Online monitoring-based methodologies to assess microaerobic 2,3-Butanediol production

Biotechnological production of platform chemicals from renewable carbon sources is a promising measure to decrease the consumption of fossil resources in the chemical industry and the fuel sector. Reduced platform chemicals like 2,3-butanediol are often produced in microaerobic cultivation processes. Under these oxygen-limited conditions, the precise adjustment of the oxygen availability is crucial to obtain the desired metabolic activity. In this thesis, 2,3-butanediol production with Bacillus licheniformis was used as model bioprocess to develop online monitoring-based methodologies to assess microaerobic cultivations. A fast and reliable shake flask methodology is presented to determine the optimum oxygen availability. Comparable results and well-characterized experimental conditions were obtained by application of a defined two-stage cultivation profile and a novel technique to determine the maximum 2,3-butanediol concentration. As the obtained results are applicable to stirred tank reactor cultivations, the product and by-product spectrum can be optimized at reduced effort and costs. Still, a drawback for microaerobic process development is the limited availability of online monitoring techniques to determine the metabolic activity with respect to oxygen availability. Therefore, online monitoring of the respiratory quotient was combined with stoichiometric analyses to reveal the metabolic activity during distinct cultivation phases. Furthermore, monitoring of the redox potential was used to monitor the dissolved oxygen tension (DOT) at trace levels that cannot be resolved with conventional DOT probes. The influence of DOT, pH and media components on the redox potential was characterized and quantified in abiotic experiments. On this basis, a corrected redox potential is obtained that solely reflects changes of the DOT, even if the pH strongly fluctuates during the cultivation. Overall, the developed methodologies are transferable to other microorganisms or products and increase the information content from online monitoring during microaerobic cultivations. Respiratory quotient and redox potential provide detailed and complementary information on the metabolic activity during microaerobic cultivations. Together with the presented approach to investigate the effects of oxygen availability, this contributes to the development of improved microaerobic cultivation processes for the production of diverse bio-based compounds

Shake flask methodology for assessing the influence of the maximum oxygen transfer capacity on 2,3-butanediol production

Additional file 3. 2,3-Butanediol formation and subsequent consumption during the cultivation of Bacillus licheniformis DSM 8785. As indicated by the vertical dotted line, the shaking frequency was reduced from 350 to 100 rpm after 3 h. Data on oxygen transfer rate (OTR) and respiratory quotient (RQ) (a), and total 2,3-butanediol and glucose concentration (b) are depicted. In addition to offline samples (open symbols), the concentrations upon glucose depletion were calculated as illustrated in Fig. 4 (closed symbols). For clarity, the connection between the measured and calculated values is shown as dotted line. 2,3-Butanediol is shown as sum of the stereoisomers. The RQ is only shown for OTR > 1 mmol/L/h. To account for the increase of metabolite concentrations due to evaporation of water, all concentrations were corrected accordingly and referred to the initial filling volume. Cultivation conditions: 250 mL unbaffled shake flasks, temperature: 37 °C, shaking frequency: 350/100 rpm, shaking diameter: 50 mm, filling volume: 30 mL, Nakashimada medium with 100 mM MES buffer