The sensitivity of the requirement of Methanobacterium ruminantium strain Mlto a newcoenzyme, 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 nMHS-CoM. Coenzyme activity could be replaced by 2,2′-dithiodiethanesulfonic acid at a half-molar equivalent of the HS-CoM concentration, orby 2-(methylthio)ethanesulfonic acid on an equimolar basis. These data reveal a very sensitive and precise requirement for HS-CoMin the nutrition of this fastidious anaerobe.
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