Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H-2) production than untreated waste. Nitrogen (N-2) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H-2 partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 +/- 0.06 to 1.98 +/- 0.1 mol-H-2 mol(-1) glycerol(consumed) by optimising N-2 sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N-2 sparging, the H-2 yield increased to 2.73 +/- 0.14 mol-H-2 mol(-1) glycerol(consumed), which was 2.22-fold higher than the non-N-2 sparged H-2 yield (1.23 +/- 0.06 mol-H-2 mol(-1) glycerol(consumed)). Copyright (C) 2010, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.