Corrado, Iolanda (2021) Microbial polyhydroxyalkanoates: converting renewable resources into bioplastics. [Tesi di dottorato]

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This thesis is focused on microbial Polyhydroxyalkanoates (PHA): polyesters produced by a wide range of microorganisms as intracellular carbon and energy reserve. In this PhD thesis the valorisation of inulin-rich biomasses for PHA production was investigated. The use of inulin as carbon source for polymer production requires the integration of inulinases production, its hydrolysis and microbial fermentation step. Penicillium lanosocoeruleum was identified as inulinase producer. Hydrolytic enzymes production by the selected fungus, was optimized reaching up to 28 U mL-1 after 4th day of growth. The enzymatic mixture PlaI was characterized in terms of isoenzymatic composition, stability, and activity profile. Optimization of inulin hydrolysis by PlaI was performed through a statistical approach using a Central Composite Rotatable Design (CCRD). In the optimized condition (T=45.5 °C, pH=5.1, substrate concentration=60 g L-1, enzyme loading=50 U gsubstrate -1) up to 97% of inulin conversion in fructose was achieved in 20 h. The integration of PlaI in a process for the conversion of inulin into PHA by Cupriavidus necator pursued two process strategies: Separated Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). A maximum of 2.2 and 3.2 g L-1 of PHB accumulation, corresponding to 60% and 82% polymer content, was achieved in the SHF and SSF processes, respectively. Another strategy for PHA production from inulin involved the use of a microbial "substrate facilitator" consortium (SFC). Bacillus gibsonii (RHF15) was selected as inulinases producer (15U/mL after 15 h of growth). RHF15 was co-coltured with C. necator DSM 428. Optimization of co-culture growth was carried out thought response surface methodology and led to a production of 1.9 g L-1 of PHB after 96 h of growth. Another part of this work was focused on the production of PHA-based nanoparticles (NPs-PHA) to be used as additive for protein-based films. Addition of PHBHHx-NPs to WP-based FFSs resulted in a plasticizing effect on the biobased material, producing a resistant and more extensible biofilm, characterized by an enhanced barrier property towards O2. A second approach for NPs-PHA application involved the encapsulation of oregano essential oils into NPs. The encapsulation protocol was optimized on two classes of polymers: PHB and P(PHBHHx). Both formulations of NPs, showed better or comparable properties to inactivate the tested microorganism respect to free EOs, preventing their volatility, stability issues and poor water solubility, increasing their bioavailability and thus their antimicrobial activity.

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