Escherichia coli produces molecular hydrogen (H-2) during glucose or mixed carbon (glucose and glycerol) fermentation. Dependence of H-2 production rate (V-H2) on glucose at different pHs was studied in a concentration dependent manner. During growth of wild-type on glucose, increasing glucose concentration from 0.05% to 0.2% resulted in the marked inhibition of V-H2. Inhibitory effect of glucose was shown at pH 7.5 and 6.5 but not pH 5.5. However, glycerol added in the growth medium with 0.1% glucose significantly increased VH, but different effects at different pHs were established upon glucose or glycerol assays. The results indicate that H-2 production is inhibited by glucose in a concentration dependent manner during glucose fermentation but glucose in combination with glycerol might enhance H-2 production during mixed carbon fermentation. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.