Adding plant biomass to a biogas reactor, hydrolysis is the first reaction step in the chain of biological events towards methane production. Maize silage was used to enrich efficient hydrolytic bacterial consortia from natural environments under conditions imitating those in a biogas plant. At 55–60°C a more efficient hydrolyzing culture could be isolated than at 37°C. The composition of the optimal thermophilic bacterial consortium was revealed by sequencing clones from a 16S rRNA gene library. A modified PCR‐RFLP pre‐screening method was used to group the clones. Pure anaerobic cultures were isolated. 70% of the isolates were related to Clostridium thermocellum. A new culture‐independent method for identification of cellulolytic enzymes was developed using the isolation of cellulose‐binding proteins. MALDI‐TOF/TOF analysis and end‐sequencing of peptides from prominent protein bands revealed cellulases from the cellulosome of C. thermocellum and from a major cellulase of Clostridium stercorarium. A combined culture of C. thermocellum and C. stercorarium was shown to excellently degrade maize silage. A spore preparation method suitable for inoculation of maize silage and optimal hydrolysis was developed for the thermophilic bacterial consortium. This method allows for concentration and long‐term storage of the mixed culture for instance for inoculation of biogas fermenters.

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