During the past year we have concentrated on two objectives. The first objective is ongoing and is to define the experimental parameters that are necessary to conduct autonomously a mineralogical analysis of the Martian surface in situ using differential thermal analysis coupled with gas chromatography (DTA/GC). The rationale in support of this objective is that proper interpretation of the mineralogical data from the DTA/GC can be used to better describe the present and past environments of Mars, leading to a better assessment of the probability of life evolving on Mars. To meet these objectives we have analyzed a number of samples collected from nature using the DTA/GC. One of the more significant findings was that in samples of desert varnish we detected magnetite and maghemite that may serve as potential biomarkers applicable to DTA/GC analyses of Martian surface material during landed missions. The second objective follows from the first and is to better understand microbe-environment interactions by determining the response of microbes to changes in their environment, including extreme desiccation and solar UV-radiation. The rationale behind this is to develop hypotheses regarding what may have happened to life that may have arose on Mars, and microbial life that may get to the surface of Mars via spacecraft, or meteors from Earth. To accomplish this objective we have exposed microbes, collected from NaCl and gypsum-halite crystals, to the space environment aboard the ESA-German Biopan facility for 15 days. The most significant finding was that these microbes survived the exposure better than others

Mancinelli, Rocco L.

English

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NASA-CP-$COZh?
Definition of Exobiology Experiments for Future Mars
I _ -:1-_'/_
7 _Th_
Missions /
Rocco L. Mancinelli, Principal Investigator
NASA Cooperative Agreement NCC 2-479
Progress Report
January 15, 1995 -January 14, 1996
https://ntrs.nasa.gov/search.jsp?R=19960014814 2020-06-16T04:41:26+00:00Z
Definition of Exobiology Experiments for Future Mars Missions
Rocco L. Mancinelli, Principal Investigator
NASA Cooperative Agreement NCC 2-479
Summary: During the past year we have concentrated on two objectives. The first
objective is ongoing and is to define the experimental parameters that are necessary to
conduct autonomously a mineralogical analysis of the Martian surface in situ using
differential thermal analysis coupled with gas chromatography (DTA/GC). The rationale in
support of this objective is that proper interpretation of the mineralogical data from the
DTA/GC can be used to better describe the present and past environments of Mars, leading
to a better assessment of the probability of life evolving on Mars. To meet these objectives
we have analyzed a number of samples collected from nature using the DTA/GC. One of
the more significant findings was that in samples of desert varnish we detected magnetite
and maghemite that may serve as potential biomarkers (see below for discussion) applicable
to DTA/GC analyses of Martian surface material during landed missions. The second
objective follows from the first and is to better understand microbe-environment
interactions by determining the response of microbes to changes in their environment,
including extreme desiccation and solar UV-radiation. The rationale behind this is to
develop hypotheses regarding what may have happened to life that may have arose on
Mars, and microbial life that may get to the surface of Mars via spacecraft, or meteors from
Earth. To accomplish this objective we have exposed microbes, collected from NaC1 and
gypsum-halite crystals, to the space environment aboard the ESA-German Biopan facility
for 15 days. The most significant finding was that these microbes survived the exposure
better than others.
Microbe Environment Interactions. The overall objective of the Biopan study is to
understand the responses of osmophilic/halophilic microbes to space vacuum and solar
UV-radiation. Nucleic acid bases absorb UV-radiation strongly, with maximum absorbance
at 254 nm. Proteins also absorb UV-radiation with a peak at 280 nm. It has been well
established that cell death by UV radiation is due primarily to its action on DNA. Among
the several defects in DNA caused by radiation, the formation of thymidine dimers
followed by strand breaks are the most prominent. Cellular death due to anhydrobiosis
(extreme desiccation) results from lipid, protein and nucleic acid irreversible phase changes
such as denaturation and structural breakage (Cox, 1993; Cox 1987; Dose et al., 1992).
Oneof thespecificreportedmechanismsof death due to anhydrobiosis in prokaryotes is
the dehydration of DNA causing strand breaks (Dose et al., 1992).
Few studies have been conducted on the effects of the space environment on microbes.
These studies reflect the effects of the space environment on representatives of a small
number of microbial species. Microbes tested in the space environment to date include
Bacillus subtilis spores, bacteriophage T-l, and Tobacco Mosaic Virus (reviewed in
Horneck and Brack, 1992, and Horneck, 1993), and diatoms. The results of these studies
revealed that Bacillus subtilis spores will survive for years in space if protected against
high solar UV-radiation flux (Horneck, 1993), whereas viruses lose viability on the order
of weeks to months (reviewed in Horneck and Brack, 1992). We have recently discovered
that microorganisms inhabiting gypsum-halite crusts and NaC1 crystals are extremely
osmophilic and can withstand extreme desiccation (Rothschild et al., 1994). Because of
this, and the fact that gypsum-halite and NaC1 attenuate UV-radiatlon, we hypothesize that
these organisms are good candidates for survival in the space environment. To that end,
we exposed these organisms, in a dried state, to the space environment in Earth orbit
aboard a satellite equipped with the European Space Agency's Biopan facility. Data from
the Biopan experiments indicates that the osmophiles can survive in the space environment,
whereas most others die within minutes. The organisms were exposed to the space
environment for 2 weeks while in earth orbit aboard the ESA Biopan facility. As controls,
organisms were kept in the dark and only exposed to space vacuum. Ground control time
course experiments (vacuum dark, vacuum UV, and UV only exposed samples) were
conducted in a space simulation facility. All samples were compared to unexposed
samples. Survivability was determined by plate counts and the most probable number
technique. DNA breakage was determined by labeling breaks in the DNA with 32p
followed by translation.
During the Biopan flight the total radiation dose to the organisms was 104 KJm -2.
Haloarcula- G and Synechococcus survived exposure to the space environment with the
number of breaks in the DNA of the UV exposed samples greater than the vacuum only
exposed sample. Results of the ground time course studies revealed that Haloarcula- G
(Figure 1) and Synechococcus (data not shown) survived over 100 hours of exposure to
UV radiation from a deuterium lamp (Intensity = 0.07 W m -2 at 200 nm and 0.03 Wm -2 at
300 nm).
The number of breaks in the DNA ofHaloarcula- G increases with increased exposure time
to UV and vacuum, with exposure to UV exhibiting a greater number of breaks than
exposure to vacuum alone (Figure 1). From these data we conclude that Haloarcula- G
isolated from a NaC1 crystal & Synechococcus (Ntigeli) inhabiting gypsum-halite crusts
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Figure 1. Survival (N/No solid symbols) and levels of Up incorporation into DNA
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UV in air, or Vacuum only.
have a better rate of survival when exposed to the space environment than any organism
tested to date. The data also suggest that DNA damage due to UV is greater than that
caused by anhydrobiosis.
Rates of nitrogen fixation, denitrification, carbon fixation and phosphate incorporation
into DNA were determined in situ on a microbial community inhabiting an area one meter
from the source of Octopus Spring, a thermal (86 _ alkaline (pH=8.2) spring in
Yellowstone National Park, Wyoming. Nitrogen fLxation rates were determined using the
acetylene reduction method. Rates of denitrification were determined similarly using
acetylene to block the reduction of nitrous oxide to dinitrogen. Nitrogen assimilation rates
were determined using 15NO 3- and 15NH4+. Carbon assimilation rates were determined
using 14C-acetate. Carbon fixation rates were determined using 14C labeled CO2.
Phosphate incorporation was determined using 33po43-. The mean nitrogen fixation rate
was 0.671 nmoles ethylene produced mg -1 protein hr -1. The mean denitrification rate was
0.158 nmoles N20 denitrified mg -1 protein hr -1. This community assimilated NI-I4 + at a
rate of 0.88 l.tg hr -1, but could not assimilate NO3-. Results of water analyses combined
with these data indicate that the community relies on nitrogen fixation for its source of fixed
nitrogen. The carbon fixation and acetate assimilation studies show that the community is
comprised of primarily phototrophs, secondarily chemoautotrophs and lastly heterotrophs.
This is surprising because no chlorophyll containing pigments were found in extracts of the
community, but a new type of carotenoid pigment appears to exist in these organisms and
may be part of a here-to-fore undiscovered photo-pigment system.
Samples of soda-nitre have been collected from the Atacama desert in Chile and are
currently being analyzed in the laboratory for viable microbes as well as by DTA/GC.
DTA/GC Measurements. We have analyzed a variety of substances important to
exobiology using DTA/GC. The substances tested included organic compounds (proteins,
amino acids), evaporites (NO3", CO32", and NO2- salts), clays (montmorillonite, kaolinite,
nontronite), Fe-enriched clays (Banin et al., 1993), non-clays (palagonite), and various
mixtures of these substances. In addition, we have analyzed samples collected from
ecosystems ranging from Antarctic endolithic rocks, evaporitic deposits occurring near
thermal alkaline and acid springs in Yellowstone National Park, Wyoming, to gypsum
halite evaporitic crusts that form along the intertidal. We compared the DTA/GC with
several other techniques and found it to be the most appropriate as a flight instrument for
the in situ determination of the mineralogy of the Martian surface material.
Desert varnish, a coating of manganese and iron oxides held in a clay matrix on the
surface of rocks, is widespread on Earth's deserts. Its formation is thought to involve,
weathering processes acting in conjunction with microbial activity. Consequently, if desert
varnish similar to that found on earth is found on Mars, then the probability for life to have
arisen on Mars increases.
Data from a typical analysis of a sample of desert varnish collected from Nevada is
depicted in figure 2. Analyses were conducted using 30 mg of aluminum oxide as the
reference material, and 30 mg of sample. Samples were removed from rocks covered with
varnish with aluminum oxide grit (this was used because it would not give a DTA thermal
signature nor would it react with the varnish). Samples were analyzed under vacuum using
a Dupont model 1600 high temperature DTA oven equipped with a model 910 cell base.
The heating rate was 10 oc min -1. The system is controlled by a Sun Sparc II
workstation. The system was tested and calibrated with pure samples of the suspected
unknown minerals in the sample, as well as mixtures of these minerals. Gas evolution
during sample heating is sensed by a pressure sensor which triggers a valve allowing the
evolved gas to expand from the oven chamber into a GC sample loop.
Thefirst endotherm(-100 oC)depictedin figure2 is accompaniedby the productionof
watervaporandcanbeattributedto thevaporizationof wateradsorbedto thesample,while
the second(-120-130 °C) is due to Illite. The exothermsand endothermsoccurring
between140and320°C aremostlikely dueto theoxidationandtransformationsof oxides
andoxyhydroxidesof manganese.The exothermicpeakobservedbetween320 oc and
360 oC is due to the transitionof magnetite(Fe304) to maghemite(_,-Fe203).The
endothermcenteredaround400oCis accompaniedby theformationof waterandis dueto
theoxidationof goethite(a-FeOOH)to hematite(a-Fe203). Thisexothermmaybedueto
the transitionof _,-Fe203 -o a-Fe203 (hematite). The next thermalevent is a large
exothermbetween420and560oCandcanbeattributedto thetransitionof ,/-Fe203 -o a-
Fe203. This exothermis followedimmediatelyby what appearsto be two overlapping
endothermsthat are accompaniedby the formation of water and are probably due to
dehydroxylationof the lllite andkaolinite. Usually clay dehydroxyaltionendothermsare
muchbroader,but in this casethey arebeing somewhatmaskedby the _-Fe203 _ o_-
Fe203 exotherm.Thenext thermalevent(- 640oC)is a smallendothermthatis probably
dueto achangein themagneticpropertiesof hematite(Mackenzie,1970). Theendotherm
occurringbetween690and725oCis accompaniedby theformationof waterandis dueto
thedehydroxylationof montmorillonite. Thenext threethermaleventsrepresenthehigh
temperaturetransitionreactionsof montmorillonite,illite andkaoliniterespectively.
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Figure 4. Results of analysis of 30 mg of desert varnish collected from Reno, Nevada by
DTA. 30 mg of Aluminum oxide was used as the reference. The samples and reference
were heated at 10 oC min-l.The endotherms and exotherms are labeled as to the minerals
and reactions that gave rise to each.
References
Banin, A., T. Ben-Shalom, L. Margulies, D. R. Blake, R. L. Mancinelli, and A. U.
Gehring. 1993. The nanophase iron mineral(s) in Mars soil. J. Geophys.
Res.(Planets). 98:20831-20853.
Cox, C. S. 1987. The Aerobiological Pathway of Microorganisms. J. Wiley and Sons,
Chichester, U.K.
Cox, C. S. 1993. Roles of water molecules in bacteria and viruses. Origins of Life. 23:29-
36.
Dose, K., A. Bieger-Dose, M. Labusch, and M. Gill. 1992. Survival in extreme dryness
and DNA-Single strand Breaks. Adv. Space Res. 12(4):221-229.
Horneck, G. 1993. Responses of Bacillus subtilis spores to space environment: results
from experiments in space. Origins of Life. 23:37-52.
Homeck, G. and A. Brack. 1992. Study of the origin, evolution and distribution of life
with emphasis on exobiology experiments in earth orbit. Adv. Space Biol. Med.
2:229-262.
Rothschild, L. J., L. J. Giver, M. R. White and R. L. MancineUi. 1994. Metabolic activity
of microorganisms in gypsum-halite crusts. J. Phyc. 30:431-438.
Followingis a list of presentationsandpublicationsresultingfrom thisprojectduringthe
past year:
Invited Presentations
1995, Survival of Osmophilic Microbes Exposed to the Space Environment,
Institute for air and space medicine, Cologne, Germany
1995, The Nature of Nitrogen in the Environment, as part of the NASA sponsored
Workshop on Nitrogen Cycling in Closed or Controlled Environments,
University of California-Berkeley, Berkeley, CA.
1995, Exposure of Osmophilic Microbes to the Space Environment: Application to
the Moon, Annual Meeting of the European Geophysical Society, Hamburg,
Germany.
Publications
Mancinelli, R. L. 1995. The regulation of methane oxidation in soil. Ann. Rev.
Microbiol. 49:581-605.
Bishop, J. L, C. M. Pieters, R. G. Bums, J. O. Edwards, R. L. Mancinelli, and
H. Fr6schl. 1995. Reflectance spectroscopy of ferric sulfate - doped
montmorillonites as Mars soil analog materials. Icarus. 117:101-119.
Banin, A., and R. L. Mancinelli. 1995. Life on Mars? I. The chemical
environment. Adv. Space Res. 15:(3)163-170.
Mancinelli, R. L. and A. Banin. 1995. Life on Mars? II. Physical Restrictions.
Adv. Space. Res., 15:(3)171-178.
Schwartz, D. E., R. L. Mancinelli, and M. R. White. 1995. Search for life on
Mars: Evaluation of techniques. Adv. Space Res., 15:(3)202-208.
Publish¢d Abstracts
Mancinelli, R. L., M. R. White, and L. J. Rothschild. 1995. DNA integrity and
survival of Synechococcus (N/igeli) exposed solar irradiation and vacuum
in earth orbit. PSA Abt. 54:11.
Rothschild, L. J., and R. L. Mancinelli. 1995. Field analyses of carbon, nitrogen,
and DNA metabolism in algal mats. PSA Abt. 82:16.
Mancinelli, R. L., M. R. White, and L. J. Rothschild. 1995. Exposure of
osmophilic microbes to the space environment. ASM Abt. 1-38:323.
Mancinelli,R.L., andM. R. White. 1995.Effectsof nitrateandammoniumon
methaneoxidation,anddenitrificationin asanitarylandfill. ASM Abt. N-
146:357.


Definition of exobiology experiments for future Mars missions

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