The objective of this study was to enhance the hydrogen production rate of dark fermentation in batch operation. For the first time, the hyperthermophilic pure culture of Thermotoga neapolitana cf. Capnolactica was applied at elevated biomass concentrations. The increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight/L led to a general acceleration of the fermentation process, reducing the fermentation time of 5 g glucose/L down to 3 h with a lag phase of 0.4 h. The volumetric hydrogen production rate increased from 323 (��11) to 654 (��30) mL/L/h with a concomitant enhancement of the biomass growth and glucose consumption rate. The hydrogen yield of 2.45 (��0.09) mol H2/ mol glucose, the hydrogen concentration of 68% in the produced gas and the composition of the end products in the digestate, i.e. 62.3 (��2.5)% acetic acid, 23.5 (��2.9)% lactic acid and 2.3 (��0.1)% alanine, remained unaffected at increasing biomass concentrations.