We had developed a new pretreatment system using cow rumen fluid to improve the methane production from lignocellulosic substrates. However, the pretreatment conditions differ from the in-situ rumen environment, therefore different microbes may be involved in plant cell wall decomposition. In the current study, shotgun metagenomic analysis using MiSeq platformwas performed to elucidate the bacteria which produce cellulase and hemicellulase in this pretreatment system. The rumen fluid which contained waste paper pieces (0.1% w/v) were incubated at 37 degrees C during 120 h. The fluid samples were collected from the reactor at each time-point and analyzed for chemical properties. Rumen microbial DNA was extracted from 0-h and 60-h samples and subjected to shotgun-metagenomic analysis. After pretreatment, approximately half of cellulose and hemicellulose contents of the waste paper were decomposed and some volatile fatty acids were accumulated. Clostridia (e.g., Ruminococcus and Clostridium) were the predominant bacteria before and after 60-h pretreatment, and their relative abundance was increased during pretreatment. However, Prevotella and Fibrobacter, one of the most dominant bacteria in-situ rumen fluid, were observed less than 3% before incubation and they were decreased after pretreatment. Genes encoding cellulase and hemicellulase were mainly found in Ruminococcus, Clostridium, and Caldicellulosiruptor. Calicellulosiruptor, which had not been previously identified as the predominant genus in lignocellulose decomposition in in-situ rumen conditions, might be considered as the main fibrolytic bacterium in this system. Thus, this study demonstrated that the composition of fibrolytic bacteria in this system was greatly different from those in the in-situ rumen. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.