Oxygen Tolerance in Methanogens

1
Oxygen Tolerance in Methanogens
Jill K. Jackson1,2, Dr. Timothy Kral2,3
1William Jewell College Depts. of Biology and
Chemistry, 2University of Arkansas Center for
Space and Planetary Sciences, 3University of
Arkansas Dept. of Biological Sciences
Introduction
Objectives
Results
Exposure to oxygen has not thus far shown a clear
effect on methanogen growth, although in many
cases, cultures exposed to atmospheric O2 in the
absence of Na2S have recovered just as well as
their counterparts receiving treatment with the
reducing agent. Three trials using M. wolfeii and
two using M. barkerii have been performed, with
methane production being analyzed daily following
O2 exposure. Preliminary results from M. wolfeii
are given in Figures 3 and 4 however, because
this procedure is still being perfected and many
more trials are upcoming, no conclusions have
been drawn.

• To analyze the recovery of three strains of
  methanogens, M. barkeri, M. wolfeii, and M.
  formicicum, upon exposure to oxygen both in the
  presence and absence of the commonly used
  reducing agent Na2S.
• To analyze whether the production of methane
  following oxygen exposure is due to the fact that
  only a few surviving organisms are present or to
  many surviving organisms being present but
  hindered by atmospheric O2.

When analyzing the possibility of life beyond
Earth, an important organism to consider is the
methanogen. Methanogens are highly anaerobic and
can reduce CO, CO2, formate, methanol,
methylamines, or acetate to methane (Reeve, Annu.
Rev. Microbiol., 1992). Mars is one location
within the solar system which could potentially
be conducive to this form of life, and the
implications of these organisms existing on Mars
would be far-reaching. However, to test the
viability of methanogens under Martian
conditions, it is necessary to perform thorough
analyses in the laboratory. Maintenance of a
highly anaerobic environment is somewhat of a
hindrance to the advancement of methanogenic
studies. However, researchers have shown that
oxygen exposure does not necessarily have a
lethal effect rather, some strains of
methanogens can endure exposure to high levels of
oxygen (Kato et. al., Braz. J. Chem. Eng., 1997).
Experimental results indicating high oxygen
tolerance in methanogens are important in the
field of exobiology. Such a discovery could lead
to increased expediency in experimentation and
thereby to increased understanding of methanogens
and their possible habitats.
Table 1. Ideal growth conditions for the strains
used in these experiments.
Materials and Methods
These experiments utilize the strains of
Methanogens listed in Table 1. Optical density
measurements were taken prior to each experiment
to determine that cultures were actively growing.
Cells were centrifuged, washed and suspended in
sterile buffer to remove residual Na2S both prior
to and following oxygen exposure. This buffer
solution was used to inoculate tubes of media
both containing and lacking Na2S. All tubes
except the controls were exposed to atmospheric
oxygen, and a pair of tubes (one containing and
one lacking Na2S) were removed from oxygen
contact at designated time intervals. Tube
contents were centrifuged and the supernatant
discarded to remove any residual oxygen present
in the exposed media from contact with cells.
Cells were re-suspended in sterile buffer and
then introduced into ideal conditions and allowed
to recover. For several days following oxygen
exposure, analysis using Gas Chromatography (GC)
was performed to quantify methane composition of
the headspace, a characteristic indicative of
methanogen growth.
Figure 3. Methane Production of M. wolfeii six
days following O2 exposure in the presence of
Na2S.
Figure 1. Dr. Tim Kral working in the anaerobic
chamber in the exobiology laboratory.
Figure 4. Methane Production of M. wolfeii six
days following O2 exposure in the absence of
Na2S.
Future work
The remainder of the summer will be devoted to
perfecting the procedure and performing more
trials with all three organisms to attain
reliable, repeatable data.
Acknowledgements
Figure 2. MM and MS media, respectively,
including control tubes and tubes exposed to O2
in the presence and absence of Na2S.
A big thanks to Dr. Tim Kral for his expert
guidance and support, and to NASA for funding the
REU program.