Production of secondary metabolites of pharmaceutical interest at late stationary phase: biological processes, fermentation strategies and mathematical modelling

In recent years, we assisted to a concerning spread of bacterial strains resistant to antibiotic therapies. In this context, a microorganism of significant importance is Streptomyces clavuligerus, a filamentous, Gram-positive bacterium capable of synthesizing a wide range of secondary metabolites, including clavulanic acid. This molecule plays a crucial role in modern medicine, as it is an inhibitor of β-lactamases, enzymes responsible for the hydrolysis of the β-lactam ring, the reactive centre of many commonly used antibiotics. This thesis project aims to achieve three main objectives: the development and improvement of a mathematical model capable of simulating the fermentation process for clavulanic acid production, the comparison of different fermentation strategies in terms of productivity, and the optimization of the nutritional profile of the culture medium used for the bacterial strain’s fermentation. The mathematical model was developed based on other models used for biobased pharmaceuticals. Model parameters were retrieved by fitting several datasets using a constrained optimization technique. This model can predict trends of biomass, product and substrates concentrations during the course of a fed-batch fermentation process, allowing also the estimation of substrates consumption at various batch times, representing a supporting tool for nutrients feeding-strategy optimization. Objectives two and three were addressed through laboratory scale experiments based on measures of biomass and product concentrations. These experiments revealed that working in a semi-continuous mode over batch and fed-batch modes, leads to higher yield of clavulanic acid, minimizing product inhibition. Also, an interesting relationship between the two carbon sources used for fermentation was observed, i.e. glycerol and refined soybean oil. In fact, clavulanic acid productivity was noticed to increase along with the ratio between soybean oil and glycerol concentration in the fermentation media.