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All Graduate Theses and Dissertations
Graduate Studies
12-2009
Designing an Instrument Based nn Native
Fluorescence to Determine Soil Microbial Content
at a Mars Analog Site
Heather D. Smith
Utah State University
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DESIGNING AN INSTRUMENT BASED ON NATIVE FLUORESCENCE TO
DETERMINE SOIL MICROBIAL CONTENT AT A MARS ANALOG SITE
by
Heather D. Smith
A dissertation submitted in partial fulfillment
of the requirements for the degree
of
DOCTOR OF PHILOSOPHY
in
Biological Engineering
Approved:
___________________________
Dr. Ronald C. Sims
Major Professor
____________________________
Dr. Anhong Zhou
Committee Member
____________________________
Dr. Anne J. Anderson
Committee Member
____________________________
Dr. Paul R. Grossl
Committee Member
____________________________
Dr. Christopher P. McKay
Committee Member
____________________________
Dr. Byron R. Burnham
Dean of Graduate Studies
UTAH STATE UNIVERSITY
Logan, Utah
2010
ii
Copyright © Heather D. Smith 2010
All Rights Reserved
iii
ABSTRACT
Designing an Instrument Based on Native Fluorescence to Determine Soil
Microbial Content at a Mars Analog Site
by
Heather D. Smith, Doctor of Philosophy
Utah State University, 2010
Major Professor: Dr. Ronald C. Sims
Department: Biological and Irrigation Engineering
For this research project we designed an instrument to detect bacteria via
biomolecular fluorescence. We introduce the current understanding of
astrobiology, our knowledge of life beyond Earth, and the commonality of Earth
life as it pertains to the search for life on Mars. We proposed a novel technique
for searching for direct evidence of life on the surface of Mars using fluorescence.
We use the arid region of the Mojave Desert as an analog of Mars. Results
indicate the fluorescence of the biotic component of desert soils is approximately
as strong as the fluorescence of the mineral component. Fluorescence laboratory
measurements using the portable instrument reveal microbial concentration in the
Mojave Desert soil is 107 bacteria per gram of soil. Soil microbial concentrations
iv
over a 50 meter area in the Mojave Desert, determined in situ via fluorescence,
show that the number varies from 104 to 107 cells per gram of soil. We then
designed an instrument for detection of biomolecular fluorescence, and
considered also fluorescence from polycyclic aromatic hydrocarbons and minerals
on the Martian surface. The majority of the instrument is designed from Mars
surface operation flight qualified components, drastically reducing development
costs. The basic design adapts the ChemCam instrument package on-board Mars
Science Laboratory rover Curiosity to detect organics via fluorescence. By
placing frequency multipliers in front of the 1064 nm laser, wavelengths suitable
for fluorescence excitation (266 nm, 355 nm, and 532 nm) will be achieved. The
emission system is modified by the addition of band pass filters in front of the
existing spectrometers to block out the excitation energy. Biomolecules and
polycyclic aromatic hydrocarbons are highly fluorescent at wavelengths in the
ultra violet (266 nm, 355 nm), but not as much in the visible 532 nm range.
Preliminary results show minerals discovered, such as perchlorate, fluoresce
highest when excited by 355 nm. Overall, we conclude the fluorescent instrument
described is suitable to detect soil microbes, organics, biomolecules, and some
minerals via fluorescence, offering a high scientific return for minimal cost with
non-contact applications in extreme environments on Earth and on future missions
to Mars.
(103 pages)
v
ACKNOWLEDGMENTS
I am grateful to the Graduate Student Research Program of the National
Aeronautics and Space Administration for funding this research and my salary
through a three-year fellowship administered by Ames Research Center with Dr.
Chris McKay as the technical monitor.
I would like to thank Dr. Linda Powers, Shayne Rich, Patrick Neary, Dr.
Walter Ellis, Dr. Jenny Norton, Dr. Joan Hevel, and Dr. Fred Rainey for valuable
discussions regarding the initial research conception, and instrument use.
I am appreciative to Dr. Charlie Miller for use of the fluorometer, which
without, this project would not have concluded. To Dr. John Shervais for use of
the USU Geology Departments mineralogy and petrology collection and helpful
discussions, Dr. Pete Kolesar for use of the X-ray diffractometer, Tiffany Evans
for the soil analysis, and Dr. Janis Boettinger for helpful discussions.
I’d like to acknowledge the Utah State University labs, facilities, and
faculty, for support and all of my insightful professors here at Utah State
University for passing the knowledge along.
I thank Dr. Chris Lloyd and Andrew Duncan for the continued access to
and support of the field portable prototype instrument as well as for insightful
discussions.
My committee members, Drs. Ron Sims, Anne Anderson, Anhong Zhou,
Paul Grossl, and Chris McKay, for the many hours of discussions,
encouragement, and support throughout the entire process.
vi
CONTENTS
Page
ABSTRACT…………………………………………………………...................iii
ACKNOWLEDGMENTS………………………………………………………..v
LIST OF TABLES……………………………………………………………...viii
LIST OF FIGURES………………………………………………………………ix
CHAPTER
I. INTRODUCTION—LITERATURE REVIEW………………………………1
Astrobiology………………………………………………1
Astrobiology in Space……………………………..2
Planetary Astrobiology……………………………3
Prebiotic Biology………………………………….5
Biological Continuum…………………………….7
Organics in Space…………………………………8
The Basics of Life………………………………………..12
Carbon Source……………………………………13
Energy Source……………………………………17
Liquid Water……………………………………..20
The Search for Life……………………………………....20
Fluorescence.……………………………………………..23
Footnotes…………………………………………………23
References………………………………………………..24
II. IN SITU SOIL MICROBIAL DETECTION IN THE MOJAVE DESERT.....33
Abstract………………………………………….33
Introduction……………………………………...34
Materials and Methods…………………………..39
vii
Results …………………………………………...42
Soil versus Baked Soil…………….……...42
Microbial Amended Soil…………………46
In Situ Results…………….………………53
Conclusions………………………………………56
References………………………………………..57
III. DETECTING ORGANICS USING FLIGHT QUALIFIED HARDWARE...60
Abstract…………………………………………..60
Introduction………………………………………61
Materials and Methods…………………………...65
Results……………………………………………69
Discussion………………………………………..75
Conclusions……………………………………....76
References………………………………………..76
IV. RESEARCH SUMMARY AND FUTURE DIRECTION………………….81
APPENDIX……………………………………………………………................84
CURRICULUM VITA…………………………………………………………..91
viii
LIST OF TABLES
Table
Page
1.1
Summary of Steps in the Kreb Cycle…………………………………….19
1.2
Payload Instruments……………………………………………………...22
2.1
Biomolecular Excitation and Emission Wavelengths…………………....36
2.2
Soil Versus Baked Soil…………………………………………………..43
2.3
Mojave Soil Characterization……………………………………………45
2.4
Mojave Soil Mineralogy…………………………………………………47
2.5
Fluorescence of Bacteria Spiked Soil……………………………………51
3.1
Organic Excitation and Emission Wavelengths………………………….63
3.2
Summary of Distinguishing Spectra...…………………………………...73
3.3
Detectable Material at 266 nm, 355 nm, and 532 nm…………………....75
4.1
Summary of Detectable Material at 266 nm, 355 nm, and 532 nm……...82
A.1
Fluorometer Mineral List………………………………………………...85
ix
LIST OF FIGURES
Figure
Page
1.1
Astrobiology Understanding………………………………………………1
1.2
Tree of Life…………………………………………………..……………7
1.3
Organics in the Solar System…………………………………………….11
1.4
Amino Acid Structure……………………………………………………13
1.5
Nucleic Acids Structure and Function…………………………………...15
1.6
The Cell Membrane……………………………………………………...16
1.7
Catabolism Pathways…………………………………………………….18
2.1
Biomolecular Fluorescence Principle……………………………………38
2.2
Mojave Soil Mineral Emission Spectra…………………………………48
2.3
Mojave Spiked Soil Fluorescence Response…………………………….49
2.4
Fluorescence Response to Increased Microbial Load…………………...52
2.5
Mojave Microbial Community…………………………………………..53
2.6
In Situ Field Site…………………………………………………………55
3.1
Instrument System Block Diagram………………………………………66
3.2
Emission System Block Diagram………………………………………..67
3.3
Polycyclic Aromatic Hydrocarbons Emission Spectra…………………..71
3.4
Hydroxy Emission Spectra………………………………………………72
3.5
Mineral Emission Spectra………………………………………………..74
A.1
Varied Silica Content Emission Spectra…………………………………87
A.2
Sulfates Emission Spectra………………………………………………..88
x
A.3
Oxides Emission Spectra………………………………………………...89
A.4
Same Composition Emission Spectra……………………………………90
CHAPTER 1
INTRODUCTION-LITERATURE REVIEW
1.1 Astrobiology
Since ancient times humans have pondered our place in the universe, the origin of
life, and the existence of life beyond our Earthly realm. These philosophical questions are
at the forefront of scientific discovery. The most fundamental and notable of these
discoveries are discussed below and depicted in Figure 1.1.
Figure 1.1 Astrobiology Understanding. The top astrobiology discoveries
categorized by relative place within the Universe. These discoveries support the
possibility of Life elsewhere as described below. Image created by author.
2
1.1a Astrobiology in Space
Our Galaxy alone contains 300 billion stars with an estimate of millions of Sunlike stars suitable of supporting planetary systems capable of harboring life.1 Within our
local neighborhood (200 parsecs), the technical boundaries of direct measurement, over
1800 sun-like stars have been discovered (Lineweaver and Grether, 2003). Sun-like stars
are classified by indentifying one or more stellar properties such as temperature, density,
mass, composition, magnetic field, and spectra similar to the Sun (Hardorp, 1978). Sunlike stars have been discovered using techniques ranging from Doppler measurements,
triangulation, gravitational micro-lensing, and high resolution spectroscopic
measurements using NASA’s Great Observatories (Cayrel de Strobel, 1996). Scheduled
to launch in 2011 European Space Agency’s (ESA’s) Gaia mission is aimed at
determining a galactic census of Sun-like stars (Perryman, 2002). Even though a
dedicated mission to detect sun-like stars is forthcoming, thousands of Sun-like stars have
already been detected within our Milky Way Galaxy (Lineweaver and Grether, 2003).
Sun-twin, G class main sequence stars, representing 7.6 % of the stars studied in our local
neighborhood, have nearly identical Radius, Mass, Luminosity, and Age of the star
compared with the Sun (LaDrew, 2001). With many similar features to our Sun, these
Sun-twins are likely candidates to support planets or even planetary systems similar to
our solar system.
3
1.1b Planetary Astrobiology
Methods for detecting planets and planetary systems were proposed as early as the
1970’s (Rosenblatt, 1971). It was not until 1995 that research paid off when a Jupiter –
sized planetary body was detected orbiting 51 Pegasi, a sun-twin star in the local
neighborhood (Mayor and Queloz, 1995). Mayor and Queloz discovered the Jupiter-sized
planet by measuring periodic variations in the radial velocity of the parent star using the
Doppler affect. Since then hundreds of (350 as of this publication) exoplanets, Jupitersized or larger, have been discovered within the local neighborhood (Butler et al., 2006).
Although most of the exoplanets discovered are Jupiter-sized planets, it may not be the
dominant planet size, it just happens to be at the lower end of the detection limit (15 m/s)
using Doppler studies.2
In addition to individual exoplanets orbiting sun-like stars, over 152 extrasolar
planetary systems have been detected within the local neighborhood (Jones et al. 2006).
As favored by the detection technique, the majority of these planets are Jupiter-sized.
Jupiter-sized planets are interesting, but have less astrobiological potential since all the
known worlds that harbor life (Earth) or water (Mars, Europa, and Encleadus ) are
smaller than Jupiter (McKay, 2009). It is estimated that 60% of extrasolar planets lie
within the habitable zone of their parent star (Jones et al., 2006). Using several
telescopes simultaneously thereby increasing the overall sensitivity, Mayor et al. (2009)
detected a 1.9 Earth mass planet orbiting within the habitable zone of the nearby star
Gliese 581.
4
In the next five years our understanding of exoplanets, planetary systems, and
habitable worlds will be revolutionized by current and near term NASA missions. The
most significant of these missions is the Kepler discovery class mission launched in
March 2009. The NASA Kepler mission will stare at a portion of the sky for three years
searching for terrestrial, Earth –sized planets orbiting within the habitable zone of sunlike stars by detecting the dimming starlight as the planet transits the star (Borucki et al.,
2003). Kepler’s gaze is planned for three years to ensure valid results. By 2012, Kepler
results might possibly present thousands of Earth- sized planets bound within the
habitable zone of their star (Borucki et al., 2003). The Spitzer Space Telescope, launched
in 2003, uses infrared light to scan the sky from an Earth-trailing solar orbit, giving us a
new perspective and insight into the universe (Werner et al., 2005). The Spitzer Space
Telescope detected a Neptune-sized planet and has the capability to characterize the
transit time of exoplanets detected by Kepler (Deming et al., 2007). The Hubble Space
Telescope (HST) has detected exoplanets using reflected starlight (Brown and Burrows
1990), the fine guidance sensor on-board HST (Benedict et al., 2002), and spectra of
planetary atmospheres captured by the imaging spectrograph on-board HST (Carollo et
al., 2001). Once decommissioned, the HST can still contribute to the search for
exoplantes by revisiting archived images. Using newly developed algorithms and image
processing software, Marois et al. (2008) analyzed a Hubble Space Telescope image
taken in 1998 of HR 8799, a star 130 light years away, exposing an exoplanet previously
overlooked. Processing archived Hubble images of areas likely to contain exoplanets
may reveal a plethora of exoplanets previously missed. Replacing HST, the James Webb
5
Space Telescope (JWST), scheduled to launch in 2014, will peer into the beginning of
galaxy formation studying along the way planetary formation (physics and chemistry of),
analog solar systems, and the establishment of habitable zones using sophisticated
infrared imagers (Gardner et al., 2006).
1.1c Prebiotic Biology
In 1953 Stanley Miller and Harold Urey synthesized amino acids, a fundamental
biomolecule, from a primordial early earth atmospheric abiotic mixture. A voltage
(simulating lightening) was applied to the abiotic mixture and allowed to brew. In a
week, amino acids, the basic building blocks to life as we know it, had formed within the
abiotic mixture (Miller, 1953). This experiment had profound implications by suggesting
life was created from abundant, earthly abiotic molecules and is the foundation of the
genesis of life originating on Earth theory.
The synthesis of amino acids from abiotic chemicals is a ground-breaking clue to
the genesis of life on Earth, however it does not paint the entire picture for the evolution
of life as we know it. All life as we know it contains amino acids, but amino acids are
also common in the absence of life. Furthermore, modern Earth life is deoxyribonucleic
acid (DNA) information based, not an amino acid based milieu. Gilbert (1986) postulated
a theory to explain the establishment of life from amino acids and the subsequent
transition to DNA, with ribonucleic acid (RNA) as an intermediate step, assuming that
RNA has retained most of its capabilities over millions of years. Gilbert proposed that
amino acids synthesized in the experiment described above were formed into RNA to
transcribe the code of life. During this period, early earth life was based on ribonucleic
6
acid (RNA) with RNA performing the duties of transcription, and replication. The early
earth ecosystem consisted of an RNA genetic based world, similar to modern viruses.
The RNA transcription template evolved to DNA as the need for efficiency increased as
organism complexity progressed. This RNA world is an intermediate step from amino
acids to DNA, and suggests that complex life likely formed from abundant amino acids
(Gilbert, 1986).
Stromatolites, fossilized cyanobacteria, off the coast of Australia confirm life
existed on Earth shortly after the planets had formed 4.3 billion years ago. Analysis of the
stromatolites indicated the presence of cyanobacteria 3.8 billion years ago (Logan et al.,
1964). This suggests that approximately at or prior to 3.8 billion years ago, life emerged
on Earth and could have emerged on Mars and other similar planetary bodies.
Using 16s RNA, Carl Woese in 1987 linked the similarity of all life on Earth to a
common ancestor. Every being that is, has ever been, and will ever be stemmed from one
microbial species approximately 3.8 billion years ago (Gya) ( see Figure 1.2). This Last
Universal Common Ancestor (LUCA) has yet to be found, but reason leads to a plausible
anaerobic, RNA genetic based organism capable of surviving current planetary surfaces
similar to early earth conditions.
7
Figure 1.2 The Tree of Life. The Tree of life based on 16sRNA sequence. The root
(common ancestor LUCA) of all life as we know it was determined using the EFTU/EFG gene coupling. Image adapted from Woese (1987).
1.1d Biological Continuum
Despite several catastrophic mass extinction events, atmospheric changes, and the
ageing of Earth, life has remained relatively constant using the same few molecules and
processes to build billions of organisms for billions of years. Amino acids were, and still
are, the fundamental building blocks for life. Despite the great diversity of habitats and
species, the fundamental units have not changed, only the way they are specifically
employed to adapt an organism for a particular environmental niche. The same
8
biochemicals have remained constant throughout the development of our solar system
through space and time. It is likely that if life developed on other planets it also
employed the same strategy, if not the same molecules (McKay 2006).
Life has been found every place we have looked for it on Earth, every place there
is a molecule of water available. Although organisms only consist of a few types of
molecules, the use and adaptability of life ranges infinitely. From the bottom of the
ocean, to the top of the clouds, and everywhere in between life favors survivability and
adaptability. With the range of extremophile habitats on Earth, it’s reasonable to consider
the lunar and Martian environments harboring life.
1.1e Organics in Space
Organic chemicals were detected in the interstellar medium (ISM) (Pendleton,
1997; Pendleton et al., 1994) and at the edge of our Solar System (Cruikshank, 1997).
Using spectroscopy, over distances as far as telescopes have peered, organic matter has
been identified within the stardust of our Sun. In the interstellar medium, polycyclic
aromatic hydrocarbons (PAHs) are abundant, the elements H, N, O, C, S, are common
molecules, and carbonaceous compounds were discovered in several of the observed
Kuiper Belt objects (Pendleton, 1997; Cruikshank, 1997; Henning and Selma, 1998).
The outer planetary objects of the solar system (Pluto, Neptune, comets) are comprised
mainly of ice and carbonaceous compounds (Pendleton et al., 1994). Results from the
New Horizon Spacecraft will greatly enhance our knowledge of the Kuiper Belt and Pluto
as it measures the atmospheric and mineral composition of our solar system beyond
Neptune (Fountain et al., 2008).
9
As you move inward, moons of the gas giants (Saturn and Jupiter) present even
more evidence of the ubiquitous nature of organic matter within our solar system. Ground
based and Voyager 1 spacecraft observations detected organic matter on Europa and
Titan. Radar studies indicate a liquid ocean beneath the water ice surface of Europa (Carr
et al., 1998). The Gas Chromatograph/Mass Spectrometer (GC/MS) on-board the
Huygens probe confirmed a thick organic rich hydrocarbon atmosphere dominated by
nitrogen and methane (Niemann et al., 2005). The Cassini spacecraft detected organic
volatiles from a cryovolcano on the surface of Titan (Sotin et al., 2005), a water vapor
plume from Enceladus (Hansen et al., 2006), and salt in Saturn’s E ring suggesting a
subsurface briny ocean on Enceladus. The Cassini spacecraft continues to monitor
Saturn and its moons. Scheduled to launch in 2011, the JUNO mission to Jupiter will give
us detailed information on Jupiter and its moons including Europa (Matousek, 2007). The
modern Planetary Era of space research is sure to resolve the curiosity of the presence of
life beyond Earth.
Liquid water is a known requirement for life as we know it. In the outer solar
system beyond the previously defined habitable zone we have found evidence of liquid,
possibly water, on Europa, Encleadus, and Titan (Waite et al., 2006). The discovery of
liquid areas makes these planetary bodies likely candidates to support life.
Ancient rocks from Mars have been found on Earth Environments ideal to
preservation, in particular arid regions (Nyquist et al., 2001). Based on the volatile
content of these meteorites, their source has been traced to Mars. Ejected from Mars as an
impact crater forms, this rock ejecta travels for millions of kilometers for possibly
10
billions of years preserving the volatile content of the rock, organic matter, and possibly
life. One particular Martian meteorite, Allen Hills 8401 meteorite was thought to contain
magnetotactic bacteria based on Scanning Electron Microscope (SEM) images of
symmetrical magnetite grains (McKay et al., 1996). Further tests revealed that
symmetrical magnetite grains can be produced abiotically as well as biotically.
Conclusive evidence distinguishing the magnetite grain source as abiotic or biotic is
undeterminable at this time.
Using ion chromatograph mass spectroscopy and isotope ratios, biogenic
hydrocarbons were found in the Orgueil meteorite (Nagy et al., 1961) and extraterrestrial
formed amino acids were discovered in the Murchison meteorite (Kvenvolden et al.,
1970). The discovery of organic material in the carbonaceous chondrite meteorites
fueled the Panspermia origin of life theory. Panspermia proposes that life originated in
outer space and subsequently deposited the seeds of life through meteoritic impacts on
planetary surfaces such as Earth and Mars (Melosh and Tonks, 2003). Figure 1.3
summarizes the current understanding of organic material associated with life in our solar
system.
11
Figure 1.3 Organics in the Solar System. The current understanding of organic
matter in our solar system. Image courtesy of the National Aeronautics and Space
Administration (NASA) 2009.
Planetary bodies within the habitable zone, the zone in our solar system where
liquid water can exist on the surface, have the potential to harbor Life. Mars and Titan
are good examples. Mars does not harbor direct, obvious signs of life. However recent
detection of methane (Formisano et al., 2004), with a half-life of 300 years, implies a
modern source of methane on Mars and possible indirect evidence of bacteria. On Earth
methane is formed both abiotically, under hydrothermal conditions (Horita and Berndt,
1999) and biotically through metabolic processes (microbial methanogens, higher
organisms, humans).
In the process of studying our solar system, we have brought life to outer space.
Bacteria (Streptococcus mitis) was found inside Surveyor 3 after spending 2.5 years on
12
the lunar surface (Mitchell and Ellis, 1972). Bacteria ( Bacillus subtilis) also survived
five years on the outside of the International Space Station exposed to the vacuum of
space (Horneck and Rabbow, 2007). Life may not have originated in outer space, but
some Earthly creatures have adapted to survive.
Traces of life and experimental results suggest that life is possible beyond Earth
and may have originated in a distant place. Within the past decades scientific
advancements in Astrobiology have begun to unravel the answers to life’s fundamental
questions. While evidence for life is surmounting, life beyond Earth has not been
detected. Therefore everything we know of life stems from our understanding of one
data set, Earth biology. The following section explains our current understanding of Earth
life, life as we know it.
1.2 The Basics of Life
While there is no formal definition of “life,” there are certain commonalities that
all life as we know it share. All life on Earth has the same biochemical composition
(Lederberg, 1960). Life is built from a particular set of available ingredients such as
amino acids, nucleic acids, fatty acids, and phosphates. Similarly certain bioprocesses are
universal to all life from the smallest microorganism to the largest multi-cellular
organism. These bio molecules (ATP,Tryptophan), common to all organisms, can be
detected using signature native fluorescence. For biology to exist three requirements must
be met: 1) carbon source (nitrogen and phosphorus too), 2) an energy source such as
reducing power, and 3) liquid water.
13
1.2a Carbon Source
Carbon is the only biological building material (carbon –based life) found on
Earth. While other materials such as silica for silica- based life are plausible, carbon due
to its chemical versatility, is ubiquitous to all life as we know it (Pace, 2001). Earth
contains both inorganic and organic forms of carbon sources derived from high-energy
stellar processes. Both organic and inorganic carbon sources are employed to construct
amino acids.
Amino acids constitute the building blocks of all carbon-based life by providing a
source of carbon and nitrogen. Twenty amino acids are common to all known life (Berg
et al., 2002). Figure 1.4 shows the general structure of the twenty amino acids. These
twenty amino acids combine to form thousands of proteins, enzymes, and metabolic
intermediates.
Figure 1.4 Amino Acid Structure. General structure of amino acids, the
fundamental unit of life. The R group varies with each amino acid. Schematic
modified from Berg et al., 2002.
14
An interesting use of amino acids is the chiral specificity preferred by organisms.
Organisms use L-amino acids to synthesis proteins and D-amino acids to construct the
cell wall (McKay, 2006). The amino acids tryptophan, phenylalanine, and tyrosine
possess alternating double-bonds (Berg et al., 2002) yielding fluorescent properties that
are useful as identification markers of extraterrestrial life using appropriate
instrumentation that is described in this dissertation proposal.
An organism’s molecular nature is controlled by nucleic acids, a distinctive
pattern of repeating sugar-phosphate units with either a purine (Adenine, Guanine) or
pyrimidine (Cytosine, Thymine, Uracil) side group attached (Lederberg, 1960). These
nucleic acids, joined by hydrogen bonds comprise an organisms genetic code as
Ribonucleic Acid (RNA) in viruses or Deoxyribonucleic Acid (DNA) in all other
organisms. Adenine always pairs with Thymine in DNA and Uracil in RNA. Likewise,
Cytosine always pairs with Guanine. Factors and enzymes associated with transcription,
translation, and destruction (RNases and DNases) of RNA and DNA are only produced
biologically. There is no known abiotic source for nucleic acids and associated factors
and enzymes. Figure 1.5 illustrates the DNA, RNA, and DNA double helix structure
along with the process by which genetic information is put into action.
15
Figure 1.5 Nucleic Acids Structure and Function. General structure of DNA, RNA,
and the double helix along with the nucleic acid decoding scheme.
All life as we know it is either a single cell or a conglomerate of cells. Bacterial cell walls
have membranes that contain fatty acids the composition of which varies from one
species to another. Figure 1.6 diagrams the fatty acids in the phospholipid bi-layer of the
cell membrane.
16
Figure 1.6 The cell membrane. The cell membrane and phospholipid fatty acids
located within the cell membrane. The exact combination of phospholipids
distinguishes one species from another. Illustration adapted from Berg et al., 2002.
When environmental conditions can no longer support life, some bacteria (e.g. B.
subtilis) and fungi (e.g. Cladosporium) survive by entering into a dormant state forming a
spore. For protection, organisms coat the spore cell wall with extra fatty acids to prevent
desiccation and increase environmental tolerance. Spores have been known to survive for
17
thousands of years becoming vegetative when environmental conditions are favorable
again (Haselwandter and Ebner, 1994).
1.2b Energy Source
There are three fundamental sources of energy (1) Sunlight in the form of
photons, (2) organic matter (decayed cells, methane) and (3) inorganic reduced
compounds (ammonium, hydrogen sulfide). The Sun and the Earth’s latent heat are the
primary sources of energy. Organisms on and near the surface obtain their energy through
photosynthesis directly or by eating those that do. In the Earths crust, chemosynthetic
organisms (Pace, 2001) obtain their energy through photochemical reactions fueled by
the latent heat in the Earth’s asthenosphere. On a molecular level, an organism obtains
energy through the metabolic breakdown of food in processes such as the Krebs cycle.
Many of the metabolic intermediates, ATP, NADH, acetyl-CoA for example, have a
distinguishing characteristic native fluorescence that can be used to identify molecules
associated with life in extraterrestrial/ Martian environments. Despite the great diversity
of organisms there are common processes used by all cells to obtain the necessary
requirements for growth, maintenance, and sustainability. Figure 1.6 is a schematic of the
process by which cells utilize food, providing the necessities of life for metabolic
respiration and growth.
18
Figure 1.7 Catabolic Pathways. Schematic of catabolic pathways common to all
cellular life. Fatty acids are degraded to acetyl Co-A by beta oxidation. Amino
acids are degraded or synthesized by a few pathways using intermediates from the
TCA cycle. Sugars are degraded through the glycolytic pathway to pyruvate. The
energy rich TCA cycle coupled with oxidative or photo phosphorylation is the final
processes in the generation of energy from food molecules. Fluorescent biomolecules
are circled in orange. Figure based on Lehninger, 2004.
19
Table 1.1 Summary of Steps in the Kreb Cycle. Note step 1 and 9 are the same.
Fluorescent biomolecules are highlighted in yellow. Table produced by author from
Berg et al., 2002.
Step
Reaction
Action Taken
Enzyme
Product
1
Acetyl-CoA +
Oxaloacetate +
H2O
Claisen condensation of
Acetyl-CoA and
Oxalacetate
Citrate Synthase
Citrate
2
Citrate
Isomerization of citrate
by dehydration/
rehydration
Aconitase
Isocitrate
3
Isocitrate +
NAD(P)
Oxidative
decarboxylation of
isocitrate
Isocitrate
dehydrogenase
α- Ketoglutarate +
CO2
4
α- Ketoglutarate +
CoA-SH + NAD+
Oxidative decarboxyl αKetoglutarate ation of
α- Ketoglutarate
dehydrogenase
complex
Succinyl-CoA +
NADPH + H+ + CO2
5
Succinyl-CoA +
GDP + Pi
Substrate level
phosphorylation
Succinyl-CoA
synthetase
Succinate + GTP +
CoA-SH
6
Succinate + FAD
Dehydration of succinate
Succinate
dehyrdogenase
Fumarate + FADH2
7
Fumarate + H20
Hydration of fumarate
Fumarase
L- Malate
8
L-Malate + NAD+
Dehydration of L-Malate
Malate
dehydrogenase
Oxaloacetate +
NADH + H+
9
Acetyl-CoA +
Oxaloacetate +
H2O
Claisen condensation of
Acetyl-CoA and
Oxalacetate
Citrate Synthase
Citrate
20
All behaviors of every organism on Earth are regulated at the cellular level.
Biologically distinct building blocks combine to form the macromolecules that run
cellular functions, and hence life. Table 1.1 summarizes the steps, listing the metabolic
intermediates formed during the TCA/ Krebs cycle diagramed above.
Fluorescent biomolecular metabolites of the Krebs cycle and associated oxidative
phosphorylation include NADH, FAD, and ATP.
1.2c Liquid Water
On Earth, liquid water is essential for life. The majority (~ 70%) of an organism is
comprised of liquid water. Water is considered the "universal solvent" as at neutral pH it
dissolves more substances than any other liquid providing organisms with necessary
chemicals, minerals, and nutrients to survive. On Earth the only limitation to biology is
the absence of liquid water. If there is water, there is life.
1.3 The Search for Life
The Martian surface contains evidence of a previously active fluvial system
(Greeley and Guest, 1987; Malin and Edgett, 2003). Similarities between Martian surface
features and Earth fluvial features suggest the Martian surface was once a wetter, more
hospitable environment with lakes and flowing water. Current images of the Martian
surface show water bound as ice at the poles, and frost patches were seen by the imager
on-board the Mars Pathfinder in 1998 (Bell et al., 2000) and the Phoenix Lander in 2008
(Smith et al., 2009). Given the connection between water and life, Mars was likely to
harbor life in the past. Evidence of that life will likely be in the form of spores just below
21
the Martian surface (McKay, 2006). Astrobiologists are searching for clues of past life
and current Martian environments able to support life, using a variety of instruments and
techniques based on the commonalities of life described above. The first missions
dedicated to searching for life were the Viking rovers, these rovers were equipped with
three life detection experiments 1) the label release experiment and 2) Gas exchange
experiment and 3) the Gas Chromatograph / Mass Spectrometer. The label release
experiment measured the chirality of amino acids to determine if amino acids were from
a biological source (favors a particular chirality) or from a chemical source (same number
of each chiral molecule). The GC/MS looked for traces of organics in the Martian soil
with the sensitivity parts per billion (ppb). Viking results were inconclusive, the label
release and gas exchange experiments indicated reactive soil, but no organics were
detected by the GC/MS (Kline, 1977). Subsequent Mars missions have focused on
indirectly detecting “life” on Mars by searching for evidence of environments capable of
hosting life, in particular water.
The Mars Exploration Rovers (MER) were designed as field geologists with
instruments capable of determining past conditions conducive to water and evidence of
water based on mineralogy. The upcoming Mars Science Laboratory (MSL) mission,
designed as a field chemist, will search for life by searching for organics. Instruments
slated to fly on MSL, Phoenix, and proposed to fly on a future field biologist mission are
summarized in Table 1.2.
22
Table 1.2 Payload Instruments. Instruments selected for current and near future
NASA Mars missions. * denotes proposed instrument for the field biologist mission.
Instrument
Measurement Description
Thermal and Gas Analyzer (TEGA)
Combined oven and mass spectrometer for detection of
organics at levels of 10 ppb.
MECA: Wet Chemistry Lab
Measures soil pH, cations, anions, conductivity, and Eh.
MECA: Atomic Force Microscope
Creates a very small-scale "topographic" map showing the
detailed structure of soil and ice grains.
MECA: Thermal and electrical conductivity probe
Indicates any transient wetness that might result from soil
excavation.
Meterological Station (LIDAR)
Measures current state of the polar atmosphere and Mars
water cycle using a thermocouple, pressure transducer and
lidar for particle distribution.
ChemCam (LIBS and RMI)
Remote elemental chemical analysis with no sample
preparation can identify hydrated minerals . High resolution
imager for Soil and pebble composition surveys.
Alpha Particle X-ray Spectrometer (APXS)
Elemental chemical analysis using a combination of
particle-induced X-ray emission (PIXE) and X-ray
fluorescence (XRF). Mounted on the robotic arm.
Mars Hand Lens Imager (MAHLI)
Focusable color camera on the robotic arm.
ChemMin (XRD and XRF)
Mineralogy of sampled rocks and soil.
SAM: Quadrupole Mass Spectrometer (QMS)
Identification of organic compounds.
SAM: Gas Chromatograph (GC)
Separation of organic prior to QMS analysis.
SAM: Tunable Laser Spectrometer (TLS)
Measures isotope ratios for C and O in carbon dioxide and
measures trace levels of methane and its carbon isotope.
Radiation Assessment Detector (RAD)
Measures the full spectrum of energetic particle radiation at
the surface of Mars, including both charged particles and
neutral particles (neutrons, gamma rays).
Dynamic Albedo of Neutrons (DAN)
An active/passive neutron spectrometer that measures the
abundance and depth distribution of H- and OH-bearing
materials.
Life Marker Chip (LMC)*
Antibody-based assay to detect present and past biomarkers.
Mars Organic Detector (MOD)*
Detects organics, amino acids, PAH's with ppt sensitivity
after sample preparation using laser induced fluorescence.
23
1.4 Fluorescence
Four of the instruments listed in Table 1.2, are designed to search for organics or
biomolecules (SAM: QMS, TEGA, MOD, and LMC). Of these instruments, MOD, has
the greatest sensitivity of parts per thousand (ppt). This sensitivity is achieved using laser
induced fluorescence.
Fluorescence is a phenomenon characteristic of many substances and may be used
to identify minerals, organic matter, and biological material. Fluorescence occurs when a
photon of the appropriate wavelength reaches the surface, exciting the molecule, and
raising the energy state of an electron. As the energy dissipates the electron cascades
down to lower energy states emitting photons. In general the emission wavelength is 50
to 100 nm longer than the excitation wavelength. The emission intensity is related to the
amount of the specific mineral, organic, or biological material present. Fluorescence is a
non-contact method of detecting and identifying fluorescent materials remotely (several
meters) and is considered here as a method for detecting organics, minerals, and
biomolecules on the surface of Mars.
1.5 Footnotes
1. The estimate of 300 billion stars is based on the following assumptions: 1)
Solar Mass is the average mass of a star. (80% of stars are lower mass than the
Sun. So this could be a low estimate). 2) 90% of the galactic mass is “dark
matter/ energy”, with 10% of the mass of stellar origin. (Massey, R. Royal
Astronomical Observatory, 2009). 3) Using NSF’s Very Long Baseline Array
(VLBA) in January 09 Mark Reid calculated the mass of the Milky Way to be 3
trillion solar masses based on velocity measurements. The number of stars in the
galaxy= (Galactic Mass, 3 x 10 12) times (% of stellar mass in the galaxy, 10%) =
300 billion stars in the galaxy. Estimating the number of Sun-like stars in the
galaxy: 1) Assume the local neighborhood is subset representative of our Galaxy.
24
2) 7.6 % of the stars in our local group are Sun-twin, scale this % to the estimated
number of stars in our galaxy (300 Billion). Number of Sun-like Stars in our
galaxy = (Galactic Census, 3 x 109 stars) times ( % of measured Sun-twin stars,
7.6%) = 22.8 Million Sun-like stars in our galaxy. Harboring “Life” refers to
micro-organisms in the vegetative or spore state as well as higher beings.
2. The reflex motion of the Sun due to Jupiter is 13 m/s. The sensitivity of
Spectrographs used in Doppler Studies in 1995 is 15 m/s. Hence all current
searches are limited to the detection of objects Jupiter mass or greater (Mayor
and Queloz 1995).
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33
CHAPTER 2
IN SITU SOIL MICROBIAL DETECTION IN THE MOJAVE DESERT USING
NATIVE FLUORESCENCE
2. 1 Abstract
The use of fluorescence techniques for microbial detection and enumeration in
desert soils on Earth and possibly Mars has been evaluated. The use of a portable
instrument employing four excitation wavelengths combined with four emission
wavelengths to analyze the Mojave Desert soil as an analog for Mars soil is described.
Known numbers of bacteria were added to soil collected from the arid part of the Mojave
Desert and used as a control to determine the sensitivity of the instrument. The
fluorescence of the biotic component of desert soils is approximately as strong as the
fluorescence of the mineral component of these soils. Using processing algorithms
developed to separate the biological fluorescence signal from the mineral fluorescence
signal, we improved detection limits for soil microorganisms by a factor of 10 to 1000.
Fluorescence laboratory measurements using the portable instrument reveal the microbial
concentration in the Mojave Desert soil is 107 bacteria per gram of soil, a level confirmed
by phospholipid fatty acid analysis. Soil microbial concentrations over a 50 meter2 area in
the Mojave Desert determined in situ using fluorescence show that the number of bacteria
varies from 104 to 107 cells per gram of soil. We suggest that this variability is due to
self-organized patchiness by analogy with variability in plants in desert environments.
Results of this research indicate that fluorescence is a practical method for detecting soil
34
microbes in non-contact applications in extreme environments on Earth and on future
missions to Mars.
2.2 Introduction
A key goal for astrobiology is the search for evidence of life on other worlds – in
particular Mars. In the near term such a search will be conducted by rovers. Experience
with the Mars Exploration Rovers, Spirit and Opportunity, shows that it is difficult for the
rovers to obtain samples. For this reason, a method for detecting organic chemicals
and/or microorganisms without collecting a sample is needed and UV fluorescence is a
strong candidate. Indeed non-contact determination of microbial content using native
fluorescence is already in use in the food, pharmaceutical, and water quality industries
(Lloyd et al., 2003). Non-contact fluorescence-based methods would also be of use in
extreme environments on Earth. Traditional methods for determining microbial content
in such environments require sample collection followed by laboratory analysis. This
procedure disturbs the soil environment, requires time for analysis, and alters the sample
as it is analyzed possibly rendering the soil useless for further studies. The capability to
directly determine soil microbial content in situ without contacting or disturbing the soil
would be useful when studying fragile or extreme ecosystems on Earth, such as
cryptobiotic crusts.
Many molecules found in living cells exhibit characteristic fluorescence (See
Table 2.1). Characterization of fluorescence involves both an excitation wavelength and
an emission wavelength (Estes et al., 2003). The excitation that results in fluorescence
occurs when a photon of the appropriate wavelength reaches, or excites, the biomolecule
35
and the energy state of an electron is raised. As the energy dissipates, the electron
cascades down to lower energy states. The energy released in this cascade is emitted as
photons of wavelengths longer than the excitation wavelength due to the loss of energy
required to raise the electron state. In biological systems, the emission wavelength is
generally 50 to 100 nm longer than the excitation wavelength. The emission intensity is
directly related to the amount of the specific biomolecule present. Table 2.1 lists common
biomolecules and their related fluorescence properties at physiological pH.
As seen in Table 2.1 there are both defined excitation and emission wavelengths
for each biomolecule. Thus it is useful to consider fluorescence response as a surface in a
two dimensional phase space with the axes of excitation wavelength and emission
wavelength. The biomolecules listed in Table 2.1 represent points in this phase space.
Estes et al. (2003) and Lloyd et al. (2003) developed instrument concepts and
algorithms for UV detection of low levels of microorganisms primarily for testing of
waste water and foodstuffs. These applications have a low mineral component and the
target surface is relatively uniform with minimal interference from background
fluorescence. In soils, fluorescence also occurs from extra-cellular organic material and
inorganic minerals in the soil. This background fluorescence often overlaps with the
biological signal and in most applications prevents the direct detection of organisms by
fluorescence techniques, except at very high biological concentrations. Careful use of
excitation and emission wavelength combinations can separate these components, and in
principal, allow the identification of microbes despite a high background fluorescence
due to soil minerals.
36
Table 2.1 Biomolecular Excitation and Emission Wavelengths. Biomolecular
Excitation and Emission wavelengths along with a brief description of common
biomolecules possessing native fluorescence properties and the reference. The
fluorescent biomolecules used in this study are highlighted in red and blue.
Biomolecule Excitation λ Emission λ Brief Description Reference ATP 280 nm 420 nm common to all cells, metabolite Katayama et al. 2001 Chlorophyll‐a 430 nm 669 nm in photo synthetic organisms Hendrix 1983 Dipicolinic acid 340 nm 410 nm 10% dry weight of spores Alimova et al. 2003 Dipicolinic acid 345 nm 410 nm in Bacillus and Clostridium Paidhungat et al. 2000 F420 420 nm 460 nm co‐enzyme in methanogens Tung et al. 2005 FAD 450 nm 535 nm universal electron receptor Richards‐Kortum 1996 FMN 450 nm 535 nm universal electron receptor Richards‐Kortum 1996 NADH 350 nm 460 nm universal co‐factor Richards‐Kortum 1996 Phenylalanine 260 nm 282 nm amino acid common to live cells Seaver 1999 Tryptophan 280 nm 353 nm amino acid common to live cells Alimova et al. 2003 Tyrosine 275 nm 304 nm amino acid common to live cells Nevin et al. 2007 To use these methods in natural environments on Earth or on future Mars
missions, the challenge of separating the biological signal from the mineralogical
background fluorescence must be overcome. Thus, there is a need to adapt the methods of
Estes et al. (2003) to the difficult situation of the complex matrix of soils. Figure 2.1
shows a conceptual diagram of an instrument based on a combination of discrete
excitation and emission wavelengths. Available Light-Emitting-Diode (LED) excitation
37
sources closest to the excitation peak listed in Table 2.1 are used in Figure 2.1. For
instance, a 365 nm LED was the lowest frequency, most cost effective wavelength
available at the time of instrument design therefore a 365 nm LED was substituted for a
350 nm excitation source. Because the 365 nm LED is at a longer wavelength than the
optimal 350 nm, multiple 365 nm LED sources are used to compensate. Light from lightemitting-diodes pass through band-pass-filters (10 nm full width half maximum) set at
four specific wavelengths to illuminate the soil. The light hits the soil stimulating
fluorescence that is detected by eight photomultiplier tubes (PMT), two for each emission
wavelength, with band-pass filters (10 nm full width half maximum) in front of them.
The PMTs are compact, high-sensitivity, low-power Hamamatsu (model H6780 series)
photomultiplier tubes. The direct signal is processed by custom electronics (described in
Estes et al., 2003) to give a relative intensity of light. This represents one output of the
system, and further algorithms process this output to yield a collective fluorescent
intensity.
In this chapter we present results for in situ fluorescence analysis of natural desert
soil and desert soil that has been modified to contain a defined bacterial load. To obtain
these data we used a semi-portable prototype instrument designed for lab and field
experiments. This instrument was used both in the lab environment, with known
concentrations of microbes, and in situ in the Mojave Desert to determine the distribution
of microbes in their natural environment. We use the desert soils as a mimic of Mars soil.
38
Figure 2. 1. Biomolecular Fluorescence Principle. Biomolecular fluorescence can be
detected using wavelength combinations, similar to those listed in Table 2.1. In the
diagram above, the 365 nm Excitation/ 440 nm Emission wavelength combination
measures fluorescence from NADH and Dipicolinic Acid. The 455 nm
Excitation/540 nm emission wavelength combination measures fluorescence from
FAD and FMN, and so forth. Custom electronics delivers a raw output for each
PMT. Algorithms taking the gain and other factors into account determine a
collective fluorescence intensity for each excitation and emission wavelength
combination. The data are then calibrated with wavelength specific microbial
concentration data to yield an overall soil microbial concentration.
39
The native fluorescence from these cells is used to quantitatively determine biological
concentrations in the soil and how the effects of mineral fluorescence can be minimized.
Both the direct output from the detectors and the output of algorithmic processing are
discussed. In principle, it allows the resolution of 104 bacteria per gram soil.
2.3 Materials and Methods
The Mojave Desert, an established Mars test bed, with its windy, arid climate,
playas, sand dunes, and general Mars-like desert terrain (Farr 2004), was chosen as the
field site for ease of logistics and test bed heritage. In situ measurements were taken east
of Silver Dry Lake Bed (N 35o23' 14", W 116o 8' 32"). Soil collected to the north of
Silver Dry Lake Bed (N 35o 23' 11", W116o 15' 50"), was used for laboratory tests and
instrument algorithm training. The soil was collected under sterile conditions (wearing
gloves, sterile scoopula), placed in sterile Whirl-pak bags and stored at room temperature
until use.
In order to determine the soil background fluorescence, we baked Mojave soil at
500°C for 1 hour to destroy any carbon-based, biological material. We then compared the
baked soil fluorescence to the native soil fluorescence to determine the biological
component of fluorescence (see Table 2.2). The next step was to calibrate and determine
the biological sensitivity of the portable instrument by adding known quantities of
bacteria to soil as described below.
The Mojave soil was spiked with a mixture of three hardy sporulating Bacillus
isolates which were cultured from arid desert soils. The Bacillus isolates selected (B.
vallismortis, B. strain SSA3, and B. atrophaeus SCH0408, indentified from 16sRNA
40
sequencing by the BLAST database ) were grown at room temperature for one week on
Plate Count Agar (0479-17 Becton Dickinson(BD) , Mountain View, California)
containing 23.5 g/L of pancreatic digested casein (5 g), yeast extract (2.5 g), dextrose (1
g), and agar (15 g) before harvesting from the agar surface with a cell scraper. The cells
were suspended in a liquid sporulation medium (10 g potassium acetate, 1 g yeast extract,
and 0.5 g dextrose, and 2 g of desert soil/L) for 1 day and an aliquot of 0.1 mL was added
to each plate of agar sporulation media (10 g potassium acetate, 1 g yeast extract, 0.5 g
dextrose, 20 g of Bacto agar, 1 pellet of NaOH, ca. 0.1 g/L). After incubation in a
controlled chamber for two weeks at 25oC and 100% humidity, cells were resuspended in
a liquid sporulation medium incubated with shaking for two weeks. The cultures were
centrifuged (3,300 x g for 15 minutes) and the pellet washed with sterile water before
resuspension and repeating the washing process. The washed pellet containing the spores
were lyophilized, and milled in a surface-sterilized agate mortar and pestle. Serial
dilutions of a water suspension of these spores were plated onto yeast peptone
dextrose(YPD) medium containing in 1 L: yeast extract (10 g), peptone (20 g), dextrose
(20 g), and Bacto Agar (20 g) to determine the culturable colonies (colony forming units
(cfu)/g) originating from the bacteria spores after 24 hours of growth at ambient
conditions.
A known weight of the above mentioned (Bacillus) spore powder was added to a
known weight of unmodified Mojave soil to provide 108 cfu/g soil. A calibration soil
series was created by ten-fold dilutions by weight by taking 6 grams of the previous soil
dilution and adding it to 54 grams of un-modified soil to create the next dilution in the
41
series. For example: 107cfu/gram of soil = 6 grams of 108 cfu/gram of soil + 54 grams of
unmodified Mojave soil; 106 = 6 grams of 107 cfu/gram of soil + 54 grams of unmodified
Mojave soil, etc., 100 = 6 grams of 101 cfu/gram of soil + 54 grams of unmodified
Mojave soil. A calibration series was created so that soils from 108 to 1 cfu/g were
generated.
Estes et al. (2003) and Lloyd et al. (2003) have shown that they can increase the
detection sensitivity for microbes by a factor of 10 to 1000 in samples with high mineral
fluorescence. Their method makes use of the fact that metabolites have a different
emission and adsorption spectra than minerals. The procedure involves adjusting the
PMT and amplifier gains, as well as combining the signals from diverse sets of excitation
and emission pairs chosen to center on certain biological features (Table 2.1).
To confirm fluorescent-based microbial quantification, the Mojave soil was sent
to Microbial Insights (Rockford, TN, www.microbe.com). The microbial content was
determined using phospholipid fatty acid analysis (PLFA) to independently determine the
cell count. PFLA coupled with peptidoglycan comprise a large portion of a cell wall,
consequently, the amount of phospholipids in soil is related to the microbial
concentration. Since phospholipids break down quickly upon death (White et al. 1979),
PLFA biomass calculations are based only on viable cells. The exact combination of fatty
acids within the cell wall distinguishes one microbial group from another. The PLFA
assay can be used to test for eight lipid categories ranging from genus-specific lipids to
general ubiquitous lipids. Thus the quantity and diversity of PLFAs in the soil is
dependent upon the abundance and diversity of the microbial community.
42
2. 4 Results
2.4a Soil Versus Baked Soil
The first set of data considered are the direct PMT outputs for Mojave soil
compared with the same Mojave soil after heating to remove cells and organic molecules.
This comparison provides an indication of the extent of the biological signal in the
samples. The data are summarized in Table 2.2 which shows the average signal, relative
fluorescent units, from each PMT for the natural soil and for the same soil after heating to
500°C. Each excitation/emission combination appears twice corresponding to the two
duplicate PMT’s for each emission channel.
For most of the excitation/emission combinations in Table 2.2 the fluorescence
output drops when the sample is heat sterilized. Lack of culturable microbes was
confirmed in the heated soils by plating. The ratios of fluorescent units from the natural
soil to the heat-treated soil varies from essentially unity to a maximum of two. These
results show that the contribution to the fluorescence by the biological component is
significant. There were exceptions to this pattern. For both channels that correspond to
the (590 nm/770 nm) excitation/emission combination, the heated soil had a higher
fluorescence. This cannot be explained by removal of the biological material. We
speculate that this reflects the production of either a mineral phase or the alteration of an
existing phase resulting in fluorescence enhancement. On a technical note, the channels
that correspond to the excitation/emission combination of 440 nm/540 nm shows zero
43
standard deviation indicating that the detector was saturated and the data were not
meaningful.
Table 2.2 Soil versus Baked Soil. An average of the raw output and the % standard
deviation from 20 measurements of Mojave natural soil compared with baked
(500°C for 1hr) Mojave soil as measured by four emission wavelengths (770, 675,
540, and 440 nm) when excited by four wavelengths (365, 455, 590, and 635 nm).
Soil Baked Soil Wavelength (Fluorescent Units) LED 365 nm Std Dev, % (Fluorescent Units) Ratio of Soil/ Std Dev, % Baked Soil 770 nm 399.85 0.89
310.35
0.98 1.29
770 nm 241.15 0.66
205.22
1.42 1.18
675 nm 348.75 0.69
265.35
1.04 1.31
675 nm 193.6 4.17
145.30
5.37 1.33
540 nm 2261.95 0.02
1407.91
0.89 1.61
540 nm 1539.35 0.07
945.91
0.15 1.63
440 nm 711.8 0.03
442.96
0.82 1.61
440 nm 667.95 0.61
489.35
0.97 1.36
LED 455 nm 44
770 nm 1060.25 0.35
810.78
0.93 1.31
770 nm 542.8 0.35
387.91
1.03 1.40
675 nm 206.9 0.35
108.87
1.12 1.90
675 nm 136.75 0.40
71.09
1.03 1.92
540 nm 2772 0 *
1380.09
1.16 2.01
540 nm 1835 0 *
913.13
1.03 2.01
LED 590 nm 770 nm 781.6 0.78
806.00
0.77 0.97
770 nm 349.65 0.66
375.04
0.77 0.93
675 nm 254.1 0.70
195.43
0.97 1.30
675 nm 198.05 0.42
150.13
0.86 1.32
LED 635 nm 770 nm 749.55 0.61
776.87
0.60 0.96
770 nm 402.15 0.57
422.39
0.94 0.95
* Std Deviation of 0 indicates possible PMT saturation on this channel. Actual value may be higher. Due to the high fluorescence background in the soil sample as revealed by the
data in Table 2.2, soil characterization (chemical, mineral, and physical) techniques were
employed to describe the soil. Table 2.3 lists the chemical and physical soil properties
45
analyzed at Utah State University Soil Analytical lab using a variety of standard methods.
The Mojave soil is classified as a sandy loam based on particle size and has relatively low
nitrogen, phosphorus, sulfur, and carbon, elements sustaining life, compared to a non
desert soil. Particle size, as altered by grinding the soil to a finer consistency, did not
significantly alter the soil fluorescent profile (data not shown).
Table 2.3 Mojave Soil Characterization. Chemical and Physical properties of topsoil
in the arid region of the Mojave. Chemical analysis was obtained via ICP-Optical
Emission Spectra, C & N via LECO employing the Dumas method. Analysis was
performed at the Utah State University Soil Analytical Lab.
Element
MJ Soil
Al %
0.72
Mg %
MJ Soil
pH
7.89
0.61
EC dS/m
0.53
Chloride
Ca %
2.14
Mn mg/kg
320
mg/L
Co mg/kg
6.64
Na %
0.02
Total N
<0.01%
Cr mg/kg
9.21
P%
0.05
Total C
0.19% - 1.22%
Cu mg/kg
15.3
S%
0.01
Texture
Sandy Loam
Fe %
3.26
Sr mg/kg
55
Sand
75
K%
0.14%
Zn mg/kg
32.6
Silt
14
Clay
10
* B, Cd, Mo, Ni, and Pb were below the detection limit.
49.9
46
Because mineral fluorescence is a key background contributor for the Mojave soil
under test, X-ray diffraction (XRD) was conducted with an XPERT-Pro X-ray
Diffractometer by the USU Geology Department for both native and heated soils. Soil
collected east, at the in situ location, and north of Silver Dry Lake bed were compared.
The data summarized in Table 2.4 illustrate that the soil at both locations consisted
primarily of quartz, two feldspars (albite, microcline), and phyllosilicates (phlogopite,
koninckite) typical of a hot, arid desert. Based on preliminary data (not shown),
fluorescence is likely due to microcline and the clayey phyllosilicates. This analysis is
consistent with the finding that baking the soil at 500°C for 1hr did not change the
composition significantly. There is a reported increase in the mineral Koninckite (7 to
12) when heated. However with only 0.05% Phosphate in the soil, Koninckite, a
phosphate mineral, would be undetectable in the XRD at this concentration. The change
in mineralogy and increase in fluorescence is likely due to phyllosilicate alteration when
heated.
2.4b Microbially-Amended Soil
To determine the relationship between measured fluorescence and bacterial cell
number, we performed studies with soils containing known bacterial amendments.
Bacteria were added at levels between 1 to 108 bacteria per gram of soil. We explored the
use of the algorithms developed by Estes et al. (2003) and Lloyd et al. (2003) to translate
the raw data from a combination of excitation/emission channels into a single fluorescent
unit related to microbial concentration. These algorithms minimized contributions due to
mineral fluorescence.
47
Table 2.4 Mojave Soil Mineralogy. Mineral composition of un-modified Mojave soil
at the in situ location compared with baked Mojave soil (500°C for 1 hr). Baking the
soil did not modify the mineral composition significantly. Scale and Score Factor are
relative indices unique to the XRD software package and are used here for
comparative analysis. XRD analysis was conducted at Utah State University
Geology Department.
In situ Mojave Soil Baked Mojave Soil Mineral Mineral Class Composition Score, Scale factor Score, Scale factor Analysis Quartz Quartz SiO2 67, 0.974 68, 0.997 Albite / Carich Feldspar (Na, Ca) Al, / Calcican (Si, Al)3O8 Microcline Feldspar K Al Si3O8 XRD XRD 42, 0.653 37, 0.581 24, 0.200 Phlogopite, Iron‐rich clay/ K(Mg ,Fe)3 , 1M ferroan mica (Al ,Fe )Si3 O10 , (OH,F)2 Koninckite Phosphate (Fe, Al)PO4 ∙3 H2O 7, 0.067 Calcite Carbonate CaCO3 19, 0.528 XRD 12, 0.176 11, 0.235 XRD 12, 0.065 XRD strongly effervescent strongly effervescent 1%M HCl
To determine the fluorescence contribution of each particular mineral to the overall
mineral background fluorescence, emission spectra of each of the minerals listed (with
the exception of koninckite) was taken as shown in Figure 2.2.
48
Figure 2.2 Mojave Soil Mineral Emission Spectra. Fluorescence of minerals found
in the Mojave soil. Phlogopite, a phyllosilicate, contributes a significant portion of
the mineral fluorescence when excited at 532 nm.
49
For each of the soil samples described above we obtained a complete set of fluorescence
measurements using the instrument described previously. Duplicate wavelengths were
summed to yield a collective fluorescent unit. The results are shown in Figure 2.3 and
Table 2.5 for all combinations of excitation and emission wavelengths available in this
instrument.
Figure 2.3 Mojave Spiked Soil Fluorescence Response. Fluorescence response for
each excitation/emission combination of Mojave spiked soil.
Figure 2.3 and Table 2.5 show a large increase in fluorescent units along all
channels between 107 and 108 bacteria / g soil. Especially noticeable are the excitation
50
and emission channels related to the spore forming biomolecule dipicolinic acid (365
excitation/ 440 emission). This increase in fluorescent units signals the triggering of
biological fluorescence surpassing the mineralogical fluorescence. In Figure 2.3, the 455
excitation/540 emission combination, illustrates an increase in fluorescent units
beginning at 105 bacteria/g soil suggesting a lower mineralogical background for that
wavelength combination or metabolic activity within the soil sample.
Table 2.5 shows the instrument working consistently with a relatively low
standard deviation over several measurements. Figure 2.4 compares the raw output signal
for one 440 nm emission channel (365 nm excitation) measured for the heat treated
sample and the samples with microbial additions. Several interesting features can be seen
in Figure 2.4. First the fluorescence of the heat-treated sample is about half that of the
natural samples with low additions of microorganisms. This is consistent with the results
shown in Table 2.2 comparing the heat treated samples to unaltered samples. Another
important point shown in Figure 2.4 is that the fluorescence signal is approximately
constant for microbial addition levels below 107 bacteria per gram soil. We interpret this
to indicate that the natural concentration of bacteria is about 107 per gram soil so that
additions of microbes in quantities less than that would not alter the fluorescence signal.
Only when the number of bacteria added to the sample equals or exceeded the initial
amount does the fluorescence signal increase. This is seen in an increase in the signal for
additions of 107 bacteria per gram of soil. This general pattern is seen in the other
combinations of excitation and emission wavelengths in Table 2.5 and Figure 2.3.
51
Table 2.5. Fluorescence of Bacteria Spiked Soil. Collective Fluorescent Intensity
for the Mojave bacteria spiked soil calibration set (1 to 108 cfu/g) and the percent
standard deviation over 20 measurements. The values are the sum of duplicate
emission channels.
770 nm
Em
Std. Dev.,
%
675 nm
Em
Ex 365 nm
1 cfu/ g of soil
10 cfu/ g of soil
495.03
497.58
3.34
3.71
518.38
528.37
102 cfu/ g of soil
488.81
0.91
103 cfu/ g of soil
473.46
4.04
104 cfu/ g of soil
485.57
105 cfu/ g of soil
Std.
Dev., %
540 nm
Em
Std. Dev.,
%
440 nm
Em
Std.
Dev., %
2.52
2.3
3227.63
3324.03
1.72
1.77
3071.65
3256.10
2.03
4.47
524.97
0.3
3297.27
0.44
3140.55
0.87
509.85
3.73
3251.21
3.63
3114.33
5.41
3.64
519.47
0.98
3236.56
2.11
3057.22
3.66
488.38
0.63
523.15
0.92
3351.00
1.88
3326.70
6.39
106 cfu/ g of soil
563.92
2.25
919.63
12.14
4217.30
10.63
3866.62
7.61
107 cfu/ g of soil
731.85
11.76
1234.30
6.24
8136.38
4.01
6588.13
1.89
108 cfu/ g of soil
Ex 455 nm
1 cfu/ g of soil
10 cfu/ g of soil
1771.22
2.59
2816.48
2.48
22444.75
2.89
15560.32
4.03
1328.83
1383.45
2
1.89
328.37
331.82
2.1
8.3
16946.47
17570.83
5.52
9.21
102 cfu/ g of soil
1355.00
0.82
324.85
3.04
16339.63
6.6
103 cfu/ g of soil
1345.40
1.29
332.42
6.04
18532.14
11.42
104 cfu/ g of soil
1372.22
4.34
331.03
8.56
19150.05
9.01
105 cfu/ g of soil
1363.57
1.65
994.00
2.67
19760.70
6.61
106 cfu/ g of soil
1533.65
2.97
1170.40
6.27
37213.95
10.46
107 cfu/ g of soil
2030.95
15.93
2156.92
8.96
64600.42
5.79
108 cfu/ g of soil
Ex 590 nm
1 cfu/ g of soil
10 cfu/ g of soil
5436.78
2.64
7427.13
1.46
278089.78
55.78
869.38
870.42
6.63
1.57
377.72
370.38
6.04
1.89
102 cfu/ g of soil
875.70
1.3
371.93
1.36
103 cfu/ g of soil
802.28
7.2
335.08
6.42
104 cfu/ g of soil
863.67
3.25
383.57
3.82
105 cfu/ g of soil
843.72
2.02
371.13
2.73
106 cfu/ g of soil
928.22
8.75
409.47
9.2
107 cfu/ g of soil
1021.95
3.03
500.53
5.12
108 cfu/ g of soil
Ex 635 nm
1 cfu/ g of soil
10 cfu/ g of soil
1875.57
3.86
1921.55
0.86
944.60
938.55
2.77
3.21
102 cfu/ g of soil
960.23
0.81
103 cfu/ g of soil
875.86
0.96
104 cfu/ g of soil
1012.35
1.59
105 cfu/ g of soil
950.60
4.11
106 cfu/ g of soil
1137.87
5.38
107 cfu/ g of soil
1293.58
10.07
108 cfu/ g of soil
2613.42
6.9
52
Based on the fluorescence reading from the native soil we conclude that the native
Mojave soil sample possessed about 107 spore equivalents/g.
Figure 2.5 Fluorescence Response to Increased Microbes. Fluorescence response to
increasing bacterial concentration of Bacillus-spiked Mojave soil.
To confirm the levels of biomass we used a commercial service to evaluate the
PLFA content of the soil. This analysis was consistent with a load of about 107 cells/g.
Assessment of the different types of PLFA (Figure 2.5) revealed several spore forming
bacteria inhabiting the soil microbial community within the arid region of the Mojave
Desert. In the phylum Firmicutes, terminally branched saturates are indicative of the
genus Desulfosporosinus which are spore-forming, sulfate-reducing bacteria (Church et
al. 2007). The Proteobacteria monoenoic, Anaerobic metal reducing branched
53
monoenoic, General ubiquitous normal saturates, and Eukaryotic polyenoic lipid
structure groups, consisting of 74 % of the total population, contain spore-forming
organisms.
Figure 2.3 Mojave Microbial Community. Microbial soil community of the topsoil
in the arid region of the Mojave Desert. Phospholipid Fatty Acid (PLFA) analysis
classifies organisms based on the phospholipid composition of the cell wall. PLFA
analysis was performed by Microbial Insights, Rockford, TN.
2.4c In Situ Results
We used the portable instrument and the algorithm package developed by Estes et
al. (2003) and Lloyd et al. (2003) in an in situ field study of Mojave Desert soils. Results
from the soil dilution series lab measurements (Table 2.5) were analyzed using a least
means square method to develop a Mojave Sensitivity Algorithm. The Mojave Sensitivity
54
Algorithm yielded a positive identification with the sensitivity of 104 cells/ gram of soil
in the lab. This algorithm was employed to determine the microbial concentration in realtime in the field. In situ measurements were taken in a pinwheel pattern on the soil
surface (0-5 cm), at 30 cm and 60 cm at the center site. Shown in Figure 2.5 is the
sampling site with the center of the sampling pinwheel marked as an X.
The results in Figure 2.5 show an interesting pattern of variability. The
concentration of bacteria per gram soil varies by three orders of magnitude (from 104 to
107 per gram soil). This level of variation is unexpected in soils and may be due to selforganized biological patchiness resulting from a difference in clay content from the
nature of extreme desert environments. Rietkerk et al. (2004) pointed out that for grasses
and trees in arid environments there is a self organizing patchiness due to the positive
feedback between the presence of the organisms and the limiting resource in the
environment – water. When trees are present in an arid landscape they enhance the
habitability of the small area around them due to the retention of water by their root
systems. This in turn encourages the growth of other trees further enhancing the local
habitability compared to the ambient conditions. This positive feedback creates bistable
states and characteristic patterns in the growth of the grasses and trees.
Self organized patchiness has not been considered for microbial ecosystems, but
the same logic could apply here. In an extreme desert environment the presence of
microorganisms could enhance water retention by the soil due to the release of
extracellular polymeric substances (EPS). Thus a positive feedback would exist between
the presence of microbes and the local enhancement of habitability.
55
Figure 2.5 In Situ Field Site. Picture of the Mojave Desert in situ sampling site, east
of Silver Dry Lake Bed. The center of the sampling site, located at GPS
coordinates: N 35° 23` 13 ``, West 116° 8` 33``, is marked by X at the site and is also
depicted at the center of the clock (107). The scale bar is 25 m corresponding to the
distance from the center of the sampling site to locations sampled over a 50 m2 area.
The table lists the in-situ microbial content measured employing the Mojave
Sensitivity Algorithm as diagramed in a clock pattern. Microbial concentrations
were calibrated to relative fluorescent units. In-situ measurements were taken in a
clock wise pattern. Samples at the center (X) were taken to a depth of 60 cm. GPS
coordinates: N 35° 23` 13 ``, West 116° 8` 33``, is marked by X at the site and is also
depicted at the center.
56
As in this case of grasses and trees this positive feedback should result in self-organized
patchiness. Such patchiness would not be apparent from looking at the soil but
measurements of bacterial concentrations could give values that vary considerably over
small spatial scales.
2.5 Conclusions
We have reviewed the use of fluorescence techniques for microbial detection and
enumeration in desert soils on Earth and possibly Mars. We have used these methods to
determine the microbial content of Mojave Desert soils by a dilution series in which
known numbers of bacterial spores were added to the soil samples. We have also
conducted in situ measurements of microbial concentrations over a 50 m2 area in the
Mojave Desert to determine if there are variations in microbial concentrations consistent
with self-organized patchiness. Our results suggest the following conclusions:
1. Fluorescence of the biotic component of desert soils is approximate as strong as
the fluorescence of the mineral component of these soils.
2. The main challenge in the quantitative determination of bacterial numbers from
fluorescence is overcoming mineral fluorescence.
3. Processing algorithms developed to separate the biological fluorescence signal
from the mineral fluorescence signal can improve detection limits for soil
microorganisms by 10 to 1000.
57
4. Microbial concentration in a typical Mojave Desert soil determined from
fluorescence is 107 bacteria per gram soil. A non-culture dependent method,
PLFA, gives the same concentration of soil microorganisms.
5. Soil concentrations in the Mojave Desert determined in-situ using fluorescence
show that the number of bacteria varies from 104 to 107 per gram of soil over a 50
meter squared area. This suggests that the variability is due to self-organized
patchiness by analogy with variability in plants in desert environments.
6. Fluorescence is a practical method for detecting soil microbes in non-contact
applications in extreme environments on Earth and on future missions to Mars.
2.6 References
Alimova, A., Katz, A., Savage, H.E., Shah, M., Minko, G., Will, D.E., Rosen, R.B.,
McCormick, S.A., and Alfano, R.R. 2003. Native fluorescence and excitation
spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria
subjected to conditions of starvation. Applied Optics 42, p 19.
Church, C.D., Wilkin, R.T., Alpers, C.N., O Rye, R., and McClesky, R.B. 2007.
Microbial sulfate reduction and metal attenuation in pH 4 acid mine water.
Geochemcial Trans.8,10-24. DOI:10.1186/1467-4866-8-10
Estes, C., Duncan, A., Wade, B., Lloyd, C., Ellis, W., and Powers, L. 2003. Reagentless
Detection of Microorganisms by Intrinsic Fluorescence. Biosensors and
Bioelectronics 18, 511-/519.
58
Farr, T.G. 2004. Terrestrial analogs to Mars: The NRC community decadal report.
Planetary and Space Science 52, 3-10.
Hendrix, J.L. 1983. Porphyrinsand chlorophylls as probes for fluoroimmunoassays.
Clinical Chemistry 29 (5), 236.
Katayama, M., Matsudaa, Y., Shimokawaa, K.I., Tanabea, S., Kanekob, S., Harac, I., and
Satoc., H. 2001. Simultaneous determination of six adenyl purines in human
plasma by high-performance liquid chromatography with fluorescence
derivatization. J. Chromatography B: Biomedical Sciences and Applications. 760
(1),53-58.
Lloyd, C.R., Cleary, F.C., Kim, H.Y., Estes, C.R., Duncan, A.G., Wade, B.D., Ellis,
W.R., and Powers, L.S. 2003. Is what you eat and drink safe? Detection and
identification of microbial contamination in foods and water. Proceedings of the
IEEE 91, 908-914.
Nevin, A., Cather, S., Anglos, D., and Fotakis, C. 2007. Laser-Induced Fluorescence
Analysis of Protein-Based Binding Media. In Lasers in the Conservation of
Artworks, edited by J. Nimrichter, W. Kautek, and M. Schreiner, Springer
Proceedings in Physics, Vol. 116, pp 399-406.
Paidhungat, M., Setlow, B., Driks, A., and Setlow, P. 2000. Characterization of spores
of Bacillus subtilis which lack dipicolinic acid. J. of Bacteriology, 182 (19),147153.
Richards-Kortum R. and Sevick-Muraca, E. 1996. Quantitative optical spectroscopy for
tissue diagnosis, Annu. Rev. Phys. Chem. 47, 555-606.
59
Rietkerk, M., Dekker, S.C., de Ruiter, P.C., and van de Koppel, J. 2004. Self-organized
patchiness and catastrophic shifts in ecosystems. Science 305, 1926-1929.
Seaver,M. 1999. Size and fluorescence measurements for field detection of biological
aerosols', Aerosol Science and Technology 30 (2), 174-185.
Tung, H.C., Bramall, N.E., and Price, P.B. 2005 Microbial origin of excess methane in
glacial ice and implications for life on Mars. PNAS 102 (51), 1048-1055.
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Determination of the sedimentary microbial biomass by extractable lipid
phosphate. Oecologia 40, 51-62.
60
CHAPTER 3
DETECTING ORGANICS USING FLIGHT QUALIFIED HARDWARE
3.1 Abstract
In this chapter we propose to detect organics, polycyclic aromatic hydrocarbons,
biomolecules, and minerals on the surface of Mars. Our instrument is designed such that
almost all components are already flight qualified for Mars surface operations, drastically
reducing development costs. We present the instrument concept, report on preliminary
results, and discuss the feasibility of such an instrument as a site survey tool. The basic
design adapts the ChemCam instrument package on-board Mar Science Laboratory
(MSL) rover Curiosity to detect organics via fluorescence. By placing frequency
multipliers in front of the 1064 nm laser, wavelengths suitable for fluorescence excitation
(266 nm, 355 nm, and 532 nm), will be achieved. The emission system is modified by the
addition of band pass filters in front of the existing spectrometers, to block out the
excitation energy. Materials fluorescing in the visible (532 nm) could be detected by a
camera rather than filtered spectrograph. Preliminary results show minerals already
discovered on Mars, such as perchlorate, fluoresce highest when excited by 355 nm.
Also we demonstrate that the polycyclic aromatic hydrocarbons, such as those present in
Martian Meteorites, are highly fluorescent at wavelengths in the ultra violet (266 nm, 355
nm), but not as much in the visible (532 nm). We conclude the instrument described in
this paper is suitable to detect organics, biomolecules, and some minerals via
fluorescence offering a high scientific return for minimal cost.
61
3.2 Introduction
The Viking Label Release (LR) and Gas exchange (GX) experiments, searching
for organics and life, indicated the Martian soil was reactive. However, the Viking Gas
Chromatograph / Mass Spectrometer (GC/MS), with a sensitivity of parts per billion
(ppb), found no organics (Kline, 1977). This led to the thought that the LR and GX
experimental results were due to chemical oxidants (Kline,1977). However the discovery
of perchlorate at concentrations of 1 % by wt. at the Phoenix landing site (Hecht et al.,
2009) implies a reassessment of the Viking results. The Viking GC/MS results were
based on pyrolysis of soil to 350oC to 500oC followed by analysis. At these temperatures
the perchlorate would be reactive and destroy any organics at a level of part per thousand
(ppt). Therefore the upper limit of organics on Mars is not the ppb limit, that has been
considered for many years, but probably at ppt. Perchlorate is not toxic to all organisms,
some organisms consume perchlorate using it as an oxidizing source (Tipton et al. 2003);
only when it is heated is it highly reactive. With the recent Phoenix results, and the
consideration that microorganisms may be present within the Mars polar ice cap, as
suggested by Smith and McKay (2005), it is likely that future missions to Mars will
renew the search for organics and microorganisms.
A practical difficulty with organic and biological analysis on Mars is getting to
and collecting the sample. Rovers remotely operated from Earth take many days to drive
to and to collect a sample. For this reason there is considerable interest in selection of
target rocks remotely at a distance of several meters. Detection of biomolecular
fluorescence at a distance of a few meters (Storrie-Lombardi et al. 2008) enables the
62
remote detection of organics and biomolecules of a target a few meters away.
Furthermore, non-contact analytical methods do not require reagents and can therefore
analyze an unlimited number of samples providing a useful screening for more detailed
reagent limited assays.
Fluorescence is a phenomenon characteristic of many substances and may be used
to identify minerals, organic matter, and biological material. Fluorescence occurs when a
photon of the appropriate wavelength reaches the surface, exciting the molecule, and
raising the energy state of an electron. As the energy dissipates the electron cascades
down to lower energy states emitting photons. In general the emission wavelength is 50
to 100 nm longer than the excitation wavelength. The emission intensity is related to the
amount of the specific mineral, organic, or biological material present. Fluorescence is a
non-contact method of detecting and identifying fluorescent materials remotely (several
meters) and is considered here as a method for detecting organics, minerals, and
biomolecules on the surface of Mars.
Organics, particularly polycyclic aromatic hydrocarbons (PAHs) fluoresce (see
Table 3.1). Polycyclic aromatic hydrocarbons have been found in Martian Meteorites,
and are the primary constituent of organic matter found in all Meteorites classes (McKay
et al., 1996). Amino acids have also been detected in the Interstellar Medium (ISM)
(Kuan et al., 2003). Hence, PAHs and amino acids are plausible candidates for organics
on Mars. Storrie-Lombardi et al. (2008) detected fluorescence, using a camera and filter
wheel, of the three- ring-PAH anthracence, the four ring pyrene, and the five-ring
perylene when excited at 365 nm. The fluorescence characteristics of several PAH’s are
63
listed in Table 3.1. Also listed are the fluorescent characteristics of the aromatic amino
acids, phenylalanine, tyrosine, and tryptophan. Although likely less abundant on Mars
than organics, other biomolecules can be detected and identified using fluorescence as
shown for ATP and NADH in Table 3.1. For a more detailed list of fluorescence of
common biomolecules and detection of biomolecules by fluorescence refer to Smith et al.
(2009b).
Table 3.1 Organic Excitation and Emission Wavelengths. Common organics
(polycyclic aromatic hydrocarbons, PAH) of extraterrestrial origin (meteorites,
interstellar medium (ISM)), and potiential biomolecules that could be on the surface
of Mars, excitation and emission wavelength peaks, and the reference.
Organics
Benzopyrene
Chrysene
Napthalene
Phenanthrene
Phenanthrene
Phenylanine
Pyrene
Pyrene
Perylene
Tryptophan
Tyrosine
Biomolecules
ATP
NADH
Description
PAH in Mars meteorites
PAH in Mars meteorites
PAH in Mars meteorites
PAH in Mars meteorites
amino acid in ISM
PAH in Mars meteorites
PAH in Mars meteorites
amino acid in ISM
amino acid in ISM
ubiquitous cellular
metabolite-Earth
ubiquitous cellular
metabolite-Earth
Excitation
λ
383 nm
307 nm
275 nm
280 nm
306 nm
260 nm
347 nm
342 nm
440 nm
280 nm
275 nm
Emission λ
435 nm
370 nm
344 nm
410 nm
361 nm
282 nm
387 nm
376, 396 nm
472 nm
353 nm
304 nm
Reference
Kuijt et al., 2001
Kuijt et al., 2001
Kuijt et al,. 2001
Pujari et al., 2002
Kuijt et al., 2001
Seaver et al., 1999
Kuijt et al., 2001
Wilson et al., 2007
Wilson et al., 2007
Alimova et al., 2003
Nevin et al., 2007
280 nm
420 nm
Katayama et al,. 2001
350 nm
460 nm
Richards-Kortum, 1996
64
In addition to detecting organics and biomolecules, fluorescence can also be used
as a tool for mineralogical identification to aid in target selection. Many minerals have
unique fluorescence, for example, the mineral fluorite derives its name after this
characteristic property. Mineral and rock species identified on the surface of Mars include
hematite, jarosite, olivine, phylosilicates, carbonates, perchlorate (ClO-4), basalts, and ice
among others (Squyres et al., 2004, Smith et al., 2009a). The current non-contact remote detection method on-board Mars Science
Laboratory (MSL), ChemCam employing Laser-Induced Breakdown Spectrometer
(LIBS), can only do elemental chemical determination (Weins et al, 2009). ChemCam
consists of two instruments 1) a remote micro-imager (RMI) capable of mm resolution
from meters away and 2) a laser-induced breakdown spectrograph (LIBS) capable of
determining elemental abundance as low as 10 ppm (Weins et al., 2009). The ChemCam
instrument sits on the Curiosity rover mast 1.8 meters above the ground allowing for
remote analysis at distances from 1.8 meters to 13 meters. The ChemCam
instrumentation has achieved several technical breakthroughs including the first flight
qualified laser. All of the ChemCam hardware, including the excitation and emission
systems, have achieved flight qualification, and will be at Technical Readiness Level
(TRL) 9 once MSL surface operations commence. The next generation remote detection
methods use LIBS combined with Raman Spectroscopy. While these methods have a
high fidelity for identification, the sensitivity is too low to identify organics and
biomolecules at the Mars ppt predicted concentrations (Sharma et al., 2007, Courreges-
65
Lacoste et al., 2007). In this paper we present an alternative design to satisfy the need of
remote detection of biological and mineralogical assays using fluorescence.
The LIBS that is part of ChemCam could be used as an excellent high sensitive
mineral, organic, and biomolecular survey instrument with minor modifications. Laser
induced breakdown spectroscopy operates by heating/energizing the target surface
producing a characteristic plasma. The plasma emission spectrum is specific and unique
for each molecule so that it could identify minerals. ChemCam uses a 5 ns pulsed 1064
nm laser with a 15 hz firing rate drawing 30 mJ per pulse to excite electrons producing
plasma. To capture the emission spectrum of the plasma, ChemCam has a Schmidt
telescope to increase the light gathering power, and hence the emission spectra signal,
before feeding the signal into three spectrometers. The spectrometers are customized
versions of off-the-shelf Ocean Optics HR2000 spectrometers, each designed to measure
specific wavelengths of the emission spectra (240-336 nm, 380-470 nm, 500-800 nm
respectively), integrating the signal over 75 laser pulses. The combined spectrometer
detection ranges from ultra-violet (UV) 240 nm to red 800 nm (Weins et al., 2009).
3.3 Materials and Methods
The primary component of the ChemCam instrument that enables fluorescence
assessment is the 1064 nm laser. This wavelength (1064 nm) is too long / low –energy to
excite electrons and induce fluorescence. However with the aid of a frequency multiplier,
the frequency can be multiplied which in turn reduces the wavelength, and increases the
energy therefore allowing fluorescence. The frequency multiplier allows for
multiplication at three harmonic wavelengths, double, triple, and quadruple of the
66
original 1064 nm wavelength. This results in the possibility of three excitation
wavelengths, 266 nm, 355 nm and 532 nm. Biological material, organics, and minerals
fluoresce when excited at 266 nm and 355 nm. The only modification of the ChemCam
excitation system is the addition of three interchangeable frequency multipliers. The
flight-qualified mechanical drive train and control box used for the camera filter wheel on
board the Beagle 2 Lander could rotate the frequency multipliers into position. Figure 3.1
is a system level block diagram of the new instrument.
Figure 3.1 Instrument System Block Diagram. System level block diagram of the
modified ChemCam instrument. Frequency multipliers are added to the excitation
system for optimization of organic, mineral, and biomolecular fluorescence
detection. Diagram adapted from Weins et al. 2009.
The emission collection system has one main modification, the placement of a
band pass notch filter in front of each of the three spectrometers. These filters will block
67
the excitation wavelengths allowing the natural fluorescence to pass through to the
spectrograph. The emission collection system (Figure 3.2) will employ the Schmidt
telescope, the original three ChemCam spectrographs, and the addition of three notch
narrow band pass filters (266 nm, 355 nm, and 532 nm). The only non-flight qualified
hardware of the entire fluorescence instrument design are the frequency multipliers and
the non-moving band pass notch filters, every other system component has been flight
qualified.
Figure 3.2. Emission System Block Diagram. Emission Collection System enabling
organics, minerals, and biomolecular detection by specific wavelength fluorescence
emission showing the appropriate filter blocks.
68
To ascertain fluorescence response at the constrained excitation wavelengths (266
nm, 355 nm, and 532 nm), we measured the emission spectra for five PAHs (pyrene,
phenanthrene, naphthalene, naphthol, and cresol) individually and when combined with a
mineral in a 50/50 mixture by volume. Powdered dolomite was used as the mineral since
it is a magnesium carbonate formed under aqueous conditions and possibly on the
Martian surface (Squyres et al. 2004). Even though the primary task of the fluorescence
instrument is to identify organics, PAHs in particular, the identification of minerals,
especially water-associated minerals, would enhance the scientific return without adding
to the cost. To determine the feasibility of identifying minerals using the fluorescence
instrument, emission spectra of several rock minerals known to be on the surface of Mars
was captured for each of the three excitation wavelengths proposed for this instrument.
Additionally emission spectra for rock minerals found at Mars Analog environments on
Earth and potentially on Mars were measured.
The polycyclic aromatic hydrocarbons, (pyrene, phenanthrene, naphthalene,
cresol, and naphthol), powdered dolomite, and Mg perchlorate were from Sigma Aldrich
(St. Louis, MO). The rock mineral specimens (except for jarosite) were from the Utah
State University Geology Department mineralogy and igneous petrology collection.
Jarosite was collected from Panoche Valley in California by the research team.
All of the data presented in this paper were taken with a Shimadzu 1501
Fluorometer. A custom angled cuvet for powdered mixtures and a sample holder able to
hold rocks was positioned in the fluorometer to obtain optimal emission spectra. Each
specimen was excited by the fluorometer xenon flash lamp, at 266 nm, 355 nm, and 532
69
nm and the emission spectra scanned from 220-900 nm, 350-900 nm, and 530-900 nm
respectively.
3.4 Results
Fluorescence and quenching results for three polyaromatic hydrocarbons likely to
be on Mars, pyrene, phenanthrene, and naphthalene, are shown in Figure 3.3 and
described in Table 3.2. The emission spectra for the individual PAH is compared with the
50/50 by volume dolomite and PAH mixture and the spectrum for powdered dolomite.
The fluorescence from pyrene when excited at stimulated by 266 nm pyrene is too high
for instrument readings. The powdered dolomite and the 50/50 mixture have an emission
peak at 340 nn. The pyrene did not enhance the dolomite signal, but the dolomite
quenched the pyrene emission. The dolomite peak from 498 to 532 nm, and the peak
from 770 to 810 nm are likely due to frequency resonance (2λ, 3λ, respectively) rather
than fluorescence. With 355 nm excitation the readings are railed until 590 nm due to the
lower energy exerted by the laser. The 440 nm emission from the 50/ 50 mixture both
enhances the dolomite peak and quenches the pyrene emission. The 710 nm emission
peak (2λ) should be regarded as an artifact of the spectrometer grating instead of
fluorescence. As the excitation energy decreases, so does the variation in emission
spectra. At 532 nm excitation, pyrene has a small emission peak at 615 nm and the
dolomite 1.5 λ (790 nm) emission is quenched compared to the excitation intensity,
pyrene, and the 50/50 mixture.
The three-ringed phenanthrene displays distinguishing emission spectra at each
excitation wavelength. Only phenanthrene has an emission peak at 861 nm (266 nm ex)
70
and quenches the emitted energy from 291 to 351 m. At 355 nm excitation the 50/50
mixture and phenanthrene both have an emission peak at 818 nm. When phenanthrene is
excited at 532 nm, the 818 nm peak seen at 355 ex disappears and the 1.5 λ (790 nm)
emission peak is quenched. Naphthalene also has an identifiable emission spectrum as
pronounced by the 683 nm emission peak at 532 nm excitation. At 266 nm excitation,
naphthalene and phenanthrene both quench the signal around 291 nm and 2 λ, but the
quenching band is narrower for naphthalene in both cases. Naphthalene and phenanthrene
have similar spectrum profile at 355 nm excitation, the higher peak intensity at 818 nm
distinguishes phenanthrene from naphthalene.
The Martian surface contains superoxides (Kline, 1977) because of this, oxidized
polycyclic aromatic hydrocarbons (naphthol and cresol) were measured and compared to
species without the addition of a hydroxyl. As shown in Figure 3.4, naphthol, powdered
dolomite, and the 50/50 mixture have relatively similar emission spectra. Some
differences to note are the relative fluorescent units at 266 nm excitation and the naphthol
emission peak at 355 nm excitation. Cresol on the other hand, has a distinct emission
spectrum for each excitation wavelength. The low intensity of the cresol spectrum at 266
nm excitation suggests quenching. At 355 nm cresol has an enhanced fluorescence
followed by quenching of the 2λ emission anomaly. At 532 nm the 50/50 mixture
quenches the dolomite emission seen at the 1.5 λ peak. The hydroxyl radical alters the
emission spectrum of the PAHs. With the exception of Cresol at 355 excitation, the
hydroxyl radical aids in quenching.
71
Figure 3.3 Polycyclic Aromatic Hydrocarbons Emission Spectra. Each graph is the
emission spectra of individual polycyclic aromatic hydrocarbons, powdered
dolomite, and a 50/50 by volume mixture of dolomite and the PAH when excited at
the proposed excitation wavelengths (266 nm, 355 nm, and 532 nm). The molecular
structure and formula are shown on the 532 nm emission graph for each molecule.
72
Figure 3.4. Hydroxy Emission Spectra. Emission spectra of polyaromatic
hydrocarbons, powdered dolomite, and a 50/50 mix for each molecule when excited
at the proposed excitation wavelengths (266 nm, 355 nm, and 532 nm). We also
compare Naphthlene and Naphthol to distinguish the effect of a hydroxy on the
emission spectra. The molecular structure and formula is shown on the 532 nm
emission graph for each molecule.
73
Table 3.2 Summary of Distinguishing Spectrum. Summary of the distinguishing
spectrum characteristics for each of the polycyclic aromatic hydrocarbons shown
above as measured for this study using a Shimadzu 1501 Fluorometer.
PAH
266 nm Ex Spectrum Comparison
355 nm Ex Spectrum Comparison
532 nm Ex Spectrum Comparison
Fluorescence(nm)
Fluorescence(nm)
Fluorescence(nm) Quenching(nm)
Quenching(nm)
Pyrene
260 to 650
Phenanthrene
352 to 500, 861
291 to 351, 501
377 to 507, 818
Naphthalene
315 to 557,
291 to 314
355 to 506, 766, 818
640 to 740
558 to 640
321 to 400
291 to 320
Naphthol
Cresol
380
Quenching(nm)
355 to 590
532 to 590
376
550 to 650
683
410
355 to 450, 770
790
710
790
We measured minerals likely and known to be on the surface of Mars. The
ability of remote mineral identification would improve the scientific merit of the
instrument. Also, as reported by Smith et al. (unpublished data) the detection of
biomolecular fluorescence was influenced by mineral fluorescence. The data set in
Figure 3.5 shows the emission spectra of carbonates (calcite, limestone, travertine,
rhodochrosite, siderite, and dolomite) and some water associated minerals (perchlorate,
hematite (red and black), serpentine, and jarosite), known to be on the surface of Mars,
when excited at 266 nm, 355 nm, and 532 nm. The portion of the spectra graphed is from
the tail end of the excitation peak to the beginning of the 2 λ (twice the wavelength) peak.
Fluorescence beyond the 2 λ peak is unexpected due to the large Stokes shift and lower
energy output. At 266 nm excitation rhodochrosite, siderite, hematite, and perchlorate
have distinct emission peaks. At 355 nm excitation, most minerals have a small
74
fluorescence peak between 400 and 410 nm with the exception of perchlorate which
fluoresces about fifty times brighter at 538 nm and 589 nm. At 532 nm the peaks are
likely due to scattering of light or a grating effect at 1.5 λ rather than fluorescence, except
for the small emission peak at 726 nm from perchlorate.
Figure 3.5 Mineral Emission Spectra. Emission Spectra for a variety of carbonates
and water-associated minerals, emission peaks are labeled with the corresponding
emission wavelength. The spectrum on the left side of the graph is the tail end of the
excitation peak. The rising spectrum on the right side of the graph is the beginning
of the 2 λ peak. The 2 λ emission peak is an artifact of the grating within the
Shimadzu Fluorometer and is not likely due to mineral fluorescence. Furthermore a
1.5 λ emission peak can be seen for Mg perchlorate and the carbonates. This also is
likely due to the instrument conditions rather than mineral fluorescence.
75
3.5 Discussion
The survey of mineral and PAH fluorescence performed in this study illustrates
that the 1064 nm laser available in the flight ready ChemCam instrument modified with
the addition of the three frequency multipliers (266 nm, 355 nm, 532 nm) and band pass
filters would provide an excellent site survey tool in near real time based on fluorescence
measurements. This instrument should obtain readings from minerals, PAHs, and
biomolecules. Table 3.3 lists the material identifiable at the specified wavelengths.
Table 3.3 Detectable Material at 266 nm, 355 nm, and 532 nm. Biomolecules,
organics, and minerals detectable by fluorescence, due to the unique emission
spectra, when excited at the corresponding wavelength.
266 nm Excitation
355 nm Excitation
532 nm Excitation
Biomolecules: ATP,
Phenylalanine, Tyrosine,
Biomolecules: Dipicolinic acid,
NADH,
Organics: Naphthalene
Organics: Pyrene,
Phenanthrene, Naphthalene
Organics: Phenanthrene, Naphthol,
Cresol,
Minerals: Mg Perchlorate,
Hematite, Calcite, Siderite
Minerals: Mg Perchlorate, Siderite,
Rhodochrosite, Hematite
Minerals: Jarosite,
Rhodochrosite, Dolomite,
Measurements using the fluorescence instrument should be taken during the Mars
night to avoid complications with ambient light interference. With the operational
flexibility of remote measurements, an outcrop could be surveyed a few meters away
76
before risking the drive. This greatly reduces the chance of mechanical failure from the
rover getting stuck and expands the range of the target selection area. Future research
will focus on expansion of the target database to include meteorites and tektites and on
taking fluorescent measurements under conditions that are similar to those anticipated on
Mars to determine the distances at which fluorescence detection is feasible.
3.6 Conclusions
1) Fluorescence can be used to detect organics (PAH), and minerals, adding to its
use in detecting specific biomolecules.
2) The instrument proposed is comprised almost entirely of flight-qualified Mars
surface operational hardware reducing the risk and cost.
3) Feasibility of the instrument design offers a high scientific return (detection of
organics) while expending minimal resources (time, development costs).
4) This instrument should be considered for the next mission to Mars as a site
survey tool.
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79
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McKay, C.P. 2009b. In situ microbial detection in the Mojave Desert using
native fluorescence. Unpublished manuscript.
Storrie-Lombardi, M.C., Muller, J.P., Fisk, M.R., Griffiths, and Coates, A.J., 2008.
Potiential for non-destructive astrochemistry using the ExoMars PanCam.
Geophysical Res. Letters 35, L12201.
Squyres, S.W., Grotzinger, J.P., Arvidson, R.E., Bell, J.F., Calvin, W., Christensen, P.R.,
Clark, B.C., Crisp, J.A., Farrand, W.H., Herkenhoff, K.E., Johnson, J.R.,
Klingelhofer, G., Knoll, A.H., McLennan, S.M., McSween, H.Y., Morris, R.V.,
Rice, J.W., Rieder, R., and Soderblom, L.A. 2004. In situ evidence for an ancient
aqueous environment at Meridiani Planum, Mars. Science 306, 1709-1714.
Tipton, D.K., Rolston, D.E., and Scow, K.M. 2003. Transport and biodegradation of
perchlorate in soils. J. Environ. Qual. 32, 40-46.
Weins, R.C., Clegg, S., Bender, S., Lanza, N., Barraclough, B., Perez, R., Maurice, S.,
Dyar, M.D., Newsom, H., and the ChemCam Team. 2009. Initial calibration of
the ChemCam LIBS instrument for the Mars Science Laboratory(MSL) rover.
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40th Lunar and Planetary Science Conference, 13-20 March, League City, Texas,
USA.
Wilson, J.N., Gao, J., and Koo, E.T. 2007. Oligodeoxyfluorosides: Strong sequence
dependence of fluorescence emission. Tetrahedron. 63(17), 3427–3433.
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CHAPTER 4
RESEARCH SUMMARY AND FUTURE DIRECTION
For this research project we designed an instrument to detect bacteria via
biomolecular fluorescence. First we introduced the current understanding of
astrobiology, our knowledge of life beyond Earth, and the commonality of Earth life as it
pertains to the search for life on Mars. We proposed a novel technique for searching for
direct evidence of life on the surface of Mars. Our research concept was validated in the
lab and tested in situ at a Mars analog environment, The Mojave Desert. We concluded
that fluorescence of the biotic component of the desert soils is approximate as strong as
the fluorescence of the mineral component of these soils by measuring unmodified soil
and soil that had been baked to remove the organics. The main challenge in the
quantitative determination of bacterial numbers from fluorescence is overcoming the high
mineral fluorescence. Algorithms developed to separate the biological fluorescence signal
from the mineral fluorescence signal can improve detection limits for soil
microorganisms by 10 to 1000. Microbial concentration in a typical Mojave Desert soil
determined from fluorescence is 107 bacteria per gram soil. A standard, non-culture
dependent method gives the same concentration of soil microorganisms. Soil
concentrations in the Mojave Desert determined in situ using fluorescence show that the
number of bacteria varies from 104 to 107 per gram of soil over a 50 meter area.
Fluorescence is a practical method for detecting soil microbes in non-contact applications
in extreme environments on Earth and on future missions to Mars. While the concept was
validated the instrument sensitivity of 104 cells/ g of soil could be improved with the
82
addition of a higher energy source, below 300 nm, to stimulate a wider selection of
biomolecules and by additional algorithm development employing neural network
techniques.
After a successful proof of concept, we designed a fluorescence instrument based
on flight qualified components to increase the chance of mission selection due to minimal
development cost and low risk. In addition to biomolecular fluorescence, we also
examined the feasibility of detecting polycyclic aromatic hydrocarbons and known mars
minerals using the fluorescence instrument. Table 4.1 summarizes the detectable matter
by excitation wavelength. Emission spectra for minerals not presented in the above paper
can be found in the Appendix.
Table 4.1 Summary of Detectable Material at 266 nm, 355 nm, and 532 nm.
Biomolecules, organics, and minerals detectable by fluorescence, due to the unique
emission spectra, when excited at the corresponding wavelength.
266 nm Excitation 355 nm Excitation 532 nm Excitation ATP, Tryptophan, Tyrosine, Dipicolinic acid, NADH, Naphthalene, Pyrene, Phenanthrene, Phenanthrene, Naphthol, Jarosite, Rhodochrosite, Naphthalene , Cresol, Dolomite, Sulfur, Jarosite, Mg Perchlorate, Hematite, Mg Perchlorate, Siderite, Gypsum, Calcite, Siderite, Andesite, Rhodochrosite, Hematite, Calcite, Travertine Dacite, Sulfur, Ilmenite, Calcite Ilmenite, Calcite, Travertine 83
The suite of biomolecules, minerals, and PAH fluorescence performed in this
study illustrates that the 1064 nm laser available in the flight ready ChemCam instrument
modified with the addition of the three frequency multipliers (266 nm, 355 nm, 532 nm)
and band pass filters would provide an excellent site survey tool in near real time using
fluorescence. This instrument should enable readings from minerals, PAHs, and
biomolecules as listed in Table 4.1. The instrument design offers a high scientific return
(detection of organics, minerals, biomolecules) while expending minimal resources (time,
development costs).
Future research will focus on taking fluorescent measurements similar to Mars
operational conditions to determine detection limits at select distances. Expansion of the
target database to meteorites and additional Mars analog environments would increase
the scientific merit of fluorescence detection of biomolecules, organics, and minerals on
the Martian surface.
84
APPENDICES
85
Fluorescence measurements were taken on a suite of minerals in addition to those
reported in the above papers. Table A.1 lists the minerals by name, the mineral class, and
the rock color. Figures A.1 to A.5 compare some of these minerals and their fluorescence
properties.
Table A.1 Fluorometer Mineral List. Rocks analyzed in the fluorometer excited at
266 nm, 355 nm, and 532 nm.
Mineral Class
Rock Color Albite Plagioclase(Na)
White
Andesite Ingneous (52‐63%)
Black and gray Apatite Phosphate (Ca)
Green
Augite Pyroxene(Ca‐Na)
Black and green Basalt Ingneous (<52%)
Black
Biotite Phylosilicate (K)
Black mica Calcite Carbonate (Ca)
Clear
Dacite Ingneous (>63%)
Gray and white Dolomite (Crystal) Carbonate+ Mg
White crystals Dolomite (Green)
Carbonate +Mg
Green planar Dolomite (White)
Carbonate+ Mg
White planar Enstatite Silicate (Mg)
Green
Fluorite Halide (CaF)
Clear
Garnet Silicate Deep Red Gypsum (Clear) Halide/Sulfate
Clear
Halite Halide (NaCl)
Clear
86
Hematite (black) Oxide (FeO3)
Black
Hematite (red) Oxide (FeO3)
Red more Fe Hornblend Amphibole/ silicate
Black
Imenite Oxide (FeTiO3)
Black
Jasper Silicate
Red Jarosite Sulfate (KFe3(SO4)2(OH)6
Yellow, brown, red Limestone Carbonate (Ca)
White
Magnetite Oxide (FeO4)
Black
Microcline Feldspar (K)
White
Muscovite Phylosilicate (K, Al)
Clear Mica Olivine Silicate(Fe)
Green
Perchlorates Oxide (XClO4)
White grains Phlogopite Phylosilicate (K Mg)
Brown mica Pyrolusite Oxide (MnO2)
Black branching pattern Quartz(clear) Silicate
Clear
Quartz (smoky) Silicate(K, Mg,Fe)
Gray Rhodochrosite Carbonate (Mn)
Pink
Serpentine Phylosilicate (Mg)
Green, reddish intrusions
Siderite Carbonate (Fe)
Brown
Sulfur Sulfate (S)
Bright Yellow Titanite (Spene) Silicate(CaTiO)SiO4)
Black
Travertine Carbonate (Ca)
Milky white to brown 87
Figure A.1 Varied Silica Content Emission Spectra. Igneous Rocks classified by
silica content. Basalt has the lowest silica content, while Dacite has the highest. The
middle silica content (Andesite) has the highest fluorescence.
88
Figure A.2 Sulfates Emission Spectra. Elemental sulfur compared with sulfate
compounds.
89
Figure A.3 Oxides Emission Spectra. Ilmenite an TiO abundant at impact sites and
on the Moon, compared with Pyrolusite a mineral that branches similar to trees,
and Magnetite a known Mars mineral.
90
Figure A.4 Same Composition Emission Spectra. Fluorescence from three minerals
with the same composition, but formed under different conditions.
91
92
Curriculum Vitae
Heather D. Smith
Education:
Ph.D, Biological Engineering, Utah State University, Logan, Utah (anticipated 2010)
“An Instrument Design for non-contact, in-situ detection of microbes in a Mars
Analog Soil”
M.Sc, Applied Environmental Geoscience, Utah State University (anticipated 2010)
“Structure and stratigraphic controls on cave systems at Tony Grove Lake Area,
Utah”
M.Sc, Space Studies, International Space University, Strasbourg, France (July 2004)
“Analysis of the flight check outs for Huygens Surface Science Package on-board
Cassini”
B.A. Physics, The Evergreen State College, Olympia, Washington (June 2001)
B.A. Psychology, Physics Minor, University of North Texas Denton, Texas (August
2000)
Relevant Career Experience:
Graduate Research Associate, NASA GSRP funded, Utah State University (Jan. 2008Current)
Design of a non-contact instrument for detection of microbes in soil in the Mojave Desert
Collected soil for testing and calibrating the instrument from local field research sites
including volcanic areas in Southern Utah, Craters of the Moon NM, Idaho, and the
Moab Desert
Research Associate, National Center for the Design of Molecular Function (Jan. 2005Dec. 07)
Research project studying soil microbiology in Mars analog environments such as
Mojave, Atacama, and Moab Deserts
Developed and Tested algorithms for native fluorescent instrument for use in soil
microbe detection.
Research Assistant, SETI Institute/ NASA ARC (Sept.2001-Dec. 2004)
Kamchatka Russian Expedition worked on ice radiation and contamination studies,
September 2004
Mars Drilling Project Science Team worked on in situ contamination studies,
Eureka, Canada, May 2003
Atacama Desert Field Season Project Manager and field assistant for soil studies
Oct. 2002 & April 2003
Mars Scout Mission Proposal Team (non-selected mission), August 2002
Deployment and maintenance of Meteorological data loggers (Campbell
Scientific and Onset)
Collection and Analysis of Hypoliths & Endoliths, U. S., Chile, and Peru
U.S. Space Camp Counselor/Mission Director (June 2000-January 2002)
Operated and trained a crew of 12 for a simulated S.T.S. Mission.
Instructed a team of 4th to 7th graders on the history and future of manned
space flight, including briefings, simulators, rocketry, and team building skills
Astronomy Lab Assistant, University of North Texas (Nov.1997 - May 2000)
Administrative Assistant to Lab Coordinator
Trained new staff.
Instructed students in celestial navigation skills.
Taught students how to operate a telescope.
Publications:
Smith, H.D., Powers, L.S., Duncan, A.G., Neary, P.L., Anderson, A.J., Sims,
R.C., and McKay, C.P. 2009. In Situ Microbial Detection in the Mojave Desert
Using Native Fluorescence. Astrobiology, In review.
Smith, H.D., McKay, C.P., Duncan, A.G, Sims, R.C., Anderson, A.J., and Grossl,
P.R. 2010. Detecting Organics Using Flight Qualified Hardware. Journal of
Biological Engineering, In review.
Smith, H.D., Sims, R.C., McKay. C.P. Anderson, A.J., Grossl, P.R. and Duncan,
A.G., 2010. Mineral Fluorescence of Mars Analog Minerals, In progress.
Smith, H.D., Liddell, W.D., Shervais, J.W., Lowry, A.R., Dickey, L.A., Barr,
K.A., and Zoric, M.A. Structural and Stratigraphic Controls on Cave Systems at
Tony Grove Lake Area, Utah. Journal of Cave and Karst Studies, In progress.
Smith, H.D., Shervais, J.W., Winters, G. A., Liddell, W.D., and Lowry, A.R.,
Geochemistry of Tektites from the Indo-China Strewn Field, In progress.
Smith, H.D., and McKay, C.P. 2005. Drilling in Ancient Permafrost on Mars for
Evidence of a Second Genesis of Life. Planetary and Space Science 53(12), 13021308.
McKay, C.P., and Smith H.D., 2005. Possibilities for Methanogenic Life in Liquid
Methane on the Surface of Titan. Icarus, 178(1), 274-276.
DOI:101016/j.icarus.2005.05.018
Scholarships and Awards:
NASA International Year of Astronomy, Astronomy Student Ambassador for the State
of Utah, January 2009 to January 2010
NASA Graduate Student Research Program Participant, September 2006 to December
2009
Mars Desert Research Station (MDRS) Spaceward Bound Crew 54_ Commander and
MDRS Crew 10_ Biologist. Hanksville, Utah, December 2006 to January 2007 and
December 2002 to January 2003
NASA Planetary Science Summer School Participant. Mars Mission Comparison (MER,
MSL, ExoMars, and AFL), Jet Propulsion Laboratory, July 24 to July 28 2006
Mars Program office student travel grant for Abscicon (Astrobiology Science
Conference) 2004.
International Space University 1 of 3 out of 50 applicants awarded a fully funded
scholarship for the Masters of Space Studies Program, July 2003