It is necessary to preserve rumen fluid for transport from slaughterhouses to the pretreatment facilities for use in treating lignocellulosic biomass. In this study, we investigated how the preservation of rumen fluid at various temperatures affects its use in hydrolysis of waste paper. Rumen fluid was preserved anaerobically at 4, 20, and 35 degrees C for 7 days. The number of protozoa and fibrolytic enzyme activity after preservation at 4 degrees C were significantly higher than that after preservation at either 20 or 35 degrees C. Waste paper was subsequently treated with preserved rumen fluid at 37 degrees C for 48 h. Preservation at 20 degrees C remarkedly decreased the hydrolysis of waste paper. Xylanase activity in rumen fluid preserved at 35 degrees C increased during the treatment, which enhanced the solubilization of waste paper as comparable to the control and preservation at 4 degrees C. Pretreatment of waste paper with rumen fluid preserved at 4 degrees C showed that the fluid retained high fibrolytic activity, and reduced the loss of organic carbon as substrate for methanogens. Our results suggest that preservation of rumen fluid at 4 degrees C is most suitable for efficient pretreatment and methane fermentation of waste paper. (C) 2019 Published by Elsevier Ltd.