Methanogens in a freshwater pond

All methanogens belong to the domain Archaea and are strict anaerobes. They are called methanogens because they degrade organic matter to methane by feeding on those components. They thrive in a large variety of habitats among which are anoxic sediments of freshwater lakes. As methanogens grow quite slowly a lot of patience is needed to cultivate them in the lab.

The Flötenteich, a small freshwater pond in Oldenburg, Germany, on a cold day in April (air temperature: 11°C, water temperature: 8°C).

To cultivate methanogens, mud from the pond is collected into a cleaned jar of marmalade. To keep air out, the jar is filled up to the top ans the lid is sealed with cling film. The sample is incubated at 30°C in the lab.

Left side: A mud sample was collected from the small hole into the jar. Location: Flötenteich in Oldenburg, Germany. Right side: The jar containing the mud sample behind the hole it originates from.

To enrich methanogens from collected sediments, anoxic medium in serum bottles is inoculated with the mud. The headspace is gassed with a mixture of CO₂ & N₂ or CO₂ & H₂ to keep oxygen out. To give methanogens time to grow, the serum bottles are incubated at 30°C for about 20 days.

Serum bottles inoculated with sediment to enrich methanogens.

Methanogens fluoresce blue when excited with UV-light under an epifluorescence microscope. Thus they can be easily detected in samples without the need to stain any cells.

Epifluorescence microscopy of methanogens. Cells show characteristic blue autofluorescence.

Methanogenic Archaea possess the red-ox-carrier F₄₂₀ and thus show blue autofluorescence when excited with UV-light. As this cofactor is unique to them, they can be easily identified using epifluorescence microscopy.

Autofluorescent cells of methanogens in epifluorescence microscopy.

A different method for cultivating methanogens is the deep agar dilution technique. Here cells are mixed with liquid agar medium in a test tube and the headspace is gassed with a mixture of CO₂ & N₂ or CO₂ & H₂ to keep oxygen out. When the agar solidifies, test tubes are kept at 30°C and colonies start to grow over time. The produced methane forms large gas bubbles in the agar.

Colonies of methanogens in a test tube from a deep agar dilution series. Small black colonies grow between large bubbles of methane.

References:

• Cheeseman, P., Toms-Wood, A. and Wolfe, R. S. (1972). Isolation and properties of a fluorescent compound, Factor420, from Methanobacterium strain M.o.H. J. Bacteriol., 112:527–531.
• Madigan, M., Martinko, J. and Parker, J. (2003). Brock – Biology of microorganisms. Prentice Hall, Pearson Education, Inc., Upper Saddle River, NJ, USA, 10th edition.
• Mink, R. W. and Dugan, P. R. (1977). Tentative identification of methanogenic bacteria by fluorescence microscopy. Appl. Environ. Microbiol., 33:713–717.

Photos:

• All photos by me (Thalassiosira).