Synthesis of co-polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) P(3HB-co-4HB) from agro-industrial solid and gaseous wastes by engineered microbes

Plastic pollution represents a tremendous threat for the environment. Accordingly, besides re-designing the waste management system in favor of recycling, the development and commercialization of alternative biodegradable and bio-based polymers becomes of primary importance. Amid the most studied and attractive bioplastics, polyhydroxyalkanoates (PHAs) are biopolymers that can be biotechnologically produced from renewable sources like biomasses. Addittionally, PHAs are then able to biodegrade quickly in environment and comply with compostability standards. What constrains the diffusion of PHAs on the market is the too elevated manufacturing costs of these materials. As a matter of fact, the feestock price alone to produce PHAs makes the process usually economically unfeasible. On top of that, poly(3-hydroxybutyrate) which is the most widespread PHA polymer, displays limited polymer properties, being brittle and stiff. To obtain performances comparable with traditional plastics derived from the oil industry, copolymers units with the polymeric chain are needed. Considering these assumptions, researchers’ endevours focus on the genetic improvement of the microorganisms that accumalte PHAs granules. Additionally, great attention is given to the quest of cheap and alternative substrates, including the adoption of various wastes and even CO2. This PhD project finds between the major goals the screening of novel feedstocks to produce PHAs. In this sense, food wastes and gaseous substrates, specifically CO2 and H2, have been found to be low-cost and effective substrates that fostered the production of both P(3HB) and P(3HB-co-3HV) a copolymer comprising the 3-hydroxyvalerate unit that confers interesting polymer properties. Among the large set of food wastes tested, melon and red apple were observed to promote a very significant accumulation of PHAs in Cupravidus necator 545. For what concerns the conversion of gases into PHAs, throughout autotrophic fermentation, a completely novel approach has been proposed. A two-step fermentation was conceived: during the first phase, the anaerobic digestion of the pre-selected melon occurred and stopped at the acetogenetic phase. In this way, large amounts of CO2 and H2 were produced. Afterwards, in a second bioreactor, the gases were converted into P(3HB) by C. necator 545. Moreover, the production of 4-hydroxybutyrate has been addressed. With C. necator, and the other major PHA-accumulating microbes, 4HB biosynthesis is achievable only by supplementing expensive structurally related precursors. Instead, to cut the costs, the strain has been genetically modified to express a package of genes that deivate the TCA-cycle towards the direct anabolism of 4HB. As a matter of fact, glucose, a largely available sugar that can more importantly be obtained from several biomasses, was directly converted into 4HB by the recombinant C. necator 545 strain. Overall, the adoption of selected low-cost wastes, the design of novel methodologies to operate in autotrophic conditions, unitedly to the creation of a recombinant C. necator that converts glucose into 4HB, move all together in the same direction: largely available, cheap, and renewable substrates, which would represent a burden for society, are quickly and efficiently converted into highly-valuable materials that can biodegrade efficiently. The evidences described in this doctorate thesis support the implementation of PHAs, opening the door to higher scale studies.