1.  Methanogens Manish Patel

2. What are they?  Taxonomy  Classified as archaebacteria  Over 50 species, in 5 orders and 9 families  Typically appear coccoid or bacilli  Some have cell wall made of pseudopeptidoglycan or a layer of proteins  Obligate Anaerobes – O 2 is poisonous

3. Where are they on Earth?  Found in environments depleted of electron acceptors (O 2, NO 3, Fe 3+, SO 4 ):  Swamps and marshes  Largest natural methane source – 250 Tg  Sediments at the bottom of rivers and lakes/oceans  Digestive tracts of animals  Cattle livestock by humans - ~150 Tg  Extremophiles  Geothermal environments  Found in ice cores from 3 km below Greenland and 4-5 km below the surface in South African Gold Mines  Found in sediments from “White Smoker” chimneys, 2600m deep in the East Pacific Rise (Temp: 48-95˚C; Pressure: 200 atm)  Can reproduce in temperatures ranges of 15-100˚C

4. How do they survive?  Methanogenesis:  Formation of methane by microbes.  Use carbon dioxide, formate, methanol, methylamines, methylthiols and acetate for carbon source.  H 2 is their electron source.  Not to be confused with methano trophs

5. General Overview for CO 2 

6. Actual ∆G’ is ~ +40kJ/mol because of low H 2 (electron source) partial pressure. Catalyzed by formylmethanofuran dehydrogenase Reduction driven by transmembrane electrochemical Na + potential

7. Formyl transfer catalyzed by formyl transferase Tetrahydromethanopterin (H 4 MPT) is analogous to tetrahydrofuran (H 4 F) as a methyl- carrier Reversible hydrolysis of N 5,N l0 -methenyl- H 4 MPT to N 5 -formyl-H 4 MPT, is catalyzed by N 5,N 10 -methenyl-H 4 MPT cyclohydrolase

8. Catalyzed by methylene H 4 MPT dehydrogenase twice. Utilizes a reduced coenzyme F 420, a deazaflavine derivative and hydride acceptor (from H 2 ). Alternative enzymes use H 2 directly. F 420 is unique to methanogens.

9. F 420 H 2 is regenerated by an F 420 -dependent hydrogenase. All of these F 420 -dependent hydrogenases contain flavin, Ni, and Fe-S clusters.

10. Catalyzed by a transmembrane methyltransferase, which also acts as a sodium ion pump. Na + gradient could be used as mentioned before in MF formation or in a Na + -driven ATP synthase. Coenzyme M is also exclusive to methanogenic microbes.

11. Catalyzed by methyl-coenzyme M reductase Utilizes 2 coenzymes: 7-mercaptoheptanoylthreonine phosphate (HS-HTP) reduces Coenzyme M and displaces methane F 430, a nickel porphinoid, coordinates the displacement.

12. A F 420 -nonreactive hydrogenase activates H 2 and channels the electrons into the electron transport chain, and the electrons are transferred to CoM-S-S-HTE In some methanogens, this is coupled with a Na + pump instead of a H + pump. In one species, this complex contains nickel, FAD, non-heme iron, and acid-labile sulfur. Also, b -type cytochromes are present for use in an electron transport chain.

14. Alternative Carbon Sources

15. How does Methanogenesis play into the whole metabolism?

16. We know how they work. How can we find them in space?

19. Not what we imagined.

20. Looking for Methanogens  When looking for potential extraterrestrial methanogenic life, the presence of methane alone does not indicate life.  Considering the flux of methane helps to determine whether methane biogenic or abiogenic.  Focus on atmospheric methane at the surface.  The observed non-uniformity in methane could be due to localized microbial colonies or localized hydrogeochemical sources.  Also determine whether to look for past life or current life. One factor could be where liquid water was or is currently.

21. Methane Sources and Sinks  Methane on Mars could be produced from any one of the four potential sources:  1. Internal, such as volcanoes or hydrothermal sources  2. Exogenous, such as meteorites, comets, or interplanetary dust particles.  3. Internal, such as a hydrogeochemical process involving serpentinization.  4. Biological. On Earth:

22. Methane Sources and Sinks  Loss of methane on Mars  Primarily from photolysis, giving Mars’ methane a photochemical lifetime of about 300 years.  Reacting with other trace chemcials/oxidants (peroxide)  Diffusion into the regiolith  Significant sinks could be responsible for non-uniform CH 4 distribution.

23. Summing Up Methane Flux

24.  Spectroscopic studies  Taking into account doppler shifts and to find CH 4 signatures  Fourier Transform Spectrometer at the Canada–France–Hawaii Telescope  Infrared Telescop Facility (IRTF)  Gemini Telescope  Robots  Take soil samples for spectroscopy Techniques

25. Life Tests:  Carbon Isotopes:  Less energy to form a bond between 12 C and 12 C elements than between 12 C and 13 C elements.  It is a good indicator of life, as we know it on Earth.  PDB (Pee Dee Belemnite) Inorganic Standard value, R std = 13 C/ 12 C = 0.01123722  Requires a measureable quantity in the atmosphere. Mars requires instruments capable of measuring 0.01ppbv

26. Life Tests:  Chirality  Organic molecules are mostly left-handed on Earth, but amino acids from meteorites have been found to also be left-handed.  Overtime, organic molecules will tend to form a racemic mixture, so the presence of chiral organic molecules could indicate present life.  Fluorescence  F 420 detected with excitation-emission wavelengths of 400-468nm  Potential test if core samples can be obtained from deep enough, where methanogens would reside, past the permafrost.

27. References:  Blaut, M. “Metabolism of Methanogens” Antonie van Leeuwenhoek. 1994. 66. 187-208.  Thauer, R. K. “Biochemistry of Methanogenesis” Microbiology. 1998. 144. 2377-2406.  Atreya, S. K.; Mahaffy, P. R.; Wong, A-S. “Methane and related trace species on Mars: Origin, loss, implications for life, and habitability.” Planetary and Space Science. 2007. 55. 358-369.  http://microbewiki.kenyon.edu/index.php/Methanococcus_jannasc hii  Methanogens Wiki