Abstract
Over the past few years, the interest in lignocellulose biomass biorefineries has shifted from the production of biofuels to the development of processes for high-added value chemical synthesis. The present work attempts to develop processes for the valorization of lignocellulosic substrates, namely beechwood, pine and corn stover, with the aim to produce fructose and 5-hydroxymethylyfurfural (HMF). Additionally, this thesis examines the discovery and production of a galactose oxidase from the filamentous fungus Fusarium oxysporum as well as its ability to catalyze oxidation reactions on HMF for the production of furan monomers. In the first stage of the thesis, beechwood biomass was studied as the main feedstock for the production of HMF. Initially, these samples were subjected to an oxidative pretreatment (OxiOrganosolv) with the use of aqueous solutions of ethanol (EtOH), acetone (ACO) and tetrahydrofuran (THF) as the solvent. Subsequently, enzymatic saccharification and fermentatio ...
Over the past few years, the interest in lignocellulose biomass biorefineries has shifted from the production of biofuels to the development of processes for high-added value chemical synthesis. The present work attempts to develop processes for the valorization of lignocellulosic substrates, namely beechwood, pine and corn stover, with the aim to produce fructose and 5-hydroxymethylyfurfural (HMF). Additionally, this thesis examines the discovery and production of a galactose oxidase from the filamentous fungus Fusarium oxysporum as well as its ability to catalyze oxidation reactions on HMF for the production of furan monomers. In the first stage of the thesis, beechwood biomass was studied as the main feedstock for the production of HMF. Initially, these samples were subjected to an oxidative pretreatment (OxiOrganosolv) with the use of aqueous solutions of ethanol (EtOH), acetone (ACO) and tetrahydrofuran (THF) as the solvent. Subsequently, enzymatic saccharification and fermentation experiments were carried out on the cellulose-rich pulps that derived from the pretreatment with the aim of evaluating them as the raw material for fructose production. The best results were observed for the sample pretreated at 175 oC for 120 min, leading to the production of 55.2 g fructose/ 100 g pretreated biomass. As a result, this sample was used for a scale-up saccharification and isomerization experiment that led to the production of a hydrolysate with a concentration of 104.5 g/L fructose and 25.0 g/L glucose. This hydrolysate was then used to study the chemical dehydration of sugars to HMF using acid solvents. Out of the all the solvents studied, formic acid (3.5% w/w) proved to be the best performing one leading to the production of HMF with a 55.8% selectivity. In the next stage of the thesis, beechwood and pine biomass were subjected to OxiOrganosolv pretreatment with the addition of polyoxometalate catalysts in the solvent, with the aim to examine both the effect of the nature of the biomass used as a feedstock, and the addition of the catalysts in a variety of organic solvents. In specific, the organic solvents in these experiments were EtOH, ACO, THF, as well as isobutanol (iBuOH), while the polyoxometalate catalysts were the commercially available phosphomolybdic acid H3PMo12O40 x H2O (HPMo), as well as the custom- made metal substituted catalysts FePMo and CuPMo. The best performing beechwood sample led to the production of 54.9 g fructose/ 100 g pretreated biomass, while the best performing pine sample 53.4 g fructose/ 100 g pretreated biomass, respectively, both for pretreatment with iBuOH at 175 oC for 120 min in the absence of catalyst. Subsequently, the best performing samples were subjected to chemical dehydration experiments with formic acid (3.5% v/v) leading to the production of HMF with 49.9% selectivity. The third stage of the thesis included the pretreatment of corn stover biomass using microwave heating in order to compare this heating mechanism with the resistance mediated heating of the traditional autoclave pretreatment. These experiments took place at 150 oC with a mixture of water: ACO as the solvent with the addition of acetic acid as the catalyst. Ten different runs were carried out in order to discover the effect of acetic acid concentration and pretreatment duration on the biomass, while the evaluation of the samples was performed by the results of enzymatic saccharification experiments. The results showed that the best performing material was the one pretreated with an acetic acid concentration of 0.5% v/v for 120 min. For the sake of comparison, the same material was subjected to autoclave pretreatment under the same conditions. The samples of the microwave and autoclave pretreatment were also subjected to saccharification and isomerization experiments yielding 28.4 and 28.7 g fructose/ 100 g pretreated biomass, respectively. However, the total pretreatment times of the two processes exhibited a discrepancy of 1 h highlighting the potential of microwaves in achieving the same results in shorter amounts of time. Finally, with the aim of discovering novel biocatalysts for the bioconversion of furans into high added value monomers, the last stage of the thesis included the examination of a protein with galactose oxidase activity from F. oxysporum. This enzyme was selected after searching for oxidative enzymes with activity on furans that belong to the AA5_2 family of the CAZy database. The enzyme encoding gene was isolated from the DNA of the microorganism using molecular techniques and produced by means of heterologous expression in Pichia pastoris X33 cells. After the determination of its optimal conditions, the enzyme was able to efficiently oxidize HMF as well as its oxidative derivatives, proving that an integrated chemoenzymatic process for the production of oxidative furan monomers from lignocellulose is possible.
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