Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 was investigated in an up-flow anaerobic sludge blanket (UASB) reactor. The reactor was operated under non-sterile conditions at 40(degrees)C and initial pH 8.0 at different hydraulic retention times (HRTs) (2-12 h) and glycerol concentrations (10-30 g/L). Decreasing the HRT led to an increase in hydrogen production rate (HPR) and hydrogen yield (HY). The highest HPR of 242.15 mmol H-2/L/d and HY of 44.27 mmol H-2/g glycerol consumed were achieved at 4 h HRT and glycerol concentrations of 30 and 10 g/L, respectively. The main soluble metabolite was 1,3-propanediol, which implies that Klebsiella sp. was dominant among other microorganisms. Fluorescence in situ hybridization (FISH) revealed that the microbial community was dominated by Klebsiella sp. with 56.96, 59.45, and 63.47% of total DAPI binding cells, at glycerol concentrations of 10, 20, and 30 g/L, respectively. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.