Ethanol Production from Lignocellulosic Materials: Potential of Continuous Cultivation, Immobilisation, and Zygomycetous Fungi

Doctoral thesis, 2005

This thesis deals with the production of bio-ethanol, a sustainable alternative to fossil fuels, from dilute-acid lignocellulosic hydrolysate in a continuous immobilised cell system and fermentation of the hydrolysate by a zygomycete organism, M. indicus. An immobilised cell system was applied in order to increase the ethanol productivity in continuous cultivation. While the traditional continuous cultivation on the hydrolysate could only be carried out with a dilution rate of less than 0.2 h-1, the immobilised cells allowed up to 0.6 h-1. Immobilised cells in continuous cultivation could also accomplish in situ detoxification against the effects of the inhibitors. Of known inhibitors, almost all furfural and 60% of HMF were converted. Yet continuous cultivation of severely inhibiting hydrolysate was not successful without detoxification. Detoxification with overliming was intended to remove the inhibitors. The effects of different variables in overliming were investigated for the severely inhibiting hydrolysates. The process involves increasing pH with Ca(OH)2 to 10, 11 or 12 at temperature 25°C and 60°C, and holding the alkali pH and the temperature at constant values for times up to 170 h. The most significant effect of overliming was a sharp decrease in the concentration of furfural and HMF, whereas the concentration of acetic acid remained unchanged and the decrease in the total phenolic compounds was less than 30%. The fermentability was improved at the expense of sugar loss to different degrees according to the conditions applied. In a study of alternative organisms from the class of zygomycetes for fermentation of dilute-acid lignocellulosic hydrolysate, three genera of Rhizopus, Mucor and Rhizomucor were tested. M. indicus showed promising characteristics as an ethanol producer. The degree of aeration was found to affect the broad range of sugar consumption and the ethanol yield. Under aerobic conditions, M. indicus could consume glucose, mannose, galactose, xylose, arabinose and sugars within the dilute-acid hydrolysate. Under anaerobic conditions, xylose was not consumed, while hexoses were comsumed and yielded up to 0.46 g/g ethanol. On the other hand, aeration allowed xylose utilisation by M. indicus, but its ethanol yield depended on the rate of aeration, with the highest yield of 0.18 g/g. Cultivation of the fungus in dilute-acid hydrolysate including hexoses and xylose resulted in ethanol yield of 0.44 g/g under anaerobic and oxygen-limited conditions. M. indicus produced metabolites similar to those of baker's yeast, S. cerevisiae, with ethanol as the main metabolite as well as glycerol and trace amounts of several carboxylic acids as the by-products.