Polyhydroxyalkanoates (PHAs), as biodegradable polymers, are considered promising alternatives for reducing the environmental impacts of waste. These compounds are accumulated intracellularly by microorganisms under stress conditions caused by nutrient limitation in the presence of excess carbon sources. Among them, hydroxybutyrate copolymer is the most well-known member of this family. Their production is generally based on the consumption of soluble organic substrates such as volatile fatty acids, which lead not only to hydroxybutyrate formation but also to hydroxyvalerate production. This study aimed to evaluate the effect of gradual fatty acid addition on improving production yield and reducing carbon source consumption. The strain Alcaligenes eutrophus was obtained from a microbial culture collection, and carbon sources included glucose and fatty acids (acetic acid, propionic acid, and their equal-weight mixture). To assess the impact of carbon sources, batch and fed-batch processes were compared. Experimental design was conducted using Minitab software with a two-way ANOVA approach. Cultures were grown in shake flasks containing 50 mL of medium, with 5 mL of inoculum added. In the fed-batch process, fatty acids were added stepwise at 24, 45, and 50 hours to prevent substrate inhibition. Incubation conditions were 32°C, 120 rpm, and 72 hours. Results showed maximum hydroxybutyrate (3.486 g/L) and hydroxyvalerate (0.794 g/L) production under acetic acid and fed-batch conditions. This approach increased hydroxyvalerate production by approximately 17% and improved resource efficiency.