POLYHYDROXYALKANOATES (PHAS) PRODUCTION FROM AGRO-INDUSTRIAL AND URBAN WASTES, CO2 AND ENERGY CROPS BY MIXED MICROBIAL CULTURES

Modern society is forced to deal with two main problems: a growing scarcity of resources and a growing production of waste. The unsustainability of the current linear economic model “make, use, dispose” originated the concept of “circular economy” that aims to reduce the environmental impact through measures in favour of a more sustainable development, such as the use of renewable energy and waste recycling. Unlike the traditional linear model, the circular economy keeps resources in use for as long as possible, extracts the maximum value from them whilst in use, then recovers and regenerates products and materials at the end of each service life. The basis of the circular economy could be represented by the biorefinery: a productive system aimed at the sustainable conversion of biomass, including energy crops, food industry by-products and organic wastes, into marketable bio-based products (i.e. chemicals, polymers, food and animal feed) and bioenergy (i.e. liquid and gaseous fuels, such as bioethanol, biodiesel and biogas). Among the wide varieties of bio-based products of biorefineries are the polyhydroxyalkanoates (PHAs), a class of bioplastics with microbial origin, which are completely biodegradable and obtainable from renewable carbon sources. PHAs are now earning great interest since they offer, differently from common bioplastics, a wide range of physico-chemical properties that make them comparable and in several applications possible substitute of traditional plastics, moreover they do not determine environmental damages after their service life as a result of their biodegradability. Nowadays, the main limit that slows down PHAs production is the high production cost that does not make them competitive on the market. In this PhD thesis the production of polyhydroxyalkanoates-PHAs was integrated as a step into simple or more complex biorefinery approaches in which cheese whey, the organic fraction of municipal solid waste (OFMSW), Arundo donax and CO2 were used as feedstocks. Currently PHAs production is accomplished by using pure microbial strains, requiring sterility and a close monitoring of the culture growing conditions, thus increasing the production costs. However, it remains the most widespread method for the highest process yields and volumetric productivities. The use of mixed microbial cultures (MMCs) has started to be investigated as an economically viable option for PHAs production since they do not require sterility, do not need restricted growing conditions, resulting in both energy and economic savings during the fermentation process. Moreover, MMCs are more able than pure cultures to grow on complex substrates, such as low-cost agro-food industry wastes, giving a further possibility to reduce PHAs production costs. For this reason in this PhD thesis MMCs were used as starting inoculum to select PHAs storing bacteria by adopting the four feedstocks above mentioned as carbon sources. Before using these feedstocks for PHAs production, they were submitted to different pre treatments aimed to the production of liquid substrates rich in organic acids, the direct metabolic precursors of PHAs in MMCs. All of these substrates were then used as carbon sources to successfully select MMCs with good PHAs storing ability, and for the massive biopolymer production by using the selected MMCs. The results obtained in terms of PHAs yield and PHAs content on cell dry weight were promising and comparable with the literature related to PHAs production from MMCs and complex feedstocks. In particular, during the accumulation tests, PHAs yield on substrate consumed resulted up to 0.75 ± 0.1 g CODPHA g-1 COD by using fermented cheese whey as carbon source, 0.52 ± 0.1 g CODPHA g-1 COD by using a percolate of the OFMSW, 0.85 ± 0.2 g CODPHA g-1 COD by using fermented hydrolysed Arundo donax, and up to 1.06 ± 0.2 g CODPHA g-1 COD by using a mixture of organic acids produced via microbial electrosynthesis (MES) from CO2. The lowest yield was detected in PHAs production from a percolate of the OFMSW, probably because of the complexity of the carbon source that acted as limiting factor during the selection of a PHAs storing MMC; in facts, in the last work performed, by using the mixture of organic acids produced via MES as substrate, the highest PHAs yield was obtained, due to the purity of the carbon source fed to the MMC that led to a selection of a MMC with high PHAs storing ability. Depending mainly on the composition in organic acids of the substrates fed to the mixed microbial consortia, different biopolymers were produced: in particular, by using substrates rich in butyric, acetic and lactic acids (fermented cheese whey from lactic fermentation, fermented hydrolysed Arundo donax and the mixture of butyric and acetic acids obtained via MES) biopolymers made mainly of hydroxybutyrate (HB) were produced (HB > 96 %); while using substrates containing also propionic and valeric acids (fermented cheese whey from mixed acid fermentation and the percolate of the OFMSW), copolymers made of hydroxybutyrate and of hydroxyvalerate (HV) in different proportion were produced (namely P(HBcoHV)), 60:40 (HB:HV, %) and 55:45 (HB:HV, %), respectively for fermented cheese whey and for the percolate of the OFMSW. Since the traditional downstream processes play a key role in the profitability of the fermentation system, counting for about the 30-50 % of the total production costs, in the last part of this study a novel PHAs extraction method from mixed microbial biomass was developed with encouraging results. In particular, by coupling the use of organic non-ionic surfactants to act on the non-PHA cell mass with the use of the green organic solvent dimethyl carbonate to extract PHAs, it was possible to reach biopolymer recovery yields not so far from the one obtained adopting traditional extraction methods based on the use of chloroform. Excluding food contact, the extracted biopolymers could be used to produce packing materials, shopping bags and biodegradable mulching films to be applied in agriculture.