Institut für Didaktik der Naturwissenschaft und Informatik

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Utilizing process waters from conversion processes based on regenerative resources for microbial production of platform chemicals
(2024) Merkel, Manuel; Hausmann, Rudolf
A future bioeconomy relying on biotechnological production processes makes it necessary to find a replacement for sugars as microbial carbon source to prevent ethically questionable competition with the food and feed sector. An attractive alternative is acetic acid, as it is inexpensive, available in high quantities and utilized equally well as glucose by some bacteria. Still, the application of acetic acid as sole carbon source in bioproduction processes offers several challenges. In its protonated form acetic acid leads to medium acidification, when applied in high concentrations, while as salt it leads to salinization causing osmotic stress for the production organisms. As such, established process strategies are difficult to apply with acetic acid, making specialized strategies necessary for production processes. Therefore, an efficient fed-batch strategy was developed and is presented in the 1st publication utilizing acetic acid as sole carbon source for production of itaconic acid with a genetically modified strain C. glutamicum ICDR453C (pEKEx2-malEcadopt). An earlier published pH-coupled feeding strategy for addition of glacial acetic acid was adapted to obtain the nitrogen limited conditions necessary for itaconic acid production. It was found that the consumption of ammonia at high carbon to nitrogen ratios of the feeds, which was necessary to achieve nitrogen limited conditions, caused acidification of the medium. This countered the increase of pH-value caused by acetic acid consumption and led to early acetate depletion. Thus, an additional DO-coupled feeding of sodium acetate was started once acetic acid in the medium was depleted. Sodium acetate did not directly effect the pH-value, but its consumption again led to an increase of the pH-value and continuation of the pH-coupled feeding. With this combined strategy an itaconic acid production process was developed with separate growth and production phases. In the end a titer of 29.2 g/L and a volumetric productivity of 0.63 g L−1h−1 were achieved. These were comparable to bacterial production processes using glucose as sole carbon source, with the only drawback being a lower yield. Still, the results demonstrated that C. glutamicum is suited as production organism on acetate as alternative carbon source. Another challenge is, that biobased acetic acid, for example produced by thermochemical conversion of lignocellulose, is often available only in dilute concentrations up to 50 g/L or in complex solutions containing potentially harmful substances. Therefore, conventional fed-batch processes are not applicable, because of strong dilution of the product and accumulation of inhibitors. Perfusion bioreactors can be a solution to these problems. They allow application of dilute substrate concentrations, as the bacteria are retained in the reactor. Furthermore, the continuous flow through the system prevents accumulation of inhibitors. Thus, the 2nd publication presented a newly developed, lab scale perfusion bioreactor, that was manufactured via 3D-printing using the fused filament fabrication method. Hydrophilic flat sheet membranes in the main bioreactor module were used for cell retention. A circulation flow was applied for diffusive oxygen supply via an oxygen transfer module that contained hydrophobic membranes and for temperature control via a heat exchanger module. The bioreactor system was characterized regarding oxygen transfer rates and mixing time. Finally, a proof-of-concept cultivation with C. glutamicum ATCC13032 on glucose utilizing a dilute feed solution resulted in 18.4 g/L biomass after 53 h of cultivation and a maximum specific growth rate of 0.34 1/h. Until the end of the process no membrane blockage occurred. This showed that the reactor system was suited for bacterial cultivation as well as for application of dilute substrates. To sum it up, it was shown that acetic acid can be efficiently used as alternative carbon source for bioproduction with C. glutamicum as model organism. Still, in case of itaconic acid production further genetic modifications are necessary in future works to increase the product yield. Regarding process strategies for utilization of biobased acetic acid in dilute solutions, a new 3D-printed perfusion bioreactor was successfully developed, and its function proven. Future works can focus on the application of the perfusion system for production processes and evaluate its suitability for bioprocesses with sustainably produced acetic acid

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