Utilizzo di acque reflue agroindustriali per aumentare le rese di molecole antiossidanti e antinfiammatorie in Arthrospira Platensis

This doctoral research thesis investigates mixotrophic cultivation strategies for the cyanobacterium Arthrospira platensis to enhance biomass productivity and improve the yield and purity of C‑phycocyanin (C‑PC), a pigment with high nutraceutical and pharmaceutical potential. Within a circular bio‑economy framework, the work exploits agro‑industrial effluents as low‑cost carbon sources, aligning waste valorization with microalgal processes. Cultivations were conducted in sterilized closed photobioreactors supplied by Teregroup, which reduce contamination risks and enable tight control of operating parameters, supporting higher productivity than traditional open systems. The first experimental phase evaluated cheese whey (CW), a lactose‑rich dairy byproduct, as a carbon source for mixotrophic growth. CW markedly enhanced biomass productivity compared with photoautotrophic controls, exceeding values reported for Spirulina grown in generic dairy wastewaters. It also increased pigment accumulation, providing C‑PC with analytical purity compatible with pharmaceutical use. A second phase compared buffalo dairy byproducts from a DOP‑certified supply chain—scotta whey (SW), buttermilk wastewater (BMW) and dairy wastewater (DWW)—to identify effluents suitable for scale‑up. Cultures supplemented with 1% BMW showed the highest C‑PC levels, suggesting that its balanced content of residual sugars, lipids and proteins favours phycobiliprotein biosynthesis. In contrast, SW yielded the lowest pigment levels. Overall, reduced effluent concentrations (0.5–2%) were sufficient to stimulate C‑PC synthesis without impairing growth. A third line of work investigated brewery wastewaters (BWW), characterized by high concentrations of fermentable carbon and nitrogen. Because elevated organic loads can negatively affect microalgal cultures, BWW with a TOC of about 11.2 g/L were used at 2%, in line with recommendations to avoid inhibitory effects. Under these conditions, mixotrophic cultivation produced roughly 50% more biomass than photoautotrophic controls, confirming the suitability of appropriately diluted brewery effluents as substrates for microalgal production. The addition of seawater induced moderate salinity stress, which further enhanced both accumulation and purity of C‑PC, consistent with evidence that salt stress in mixotrophic Arthrospira cultures can improve phycocyanin content and modulate metabolite profiles. Building on these optimized cultivation protocols, the thesis then focused on the functional characterization of the resulting biomass, with emphasis on antioxidant and anti‑inflammatory activities. Phycocyanin‑rich extracts from Spirulina are known to exert strong antioxidant and cytoprotective effects, and this research aimed to link culture conditions with biological performance. Aqueous and lyophilized extracts from the same mixotrophic cultures were evaluated at the University of Pisa using standard DPPH and ABTS assays. Both extract types showed comparable phycobiliprotein content and C‑PC purity, while aqueous extracts tested negative in polyphenol detection assays, indicating that radical‑scavenging activity arises predominantly from proteinaceous pigments rather than phenolic compounds. These results support a synergistic contribution of different phycobiliproteins to antioxidant activity and confirm that salinity stress combined with mixotrophic growth promotes the accumulation of secondary metabolites with enhanced antioxidant potential. Finally, the thesis included a comprehensive evaluation of polysaccharide‑enriched extracts obtained from Spirulina cultures grown under photoautotrophic and mixotrophic conditions, using mammalian cell models to assess their protective effects. Experiments were performed on human keratinocytes (HaCaT cells) exposed to lipopolysaccharide (LPS) and tumour necrosis factor‑alpha (TNF‑α), which induce oxidative and inflammatory stress. Polysaccharide extracts obtained from both cultivation methods showed marked antioxidant and anti-inflammatory activity, while those derived from mixotrophic cultures showed comparable or superior efficacy in attenuating LPS- and TNF-α-induced cell damage. Pretreatment with these extracts significantly reduced IL-1β and IL-6 secretion from basal levels, while the mixotrophic polysaccharide fraction consistently outperformed the photoautotrophic one. Collectively, the findings demonstrate that integrating agro‑industrial effluents into controlled mixotrophic systems not only improves C‑PC production and purity but also yields bioactive pigment and polysaccharide fractions with promising therapeutic potential, reinforcing Arthrospira platensis as a versatile platform for sustainable health‑oriented bioproducts.