Enzymatic conversion of waste paper to mono- and oligosaccharides

M-BIOTEK

English. Efficient utilization of waste products is a key step in the transition into a sustainable society. Recalcitrant lignocellulosic biomass is a major constituent of industrial and household waste, and its conversion into fuels represents a promising green alternative to fossil sources.Lignocellulosic biomass is a complex composite structure, containing mainly cellulose,hemicelluloses and lignin; this intricate matrix provides tensile strength in the plant cell wall and resistance against enzymatic degradation.There is a high demand for paper products in the world, and consequently, it is a major part of waste products. Cellulose represents the dominant component in paper and its conversion into monosaccharides and oligosaccharides could be a first step in generating value-added products such as fuels. Enzymatic cellulose degradation involves four main types of enzymes:endoglucanases (EGs), cellobiohydrolases (CBHs), lytic polysaccharide monooxygenases (LPMOs) and β-glucosidases (BGs).This thesis focuses on the conversion of waste paper to monosaccharides with a commercial enzyme cocktail (Cellic CTec 2), as well as conversion of oligosaccharides using three purified endoglucanases. Experiments were conducted on shredded office paper (SOP), cut cardboard (CCB) and shredded newspaper (SNP), that were all pretreated by steam explosion. A compositional analysis of SOP, CCB and SNP was performed in order to identify the ratios of the structural components. This analysis also enabled accurate determination of the conversion yields after enzymatic hydrolysis. The cellulose-hemicellulose-lignin-ash-others ratios were (41.4%-8.4%-2.6%-21.2%-26.0%), (58.7%-10.1%-16.3%-11.2%-3.5%), (31.6%-3.5%-39.9%-6.6%-24.1%) for SOP, CCB and SNP, respectively pretreated with steam explosion at 210°C for 14 min. An additive in paper proved a challenge in the conversion using Cellic CTec2 because of its alkaline properties; experiments where the acid loading was varied were therefore performed, in order to achieve the optimal pH for the enzymatic reaction. Following the acid-loading test, a 24-hour hydrolysis was performed, 54%, 46% and 29% of theoretical maximum yields was observed with enzyme loadings of 5 mg/g dry mass (DM) over 24 hours for SOP, CCB and SNP respectively. Furthermore, optimization experiments achieved 89.6%, 55.2% and 56.3% conversions with enzyme loadings of 25mg/g DM and 72-hour incubation.Three endoglucanases were produced using the eukaryotic expression host Pichia pastoris. The purified enzymes were able to produce a range of soluble cellodextrines from both pure cellulose and the waste paper substrates. Yields between 0 to 10% of the theoretical maximum yields were observed. Avicel, which contains highly crystalline cellulose, resulted in yields below 1.3%. Steam exploded SOP, CCB and SNP, on the other hand, resulted in yields of 7.3% in SOP with MaCel45A, 4.1% in CCB with MaCel45A and 13.5% in SNP with TaCel5A In addition to studying the release of soluble products, the effect of the enzymes oninsoluble cellulose was tested to better understand how the endoglucanases work. The results demonstrate how the endoglucanases reduce the degree of polymerization by introducing cuts on the insoluble cellulose chains. It thus seem that the endoglucanases hydrolyze the accessible regions of cellulose chain but not to an extent that leads to a high degree of conversion of soluble material.