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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.

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Dihydroxyacetone production from glycerol has been studied. Cultures of Gluconobacter oxydans ATCC 621, a promising microorganism that is able to convert glycerol into dihydroxyacetone, has been employed. In this work, the influence of oxygen transport rate and the fluid dynamic conditions have been studied working with resting cells cultures. Several experiments were carried out at two different scales: 250 mL Erlenmeyer flasks and a 2 L stirred tank bioreactor, varying the agitation speed. Product and substrate concentration were determined employing high-performance liquid chromatography. Additionally, oxygen concentration was measured in the runs carried out in stirred tank reactors. Taking into account the results obtained in these experiments, three different behaviors were observed, depending on the mass transfer and chemical reactions rates. For experiments with low stirring speed (below 200 rpm for shake flasks and 300 rpm for reactors), the oxygen transport rate is the controlling step, while at high stirring speed (over 300 rpm in shake flasks and 560 rpm in the bioreactor), the chemical reaction is controlling the overall process rate. In some runs conducted at medium agitation, a mix control was found. All the kinetic models were able to reproduce experimental data and fulfill thermodynamic and statistical criteria, highlighting the importance of the mass transfer rate upon this system.