The effect of superheated steam pretreatment of wheat straw on fermentation. Biomass recalcitrance to bioconversion for microbial production of fuels and co-products is directly related to the structure and composition of lignocellulosic biomass. The effect of superheated steam (SS) pre-treatment on biomass fermentation by Clostridium thermocellum was investigated. Wheat straw was ground to < 355 µm and exposed to three methods of pretreatment of various severities: i) soaking in 119 °C boiling water (BW) under absolute pressure of 193 kPa for 15 minutes, which corresponded to a severity factor of 1.73; ii) processing with SS at atmospheric pressure for 15 minutes; and at 180, 200 and 220° C, which corresponded to a severity factor ranging from 3.53 to 4.70 and iii) soaking in 119°C BW under absolute pressure of 193 kPa for 15 minutes, followed by processing with SS at atmospheric pressure at 180, 200, and 220 °C, with a corresponding severity factor of 5.26 to 6.44. Processing with SS was conducted at three temperatures: 180, 200, and 220 °C. The intensity of pre-treatment was expressed through a 'treatment severity factor'. Wheat straw samples were then used as substrates in fermentation reactions with C. thermocellum. The most pronounced effects were observed at the highest severity of 6.44, corresponding to treatment in BW followed by SS at 220°C. This resulted in structural changes in the material reflected in an increased 'contribution of amorphous cellulose' (CAC) of 22%, an increase of hydrogen production of 94.2%, an increase of carbon dioxide production of 221.5% and a 160% increase in ethanol production. The combinatorial effect of BW and SS pre-treatment was more effective in improving substrate conversion than either BW or SS pre-treatment alone.