Analysis of Substrates for Methanogenesis in Anaerobic Sludges Using Specific Inhibitors

We examined what kind and how much of the specific inhibitor is effective for the estimation availabilities of substrates for methanogens in anaerobic sludges. The chloroform (0.1%, v/v) inhibited 90% of the methanogenesis in an anaerobic sludge (LD), which was cultivated with glucose, and was more effective than 2-bromoethanesulfonic acid (BES, 0-200 mM). Hydrogen, acetate and propionate were linearly accumulated when the methanogenesis was inhibited by 0.1% of chloroform. The propionate accumulation rate in the presence of chloroform was identical with that in the presence of high partial pressure of hydrogen (H₂:CO₂ = 20:80) which inhibits syntrophic H₂- producing fatty-acids-oxidizing bacteria. This shows that the methanogens utilized hydrogen and acetate converted by syntrophic hydrogen-producing propionate-oxidizing bacteria through interspecies hydrogen transfer. Using chloroform (0.1%) as the specific inhibitor, in the LD sludge 49% of methane was estimated to be derived from propionate. The H₂ + CO₂ and acetate from the other pathway (NS), contributed 2 and 34% of the methanogenesis, respectively. This method was also applied to a municipal sewage digester sludge (MD). In the MD sludge, methanogenesis from propionate, H₂ + CO₂ and acetate via the NS pathway were 16, 2 and 78%, respectively. Major substrates for methanogenesis, therefore, could be estimated in anaerobic sludges.