Lignocellulosic biomass is a renewable resource that can be utilized for the production of biofuels, chemicals, and bio-based materials. Biochemical conversion of lignocellulose to advanced biofuels, such as cellulosic ethanol, is generally performed through microbial fermentation of sugars generated by thermochemical pretreatment of the biomass followed by an enzymatic hydrolysis of the cellulose. The aims of the research presented in this thesis were to address problems associated with pretreatment by-products that inhibit microbial and enzymatic biocatalysts, and to investigate the potential of utilizing residual streams from pulp mills and biorefineries to produce hydrolytic enzymes.
A novel method to detoxify lignocellulosic hydrolysates to improve the fermentability was investigated in experiments with the yeast Saccharomyces cerevisiae. The method is based on treatment of lignocellulosic slurries and hydrolysates with reducing agents, such as sodium dithionite and sodium sulfite. The effects of treatment with sodium borohydride were also investigated. Treatment of a hydrolysate of Norway spruce by addition of 10 mM dithionite resulted in an increase of the balanced ethanol yield from 0.03 to 0.35 g/g. Similarly, the balanced ethanol yield of a hydrolysate of sugarcane bagasse increased from 0.06 to 0.28 g/g after treatment with 10 mM dithionite. In another study with a hydrolysate of Norway spruce, addition of 34 mM borohydride increased the balanced ethanol yield from 0.02 to 0.30 g/g, while the ethanol productivity increased from 0.05 to 0.57 g/(L×h). While treatment with sulfur oxyanions had a positive effect on microbial fermentation and enzymatic hydrolysis, treatment with borohydride resulted in an improvement only for the microbial fermentation. The chemical effects of treatments of hydrolysates with sodium dithionite, sodium sulfite, and sodium borohydride were investigated using liquid chromatography-mass spectrometry (LC-MS). Treatments with dithionite and sulfite were found to rapidly sulfonate inhibitors already at room temperature and at a pH that is compatible with enzymatic hydrolysis and microbial fermentation. Treatment with borohydride reduced inhibitory compounds, but the products were less hydrophilic than the products obtained in the reactions with the sulfur oxyanions.
The potential of on-site enzyme production using low-value residual streams, such as stillage, was investigated utilizing recombinant Aspergillus niger producing xylanase and cellulase. A xylanase activity of 8,400 nkat/ml and a cellulase activity of 2,700 nkat/ml were reached using stillages from processes based on waste fiber sludge. The fungus consumed a large part of the xylose, the acetic acid, and the oligosaccharides that were left in the stillages after fermentation with S. cerevisiae. In another study, the capability of two filamentous fungi (A. niger and Trichoderma reesei) and three yeasts (S. cerevisiae, Pichia pastoris, and Yarrowia lipolytica) to grow on inhibitory lignocellulosic media were compared. The results indicate that the two filamentous fungi had the best capability to utilize different nutrients in the media, while the S. cerevisiae strain exhibited the best tolerance against the inhibitors. Utilization of different nutrients would be especially important in enzyme production using residual streams, while tolerance against inhibitors is desirable in a consolidated bio-process in which the fermenting microorganism also contributes by producing enzymes.