Kluyvera species strain OM3 isolated from spent mushroom substrate could produce a high level of cellulase-free xylanase (5.12 U/mL). This xylanase showed maximum activities at 70 degrees C and pH 8.0, which could retain 100% and 71% activity after 1 h incubation at 60 degrees C and 70 degrees C, and maintain stability over a wide range of pHs (5.0-9.0), indicating its thermal and pH stability. Moreover, the xylanase could hydrolyze untreated lignocellulosics (e.g., palm oil fiber) to reducing sugars with a yield of 27.1-46.9 mg/g. A co-culture consisting of Kluyvera sp. strain OM3 and Clostridium sp. strain BOH3 could directly convert birchwood xylan to 1.2 g/L butanol, which was comparable to the amount of butanol (1.7 g/L) generated via separate hydrolysis by the xylanase and fermentation by Clostridium sp. strain BOH3. This is the first report on the production, characterization of a xylanase from genus kluyvera and its application for butanol production directly from hemicelluloses. (C) 2012 Elsevier Ltd. All rights reserved.