Novel biocatalytic approaches for the valorization of furfural and 5-hydroxymethylfurfural

Resumo

The current dependence on fossil resources to produce energy and commodity chemicals poses a major concern to the environment. Finding renewable alternatives is crucial to support and sustain future life on planet Earth. Biomass, specifically in the form of lignocellulose, presents as a promising alternative because it is abundant as waste, it does not compete for food supplies, and it is the most copious carbon feedstock of the planet, containing high amounts of sugars in the forms of cellulose and hemicellulose. Monosaccharides can be released through pretreatment and hydrolysis of the lignocellulosic material and further transformed into fuels such as ethanol. Furfural (FF) and 5-hydroxymethylfurfural (HMF) are furaldehydes generated by dehydration of sugars during the pretreatment of lignocellulose and are inhibitors for the sugar fermenting strains. The study of the transformation pathways that lead to their fewer toxic derivatives has opened a new line of research on the upgrade of FF and HMF, given the value of their derivatives at biorefineries. The main objective of this thesis is to contribute to the biocatalytic valorization of FF and HMF. First, an optimization of the enzymatic oxidation of HMF to 2,5-diformylfuran (DFF) via galactose oxidase, catalase, and horseradish peroxidase was attempted. A high yield (>90 %) was achieved by adjusting the ratio between galactose oxidase and catalase and omitting the addition of horseradish peroxidase. However, it was concluded that the process had some limitations and that whole cells would be a more feasible (and yet not described) approach. In the search for whole-cell biocatalysts, a screening of seven Fusarium species, natural producers of the enzyme galactose oxidase, was considered. Five of the species evaluated showed a high capability to reduce HMF to 2,5-di(hydroxymethyl)furan (DHMF), and two of them showed the capability to oxidize HMF to DFF with low yields and selectivities. The whole-cell production of DHMF was considered of interest and studied using F. striatum. It showed high tolerance towards HMF when using small inoculum sizes, transforming 75 mM HMF within 24 h with a high yield and selectivity. A fed-batch approach allowed a higher concentration of DHMF in the media. Finally, the feasibility of the scale-up of the process was evaluated, and a quantitative DHMF yield (95 %) and selectivity (98 %) were obtained in a bench-scale bioreactor (1.3 L), concluding that F. striatum is a promising candidate for DHMF production. Among the two Fusarium species that showed HMF oxidation to DFF (F. culmorum and F. sambucinum), the former showed more encouraging preliminary results and was selected to optimize the process further. It was found that the nitrogen source and the concentration of glucose and peptones in the media highly influenced the redox capability of the strain. The concentration of both nutrients was carefully optimized through Response Surface Methodology by building two successive Central Composite Designs. A high DFF yield (92 %) and selectivity (94 %) were obtained under the optimized conditions starting from 50 mM HMF, thus describing for the first time an efficient whole-cell production of DFF and opening a new line of investigation. Finally, the last Chapter of this thesis was focused on the use of F. striatum as a biological detoxification method in bio-based ethanol production from lignocellulosic hydrolysates containing high concentrations of FF and HMF. A co-culture between F. striatum and a xylose-consuming S. cerevisiae showed better performance than other biological detoxification methods previously reported in the literature. Moreover, it overcame the main drawbacks of biological detoxification: it was able to handle significantly higher inhibitor concentrations with higher degradation rates, the detoxification and fermentation steps were performed simultaneously, there was complete detoxification of the inhibitors, and there was no consumption of sugars during the detoxification process, meaning that the presence of F. striatum did not influence the ethanol yield. A high ethanol yield (0.40 g/g) and productivity (0.46 g/L/h) were obtained in a bench-scale bioreactor in the presence of 2.5 g/L FF and 3.5 g/L HMF, a concentration of furaldehydes that inhibited the fermentation in the absence of F. striatum. Moreover, the added-value alcohol derivatives of FF and HMF were produced during the detoxification process with high yields, adding more value to the lignocellulosic hydrolysate. The biocatalytic approaches developed throughout this thesis provide novel sustainable methods to produce high-value compounds from biomass and open new lines of investigation in the biocatalytic valorization of FF and HMF.