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The focus of this study was to determine the effects of pretreatment of wheat straw by superheated steam (SS) alone or in combination with boiling water (BW) on biomass structure and yields of fermentation products (cell mass and fermentation end-products) by Clostridium thermocellum. Different cultivars of wheat straw were ground to a particle size less than 355 µm, and exposed to the following methods of pretreatment: i) 15 min soaking in 119 °C boiling water under absolute pressure of 193 kPa, followed by processing with SS at atmospheric pressure at different temperatures and retention times; ii) 15 min processing with SS at atmospheric pressure; and iii) 15 min soaking in 119 °C boiling water under absolute pressure of 193 kPa. Processing with SS was conducted at a variety of temperatures in the range of 180-220 °C. The severity of pretreatment was expressed through a treatment severity factor as a measure of harshness of treatment. Pretreatment combinations of boiling water with superheated steam at different retention times inside the SS chamber were also investigated. Wheat straw samples were then used as substrates in fermentation reactions with C. thermocellum. The most noticeable effects on biomass structure and fermentation were observed at the highest severity factor of 6.5, corresponding to 15 min pretreatment with boiling water followed by 15 min treatment with SS at 220˚C. This pretreatment provided the maximum increase in percentage of contribution of amorphous cellulose (% CAC), and the highest fermentation yield in terms of hydrogen, carbon dioxide, and ethanol production. / October 2015