Wet oxidation pretreatment, enzymatic hydrolysis and simultaneous saccharification and fermentation of clover–ryegrass mixtures

The potential of clover (Trifolium repens) and ryegrass (Lolium perenne) mixtures as raw materials for ethanol production was investigated. Wet oxidation, at 175, 185 or 195 °C during 10 min at two different oxygen pressures and with either addition or no addition of sodium carbonate, was evaluated as pretreatment method for clover–ryegrass mixtures. The enzymatic hydrolysis of cellulose was significantly improved after pretreatment. The highest conversion efficiency, 93.6%, was achieved for the sample pretreated at 195 °C, 10 min, 1.2 MPa and no addition of Na2CO3. For that sample, the overall glucose yield after pretreatment and hydrolysis was 75.5%. No inhibition of cellulose enzymatic conversion by the filtrates was observed. The simultaneous saccharification and fermentation of the pretreated material yielded cellulose conversions of 87.5 and 86.6%, respectively, with Saccharomyces cerevisiae and the filamentous fungus Mucor indicus, and revealed that no addition of nutrients is needed for the fermentation of clover–ryegrass hydrolysates.