New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressureized atmosphere

TLDR

The authors investigated the sensitivity of Methanobacterium ruminantium strain M1 to the coenzyme 2‑mercaptoethanesulfonic acid (HS‑CoM) using newly developed rapid culture handling techniques. They employed sealed tubes containing 80 % H₂/20 % CO₂ at 2–3 atm, a modified Hungate system that reduces variability and simplifies media handling for methanogenic bacteria. The assay detected HS‑CoM sensitivity down to 5 nM, with half‑maximal growth at 25 nM, and showed that its activity could be substituted by dithiodiethanesulfonic acid or methylthioethanesulfonic acid, demonstrating a highly precise requirement for HS‑CoM in this fastidious anaerobe.

Abstract

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.