Salzano, Flora (2024) An insight into the eco-friendly valorization of lignocellulosic biomass. [Tesi di dottorato]

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Abstract

The transition from fossil-based economies to a circular bioeconomy is necessary to mitigate climate change by adopting more sustainable strategies. Replacing fossil resources and reducing greenhouse gas emissions is possible by biorefining renewable lignocellulosic biomass into biofuels and high-value products. In this context, thermophilic microorganisms have evolved to colonize extreme environments on Earth, and their thermozymes are regarded as powerful and ecofriendly tools in biorefinery to promote biomass valorization. They help maintain high operating temperatures that are associated with higher yields of lignocellulose transformation. Therefore, the research of new thermophilic enzymes that act on the carbohydrate matrix of biomass is needed to make the enzymatic process cost-competitive, reduce the enzyme load dosage, improve substrate and product solubility, and minimize the risk of microbial contaminations. The first section of my doctoral project focuses on the characterization of two novel hemicellulolytic enzymes from the thermo-acidophilic bacterium Alicyclobacillus mali FL18, isolated from a hot spring of Pisciarelli, near Naples, Italy. The genome of A. mali FL18 was sequenced and annotated using the NCBI and RAST database [1]. Among several glycosyl hydrolases (GHs) retrieved from the genome, we selected a b-xylosidase (AmbXyl) annotated as GH3 and an endo-b-1,4-xylanase (AmXyn) annotated as GH10. The synthetic genes were cloned into pET-30 (a+) expression vectors and the two enzymes were produced recombinantly in Escherichia coli BL21 (DE3) RIL strain and subsequently biochemically characterized. AmbXyl showed a dimeric structure and an optimal catalytic activity at 80 °C and pH 5.6. It exhibited not only an excellent tolerance to organic solvents DMSO, ethanol, and methanol and monosaccharides xylose, arabinose, and glucose, but was also activated by 0.75 M xylose and up to 1.5 M by both arabinose and glucose. With the aim of developing a new enzymatic cocktail acting on hemicellulose, the recombinant AmXyn was also biochemically characterized. Molecular exclusion chromatography coupled with light scattering showed a monomeric structure of the protein with a molecular weight of ~ 40 kDa. The enzyme displayed peculiar biochemical properties like an optimal hydrolytic activity at 80 °C and pH 6.6, a good thermostability at 60 °C, and a high catalytic efficiency on beechwood xylan, compared to other thermophilic 2 xylanases. AmXyn hydrolyzed exclusively xylan and not cellulose, that makes it very suitable in the paper and pulp industry for bio-bleaching process. The two enzymes from the same microorganism shared optimal activity at similar pH and temperature and a good stability at 60 °C and were used to develop a new cocktail for xylan hydrolysis. They exhibited a high degree of synergy in both sequential and simultaneous assays at 60 °C. According to TLC and HPAECPAD analyses, the combination of AmXyn and AmbXyl resulted in the complete degradation of xylan to xylose, with a conversion yield of 22.62 %. With the aim of valorizing lignocellulosic wastes as sources of fermentable sugars, AmXyn and AmbXyl were assayed on ultrasound-treated wheat bran (WB), showing a conversion yield into xylose of 91.56 %. The results obtained demonstrated the capability of these enzymes to degrade xylan substrates into fermentable sugars, important feedstocks in food, biofuel, and biochemical production. The second section of this thesis concerns the assessment of antioxidant, antimicrobial and antiproliferative properties of selected agri-food residues after an enzyme-assisted extraction (EAE) method. Different associations of commercial enzyme cocktails from Novozyme were employed for the hydrolysis of spent coffee grounds (SCGs), sunflower and citrus wastes: Viscozyme L (V), Cellulase (C) and V + C. SCGs extract showed the highest total phenolic content (TPC) (~ 30.00 mg GAE/g biomass) in all the enzymatic conditions, followed by sunflower and citrus extracts (~ 20 and ~ 15 mg GAE/g biomass). In accordance with TPC, DPPH and ABTS results indicated that SCGs and sunflower have the highest antioxidant capacity in all the enzymatic conditions, compared to citrus extract. On the other hand, DNS assay was performed to measure the reducing sugar content of each extract, and the most favourable outcome was achieved from the citrus subjected to V + C treatment (~ 400 mg reducing sugars/g biomass). Studies of the antimicrobial effect on different microorganisms highlighted that citrus extract obtained through V + C combined treatment was the most promising, reducing the development of microbial biofilms at the sub- MIC concentration of 2.5 mg/mL. It reached up to 65 % inhibition for Klebsiella pneumoniae and up to 50 % for Staphilococcus aureus, Candida albicans, and Candida parapsilopsis. Lastly, the antiproliferative effect of all residue extracts was assessed on human dermal fibroblasts (HDF). Based on preliminary results, the citrus extract obtained through V + C treatment did not affect HDF viability. Further experiments are underway to assess its antiproliferative effect on metastatic melanoma cells (WM266).

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