Production of Poly(3-hydroxybutyrate) by Pseudomonas sp. phDV1 Strains Using Second Cheese Whey Effluent

The aim of the circular economy for plastics is to replace some of them with bio-based polymers in the future. In this work, second cheese whey (SCW) was used as a low-cost substrate for the production of the natural polyester poly(3-hydroxybutyrate)-hydroxybutyrate (PHB) by three <i>Pseudomonas</i> sp. phDV1 strains, namely, the wild type, a depolymerase PhaZ and PhaR knockout mutants. SCW has high polluting loads, characterized by high levels of lactose, phosphorus, nitrogen and salinity, as well as high turbidity due to the presence of whey solids. Initially, SCW was evaluated as the sole carbon source for the growth of the bacterial strains and the production of PHB. Fermentation conditions were screened to maximize polymer synthesis. Small-scale experiments showed that the strains could grow and produce PHB in SCW with and without enzymatic treatment. The formation and intracellular localization of PHB were determined with fluorescence microscopy, using Nile Red staining. Analytical HPLC was also used to quantify the PHB content in the cells and to optimize production. This study demonstrates the efficacy of <i>Pseudomonas</i> sp. phDV1 in SCW treatment and biomass valorization, providing a sustainable solution for dairy wastewater management while producing valuable resources.