energy-yielding under those conditions (Amend &
LaRowe, 2019). While an energy yield is meaningless for determining whether or not the
reaction will proceed, it is meaningful to identify unoccupied niches that could be
advantageous for certain microbes.
2.1 Gibbs Energy and Predicting Novel Metabolisms
The Gibbs Energy of a reaction is the combination of the entropy and enthalpy of
transforming the reactants into the products of a reaction. Overall, it represents the energy
yield of a reaction: if negative, the reaction is exergonic and if positive, the reaction is
endergonic. Of note, the calculation of the Gibbs energy of a reaction requires specific
environmental parameters (temperature, ionic strength, activity of relevant species, etc.), as
such the energy yield of a metabolic reaction will vary based on the environment it is
occurring within. Calculating the energy yield of different metabolisms has proven to be a
useful framework for exploring potential microbial activity in a range of isolated and/or
extreme environments on Earth (e.g. Amend & Shock 2001; Bradley et al. 2020; LaRowe &
Amend 2019, 2015; Lu et al. 2021) and certain planetary bodies (e.g. Mars, Europa, and
Enceladus, Jameson et al. 2019; McCollom 1999; Sauterey et al. 2022; Taubner et al. 2018).
It is worthwhile to apply these calculations to extraterrestrial environments in order to
constrain the possible life that could exist therein.

10
Gibbs energies of a reaction, ∆𝐺𝑟
, are calculated using
∆𝐺𝑟 = ∆𝐺𝑟
0 − 𝑅𝑇ln(𝑄𝑟
) (3)
where ∆𝐺𝑟
0
represents the standard state Gibbs energy of reaction, R denotes the gas
constant, T stands for temperature in Kelvin and Qr corresponds to the activity product,
which is defined by
𝑄𝑟 = ∏ 𝑎𝑖
𝜈𝑖
𝑖
(4)
The symbols ai and vi refer to the activity and stoichiometric coefficient of the ith species in
the chemical reactions of interest. Values of activity are calculated from
𝑎𝑖 = 𝐶𝑖/𝐶𝑖
𝜃
∗ 𝛾𝑖
(5)
where Ci and 𝐶𝑖
𝜃
stand for the concentration and standard state concentration (one molal
referenced to infinite dilution) of the ith species, respectively, in the reaction of interest, and
𝛾𝑖 designates the activity coefficient of the ith species.
Values of ∆𝐺𝑟
0 must be calculated for the specific temperature and pressure of the
environment of interest using the revised Helgeson-Kirkham-Flowers (HKF) equations of
state (Helgeson, 1981; Shock et al., 1992; Tanger and Helgeson, 1988). Thermodynamic
data is required for each component of the reaction and is often sourced from large databases
informed by empirical data or extrapolations of empirical data for conditions that cannot be
mimicked in a lab (Gurvich et al., 1989; Johnson et al., 1992; Leal et al., 2016; Pennington
and Kobe, 1954; Wagner and Pruß, 2002). A large library of ∆𝐺𝑟
0
values can be found in
Amend and Shock, 2001. Calculated ∆𝐺𝑟
0 values were sourced from the reaktoro Python
package, although other packages such as WORM and CHNOSZ can also calculate these
values in R or Python (Leal et al., 2016).
Calculating the energetics of reactions across various environmental conditions can be a

11
useful tool: there is a history of identifying energy-yielding reactions, investigating the
environments that have those energy-yielding conditions, and discovering novel microbes or
novel metabolisms within those environments. In 1977, Engelbert Broda hypothesized
anaerobic ammonia oxidation (anammox) based on thermodynamic energy yield
calculations (Broda, 1977). He believed there must be microorganisms using these
metabolisms, yet it took nearly 20 years before those novel microbes were identified (van de
Graaf et al., 1995). Now, 47 years later we understand that anammox is critical to global
nitrogen cycling (Kuypers et al., 2018). Energetics can highlight a metabolism of interest
that may have otherwise been undiscovered.
I calculated the energetics of the acetylenotrophy’s catabolic reaction (Reaction 2) and
the catabolic reaction for hydrogenotrophic methanogenesis (Reaction 6) for two ocean
world moons of Saturn, Titan and Enceladus. My goal was to provide a zero-order metric to
consider different metabolisms within the same extraterrestrial environments, given our low
resolution data of those locations.
2.2. Acetylenotrophy & Methanogenesis: A Quick Comparison
Enceladus is a moon of Saturn that, like Titan, also has a large subsurface liquid water
ocean, shielded beneath a thick ice shell. Of distinction from Titan, Enceladus has a plume
of jets emanating from its south pole that ejects ocean contents directly into space (Waite et
al., 2009). The Cassini mission visited Enceladus with flybys as it did Titan, however the
spacecraft was able to pass through the water ice jets and sample the composition. As such,
the data suggest acetylene is a constituent of Enceladus’ ocean (Waite et al., 2017, 2009).
Both Titan and Enceladus are astrobiology targets, (Mousis and Schmitt, 2008; Sohl et al.,
2014; Vance et al., 2018) and these oceans could source the crucial elements and energy

12
required for life given the colocation of liquid water, organic molecules, and potential waterrock interactions (Artemieva and Lunine, 2003; Cable et al., 2021; Des Marais et al., 2008;
Glein and Shock, 2013; Hedgepeth et al., 2022; Hemingway and Mittal, 2019; Hendrix et
al., 2019; Iess et al., 2014; National Academies of Sciences, 2022; Nixon et al., 2018;
Postberg et al., 2023; Thomas et al., 2016; Waite et al., 2017, 2009, 2006). Cassini-Huygens
and supplemental ground-based observations have identified hydrogen (H2), acetylene
(C2H2), and methane (CH4) on these worlds (Singh et al., 2016; Vuitton et al., 2019; Waite
et al., 2017, 2009). The detection of hydrogen and methane, in particular, has prompted
many astrobiologists to propose hydrogenotrophic methanogenesis, Reaction 6, as a
possible metabolic (energy-yielding) reaction on Titan, Enceladus, and other worlds
(Affholder et al., 2021; Hoehler, 2022; McKay, 2016; McKay and Smith, 2005; Porco et al.,
2006; Ray et al., 2021; Sauterey et al., 2022; Steel et al., 2017; Taubner et al., 2018; Vance
et al., 2016). In particular, evidence suggests a possible hydrothermal environment in
Enceladus’ ocean that could support methanogenesis (Hsu et al., 2015; Waite et al., 2017).
However, no astrobiological study has considered the microbial fermentation of acetylene
(Reaction 2) as a potential source of energy, prior to the work presented here (Yanez et al.,
2024).
4H2 + CO2 → CH4 + 2H2O (6)
On Earth, acetylenotrophy and the corresponding microorganisms, acetylenotrophs, have
been found in bioremediation sites, abandoned gold mines, and fresh water and marine
sediments, despite low environmental concentrations of acetylene (Akob et al., 2018;
Jameson et al., 2019; Lovley et al., 1995; Madrid et al., 2001; Mao et al., 2017; Oremland
and Voytek, 2008; Schink, 1985; Xiao et al., 2007). Although the ocean compositions on

13
Titan and Enceladus are not well-known, I can constrain the plausible environmental
conditions and determine the potential energy yields for Reactions (2) and (6). Here, I utilize
best estimates of Titan and Enceladus oceans’ physical and chemical properties to calculate
values of Gibbs energy for acetylenotrophy. To provide context for comparison, I also
calculate the energy yields of hydrogenotrophic methanogenesis.
I calculated the energy yields of acetylenotrophy varying from 72-96 kJ/mol C,
comparable to the widely considered, hydrogenotrophic methanogenesis, at 25-200 kJ/mol C.
Figure 2.1 displays these energy yields as a function of temperature, substrate concentration,
at the unique subsurface ocean conditions of Titan and Enceladus. Energy yields were
calculated at a pressure of 250 MPa for Titan (Figure 2.1a,b) and 10 MPa for Enceladus
(Figure 2.1c,d) (Glein et al., 2015; Journaux et al., 2020; Postberg et al., 2023; Vance et al.,
2018). In both cases, the ocean temperature was set at 1C, similar to that predicted by Vance
et al. (2018). The pH of Titan’s ocean is hard to constrain; here I assumed neutrality (7.5 at
1C). A slightly alkaline pH (8.5) was used for Enceladus (Glein et al., 2015; Glein and
Waite, 2020; Hoehler, 2022; Postberg et al., 2023). Values of ai were calculated as described
in Amend & LaRowe (2019) for an ionic strength of 0.7 molal (m). Concentrations of H2 and
C2H2 were varied from 10-8
to 1m; the concentration of all other aqueous species were set at
10µm. For Titan, our best estimates of aqueous H2 (1.5 µm) and C2H2 (0.4 µm)
concentrations were based on solubilities of atmospheric H2 (0.1%) and C2H2 (5ppm)
(Lorenz et al., 2019; Vuitton et al., 2019). For Enceladus, assumed concentrations of
hydrogen and acetylene were measured by Cassini mission data. Based on material ejected
into space through plumes, (Dougherty et al., 2006; Hansen et al., 2006; Nixon et al., 2018;
Porco et al., 2006; Spahn et al., 2006; Tokar et al., 2006; Waite et al., 2006) an Enceladus

14
ocean model and mixing ratios (Waite et al. 2009; 2017) yielded levels of 0.1 mm H2 and 20
µm C2H2. Energy yields for acetylenotrophy (Reaction 2) are 72-81 kJ/mol C in a Titan-like
ocean, and 72-84 kJ/mol C in an Enceladus-like ocean.
At the most likely conditions (white stars in Figure 2.1), acetylenotrophy yields 78 and
82 kJ/mol C in the oceans of Titan and Enceladus, respectively. Energy yields for
methanogenesis, by contrast, vary from very low (<25 kJ/mol C) to well above 150 kJ/mol
C for the conditions considered in this study. At the most likely conditions (white stars),
energy yields for methanogenesis in the oceans of Titan and Enceladus are 88 and 112
kJ/mol C, respectively. It should be noted that while the range of energy yields is much
wider for methanogenesis, at the most likely ocean conditions for Titan, acetylenotrophy is
comparably exergonic. While methanogenesis is more exergonic, especially at Enceladus’
ocean conditions, in general these values are similar to one another given the range of
possible values shown in Figure 2.1. I conclude that acetylenotrophy is a viable catabolic
strategy on those ocean worlds and may be a more tantalizing astrobiological target than
methanogenesis, depending on the available substrate. For example, at hydrogen and
acetylene concentrations equal to 0.4µm, acetylenotrophy would be more exergonic than
methanogenesis on both worlds: 78 vs 76 kJ/mol C on Titan and 77 vs 62 kJ/mol C on
Enceladus.
These results illustrate the importance of determining species concentration to calculate
reaction energetics, which in turn is critical for assessing the habitability of ocean worlds.
Within a global subsurface ocean, the concentration of the species of interest would be
diluted, limiting the plausible locations for microbes to thrive. However, even very low
acetylene activities lead to relatively large energy yields, so most any colocation of microbes

15
and the species of interest ought to be sufficient for them to use that substrate. The same is
not true for low activities of hydrogen, where the Gibbs energy of methanogenesis could
represent an endergonic reaction.
Previous studies have calculated the energy yield of hydrogenotrophic methanogenesis
for ocean worlds. Values determined for Enceladus include 40-125 kJ/mol CH4 (Hoehler
2022; Porco et al. 2017; Ray et al. 2021; Steel et al. 2017; Taubner et al. 2018). One study
explored a variety of Enceladus ocean conditions where methanogenesis would be exergonic
(Affholder et al., 2021). The range of reported values represents different presumed ocean
environments using carbonate disequilibrium (or other) models. The key distinguishing
factor that would lead to a higher energy yield is the presumed concentration/activity of H2
available within these modeled oceans, although the presumed pH and temperature of the
ocean also varies across these models with less of an overall change. One ocean model was
distinct: broad enough to predict endergonic as well exergonic methanogenesis, 0-150
kJ/mol (Higgins et al. 2021). Methanogenesis has been considered for other ocean worlds as
well: a maximum energy yield of 125 kJ/mol C for Europa was computed, though the range
varied depending on the model being used (McCollom 1999). Additional energetics studies
on Europa considered many possible metabolisms, including methanogenesis, but not via
Reaction (6) (Zolotov & Shock 2004). Acetylenotrophy has not been considered in these
studies previously. I advocate that it ought to be included in these astrobiology analyses of
other worlds, especially given it could be appreciably exergonic in ocean worlds that are
targeted for astrobiology missions.
Here, I provide the first analyses of energy yields for acetylenotrophy under specific
environmental conditions. I demonstrate that this metabolism is as exergonic as the much-

16
considered hydrogenotrophic methanogenesis at the conditions applicable to Titan and
Enceladus’ oceans. I therefore suggest that future astrobiology missions, including
Dragonfly to Titan (Barnes et al., 2021) and a future Enceladus flagship mission
(MacKenzie et al., 2021b), consider acetylenotrophy in their investigations in addition to
other relevant metabolisms. Acetylenotrophy may have held a crucial role on early Earth,
especially as acetylene may have been over a million times more abundant at that time
(Oremland, 1989; Oremland and Voytek, 2008). The end products of acetylenotrophy
(ethanol, acetate, and hydrogen gas) can be scavenged by methanogens, sulfate reducers and
others for their own metabolisms (Miller et al., 2013). Acetylene also inhibits methanogens
and sulfate reducers since it binds to their key catabolic enzymes (Mao et al., 2017). Early
Earth’s potential abundance of acetylene, and Titan’s current abundance, could have
impeded methanogens and sulfate reducers that were present and attempting to colonize the
soil environments; therefore acetylenotrophs would be needed to reduce the acetylene
concentration and provide for a primordial microbial community (Oremland, 1989;
Oremland and Voytek, 2008). If acetylene is abundant on an ocean world, acetylenotrophy
may be a critical catabolic strategy in establishing microbial communities there. As we
continue to understand the subsurface environments of ocean worlds and substrates available
therein, we can better constrain energy yields for putative microbial lifestyles beyond Earth.
Using the knowledge gained from these energetics calculations, I studied acetylenotrophs
under a laboratory setting in order to estimate the power cost per cell for an acetylenotroph,
so that I might provide predictions that could act as parameters for spacecraft
instrumentation.

17
Figure
Figure 2.1: Filled contour plots of the Gibbs energies, -∆𝐺𝑟
, for hydrogenotrophic methanogenesis (a, c)
and acetylenotrophy (b, d) as a function of temperature and substrate concentration (H2 for panels a and
c and C2H2 for panels b and d). Energy yields are shown in kJ/mol C. The top two panels represent Titan
subsurface ocean pressure, 250 MPa, and neutral pH and the bottom two represent Enceladus’ ocean
pressure, 10 MPa, and pH = 8.5. The starred locations are representing the most likely substrate
concentration for each ocean world considered, at a temperature of 1C and assumed water activity =
1.0. Contour lines for panels a and c represent 25 kJ differences and those for panels b and d represent 3
kJ increments.

18
Chapter Three: Acetylenotrophy on Titan
Laboratory culturing is critical to understanding microbial life. Acetylenotrophs are
relatively understudied and there are few species capable of consuming acetylene. Yet there is a
strong energetics argument for acetylenotrophy to occur under Titan-like conditions. Culturing a
microbe under the conditions of extraterrestrial worlds can provide empirical data to use in future
models and for use in calculations to set detection limits of instrumentation. Here, I cultured an
acetylenotroph and characterized its growth under temperature and salinity conditions. In further
experiments, I coupled cell growth measurements with acetylene measurements to determine the
energy cost of making an acetylenotroph cell. Using my calculated energy costs and those
available from other models, I predict the maximum cell densities possible in Titan-like
environments. The energy cost is a critical variable to the number of cells possible, but the
acetylene activity is also crucial.
3.1 Introduction & Background
In most environments on Earth, 70-97% of the microorganisms present have never been
cultured in the lab (Steen et al., 2019). This could be due to the differences between the
laboratory and the in situ environments: lab environments are well-defined but may not provide
critical conditions for uncultured microbes creating a bias towards some organisms (e.g.
temperature and substrate conditions, Rivera-Yoshida et al., 2020). Furthermore, most microbes
live in nutrient-limited and energy-limited environments (Bradley et al., 2020; Jackson and
McInerney, 2002) and find themselves inundated with nutrients and substrates, etc. in the lab,
which may induce a stress response causing the microbes to not grow. Yet laboratory culturing is
a critical mechanism for studying microbes, understanding their growth limits, and providing
empirical data to refine models.

19
Acetylenotrophy is found in roughly a dozen different species, yet is understudied and has
been the subject of too few culturing experiments. The type strain is Syntrophotalea
acetylenica—first isolated in 1985 from freshwater and marine sediments, has been found in a
variety of global environments since. S. acetylenica belongs to the Deltaproteobacteria and is a
rod-shaped gram-negative bacterium of approximately 1µm in length (Figure 3.1). During its
original isolation, no growth was observed with common substrates such as glucose, succinate,
pyruvate, or many others (Schink, 1985). It will consume acetoin (C4H8O2) via Reaction 7,
C4H8O2 + H2O → CH3CH2OH + CH3COO
− + H
+ (7)
which is akin to the acetylenotrophy reaction, Reaction 2, as it takes either acetylene or acetoin
to form two molecules of the intermediary acetaldehyde (CH3CHO), and one molecule of
acetaldehyde becomes ethanol and the other becomes acetate. The energy yields for an
acetylenotroph using acetoin or acetylene as its substrate are shown in Figure 3.2. Acetoin as the
substrate has energy yields from 21-37 kJ/mol C via Reaction 7, whereas using acetylene as the
substrate provides energy yields from 72-93 kJ/mol C from Reaction 2. Consuming acetylene as
the substrate is calculated to be over three times as exergonic than consuming acetoin under the
conditions of laboratory culturing (see 3.2 Methods for details on conditions).
Two strains of S. acetylenica are available from the German Collection of Cultures and
Microorganisms (DSMZ): DSMZ 3246 which is the freshwater strain, WoAcy1, and DSMZ
3247 which is the marine strain, GhAcy1. These strains were isolated and described in the same
study. The only distinction is the salinity of their respective environments, marine and
freshwater sediments, however, no further description of their natural environment is provided.
In the years following its initial isolation, WoAcy1 has been used consistently in studies and
GhAcy1 has not. Furthermore there is a strain, GhAcy3, that is discussed in the isolation study,

20
but is not available in any culture collection (Schink, 1985). Schink 1985 does not show growth
curves, growth rates, or other data regarding growth of GhAcy1. A growth curve for WoAcy1
under optimal conditions with acetylene as the substrate was shown. They report temperature
and pH limits for WoAcy1. Temperatures of 30-34C with acetylene as the substrate showed
growth,
“which was the optimum temperature range. The same growth rates were found with
acetoin. Growth limits with acetoin were at 15 and 45C. The optimum pH was 6.5-7.5;
no growth was found at pH 6.0 and pH 8.0. […] Strain WoAcy1 grew in freshwater
medium as well as in saltwater medium, and the same was true for strain GhAcy3. […]
Contrary, strain GhAcyl did not grow in freshwater medium and only weakly in brackish
water” (Schink, 1985).
Given uncertainties about the salinity of possible habitats on Titan, I carried out
experiments with both the fresh and marine strains of S. acetylenica. Titan hosts two putative
aqueous environments where acetylene might be co-located with other necessary nutrients to
foster life: the subsurface ocean of significantly greater volume than the ocean water here on
Earth (MacKenzie et al., 2021b), and possible impact melt ponds on the surface. I will describe
these environments and the potential availability of acetylene (energy source) and nutrients
(Sulfur, Phosphorous, Oxygen, Nitrogen, Carbon, and Hydrogen).
3.1.1 Subsurface Ocean
The observations and data available for Titan’s ocean limit our understanding to simply infer
that the ocean exists, is comprised of liquid water with some solute or salt composition, and its
volume is roughly 18 times that of Earth’s ocean water. As the age of Titan’s ocean is
undetermined (thousands to millions of years old), perhaps life had sufficient time to originate
and evolve within the ocean. Some ocean components are theorized to be present like ammonia
or methanol given different possible origins of Titan’s ocean (Deschamps et al., 2010;
MacKenzie et al., 2021a; Vance et al., 2016). Magnesium sulfate has also been considered

21
(Vance et al., 2018) as well as hydrogen cyanide given its prevalence on the surface, but neither
appear to be included in models (Hedgepeth et al., 2022; Hörst, 2017; Vance et al., 2016).
Furthermore, the heating caused by the tidal friction with Saturn could also contribute to
maintaining an ocean (Hay and Matsuyama, 2017; Perkins, 2012).The ocean is likely near 0C
but possibly even lower; liquid water is maintained by the high pressure environment (650-
850MPa), tidal heating, and a freezing-point depression could be due to the high concentration of
ammonia or methanol (Vance et al., 2018). At ocean formation and prior to the deeper ice-shell
formation sequestering the ocean from the silicate core, water-rock interactions were possible
allowing for salts and metals to enter the ocean. Carbon-, Hydrogen-, Oxygen-, and Nitrogenbearing species are detected in Titan’s atmosphere and on its surface (MacKenzie et al., 2021b;
Nixon et al., 2018). I postulate that phosphorous and sulfur could be introduced into its ocean via
water-rock interactions, given that the materials that Titan coalesced from are likely represented
by carbonaceous chondrites (e.g. CM chondrites that likely formed phosphorus- and sulfurbearing species during solar system formation, Nazarov et al., 2009). While current surfacesubsurface material exchange on Titan is unconstrained, at some point since ocean formation,
surface material was likely incorporated into the ocean. Thus acetylene formed in the atmosphere
and deposited onto the surface of Titan could be incorporated into this aqueous environment
collocated with necessary nutrients. Given that cryovolcanism is a possibility on Titan’s surface,
perhaps cells/life formed in the ocean are transported to the surface or otherwise embedded into
the water ice crust.
3.1.2 Surficial Impact Melt Ponds
Theorized impact melt ponds on Titan’s surface are exciting potential oases for life. An
impactor colliding with the surface of a planetary body produces sufficient heat to melt the

22
surrounding material; for Titan, this is water ice and surface organics. At the -179.15C surface
temperatures of Titan, a cap will immediately freeze insulating a pool of liquid water that can
remain for 10s to 10000s of years (depending on the size of the crater and model used,
Artemieva and Lunine, 2003; Hedgepeth et al., 2022; Kalousová et al., 2024; O’Brien et al.,
2005). There are several possibilities for how life could be introduced or originate in such an
ephemeral environment. While pangermia may be a less supported origin of life theory for Earth,
it is one still under consideration for other worlds (Yamagishi et al., 2019). The possible delivery
of cell matter via the impactor could introduce life to the impact melt. Life could be endogenous
to Titan as well. Given the possibility of subsurface ocean material being delivered to the surface
of Titan via cryovolcanism (for example), perhaps cells have been emplaced and preserved (i.e.
sporulation) in the icy surface for some time, and are able to germinate under the impact melt
conditions.
One of the reasons acetylenotrophy is such a promising candidate for Titan is the abundance
of acetylene. Acetylene and many other organic molecules are formed in Titan’s atmosphere via
photochemistry. These hydrocarbons are deposited on the surface to form a regolith, lying above
a water-ice bedrock. Other than water ice and the composition of the impactor itself, these
organics would become part of the impact melt and could create slightly different habitats
depending on relative concentrations, salts, etc. The likely pH range of these ponds would be
basic given the pKa values of known surface components. For example, hydrogen cyanide would
have a pKa of 9.96 under Titan’s surface conditions (Hedgepeth et al., 2022). The impactor and
any minerals, metals, nutrients (amino acids), etc. enclosed within it are also incorporated into
the impact melt pond. As such, these ponds would likely have sufficient Carbon, Hydrogen,
Nitrogen, Oxygen, Phosphorous, and Sulfur to support life, as well as trace metals that may not

23
be found on Titan’s surface (Aléon et al., 2002; Alexander et al., 2008; Connolly et al., 2001;
Cronin and Pizzarello, 1983). Although salts could be incorporated from the impactor, I consider
impact melts to be a freshwater environment on Titan as the icy bedrock melted to make up the
pond would likely be comparable to sea ice on Earth. During formation of sea ice, two thirds of
the water’s salt content is rejected from the ice. The remaining brine, at cold temperatures <0C,
form solid salt inclusions within the ice. Salts and brines are lost with time, and after a million
years, sea ice has a salinity of 3 parts per thousand (Weeks and Schroeter, 2019). Since the
surface ice lies ~100km above the ocean and could have formed millions of years ago, I assume
sufficient time has passed that most salt has left the ice. Additionally, the regular deposition of
1500 mols of acetylene per second globally ought to make it readily available within the impact
melt given that surficial organics and water-ice would form the bulk of the melt.
These environments will be the focus of Titan-like experiments. I report the Gibbs energies
for acetylenotrophy (Reaction 2) under these different environmental and laboratory conditions
in Table 3.1. The environmental conditions, the metabolic energy yields of acetylenotrophy, and
the energy cost of producing an acetylenotroph cell were determined and used to predict putative
cell densities on Titan. I cultured an acetylenotroph in the lab to parameterize cell density models
on Titan.
3.2 Methods
I characterized the growth of the GhAcy1 (marine) and WoAcy1 (fresh) strains of
acetylenotrophs under different temperature, pH, and salinity (NaCl concentrations) conditions.
Additionally, I performed experiments under conditions that overlapped between the aqueous
environments on Titan and the growth limits identified. Finally, using the data gathered over
these experiments and energy yield calculations, I was able to approximate the energy cost to

24
produce an acetylenotroph cell which in turn can be used to predict the maximum cell densities
within those environments on Titan.
To begin culturing S. acetylenica in the lab, I acquired pure cultures of the fresh and marine
strains from the DSMZ. Using DSMZ recipes for Media 293, for GhAcy1 (Table 3.2), Media
298, for WoAcy1 (Table 3.3), and trace metal solution SL-10 (Table 3.4) for both, I prepared the
media in either 10mL Balch tubes or 50mL serum bottles. Media 298 had a final NaCl
concentration of 1 g/L, used Na2CO3 as its buffer with an 80:20 N2/CO2 headspace to maintain a
7.2-7.5 pH range. Media 293 had a final NaCl concentration of 20g/L, used a NaHCO3 buffer
with an 80:20 N2/CO2 headspace to maintain a pH of 7.2-7.5. A final concentration of 1.5mM
Na2S x 9H2O was used to keep both media reduced and scrub any oxygen that inadvertently
entered the bottles. Resazurin was used as a redox indicator in order to indicate any oxidation of
the media after being reduced. Tables 3.2, 3.3, and 3.4 detail the media recipes as followed from
the DSMZ. All media preparation was done anaerobically, either sparged with 80:20 N2/CO2 or
100% N2, and nearly all constituents were autoclaved. The sulfide, the sodium carbonate and
bicarbonate, and the acetoin stock solutions were filter sterilized. These filter sterilized stocks
used autoclaved and filtered MilliQ water to minimize introduction of other biological material.
Growth characterization experiments considered on the two strains were carried out over a
range of temperatures, pH values, and salinities. The optimal conditions were determined to be
30C, pH=7.5, at preferred salinities (1g/L, fresh and 20 g/L, marine), and acetoin provided as
the substrate. As temperature was altered, salinity and pH were kept optimal. The same
framework applied to the experiments varying salinity and pH as well. Growth rates were
determined for each set of conditions. Temperature experiments tested growth from 15C to
50C. Growth rates for salinity conditions were determined for NaCl concentrations between

25
1g/L to 35 g/L. Growth was tested under various pH from 6.0-9.0 using buffers used for other
acetylenotroph studies (Baesman et al. 2021); unfortunately, no growth was observed outside of
the optimal range from, pH 7.2-7.5.
Growth was measured via cell counts using fluorescence microscopy and nucleopore
filters (Hobbie et al., 1977). A typical image of S. acetylenica cells are shown in Figure 3.1.
Samples were taken with needles and syringes flushed with N2 gas to dispel any oxygen. A
sample volume of 0.5mL was collected at each timepoint and diluted if necessary with filtered
MilliQ H2O to maintain a volume of 0.5mL. The samples were added to Eppendorf tubes
containing 25 µL of 25% Glutaraldehyde Microscopy solution and immediately vortexed in
order to fix the cells and prevent lysing. Cell count samples were stored at 4C if not counted
immediately. When ready to be counted, 125µL of 10x SYBR Green I nucleic acid stain was
added to each sample, vortexed, and then incubated in the dark for at least 10 minutes (MartensHabbena and Sass, 2006). Samples were then filtered onto 25mm, black nucleopore filters, and
counted immediately using fluorescence microscopy (Hobbie et al., 1977).
After the growth characterization experiments, I performed “Titan-like” experiments using
the conditions informed by the acetylenotroph growth limits. I considered the fresh strain under
the conditions of a freshwater environment (surficial impact melt ponds) and the marine strain
under the conditions of a more marine environment (subsurface ocean). In addition, these
experiments feature acetylene as the substrate instead of acetoin to better simulate Titan.
Previous studies added up to 10mL of acetylene gas to the headspace (Seitz et al., 1990), but
since this amount approaches toxic levels even for microbes adapted to eating acetylene
(~40µM, Fulweiler et al., 2015; Yoshinari and Knowles, 1976), I performed two sets of freshwater experiments and two sets of marine experiments: each done in triplicate, an abiotic

26
control, and differed by the addition of either 1mL or 2mL of gaseous acetylene at inoculation.
The equilibrium aqueous acetylene concentration was ~31µm with 1mL and ~62µm with a 2mL
addition. Both the fresh and marine experiments were performed at 20C, the coldest growth
that was observed (Figure 3.4), pH = 7.5, the most basic pH where growth was observed (data
not shown), and using acetylene gas in the headspace as the substrate. All media was prepared
as previously described. Cell counts were performed using the aforementioned protocol as well.
At each timepoint, 1mL of headspace was sampled with air-tight syringe and immediately
injected into the gas chromatograph (GC) with a Flame Ionization Detector (FID). The FID is
used to detect acetylene with a retention time of ~5.8 minutes. To maintain pressure, an
equivalent volume of pure N2 gas was added at each timepoint to replace the volume removed.
Acetylene used in these experiments was produced in lab via Reaction 8, calcium carbide plus
water generated acetylene gas and solid calcium hydroxide.
CaC2 + 2H2O → C2H2 + Ca(OH)2
(8)
The acetylene-generating protocol and apparatus used were designed and shared by the Reston
Microbiology Laboratory at the USGS in Reston, VA, and have been used in other
acetylenotrophy studies (Akob et al., 2018; Baesman et al., 2021). As acetylene is highly
flammable and may explosively polymerize if stored/transported in standard gas tanks, I used the
generated acetylene to create standards from 25% down to 0.1% for use on the GC. The standard
curve yielded an R2 = 0.998.
By measuring a sample of the headspace for acetylene on the GC in addition to cell count
measurements at each timepoint, I calculated energy costs per cell (Table 3.5). Each of the
experiments using acetylene had roughly the same energy yield (166-168 kJ/mol C2H2, Table
3.1) as the only different variables were ionic strength across the marine and fresh experiments,

27
and the concentration of acetylene between the 1mL and 2mL experiments. The total mols of
acetylene consumed were derived from the change in acetylene with time. The energy yield of
acetylenotrophy was used to calculate the average energy cost per cell, Table 3.5, assuming all
the energy produced goes to making cells.
Predicted cell densities on Titan were calculated using the total volume of acetylene
available in the environment as well as the energy yield for acetylenotrophy under the prevailing
environmental conditions (Table 3.1). The cell densities represent the maximum number of cells
that could be produced, as all available acetylene in the environment is used for energy, and all
energy is taken to be used for cells. For the subsurface ocean environment, I assumed a marine
environment with an ionic strength similar to Earth’s, I = 0.7m. It is likely that the pH of the
subsurface ocean would be rather basic given the possible components. For example, ammonia
has a pKa of 10.06 in the conditions of Titan’s subsurface ocean (Deschamps et al., 2010). The
current rate of acetylene production in the atmosphere is treated as constant since the ocean
formed (up to 10 Mya), 4.6x10-14 g/cm2
s (Vuitton et al., 2019). In order to determine acetylene
concentration in the ocean, I assume there was sufficient time for the atmosphere and ocean to
achieve equilibrium, or otherwise transfer a large delivery of acetylene to the subsurface ocean.
With the observed atmospheric concentration and solubility, this leads to a possible acetylene
concentration of 0.4µm, as used previously in Chapter 2 and Figure 2.1. For the surficial impact
melt ponds, a basic pH is also likely given the surface organics incorporated into the melt. And
as described in 3.1, I consider this environment to be low salt content akin to the 1g/L NaCl
concentration. Using the current rate of acetylene production and deposition observed today
(Vuitton et al., 2019) as constant for 10,000 years prior to impact, I could determine the volume
of acetylene deposited on the surface. Models suggest ~5-10% of the volume of the crater is

28
retained as melt (Artemieva and Lunine, 2003; Hedgepeth et al., 2022; Kalousová et al., 2024;
O’Brien et al., 2005), and assuming Selk Crater, the Dragonfly landing site, is host to an impact
melt pond, the 84km in diameter crater would have an acetylene concentration of 1-3µM.
The energy cost per cell of an acetylenotroph was determined from four different
experiments. The marine and fresh experiments with 1mL of acetylene yielded similar values, as
such the cell density predictions were indistinguishable. I averaged the energy cost of the 1mL
experiments, used the two unique energy costs determined from the marine and fresh
experiments with 2mL of acetylene, and considered a modeled energy cost for an E. coli cell
(Ortega-Arzola et al., 2024). These four different energy costs were used to predict maximum
cell densities for different environments on Titan, and Enceladus’ subsurface ocean for
comparison (Figure 3.8).
3.3 Results
Figure 3.3 shows growth of both strains of S. acetylenica in triplicate under optimal
conditions at 20C. Over the course of one week, nearly 200 hours, cell densities would increase
by four orders of magnitude. The growth rates of S. acetylenica for different temperatures are
shown in Figure 3.4. As compared to previous reports (Schink, 1985), I observed zero growth
(and even cell death in some replicates) at 15C and 50C for both strains. The optimum
temperature is in the 30-40C range consistent with the previous study (Schink, 1985). Strong
growth was observed at 20C (Figure 3.3 and 3.4). As such, later experiments designed to be
Titan-like were cultured at 20C so that sufficient growth would occur.
Figure 3.5 shows the growth rates as a function of NaCl concentrations for both strains of S.
acetylenica. The marine strain demonstrated more tolerance to freshwater conditions, growing
from 15 g/L to 35 g/L NaCl, whereas the fresh strain had significantly lower growth rates for all

29
NaCl concentrations greater than 1 g/L. The optimal salinity is 20g/L NaCl for the marine strain
and 1g/L NaCl for the fresh strain. All experiments were completed in triplicate and in some
cases (5, 10, and 15 g/L) were performed multiple times to ensure consistency of data. Growth
rate experiments were attempted from pH = 6.0 to 8.5, but did not significantly indicate growth
out of the 7.2-7.5 range (data not shown).
For those experiments performed with acetylene gas in the headspace, I observed less total
growth than when acetoin was provided. At 20C with acetylene gas, Figure 3.6, the cell
densities increase nearly three orders of magnitude over the same amount of time where those
grown at 20C and acetoin (Figure 3.3) increased four orders of magnitude. Acetylene
consumption was monitored each day. The abiotic controls showed some decrease in acetylene
concentration initially, due to the headspace equilibrating with the media and pressure changes
with sampling, but always plateaued with time (Figure 3.7). Acetylene concentrations dropped
substantially in each experiment as it was consumed by the cells present. In the marine
experiment with 1mL of acetylene added, acetylene gas was consumed entirely (i.e. 0% at end of
experiment), whereas the abiotic control dropped to ~1.4% and held there (Figure 3.7). Cell
growth was observed in all cases, yet was less in the experiments with 2mL of acetylene added
(Growth curves shown in Appendix A.1).
As previously described (Sec. 3.2), I calculated an energy cost per cell for each experiment.
These values varied from 7 x 10-10 kJ/cell to 285 x 10-10 kJ/cell (Table 3.5), but the marine and
fresh experiments using 1mL of acetylene produced very similar values (7.3 x 10-10 and 7.6 x 10-
10 kJ/cell). Therefore, I averaged those two values and I report three unique energy costs that are
empirically derived. The 1mL acetylene experiments yielded the most optimistic energy cost of
7.48 x 10-10 kJ/cell. The 2mL acetylene fresh experiment yielded an energy cost of ten times

30
greater, 76 x 10-10 kJ/cell. The 2mL acetylene marine experiment yielded an even greater energy
cost of 285 x 10-10 kJ/cell. Potential explanations for the difference in energy costs are in Sec.
3.4. I calculated predicted cell densities for these values and the minimum energy cost reported
in Ortega-Arzola et al. 2024 for a typical Escherichia coli cell (0.000954 x 10-10 kJ/cell).
Regardless of the energy cost used, cell densities varied across the environment considered.
For the two environments on Titan, an impact melt pond in Selk Crater could support more cells
than the subsurface ocean at all energy costs. However, I also considered Enceladus’ subsurface
ocean as an environment of interest. At all energy costs, higher cell densities could be supported
in Enceladus’ ocean than any other environment. For comparison, hydrothermal vent fluid on
Earth is known to have cell densities of ~102
cells/mL. At the energy cost of the 1mL acetylene
experiments, Selk Crater could support more cells than hydrothermal vent fluid. At the minimum
energy cost per cell (0.000954 x 10-10 kJ/cell), Selk Crater could support greater than 106
cells/mL, which is equal to the cell density found in any natural aquatic environment on Earth.
Titan’s subsurface ocean could also support a population near 106
cells/mL at the same energy
cost. However, cell densities in Enceladus’ subsurface ocean do not fall beneath 102
cells/mL at
any energy cost. If acetylenotroph cells operate efficiently, the minimum energy cost per cell
may be more representative. Otherwise, under times of stress, microbes may require more energy
per cell produced and a lower cell density would result. These cell densities also represent the
maximum number of cells created given all available acetylene is consumed—a snippet of time
in the putative history of life on Titan.
3.4 Discussion
As discussed in Chapter 1, acetylenotrophy is an understudied metabolism and there have
been relatively few laboratory culturing campaigns to characterize S. acetylenica among other

31
acetylenotrophs. The growth rates of S. acetylenica reported in Figures 3.4 and 3.5 are
somewhat contradictory to the reported observations of the original isolation study for these
strains (Schink, 1985). For example, I observe the opposite trend between the two strains in
regards to salinity tolerance: Schink 1985 reports that the fresh strain WoAcy1 could grow in
marine media which I did not observe. They also report that the marine strain GhAcy1 could not
grow on fresh or brackish media, yet I observed this growth. The disparities between my studies
and the isolation study of S. acetylenica are intriguing: the field of microbiology has bloomed in
the decades since, and the lack of data provided in Schink 1985 limits are ability to
contextualize the current study’s results. However, continuing laboratory culturing campaigns
on acetylenotrophs could continue to refine the knowledge of S. acetylenica and other
acetylenotrophs.
When performing the acetylene (Titan-like) experiments, slightly less growth was observed
than those experiments where acetoin was the substrate. As such, this was reflected with much
higher energy costs per cell. One possible explanation would be that a high acetylene
concentration (>40µm) is inevitably toxic to acetylenotrophs, as is true for microbes that
perform sulfide reduction, methanogenesis, or diazotrophy (Mao et al., 2017; Yoshinari and
Knowles, 1976). Translating Titan-like environments to laboratory conditions is an imperfect
practice: since Earth life is the only example for us to study, we cannot subject it to all Titanlike conditions as it simply would not be able to grow. I altered the conditions that are
reasonable to reflect these environments but not so unreasonable that life as we know it cannot
grow. For example, attempting to mimic the extreme pressure environment of Titan’s
subsurface ocean is beyond the scope of these experiments, although other groups focus on such
endeavors (Malas et al., 2024).

32
Nonetheless, the energy costs derived from the acetylene experiments spanned three orders
of magnitude. And the modelled minimum energy cost was three to six orders of magnitude
smaller than those costs, however this energy cost is modelled for a different bacterial cell:
Escherichia coli. E. coli strains are considered model organisms for many microbiology studies,
due to its quick doubling time (minutes to hours for some strains). As the subject of many
studies, the cellular processes of E. coli are well-defined and understood. Therefore, it is
possible to incorporate it as an ideal bacterial cell for this energy cost model with fewer
assumptions than other bacteria. The energy cost for such an ideal organism may not be
representative of most bacteria, especially those anaerobic and slower-growing organisms like
S. acetylenica. Although, E. coli and S. acetylenica are extremely comparable cells in size and
shape, as well as being gram-negative, so perhaps the building blocks of the cell are more
similar. The model does intentionally estimate the minimum energy cost to build a bacterial cell
and represents the most optimistic case in the cell density predictions. Other cell energy cost
estimates yield values in line with the robust model of Ortega-Arzola et al. 2024: 0.0000198 x
10-10 to 0.192 x 10-10 kJ/cell (LaRowe and Amend, 2015) as compared to 0.000954 x 10-10
kJ/cell. Modelled energy costs were significantly lower than those calculated from laboratory
experiments.
Most microbes on Earth are located within energy or nutrient limited conditions. The stress
of those environments is not mimicked in laboratory environments. Under the 2mL acetylene
experiments (as compared to the addition of 1mL), the microbes were still consuming acetylene
for energy, but less of that energy was being used to make cells, hence the increased power cost.
Under stressed conditions, bacteria and other microbes will not necessarily make more cells
until conditions improve (Hamill et al., 2020). Given the still limited understanding of

33
acetylenotrophs, using the minimum energy to build a cell derived from intracellular processes
of other bacteria may be more representative of the energy requirement to build biomass under
in situ environmental conditions. However, the ideal process would be to continue to observe
substrate consumption coupled with biomass production for different bacteria performing
various metabolisms (i.e. redox and fermentative metabolisms).
Previous work has predicted cell densities on Enceladus for methanogenesis (Reaction 6)
using maintenance energy, substrate flux, and energy yields (Cable et al., 2021). Maintenance
energy reflects the energy expended by microbial cells for maintenance, not for producing more
biomass. Unfortunately, maintenance energy is extremely difficult to determine empirically and
has not been well approximated for in situ environments (Hoehler and Jørgensen, 2013; Tijhuis
et al., 1993). A comparable value would be the minimum power cost per cell—using
environmental data where cells are observed to live and grow, one can determine what the
minimum power available to cells would be (Bradley et al., 2020). In addition, recent modeling
has incorporated empirical data and kinetics of known intracellular processes to predict the
minimum energy cost to produce a cell depending on individual inputs and specific cell types
(Ortega-Arzola et al., 2024). Ultimately, the energy cost required to build a cell is easier to
constrain and model than the energy cost of maintaining a cell. Acetylenotrophs may be wellsuited candidates to inhabit Titan, however continued studies on the growth characterization and
coupled biomass and acetylene consumption experiments are needed to better constrain our
predictions.
3.5 Outlook
Predicting cell densities can provide detection limits for spacecraft instrumentation such as
future missions to the surface of Titan to search for life. While the predicted cell densities range

34
from 10-106
cells/mL, spacecraft instrumentation may be able to detect between 102
and 106
cells/mL given that they are often field tested in natural environments on Earth. Although these
cell densities are approximations and assume that all available acetylene is consumed for energy,
and all energy goes into making cells, it is exciting to note that an impact melt pond within Selk
Crater could support more cells than Titan’s subsurface ocean at all energy costs. Selk Crater will
be the Dragonfly landing site in the 2030s and will be host to a variety of measurements and
analyses in the search for life as we do or do not know it. Energetics forms the basis of predicting
these cell densities and remains a useful tool to probe these ocean world environments where our
data is limited. While optimal data is gathered by in situ missions and observations, utilizing the
tools at our disposal on Earth prepares missions to push the limits of instrumentation in order to
gather multiple lines of robust evidence and discover life elsewhere.

35
Figures and Tables
Table 3.1: A summary of the conditions and Gibbs energies calculated for different environments and
those replicated in the lab. All other concentrations were held at 10µm.
Case Temp. (C) Pressure [C2H2] µm Location ∆𝑮𝒓
kJ/mol C2H2
FreshwaterTitan 1C 1.5 bar 2.1 Impact Melt Pond -155
MarineTitan 1C 250MPa 0.4 Subsurface Ocean -157
Enceladus
Ocean 1C 10MPa 20 Subsurface Ocean -163
Lower
acetylene 20C 0.1 MPa 340 Laboratory -167
Higher
acetylene 20C 0.1 MPa 680 Laboratory -168

36
Table 3.2: A list of the components of DMSZ Media 293 used for the marine strain GhAcy1 and used for
marine-like experiments.
DSMZ Media 293
Media Component Amount added to 1L
KH2PO4 0.20 g
NH4Cl 0.25 g
NaCl 20.00 g
MgCl2 x 6 H2O 3.00 g
KCl 0.50 g
CaCl2 x 2 H2O 0.15 g
Trace element solution SL-10 1.00 ml
Na-resazurin solution (0.1% w/v) 0.50 ml
NaHCO3 2.50 g
Acetoin 1.00 g
Na2S x 9 H2O 0.36 g

37
Table 3.3: A list of the components of DMSZ Media 298 used for the freshwater strain WoAcy1 and used
for freshwater experiments.
DSMZ Media 298
Media Component Amount added to 1L
KH2PO4 0.20 g
NH4Cl 0.25 g
NaCl 1.00 g
MgCl2 x 6 H2O 0.40 g
KCl 0.50 g
CaCl2 x 2 H2O 0.15 g
Trace element solution SL-10 1.00 ml
Na-resazurin solution (0.1% w/v) 0.50 ml
Na2CO3 1.50 g
Acetoin 1.00 g
Na2S x 9 H2O 0.36 g

38
Table 3.4: A list of the components of DMSZ Trace element solution SL-10 used in all laboratory
experiments to provide various trace metals.
DSMZ Trace Element Solution SL-10
Component Amount added to 1L
HCl (25%) 10.00 ml
FeCl2 x 4 H2O 1.50 g
ZnCl2 70.00 mg
MnCl2 x 4 H2O 100.00 mg
H3BO3 6.00 mg
CoCl2 x 6 H2O 190.00 mg
CuCl2 x 2 H2O 2.00 mg
NiCl2 x 6 H2O 24.00 mg
Na2MoO4 x 2 H2O 36.00 mg

39
Table 3.5: A list of the energy costs per cell used to predict cell densities shown in Figure 3.8. The first
three rows were calculated from experiments. The final one is taken from the reference.
Experiment Energy Cost per Cell
(10-10 kJ/cell)
Average of Fresh and Marine 1mL
C2H2 experiments 7.48
Marine Experiment w/ 2mL C2H2 285
Fresh Experiment w/ 2mL C2H2 76
Ortega-Arzola, et al. 2024—E.coli cell 0.000954

40
Figure 3.1: An image of Syntrophotalea acetylenica marine strain GhAcy1. Scalebar represents 5µm.
Cells are stained using SYBR-Green I nucleic acid stain and viewed using fluorescence microscopy. The
picture contrast is increased to better visualize the cells.

41
Figure 3.2: Gibbs energy of A) acetoin and B) acetylene fermentation, Reactions 7 and 2, as a function of
temperature and substrate concentration. The white stars refer to the conditions prevailing in the
laboratory experiments noted in Section 3.2 as optimal. Acetoin thermodynamic properties were
extrapolated from other known data and calculated using equations of state.
B)
A B

42
Figure 3.3: Growth curves for the marine and fresh strains of S. acetylenica at 20C and optimal
conditions as described by the DSMZ. These represent the mean of three cultures.

43
Figure 3.4: Growth rates as a function of temperature for the marine and fresh strains of S. acetylenica
at pH = 7.5, [NaCl] = 20 g/L for the marine strain, and [NaCl] = 1 g/L for the fresh strain, optimal
conditions as described by the DSMZ.

44
Figure 3.5: Growth rates as a function of sodium chloride (salinity) concentrations for the marine and
fresh strains of S. acetylenica at 30C and pH = 7.5, optimal conditions as described by the DSMZ

45
Figure 3.6 Growth curve of the marine strain of S. acetylenica coupled with measurements of the
acetylene in the headspace. This experiment was performed at 20C, pH = 7.5, and instead of acetoin,
had 1mL of gaseous acetylene added to the headspace prior to inoculation.

46
Figure 3.7: Consumption of acetylene with time as measured via gas chromatography. The blue line
represents a control containing no cells; whereas the red, green, and orange lines represent the culture in
triplicate. Acetylene can be seen to be completely consumed in the cultures and not in the control.

47
Figure 3.8: Predicted cell densities for acetylenotrophs across three environments: An impact melt pond
in Selk Crater on Titan (green), Titan’s subsurface ocean (blue), and Enceladus’ subsurface ocean
(purple). Four energy costs were used for these predictions; 1mL-C2H2 (leftmost) represent the average
energy cost per cell of the marine and fresh experiments where 1mL of acetylene was added. Marine
2mL-C2H2 and Fresh 2mL-C2H2 represent the same but where 2mL of acetylene was added. The right
most energy cost represents the minimum energy cost to produce a cell as modelled for E. coli published
in Ortega-Arzola, et al. 2024.

48
Chapter Four: The Intersection of Scientific Research & Education
4.1 Philosophy on Scientists’ Role in Education
Throughout my career, I want to give back and motivate younger generations of scientists
and scholars. Despite attending many schools that were underfunded and therefore underachieving, I found incredible educators that stood in my corner, allowed me to explore my
interests, and fostered a can do energy. Now as a scientist and educator myself, I recognize the
rarity of resources available to those schools and educators. In a post-COVID society, remote
learning tools are abundant and there are many applications that can foster curiosity amongst
students. However, there are too few content/subject matter experts to adapt these tools to meet
educator’s needs.
Under the advisement of Dr. Dieuwertje Kast while acting as the STEM Education
Fellow for the Joint Education Project (JEP) at USC, I designed and tested lesson plans aimed at
K-5 students that were aligned to the Next Generation Science Standards, federal and state
supported content standards for science and engineering across K-12 education. These lessons
are meant to be direct translations of the research I report in this thesis. The aim is to provide
tools to introduce astrobiology in the elementary classroom. The lessons can be taught stand
alone or together as a unit; they highlight astronomy, planetary formation, mission development,
mission limitations, microbial energetics, and laboratory culturing of microbes.
Once the lessons were completed, I piloted the lessons in afterschool programs both
virtually and in-person through the JEP. After revision from those experiences, I hosted a teacher
professional development workshop where teachers could earn a salary credit by attending and
using the provided supplies to teach these lessons in their own classrooms. Ultimately, the lesson
plans were submitted to the National Earth Sciences Teaching Association’s peer-reviewed

49
publication The Earth Scientist (TES). TES reaches thousands of Earth Sciences teachers across
the country. Furthermore, these lesson plans are provided for free to anyone who requests them.
The GoogleDrive containing the lesson plans has additional resources for teachers to prepare
themselves for delving into astronomy and microbiology. A reproduction of the article submitted
to TES is included in this chapter.
Scientists are in a critical and empowering role in society: we are lucky to be voices of
authority in the fields that we build our expertise. I believe more unique lesson plans directly
adapted from published research is a pathway to exciting more students about STEM. A path that
only scientists can forge and one that should be heavily encouraged. Furthermore, the inclusion
of more diverse representation and exposure to more folks and content within STEM has been
documented to support the entrance and retention of underrepresented students in STEM. The
best way scientists can help in education is to become an active force that provides new content
and works alongside educators to implement the content in the most fruitful manner.
Lesson Plans can be found at the GoogleDrive below and linked in the USC Digital Library
alongside this dissertation:
https://drive.google.com/drive/folders/10Ybtt26Nme_3FqWWbFpY8LDt3mO4pyGy?usp=drive
_link
The manuscript below was published in 2024 as:
Yanez, M., Stellmann, J., LaRowe, D., Amend, J., Kast, D.J. (2023) “Astrobiology: Bringing the Search
for Aliens to Elementary Classrooms.” The Earth Scientist. National Earth Science Teaching Association.
Volume XL, Issue 3, pages 11-19.

50
4.2 Astrobiology: Bringing the Search for Aliens to Elementary Classrooms
Maya D. Yanez1*, Jessica Stellmann2
, Doug LaRowe1
, Jan Amend1,3 & Dieuwertje Kast2
Abstract:
Astrobiology is an interdisciplinary field focusing on the origin of life and search for life beyond
Earth. It uses key methods from STEM fields already aligned with Next Generation Science
Standards (NGSS): Biology, Chemistry, Geology, Math, and Physics. We translated the doctoral
research of an astrobiologist into four lesson plans aimed at elementary students (K-5) for the
University of Southern California’s Joint Educational Project’s (JEP) STEM education
programs. These lesson plans were piloted in two forums: a virtual JEP STEM afterschool
program for K-5 students and a teacher professional development workshop. In the afterschool
program, we observed an increase in comprehension of astrobiology concepts in just two lessons.
The teacher workshop provided feedback that was utilized to enhance the lessons. The use of
interdisciplinary lesson plans offers educators the freedom to incorporate them where they best
align with standards and other curricula. Furthermore, the lessons can be expanded on or
adjusted to be better suited for different grade levels. Scientists summarizing their work and
designing lesson plans from their research data are crucial in bringing a variety of relevant and
current educational information to our schoolchildren. STEM fields are exciting, complex, and
often interdisciplinary; hence they can be less accessible, but this standard can be challenged by
the implementing lessons designed by scientists for elementary-aged children.
Introduction/ Background Information:
Astrobiology is an interdisciplinary field that focuses on the study of and search for life
and its origins on and beyond Earth. Studying the various ways life has adapted to unique
environments on Earth is critical to our understanding and exploration. With limited data about

51
the environments of ocean worlds (planets or moons with vast subsurface oceans) astrobiologists
make predictions using models based on Earth organisms or systems. To create such models,
astrobiologists utilize many STEM fields. For example:
• Physics describes the formation and structure of planetary bodies.
• Geology explains the surface processing or ways that the subsurface and surface of a
planetary body exchange material.
• Chemistry explains the atmospheres or lack thereof, the potential reactions that life could
use, as well as ocean composition.
• Biology explores the potential for life and identifying whether the requirements for life
are met on these worlds.
• Astronomy and Engineering provide the spacecraft and observational data that other
fields rely on to make inferences and draw conclusions.
• Mathematics is used to build models and explore possible conditions on planetary bodies
that cannot be measured with our current data.
Space exploration and the search for life have excited and inspired humans for generations and
continue to do so. The cross-cutting ideals of physics, geology, chemistry, and biology at play in
astrobiology can be aligned to different NGSS and build on the knowledge gained over several
elementary grade levels. The diverse content within the astrobiology lesson plans provided here
allow educators flexibility to incorporate and administer lessons to best fit their curricula at
various grade levels.
Scientists are in the best position to summarize, condense, and translate their research for
the greater public. By providing the astrobiology lesson plans we created and the framework

52
used to bring them to underrepresented populations, we hope to empower other scientists to
translate their research into accessible activities for elementary students.
Program Description
Over the 2022-2023 academic year, Maya D. Yanez was the STEM Education Fellow for
the University of Southern California’s (USC) Joint Educational Project (JEP). In this role, she
designed, managed, and taught the virtual WonderKids afterschool program, one of several
STEM-focused programs in JEP. WonderKids focuses on providing representation of
traditionally underrepresented identities in STEM and exposure to the variety of STEM fields
that exist to the students it serves. Each of our six weeks of programming is themed around a
specific STEM field; students are shipped supplies at the beginning of the semester for 1-2
activities per week. After the initial lesson/activity for the week, a guest speaker, an expert in
that STEM field, joins the students for a Q&A period.
WonderKids participants are kindergarteners through fifth graders from South Los
Angeles that attend Title I schools. JEP serves a community that is upwards of 80%
Hispanic/Latino, meaning most student participants are members of underrepresented identities
in STEM. The Fall 2022 program had 58 students registered and the Spring 2023 had 45
registered. Across both semesters, 100% of our students identify as BIPOC (Black, Indigenous
and people of color). A priority of the Wonderkids program is to bring in speakers who represent
the students we teach (Kast, 2021). In our efforts to do so, we recruit STEM experts from
marginalized identities if possible. In the Fall 2022 semester, our speakers were 83% women,
with 33% self-identifying as BIPOC. In the Spring 2023 semester, our speakers were 86%
women with 71% identifying as BIPOC. Representation and mentorship are critical to the
retention of BIPOC students in STEM (e.g. Dickens et al., 2021, Edwards & Thomas, 2015). We

53
aim to provide a jumping off point where students are exposed to the wide breadth of
opportunities within STEM. To determine the success of our efforts, we measured science
comprehension of the students through pre- and post-tests administered before introducing the
topic of the week and again after the guest speaker engagement.
In Fall 2022, Yanez tested Lessons 1 and 4 from the described astrobiology lessons in
WonderKids. She was also the guest speaker for that week, using Spanish and Latino cultural
references to engage more effectively with the students. Figure 4.1 provides example responses
to these pre- and post-tests which were used to assess comprehension. The kindergarten through
second grader group demonstrated an increased comprehension of 25% over the course of the
two lessons, and the third through fifth grade group had a 50% increase in comprehension.
Through the JEP, we administered a professional development workshop for teachers on
these lessons. Teachers were provided with lectures on background material for Yanez’ research,
they performed the activities of each lesson plan, and received the materials to teach these
lessons for their students. Teachers were eligible to receive a salary credit if they attended the
workshop, provided feedback at the workshop, implemented the lessons in their class, and then
provided feedback on their own implementation of the lessons. Teachers responded
overwhelmingly positively to these lessons and the workshop---100% indicated that 1) the
workshop made them feel more comfortable teaching astrobiology to their students and 2) they
would share it with a colleague.
Lesson Description
Yanez’ doctoral dissertation research on the potential habitability of Saturn’s moon Titan
for acetylenotrophs (microbes that feed on acetylene as their diet), as summarized in Figure 4.2,

54
formed the base concepts of our lesson plans. Four lessons were created to represent this research
(Figure 4.3) to provide answers to the following questions:
1. Edible Saturn Model—How do planets and moons form? Are there differences when they
are made from the same materials?
2. Spacecraft Payload—How do we learn about planets and moons? What do we need to
consider when building a spacecraft?
3. Competing Energetics—How might the same environment be better or worse for a
microbe depending on the microbe’s diet?
4. Bacteria Soup—How can we provide everything that a microbe needs to live in a single
bottle in the lab?
Lesson One is the edible Saturn model activity. Saturn and its moons are very different from one
another: If these objects were formed from the same building blocks (materials) why did they
end up so different? The ratio of rare to not-so-rare materials in these building blocks means that
the first object to be made, especially if larger than the other objects to be made, will contain
more of the rarer materials. Since Saturn forms first and is much bigger than its moons, then we
would expect Saturn to have more rare materials. Students learn this by “accreting” mass onto
their baby planet/Saturn (rolling a marshmallow cereal treat through a mix of building blocks:
rare (sprinkles) and not-so-rare (graham crackers) materials. This follows the basic principles of
planet/solar system formation where objects go through accretion—collecting more mass to get
bigger and rounder. Students then make rings to go around Saturn out of fruit snacks that also
accrete some mass, and then compare which object (the planet or the rings) has more of the rare
materials (sprinkles).

55
Lesson Two is the Spacecraft Payload activity. Throughout the lessons we discuss environments
out in space and primarily on two of Saturn’s moons, Titan and Enceladus. Lesson two aims to
provide context for students about the limitations of space exploration and the exceptional feats
performed to gather data—we must send spacecraft out to visit planets and moons and those
spacecraft use different instruments to gather different types of data. Table 4.1 is recreated here
from the lesson plans; it describes in simpler terms what these different instruments can tell us
from the data they gather. Students construct their own spacecraft payload that must fit in the
cargo fairing of the next generation rockets NASA has underdevelopment, specifically the Space
Launch System Block 2 Cargo.
Lesson Three focuses on microbial diets based on energetics. Yanez calculates the energy yield
for two types of metabolisms (or diets) which we call pink-eaters and blue-eaters in the lesson.
Pink-eaters represent methanogens and blue-eaters represent acetylenotrophs. These diets require
eating two different molecules and have different energy yields based on the environmental
conditions. Figure 4.4 is recreated from Yanez et al. (2024), representing the energy yields for
methanogens and acetylenotrophs as well as how those were translated into the environments
used in this lesson. Theoretical environments were used to determine possible energy yields of
pink- and blue-eaters: a realistic case (best estimates from our current data) and an optimistic
case (much more food available) for both Titan and Enceladus (environments 1-4). The fifth
environment represents an imaginary environment where pink-eaters and blue-eaters are equally
viable. All five environments are set up in trays and students are paired up and given clips. In 30
second rounds, students must first select whether they are a pink-or blue-eater and compete to
“eat” as many marbles of that color as they can to get energy. After 30 seconds, they stay in the
same environment, but swap colors. They compete in two rounds at each environment before

56
rotating to the next one. Students should note that not all environments are equal; it is more
difficult or easier to be a pink-eater or blue-eater depending on the environment, and more
specifically by the food available. This lesson provides opportunities to connect to ecology and
ecosystem studies that may already be a part of the curricula.
Lesson 4 is the Bacteria Soup lesson. In order to study microbes efficiently and determine what
they might be doing in their environments, we must culture or grow them in the lab. To grow
microbes/bacteria, we must provide the components that they need to survive. This lesson has
students help the instructor make Bacteria Soup by providing the ingredients, in the correct
order, to build our media or “soup” that we then use to culture (or grow) bacteria in the
laboratory. It begins by teaching students the six elements that every lifeform on Earth needs to
survive (Sulfur, Phosphorous, Oxygen, Nitrogen, Carbon, and Hydrogen or SPONCH) and
identifies how we as humans get our doses of SPONCH. We prefer the use of SPONCH over
CHNOPS (a more widely used term) for this abbreviation as SPONCH shares a name with a
snack common to our Latino community. As the instructor adds ingredients to the soup, there are
many opportunities to engage with students about nutrition, the environment, other types of
organisms, and their own bodies, as relevant to other curricula.
Conclusion
The Astrobiology lessons cover physics, geology, chemistry, and biology that can be
expanded as relevant to each educator’s classroom. These lessons can be taught as one unit or
incorporated into already existing NGSS-aligned curricula or others. Table 4.2 describes the
NGSS alignment of the lesson plans. The JEP WonderKids program provided a fruitful chance to
pilot the lessons to a varied grade level audience where they were successful. We saw an
increase in comprehension of astrobiology, as well as observing students placing women and

57
themselves in the roles of an Astrobiologist. Interdisciplinary lessons not only provide flexibility
to educators, but also to the students. Students retain different aspects of lessons: some remember
astrobiology as A) the search for aliens, B) what Ms. Maya did, or C) making Saturn and getting
to eat it! Astrobiology content is not included by default in standard elementary curricula. Yet it
can prove to be a successful media for complex interdisciplinary topics if experts in the field are
open to making their research accessible for an elementary audience. We encourage other
scientists to find ways to translate their research into interdisciplinary lesson plans, to provide
professional development opportunities to teachers to share these lesson plans, and to work with
service learning and community initiatives to incorporate these lesson plans in a way that can
reach all students, especially those traditionally underrepresented in STEM fields.
Acknowledgements
USC WonderKids Program would like to thank our funders, the USC Good Neighbors
Campaign, guest speakers (Tiffany James, Angelica Saenz-Trevizo, Adam Maclean, Maya
Gomez, Zhilei (Julie) Shen, Kyle Russell, Sasha Sproch, Madison Aubey, Nicole Smith, Sonal
Sharda, Saeima Marium), and our other three teaching staff, Eduardo Lopez, Nandini Patel, and
Preyashi Poddar, for their contributions to the program.
References
Dickens, Danielle D.; Ellis, Valeisha; and Hall, Naomi M. (2021) Changing the Face of STEM:
Review of Literature on The Role of Mentors in the Success of Undergraduate Black Women in
STEM Education, Journal of Research Initiatives. Vol. 5 : Iss. 3
Edwards, C. & Thomas, K. (2015). The Role Mentorship in Supporting African-American
Students Entry into STEM Careers. CUNY Academic Works.
Kast, Dieuwertje J., (2021) Authentic Marine Research Experiences for Low-Income and First
Generation High School Students Used to Level the Playing Field and Decrease Attrition in
STEM. The Journal of Marine Education. Vol. 35 Iss. 1 pg16-30. DOI: 10.5334/cjme.56
Yanez, M. D., LaRowe, D., Cable, M., Amend, J. (2024) Energy Yields for Acetylenotrophy on
Enceladus and Titan. Icarus. https://doi.org/10.1016/j.icarus.2024.115969

58
Figures & Tables
Table 4.1: Recreated here from the text of Lesson Two; this table highlights the true names of the
instruments used by spacecraft, a more simplified name, and a brief simple description of what the
instrument does. Imaging Science Subsystem is the name used on the Cassini mission.
Instrument
Name
Simple
Name
What it does!
Mass
Spectrometer
The Life
Materials
Detector
Measures how heavy and light different materials are to detect
what they are made of and if they came from life!
Gas
Chromatograph
The
What’sStuffMade-Of
Detector
Often it heats stuff up to vaporize it (to turn it into a gas)!
Then zaps the gas to figure out what it’s made from; can
measure stuff like water, hydrogen, oxygen, methane etc.
Magnetometer The
Compass
Compasses get pulled towards metal (and the North Pole!) on
Earth because it feels the magnet pull of those metals–this
instrument measures the magnetic field (or pull!) around the
spacecraft.
Radar The
Mapmaker
Helps scientists see through the thick atmosphere and measure
the highs and lows of other worlds, like mountains and seas.
"Imaging
Science
Subsystem”*
Cameras! Takes pictures and images in different types of light to send
back to Earth for science and the public!
Radio Dish The Phone Helps the spacecraft talk to Earth or other spacecraft!
Power Source The
Battery
All spacecraft need to take their power with them–there are no
plug ins in space! Some are literally batteries and others are
much more complex, like solar panels.
Storage Drives The Brain Stores all the data, information, and images that the spacecraft
collects, so that The Phone can send it back to Earth.

59
Next Generation Science Standards Alignment
Performance Expectations
● Lesson #1:
○ Earth’s Place in the Universe
● Lesson #2: Engineering Design
○ 3-5-ETS1-1
○ 3-5-ETS1-2
○ 3-5-ETS1-3
● Lesson #3:
○ Biological Evolution: Unity and Diversity
○ Ecosystems: Interactions, Energy, and Dynamics
● Lesson #4:
○ Ecosystems: Interactions, Energy, and Dynamics
Dimension Classroom Connections
Science and Engineering Practice:
Developing and using models
Modeling in 3–5 builds on K–2
experiences and progresses to building
and revising simple models and using
models to represent events and design
solutions.
Lesson #1: Students create a model of how Saturn
was formed to understand the accretion process
Lesson #2: Students create a model of a spacecraft
through a tangram puzzle to figure out which
instruments would be worth the payload in
exploring Saturn.
Lesson #3: Students create a model of energy yields
in microbes that mimic microbes found in space
Lesson #4: Students create a model to represent all
of the variables and conditions that microbes need
to thrive
Disciplinary Core Ideas
Lesson #1:
● ESS1.B: Earth & the Solar System
● ESS1.C: The History of Planet
Earth
Lesson #2:
● ETS1.B: Developing Possible
Solutions
Lesson #3:
● LS4.D: Biodiversity & Humans
● LS4.C: Adaptation
● PS3.D: Energy in Chemical
Processes & Everyday Life
● LS2.B: Cycles of Matter & Energy
Transfer in Ecosystems
Lesson #4:
● LS2.B: Cycles of Matter & Energy
Transfer in Ecosystems
Lesson #1: Students will become familiar with the
process of accretion by creating a model of Saturn
following the steps of how planets form.
Lesson #2: Students create a model and design a
way to measure payloads for spacecraft with nonstandard measurements. They are creating a
possible solution and designing to make the
spacecraft be able to take-off and accomplish its
mission.
Lesson #3: Students will learn how microbes in
different environments compare based on their
energy yields.
Lesson #4: Students will learn about microbes
combining all the necessary ingredients to grow
bacteria in the lab and how that compares to the
natural world.

60
Cross Cutting Concepts
● Lesson #1:
○ Stability and change
○ Patterns
● Lesson #2:
○ Systems and system
modeling
○ Interdependence of
Science, Engineering,
and Technology
● Lesson #3:
○ Scale, Proportion, and
Quantity
● Lesson #4:
○ Cause & Effect
○ Patterns
Lesson #1: Planets and moons have the same
composition but in different ratios that are stable
from origin.
Lesson #2:
● A system can be described in terms of its
components and their interactions similar to
the payload system created for the space
craft
● Knowledge of relevant scientific concepts
and research findings is important in
engineering
Lesson #3: Bacteria are tiny in scale
Lesson #4: All of life requires some of the same
materials. If we miss an important ingredient, then
life will not grow.
Table 4-2: Grade level specific NGSS alignment from The Next Generation Science Standards (NGSS,
2013).

61
Figure 4.1: Images from the Fall 2022 WonderKids Program. A Highlights of post-test results from the
3-5 grade group, noting specific facts covered in the lessons, indicating comprehension. B Pre-test results
from the K-2 group: Aryana and Zelda (top B) and Allison (bottom B) represent a lack of comprehension.
Zhanel’s response (middle B) represents comprehension as it is related to astrobiology. Students explain
their drawings to aid in these determinations. C Post-test results from the K-2 group. Victoria and
Giovanna’s answers feature either Ms. Maya (the expert for the week) or themselves doing science, which
we consider to demonstrate comprehension. Allison’s inclusion of aliens is also comprehension of
astrobiology. Photo Credit: M. D. Yanez

62
Figure 4.2: A summary figure representing Yanez’s research.

63
Figure 4.3: Depictions of each lesson are shown. 1 is an image of the final product of Lesson 1, an edible
Saturn model. 2 is the answer key to Lesson 2 showing all instruments fitting in the cargo fairing. 3 is
from the teacher professional development workshop where teachers are actively playing the microbe
energy game in Lesson 3. 4 is an image of the final “bacteria soup” bottle from Lesson 4, also from the
teacher professional development workshop. Photo Credit: 1, 2, M. D. Yanez and 3, 4, Kathrin Rising

64
Figure 4.4: Energy yields reproduced from Yanez’ research. These energy yields (color bar) are a
function of how much food is available for each diet (y-axis) and the temperature of the environment (xaxis). The left plot represents pink-eaters or methanogens and the right plot represents blue-eaters or
acetylenotrophs (see Figure 2). Values used in Lesson 3 are denoted by 1-4. Bottom boxes 1-5 represent
the pink and blue marbles in each environment of Lesson 3.

65
Chapter Five: Conclusion
5.1 Holistic, Interdisciplinary Habitability
Acetylenotrophy is a fascinating metabolism: as far as we know, there is one enzyme
capable of breaking down acetylene to an intermediary. Yet acetylenotrophy is understudied on
Earth and has not been considered in most astrobiology research. Syntrophotalea acetylenica
continues to prove to be a quixotic bacterium; while it remains the type strain for
acetylenotrophy, the growth rates under various conditions shown here differ from those
previously reported. Perhaps there exist undiscovered acetylenotrophs with growth limits beyond
the ranges presented here, especially considering the range of environmental conditions where S.
acetylenica has been found on Earth; although that is only speculation for now. Even under Earth
conditions, acetylenotrophy remains interesting. Assessing the habitability of a distant world
such as Titan can appear nearly impossible. However, with limited information comes ingenuity.
Any advancement and contribution to the database of knowledge used to compare and
contextualize results from the search for life is fruitful. We can only search for that which we are
prepared to seek, whether like life on Earth or vastly different from it. Observations, calculations,
and laboratory experiments are our only opportunities to contextualize spacecraft data and
expand what constitutes life on another world. The abundance of acetylene on other worlds could
indicate acetylenotrophy may play a crucial role in establishing life or microbial communities
beyond Earth. In fact this extends beyond our Solar System, young exoplanets have acetylene in
their atmospheres (Rimmer et al., 2019). Energetics show that acetylenotrophy would be highly
exergonic in the oceans of Titan and Enceladus (Figure 2.1). Acetylenotrophy could support cell
densities within the ranges of aquatic environments on Earth within Selk Crater on Titan (Figure
3.8). Back on Earth, translating this research into elementary lesson plans as a mechanism of
disseminating new and exciting science information could be critical in recruiting and retaining

66
the next generation of scientists. Embedding interdisciplinary questions and approaches early on
in K-12 education ought to curate scholars capable of bridging these fields and developing a
holistic understanding of life, on Earth and elsewhere. Focusing on a single microbe or
metabolism in the search for life is ill-advised, but this must occur in experiments and
calculations on Earth such that each extraterrestrial environment can be probed for the life most
likely to be found there. Microbes do not exist alone—acetylenotrophs could form the basis of a
microbial community that includes methanogens. And perhaps other species exist that could
contribute and benefit from such a community as well. I eagerly await clarity on these questions
following the next generation of spacecraft searching for life in our Solar System.
At risk of including far more exposition than is usual in a thesis, but usual of me, I would
like to share my personal goals for this journey. One should not pursue a PhD if they are not
infatuated with their chosen discipline. I was propelled by: Are we alone? And subsequently,
Can I find out? I wanted to become an interdisciplinary scientist. There is knowledge to be
gained in specific expertise. But the world is bereft of those wishing to pursue questions that
cross fields—not that these scientists do not exist, but that we need many more of them to tackle
the global questions ahead of us, whether about climate change or our place in the Universe. The
search for life is inherently one of many disciplines. I am happy to have many scientific skills
under my belt after 10 years of training. I am immensely lucky to be on the path I find myself
completing. And I could not have done it alone. I am grateful to the incredible and intelligent
people I find myself surrounded by: microbiologists, geochemists, geomicrobiologists,
astronomers, planetary scientists, and chemists, have all provided me with tidbits and expertise
that allowed me to explore and answer these questions. Together, in some near or distant future,
we will discover whether or not we are alone.

67
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Appendix A
A.1 Additional Growth Curves
Figure A.1: Plots of cell densities over time for all four acetylene experiments: Fresh Conditions
+ 1mL of C2H2 (F1), Marine Conditions + 1mL of C2H2 (M1), and those conditions repeated
with 2mL of C2H2 (F2 and M2). Each point represents the mean and standard deviation of a
triplicate of cultures.

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A.2 Acetate Data
As the products of acetylenotrophy (Reaction 2), ethanol and acetate were analyzed during those
experiments containing acetylene gas. However, acetate is readily used by the cells to build
biomass and therefore a consistent increase in acetate was not observed. Below is a summary of
the acetate concentrations for all replicates. Data is included here, yet no strong trend was
observed for use in the cell density calculations. Concentrations are reported in millimolar of
acetate.
Acetate could not be analyzed at the University of Southern California. Filtered samples of
bacterial media were stored at -20C until being shipped over dry ice to Berkeley, California. My
collaborator Dr. Heidi Aronson was able to obtain this data using an HPLC and the addition of a
known concentration of acetate to the samples. These plots were made by Dr. Aronson.

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A.3 Astrobiology Lesson Plans
The text of the four lesson plans described in Chapter 4 are provided beginning on the next page.
Additional text, video, and visual resources are available online alongside this document. Links
in the text are to an external GoogleDrive. In the event those links cease to work, please refer to
the USC Digital Library page where this dissertation was accessed to see additional documents.

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Astrobiology Lesson Plans
Maya D. Yanez ◼ mdyanez310@gmail.com ◼ planetarymaya.wordpress.com
Supply Sheet: Astrobio Lesson Supplies
Lesson Plans, additional documents, videos, etc. that are related to these lesson
plans can be viewed here: Yanez Astrobiology GoogleDrive
NGSS Disciplinary Core Ideas Applicable to These Lessons:
Lesson 1
ESS1.C: The History of Planet Earth: Some events happen very quickly; others occur very slowly, over a
time period much longer than one can observe. (2-ESS1-1)
ESS1.B: Earth & the Solar System: The orbits of Earth around the sun and of the moon around Earth,
together with the rotation of Earth about an axis between its North and South poles, cause observable
patterns. These include day and night; daily changes in the length and direction of shadows; and different
positions of the sun, moon, and stars at different times of the day, month, and year. (5-ESS1-2)
Lesson 2
ETS1.B: Developing Possible Solutions: Designs can be conveyed through sketches, drawings, or
physical models. These representations are useful in communicating ideas for a problem’s solutions to
other people. (K-2-ETS1-2)
Lesson 3
LS4.D: Biodiversity & Humans: There are many different kinds of living things in any area, and they
exist in different places on land and in water. (2-LS4-1)
LS4.C: Adaptation: For any particular environment, some kinds of organisms survive well, some survive
less well, and some cannot survive at all. (3-LS4-3)
PS3.D: Energy in Chemical Processes & Everyday Life: The expression “produce energy” typically
refers to the conversion of stored energy into a desired form for practical use. (4-PS3-4)
LS2.B: Cycles of Matter & Energy Transfer in Ecosystems: Matter cycles between the air and soil and
among plants, animals, and microbes as these organisms live and die. Organisms obtain gasses, and
water, from the environment, and release waste matter (gas, liquid, or solid) back into the environment.
(5-LS2-1)
Lesson 4
LS2.B: Cycles of Matter & Energy Transfer in Ecosystems: Matter cycles between the air and soil and
among plants, animals, and microbes as these organisms live and die. Organisms obtain gasses, and
water, from the environment, and release waste matter (gas, liquid, or solid) back into the environment.
(5-LS2-1)
Feel free to reach out to Maya Yanez with any questions or comments!
Astrobiology Lesson #1–Edible Saturn Model–Planet Formation
Lesson Summary: Students will become familiar with the process of accretion by creating a
model of Saturn following the steps of how planets form.
• Relevant Books:
Saturn Book
Additional Saturn Book
Both contain similar information!

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Vocabulary:
• Solar system: all the planets, moons, rings, gas and everything that orbits our star, the
Sun!
• Orbit: when objects move in a circle or oval around another object; caused by gravity.
• Saturn: the sixth planet from our Sun. It has many moons and rings that orbit it.
• Planets: Big rocks that orbit the Sun; you must be round, go around the Sun, and stay in
your lane to be a planet! (Because someone will ask: Pluto does not stay in its lane, so it
cannot be a planet. It sometimes travels in Neptune’s lane :) )
• Moons: Smaller* rocks that orbit planets in our solar system. (*They are always smaller
than the planets they orbit, but some moons are quite big! In fact two moons in our solar
system, Jupiter’s moon Ganymede and Saturn’s moon Titan, are both so large that they
are larger than the planet Mercury! But because they orbit Saturn and not the Sun, they
are moons and would never be considered planets!)
• Rings: Very tiny rocks and ice (compared to planets that is! Their average size is that of
a refrigerator!) that orbit planets in our solar system. Uranus also has rings but they are
much harder to see than Saturn.
• Accretion: When a planet or ring is forming, it collects all the other rocks, ice, and gas
around it by running into them and picking them up! As this baby planet starts to get
bigger (or accrete more stuff), gravity forces those gas/dust/ice grains to go towards the
heaviest object which is the baby planet that’s still forming. This collecting of stuff to get
bigger due to gravity is called accretion.
• Titan: Saturn’s biggest moon; has skies and weather, and lakes and seas. But those lakes
and seas are filled with liquid that is icky and poisonous, not water.
• Enceladus: Another of Saturn’s moons; it has a very icy surface so it is very bright; it
has tiger stripes that shoot jets of water into space.
• Astrobiologists: scientists that search for life (bio-) in space (astro-)!
Materials:
• Rice krispy treats, 1 per student, represents the initial accreted mass (little rice krispies
are stuck to each other to make a big mass)
• Sprinkles, 1 cup for the class, represent rare materials, things like metals out in space
• crushed graham crackers, 1 square per student, represent the not-so-rare materials, things
we have a lot of in space like gas and dust
• Fruit by the foot, 1 per student, represents the plane of the rings, since we can’t have little
chunks float around our planet like what happens in space, this is just an area to build the
rings on,
• Toothpicks, 2 per student
NGSS DCI Link:
ESS1.C: The History of Planet Earth: Some events happen very quickly; others occur very slowly, over a
time period much longer than one can observe. (2-ESS1-1)
ESS1.B: Earth & the Solar System: The orbits of Earth around the sun and of the moon around Earth,
together with the rotation of Earth about an axis between its North and South poles, cause observable
patterns. These include day and night; daily changes in the length and direction of shadows; and different
positions of the sun, moon, and stars at different times of the day, month, and year. (5-ESS1-2)
Math Link: Shape recognition

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Background Information:
You have probably been outside at night. You’ve likely looked up at the sky and seen
stars and the Moon, perhaps you’ve seen planets or shooting stars. Maybe you have wondered if
aliens are real! Some scientists spend their time studying everything out in space to see if life
may exist somewhere other than here on Earth (aliens!). The scientists that search through space
searching for life are known as ASTROBIOLOGISTS! Astro- for space and biology for
studying life. Over the next few lessons, we are going to learn about a special planet with special
moons in our solar system and how some scientists look at these same moons and search for life!
**I suggest breaking here and sharing images of Saturn and its moons!**
Our solar system is all of the rocks, planets, moons, and gas that orbit our star, the Sun.
When astrobiologists are researching space and the different planets and moons in our Solar
System, they are searching for life. Saturn, the sixth planet from the Sun, has somewhere
between 53 and 82 moons! And two of Saturn’s moons are very interesting places where
astrobiologists want to search for life. Saturn’s moon Titan has liquid on its surface and water
trapped inside of it--an underground ocean the size of the whole planet! The moon Enceladus
also has an underground ocean, but it also has tiger stripes on its surface that are cracks where
the water can escape as geysers! Astrobiologists follow the water and other clues to see whether
life could exist on Saturn’s moons.
Saturn also has rings, many many rings. Rings are not composed of thin flat rock, but
rather hundreds and thousands of bits of rock and ice. When Saturn’s moons are hit by asteroids,
they get broken up into chunks of rock and ice, and the chunks begin to orbit Saturn as rings!
Procedure:
1. Tell students, “Today we are going to learn about Saturn!”
2. Read the background information to the students.
3. Share the images of Saturn and Cassini from the books or NASA sites.
4. Read the recommended book with the students and/or:
1. The key takeaways here are mainly the visuals. Space is beautiful! But it’s also hard
to understand without all the pictures and analogies.
If you have access, show students the “Saturn 101” video:
Saturn 101 | Planet With Rings | The Dr Binocs Show | Peekaboo Kidz
5. Tell students that today they will create a model of Saturn, following the steps for how
Saturn and its rings were made
1. WARNING: Remind students that while we are using food to make our Saturn
model, we are doing science first. So we cannot eat our models until the very
end!
6. Have students open up their rice krispy treat. Point out that our rice krispy treat is already an
object that was formed by accretion! A bunch of tiny stuff (rice krispies) have been stuck
together to form our treat! A lot like how gravity causes tiny stuff in space to stick together
and form bigger objects.
7. Squeeze the rice krispy treat in your hand to form it into a sphere/ball. Place the sphere off to
the side.

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8.
In
their
work
area/bowl/plate, have students spread out the bag full of crushed graham crackers or have
them smash them:
1. Place a single square down on the plate, lay a plastic bag or piece of cling wrap over
the graham cracker, and have students use their fist to break it into pieces. Then use
just a single finger on each small piece, to break into crumbs.A pinch of sprinkles
should be incorporated into the crushed graham crackers.

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9. Have students take their rice krispy sphere (their baby planet!), and roll it around in the
graham cracker crumbs and sprinkles (this is the planet accreting more mass). They might
need to apply pressure as they roll so that the crumbs/sprinkles stick. Place the newly
accreted planet off to the side.
10. Tell students: “You have now made a planet! But it doesn’t look like Saturn yet, and you still
have building blocks of planets leftover” The building blocks being crumbs/sprinkles
11. To create the rings, students should now open their Fruit By the Foot and measure out some
length of it before tearing it off. While the fruit snack is still on the wax paper, turn it paper
side up and press it into the remaining crumbs and sprinkles.
12. Then flip it over and use a toothpick to form it into a circle. Stick that toothpick into one half
of the planet. Then take a second toothpick to stick into the other half of the planet. You have
now made Saturn and its rings!

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13. To learn more about planet formation, answer these questions as a group:
i) Which has more sprinkles: the planet or the rings? Why?
ii) How are your models of Saturn and the planet Saturn the same? How are they
different?
14. The big takeaway of the lesson is:
When planets are forming even though all parts of it (the planet, the moons, the rings)
start with the same stuff (the same building blocks of graham crackers and sprinkles),
the different parts can have different amounts of stuff (sprinkles for example) because
of the order in which they are made! So the rings have less precious/rare materials
than the planet since it was made with the leftover bits. Sprinkles represent rarer
materials like metals because those tend to get mostly used up by the BIG planet
forming first.

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Astrobiology Lesson #2–Titan, Enceladus, and Spacecraft (How we learn
about our solar system)
Lesson Summary: Students will construct a payload to visit other worlds that fits the restrictions
of the Space Launch System.
Relevant Books:
Saturn Book
Additional Saturn Book
Both contain similar information!
Vocabulary:
• NASA: The National Aeronautics and Space Administration; the US government agency
in charge of all scientific exploration of space
• Enceladus: an icy moon of Saturn with an underground ocean and jets!
• Titan: an icy moon of Saturn with thick clouds, toxic liquid on the surface, and an
underground ocean!
• Cassini-Huygens: A NASA mission that visited Saturn, its moons, and the Huygens
probe even landed on Titan!
• Space Launch System (SLS): New shuttle system; a rocket that will send future
spacecraft out to space! The biggest one they plan to build is the SLS Block 2 Cargo!
• Cargo fairing: The place on the rocket that stores the payload
• Payload: The items (spacecraft or satellites) or people (astronauts) that the rocket is
carrying to space!
• Instruments: These are the different parts of a spacecraft that gather information about
the places we send spacecraft
• Environmental Conditions: These are the facts about an environment. Spacecraft and
instruments help us learn these conditions from the measurements they make.
Materials:
• Lesson 2 CargoFairing_Worksheet.pdf
• (For Teacher): Lesson 2 CargoFairing_TangramKey.pdf
• Tape/Glue Sticks
• Videos on USB/downloads in the GoogleDrive: Cassini's Fatal Crash | Mission Saturn
Titan Touchdown
NGSS DCI Link:
ETS1.B: Developing Possible Solutions: Designs can be conveyed through sketches, drawings, or
physical models. These representations are useful in communicating ideas for a problem’s solutions to
other people. (K-2-ETS1-2)
Math Link: Reason with shape and their attributes
Background Information:
How do astrobiologists learn about space and search for life? NASA (the National
Aeronautics and Space Administration) helps thousands of scientists across the world explore
space. They launch and manage spacecraft missions like the Cassini-Huygens mission that
visited the Saturnian System from 2004-2017. These missions use spacecraft to explore these

90
places! Spacecraft are robots that have instruments on them that help them gather data and
make observations about the places they visit. Spacecraft are the payload of big rockets like the
Space Launch System! We can’t learn everything we need to know about space from down on
Earth. Launching our payloads/spacecraft into space helps us visit places other than Earth and
learn about them. The Cassini-Huygens mission had two parts: one that orbited Saturn and was
able to fly by many of its moons, including when it flew through water jets that the moon
Enceladus was shooting into space. The other part of the mission, the Huygens probe, actually
landed on the surface of the moon Titan, and took a video the whole time! Today you will learn
a bit more about Enceladus, Titan, and Cassini while you build your own spacecraft to explore
Saturn’s moons!
Procedure:
1) Tell students, “Today we are going to learn how astrobiologists learn what they do about
Saturn!”
2) Use the associated book to introduce Titan and Enceladus! Play the Cassini Final Crash video
from the USB to learn more about the Cassini mission and its finale.
a) Key takeaway here: Visuals of Titan and Enceladus! These are weird places. Identifying
Enceladus’ jets in the images too. We only have these pictures and this information about
these moons because of spacecraft, specifically Cassini. Thinking about all the
instruments included, Cassini is really small! All the instruments had to fit very snugly
together to be able to fit in their rocket and launch.
3) A lot of the spacecraft we send to planets are orbiters like Cassini. We have only landed
probes (or rovers–so moving robots!) on 4 bodies in the whole Solar System: the Moon,
Mars, Venus (by Russia not NASA), and Saturn’s moon Titan! Next show the Huygens
probe video which is a collection of images turned into a video from the probe’s perspective
as it landed on Titan: Titan Touchdown
4) Have students discuss as a group the following questions about the moons:
a) Introduce environmental conditions (temperature or the amount of stuff there, get
students to think about soil vs ice, water vs air, etc.). How are deserts and oceans
different? Do you look for the same types of things/life in deserts as you do the ocean?
b) Ask students what environmental conditions might be different between Enceladus and
Titan. Use the pictures (lakes and seas on Titan and jets on Enceladus!)
c) How did we learn about the environmental conditions on Titan and Enceladus?
d) Do you think life is more likely on Enceladus or Titan?
5) Discuss the table below to answer the question: how do we learn this information. These are
just a couple examples of the types of instruments that can be used to explore planets and
what they help us understand. The final one is actually the name of the camera system on
Cassini!
6) Remind students that part of Cassini landed on Titan and the whole spacecraft flew through
Enceladus’ jets: So these instruments were able to gather data on the jets too!
https://solarsystem.nasa.gov/missions/cassini/the-journey/the-spacecraft/
7) Project images of the SLS Block 2 Cargo (or any of the SLS rockets!). These are the next
generation rockets/shuttles that are being built by NASA to explore worlds like Saturn and
Titan!
https://www.nasa.gov/exploration/systems/sls/fs/sls.html
https://www.nasa.gov/sites/default/files/thumbnails/image/sls_lift_capabilities_configurat
ions_12022021_woleo.jpg

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8) Point out the cargo fairing in the images (just the top white portion!) and how most of the
rocket is fuel(the orange portions)! When you build a spacecraft you don’t get to use the
whole rocket for space, but only the tiny cargo fairing. It takes a lot of energy and power to
get off of Earth, so rockets have to be mainly fuel!
9) Now we will all make our own spacecraft that can fit in the cargo fairing of the SLS Block 2
Cargo! Pass out Cargo Fairing worksheets. Have students identify their shapes as specific
instruments as they build up their spacecraft. They can fit all the shapes inside their cargo
fairing (see the Tangram Key), but they can also use fewer shapes to build a spacecraft that
fits inside the cargo fairing.
10) Afterwards, ask students which instruments they chose. Why those ones? What do they hope
to learn about a place like Titan or Enceladus with their spacecraft?
Correct Instrument
Name
Simple Name What it does!
Mass
Spectrometer
The Life
Materials
Detector
Measures how heavy and light different materials are
to detect what they are made of and if they came from
life!
Gas
Chromatograph
The What’sStuff-Made-Of
Detector
Often it heats stuff up to vaporize it (to turn it into a
gas)! Then zaps the gas to figure out what it’s made
out of; can measure stuff like water, hydrogen,
oxygen, methane etc.
Magnetometer The Compass Compasses get pulled towards metal (and the North
Pole!) on Earth because it feels the magnet pull of
those metals–this instrument measures the magnetic
field (or pull!) around the spacecraft.
Radar The Mapmaker Helps scientists see through the thick atmosphere and
measure the highs and lows of other worlds, like
mountains and seas.
"Imaging Science
Subsystem"
Cameras! Takes pictures and images in different types of light to
send back to Earth for science and the public!
Radio Dish The Phone Helps the spacecraft talk to Earth or other spacecraft!

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Power Source The Battery All spacecraft need to take their power with them–
there are no plug ins in space! Some are literally
batteries and others are much more complex, like solar
panels.
Storage Drives The Brain Stores all the data, information, and images that the
spacecraft collects, so that The Phone can send it back
to Earth.

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Astrobiology Lesson #3–Microbial Competition based on Energetics
Lesson Summary: Students will become familiar with how microbes in different environments
compare based on their energy yields.
Book: Microbe Book
Microorganisms | The Dr. Binocs Show | Educational Videos For Kids
Vocabulary:
• Microbes: The tiniest type of life on Earth! Any organism that is too tiny to see with our
eyes is microscopic and therefore a microorganism or a microbe!
• Energy: All life needs energy! We get our energy from food–but different kinds of food
give us different amounts of energy or energy yields. Microbes get different energy in
different environments.
• Environmental Conditions: These are the facts about an environment that affect how
much energy microbes can get from their diets–things like temperature, or how much of
different kinds of food is available. For places on Earth, this can be like sand in a desert
and water in an ocean.
• Astrobiologists
Materials:
• Pink (or Green) marbles–2lbs for group of 10 students (color depends on availability
when ordering)
• Blue marbles–2lbs for group of 10 students (5 different trays, each with their own set of
‘environmental conditions’ reflected in relative numbers of marbles)
• Clips, 1 for each student
• 9x13 Aluminum trays-5 per group of 10 students, (5 different trays, each with their own
set of ‘environmental conditions’ reflected in relative numbers of marbles)
• Small bowls–1 for each student
• Timers
NGSS DCI Link:
LS4.D: Biodiversity & Humans: There are many different kinds of living things in any area, and they
exist in different places on land and in water. (2-LS4-1)
LS4.C: Adaptation: For any particular environment, some kinds of organisms survive well, some survive
less well, and some cannot survive at all. (3-LS4-3)
PS3.D: Energy in Chemical Processes & Everyday Life: The expression “produce energy” typically
refers to the conversion of stored energy into a desired form for practical use. (4-PS3-4)
LS2.B: Cycles of Matter & Energy Transfer in Ecosystems: Matter cycles between the air and soil and
among plants, animals, and microbes as these organisms live and die. Organisms obtain gasses, and
water, from the environment, and release waste matter (gas, liquid, or solid) back into the environment.
(5-LS2-1)
Math Link: Number comparison and data interpretation
Background Information:
When astrobiologists are researching space and the different planets and moons in our
Solar System, they are searching for life. But what are they searching for and how do they
decide? You might hope that it is very easy: you point a telescope at a planet and see if a giraffe

94
or other big creatures are walking around, right? NO! Unfortunately, it’s much more difficult
than that. In fact, it’s more likely that the life that we would find would be microscopic or
microbes! So instead, astrobiologists make predictions about how much energy life could get on
that moon or planet, based on the environmental conditions there! If there is more than one
type of life that could survive those conditions, then we compare their energy yields to see
which microbe might be able to survive better in that environment! We will compare pinkeaters** and blue-eaters and see how they compete in different environmental conditions based
on energy. **Note: Due to supply constraints while pink is mentioned throughout, the
marbles may be any non-blue color such as green**
Procedure:
1) Tell students, “Today we are going to learn about super tiny life and how astrobiologists
predict where they might live!”
2) Share the book so that students get an idea of how many microbes surround us.
a) Key Takeaway here: Microbes surround us! They are everywhere and they are both good
and bad. Generally, most students have only ever heard about microbes since they can
make us sick. Environmental microbiology is more about how important microbes are in
the environments they are found. The book and video aim to introduce microbes to the
students and give them some idea about how prevalent and abundant they are, with
examples that are not just illnesses.
b) If you have access, show students the “Microbes” video: Microorganisms | The Dr.
Binocs Show | Educational Videos For Kids
3) Explain to students that microbes are the most common type of life on Earth. They are some
of the oldest life on Earth too! They were probably the first life forms to show up on Earth,
which means they might be the first ones to show up on other planets or moons too. But
looking for microbes is really difficult! You need a microscope. But you can’t just put a
microscope on a robot to look around for microbes on another planet! So instead,
astrobiologists have to make predictions using the information they have available. So they
look at the environmental conditions on moons and planets to predict how much energy
microbes can get. To determine how much energy is there, astrobiologists look at how much
of their food is available. Today, you will pretend to be two different types of microbes:
pink-eaters and blue-eaters! ***Pink marbles were not always available so green were
used instead. Any color that is not blue is fine!***
4) Following the handout “Pink v Blue Setups” set up five trays of pink and blue marbles for
the students.
5) Pair off students. 5 pairs of students can work simultaneously. One pair at each of the 5
stations.
6) At each station, students should fill out the “Survival Predictions” worksheet. They circle
the color of marble they are eating, and then write the number of marbles that they ate.
7) There are instructions for how to play in the “Pink v Blue Setups” handouts. Reiterated here:
a) The teacher will be in charge of the timer and starting and ending each 30 second round
b) There will be two rounds at each station, so that students can be both pink and blue at
each station. The students in pairs will each choose a color and during the 30 seconds,
they must use their binder clip to eat the pink or blue marbles based on their choice.
Students are only allowed to “eat” the marbles they can gather with their binder clips. NO
SCOOPING OR SLIDING. Your binder clip is like a mouth, so you have to get the
marble fully inside the mouth to eat it! And because you are EATING, you must pick up

95
the marble with the binder clip mouth and drop the marble in your tiny bowl that
resembles your stomach or the inside of a bacterium’s cell.
c) After the timer goes off, have each student count how many marbles (of the correct
color!) they “ate.”
d) Then have the students switch colors from before and play another 30 second round.
Record the results. And then rotate to the next station until students have completed all
five stations.
8) Afterwards, ask students questions about the different stations:
a) Was it easier to be a pink eater or a blue eater? Did it depend on the station?
b) If you had to guess which bacteria would do better, what would you look for? (Hint: were
there times where there was a lot more of one kind of food?)
c) How do you think astrobiologists make these predictions? (call back to the previous
lesson where they should learn that Cassini helped us learn about the environmental
conditions of different moons of Saturn!)
Worksheets/Attachments:
Pink v. Blue SetUp and Instructions
Survival Predictions

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Astrobiology Lesson #4–Culturing Bacteria
Lesson Summary: Students will learn about microbes combining all the necessary ingredients to
grow bacteria in the lab
Book: Microbe Book Saturn Book
Vocabulary:
• Bacteria: One type of microbe. It is the most common type of microbe. They are in
yogurt and help us stay healthy! But some of them are not as good and can make us sick.
• Media: The Bacteria Soup! This is the liquid that we make and put into bottles that we
culture (or grow!) bacteria in while doing experiments in the lab.
• Vitamins & Minerals & Metals: Special chemicals that all of life needs to survive!
Things like iron, Vitamin D or B, and minerals like salt are included.
• SPONCH/CHNOPS: Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorous, Sulfur–these
six elements are required by every single living organism on Earth! So it is really
important that we add them to our media and even we as humans get these elements from
what we eat, drink, and breathe!
Materials:
• Large Serum Bottle Cut-Out with hidden pink O2
• Tape
• Pre-Cut Bacteria Soup Ingredients
Math Link: Compare numbers to determine which is larger or smaller
Background Information:
You have learned about Saturn and its moons Titan and Enceladus. You have learned
about astrobiologists and how we make predictions about where life can survive on Titan and
Enceladus based on how much energy is available. Now it’s time for us to put it all together and
learn about what we do here on Earth, with Earth life known as bacteria. Bacteria are a type of
microbe (or microorganism) that are some of the most abundant creatures on Earth! If we want to
grow bacteria in different environmental conditions (like those on Titan and Enceladus!), then
we must change the media. The media is kind of like the soup that we make for bacteria to live
in and eat. The media has to contain all of the vitamins and minerals and metals that life needs
to survive and grow. All life on Earth requires SPONCH to survive; this stands for Sulfur,
Phosphorous, Oxygen, Nitrogen, Carbon, and Hydrogen! Today we will make a bacteria soup!
Procedure:
1) Ask students to list off what life needs to survive. Make a list on the board.
2) Then introduce SPONCH again: these 6 chemicals or elements are required by ALL OF
LIFE. Just like water, these are just as important. Where do you think you all get your
SPONCH from?
a) Eggs = Sulfur
b) Milk = Phosphorous
c) Air = Oxygen
d) Fruits and Vegetables = Nitrogen

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e) Sugar, Bread, Fruit = Carbon
f) Water = Hydrogen
3) When scientists grow bacteria in the lab, they have to provide everything for the bacteria to
survive and grow. All of SPONCH, the water, their food, vitamins and everything else! So
we are going to build up our bacteria soup today to see what scientists give bacteria to help
them grow and survive!
4) Hang up our serum bottle on the board. Pass out the various soup ingredients to the students.
Make sure that there is some O_2 in our serum bottle!
5) The order that we add ingredients to the soup is very important!
a) Let’s begin with water! Multiple students may have water and will need to add it to the
bottle.
b) We can go ahead and begin adding salts. Salts are super important! We don’t think about
it because we get plenty but salts are very important for our body or bacteria cells to
work! Sometimes when we exercise a lot and get sweaty, we need to drink something like
gatorade or juice to replenish our salts as well as water!
i) Naii) Mgiii) K- (KH2PO4–Potassium Phosphate–P source!)
iv) Cav) Cl- (NH4Cl–Ammonium Chloride–N source!)
c) Now that we have our salts we can add in all of our metals:
i) Iron
ii) Zinc
iii) Manganese
iv) Boron
v) Copper
vi) Nickel
vii)Molybdenum
d) Now comes something tricky. We need oxygen to breathe right? But for some bacteria
like the ones that might live on Titan and Enceladus, having oxygen around in the air will
kill them! So let’s add an Oxygen-indicator to our media so we can tell if we have
oxygen gas (like that in our air!) inside
i) Have student with the O2 indicator add it to the bottle, after this happens, the teacher
should reveal some pink O2 in the bottle!
e) Oh no! It looks like there’s oxygen trapped in the bottle. We have to remove that before
we add the rest of the ingredients!
i) First let’s add our Oxygen Scrubber, or the chemical that removes the oxygen. Have
the student add the Na2S and then have the teacher remove the O2 from the bottle–
this is also our source of S!
ii) Then we have to add N2 gas so that we can replace the gas we removed.
f) Whoo! We got rid of the oxygen. Now we can add the last few ingredients to our media
or bacteria soup! Have we added all of our SPONCH ingredients? I would suggest
writing the letters on the board and crossing each one off as we add them to the bottle.
Water provides the hydrogen and oxygen needed by our microbes since they can’t use the
oxygen gas in the air.

98
g) Add the buffer; a special chemical that helps keep the media the same environmental
conditions that we set!
h) Now we get to add the Carbon Source or the food for the bacteria
i) Add acetylene to the media
6) Alright we added all the ingredients for Bacteria soup is it ready yet??
a) No! We haven’t added any bacteria :)
7) Now that we know everything we have to add to make bacteria grow, add Wolly & Ghary
8) Use relevant images to show students what real microbes (bacteria) look like and see videos
for more visuals.
Worksheets:
BacteriaSoup Ingredients PrintOuts.docx

Abstract (if available)

Abstract The search for life beyond Earth has been closely tied to the presence of liquid water. Often, astrobiologists focus on three key criteria for life: liquid water, nutrient availability, and metabolic energy sources. The putative energy sources are defined by whether the reactants for known metabolisms on Earth are found in abundance on those other worlds, in environments collocated with available nutrients and liquid water. Our observations of Saturn’s moon Titan describe a world comprised of chemicals and bedrock layers so different from Earth, yet still likely environments to meet the aforementioned criteria. Titan is comprised of a surface shrouded by a thick atmosphere with stable liquids (albeit not liquid water) in lakes and seas; fluvial and aeolian erosion that are altering a changing landscape; and a deep subsurface liquid water ocean bounded by shells of thick water ice. Utilizing methods that have been proven to work on Earth for exploring environments in search of novel microbes or unique metabolisms, could prove fruitful for those extraterrestrial environments with limited access and information, such as the subsurface ocean on Titan. In the coming years, missions like NASA’s New Frontiers mission Dragonfly will arrive on Titan’s surface and complete measurements aimed at identifying any possible life found therein, and in turn provide a more detailed view of surface environments. However, Dragonfly’s instrument capabilities and possible measurements will be constrained by the type of life expected to be found on Titan. As environmental microbiology research has bloomed in recent decades, there is an ever-growing list of possible microbial metabolisms. On Earth, we predict undiscovered metabolisms given the energy yields of novel catabolic reactions and search for environments where those energy yields are possible.
Our search for life elsewhere can only be informed by our understanding of life’s origins and evolutions on Earth. One plausible source of metabolic energy is acetylene (C2H2), a simple organic compound that is the second most abundant photochemical product in Titan’s atmosphere. Acetylenotrophy, or the microbial fermentation of acetylene, is utilized by microbes on Earth in a number of environments. Here I provide an energetics analysis of acetylenotrophy for Titan including predicted cell densities based on laboratory culturing of Syntrophotalea acetylenica, the type strain of acetylenotrophs. In addition, I advocate for the translation of these research projects into lesson plans for elementary students to foster excitement and engagement in the field of astrobiology. Overall, I describe the potential habitability of Titan for acetylenotrophs as a small contribution to the knowledge needed to guide future missions aiming to search for life on ocean worlds.

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Habitability of Saturn's moon Titan for acetylenotrophy: laboratory culturing and energetics as tools for the search for life

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Asset Metadata

Creator Yanez, Maya Danielle (author)

Core Title Habitability of Saturn's moon Titan for acetylenotrophy: laboratory culturing and energetics as tools for the search for life

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Geological Sciences

Degree Conferral Date 2024-08

Publication Date 08/29/2024

Defense Date 05/22/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag acetylene,acetylenotrophy,anaerobic culturing,astrobiology,Dragonfly Mission,education,enceladus,energetics,gibbs energy,habitability,OAI-PMH Harvest,Syntrophotalea acetylenica,titan

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Amend, Jan (committee chair), Cable, Morgan (committee member), Corsetti, Frank (committee member), Finkel, Steven (committee member), LaRowe, Doug (committee member)

Creator Email mdyanez@usc.edu,mdyanez310@gmail.com

Unique identifier UC113999ZI9

Identifier etd-YanezMayaD-13451.pdf (filename)

Legacy Identifier etd-YanezMayaD-13451

Document Type Dissertation

Format theses (aat)

Rights Yanez, Maya Danielle

Internet Media Type application/pdf

Type texts

Source 20240830-usctheses-batch-1204 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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