Cultivation of Methanogens

Abstract

Methanogens are methane-producing archaea that thrive only under strictly anaerobic conditions where electron acceptors (sulfate, nitrate, oxidized forms of metals, etc.) other than CO₂ are depleted. Because of their nature, not only anoxic but highly reduced conditions with a redox potential as low as −300 mV must be retained during all processes of cultivation by the following essential steps: flushing out air from medium with O₂-free gases, sealing with gas-tight closures, and supplying reducing agents. The medium used for methanogens should have CO₂/bicarbonate/carbonate buffering system and is composed of minerals, trace metals, vitamins, and methanogenic substrates. Based on the limited number of substrates known, unless otherwise specified, H₂, acetate, or methanol is generally supplied to the medium as energy source. The protocol described here introduces the basic techniques for cultivation (pure culture and enrichment) of methanogens including medium preparation, sample inoculation, and colony isolation using Hungate roll tube, deep agar slant, and recently developed six-well plate method and coculture methods. The protocols are simple and easy and do not require an anaerobic box, the most costly equipment.