BIOREMEDIATION OF LUNAR REGOLITH VIA MICROBIAL METABOLISM
Sophie Claire Milam
Department of Physics and Astronomy
University Of Hawai`i at Hilo
Hilo, HI 96720
ABSTRACT
Bioremediation is the process by which microbial and plant life work to break down
harmful compounds in soil and leave it more habitable than it originally was. Such a process has
been used in oil spills and other less extreme pollution situations here on Earth and there is a
good deal of hope that it can also be used to transform lunar regolith into a life sustaining
substrate. This experiment used JSC-1A lunar regolith simulant and a combination of Bacillus
Cereus and Bacillus Megaterium to create a soil capable of sustaining Arabidopsis Thaliana
plants. The results show that the regolith is very capable of sustaining life under the proper
conditions, however, it also shows that those conditions are difficult to create and could use
further research.
INTRODUCTION
It has been over 40 years since NASA and the Apollo program sent the first men to the
moon. Since then there has been constant debate in the scientific world regarding the risk to gain
ratio of those missions and whether it is small enough to warrant a return trip. The last two
administrations have promised a return to manned space missions beyond Low Earth Orbit
(LEO) and the main question brought up by scientists and laymen alike has been “why go back
to the moon?”
The Moon and Mars are stepping stones toward the inevitable future of humans
becoming a spacefaring species. Our star, Sol, is middle aged which means that the human race
has relatively little time to evolve before our planet is destroyed in Sol’s final millennia. In order
to live without a planet we have to understand the dangers of space, its pros and cons, how to
protect ourselves and how to sustain ourselves. It is a combination of these concerns that has led
scientist toward the field of In-Situ Resource Utilization (ISRU).
Major Element Composition
Silicon Dioxide (SiO2)
Titanium Dioxide (TiO2)
Aluminum Oxide (Al2O3)
Ferric Oxide (Fe2O3)
Iron Oxide (FeO)
Magnesium Oxide (MgO)
Calcium Oxide (CaO)
Sodium Oxide (Na2O)
Potassium Oxide (K2O)
Manganese Oxide (MnO)
Chromium III Oxide (Cr2O3)
Diphosphorus Pentoxide (P2O5)
% by Wt.
46-49
1-2
14.5 – 15.5
3-4
7 – 7.5
8.5 – 9.5
10 – 11
2.5 – 3
0.75 – 0.85
0.15 – 0.20
0.02 – 0.06
0.6 – 0.7
Figure 1: table describes the composition of JSC1A, picture of the microscopic grain of regolith.
Lunar regolith composition, as seen in Figure 1, can be duplicated here on Earth with a
good deal of precision and it is this simulant that allows modern ISRU technology to be
calibrated to work on the Moon. Certain bacteria are ground dwelling and able to metabolize
harmful compounds from the regolith and excrete benign, and sometimes even helpful, waste
products. This experiment used B. Cereus to inoculate a JSC-1A slurry and B. Megaterium to
inoculate Arabidopsis seeds.
B. Cereus is a soil dwelling bacteria that is rod shaped and forms chains. because of the
endospore that is creates it is capable of adapting to very extreme environments and is resistant
to heat, cold, and radiation (Mignot et al). It has respiration processes that are both aerobic and
anaerobic and metabolizes carbohydrates, proteins, peptides and amino acids while producing Llactate, acetate, formate, succinate, ethanol, and carbon dioxide (Mols, et al). It is also a nitrogen
fixer for the soil.
B. Megaterium is also soil dwelling, rod shaped, and forms chains; it is very hardy,
motile, and is a relatively large bacterium (~60um). It was used as the seed inoculant in this
experiment because the protective endospores it forms can be used as armor for the fragile roots
of the Arabidopsis plant. The Arabidopsis Thaliana plant was used because of its fully explored
genome, hardiness, and its ideal growth compared well with those of the bacteria.
The regolith simulant, JSC-1A, compares with real lunar regolith up to 99% accurate
compositionally, however there is one aspect of the dirt that we aren’t capable of recreating and
that is its physical properties. The dust on the moon was created through giant impacts in the
vacuum and cold of space, this gave each microscopic grain razor sharp edges that can slice
through even bacteria. It is for this reason that the Bacillus chosen for this study were able to
produce those protective endospores that would preserve their genetic material from physical
harm as well as radiation.
Ultimately, this experiment had two goals: 1) for the simulant to be directly chemically
altered by the metabolism of the microbes in the soil and on the seeds; 2) for the microbes to be
able to grow well enough to become and integrated organic part of the regolith and support plant
growth.
METHODS
Using sterile lab technique the regolith simulant was divided in half; water was added to
one half to create a slurry and a 50% beef broth nutrient agar was mixed with the other half to
create a nutrient slurry. Enough liquid was added to entirely moisten the sample of simulant
without having standing water when it settled. Approximately 40mL of the slurry was added to
each of 13 beakers; 4 beakers were given the water slurry, and the remaining 9 were given the
nutrient slurry. A break down of the trial and control sample contents is given in the following
Figure 2.
Half of the seeds were inoculated with the B. Megaterium and the other half were coldsnapped in distilled water, both sets were allowed to sit for 2 days before being planted in the
slurry beakers. Once the seeds had be deposited on the surface of the slurry, the beakers were set
under a 60W natural sunlight lamp where they received “sunlight” continuously and maintained
a steady 23 C temperature. The plants were watered automatically and individually via gravity
drip system from either a distilled water reservoir or a 50% beef broth nutrient agar reservoir for
2 hours a day to maintain an appropriate level of moisture in the sample beakers. The
experiment was conducted for a full 2 week growth period for the Arabidopsis plant after which
the samples were investigated for changes in soil composition, plant growth, and microbe
survival.
There were several elements of control that needed to be tested:
Control 1: Tested unaided growth of seeds in simulant
Control 2: Tested the growth of inoculated seeds grown in water slurry
Control 3: Tested the effect Bacillus Cereus in the soil had on non-inoculated seeds
Control 4: Tested the non-inoculated seeds’ reaction to being watered with a 50% beef broth
nutrient mixture
Control 5: Tested how the Bacillus Cereus in the soil affected the non-inoculated seeds when
watered with the nutrient mix
Control 6: Tested how the nutrient mixture affected the inoculated seeds.
Control 7: Tested how the Bacillus Cereus in the soil in the inoculated seeds fared without the
nutrient mixture to fuel their metabolism
Seed
inoculation
B. Cereus in
slurry
Seeds coldsnapped in
water
Y
Y
Slurry made
with water
only
Y
Y
Daily H2O
watering
Test
designation
N
N
Nutrient mix
daily
watering
N
N
N
Y
Y
Y
Control 1
Control 2
N
N
N
Y
N
Y
N
Y
Y
Y
Y
Y
N
Y
N
Y
N
N
Control 3
Control 4
Control 5
Y
Y
Y
N
Y
Y
Y
N
Y
Y
Y
N
Y
N
N
N
Y
N
Control 6
Control 7
Trials 1-6
Figure 2: Table describing the set up for the experiment samples, Y=yes N=no.
The tests designated “Trials 1-6” were the theoretical ideal states for the experiment
where the bacteria in the soil and on the seeds would work together metabolizing the nutrition
agar and creating a more habitable soil. Because the B. Cereus wasn’t being forced to
metabolize only the simulant it was thought that this would yield the best results for plant growth
as well as soil composition change.
RESULTS
Plant growth during this project did not reach over 0.75 cm of vertical growth and
maturation of the Arabidopsis thaliana only progressed as far as 3 leaves per stalk. Arabidopsis
thaliana usually reaches this level of growth 3-4 days after sprouting; the subjects in the
experiment reached this stage approximately one week after sprouting. Additional growth
beyond this phase did not occur, instead the plants turned yellow and the survival rate dropped
by about half in most cases as shown in Figures 4 and 5.
Days after
planting
Control 1
Control 2
Control 3
Control 4
Control 5
Control 6
Control 7
1
4
0
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6
8
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9 10
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11 12 13 14
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Figure 3: Table of plant growth value from experiment
Figure 5: graph of plant growth in Controls 1, 3, 4, 5 over the 2-week growth period
Plant growth in the “ideal” trials was nonexistent with little to no reliable information on
the status of the microbes in them. Inoculated seeds showed a germination period twice that of
the H2O cold-snapped seeds and in all cases produced weaker plants with lower survival rates.
There was only one case of soil irregularity, which resulted in a clay-like layer covering
half of one of the samples (see Figure 6 right). Upon closer inspection it was revealed that this
layer was made up of smaller particles banding together resisting the flow of water causing little
moisture or life to accumulate beneath it.
DISCUSSION AND CONCLUSION
This project is actually a follow up to a project that was done in the summer of 2009 by the
NASA Ames Academy group that I was a part of. The project done that summer used different
bacteria to inoculate the simulant and the Arabidopsis seeds, however there was no plant growth
from that experiment. Upon closer inspection using a flouromicroscope there appeared to be
strands of Nostoc bacteria living among the grains of regolith. It was that success that allowed
my current project to be funded.
The results of this project could not have been more different from the original NASA
Academy results and the actual growth of plants suggests a couple of different options, both of
which warrant further investigation. First and more optimistically, the interactions between the
bacteria and the plants are more communal than those in the Academy’s experiment leading to a
balanced growth of both. Secondly, the pathogenic bacteria in the simulant, B. Cereus, could be
growing too rapidly for the plants defenses and retarding the growth until it eventually kills it.
Of one thing we can be sure and that is that the combination of B. Cereus and B. Megaterium
with a nutrient mix does not promote plant growth at all. The stunted growth of the plants and
inability to mature in the normal growth period most likely indicates a rivalry for resources
between the Arabidopsis and the bacteria.
Figure 6: left visible plant growth, <4mm tall; right odd clay-like layer on Control 6
Very little usable quantitative data was obtained from the soil analysis due to
contamination of the samples at the lab. However, it did show a lack of plant matter and a
noticeably higher concentration of bacteria in the “ideal” samples than in the controls. Being
unable to tell which types of bacteria were in the samples makes it hard to say with certainty that
the bacteria were the B. Cereus and B. Megaterium but no sign of fungus or spores means that
there was probably not any contamination during the growth phase.
In conclusion, there are many different types of bacteria that can form communal
relationships with plant life to the point that a co-dependent ecosystem can evolve. However,
those used in this experiment are probably not the most efficient and further research into the
metabolism of other soil dwelling bacteria could provide better insight into the possible
ecosystems necessary to create a self sustaining habitat on the Moon.
ACKNOWLEDGEMENTS
I would like to acknowledge all of the members of the 2009 NASA Ames Academy:
Carolyn Belle, Heather Duckworth, Laura Simurda, Nicholas de Leon, Misha Berger, David
Ottesen, John Ferriera, Dustin Kendrick, Jake Gamsky, Naiki Takamasa, and Joe Starek for their
hard work and patience to complete the fundamental work that made this project so much easier.
Brad Bailey, Kristina Gibbs, Douglas O’Handley, Matt Reyes, Anita Mantri, and Nathalie
Cabrol we among those who nurtured and encouraged my interest in this project while teaching
me essential skills for a scientist to have. Additionally, I’d like to thank my mentors at the
University of Hawaii at Hilo, John Hamilton and Christian Andersen as well as Debbie Scott of
the Biology department who helped me with everything I needed there. Thank you to the Hawaii
Space Grant Consortium, Mars Rei Sistoso for all her help during this and previous HSGC work,
and Ken Hon for his insight, encouragement, advice, and friendship.
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