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THE ANATOMICAL RECORD 268:180 185 (2002)

Chemical Markers for Bacteria in


Extraterrestrial Samples
ALVIN FOX*
Department of Microbiology and Immunology, School of Medicine, University of
South Carolina, Columbia, South Carolina

ABSTRACT
Interplanetary missions to collect pristine Martian surface samples for
analysis of organic molecules, and to search for evidence of life, are in the
planning phases. The only extraterrestrial samples currently on Earth are
lunar dust and rocks, brought back by the Apollo (U.S.) and Luna (Soviet
Union) missions to the moon, and meteorites. Meteorites are contaminated
when they pass through the Earths atmosphere, and during environmental
exposure on Earth. Lunar fines have been stored on Earth for over 30 years
under conditions designed to avoid chemical but not microbiological contamination. It has been extremely difficult to draw firm conclusions about
the origin of chemicals (including amino acids) in extraterrestrial samples.
Of particular concern has been the possibility of bacterial contamination.
Recent work using state-of-the-art gas chromatography tandem mass spectrometry (GC-MS/MS) has dramatically lowered the chemical background,
allowing a clear demonstration that lunar fines are remarkably different
from terrestrial dust in that they generally lack certain chemical markers
(muramic acid and 3-hydroxy fatty acids) characteristic of Earths bacteria.
Thus, lunar dust might be used as a negative control, in conjunction with
GC-MS/MS analyses, in future analytical studies of lunar dust and meteorites. Such analyses may also be important in studies designed to search
for the presence of life on Mars. Anat Rec 268:180 185, 2002.

2002 Wiley-Liss, Inc.

Key words: bacteria; mass spectrometry; exobiology

There is a long history of analyses of meteorites and


lunar dust for their organic chemical content. A major
focus of these studies has been to determine whether these
chemicals were innately derived from the samples or were
derived from biological or chemical contamination. A secondary goal has been to determine whether the compounds, if indeed they are innate to the extraterrestrial
samples, are biotic or abiotic in origin. The purpose of the
current review is to summarize where we are and what
needs to be done, from the perspective of a bacteriologist
experienced in trace analysis of chemical markers for bacteria. This perspective is rather unusual, since previous
work has been almost entirely performed by geochemists
and analytical chemists.

EXTRATERRESTRIAL SAMPLES
AVAILABLE FOR ANALYSIS
The only extraterrestrial samples currently on Earth
are lunar dust and meteorites. The lunar samples were
brought back by the Apollo (U.S.) and Luna (Soviet Union)
missions to the moon (Heiken et al., 1991). Many meteor

2002 WILEY-LISS, INC.

ites have landed here naturally. Unfortunately, they were


invariably contaminated as they passed through the
Earths atmosphere, and during their long residence here.
Viable bacteria were not found in lunar material (collected
on the Apollo missions) when they were tested by classical
microbiological culture (Oyama et al., 1970). However,
until recently it was unclear whether they remained pristine during their storage on Earth (Kozar et al., 2001).
The claim made by McKay et al. (1996) that they identified ancient extraterrestrial life in carbonate formations
in the Martian meteorite ALH84001, believed to have

*Correspondence to: Alvin Fox, Department of Microbiology


and Immunology, School of Medicine, University of South Carolina, Columbia, SC 29208. Fax: (803) 733-3275.
E-mail: afox@med.sc.edu
Received 5 December 2001; Accepted 21 February 2002
DOI 10.1002/ar.10152
Published online 00 Month 2002 in Wiley InterScience
(www.interscience.wiley.com).

CHEMICAL MARKERS FOR BACTERIA

arrived on Earth 13,000 years ago, has had a great impact


on the field of exobiology. However, it is unclear whether
these carbonate formations are the result of past terrestrial microbiological activity or they were formed by inorganic processes (Buczynsik and Chafetz, 1991; Anders,
1996). The presence of polyaromatic hydrocarbons (PAHs)
may represent the presence of indigenous fossilized bacteria. Isoprenoids and chlorophyl (from bacterial and
plant sources) can generate such compounds by loss of
oxygen, and PAHs are common in fossil fuels (Volkman
and Maxwell, 1964; Kissin, 1993). ALH84001 could also
have been naturally contaminated with PAHs during
long-term exposure on Earth. Magnetite, which may be
derived from magnetotactic bacteria (Friedmann et al.,
2001), is also present. However, a recent review (Gibson et
al., 2001) noted that it is likely that there is terrestrial
contamination in ALH84001, and it may be necessary to
await the return of pristine samples from Mars to determine whether life ever existed there.

AMINO ACIDS AND LIFE DETECTION


Amino acids are found in meteorites, but at only slightly
lower levels than in the surrounding terrestrial terrain
(Kvenholden et al., 1970; Engel and Nagy, 1982; Bada et
al., 1983; Engel et al., 1990; Cronin and Pizzarello, 1997;
Engel and Macko, 1997; Glavin et al., 1999). Amino acids
are present at much lower levels in lunar dust. However,
lunar samples are readily distinguished from blanks by
amino acid levels, which suggests that the source of the
amino acids is not the analytical procedure (Ponnamperuma et al., 1970; Harada et al., 1971; Nagy et al., 1971;
Brinton and Bada, 1996). In any event, the presence of
detectable levels of amino acids in blanks does complicate
data interpretation.
Whether amino acids in meteorites originate from extraterrestrial/terrestrial or biotic/abiotic sources remains
controversial (Engel and Nagy, 1982; Bada et al., 1983;
McKay et al., 1996; Ehrenfreund et al., 2001). As noted
above, meteorites are invariably contaminated on passing
through the Earths atmosphere, and during their long
residence here. Thus it is difficult to rule out contamination by terrestrial life as the source of these amino acids.
Several attempts have been made to prove the uniqueness
of amino acids found in extraterrestrial samples by assaying enantiomeric excesses, by determining the presence of
nonprotein amino acids, and by stable isotope enrichment.
However, all of these findings could be explained by bacterial contamination.

Enantiomeric Excess
Meteorites contain both L- and D-amino acids, with the
former in excess. It has been suggested that amino acids
on Earth are generally derived from proteins that are of
the L- configuration. It has also been postulated that abiotic (chemical) processes should generate amino acids
with an equal ratio of D- and L-amino acids (Bada, 1997;
Cronin and Pizzarello, 1997). Thus a D-amino/L-amino
acid ratio not equal to one was suggested to be evidence of
terrestrial contamination. What was not discussed is that
there is a major source of D-amino acids in nature: bacterial cell walls. Indeed, there are extremely high concentrations in terrestrial dust; at the one part per 10,000 level
(Sonesson et al., 1988). The primary D-amino acids are
D-glutamic acid and D-alanine, which are commonly

181

found in meteorites. Determining the source of D-amino


acids in extraterrestrial samples clearly requires further
investigation.

Presence of Nonprotein Amino Acids


Some unusual amino acids (e.g., 2-amino-2,3-dimethylpentanoic acid) have been reported to be unique to meteorites (Cronin and Pizzarello, 1997). However, the distribution and concentration of 2-amino-2,3-dimethylpentanoic acid in terrestrial samples has not been exhaustively studied. Other nonprotein amino acids, e.g., ornithine and diaminopimelic acid, are widely found in bacterial peptidoglycan (PG) (Schleifer and Kandler, 1972) and
are readily detected in organic dust as a consequence of
bacterial contamination (Sonesson et al., 1988; Ueda et
al., 1989).

Stable Isotope Enrichment


Differences in stable isotope concentrations in meteorites relative to their terrestrial counterparts may be an
indicator of extraterrestrial origin (Engel and Macko,
1997). However, bacterial growth on a meteorite might
incorporate carbon, e.g., from endogenous carbonate
present in the meteorite (Tull et al., 1998). Thus the amino
acid would appear to have originated from space, but may
actually have been synthesized from simpler precursors
on Earth.
In contrast to meteorites, lunar dust samples have been
stored under strict isolation conditions (Allton et al.,
1998). Therefore, it is less likely that the amino acids
present in lunar fines represent terrestrial contamination.
However, it is still possible that contamination occurred
during transport to Earth or during curation. The spaceships used in the Apollo missions were documented to be
contaminated with bacteria (Puleo et al., 1970, 1973,
1977). Furthermore, the lunar curation facility was designed to avoid chemical but not microbiological contamination. If amino acids in lunar dust are not derived from
terrestrial sources, they must be from extraterrestrial
sources (whether biotic or abiotic).

PYROLYSIS GAS CHROMATOGRAPHY-MASS


SPECTROMETRY STUDIES
Experiments employing pyrolysis (pyr) gas chromatography-mass spectrometry (GC-MS), one of the first analytical chemical techniques used for studies of lunar dust,
have demonstrated the presence of trace levels of organic
carbon in the lunar samples (Burlingame et al., 1970).
However, it proved difficult to categorically state that
these compounds are of lunar origin as opposed to being
formed by terrestrial contamination (e.g., from microbes).
Pyr GC-MS involves heating a sample in the range of
400-1,000C in the absence of oxygen. Complex organic
monomers, oligomers, and polymers are converted into
simpler molecules (e.g., CO and CH4 ) that are separated
by gas chromatography and structurally identified by
mass spectrometry (Nagy et al., 1971). Unfortunately, pyr
results in a complex series of reactions, including scission,
rearrangement, and dehydration of chemical bonds. Thus
it is often difficult to relate the structure of pyr products to
the parent molecules present in bacteria and other life
forms (Simmonds, 1970; Eudy et al., 1985; Smith et al.,
1987; Watt et al., 1991; Knabner-Ko gel, 2000).
It has proven tremendously difficult to perform pyrGC-MS studies on the surface of Mars (Biemann et al.,

182

FOX

1977). Indeed, the initial results were widely interpreted


as indicating the absence of organic carbon or life. However, a recent reexamination of these data suggests that
bacteria could have been present and missed (Glavin et
al., 2001).

CHEMICAL MARKERS FOR BACTERIA


Standard culture techniques are able to detect bacteria
when the nutritional requirements and culture conditions
are known. However, most environmental organisms are
non-culturable and remain undetected. Analytical microbiology techniques (primarily GC-MS) detect not only viable bacteria, but also their nonviable cell envelope remnants when present in complex biological matrices
(Maitra et al., 1978; Fox et al., 1980; Findlay et al., 1983;).
More recently, gas chromatography tandem mass spectrometry (GC-MS/MS) has been proven to be capable of
detecting terrestrial bacterial markers at minute concentrations in environmental and clinical samples (Fox et al.,
1995, 1996; Saraf and Larsson, 1996; Saraf et al., 1999;
Bal and Larsson, 2000; Kozar et al., 2000). GC-MS/MS
was only recently used for the first time in studies of
material of extraterrestrial origin (Kozar and Fox, 2001).
There are a number of substances present in the cell
envelope of bacteria that are of limited distribution in
nature, including muramic acid (3-O-lactyl, 2-deoxy-2amino glucose) and certain 3-hydroxy fatty acids (3-OH
FAs). Muramic acid (Mur) is both an amino acid and an
amino sugar, and is a constituent of the cell wall PG
backbone of Gram-positive (Gm) and Gram-negative
(Gm) bacteria. PG consists of a backbone of repeating
alternating subunits of Mur and glucosamine. There are
attached tetra- or penta peptide side chains and
crosslinks. Unlike proteins (which contain exclusively Lamino acids) there are both D- and L-amino acids in PG.
The most common amino acids in PG are D-glutamic acid,
L-alanine, and D-alanine (Schleifer and Kandler, 1972),
which are among the major amino acids found in meteorites (Engel and Nagy, 1982). Since PG is the primary
source of both Mur and D-amino acids in terrestrial samples, they generally are present in similar concentrations
(at the 10 100 parts per million (ppm) level) (Sonesson et
al., 1988; Fox et al., 1993).
Lipopolysaccharide (LPS) is a component of the Gm
bacterial cell envelope. LPS is found in the surface layer of
the outer membrane, but not in the cytoplasmic membrane. The structure of LPS is similar among all Gm
bacteria and is composed of an outer O antigen, a middle
core region covalently bound to a glycolipid termed lipid A.
The lipid A portion of LPS is composed of a disaccharide of
glucosamine with covalently bound 2- and 3-OH FAs (Rietschel, 1984).
In a recent study using GC-MS/MS, levels of Mur and
3-OH FAs were assayed in lunar samples that have been
stored at the Johnson Space Center (JSC) under strict
isolation conditions. This was the first study of chemical
markers, specific for bacteria, in lunar material (or indeed
any other sample of extraterrestrial origin). It was noted
that lunar and terrestrial samples are strikingly different
in that chemical markers are absent in the former. The
ability to measure trace levels of chemical markers for
Earth bacteria in extraterrestrial samples would greatly
aid future studies designed to confirm the extraterrestrial
origin of amino acids. The absence of chemical markers for
Earth bacteria would indicate that samples are not con-

taminated, and, consequently, if organic chemicals are


present, they could not be from this source (Kozar et al.,
2001).

ANALYTICAL MICROBIOLOGICAL
CONSIDERATIONS
There have been extensive chemical, and some biological, characterizations of lunar samples and meteorites.
For example, as noted above, amino acids have been detected at trace levels. In recent years GC-MS and highperformance liquid chromatography with fluorescence detection (LC-fluor) have primarily been used for identifying
amino acids in lunar dust and meteorites. GC-MS in total
ion mode allows identification when compounds are
present at relatively high concentrations. However, coeluting peaks are often present which may confound identification (Pizarello and Cronin, 2000). Alternatively, selected ion monitoring (SIM) can home in on characteristic
features in a mass spectrum, thereby eliminating much of
this background. This dramatically lowers the detection
limit for trace analysis. Unfortunately, definitive identification is lost in SIM mode.
In LC-fluor analysis, amino groups, which are present
in amino acids and other basic organic materials, are
converted to fluorescent derivatives prior to analysis. This
technique is the most sensitive approach available for
screening for such organic materials. Unfortunately, any
compound with an amino group is labeled, and thus background interferences are high. As noted by Glavin et al.
(1999), for definitive identification it is vital that such
results be confirmed by mass spectrometry. Unfortunately, the mass spectrometric procedures used in that
work were too insensitive to corroborate LC-fluor analyses.
GC-MS/MS for trace analysis of chemical markers for
bacteria in environmental matrices has been used in the
U.S. and Sweden for the past 6 years. GC-MS/MS, like
GC-MS, employs a high-resolution GC separation. However, the exquisite selectivity of MS/MS detection dramatically reduces background, thereby lowering detection limits approximately two orders of magnitude over GC-MS
(employing SIM). Furthermore, the product ion spectrum
(MS/MS chemical fingerprint) allows for categorical identification of chemical identity at parts per billion (ppb)
levels (Kozar et al., 2001).
LC-fluor and GC-MS/MS are complementary techniques. LC-fluor will detect all compounds that have
amino groups, including amino acids, with high sensitivity. GC-MS/MS (which has comparable sensitivity but
much greater specificity) allows definitive analysis, but
must target specific chemicals that are likely to be
present. Thus, LC-fluor is a useful screening method to
target more definitive analysis by GC-MS/MS.
Classical microbiology techniques focus on the detection
of live bacteria. The LC-fluor, GC-MS, and GC-MS/MS
methods alternatively detect both viable bacteria and
their nonviable cell wall remnants. The most unconventional technique, GC-MS/MS, has proven capable of detecting terrestrial bacterial markers at minute concentrations (100 ppb) in a variety of complex environmental
and clinical matrices (Fox et al., 1996; Krahmer et al.,
1998, 1999; Saraf et al., 1999; Kozar et al., 2000; Liu et al.,
2000). However, GC-MS/MS has been used in only one
extraterrestrial study to date (Kozar et al., 2001). In that
work it was demonstrated that pristine lunar dust does

183

CHEMICAL MARKERS FOR BACTERIA

not contain chemical markers for bacteria, and therefore it


can be used as a negative control in studies of other
samples of extraterrestrial origin. In view of the long
history of difficulties in interpreting results due to chemical and biological contamination (Tasch, 1964), this result
could be a highly significant observation.

PLANS TO VISIT MARS TO COLLECT


SAMPLES FOR LIFE DETECTION STUDIES
In order to directly address the issue of whether life
exists beyond the Earth, NASA is planning a series of
missions to retrieve Martian surface samples for analysis.
Contamination of these samples with terrestrial material
during passage to Earth, or upon subsequent curation,
would be a catastrophic loss for the scientific community.
These studies will be particularly important since experiments conducted by the Viking expeditions to Mars were
generally interpreted as negative for organic matter and
detection of life (Biemann et al., 1977). This does not rule
out the possibility of organic matter and/or life existing
elsewhere on Mars, especially below the ultraviolet-irradiated surface of the planet (Weiss et al., 2000). Recent
reports of detection of organic molecules in meteorites
(Glavin et al., 1999), simple sugars in interstellar clouds
(Hollis et al., 2000), and the probability of liquid water on
Jupiters moon, Europa (Kivelson et al., 2000), serve to
intensify the search for extraterrestrial life. The discovery
of planets revolving around stars has also intensified the
debate about the possibility of life on other planets
(Lunine, 1999).
As noted above, the only extraterrestrial samples currently on the Earth are lunar dust, collected on the Apollo
and Luna missions to the Moon, and meteorites. Unfortunately, meteorites are contaminated on passing through
the Earths atmosphere and during environmental exposure on earth. Thus, it has been difficult to interpret the
biological or chemical origin of compounds found in meteorites (Kvenholden et al., 1970, 1971; Engel and Nagy,
1982; Bada et al., 1983; Engel et al., 1990; McKay et al.,
1996; Cronin and Pizzarello, 1997; Engel and Macko,
1997; Bada et al., 1998; Glavin et al., 1999; Ehrenfreund
et al., 2001;). Organic substances have also been detected
in lunar dust, but at much lower (trace) levels (Burlingame et al., 1970; Ponnamperuma et al., 1970; Harada et
al., 1971; Nagy et al., 1971; Brinton and Bada, 1996).
The spacecraft used in the Apollo missions were contaminated with terrestrial bacteria (Puleo et al., 1977).
However, viable bacteria were not detected (using classical microbiological culture) in the lunar material when it
was first brought by the Apollo missions (Oyama et al.,
1970). Unfortunately, chemical methods that detect dead
bacterial remnants, or non-culturable bacteria, were in a
primitive stage of development at that time (Maitra et al.,
1978; Fox et al., 1980, 1993, 1995, 1996; Findlay et al.,
1983; Saraf and Larsson, 1996; Saraf et al., 1999; Bal and
Larsson, 2000; Kozar et al., 2000).
The lunar samples have been stored under strict isolation conditions for the past 30 years (Allton et al., 1998). It
is possible that terrestrial contamination could have occurred during this extended curation period. Although it
was hypothesized that pristine lunar dust lacks chemical markers for terrestrial bacteria, this remained to be
demonstrated experimentally until only recently. The experimental test of this hypothesis was the primary subject
of a recent study (Kozar et al., 2001). That work also

provided an important negative baseline for future studies


of extraterrestrial samples (e.g., from Mars).

CONCLUSIONS
Despite a great deal of research, the origin of organic
chemicals (including amino acids) in samples of extraterrestrial origin is still far from clear. Part of the problem
stems from the difficulty of obtaining definitive analyses of
trace organics in complex environmental matrices. Another problem is the ubiquitous contamination of meteorites with terrestrial bacteria.
Kozar et al. (2001) proposed a null hypothesis, as follows: Pristine lunar samples, since they come from a satellite believed to be void of life, should not contain markers
for terrestrial bacteria. This has been confirmed experimentally (Kozar et al., 2001). As noted above, to our
knowledge this is the first study of the levels of specific
chemical markers (e.g., Mur and 3-OH FAs) for terrestrial
bacteria in a curated lunar sample (or any other sample of
extraterrestrial origin). State-of-the art GC-MS/MS methodology was employed to eliminate the background that
often complicates interpretation of data at trace levels.
If, in future studies, amino acids are detected in pristine
lunar samples (as shown by a lack of Mur and 3-OH FAs),
this would suggest that they are not of terrestrial origin.
Interpretation of the amino acid content of meteorites will
be more difficult. Finding that D-amino acid levels are
similar to those of 3-OH FAs and Mur would indicate that
all are primarily of bacterial origin. This will reinforce the
need to collect uncontaminated samples from Mars.
Other difficulties stem from limited communication between geochemists and microbiologists. The former have a
great deal of knowledge about space science, but less experience with microbial chemistry. The classical microbiologist, on the other hand, has not spent a great deal of
time speculating on the origin of life or how to study it
experimentally. It is hoped that this review may play a
part in encouraging further interdisciplinary interactions,
which will be extremely important as we begin to draw
conclusions about the presence of life in a previously unstudied world.
Contamination of samples can occur during their transport and/or long-term curation. In future interplanetary
missions, assessing the levels of chemical markers, both
pre- and post-mission, would be advisable in life-detection
studies. Current protocols for sample storage in the Lunar
Curation Facility at JSC appear to have been successful at
safeguarding the lunar sample collection from terrestrial
bacterial contamination. However, this facility has been
primarily concerned with keeping the collection clean
from chemical contamination. It may be necessary in future planetary missions to store subsets of samples during
both the return mission and curation to minimize biological contamination, using standard microbiological approaches (e.g., storage at 20 to 70C).

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