In the current context of global warming, the substitution of conventional plastics with bioplastics is a challenge. To take up this challenge, we must meet different technical and economic constraints. In the case of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the technical properties can be modulated by varying the 3-hydroxyvalerate content. 3-Hydroxyvalerate (3-HV) enhancement is an issue; therefore, simultaneous evaluation of several 3-hydroxyvalerate-enhancing substrates through fractional factorial design of experiments is described. Eight substrates citric, valeric, propionic, and levulinic acids; propanol; pentanol; and sodium propionate were studied for 3-HV enhancement, and sodium glutamate was studied for biomass and polyhydroxyalkanoate (PHA) enhancement. The most efficient 3-hydroxyvalerate-enhancing factors were levulinic acid, sodium propionate, and pentanol; however, pentanol, at a concentration of 1 g/L, had an extremely negative influence on biomass production and the PHA content of cells. The effect of the inoculum nutrient composition on the final 3-HVcontent was also evaluated. These results showed that the most efficient combination for the production of high 3-HVcontent in PHBV was primary inoculum growth on mineral medium followed by fermentation for 48 h with levulinic acid and sodium propionate (at 1 g/L) as the only carbon sources. This allowed us to produce PHBV with a 3-HVcontent of 80 mol % and overall volumetric and specific productivities of 2 mg/L/h and 3.9 mg/gCDW/h, respectively, with the addition of only 2 g/L of inducing substances.