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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.
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
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
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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
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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).
LITERATURE CITED
Allton JH, Bagby JR, Stabekis PD. 1998. Lessons learned during
Apollo sample quarantine and sample curation. Adv Space Res
22:373382.
Anders E. 1996. Evaluating the evidence for past life on Mars. Science
274:2119 2121.
Bada JL, Cronin JR, Ho MS, Kvenholden K, Lawless JG, Miller SL,
Oro Steinberg JS. 1983. On the reported optical activity of amino
acids in the Murchison meteorite. Nature 301:494 497.
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