R. S. Wolfe

The oxidation of ethanol to acetate by cultures of Methunobacillus omelianskii was found by Barker to be dependent upon carbon dioxide (1) ; using C140Z, Stadtman and Barker demonstrated conclusively that carbon dioxide was the precursor of methane (2). Recently Johns and Barker have reported hydrogen formation from ethanol by resting cells of M. omelianskii in the absence of carbon dioxide (3). The mechanism of carbon dioxide reduction to methane by M. omelianslcii as well as the mechanism of methane formation by other organisms is unknown. Current knowledge of the nutrition and fermentative patterns of the methane bacteria has been reviewed by Barker (4). The present communication is concerned with the formation of methane by cell-free extracts of &!f. omeliunskii; a brief report of some of these findings has appeared (5).

The sensitivity of the requirement of Methanobacterium ruminantium strain M1 to a new coenzyme, 2-mercaptoethanesulfonic acid (HS-CoM) was examined by use of new techniques that were developed for rapid and efficient handling of large numbers of cultures of methanogenic bacteria. The system uses sealed tubes that contain a gas mixture of 80% hydrogen and 20% carbon dioxide under a pressure of 2 to 3 atm. This modification of the Hungate technique reduces variability among replicate cultures and simplifies the dispensing, sterilization, and storage of liquid media as well as the transfer and maintenance of methanogenic bacteria. Results indicate a limit of sensitivity of the assay at 5 nM HS-CoM, with half-maximal growth at 25 nM HS-CoM. Coenzyme activity could be replaced by 2,2'-dithiodiethanesulfonic acid at a half-molar equivalent of the HS-CoM concentration, or by 2-(methylthio)ethanesulfonic acid on an equimolar basis. These data reveal a very sensitive and precise requirement for HS-CoM in the nutrition of this fastidious anaerobe.