Lignocellulosic biomass as co-substrate enhances the 1,3-propanediol (1,3-PD) production of anaerobic fermenters by increasing their conversion yield from glycerol. To improve 1,3-propanediol (1,3-PD) production by this efficient approach, Clostridium butyricum 15-42 was supplemented with lignocellulosic biomasses (starch free fiber (CPF) from cassava pulp and xylan) as co-substrates. The 1,3-PD production and growth of C. butyricum were considerably higher in glycerol plus CPF and xylan than in glycerol alone, whereas another major polysaccharide (cellulose co-substrate) failed to improve the 1,3-PD production. C. butyricum 15-42 showed no degradation ability on cellulose powder, and only weak activity and slight growth on xylan. However CPF supplemented with xylan strongly enhanced the transcription levels of the major enzymes of 1,3-PD production (glycerol dehydratase, 1,3-propanediol dehydrogenase, and glycerol dehydrogenase). The intracellular redox reactions maintained equal balance in the supplemented media, suggesting that CPF plus xylan promotes 1,3-PD production in the reductive pathway. This promotion is probably mediated by NADH, which is effectively regenerated by small amounts of released oligosaccharides and subsequent activation of the glycerol oxidative pathway. Both supplements also improved the 1,3-PD production at high glycerol concentration. Therefore, supplementation with lignocellulolytic polysaccharides such as xylan can improve the production and productivity of 1,3-PD from glycerol in C. butyricum. Direct supplementation of CPF with xylan in 1,3-PD production has not been previously reported. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.