UNIT IV

Space Microbiology - Aim and objectives of space research. Life detection

methods a) Evidence of metabolism (Gulliver) b) Evidence of photosynthesis
(autotrophic and heterotrophic) c) ATP production d) phosphate uptake e)
sulphur uptake.
UNIT V
• Martian environment (atmosphere, climate and other details). Antartica as a
model for Mars. Search for life on Mars, Viking mission, Viking landers, and
Biology box experiment. Gas exchange, label release and pyrolytic release
experiments. Monitoring of astronauts microbial flora: Alterations in the load
of medically important microorganisms, changes in mycological and bacterial
autoflora.
 SPACE RESEARCH
• Space research is scientific study carried out in outer space, and by studying outer space.
• From the use of space technology to the observable universe, space research is a
wide research field
• . Earth science, materials science, biology, medicine, and physics all apply to the space
research environment.
• Indian space programme encompasses research in areas like astronomy, astrophysics,
planetary and earth sciences, atmospheric sciences and theoretical physics.
• A series of sounding rockets are available for atmospheric experiments. Several scientific
instruments have been flown on satellites especially to direct celestial X-ray and gamma-
ray bursts.
Aim and Objectives of Space Research
• Space research seeks to understand the origins, evolution, and destiny of the
universe and the strange phenomena that shape it. Included in SMD(Science
Mission Directorate) goals are to understand the following:
• The nature of life in the universe and what kinds of life may exist beyond
Earth;
• The solar system, both scientifically and in preparation for human
exploration;
• The Sun–Earth system, changes to the system, and the consequences for life
on Earth;
• The birth of the universe, the edges of space and time near black holes, and
the darkest space, between galaxies; and
• The relationship between the smallest subatomic particles and the vast
expanse of the cosmos.
Fundamental research activities in Space
• Included in these fundamental research activities are the following:
• Understanding the history of Mars and the formation of the solar
system;
• The search for Earth-like planets and habitable environments around
other stars; and
• Support for the safety of robotic and human exploration of space by
predicting potentially harmful conditions in space, such as space
radiation.
• Responsibility for the defining, planning, and overseeing of NASA space and
Earth science goals lies in the four divisions of the SMD(Science Mission
Directorate), which have the following objectives:
• Earth science. Study planet Earth from space to advance scientific
understanding of it and to help meet societal needs;
• Planetary science. Advance the scientific knowledge of the origin and history
of the solar system, the potential for life elsewhere, hazards faced by
humans as they explore space, and the resources that they present;
• Heliophysics. Understand the Sun and its effects on Earth and the solar
system
• Astrophysics. Discover the origin, structure, evolution, and destiny of the
universe, and search for Earth-like planets.
Significant milestones in space exploration
date accomplished event details country or agency

Oct. 4, 1957 first artificial Earth satellite Sputnik 1 U.S.S.R.

first animal launched into

Nov. 3, 1957 dog Laika aboard Sputnik 2 U.S.S.R.
space

first spacecraft to hard-land on

Sept. 14, 1959 another celestial object Luna 2 U.S.S.R.
(the Moon)

first pictures of the far side of

Oct. 7, 1959 Luna 3 U.S.S.R.
the Moon

first applications satellite TIROS 1 (weather

April 1, 1960 U.S.
launched observation)

Discoverer 13 (part of Corona

first recovery of a payload
Aug. 11, 1960 reconnaissance satellite U.S.
from Earth orbit
program)

April 12, 1961 first human to orbit Earth Yury Gagarin on Vostok 1 U.S.S.R.

first data returned from another

Dec. 14, 1962 Mariner 2 U.S.
planet (Venus)

Valentina
June 16, 1963 first woman in space U.S.S.R.
Tereshkova on Vostok 6

first satellite to operate Syncom 2

July 26, 1963 U.S.
in geostationary orbit (telecommunications satellite)
Space Microbiology
• The launching of artificial earth satellites, space shuttles and rockets has now opened new avenues in almost all
branches of knowledge.
• One such exceptionally interesting area which got huge impetus from such developments in space technology is the area
of ‘space microbiology’.
• Aim of this discipline(Space Microbiology) is to study the biological responses of microorganisms in response to the
harsh and inhospitable conditions (intense solar UV radiation, space vacuum, thermal extremes and microgravity) of
outer space environment and also to use microbes for studying the conditions of life there.
• The ‘spaceflight microbes’ hold great potential for the development of novel therapeutics and vaccines against infectious
diseases.
• Moreover, for the manned spaceflight missions, understanding human biological changes and microbial responses while
living in the closed quarters in space is important to the health, safety and performance of crewmembers.
• For instance, prolonged exposure to cosmic radiation and microgravity is believed to have a negative effect on the
human immune system; hence, microorganisms have been studied as radiobiological model systems in space for
assessing radiation risks to humans in space
• Once the microbes return from space, their responses to selected space parameters like
varying gravity conditions are studied and compared with those obtained on ground.
• In order to carry out these microbiological studies in space, special facilities simulating
the conditions of outer space like microgravity or ‘weightlessness’ are used.
• A wide variety of payloads have been developed by numerous international teams like
National Aeronautics and Space Administration (NASA) and European Space Agency
(ESA) to carry out cellular and molecular biology studies inside the pressurized
environment of the spacecraft.
• With this beginning, the space research is increasingly booming and is likely to provide
further momentum towards commercial pharmaceutical applications like secondary
metabolite (antibiotic) production, controlling the spread of multidrug resistant pathogens
and most importantly vaccine development.
• So, it would be appropriate to mention that all the experimental data obtained from such
space studies can serve as a ‘time-machine’ for predicting the feasibility as well as
sensitivity of life in the space.
• Life detection methods
 Gulliver Experiment
Gulliver” is an experiment designed to detect extraterrestrial life and to begin a study of its metabolism.
Any first attempt to find extraterrestrial life must be based upon certain assumptions.
the Gulliver experiment assumes that:

A. Extraterrestrial life will be of an aqueous, carbonaceous nature.

B. Its biochemistry at the cellular level will be similar to that on earth.
C. If any life exists on an alien planet, the widespread existence of microorganisms is likely.

• There seems to be general agreement that the planet in our solar system most likely to support extraterrestrial life is
Mars.

• Environmental considerations make it likely that, if life similar to ours does exist on Mars, the number of organisms
per unit of surface area is less than on earth.

• Both of these considerations make it imperative that a life detection test be highly sensitive.
• Radioisotopes fulfill the requirement for sensitivity. Moreover, they can
probe metabolic reactions at the molecular level. These advantages combine
to offer a technique that can provide very rapid detection of fundamental
metabolic processes.
• Furthermore, radioisotope techniques can be employed with relatively simple
instrumentation which can readily be miniaturized. Power requirements for
operation are small.
• Essential to the radioisotope approach is the development of a medium, or
media, containing appropriately labeled compounds.
• The ease with which microorganisms can be detected by collecting and
counting C14O2 evolved by them from substrates containing C14 has been
demonstrated.
• The production of gases is common among earth microorganisms, and
probably all species produce carbon dioxide.
• Other metabolic gases which could be readily labeled in one or more elements
are methane, ammonia, hydrogen sulfide and molecular hydrogen.
• At present, only C14 is being incorporated into experimental media because it
has served successfully with a wide range of microorganisms.
TYPES OF MEDIA
• Two types of media are currently being developed.
• The first is a complex medium incorporating essential inorganic salts and organic extracts and compounds.
• This medium is being designed to support as wide a variety of microbial species as possible.
• Aerobes, anaerobes, facultative anaerobes, thermophiles, mesophiles, psychrophiles, heterotrophs,
autotrophs (including phototrophs and chemotrophs), spore formers and nonspore formers have been
successfully detected with it.

• However, some species of microorganisms are known to be inhibited by various organic compounds
present in complex media.
• Therefore, a parallel effort is underway to develop a simple medium which, except for the labeled
compounds, contains no organic constituents.
• The selection of radioactive compounds for incorporation into the media is based upon their ready
metabolic conversion to radioactive gas.
• The widespread importance of the Krebs cycle in aerobic metabolism and of the Embden-Meyerhof
pathway in anaerobic metabolism makes the use of C14-metabolites related to them highly attractive since both
sequences of reactions produce carbon dioxide.
• To date, best results have been obtained with a combination of C14-sodium formate and uniformly labeled
C14-glucose. However, various C14-labeled amino acids, microbial extracts and other compounds are being
tried.
• The concept of an extraterrestrial microbial life detection instrument which can
serve either as a minimum biological payload or a sub-system for a fully automated
biological laboratory is described.
• Five separate biological experiments look for life through distinct, but related,
“metabolic windows.”
• The experiments are conceived as ranging from minimally geocentric to moderately
geocentric.
• Depending on the nature of the responses obtained, positive results would yield
some degree of information concerning the biochemistry of the living processes
encountered.
• The specific metabolic experiments are based on:
• (1) the metabolism of radioactive substrates with the evolution of labeled gases,
• (2) the detection of photosynthesis in a heterotrophic-autotrophic system,
• (3) the detection of photosynthesis in strict autotrophs,
• (4) the detection of intracellular adenosinetriphosphate(ATP) and
• (5) the metabolic uptake of phosphorus.
• (6) the metabolic uptake of sulphur
Function of GULLIVER EXPERIMENT
• The experiment will function in the following manner.
• At least two instruments, one a test and the other a control, will be incorporated into a capsule
destined to land on Mars.
• Sealed ampoules contain the radioactive medium.
• The entire instrument is being designed to withstand heat sterilization.
• When the capsule lands on Mars, squibs will fire the projectiles. Squib is a miniature explosive
device and it generally consists of a small tube filled with an explosive substance.
• Each will deploy a 25 foot length of silicone grease-impregnated retrieval line over the surface of
the planet.
• The motor will then reel the line, together with adhering particulate matter, into the incubation
chamber.
• During this period, the culture chamber will achieve equilibrium with the Mars atmosphere.
• After the line is retrieved, the incubation chamber will be sealed and the ampoule will be broken,
releasing the radioactive medium onto the line.
• Attached to the outside of the chamber is a thermostatically controlled heater to prevent freezing.
• If organisms are present on the soil particles and are able to metabolize any of the labeled
substrates, radioactive gas will be produced.
1. Metabolism of Radioactive Substrates and Evolution of Labeled Gases
• This experiment called “Gulliver,” offers radioactive substrates containing
C14 and S35 in aqueous solution to the samples suspected of containing
microorganisms.
• If organisms are present and can metabolize one or more of the labeled
substrates, the production of radioactive gas is likely.
• If growth or reproduction takes place, this is indicated by an exponential
increase in radioactive gas produced.
• In the event metabolism occurs without growth or reproduction, this is also
evident. The curve produced by the test is compared to that obtained from
an inhibited control.
• A thin window Geiger tube is mounted directly above the culture chamber.
• It is prevented from “seeing” the radioactive medium by metal and solid
foam baffles.
• However, gas produced by the culture is free to travel through the baffles.
• The window of the Geiger tube is thinly coated with barium hydroxide
which traps carbon dioxide.
• As C14O2 collects on the Geiger tube window, it is detected by a rise in
measured radioactivity.
• Simultaneously with the inoculation of the test instrument as just described,
the control instrument will also be inoculated.
• However, shortly after inoculation, its culture chamber will be injected with
a broad spectrum anti-metabolite (also under development).
• All data will be transmitted to Earth by radio.
• The production of a typical biological growth curve from the test instrument
and a negative, or materially reduced, response from the control instrument
will constitute evidence for microbial life on Mars.
• In the event reproduction does not occur during the course of the
experiment, respiration of the metabolizing culture could still be detected.
• NASA formulated what would become the Viking missions to land on
Mars with life-detection experiments.
• The Viking landers reached the surface of Mars in 1976 and performed
a suite of carefully designed and tested experiments seeking signs of
active metabolism in martian regolith samples(martian soil).
2Detection of Photosynthesis, Heterotrophic-Autotrophic System

In this experiment, carbon labeled substrates are supplied in aqueous form to the soil sample.

Organisms capable of heterotrophic assimilation of one or more of the substrates and capable of photosynthesis can be
detected by monitoring the production of radioactive carbon dioxide when the sample is alternately exposed to light and
dark.
Algae tested in this experiment evolved C14O2 in the dark, but not when photosynthesizing in the light.

Fig. 3 shows the rapidity with which the response follows the manipulation of light and dark periods.
3. Detection of Photosynthesis, Autotrophic System

A modification of the above experiment makes it possible to detect strict phototrophs.

In this experiment the sample is simultaneously exposed to light and C14O2.

After a suitable period, the light is excluded and the unassimilated C14O2 replaced with planetary atmosphere.

The sample is then maintained in the dark and monitored for the evolution of C14O2.

If photosynthetic organisms had fixed C14O2 in the light, when placed in the dark they would be expected to consume
energy compounds recently photosynthesized and thereby release C14O2 in a process paralleling endogenous
respiration.
3.
4. Firefly Bioluminescent Assay for Microbial Adenosinetriphosphate (ATP)

• The ATP detection experiment assumes a higher order of geocentricity than any of the other experiments proposed for the
life detection subsystem.

• This life detection experiment is based upon the sensitivity and specificity of the firefly lantern bioluminescent system for
ATP and upon the ubiquity of ATP in all known biological forms.

• Luciferases are enzymes that produce light when they oxidize their substrate. The gene for the most common luciferase
comes from the firefly.

• The firefly luciferase reaction requires its substrate luciferin, plus adenosine triphosphate (ATP), O2, and Mg2+.

• The bioluminescent reactants of the firefly lantern - luciferase, luciferin, magnesium and oxygen – are readily extracted in
usable form.

• When ATP is injected into this system, light is instantaneously produced.

• In a life detection test, a sample suspected of containing microorganisms is treated in a manner to extract ATP.

• An aliquot of this extract is injected into a cuvette containing the firefly lantern extract preparation.

• If ATP is present in the sample, light is emitted, reaching a maximum intensity proportional to the quantity of ATP in less
than one second.
• In the development of these experiments, thin window geiger tubes
have been used as the sensors.
• It is possible, however, through the use of plastic scintillators, to
convert the beta particle radiation of these experiments into photons
which can be measured by a photomultiplier tube.
• Such a photomultiplier system is used to measure ATP content by
the firefly bioluminescent assay.
5. Metabolic uptake of phosphorus
• Phosphorus (P) is important because it is an essential ingredient of
the energy metabolism of all forms of life.
• All organisms require P for cell division, energy
transformations, and cell maintenance as phosphorus is a
critical component of nucleic acids, ADP, and phospholipids.
• The P requirement for photosynthetic organisms, bacteria,
and fungi is met completely or in part by the uptake of
dissolved P.
• Every known biological reaction is ultimately dependent upon
phosphorus for energy conversion and transfer.
• phosphorus may be accepted from the environment, as
orthophosphate
• The dissolved inorganic orthophosphate, were supplemented with succinate
and glucose and then aerated.
• One of the flasks also received 2,4-dinitrophenol, known to uncouple
oxidative phosphorylation.
• At intervals over a five-hour period, aliquots of the wild cultures were
filtered to remove the microorganisms and the filtrate was analyzed for
orthophosphate. The depletion of orthophosphate in the organism-free
filtrate represents the amount of orthophosphate taken up by the
microorganisms.
• Within the experimental period, roughly 85% of the approximately 3.5 mg/L
of phosphate phosphorus available to the organisms was taken up by the
uninhibited culture.
• In the culture containing the uncoupling agent, the phosphate uptake was
less.
6) Metabolic uptake of sulphur
• To all organisms, sulfur is an essential and important element.
• The assimilation of inorganic sulfur molecules such as sulfate and thiosulfate into organic sulfur compounds
such as L-cysteine and L-methionine (essential amino acid for human) is largely contributed by
microorganisms.
• Of these, special attention is given to thiosulfate (S2O32-) assimilation, because thiosulfate relative to often
utilized sulfate (SO42-) as a sulfur source is proposed to be more advantageous in microbial growth and
biotechnological applications like L-cysteine fermentative overproduction toward industrial manufacturing.
• An experiment very similar to that described above for phosphorus uptake will seek to detect the metabolic
uptake of sulfur as an index of life.
• This vital element can be supplied as the radioisotope s35 since the half life is sufficiently long for a Mars
voyage and experiment.
• Various compounds of sulfur could be supplied to the sample.
• The importance of sulfur uptake as an exobiological experiment is emphasized not only by its occurrence in
all terrestrial life, but also by the fact that high-energy sulfur bonds could conceivably mediate
extraterrestrial biological energy transfers in a manner analogous to the role of high-energy phosphate bonds.
Instrumentation
• A high degree of compatibility in other experimental systems is attainable for the life
detection subsystem.
• Of the six biological experiments proposed, only three require sample acquisition, the
ATP assay and the phosphate and sulfur uptake experiments.
• If the life detection subsystem becomes a part of a fully automated biological
laboratory, a single sample from the central sampling system is all that is required.
• Should the life detection subsystem constitute the entire biological payload of the
capsule, it will have to obtain its own sample.
• Again, a single sample will satisfy the requirements. The ATP assay and phosphate and
sulfur uptake experiments can be accommodated by a single culture chamber.
• The ATP, phosphate and sulfur assays can be made from aliquots taken from this
culture. The mechanical handling and transfer operations associated with these three
experiments can also be combined.
• The remaining three biological experiments are performed in situ.
• Of these, the experiments monitoring the metabolism of radioactive substrates and
photosynthesis detection through the use of radioactive substrates can probably be
combined into a single physical unit.
• The final transport system in this preliminary concept consists of a revolving
ring containing an array of test chambers. This ring accommodates each assay
to be made and positions each, including those from the in situ experiments,
in front of the readout station.
• Once a readout is obtained, the revolving ring indexes and a fresh test
chamber is positioned in front of the readout station.
• The entire operation is programmed so that the ATP, PO4. and sulfur
experiments are read at desired intervals as are the in situ experiments.
Readings for the latter are accommodated by a special chamber in the
revolving ring.
• This chamber effects an optical coupling between the plastic scintillator and
the photomultiplier tubes. The in situ experiments are located below the
readout chamber for this reason.
The following biologically important environmental parameters could be determined by the life detection subsystem.

1. Background Radiation

The planetary surface background at the sampling site can be determined by the radiation detection system used in the tracer
experiments through the incorporation of appropriate scintillators.

2. Temperature

Temperature will be monitored in conjunction with all metabolism, growth, or reproduction experiments to permit better
interpretation of the data. The same temperature sensor can be used to determine surface temperature.

3. Oxygen

If photosynthesis is detected, it is important to know whether oxygen is produced, as in the case with algae, or whether it is not as
in the case of photosynthetic bacteria. To determine this, an oxygen electrode would be incorporated into the photosynthesis
experiments. This electrode can be used to determine the oxygen concentration in the planetary atmosphere.

4. Ambient Light Intensity

Photomultiplier tubes are proposed as the primary sensors for the biological experiments. A relatively simple system utilizing an
appropriate range of neutral density filters might be used in conjunction with the photomultiplier tubes to make periodic readings
of the light intensity at the surface of the planet.
5. Soluble Orthophosphate Content of the Soil

In the phosphate uptake experiment, the phosphate concentration of the medium

will be known. If a known volume of the soil is introduced into the medium in
starting the experiment, and the soil and medium are mechanically mixed, any
orthophosphate present in the sample will go into solution. A “zero-time”
determination of the orthophosphate concentration in the liquid phase
immediately after mixing would reveal the amount of dissolved orthophosphate
contributed by the sample.

6. pH of Soil

In any experiment involving culturing of microorganisms, the pH of the culture is

important to the interpretation of the results. A pH electrode can be incorporated
for this purpose. If so, the “zero-time” determination of pH in the phosphate
uptake experiment would provide information on the pH of the soil sample.