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Polyhydroxyalkanoates (PHAs) are polyesters intracellularly accumulated by various microorganisms as carbon and energy storage materials. The most common representative is poly-β-hydroxybutyrate (PHB). These biopolymers have been considered to be good substitutes for petroleum-derived synthetic plastics because of their similar properties to synthetic polymers and its complete biodegradability after disposal. Nevertheless a major drawback to the commercialisation of PHAs is their much higher production cost compared with petrochemical-based synthetic plastic materials. Optimization of fermentation process is one of the strategies more used to reducing high production costs of PHB. The aims of this work were to determine the optimal initial pH value for PHB production, as well as to optimize the agitation-aeration conditions of the fermentation process for product-biomass yield and for PHB accumulation by Rhizobium tropici strain 3. Optimization of initial pH was carried out in an orbital shaker. The pH values were adjusted between 6 and 8. The relation between pH and PHB concentration (g ⋅ L-1 ) was modelled by using orthogonal polynomials. The optimal pH that assures the biggest PHB production is 6.58 ± 0.2. As statistical tools, a central compose design Box-Wilson was used to search the optimal values of two important variables in fermentation process: agitation-aeration rates, for PHB production and for product-biomass yield. The fermentations were carried out in a bioreactor with 7.5 L capacity with 5 L of optimum culture medium. It was determined that optimal values to obtain a maximum PHB production are 500 r ⋅ min-1 and 1 vvm (volumes of air per minute per volume of batch) for agitation and aeration respectively and for product-biomass yield optimal values are 472 r ⋅ min-1 and 1.55 vvm. [ABSTRACT FROM AUTHOR]