Browsing by Author "Kruus, Kristiina, Research Prof., VTT Technical Research Centre of Finland, Espoo, Finland"

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Development of pretreatment technology and enzymatic hydrolysis for biorefineries
(2014) Kallioinen, Anne
School of Chemical Technology | Doctoral dissertation (article-based)
The growing demand for energy, materials and food, depletion of fossil raw material reservoirs and increasing environmental concerns have all increased interest in renewable resources. Lignocellulosic biomass is an alternative for replacing fossil raw materials in the production of fuels, materials and various chemicals. Lignocellulose present in plant cell walls consists mainly of polysaccharides, cellulose and hemicellulose, and aromatic lignin. These major components form a complex structure that is resistant to microbial and enzymatic activity. Due to the recalcitrant structure of plant cell walls, lignocellulosic raw materials must be pretreated before their enzymatic hydrolysis to monosaccharides. Various pretreatment methods; chemical, physical, biological or their combinations, have been developed. After pretreatment, polysaccharides can be hydrolysed enzymatically to monosaccharides, which in turn can be fermented to different products such as ethanol. Currently the first commercial scale lignocellulosic ethanol plants have started production. A secure supply of biomass is one of the key factors for a feasible biorefinery, and new alternative feedstocks are still required especially in northern climates in order to fulfil the raw material demands of biorefineries in a sustainable way. In addition, development of new pretreatment technologies and more efficient enzymatic hydrolysis are needed. New lignocellulosic feedstocks and improved pretreatment methods were studied in the work described in this thesis. Reed canary grass and barley straw were found to be interesting carbohydrate-rich raw materials that could be pretreated by steam explosion and hydrolysed enzymatically with yields comparable to those obtained from wheat straw. Selection of the most favourable harvest time for reed canary grass, autumn or spring, was studied in relation to pretreatment and hydrolysis yields. Spring harvested reed canary grass was found to be the more suitable raw material as it had a higher cellulose content and the pretreated fibre was hydrolysed more efficiently compared to autumn harvested material. A new pretreatment method using sodium carbonate and oxygen pressure was developed. The alkaline oxidation method fractionated biomass into a carbohydrate-rich fibre and a dissolved fraction containing most of the lignin. The produced carbohydrate-rich fibre could be efficiently hydrolysed by enzymes and the hydrolysis was also efficient at 12% dry matter content. Compared to the 52% total glucose yield obtained in enzyme hydrolysis of spruce after pretreatment by steam explosion, a significantly higher glucose yield of 84% was obtained in hydrolysis after alkaline oxidation. Different kinds of raw materials, such as spruce, birch and sugar cane bagasse, could be efficiently pretreated by alkaline oxidation. The main effects of alkaline oxidation pretreatment were dissolution and partial degradation of lignin and hemicellulose. Some galactoglucomannan and xylan was solubilised and further oxidised to other products, and therefore relatively low yields of hemicellulose were obtained. Organic acids were formed as degradation products of lignin and carbohydrates. Process conditions were partially optimized using spruce as raw material in order to improve the efficiency of alkaline oxidation. The pretreatment could be accelerated by increasing the treatment temperature, by the use of copper-phenanthroline catalyst, and by decreasing the particle size of the raw material. Further optimization of e.g. alkali dosage and the solid to liquid ratio is still required to improve hemicellulose yield and economical feasibility. Fibre fractions of alkaline oxidation could be hydrolysed by low enzyme dosages, 2–4 FPU/g dry matter. Significantly higher enzyme dosages were required in the hydrolysis of steam exploded materials, probably due to the inhibitory effect of the high residual lignin content after the pretreatment. The efficient hydrolysis of alkaline oxidised materials by low enzyme dosages can decrease enzyme costs or enable shorter hydrolysis time. In order to further improve the hydrolysis efficiency and decrease the required enzyme dosage, enzyme mixtures were optimized regarding the major enzymes needed in biomass hydrolysis. Optimized mixtures of thermostable enzymes were found to have significantly different proportions of cellobiohydrolases, endoglucanases and xylanase than the optimized mixtures of Trichoderma reesei enzymes. Although different, the significant role of cellobiohydrolases was demonstrated in both types of mixtures. The results also indicated that high xylanase activity was required in the hydrolysis of pretreated materials having decreased enzyme accessibility to cellulose due to high xylan content or possibly due to drying of the substrate. The hydrolysis performance of optimized enzyme mixtures of five thermostable enzyme components was shown to be close to that of state-of-the-art commercial mixtures.
Enzymatic hydrolysis of cellulose in aqueous ionic liquids
(2014) Wahlström, Ronny
School of Chemical Technology | Doctoral dissertation (article-based)
Total enzymatic hydrolysis of the polysaccharides in lignocellulosic biomass to monosaccharides is currently a focus research area. The monosaccharides obtained from lignocellulose hydrolysis can be used for the production of platform chemicals and biofuels, most notably ethanol. One major challenge in the commercialization of lignocellulosic ethanol production is the recalcitrance of lignocellulosics towards enzymatic hydrolysis, necessitating efficient pretreatment of the lignocellulosic feedstock. Certain ionic liquids (ILs, salts with melting points below 100 °C) dissolve cellulose and even lignocellulosic biomass and are as such interesting candidates for pretreatment technology. However, cellulose-dissolving ILs have been found to severely inactivate the hydrolytic enzymes (cellulases) employed in cellulose hydrolysis. This work focuses on elucidating how certain ILs affect the action of cellulases in cellulose hydrolysis. The main emphasis was on the action of purified monocomponent Trichoderma reesei cellulases, but some commercial cellulase preparations were also studied in IL matrices. Hydrolysis experiments were made in solutions containing up to 90% of the two cellulose-dissolving ILs 1-ethyl-3-methylimidazolium acetate ([EMIM]AcO) and 1,3-dimethylimidazolium dimethylphosphate ([DMIM]DMP). The presence of increasing amounts of IL led to decreasing yields of solubilised saccharides in enzymatic hydrolysis. Depending on the IL and cellulase, no soluble saccharides were released in hydrolysis matrices containing over 40–50% IL. There were clear differences in the severity of the effects of different cellulose-dissolving ILs on cellulase action. [EMIM]AcO was generally more harmful for cellulase action than [DMIM]DMP. Pure [EMIM]AcO completely inactivated T. reesei endoglucanase in 4 h in residual activity measurements, whereas pure [DMIM]DMP supported considerable cellulase activity for at least three days. These results were confirmed by time curves of microcrystalline cellulose (MCC) hydrolysis in matrices containing the two ILs. Cellulose-dissolving ILs based on carboxylate salts of the organic superbases 1,1,3,3-tetramethylguanidine (TMG) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) have recently become available. These compounds are distillable under relatively mild conditions and are thus recyclable. However, these ILs were found to be at least as harmful for cellulase action as the studied imidazolium-based ILs and did thus not offer any benefits in terms of enzyme compatibility. T. reesei endoglucanases were unable to reduce the molecular weight of MCC in buffer or in any aqueous matrix containing IL, except in 90% (v/v) [DMIM]DMP in which the MCC was partially dissolved. Cellulose-dissolving ILs were found to be basic in aqueous solution. According to the results in this work, the pH increase caused by IL basicity was not the main reason for the observed cellulase inactivation. Cellulases with confirmed activity at high pH did not perform better than acidic or neutral cellulases in IL solutions. Some indications were however obtained that cellulase thermostability may be associated with better activity in cellulose-dissolving ILs. The studied ILs were found to have very detrimental effects on saccharide analytics. A capillary electrophoresis (CE) method was developed for the analysis of mono- and oligosaccharides in matrices containing ILs. With this CE method, the yields and product distribution of cello-oligomers produced in the hydrolysis experiments could be determined. It was found that the presence of ILs shifted the product distribution to larger cello-oligomers for some cellulases. The CE method was also used to monitor the hydrolysis of cello-oligomers with Aspergillus niger β-glucosidase in IL matrices. This β-glucosidase was found to be very IL sensitive. ILs were found to affect the cellulose binding of T. reesei cellulases. The cellulase binding to MCC in solutions with [DMIM]DMP and [EMIM]AcO was studied with radiolabeled T. reesei Cel5A (endoglucanase II) and Cel7A (cellobiohydrolase I) and their respective core domains. Cel7A was able to bind to MCC with its core domain, whereas it was shown that Cel5A was very dependent on its CBM for efficient substrate binding. High cellulose binding affinity was not necessary for all the cellulases in order for them to be hydrolytically active. [EMIM]AcO interfered more with cellulase substrate binding than [DMIM]DMP. The binding ability of the T. reesei carbohydrate-binding modules (CBMs) was very IL sensitive.