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The production of dihydroxyacetone from glycerol employing aerobic cultures of Gluconobacter oxydans is studied. Dihydroxyacetone is one of the most important value‐added products obtained from glycerol, a by‐product of biodiesel production. The effect of organic nitrogen source and initial substrate concentrations has been studied together with the possibility of product inhibition. Afterward, the influence of the main operating conditions (temperature, shaking speed, and initial biomass concentration) on in vivo glycerol dehydrogenase activity has also been considered. The results show no evidence of glycerol inhibition, but an important product inhibition was detected, which has been taken into account in a kinetic model for enzymatic activity description. In terms of operating conditions, pH was found to exert a great impact on glycerol conversion, being necessary to keep it above 4 to ensure complete glycerol conversion. The minimum temperature that maximized enzymatic activity was found to be 30°C. In addition, a surprising decoupling between biomass concentration and dihydroxyacetone production rate was observed when adding increasing nitrogen source concentrations at a fixed shaking speed. Glycerol dehydrogenase activity remains constant despite the increase in biomass concentration, contrary to what would be expected. This fact revealed the existence of a rate limiting factor, identified subsequently as oxygen transfer rate depending on the biomass concentration.

Isobutanol is a promising gasoline additive and could even be a potential substitute used directly as combustible. In this work, the production of isobutanol from glucose by Shimwellia blattae (p424IbPSO) in resting cell cultures is studied. This production has two stages, involving a resting cell phase that has not been studied before. The cell growth was carried out under different operating conditions: temperature and medium composition (YE, ammonium, and IPTG concentrations), looking for the highest isobutanol production. Moreover, the cells were collected at three different growth times checking their isobutanol production capacity. The best operating conditions have been determined as: 30°C of temperature, a medium containing 1.5 g L−1 YE and 1.4 g L−1of ammonium as nitrogen sources, adding 0.5 mM IPTG as inducer. The cells collected at early growth times are significantly more active. The use of S. blattae (p424IbPSO) in resting cells is a good strategy for the production of isobutanol from glucose yielding better results than in batch growth cultures, a yield of 60% attainment of theoretical maximum yield is obtained under optimal conditions. In addition, it has been demonstrated that if the cells are cultured at higher temperatures and with high IPTG concentrations, inclusion bodies are formed in the cytoplasm inhibiting the isobutanol production in the resting cell stage.